AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 7, 1992 Mary Ann Liebert, Inc., Publishers
Detection of HIV-1 Genome in Leukocytes of Human Colostrum from Anti-HIV-1 Seropositive Mothers NICOLETTA VONESCH,1 ELENA STURCHIO,1 ANDREA CASTIGLIONE HUMANI,3 PAOLA CORDIALI FEI,2 DANILA COSENZA,1 FEDERICO CAPRILLI,2 MARIO PEZZELLA'
ABSTRACT In order to obtain more information about the presence of HIV-1 in mononuclear cells of colostrum, research was carried out on both the HIV-1 genome in the cellular fraction of colostrum and the viral antibody in cell-free colostrum of eight anti-HIV-1 seropositive asymptomatic mothers. In five cases cell fractions of the colostrum harbored HIV-1 genome by DNA-DNA and DNA-RNA in situ hybridization, whereas viral antibody were detected in all cell-free colostrum specimens. The data confirms the colostrum as a possible route of HIV-1 infection.
INTRODUCTION
SEVERAL hypothesis breastfeeding
EP1DEMIOLOGICAL AND clinical observations support the that breast milk can be a vehicle for HIV-1 is an unusual and not predom¬ infection. Although inant way of HIV-1 transmission, several cases have been reported of breastfed babies infected by mothers who became ' infected after delivery via postpartum blood transfusion. This is a problem in both public health and medical ethics especially in developing countries where breastfeeding may have the greatest benefit, and safe and effective alternatives cannot be used. The aim of our investigation was to detect HIV-1 genome in cellular fraction of colostrum from eight anti-HIV-1 seropositive mothers. Moreover, we carried out research of viral antibody in both serum and cell-free colostrum samples of all infected mothers.
MATERIALS AND METHODS
defined according to the Centers for Disease Control (CDC) surveillance criteria. Their regular sexual partners were also anti-HIV-1 seropositive.
Cells In order to eliminate as much as possible the fatty substances of colostrum samples, which could have interfered in both cell separation and in hybridization reaction, colostrum samples were diluted 1:2 with phosphate-buffered saline solution. Mono¬ nuclear cells were then separated by Ficoll-Hypaque gradient centrifugation. The cells were removed at the serum-density gradient interface, washed three times in RPMI-1640 and resuspended at a concentration not exceeding 1 X 106/ml. Sam¬ ples of 105 cells were cytocentrifuged on to microscope slides, air-dried, fixed in acetone for 5 minutes, and then stored at 20°C until used for hybridization. Under the above operative conditions, fatty substances were always found on the slides, although never in any amount great enough to interfere with the hybridization reaction. HIV-1-infected and uninfected MT4 cells and HIV-1-infected and uninfected H9 T-cell culture lines were cytocentrifuged and fixed as described above. —
Patients From October 1988 to May 1990 we selected and included in study 8 anti-HIV-1 seropositive asymptomatic women (ELISA test confirmed by Western blot). The clinical status was
our
Serological analysis The presence of antibody to HIV-1 was investigated in serum and cell-free fraction of colostrum specimens by ELISA test
'Institute of Infectious Diseases, University of Rome "La Sapienza", Rome, Italy.
2Dermosyphilopatic Institute S. Maria and S. Gallicano Rome. Italy. 3Nuovo Regina Margherita Hospital, Rome, Italy.
1283
1284
VONESCH ET AL.
FIG. 1. (A) lane 10: Serum from uninfected mother; 20: serum HIV-1 positive control; 5: blood serum from the infected mother; 6: blood serum from the newborn; 9: cell-free fraction of colostrum from the infected mothers; 1 : cell-free fraction of colostrum from healthy mother; 8: cell-free fraction of colostrum infected with HIV-1. (B) lane 29: Blood serum from the infected mother; 20: blood serum from the newborn; 34: cell-free fraction of colostrum from the infected mothers.
(ELAVIA Ac-Ab-Ak-I Pasteur). The antibody
to HIV-1 were
confirmed by Western blot (LAV BLOT I Pasteur).
In situ The
hybridization hybridization
reaction
was
carried out
described.2 Briefly, all the cytocentrifuged cells
probes: SP64 plasmid containing the
as
previously hybrid¬
were
ized with two
1. The 9 kb Sstl-SstI viral insert from lambda BH 10 recombinant clone which corresponds to almost the entire genome and lacks only 180 base pairs of the 5'long terminal repeat (LTR) 2. The pBR322 plasmid lacking viral insert ·
samples were also hybridized with the hybridization mixture lacking probes. Moreover, an inhibition hybridization reaction was performed with non labelled probes. The heat-denaturated DNA probes were labelled by inserting an antigenic sulfone group into cytosine moieties (Chemiprobe, Orgenics). The visualization reaction was performed by a double antibody system using a monoclonal mouse anti-sulfone group antibody and then an alkaline phosphatase-conjugated anti-mouse antibody. Nitroblue tetrazolium containing 5-bromo-4-chloro-3 indolylphosphate was the chromogenic All
substrate used. The specificity of the hybridization reaction positive and negative controls.
was
proved by
HIV-1 GENOME IN CELL FRACTION OF HUMAN COLOSTRUM
1285
FIG. 2. (A) Leukocytes from colostrum of healthy mother treated with hybridization mixture containing pBR322 plasmid lacking viral insert (negative control). All cells are unstained (arrows) (X500). (B, C, D) Cell fraction of colostrum from HIV-1-infected mothers hybridized with the SP64 plasmid containing HIVBH10 viral insert. A strong intracytoplasmatic well-defined granular staining indicates the presence of HIV-1 virus (arrows). In one cell (C, arrow) nuclear-positive granules are also demonstrated (B,
x500;C, D X1000).
Positive controls: Cytocentrifuged H9 T-cell culture lines infected with HIV-1 and MT4 cells 3 h after infection with HIV-1 hybridized with SP64 plasmid containing HIVBH10 viral insert.
Negative controls: HIV-1-infected H9 T-cell line hybridized with the pBR322 plasmid lacking viral insert Uninfected MT4 and H9 T-cell lines hybridized with the two
probes Peripheral blood mononuclear cells from six negative selected blood donors hybridized with the two probes Leukocytes obtained by colostrum from two healthy women RESULTS The cell-free fraction of colostrum specimens and blood samples from all the infected mothers and their newborns
serum
showed substantially the same pattern of positivity for antiHIV-1 antibody at Western blot. Figures 1 and 2 showed a pattern of reactivity of blood serum from infected mothers, their newborns. and cell-free fraction of colostrum specimens. The cell-free fraction of colostrum spec¬ imens studied showed different reactivity at Western blot. Actually lane 9 (Fig. 1A) shows low reactivity, whereas lane 34 (Fig. IB) shows high reactivity. Negative and positive controls of the serum are shown in Figure 1A (Lanes 10 and 20, respectively). Colostrum from healthy mothers and colostrum infected with HIV-1 are dis¬ played in Figure 1A (lanes 1 and 8). The specificity of the in situ hybridization reaction was indicated by positive and negative controls. Both cytocentri¬ fuged H9 T-cell culture lines and MT4 cells infected with HIV-1 in situ hybridized with the SP64 plasmid containing the HIVBH10 viral insert showed a strong intracytoplasmic granu¬ lar staining. Actually in MT4 cells three hours after infection viral DNA appears only in the cytoplasm.5 Peripheral blood
1286
VONESCH ET AL.
mononuclear cells from healthy blood donors, uninfected H9 T-cell culture lines, and MT4 cells were consistently negative, irrespective of the probes used. A negative pattern was also observed in HIV-1 chronically infected H9 T-cell culture lines and MT4 cells 3 h after infection when treated with: (a) the hybridization mixture lacking probe, (b) the pBR322 plasmid lacking viral insert, (c) the SP64 plasmid containing the lambda BHIO viral insert after inhibition reaction performed with non labelled probes. A negative pattern was observed in the leukocytes from the colostrum samples from the two healthy mothers and from 3 out of 8 colostrum specimens of HIV-1-infected mothers. A strong intracytoplasmic granular staining was visualized in the cyto¬ plasm of mononuclear cells in 5 out of 8 cell fractions of colostrum from seropositive mothers (Fig. 2 A.B.C.D). More¬ over, the signal intensity was different from cell to cell. In some cells a strong staining reaction was found suggesting the pres¬ ence of many viral genome copies, whereas in other cells there was only a very low signal intensity.
samples. In developed countries seropositive mothers should be counselled about possible risk of HIV-1 transmission and should be discouraged from breastfeeding, in order to avoid postnatal transmission to a child who may not yet be infected. Seronega¬ tive at high-risk women (i.e., drug addicts, prostitutes, regular sexual partners of seropositive subjects)14 should also be dis¬ couraged from breastfeeding as they could still be seroconverting. During silent infection they may represent a potential source of transmission of HIV-1 infection, even though the additional risk of breastfeeding for infants is not clear. ' Two strategies are available to reduce contamination of banked breast milk: (a) sensible selection of donors especially in areas where the prevalence of HIV-1 infection is known to be high; (b) pasteur¬ ization to inactivate the virus.'5'7 In developing countries, where alternative means cannot safely and effectively be used, breastfeeding should continue to be supported, protected, and encouraged for all children. ' ACKNOWLEDGMENTS
DISCUSSION
Epidemiological and pothesis that the HIV-1
clinical observations support the hy¬ infection could be transmitted through breastfeeding/breast milk, although such a route is of small importance, in terms of absolute numbers, when compared with in utero and intrapartum transmission. The detection of HI V-1 in leukocytes of human colostrum was not completely unexpected, as HIV-1 has already been isolated from cell-free fraction of colostrum, vaginal secretions, saliva, tears, urine, cerebrospinal fluid, and alveolar fluid.6~x The maternal infant transmission rate of HIV-1 in breastfed subjects is comparable with that in non-breastfed subjects. This suggests that although the viral genome can be detected in colostrum from HIV-1 seropositive mothers, breastfeeding may not be a major route of maternalinfant transmission. Actually, the risk of perinatal infection is substantially greater when the infected mother has symptoms of HIV-1 infection or is in an advanced stage of disease in
pregnancy.9 Studies performed on breastfed infants—whose mothers were infected postpartum and without risk factors other than breast milk—showed that HIV-1 transmission via breastfeeding may be a small and identifiable mode of transmitting infection. I0~12 Van de Perre et al. found colostrum and breast milk as a route for the transmission of HIV-1 infection. Actually, they proved recently infected postnatal transmission of HIV-1 infection from '3 mothers to their infants by breastfeeding. Our probe lambda BHIO is able to recognize both mRNA and proviral DNA. However, since our aim was not to discriminate between DNA and mRNA, we have focused our research only on the detection of HIV-1 virus in leukocytes of colostrum. Moreover, the in situ hybridization testing is unique because it reflects the distribution of HIV-1 genome within cells. The presence of viral antibody in cell-free fraction of colostrum does not necessarily mean that its mononuclear cell fraction harbors HIV-1 virus. Actually, by Western blot we have found patterns of positivity in both blood serum and in cell-free colostrum specimens in all the mothers studied, whereas HIV-1 genome was present only in 5 out of 8 cellular fractions of colostrum
This work was supported by Ministero Pubblica Istruzione, Fondi Ricerca Scientifica (MP) 1989 and Ministero SanitàIstituto Superiore Sanità, III Progetto AIDS. We are grateful to Mr. V. Panosetti and E. Crescimbeni for excellent technical assistance.
REFERENCES 1. 2.
3.
4.
5.
6. 7.
Logan S. Newell ML, Ades T, and Peckham CS: Breast-feeding and HIV infection. Lancet 1988; 1:1346. Pezzella M, Pezzella F, Galli C, Macchi B, Verani P, Sorice F, and Baroni CD: In situ hybridization of human immunodeficiency virus (HTLV-III) in cryostat sections of lymph nodes of lymphadenopathy syndrome patients. J Med Virol 1987;22:135-142. Hahn BH, Shaw GM, Arya SK, Popovic M, Gallo RC, and Wong-Staal F: Molecular cloning and characterization of the HTLV-III virus associated with AIDS. Nature 1984;312:166-169. Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC, and Wong-Staal F: Molecular characterization of human T-cell leuke¬ mia (lymphotropic) virus type III in the acquired immune defi¬ ciency syndrome. Science 1984;226:1165-1171. Stevenson M, Haggerty S. Lemonica CA, Meier CM, Welch SK, and Wasiak A: Integration is not necessary for expression of human immunodeficiency virus type 1 protein products. J Virol 1990; 64:2421-2425. Friedland GH and Klein RS: Transmission of the human immuno¬ deficiency virus. Engl J Med 1987;317:1125-1135. Ziegler JB, Cooper DA, and Gold J: Postnatal transmission of AIDS associated retrovirus from mother to infant. Lancet 1985;1:896-899.
8.
Thiry L. Sprecher-Goldberger S, Jonckheer T, Levy J, Van De Perre , Henrivaux P. Cogniaux-LeClerc J, and Clumeck N: Isolation of AIDS virus from cell-free breast milk of three healthy
virus carriers. Lancet 1985;2:891-892. 9. Mok JQ, Giaquinto C, De Rossi A, Grosch-Worner I, Ades AE, and Peckhan CS: Infants born to mothers seropositive for human immunodeficiency virus. Lancet 1987;1:1164-1168. 10. Weinbreck F, Loustand V, Denis F, Viudal B, Mouinier M, and DeLumley L: Postnatal transmission of HIV infection. Lancet 1988:1:492.
HIV-1 GENOME IN CELL FRACTION OF HUMAN COLOSTRUM 11. Colebunders R, Kapita B, Nekwei W, Bahwe Y, Lebughe I, Oxtoby M, and Ryder R: Breastfeeding and transmission of HIV. Lancet 1988:2:1487. 12. Canosa CA: SIDA pediatrico. Rev Esp Pediatyr 1989;45:1-25. 13. Van de Perre P. Simonon A, Msellati P. Hitimana D-G, Vaira D, Bazubagira A, Van Goethem C. Stevens -M, Karita E. SondagThull D, Dabis F. and Lepage P: Postnatal transmission of human immunodeficiency virus type 1 from mother to infant. Engl J Med 1991;325:593-598. 14 Pezzella M. Rossi P. Lombardi V, Gemelli V. Mariani Costantini R. Mirólo M. Fundarò C, Moschese V, and Wigzell H: HIV viral sequences in seronegative people at risk detected by in situ hybrid¬ ization and polymerase chain reaction. Br Med J 1989:298:713716.
1287
15. Lucas A: AIDS and human milk bank closures. Lancet 1987:1:1092-1093. 16. Mortimer and Cooke AM: HIV infection, breastfeeding, and human milk banking. Lancet 1988:2:452-453. 17. Eglin RP and Wilkison AR: HIV infection and pasteurization of breastmilk. Lancet 1987; 1:1903.
Address
reprint requests to: Prof. Mario Pezzella
Institute
Infectious
Diseases Policlinico Umberto I University "La Sapienza" 00161 Rome, Italy
Detection of HIV-1 Genome in Leukocytes of Human Colostrum from Anti-HIV-1 Seropositive Mothers NICOLETTA VONESCH,1 ELENA STURCHIO,1 ANDREA CASTIGLIONE HUMANI,3 PAOLA CORDIALI FEI,2 DANILA COSENZA,1 FEDERICO CAPRILLI,2 MARIO PEZZELLA'
ABSTRACT In order to obtain more information about the presence of HIV-1 in mononuclear cells of colostrum, research was carried out on both the HIV-1 genome in the cellular fraction of colostrum and the viral antibody in cell-free colostrum of eight anti-HIV-1 seropositive asymptomatic mothers. In five cases cell fractions of the colostrum harbored HIV-1 genome by DNA-DNA and DNA-RNA in situ hybridization, whereas viral antibody were detected in all cell-free colostrum specimens. The data confirms the colostrum as a possible route of HIV-1 infection.
INTRODUCTION
SEVERAL hypothesis breastfeeding
EP1DEMIOLOGICAL AND clinical observations support the that breast milk can be a vehicle for HIV-1 is an unusual and not predom¬ infection. Although inant way of HIV-1 transmission, several cases have been reported of breastfed babies infected by mothers who became ' infected after delivery via postpartum blood transfusion. This is a problem in both public health and medical ethics especially in developing countries where breastfeeding may have the greatest benefit, and safe and effective alternatives cannot be used. The aim of our investigation was to detect HIV-1 genome in cellular fraction of colostrum from eight anti-HIV-1 seropositive mothers. Moreover, we carried out research of viral antibody in both serum and cell-free colostrum samples of all infected mothers.
MATERIALS AND METHODS
defined according to the Centers for Disease Control (CDC) surveillance criteria. Their regular sexual partners were also anti-HIV-1 seropositive.
Cells In order to eliminate as much as possible the fatty substances of colostrum samples, which could have interfered in both cell separation and in hybridization reaction, colostrum samples were diluted 1:2 with phosphate-buffered saline solution. Mono¬ nuclear cells were then separated by Ficoll-Hypaque gradient centrifugation. The cells were removed at the serum-density gradient interface, washed three times in RPMI-1640 and resuspended at a concentration not exceeding 1 X 106/ml. Sam¬ ples of 105 cells were cytocentrifuged on to microscope slides, air-dried, fixed in acetone for 5 minutes, and then stored at 20°C until used for hybridization. Under the above operative conditions, fatty substances were always found on the slides, although never in any amount great enough to interfere with the hybridization reaction. HIV-1-infected and uninfected MT4 cells and HIV-1-infected and uninfected H9 T-cell culture lines were cytocentrifuged and fixed as described above. —
Patients From October 1988 to May 1990 we selected and included in study 8 anti-HIV-1 seropositive asymptomatic women (ELISA test confirmed by Western blot). The clinical status was
our
Serological analysis The presence of antibody to HIV-1 was investigated in serum and cell-free fraction of colostrum specimens by ELISA test
'Institute of Infectious Diseases, University of Rome "La Sapienza", Rome, Italy.
2Dermosyphilopatic Institute S. Maria and S. Gallicano Rome. Italy. 3Nuovo Regina Margherita Hospital, Rome, Italy.
1283
1284
VONESCH ET AL.
FIG. 1. (A) lane 10: Serum from uninfected mother; 20: serum HIV-1 positive control; 5: blood serum from the infected mother; 6: blood serum from the newborn; 9: cell-free fraction of colostrum from the infected mothers; 1 : cell-free fraction of colostrum from healthy mother; 8: cell-free fraction of colostrum infected with HIV-1. (B) lane 29: Blood serum from the infected mother; 20: blood serum from the newborn; 34: cell-free fraction of colostrum from the infected mothers.
(ELAVIA Ac-Ab-Ak-I Pasteur). The antibody
to HIV-1 were
confirmed by Western blot (LAV BLOT I Pasteur).
In situ The
hybridization hybridization
reaction
was
carried out
described.2 Briefly, all the cytocentrifuged cells
probes: SP64 plasmid containing the
as
previously hybrid¬
were
ized with two
1. The 9 kb Sstl-SstI viral insert from lambda BH 10 recombinant clone which corresponds to almost the entire genome and lacks only 180 base pairs of the 5'long terminal repeat (LTR) 2. The pBR322 plasmid lacking viral insert ·
samples were also hybridized with the hybridization mixture lacking probes. Moreover, an inhibition hybridization reaction was performed with non labelled probes. The heat-denaturated DNA probes were labelled by inserting an antigenic sulfone group into cytosine moieties (Chemiprobe, Orgenics). The visualization reaction was performed by a double antibody system using a monoclonal mouse anti-sulfone group antibody and then an alkaline phosphatase-conjugated anti-mouse antibody. Nitroblue tetrazolium containing 5-bromo-4-chloro-3 indolylphosphate was the chromogenic All
substrate used. The specificity of the hybridization reaction positive and negative controls.
was
proved by
HIV-1 GENOME IN CELL FRACTION OF HUMAN COLOSTRUM
1285
FIG. 2. (A) Leukocytes from colostrum of healthy mother treated with hybridization mixture containing pBR322 plasmid lacking viral insert (negative control). All cells are unstained (arrows) (X500). (B, C, D) Cell fraction of colostrum from HIV-1-infected mothers hybridized with the SP64 plasmid containing HIVBH10 viral insert. A strong intracytoplasmatic well-defined granular staining indicates the presence of HIV-1 virus (arrows). In one cell (C, arrow) nuclear-positive granules are also demonstrated (B,
x500;C, D X1000).
Positive controls: Cytocentrifuged H9 T-cell culture lines infected with HIV-1 and MT4 cells 3 h after infection with HIV-1 hybridized with SP64 plasmid containing HIVBH10 viral insert.
Negative controls: HIV-1-infected H9 T-cell line hybridized with the pBR322 plasmid lacking viral insert Uninfected MT4 and H9 T-cell lines hybridized with the two
probes Peripheral blood mononuclear cells from six negative selected blood donors hybridized with the two probes Leukocytes obtained by colostrum from two healthy women RESULTS The cell-free fraction of colostrum specimens and blood samples from all the infected mothers and their newborns
serum
showed substantially the same pattern of positivity for antiHIV-1 antibody at Western blot. Figures 1 and 2 showed a pattern of reactivity of blood serum from infected mothers, their newborns. and cell-free fraction of colostrum specimens. The cell-free fraction of colostrum spec¬ imens studied showed different reactivity at Western blot. Actually lane 9 (Fig. 1A) shows low reactivity, whereas lane 34 (Fig. IB) shows high reactivity. Negative and positive controls of the serum are shown in Figure 1A (Lanes 10 and 20, respectively). Colostrum from healthy mothers and colostrum infected with HIV-1 are dis¬ played in Figure 1A (lanes 1 and 8). The specificity of the in situ hybridization reaction was indicated by positive and negative controls. Both cytocentri¬ fuged H9 T-cell culture lines and MT4 cells infected with HIV-1 in situ hybridized with the SP64 plasmid containing the HIVBH10 viral insert showed a strong intracytoplasmic granu¬ lar staining. Actually in MT4 cells three hours after infection viral DNA appears only in the cytoplasm.5 Peripheral blood
1286
VONESCH ET AL.
mononuclear cells from healthy blood donors, uninfected H9 T-cell culture lines, and MT4 cells were consistently negative, irrespective of the probes used. A negative pattern was also observed in HIV-1 chronically infected H9 T-cell culture lines and MT4 cells 3 h after infection when treated with: (a) the hybridization mixture lacking probe, (b) the pBR322 plasmid lacking viral insert, (c) the SP64 plasmid containing the lambda BHIO viral insert after inhibition reaction performed with non labelled probes. A negative pattern was observed in the leukocytes from the colostrum samples from the two healthy mothers and from 3 out of 8 colostrum specimens of HIV-1-infected mothers. A strong intracytoplasmic granular staining was visualized in the cyto¬ plasm of mononuclear cells in 5 out of 8 cell fractions of colostrum from seropositive mothers (Fig. 2 A.B.C.D). More¬ over, the signal intensity was different from cell to cell. In some cells a strong staining reaction was found suggesting the pres¬ ence of many viral genome copies, whereas in other cells there was only a very low signal intensity.
samples. In developed countries seropositive mothers should be counselled about possible risk of HIV-1 transmission and should be discouraged from breastfeeding, in order to avoid postnatal transmission to a child who may not yet be infected. Seronega¬ tive at high-risk women (i.e., drug addicts, prostitutes, regular sexual partners of seropositive subjects)14 should also be dis¬ couraged from breastfeeding as they could still be seroconverting. During silent infection they may represent a potential source of transmission of HIV-1 infection, even though the additional risk of breastfeeding for infants is not clear. ' Two strategies are available to reduce contamination of banked breast milk: (a) sensible selection of donors especially in areas where the prevalence of HIV-1 infection is known to be high; (b) pasteur¬ ization to inactivate the virus.'5'7 In developing countries, where alternative means cannot safely and effectively be used, breastfeeding should continue to be supported, protected, and encouraged for all children. ' ACKNOWLEDGMENTS
DISCUSSION
Epidemiological and pothesis that the HIV-1
clinical observations support the hy¬ infection could be transmitted through breastfeeding/breast milk, although such a route is of small importance, in terms of absolute numbers, when compared with in utero and intrapartum transmission. The detection of HI V-1 in leukocytes of human colostrum was not completely unexpected, as HIV-1 has already been isolated from cell-free fraction of colostrum, vaginal secretions, saliva, tears, urine, cerebrospinal fluid, and alveolar fluid.6~x The maternal infant transmission rate of HIV-1 in breastfed subjects is comparable with that in non-breastfed subjects. This suggests that although the viral genome can be detected in colostrum from HIV-1 seropositive mothers, breastfeeding may not be a major route of maternalinfant transmission. Actually, the risk of perinatal infection is substantially greater when the infected mother has symptoms of HIV-1 infection or is in an advanced stage of disease in
pregnancy.9 Studies performed on breastfed infants—whose mothers were infected postpartum and without risk factors other than breast milk—showed that HIV-1 transmission via breastfeeding may be a small and identifiable mode of transmitting infection. I0~12 Van de Perre et al. found colostrum and breast milk as a route for the transmission of HIV-1 infection. Actually, they proved recently infected postnatal transmission of HIV-1 infection from '3 mothers to their infants by breastfeeding. Our probe lambda BHIO is able to recognize both mRNA and proviral DNA. However, since our aim was not to discriminate between DNA and mRNA, we have focused our research only on the detection of HIV-1 virus in leukocytes of colostrum. Moreover, the in situ hybridization testing is unique because it reflects the distribution of HIV-1 genome within cells. The presence of viral antibody in cell-free fraction of colostrum does not necessarily mean that its mononuclear cell fraction harbors HIV-1 virus. Actually, by Western blot we have found patterns of positivity in both blood serum and in cell-free colostrum specimens in all the mothers studied, whereas HIV-1 genome was present only in 5 out of 8 cellular fractions of colostrum
This work was supported by Ministero Pubblica Istruzione, Fondi Ricerca Scientifica (MP) 1989 and Ministero SanitàIstituto Superiore Sanità, III Progetto AIDS. We are grateful to Mr. V. Panosetti and E. Crescimbeni for excellent technical assistance.
REFERENCES 1. 2.
3.
4.
5.
6. 7.
Logan S. Newell ML, Ades T, and Peckham CS: Breast-feeding and HIV infection. Lancet 1988; 1:1346. Pezzella M, Pezzella F, Galli C, Macchi B, Verani P, Sorice F, and Baroni CD: In situ hybridization of human immunodeficiency virus (HTLV-III) in cryostat sections of lymph nodes of lymphadenopathy syndrome patients. J Med Virol 1987;22:135-142. Hahn BH, Shaw GM, Arya SK, Popovic M, Gallo RC, and Wong-Staal F: Molecular cloning and characterization of the HTLV-III virus associated with AIDS. Nature 1984;312:166-169. Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC, and Wong-Staal F: Molecular characterization of human T-cell leuke¬ mia (lymphotropic) virus type III in the acquired immune defi¬ ciency syndrome. Science 1984;226:1165-1171. Stevenson M, Haggerty S. Lemonica CA, Meier CM, Welch SK, and Wasiak A: Integration is not necessary for expression of human immunodeficiency virus type 1 protein products. J Virol 1990; 64:2421-2425. Friedland GH and Klein RS: Transmission of the human immuno¬ deficiency virus. Engl J Med 1987;317:1125-1135. Ziegler JB, Cooper DA, and Gold J: Postnatal transmission of AIDS associated retrovirus from mother to infant. Lancet 1985;1:896-899.
8.
Thiry L. Sprecher-Goldberger S, Jonckheer T, Levy J, Van De Perre , Henrivaux P. Cogniaux-LeClerc J, and Clumeck N: Isolation of AIDS virus from cell-free breast milk of three healthy
virus carriers. Lancet 1985;2:891-892. 9. Mok JQ, Giaquinto C, De Rossi A, Grosch-Worner I, Ades AE, and Peckhan CS: Infants born to mothers seropositive for human immunodeficiency virus. Lancet 1987;1:1164-1168. 10. Weinbreck F, Loustand V, Denis F, Viudal B, Mouinier M, and DeLumley L: Postnatal transmission of HIV infection. Lancet 1988:1:492.
HIV-1 GENOME IN CELL FRACTION OF HUMAN COLOSTRUM 11. Colebunders R, Kapita B, Nekwei W, Bahwe Y, Lebughe I, Oxtoby M, and Ryder R: Breastfeeding and transmission of HIV. Lancet 1988:2:1487. 12. Canosa CA: SIDA pediatrico. Rev Esp Pediatyr 1989;45:1-25. 13. Van de Perre P. Simonon A, Msellati P. Hitimana D-G, Vaira D, Bazubagira A, Van Goethem C. Stevens -M, Karita E. SondagThull D, Dabis F. and Lepage P: Postnatal transmission of human immunodeficiency virus type 1 from mother to infant. Engl J Med 1991;325:593-598. 14 Pezzella M. Rossi P. Lombardi V, Gemelli V. Mariani Costantini R. Mirólo M. Fundarò C, Moschese V, and Wigzell H: HIV viral sequences in seronegative people at risk detected by in situ hybrid¬ ization and polymerase chain reaction. Br Med J 1989:298:713716.
1287
15. Lucas A: AIDS and human milk bank closures. Lancet 1987:1:1092-1093. 16. Mortimer and Cooke AM: HIV infection, breastfeeding, and human milk banking. Lancet 1988:2:452-453. 17. Eglin RP and Wilkison AR: HIV infection and pasteurization of breastmilk. Lancet 1987; 1:1903.
Address
reprint requests to: Prof. Mario Pezzella
Institute
Infectious
Diseases Policlinico Umberto I University "La Sapienza" 00161 Rome, Italy
