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RHEUMATISM OFFICIAL JOURNAL O F T H E AMERICAN RHEUMATISM ASSOCIATION SECTION O F T H E A R T H R I T I S FOUNDATION
MONOZYGOTIC TWINS DISCORDANT FOR SYSTEMIC LUPUS ERYTHEMATOSUS COMPARISON OF IMMUNE RESPONSE, AUTOANTIBODIES, VIRAL ANTIBODY TITERS, GAMMA GLOBULIN, AND L I G H T CHAIN METABOLISM MICHAEL W. YOCUM, JAY GROSSMAN, CHRISTINE WATERHOUSE. GEORGE N. ABRAHAM, ALLYN G. MAY, and J O H N J. CONDEMI
A pair of monozygotic twins discordant for systemic lupus erythematosus (SLE) were studied and no differences noted in their immune response to tetanus toxoid, keyhole lympet hemocyanin, DNCB, delayed sensitivity, or antibody titers to viruses. Both were noted to have biologically false positive serology at an early age, but only one twin developed SLE. The clinically unaffected twin underwent castration at an early age, suggesting that ovarian hormones
may play an important role in the development of SLE.
From the Departments of Medicine and Surgery, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. Supported in part by United States Public Health Service Research Grants RR-00044 from the Division of Research Facilities and Resources, National Institutes of Health; AI11550-01 from the National Institutes of Health: United States Public Health Service Training Grant AI-00028 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health: and the Monroe County Chapter of T h e Arthritis Foundation, Rochester, New York, and the New York State Kidney Disease Institute. Michael W. Yokum. M.D.: Instructor and Trainee in Medicine (Immunology), University of Rochester School of Medicine and Dentistry, and Naval Hospital NNMC. Box 302, Physician’s Mail, Bethesda. Maryland; Jay Grossman, M.D.: Instruc-
tor and Trainee in Medicine (Immunology), University of Rochester School of Medicine and Dentistry; Christine Waterhouse, M.D.: Professor of Medicine, University of Rochester School of Medicine and Dentistry; George N. Abraham, M.D.: Assistant Professor of Medicine and Microbiology, University of Rochester School of Medicine and Dentistry, and the recipient of a USPHS allergic disease academic award: Allyn G. May, M.D.: Associate Professor of Surgery, University of Rochester School of Medicine and Dentistry: and John J. Condemi, M.D.: Professor of Medicine, University of Rochester School of Medicine and Dentistry. Address reprint requests to John J. Condemi, M.D., Department of Medicine, University of Rochester School of Medicine, 260 Crittenden Boulevard, Rochester, New York 14642. Submitted for publication March 15, 1974: accepted August 8, 1974.
Arthritis and Rheumatism, Vol. 18, No. 3 (May-June 1975)
The various proposed etiologies of systemic lupus erythematosus (SLE) include viral infection (1). endocrine abnormality (2), genetic predisposition (3), and hypersensitivity to self with loss of immunologic
194
YOCUM ET AL
homeostasis (4). A genetic predisposition is supported by reports that describe a n increased incidence of SLE a n d related collagen diseases in relatives of probands with SLE (5,6) a n d reports of monozygotic twins concordant for SLE (7,8). W h e n discordance is present i n monozygotic twins, etiologic mechanisms other t h a n a genetic predisposition may be responsible for a disease. A t present there are only three published reports describing monozygotic twins discordant for SLE (9-1 1). In this paper, a set of monozygotic twins, discordant for SLE, a n d their viral antibody titers, autoantibodies, humoral a n d cellular i m m u n e response to antigenic challenge, a n d gamma globulin a n d light chain metabolism, are described.
CASE REPORT 1 ML,* a 44-year-old married white female, demonstrated a biologic false positive test for syphilis without systemic illness during her first pregnancy, at 25 years of age. At age 32, a left oophorectomy and salpingectomy were performed for an ovarian cyst. At age 37, morning stiffness, digital arthralgia, Raynaud's phenomenon, and intermittent bilateral calf pain without phlebitis was noted. At age 40, in addition to the above, progressive fatigue was noted. LE cell and antinuclear antibody (ANA) tests were negative but serum IgG and IgM were elevated to 1840 and 230 mg%, respectively. I n 1971 at age 43, because of more severe morning stiffness with swelling and pain of both hands, knees, ankles, and shoulders, she was admitted to Strong Memorial Hospital. A systems review revealed that she had normal menstrual periods and was not menopausal. Family history revealed a monozygotic twin. On physical examination, there was warmth, swelling, and tenderness of all PIP joints and of the second and third MC-P joints of both hands, and tenderness on rotation of the humerus and of the metatarsal heads in both feet. A mild sensory neuropathy with decreased pinprick and vibration sensation below the ankles and wrists, bilaterally, was present. The remainder of the physical examination, as well as the CBC, urinalysis, BUN, and creatinine clearance, was normal. LE cell and ANA tests were now positive and a Westergren erythrocyte sedimentation rate was 35 mm/hr. Roentgenograms of the hands revealed soft tissue swelling without joint abnormalities. Chest x-ray and EKG were normal. A punch biopsy of normal-appearing skin showed focal lymphocytic infiltration and, by immunofluorescence, IgG deposits at the epidermal-dermal basement membrane area. A diagriosis of systemic lupus erythematosus was made. The patient was taking salicylates and her arthritis had improved markedly by the time she was discharged. * M d t w i n with systemic lupus erythematosus.
CASE REPORT 2 MC, the 44-year-old monozygotic twin of ML, was well until the age of 21 when she developed vaginal bleeding. A pelvic examination revealed a right adnexal mass. A biologic false-positive test for syphilis was found. An adenocarcinoma of the right ovary, metastatic to the uterus, required an hysterectomy, bilateral oophorectomy, and salpingectomy. She was followed for 23 years during which time she did not receive ovarian hormone replacement and the tumor did not recur. In January 1972 she and her sister were admitted to the Clinical Research Center. She had no complaints, and physical examination revealed slight hirsuitism of the upper lip and pubic region and a more muscular physique than her twin. Blood pressure was 150/100 mm Hg. Mild arteriolar narrowing on funduscopic examination and a presystolic gallop without cardiomegaly were noted. The remainder of the physical examination and the CBC, urinalysis, BUN, and creatinine clearance were normal. Chest xray revealed mild osteoporosis and a normal size heart; the electrocardiogram revealed a mild left ventricular hypertrophy.
MATERIALS AND METHODS Laboratory Data. Leukocyte chemotaxis was performed by Baum, as previously described (12). Follide-stimulating hormone (FSH) and luteinizing hormone (LH) were determined by radioimmunoassay. Immunoglobulin levels were determined by quantitative radial immunodiffusion. Antibodies to native and heat-denatured deoxyribonucleic acid were measured by a modification of the method of Pincus et a1 (13). Other antitissue and antiorganelle antibody titers were performed by hemagglutination or the indirect immunoflourescence techniques. Serologic Studies. Tetanus and diphtheria titers were determined by hemagglutination on serum obtained 10 days after the administration of tetanus toxoid. Keyhole lympet hemocyanin (KLH) titers were performed by hemagglutination. Influenza A, Hong Kong titers were determined by viral neutralization. Other viral titers were performed by standard complement fixation assays. Delayed Hypersensitivity. The following antigens were used for skin testing: trichophyton 1 :500, monilia 1:500, mosquito 1000 PNU/ml (Hollister-Stier), mumps (Lilly), staphylococcal toxoid dilution 1, streptokinase 10 units/ml and streptodornase 2.5 units/ml (Lederle), and intermediate strength PPD (MSD). Dinitrochlorobenzene sensitization was performed as described by Catalona et a1 (14) and mixed lymphocyte cultures and phytohemagglutinin stimulation by the technique of Bach et al (15,16). Monozygotic Data. Lymphocytes of each patient were tested for HL-A 1, 2, 3, 5, 7, 8, 9, 10, 12, and 13 with a total of 40 antisera by means of the standard NIH microlymphocytotoxicity method. Gamma Globulin Metabolism. Gamma globulin and light chain metabolism were studied in detail, as previously described (1 7). DEAE-purified IgC, obtained as Cohn fraction 11, and light chains obtained from this preparation
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MONOZYGOTIC TWINS DISCORDANT FOR SLE
Table 3. Autoantibodies
Table 1. Monozygosity Data
Blood type ABO Rhesus Lewis MNSs Duffy Kidd Kell HL-A-type Mixed lymphocyte culture
M L*
MC
AB R/r DCce LeaNSs Fy" Fyb JK8 JKb K KPb JSa23.12 Negative for 7,8,9,13 No transformation with MC lymphocytes Transformation with other donor lymphocytes
AB R/r DCce LeaNSs Fy" Fyb JK' JKb K KPb JS2.3.12 Negative for 7,8,9,13 No transformation with ML lymphocytes Transformation with other donor lymphocytes
ML*
*Twin with systemic lupus erythematosus. were utilized for turnover studies. T h e proteins were radiolabeled, as described by Bale et a1 (18), freed of aggregates a n d subjected to ultrafiltration t o ensure sterility. Approximately 1 mg of IgGI-131 a n d L chain'-125 containing 5 x 10s cpm of specific activity was simultaneously injected intravenously. Blood samples were obtained a t frequent intervals beginning within 2 minutes of injection and for 30 days after administration. Serum IgC a n d L-chain levels were determined by radial immunodiffusion a n d complement fixation assays, respectively (17).
RESULTS Monozygosity Data. These twins appeared monozygotic and their blood typing as well as HL-A
VDRL titer FTA-ABS LE prep ANA titer-pattern
Normal Values ML*
4
Neat 2+ rim 1:lO 1+ 1:lOO -
Neat 14- speckled
-
1:80 1:5
-
-
-
-
-
1:40
-
-
-
1:40
-
-
-
24% binding
4% binding
17% binding
2% binding
typing was identical (Table 1). Neither twin's lymphocytes transformed when cultured with the other's but they did transform when cultured with nonrelated donor lymphocytes. Laboratory Data. As shown in Table 2, both patients had an elevated erythrocyte sedimentation rate. Twin MC showed evidence of menopause with an elevated FSH, and her IgM was markedly elevated but polydispersed on agar zone electrophoresis. Autoantibodies are compared in Table 5. A
Table 4. Serologic Studies MC
20-30
Normal 13.1 6,400 200,000 52
Normal 13.8 6,300 200,000 68
(> 450) 10-20 0.8-1.2 80-140 70-100 2-7 3.5-14 6-16 600-1,700 50-250 50-200 > 80
1,252 19 0.8 104 80 3.1 6.9 4.5 1,365 58 275 80
1,400 20 0.8 111 ND 5.4 21.6 7.7 990 62 414 111
12.0-15.4 4,000-1 1,000 > 150,000
-
+Twin with systemic lupus erythematosus.
Test ~
Urinalysis Hemoglobin (g%) WBC/mma Platelets/mms Westergren sedimentation (mm/ hr) WBC chemotaxis index @%Yo) Creatinine (mgyo) Ccr (ml/min) Prothrombin time (yo) Uric acid (mgyo) FSH (mpg/ml) LH (mpg/ml) I& (mg%) IgA (mg%) IgM (mg%) CH, units
4
-
+
ANP latex titer Anti-IgG titer Antithyroglobulin titer Antismooth muscle titer Antiautochondrial titer Antihuman ovary titer Antithyroid microsome titer Antigastric mucosa titer Antiesophageal mucosa titer Coombs gamma and nongamma Antinative DNA (normal < 10%) Antiheat denatured DNA (normal < 8y0)
Table 2. Laboratory Data Test
MC
~
_
_
ML*
MC
1:20,480 1:160
1:40,960 1:80
1:4, 1:s 1:256 1: 128 1:128
1:2, 1:4 1:64, 1:128 1 :32 132
1:16
1:64
< 1:8 < 1:8
c 1:s
_
Hemagglutination titers Tetanus (10 days after booster) Diphtheria Keyhole lympet hemocyanin Baseline 10 days 20 days 30 days KLH delayed skin test Viral neutralization titers Influenza A, H K Complement fixation titers Mumps Rubeola Parainfluenza 1,2, 3 Respiratory syncytial virus Herpes simplex Cytomegalovirus
1:40 1:lO < 1:4 1: 16
~~
*Twin with systemic lupus erythematosus; ND = not done.
*Twin with systemic lupus erythematosus.
C 1:8
1:5
> 1:40 1:16
C 1:4
YOCUM E T AL
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Table 5. Delayed Hypersensitivity MLf Mumps Trichophyton Monilia Streptokinase-streptodornase Staph toxoid Mosquito IPPD DNCB PHA
15 X 15 mmf 0 30 X 30 mmf 7 mmt 0 0
MC
10
X
30
X
0
3+ > 71,000 cpm
>
10mmt 0 30 mrnf 0 0 0
L chains were strikingly different. ML had serum kappa and lambda L-chain levels of 28 and 6 ,N/ml, respectively; the unaffected twin, MC, had normal levels of 5.6 (kappa) and 2.2 (lambda). Thus, the calculated production of L chains was 4.51 g per day in ML, a figure about five times greater than the normal production rate of 0.875 g per day in MC.
0 4+ 26,000 cpm
*Twin with systemic lupus erythematosus. tMillimeters of induration at 48 hr.
positive VDRL test in low titer with a negative fluorescent treponemal antibody absorption (FTA-ABS) test was noted in both. T h e affected twin, ML, had a positive LE cell preparation and persistent and significant rim-positive antinuclear antibody (ANA) test. Of six ANA tests in the unaffected twin, only one was positive in low titer, with a speckled pattern. ML had weakly positive titers of anti-IgG and antithyroglobulin and significant titers of antihuman ovary and antigastric mucosa antibodies. Serum from ML also bound 24% native DNA and 17% single-stranded DNA. Serologic Studies. Table 4 shows that tetanus toxoid immunization and keyhole lympet hemocyanin (KLH) sensitization resulted in a normal production of hemagglutinating antibody in both twins. Neither exhibited a delayed skin test to KLH, but each was not rechallenged with antigen after 30 days. Parainfluenza antibody titers were elevated in twin M L and respiratory syncytial virus titers were elevated in MC. Evidence of past infection with cytomegalovirus in ML and with herpes simplex virus in MC was found. Assessment of delayed hypersensitivity (Table 5) did not reveal significant differences. Both M L and MC had positive delayed skin tests to mumps and monilia, were able to be sensitized to dinitrochlorobenzene, and were able to respond to phytohemagglutinin stimulation. Gamma Globulin Metabolism. Labeled IgG globulin decay curves showed a rapid final disappearance in ML (t,,2 = 9 days), and a normal rate (tl,2 = 15 days) in MC. T h e more rapid decay rate and slightly higher IgG level of M L indicate an increased disappearance rate of IgG globulin, confirming previous data (19). T h e labeled (L) chain decay curves of the two subjects were similar and within our normal range, but absolute levels of free serum kappa and lambda
DISCUSSION Cited reports of an increased incidence of autoantibodies and rheumatic diseases in relatives of patients with SLE, plus an increased frequency of SLE within families, and SLE in monozygotic twins, imply a significant genetic predisposition toward the development of this disease. Recent etiologic hypotheses for SLE suggest that, given a predisposition, an environmental factor, such as a drug or a viral infection, may elicit manifest disease (20,21). T h e study of monozygotic twins discordant for SLE thus provides a unique opportunity to study environmental influences that might explain the development of disease in only one of two individuals of identical genotype. Of the three published reports of monozygotic twins discordant for SLE, only in the case report by Brunner (1 1) is the identity of the twins documented as well as ours. By studying the unaffected twin, we hoped to separate those abnormalities predating the onset of SLE. I n addition, it was postulated that the immune response of the twin with definite but mild SLE would not be markedly suppressed, as can occur in severe systemic disease or renal failure. T h e biologically false-positive serologic test for syphilis (BFP-STS) in both patients and the one faintly positive ANA test in the healthy twin suggest that both had the potential to develop SLE. Hereditary BFP-STS (22) was ruled out by a negative Wasserman test in the parents and a brother of the twins. Previous reports describing the immune response i n patients with SLE have shown, in studies with matched controls, similar primary and secondary responses to tetanus toxoid (23), an increased 7s but decreased 19s antibody response to bacterial antigens (24), and, i n monozygotic twins discordant for SLE, a higher antibody response to KLH protein in the twin with SLE (11). These twins both developed an adequate secondary response to tetanus toxoid, and ML developed a higher and more sustained antibody titer to KLH immunization. Because of the reports of a viral illness preced-
MONOZYGOTIC TWINS DISCORDANT FOR SLE
ing the onset of SLE (1,25), viral-like particles in tissues from patients with SLE (26), and the elevated viral antibody titers in patients with SLE (21,25), this parameter was also tested. T h e only difference observed was increased antibody titers to parainfluenza 1, 2, 3, in ML. Several reports (1 1,2327) have demonstrated decreased cell-mediated immunity in patients with SLE, as evaluated by delayed skin test antigens and, as this may predispose to viral infection, we studied this aspect of the immune response. No differences in response to seven delayed skin test antigens, DNCB sensitization, PHA stimulation, or mixed lymphocyte cultures were demonstrated in these twins. I n several reports demonstrating decreased delayed skin tests in SLE, patients acutely ill with SLE and frequently on corticosteroids were compared with healthy normal adults. I n a study performed by skin testing patients on a general medical ward, we noted a n absence of positive delayed skin tests in 387, (28). I t is therefore important that in all studies evaluating delayed immunity the effect of severe illness be considered. Certain murine diseases have been linked to the H-2 histocompatibility locus, and an increased incidence of HL-A antigens has been noted in humans with SLE (3). I n these twins a n identical genotype was demonstrated; therefore, the discordance for SLE cannot be related solely to the immune response genes. Increased IgG turnover has been reported in SLE (19). Two known causes for an increased disappearance rate of IgG are excessive gastrointestinal loss (29) and an elevated serum IgG level. I n ML a high serum level was not a factor, but some intestinal loss cannot be ruled out. Elevated serum L chains have been previously described in active SLE (39). A recent report suggests that increased urinary excretion of free L chains indicates activity of the disease process (31) and is a manifestation of tubular disease and inability to catabolize L chains. Because no trichloroacetic acid precipitable radioactivity was noted in either ML or MC, free urinary L chains were not measured. T h e high production rate of 4.5 g of L chain per day in ML is similar to that in multiple myeloma and implies a well-preserved catabolic rate. It is clear that L-chain production in SLE is not tightly coupled to IgG synthesis and a normal feedback control seems inoperative as in multiple myeloma. Of all patients with SLE, 90% are female and usually of childbearing age. However, the etiologic
197
or permissive role of ovarian hormones in this disease is controversial. Nonetheless, certain facts are known that associate SLE with ovarian hormones and must be explained by any theory of the pathogenesis of this disease. Walker and Bole (32) studying NZB/NZW mice showed that mesiranol accelerated the development of ANA but not autoimmune nephritis in male mice and that neither mestranol nor early oophorectomy had an observable effect on these parameters in female mice. Studies of patients with SLE who become pregnant have demonstrated a n exacerbation of disease activity most frequently during the postpartum period (33,34). SLE patients may exacerbate if given oral contraceptives, and recently a report of normal females developing a n SLE-like syndrome while using contraceptive pills has appeared (2). T h e pregnant state, or ovarian or placental hormones, can depress cell-mediated immunity. Andresen and Monroe (35) found that both the first and second set skin homograft rejection times were prolonged by a factor of 2 in pregnant women during the third trimester compared to nonpregnant age-matched controls. Purtilo (36) found that lymphocyte response to PHA is decreased at all stages of pregnancy but most markedly during the third trimester and postpartum period. Munroe (37) found that newborn monkeys, monkeys pretreated with progesteroids, and pregnant monkeys were all highly susceptible to Rous chicken sarcoma virus infection, while normal juvenile and adult monkeys were highly resistant to this virus. He further demonstrated that intravenous progesterone decreased the number of circulating lymphocytes and prolonged skin graft rejection. I t was concluded that progesteroids significantly decreased cell-mediated immunity and resistance to viral infection. Thus, there is evidence that pregnancy or ovarian hormones may exacerbate existing SLE, create an SLE-like syndrome, depress cell-mediated immunity, and increase susceptibility to viral infection. This present report of monozygotic twins discordant for SLE reveals the following data. Both patients demonstrated an abnormal immune response at an early age as manifested by a BFP-STS and a positive ANA in later life which to us suggests that both had the genetic potential to develop SLE. T h e subsequently unaffected twin, MC, underwent surgical castration at 21 years of age without ovarian hormone replacement, while the affected twin, ML, developed mild but definite SLE with an immune response not blunted by uremia or severe systemic disease activity.
YOCUM ET AL
Humoral and cellular i m m u n e responses a n d viral antibody titers revealed no striking differences, a fact suggesting that their exposure to the viruses studied was quite similar. T h e antibodies to DNA, antitissue antibodies, decreased half life of IgG globulin, and increased synthesis of L chains in the affected twin, ML, can be explained by her SLE. The major difference between this twin pair that cannot be explained by SLE was therefore the absence of ovarian hormones i n the unaffected twin. T h i s difference raises the question as to whether ovarian hormones may allow environmental factors, such as viral infection, to induce development of SLE and, we hope, stimulates further investigation i n the role of ovarian hormones in SLE.
REFERENCES 1. Ziff M: Viruses and the connective tissue diseases. Ann
Intern Med 75:951-958, 1971 2. Bole GG Jr, Friedlaender MH, Smith CK: Rheumatic symptoms and serological abnormalities induced by oral contraceptives. Lancet 1:323-328, 1969 3. Grumet FC, Coukell A, Bodmer JG, et al: Histocompatibility (HL-A) antigens associated with systemic lupus erythematosus. N Engl J Med 285:193-196, 1971 4. Rowel1 NR: Etiology of certain connective tissue diseases. Clin Exp Immunol 2:813, 1967 5. Leonhardt ETG: Family studies in systemic lupus erythematosus. Clin Exp Immunol 2:743-759, 1967 6. Morteo OG, Franklin EC, McEwen C, et al: Studies of relatives of patients with systemic lupus erythematosus. Arthritis Rheum 4:356-363, 1961 7. Davis MW, Gutridge GN: Disseminated lupus erythematosus in identical twin sisters associated with diabetes mellitus in one case. J Missouri Med Assoc 48: 446450, 1951 8. Joseph RR, Zarafonetis CJD: Fatal systemic lupus erythematosus in identical twins: case reports and review of the literature. Am J Med Sci 249:190-199, 1965 9. Holmes FF. Stubbs DW, Larsen WE: Systemic lupus erythematosus and multiple sclerosis in identical twins. Arch Intern Med 119:302-304, 1967 10. Larsson 0, Leonhardt T: Hereditary hypergammaglobulinemia and systemic lupus erythematosus. I. Acta Med Scand 165: 371-393, 1959 11. Brunner CM, Horwitz DA, Shann MG, et al: Clinical and immunologic studies in identical twins discordant for systemic lupus erythematosus. Am J Med 55:249254. 1973 12. Baum J, Mowat AG, Kirk JA: A simplified method for the measurement of chemotaxis of polymorphonudear leukocytes from human blood. J Lab Clin Med 77: 501-509, 1971
13. Pincus T, Schur PH, Rose JA, et al: Measurement of serum DNA-binding activity in systemic lupus erythematosus. N Engl J Med 281:701-705, 1969 14. Catalona WJ, Taylor PT, Rabson AS, et al: A method for dinitrochlorobenzene contact sensitization. N Engl J Med 286:39H02, 1972 15. Bach FH, Voynow NK: One-way stimulation in mixed leukocyte cultures. Science 153:545-547, 1966 16. Bach FH, Hirschhorn K: T h e in vitro response of penpheral blood lymphocytes. Semin Hematol 2:68, 1965 17. Waterhouse C, Abraham G, Vaughan J: The relationship between L-chain synthesis and gamma globulin production. J Clin Invest 52: 1067-1077, 1973 18. Bale WF, Helmkamp RW, Davis TP, et al: High specific activity labeling of protein with 1131 by the iodine monochloride method. Proc SOCExp Biol Med 122:407414, 1966 19. Birke G, Liljedahl SO, Olhagen B, et al: Catabolism and distribution of gamma globulin. A preliminary study with 131I-labeled gamma globulin. Acta Med Scand 173:589-603, 1963 20. Blomgren SE, Condemi JJ, Vaughan JH: Procainamide induced lupus erythematosus. Am J Med 52338-348, 1972 21. Hollinger FB, Sharp JT, Lidsky MD, et al: Antibodies to viral antigens in systemic lupus erythematosus. Arthritis Rheum 14:l-11, 1971 22. Kostant GH: Familial chronic biologic false positive seroreactions for syphilis. JAMA 219:45-48, 1972. 23. Abe T , Homma M: Immunological reactivity in patients with systemic lupus erythematosus. Acta Rheumatol Scand 17:35-46, 1971 24. Baum J, Ziff M: Decreased 19s antibody response to bacterial antigens in systemic lupus erythematosus. J Clin Invest 48:758-767, 1969 25. Koivisto 0, Sotaniemi E, Vesikari T: Recurrent virus infections in identical twins with systemic lupus erythematosus. Acta Rheumatol Scand 17304-311, 1971 26. Fresco R: Tubular (myxovirus-like) structures in glomerular deposits from a case of lupus nephritis. Fed Proc 27:246, 1968 (abstr) 27. Horwitz DA: Impaired delayed hypersensitivity in systemic lupus erythematosus. Arthritis Rheum 15:353359, 1972 28. Grossman J, Baum J, Gluckman J, et al: The effect of aging and acute illness on delayed hypersensitivity. J Allergy Clin Immunol 51:127, 1973 (abstr) 29. Waldmann TA, Broder S, Strober W: Protein-losing enteropathies in malignancy. Ann NY Acad Sci 230: 306-317, 1974 30. Epstein WV, T a n M: Increase of L-chain proteins in the sera of patients with systemic lupus erythematosus and the synovial fluids of patients with peripheral rheumatoid arthritis. Arthritis Rheum 9:713-719, 1966 31. Epstein WV: Immunologic events preceding clinical
MONOZYGOTIC TWINS DISCORDANT FOR SLE
exacerabation of systemic lupus erythematosus. Am J Med 54:631-636, 1973 32. Walker SE, Bole GG Jr: Influence of natural and synthetic estrogens on the course of autoimmune disease in the NZB/NZW mouse. Arthritis Rheum 16:231-239, 1973 33. Garsenstein M, Pollak VE, Kark RM: Systemic lupus erythematosus and pregnancy. N Engl J Med 267:165169, 1962 34. McGee CD, Makowski EL: Systemic lupus erythema-
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tosus in pregnancy. Am J Obstet Gynecol 107:10081012, 1970 35. Andresen RH, Monroe CW: Experimental study of the behavior of adult human skin homografts during pregnancy. Am J Obstet Gynecol 83:1096-1101, 1962 36. Purtilo DT, Hallgren HM, Yunis EJ: Depressed maternal lymphocyte response to phytohaemagglutinin in human pregnancy. Lancet 1:769-771, 1972 37. Munroe JS: Progesteroids as immunosuppressive agents. J Reticuloendothel SOC9561-375, 1971
On July 1, 1975
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193
RHEUMATISM OFFICIAL JOURNAL O F T H E AMERICAN RHEUMATISM ASSOCIATION SECTION O F T H E A R T H R I T I S FOUNDATION
MONOZYGOTIC TWINS DISCORDANT FOR SYSTEMIC LUPUS ERYTHEMATOSUS COMPARISON OF IMMUNE RESPONSE, AUTOANTIBODIES, VIRAL ANTIBODY TITERS, GAMMA GLOBULIN, AND L I G H T CHAIN METABOLISM MICHAEL W. YOCUM, JAY GROSSMAN, CHRISTINE WATERHOUSE. GEORGE N. ABRAHAM, ALLYN G. MAY, and J O H N J. CONDEMI
A pair of monozygotic twins discordant for systemic lupus erythematosus (SLE) were studied and no differences noted in their immune response to tetanus toxoid, keyhole lympet hemocyanin, DNCB, delayed sensitivity, or antibody titers to viruses. Both were noted to have biologically false positive serology at an early age, but only one twin developed SLE. The clinically unaffected twin underwent castration at an early age, suggesting that ovarian hormones
may play an important role in the development of SLE.
From the Departments of Medicine and Surgery, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. Supported in part by United States Public Health Service Research Grants RR-00044 from the Division of Research Facilities and Resources, National Institutes of Health; AI11550-01 from the National Institutes of Health: United States Public Health Service Training Grant AI-00028 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health: and the Monroe County Chapter of T h e Arthritis Foundation, Rochester, New York, and the New York State Kidney Disease Institute. Michael W. Yokum. M.D.: Instructor and Trainee in Medicine (Immunology), University of Rochester School of Medicine and Dentistry, and Naval Hospital NNMC. Box 302, Physician’s Mail, Bethesda. Maryland; Jay Grossman, M.D.: Instruc-
tor and Trainee in Medicine (Immunology), University of Rochester School of Medicine and Dentistry; Christine Waterhouse, M.D.: Professor of Medicine, University of Rochester School of Medicine and Dentistry; George N. Abraham, M.D.: Assistant Professor of Medicine and Microbiology, University of Rochester School of Medicine and Dentistry, and the recipient of a USPHS allergic disease academic award: Allyn G. May, M.D.: Associate Professor of Surgery, University of Rochester School of Medicine and Dentistry: and John J. Condemi, M.D.: Professor of Medicine, University of Rochester School of Medicine and Dentistry. Address reprint requests to John J. Condemi, M.D., Department of Medicine, University of Rochester School of Medicine, 260 Crittenden Boulevard, Rochester, New York 14642. Submitted for publication March 15, 1974: accepted August 8, 1974.
Arthritis and Rheumatism, Vol. 18, No. 3 (May-June 1975)
The various proposed etiologies of systemic lupus erythematosus (SLE) include viral infection (1). endocrine abnormality (2), genetic predisposition (3), and hypersensitivity to self with loss of immunologic
194
YOCUM ET AL
homeostasis (4). A genetic predisposition is supported by reports that describe a n increased incidence of SLE a n d related collagen diseases in relatives of probands with SLE (5,6) a n d reports of monozygotic twins concordant for SLE (7,8). W h e n discordance is present i n monozygotic twins, etiologic mechanisms other t h a n a genetic predisposition may be responsible for a disease. A t present there are only three published reports describing monozygotic twins discordant for SLE (9-1 1). In this paper, a set of monozygotic twins, discordant for SLE, a n d their viral antibody titers, autoantibodies, humoral a n d cellular i m m u n e response to antigenic challenge, a n d gamma globulin a n d light chain metabolism, are described.
CASE REPORT 1 ML,* a 44-year-old married white female, demonstrated a biologic false positive test for syphilis without systemic illness during her first pregnancy, at 25 years of age. At age 32, a left oophorectomy and salpingectomy were performed for an ovarian cyst. At age 37, morning stiffness, digital arthralgia, Raynaud's phenomenon, and intermittent bilateral calf pain without phlebitis was noted. At age 40, in addition to the above, progressive fatigue was noted. LE cell and antinuclear antibody (ANA) tests were negative but serum IgG and IgM were elevated to 1840 and 230 mg%, respectively. I n 1971 at age 43, because of more severe morning stiffness with swelling and pain of both hands, knees, ankles, and shoulders, she was admitted to Strong Memorial Hospital. A systems review revealed that she had normal menstrual periods and was not menopausal. Family history revealed a monozygotic twin. On physical examination, there was warmth, swelling, and tenderness of all PIP joints and of the second and third MC-P joints of both hands, and tenderness on rotation of the humerus and of the metatarsal heads in both feet. A mild sensory neuropathy with decreased pinprick and vibration sensation below the ankles and wrists, bilaterally, was present. The remainder of the physical examination, as well as the CBC, urinalysis, BUN, and creatinine clearance, was normal. LE cell and ANA tests were now positive and a Westergren erythrocyte sedimentation rate was 35 mm/hr. Roentgenograms of the hands revealed soft tissue swelling without joint abnormalities. Chest x-ray and EKG were normal. A punch biopsy of normal-appearing skin showed focal lymphocytic infiltration and, by immunofluorescence, IgG deposits at the epidermal-dermal basement membrane area. A diagriosis of systemic lupus erythematosus was made. The patient was taking salicylates and her arthritis had improved markedly by the time she was discharged. * M d t w i n with systemic lupus erythematosus.
CASE REPORT 2 MC, the 44-year-old monozygotic twin of ML, was well until the age of 21 when she developed vaginal bleeding. A pelvic examination revealed a right adnexal mass. A biologic false-positive test for syphilis was found. An adenocarcinoma of the right ovary, metastatic to the uterus, required an hysterectomy, bilateral oophorectomy, and salpingectomy. She was followed for 23 years during which time she did not receive ovarian hormone replacement and the tumor did not recur. In January 1972 she and her sister were admitted to the Clinical Research Center. She had no complaints, and physical examination revealed slight hirsuitism of the upper lip and pubic region and a more muscular physique than her twin. Blood pressure was 150/100 mm Hg. Mild arteriolar narrowing on funduscopic examination and a presystolic gallop without cardiomegaly were noted. The remainder of the physical examination and the CBC, urinalysis, BUN, and creatinine clearance were normal. Chest xray revealed mild osteoporosis and a normal size heart; the electrocardiogram revealed a mild left ventricular hypertrophy.
MATERIALS AND METHODS Laboratory Data. Leukocyte chemotaxis was performed by Baum, as previously described (12). Follide-stimulating hormone (FSH) and luteinizing hormone (LH) were determined by radioimmunoassay. Immunoglobulin levels were determined by quantitative radial immunodiffusion. Antibodies to native and heat-denatured deoxyribonucleic acid were measured by a modification of the method of Pincus et a1 (13). Other antitissue and antiorganelle antibody titers were performed by hemagglutination or the indirect immunoflourescence techniques. Serologic Studies. Tetanus and diphtheria titers were determined by hemagglutination on serum obtained 10 days after the administration of tetanus toxoid. Keyhole lympet hemocyanin (KLH) titers were performed by hemagglutination. Influenza A, Hong Kong titers were determined by viral neutralization. Other viral titers were performed by standard complement fixation assays. Delayed Hypersensitivity. The following antigens were used for skin testing: trichophyton 1 :500, monilia 1:500, mosquito 1000 PNU/ml (Hollister-Stier), mumps (Lilly), staphylococcal toxoid dilution 1, streptokinase 10 units/ml and streptodornase 2.5 units/ml (Lederle), and intermediate strength PPD (MSD). Dinitrochlorobenzene sensitization was performed as described by Catalona et a1 (14) and mixed lymphocyte cultures and phytohemagglutinin stimulation by the technique of Bach et al (15,16). Monozygotic Data. Lymphocytes of each patient were tested for HL-A 1, 2, 3, 5, 7, 8, 9, 10, 12, and 13 with a total of 40 antisera by means of the standard NIH microlymphocytotoxicity method. Gamma Globulin Metabolism. Gamma globulin and light chain metabolism were studied in detail, as previously described (1 7). DEAE-purified IgC, obtained as Cohn fraction 11, and light chains obtained from this preparation
195
MONOZYGOTIC TWINS DISCORDANT FOR SLE
Table 3. Autoantibodies
Table 1. Monozygosity Data
Blood type ABO Rhesus Lewis MNSs Duffy Kidd Kell HL-A-type Mixed lymphocyte culture
M L*
MC
AB R/r DCce LeaNSs Fy" Fyb JK8 JKb K KPb JSa23.12 Negative for 7,8,9,13 No transformation with MC lymphocytes Transformation with other donor lymphocytes
AB R/r DCce LeaNSs Fy" Fyb JK' JKb K KPb JS2.3.12 Negative for 7,8,9,13 No transformation with ML lymphocytes Transformation with other donor lymphocytes
ML*
*Twin with systemic lupus erythematosus. were utilized for turnover studies. T h e proteins were radiolabeled, as described by Bale et a1 (18), freed of aggregates a n d subjected to ultrafiltration t o ensure sterility. Approximately 1 mg of IgGI-131 a n d L chain'-125 containing 5 x 10s cpm of specific activity was simultaneously injected intravenously. Blood samples were obtained a t frequent intervals beginning within 2 minutes of injection and for 30 days after administration. Serum IgC a n d L-chain levels were determined by radial immunodiffusion a n d complement fixation assays, respectively (17).
RESULTS Monozygosity Data. These twins appeared monozygotic and their blood typing as well as HL-A
VDRL titer FTA-ABS LE prep ANA titer-pattern
Normal Values ML*
4
Neat 2+ rim 1:lO 1+ 1:lOO -
Neat 14- speckled
-
1:80 1:5
-
-
-
-
-
1:40
-
-
-
1:40
-
-
-
24% binding
4% binding
17% binding
2% binding
typing was identical (Table 1). Neither twin's lymphocytes transformed when cultured with the other's but they did transform when cultured with nonrelated donor lymphocytes. Laboratory Data. As shown in Table 2, both patients had an elevated erythrocyte sedimentation rate. Twin MC showed evidence of menopause with an elevated FSH, and her IgM was markedly elevated but polydispersed on agar zone electrophoresis. Autoantibodies are compared in Table 5. A
Table 4. Serologic Studies MC
20-30
Normal 13.1 6,400 200,000 52
Normal 13.8 6,300 200,000 68
(> 450) 10-20 0.8-1.2 80-140 70-100 2-7 3.5-14 6-16 600-1,700 50-250 50-200 > 80
1,252 19 0.8 104 80 3.1 6.9 4.5 1,365 58 275 80
1,400 20 0.8 111 ND 5.4 21.6 7.7 990 62 414 111
12.0-15.4 4,000-1 1,000 > 150,000
-
+Twin with systemic lupus erythematosus.
Test ~
Urinalysis Hemoglobin (g%) WBC/mma Platelets/mms Westergren sedimentation (mm/ hr) WBC chemotaxis index @%Yo) Creatinine (mgyo) Ccr (ml/min) Prothrombin time (yo) Uric acid (mgyo) FSH (mpg/ml) LH (mpg/ml) I& (mg%) IgA (mg%) IgM (mg%) CH, units
4
-
+
ANP latex titer Anti-IgG titer Antithyroglobulin titer Antismooth muscle titer Antiautochondrial titer Antihuman ovary titer Antithyroid microsome titer Antigastric mucosa titer Antiesophageal mucosa titer Coombs gamma and nongamma Antinative DNA (normal < 10%) Antiheat denatured DNA (normal < 8y0)
Table 2. Laboratory Data Test
MC
~
_
_
ML*
MC
1:20,480 1:160
1:40,960 1:80
1:4, 1:s 1:256 1: 128 1:128
1:2, 1:4 1:64, 1:128 1 :32 132
1:16
1:64
< 1:8 < 1:8
c 1:s
_
Hemagglutination titers Tetanus (10 days after booster) Diphtheria Keyhole lympet hemocyanin Baseline 10 days 20 days 30 days KLH delayed skin test Viral neutralization titers Influenza A, H K Complement fixation titers Mumps Rubeola Parainfluenza 1,2, 3 Respiratory syncytial virus Herpes simplex Cytomegalovirus
1:40 1:lO < 1:4 1: 16
~~
*Twin with systemic lupus erythematosus; ND = not done.
*Twin with systemic lupus erythematosus.
C 1:8
1:5
> 1:40 1:16
C 1:4
YOCUM E T AL
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Table 5. Delayed Hypersensitivity MLf Mumps Trichophyton Monilia Streptokinase-streptodornase Staph toxoid Mosquito IPPD DNCB PHA
15 X 15 mmf 0 30 X 30 mmf 7 mmt 0 0
MC
10
X
30
X
0
3+ > 71,000 cpm
>
10mmt 0 30 mrnf 0 0 0
L chains were strikingly different. ML had serum kappa and lambda L-chain levels of 28 and 6 ,N/ml, respectively; the unaffected twin, MC, had normal levels of 5.6 (kappa) and 2.2 (lambda). Thus, the calculated production of L chains was 4.51 g per day in ML, a figure about five times greater than the normal production rate of 0.875 g per day in MC.
0 4+ 26,000 cpm
*Twin with systemic lupus erythematosus. tMillimeters of induration at 48 hr.
positive VDRL test in low titer with a negative fluorescent treponemal antibody absorption (FTA-ABS) test was noted in both. T h e affected twin, ML, had a positive LE cell preparation and persistent and significant rim-positive antinuclear antibody (ANA) test. Of six ANA tests in the unaffected twin, only one was positive in low titer, with a speckled pattern. ML had weakly positive titers of anti-IgG and antithyroglobulin and significant titers of antihuman ovary and antigastric mucosa antibodies. Serum from ML also bound 24% native DNA and 17% single-stranded DNA. Serologic Studies. Table 4 shows that tetanus toxoid immunization and keyhole lympet hemocyanin (KLH) sensitization resulted in a normal production of hemagglutinating antibody in both twins. Neither exhibited a delayed skin test to KLH, but each was not rechallenged with antigen after 30 days. Parainfluenza antibody titers were elevated in twin M L and respiratory syncytial virus titers were elevated in MC. Evidence of past infection with cytomegalovirus in ML and with herpes simplex virus in MC was found. Assessment of delayed hypersensitivity (Table 5) did not reveal significant differences. Both M L and MC had positive delayed skin tests to mumps and monilia, were able to be sensitized to dinitrochlorobenzene, and were able to respond to phytohemagglutinin stimulation. Gamma Globulin Metabolism. Labeled IgG globulin decay curves showed a rapid final disappearance in ML (t,,2 = 9 days), and a normal rate (tl,2 = 15 days) in MC. T h e more rapid decay rate and slightly higher IgG level of M L indicate an increased disappearance rate of IgG globulin, confirming previous data (19). T h e labeled (L) chain decay curves of the two subjects were similar and within our normal range, but absolute levels of free serum kappa and lambda
DISCUSSION Cited reports of an increased incidence of autoantibodies and rheumatic diseases in relatives of patients with SLE, plus an increased frequency of SLE within families, and SLE in monozygotic twins, imply a significant genetic predisposition toward the development of this disease. Recent etiologic hypotheses for SLE suggest that, given a predisposition, an environmental factor, such as a drug or a viral infection, may elicit manifest disease (20,21). T h e study of monozygotic twins discordant for SLE thus provides a unique opportunity to study environmental influences that might explain the development of disease in only one of two individuals of identical genotype. Of the three published reports of monozygotic twins discordant for SLE, only in the case report by Brunner (1 1) is the identity of the twins documented as well as ours. By studying the unaffected twin, we hoped to separate those abnormalities predating the onset of SLE. I n addition, it was postulated that the immune response of the twin with definite but mild SLE would not be markedly suppressed, as can occur in severe systemic disease or renal failure. T h e biologically false-positive serologic test for syphilis (BFP-STS) in both patients and the one faintly positive ANA test in the healthy twin suggest that both had the potential to develop SLE. Hereditary BFP-STS (22) was ruled out by a negative Wasserman test in the parents and a brother of the twins. Previous reports describing the immune response i n patients with SLE have shown, in studies with matched controls, similar primary and secondary responses to tetanus toxoid (23), an increased 7s but decreased 19s antibody response to bacterial antigens (24), and, i n monozygotic twins discordant for SLE, a higher antibody response to KLH protein in the twin with SLE (11). These twins both developed an adequate secondary response to tetanus toxoid, and ML developed a higher and more sustained antibody titer to KLH immunization. Because of the reports of a viral illness preced-
MONOZYGOTIC TWINS DISCORDANT FOR SLE
ing the onset of SLE (1,25), viral-like particles in tissues from patients with SLE (26), and the elevated viral antibody titers in patients with SLE (21,25), this parameter was also tested. T h e only difference observed was increased antibody titers to parainfluenza 1, 2, 3, in ML. Several reports (1 1,2327) have demonstrated decreased cell-mediated immunity in patients with SLE, as evaluated by delayed skin test antigens and, as this may predispose to viral infection, we studied this aspect of the immune response. No differences in response to seven delayed skin test antigens, DNCB sensitization, PHA stimulation, or mixed lymphocyte cultures were demonstrated in these twins. I n several reports demonstrating decreased delayed skin tests in SLE, patients acutely ill with SLE and frequently on corticosteroids were compared with healthy normal adults. I n a study performed by skin testing patients on a general medical ward, we noted a n absence of positive delayed skin tests in 387, (28). I t is therefore important that in all studies evaluating delayed immunity the effect of severe illness be considered. Certain murine diseases have been linked to the H-2 histocompatibility locus, and an increased incidence of HL-A antigens has been noted in humans with SLE (3). I n these twins a n identical genotype was demonstrated; therefore, the discordance for SLE cannot be related solely to the immune response genes. Increased IgG turnover has been reported in SLE (19). Two known causes for an increased disappearance rate of IgG are excessive gastrointestinal loss (29) and an elevated serum IgG level. I n ML a high serum level was not a factor, but some intestinal loss cannot be ruled out. Elevated serum L chains have been previously described in active SLE (39). A recent report suggests that increased urinary excretion of free L chains indicates activity of the disease process (31) and is a manifestation of tubular disease and inability to catabolize L chains. Because no trichloroacetic acid precipitable radioactivity was noted in either ML or MC, free urinary L chains were not measured. T h e high production rate of 4.5 g of L chain per day in ML is similar to that in multiple myeloma and implies a well-preserved catabolic rate. It is clear that L-chain production in SLE is not tightly coupled to IgG synthesis and a normal feedback control seems inoperative as in multiple myeloma. Of all patients with SLE, 90% are female and usually of childbearing age. However, the etiologic
197
or permissive role of ovarian hormones in this disease is controversial. Nonetheless, certain facts are known that associate SLE with ovarian hormones and must be explained by any theory of the pathogenesis of this disease. Walker and Bole (32) studying NZB/NZW mice showed that mesiranol accelerated the development of ANA but not autoimmune nephritis in male mice and that neither mestranol nor early oophorectomy had an observable effect on these parameters in female mice. Studies of patients with SLE who become pregnant have demonstrated a n exacerbation of disease activity most frequently during the postpartum period (33,34). SLE patients may exacerbate if given oral contraceptives, and recently a report of normal females developing a n SLE-like syndrome while using contraceptive pills has appeared (2). T h e pregnant state, or ovarian or placental hormones, can depress cell-mediated immunity. Andresen and Monroe (35) found that both the first and second set skin homograft rejection times were prolonged by a factor of 2 in pregnant women during the third trimester compared to nonpregnant age-matched controls. Purtilo (36) found that lymphocyte response to PHA is decreased at all stages of pregnancy but most markedly during the third trimester and postpartum period. Munroe (37) found that newborn monkeys, monkeys pretreated with progesteroids, and pregnant monkeys were all highly susceptible to Rous chicken sarcoma virus infection, while normal juvenile and adult monkeys were highly resistant to this virus. He further demonstrated that intravenous progesterone decreased the number of circulating lymphocytes and prolonged skin graft rejection. I t was concluded that progesteroids significantly decreased cell-mediated immunity and resistance to viral infection. Thus, there is evidence that pregnancy or ovarian hormones may exacerbate existing SLE, create an SLE-like syndrome, depress cell-mediated immunity, and increase susceptibility to viral infection. This present report of monozygotic twins discordant for SLE reveals the following data. Both patients demonstrated an abnormal immune response at an early age as manifested by a BFP-STS and a positive ANA in later life which to us suggests that both had the genetic potential to develop SLE. T h e subsequently unaffected twin, MC, underwent surgical castration at 21 years of age without ovarian hormone replacement, while the affected twin, ML, developed mild but definite SLE with an immune response not blunted by uremia or severe systemic disease activity.
YOCUM ET AL
Humoral and cellular i m m u n e responses a n d viral antibody titers revealed no striking differences, a fact suggesting that their exposure to the viruses studied was quite similar. T h e antibodies to DNA, antitissue antibodies, decreased half life of IgG globulin, and increased synthesis of L chains in the affected twin, ML, can be explained by her SLE. The major difference between this twin pair that cannot be explained by SLE was therefore the absence of ovarian hormones i n the unaffected twin. T h i s difference raises the question as to whether ovarian hormones may allow environmental factors, such as viral infection, to induce development of SLE and, we hope, stimulates further investigation i n the role of ovarian hormones in SLE.
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Intern Med 75:951-958, 1971 2. Bole GG Jr, Friedlaender MH, Smith CK: Rheumatic symptoms and serological abnormalities induced by oral contraceptives. Lancet 1:323-328, 1969 3. Grumet FC, Coukell A, Bodmer JG, et al: Histocompatibility (HL-A) antigens associated with systemic lupus erythematosus. N Engl J Med 285:193-196, 1971 4. Rowel1 NR: Etiology of certain connective tissue diseases. Clin Exp Immunol 2:813, 1967 5. Leonhardt ETG: Family studies in systemic lupus erythematosus. Clin Exp Immunol 2:743-759, 1967 6. Morteo OG, Franklin EC, McEwen C, et al: Studies of relatives of patients with systemic lupus erythematosus. Arthritis Rheum 4:356-363, 1961 7. Davis MW, Gutridge GN: Disseminated lupus erythematosus in identical twin sisters associated with diabetes mellitus in one case. J Missouri Med Assoc 48: 446450, 1951 8. Joseph RR, Zarafonetis CJD: Fatal systemic lupus erythematosus in identical twins: case reports and review of the literature. Am J Med Sci 249:190-199, 1965 9. Holmes FF. Stubbs DW, Larsen WE: Systemic lupus erythematosus and multiple sclerosis in identical twins. Arch Intern Med 119:302-304, 1967 10. Larsson 0, Leonhardt T: Hereditary hypergammaglobulinemia and systemic lupus erythematosus. I. Acta Med Scand 165: 371-393, 1959 11. Brunner CM, Horwitz DA, Shann MG, et al: Clinical and immunologic studies in identical twins discordant for systemic lupus erythematosus. Am J Med 55:249254. 1973 12. Baum J, Mowat AG, Kirk JA: A simplified method for the measurement of chemotaxis of polymorphonudear leukocytes from human blood. J Lab Clin Med 77: 501-509, 1971
13. Pincus T, Schur PH, Rose JA, et al: Measurement of serum DNA-binding activity in systemic lupus erythematosus. N Engl J Med 281:701-705, 1969 14. Catalona WJ, Taylor PT, Rabson AS, et al: A method for dinitrochlorobenzene contact sensitization. N Engl J Med 286:39H02, 1972 15. Bach FH, Voynow NK: One-way stimulation in mixed leukocyte cultures. Science 153:545-547, 1966 16. Bach FH, Hirschhorn K: T h e in vitro response of penpheral blood lymphocytes. Semin Hematol 2:68, 1965 17. Waterhouse C, Abraham G, Vaughan J: The relationship between L-chain synthesis and gamma globulin production. J Clin Invest 52: 1067-1077, 1973 18. Bale WF, Helmkamp RW, Davis TP, et al: High specific activity labeling of protein with 1131 by the iodine monochloride method. Proc SOCExp Biol Med 122:407414, 1966 19. Birke G, Liljedahl SO, Olhagen B, et al: Catabolism and distribution of gamma globulin. A preliminary study with 131I-labeled gamma globulin. Acta Med Scand 173:589-603, 1963 20. Blomgren SE, Condemi JJ, Vaughan JH: Procainamide induced lupus erythematosus. Am J Med 52338-348, 1972 21. Hollinger FB, Sharp JT, Lidsky MD, et al: Antibodies to viral antigens in systemic lupus erythematosus. Arthritis Rheum 14:l-11, 1971 22. Kostant GH: Familial chronic biologic false positive seroreactions for syphilis. JAMA 219:45-48, 1972. 23. Abe T , Homma M: Immunological reactivity in patients with systemic lupus erythematosus. Acta Rheumatol Scand 17:35-46, 1971 24. Baum J, Ziff M: Decreased 19s antibody response to bacterial antigens in systemic lupus erythematosus. J Clin Invest 48:758-767, 1969 25. Koivisto 0, Sotaniemi E, Vesikari T: Recurrent virus infections in identical twins with systemic lupus erythematosus. Acta Rheumatol Scand 17304-311, 1971 26. Fresco R: Tubular (myxovirus-like) structures in glomerular deposits from a case of lupus nephritis. Fed Proc 27:246, 1968 (abstr) 27. Horwitz DA: Impaired delayed hypersensitivity in systemic lupus erythematosus. Arthritis Rheum 15:353359, 1972 28. Grossman J, Baum J, Gluckman J, et al: The effect of aging and acute illness on delayed hypersensitivity. J Allergy Clin Immunol 51:127, 1973 (abstr) 29. Waldmann TA, Broder S, Strober W: Protein-losing enteropathies in malignancy. Ann NY Acad Sci 230: 306-317, 1974 30. Epstein WV, T a n M: Increase of L-chain proteins in the sera of patients with systemic lupus erythematosus and the synovial fluids of patients with peripheral rheumatoid arthritis. Arthritis Rheum 9:713-719, 1966 31. Epstein WV: Immunologic events preceding clinical
MONOZYGOTIC TWINS DISCORDANT FOR SLE
exacerabation of systemic lupus erythematosus. Am J Med 54:631-636, 1973 32. Walker SE, Bole GG Jr: Influence of natural and synthetic estrogens on the course of autoimmune disease in the NZB/NZW mouse. Arthritis Rheum 16:231-239, 1973 33. Garsenstein M, Pollak VE, Kark RM: Systemic lupus erythematosus and pregnancy. N Engl J Med 267:165169, 1962 34. McGee CD, Makowski EL: Systemic lupus erythema-
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tosus in pregnancy. Am J Obstet Gynecol 107:10081012, 1970 35. Andresen RH, Monroe CW: Experimental study of the behavior of adult human skin homografts during pregnancy. Am J Obstet Gynecol 83:1096-1101, 1962 36. Purtilo DT, Hallgren HM, Yunis EJ: Depressed maternal lymphocyte response to phytohaemagglutinin in human pregnancy. Lancet 1:769-771, 1972 37. Munroe JS: Progesteroids as immunosuppressive agents. J Reticuloendothel SOC9561-375, 1971
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