Special THE
HIV/AIDS
Lectures
EPIDEMIC: A CURRENT PICTURE
Novelle», M.D., Surgeon General
Antonia C.
M.P.H.
Public Health Service Washington, D.C. 20201
Thank you Dr. Gallo. It is a pleasure to be here today and honor to speak before such a distinguished group of scientists. I would like to talk to you about one of the most important problems we face today—and one I am deeply concerned about—HIV infection and AIDS. Of course, many of you work in this area, and we could spend quite a bit of time on this subject; but I'd like to try to give you some perspective on the overall picture of the HIV/AIDS epidemic and particularly its impact on women and adolescents, as seen through the eyes of the Surgeon General. I chose this topic because I believe the increasing impact of AIDS on women, their children, and consequently on their entire families will continue to have a great impact on societies. AIDS is no longer a disease of men or women AIDS is a disease of families, and as such we must start dealing with this. Why do we talk about women then? Well, because women are usually the cornerstone of the family, the substance that holds the family together, and they are increasingly suffering the effects of AIDS—as patients and as caregivers. Women who are themselves infected with HIV; women whose children, from infants to adults, are infected; women whose partners are HIV-infected and women who care for HIV-infected patients, family members, or friends are going to need our help. We must realize that this is the true picture of HIV infection and AIDS of the future, and deal with it accordingly. I do not have any new science that you already do not know, to bring to you today. But, I am here t show you a picture of the HIV/AIDS epidemic, as it exists today from the societal and beaucratic point of view in hopes that we can work together to find an end to this tragedy. an
-
I will talk
today
about science and statistics—
epidemiology, surveillance, and
costs. But we must keep in mind the faces behind the statistics—the faces of men, women, and Dr. Gerald Friedland, former Codirector of the AIDS children. Center at Montefiore Medical Center and Professor of Medicine at the Albert Einstein College of Medicine in New York and now at Yale University, has said, "The statistics of epidemiology are human beings with the tears removed. They capture the number of sufferers but not the quality or the weight of the suffering that
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is
being endured, for the fundamental reality of slowly, prematurely, and with great cruelty robs women of their function, their plans and dreams,
AIDS is that it
young men and and ultimately,
their lives." The human immunodeficiency virus (HIV) that causes AIDS has been around for a lot longer than most people realize. By testing blood samples that were frozen for research purposes many years ago, HIV was found in human blood collected in Africa in 1959, and in the United States, from the blood of a 15-year-old sexually active male, in 1968. The AIDS epidemic that we observe today is the result of all those years of HIV infection spreading silently through populations of ordinary people who had absolutely no idea their lifestyle choices could hurt anyone
else.
HIV infection and AIDS are found on virtually every continent on Earth. (The only exception is Antarctica.) Experts at the Centers for Disease Control and at the World Health Organization have used existing data and mathematical models to make projections of future numbers of expected AIDS cases. According to some estimates, there are approximately 1 million HIV-infected persons in the United States, and the World Health Organization (WHO) estimates 8 to 10 million people may be infected around the world. Based on the number of estimated HIV infections and other data, by the end of this year WHO expects more than one million adult AIDS cases will be reported worldwide. Total AIDS cases are expected to triple in Africa and the Americas this year alone. In Thailand, where the epidemic is still in a very early stage, AIDS cases will perhaps increase from 50 reported in 1990 to more than 10,000 by 1995. In the next decade of AIDS, WHO expects 5 to 6 million cumulative AIDS cases, compared with one million through the 1980s; in Africa, 15 to 20 percent of the continent's workforce could die. Long-term projections about HIV infection are harder to make, but the World Health Organization predicted 3 years ago there could be 15 to 20 million infected individuals by the year 2000; today, they say that may have been a conservative estimate. The WHO now believes that number may be reached by the mid-1990s, with as many as 40 million infections among the world's men, And the most frightening wo:nen, and children by the year 2000. can't those 40 million people, tell at thing is, you by looking and they may not know either, that they are HIV infected. During this first decade of AIDS, we have seen the number of AIDS cases and deaths increase dramatically each year, from 132 deaths in 1981 to more than 25,000 deaths in 1990. According to projections, the cumulative number of deaths from AIDS will approach 200,000 by the end of 1993. CDC estimates that approximately 1 million people are currently infected with HIV in this country alone. This represents approximately 1 in 100 adult males and 1 in 600 adult
females in the United States. In 1988, AIDS was the third leading cause of death among U.S. men 25-44 years of age, and in 1989 it had moved to second place, causing 14 percent of all deaths among men in this age group-surpassing heart disease, cancer, suicide, and homicide, in fact AIDS surpassed all causes except unintentional injuries. 696
women 25-44 years of age, AIDS was the eighth leading of death in 1988 and 1989. Estimates based on current trends indicate that, in 1991, AIDS will be one of the five leading causes of death for women in this age group. More than 19,000 women in the United States have been reported with AIDS thus far, and over half of these have been reported since January 1989. More than 10,000 of these women Almost 12 percent of all AIDS cases are now reported have died. among women, up from 6 percent in 1984. Although most AIDS cases in the United States still occur in men, HIV infections are spreading rapidly to women. Based on cases reported in 1988 and 1989, women and perinatally infected children were the fastest growing groups, increasing 23 percent and 34 percent, respectively, compared with 13 percent for men. While the majority of AIDS cases in U.S. women have been reported in injecting drug users, and these cases continue to increase, AIDS cases attributed to heterosexual transmission are increasing faster than any other exposure category. They now account for more than one-third of all cases among U.S. women and for three quarters of all worldwide cases. In addition, many of the cases having "no identified risk" will most likely be reclassified following investigation as cases of heterosexual transmission. Only 6 percent of cases are currently attributed to transfusion or other blood product exposures, and the number of such cases has stopped increasing. In 1990, 64 percent of women who acquired HIV infection from an infected man reported that the man was an injecting drug user. However, an increasing number and proportion of AIDS cases are being attributed to sexual contact with an HIV-infected man whose risk of being infected was unknown to the woman. This category has increased from 6 percent in 1985 to approximately 20 percent in 1990. Also increasing, though less rapidly than other categories, is the number of cases among women who report sexual contact with a bisexual man; this currently accounts for about 10 percent of heterosexual AIDS cases among women. Clearly, most AIDS cases among U.S. women are occurring in young women, but it is interesting to note that women who report sex with a bisexual man are slightly older, with the largest numbers of women between 30 and 49 years of age. The number of infections recorded among racial and ethnic minority women confirm the stark reality that the HIV epidemic disproportionately affects them. Black and Hispanic women make up only 17 percent of all U.S. women, but represent 7 3 percent (52 and 21 percent, respectively) of all reported AIDS cases However, the number of women affected in every race among women. and ethnic group is continuing to climb. Early in the epidemic, most women (70 percent) with AIDS lived in the Northeast. In contrast, in 1990, slightly fewer than half of the women with AIDS were reported to reside in the Northeast, with a striking increase in the proportion of cases coming from the South, particularly from the South Atlantic To date this year, Florida has reported more coastal states. cases of AIDS in women than any state except New York. Most women with AIDS reside in large urban areas with at least 1 million population, although this is changing, with an
For
cause
697
increase in cases outside large cities, particularly in the Southeast. As I mentioned earlier, HIV infections are spreading rapidly to women, and I am particularly alarmed by this. One of the reasons this is occurring is because so many women are unaware of The fact that many HIV-positive women cannot or deny their risk. identify a risk for their HIV infection is very disturbing. If women continue to be unaware of their risks, and recent trends continue, thousands of women will die prematurely each year. I am also particularly concerned about the fact that the incidence of AIDS among young women, and young men, high indicates that many of these individuals became infected as teenagers. Adolescents are increasingly vulnerable—surveys indicate that the majority of them in the United States have engaged in sexual intercourse. In a 1988 survey of teenagers between the ages of 15 and 19, 52 percent of women and 60 percent of men reported they had already engaged in premarital sexual intercourse. The average age of first sexual experience among U.S. adolescents is 16, but in some large urban areas, it may be as young as 12. In 1989, 21 percent of high school students said had they already engaged in sex with four or more partners. Keep in mind that this survey was conducted among high school students, and the data do not reflect information on youth outside the school system, such as street youth or runaways, who as a group must be considered to be at even greater risk. As a result of the increases in adolescent sexual activity, many teenagers are being diagnosed with sexually transmitted diseases, or STDs. Every year in the United States, 12 million and two-thirds of those, or 8 cases of STDs are diagnosed million cases, occur in persons under the age of 25. To put it more succinctly, a U.S. teenager gets a sexually transmitted disease every 30 seconds of every single day. Keep in mind, too, that one recent study from the Centers for Disease Control found that 50 percent of U.S. adolescent females who were diagnosed with AIDS in 1990 contracted the virus through heterosexual contact. Surveys have shown that adolescents know how HIV is transmitted, but 63 percent of those who are sexually active still do not consistently use condoms. In one survey, 40 percent reported "sometimes" using condoms, and 23 percent reported "never" using them. Because many people see them primarily as a means to prevent pregnancy, teenagers may be less likely to use condoms when their sexual activity will not result in pregnancy. The problem with that is, according to studies recently conducted in Europe and the United States, the sexual activities most likely NOT to result in pregnancy, such as anal intercourse or sex during menses, may even put people at even greater risk of acquiring HIV infection. These behaviors may be more common than many people suspect. One recent review indicated that between 10 and 26 percent of all U.S. adolescents practice anal intercourse. Another study conducted among University of Puerto Rico students found that 31 percent of women and 40 percent of men practice anal sex, both to prevent pregnancy and to preserve virginity. Every year, more and more people seek counseling and --
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In the United States and its territories, at publicly funded clinics alone, 892,714 people were tested in 1989 and 1990. Of those, 125,900 were young people aged 13 to 19, and 766,814 were adults (age 20 and above). In every age, even among the 13-year-olds, the place most often tested for HIV infection was at a sexually transmitted disease clinic. This tells us a couple of things, but most importantly, it demonstrates better than any other fact that teenagers are not protecting themselves from disease. And since we also know that having a sexually transmitted disease may actually increase a person's chances of getting the AIDS virus from his or her sex partners, the fact that teenagers are getting sexually transmitted diseases is doubly troubling. The ratio of HIV-infected males to females is currently 1.7 to 1 in the U.S. adolescent population, as compared with 9 to 1 in the adult population. Among military recruits in 1990, the This should ratio was found to be nearly identical .9 to 1. be another "red flag," as it is one more indication that heterosexual contact is becoming a primary HIV transmission route among teens. We must make an effort to better educate our youth about STDs, especially HIV infection. We have too much to lose to ignore these statistics. Since infections that occurred 5-10 years ago are reflected in today's AIDS case reports, the actual extent of the problem is better defined by HIV infection trends since large increases in female HIV and AIDS rates in this country are relatively recent. CDC is conducting HIV serosurveys to supplement our knowledge of The childbearing the geographic dispersion of the epidemic. women's survey, now ongoing in 44 states, Washington, D.C., and Puerto Rico, tests newborn blood samples routinely collected on filter paper for other purposes. Detection of antibody to HIV reflects maternal antibody status; thus, the survey measures the prevalence of HIV infection in women who deliver live born It is the largest population-base HIV serosurvey being children. conducted in the United States. HIV seroprevalence rates in 1988 and 1989, for selected metropolitan areas, indicate that the highest seroprevalence rates per 1,000 childbearing women are in Miami, New York City, It is interesting to note the relatively small and Newark. spread between rates in Atlanta and other parts of Georgia—this reflects the dispersion of the epidemic into the South outside of
testing.
—
the
large metropolitan areas. Epidemiologie studies have suggested
a number of possible cofactors in heterosexual transmission. Clearly, host infectiousness may vary. We believe that persons late in the course of illness when T-helper cell counts decline are more infectious. Early HIV infection, before the development of HIV antibodies, may also be transiently associated with a higher level of infectivity. Many studies have recently demonstrated that both ulcerative and nonulcerative sexually transmitted diseases increase the risk of HIV transmission by several-fold. Given that the number of cases of syphilis among women has more than doubled since 1985, especially in the South where there are already high rates of heterosexually transmitted AIDS, we are presented with a
699
frightening forecast for the future of heterosexual transmission If current trends continue, of HIV in the United States. heterosexual transmission of HIV will become the predominant mode of transmission among U.S. women by the middle of this decade. As I just mentioned, genital ulcer disease and nonulcerative sexually transmitted infections, as well as lack of circumcision, The exact at least in Africa, appear to influence transmission. mechanisms by which lack of circumcision and STDs influence transmission remain uncertain. Presumably genitalulcers provide a portal for virus entry and inflammatory cells as targets for The role of nonulcerative STD is less obvious. In a infection. recent study conducted in Nairobi and reported in Florence, Dr. Willerford working with Drs. Kreiss and Plummer noted a fourfold increased risk of cervical shedding of HIV, as measured by polymerase chain reaction (PCR), associated with cervical inflammation and cervicitis. The role of other cofactors as determinants of transmission is less well understood but new data are beginning to shed some additional light. For example, in Florence we heard a report of a study which used in situ hybridization to identify HIVinfected cells in the reproductive tracts of experimentally infected rhesus macaques. Lymphocytes and macrophages in the and and cells which appear to be Langerhans cells cervix, vagina in the dermis and epidermis of the penis were positive for SIV. These data, from Dr. Chris Miller and Dr. Preston Marx, suggest that Langerhans cells may be a cellular target for SIV and, by inference, HIV in the female reproductive tract. At the same conference, Dr. Eric Langhoff from Dr. William Haseltine's group presented data indicating that primary human dendritic cells support replication of HIV to a much greater The data from level than infected macrophages or lymphocytes. these two groups taken together suggest that this cell population may play an important role in the sexual transmission of HIV. Further reports from Florence raise questions about the appropriate use of spermicides as virucidal agents. Researchers from Thailand working with Family Health International found that prostitutes using nonoxynol-9 were 60 percent more likely than placebo users to have signs or symptoms of vulvovaginal mucosal irritation. In a randomized, placebo-controlled study in among prostitutes Nairobi, Kreiss et al. found that nonoxynol9 impregnated sponge use was associated with a 7 0 percent increased risk of HIV seroconversion. Because nonoxynol-9 is a we to need whether its application, detergent, question its particularly frequent application, may cause sufficient inflammation and microulceration to adversely affect the risk of These provocative preliminary observations mandate transmission. further the need for investigation in light of nonoxynol-9's antiviral proven activity in vitro. Although the role of spermicides for HIV prevention remains uncertain, it is clear that latex condoms provide an effective mechanical barrier to HIV. Evidence for this includes a study using scanning electron microscopy on 50 samples of stretched and unstretched latex condoms; no pores were found in any condoms even when viewed at a magnification of 2000X. Furthermore, in vitro data indicate that latex condoms are impermeable to hepatitis B virus surface antigen, which at 22 nm (nanometers) in 700
diameter is appreciably smaller than HIV (120 nm in diameter). These data indicate that latex condoms can serve as an effective mechanical barrier to HIV transmission. But condom effectiveness in the laboratory cannot be directly translated into clinical effectiveness. Behavioral determinants clearly play a large role, as condoms must be used consistently and correctly if they are to be clinically The clinical efficacy of condoms is demonstrated by effective. from the European Community Study of Heterosexual recent data HIV Transmission. In this study, 251 discordant couples were followed prospectively for an average of 14 months. Data clinical of in Florence the condoms, support presented efficacy when used consistently and correctly, for the prevention of HIV transmission. Ten seroconversions to concordance were observed, all among couples who did not use condoms consistently; there were no seroconversion events among systematic condom users. Unfortunately, a factor in correct use of condoms may be the clarity of instructions that accompany them. In a recent study, written instructions for condom use for 14 brands of condoms were evaluated. According to the study, all instruction brochures required at least 10 years of education, and some required greater than a high school degree. How, then, are women to protect themselves? Providing education on safer sex may be helpful, but it is not enough. It is easier for man to protect themselves against sexual For women, protection transmission of HIV by wearing a condom. is more of a problem, especially in cultures or subcultures where women have a subordinate role in society. It may be difficult, if not impossible, for women to influence their partners' use of condoms because of cultural, societal, or religious restraints Even the female condom, not yet available in and perceptions. the United States, may not be a viable option for women for many of the same reasons. We still have much to learn about behavioral factors which affect education and prevention efforts for women, but studies are underway which should help to shed some light on this issue. Studies are also underway to better define the spectrum of disease in both men and women. HIV Scientists are comparing the kinds of opportunistic infections that occur in HIV-infected men and women. Many of the opportunistic infections in women are similar to those of men, although some differences are evident. Pneumocystis pneumonia is diagnosed in the majority of women and men with AIDS. Kaposi's sarcoma is reported among less than 2 of with AIDS, although it is reported in more than women percent 15 percent of men, predominantly among homosexual/bisexual men. In addition, researchers are trying to evaluate what specific gynecologic manifestations occur in HIV-infected women and whether gender-specific exposures, such as pregnancy, influence the progression of HIV disease in women. It is also possible that HIV disease will influence the prevalence of, or complicate the course of, gynecologic diseases which also occur in uninfected women. One possible example is of 21 studies of HIV infection cervical neoplasia. A review and cervical neoplasia found that the prevalence of cervical neoplasia ranged from 8 percent to 100 percent, and that women
701
with HIV infection were almost 5 times more likely to have cervical neoplasia than uninfected women. What we still need to know is whether that association is attributable to HIV infection, per se, or to a high prevalence of conventional risk factors among HIV-infected women. Researchers are also investigating whether cervical dysplasia progresses more rapidly to cervical cancer in HIV-infected women, and whether progression is attributable to immunosuppression or to other factors. What about Pelvic Inflammatory Disease, or PID? Is the apparent association between PID and HIV infection attributable to the fact that both are sexually transmitted conditions? Further, is any observed association between HIV infection and severe PID attributable to immunosuppression or to other factors? So far, two studies have demonstrated a high prevalence of HIV infection among women with PID. Another study found that PID among HIV-infected women was clinically more severe and more likely to require surgical intervention. It is still unclear whether this reflects more severe PID in HIV-infected women by virtue of immunosuppression or other HIV-related causes, or if these differences can be explained solely by differential access to health care or related issues. Finally, what is the association between HIV infection and candidal vulvovaginitis? One study found that 38 percent of 140 symptomatic women had recurrent candidal vaginitis. What we still need to know is whether the frequency and severity of candidal vaginitis is related to immunosuppression, to prior antimicrobial therapy, or to other factors. When all the findings are in, more decisions will have to be made about managing HIV disease in women. For example, how frequently should HIV-infected women be screened for cervical neoplasia? Should HIV-infected women found to have cervical neoplasia receive more aggressive treatment than noninfected women? Sometimes it seems that all we have are questions, but every year, every month, we find more answers. CDC has recently tried to address some of these questions and issues in examining its AIDS surveillance definition. As you may know, CDC developed the AIDS surveillance case definition to measure trends in conditions that are highly specific to HIV The AIDS surveillance case definition has provided a infection. very useful measure of severe and life-threatening illness associated with HIV infection by (1) allowing accurate tracking of the epidemic geographically, demographically, and temporally; (2) identifying and ruling out modes of transmission; and (3) providing the foundation for projecting the future course of the
epidemic.
CDC is in the process of implementing a broad change in the definition for surveillance of AIDS in adolescents and adults in the United States. The planned change would add to the all case AIDS current definition persons with documented HIV infection and a CD4 lymphocyte count less than 200/mm This modification is being made to include those HIV-infected individuals with severe immunosuppression who may not have a specific AIDS indicator disease listed in the 1987 revision of the AIDS case definition. case
.
702
HIV-infected persons with CD4 lymphocyte counts of less than 200/mm are most likely to be severely ill or disabled and in greatest need of medical and social services. Data indicate that as CD4 levels decline, the incidence of AIDS, as currently defined, increases. When researchers looked at the CD4 data for people with AIDS, approximately 80 percent had a CD4 count under
200.
It is estimated that there may be 150,000 to 200,000 persons currently infected with HIV who have a CD4 count less than 200 who are not currently reported with AIDS. However, we do not that all of these will be expect reported, since half or more of these HIV-infected persons may not know their HIV infection status or their CD4 count. Although such a broad expansion of the AIDS case definition
will make interpretation of trends in incidence and characteristics of cases more difficult, this expansion will provide a better estimate of the health care needs of severely ill persons with HIV infection. Using the CD4 count will help at severe illness and most in those risk for greatest identify need of medical follow-up without adding unneeded complexity to the surveillance process. CDC is working with the Council of State and Territorial
Epidemiologists and state health departments to plan for the change in the AIDS case definition, and expects to publish the new definition by next spring. At this point, I would like to talk a little more about he cost and availability of services for individuals with HIV
infection and AIDS. The cost of treatment for HIV-infected and AIDS patients is At the AIDS Conference in Florence tremendous and it is rising. this June, Fred Hellinger of the Agency for Health Care Policy and Research estimated that the cost of treating all HIVseropositive persons in the United States in 1991 will be $5.8 billion; this figure may rise to $10.4 billion by 1994. Some previous estimates have understated the costs of care due to the HIV epidemic because they have considered only those cost occurring after a diagnosis of AIDS. Dr. Hellinger estimates the average cost of treating a person with HIV who does not have AIDS is about $5,150 a year, and someone with AIDS, $32,000 a year, of which $24,000 is for inpatient hospital care and $8,000 for other He estimates the lifetime costs for a person with AIDS services. to be more than $85,000. These figures are higher than those from other recent studies, which estimated the lifetime costs of the medical care of someone with AIDS from the time of diagnosis at $40,000 to $75,000, because AIDS patients are living longer and the drugs used for treatment are increasingly costly. Dr. Hellinger based his estimates on case projections similar to CDC's, assuming that 63,000 AIDS cases will be diagnosed in 1991, 74,000 in 1992, 87,000 in 1993, and 101,000 in 1994. He estimated that the number of HIV-infected asymptomatic persons receiving care each year is twice the annual number of AIDS cases.
We can also assume that the cost of treating children with AIDS will continue to rise. However, children have additional nonmedical costs which usually are not covered by existing financial resources. Estimates of lifetime cost for children
703
with HIV infection and AIDS range from
$90,000.
$40,000
to
more
than
What then can be done to increase the availability of services for individuals with or at risk for HIV infection and AIDS? The Health Resources and Services Administration (HRSA) and CDC have shown that community-based primary care facilities can be effective components of HIV treatment and prevention
high incidence areas. HRSA's Community and Migrant Health Center Program is the largest community-based primary care system serving low-income Facilities are located in highpersons in the United States. need areas and offer comprehensive primary care services on a sliding-fee scale. In 1990, HRSA provided $496 million to 554 grantees with over 2,000 health care delivery sites. More than million low-income patients received care under this program, programs in
6
most of whom are members of racial/ethnic minorities. In a two-phase study of the program's HIV/AIDS-related services, HRSA found that about one-third of the centers were providing prevention and treatment services to a substantial number of patients. This study confirmed the high costs associated with HIV infection and AIDS it found that HIV/AIDS have visits the centers than other to more patients significantly —
with an average of 16.2 visits per year (versus 3.2 visits/year for non-HIV/AIDS patients). In addition, the average HIV-related visit required 29 minutes, compared with 13 minutes for non-HIV illnesses. And the average HIV-infected patient more required significantly laboratory services than other infected with not HIV. patients We must all continue in our efforts to seek solutions to the problem of HIV infection and AIDS. Much more needs to be done in the area of prevention and treatment. Research must continue with increased vigor. As we develop promising treatment protocols and we ensure that clinical trials are open to women must vaccines, and children. We must also exercise leadership in preventing HIV infection among women and children by addressing the cultural and societal barriers which deny them access to prevention. And until better treatment protocols or a vaccine are available, education on the ways in which HIV infection is spread and how people can protect themselves, and knowledge of serostatus through readily available counseling and testing services, are the keys to stopping the spread of HIV infection. Although some of you may not work in a field directly related to HIV infection and AIDS, on a personal level you can You can try in your own lives to be still have an impact. and compassionate caring toward people with AIDs. You can educate others about HIV infection and AIDS, helping them to understand the role of risk behaviors in transmission and the need for compassion toward HIV-infected individuals. Those of who are have an you parents important responsibility to educate HIV children about infection and AIDS. As I mentioned your adolescents are earlier, having sex earlier and with more than 10 were years ago--this makes them partners they to vulnerable particularly sexually transmitted diseases,
patients,
including
HIV infection.
The magnitude of this epidemic and the urgent need to find better treatments or a cure represent an enormous challenge for 704
Much has been accomplished during this first decade of still have along way to go. As Margaret Hamburg and Fauci have said so succinctly in their article AIDS: The Tony to Biomédical than more Research, disease, "Perhaps any Challenge AIDS has raised a host of complex questions, from the most fundamental nature of biological systems to the role of scientific research in society at large. And beyond the
science.
AIDS, yet
we
challenge to implications
biomédical research, there are profound and broader of the AIDS crisis for many aspects of health care, law, ethics, and society. The needs are tremendous and the answers difficult, but we are moving forward and new solutions The benefits will be far-reaching—not are within our grasp. but for for AIDS, many other areas of health and disease as just
well.
Indeed, AIDS has been
mighty challenge to science. But we challenge. Science has brought complex questions, and it will play an essential role in stemming this tragic epidemic. We have some of the finest minds in science in this room here today, and my challenge to you is to work within your disciplines, and your personal lives, to conquer this disease. As you do this, however, remember the human impact of this epidemic, particularly its toll on women. Remember that, as women contract this disease and pass it on to their children in ever increasing numbers, families will suffer, societies will suffer, and the world community will suffer. You can make a difference—you can help stop this suffering. Let us move forward together to find these solutions—if we work together, we will surely succeed. Thank you. a
must continue to respond to this us answers to many difficult and
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Vogel P, Dandekar S, Hendrickx A, Alexander N, Localization of SIV in the reproductive tract of rhesus macaques: relationship to the distribution of T cells and macrophages. Abstract No. WA 1119. Seventh International Conference on AIDS, Florence, Italy. June 16-21, 1991.
Marx P.
Langhoff E, Terwilliger EF, Poznansky MC, Bos H, Kalland KH, Haseltine WA. Prolific HIV-1 growth in human dendritic cells. Abstract No. WA 70. Seventh International June 16-21, 1991. Conference on AIDS, Florence, Italy.
Roddy RE, Fortney JA, Chutivongse S. comparative trial of mucosal irritation with nonoxynol-9 or placebo. Abstract No. WC 3084. Seventh International Conference on AIDS, Florence, Italy. June 16-21, 1991. Niruthisard S,
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Kreiss J, Ruminjo I, Ngugi E, Roberts P, Ndinya-Achola J, Plummer F. Efficacy of nonoxynol-9 in preventing HIV transmission. Abstract No. MAO 36. Fifth International Conference on AIDS, Montreal. June 4-9, 1989.
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Abulafia 0, Sedles A, Feldman J, Des Jaríais D, Landesman S, Minkoff H. Sexually transmitted diseases and human immunodeficiency virus infection among women with pelvic inflammatory disease. Am J Obstet Gynecol 163:1135, 1990.
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200 North American women. Seventh International Conference June 16-21, 1991.
on
AIDS, Florence, Italy.
PHARMACOLOGIC REGULATION OF HIV EXPRESSION
Anthony
S. Fauci
Laboratory of Immunoregulation National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland 20892
My presentation today will encompass the broader concept of the immunopathogenic mechanisms of HIV infection with a slant towards the pharmacologie modulation of HIV expression. I will focus on several aspects of the pathogenesis of HIV infection: the initial infection, the acute syndrome, the period of clinical latency which is associated with some degree of microbiological latency, the concept of viral burden and tissue distribution, and the induction of HIV expression to active viral replication, cytopathicity and ultimately clinical disease. During the typical course of infection with HIV, there is a precipitous in CD4+ T cells during the acute syndrome, a burst of viremia and the drop generation of an immune response, which apparently curtails the burst of viremia.
There follows a period of time ranging in years in which an individual is in a clinically latent stage, which in many situations is misinterpreted as a microbiologically latent stage. During this time period which is generally measured in years, the HIV-infected individual experiences a now well-recognized progressive diminution of CD4+ T cells to a level at which opportunistic infections occur. The acute syndrome has been much under-appreciated since the beginning of the AIDS epidemic. We know now from a number of groups that up to 70% of individuals experience an acute HIV syndrome. Within a few weeks of the initial infection with HIV, there is a burst of viremia and an extraordinary retrafficking of lymphocytes associated with a clinical syndrome that resembles acute mononucleosis. Re-trafficking of lymphocytes into the peripheral lymphoid
707
tissue occurs and an immune response to the virus is generated which results in diminution of plasma viremia. However, a number of studies have shown that the immune response is not completely effective despite the fact that the viremia seems to be curtailed. This is evident from the fact that there is a progressive diminution of CD4+ T cells during the period of clinical latency. Thus, it appears that the events which occur during the acute infection, particularly in the area of lymphocyte retrafficking, are very important. It is my opinion, and the opinion of a number of other people, that a substantial amount of damage I will return to this concept in a moment. occurs very early in HIV infection. I would like to talk about viral burden and its relationship to latency. Last year, we had mentioned the relationship of CD4+ T lymphocyte count, disease progression and viral burden. It is very clear that there is a significant increase in viral burden over time per given number of CD4+ T cells. By examining highly purified T cells at different points in time in individuals who deteriorate immunologically and clinically to develop full-blown AIDS, we see a very substantial increase in viral burden per given number of CD4+ T cells. For individuals who do not progress to AIDS, the viral burden in CD4+ T cells remains a
relatively
constant.
also interested in delineating the course of active viral HIV-infected in It had previously been thought that individuals. replication active viral replication generally occurs during the later stages of the disease. We know now from studies from a number of laboratories that this is, in fact, not Active viral replication occurs at all stages of disease, as the case. determined by plasma viremia, and most recently by RNA PCR. We have examined HIV-infected individuals with nearly normal CD4 +T cells, individuals with declining CD4+ T cell levels and individuals with less than 200 CD4+ T cells per cubic mm. We found that there was a degree of active replication as measured by RNA PCR in all stages of disease. Thus, a quiescent, microbiologically truly latent state does not exist even in asymptomatic individuals. Although, at any given time, there are approximately 10 infected cells that are not expressing virus for every infected cell that does express viral proteins and/or RNA, there seems always to be some cells expressing virus. I would like to focus now on the tissue distribution of HIV in infected individuals. We had reported last year that a number of immune competent cell compartments could be infected by HIV. The intrathymic T cell precursor, the socalled triple-negative (CD3-CD4-CD8-) cell, is in fact not triple negative but expresses a low level of surface CD4, and can be infected by HIV. We have also demonstrated that CD34+ bone marrow precursor cells can be infected with HIV in vitro and in vivo. We know now from studies from a number of laboratories of the degree of active viral replication that occurs in other lymphoid tissues, such as in the lymph nodes, at all stages of disease. As I mentioned earlier, re-trafficking We know that there is of lymphocytes occurs during the HIV acute syndrome. virus cells in the lymph dendritic filtering of antibody-coated by the follicular node germinal centers. infect virus can T cells that The filtered very likely the node. coated virus In travel through addition, antibody trapped in the lymph well HIV-infected cells that home nodes activate as as center to T germinal lymph B cells in what was thought to be a polyclonal, but now is known to be an oligoclonal, HIV-envelope specific response. Uninfected CD4+ cells are also at risk of infection within the microenvironment of the lymph node. By examining paired lymph node and peripheral blood samples from HIVinfected individuals at all stages of disease, we found that the viral burden at any given time in the lymph node per given number of CD4+ cells was at least a log higher than in the peripheral blood. We performed quantitative analysis using the ACH-2 cell line and compared the strength of the signal in the ACH-2 cells with the signal in the peripheral blood and lymph node. A stronger signal We
were
708
observed per given number of CD4+ T cells in the lymph nodes with all primer pairs tested. From work done in our laboratory by Cecil Fox, it is apparent that virus accumulates in the germinal center of the lymph node. The in situ hybridization studies of the lymph node tissue demonstrate that HIV-infected cells are located adjacent to cells that are activated and secreting various cytokines. This observation becomes relevant for what I am going to discuss next, namely the induction of HIV expression by cells secreting cytokines. We have been very interested in the mechanisms of induction of HIV expression in a chronically or latently infected cell, be it of the T cell or monocyte lineage. We, as well as a number of other laboratories, have published over the last several years on a variety of mechanisms that may be involved in the upregulation of HIV expression. These mechanisms involve heterologous viruses, other co-infecting microbes, such and a number of endogenous cellular factors. Because of our as mycoplasmas, immune on work the of the normal human previous regulation response antedating the AIDS epidemic, we have been interested in immunoregulatory cytokines. The immunoregulatory network in man is a very complex, highly redundant, paracrine and autocrine system, which keeps the immune system in a state of homeostatic regulation. We have demonstrated over the past few years that the mechanisms that are responsible for normal homeostasis can in fact upvery and We have demonstrated that down-regulate HIV expression. regulate such and as can PMA TNF transcriptional activators, trigger HIV expression by the factor In NFkB. contrast, IL-6 and GMCSF transcription activating inducing or are in activators of HIV expression. alone combination post-transcriptional in normal immune homeostasis have Thus, these cytokines which are very important been clearly shown to use the molecular and cellular mechanisms that are involved in the normal immune response to induce HIV expression. The physiological relevance of cytokine-induction of HIV expression is We investigated the role of illustrated by our work with activated B cells. activated B cells in the induction of HIV expression. We demonstrated some time It ago that HIV-infected individuals have a high degree of B cell activation. has subsequently been shown that a substantial proportion of these activated B cells are specific for HIV. We have recently discovered that activated normal tonsillar B cells stimulated with S_^ aureus Cowan and IL-2 secrete substantial amounts of TNF and IL-6, which becomes highly relevant for pathogenesis. We have also demonstrated that unstimulated B cells (which are in fact activated in vivo) from HIV-infected individuals, but not from uninfected controls, can induce the expression of HIV in infected T cells and monocytes. In the autologous system with peripheral blood T cells and B cells from an HIVinfected individual, we observed the spontaneous induction of HIV, which can be blocked by antibodies to the relevant cytokines. Thus, B cells actively making inductive cytokines in the presence of HIV-infected cells is physiologically relevant for the induction of HIV expression in the lymph node. Since the end result of induction of HIV expression in vivo is clinical disease, we are interested is developing therapeutics that can modulate this inexorable inductive chain, which is mediated by normal immunoregulatory cytokines. This interest brings me to the topic of my talk the pharmacological Because of work done by John Kehrl in my regulation of HIV expression. laboratory on TGF-/3, we examined the role of TGF-/3 in the regulation of HIV expression. We found that TGF-/3 was a potent blocker of the induction of HIV expression, both transcriptionally by PMA and also post-transcriptionally, by IL6 and GMCSF. I must point out that TGF-/3 is clearly bifunctional and, depending on the circumstances and the conditions, can either up- or down-regulate HIV induction of HIV expression by with the to However, expression. regard is a TGF-/3 cytokines, potent down-regulator. Since TGF-/3 cannot feasibly be used therapeutically, we began searching for was
-
709
molecules that might mimic the suppressive effect of TGF-/3 on cytokine-induced HIV expression. We found that retinoic acid which has been used clinically in other diseases is one such agent. Over the past year, there have been a number of studies indicating that retinoic acid is capable of inducing complete and partial remissions in a number of neoplastic states, such as a variety of leukemias. Retinoic acid is required to maintain normal differentiation and proliferation of virtually all cells in the mammalian organism. The receptors for retinoids belong to the super-family of intercytoplasmic steroid receptors. Functionally, there is a clear-cut relationship between retinoids and TGF-/3. We found that retinoic acid suppressed the induction of HIV in the chronically infected cell lines with a pattern strikingly similar to that of TGF-/3. Retinoic acid and TGF-/3 block transcriptional activation by PMA but not TNF, and block post-transcriptional-activation by IL-6 and GMCSF. We also have demonstrated that retinoic acid and TGF-/3 can block the induction of HIV expression by TNF plus dexamethasone and IL-6 plus dexamethasone. Using western blot analysis of chronically infected promonocytic cells, we have shown that treatment of the cells with retinoic acid completely blocks the production of HIV proteins. Using nuclear run-on analysis, treatment with retinoic acid blocks the HIV transcription induced by PMA. As I mentioned, retinoic acid does not block TNF-induced transcription of HIV but it does block the post-transcriptional induction of HIV by IL-6 and GMCSF. Because of the relationship between TGF-/3 and retinoic acid, it may be possible that retinoic acid is inducing TGF-/3. To address this problem, we treated PMA-activated chronically HIV-infected cells with TGF-/3 or retinoic acid and then exposed the cells to anti-TGF-/3 antibody. The anti-TGF-/3 antibody was able to block TGF-/3 mediated suppression of HIV in PMA treated cells. However, anti-TGF-/3 antibody had no effect on retinoic acid suppression of HIV in the PMA treated cells. Similar results were obtained when the cells were activated by IL-6 and dexamethasone. In addition, unlike PMA, retinoic acid does not increase the accumulation of TGF-
ß.
found that retinoic acid can suppress HIV at we have Retinoic acid exerts its effect with a pattern closely resembling TGF-/3, and the suppression of expression of HIV does not seem to be We are currently in the correlated with the induction of endogenous TGF-/3. process of trying to dissect out the molecular mechanisms of this effect because of its potential as a therapeutic agent. We have also been examining another which has been shown by Leonard Herzenberg as (NAC), agent, n-acetyl cysteine well as ourselves to block the induction of HIV expression. In closing, it is clear that without the decades of prior research on the complexity of the immune system, we could not have begun to understand the mechanisms of HIV pathogenesis nor to attempt to modulate the replication of HIV pharmacologically. In addition, what we are learning about the regulation of the immune system through the study of HIV will certainly have a substantial impact on our understanding of other diseases of the immune system which are unrelated to HIV. In
multiple
conclusion, levels.
710
SPECIAL LECTURES VIII Jean-Claude CHERMANN, Ph.D, Jacques FANTINI, Ph.D., Valérie CALENDA, Françoise SILVY and Ivan HIRSCH, PH.D. UNITE DE RECHERCHES SUR LES RETROVIRUS ET MALADIES ASSOCIEES
(INSERM U 322) Campus Universitaire de Luminy, BP 33 -13273 MARSEILLE Cedex 9
France -
/ would Tike to thank Dr. Robert GALLO for Inviting me to speak in the lab meeting.
The etiological agent of AIDS is the human immunodeficiency virus which infects and kills CD4 lymphocytes. It has been first isolated in 1983 (1), then characterized and associated with AIDS (2,3). It is a lentivirus (4) going a slow and progressive disease. HIV is CD4 lymphotropic (5) and cytopathic for the CD4+ lymphocytes (2). TABLE 1
:
HIV TARGET CELLS INTERACTION
CD4
lymphocytes Macrophages
DIRECT ACTION
Intestinal cells
=
=
=
>
AIDS
=
=
=
>
Associated diseases
=
=
=
>
(pneumonia dementia...) Malabsorption Diarrhea
Bone marrow precursors
= = =
>
Lymphopenia Thrombocytopenia Anemia
INDIRECT
Kaposi sarcoma B lymphoma ? Other malignancies ?
AIDS is characterized
by an important decrease of immunological defense and more especially by a profound progressive depletion of the CD4+ lymphocytes. HIV1 and HIV2 show a selective tropism for the CD4 lymphocytes and on the envelope gp120 is localized a CD4 binding domain (6) and a V3 loop (7) for the production of neutralizing antibodies. 711
HIV prototype (LAV/HTLV|n) entry is blocked by soluble CD4 or by the preincubation of CD4+ cells with OKT4A or Leu A monoclonal antibodies (8,9). HIV is strictly restricted to CD4 lymphocytes and does not replicate in CD8 cells (5).
Activation of CD4 lymphocytes produces a nuclear factor (NFkB) that permits the replication of latent proviral DNA (10). 1) Virus isolation in AIDS patients : as indicated in Table 2 virus can be isolated in 100 p. cent of AIDS patients. TABLE 2 : AIDS PATIENTS PATIENTS
DIRECT CULTURE
ANTIGENEMIA
COCULTURE
BIASE
337,996
33,664
+
ROUAL
33,926
80,752
+ + +
PEDMA
16,316
38,412
+
43,29 pg/ml
+
22,20 pg/ml
LOP JA
73,684
FOR AL
52,290
AMA MO
173,574
BOU BE
13,748
35,085
LAM MA
57,548
35,590
GILBE
95,306
553,518
AOULA
328,212
RESMU
227,634
STEGI
8,88 pg/ml
13,32 pg/ml
5,55 pg/ml
163,532
16,446
+ +
57,72 pg/ml
Some cocultures are negative like AMA MO in table 2, and this fact can be explained by the high amount of virus present in PBL (direct culture or CD4 fractionated cell) with a high cytopathic effect for all susceptible cells for added first PBL by killing in one cycle all the susceptible cells. In some cases virus cannot be isolated from PBL, not enough CD4 cells for direct cultivation of these fractionated CD4 cells like GAG, table 3 for instance.
These results confirm that the decline of CD4 lymphocytes in HIV is due to the presence of HIV and his cytopathic effect.
712
patients
infected with
2) Virus isolation correlates with a depletion of CD4 lymphocytes as shown virus is constantly isolated from PBL in AIDS patients with low T4 count.
in table 3,
TABLE 3 : VIRUS ISOLATION CORRELATES WITH A DEPLETION OF CD4+ LYMPHOCYTES CD4 COUNT
VIRUS ISOLATION
JAR
172
Posit ve
TAF
291
Posit ve
SAN
527
Posit ve
MAR
208
Posit ve
PRA
330
Posit ve
TIS
396
Posit ve
RES
237
Posit ve
IGL
396
Posit ve
BLA
151
Posit ve
LAT
462
Posit ve
GOM
504
Posit ve
STE
375
Posit ve
GAG
12
Posit ve
GAR
25
Posit ve
SAR
383
Posit ¡ve
HIVAgP24
3) When a virus has been isolated once in HIV infected patients, it is always found in subsequent virus isolation. 19 patients were followed several months in an antiviral clinical trial and they were included in a group receiving a placebo. In table 4 it is shown that the presence of HIV by isolation using the PBL coculture method is 100 p. cent positive and more sensitive than the direct culture. Two strains were used in this study : HIV1-BRU prototype (1) as reference strain and the Zairian highly cytopathic HIV1-NDK (11). The characteristics of the two strains are given in table 5 (12, 13). The second target for HIV is the macrophage (14, 15, 16). HIV infects macrophages
713
TABLE 4 : WHEN A VIRUS HAS BEEN ISOLATED ONCE IN PBL, IT IS ALWAYS FOUND IN
SUBSEQUENT VIRUS ISOLATIONS. PATIENT
(A)
(B)
DIRECT CULTURE
(C)
COCULTURE
(C)
FOR
(5)
(12)
Positive
(1)
Positive
(5)
TEX
(3)
(12)
Negative
(0)
Positive
(3)
TOR
(7)
(13)
Positive
(6)
Positive
(7)
TIM
(12)
(7)
Positive
0)
Positive
(12)
ANS
(8)
(4)
Positive
(6)
Positive
(8)
LEE
(4)
(2)
Positive
(3)
Positive
(4)
CAL
(5)
(2,5)
Positive
(4)
Positive
(5)
MES
(3)
(2,5)
Positive
(2)
Positive
(3)
POU
(10)
(5)
Positive
(10)
Positive
(10)
PHA
(2)
(2)
Positive
(2)
Positive
(2)
CAR
(5)
(2)
Positive
(5)
Positive
(5)
KIE
(2)
(1)
Positive
(2)
Positive
(2)
REN
(4)
(2)
Positive
(3)
Positive
(4)
CAM
(2)
(1)
Positive
(2)
Positive
(2)
GRE
(11)
(5)
Positive
(11)
Positive
(11)
GRA
(3)
(1)
Positive
(3)
Positive
(3)
CAL
(5)
(3)
Positive
(4)
Positive
(4)
BRE
(4)
(2,5)
Positive
(1)
Positive
(4)
MES
(3)
(2,5)
Positive
(2)
Positive
(3)
(A) Number of virus isolation. (B) coculture positive.
through
receptor and replicates without
a
becomes
In months time of follow-up of the
a
reservoir for the virus and is
(dementia (17, 18), pneumonia (14). cerebrospinal fluid during
acute
a
patient. (C)
Number of culture
or
cytopathic effect. This infected cell
responsible
The infected
for HIV associated diseases
macrophage is isolated from the encephalopathy in asymptomatic or AIDS patients 714
TABLE 5 : DIFFERENCES BETWEEN HIV1-BRU AND HIV1-NDK
Cytopathic effect inMT4
Day of detection of 1 TCIU
HIV1-BRU
HIV1-NDK
10-
10-
10
% of infected CEM cells at day 7
0.005
100
Block with OKT4A Leu 3A
Yes
No
or
Block with sCD4
Yes 0.5 m g
Neutralization with monoclonal antibodies directed to V3 loop
(17). Various isolates
(or
more
No
Yes
can
virus, macrophage tropic
be divided at least in three
or
dual
No than 100 ¿¿g)
categories
CD4
tropic (17, 19, 20). Certain patients
lymphotropic
can
present the
HIV associated diseases without immunodeficiency (17). The third target for HIV is the intestinal cells. The gastrointestinal tract is a
putative site of entry for HIV. The ability of HIV to infect rectal or colonie epithelial cells was previously shown in vivo by in situ hybridization (21, 22) and in vitro by using normal (23) or tumoral (24) epithelial cells of colonie origin which are permissive for HIV infection.
sensitivity of human colon epithelial cells to HIV infection has been documented (25, 26, 27, 28). We recently established that viral replication in a well characterized colon cell line (HT29) was a strain-dependent phenomenon, and that producer HT29 cells displayed numerous ultrastructural abnormalities, including altered secretion and impaired differentiation (29). HT29 cells were successfully infected by HIV1-BRU and HIV1-NDK. For NDK, the virus was able to perform its complete cycle of replication and the infected cells could be established as a continuous producer cell line (mean RT activity in the cell-free supernatants around 3x105 cpm/ml). Infection of HT29 cells by HIV1-BRU appeared not to be productive but the virus could be rescued by co-cultivation with lymphoid indicator cells (30, 31). The presence of viral particle in the colorectal lumen could explain the route of
The
715
transmission of HIV in the case of anal intercourse (31) for replicating virus and a silent reservoir, viral expression being under the control of underlying intestinal immune system. Fully replicative HIV in colon epithelial cells may elicit a fundamental disturbance of intestinal function, including a defect of brush border assembly and an increased secretion activity controlled by neurohormonal agents. This functional perturbation caused by HIV replication in epithelial cells, may account for malabsorption and diarrhea occuring in AIDS patients (29).
Hematopoietic cells are also important targets for HIV and some marrow T precursors cells (32), myeloid stem cells (33) and megacaryocytes (34) have been shown to be infected by HIV. To date, the mechanisms responsible for the dysregulated hematopoiesis in AIDS patients are still not clear. We have investigated a possible direct or indirect effect of HIV-1 and HIV-2 on the continued production and differentiation of hematopoietic progenitors maintained in long term liquid culture. We have studied the in vitro effects of HIV on bone marrow hematopoietic progenitor cells (BMHPC). In an effort to standardize our methods of BMHPC infection, we have first studied the replication of HIV in infected normal bone marrow mononuclear cells (BMMNC) depleted of adherent cells and mature T cells, as it was previously performed by Y. Lunardi-lskandar et al. (32). After having reproduced the HIV-1 infection of immature bone marrow T cells, we have studied the susceptibility of hemopoietic progenitors to HIV-1 and HIV-2 infection. For this purpose, we have performed long term bone marrow cultures (LTBMC) after 24 hours incubation with two isolates of HIV-1 (HIV-1 BRU and HIV-1 NDK) and two isolates of HIV-2 (HIV-2 ROD and HIV-2 MEND) at different multiplicities of infection (MOI) (35, 36). Purified hematopoietic progenitors from LTBMC are non producer cells and the virus can be rescued by cocultivation with HIV permissive cells. Bone marrow derived stroma cells are non producer after HIV-1 infection but weakly replicate the virus after HIV-2 infection. HIV-1 and HIV-2 are clearly not cytopathic for hemopoietic precursor cells.
Following production and growth of erythroid burst forming unit (BFU-E), erythroid colony forming unit (CFU-E) and granulo-monocytic progenitors (CFU-GM), for at least 6 weeks after HIV-1 infection, colony assays displayed the same profile of CFUE formation for the two isolates : the production of these progenitors was inhibited by 80 to 100 % after four weeks of LTBMC. On the contrary, BFU-E production in HIV-1 infected LTBMC is depending on HIV strain. HIV-1 LAV gave a 150 % stimulation of BFU-E production during the first week of LTBMC followed by a 50 % inhibition while HIV-1 NDK induced a delayed stimulation (day 24). CFU-GM production was 716
inhibited in LTBMC infected with HIV-1 LAV and HIV-1 NDK, the degree of inhibition ranging from 50 % the first week of LTBMC to about 100 % the last week. Different patterns of progenitors production have been observed after HIV-2 infection. On the contrary to HIV-1, HIV-2 induced a drastic inhibition of erythropoiesis in LTBMC infected with the two HIV-2 strains. Concerning the CFU-GM production, a significant stimulation was first observed during 4 weeks of LTBMC, then, followed by a strong inhibition.
gradually
Stimulating and inhibiting activities were recovered from supernatant of infected LTBMC, used as conditioned media (CM) in clonogenic assay for normal BMMNC. CM from HIV infected CEM cell line displayed stimulating and inhibiting activities in the same fashion as CM from infected LTBMC did. Consequently, we can argue that HIV induces release of (a) humoral factor(s) responsible for disruption of hemopoietic progenitor cells in vitro (37). REFERENCES 1.
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718
Symposium
on
Molecular
Biology
of
IMMUNOPATHOGENESIS OF HTLV MYRON ESSEX, D.V.M., Ph.D.
Chairman, Department of Cancer Biology Harvard School of Public Health 665 Huntington Avenue Boston, MA. 02115
The next speaker will be ¡mmunopathogenesis of HTLVs.
MODERATOR:
Max
Essex, discussing
ESSEX: Thank you, Tony. What I'm going to talk about is exclusively HTLV, and most of the emphasis will be on TAX of HTLV-1. I should mention in advance that most of this work was done by Akihiko Okayama, Betty Korber and Arthur Chen in my lab, or formerly in my lab. There was also collaboration with Tun-Hou Lee and Nobuyashi Tachibana in Japan. And unless I say otherwise, all other human serum and tissue materials to which I'll refer were from Miyazaki, Japan, and the island of Kyushu, a highly endemic region for HTLV-1. HTLV-2 has not been identified there. For diagnostic purposes, proteins encoded by the GAG gene or the ENV gene quite regularly induce antibodies.1 They are therefore useful for blood screening and seroepidemiological studies. As opposed to HIV, the polymerase of HTLV very rarely induces antibodies. Only a small fraction of people make antibodies to the reverse transcriptase but it does occur. The TAX protein and the REX protein are more interesting in that they induce antibodies in some people and not in others. About half to twothirds of HTLV-1 infected people have antibodies to TAX. This proportion varies with health status and according to route of exposure. As analyzed by radioimmunoprecipitation, virtually everybody has antibodies to the envelope, and about half of the healthy HTLV-1 carriers have antibodies to TAX. This study was done with some particular samples from HTLV-infected families. We had access to samples from a large number of individuals who were in leukemic families. In such families there was a patient with adult T cell leukemia, and between 40 and 50% of the other healthy individuals scored as antibody positive by conventional structural protein antibody assays for HTLV-1 structural protein antigens. We also showed that people who had TAX antibodies had a much greater risk for excreting the virus, and infecting their spouses, so that in a seroconversion cohort in Miyazaki, Japan, all of the partners who infected their previously uninfected spouses had such antibodies.2 Only a quarter of the spouses who were infected but did not infect their spouses over the same period of time had such antibodies. Beyond the correlation of TAX antibodies with infectiousness or transmissibility by sex, there's also a clear positive correlation with the presence of such antibodies in HTLV-1 positive children of ATL mothers.3 Children of ATL positive mothers are more likely to have TAX antibodies in younger age categories as compared to age-matched HTLV-positive blood donors. Conversely, older age groups of HTLV-positive blood donors are more likely to 719
have TAX antibodies than age-matched children of ATL mothers. In either case, the presence of TAX antibodies seems to be associated with infectability and with the proximity to time of infection. We and others, such as Slamon and Chen, and Poiesz and colleagues had noticed on various occasions in screening blood bank samples that a few rare samples scored as negative for antibodies to whole virus, but positive for antibodies to TAX. Nobody knew quite what significance to put on this. To address that question more specifically, we looked at the samples from ATL households in southwestern Japan, in Miyazaki, and specifically looked at seronegative individuals in the HTLV cluster households. We found that under these conditions, the rate of TAX-only antibody people, who scored positive for TAX antibody after scoring negative for structural protein antibodies, as verified by radioimmunoprecipitation and Western blot and other techniques, was quite significant. It was about 8 or 9% of the HTLV-1 seronegative individuals who were family members of ATL To verify specificity, we showed that reactivity occurred with native TAX in the absence of any antibody to structural proteins, and that this reactivity could be blocked very nicely with the recombinant TAX protein. The TAX antibodies could be detected by a variety of assays, using different sources of TAX proteins, whether naturally made in the cell, or made in E.coli by recombinant technology. Either way they cross-reacted very well to block the reactivity. We then screened other serum collections to see if individuals with antibodies to TAX but not to other HTLV antigens were found in association with exposure to HTLV. Samples from the general adult population in the high-risk city of Miyazaki revealed 11 of 700 that were negative for antibodies to HTLV structural antigens, but were positive for antibodies to TAX. Similarly, in a group of drug abusers at Bronx Lebanon Hospital in New York, at high risk for HTLV exposure, the same general result was obtained. Of 114 HTLV seronegatives that happened to be HIV seropositive, 4 had antibodies to TAX, as did 2 of 113 HIV seronegative people from the same population. Similar results were obtained from a group of Israeli hemophiliacs. There appeared to be several different ways that people would be infected so that they would have antibodies only to TAX. One would be the presence of the virus with a defective REX gene. Another would be the presence of an HTLV with a deleted genome. Whether or not either of these would be biologically important is unknown. We then undertook an extensive PCR study of fresh adult T cell leukemia cells, again from patients residing in the HTLV-endemic area of Miyazaki in southwestern Japan. We used a number of primers to evaluate the pattern of the proviral genome in these cells. Cells were examined from 23 different patients assumed to have ATL. Except for one, all scored positive quite well with the LTR primer. Many of them scored negative or almost negative with primers for GAG and protease. These patients were particularly interesting. They represented adult T cell leukemia patients with very high circulating abnormal cell counts, where the abnormal leukemic cells must have accounted for almost all of the nucleated cells in the circulation. A significant fraction of such patients scored very high for TAX and LTR, but were essentially negative or close to negative for protease, GAG, and sometimes for envelope, when primers specific for those regions were used. This result was obtained with 30-40% of the samples. For 50-60% of the individuals, all of the PCR primers scored highly positive, including gag and protease, representing all different regions of the viral genome. The latter
patients.^
720
individuals presumably had one or more copies of complete provirus in each leukemic cell. There were also a few unusual individuals whose cells we examined who did not have a diagnosis of conventional ATL. Some of these would fit the same pattern of positivity for TAX and LTR and negativity for GAG and protease. Some would fit the pattern of positive for everything, and some would fit the pattern of negative for everything. The latter were probably not ATL at all, but some other type of leukemia that happened by chance to occur in an HTLVinfected person. The three classes of results are summarized in Table 1 and will be published soon.5 TABLE 1. Classes of PCR Reactivity in Leukemic Cells from HTLV-1 Positive Patients
Category
GAG
PRO
PCR Primers POL ENV TAX
LTR
Antibody
Interpretation_
1
+/-
Selectively deleted provirus present.
2
+
Complete provirus present.
No provirus in most leukemic cells.
3
The
possibility that the defective provirus might be made by reverse transcription of a spliced message was then examined using a primer for the splice junction. This primer gave negative results when leukemic cells with the deleted provirus pattern were examined. By southern blotting procedures, the defective deleted genomes gave predicted results, a profile of a partial genome without all the expected bands present when PST-digested DNA was probed. There have been previous reports from the Yoshida and Gallo labs, describing defective genomes in ATL.6.7 However, such defective genomes were usually considered an unusual occurrence or an artifact of in vitro cultivation rather than a frequent event in the natural history and evolution of the
leukemic cells in their host. The current results show that in at least a third, and in probably more than a third, the tumor cell itself has a defective provirus as the predominant, or only major provirus form present. One hypothesis to explain how this might occur is ¡mmunoselection. With HTLV-1 infection there is an active and efficient immune response under most circumstances, unlike the case with HIV infection. With HTLV-1, most people do not develop a lethal terminal disease, and the virus is more sufficiently suppressed in a non-replicative form than is HIV. The HTLV-1 infection is then activated by antigenic or mitogenic stimulation. When an immortalizing transformation type of event occurs in a cell that is infected with complete copies of provirus, virus production should occur. This might result in elimination of that cell by an immune mechanism such as CTL's or antibodies directed to virus structural antigens on or in the tumor cells themselves. That would then allow for selection of cells that were immortalized,
721
but had only retained partial genomes. Such genomes might express only TAX and LTR. Those cells that fail to express structural proteins might have a selective advantage to grow out as malignant clones. Now, one could also imagine that exactly the same type of mechanism occur in other systems that haven't been adequately explained, such as might B virus hepatitis hepatoma, for example, where an immune lysis of virus infected hepatocytes occurs as hepatitis before the hepatoma develops. Most of the liver damage appears to be immune-mediated, related to activation of immune effector mechanisms against viral proteins. We know that when tumors emerge as hepatomas later in the disease, they occur in such a way that the viral DNA in the tumor cells has undergone extensive deletion. However, if any of the hepatitis B virus DNA is retained, it is most often the X gene; the gene that is analogous to TAX of HTLV. Although we know very little about the immune response that occurs following infection with human papilloma 16 or 18, the associated cervix cancers that emerge also appear to have deleted defective genomes and retention of transactivation type genes. Thus, in a wildly speculative way I could hypothesize that most virus-associated human cancers evolve in a host environment of immunoselection that favors the loss of viral structural proteins and genes from the tumor cells, if not from the entire host. I should also refer to the very recent report by Hall et al.8 describing the analysis of several cases of HTLV antibody negative mycosis fungoides using PCR. They found defective deleted HTLV provirus in several cases and cloned out a virus in at least one case. It was the same general type of defective provirus that we now see in at least a third of the ATLs in Japan. One logical conclusion that follows from all of these observations is that at least some human cancers may be virus associated and virus-caused but lose virus replication, virus structural proteins, and virus structural protein genes by the time the tumor grows out. Logically, then, searches to link other human cancers to viruses should include probes for transactivator genes. Another question came to mind. Could such defective viral genomes be passed to another host? If so, maternal-infant transmission would seem to be most likely, where lymphocyte cell contact might occur.. It is intriguing that the highest rate of the TAX-only antibody profile was found in children born in ATL families. That of course, could be another and much more exciting explanation for why some people might have antibodies only to the TAX gene product.
Thank you.
MODERATOR: Questions
or
comments?
QUESTION: We are finding messages for the PX proteins in the majority of the cases of ATL. We don't know yet whether leukemic cells are producing it, or non-leukemic cells or both. find
ESSEX: That would make sense, but I suspect that it would be harder to expression of virus or viral structural proteins in many cases.
QUESTION: PCR, you have to go to PCR to be able to find messages. Because... in ATL you don't have the expression of messages, and you don't find any proteins. But it might be there at a low level. You also find messages with the classical spliced donor, or classical spliced acceptor sites. We don't, 722
haven't analyzed yet extensively the integrity of the genomes. But the messages we find in other cases, and we've cloned them, conformed to the classical splice acceptor sites, and the classical spliced donor sites. we
some
ESSEX: Yes, I think that would be an important experiment to do with tumor cell preparations that were specifically deleted in GAG and
polymerase. In fact, see sitting here.
I discussed that at
one
point
with
George
Pavlakis who I
Perhaps I could just make one comment on the mycosis In addition to the American, or sorry the Swedish mycosis we looked fungoides. have started look at some Japanese HTLV seronegative mycosis but we to at, we haven't finished these studies yet. I'll describe them a little bit on Saturday, but what we do know in those, in the ones that have HTLV-1 involvement they all don't but the PX is also retained in those, whereas other regions appear to QUESTION:
-
be
1
)
-
missing.
Lee, T.H., Coligan, J.E., Homma, T., McLane M.F., Tachibana, N.., and Essex, M. Human T-cell Leukemia Virus Associated Membrane Antigens
(HTLV-MA): Identity of the Major Antigens Recognized Following Virus Infection. Proc. Nati. Acad. Sei. 81:3856-3860 (1984).
2)
Chen, Y.A., Okayama, A., Lee, T.H. Tachibana, N., Mueller, N. and Essex,
Sexual Transmission of Human T-Cell Leukemia Virus Type 1 Associated with the Presence of Anti-Tax Antibody. Proc. Nati. Acad. Sei., M.
8_8_:1182-86 (1991).
A. Chen, Y.A., Tachibana, Shioiri, S., Lee, T.H., Tsuda, K. and Essex, M. High Incidence of Antibodies to HTLV-1 TAX in Blood Relatives of Adult T-cell Leukemia Patients. J. Infect. Dis., 163:47-52 (1991 ).
3)
Okayama,
4)
Okayama, A., Korber, B., Chen, Y.A., Allan, J., Lee, T.H., Shioiri, S., Tachibana, N., Tsuda, K., Mueller, N., McLane, M., Mayan, S. Marlink, R., Orgad, S. amd Essex, M. Unusual Pattern of Antibodies to HTLV-1 in Family Members of Adult T-cell Leukemia Patients. Blood. 7.8:3323-3329 (1991).
5)
Korber, B., Okayama, A., Donnelly, R., Tachibana, N. and Essex, M.
Chain Reaction Analysis of Defective Human T-Cell Leukemia Virus Type 1 Proviral Genomes in Leukemic Cells of Patients with Adult T-Cell Leukemia. J. Virol., £5_: 5471-76 (1991).
Polymerase
6)
Yoshida, M. Seiki, M. Hattori, S., and Watanabe, T. 1984.
7)
Aldovini, A. DeRossi, A, Feinberg, M., Wong-Staal, F., and Franchini, G.
Genome Structure of Human T-cell Leukemia Virus and Its Involvement in the Development of Adult T-cell Leukemia/Lymphoma. p 141-148. In: R. Gallo, M. Essex and L. Gross (ed.), Human T-cell Leukemia/Lymphoma Virus, Cold Spring Harbor. 1986. Molecular Analysis of a Deletion Mutant Provirus of Type 1 Human T-cell Lymphotropic Virus: Evidence of a Double Spliced X-lor mRNA. Proc. Nati. Acad. Sei. USA, 83:38-42. 723
8)
Hall, W.W., Liu, C.R., Scheerwind, O., Takahashi, H., Kaplan, M. H., Röupe, G. and Vahlne, A. Deleted HTLV-1 Provirus in Blood and Cutaneous Lesions of Patients with Mycosis Fungoides. Science, 253:317-320
(1991).
SPECIAL LECTURES IX
"EXTRACELLULAR TAX, PROTEIN STIMULATES NF-kB AND EXPRESSION OF NF-kB-RESPONSIVE Ig KAPPA and TNFB GENES IN LYMPHOID CELLS"
John N. Brady, Ph.D. Laboratory of Molecular Virology National Cancer Institute National Institutes of Health Bethesda, MD 20892
HTLV-I Tax1 plays an important role in virus replication and cellular transformation. As a nuclear protein, Tax1 clearly has the ability to regulate both viral and cellular gene expression. Today, I would like to discuss a series of experiments performed by Paul Lindholm that suggest the possibility that Tax, also works as an extracellular cytokine, activating transcription factors and expression of specific cellular genes in noninfected cells. We have been able to define two functions for the extracellular Tax, protein. First, in pre-B lymphocytes Tax, activates the nuclear accumulation of the NF-kB transcription factor. This activation apparently involves either the release of the cytoplasmic IkB inhibitors or processing of the NF-kB pl05 precursor protein. The level of NF-kB induction is sufficient to activate expression of endogenous cellular genes. Second, Susan Marriott has demonstrated that extracellular Tax, stimulates proliferation of peripheral blood lymphocytes. The PBL proliferation is accompanied by an increase in the expression of the IL-2 receptor. Before we begin our discussion of the functions of extracellular Tax,, it is important to consider the evidence that Tax, is, in fact, secreted into the tissue culture media of cells transformed or infected with HTLV-I. We have used two experimental approaches to test this possibility. First, HTLV-I transformed cells were pulsed with S and then the accumulation of Tax, in the cell or the extracellular media was assayed by immunoprecipitation with Tax,-specific antibody. As expected, Tax, is easily detected in the nucleus of the transformed C81 cells. We estimate that the half-life of the cellular Tax, is about 2-3 hours, consistent with observations from other laboratories. In the extracellular media, Tax, can be detected by one hour of chase incubation in fresh culture media. The level of extracellular Tax, remains constant for about six hours and then declines gradually. By western blot analysis, using purified Tax, as a standard, we estimate that the concentration of Tax, in the extracellular fluid ranges from 0.5 to 1 nanomolar. This compares with a concentration of 1 to 5 micromolar Tax, in the infected cell. Tax, does not contain a distinguishable signal peptide sequence. Thus, the mechanism by which Tax, is released from the cell remains to be established. The Tax, protein that we have used in the studies I will be discussing today is purified from E. coli. Using a purification protocol developed in the laboratory, the Tax, has been been purified to homogeneity and is a single band on a SDS polyacrylamide gel. If 724
you label this protein with 125I and monitor its very rapid accumulation of Tax, in the cells.
uptake in pre-B lymphocytes, you see a Initially, the majority of the Tax, protein about 20 cytoplasm. By hours, you see that 80% of the Tax, is in the
is located in the nucleus of the cell. Following the uptake of the Tax, protein by these cells, the level of NF-kB binding activity is significantly increased. You see in this gel shift assay that the control level of NF-kB in these pre-B cells is very low. If the cells are treated with control bacterial extract, you see no increase in the level of NF-kB binding activity. In contrast, when we add Tax, to the extracellular media, at a concentration of 2.5 nanomolar, you see a quantitative induction of the NF-kB transcription factor in the nuclear fraction. The specificity of the gel shift complex is shown by competition with a wild type, but not
mutant, oligonucleotide. Because the Tax, was purified from E. coli, it was important for us to control for the possible presence of endotoxin in these extracts. Two lines of evidence demonstrate that the NF-kB induction is not due to endotoxin, but is due to the Tax, protein. Chloroform extraction of soluble extracts removes protein, but not endotoxin, from the sample. Chloroform extraction of the Tax, protein removes the protein from the sample. If we take the control and chloroform-extracted Tax, and analyze it for the ability to induce NF-kB, only the control non-extracted Tax, will induce binding activity. No induction with the chloroform-extracted Tax, was observed. This same experiment was done on bacterial endotoxin. No significant change in the ability to induce NF-kB was observed. The other experiment that we have done is to clear the Tax, protein sample with antiTax, serum. When we take the supernatants from immunoprecipitations done either with normal rabbit serum or anti-Tax, serum, only the latter neutralized the ability of Tax, to induce NF-kB binding activity. These two lines of evidence strongly suggest the a
biological activity is due to the Tax, protein. The ability of Tax, to activate NF-kB is dose-dependent. If we add increasing amounts of the Tax, protein to the extracellular media, we get a quantitative increase in the level of NF-kB. Within the detection limit of the gel shift assay, we need about 750 picomolar extracellular Tax, to induce detectable NF-kB binding activity. This concentration is in
the range that we see in the extracellular media of HTLV-I transformed cells. What interests us about the ability of Tax, to induce the NF-kB transcription factor is its importance in regulation of a number of cellular genes. These include genes of the immunoreceptor family, such as IL-2Ro, and important cytokines, such as IL-2, TNFa, TNFß, GMCSF and IL-6. Several of these genes, including IL-2Ra, IL-2, TNFß, GMCSF and IL-6 are induced by Tax,. What I will show you today is an analysis of two of these genes, TNFß and the immunoglobulin kappa light chain. We are particularly interested in the TNFß, or the lymphotoxin gene, because in addition to its cytotoxic effects, it also has the ability to act as an osteoclast activating factor and stimulate bone résorption. This may result in elevated levels of calcium in the circulatory system and might be instrumental in the hypercalcemia that one sees in ATL patients. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of either the TNFß or the immunoglobulin kappa light chain indicates that Tax, stimulation elevates the level of RNA expression from these genes. In the case of the TNFß, we see a very quick induction. By three hours after the addition of the Tax,, you see an increase in the level of RNA synthesis. This elevated level of TNFß RNA is maintained over the first 24 hours. We also see the induction for the immunoglobulin kappa light chain, although the kinetics are a little different. It takes about eight hours to see a significant accumulation of kappa light chain RNA. The elevated level of mRNA is maintained for twenty-four hours. We have used actin as a RT-PCR control. No change in the level of actin RNA synthesis was observed between the control cells and the Tax,-activated cells. 725
Because of the complexity of NF-kB transcription factor family and the difference in the kinetics of the activation of the TNFß and the immunoglobulin kappa light chain, we were
interested in
determining
if
Tax,
turned
on
these two genes by the
same
proteins.
The sequence of the NF-kB binding sites in the immunoglobulin kappa enhancer and the TNFß promoter are as follows: Igk 5'-GATCCAGAGGGGACTTTCCGAGAG-3' and TNFß 5'-GATCCAGAGGGGCTTCCCCGAGAG-3\ The immunoglobulin kappa enhancer element is a nonpalindromic sequence which is purine-rich on the 5' side and rich on this 3' side. In contrast, the TNFß promoter has a palindromic
pyrimidine sequence.
The induction of NF-kB binding to the kappa light chain and TNFb has been analyzed in detail following Tax, induction. With the kappa light chain oligonucleotide, you see one gel shift complex. This complex increases by one to three hours after the addition of Tax,. The level of NF-kB binding continues to increase over the first seven hours of stimulation and then starts to decrease. In contrast, what you see with the TNFß probe is two distinct complexes. The C-l complex, which migrates to a similar position in the gel as the kappa light chain probe, and a faster migrating C-2 complex. The C-2
complex is induced by one hour, decreases in intensity between three and seven hours and then increases by twenty-four hours of incubation. The C-l complex follows more or less the kinetics of what one sees for the immunoglobulin kappa light chain. We next UV cross-linked these gel shift complexes and then analyzed the products on protein gels. With the immunoglobulin kappa light chain probe, you get one predominant protein species of 75 kD (p65) in the Cl complex. In addition, a minor protein species of 59 kD (p50) is detected in this complex. With the TNFß probe, the major protein in both the Cl and C2 complex is the 59 kD (p50) protein. The Cl and C2 complex also show a difference in sensitivity to NF-kB inhibitor, IkB. Addition of IkB to nuclear extracts inhibits formation of the Cl gel shift complex. No effect on formation of the C2 complex was observed. Our analysis suggests that the immunoglobulin kappa light chain Cl complex consists of two proteins, the p50 NF-kB transcription factor and the p65 transmodulator. This complex is sensitive to inhibition by IkBa and MAD 3. In contrast, the TNFß C2 complex seems to be comprised of two p50 NF-kB proteins. The p65 transmodulator is apparently not found in this complex. This complex would be sensitive to different inhibitors in the cell, IkBß or the carboxy terminal fragment of the P-105 precursor, PDI. How does
transcription factors? One of the most common cytoplasmic form of NF-kB is to phosphorylate the inhibitor. This modification causes the release of the inhibitor, allowing the p50/p65 complex to migrate to the nucleus, bind to the DNA and activate transcription. We have investigated whether this is due to the protein kinase C pathway. Using staurosporin inhibition and TPA depletion studies, we conclude that the protein kinase C pathway is not involved in the Tax, activation. The possibility that Tax, activates other protein kinases in the cell, either through the protein kinase A pathway or other protein kinases remains to be
pathways
Tax,
activate the NF-kB
to activate the
established. Tax, activation of NF-kB is not dependent upon protein synthesis. If you add Tax, to the extracellular media in the presence of cycloheximide, no significant reduction in the ability of Tax, to induce NF-kB is observed. Consistent with these results, using RTPCR analysis no increase in the level of either c-rel or NF-kB RNA synthesis was detected in the Tax,-treated cells. Thus, whatever the mechanism of Tax, induction, it seems to be dependent upon activation of pre-existing protein complexes or precursor
proteins.
we have investigated is to determine whether Tax, could directly release the inhibitor from the inactive cytoplasmic complex. In this analysis cytoplasmic
The other pathway that
726
cells, which contain very little or low activity of the NF-kB have been treated with either DOC/NP40 or Tax,. If you treat the transcription factor, inactive p50/p65/IkBa complex with DOC/NP40, an induction of gel shift activity is observed. In contrast, the addition of Tax, protein to the p50/p65/IkBa containing extract did not increase the level of gel shift activity. These results suggest that Tax, does not directly dissociate the IkB inhibitor from the complex. The results of a related series of experiments suggest that Tax, does not effect the ability of the IkBa inhibitor to reassociate with the p50/p65 complex. extracts from untreated
In summary, I would draw the following conclusions from our studies: 1. Tax, is rapidly taken up by pre-B lymphocytes and peripheral blood lymphocytes. Cellular f ractionation demonstrates that approximately 80% of the cell-associated
2. 3
4.
5. 6. 7. 8.
Tax, protein is transported to the nucleus of the uninfected cell. Tax, protein stimulates nuclear NF-kB binding activity in pre-B lymphocytes in a rapid, transient and dose-dependent manner. Tax, stimulation of NF-kB does not require an active protein kinase C system. It may involve either the protein kinase A pathway or other kinases. Tax, was unable to dissociate the NF-kB/IkB complex directly. Tax, did not prevent reassociation of IkB with NF-kB. Tax, stimulation of NF-kB binding activity was not inhibited by cycloheximide. Tax, stimulation of pre-B lymphocytes caused an increase in RNA expression from the immunoglobulin kappa light chain and the TNFß genes, as determined by RT-PCR analysis. The TNFß and immunoglobulin light chain NF-kB elements show differential binding to NF-kB proteins by gel shift and UV cross-linking analysis. Purified IkB inhibits binding of the p50/p65 (Cl) complex but not the p50/p50
(C2) complex.
References:
Lindholm, P. F., S. J. Marriott, S. D. Gitlin, C. A. Bohan, and J. N. Brady: Induction of nuclear NF-kB DNA binding activity after exposure of lymphoid cells to soluble Tax, protein. The New Biologist 2:1034-1043, 1990. Lindholm, P. F., S. J. Marriott, S. D. Gitlin, and J. N. Brady: Differential precipitation and zinc chelate chromatography purification of biologically active HTLV-I Tax, expressed in E. coli. J. Biochem. and Biophys. Methods 22:233-241,1991.
Marriott, S. J., P. F. Lindholm, R. L. Reid and J. N. Brady: Soluble HTLV-I Tax, stimulates proliferation of human peripheral blood lymphocytes. The New Biologist 3:678-686, 1991. Lindholm P. F., R. L. Reid and J. N. Brady: Extracellular necrosis factor
(In press)
e
and
immunoglobulin kappa light
727
chain in
Tax, protein stimulates tumor
lymphoid cells. J. Virology
NEW ADVANCES IN THE MANAGEMENT OF AIDS ASSOCIATED OPPORTUNISTIC INFECTIONS
Henry Masur, M.D. Chief, Critical Care Medicine Department National Institutes of Health Bethesda, MD 20892
September 1991 issue of Medicine, our group at NTH has published its cumulative with experience patients who have been followed for their entire course and who ultimately came to at NTH. The frequencies of various infections and tumors are demonstrated in this review. autopsy It's important to focus on the fact that there is really a wide diversity of infections that cause morbidity and mortality. It is also important to try to focus on those processes that really cause the most morbidity, and those that cause mortality. In this list, there are processes like pneumocystis, CMV, cryptococcus and toxoplasma which cause very substantial morbidity, and other processes like candida or Herpes simplex, which can certainly be annoying, but which do not usually have such a substantial impact on survival. Thus, opportunistic infections can have a wide range of implications for patients: some are life threatening, others are not. With some opportunistic processes, our progress in improving diagnosis, therapy and prevention has been more substantial than in others. We have made particular progress in our ability to manage pneumocystis pneumonia and cerebral toxoplasmosis. For other organisms like CMV and perhaps Mycobacterium tuberculosis, however, there is an increasing impact due either to our lack of advances in diagnostics and therapeutics, or for tuberculosis, due to changing socioeconomic trends. Some of the organisms that we first recognized at the beginning of the 1980s, like Mycobacterium avium-intracellulare are having an increasing impact. Patients are surviving longer due to improved antiretroviral therapy and improved management of other previously fatal processes. Thus, patients are surviving longer in a very immunosuppressed state. Mycobacterium avium-intracellulare is becoming more important because we haven't made as much progress in terms of understanding when to treat it, or what drugs to use. There are a number of newer organisms which we didn't know how to recognize previously, which we're now recognizing. These include cryptosporidia, microsporidia, and Rochalimaea. We are just learning how to diagnose these agents. We have no therapy for them. I think that one of the issues for the 1990s is that as HIV moves to new geographic areas and as we're more successful in preventing death due to some of these processes, we are going to see new processes emerge which are going to cause increasing morbidity and mortality as patients survive longer. There are a number of reasons why we're more successful in dealing with some of the classic opportunistic processes. First, we know a lot more about the natural history of HIV disease. A number of studies that have been reviewed this week focus on the natural history and the immunopathogenesis of HIV and have helped delineate the time when opportunistic processes occur in the life history of an HIV infected patient. To a large degree, this has helped clinicians focus on a time when they can look for specific opportunistic processes. Thus, understanding the natural history has been a very important advance in terms of more effective management of patients. We know that the CD4 counts inevitably decline over time despite the persistence of AZT therapy. As an example, we know that pneumocystis pneumonia generally occurs in patients with CD4 cell counts below 200/mm3. Thus, when a cough occurs in patients with CD4 counts greater than 300-400 /mm3, the likelihood of pneumocystis pneumonia is low. In contrast, in a patient with a CD4 count below 100/mm3, there is considerable likelihood that cough or dyspnea could be caused by pneumocystis. Understanding when opportunistic processes occur in the natural history of HIV is very important for clinicians in developing effective management strategies. Patients who have more than 500 CD4 cells/mm3 are generally asymptomatic, although they may develop Kaposi sarcoma or lymphoma. Those in the 200-500/mm3 range may have fevers, sweats, and weight loss, which In the
728
might be due to HIV, CMV, or some other process. They may develop some of the less lifethreatening processes like hairy leukoplakia, oral or esophageal candidiasis, or tuberculosis. Only when a patient gets to the lower CD4 levels do you start developing pneumocystis, cryptococcus or dementia, and it's only when your CD4 count is extremely low, that you develop disseminated mycobacteria or significant CMV disease. As Bob Yarchoan has shown recently in the Annals of Internal Medicine, most of the patients who die despite aggressive management are
those whose CD4 cells are less than 50/mm3. Armed with this knowledge, one can decide when to do various diagnostic tests when assessing different clinical syndromes. I'd like to provide an example of the progress that we've made both in understanding when in the natural history of HIV disease opportunistic processes occur, and in knowing how to diagnose the process when we know that a patient is susceptible. I'd like to take pneumocystis pneumonia as a specific example. We've not made as much progress for certain other opportunistic processes like CMV and perhaps MAI in terms of knowing when and how to treat these processes. For pneumocystis, we know that any time we can find pneumocystis organisms in pulmonary secretions or tissue, that the patient likely has active disease and needs to be treated. One of the major advances over the last decade has been that we not longer have to do an invasive procedure like open lung biopsy in order to make the diagnosis. And actually, we don't even need to do an expensive, and time-consuming procedure like bronchoscopy, which may cost $300-600 to perform, and can be uncomfortable. Now with an induced sputum technique, we can noninvasively make the diagnosis of pneumocystis in over 90% of patients who are not receiving aerosol pentamidine prophylaxis. The ability to diagnose a life-threatening process by a non-invasive, inexpensive technique is both a major economic advance, and it's a major advance in terms of our ability to improve survival. Patients are much more likely to come forward and have this test when their symptoms are mild. And we know that the milder the disease at the time we start therapy, the more likely to a outcome. to is have patient good Improved diagnostic techniques for pneumocystis have been a major advance in our management over the last 10 years. I think that now, at least in the developed world, having induced sputums available is a major opportunity to improve the quality of patient survival. In addition to the ability to make a diagnosis of pneumocystis, we also understand a lot more about the pathophysiology of pneumocystis. Just as understanding pathophysiology's been important for HIV, it's been very important in improving our management of pneumocystis. In terms of understanding pathogenesis, we've made some real advances. A feature of pneumocystis pneumonia that was not widely appreciated before the era of AIDS has been that, when you start therapy, you appear to increase the inflammatory reaction in the lung, and the pulmonary physiology deteriorates. If one looks at the hypoxemic ratio in patients who are on standard therapy, generally the patient will become substantially more hypoxemia during the first 3 days one starts therapy. This is presumably because lysis of organisms is initiating an inflammatory response and thus inflammatory responses exacerbate gas exchange. We are just beginning to understand which mediators are responsible for this. But if you administer corticosteroids to the patient, you can prevent this deterioration in respiratory physiology from occurring. This means that as we give standard therapies, we will want to avoid this drop because often a patient will either die because of this drop, or they'll need ventilatory support in the ICU during this drop. By understanding that this event occurs, we can intervene with corticosteroids. Hopefully in the years to come, as we understand specifically about what cells and what cytokines are responsible for this drop, we can intervene with drugs tha,t don't have some of the deleterious effects that corticosteroids may have. We may be able to intervene in a much more specific, and much less harmful way, and further improve the prognosis and quality of life of patients with pneumocystis pneumonia. Specifically, if we take patients with moderate or severe pneumocystis, and add adjunctive corticosteroids to standard therapy, that we can decrease mortality from about 22% to about 11%. Especially for patients with P02's under 70mmHg at the time therapy is instituted, understanding that this hypoxemic event occurs during the first 72 hours, and understanding that intervening with steroids can be beneficial, has been a major advance. As we understand more about what the specific immunologie events responsible for deterioration are, we 729
intervene with have.
something that is less likely to have the deleterious side effects that steroids Another issue of note is that a number of funding agencies, including the Division of AIDS, NIAID, have put major resources into identifying new drugs for the therapy and prophylaxis of pneumocystis. I think that many house officers in 1990 can't remember the era in the early 1980s when we only had two drugs that were effective against pneumocystis, i.e. trimethoprim-sulfamethoxazole and parenteral pentamidine. By using a variety of approaches, including an empiric approach looking for agents that are active against taxonomically similar organisms to pneumocystis, or using in vitro cultivation techniques, or using metabolic approaches which have used cell-free enzyme systems derived from pneumocystis, we've identified a number of new agents, including macrolides, primaquine analogs, some pentamidine analogs, and some other drugs. One of the most interesting developments just came to light in this past week. A drug that Burroughs-Wellcome had developed, 566C80, had been known in an animal model to have activity against pneumocystis. In an article recently published in The New England Journal of Medicine, this hydroxynaphoquinone, which is an entirely novel compound, has been shown in an open trial to possess a high degree of efficacy, with very little toxicity. In addition, BurroughsWellcome recently completed a multicenter trial looking at 566C80 versus standard therapy with trimethoprim-sulfamethoxazole. It will be very interesting to see how this new drug compares to standard therapy in terms of efficacy, toxicity, and ability to prevent relapse. Not only do we understand more about the immunopathogenesis of pneumocystis, not only do we have more drugs available for treatment, but we've also made substantial progress in terms of preventing pneumocystis. The number of cases of pneumocystis that occurred in the pre-AIDS era was quite small and has dramatically increased from about 60 a year in the pre-AIDS era to about 60,000 a year that are projected in the post AIDS years of the late 1980s and early 1990s. The number of cases of pneumocystis appears to be declining despite the increase in size of the population at risk. Clearly, we have been much more effective in developing prophylactic strategies recently. I think the first development in the 1980's that was impressive was the use of a novel method for drug delivery, namely aerosolization with regard to pentamidine. Aerosol pentamidine delivered by the route recommended from the San Francisco Community Study clearly showed efficacy in reducing secondary cases. There has been considerable amount of enthusiasm for aerosol pentamidine as a means to substantially reduce the dramatic number of cases of pneumocystis that occurred, but there's been a growing amount of data suggesting that oral trimethoprim-sulfamethoxazole may be more effective. In data that have just been announced in a press release yesterday, the ACTG study 021 has shown that in fact, trimethoprim-sulfamethoxazole is about 3 1/2 times more effective than aerosol pentamidine, and that its toxicity, although somewhat greater than aerosol pentamidine, may not be as great as one might think. Thus, pneumocystis is a good example of an organism for which we really have made a lot of progress, because 1) we understand when in the natural history of HIV it occurs, 2) we understand how we can prevent the deterioration in respiratory physiology associated with early therapy, 3) we know how to diagnose it more readily, and 4) we've made some real advances in therapy and in prophylaxis. There are other processes that we've known how to recognize where, unfortunately, we've made a lot less progress. CMV is a disease which is very common in HIV-infected patients. We've recognized that CMV can cause very obvious disease like retinitis, we have seen that a very large number of patients, 73% in one series, have evidence of disease, not just infection, due to CMV at autopsy. And as we're able to handle pneumocystosis, toxoplasmosis, and cryptococcosis better, we are not able to manage CMV in many situations with much better results than we were probably in the middle part of the 1980s. The reason for this is that, while we do have some drugs that are finally effective against CMV, we don't really know precisely when CMV is actually causing fever, weight loss, or inanition that often occurs when a patient has an occult gastrointestinal or pulmonary disease. One of the advances that we need for CMV that we have for pneumocystosis is a mechanism for deciding when patients who are infected with CMV in fact need treatment. One of the very cost-ineffective things that we do for CMV is that we culture blood and urine for CMV, but don't know how to use the resulting data. There is a very clear relationship between viremia can
730
and the CD4 count, which is not surprising. What is surprising is that when we've looked at the prognostic implications of being blood culture positive, we find that those who are blood culture positive are not much more likely to have a premature death than whose who are blood culture negative, and that having a positive blood culture does not substantially predict who's going to develop retinitis, pneumonitis, or colitis in the ensuing six months. If you look at the other side of the coin, i.e. those who already have retinitis, or pneumonitis or colitis, we find that many of those individuals are in fact blood culture negative, at least as assessed by single cultures of blood. So we don't have a good way, short of getting a large piece of tissue, to decide who has CMV disease. If we want to make progress in the next few years in terms of decreasing the number of patients who have fever, inanition, or who ultimately die of hypotension or failure, much of which is probably caused by CMV, we are going to have to figure out which ones will benefit from treatment for CMV rather than MAI or some other process. However, the good news about CMV disease is that we do have effective drugs. I think that everybody's aware of the fact that there are two parenteral alternatives, ganciclovir and foscarnet. There are unfortunately no oral agents. An exciting development about foscarnet has recently been published in the Annals of Internal Medicine. This study shows that foscarnet patients with CMV retinitis will have a high degree of efficacy in reducing the activity of CMV retinitis and in delaying recurrences. There are several studies suggesting that ganciclovir and foscarnet have comparable efficacy although markedly different toxicities. Unfortunately, neither has an especially promising formulation. In closing, it is clear that the ultimate solution to patients with HIV is understanding more about the pathogenesis of HrV and finding better therapy for the underlying retroviral disease. Until that time comes, when the retroviral process can be stabilized or reversed, we need to make more progress with the management of opportunistic infections. While we have made major advances, particularly in the fields of pneumocystosis and toxoplasmosis, there are areas like CMV and MAI where we need to understand a lot more about when to intervene with therapy, and we need to develop more cost-effective, less toxic therapies.
Thank you.
MOLECULAR PATHOGENESIS OF HIV-ASSOCIATED LYMPHOMAS Paola Ballerini, M.D., Gianluca Gaidano, M.D Jerry Gong, M.D., Vittorio Tassi, M.D., Giuseppe Saglio*, M.D., Daniel M. Knowles, M.D., and Riccardo Dalla-Favera, M.D. ,
Department of Pathology and Cancer Center, College of Physicians & Surgeons, Columbia University, New York, NY 10032; *Clinica Medica, Universita' di Torino, Torino, Italy The incidence of malignant Non Hodgkin Lymphomas (NHL) in HIV-infected patients is increasing, possibly as a consequence of the increasing life span of AIDS patients treated with antiretroviral therapy (1). According to the Working Formulation classification, about 70% of AIDS-associated NHL (AIDS-NHL) are high grade, small non cleaved cell (SNCC) or large cell immunoblastic- plasmocytoid QLC-IBP), while 30% are represented by intermediate grade, diffuse large non cleaved cell (LNCC). Distinctive features of AIDS-NHL include the frequent involvement of the central nervous system (CNS) often as a primary site (2) and the association with a polyclonal B-cell proliferative lymph node expansion (Lymphadenopathy Syndrome, LAS) often preceding the onset of the lymphoma (3-4). Little is known about the genetic alterations underlying the pathogenesis of AIDS731
NHL. Observations involving a limited number of cases have indicated an associations with chromosomal translocations involving the c-myc oncogene (5-6). Epstein-Barr virus (EBV) infection of the tumor cells has also been observed in 30-40% of cases, and it has been suggested to play a pathogenetic role based on the observation that viral infection precedes clonal expansion (5-7). Although EBV infection and constitutive c-myc oncogene expression are sufficient for the malignant transformation of human B cell in vitro (8), their combined presence does not appear to be required for B cell lymphomagenesis in vivo and the presence of other alterations has not been investigated. In the present study, we have redefined the frequency of c-myc activation and EBV infection in a large panel of tumors and investigated the possible roles of other genetic alterations, such as activation of ras oncogene and loss/inactivation of the tumor suppressor genes p53 and RBI, which are frequently associated with the development of various types of neoplasia but whose association with lymphomagenesis in AIDS has not been explored. Histology. Immunophenotype, Immunogenotype. We have analyzed a panel of 27 cases of systemic AIDS-NHL which were diagnosed upon analysis of histopathology and cell surface markers (9). All cases were diagnosed as B cell AIDS-NHL; 16 were shown to be SNCLL, 6 LNCCL, and 5 LC-IBP. In addition, immunoglobulin gene rearrangement analysis (10) indicated that all the biopsies contained a single predominant clonal B cell
population accounting for at least 60% of the sample. EBV infection. In order to assess the possible etiologic contribution of EBV to malignant transformation the presence of EBV genomes within AIDS-NHL cells was investigated by Southern blot hybridization using a probe specific for the genomic termini of EBV (11). This assay allows to determine whether EBV infection has preceded and, thus, possibly contributed to clonal expansion or whether infection has occurred after clonal expansion, thus representing an unlikely pathogenetic element (12). As summarized in Table 1, EBV sequences were detectable in -40% of AIDS-NHL, although the frequency appears to vary among different subtypes, the highest frequency being in the LC-IBP lymphoma subtype. All the positive cases displayed a single intense band suggesting that EBV infection has preceded and, thus, possibly contributed to clonal expansion in these malignancies. c-myc oncogene activation. Chromosomal translocations leading to c-myc oncogene activation can be detected as: i) truncations of the c-myc gene within the first exon, first intron or 5' flanking sequences which can be identified as rearrangements by Southern blot hybridization analysis and are characteristic of sporadic-type Burkitt lymphoma (BL) carrying (8; 14) translocations (13); or ii) mutations within sequences spanning the first exon/first intron border which are detectable by direct sequencing and are characteristics of t(8;14) in endemic-type BL and variant, t(2;8) and t(8;22), translocations in all types of BL (14). In our panel of AIDS-NHL, rearrangements of the c-myc gene were detectable in 66% of the cases by Southern blot hybridization, whereas the remaining cases appeared normal also by PCR/sequencing analysis for mutations (table 1). These results confirm that c-myc oncogene activation represents a frequent event in AIDS-NHL particularly in the SNCCL subtype. The mechanism of activation appears analogous to the one typical of Frequency and distribution of molecular lesions in HIV- associated lymphomas according to the histopathological classification
Table 1
Histotype
# of cases
EBV
c-myc
p53
ras
RBI
SNCCL LNCCL LC-IBP
16 6
5/16 1/4 4/4
14/16 1/4 1/4
10/16 0/6 0/5
3/16 0/6 1/5
0/16 0/6 0/5
5
732
sBL, where c-myc recombination with Ig switch sequences has been suggested to occur at the time of Ig isotype switching in a relatively mature B-cell (15). p53 loss/mutation. The p53 gene encodes a nuclear phosphoprotein that appears to negatively regulate cell proliferation (16). Exons 5 to 9, which represent the regions most affected by mutations in human tumors (17), have been investigated by means of PCR/Single Strand Conformational Polymorphisms (SSCP) analysis followed by PCR /
Direct Sequencing ( DS ) of the positive cases, as already described (18). p53 gene alterations were observed in 37% of AIDS-NHL. However, p53 lesions are not uniformely distributed among the different subtypes of NHL since they were found only in the SNCC group ( see table 1 ). In this group, 10 of 16 cases (62.5%) displayed point mutations, short sequence deletions or insertions, frequently associated with the loss of the normal p53 alíele. Ras oncogene mutation. The presence of mutations affecting codons 12, 13 and 61 of the three ras genes ( H-, K- and N- ras ) was investigated in AIDS-NHL DNA by PCR/SSCP and PCR/DS analysis. As shown in the Table 1, 4 cases ( 3 SNCC and 1 LCIBP of NHL ) scored positive. The mutations were found in N-ras codon 61 (2 cases), Nras 12(1 case) and K-ras 12(1 case). Previous evidence had shown that among lymphoid malignancies ras mutations are specifically restricted to Multiple Myeloma (19) and Acute Lymphoblastic Leukemias, while a large panel of NHL was found completely negative (20). Thus, the finding of Ras mutations among AIDS-NHL might represent a distinctive molecular feature of these lymphomas versus non AIDS-related NHL. RB1 gene inactivation. RBI gene encodes for an ubiquousely expressed 110-115 KD phosphoprotein endowed with growth suppressor activity (21). Alterations, i.e loss or mutations, of the RB 1 gene, initially found associated with Retinoblastoma, have been recently identified in other tumors suggesting a wider role of this gene in tumorigenesis (21). The molecular mechanisms of RBI inactivation are heterogeneous, including complete loss of the locus, partial deletions, rearrangements or splicing mutations (21). The structure of the RBI gene in AEDS-NHL DNA was studied by Southern blot analysis and PCR/SSCP analysis of exons 10 through 27, which span the domains of the gene in which most of the tumor-associated mutations have been found. Although the gene was found rich in polymorphic sequences which were originally thought to reflect somatic mutations, no evidence of tumor-specific rearrangements or mutations were detected in the exons examined (Table 1) when AIDS-NHL DNA was compared to a panel of normal DNAs. SUMMARY AND CONCLUSIONS The data presented here indicate that the pathogenesis of AIDS-NHL is variably associated with multiple genetic alterations including monoclonal EBV infection, oncogene activation ( c-myc, N-, Ki-ras ) and tumor suppressor gene ( p53 ) inactivation. Up to three ( 3 cases ) or four ( 1 case ) different lesions have been observed in the same tumor The distribution of these lesions among the various histotypes is heterogeneous, although some preferential associations have been found either between lesion and histotype or between lesions. The most notable case involves p53 mutations/loss that is exclusively associated with the SNCC lymphoma subtype. Since alterations of the c-myc gene occur at very high frequency in this same histotype it is possible that both lesions may be required for the pathogenesis of the BL phenotype. The consistent negativity of p53 lesions in other NHLs associated or non associated with HIV infection (18) reinforces this hypothesis. Finally, we note that the frequency of p53 mutations is significantly higher in AIDS-BL than in non HIV-related BL (18), although the significancy of this difference remains to be assessed. This study confirms the relatively low frequency of EBV infection in systemic AIDS-NHL in general, but reinforces the notion that EBV may be required for the pathogenesis of AIDS-LC-IBP, as recently suggested by the high frequency of EBV positivity in primary CNS AIDS-NHL which are mostly represented by LC-IBP (2). Conversely, the low frequency of EBV sequences in the AJJDS-SNCC lymphomas appears similar to that observed in sBL. 733
Only in a small minority of cases were ras oncogene mutations found, mostly associated with the BL type. Since ras mutations have never been found in non HIVrelated NHL, we examined whether these mutations could be related to the potential mutagenic action of antiretroviral agents, namely AZT. However, none of the patients included in our survey had received previous antiviral therapy. The presence of multiple lesions in some AIDS-NHL is perhaps surprising considering the relatively short time involved in the development of these tumors (4-5 years ) (22), and the fact that, since only a few common lesions have been studied, additional presently unknown alterations are likely to be present. While numerous studies have defined the multistep nature of tumorigenesis (23), the example of the multi-lesion pathogenesis of colon carcinoma has been taken to suggest that the long time (30-40 years) involved in tumor development is related to the number of independent genetic steps/lesions required (24). However, the example of some cases of AJDS-NHL suggests that genetic lesions may accumulate more rapidily at least in some tissues. ACKNOWLEDGMENTS This work has been supported by National Institutes of Health grants CA 37295 (to R.D.F.), and CA 40236 (to D.M.K.). P.B. and V.T. have been supported by fellowships of the Associazione Italiana per la Ricerca sul Cancro. G.G. has been partially supported by Gigi Ghirotti Foundation. REFERENCES
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J.M.Pluda, R.Yarchoan, E.SJaffe, I.M. Feuerstein, D. Salomon, S.M. Steinberg, K.M. Wyvill, A. Raubitschek, D. Katz, S. Broder : Development of non Hodkin
lymphoma in a cohort of patients with severe immunodeficiency virus infection on long term retroviral therapy. Ann. Int. Med. 113, 4, 276-282, 1990 E. M. E. MacMahon, J. D. Glass, S. D. Hay ward, R. B. Mann, P. Scott Becker, P. Charache, J. C. McArthur, R. Ambinder : Epstein-Barr virus in AIDSrelatedprimary central nervous system lymphoma. The Lancet, vol. 338: oct 19, 969-973, 1991. PG. Pelicci, D. M. Knowles, Z. A. Arlin, R. Wieczorek, P. Luciw, N. Dina, C. Basifico, R. Dalla-Favera : Multiple monoclonal B-cell expansions and c-myc
acquired immune deficiency syndrome-related lymphoproliferative disorders. J. Exp. Med. 164, 2049-2076, 1986. D. Shibata, L. M. Weiss, B. N. Nathwani, R. K. Brynes, A. M. Levine : EpsteinBarr virus in benign lymph node biopsies from individuals infected with the human immunodeficiency virus is associated with concurrent or subsequent development of non-Hodgkin lymphoma. Blood, 77, 1527-1533, 1991. M. Subar, A. Neri, G. Inghirami, D. M. Knowles, R. Dalla-Favera : Frequent cmyc oncogene activation and infrequent presence of Epstein-Barr virus genome in AIDS-associated lymphoma. Blood, vol.72, 2, 667-671, 1988. J. Whang-Peng, E. C. Lee, H. Sieverts, I. T. Magrath : Burkitt's lymphoma in AIDS : cytogenetic study. Blood, 63, 4, 818-822, 1984. A. Neri, F. Barriga, D. M. Knowles, J. Neequaye, I. T. Magrath, R. Dalla-Favera : Epstein-Barr virus infection precedes clonal expansion in Burkitt's and AIDSassociated lymphoma. Blood, 77, 5, 1092-1095, 1991. L. Lombardi, E. W. Newcomb, R. Dalla-Favera : Pathogenesis of Burkitt Lymphoma : expression of an activated c-myc oncogene causes the tumorigenic conversion of EBV- infected human B lymphoblasts. Cell, 49, 161-170, 1987. D. M. Knowles, G. A. Chamulak, M. Subar, J. S. Burke, M. Dugan, J. Wernz, C. Slywotzky, PG. Pelicci, R. Dalla-Favera, B. Raphael : Lymphoid neoplasia oncogene rearrangements in
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associated with the acquired immunodeficiency syndrome (AIDS). Ann. Int. Med. 108: 744-753, 1988. S. J. Korsmeyer, P. A. Hieter, J. V. Ravetch, D. G. Poplack, T. A. Waldmann, P. Leder : Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells. Proc. Naü. Acad. Sei. USA, 78, 7096-7100, 1981. N. A. Brown, C. R. Liu, Y. F. Wang, C. R. Garcia : B-cell lymphoproliferation and lymphomagenesis are associated with clonotypic intracellular terminal regions of the Epstein-Barr virus. J.Virol. 62 (3), 962-970, 1988 N. Raab-Taub, K. Flynn : The structure of the termini of the Epstein-Barr virus as a marker of clonal cell proliferation. Cell 47, 883- 889, 1986. PG. Pelicci, D. M. Knowles, I. T. Magrath, R. Dalla-Favera : Chromosomal breakpoints and structural alterations of the c-myc locus differ in endemic and sporadic forms of Burkitt's lymphoma. Proc. Nati. Acad. Sei. USA, 83 29842988, 1986. E. Cesarman, R. Dalla-Favera, D. Bentley, M. Groudine : Mutations in the first exon are associated with altered transcription of c-myc in Burkitt lymphoma. Science, 238, 1272-1275, 1987. Dalla-Favera, R. Chromosomal translocations involving the c-myc oncogene and their role in the pathogenesis of B cell neoplasia. Origin of Human Cancer. Brugge, J, Curran, T., Harlow, E., McCormick, F. (eds.). Cold Spring Harbor Laboratory (publ.), in press. E. J. Stanbridge : Human tumor suppressor genes. Annu. Rev. Genet. 24 : 615657, 1990. M. Hollestein, D. Sidransky, B. Vogelstein, C. C. Harris : p53 mutations in human cancers. Science, 253, 49-53, 1991. G. Gaidano, P. Ballerini, J. Z. Gong, G. Inghirami, A. Neri, E. W. Newcomb, I. T. Magrath, D. M. Knowles, R. Dalla-Favera : p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc. Nati. Acad. Sei. USA, 88, 5413-5417, 1991. A. Neri, J. P. Murphy, L. Cro, D. Ferrero, C. Tarella, R. Dalla-Favera : Ras oncogene mutations in multiple myeloma. J. Exp. Med. 1715-1725, 1989. A. Neri, D. M. Knowles, A. Greco, F. McCormick, R. Dalla-Favera : Analysis of Ras oncogenes mutations in human lymphoid malignancies. Proc. Nati. Acad. Sei. USA, 85: 9268-9272, 1988 D. W. Goodrich, W. H. Lee : The molecular genetics of retinoblastoma. Cancer surveys 9, 529-263, 1990. V. Beral, T. Peterman, R. Berkelman, H. Jaffe : AIDS-associated non-Hodgkin lymphoma. The Lancet, vol.337, apr 6, 805-809, 1991. R. A. Weiberg : Oncogenes, antioncogenes and the molecular bases of multistep carcinogenesis. Cancer Res. 49, 3713-3721, 1989. E. R. Fearon B.Vogelstein : A genetic model for colorectal tumorigenesis. Cell, ,
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EBV-INDUCED HUMAN B CELL LYMPHOMAS IN hu-PBL-SCID MICE D.E. Mosier, Ph.D., M.D.1, G.R. Picchio, M.S.1, M.B. Kirven1, J.L Gamier, M.D.1, B.E. Torbett, Ph.D.1, S.M. Baird, M.D.2, R. Kobayashi, Ph.D.2 & T.J. Kipps, M.D.2 1 Medical
Biology Institute Pines Road; La Jolla, CA 92037 and 2University of California, San Diego 9500 Gilman Drive; La Jolla, CA 92093
11077 North
Torrey
735
ABSTRACT infection is associated with Burkitt's lymphoma (BL) in normal and immunoblastic B cell lymphomas in immunosuppressed or HIVindividuals infected individuals. SCID mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) from EBV-seropositive donors also may develop spontaneous B cell lymphomas which histologically and phenotypically resemble post-transplant tumors, and are distinct from BL. These tumors always contain EBV DNA. We have noted three different reproducible outcomes depending upon the EBV-seropositive donor used for generation of hu-PBL-SCID mice: (i) no tumors appear; (¡i) tumors appear in a fraction of hu-PBL-SCID mice with a 10-20 wk. latent period; or (iii) tumors appear in all hu-PBL-SCID mice within 6-10 wk. Southern blot analysis of late versus early tumors using a probe specific for the EBV terminal repeat sequences (BamNJ), which allows distinction between circular latent and linear replicating genomes, shows that late tumors do not involve active EBV replication but that early tumors do show replicating genomes. In addition, EBV genomes were monoclonal in late tumors but polyclonal in early tumors. These data suggest two mechanisms for EBV lymphomagenesis, slow outgrowth of rare latently-infected B cells, and more rapid transformation of uninfected bystander B cells by replicating virus. The latter process may by highly amenable to therapy in patients at risk for EBV-related lymphomas. In addition, prospective screening of EBV-seropositive transplant recipients in the huPBL-SCID model may predict the risk of post-transplant lymphoma development.
Epstein-Barr virus (EBV)
INTRODUCTION EBV is a common DNA herpesvirus that infects the majority of the human population. It is associated with several benign clinical manifestations in healthy (infectious mononucleosis) or immunocompromised hosts (lymphocytic interstitial pneumonitis, oral hairy leukoplakia), and three malignant diseases (endemic Burkitt's lymphoma [1], nasopharyngeal carcinoma [2], and immunoblastic B cell lymphomas [3, 4]). The B cell lymphomas occur with high frequency in patients with primary, secondary, or iatrogenic immunodeficiency (5-7), and are thought to reflect inadequate immune surveillance of B cells latently infected with EBV.
Acute clinical infection with EBV results in active viral replication (lytic cycle) in both B cells and epithelial cells of the oropharynx. Following the development of EBVspecific immunity, there is low level persistent viral replication in the oropharynx, and small numbers of latently infected B cells persist in the peripheral blood and lymphoid organs. In addition, normal B lymphocytes can be transformed in vitro by EBV, and only latent gene expression is required for transformation. These latent genes include several EBV nuclear antigens (EBNA-1, -2, -3a, -3b, -3c, -4 or leader protein, LP) and two membrane proteins (LMP-1, LMP-2) (8). Two animal models for EBV infection exist. Infection of cottontop tamirins with high dose EBV leads to the acute development of B cell lymphomas (9). In a more recently developed model, xenotransplantation of human peripheral blood lymphocytes derived from EBV-seropositive donors to SCID (severe combined immune deficient) mice leads to the onset of human B cell immunoblastic lymphomas (10-14). Using the hu-PBL-SCID model, we have performed studies to assess the risk of these lymphomas arising from different donors and to evaluate the risk factors.
736
RESULTS AND DISCUSSION
Tumors generated in hu-PBL-SCID mice appear within 20 weeks of transferring 25-50 x 106 PBL from EBV-seropositive donors i.p. to SCID recipients. We analyzed the overall incidence of tumor formation and the latency period from PBL injection to tumor detection in 85 hu-PBL-SCID mice derived from 10 healthy donors with IgG anti-viral capsid antigen (VCA) titers ranging from 1:80 to 1:640. Two of 10 donors failed to produce any tumors in SCID mice. The results for the remaining 8/10 donors are shown in
Figure
1.
1. Mean tumor incidence (% of injected hu-PBL-SCID mice developing tumors) plotted versus mean latent period to tumor detection (in weeks) in 4-9 mice/donor. Each symbol represents the mean values for 1 of 8 donors. Virtually identical results were obtained in a second experiment utilizing
Fig.
donors
category. These
representing
each
incidence
donors
fell into three categories: high incidence donors who gave tumors in 100% of SCID recipients within 10 weeks, intermediate incidence donors who gave rise to tumors in 40-80% of SCID mice in 10-13 weeks, and low incidence donors who produced tumors in —
(B
.E E
r"Tr¥+-HE
DONOR TUMOR INCIDENCE
Fig. 2. Southern blot analysis of EBV genomes recovered from hu-PBL-SCID tumors. BamH1 restricted DNA was probed with the Bam NJ probe, which distinguishes between circular episomal DNA and linear replicating DNA, as illustrated on the left side of the figure. Results are summarized schematically on the right side of the figure. 738
studies may be useful in predicting risk for the associated B cell lymphomas.
development
of
post-tranplant or AIDS-
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13. 14.
Henle, G., W. Henle and V. Diehl. 1968. Relationship of Burkitt's tumor-
associated herpes-type virus to infectious mononucleosis. Proc. Nati. Acad. Sei. USA 59:94. Raab-Traub, N., K. Flynn, G. Pearson, A. Huang, P. Levine, A. Lainer and J. Pagano. 1987. The differentiated form of nasopharyngeal carcinoma contains Epstein-Barr virus DNA. Int. J. Cancer 39:25. Hanto, D.W., G. Frizzera, K.J. Gajl-Peczalska and R.L. Simmons. 1985. EpsteinBarr virus, immunodeficiency, and B-cell lymphoproliferation. Transplantation 39:461. Cleary, M.L, M.A. Nalesnik, W.T. Shearer and J. Sklar. 1988. Clonal analysis of transplant-associated lymphoproliferations based on the structure of the genomic termini of the Epstein-Barr virus. Blood 72:349. Cleary, M.L., R.F. Dorfman and J. Sklar, Failure in immunological control of the virus infection: Post-transplant lymphomas, in The Epstein-Barr Virus: Recent Advances, M.A. Epstein and B.G. Achong, Editor. 1986, William Heinemann Medical Books: London, p. 163. Beral, V., T. Peterman, R. Berkelman and H. Jaffe. 1991. AIDS-associated nonHodgkin lymphoma. Lancet 337:805. Harnly, M.E., S.H. Swan, E.A. Holly, A. Keltner and N. Pardian. 1988. Temporal trends in the incidence of non-Hodgkin's lymphoma and selected malignancies in a population with a high incidence of acquired immunodeficiency syndrome (AIDS). Am. J. Epidemiol. 128:261. Young, L., C. Alfieri, K. Hennessey, H. Evans, C. O'Hara, K.C. Anderson, J. Ritz, R.S. Shapiro, A. Richinson, E. Kieff and J.I. Cohen. 1989. Expression of EpsteinBarr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease. N. Engl. J. Med. 321:1080. Young, L.S., S. Finerty, L. Brooks, F. Scullion, A.B. Rickinson and A.J. Morgan. 1989. Epstein-Barr virus gene expression in malignant lymphomas induced by experimental virus infection of cottontop tamarins. J. Virol. 63:1967. Mosier, D.E., R.J. Gulizia, S.M. Baird and D.B. Wilson. 1988. Transfer of a functional human immune system to mice with severe combined
immunodeficiency. Nature (London) 335:256. Mosier, D.E., S.M. Baird, M.B. Kirven, R.J. Gulizia, D.B. Wilson, R. Kubayashi, G. Picchio, J.L. Garnier, J.L. Sullivan and T.J. Kipps, EBV-associated B cell lymphomas following transfer of human peripheral blood lymphocytes to mice with severe combined immune deficiency, in Current Topics in Microbiology and Immunology: Mechanisms in B-Cell Neoplasia 1990, M. Potter and F. Melchers, Editor. 1990, Springer-Verlag: Berlin-Heidelberg, p. 317. Okano, M., Y. Taguchi, H. Nakamine, S.J. Pirruccello, J.R. Davis, K.W. Beisel, K.L. Kleveland, W.G. Sanger, R.R. Fordyce and D.T. Purtilo. 1990. Characterization of Epstein-Barr virus-induced lymphoproliferation derived from human peripheral blood mononuclear cells transferred to severe combined immunodeficient mice. Am. J. Pathol. 137:517. Cannon, M.J., P. Pisa, R.I. Fox and N.R. Cooper. 1990. Epstein-Barr virus induces aggressive lymphoproliferative disorders of human B cell origin in SCID/human chimeric mice. J. Clin. Invest. 85:1333. Rowe, M., L.S. Young, J. Crocker, H. Stokes, S. Henderson and A.B. Rickinson. 739
virus (EBV)-associated lymphoproliferative disease in the model: Implications for the pathogenesis of EBV-positive lymphomas in man. J. Exp. Med. 173:147. Raab-Traub, N. and K. Flynn. 1986. The structure of the termini of the EpsteinBarr virus as a marker of clonal cell proliferation. Cell 47:883.
1991. SCID 15.
Epstein-Barr mouse
ASSOCIATION OF EPSTEIN-BARR VIRUS WITH PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA IN AIDS EITHNE M.E. MACMAHON MB MRCPI, JONATHAN D. GLASS MD, S. DIANE HAYWARD, PhD, RISA B. MANN, PATRICIA CHARACHE MD, JUSTIN C. MCARTHUR MB, RICHARD F. AMBINDER, MD PhD
JOHNS HOPKINS SCHOOL OF MEDICINE
BALTIMORE, The
MD 21205
of primary brain lymphoma in the acquired immunodeficiency syndrome was recognized early in the epidemic. Subsequently systemic non-Hodgkin's lymphomas were also designated as AIDS-defining illnesses. Epstein-Barr virus (EBV)associated lymphoproliferative disease and lymphoma is well recognized in the in the postorgan transplant setting. These tumors are uniformly associated with Epstein-Barr virus and such an association might have been anticipated for AIDS-associated lymphomas. However, less than half of these tumors were found to contain EBV. EBV has been associated with primary central nervous system (CNS) lymphoma both in patients with and without a history of immune deficit. In order to better characterize the association of EBV with primary CNS lymphoma in AIDS we examined tumor tissue from a consecutive series by in situ hybridization (3). Epstein-Barr virus is characteristically latent in host B-lymphocytes, the genomic DNA existing as nuclear plasmids with limited antigen expression. The products of two EBV genes, the EBERs 1 and 2, are pol DI RNA transcripts which exist as nucleoproteins, present in great abundance in the nuclei of latently infected B-lymphocytes (2). We used EBER 1 as a target for in situ hybridization with labeled riboprobes (5). The technique was first refined in formalin-fixed paraffin-embedded pelleted EBV negative and positive cell lines before application to brain sections. The hybridization signal was nuclear with nucleolar sparing. No signal was detected with the sense probe. Examination of a consecutive autopsy series of 19 AIDS-associated primary CNS lymphomas showed expression of EBER 1 RNA in all 19 cases, indicating the presence of EBV. The strong positive signal was confined to tumor cells. The specificity of the technique was confirmed by examination of a wide range of brain tissue controls. No evidence of cross hybridization was seen with primary HIV infection, cytomegalovirus encephalitis, toxoplasmosis, progressive multifocal leukoencephalopathy or other opportunistic CNS infections arising in AIDS. Nor was signal detectable in herpes simplex encephalitis or in normal brain. We used this technique to examine systemic lymphomas in AIDS patients and detected EBV in just 3 of 7 cases. Primary brain lymphoma tissue from 15 patients without known occurrence
740
Fig. 1. AIDS-associated primary brain lymphoma. High power views of serial sections, (a) hematoxylin and eosin (b) in situ hybridization with digoxigenin-labeled antisense EBER1 riboprobe and counterstained with eosin. immune
deficiency were also tested, of which just one was EBV positive. A single case of CNS primary lymphoma occurring after kidney transplantation was positive. The detection of EBV in all cases of AIDS-associated CNS lymphoma examined suggests that this virus may well play a part in the pathogenesis of these tumors. To further examine the role of EBV we looked for expression of the Epstein-Barr latency membrane protein (LMP). Immunostaining with pooled anti-LMP monoclonal antibodies (4) showed LMP expression in half of the cases. Of the handful of EBV proteins expressed in latency, two have known oncogenic potential-latency membrane protein (LMP) and EB nuclear antigen 2 (EBNA-2). In vitro, LMP expression can transform immortalized rodent cells. Recently LMP has been shown to protect B-cells from apoptosis by induction of the BCL-2 protein (1). LMP is expressed in several other EBV-associated tumors including nasopharyngeal carcinoma, Hodgkin's disease, and post-transplant lymphoma but not African Burkitt's lymphoma. The identification of LMP in this series provides further evidence that EBV may have a role in tumorigenesis. Detection in only a proportion of the tumors might reflect variable quality of fixed tissue and/or variable LMP expression. The association of EBV with primary CNS lymphomas in AIDS has implications not only for understanding their pathogenesis but also for diagnosis and management. The characteristic periventricular location of tumors suggests that EBV DNA might spill into the spinal fluid from dying tumor cells even in the absence of lymphomatous meningitis. In a pilot study to test this hypothesis, PCR amplification for EBV was applied to CSF specimens from HIV- positive patients. EBV was detected in three of seven specimens from patients with primary CNS lymphoma and from one of 42 patients without lymphoma. The observation that EBV DNA is not usually present in the CSF of HIV-positive patients but may be detected in the CSF of those with CNS lymphoma, has led to the ongoing prospective evaluation of EBV as a tumor marker in this setting. References 1. Henderson, S., M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, and A. Rickinson. 1991. Induction of bd-2 expression by Epstein-Barr virus 741
latent membrane 65:1107-1115.
protein 1 protects
infected B cells from
programmed cell death.
Cell
2. Howe, J.G. and J. A. Steitz. 1986. Localization of Epstein-Barr virus-encoded small RNAs by in situ hybridization. Proc. Nati. Acad. Sei. USA 83:9006-9010.
3.
MacMahon, E.M.E., J. D. Glass, S. D. Hayward, R. B. Mann, P. S. Becker, Charache, J. C. McArthur, and R. F. Ambinder. 1991. Epstein-Barr virus in AIDS-related primary central nervous system lymphoma. Lancet 338:969-973.
P.
4. Rowe, M., H. S. Evans, L. S. Young, K. Hennessy, E. Kieff, and A. B. Rickinson. 1987. Monoclonal antibodies to the latent membrane protein of Epstein- Barr virus reveal heterogeneity of the protein and inducible expression in virus-transformed cells. J. Gen. Virol. 68:1575-1586. 5. Wu, T-C, R. B. Mann, P. Charache, S. D. Hayward, S. Staal, B. C. Lambe, and R. F. Ambinder. 1990. Detection of EBV gene expression in Reed-Sternberg cells of Hodgkin's disease. Int. J. Cancer 46:801-804.
STUDIES ON A TYPE D RETROVIRUS ISOLATED FROM AN AIDS PATIENT LYMPHOMA Richard J. Ford1 Lawrence A. Donehower2, and Robert C. Bohannon1'2 ,
^.D. Anderson Cancer Center 2Baylor College of Medicine, Houston, TX 77030 ABSTRACT The development of AIDS-related lymphomas (ARL) has been on the rise in recent years. During an analysis of ARL from AIDS patients, one individual developed atypical syncytial variants of high-grade Burkitt'sHowever, the type B-cell lymphomas, which prompted further study. search for a HIV-1 retrovirus, which we hypothesized was infecting these cells, led to the subsequent discovery of a type D retrovirus in two earlyNucleotide and passage lymphoma cell lines derived from this patient. amino acid sequence analysis, as well as immunologie reactivity, indicated that the virus was closely related to Mason-Pfizer monkey virus (MPMV) simian retrovirus type 1 (SRV-1). or MPMV and SRV-1 are immunosuppressive type D retroviruses that cause an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by the polymerase chain reaction generated MPMV-specific fragments indicative of infection by a retrovirus similar to 742
MPMV.
Additionally,
the patient's serum contained antibodies that recognized type D retroviral env proteins (gp20 and gp70) and gag proteins (p27 and pl4) as assayed by immunoblot and radioimmunoprecipitation techniques. Although there have been reports of human cell lines infected with type D retroviruses and of type D reactive human sera, this is the first report of a type D retrovirus infection in a human confirmed by virus isolation, serum immunoreactivity, and viral DNA identification in tumor tissue.
INTRODUCTION Recent reports indicate that there is an increasingly high incidence of non-Hodgkin's lymphomas among HIV-1 positive individuals (1). AIDS related lymphomas (ARL) have been found to consist primarily of the Burkitt's-type (small non-cleaved cell) or immunoblastic, high grade lymphomas (2). Although the HIV-1 virus has been hypothesized to be involved in lymphomagenesis in these patients, repeated attempts to isolate HIV-1 from ARL cells have been unsuccessful (3). Therefore, it was of particular interest to us when an ARL patient presented with a previously unrecognized morphologic syncytial variant of Burkitt's lymphoma that we originally thought might represent infection by HIV-1 A retrovirus was subsequently isolated from two Bor an HIV-1 variant. cell lymphoma cell lines derived from the patient's original biopsy material. Further analysis indicated that the retrovirus was not HIV-1, as originally hypothesized, but rather a type D retrovirus closely related to the Mason-Pfizer monkey virus (MPMV), a causative agent of simian AIDS
(4).
MATERIALS AND METHODS Patient. The patient studied was a 32-year-old homosexual male hospital administrator who complained of abdominal pain, fever, and night sweats prior to admission to an outside hospital. He was admitted to the University of Texas M. D. Anderson Cancer Center with generalized lymphadenopathy and the presumptive diagnosis of AIDS-related lymphoma (ARL). The patient' was diagnosed as having AIDS and a serum A bone sample obtained at admission showed HIV-1 seropositivity. marrow biopsy revealed extensive involvement with a high grade A malignant lymphoma of the small non-cleaved (Burkitt's) type. combination chemotherapy regimen was begun and a partial remission In spite of this treatment, however, the patient later was achieved. developed B-cell lymphomas at multiple sites and subsequently died of progressive lymphoma and sepsis approximately 9 months after admission. Permission to perform an autopsy was denied and no other tissues could be obtained for further analysis. Cell lines were established 743
from lymphoma tissue obtained from three distinct sites of lymphoma involvement that occurred during the course of the patient's disease. Further clinical and pathologic information on this patient is described elsewhere (5).
Molecular and sérologie and sérologie analysis were
analysis. The techniques recently described (6).
used for molecular
RESULTS
Upon admission, patient revealed that
diagnostic bone marrow biopsy (fig. 1) from the a monomorphous non-Hodgkin's lymphoma had infiltrated the normal marrow. Histopathologic examination of this lymphoma and of a subsequent lymphoma isolated from the patient's kidney at a later date (fig. 2) revealed that multinucleate syncytial-like cells were present in the original biopsies. Cell lines were established from these lymphomas and electron microscopic examination revealed the presence of retrovirus-like particles both within intracellular vesicles (fig. 3), as well as extracellularly (6). A reverse transcriptase assay performed on twice glycerol gradient purified virus from these cell lines using a poly r(A):oligo d(T) template-primer showed a low level of activity (fig. 4) as compared to HIV-1 ihr, even though an estimated 108 viral particles per ml A screen for the presence of HIV-1 p24 by an antigen were present. method (Abbott) failed to detect this HIV-1 diagnostic core capture a
Fig. 1. Patient diagnostic bone marrow biopsy. A diagnostic bone marrow biopsy was taken from patient RC, formalin fixed, and paraffin embedded. The low-power photomicrograph (lOx) of the H&E stained, diagnostic bone marrow biopsy specimen shows replacement of normal marrow (between bony spicules) with monomorphous lymphoma cells (arrow). 744
Fig. 2. Photomicrograph of kidney aspirate of patient RC. A high-power photomicrograph (lOOOx) of the original aspirate of a kidney lymphoma (RC^) from patient RC. Large syncytial cells are indicated by arrows and are in contrast to the usual Burkitt's lymphoma cells surrounding these atypical cells. detect HIV-1 by Southern and PCR analysis characterization of the reverse transcriptase of the viral isolate revealed that there was a preference for poly r(C):oligo d(G) over poly r(A): oligo d(T) (Table 1). The patient's serum recognized many of the proteins of the viral isolate as compared to most other HIV-1 Cell specific infectivity of primary B cells, seropositive sera tested (fig. 5). T cells, and macrophage/monocytes revealed the isolate could infect both B In and T cells, as assayed by reverse transcriptase activities (Table 2). in the isolate caused syncytia formation addition, primary peripheral blood B-cells and in Raji cells similar to that seen in the original patient diagnostic biopsies taken at the time of admission (6). In order to determine what type of virus was present in the lymphoma cells, we utilized degenerate primers to an amino acid region within the pol gene that is conserved for all known retroviruses and amplified a 135 bp pol fragment from the viral isolate (6,7). The amplified fragment was cloned, sequenced, and compared to known sequences in GenBank. The sequences proved to be related to the MasonPfizer monkey virus (MPMV) and to simian retrovirus type 1 (SRV-1), a type D retrovirus responsible for causing simian AIDS in monkeys. Amino
protein. All attempts to proved negative. Further
745
r,
,.
«. "
Fig. 3. Electron Micrograph of cell line RCBM. Electron microscope examination (20,000x) of the bone marrow derived cell line (RCBM) revealed type D retroviral like particles present in both intracytoplasmic vesicles as well as outside the plasma membrane. An arrow points to one of the extracellular retroviruses that has a barrel shaped core indicative of a type D retrovirus. 800
30000
20000
CPM for HIV
CPM for CB
(rA):(dT)
(rA): receptor. In contrast, interferon gamma interacts with a distinct receptor (IFN-yR). I would like to focus on the interferon gamma receptor (IFN-yR). Fig. 3 provides a perspective of that receptor with respect to the other cloned receptors. We and others have cloned the interferon gamma receptor (IFN-yR) shown here, and also the interferon
777
INTERFERON ACTION
Fig.
2. Illustration of interferon action.
alpha receptor (IFN-aR) shown schematically in perspective with the LDL, EGF, PDGF, insulin, LDL, IL-2 (p55) and CD-4 receptors. As I noted, I shall focus on the interferon gamma receptor. Although I shall not have time to discuss the interferon alpha receptor, I would speculate that the principles determining how both of these receptors function are probably going to be very similar. There are some unique features of the interferon receptors that are not present or exhibited to the same degree in most other cytokines. In the first place the interferons demonstrate high species specificity, particularly interferon gamma. Human IFN-y (HuIFN-y) is not active on mouse or hamster cells, for example; and mouse IFN-y (Mu-IFN-y) is not active on human cells, even at enormous concentrations. You will see shortly that the species specificity even goes beyond the surface receptors. f800
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LDL
IFN-y IFN-oc
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CD4
(p55)
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Jh20 kb appear (Makos et al., 1992). What is perhaps most important about this methylation abnormality, is that it appears in the benign adenomas which are thought to be precursor lesions for colon cancers. In the adenoma DNA, the methylation of the YNZ22 region is not as extensive as in the Six to 8 kb Not I fragments are seen instead of the normal 5 kb, but cancers. the >20 kb fragments are not present. Thus, abnormal methylation of YNZ22 regions begins in benign colon adenomas and increases in extent with the transition of these lesions to malignant tumors. An important aspect of the above timing for YNZ22 region methylation is that they precede the structural alterations of chromosome 17p which In 6 of 7 adenomas examined, the are characteristic of colon cancers. abnormal 17p events were found in the absence of 17p allelic loses or p53 gene mutations (Makos et al., 1992). In contrast, the more extensive methylation changes in the cancers were all associated with loss of one 17p YNZ22 and one p53 alíele and with point mutations of the remaining p53 alíele. As we have previously reviewed elsewhere (Baylin et al., 1991), these dynamics for appearance of abnormal methylation of chromosome 17pl3.3 in colon cancer evolution raise at least two possibilities for functional consequences. First, the location of CpG islands often denotes the position of associated genes. The potential for CpG island methylation to inhibit, directly, expression of such genes has been reviewed earlier in this talk. Although, the p53 gene at chromosome 17pl3.1 is the only well defined tumor suppressor gene on 17p, increasing evidence for additional, more telomerically located, It will be of interest to determine if the defined genes has been accruing. pYZ22 methylation abnormalities may participate in decreased function of such genes. events
Second, the above data illustrate an additional potential consequence of abnormal CpG island methylation in tumor cell DNA that we have also reviewed elsewhere (Baylin et al., 1991). Such methylation has been temporally associated in the genetic mental retardation syndrome, Fragile-X, with instability and structural changes of the putative gene for this disorder (see Oberlé et al., 1991). Methylation induced delays of replication of DNA have been postulated to enhance the instability of DNA fragile sites. Thus, extending abnormal methylation of a region such as YNZ22 during tumor progression could be closely linked to the allelic losses, rearrangements, and other chromosomal structure changes which are critical to cancer evolution. With respect to this discussion, it is important to note that patterns of abnormally increased DNA methylation may be non-randomly associated with chromosome changes specific to individual types of human tumors. We have seen 815
examples of this cell lung cancer
on
DNA
regions
(Makos
of chromosome et
al., 1992).
3p consistently deleted
in small
Now very briefly, I wish to discuss the third component of DNA methylation imbalance in neoplasia, overexpression of the DNA MTase gene. Time precludes discussing the fascinating events accompanying evolution of this gene from prokaryotes to higher order eukaryotes (reviewed in Bestor, 1990). The eukaryotic genes have been cloned from rodents (Bestor, 1990), and recently by our lab, from humans (Yen et al., submitted for publication). Each gene expresses a 5 kb transcript. In general, steady state levels of this mRNA are very low in normal compared to neoplastic tissues and cell lines (for review Baylin et al., 1991). Perhaps, our most interesting observation regarding DNA MTase gene expression again goes back to the colon cancer progression model. Using a PCR assay, we have found increased expression of this gene at the steady state transcript level in normal appearing colonie mucosa from patients with benign adenomas or colon cancers. Thus, in DNA MTase DNA of the changes overexpression methylation may precede patterns that first appear in the DNA of benign adenomas and become more
extensive in the colon cancers. The levels of DNA MTase gene expression continue to increase in the adenomas and colon cancers (El-Deiry et al., 1991) and thus may play a key role at each stage of tumor progression. SUMMARY Let
summarize by reviewing a model which is meant to raise as many it answers (Fig. 2). What I have discussed today are data questions that suggesting during progression of solid tumors, like colon cancer, an increased cellular DNA methylating capacity characterizes the initial stages of multi-clonal hyperplasia. Despite this increase, the altered pattern of DNA methylation which subsequently emerges is largely manifest by a widespread hypomethylation of DNA. However, on a more regional basis, areas of me as
hypermethylation appear which can affect strategic areas such as normally unmethylated CpG islands. These shifted DNA methylation patterns have the capacity to both follow, or cause, chromatin changes that can both directly
silence genes critical for normal cell maturation and/or participate in the structural chromosome changes which constitute genetic instability during -
Clonal
Multiclonal
O
Hyperplasia
Clonal Cancer
Benign
Lesion
Loss of Gene Function anci
~g
T
"V
DNA
\
Normal Maintenance
Hypomethylation +
Regional Hypermethylation
DNA
ethylation
Imbalance
Fig. 3. A working model for the molecular mechanisms underlying the evolution of abnormal DNA methylation patterns in neoplastic cells. The top panel depicts the regulation of DNA methylation in a normal cell. This diagram, adapted from Holliday (23), shows the progressive fate of a methylated CpG dinucleotide (*) as it passes through a DNA replication fork. A molecule of the DNA-MT enzyme is strategically located at the fork, and enzyme activity is activated, as shown by the + arrow, with onset of DNA replication. This structural and biochemical halance ensures methylation of the CpG dinucleotide on the newly synthesized daughter strand and thus preserves the normal methylation pattern for the cell type. A feedback repression of DNA-MT activity, shown by the arrow, could occur either via a signal from methylated DNA beyond the replication fork or from cessation of DNA replication activity. The bottom panel depicts the possible dynamics underlying the DNA methylation imbalance characteristic of neoplastic cells. Abnormal numbers of DNA replication events, indicated by the heavy bars on each DNA strand at the replication fork, may provide pressure for increased DNA-MT activity as shown by the heavy shaded + arrow at the replication fork. The critical balance between the DNA-MT enzyme and the replication fork could be disrupted either by a change in chromatin structure or a change in shape of the nuclear matrix. In principle, these dynamics would have three major effects on methylation. First, the maintenance methylation function would be disrupted, as seen on the upper post-replication fork strand, resulting in "missed sites" and widespread hypomethylation. Second, the altered position and increased activity of the DNA-MT would lead to new sites of methylation, as depicted on the bottom strand, resulting in regional hypermethylation. Finally, there would be a block to the feedback loop that normally controls DNA-MT levels (depicted by disruption of the arrow), resulting in increased synthesis of the enzyme. This block might arise because of the continued DNA replication or because of failure of a normal maintenance methylation pattern to be achieved. -
-
progression (Fig. 2). I suggest that one must view these changes as an interchangeable cycle of events during tumor progression. The chromatin changes and abnormal methylation patterns can drive one another with increasingly deleterious effects as the malignant phenotype emerges (reviewed in Baylin, 1991). tumor
What
working
A are the molecular events that would initiate the above dynamics? As discussed for the normal adult construct model is shown in Fig. 3.
cell, there
is
a
strategic location of DNA MTase, synthesis at replication forks (top cancer cells, perhaps failure of cells
delicate balance between the
regulation of this enzyme, and rate of panel, Fig. 3). In pre-neoplastic and
DNA
817
exit the cell cycle and halt DNA replication, facilitates some sort of pressure to increase cellular DNA methyltransferase activity (bottom panel, Fig. 3). This increase may involve loss of feedback inhibition of the enzyme during the post DNA replication phase. There are also probable structural alterations in the nucleus which may alter the geographic relationship between In consequence, many DNA areas that the DNA replication fork and DNA MTase. should be getting methylated do not, and novel areas of methylation also arise. This cycle of events leads to the imbalance of DNA methylation that I Future investigations of these possibilities, and of their have talked about. specific consequences for alterations of gene expression and chromosome structure, may reveal a key molecular step underlying virtually all stages of to
tumor
progression. Thank you.
MODERATOR : Yes, thank you very much, Dr. Baylin. May I perhaps open the discussion by saying that perhaps you have a hypothesis that applies to some genes and doesn't apply to others. Can you make any predictions then as to which genes it would apply to and which ones not? That is, whether methylation precedes, or hypermethylation precedes or follows other events. events I have discussed may and both of at in least two ways affect the specific expression of genes these probably have to do, primarily, with a loss of gene expression. Despite the widespread hypomethylation in tumor DNA, I'm not aware of any documentation that this has caused expression of a specific gene to be activated. I think this is expected because hypomethylation of a gene, especially in the regulatory region, is only a permissive event for gene transcription. Specific activation obviously depends on other factors including changes in transcription factors. If the hypomethylation is functionally important, I would predict that it participates in abnormalities of chromosome and chromatin structure which may actually contribute to gene loss. What I have also tried to suggest is that it is even more likely that the regional hypermethylation events I described are the most critical for silencing certain genes and that the involved genes would be tumor suppressor In this genes. The myo-D gene example I gave is illustrative of this point. a case, transcription factor important to cell differentiation is hypermethylated in fibroblasts when reactivated by treating the cells with the demethylating agent 5-Azacytidine, the cells demonstrate myoblast features. I think, then, it's going to be these kinds of genes which, when involved in hypermethylation events during tumor progression, contribute to
BAYLIN:
I think that the abnormal
methylation
-
-
the tumor
phenotype.
The other thing I stressed is that allelic loss and instability, which also results in the loss of functional genes during tumor progression, could be a major consequence of abnormal DNA methylation in CpG rich areas. As discussed, the CpG islands around genes normally are positioned in early replication areas. When methylated, the replication of these regions would be delayed and this could enhance fragility of such sites. This might then play some role in allelic losses. MODERATOR:
QUESTION:
Yes, go ahead. What is known about the
methyltransferase genes?
transcriptional,
818
or
other
regulation
of DNA
BAYLIN:
Are you
The
QUESTION:
asking
me
what is known about the
transcription?
regulation.
BAYLIN: The regulation. Two things are known. DNA MTase activity and steady state mRNA levels rise sharply when cells enter proliferation and the S phase of the cell cycle. The early evidence is that much of this regulation may be Little else is known about regulation of at the post-transcriptional level. and the eukaryotic DNA MTase the transcriptional region of the gene has not been studied in detail. told me that we have more time available than therefore, with your permission, Dr. Feldman, I will ask another question. I don't think there are any other questions that I see. With respect to your answer to my question, of course you know that 5-azacytidine will cause further gene expression, but I didn't understand your statement that you only expect to find genes turned off, not turned back on. MODERATOR: I thought,
BAYLIN:
Dr. Feldman has
just
so
The
azacytidine
point
is very well taken.
However, in
most instances
where 5-
expression, particularly in cultured, immortalized be doing it by reducing abnormally increased regional and Robin Holliday and others pointed out some years ago, that genes in cultured group has more recently emphasized this
turns on gene
cells, it may
methylation.
Adrian Bird's
-
cells can be silenced by increased methylation. There are well described instances where cell auxotrophy for metabolic pathways is based on silencing of at least one copy of a gene by this mechanism. There are a number of examples where 5-azacytidine will relieve auxotrophy for such cells. Again, I return to the myo-D gene story, where abnormally increased methylation is being relieved by 5-azacytidine to reactivate gene expression. -
MODERATOR:
Well, thank you very much,
Dr.
Baylin.
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DNA
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M.
Makos,
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Wu, R.-W.C. Yen, A. de Bustros, P. Vertino and B.D.
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Bestor, T.H. 1990. DNA methylation: evolution of
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A
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Latif,
chromosome inactivation from studies of and replication, and vice versa. Genet.
Oberlé, I., F. Rousseau, D. Heitz, C. Kretz, D. Devys, A. Hanauer, J. Boue, M.F. Bertheas and J.L. Mandel. 1991. Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 24:1097-1102. Tazi, J. and
A. Bird. 1990. Alternative chromatin structure at
Cell 60:909-920.
CpG islands.
"A TRANSGENIC MOUSE MODEL FOR MEN 2"
Nicholas Schulz and George F. Vande Woude ABL-Basic Research Program NCI-Frederick Cancer Research & Development Center P.O. Box B Frederick, MD 21702-1201
MODERATOR: Dr. Gallo us go to the next Schulz is speaking
then, let Nick
points out we are way behind, way over time. Okay, speaker. George Vande Woude could not be here, and on his behalf about oncogenes, cell cycle and
antineoplastic drugs. Nick?
SCHULZ: Hello. Dr. Vande Woude could not come this afternoon, and instead I'd like to let you know about the characterization of a model system involving transgenic mice that bears remarkable resemblance to a familial neoplastic syndrome, namely MEN Type 2. Dr. Vande Woude's laboratory, has been interested in determining the function of the Mos proto-oncogene. To refresh your memory, c-Mos is the cellular homologue of the viral mos oncogene of the Moloney Murine sarcoma virus, and has been isolated from several vertebrate species. It is always present as a single coding exon that has amino acid homology with the sre family of protein kinases. The transforming activity of this oncogene is manifest when constitutively expressed at very low levels in NIH3T3 fibroblasts. In fact, to display that it is expressed in transformed cells, it is necessary to demonstrate this via RNase protection assays, or SI nucleus protection assays. As I said, it consists of one exon, usually between 800 base pairs and 1 KB, and the expression of this proto-oncogene is present in low levels in brain, kidney and mammary gland, as well as epididymis, heart and lung. It had for the first time shown to have been present in high levels in embryo, and most high in germ cells. The initial 820
characterization of the activity of this proto-oncogene showed that it is necessary for maturation of both sperm and oocytes. Because of c-Mos's potential role in cell cycle regulation, we sought to define what aberrant expression of this proto-oncogene would have on transgenic mice. A Moloney virus, LTR, in tandem with the murine c-Mos proto-oncogene, was introduced by conventional techniques into one-cell embryos. The most obvious characteristic, or phenotype of these mice is that they won't hold still when you take their picture. That is to say, here is one such mouse, trying to get away sometimes the way from the camera, and only succeeding in chasing its tail some of us feel while we have been trying to characterize this oncogene. --
The other manifestation the obvious manifestation within a few weeks of these mice's life is they have remarkable development of cataracts. In normal development of lens tissue, one has the orderly differentiation of lens fibers along the periphery. They migrate to the center, lose their nuclei, and become absolutely crystalline, and produce a functional lens. In the transgenic mice, In fact, the cells continue to proliferate, and at one has no loss of nuclei. about 2-4 weeks they'll rupture the lens, and eventually fill the entire cavity of the eye with tissue that has failed to differentiate. --
Currently we have four lines of mice generated on two different backgrounds. The lines used were the FVB/N background, and the Black 6 background. The FVB/N background was derived in conjunction with Dr. Heiner Westphall. One interesting point is that when these mice, despite the severe neurological phenotype age to about 8-12 months and are sacrificed, one has a large number of pheochromocytomas, which are tumors of the adrenal medulla, and medullary thyroid neoplasms. These two tumors are hallmark lesions of multiple endocrine neoplasia Type 2. It is of Pheochromocytomas originate in the adrenal medulla. neuroendocrine origin and secretes the hormones adrenalin and noradrenalin. The outer portion, the cortex is responsible for the secretion of mineralocorticoids and glucocorticoids. the
The adrenals of older animals contain presentation of these tumors most often
than one tumor with being bilateral.
more
If one takes these adrenal glands and stains them with neuron-specific this work is done in markers; in this case, neuron-specific enolase with Linnoila of the branch of Dr. NCI one sees again an conjunction Navy identical histological phenotype as in human tumors. --
--
The pheochromocytomas are not the only tumor found in the transgenic mice. About 55-60% of mice in the second line studied will present with either medullary thyroid carcinoma, or C cell hyperplasia of the thyroid. Again, these tumors are of neural crest tissue origin. Like the pheochromocytomas of the line 1 animals, the thyroid lesions of the line 2 animals are multifocal and the tumors are bilateral, indicating that they also arise in a polyclonal fashion. Line 4 Mos transgenic animals develop multifocal pheochromocytomas as well as medullary thyroid neoplasms, with animals quite often having both types of tumors (Table 1).
In contrast to line 1, 2, and 4 mice, the line 3 Mos transgenic mice fail develop pheochromocytomas or C-cell thyroid neoplasms above the sporadic background level observed in the nontransgenic control animals (Table 1), even though their lens-fiber development defect and neuropathology are indistinguish-
to
able from the other lines.
821
Table 1
Tumor incidence in
transgenic and control mice Percentage of mice with tumors
Transgenic mouse
Background
line
1
Number of animals
79/75A
Medullary thyroid neoplasia only 0%
0/0
4%
1/5
62%
40/56
39/44
0%
0/0
63%
19/33
4%
0/3
66%
19/36
89
48/41
2%
1/1
0%
0/0
0%
2%
1/1
B6C3
62
29/33
23%
7/7
13%
4/4
32%
0/0 8/12
68%
19/23
FVB/N
46
21/25
2%
1/0
0%
0/0
0%
0/0
2%
1/0
154
2
83
3
FVB/N
4
Control
58%
number of mal es/females.
To demonstrate that the tumors observed in these animals
Mos
Total
Both
39/51
FVB/N FVB/N
AActual
Pheochromocytomas only
were
related to
expression, we examined the adrenal and thyroid tumors from line 4 Mos transgenic animals for Mos RNA expression by Northern analysis. We found that the adrenal and thyroid tumors, like the affected brains, express high levels of Mos RNA. The unaffected organs, like liver and kidney, however, are negative by Northern analysis, and the tissues from nontransgenic control litter mates show little or no expression of Mos RNA. When the thyroid tumors are examined via in situ hybridization for mos expression, one sees high level expression of the transgene specifically over areas of tumor involvement. The tumor presentation pattern was line-dependent (Table 1), and suggested that the transgene integration site or background of the animal played an important role. To evaluate this, the three Mos transgenic FVB/N lines were crossed with BALB/c mice (Table 2). The same disease phenotype was seen in the Fj animals of the first two lines. The f1 animals of the line 3 X BALB/c cross, however, display a high frequency of both pheochromocytomas and medullary thyroid C-cell carcinomas. Neither neoplasm is found in the parental line. Thus, it is the integration site and/or background which affects the penetrance of the transgene on phenotype. Table 2
Tumor incidence in
offspring
of
transgenic mice crossed
to BALB/c mice
_Percentage of mice with tumors_ Number of animals
Cross 1 X
49
21/29A
2
BALB/c X BALB/c
31
BALB/c
15
16/15 7/8
3 X
AActual
Pheochromocytomas only 51%
Medullary thyroid neoplasia only 2%
0%
12/13 0/0
52%
7%
1/0
33%
0/1
Both
Total 65%
7/9
1/5 16% 1/4
68%
13/19 8/13
3/2
20%
60%
5/4
12%
1/2
number of mal es/ females.
The variation of tumor presentation pattern between the transgenic lines is similar to that observed in humans with MEN 2. In the human syndrome, the tumor presentation pattern is consistent within a kindred. The most commonly observed pattern of tumor formation in humans with MEN 2 is the presence of both medullary thyroid carcinomas and pheochromocytomas, as we observe in the line 4 animals. Staining the thyroids of line 1 animals for calcitonin revealed 822
evidence for C-cell hyperplasia, the precursor lesion of medullary thyroid carcinoma. The study of these two lines could reveal mechanisms whereby there is preference given to the predisposition towards one tumor type or the other. For example, it is possible that the differences are due to the time that the transgene is expressed during development. A far less common form of MEN 2 is exhibited by patients who present solitary medullary thyroid carcinomas. These patients never develop pheochromocytomas, which is very similar to the presentation observed in the line 2 Mos transgenic animals.
The differences in tumor presentation patterns in the various lines may be attributable to variations in the level of transgene expression or may reflect earlier activation of expression in the target organ of the tumor-bearing lines. This could be due to the transgene integration site or the genetic background used. As illustrated in Table 2, the interaction of transgene with genetic background can influence the penetrance of the phenotype. The tumor phenotypes of the Fj progeny of the FVB/N Mos transgenic lines crossed with BALB/c may change because of alterations in transgene expression. Alternatively, it may be that a second genetic event (as suggested by the long latent period before disease is observed in these animals) may be suppressed in line 3 or predisposed to in the F1 progeny. These transgenic lines provide a valuable opportunity to study the genetic and molecular basis of tumor induction and may help in
elucidating
the mechanism of tissue
MODERATOR:
Thank you.
QUESTION:
Could you go
in these tumors?
over
targeting
in the human
again the correlation
syndrome.
with
expression of
Mos
The correlation is such that if you take a look at any one in the animal selected for a malignant clone, and they do consistently express high levels of Mos. We have some preliminary data. We've taken tumors from humans with MEN 2. Out of nine primary tumors, only one has had a low level of Mos expression. We've looked at sporadic pheochromocytomas I've not been able to demonstrate sporadic pheochromocytomas, out of ten cases Out of ten primary neural blastomas similarly, no Mos any Mos expression. expression. One thing I should bring up is the MEN 2 has been mapped to chromosome 10. Mos in the human is on chromosome 8. SCHULZ:
tumor, you
--
-
-
MODERATOR:
Any other questions? Thank
you.
ACKNOWLEDGMENTS
Hopkins for help in preparing the manuscript. Research sponsored in part by the National Cancer Institute, DHHS, under contract No. NO1-CO-74101 with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. We wish to thank J.
823
"ONCOGENES AS PROBES FOR CELLULAR SIGNAL PROCESSES: THE FAMILY OF ETS GENES" Takis S. Papas
Laboratory
of Molecular
Oncology
National Cancer Institute
Frederick,
MD 21702-1201
Our laboratory over the past five years, has been studying the ets family of genes. The human ETS1 and ETS2 genes, were the first identified and molecularly characterized members of this gene family, all members of which are related to the E2 6 virus oncogene, v-ets. The oncogene v-ets was originally discovered as a component of the chimeric genome, along with a truncated c-myb gene, of the avian leukemia virus, E2 6. This virus transforms myeloblasts and erythroblasts in vitro, and causes mixed erythroid-myeloid The E2 6 virus expresses a 5.7 kb RNA that leukemias in vivo. encodes a 135 Kd tripartite gag-myb-ets phosphoprotein that localizes in the nucleus. Questions that we asked initially were: How did the E2 6 virus acguire the cellular sequences; what are the differences between the normal c-ets and v-ets and their gene products, as well as something of the nature of their functions, and finally, how are the normal genes regulated. To address these questions we employed several different techniques of molecular biology including cloning, sequencing and construction of vectors expressing the ets gene products making useful for making specific polyclonal and monoclonal antibodies.
E26
sequences
is
a
replication
transduced
proto-oncogenes, myb
defective
from two and ets.
retrovirus that carries portions of the chicken cellular To deduce how the virus captured
these sequence and to determine the structural relationship between the ets sequence of E26 and its cellular progenitor, the genetic organization of the chicken locus related to v-ets was examined. Analysis revealed that the chicken c-ets locus contained nine presumptive exons homologous to the v-ets. The v-ets homologous sequences (142 6 bp) were found to be spread over 60 kb of genomic DNA. As expected, since v-ets was transduced from the chicken genome, the chicken c-ets gene was found to be nearly identical to the viral sequences. A major change was found at the 3' end of the v-ets and c-ets genes where 85 nucleotides of v-ets were totally different from the 3' end of c-ets ; thus, the chicken and viral ets genes are not co-terminal.
During the process of transducing of cellular sequences by the virus, two cellular genes were truncated. While the major transcripts of chicken myb and ets are 4.0 and 7.5 kb, respectively, the virus captured only 0.85 kb and 1.5 kb of these cellular genes. Therefore what possible transduction mechanism(s) E26
The fusion of myb and ets sequences gave rise to the E2 6 virus? with retroviral gag and env sequence required a minimum of three
824
recombinational events. Alignment of gag, env, cellular and viral myb and cellular and viral ets sequence failed to reveal any significant stretches of identical nucleotides. Therefore, to be consistent with this information, we suggested that the cellular myb and ets genes was acquired by non-homologous, or illegitimate
recombination.
Sequences related to ets have been identified and cloned from humans. Initially two loci designated ETS1 and ETS2 were analyzed and characterized. The human ETS1 gene is located on chromosome llq23-24; this locus has also been implicated in a number of monocytic and certain childhood associated leukemias. Recently, we have characterized another ets-related gene, which we have named ERGB (see below); this gene too is on chromosome 11, and cotranslocates with ETS1 in the t(4;ll) (q21;q23) translocation noted for Acute nonlymphocytic leukemias (ANLL). Similarly the human ETS2 and ERG genes we found, were co-localized on chromosome 21q22.3, and are part of a group of genes that are known to be amplified in Down Syndrome (DS), as well as being associated with known non-random translocation characteristic of certain types of acute myelogenous leukemias (AML). The fact that there is a higher predisposition of individuals with DS to leukemias and transient myelodysplasias may not be coincidental, given the amplification of several known ETS genes in the critical area of chromosome 21, which is known to be associated with certain DS phenotypic characteristics.
addition to the above four human ETS family members, other related members have been and identified The ELK-1 gene has been localized on Ch.Xpll.2; characterized. this gene may be involved in a type of synovial sarcoma that nonrandomly translocates the ELK gene from the X chromosome to chromosome 18. Also, another human ETS family gene, ELK-2, has been localized onto Ch. 14q32.3; this gene has been implicated in certain T-cell malignancies. A number of chromosomal breakpoints have been known to occur near the ELK-2 locus, and such translocation are characteristically associated with T-cell diseases. The human SPI-1 gene has been identified by homology to the mouse Spi-1 (PU. 1) gene and has been localized to human In
several
chromosome
llpll.22.
The chromosomal site of the ets genes and their relationship malignancy and tumor progression in animals has proved to be quite interesting. It has been frequently observed that malignant transformation by retroviruses results in the integration of proviral sequences nearby or within specific cellular protooncogenes, activating gene expression. Recently, activation of an ets-related gene Spi-1. has been observed in a collection of murine erythroblastic tumors induced by spleen focus forming virus (SFFV). In fact 21 of 22 such erythroid malignancies studied, induced by SFFV, had proviral insertional rearrangements near the Spi-1 gene. In rat thymomas induced by the Moloney strain of the Murine leukemia virus (Mo-MuLV), it has been found that proviral insertion events occurred at common integration loci thought to be associated
to
825
progression, termed Tpl-1. These sites have now been shown to result in the rearrangement or activated expression of the ets-1 gene. Another study has recently shown that erythroleukemias induced by Friend-MuLV activated the Fli-1 locus which has been shown to be an ets related family member. The Fli-1 gene is the murine homolog of the human ERGB gene that we have identified in The occurrence of our laboratory by sequence homology to ETS2. three types of hematopoietic malignancies as a consequence of retroviral insertions are significant, and are thought to be due to the fact that these events have in common the activation of an ets gene or its related family member. with tumor
Members of the ets gene family have been cloned and sequenced from a variety of species ranging from human to Drosophila; their encoded products are highly conserved, more so than the other known nuclear protooncogene products. Comparison of all the predicted proteins of the ets-gene family was made with respect to the chicken ets-1 gene (shown in Figure 1) On the basis of their amino acid sequence homology we have identified three regions; A, B and C; however, not all of these regions are found identically distributed among the family of ets related proteins. The 'A' region is localized at the amino or N-terminus, and is a moderately .
The ets Gene
Family B
Ch 8ls-2
nnrjiwmniiiiniiBnzi nrauiiiDiniiDiii^ri^ ZD3H1IHIMIIII
Xe efs-2
mrimmTiiiniiiiiiiMii i
I- Hu/Mo
efs-2
I i i
i
ii 11 h mmllll II I1HM rriirni llll! I IIHM il mi in usa 11 ii i mu m
mill! Him: mi nun
Hu/Mo/Ch/Xe els-1
IIIIHUIIIIH Su ets Dm ets
OHnimn MTJIIIttllllllMa
Hu erg -c Dm elg
Ç
aaiiiiiiiiBinEiz
Hi, elk
mi «ni im
Mo PU.1 Dm 74E
rji Hu Xe
-
Human
Mo
Xenopus
Su
-
4,500 4,000 3,000 -J_I_
-
Mouse
Ch
Sea Urchin
Dm
-
Earth
of
Drosophila
-
1,000 600
2,000
__J-L_/iL_
Meso- I CenoIQlC zoic
Paleozoic
Origin
Chicken -
lyl
| First
Eukaryotic Fossils
|
I
Oldest
Mammals
Invertebrates and Vertebrates
Appear
-
Comparison of the different members of the ets gene family. three regions: A, B, and C; based upon their homology to the human Fig.
1.
826
The ets gene is divided into ETS1 gene.
homologous domain when compared to each of the members of the ets family. By contrast, the 'B' region, located in the middle, is only weakly homologous to one another; this region appears to be dispensable, since it is known to be deleted in the predicted sequences of several of the ets-related gene members (e.g., ERG. The 'C region on the other hand, is located at the carboxy elg) or C-terminus and is a very highly conserved protein region .
encoding the DNA binding domain and found in common among all the members of ets-family. This yC region is the only one that is present in the predicted sequences of every ets- related gene characterized thus far, and therefore it forms the basis of defining membership in the ets-family. These three distinctive regions, enable us to group the ets genes into three classes: I, II, and III; classification was determined on the basis of homology to ETS1. the human homolog of the chicken proto-oncogene c-ets-1 Class I members contain all three of the above-mentioned (31) homologous regions 'A', 'B', and ' C (ets-1, v-ets); class II members contain the homologous 'A' and 'C regions (ets-2, erg). while class III members contain only the homologous 'C region (i.e., elk. PU.l, and Drosophila E74A). .
Representative members of ets gene family are distributed throughout metazoans, and are related to one another by their homology to the carboxy terminal portion of the v-ets oncogene. Given that the ets family proteins appear to play a role as important transcription factors that control multiple gene cellular functions in and differentiation proliferation, development; any alteration of the ets genes would markedly alter the cell's physiology and have a profound pleiotropic effect. of sequence divergence allowed a prediction of the of the ets-gene family; assignment of the members into the three classes, demonstrates that all the ets-1 and ets-2 members each formed a tightly clustered grouping. However other ets-family products, (e.g., human and sea urchin erg genes, the human ELK1, Drosophila 74E and the murine PU.l gene) form a somewhat looser clustered grouping. A plausible explanation of the origins of these groupings and classes of ets-family members could be due to an ancestral gene duplication (e.g., class I) occurring, followed by genetic recombination(s) or divergence yielding the other classes (I and II) However, since class I and III members are represented by both invertebrates as well as vertebrates, this ancestral duplication event must predate the emergence of chordates, i.e, prior to 500 million years ago.
Analyses
phylogenetic history different ets-family
.
All the ets and related genes
are
transcriptionally
active and
In adult expressed in a wide variety of cells and tissues. murine tissue, the Ets-1 message is expressed preferentially in thymus, lung and spleen whereas Ets-2 product is found expressed in almost every type of tissue tested. The human ETS1 gene encodes a unique 6.8 Kb RNA. The ETS2 gene is known to be expressed as three distinct messages sized 4.7, 3.2 and 2.7 Kb, this gene does not The generation of the three appear to be differentially spliced. ETS2 messages is due to the preferential utilization of its three are
827
different poly A signals. Since all the three ETS2 messages contain complete coding sequences, it is not yet clear which of the three messages is being translated to produce the ETS2 product. The ERG message remains the most restrictive product expressed by the
ets-gene family.
The ETS1 gene encodes a set of phosphoproteins ranging in size from 42 kd to 52 kd (Figure 2) The p51ETS1 isoform is the product of the full length MRNA, while the p42ETS1 isoform results from the ETS1 protein product of a spliced MRNA lacking exon VII. phosphorylation is Ca++-dependent and treatment of cells with calcium ionophore, as well as by complexing the T-cell receptor with antibody, increases the level of phosphorylation of the ETS1 protein. The ets-gene products and its related family of proteins contain the calmodulin dependent protein kinase II consensus site (RXXS/T). We have been able to demonstrate that the ETS1 protein is phosphorylated by the calmodulin-dependent protein kinase II, Since the p42ETS1 product is using an in vitro labeling assay. encoded by the MRNA lacking exon VII it does not become .
phosphorylated; this characteristic implicates splicing as important for the differential regulation of ETS1 proteins via Ca++-mediated phosphorylation. The ETS2 gene encodes
a
p54 kd protein that is phosphorylated
Ca++-dependent mitogenic signal process (Figure 2) The stability of the ETS2 protein is dramatically increased as a consequence of protein kinase C activation from a protein with a half-life of 20 min. to 140 min. one with a half-life. Thus this (pp56) by
a
.
ETS1 Isoforms ETS2 Isoforms
PS¡?
=
p42
pp56p54-
• «»
-
Met
NH2 ETS1
HIV/AIDS
Lectures
EPIDEMIC: A CURRENT PICTURE
Novelle», M.D., Surgeon General
Antonia C.
M.P.H.
Public Health Service Washington, D.C. 20201
Thank you Dr. Gallo. It is a pleasure to be here today and honor to speak before such a distinguished group of scientists. I would like to talk to you about one of the most important problems we face today—and one I am deeply concerned about—HIV infection and AIDS. Of course, many of you work in this area, and we could spend quite a bit of time on this subject; but I'd like to try to give you some perspective on the overall picture of the HIV/AIDS epidemic and particularly its impact on women and adolescents, as seen through the eyes of the Surgeon General. I chose this topic because I believe the increasing impact of AIDS on women, their children, and consequently on their entire families will continue to have a great impact on societies. AIDS is no longer a disease of men or women AIDS is a disease of families, and as such we must start dealing with this. Why do we talk about women then? Well, because women are usually the cornerstone of the family, the substance that holds the family together, and they are increasingly suffering the effects of AIDS—as patients and as caregivers. Women who are themselves infected with HIV; women whose children, from infants to adults, are infected; women whose partners are HIV-infected and women who care for HIV-infected patients, family members, or friends are going to need our help. We must realize that this is the true picture of HIV infection and AIDS of the future, and deal with it accordingly. I do not have any new science that you already do not know, to bring to you today. But, I am here t show you a picture of the HIV/AIDS epidemic, as it exists today from the societal and beaucratic point of view in hopes that we can work together to find an end to this tragedy. an
-
I will talk
today
about science and statistics—
epidemiology, surveillance, and
costs. But we must keep in mind the faces behind the statistics—the faces of men, women, and Dr. Gerald Friedland, former Codirector of the AIDS children. Center at Montefiore Medical Center and Professor of Medicine at the Albert Einstein College of Medicine in New York and now at Yale University, has said, "The statistics of epidemiology are human beings with the tears removed. They capture the number of sufferers but not the quality or the weight of the suffering that
695
is
being endured, for the fundamental reality of slowly, prematurely, and with great cruelty robs women of their function, their plans and dreams,
AIDS is that it
young men and and ultimately,
their lives." The human immunodeficiency virus (HIV) that causes AIDS has been around for a lot longer than most people realize. By testing blood samples that were frozen for research purposes many years ago, HIV was found in human blood collected in Africa in 1959, and in the United States, from the blood of a 15-year-old sexually active male, in 1968. The AIDS epidemic that we observe today is the result of all those years of HIV infection spreading silently through populations of ordinary people who had absolutely no idea their lifestyle choices could hurt anyone
else.
HIV infection and AIDS are found on virtually every continent on Earth. (The only exception is Antarctica.) Experts at the Centers for Disease Control and at the World Health Organization have used existing data and mathematical models to make projections of future numbers of expected AIDS cases. According to some estimates, there are approximately 1 million HIV-infected persons in the United States, and the World Health Organization (WHO) estimates 8 to 10 million people may be infected around the world. Based on the number of estimated HIV infections and other data, by the end of this year WHO expects more than one million adult AIDS cases will be reported worldwide. Total AIDS cases are expected to triple in Africa and the Americas this year alone. In Thailand, where the epidemic is still in a very early stage, AIDS cases will perhaps increase from 50 reported in 1990 to more than 10,000 by 1995. In the next decade of AIDS, WHO expects 5 to 6 million cumulative AIDS cases, compared with one million through the 1980s; in Africa, 15 to 20 percent of the continent's workforce could die. Long-term projections about HIV infection are harder to make, but the World Health Organization predicted 3 years ago there could be 15 to 20 million infected individuals by the year 2000; today, they say that may have been a conservative estimate. The WHO now believes that number may be reached by the mid-1990s, with as many as 40 million infections among the world's men, And the most frightening wo:nen, and children by the year 2000. can't those 40 million people, tell at thing is, you by looking and they may not know either, that they are HIV infected. During this first decade of AIDS, we have seen the number of AIDS cases and deaths increase dramatically each year, from 132 deaths in 1981 to more than 25,000 deaths in 1990. According to projections, the cumulative number of deaths from AIDS will approach 200,000 by the end of 1993. CDC estimates that approximately 1 million people are currently infected with HIV in this country alone. This represents approximately 1 in 100 adult males and 1 in 600 adult
females in the United States. In 1988, AIDS was the third leading cause of death among U.S. men 25-44 years of age, and in 1989 it had moved to second place, causing 14 percent of all deaths among men in this age group-surpassing heart disease, cancer, suicide, and homicide, in fact AIDS surpassed all causes except unintentional injuries. 696
women 25-44 years of age, AIDS was the eighth leading of death in 1988 and 1989. Estimates based on current trends indicate that, in 1991, AIDS will be one of the five leading causes of death for women in this age group. More than 19,000 women in the United States have been reported with AIDS thus far, and over half of these have been reported since January 1989. More than 10,000 of these women Almost 12 percent of all AIDS cases are now reported have died. among women, up from 6 percent in 1984. Although most AIDS cases in the United States still occur in men, HIV infections are spreading rapidly to women. Based on cases reported in 1988 and 1989, women and perinatally infected children were the fastest growing groups, increasing 23 percent and 34 percent, respectively, compared with 13 percent for men. While the majority of AIDS cases in U.S. women have been reported in injecting drug users, and these cases continue to increase, AIDS cases attributed to heterosexual transmission are increasing faster than any other exposure category. They now account for more than one-third of all cases among U.S. women and for three quarters of all worldwide cases. In addition, many of the cases having "no identified risk" will most likely be reclassified following investigation as cases of heterosexual transmission. Only 6 percent of cases are currently attributed to transfusion or other blood product exposures, and the number of such cases has stopped increasing. In 1990, 64 percent of women who acquired HIV infection from an infected man reported that the man was an injecting drug user. However, an increasing number and proportion of AIDS cases are being attributed to sexual contact with an HIV-infected man whose risk of being infected was unknown to the woman. This category has increased from 6 percent in 1985 to approximately 20 percent in 1990. Also increasing, though less rapidly than other categories, is the number of cases among women who report sexual contact with a bisexual man; this currently accounts for about 10 percent of heterosexual AIDS cases among women. Clearly, most AIDS cases among U.S. women are occurring in young women, but it is interesting to note that women who report sex with a bisexual man are slightly older, with the largest numbers of women between 30 and 49 years of age. The number of infections recorded among racial and ethnic minority women confirm the stark reality that the HIV epidemic disproportionately affects them. Black and Hispanic women make up only 17 percent of all U.S. women, but represent 7 3 percent (52 and 21 percent, respectively) of all reported AIDS cases However, the number of women affected in every race among women. and ethnic group is continuing to climb. Early in the epidemic, most women (70 percent) with AIDS lived in the Northeast. In contrast, in 1990, slightly fewer than half of the women with AIDS were reported to reside in the Northeast, with a striking increase in the proportion of cases coming from the South, particularly from the South Atlantic To date this year, Florida has reported more coastal states. cases of AIDS in women than any state except New York. Most women with AIDS reside in large urban areas with at least 1 million population, although this is changing, with an
For
cause
697
increase in cases outside large cities, particularly in the Southeast. As I mentioned earlier, HIV infections are spreading rapidly to women, and I am particularly alarmed by this. One of the reasons this is occurring is because so many women are unaware of The fact that many HIV-positive women cannot or deny their risk. identify a risk for their HIV infection is very disturbing. If women continue to be unaware of their risks, and recent trends continue, thousands of women will die prematurely each year. I am also particularly concerned about the fact that the incidence of AIDS among young women, and young men, high indicates that many of these individuals became infected as teenagers. Adolescents are increasingly vulnerable—surveys indicate that the majority of them in the United States have engaged in sexual intercourse. In a 1988 survey of teenagers between the ages of 15 and 19, 52 percent of women and 60 percent of men reported they had already engaged in premarital sexual intercourse. The average age of first sexual experience among U.S. adolescents is 16, but in some large urban areas, it may be as young as 12. In 1989, 21 percent of high school students said had they already engaged in sex with four or more partners. Keep in mind that this survey was conducted among high school students, and the data do not reflect information on youth outside the school system, such as street youth or runaways, who as a group must be considered to be at even greater risk. As a result of the increases in adolescent sexual activity, many teenagers are being diagnosed with sexually transmitted diseases, or STDs. Every year in the United States, 12 million and two-thirds of those, or 8 cases of STDs are diagnosed million cases, occur in persons under the age of 25. To put it more succinctly, a U.S. teenager gets a sexually transmitted disease every 30 seconds of every single day. Keep in mind, too, that one recent study from the Centers for Disease Control found that 50 percent of U.S. adolescent females who were diagnosed with AIDS in 1990 contracted the virus through heterosexual contact. Surveys have shown that adolescents know how HIV is transmitted, but 63 percent of those who are sexually active still do not consistently use condoms. In one survey, 40 percent reported "sometimes" using condoms, and 23 percent reported "never" using them. Because many people see them primarily as a means to prevent pregnancy, teenagers may be less likely to use condoms when their sexual activity will not result in pregnancy. The problem with that is, according to studies recently conducted in Europe and the United States, the sexual activities most likely NOT to result in pregnancy, such as anal intercourse or sex during menses, may even put people at even greater risk of acquiring HIV infection. These behaviors may be more common than many people suspect. One recent review indicated that between 10 and 26 percent of all U.S. adolescents practice anal intercourse. Another study conducted among University of Puerto Rico students found that 31 percent of women and 40 percent of men practice anal sex, both to prevent pregnancy and to preserve virginity. Every year, more and more people seek counseling and --
698
In the United States and its territories, at publicly funded clinics alone, 892,714 people were tested in 1989 and 1990. Of those, 125,900 were young people aged 13 to 19, and 766,814 were adults (age 20 and above). In every age, even among the 13-year-olds, the place most often tested for HIV infection was at a sexually transmitted disease clinic. This tells us a couple of things, but most importantly, it demonstrates better than any other fact that teenagers are not protecting themselves from disease. And since we also know that having a sexually transmitted disease may actually increase a person's chances of getting the AIDS virus from his or her sex partners, the fact that teenagers are getting sexually transmitted diseases is doubly troubling. The ratio of HIV-infected males to females is currently 1.7 to 1 in the U.S. adolescent population, as compared with 9 to 1 in the adult population. Among military recruits in 1990, the This should ratio was found to be nearly identical .9 to 1. be another "red flag," as it is one more indication that heterosexual contact is becoming a primary HIV transmission route among teens. We must make an effort to better educate our youth about STDs, especially HIV infection. We have too much to lose to ignore these statistics. Since infections that occurred 5-10 years ago are reflected in today's AIDS case reports, the actual extent of the problem is better defined by HIV infection trends since large increases in female HIV and AIDS rates in this country are relatively recent. CDC is conducting HIV serosurveys to supplement our knowledge of The childbearing the geographic dispersion of the epidemic. women's survey, now ongoing in 44 states, Washington, D.C., and Puerto Rico, tests newborn blood samples routinely collected on filter paper for other purposes. Detection of antibody to HIV reflects maternal antibody status; thus, the survey measures the prevalence of HIV infection in women who deliver live born It is the largest population-base HIV serosurvey being children. conducted in the United States. HIV seroprevalence rates in 1988 and 1989, for selected metropolitan areas, indicate that the highest seroprevalence rates per 1,000 childbearing women are in Miami, New York City, It is interesting to note the relatively small and Newark. spread between rates in Atlanta and other parts of Georgia—this reflects the dispersion of the epidemic into the South outside of
testing.
—
the
large metropolitan areas. Epidemiologie studies have suggested
a number of possible cofactors in heterosexual transmission. Clearly, host infectiousness may vary. We believe that persons late in the course of illness when T-helper cell counts decline are more infectious. Early HIV infection, before the development of HIV antibodies, may also be transiently associated with a higher level of infectivity. Many studies have recently demonstrated that both ulcerative and nonulcerative sexually transmitted diseases increase the risk of HIV transmission by several-fold. Given that the number of cases of syphilis among women has more than doubled since 1985, especially in the South where there are already high rates of heterosexually transmitted AIDS, we are presented with a
699
frightening forecast for the future of heterosexual transmission If current trends continue, of HIV in the United States. heterosexual transmission of HIV will become the predominant mode of transmission among U.S. women by the middle of this decade. As I just mentioned, genital ulcer disease and nonulcerative sexually transmitted infections, as well as lack of circumcision, The exact at least in Africa, appear to influence transmission. mechanisms by which lack of circumcision and STDs influence transmission remain uncertain. Presumably genitalulcers provide a portal for virus entry and inflammatory cells as targets for The role of nonulcerative STD is less obvious. In a infection. recent study conducted in Nairobi and reported in Florence, Dr. Willerford working with Drs. Kreiss and Plummer noted a fourfold increased risk of cervical shedding of HIV, as measured by polymerase chain reaction (PCR), associated with cervical inflammation and cervicitis. The role of other cofactors as determinants of transmission is less well understood but new data are beginning to shed some additional light. For example, in Florence we heard a report of a study which used in situ hybridization to identify HIVinfected cells in the reproductive tracts of experimentally infected rhesus macaques. Lymphocytes and macrophages in the and and cells which appear to be Langerhans cells cervix, vagina in the dermis and epidermis of the penis were positive for SIV. These data, from Dr. Chris Miller and Dr. Preston Marx, suggest that Langerhans cells may be a cellular target for SIV and, by inference, HIV in the female reproductive tract. At the same conference, Dr. Eric Langhoff from Dr. William Haseltine's group presented data indicating that primary human dendritic cells support replication of HIV to a much greater The data from level than infected macrophages or lymphocytes. these two groups taken together suggest that this cell population may play an important role in the sexual transmission of HIV. Further reports from Florence raise questions about the appropriate use of spermicides as virucidal agents. Researchers from Thailand working with Family Health International found that prostitutes using nonoxynol-9 were 60 percent more likely than placebo users to have signs or symptoms of vulvovaginal mucosal irritation. In a randomized, placebo-controlled study in among prostitutes Nairobi, Kreiss et al. found that nonoxynol9 impregnated sponge use was associated with a 7 0 percent increased risk of HIV seroconversion. Because nonoxynol-9 is a we to need whether its application, detergent, question its particularly frequent application, may cause sufficient inflammation and microulceration to adversely affect the risk of These provocative preliminary observations mandate transmission. further the need for investigation in light of nonoxynol-9's antiviral proven activity in vitro. Although the role of spermicides for HIV prevention remains uncertain, it is clear that latex condoms provide an effective mechanical barrier to HIV. Evidence for this includes a study using scanning electron microscopy on 50 samples of stretched and unstretched latex condoms; no pores were found in any condoms even when viewed at a magnification of 2000X. Furthermore, in vitro data indicate that latex condoms are impermeable to hepatitis B virus surface antigen, which at 22 nm (nanometers) in 700
diameter is appreciably smaller than HIV (120 nm in diameter). These data indicate that latex condoms can serve as an effective mechanical barrier to HIV transmission. But condom effectiveness in the laboratory cannot be directly translated into clinical effectiveness. Behavioral determinants clearly play a large role, as condoms must be used consistently and correctly if they are to be clinically The clinical efficacy of condoms is demonstrated by effective. from the European Community Study of Heterosexual recent data HIV Transmission. In this study, 251 discordant couples were followed prospectively for an average of 14 months. Data clinical of in Florence the condoms, support presented efficacy when used consistently and correctly, for the prevention of HIV transmission. Ten seroconversions to concordance were observed, all among couples who did not use condoms consistently; there were no seroconversion events among systematic condom users. Unfortunately, a factor in correct use of condoms may be the clarity of instructions that accompany them. In a recent study, written instructions for condom use for 14 brands of condoms were evaluated. According to the study, all instruction brochures required at least 10 years of education, and some required greater than a high school degree. How, then, are women to protect themselves? Providing education on safer sex may be helpful, but it is not enough. It is easier for man to protect themselves against sexual For women, protection transmission of HIV by wearing a condom. is more of a problem, especially in cultures or subcultures where women have a subordinate role in society. It may be difficult, if not impossible, for women to influence their partners' use of condoms because of cultural, societal, or religious restraints Even the female condom, not yet available in and perceptions. the United States, may not be a viable option for women for many of the same reasons. We still have much to learn about behavioral factors which affect education and prevention efforts for women, but studies are underway which should help to shed some light on this issue. Studies are also underway to better define the spectrum of disease in both men and women. HIV Scientists are comparing the kinds of opportunistic infections that occur in HIV-infected men and women. Many of the opportunistic infections in women are similar to those of men, although some differences are evident. Pneumocystis pneumonia is diagnosed in the majority of women and men with AIDS. Kaposi's sarcoma is reported among less than 2 of with AIDS, although it is reported in more than women percent 15 percent of men, predominantly among homosexual/bisexual men. In addition, researchers are trying to evaluate what specific gynecologic manifestations occur in HIV-infected women and whether gender-specific exposures, such as pregnancy, influence the progression of HIV disease in women. It is also possible that HIV disease will influence the prevalence of, or complicate the course of, gynecologic diseases which also occur in uninfected women. One possible example is of 21 studies of HIV infection cervical neoplasia. A review and cervical neoplasia found that the prevalence of cervical neoplasia ranged from 8 percent to 100 percent, and that women
701
with HIV infection were almost 5 times more likely to have cervical neoplasia than uninfected women. What we still need to know is whether that association is attributable to HIV infection, per se, or to a high prevalence of conventional risk factors among HIV-infected women. Researchers are also investigating whether cervical dysplasia progresses more rapidly to cervical cancer in HIV-infected women, and whether progression is attributable to immunosuppression or to other factors. What about Pelvic Inflammatory Disease, or PID? Is the apparent association between PID and HIV infection attributable to the fact that both are sexually transmitted conditions? Further, is any observed association between HIV infection and severe PID attributable to immunosuppression or to other factors? So far, two studies have demonstrated a high prevalence of HIV infection among women with PID. Another study found that PID among HIV-infected women was clinically more severe and more likely to require surgical intervention. It is still unclear whether this reflects more severe PID in HIV-infected women by virtue of immunosuppression or other HIV-related causes, or if these differences can be explained solely by differential access to health care or related issues. Finally, what is the association between HIV infection and candidal vulvovaginitis? One study found that 38 percent of 140 symptomatic women had recurrent candidal vaginitis. What we still need to know is whether the frequency and severity of candidal vaginitis is related to immunosuppression, to prior antimicrobial therapy, or to other factors. When all the findings are in, more decisions will have to be made about managing HIV disease in women. For example, how frequently should HIV-infected women be screened for cervical neoplasia? Should HIV-infected women found to have cervical neoplasia receive more aggressive treatment than noninfected women? Sometimes it seems that all we have are questions, but every year, every month, we find more answers. CDC has recently tried to address some of these questions and issues in examining its AIDS surveillance definition. As you may know, CDC developed the AIDS surveillance case definition to measure trends in conditions that are highly specific to HIV The AIDS surveillance case definition has provided a infection. very useful measure of severe and life-threatening illness associated with HIV infection by (1) allowing accurate tracking of the epidemic geographically, demographically, and temporally; (2) identifying and ruling out modes of transmission; and (3) providing the foundation for projecting the future course of the
epidemic.
CDC is in the process of implementing a broad change in the definition for surveillance of AIDS in adolescents and adults in the United States. The planned change would add to the all case AIDS current definition persons with documented HIV infection and a CD4 lymphocyte count less than 200/mm This modification is being made to include those HIV-infected individuals with severe immunosuppression who may not have a specific AIDS indicator disease listed in the 1987 revision of the AIDS case definition. case
.
702
HIV-infected persons with CD4 lymphocyte counts of less than 200/mm are most likely to be severely ill or disabled and in greatest need of medical and social services. Data indicate that as CD4 levels decline, the incidence of AIDS, as currently defined, increases. When researchers looked at the CD4 data for people with AIDS, approximately 80 percent had a CD4 count under
200.
It is estimated that there may be 150,000 to 200,000 persons currently infected with HIV who have a CD4 count less than 200 who are not currently reported with AIDS. However, we do not that all of these will be expect reported, since half or more of these HIV-infected persons may not know their HIV infection status or their CD4 count. Although such a broad expansion of the AIDS case definition
will make interpretation of trends in incidence and characteristics of cases more difficult, this expansion will provide a better estimate of the health care needs of severely ill persons with HIV infection. Using the CD4 count will help at severe illness and most in those risk for greatest identify need of medical follow-up without adding unneeded complexity to the surveillance process. CDC is working with the Council of State and Territorial
Epidemiologists and state health departments to plan for the change in the AIDS case definition, and expects to publish the new definition by next spring. At this point, I would like to talk a little more about he cost and availability of services for individuals with HIV
infection and AIDS. The cost of treatment for HIV-infected and AIDS patients is At the AIDS Conference in Florence tremendous and it is rising. this June, Fred Hellinger of the Agency for Health Care Policy and Research estimated that the cost of treating all HIVseropositive persons in the United States in 1991 will be $5.8 billion; this figure may rise to $10.4 billion by 1994. Some previous estimates have understated the costs of care due to the HIV epidemic because they have considered only those cost occurring after a diagnosis of AIDS. Dr. Hellinger estimates the average cost of treating a person with HIV who does not have AIDS is about $5,150 a year, and someone with AIDS, $32,000 a year, of which $24,000 is for inpatient hospital care and $8,000 for other He estimates the lifetime costs for a person with AIDS services. to be more than $85,000. These figures are higher than those from other recent studies, which estimated the lifetime costs of the medical care of someone with AIDS from the time of diagnosis at $40,000 to $75,000, because AIDS patients are living longer and the drugs used for treatment are increasingly costly. Dr. Hellinger based his estimates on case projections similar to CDC's, assuming that 63,000 AIDS cases will be diagnosed in 1991, 74,000 in 1992, 87,000 in 1993, and 101,000 in 1994. He estimated that the number of HIV-infected asymptomatic persons receiving care each year is twice the annual number of AIDS cases.
We can also assume that the cost of treating children with AIDS will continue to rise. However, children have additional nonmedical costs which usually are not covered by existing financial resources. Estimates of lifetime cost for children
703
with HIV infection and AIDS range from
$90,000.
$40,000
to
more
than
What then can be done to increase the availability of services for individuals with or at risk for HIV infection and AIDS? The Health Resources and Services Administration (HRSA) and CDC have shown that community-based primary care facilities can be effective components of HIV treatment and prevention
high incidence areas. HRSA's Community and Migrant Health Center Program is the largest community-based primary care system serving low-income Facilities are located in highpersons in the United States. need areas and offer comprehensive primary care services on a sliding-fee scale. In 1990, HRSA provided $496 million to 554 grantees with over 2,000 health care delivery sites. More than million low-income patients received care under this program, programs in
6
most of whom are members of racial/ethnic minorities. In a two-phase study of the program's HIV/AIDS-related services, HRSA found that about one-third of the centers were providing prevention and treatment services to a substantial number of patients. This study confirmed the high costs associated with HIV infection and AIDS it found that HIV/AIDS have visits the centers than other to more patients significantly —
with an average of 16.2 visits per year (versus 3.2 visits/year for non-HIV/AIDS patients). In addition, the average HIV-related visit required 29 minutes, compared with 13 minutes for non-HIV illnesses. And the average HIV-infected patient more required significantly laboratory services than other infected with not HIV. patients We must all continue in our efforts to seek solutions to the problem of HIV infection and AIDS. Much more needs to be done in the area of prevention and treatment. Research must continue with increased vigor. As we develop promising treatment protocols and we ensure that clinical trials are open to women must vaccines, and children. We must also exercise leadership in preventing HIV infection among women and children by addressing the cultural and societal barriers which deny them access to prevention. And until better treatment protocols or a vaccine are available, education on the ways in which HIV infection is spread and how people can protect themselves, and knowledge of serostatus through readily available counseling and testing services, are the keys to stopping the spread of HIV infection. Although some of you may not work in a field directly related to HIV infection and AIDS, on a personal level you can You can try in your own lives to be still have an impact. and compassionate caring toward people with AIDs. You can educate others about HIV infection and AIDS, helping them to understand the role of risk behaviors in transmission and the need for compassion toward HIV-infected individuals. Those of who are have an you parents important responsibility to educate HIV children about infection and AIDS. As I mentioned your adolescents are earlier, having sex earlier and with more than 10 were years ago--this makes them partners they to vulnerable particularly sexually transmitted diseases,
patients,
including
HIV infection.
The magnitude of this epidemic and the urgent need to find better treatments or a cure represent an enormous challenge for 704
Much has been accomplished during this first decade of still have along way to go. As Margaret Hamburg and Fauci have said so succinctly in their article AIDS: The Tony to Biomédical than more Research, disease, "Perhaps any Challenge AIDS has raised a host of complex questions, from the most fundamental nature of biological systems to the role of scientific research in society at large. And beyond the
science.
AIDS, yet
we
challenge to implications
biomédical research, there are profound and broader of the AIDS crisis for many aspects of health care, law, ethics, and society. The needs are tremendous and the answers difficult, but we are moving forward and new solutions The benefits will be far-reaching—not are within our grasp. but for for AIDS, many other areas of health and disease as just
well.
Indeed, AIDS has been
mighty challenge to science. But we challenge. Science has brought complex questions, and it will play an essential role in stemming this tragic epidemic. We have some of the finest minds in science in this room here today, and my challenge to you is to work within your disciplines, and your personal lives, to conquer this disease. As you do this, however, remember the human impact of this epidemic, particularly its toll on women. Remember that, as women contract this disease and pass it on to their children in ever increasing numbers, families will suffer, societies will suffer, and the world community will suffer. You can make a difference—you can help stop this suffering. Let us move forward together to find these solutions—if we work together, we will surely succeed. Thank you. a
must continue to respond to this us answers to many difficult and
References 1.
Premarital sexual adolescent women—United among States,
Centers for Disease Control.
experience MMWR Vol No. (CDC)
39,
Nos.
51 and
91-8017.
52, 1991,
1970-1988.
HHS Publication
2.
Division of STD/HIV Prevention. Sexually transmitted disease surveillance, 1990. U.S. Department of Health and Centers for Human Services, Public Health Service, Atlanta: Disease Control, July 1991.
3.
Centers for Disease Control, Division of HIV/AIDS— unpublished surveillance data.
4.
Berlin B, Hingson R, Strunin L, Heeren T. Changes in adolescent condom use, knowledge and beliefs about AIDS in Massachusetts, 1986-1989. Abstract No. SC 576. Sixth International Conference on AIDS, San Francisco. June 20-23, 1990.
5.
Kipke MD, Futterman D, Hein K. HIV during adolescence. Med Clin North
6.
Cunningham I, Rodriguez
Sanchez M.
705
infection and AIDS 74(5):1149, 1990.
Am
AIDS
knowledge
and
sexual behavior among University of Puerto Rico students. Abstract No. ThD 774. Sixth International Conference on AIDS, San Francisco. June 20-23, 1990.
Willerford D, Emonyi W, Hensel M, Bwayo J, Plummer F, Holmes K, Gallatin M, Kreiss J. Association between cervical MC 3095.
Florence,
of HIV and cervicitis. Abstract No. Seventh International Conference on AIDS, Italy. June 16-21, 1991.
shedding
Miller C,
Vogel P, Dandekar S, Hendrickx A, Alexander N, Localization of SIV in the reproductive tract of rhesus macaques: relationship to the distribution of T cells and macrophages. Abstract No. WA 1119. Seventh International Conference on AIDS, Florence, Italy. June 16-21, 1991.
Marx P.
Langhoff E, Terwilliger EF, Poznansky MC, Bos H, Kalland KH, Haseltine WA. Prolific HIV-1 growth in human dendritic cells. Abstract No. WA 70. Seventh International June 16-21, 1991. Conference on AIDS, Florence, Italy.
Roddy RE, Fortney JA, Chutivongse S. comparative trial of mucosal irritation with nonoxynol-9 or placebo. Abstract No. WC 3084. Seventh International Conference on AIDS, Florence, Italy. June 16-21, 1991. Niruthisard S,
A randomized
Kreiss J, Ruminjo I, Ngugi E, Roberts P, Ndinya-Achola J, Plummer F. Efficacy of nonoxynol-9 in preventing HIV transmission. Abstract No. MAO 36. Fifth International Conference on AIDS, Montreal. June 4-9, 1989.
data from study supported by the National Institute of Child Health and Human Development.
Unpublished
Minuk GY, Böhme CE, Bowen TJ. Condoms and Ann Intern Med 104:584, 1986. infection.
hepatitis
B virus
DeVincenzi I, Ancelle-Park R, for the European Community Study Group on heterosexual transmission of HIV. Abstract Seventh International Conference on AIDS, No. MC 3028. Florence, Italy. June 16-21, 1991.
Richwald GA, Wamsley MA, Coulson AH, Morisky DE. Are condom instructions readable? Results of a reliability study. Public Health Rep 103(4):355, 1988. Fahs M, Mandelblatt J, Garibaldi K, Senie R, Peterson HB. The association between human immunodeficiency virus infection and cervical neoplasia: implications for the
clinical care of women at risk for both conditions. Abstract No. WC 3136. Seventh International Conference June 16-21, 1991. on AIDS, Florence, Italy.
Safrin S, Dattel B, Hauer L, Sweet R. 706
Seroprevalence
and
correlates of human immunodeficiency virus infection in women with acute pelvic inflammatory disease. Obstet Gynecol 75:666, 1990.
epidemiologic 18.
Sperling R, Friedman F, Joyner M, Brodman M, Dottino P. Seroprevalence of human immunodeficiency virus in women admitted to the hospital with pelvic inflammatory disease. J Reprod Med 36:122, 1991.
19.
Abulafia 0, Sedles A, Feldman J, Des Jaríais D, Landesman S, Minkoff H. Sexually transmitted diseases and human immunodeficiency virus infection among women with pelvic inflammatory disease. Am J Obstet Gynecol 163:1135, 1990.
20.
Carpenter CJ, Leibman BD, Stein M, Mayer KH. Distinctive features of
Hoegsberg B,
Fisher A, Fiore TC, HIV infection in Abstract No. MC 3000.
200 North American women. Seventh International Conference June 16-21, 1991.
on
AIDS, Florence, Italy.
PHARMACOLOGIC REGULATION OF HIV EXPRESSION
Anthony
S. Fauci
Laboratory of Immunoregulation National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland 20892
My presentation today will encompass the broader concept of the immunopathogenic mechanisms of HIV infection with a slant towards the pharmacologie modulation of HIV expression. I will focus on several aspects of the pathogenesis of HIV infection: the initial infection, the acute syndrome, the period of clinical latency which is associated with some degree of microbiological latency, the concept of viral burden and tissue distribution, and the induction of HIV expression to active viral replication, cytopathicity and ultimately clinical disease. During the typical course of infection with HIV, there is a precipitous in CD4+ T cells during the acute syndrome, a burst of viremia and the drop generation of an immune response, which apparently curtails the burst of viremia.
There follows a period of time ranging in years in which an individual is in a clinically latent stage, which in many situations is misinterpreted as a microbiologically latent stage. During this time period which is generally measured in years, the HIV-infected individual experiences a now well-recognized progressive diminution of CD4+ T cells to a level at which opportunistic infections occur. The acute syndrome has been much under-appreciated since the beginning of the AIDS epidemic. We know now from a number of groups that up to 70% of individuals experience an acute HIV syndrome. Within a few weeks of the initial infection with HIV, there is a burst of viremia and an extraordinary retrafficking of lymphocytes associated with a clinical syndrome that resembles acute mononucleosis. Re-trafficking of lymphocytes into the peripheral lymphoid
707
tissue occurs and an immune response to the virus is generated which results in diminution of plasma viremia. However, a number of studies have shown that the immune response is not completely effective despite the fact that the viremia seems to be curtailed. This is evident from the fact that there is a progressive diminution of CD4+ T cells during the period of clinical latency. Thus, it appears that the events which occur during the acute infection, particularly in the area of lymphocyte retrafficking, are very important. It is my opinion, and the opinion of a number of other people, that a substantial amount of damage I will return to this concept in a moment. occurs very early in HIV infection. I would like to talk about viral burden and its relationship to latency. Last year, we had mentioned the relationship of CD4+ T lymphocyte count, disease progression and viral burden. It is very clear that there is a significant increase in viral burden over time per given number of CD4+ T cells. By examining highly purified T cells at different points in time in individuals who deteriorate immunologically and clinically to develop full-blown AIDS, we see a very substantial increase in viral burden per given number of CD4+ T cells. For individuals who do not progress to AIDS, the viral burden in CD4+ T cells remains a
relatively
constant.
also interested in delineating the course of active viral HIV-infected in It had previously been thought that individuals. replication active viral replication generally occurs during the later stages of the disease. We know now from studies from a number of laboratories that this is, in fact, not Active viral replication occurs at all stages of disease, as the case. determined by plasma viremia, and most recently by RNA PCR. We have examined HIV-infected individuals with nearly normal CD4 +T cells, individuals with declining CD4+ T cell levels and individuals with less than 200 CD4+ T cells per cubic mm. We found that there was a degree of active replication as measured by RNA PCR in all stages of disease. Thus, a quiescent, microbiologically truly latent state does not exist even in asymptomatic individuals. Although, at any given time, there are approximately 10 infected cells that are not expressing virus for every infected cell that does express viral proteins and/or RNA, there seems always to be some cells expressing virus. I would like to focus now on the tissue distribution of HIV in infected individuals. We had reported last year that a number of immune competent cell compartments could be infected by HIV. The intrathymic T cell precursor, the socalled triple-negative (CD3-CD4-CD8-) cell, is in fact not triple negative but expresses a low level of surface CD4, and can be infected by HIV. We have also demonstrated that CD34+ bone marrow precursor cells can be infected with HIV in vitro and in vivo. We know now from studies from a number of laboratories of the degree of active viral replication that occurs in other lymphoid tissues, such as in the lymph nodes, at all stages of disease. As I mentioned earlier, re-trafficking We know that there is of lymphocytes occurs during the HIV acute syndrome. virus cells in the lymph dendritic filtering of antibody-coated by the follicular node germinal centers. infect virus can T cells that The filtered very likely the node. coated virus In travel through addition, antibody trapped in the lymph well HIV-infected cells that home nodes activate as as center to T germinal lymph B cells in what was thought to be a polyclonal, but now is known to be an oligoclonal, HIV-envelope specific response. Uninfected CD4+ cells are also at risk of infection within the microenvironment of the lymph node. By examining paired lymph node and peripheral blood samples from HIVinfected individuals at all stages of disease, we found that the viral burden at any given time in the lymph node per given number of CD4+ cells was at least a log higher than in the peripheral blood. We performed quantitative analysis using the ACH-2 cell line and compared the strength of the signal in the ACH-2 cells with the signal in the peripheral blood and lymph node. A stronger signal We
were
708
observed per given number of CD4+ T cells in the lymph nodes with all primer pairs tested. From work done in our laboratory by Cecil Fox, it is apparent that virus accumulates in the germinal center of the lymph node. The in situ hybridization studies of the lymph node tissue demonstrate that HIV-infected cells are located adjacent to cells that are activated and secreting various cytokines. This observation becomes relevant for what I am going to discuss next, namely the induction of HIV expression by cells secreting cytokines. We have been very interested in the mechanisms of induction of HIV expression in a chronically or latently infected cell, be it of the T cell or monocyte lineage. We, as well as a number of other laboratories, have published over the last several years on a variety of mechanisms that may be involved in the upregulation of HIV expression. These mechanisms involve heterologous viruses, other co-infecting microbes, such and a number of endogenous cellular factors. Because of our as mycoplasmas, immune on work the of the normal human previous regulation response antedating the AIDS epidemic, we have been interested in immunoregulatory cytokines. The immunoregulatory network in man is a very complex, highly redundant, paracrine and autocrine system, which keeps the immune system in a state of homeostatic regulation. We have demonstrated over the past few years that the mechanisms that are responsible for normal homeostasis can in fact upvery and We have demonstrated that down-regulate HIV expression. regulate such and as can PMA TNF transcriptional activators, trigger HIV expression by the factor In NFkB. contrast, IL-6 and GMCSF transcription activating inducing or are in activators of HIV expression. alone combination post-transcriptional in normal immune homeostasis have Thus, these cytokines which are very important been clearly shown to use the molecular and cellular mechanisms that are involved in the normal immune response to induce HIV expression. The physiological relevance of cytokine-induction of HIV expression is We investigated the role of illustrated by our work with activated B cells. activated B cells in the induction of HIV expression. We demonstrated some time It ago that HIV-infected individuals have a high degree of B cell activation. has subsequently been shown that a substantial proportion of these activated B cells are specific for HIV. We have recently discovered that activated normal tonsillar B cells stimulated with S_^ aureus Cowan and IL-2 secrete substantial amounts of TNF and IL-6, which becomes highly relevant for pathogenesis. We have also demonstrated that unstimulated B cells (which are in fact activated in vivo) from HIV-infected individuals, but not from uninfected controls, can induce the expression of HIV in infected T cells and monocytes. In the autologous system with peripheral blood T cells and B cells from an HIVinfected individual, we observed the spontaneous induction of HIV, which can be blocked by antibodies to the relevant cytokines. Thus, B cells actively making inductive cytokines in the presence of HIV-infected cells is physiologically relevant for the induction of HIV expression in the lymph node. Since the end result of induction of HIV expression in vivo is clinical disease, we are interested is developing therapeutics that can modulate this inexorable inductive chain, which is mediated by normal immunoregulatory cytokines. This interest brings me to the topic of my talk the pharmacological Because of work done by John Kehrl in my regulation of HIV expression. laboratory on TGF-/3, we examined the role of TGF-/3 in the regulation of HIV expression. We found that TGF-/3 was a potent blocker of the induction of HIV expression, both transcriptionally by PMA and also post-transcriptionally, by IL6 and GMCSF. I must point out that TGF-/3 is clearly bifunctional and, depending on the circumstances and the conditions, can either up- or down-regulate HIV induction of HIV expression by with the to However, expression. regard is a TGF-/3 cytokines, potent down-regulator. Since TGF-/3 cannot feasibly be used therapeutically, we began searching for was
-
709
molecules that might mimic the suppressive effect of TGF-/3 on cytokine-induced HIV expression. We found that retinoic acid which has been used clinically in other diseases is one such agent. Over the past year, there have been a number of studies indicating that retinoic acid is capable of inducing complete and partial remissions in a number of neoplastic states, such as a variety of leukemias. Retinoic acid is required to maintain normal differentiation and proliferation of virtually all cells in the mammalian organism. The receptors for retinoids belong to the super-family of intercytoplasmic steroid receptors. Functionally, there is a clear-cut relationship between retinoids and TGF-/3. We found that retinoic acid suppressed the induction of HIV in the chronically infected cell lines with a pattern strikingly similar to that of TGF-/3. Retinoic acid and TGF-/3 block transcriptional activation by PMA but not TNF, and block post-transcriptional-activation by IL-6 and GMCSF. We also have demonstrated that retinoic acid and TGF-/3 can block the induction of HIV expression by TNF plus dexamethasone and IL-6 plus dexamethasone. Using western blot analysis of chronically infected promonocytic cells, we have shown that treatment of the cells with retinoic acid completely blocks the production of HIV proteins. Using nuclear run-on analysis, treatment with retinoic acid blocks the HIV transcription induced by PMA. As I mentioned, retinoic acid does not block TNF-induced transcription of HIV but it does block the post-transcriptional induction of HIV by IL-6 and GMCSF. Because of the relationship between TGF-/3 and retinoic acid, it may be possible that retinoic acid is inducing TGF-/3. To address this problem, we treated PMA-activated chronically HIV-infected cells with TGF-/3 or retinoic acid and then exposed the cells to anti-TGF-/3 antibody. The anti-TGF-/3 antibody was able to block TGF-/3 mediated suppression of HIV in PMA treated cells. However, anti-TGF-/3 antibody had no effect on retinoic acid suppression of HIV in the PMA treated cells. Similar results were obtained when the cells were activated by IL-6 and dexamethasone. In addition, unlike PMA, retinoic acid does not increase the accumulation of TGF-
ß.
found that retinoic acid can suppress HIV at we have Retinoic acid exerts its effect with a pattern closely resembling TGF-/3, and the suppression of expression of HIV does not seem to be We are currently in the correlated with the induction of endogenous TGF-/3. process of trying to dissect out the molecular mechanisms of this effect because of its potential as a therapeutic agent. We have also been examining another which has been shown by Leonard Herzenberg as (NAC), agent, n-acetyl cysteine well as ourselves to block the induction of HIV expression. In closing, it is clear that without the decades of prior research on the complexity of the immune system, we could not have begun to understand the mechanisms of HIV pathogenesis nor to attempt to modulate the replication of HIV pharmacologically. In addition, what we are learning about the regulation of the immune system through the study of HIV will certainly have a substantial impact on our understanding of other diseases of the immune system which are unrelated to HIV. In
multiple
conclusion, levels.
710
SPECIAL LECTURES VIII Jean-Claude CHERMANN, Ph.D, Jacques FANTINI, Ph.D., Valérie CALENDA, Françoise SILVY and Ivan HIRSCH, PH.D. UNITE DE RECHERCHES SUR LES RETROVIRUS ET MALADIES ASSOCIEES
(INSERM U 322) Campus Universitaire de Luminy, BP 33 -13273 MARSEILLE Cedex 9
France -
/ would Tike to thank Dr. Robert GALLO for Inviting me to speak in the lab meeting.
The etiological agent of AIDS is the human immunodeficiency virus which infects and kills CD4 lymphocytes. It has been first isolated in 1983 (1), then characterized and associated with AIDS (2,3). It is a lentivirus (4) going a slow and progressive disease. HIV is CD4 lymphotropic (5) and cytopathic for the CD4+ lymphocytes (2). TABLE 1
:
HIV TARGET CELLS INTERACTION
CD4
lymphocytes Macrophages
DIRECT ACTION
Intestinal cells
=
=
=
>
AIDS
=
=
=
>
Associated diseases
=
=
=
>
(pneumonia dementia...) Malabsorption Diarrhea
Bone marrow precursors
= = =
>
Lymphopenia Thrombocytopenia Anemia
INDIRECT
Kaposi sarcoma B lymphoma ? Other malignancies ?
AIDS is characterized
by an important decrease of immunological defense and more especially by a profound progressive depletion of the CD4+ lymphocytes. HIV1 and HIV2 show a selective tropism for the CD4 lymphocytes and on the envelope gp120 is localized a CD4 binding domain (6) and a V3 loop (7) for the production of neutralizing antibodies. 711
HIV prototype (LAV/HTLV|n) entry is blocked by soluble CD4 or by the preincubation of CD4+ cells with OKT4A or Leu A monoclonal antibodies (8,9). HIV is strictly restricted to CD4 lymphocytes and does not replicate in CD8 cells (5).
Activation of CD4 lymphocytes produces a nuclear factor (NFkB) that permits the replication of latent proviral DNA (10). 1) Virus isolation in AIDS patients : as indicated in Table 2 virus can be isolated in 100 p. cent of AIDS patients. TABLE 2 : AIDS PATIENTS PATIENTS
DIRECT CULTURE
ANTIGENEMIA
COCULTURE
BIASE
337,996
33,664
+
ROUAL
33,926
80,752
+ + +
PEDMA
16,316
38,412
+
43,29 pg/ml
+
22,20 pg/ml
LOP JA
73,684
FOR AL
52,290
AMA MO
173,574
BOU BE
13,748
35,085
LAM MA
57,548
35,590
GILBE
95,306
553,518
AOULA
328,212
RESMU
227,634
STEGI
8,88 pg/ml
13,32 pg/ml
5,55 pg/ml
163,532
16,446
+ +
57,72 pg/ml
Some cocultures are negative like AMA MO in table 2, and this fact can be explained by the high amount of virus present in PBL (direct culture or CD4 fractionated cell) with a high cytopathic effect for all susceptible cells for added first PBL by killing in one cycle all the susceptible cells. In some cases virus cannot be isolated from PBL, not enough CD4 cells for direct cultivation of these fractionated CD4 cells like GAG, table 3 for instance.
These results confirm that the decline of CD4 lymphocytes in HIV is due to the presence of HIV and his cytopathic effect.
712
patients
infected with
2) Virus isolation correlates with a depletion of CD4 lymphocytes as shown virus is constantly isolated from PBL in AIDS patients with low T4 count.
in table 3,
TABLE 3 : VIRUS ISOLATION CORRELATES WITH A DEPLETION OF CD4+ LYMPHOCYTES CD4 COUNT
VIRUS ISOLATION
JAR
172
Posit ve
TAF
291
Posit ve
SAN
527
Posit ve
MAR
208
Posit ve
PRA
330
Posit ve
TIS
396
Posit ve
RES
237
Posit ve
IGL
396
Posit ve
BLA
151
Posit ve
LAT
462
Posit ve
GOM
504
Posit ve
STE
375
Posit ve
GAG
12
Posit ve
GAR
25
Posit ve
SAR
383
Posit ¡ve
HIVAgP24
3) When a virus has been isolated once in HIV infected patients, it is always found in subsequent virus isolation. 19 patients were followed several months in an antiviral clinical trial and they were included in a group receiving a placebo. In table 4 it is shown that the presence of HIV by isolation using the PBL coculture method is 100 p. cent positive and more sensitive than the direct culture. Two strains were used in this study : HIV1-BRU prototype (1) as reference strain and the Zairian highly cytopathic HIV1-NDK (11). The characteristics of the two strains are given in table 5 (12, 13). The second target for HIV is the macrophage (14, 15, 16). HIV infects macrophages
713
TABLE 4 : WHEN A VIRUS HAS BEEN ISOLATED ONCE IN PBL, IT IS ALWAYS FOUND IN
SUBSEQUENT VIRUS ISOLATIONS. PATIENT
(A)
(B)
DIRECT CULTURE
(C)
COCULTURE
(C)
FOR
(5)
(12)
Positive
(1)
Positive
(5)
TEX
(3)
(12)
Negative
(0)
Positive
(3)
TOR
(7)
(13)
Positive
(6)
Positive
(7)
TIM
(12)
(7)
Positive
0)
Positive
(12)
ANS
(8)
(4)
Positive
(6)
Positive
(8)
LEE
(4)
(2)
Positive
(3)
Positive
(4)
CAL
(5)
(2,5)
Positive
(4)
Positive
(5)
MES
(3)
(2,5)
Positive
(2)
Positive
(3)
POU
(10)
(5)
Positive
(10)
Positive
(10)
PHA
(2)
(2)
Positive
(2)
Positive
(2)
CAR
(5)
(2)
Positive
(5)
Positive
(5)
KIE
(2)
(1)
Positive
(2)
Positive
(2)
REN
(4)
(2)
Positive
(3)
Positive
(4)
CAM
(2)
(1)
Positive
(2)
Positive
(2)
GRE
(11)
(5)
Positive
(11)
Positive
(11)
GRA
(3)
(1)
Positive
(3)
Positive
(3)
CAL
(5)
(3)
Positive
(4)
Positive
(4)
BRE
(4)
(2,5)
Positive
(1)
Positive
(4)
MES
(3)
(2,5)
Positive
(2)
Positive
(3)
(A) Number of virus isolation. (B) coculture positive.
through
receptor and replicates without
a
becomes
In months time of follow-up of the
a
reservoir for the virus and is
(dementia (17, 18), pneumonia (14). cerebrospinal fluid during
acute
a
patient. (C)
Number of culture
or
cytopathic effect. This infected cell
responsible
The infected
for HIV associated diseases
macrophage is isolated from the encephalopathy in asymptomatic or AIDS patients 714
TABLE 5 : DIFFERENCES BETWEEN HIV1-BRU AND HIV1-NDK
Cytopathic effect inMT4
Day of detection of 1 TCIU
HIV1-BRU
HIV1-NDK
10-
10-
10
% of infected CEM cells at day 7
0.005
100
Block with OKT4A Leu 3A
Yes
No
or
Block with sCD4
Yes 0.5 m g
Neutralization with monoclonal antibodies directed to V3 loop
(17). Various isolates
(or
more
No
Yes
can
virus, macrophage tropic
be divided at least in three
or
dual
No than 100 ¿¿g)
categories
CD4
tropic (17, 19, 20). Certain patients
lymphotropic
can
present the
HIV associated diseases without immunodeficiency (17). The third target for HIV is the intestinal cells. The gastrointestinal tract is a
putative site of entry for HIV. The ability of HIV to infect rectal or colonie epithelial cells was previously shown in vivo by in situ hybridization (21, 22) and in vitro by using normal (23) or tumoral (24) epithelial cells of colonie origin which are permissive for HIV infection.
sensitivity of human colon epithelial cells to HIV infection has been documented (25, 26, 27, 28). We recently established that viral replication in a well characterized colon cell line (HT29) was a strain-dependent phenomenon, and that producer HT29 cells displayed numerous ultrastructural abnormalities, including altered secretion and impaired differentiation (29). HT29 cells were successfully infected by HIV1-BRU and HIV1-NDK. For NDK, the virus was able to perform its complete cycle of replication and the infected cells could be established as a continuous producer cell line (mean RT activity in the cell-free supernatants around 3x105 cpm/ml). Infection of HT29 cells by HIV1-BRU appeared not to be productive but the virus could be rescued by co-cultivation with lymphoid indicator cells (30, 31). The presence of viral particle in the colorectal lumen could explain the route of
The
715
transmission of HIV in the case of anal intercourse (31) for replicating virus and a silent reservoir, viral expression being under the control of underlying intestinal immune system. Fully replicative HIV in colon epithelial cells may elicit a fundamental disturbance of intestinal function, including a defect of brush border assembly and an increased secretion activity controlled by neurohormonal agents. This functional perturbation caused by HIV replication in epithelial cells, may account for malabsorption and diarrhea occuring in AIDS patients (29).
Hematopoietic cells are also important targets for HIV and some marrow T precursors cells (32), myeloid stem cells (33) and megacaryocytes (34) have been shown to be infected by HIV. To date, the mechanisms responsible for the dysregulated hematopoiesis in AIDS patients are still not clear. We have investigated a possible direct or indirect effect of HIV-1 and HIV-2 on the continued production and differentiation of hematopoietic progenitors maintained in long term liquid culture. We have studied the in vitro effects of HIV on bone marrow hematopoietic progenitor cells (BMHPC). In an effort to standardize our methods of BMHPC infection, we have first studied the replication of HIV in infected normal bone marrow mononuclear cells (BMMNC) depleted of adherent cells and mature T cells, as it was previously performed by Y. Lunardi-lskandar et al. (32). After having reproduced the HIV-1 infection of immature bone marrow T cells, we have studied the susceptibility of hemopoietic progenitors to HIV-1 and HIV-2 infection. For this purpose, we have performed long term bone marrow cultures (LTBMC) after 24 hours incubation with two isolates of HIV-1 (HIV-1 BRU and HIV-1 NDK) and two isolates of HIV-2 (HIV-2 ROD and HIV-2 MEND) at different multiplicities of infection (MOI) (35, 36). Purified hematopoietic progenitors from LTBMC are non producer cells and the virus can be rescued by cocultivation with HIV permissive cells. Bone marrow derived stroma cells are non producer after HIV-1 infection but weakly replicate the virus after HIV-2 infection. HIV-1 and HIV-2 are clearly not cytopathic for hemopoietic precursor cells.
Following production and growth of erythroid burst forming unit (BFU-E), erythroid colony forming unit (CFU-E) and granulo-monocytic progenitors (CFU-GM), for at least 6 weeks after HIV-1 infection, colony assays displayed the same profile of CFUE formation for the two isolates : the production of these progenitors was inhibited by 80 to 100 % after four weeks of LTBMC. On the contrary, BFU-E production in HIV-1 infected LTBMC is depending on HIV strain. HIV-1 LAV gave a 150 % stimulation of BFU-E production during the first week of LTBMC followed by a 50 % inhibition while HIV-1 NDK induced a delayed stimulation (day 24). CFU-GM production was 716
inhibited in LTBMC infected with HIV-1 LAV and HIV-1 NDK, the degree of inhibition ranging from 50 % the first week of LTBMC to about 100 % the last week. Different patterns of progenitors production have been observed after HIV-2 infection. On the contrary to HIV-1, HIV-2 induced a drastic inhibition of erythropoiesis in LTBMC infected with the two HIV-2 strains. Concerning the CFU-GM production, a significant stimulation was first observed during 4 weeks of LTBMC, then, followed by a strong inhibition.
gradually
Stimulating and inhibiting activities were recovered from supernatant of infected LTBMC, used as conditioned media (CM) in clonogenic assay for normal BMMNC. CM from HIV infected CEM cell line displayed stimulating and inhibiting activities in the same fashion as CM from infected LTBMC did. Consequently, we can argue that HIV induces release of (a) humoral factor(s) responsible for disruption of hemopoietic progenitor cells in vitro (37). REFERENCES 1.
BARRE-SINOUSSI F., CHERMANN J.C., REY F. et al : Science, 1983, 220, 868-871.
2.
CHERMANN J.C., BARRE-SINOUSSI F., MONTAGNIER L : UCLA Symposia on Molecular and Cellular Biology, New Series, volume 16. Eds, Michael S. Gottlieb and Jerome E. Groopman Alan R. Liss, Inc., New York, 1984, pp. 31-46.
3.
GALLO R., SARIN P.S., GELMANN E.P. et al. : Science 1983, 220, 865-867.
4.
MONTAGNIER L, DAUGUET C, AXLER C. et al.
:
Ann. Inst. Pasteur
(Virol.) 1984, 135E,
119-
134.
D., BARRE-SINOUSSI F., NUGEYRE M.T. : Science, 1984, 225, 59-63.
5.
KLATZMANN
6.
MACDOUGAL J.S., KENNEDY M.S., SLIGH J.M. et al. : Science 1986, 231, 382-385.
7.
GOUDSMIT J., DEBOUCK C, MELOEN R.H. et al. : Proc. Nati. Acad. Sei. 4482.
(USA) 1988, 85, 4478-
8.
DALGLEISH A.G., BEVERLEY P.C.L, CLAPHAM P.R. et al. : Nature 1984, 312, 763-766.
9.
KLATZMAN D., CHAMPAGNE E., CHAMARET S. et al. : Nature 1984, 312, 767-771.
10.
NABEL G., BALTIMORE D. : Nature 1987, 326, 711-713.
11.
ELLRODT A., BARRE-SINOUSSI F., LE BRAS P. et al. : Lancet, 1984, i, 1383-1385.
12.
REY F., DONKER
13.
CHERMANN J.C, DONKER G., YAHI N. et al. : Nature, 1991, 351, 277-278.
14.
ZIZA J.M., BRUN-VEZINET F., VENETA. et al. : N.
G., HIRSCH I., CHERMANN J.C. : Virology, 1991, 181, 165-171.
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England J.
Med. 1985, 313, 183.
15.
GARTNER S., MARKOVITZ P., MARKOVITZ D.M. et al. : Science 1986, 233, 215.
16.
KOENIG S., GENDELMAN H.E., ORENSTEIN J.M. et al. : Science, 1986, 233, 1089.
17.
GOUT O., ROUQUETTE B., TOURNIER-LASSERVE E. et al. : Biomed. Pharmacother., 1988, 42, 15-20.
18.
CHERMANN J.C. : Inst.
19.
KUHNEL H., VON BRIESEN H., DIETRICH U. et al. 2383-2387.
20.
REY M.A., SPIRE B., DORMONT D. étal. : Biochem.
21.
NELSON J.A., WILEY CA., REYNOLDS-KOHLER C. et al. : Lancet, 1988, i, 259-262.
22.
MATHIJSJ.M., HING M., GRIERSONJ. étal. : Lancet, 1988, i, 1111.
23.
MOYER M.P., HUOT R.I., RAMIREZ A. et al. : AIDS Res. Human Retrovirus, 1990, 6, 1409-1415.
24.
ADACHI A., KOENIG. S., GENDELMAN H.E. et al. : J. Virol., 1987, 61, 209-213.
25.
BARNETT S.W., BARBARA A., WILCOX CM. et al.
26.
ADACHI A., KOENING S., GENDELMAN H.E. et al. : J. Virol., 1987, 61, 209-213.
27.
MOYER M.P., HUOT R.I, RAMIREZ A. et al. : AIDS Res. Human Retrovirus, 1990, 6, 1409-1415.
28.
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29.
FANTINI J., YAHI N., BAGHDIGUIAN
30.
FANTINI J., YAHI N., CHERMANN J.C. : Proc. Nati. Acad. Sei. USA, 1991, 88 : 9297-9301.
31.
FANTINI J., BAGHDIGUIAN S., YAHI N., CHERMANN J.C.
32.
Pasteur/Res. Virol., 1990, 141,
:
137-141. :
Proc. Nati. Acad. Sei.
(USA) 1989, 86,
Biophys. Res. Comm. 1984, 121,
126-133.
Virology, 1991, 182, 802-809.
S., CHERMANN J.C. : J. Virol., 1992,
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LUNARDI-ISKANDAR Y, NUGEYRE MX, GEORGOULIAS V. et al
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610-615. 33.
KITANO K., ABBOUD C.N., RYAN D.H. et al. : Blood, 1991, 77, 1699-1702.
34.
ZUCKLER-FRANKLIN D., CAO Y. : Proc. Nati. Acad. Sei. USA, 1989, 86, 5595-5599.
35.
CALENDA V., SEBAHOUN G., CHERMANN J.C.
:
AIDS Res. Hum. Retroviruses,
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presse
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CALENDA V., TAMALET C, CHERMANN J.C. : Soumis à Blood, 1991.
37.
CALENDA V., CHERMANN J.C.
:
Proc. Second Intern.
Hematopoiesis, Innsbruck, July 14-18, 1991, in press.
718
Symposium
on
Molecular
Biology
of
IMMUNOPATHOGENESIS OF HTLV MYRON ESSEX, D.V.M., Ph.D.
Chairman, Department of Cancer Biology Harvard School of Public Health 665 Huntington Avenue Boston, MA. 02115
The next speaker will be ¡mmunopathogenesis of HTLVs.
MODERATOR:
Max
Essex, discussing
ESSEX: Thank you, Tony. What I'm going to talk about is exclusively HTLV, and most of the emphasis will be on TAX of HTLV-1. I should mention in advance that most of this work was done by Akihiko Okayama, Betty Korber and Arthur Chen in my lab, or formerly in my lab. There was also collaboration with Tun-Hou Lee and Nobuyashi Tachibana in Japan. And unless I say otherwise, all other human serum and tissue materials to which I'll refer were from Miyazaki, Japan, and the island of Kyushu, a highly endemic region for HTLV-1. HTLV-2 has not been identified there. For diagnostic purposes, proteins encoded by the GAG gene or the ENV gene quite regularly induce antibodies.1 They are therefore useful for blood screening and seroepidemiological studies. As opposed to HIV, the polymerase of HTLV very rarely induces antibodies. Only a small fraction of people make antibodies to the reverse transcriptase but it does occur. The TAX protein and the REX protein are more interesting in that they induce antibodies in some people and not in others. About half to twothirds of HTLV-1 infected people have antibodies to TAX. This proportion varies with health status and according to route of exposure. As analyzed by radioimmunoprecipitation, virtually everybody has antibodies to the envelope, and about half of the healthy HTLV-1 carriers have antibodies to TAX. This study was done with some particular samples from HTLV-infected families. We had access to samples from a large number of individuals who were in leukemic families. In such families there was a patient with adult T cell leukemia, and between 40 and 50% of the other healthy individuals scored as antibody positive by conventional structural protein antibody assays for HTLV-1 structural protein antigens. We also showed that people who had TAX antibodies had a much greater risk for excreting the virus, and infecting their spouses, so that in a seroconversion cohort in Miyazaki, Japan, all of the partners who infected their previously uninfected spouses had such antibodies.2 Only a quarter of the spouses who were infected but did not infect their spouses over the same period of time had such antibodies. Beyond the correlation of TAX antibodies with infectiousness or transmissibility by sex, there's also a clear positive correlation with the presence of such antibodies in HTLV-1 positive children of ATL mothers.3 Children of ATL positive mothers are more likely to have TAX antibodies in younger age categories as compared to age-matched HTLV-positive blood donors. Conversely, older age groups of HTLV-positive blood donors are more likely to 719
have TAX antibodies than age-matched children of ATL mothers. In either case, the presence of TAX antibodies seems to be associated with infectability and with the proximity to time of infection. We and others, such as Slamon and Chen, and Poiesz and colleagues had noticed on various occasions in screening blood bank samples that a few rare samples scored as negative for antibodies to whole virus, but positive for antibodies to TAX. Nobody knew quite what significance to put on this. To address that question more specifically, we looked at the samples from ATL households in southwestern Japan, in Miyazaki, and specifically looked at seronegative individuals in the HTLV cluster households. We found that under these conditions, the rate of TAX-only antibody people, who scored positive for TAX antibody after scoring negative for structural protein antibodies, as verified by radioimmunoprecipitation and Western blot and other techniques, was quite significant. It was about 8 or 9% of the HTLV-1 seronegative individuals who were family members of ATL To verify specificity, we showed that reactivity occurred with native TAX in the absence of any antibody to structural proteins, and that this reactivity could be blocked very nicely with the recombinant TAX protein. The TAX antibodies could be detected by a variety of assays, using different sources of TAX proteins, whether naturally made in the cell, or made in E.coli by recombinant technology. Either way they cross-reacted very well to block the reactivity. We then screened other serum collections to see if individuals with antibodies to TAX but not to other HTLV antigens were found in association with exposure to HTLV. Samples from the general adult population in the high-risk city of Miyazaki revealed 11 of 700 that were negative for antibodies to HTLV structural antigens, but were positive for antibodies to TAX. Similarly, in a group of drug abusers at Bronx Lebanon Hospital in New York, at high risk for HTLV exposure, the same general result was obtained. Of 114 HTLV seronegatives that happened to be HIV seropositive, 4 had antibodies to TAX, as did 2 of 113 HIV seronegative people from the same population. Similar results were obtained from a group of Israeli hemophiliacs. There appeared to be several different ways that people would be infected so that they would have antibodies only to TAX. One would be the presence of the virus with a defective REX gene. Another would be the presence of an HTLV with a deleted genome. Whether or not either of these would be biologically important is unknown. We then undertook an extensive PCR study of fresh adult T cell leukemia cells, again from patients residing in the HTLV-endemic area of Miyazaki in southwestern Japan. We used a number of primers to evaluate the pattern of the proviral genome in these cells. Cells were examined from 23 different patients assumed to have ATL. Except for one, all scored positive quite well with the LTR primer. Many of them scored negative or almost negative with primers for GAG and protease. These patients were particularly interesting. They represented adult T cell leukemia patients with very high circulating abnormal cell counts, where the abnormal leukemic cells must have accounted for almost all of the nucleated cells in the circulation. A significant fraction of such patients scored very high for TAX and LTR, but were essentially negative or close to negative for protease, GAG, and sometimes for envelope, when primers specific for those regions were used. This result was obtained with 30-40% of the samples. For 50-60% of the individuals, all of the PCR primers scored highly positive, including gag and protease, representing all different regions of the viral genome. The latter
patients.^
720
individuals presumably had one or more copies of complete provirus in each leukemic cell. There were also a few unusual individuals whose cells we examined who did not have a diagnosis of conventional ATL. Some of these would fit the same pattern of positivity for TAX and LTR and negativity for GAG and protease. Some would fit the pattern of positive for everything, and some would fit the pattern of negative for everything. The latter were probably not ATL at all, but some other type of leukemia that happened by chance to occur in an HTLVinfected person. The three classes of results are summarized in Table 1 and will be published soon.5 TABLE 1. Classes of PCR Reactivity in Leukemic Cells from HTLV-1 Positive Patients
Category
GAG
PRO
PCR Primers POL ENV TAX
LTR
Antibody
Interpretation_
1
+/-
Selectively deleted provirus present.
2
+
Complete provirus present.
No provirus in most leukemic cells.
3
The
possibility that the defective provirus might be made by reverse transcription of a spliced message was then examined using a primer for the splice junction. This primer gave negative results when leukemic cells with the deleted provirus pattern were examined. By southern blotting procedures, the defective deleted genomes gave predicted results, a profile of a partial genome without all the expected bands present when PST-digested DNA was probed. There have been previous reports from the Yoshida and Gallo labs, describing defective genomes in ATL.6.7 However, such defective genomes were usually considered an unusual occurrence or an artifact of in vitro cultivation rather than a frequent event in the natural history and evolution of the
leukemic cells in their host. The current results show that in at least a third, and in probably more than a third, the tumor cell itself has a defective provirus as the predominant, or only major provirus form present. One hypothesis to explain how this might occur is ¡mmunoselection. With HTLV-1 infection there is an active and efficient immune response under most circumstances, unlike the case with HIV infection. With HTLV-1, most people do not develop a lethal terminal disease, and the virus is more sufficiently suppressed in a non-replicative form than is HIV. The HTLV-1 infection is then activated by antigenic or mitogenic stimulation. When an immortalizing transformation type of event occurs in a cell that is infected with complete copies of provirus, virus production should occur. This might result in elimination of that cell by an immune mechanism such as CTL's or antibodies directed to virus structural antigens on or in the tumor cells themselves. That would then allow for selection of cells that were immortalized,
721
but had only retained partial genomes. Such genomes might express only TAX and LTR. Those cells that fail to express structural proteins might have a selective advantage to grow out as malignant clones. Now, one could also imagine that exactly the same type of mechanism occur in other systems that haven't been adequately explained, such as might B virus hepatitis hepatoma, for example, where an immune lysis of virus infected hepatocytes occurs as hepatitis before the hepatoma develops. Most of the liver damage appears to be immune-mediated, related to activation of immune effector mechanisms against viral proteins. We know that when tumors emerge as hepatomas later in the disease, they occur in such a way that the viral DNA in the tumor cells has undergone extensive deletion. However, if any of the hepatitis B virus DNA is retained, it is most often the X gene; the gene that is analogous to TAX of HTLV. Although we know very little about the immune response that occurs following infection with human papilloma 16 or 18, the associated cervix cancers that emerge also appear to have deleted defective genomes and retention of transactivation type genes. Thus, in a wildly speculative way I could hypothesize that most virus-associated human cancers evolve in a host environment of immunoselection that favors the loss of viral structural proteins and genes from the tumor cells, if not from the entire host. I should also refer to the very recent report by Hall et al.8 describing the analysis of several cases of HTLV antibody negative mycosis fungoides using PCR. They found defective deleted HTLV provirus in several cases and cloned out a virus in at least one case. It was the same general type of defective provirus that we now see in at least a third of the ATLs in Japan. One logical conclusion that follows from all of these observations is that at least some human cancers may be virus associated and virus-caused but lose virus replication, virus structural proteins, and virus structural protein genes by the time the tumor grows out. Logically, then, searches to link other human cancers to viruses should include probes for transactivator genes. Another question came to mind. Could such defective viral genomes be passed to another host? If so, maternal-infant transmission would seem to be most likely, where lymphocyte cell contact might occur.. It is intriguing that the highest rate of the TAX-only antibody profile was found in children born in ATL families. That of course, could be another and much more exciting explanation for why some people might have antibodies only to the TAX gene product.
Thank you.
MODERATOR: Questions
or
comments?
QUESTION: We are finding messages for the PX proteins in the majority of the cases of ATL. We don't know yet whether leukemic cells are producing it, or non-leukemic cells or both. find
ESSEX: That would make sense, but I suspect that it would be harder to expression of virus or viral structural proteins in many cases.
QUESTION: PCR, you have to go to PCR to be able to find messages. Because... in ATL you don't have the expression of messages, and you don't find any proteins. But it might be there at a low level. You also find messages with the classical spliced donor, or classical spliced acceptor sites. We don't, 722
haven't analyzed yet extensively the integrity of the genomes. But the messages we find in other cases, and we've cloned them, conformed to the classical splice acceptor sites, and the classical spliced donor sites. we
some
ESSEX: Yes, I think that would be an important experiment to do with tumor cell preparations that were specifically deleted in GAG and
polymerase. In fact, see sitting here.
I discussed that at
one
point
with
George
Pavlakis who I
Perhaps I could just make one comment on the mycosis In addition to the American, or sorry the Swedish mycosis we looked fungoides. have started look at some Japanese HTLV seronegative mycosis but we to at, we haven't finished these studies yet. I'll describe them a little bit on Saturday, but what we do know in those, in the ones that have HTLV-1 involvement they all don't but the PX is also retained in those, whereas other regions appear to QUESTION:
-
be
1
)
-
missing.
Lee, T.H., Coligan, J.E., Homma, T., McLane M.F., Tachibana, N.., and Essex, M. Human T-cell Leukemia Virus Associated Membrane Antigens
(HTLV-MA): Identity of the Major Antigens Recognized Following Virus Infection. Proc. Nati. Acad. Sei. 81:3856-3860 (1984).
2)
Chen, Y.A., Okayama, A., Lee, T.H. Tachibana, N., Mueller, N. and Essex,
Sexual Transmission of Human T-Cell Leukemia Virus Type 1 Associated with the Presence of Anti-Tax Antibody. Proc. Nati. Acad. Sei., M.
8_8_:1182-86 (1991).
A. Chen, Y.A., Tachibana, Shioiri, S., Lee, T.H., Tsuda, K. and Essex, M. High Incidence of Antibodies to HTLV-1 TAX in Blood Relatives of Adult T-cell Leukemia Patients. J. Infect. Dis., 163:47-52 (1991 ).
3)
Okayama,
4)
Okayama, A., Korber, B., Chen, Y.A., Allan, J., Lee, T.H., Shioiri, S., Tachibana, N., Tsuda, K., Mueller, N., McLane, M., Mayan, S. Marlink, R., Orgad, S. amd Essex, M. Unusual Pattern of Antibodies to HTLV-1 in Family Members of Adult T-cell Leukemia Patients. Blood. 7.8:3323-3329 (1991).
5)
Korber, B., Okayama, A., Donnelly, R., Tachibana, N. and Essex, M.
Chain Reaction Analysis of Defective Human T-Cell Leukemia Virus Type 1 Proviral Genomes in Leukemic Cells of Patients with Adult T-Cell Leukemia. J. Virol., £5_: 5471-76 (1991).
Polymerase
6)
Yoshida, M. Seiki, M. Hattori, S., and Watanabe, T. 1984.
7)
Aldovini, A. DeRossi, A, Feinberg, M., Wong-Staal, F., and Franchini, G.
Genome Structure of Human T-cell Leukemia Virus and Its Involvement in the Development of Adult T-cell Leukemia/Lymphoma. p 141-148. In: R. Gallo, M. Essex and L. Gross (ed.), Human T-cell Leukemia/Lymphoma Virus, Cold Spring Harbor. 1986. Molecular Analysis of a Deletion Mutant Provirus of Type 1 Human T-cell Lymphotropic Virus: Evidence of a Double Spliced X-lor mRNA. Proc. Nati. Acad. Sei. USA, 83:38-42. 723
8)
Hall, W.W., Liu, C.R., Scheerwind, O., Takahashi, H., Kaplan, M. H., Röupe, G. and Vahlne, A. Deleted HTLV-1 Provirus in Blood and Cutaneous Lesions of Patients with Mycosis Fungoides. Science, 253:317-320
(1991).
SPECIAL LECTURES IX
"EXTRACELLULAR TAX, PROTEIN STIMULATES NF-kB AND EXPRESSION OF NF-kB-RESPONSIVE Ig KAPPA and TNFB GENES IN LYMPHOID CELLS"
John N. Brady, Ph.D. Laboratory of Molecular Virology National Cancer Institute National Institutes of Health Bethesda, MD 20892
HTLV-I Tax1 plays an important role in virus replication and cellular transformation. As a nuclear protein, Tax1 clearly has the ability to regulate both viral and cellular gene expression. Today, I would like to discuss a series of experiments performed by Paul Lindholm that suggest the possibility that Tax, also works as an extracellular cytokine, activating transcription factors and expression of specific cellular genes in noninfected cells. We have been able to define two functions for the extracellular Tax, protein. First, in pre-B lymphocytes Tax, activates the nuclear accumulation of the NF-kB transcription factor. This activation apparently involves either the release of the cytoplasmic IkB inhibitors or processing of the NF-kB pl05 precursor protein. The level of NF-kB induction is sufficient to activate expression of endogenous cellular genes. Second, Susan Marriott has demonstrated that extracellular Tax, stimulates proliferation of peripheral blood lymphocytes. The PBL proliferation is accompanied by an increase in the expression of the IL-2 receptor. Before we begin our discussion of the functions of extracellular Tax,, it is important to consider the evidence that Tax, is, in fact, secreted into the tissue culture media of cells transformed or infected with HTLV-I. We have used two experimental approaches to test this possibility. First, HTLV-I transformed cells were pulsed with S and then the accumulation of Tax, in the cell or the extracellular media was assayed by immunoprecipitation with Tax,-specific antibody. As expected, Tax, is easily detected in the nucleus of the transformed C81 cells. We estimate that the half-life of the cellular Tax, is about 2-3 hours, consistent with observations from other laboratories. In the extracellular media, Tax, can be detected by one hour of chase incubation in fresh culture media. The level of extracellular Tax, remains constant for about six hours and then declines gradually. By western blot analysis, using purified Tax, as a standard, we estimate that the concentration of Tax, in the extracellular fluid ranges from 0.5 to 1 nanomolar. This compares with a concentration of 1 to 5 micromolar Tax, in the infected cell. Tax, does not contain a distinguishable signal peptide sequence. Thus, the mechanism by which Tax, is released from the cell remains to be established. The Tax, protein that we have used in the studies I will be discussing today is purified from E. coli. Using a purification protocol developed in the laboratory, the Tax, has been been purified to homogeneity and is a single band on a SDS polyacrylamide gel. If 724
you label this protein with 125I and monitor its very rapid accumulation of Tax, in the cells.
uptake in pre-B lymphocytes, you see a Initially, the majority of the Tax, protein about 20 cytoplasm. By hours, you see that 80% of the Tax, is in the
is located in the nucleus of the cell. Following the uptake of the Tax, protein by these cells, the level of NF-kB binding activity is significantly increased. You see in this gel shift assay that the control level of NF-kB in these pre-B cells is very low. If the cells are treated with control bacterial extract, you see no increase in the level of NF-kB binding activity. In contrast, when we add Tax, to the extracellular media, at a concentration of 2.5 nanomolar, you see a quantitative induction of the NF-kB transcription factor in the nuclear fraction. The specificity of the gel shift complex is shown by competition with a wild type, but not
mutant, oligonucleotide. Because the Tax, was purified from E. coli, it was important for us to control for the possible presence of endotoxin in these extracts. Two lines of evidence demonstrate that the NF-kB induction is not due to endotoxin, but is due to the Tax, protein. Chloroform extraction of soluble extracts removes protein, but not endotoxin, from the sample. Chloroform extraction of the Tax, protein removes the protein from the sample. If we take the control and chloroform-extracted Tax, and analyze it for the ability to induce NF-kB, only the control non-extracted Tax, will induce binding activity. No induction with the chloroform-extracted Tax, was observed. This same experiment was done on bacterial endotoxin. No significant change in the ability to induce NF-kB was observed. The other experiment that we have done is to clear the Tax, protein sample with antiTax, serum. When we take the supernatants from immunoprecipitations done either with normal rabbit serum or anti-Tax, serum, only the latter neutralized the ability of Tax, to induce NF-kB binding activity. These two lines of evidence strongly suggest the a
biological activity is due to the Tax, protein. The ability of Tax, to activate NF-kB is dose-dependent. If we add increasing amounts of the Tax, protein to the extracellular media, we get a quantitative increase in the level of NF-kB. Within the detection limit of the gel shift assay, we need about 750 picomolar extracellular Tax, to induce detectable NF-kB binding activity. This concentration is in
the range that we see in the extracellular media of HTLV-I transformed cells. What interests us about the ability of Tax, to induce the NF-kB transcription factor is its importance in regulation of a number of cellular genes. These include genes of the immunoreceptor family, such as IL-2Ro, and important cytokines, such as IL-2, TNFa, TNFß, GMCSF and IL-6. Several of these genes, including IL-2Ra, IL-2, TNFß, GMCSF and IL-6 are induced by Tax,. What I will show you today is an analysis of two of these genes, TNFß and the immunoglobulin kappa light chain. We are particularly interested in the TNFß, or the lymphotoxin gene, because in addition to its cytotoxic effects, it also has the ability to act as an osteoclast activating factor and stimulate bone résorption. This may result in elevated levels of calcium in the circulatory system and might be instrumental in the hypercalcemia that one sees in ATL patients. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of either the TNFß or the immunoglobulin kappa light chain indicates that Tax, stimulation elevates the level of RNA expression from these genes. In the case of the TNFß, we see a very quick induction. By three hours after the addition of the Tax,, you see an increase in the level of RNA synthesis. This elevated level of TNFß RNA is maintained over the first 24 hours. We also see the induction for the immunoglobulin kappa light chain, although the kinetics are a little different. It takes about eight hours to see a significant accumulation of kappa light chain RNA. The elevated level of mRNA is maintained for twenty-four hours. We have used actin as a RT-PCR control. No change in the level of actin RNA synthesis was observed between the control cells and the Tax,-activated cells. 725
Because of the complexity of NF-kB transcription factor family and the difference in the kinetics of the activation of the TNFß and the immunoglobulin kappa light chain, we were
interested in
determining
if
Tax,
turned
on
these two genes by the
same
proteins.
The sequence of the NF-kB binding sites in the immunoglobulin kappa enhancer and the TNFß promoter are as follows: Igk 5'-GATCCAGAGGGGACTTTCCGAGAG-3' and TNFß 5'-GATCCAGAGGGGCTTCCCCGAGAG-3\ The immunoglobulin kappa enhancer element is a nonpalindromic sequence which is purine-rich on the 5' side and rich on this 3' side. In contrast, the TNFß promoter has a palindromic
pyrimidine sequence.
The induction of NF-kB binding to the kappa light chain and TNFb has been analyzed in detail following Tax, induction. With the kappa light chain oligonucleotide, you see one gel shift complex. This complex increases by one to three hours after the addition of Tax,. The level of NF-kB binding continues to increase over the first seven hours of stimulation and then starts to decrease. In contrast, what you see with the TNFß probe is two distinct complexes. The C-l complex, which migrates to a similar position in the gel as the kappa light chain probe, and a faster migrating C-2 complex. The C-2
complex is induced by one hour, decreases in intensity between three and seven hours and then increases by twenty-four hours of incubation. The C-l complex follows more or less the kinetics of what one sees for the immunoglobulin kappa light chain. We next UV cross-linked these gel shift complexes and then analyzed the products on protein gels. With the immunoglobulin kappa light chain probe, you get one predominant protein species of 75 kD (p65) in the Cl complex. In addition, a minor protein species of 59 kD (p50) is detected in this complex. With the TNFß probe, the major protein in both the Cl and C2 complex is the 59 kD (p50) protein. The Cl and C2 complex also show a difference in sensitivity to NF-kB inhibitor, IkB. Addition of IkB to nuclear extracts inhibits formation of the Cl gel shift complex. No effect on formation of the C2 complex was observed. Our analysis suggests that the immunoglobulin kappa light chain Cl complex consists of two proteins, the p50 NF-kB transcription factor and the p65 transmodulator. This complex is sensitive to inhibition by IkBa and MAD 3. In contrast, the TNFß C2 complex seems to be comprised of two p50 NF-kB proteins. The p65 transmodulator is apparently not found in this complex. This complex would be sensitive to different inhibitors in the cell, IkBß or the carboxy terminal fragment of the P-105 precursor, PDI. How does
transcription factors? One of the most common cytoplasmic form of NF-kB is to phosphorylate the inhibitor. This modification causes the release of the inhibitor, allowing the p50/p65 complex to migrate to the nucleus, bind to the DNA and activate transcription. We have investigated whether this is due to the protein kinase C pathway. Using staurosporin inhibition and TPA depletion studies, we conclude that the protein kinase C pathway is not involved in the Tax, activation. The possibility that Tax, activates other protein kinases in the cell, either through the protein kinase A pathway or other protein kinases remains to be
pathways
Tax,
activate the NF-kB
to activate the
established. Tax, activation of NF-kB is not dependent upon protein synthesis. If you add Tax, to the extracellular media in the presence of cycloheximide, no significant reduction in the ability of Tax, to induce NF-kB is observed. Consistent with these results, using RTPCR analysis no increase in the level of either c-rel or NF-kB RNA synthesis was detected in the Tax,-treated cells. Thus, whatever the mechanism of Tax, induction, it seems to be dependent upon activation of pre-existing protein complexes or precursor
proteins.
we have investigated is to determine whether Tax, could directly release the inhibitor from the inactive cytoplasmic complex. In this analysis cytoplasmic
The other pathway that
726
cells, which contain very little or low activity of the NF-kB have been treated with either DOC/NP40 or Tax,. If you treat the transcription factor, inactive p50/p65/IkBa complex with DOC/NP40, an induction of gel shift activity is observed. In contrast, the addition of Tax, protein to the p50/p65/IkBa containing extract did not increase the level of gel shift activity. These results suggest that Tax, does not directly dissociate the IkB inhibitor from the complex. The results of a related series of experiments suggest that Tax, does not effect the ability of the IkBa inhibitor to reassociate with the p50/p65 complex. extracts from untreated
In summary, I would draw the following conclusions from our studies: 1. Tax, is rapidly taken up by pre-B lymphocytes and peripheral blood lymphocytes. Cellular f ractionation demonstrates that approximately 80% of the cell-associated
2. 3
4.
5. 6. 7. 8.
Tax, protein is transported to the nucleus of the uninfected cell. Tax, protein stimulates nuclear NF-kB binding activity in pre-B lymphocytes in a rapid, transient and dose-dependent manner. Tax, stimulation of NF-kB does not require an active protein kinase C system. It may involve either the protein kinase A pathway or other kinases. Tax, was unable to dissociate the NF-kB/IkB complex directly. Tax, did not prevent reassociation of IkB with NF-kB. Tax, stimulation of NF-kB binding activity was not inhibited by cycloheximide. Tax, stimulation of pre-B lymphocytes caused an increase in RNA expression from the immunoglobulin kappa light chain and the TNFß genes, as determined by RT-PCR analysis. The TNFß and immunoglobulin light chain NF-kB elements show differential binding to NF-kB proteins by gel shift and UV cross-linking analysis. Purified IkB inhibits binding of the p50/p65 (Cl) complex but not the p50/p50
(C2) complex.
References:
Lindholm, P. F., S. J. Marriott, S. D. Gitlin, C. A. Bohan, and J. N. Brady: Induction of nuclear NF-kB DNA binding activity after exposure of lymphoid cells to soluble Tax, protein. The New Biologist 2:1034-1043, 1990. Lindholm, P. F., S. J. Marriott, S. D. Gitlin, and J. N. Brady: Differential precipitation and zinc chelate chromatography purification of biologically active HTLV-I Tax, expressed in E. coli. J. Biochem. and Biophys. Methods 22:233-241,1991.
Marriott, S. J., P. F. Lindholm, R. L. Reid and J. N. Brady: Soluble HTLV-I Tax, stimulates proliferation of human peripheral blood lymphocytes. The New Biologist 3:678-686, 1991. Lindholm P. F., R. L. Reid and J. N. Brady: Extracellular necrosis factor
(In press)
e
and
immunoglobulin kappa light
727
chain in
Tax, protein stimulates tumor
lymphoid cells. J. Virology
NEW ADVANCES IN THE MANAGEMENT OF AIDS ASSOCIATED OPPORTUNISTIC INFECTIONS
Henry Masur, M.D. Chief, Critical Care Medicine Department National Institutes of Health Bethesda, MD 20892
September 1991 issue of Medicine, our group at NTH has published its cumulative with experience patients who have been followed for their entire course and who ultimately came to at NTH. The frequencies of various infections and tumors are demonstrated in this review. autopsy It's important to focus on the fact that there is really a wide diversity of infections that cause morbidity and mortality. It is also important to try to focus on those processes that really cause the most morbidity, and those that cause mortality. In this list, there are processes like pneumocystis, CMV, cryptococcus and toxoplasma which cause very substantial morbidity, and other processes like candida or Herpes simplex, which can certainly be annoying, but which do not usually have such a substantial impact on survival. Thus, opportunistic infections can have a wide range of implications for patients: some are life threatening, others are not. With some opportunistic processes, our progress in improving diagnosis, therapy and prevention has been more substantial than in others. We have made particular progress in our ability to manage pneumocystis pneumonia and cerebral toxoplasmosis. For other organisms like CMV and perhaps Mycobacterium tuberculosis, however, there is an increasing impact due either to our lack of advances in diagnostics and therapeutics, or for tuberculosis, due to changing socioeconomic trends. Some of the organisms that we first recognized at the beginning of the 1980s, like Mycobacterium avium-intracellulare are having an increasing impact. Patients are surviving longer due to improved antiretroviral therapy and improved management of other previously fatal processes. Thus, patients are surviving longer in a very immunosuppressed state. Mycobacterium avium-intracellulare is becoming more important because we haven't made as much progress in terms of understanding when to treat it, or what drugs to use. There are a number of newer organisms which we didn't know how to recognize previously, which we're now recognizing. These include cryptosporidia, microsporidia, and Rochalimaea. We are just learning how to diagnose these agents. We have no therapy for them. I think that one of the issues for the 1990s is that as HIV moves to new geographic areas and as we're more successful in preventing death due to some of these processes, we are going to see new processes emerge which are going to cause increasing morbidity and mortality as patients survive longer. There are a number of reasons why we're more successful in dealing with some of the classic opportunistic processes. First, we know a lot more about the natural history of HIV disease. A number of studies that have been reviewed this week focus on the natural history and the immunopathogenesis of HIV and have helped delineate the time when opportunistic processes occur in the life history of an HIV infected patient. To a large degree, this has helped clinicians focus on a time when they can look for specific opportunistic processes. Thus, understanding the natural history has been a very important advance in terms of more effective management of patients. We know that the CD4 counts inevitably decline over time despite the persistence of AZT therapy. As an example, we know that pneumocystis pneumonia generally occurs in patients with CD4 cell counts below 200/mm3. Thus, when a cough occurs in patients with CD4 counts greater than 300-400 /mm3, the likelihood of pneumocystis pneumonia is low. In contrast, in a patient with a CD4 count below 100/mm3, there is considerable likelihood that cough or dyspnea could be caused by pneumocystis. Understanding when opportunistic processes occur in the natural history of HIV is very important for clinicians in developing effective management strategies. Patients who have more than 500 CD4 cells/mm3 are generally asymptomatic, although they may develop Kaposi sarcoma or lymphoma. Those in the 200-500/mm3 range may have fevers, sweats, and weight loss, which In the
728
might be due to HIV, CMV, or some other process. They may develop some of the less lifethreatening processes like hairy leukoplakia, oral or esophageal candidiasis, or tuberculosis. Only when a patient gets to the lower CD4 levels do you start developing pneumocystis, cryptococcus or dementia, and it's only when your CD4 count is extremely low, that you develop disseminated mycobacteria or significant CMV disease. As Bob Yarchoan has shown recently in the Annals of Internal Medicine, most of the patients who die despite aggressive management are
those whose CD4 cells are less than 50/mm3. Armed with this knowledge, one can decide when to do various diagnostic tests when assessing different clinical syndromes. I'd like to provide an example of the progress that we've made both in understanding when in the natural history of HIV disease opportunistic processes occur, and in knowing how to diagnose the process when we know that a patient is susceptible. I'd like to take pneumocystis pneumonia as a specific example. We've not made as much progress for certain other opportunistic processes like CMV and perhaps MAI in terms of knowing when and how to treat these processes. For pneumocystis, we know that any time we can find pneumocystis organisms in pulmonary secretions or tissue, that the patient likely has active disease and needs to be treated. One of the major advances over the last decade has been that we not longer have to do an invasive procedure like open lung biopsy in order to make the diagnosis. And actually, we don't even need to do an expensive, and time-consuming procedure like bronchoscopy, which may cost $300-600 to perform, and can be uncomfortable. Now with an induced sputum technique, we can noninvasively make the diagnosis of pneumocystis in over 90% of patients who are not receiving aerosol pentamidine prophylaxis. The ability to diagnose a life-threatening process by a non-invasive, inexpensive technique is both a major economic advance, and it's a major advance in terms of our ability to improve survival. Patients are much more likely to come forward and have this test when their symptoms are mild. And we know that the milder the disease at the time we start therapy, the more likely to a outcome. to is have patient good Improved diagnostic techniques for pneumocystis have been a major advance in our management over the last 10 years. I think that now, at least in the developed world, having induced sputums available is a major opportunity to improve the quality of patient survival. In addition to the ability to make a diagnosis of pneumocystis, we also understand a lot more about the pathophysiology of pneumocystis. Just as understanding pathophysiology's been important for HIV, it's been very important in improving our management of pneumocystis. In terms of understanding pathogenesis, we've made some real advances. A feature of pneumocystis pneumonia that was not widely appreciated before the era of AIDS has been that, when you start therapy, you appear to increase the inflammatory reaction in the lung, and the pulmonary physiology deteriorates. If one looks at the hypoxemic ratio in patients who are on standard therapy, generally the patient will become substantially more hypoxemia during the first 3 days one starts therapy. This is presumably because lysis of organisms is initiating an inflammatory response and thus inflammatory responses exacerbate gas exchange. We are just beginning to understand which mediators are responsible for this. But if you administer corticosteroids to the patient, you can prevent this deterioration in respiratory physiology from occurring. This means that as we give standard therapies, we will want to avoid this drop because often a patient will either die because of this drop, or they'll need ventilatory support in the ICU during this drop. By understanding that this event occurs, we can intervene with corticosteroids. Hopefully in the years to come, as we understand specifically about what cells and what cytokines are responsible for this drop, we can intervene with drugs tha,t don't have some of the deleterious effects that corticosteroids may have. We may be able to intervene in a much more specific, and much less harmful way, and further improve the prognosis and quality of life of patients with pneumocystis pneumonia. Specifically, if we take patients with moderate or severe pneumocystis, and add adjunctive corticosteroids to standard therapy, that we can decrease mortality from about 22% to about 11%. Especially for patients with P02's under 70mmHg at the time therapy is instituted, understanding that this hypoxemic event occurs during the first 72 hours, and understanding that intervening with steroids can be beneficial, has been a major advance. As we understand more about what the specific immunologie events responsible for deterioration are, we 729
intervene with have.
something that is less likely to have the deleterious side effects that steroids Another issue of note is that a number of funding agencies, including the Division of AIDS, NIAID, have put major resources into identifying new drugs for the therapy and prophylaxis of pneumocystis. I think that many house officers in 1990 can't remember the era in the early 1980s when we only had two drugs that were effective against pneumocystis, i.e. trimethoprim-sulfamethoxazole and parenteral pentamidine. By using a variety of approaches, including an empiric approach looking for agents that are active against taxonomically similar organisms to pneumocystis, or using in vitro cultivation techniques, or using metabolic approaches which have used cell-free enzyme systems derived from pneumocystis, we've identified a number of new agents, including macrolides, primaquine analogs, some pentamidine analogs, and some other drugs. One of the most interesting developments just came to light in this past week. A drug that Burroughs-Wellcome had developed, 566C80, had been known in an animal model to have activity against pneumocystis. In an article recently published in The New England Journal of Medicine, this hydroxynaphoquinone, which is an entirely novel compound, has been shown in an open trial to possess a high degree of efficacy, with very little toxicity. In addition, BurroughsWellcome recently completed a multicenter trial looking at 566C80 versus standard therapy with trimethoprim-sulfamethoxazole. It will be very interesting to see how this new drug compares to standard therapy in terms of efficacy, toxicity, and ability to prevent relapse. Not only do we understand more about the immunopathogenesis of pneumocystis, not only do we have more drugs available for treatment, but we've also made substantial progress in terms of preventing pneumocystis. The number of cases of pneumocystis that occurred in the pre-AIDS era was quite small and has dramatically increased from about 60 a year in the pre-AIDS era to about 60,000 a year that are projected in the post AIDS years of the late 1980s and early 1990s. The number of cases of pneumocystis appears to be declining despite the increase in size of the population at risk. Clearly, we have been much more effective in developing prophylactic strategies recently. I think the first development in the 1980's that was impressive was the use of a novel method for drug delivery, namely aerosolization with regard to pentamidine. Aerosol pentamidine delivered by the route recommended from the San Francisco Community Study clearly showed efficacy in reducing secondary cases. There has been considerable amount of enthusiasm for aerosol pentamidine as a means to substantially reduce the dramatic number of cases of pneumocystis that occurred, but there's been a growing amount of data suggesting that oral trimethoprim-sulfamethoxazole may be more effective. In data that have just been announced in a press release yesterday, the ACTG study 021 has shown that in fact, trimethoprim-sulfamethoxazole is about 3 1/2 times more effective than aerosol pentamidine, and that its toxicity, although somewhat greater than aerosol pentamidine, may not be as great as one might think. Thus, pneumocystis is a good example of an organism for which we really have made a lot of progress, because 1) we understand when in the natural history of HIV it occurs, 2) we understand how we can prevent the deterioration in respiratory physiology associated with early therapy, 3) we know how to diagnose it more readily, and 4) we've made some real advances in therapy and in prophylaxis. There are other processes that we've known how to recognize where, unfortunately, we've made a lot less progress. CMV is a disease which is very common in HIV-infected patients. We've recognized that CMV can cause very obvious disease like retinitis, we have seen that a very large number of patients, 73% in one series, have evidence of disease, not just infection, due to CMV at autopsy. And as we're able to handle pneumocystosis, toxoplasmosis, and cryptococcosis better, we are not able to manage CMV in many situations with much better results than we were probably in the middle part of the 1980s. The reason for this is that, while we do have some drugs that are finally effective against CMV, we don't really know precisely when CMV is actually causing fever, weight loss, or inanition that often occurs when a patient has an occult gastrointestinal or pulmonary disease. One of the advances that we need for CMV that we have for pneumocystosis is a mechanism for deciding when patients who are infected with CMV in fact need treatment. One of the very cost-ineffective things that we do for CMV is that we culture blood and urine for CMV, but don't know how to use the resulting data. There is a very clear relationship between viremia can
730
and the CD4 count, which is not surprising. What is surprising is that when we've looked at the prognostic implications of being blood culture positive, we find that those who are blood culture positive are not much more likely to have a premature death than whose who are blood culture negative, and that having a positive blood culture does not substantially predict who's going to develop retinitis, pneumonitis, or colitis in the ensuing six months. If you look at the other side of the coin, i.e. those who already have retinitis, or pneumonitis or colitis, we find that many of those individuals are in fact blood culture negative, at least as assessed by single cultures of blood. So we don't have a good way, short of getting a large piece of tissue, to decide who has CMV disease. If we want to make progress in the next few years in terms of decreasing the number of patients who have fever, inanition, or who ultimately die of hypotension or failure, much of which is probably caused by CMV, we are going to have to figure out which ones will benefit from treatment for CMV rather than MAI or some other process. However, the good news about CMV disease is that we do have effective drugs. I think that everybody's aware of the fact that there are two parenteral alternatives, ganciclovir and foscarnet. There are unfortunately no oral agents. An exciting development about foscarnet has recently been published in the Annals of Internal Medicine. This study shows that foscarnet patients with CMV retinitis will have a high degree of efficacy in reducing the activity of CMV retinitis and in delaying recurrences. There are several studies suggesting that ganciclovir and foscarnet have comparable efficacy although markedly different toxicities. Unfortunately, neither has an especially promising formulation. In closing, it is clear that the ultimate solution to patients with HIV is understanding more about the pathogenesis of HrV and finding better therapy for the underlying retroviral disease. Until that time comes, when the retroviral process can be stabilized or reversed, we need to make more progress with the management of opportunistic infections. While we have made major advances, particularly in the fields of pneumocystosis and toxoplasmosis, there are areas like CMV and MAI where we need to understand a lot more about when to intervene with therapy, and we need to develop more cost-effective, less toxic therapies.
Thank you.
MOLECULAR PATHOGENESIS OF HIV-ASSOCIATED LYMPHOMAS Paola Ballerini, M.D., Gianluca Gaidano, M.D Jerry Gong, M.D., Vittorio Tassi, M.D., Giuseppe Saglio*, M.D., Daniel M. Knowles, M.D., and Riccardo Dalla-Favera, M.D. ,
Department of Pathology and Cancer Center, College of Physicians & Surgeons, Columbia University, New York, NY 10032; *Clinica Medica, Universita' di Torino, Torino, Italy The incidence of malignant Non Hodgkin Lymphomas (NHL) in HIV-infected patients is increasing, possibly as a consequence of the increasing life span of AIDS patients treated with antiretroviral therapy (1). According to the Working Formulation classification, about 70% of AIDS-associated NHL (AIDS-NHL) are high grade, small non cleaved cell (SNCC) or large cell immunoblastic- plasmocytoid QLC-IBP), while 30% are represented by intermediate grade, diffuse large non cleaved cell (LNCC). Distinctive features of AIDS-NHL include the frequent involvement of the central nervous system (CNS) often as a primary site (2) and the association with a polyclonal B-cell proliferative lymph node expansion (Lymphadenopathy Syndrome, LAS) often preceding the onset of the lymphoma (3-4). Little is known about the genetic alterations underlying the pathogenesis of AIDS731
NHL. Observations involving a limited number of cases have indicated an associations with chromosomal translocations involving the c-myc oncogene (5-6). Epstein-Barr virus (EBV) infection of the tumor cells has also been observed in 30-40% of cases, and it has been suggested to play a pathogenetic role based on the observation that viral infection precedes clonal expansion (5-7). Although EBV infection and constitutive c-myc oncogene expression are sufficient for the malignant transformation of human B cell in vitro (8), their combined presence does not appear to be required for B cell lymphomagenesis in vivo and the presence of other alterations has not been investigated. In the present study, we have redefined the frequency of c-myc activation and EBV infection in a large panel of tumors and investigated the possible roles of other genetic alterations, such as activation of ras oncogene and loss/inactivation of the tumor suppressor genes p53 and RBI, which are frequently associated with the development of various types of neoplasia but whose association with lymphomagenesis in AIDS has not been explored. Histology. Immunophenotype, Immunogenotype. We have analyzed a panel of 27 cases of systemic AIDS-NHL which were diagnosed upon analysis of histopathology and cell surface markers (9). All cases were diagnosed as B cell AIDS-NHL; 16 were shown to be SNCLL, 6 LNCCL, and 5 LC-IBP. In addition, immunoglobulin gene rearrangement analysis (10) indicated that all the biopsies contained a single predominant clonal B cell
population accounting for at least 60% of the sample. EBV infection. In order to assess the possible etiologic contribution of EBV to malignant transformation the presence of EBV genomes within AIDS-NHL cells was investigated by Southern blot hybridization using a probe specific for the genomic termini of EBV (11). This assay allows to determine whether EBV infection has preceded and, thus, possibly contributed to clonal expansion or whether infection has occurred after clonal expansion, thus representing an unlikely pathogenetic element (12). As summarized in Table 1, EBV sequences were detectable in -40% of AIDS-NHL, although the frequency appears to vary among different subtypes, the highest frequency being in the LC-IBP lymphoma subtype. All the positive cases displayed a single intense band suggesting that EBV infection has preceded and, thus, possibly contributed to clonal expansion in these malignancies. c-myc oncogene activation. Chromosomal translocations leading to c-myc oncogene activation can be detected as: i) truncations of the c-myc gene within the first exon, first intron or 5' flanking sequences which can be identified as rearrangements by Southern blot hybridization analysis and are characteristic of sporadic-type Burkitt lymphoma (BL) carrying (8; 14) translocations (13); or ii) mutations within sequences spanning the first exon/first intron border which are detectable by direct sequencing and are characteristics of t(8;14) in endemic-type BL and variant, t(2;8) and t(8;22), translocations in all types of BL (14). In our panel of AIDS-NHL, rearrangements of the c-myc gene were detectable in 66% of the cases by Southern blot hybridization, whereas the remaining cases appeared normal also by PCR/sequencing analysis for mutations (table 1). These results confirm that c-myc oncogene activation represents a frequent event in AIDS-NHL particularly in the SNCCL subtype. The mechanism of activation appears analogous to the one typical of Frequency and distribution of molecular lesions in HIV- associated lymphomas according to the histopathological classification
Table 1
Histotype
# of cases
EBV
c-myc
p53
ras
RBI
SNCCL LNCCL LC-IBP
16 6
5/16 1/4 4/4
14/16 1/4 1/4
10/16 0/6 0/5
3/16 0/6 1/5
0/16 0/6 0/5
5
732
sBL, where c-myc recombination with Ig switch sequences has been suggested to occur at the time of Ig isotype switching in a relatively mature B-cell (15). p53 loss/mutation. The p53 gene encodes a nuclear phosphoprotein that appears to negatively regulate cell proliferation (16). Exons 5 to 9, which represent the regions most affected by mutations in human tumors (17), have been investigated by means of PCR/Single Strand Conformational Polymorphisms (SSCP) analysis followed by PCR /
Direct Sequencing ( DS ) of the positive cases, as already described (18). p53 gene alterations were observed in 37% of AIDS-NHL. However, p53 lesions are not uniformely distributed among the different subtypes of NHL since they were found only in the SNCC group ( see table 1 ). In this group, 10 of 16 cases (62.5%) displayed point mutations, short sequence deletions or insertions, frequently associated with the loss of the normal p53 alíele. Ras oncogene mutation. The presence of mutations affecting codons 12, 13 and 61 of the three ras genes ( H-, K- and N- ras ) was investigated in AIDS-NHL DNA by PCR/SSCP and PCR/DS analysis. As shown in the Table 1, 4 cases ( 3 SNCC and 1 LCIBP of NHL ) scored positive. The mutations were found in N-ras codon 61 (2 cases), Nras 12(1 case) and K-ras 12(1 case). Previous evidence had shown that among lymphoid malignancies ras mutations are specifically restricted to Multiple Myeloma (19) and Acute Lymphoblastic Leukemias, while a large panel of NHL was found completely negative (20). Thus, the finding of Ras mutations among AIDS-NHL might represent a distinctive molecular feature of these lymphomas versus non AIDS-related NHL. RB1 gene inactivation. RBI gene encodes for an ubiquousely expressed 110-115 KD phosphoprotein endowed with growth suppressor activity (21). Alterations, i.e loss or mutations, of the RB 1 gene, initially found associated with Retinoblastoma, have been recently identified in other tumors suggesting a wider role of this gene in tumorigenesis (21). The molecular mechanisms of RBI inactivation are heterogeneous, including complete loss of the locus, partial deletions, rearrangements or splicing mutations (21). The structure of the RBI gene in AEDS-NHL DNA was studied by Southern blot analysis and PCR/SSCP analysis of exons 10 through 27, which span the domains of the gene in which most of the tumor-associated mutations have been found. Although the gene was found rich in polymorphic sequences which were originally thought to reflect somatic mutations, no evidence of tumor-specific rearrangements or mutations were detected in the exons examined (Table 1) when AIDS-NHL DNA was compared to a panel of normal DNAs. SUMMARY AND CONCLUSIONS The data presented here indicate that the pathogenesis of AIDS-NHL is variably associated with multiple genetic alterations including monoclonal EBV infection, oncogene activation ( c-myc, N-, Ki-ras ) and tumor suppressor gene ( p53 ) inactivation. Up to three ( 3 cases ) or four ( 1 case ) different lesions have been observed in the same tumor The distribution of these lesions among the various histotypes is heterogeneous, although some preferential associations have been found either between lesion and histotype or between lesions. The most notable case involves p53 mutations/loss that is exclusively associated with the SNCC lymphoma subtype. Since alterations of the c-myc gene occur at very high frequency in this same histotype it is possible that both lesions may be required for the pathogenesis of the BL phenotype. The consistent negativity of p53 lesions in other NHLs associated or non associated with HIV infection (18) reinforces this hypothesis. Finally, we note that the frequency of p53 mutations is significantly higher in AIDS-BL than in non HIV-related BL (18), although the significancy of this difference remains to be assessed. This study confirms the relatively low frequency of EBV infection in systemic AIDS-NHL in general, but reinforces the notion that EBV may be required for the pathogenesis of AIDS-LC-IBP, as recently suggested by the high frequency of EBV positivity in primary CNS AIDS-NHL which are mostly represented by LC-IBP (2). Conversely, the low frequency of EBV sequences in the AJJDS-SNCC lymphomas appears similar to that observed in sBL. 733
Only in a small minority of cases were ras oncogene mutations found, mostly associated with the BL type. Since ras mutations have never been found in non HIVrelated NHL, we examined whether these mutations could be related to the potential mutagenic action of antiretroviral agents, namely AZT. However, none of the patients included in our survey had received previous antiviral therapy. The presence of multiple lesions in some AIDS-NHL is perhaps surprising considering the relatively short time involved in the development of these tumors (4-5 years ) (22), and the fact that, since only a few common lesions have been studied, additional presently unknown alterations are likely to be present. While numerous studies have defined the multistep nature of tumorigenesis (23), the example of the multi-lesion pathogenesis of colon carcinoma has been taken to suggest that the long time (30-40 years) involved in tumor development is related to the number of independent genetic steps/lesions required (24). However, the example of some cases of AJDS-NHL suggests that genetic lesions may accumulate more rapidily at least in some tissues. ACKNOWLEDGMENTS This work has been supported by National Institutes of Health grants CA 37295 (to R.D.F.), and CA 40236 (to D.M.K.). P.B. and V.T. have been supported by fellowships of the Associazione Italiana per la Ricerca sul Cancro. G.G. has been partially supported by Gigi Ghirotti Foundation. REFERENCES
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J.M.Pluda, R.Yarchoan, E.SJaffe, I.M. Feuerstein, D. Salomon, S.M. Steinberg, K.M. Wyvill, A. Raubitschek, D. Katz, S. Broder : Development of non Hodkin
lymphoma in a cohort of patients with severe immunodeficiency virus infection on long term retroviral therapy. Ann. Int. Med. 113, 4, 276-282, 1990 E. M. E. MacMahon, J. D. Glass, S. D. Hay ward, R. B. Mann, P. Scott Becker, P. Charache, J. C. McArthur, R. Ambinder : Epstein-Barr virus in AIDSrelatedprimary central nervous system lymphoma. The Lancet, vol. 338: oct 19, 969-973, 1991. PG. Pelicci, D. M. Knowles, Z. A. Arlin, R. Wieczorek, P. Luciw, N. Dina, C. Basifico, R. Dalla-Favera : Multiple monoclonal B-cell expansions and c-myc
acquired immune deficiency syndrome-related lymphoproliferative disorders. J. Exp. Med. 164, 2049-2076, 1986. D. Shibata, L. M. Weiss, B. N. Nathwani, R. K. Brynes, A. M. Levine : EpsteinBarr virus in benign lymph node biopsies from individuals infected with the human immunodeficiency virus is associated with concurrent or subsequent development of non-Hodgkin lymphoma. Blood, 77, 1527-1533, 1991. M. Subar, A. Neri, G. Inghirami, D. M. Knowles, R. Dalla-Favera : Frequent cmyc oncogene activation and infrequent presence of Epstein-Barr virus genome in AIDS-associated lymphoma. Blood, vol.72, 2, 667-671, 1988. J. Whang-Peng, E. C. Lee, H. Sieverts, I. T. Magrath : Burkitt's lymphoma in AIDS : cytogenetic study. Blood, 63, 4, 818-822, 1984. A. Neri, F. Barriga, D. M. Knowles, J. Neequaye, I. T. Magrath, R. Dalla-Favera : Epstein-Barr virus infection precedes clonal expansion in Burkitt's and AIDSassociated lymphoma. Blood, 77, 5, 1092-1095, 1991. L. Lombardi, E. W. Newcomb, R. Dalla-Favera : Pathogenesis of Burkitt Lymphoma : expression of an activated c-myc oncogene causes the tumorigenic conversion of EBV- infected human B lymphoblasts. Cell, 49, 161-170, 1987. D. M. Knowles, G. A. Chamulak, M. Subar, J. S. Burke, M. Dugan, J. Wernz, C. Slywotzky, PG. Pelicci, R. Dalla-Favera, B. Raphael : Lymphoid neoplasia oncogene rearrangements in
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EBV-INDUCED HUMAN B CELL LYMPHOMAS IN hu-PBL-SCID MICE D.E. Mosier, Ph.D., M.D.1, G.R. Picchio, M.S.1, M.B. Kirven1, J.L Gamier, M.D.1, B.E. Torbett, Ph.D.1, S.M. Baird, M.D.2, R. Kobayashi, Ph.D.2 & T.J. Kipps, M.D.2 1 Medical
Biology Institute Pines Road; La Jolla, CA 92037 and 2University of California, San Diego 9500 Gilman Drive; La Jolla, CA 92093
11077 North
Torrey
735
ABSTRACT infection is associated with Burkitt's lymphoma (BL) in normal and immunoblastic B cell lymphomas in immunosuppressed or HIVindividuals infected individuals. SCID mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) from EBV-seropositive donors also may develop spontaneous B cell lymphomas which histologically and phenotypically resemble post-transplant tumors, and are distinct from BL. These tumors always contain EBV DNA. We have noted three different reproducible outcomes depending upon the EBV-seropositive donor used for generation of hu-PBL-SCID mice: (i) no tumors appear; (¡i) tumors appear in a fraction of hu-PBL-SCID mice with a 10-20 wk. latent period; or (iii) tumors appear in all hu-PBL-SCID mice within 6-10 wk. Southern blot analysis of late versus early tumors using a probe specific for the EBV terminal repeat sequences (BamNJ), which allows distinction between circular latent and linear replicating genomes, shows that late tumors do not involve active EBV replication but that early tumors do show replicating genomes. In addition, EBV genomes were monoclonal in late tumors but polyclonal in early tumors. These data suggest two mechanisms for EBV lymphomagenesis, slow outgrowth of rare latently-infected B cells, and more rapid transformation of uninfected bystander B cells by replicating virus. The latter process may by highly amenable to therapy in patients at risk for EBV-related lymphomas. In addition, prospective screening of EBV-seropositive transplant recipients in the huPBL-SCID model may predict the risk of post-transplant lymphoma development.
Epstein-Barr virus (EBV)
INTRODUCTION EBV is a common DNA herpesvirus that infects the majority of the human population. It is associated with several benign clinical manifestations in healthy (infectious mononucleosis) or immunocompromised hosts (lymphocytic interstitial pneumonitis, oral hairy leukoplakia), and three malignant diseases (endemic Burkitt's lymphoma [1], nasopharyngeal carcinoma [2], and immunoblastic B cell lymphomas [3, 4]). The B cell lymphomas occur with high frequency in patients with primary, secondary, or iatrogenic immunodeficiency (5-7), and are thought to reflect inadequate immune surveillance of B cells latently infected with EBV.
Acute clinical infection with EBV results in active viral replication (lytic cycle) in both B cells and epithelial cells of the oropharynx. Following the development of EBVspecific immunity, there is low level persistent viral replication in the oropharynx, and small numbers of latently infected B cells persist in the peripheral blood and lymphoid organs. In addition, normal B lymphocytes can be transformed in vitro by EBV, and only latent gene expression is required for transformation. These latent genes include several EBV nuclear antigens (EBNA-1, -2, -3a, -3b, -3c, -4 or leader protein, LP) and two membrane proteins (LMP-1, LMP-2) (8). Two animal models for EBV infection exist. Infection of cottontop tamirins with high dose EBV leads to the acute development of B cell lymphomas (9). In a more recently developed model, xenotransplantation of human peripheral blood lymphocytes derived from EBV-seropositive donors to SCID (severe combined immune deficient) mice leads to the onset of human B cell immunoblastic lymphomas (10-14). Using the hu-PBL-SCID model, we have performed studies to assess the risk of these lymphomas arising from different donors and to evaluate the risk factors.
736
RESULTS AND DISCUSSION
Tumors generated in hu-PBL-SCID mice appear within 20 weeks of transferring 25-50 x 106 PBL from EBV-seropositive donors i.p. to SCID recipients. We analyzed the overall incidence of tumor formation and the latency period from PBL injection to tumor detection in 85 hu-PBL-SCID mice derived from 10 healthy donors with IgG anti-viral capsid antigen (VCA) titers ranging from 1:80 to 1:640. Two of 10 donors failed to produce any tumors in SCID mice. The results for the remaining 8/10 donors are shown in
Figure
1.
1. Mean tumor incidence (% of injected hu-PBL-SCID mice developing tumors) plotted versus mean latent period to tumor detection (in weeks) in 4-9 mice/donor. Each symbol represents the mean values for 1 of 8 donors. Virtually identical results were obtained in a second experiment utilizing
Fig.
donors
category. These
representing
each
incidence
donors
fell into three categories: high incidence donors who gave tumors in 100% of SCID recipients within 10 weeks, intermediate incidence donors who gave rise to tumors in 40-80% of SCID mice in 10-13 weeks, and low incidence donors who produced tumors in —
(B
.E E
r"Tr¥+-HE
DONOR TUMOR INCIDENCE
Fig. 2. Southern blot analysis of EBV genomes recovered from hu-PBL-SCID tumors. BamH1 restricted DNA was probed with the Bam NJ probe, which distinguishes between circular episomal DNA and linear replicating DNA, as illustrated on the left side of the figure. Results are summarized schematically on the right side of the figure. 738
studies may be useful in predicting risk for the associated B cell lymphomas.
development
of
post-tranplant or AIDS-
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immunodeficiency. Nature (London) 335:256. Mosier, D.E., S.M. Baird, M.B. Kirven, R.J. Gulizia, D.B. Wilson, R. Kubayashi, G. Picchio, J.L. Garnier, J.L. Sullivan and T.J. Kipps, EBV-associated B cell lymphomas following transfer of human peripheral blood lymphocytes to mice with severe combined immune deficiency, in Current Topics in Microbiology and Immunology: Mechanisms in B-Cell Neoplasia 1990, M. Potter and F. Melchers, Editor. 1990, Springer-Verlag: Berlin-Heidelberg, p. 317. Okano, M., Y. Taguchi, H. Nakamine, S.J. Pirruccello, J.R. Davis, K.W. Beisel, K.L. Kleveland, W.G. Sanger, R.R. Fordyce and D.T. Purtilo. 1990. Characterization of Epstein-Barr virus-induced lymphoproliferation derived from human peripheral blood mononuclear cells transferred to severe combined immunodeficient mice. Am. J. Pathol. 137:517. Cannon, M.J., P. Pisa, R.I. Fox and N.R. Cooper. 1990. Epstein-Barr virus induces aggressive lymphoproliferative disorders of human B cell origin in SCID/human chimeric mice. J. Clin. Invest. 85:1333. Rowe, M., L.S. Young, J. Crocker, H. Stokes, S. Henderson and A.B. Rickinson. 739
virus (EBV)-associated lymphoproliferative disease in the model: Implications for the pathogenesis of EBV-positive lymphomas in man. J. Exp. Med. 173:147. Raab-Traub, N. and K. Flynn. 1986. The structure of the termini of the EpsteinBarr virus as a marker of clonal cell proliferation. Cell 47:883.
1991. SCID 15.
Epstein-Barr mouse
ASSOCIATION OF EPSTEIN-BARR VIRUS WITH PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA IN AIDS EITHNE M.E. MACMAHON MB MRCPI, JONATHAN D. GLASS MD, S. DIANE HAYWARD, PhD, RISA B. MANN, PATRICIA CHARACHE MD, JUSTIN C. MCARTHUR MB, RICHARD F. AMBINDER, MD PhD
JOHNS HOPKINS SCHOOL OF MEDICINE
BALTIMORE, The
MD 21205
of primary brain lymphoma in the acquired immunodeficiency syndrome was recognized early in the epidemic. Subsequently systemic non-Hodgkin's lymphomas were also designated as AIDS-defining illnesses. Epstein-Barr virus (EBV)associated lymphoproliferative disease and lymphoma is well recognized in the in the postorgan transplant setting. These tumors are uniformly associated with Epstein-Barr virus and such an association might have been anticipated for AIDS-associated lymphomas. However, less than half of these tumors were found to contain EBV. EBV has been associated with primary central nervous system (CNS) lymphoma both in patients with and without a history of immune deficit. In order to better characterize the association of EBV with primary CNS lymphoma in AIDS we examined tumor tissue from a consecutive series by in situ hybridization (3). Epstein-Barr virus is characteristically latent in host B-lymphocytes, the genomic DNA existing as nuclear plasmids with limited antigen expression. The products of two EBV genes, the EBERs 1 and 2, are pol DI RNA transcripts which exist as nucleoproteins, present in great abundance in the nuclei of latently infected B-lymphocytes (2). We used EBER 1 as a target for in situ hybridization with labeled riboprobes (5). The technique was first refined in formalin-fixed paraffin-embedded pelleted EBV negative and positive cell lines before application to brain sections. The hybridization signal was nuclear with nucleolar sparing. No signal was detected with the sense probe. Examination of a consecutive autopsy series of 19 AIDS-associated primary CNS lymphomas showed expression of EBER 1 RNA in all 19 cases, indicating the presence of EBV. The strong positive signal was confined to tumor cells. The specificity of the technique was confirmed by examination of a wide range of brain tissue controls. No evidence of cross hybridization was seen with primary HIV infection, cytomegalovirus encephalitis, toxoplasmosis, progressive multifocal leukoencephalopathy or other opportunistic CNS infections arising in AIDS. Nor was signal detectable in herpes simplex encephalitis or in normal brain. We used this technique to examine systemic lymphomas in AIDS patients and detected EBV in just 3 of 7 cases. Primary brain lymphoma tissue from 15 patients without known occurrence
740
Fig. 1. AIDS-associated primary brain lymphoma. High power views of serial sections, (a) hematoxylin and eosin (b) in situ hybridization with digoxigenin-labeled antisense EBER1 riboprobe and counterstained with eosin. immune
deficiency were also tested, of which just one was EBV positive. A single case of CNS primary lymphoma occurring after kidney transplantation was positive. The detection of EBV in all cases of AIDS-associated CNS lymphoma examined suggests that this virus may well play a part in the pathogenesis of these tumors. To further examine the role of EBV we looked for expression of the Epstein-Barr latency membrane protein (LMP). Immunostaining with pooled anti-LMP monoclonal antibodies (4) showed LMP expression in half of the cases. Of the handful of EBV proteins expressed in latency, two have known oncogenic potential-latency membrane protein (LMP) and EB nuclear antigen 2 (EBNA-2). In vitro, LMP expression can transform immortalized rodent cells. Recently LMP has been shown to protect B-cells from apoptosis by induction of the BCL-2 protein (1). LMP is expressed in several other EBV-associated tumors including nasopharyngeal carcinoma, Hodgkin's disease, and post-transplant lymphoma but not African Burkitt's lymphoma. The identification of LMP in this series provides further evidence that EBV may have a role in tumorigenesis. Detection in only a proportion of the tumors might reflect variable quality of fixed tissue and/or variable LMP expression. The association of EBV with primary CNS lymphomas in AIDS has implications not only for understanding their pathogenesis but also for diagnosis and management. The characteristic periventricular location of tumors suggests that EBV DNA might spill into the spinal fluid from dying tumor cells even in the absence of lymphomatous meningitis. In a pilot study to test this hypothesis, PCR amplification for EBV was applied to CSF specimens from HIV- positive patients. EBV was detected in three of seven specimens from patients with primary CNS lymphoma and from one of 42 patients without lymphoma. The observation that EBV DNA is not usually present in the CSF of HIV-positive patients but may be detected in the CSF of those with CNS lymphoma, has led to the ongoing prospective evaluation of EBV as a tumor marker in this setting. References 1. Henderson, S., M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, and A. Rickinson. 1991. Induction of bd-2 expression by Epstein-Barr virus 741
latent membrane 65:1107-1115.
protein 1 protects
infected B cells from
programmed cell death.
Cell
2. Howe, J.G. and J. A. Steitz. 1986. Localization of Epstein-Barr virus-encoded small RNAs by in situ hybridization. Proc. Nati. Acad. Sei. USA 83:9006-9010.
3.
MacMahon, E.M.E., J. D. Glass, S. D. Hayward, R. B. Mann, P. S. Becker, Charache, J. C. McArthur, and R. F. Ambinder. 1991. Epstein-Barr virus in AIDS-related primary central nervous system lymphoma. Lancet 338:969-973.
P.
4. Rowe, M., H. S. Evans, L. S. Young, K. Hennessy, E. Kieff, and A. B. Rickinson. 1987. Monoclonal antibodies to the latent membrane protein of Epstein- Barr virus reveal heterogeneity of the protein and inducible expression in virus-transformed cells. J. Gen. Virol. 68:1575-1586. 5. Wu, T-C, R. B. Mann, P. Charache, S. D. Hayward, S. Staal, B. C. Lambe, and R. F. Ambinder. 1990. Detection of EBV gene expression in Reed-Sternberg cells of Hodgkin's disease. Int. J. Cancer 46:801-804.
STUDIES ON A TYPE D RETROVIRUS ISOLATED FROM AN AIDS PATIENT LYMPHOMA Richard J. Ford1 Lawrence A. Donehower2, and Robert C. Bohannon1'2 ,
^.D. Anderson Cancer Center 2Baylor College of Medicine, Houston, TX 77030 ABSTRACT The development of AIDS-related lymphomas (ARL) has been on the rise in recent years. During an analysis of ARL from AIDS patients, one individual developed atypical syncytial variants of high-grade Burkitt'sHowever, the type B-cell lymphomas, which prompted further study. search for a HIV-1 retrovirus, which we hypothesized was infecting these cells, led to the subsequent discovery of a type D retrovirus in two earlyNucleotide and passage lymphoma cell lines derived from this patient. amino acid sequence analysis, as well as immunologie reactivity, indicated that the virus was closely related to Mason-Pfizer monkey virus (MPMV) simian retrovirus type 1 (SRV-1). or MPMV and SRV-1 are immunosuppressive type D retroviruses that cause an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by the polymerase chain reaction generated MPMV-specific fragments indicative of infection by a retrovirus similar to 742
MPMV.
Additionally,
the patient's serum contained antibodies that recognized type D retroviral env proteins (gp20 and gp70) and gag proteins (p27 and pl4) as assayed by immunoblot and radioimmunoprecipitation techniques. Although there have been reports of human cell lines infected with type D retroviruses and of type D reactive human sera, this is the first report of a type D retrovirus infection in a human confirmed by virus isolation, serum immunoreactivity, and viral DNA identification in tumor tissue.
INTRODUCTION Recent reports indicate that there is an increasingly high incidence of non-Hodgkin's lymphomas among HIV-1 positive individuals (1). AIDS related lymphomas (ARL) have been found to consist primarily of the Burkitt's-type (small non-cleaved cell) or immunoblastic, high grade lymphomas (2). Although the HIV-1 virus has been hypothesized to be involved in lymphomagenesis in these patients, repeated attempts to isolate HIV-1 from ARL cells have been unsuccessful (3). Therefore, it was of particular interest to us when an ARL patient presented with a previously unrecognized morphologic syncytial variant of Burkitt's lymphoma that we originally thought might represent infection by HIV-1 A retrovirus was subsequently isolated from two Bor an HIV-1 variant. cell lymphoma cell lines derived from the patient's original biopsy material. Further analysis indicated that the retrovirus was not HIV-1, as originally hypothesized, but rather a type D retrovirus closely related to the Mason-Pfizer monkey virus (MPMV), a causative agent of simian AIDS
(4).
MATERIALS AND METHODS Patient. The patient studied was a 32-year-old homosexual male hospital administrator who complained of abdominal pain, fever, and night sweats prior to admission to an outside hospital. He was admitted to the University of Texas M. D. Anderson Cancer Center with generalized lymphadenopathy and the presumptive diagnosis of AIDS-related lymphoma (ARL). The patient' was diagnosed as having AIDS and a serum A bone sample obtained at admission showed HIV-1 seropositivity. marrow biopsy revealed extensive involvement with a high grade A malignant lymphoma of the small non-cleaved (Burkitt's) type. combination chemotherapy regimen was begun and a partial remission In spite of this treatment, however, the patient later was achieved. developed B-cell lymphomas at multiple sites and subsequently died of progressive lymphoma and sepsis approximately 9 months after admission. Permission to perform an autopsy was denied and no other tissues could be obtained for further analysis. Cell lines were established 743
from lymphoma tissue obtained from three distinct sites of lymphoma involvement that occurred during the course of the patient's disease. Further clinical and pathologic information on this patient is described elsewhere (5).
Molecular and sérologie and sérologie analysis were
analysis. The techniques recently described (6).
used for molecular
RESULTS
Upon admission, patient revealed that
diagnostic bone marrow biopsy (fig. 1) from the a monomorphous non-Hodgkin's lymphoma had infiltrated the normal marrow. Histopathologic examination of this lymphoma and of a subsequent lymphoma isolated from the patient's kidney at a later date (fig. 2) revealed that multinucleate syncytial-like cells were present in the original biopsies. Cell lines were established from these lymphomas and electron microscopic examination revealed the presence of retrovirus-like particles both within intracellular vesicles (fig. 3), as well as extracellularly (6). A reverse transcriptase assay performed on twice glycerol gradient purified virus from these cell lines using a poly r(A):oligo d(T) template-primer showed a low level of activity (fig. 4) as compared to HIV-1 ihr, even though an estimated 108 viral particles per ml A screen for the presence of HIV-1 p24 by an antigen were present. method (Abbott) failed to detect this HIV-1 diagnostic core capture a
Fig. 1. Patient diagnostic bone marrow biopsy. A diagnostic bone marrow biopsy was taken from patient RC, formalin fixed, and paraffin embedded. The low-power photomicrograph (lOx) of the H&E stained, diagnostic bone marrow biopsy specimen shows replacement of normal marrow (between bony spicules) with monomorphous lymphoma cells (arrow). 744
Fig. 2. Photomicrograph of kidney aspirate of patient RC. A high-power photomicrograph (lOOOx) of the original aspirate of a kidney lymphoma (RC^) from patient RC. Large syncytial cells are indicated by arrows and are in contrast to the usual Burkitt's lymphoma cells surrounding these atypical cells. detect HIV-1 by Southern and PCR analysis characterization of the reverse transcriptase of the viral isolate revealed that there was a preference for poly r(C):oligo d(G) over poly r(A): oligo d(T) (Table 1). The patient's serum recognized many of the proteins of the viral isolate as compared to most other HIV-1 Cell specific infectivity of primary B cells, seropositive sera tested (fig. 5). T cells, and macrophage/monocytes revealed the isolate could infect both B In and T cells, as assayed by reverse transcriptase activities (Table 2). in the isolate caused syncytia formation addition, primary peripheral blood B-cells and in Raji cells similar to that seen in the original patient diagnostic biopsies taken at the time of admission (6). In order to determine what type of virus was present in the lymphoma cells, we utilized degenerate primers to an amino acid region within the pol gene that is conserved for all known retroviruses and amplified a 135 bp pol fragment from the viral isolate (6,7). The amplified fragment was cloned, sequenced, and compared to known sequences in GenBank. The sequences proved to be related to the MasonPfizer monkey virus (MPMV) and to simian retrovirus type 1 (SRV-1), a type D retrovirus responsible for causing simian AIDS in monkeys. Amino
protein. All attempts to proved negative. Further
745
r,
,.
«. "
Fig. 3. Electron Micrograph of cell line RCBM. Electron microscope examination (20,000x) of the bone marrow derived cell line (RCBM) revealed type D retroviral like particles present in both intracytoplasmic vesicles as well as outside the plasma membrane. An arrow points to one of the extracellular retroviruses that has a barrel shaped core indicative of a type D retrovirus. 800
30000
20000
CPM for HIV
CPM for CB
(rA):(dT)
(rA): receptor. In contrast, interferon gamma interacts with a distinct receptor (IFN-yR). I would like to focus on the interferon gamma receptor (IFN-yR). Fig. 3 provides a perspective of that receptor with respect to the other cloned receptors. We and others have cloned the interferon gamma receptor (IFN-yR) shown here, and also the interferon
777
INTERFERON ACTION
Fig.
2. Illustration of interferon action.
alpha receptor (IFN-aR) shown schematically in perspective with the LDL, EGF, PDGF, insulin, LDL, IL-2 (p55) and CD-4 receptors. As I noted, I shall focus on the interferon gamma receptor. Although I shall not have time to discuss the interferon alpha receptor, I would speculate that the principles determining how both of these receptors function are probably going to be very similar. There are some unique features of the interferon receptors that are not present or exhibited to the same degree in most other cytokines. In the first place the interferons demonstrate high species specificity, particularly interferon gamma. Human IFN-y (HuIFN-y) is not active on mouse or hamster cells, for example; and mouse IFN-y (Mu-IFN-y) is not active on human cells, even at enormous concentrations. You will see shortly that the species specificity even goes beyond the surface receptors. f800
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IFN-y IFN-oc
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Jh20 kb appear (Makos et al., 1992). What is perhaps most important about this methylation abnormality, is that it appears in the benign adenomas which are thought to be precursor lesions for colon cancers. In the adenoma DNA, the methylation of the YNZ22 region is not as extensive as in the Six to 8 kb Not I fragments are seen instead of the normal 5 kb, but cancers. the >20 kb fragments are not present. Thus, abnormal methylation of YNZ22 regions begins in benign colon adenomas and increases in extent with the transition of these lesions to malignant tumors. An important aspect of the above timing for YNZ22 region methylation is that they precede the structural alterations of chromosome 17p which In 6 of 7 adenomas examined, the are characteristic of colon cancers. abnormal 17p events were found in the absence of 17p allelic loses or p53 gene mutations (Makos et al., 1992). In contrast, the more extensive methylation changes in the cancers were all associated with loss of one 17p YNZ22 and one p53 alíele and with point mutations of the remaining p53 alíele. As we have previously reviewed elsewhere (Baylin et al., 1991), these dynamics for appearance of abnormal methylation of chromosome 17pl3.3 in colon cancer evolution raise at least two possibilities for functional consequences. First, the location of CpG islands often denotes the position of associated genes. The potential for CpG island methylation to inhibit, directly, expression of such genes has been reviewed earlier in this talk. Although, the p53 gene at chromosome 17pl3.1 is the only well defined tumor suppressor gene on 17p, increasing evidence for additional, more telomerically located, It will be of interest to determine if the defined genes has been accruing. pYZ22 methylation abnormalities may participate in decreased function of such genes. events
Second, the above data illustrate an additional potential consequence of abnormal CpG island methylation in tumor cell DNA that we have also reviewed elsewhere (Baylin et al., 1991). Such methylation has been temporally associated in the genetic mental retardation syndrome, Fragile-X, with instability and structural changes of the putative gene for this disorder (see Oberlé et al., 1991). Methylation induced delays of replication of DNA have been postulated to enhance the instability of DNA fragile sites. Thus, extending abnormal methylation of a region such as YNZ22 during tumor progression could be closely linked to the allelic losses, rearrangements, and other chromosomal structure changes which are critical to cancer evolution. With respect to this discussion, it is important to note that patterns of abnormally increased DNA methylation may be non-randomly associated with chromosome changes specific to individual types of human tumors. We have seen 815
examples of this cell lung cancer
on
DNA
regions
(Makos
of chromosome et
al., 1992).
3p consistently deleted
in small
Now very briefly, I wish to discuss the third component of DNA methylation imbalance in neoplasia, overexpression of the DNA MTase gene. Time precludes discussing the fascinating events accompanying evolution of this gene from prokaryotes to higher order eukaryotes (reviewed in Bestor, 1990). The eukaryotic genes have been cloned from rodents (Bestor, 1990), and recently by our lab, from humans (Yen et al., submitted for publication). Each gene expresses a 5 kb transcript. In general, steady state levels of this mRNA are very low in normal compared to neoplastic tissues and cell lines (for review Baylin et al., 1991). Perhaps, our most interesting observation regarding DNA MTase gene expression again goes back to the colon cancer progression model. Using a PCR assay, we have found increased expression of this gene at the steady state transcript level in normal appearing colonie mucosa from patients with benign adenomas or colon cancers. Thus, in DNA MTase DNA of the changes overexpression methylation may precede patterns that first appear in the DNA of benign adenomas and become more
extensive in the colon cancers. The levels of DNA MTase gene expression continue to increase in the adenomas and colon cancers (El-Deiry et al., 1991) and thus may play a key role at each stage of tumor progression. SUMMARY Let
summarize by reviewing a model which is meant to raise as many it answers (Fig. 2). What I have discussed today are data questions that suggesting during progression of solid tumors, like colon cancer, an increased cellular DNA methylating capacity characterizes the initial stages of multi-clonal hyperplasia. Despite this increase, the altered pattern of DNA methylation which subsequently emerges is largely manifest by a widespread hypomethylation of DNA. However, on a more regional basis, areas of me as
hypermethylation appear which can affect strategic areas such as normally unmethylated CpG islands. These shifted DNA methylation patterns have the capacity to both follow, or cause, chromatin changes that can both directly
silence genes critical for normal cell maturation and/or participate in the structural chromosome changes which constitute genetic instability during -
Clonal
Multiclonal
O
Hyperplasia
Clonal Cancer
Benign
Lesion
Loss of Gene Function anci
~g
T
"V
DNA
\
Normal Maintenance
Hypomethylation +
Regional Hypermethylation
DNA
ethylation
Imbalance
Fig. 3. A working model for the molecular mechanisms underlying the evolution of abnormal DNA methylation patterns in neoplastic cells. The top panel depicts the regulation of DNA methylation in a normal cell. This diagram, adapted from Holliday (23), shows the progressive fate of a methylated CpG dinucleotide (*) as it passes through a DNA replication fork. A molecule of the DNA-MT enzyme is strategically located at the fork, and enzyme activity is activated, as shown by the + arrow, with onset of DNA replication. This structural and biochemical halance ensures methylation of the CpG dinucleotide on the newly synthesized daughter strand and thus preserves the normal methylation pattern for the cell type. A feedback repression of DNA-MT activity, shown by the arrow, could occur either via a signal from methylated DNA beyond the replication fork or from cessation of DNA replication activity. The bottom panel depicts the possible dynamics underlying the DNA methylation imbalance characteristic of neoplastic cells. Abnormal numbers of DNA replication events, indicated by the heavy bars on each DNA strand at the replication fork, may provide pressure for increased DNA-MT activity as shown by the heavy shaded + arrow at the replication fork. The critical balance between the DNA-MT enzyme and the replication fork could be disrupted either by a change in chromatin structure or a change in shape of the nuclear matrix. In principle, these dynamics would have three major effects on methylation. First, the maintenance methylation function would be disrupted, as seen on the upper post-replication fork strand, resulting in "missed sites" and widespread hypomethylation. Second, the altered position and increased activity of the DNA-MT would lead to new sites of methylation, as depicted on the bottom strand, resulting in regional hypermethylation. Finally, there would be a block to the feedback loop that normally controls DNA-MT levels (depicted by disruption of the arrow), resulting in increased synthesis of the enzyme. This block might arise because of the continued DNA replication or because of failure of a normal maintenance methylation pattern to be achieved. -
-
progression (Fig. 2). I suggest that one must view these changes as an interchangeable cycle of events during tumor progression. The chromatin changes and abnormal methylation patterns can drive one another with increasingly deleterious effects as the malignant phenotype emerges (reviewed in Baylin, 1991). tumor
What
working
A are the molecular events that would initiate the above dynamics? As discussed for the normal adult construct model is shown in Fig. 3.
cell, there
is
a
strategic location of DNA MTase, synthesis at replication forks (top cancer cells, perhaps failure of cells
delicate balance between the
regulation of this enzyme, and rate of panel, Fig. 3). In pre-neoplastic and
DNA
817
exit the cell cycle and halt DNA replication, facilitates some sort of pressure to increase cellular DNA methyltransferase activity (bottom panel, Fig. 3). This increase may involve loss of feedback inhibition of the enzyme during the post DNA replication phase. There are also probable structural alterations in the nucleus which may alter the geographic relationship between In consequence, many DNA areas that the DNA replication fork and DNA MTase. should be getting methylated do not, and novel areas of methylation also arise. This cycle of events leads to the imbalance of DNA methylation that I Future investigations of these possibilities, and of their have talked about. specific consequences for alterations of gene expression and chromosome structure, may reveal a key molecular step underlying virtually all stages of to
tumor
progression. Thank you.
MODERATOR : Yes, thank you very much, Dr. Baylin. May I perhaps open the discussion by saying that perhaps you have a hypothesis that applies to some genes and doesn't apply to others. Can you make any predictions then as to which genes it would apply to and which ones not? That is, whether methylation precedes, or hypermethylation precedes or follows other events. events I have discussed may and both of at in least two ways affect the specific expression of genes these probably have to do, primarily, with a loss of gene expression. Despite the widespread hypomethylation in tumor DNA, I'm not aware of any documentation that this has caused expression of a specific gene to be activated. I think this is expected because hypomethylation of a gene, especially in the regulatory region, is only a permissive event for gene transcription. Specific activation obviously depends on other factors including changes in transcription factors. If the hypomethylation is functionally important, I would predict that it participates in abnormalities of chromosome and chromatin structure which may actually contribute to gene loss. What I have also tried to suggest is that it is even more likely that the regional hypermethylation events I described are the most critical for silencing certain genes and that the involved genes would be tumor suppressor In this genes. The myo-D gene example I gave is illustrative of this point. a case, transcription factor important to cell differentiation is hypermethylated in fibroblasts when reactivated by treating the cells with the demethylating agent 5-Azacytidine, the cells demonstrate myoblast features. I think, then, it's going to be these kinds of genes which, when involved in hypermethylation events during tumor progression, contribute to
BAYLIN:
I think that the abnormal
methylation
-
-
the tumor
phenotype.
The other thing I stressed is that allelic loss and instability, which also results in the loss of functional genes during tumor progression, could be a major consequence of abnormal DNA methylation in CpG rich areas. As discussed, the CpG islands around genes normally are positioned in early replication areas. When methylated, the replication of these regions would be delayed and this could enhance fragility of such sites. This might then play some role in allelic losses. MODERATOR:
QUESTION:
Yes, go ahead. What is known about the
methyltransferase genes?
transcriptional,
818
or
other
regulation
of DNA
BAYLIN:
Are you
The
QUESTION:
asking
me
what is known about the
transcription?
regulation.
BAYLIN: The regulation. Two things are known. DNA MTase activity and steady state mRNA levels rise sharply when cells enter proliferation and the S phase of the cell cycle. The early evidence is that much of this regulation may be Little else is known about regulation of at the post-transcriptional level. and the eukaryotic DNA MTase the transcriptional region of the gene has not been studied in detail. told me that we have more time available than therefore, with your permission, Dr. Feldman, I will ask another question. I don't think there are any other questions that I see. With respect to your answer to my question, of course you know that 5-azacytidine will cause further gene expression, but I didn't understand your statement that you only expect to find genes turned off, not turned back on. MODERATOR: I thought,
BAYLIN:
Dr. Feldman has
just
so
The
azacytidine
point
is very well taken.
However, in
most instances
where 5-
expression, particularly in cultured, immortalized be doing it by reducing abnormally increased regional and Robin Holliday and others pointed out some years ago, that genes in cultured group has more recently emphasized this
turns on gene
cells, it may
methylation.
Adrian Bird's
-
cells can be silenced by increased methylation. There are well described instances where cell auxotrophy for metabolic pathways is based on silencing of at least one copy of a gene by this mechanism. There are a number of examples where 5-azacytidine will relieve auxotrophy for such cells. Again, I return to the myo-D gene story, where abnormally increased methylation is being relieved by 5-azacytidine to reactivate gene expression. -
MODERATOR:
Well, thank you very much,
Dr.
Baylin.
REFERENCES
Adams, R.L.P. 1990.
DNA
methylation.
BLochern.J. 265:309-320.
J. Boyes and A. Bird. 1990. High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 62:503-514.
Antequera, F.,
Baylin, S.B.,
M.
Makos,
J.
Wu, R.-W.C. Yen, A. de Bustros, P. Vertino and B.D.
Nelkin. 1991. Abnormal patterns of DNA methylation in human neoplasia: Potential consequences for tumor progression. Cancer Cells 3:383-390.
Bestor, T.H. 1990. DNA methylation: evolution of
a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes. Phil. Trans. R. Soc. Lona. 326:179-187.
El-Deiry, W.S., B.D. Nelkin, P. Celano, R-W.C. Yen, J.P. Falco, S.R. Hamilton and S.B. Baylin. 1991. High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer.
Proc.Nati.Acad.Sei.USA 88 : 3470 3474. -
Fearon, E.R. and B. Vogelstein. 1990.
tumorigenesis.
A
Cell 61:759-767.
819
genetic
model for colorectal
Jones, P.A. and J.D. Buckley. 1990. The role of
DNA
Adv.Cancer Res. 54:1-23.
in
methylation
cancer.
Jones, P.A., M.J. Wolkowicz, W.M. Rideout, III, F.A. Gonzales, CM. Marziasz, G.A. Coetzee and S.J. Tapscott. 1990. De novo methylation of the MyoDl CpG island during the establishment of immortal cell lines. Proc.Nati.Acad.Sei.USA 87:6117-6121. B. Zbar and S.B. Baylin. 1992. Distinct hypermethylation patterns occur at altered chromosome loci in human lung and colon cancer. Proc. Nati. Acad. Sei. USA, in press.
Makos, M., B.D., Nelkin,
M.I. Lerman, F.
Migeon, B.R. 1990. Insights into X species variation, DNA methylation
Res. 56:91-98.
Latif,
chromosome inactivation from studies of and replication, and vice versa. Genet.
Oberlé, I., F. Rousseau, D. Heitz, C. Kretz, D. Devys, A. Hanauer, J. Boue, M.F. Bertheas and J.L. Mandel. 1991. Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 24:1097-1102. Tazi, J. and
A. Bird. 1990. Alternative chromatin structure at
Cell 60:909-920.
CpG islands.
"A TRANSGENIC MOUSE MODEL FOR MEN 2"
Nicholas Schulz and George F. Vande Woude ABL-Basic Research Program NCI-Frederick Cancer Research & Development Center P.O. Box B Frederick, MD 21702-1201
MODERATOR: Dr. Gallo us go to the next Schulz is speaking
then, let Nick
points out we are way behind, way over time. Okay, speaker. George Vande Woude could not be here, and on his behalf about oncogenes, cell cycle and
antineoplastic drugs. Nick?
SCHULZ: Hello. Dr. Vande Woude could not come this afternoon, and instead I'd like to let you know about the characterization of a model system involving transgenic mice that bears remarkable resemblance to a familial neoplastic syndrome, namely MEN Type 2. Dr. Vande Woude's laboratory, has been interested in determining the function of the Mos proto-oncogene. To refresh your memory, c-Mos is the cellular homologue of the viral mos oncogene of the Moloney Murine sarcoma virus, and has been isolated from several vertebrate species. It is always present as a single coding exon that has amino acid homology with the sre family of protein kinases. The transforming activity of this oncogene is manifest when constitutively expressed at very low levels in NIH3T3 fibroblasts. In fact, to display that it is expressed in transformed cells, it is necessary to demonstrate this via RNase protection assays, or SI nucleus protection assays. As I said, it consists of one exon, usually between 800 base pairs and 1 KB, and the expression of this proto-oncogene is present in low levels in brain, kidney and mammary gland, as well as epididymis, heart and lung. It had for the first time shown to have been present in high levels in embryo, and most high in germ cells. The initial 820
characterization of the activity of this proto-oncogene showed that it is necessary for maturation of both sperm and oocytes. Because of c-Mos's potential role in cell cycle regulation, we sought to define what aberrant expression of this proto-oncogene would have on transgenic mice. A Moloney virus, LTR, in tandem with the murine c-Mos proto-oncogene, was introduced by conventional techniques into one-cell embryos. The most obvious characteristic, or phenotype of these mice is that they won't hold still when you take their picture. That is to say, here is one such mouse, trying to get away sometimes the way from the camera, and only succeeding in chasing its tail some of us feel while we have been trying to characterize this oncogene. --
The other manifestation the obvious manifestation within a few weeks of these mice's life is they have remarkable development of cataracts. In normal development of lens tissue, one has the orderly differentiation of lens fibers along the periphery. They migrate to the center, lose their nuclei, and become absolutely crystalline, and produce a functional lens. In the transgenic mice, In fact, the cells continue to proliferate, and at one has no loss of nuclei. about 2-4 weeks they'll rupture the lens, and eventually fill the entire cavity of the eye with tissue that has failed to differentiate. --
Currently we have four lines of mice generated on two different backgrounds. The lines used were the FVB/N background, and the Black 6 background. The FVB/N background was derived in conjunction with Dr. Heiner Westphall. One interesting point is that when these mice, despite the severe neurological phenotype age to about 8-12 months and are sacrificed, one has a large number of pheochromocytomas, which are tumors of the adrenal medulla, and medullary thyroid neoplasms. These two tumors are hallmark lesions of multiple endocrine neoplasia Type 2. It is of Pheochromocytomas originate in the adrenal medulla. neuroendocrine origin and secretes the hormones adrenalin and noradrenalin. The outer portion, the cortex is responsible for the secretion of mineralocorticoids and glucocorticoids. the
The adrenals of older animals contain presentation of these tumors most often
than one tumor with being bilateral.
more
If one takes these adrenal glands and stains them with neuron-specific this work is done in markers; in this case, neuron-specific enolase with Linnoila of the branch of Dr. NCI one sees again an conjunction Navy identical histological phenotype as in human tumors. --
--
The pheochromocytomas are not the only tumor found in the transgenic mice. About 55-60% of mice in the second line studied will present with either medullary thyroid carcinoma, or C cell hyperplasia of the thyroid. Again, these tumors are of neural crest tissue origin. Like the pheochromocytomas of the line 1 animals, the thyroid lesions of the line 2 animals are multifocal and the tumors are bilateral, indicating that they also arise in a polyclonal fashion. Line 4 Mos transgenic animals develop multifocal pheochromocytomas as well as medullary thyroid neoplasms, with animals quite often having both types of tumors (Table 1).
In contrast to line 1, 2, and 4 mice, the line 3 Mos transgenic mice fail develop pheochromocytomas or C-cell thyroid neoplasms above the sporadic background level observed in the nontransgenic control animals (Table 1), even though their lens-fiber development defect and neuropathology are indistinguish-
to
able from the other lines.
821
Table 1
Tumor incidence in
transgenic and control mice Percentage of mice with tumors
Transgenic mouse
Background
line
1
Number of animals
79/75A
Medullary thyroid neoplasia only 0%
0/0
4%
1/5
62%
40/56
39/44
0%
0/0
63%
19/33
4%
0/3
66%
19/36
89
48/41
2%
1/1
0%
0/0
0%
2%
1/1
B6C3
62
29/33
23%
7/7
13%
4/4
32%
0/0 8/12
68%
19/23
FVB/N
46
21/25
2%
1/0
0%
0/0
0%
0/0
2%
1/0
154
2
83
3
FVB/N
4
Control
58%
number of mal es/females.
To demonstrate that the tumors observed in these animals
Mos
Total
Both
39/51
FVB/N FVB/N
AActual
Pheochromocytomas only
were
related to
expression, we examined the adrenal and thyroid tumors from line 4 Mos transgenic animals for Mos RNA expression by Northern analysis. We found that the adrenal and thyroid tumors, like the affected brains, express high levels of Mos RNA. The unaffected organs, like liver and kidney, however, are negative by Northern analysis, and the tissues from nontransgenic control litter mates show little or no expression of Mos RNA. When the thyroid tumors are examined via in situ hybridization for mos expression, one sees high level expression of the transgene specifically over areas of tumor involvement. The tumor presentation pattern was line-dependent (Table 1), and suggested that the transgene integration site or background of the animal played an important role. To evaluate this, the three Mos transgenic FVB/N lines were crossed with BALB/c mice (Table 2). The same disease phenotype was seen in the Fj animals of the first two lines. The f1 animals of the line 3 X BALB/c cross, however, display a high frequency of both pheochromocytomas and medullary thyroid C-cell carcinomas. Neither neoplasm is found in the parental line. Thus, it is the integration site and/or background which affects the penetrance of the transgene on phenotype. Table 2
Tumor incidence in
offspring
of
transgenic mice crossed
to BALB/c mice
_Percentage of mice with tumors_ Number of animals
Cross 1 X
49
21/29A
2
BALB/c X BALB/c
31
BALB/c
15
16/15 7/8
3 X
AActual
Pheochromocytomas only 51%
Medullary thyroid neoplasia only 2%
0%
12/13 0/0
52%
7%
1/0
33%
0/1
Both
Total 65%
7/9
1/5 16% 1/4
68%
13/19 8/13
3/2
20%
60%
5/4
12%
1/2
number of mal es/ females.
The variation of tumor presentation pattern between the transgenic lines is similar to that observed in humans with MEN 2. In the human syndrome, the tumor presentation pattern is consistent within a kindred. The most commonly observed pattern of tumor formation in humans with MEN 2 is the presence of both medullary thyroid carcinomas and pheochromocytomas, as we observe in the line 4 animals. Staining the thyroids of line 1 animals for calcitonin revealed 822
evidence for C-cell hyperplasia, the precursor lesion of medullary thyroid carcinoma. The study of these two lines could reveal mechanisms whereby there is preference given to the predisposition towards one tumor type or the other. For example, it is possible that the differences are due to the time that the transgene is expressed during development. A far less common form of MEN 2 is exhibited by patients who present solitary medullary thyroid carcinomas. These patients never develop pheochromocytomas, which is very similar to the presentation observed in the line 2 Mos transgenic animals.
The differences in tumor presentation patterns in the various lines may be attributable to variations in the level of transgene expression or may reflect earlier activation of expression in the target organ of the tumor-bearing lines. This could be due to the transgene integration site or the genetic background used. As illustrated in Table 2, the interaction of transgene with genetic background can influence the penetrance of the phenotype. The tumor phenotypes of the Fj progeny of the FVB/N Mos transgenic lines crossed with BALB/c may change because of alterations in transgene expression. Alternatively, it may be that a second genetic event (as suggested by the long latent period before disease is observed in these animals) may be suppressed in line 3 or predisposed to in the F1 progeny. These transgenic lines provide a valuable opportunity to study the genetic and molecular basis of tumor induction and may help in
elucidating
the mechanism of tissue
MODERATOR:
Thank you.
QUESTION:
Could you go
in these tumors?
over
targeting
in the human
again the correlation
syndrome.
with
expression of
Mos
The correlation is such that if you take a look at any one in the animal selected for a malignant clone, and they do consistently express high levels of Mos. We have some preliminary data. We've taken tumors from humans with MEN 2. Out of nine primary tumors, only one has had a low level of Mos expression. We've looked at sporadic pheochromocytomas I've not been able to demonstrate sporadic pheochromocytomas, out of ten cases Out of ten primary neural blastomas similarly, no Mos any Mos expression. expression. One thing I should bring up is the MEN 2 has been mapped to chromosome 10. Mos in the human is on chromosome 8. SCHULZ:
tumor, you
--
-
-
MODERATOR:
Any other questions? Thank
you.
ACKNOWLEDGMENTS
Hopkins for help in preparing the manuscript. Research sponsored in part by the National Cancer Institute, DHHS, under contract No. NO1-CO-74101 with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. We wish to thank J.
823
"ONCOGENES AS PROBES FOR CELLULAR SIGNAL PROCESSES: THE FAMILY OF ETS GENES" Takis S. Papas
Laboratory
of Molecular
Oncology
National Cancer Institute
Frederick,
MD 21702-1201
Our laboratory over the past five years, has been studying the ets family of genes. The human ETS1 and ETS2 genes, were the first identified and molecularly characterized members of this gene family, all members of which are related to the E2 6 virus oncogene, v-ets. The oncogene v-ets was originally discovered as a component of the chimeric genome, along with a truncated c-myb gene, of the avian leukemia virus, E2 6. This virus transforms myeloblasts and erythroblasts in vitro, and causes mixed erythroid-myeloid The E2 6 virus expresses a 5.7 kb RNA that leukemias in vivo. encodes a 135 Kd tripartite gag-myb-ets phosphoprotein that localizes in the nucleus. Questions that we asked initially were: How did the E2 6 virus acguire the cellular sequences; what are the differences between the normal c-ets and v-ets and their gene products, as well as something of the nature of their functions, and finally, how are the normal genes regulated. To address these questions we employed several different techniques of molecular biology including cloning, sequencing and construction of vectors expressing the ets gene products making useful for making specific polyclonal and monoclonal antibodies.
E26
sequences
is
a
replication
transduced
proto-oncogenes, myb
defective
from two and ets.
retrovirus that carries portions of the chicken cellular To deduce how the virus captured
these sequence and to determine the structural relationship between the ets sequence of E26 and its cellular progenitor, the genetic organization of the chicken locus related to v-ets was examined. Analysis revealed that the chicken c-ets locus contained nine presumptive exons homologous to the v-ets. The v-ets homologous sequences (142 6 bp) were found to be spread over 60 kb of genomic DNA. As expected, since v-ets was transduced from the chicken genome, the chicken c-ets gene was found to be nearly identical to the viral sequences. A major change was found at the 3' end of the v-ets and c-ets genes where 85 nucleotides of v-ets were totally different from the 3' end of c-ets ; thus, the chicken and viral ets genes are not co-terminal.
During the process of transducing of cellular sequences by the virus, two cellular genes were truncated. While the major transcripts of chicken myb and ets are 4.0 and 7.5 kb, respectively, the virus captured only 0.85 kb and 1.5 kb of these cellular genes. Therefore what possible transduction mechanism(s) E26
The fusion of myb and ets sequences gave rise to the E2 6 virus? with retroviral gag and env sequence required a minimum of three
824
recombinational events. Alignment of gag, env, cellular and viral myb and cellular and viral ets sequence failed to reveal any significant stretches of identical nucleotides. Therefore, to be consistent with this information, we suggested that the cellular myb and ets genes was acquired by non-homologous, or illegitimate
recombination.
Sequences related to ets have been identified and cloned from humans. Initially two loci designated ETS1 and ETS2 were analyzed and characterized. The human ETS1 gene is located on chromosome llq23-24; this locus has also been implicated in a number of monocytic and certain childhood associated leukemias. Recently, we have characterized another ets-related gene, which we have named ERGB (see below); this gene too is on chromosome 11, and cotranslocates with ETS1 in the t(4;ll) (q21;q23) translocation noted for Acute nonlymphocytic leukemias (ANLL). Similarly the human ETS2 and ERG genes we found, were co-localized on chromosome 21q22.3, and are part of a group of genes that are known to be amplified in Down Syndrome (DS), as well as being associated with known non-random translocation characteristic of certain types of acute myelogenous leukemias (AML). The fact that there is a higher predisposition of individuals with DS to leukemias and transient myelodysplasias may not be coincidental, given the amplification of several known ETS genes in the critical area of chromosome 21, which is known to be associated with certain DS phenotypic characteristics.
addition to the above four human ETS family members, other related members have been and identified The ELK-1 gene has been localized on Ch.Xpll.2; characterized. this gene may be involved in a type of synovial sarcoma that nonrandomly translocates the ELK gene from the X chromosome to chromosome 18. Also, another human ETS family gene, ELK-2, has been localized onto Ch. 14q32.3; this gene has been implicated in certain T-cell malignancies. A number of chromosomal breakpoints have been known to occur near the ELK-2 locus, and such translocation are characteristically associated with T-cell diseases. The human SPI-1 gene has been identified by homology to the mouse Spi-1 (PU. 1) gene and has been localized to human In
several
chromosome
llpll.22.
The chromosomal site of the ets genes and their relationship malignancy and tumor progression in animals has proved to be quite interesting. It has been frequently observed that malignant transformation by retroviruses results in the integration of proviral sequences nearby or within specific cellular protooncogenes, activating gene expression. Recently, activation of an ets-related gene Spi-1. has been observed in a collection of murine erythroblastic tumors induced by spleen focus forming virus (SFFV). In fact 21 of 22 such erythroid malignancies studied, induced by SFFV, had proviral insertional rearrangements near the Spi-1 gene. In rat thymomas induced by the Moloney strain of the Murine leukemia virus (Mo-MuLV), it has been found that proviral insertion events occurred at common integration loci thought to be associated
to
825
progression, termed Tpl-1. These sites have now been shown to result in the rearrangement or activated expression of the ets-1 gene. Another study has recently shown that erythroleukemias induced by Friend-MuLV activated the Fli-1 locus which has been shown to be an ets related family member. The Fli-1 gene is the murine homolog of the human ERGB gene that we have identified in The occurrence of our laboratory by sequence homology to ETS2. three types of hematopoietic malignancies as a consequence of retroviral insertions are significant, and are thought to be due to the fact that these events have in common the activation of an ets gene or its related family member. with tumor
Members of the ets gene family have been cloned and sequenced from a variety of species ranging from human to Drosophila; their encoded products are highly conserved, more so than the other known nuclear protooncogene products. Comparison of all the predicted proteins of the ets-gene family was made with respect to the chicken ets-1 gene (shown in Figure 1) On the basis of their amino acid sequence homology we have identified three regions; A, B and C; however, not all of these regions are found identically distributed among the family of ets related proteins. The 'A' region is localized at the amino or N-terminus, and is a moderately .
The ets Gene
Family B
Ch 8ls-2
nnrjiwmniiiiniiBnzi nrauiiiDiniiDiii^ri^ ZD3H1IHIMIIII
Xe efs-2
mrimmTiiiniiiiiiiMii i
I- Hu/Mo
efs-2
I i i
i
ii 11 h mmllll II I1HM rriirni llll! I IIHM il mi in usa 11 ii i mu m
mill! Him: mi nun
Hu/Mo/Ch/Xe els-1
IIIIHUIIIIH Su ets Dm ets
OHnimn MTJIIIttllllllMa
Hu erg -c Dm elg
Ç
aaiiiiiiiiBinEiz
Hi, elk
mi «ni im
Mo PU.1 Dm 74E
rji Hu Xe
-
Human
Mo
Xenopus
Su
-
4,500 4,000 3,000 -J_I_
-
Mouse
Ch
Sea Urchin
Dm
-
Earth
of
Drosophila
-
1,000 600
2,000
__J-L_/iL_
Meso- I CenoIQlC zoic
Paleozoic
Origin
Chicken -
lyl
| First
Eukaryotic Fossils
|
I
Oldest
Mammals
Invertebrates and Vertebrates
Appear
-
Comparison of the different members of the ets gene family. three regions: A, B, and C; based upon their homology to the human Fig.
1.
826
The ets gene is divided into ETS1 gene.
homologous domain when compared to each of the members of the ets family. By contrast, the 'B' region, located in the middle, is only weakly homologous to one another; this region appears to be dispensable, since it is known to be deleted in the predicted sequences of several of the ets-related gene members (e.g., ERG. The 'C region on the other hand, is located at the carboxy elg) or C-terminus and is a very highly conserved protein region .
encoding the DNA binding domain and found in common among all the members of ets-family. This yC region is the only one that is present in the predicted sequences of every ets- related gene characterized thus far, and therefore it forms the basis of defining membership in the ets-family. These three distinctive regions, enable us to group the ets genes into three classes: I, II, and III; classification was determined on the basis of homology to ETS1. the human homolog of the chicken proto-oncogene c-ets-1 Class I members contain all three of the above-mentioned (31) homologous regions 'A', 'B', and ' C (ets-1, v-ets); class II members contain the homologous 'A' and 'C regions (ets-2, erg). while class III members contain only the homologous 'C region (i.e., elk. PU.l, and Drosophila E74A). .
Representative members of ets gene family are distributed throughout metazoans, and are related to one another by their homology to the carboxy terminal portion of the v-ets oncogene. Given that the ets family proteins appear to play a role as important transcription factors that control multiple gene cellular functions in and differentiation proliferation, development; any alteration of the ets genes would markedly alter the cell's physiology and have a profound pleiotropic effect. of sequence divergence allowed a prediction of the of the ets-gene family; assignment of the members into the three classes, demonstrates that all the ets-1 and ets-2 members each formed a tightly clustered grouping. However other ets-family products, (e.g., human and sea urchin erg genes, the human ELK1, Drosophila 74E and the murine PU.l gene) form a somewhat looser clustered grouping. A plausible explanation of the origins of these groupings and classes of ets-family members could be due to an ancestral gene duplication (e.g., class I) occurring, followed by genetic recombination(s) or divergence yielding the other classes (I and II) However, since class I and III members are represented by both invertebrates as well as vertebrates, this ancestral duplication event must predate the emergence of chordates, i.e, prior to 500 million years ago.
Analyses
phylogenetic history different ets-family
.
All the ets and related genes
are
transcriptionally
active and
In adult expressed in a wide variety of cells and tissues. murine tissue, the Ets-1 message is expressed preferentially in thymus, lung and spleen whereas Ets-2 product is found expressed in almost every type of tissue tested. The human ETS1 gene encodes a unique 6.8 Kb RNA. The ETS2 gene is known to be expressed as three distinct messages sized 4.7, 3.2 and 2.7 Kb, this gene does not The generation of the three appear to be differentially spliced. ETS2 messages is due to the preferential utilization of its three are
827
different poly A signals. Since all the three ETS2 messages contain complete coding sequences, it is not yet clear which of the three messages is being translated to produce the ETS2 product. The ERG message remains the most restrictive product expressed by the
ets-gene family.
The ETS1 gene encodes a set of phosphoproteins ranging in size from 42 kd to 52 kd (Figure 2) The p51ETS1 isoform is the product of the full length MRNA, while the p42ETS1 isoform results from the ETS1 protein product of a spliced MRNA lacking exon VII. phosphorylation is Ca++-dependent and treatment of cells with calcium ionophore, as well as by complexing the T-cell receptor with antibody, increases the level of phosphorylation of the ETS1 protein. The ets-gene products and its related family of proteins contain the calmodulin dependent protein kinase II consensus site (RXXS/T). We have been able to demonstrate that the ETS1 protein is phosphorylated by the calmodulin-dependent protein kinase II, Since the p42ETS1 product is using an in vitro labeling assay. encoded by the MRNA lacking exon VII it does not become .
phosphorylated; this characteristic implicates splicing as important for the differential regulation of ETS1 proteins via Ca++-mediated phosphorylation. The ETS2 gene encodes
a
p54 kd protein that is phosphorylated
Ca++-dependent mitogenic signal process (Figure 2) The stability of the ETS2 protein is dramatically increased as a consequence of protein kinase C activation from a protein with a half-life of 20 min. to 140 min. one with a half-life. Thus this (pp56) by
a
.
ETS1 Isoforms ETS2 Isoforms
PS¡?
=
p42
pp56p54-
• «»
-
Met
NH2 ETS1
