JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 15:503-508 (1995)Mary Ann Liebert, Inc., Publishers

Protective Effect of Interferon-a Against Cell-Mediated HumanImmunodeficiency Virus Transmission Resulting from Coculture

of Infected Lymphocytes with Fetal TrophoblastsALDAR S. BOURINBAIAR,1 KEITH KRASINSKI,2 WILLIAM BORKOWSKY,2 and

SYLVIA LEE-HUANG1

ABSTRACT

The hypothesis that the low transmission rate of HIV in utero may be due, in part, to the protective effect ofIFN-producing placental trophoblasts was explored in vitro. The model consisted of H9 lymphocytes, as

surrogates of maternal HIV-infected T cells, incubated for 3 h with JEG-3 trophoblasts in the presence of10-fold dilutions of leukocyte-derived IFN-a (from 1000 to 0.1 IU/ml). The dose effect was monitored eitherdirectly, by measuring the levels of proviral DNA by PCR after a single round of infection, or indirectly, bycoculturing infected JEG-3 with cord blood-derived MT-4 lymphocytes and determining the levels of p24production by ELISA. Both assays revealed a dose-dependent blocking effect of IFN-a on cell-mediated HIVtransmission. The complete inhibition of HIV infection was observed in the presence of 100 IU IFN-a. Theefficacy of such a low dose could not be attributed to insufficient viral load because up to 108 infectious particlescould be transmitted during cell-cell contact. An adhesion assay ruled out the possibility that IFN-a actsthrough prevention of lymphocyte-trophoblast contact. The results suggest that physiologic levels of IFN-a,present in the placenta! environment, may contribute to the protection of the fetus against HIV infection.

INTRODUCTION trophoblasts are resistant to infection with physiologic levels ofcell-free HIV,<lll2) they can become permissive when exposed

IT WAS RECENTLY DEMONSTRATED that during pregnancy, at to very high titers of infectious virus or when brought intoleast three species of type I interferons (IFNs), that is, IFNs physical contact with HIV-infected leukocytes."3,14' In the

a, ß, and t, are produced by trophoblasts, fetal cells lining the presence of neutralizing antibodies, the spread of HIV in theexternal surface of the placenta/1-3' The function of these IFNs body is most likely to be mediated by infected cells than byin pregnancy is not well known. They probably act as local cell-free virus."5' This implies that cell-to-cell contact is a

immunomodulating hormones and protect the fetus against viral crucial physiologic requirement for efficient viral spread, espe-infection/4-6' Women with impaired IFN production seem to daily in tissues lacking the CD4 receptor."0,1617' Because thebe prone to viral infections, and their fetuses are likely to be vast majority of in vitro studies dealt preferentially with cell-viral targets/7,8' free virus, our understanding of the cell-mediated mode of HIVThe activity of type I IFNs against the human immunodefi- spread and its control is incomplete. To the best of our knowl-

ciency virus (HIV), IFN-a in particular, is well documented, edge, there are no investigations aimed at demonstrating thealthough the precise biologic mechanism of action is still un- antiviral effect of IFN on the cell-mediated HIV infection ofclear/9' In a cell-mediated mode of HIV spread, IFN-a acts by trophoblasts.blocking simultaneously the release of viral particles from the The present in vitro model was designed to mimic the mater-surface of infected cells and the entry of virions into target nal transmission of retroviruses across an intact placenta, withcells/10' Because CD4-negative trophoblasts are the only cells emphasis on the vital role of the IFN-producing trophoblasticof fetal origin in direct contact with maternal blood, they are barrier interposed between virus-bearing maternal lymphocyteslikely to be the primary target of HIV infection. Although and cord blood lymphocytes of the fetus.

Departments of 'Biochemistry and 2Pediatrics, Division of Infectious Diseases, New York University Medical Center, 550 First Avenue, NewYork, NY 10016.

503

504 BOURINBAIAR ET AL.

MATERIALS AND METHODS

InterferonHuman natural IFN-a (generously provided by Hayashibara

Biochemical Laboratories, Inc., Okayama, Japan) was derivedfrom the supernatant of the human lymphoblastoid line BALL-1induced by Sendai virus. All concentrations of IFN used in thisstudy are expressed in international units (IU) per ml. This"natural" IFN preparation (specific activity 2 x 108 IU/mg)contains nine subspecies of affinity-purified IFN-a, as charac-terized previously/18'

Antiviral assayThe human JEG-3 (HTB 36; American Type Culture Collec-

tion, Rockville, MD) trophoblast line (105 cells/cm2) was

grown to near confluence for 2 days in a 24-well culture plate.These choriocarcinoma-derived cells do not produce detectableamounts of interferon/6' The trophoblasts were washed andexposed for 1-3 h to 106 H9 lymphocytes infected with the typeMB strain of HIV-1 in the presence or absence of 10-folddilutions of IFN ranging from 103 to 10_l IU/ml. Thereafter,donor H9 cells were removed from the plastic-adherent JEG-3by repeated rinsing with ice-cold phosphate-buffered saline.Next, virus-exposed trophoblasts were trypsinized and pas-saged into new culture dishes until tested for viral infection. Allcells were grown in RPMI1640 medium with 10% fetal bovineserum (Gemini Bioproducts, Calabasas, CA) in humidified 5%C02 at 37°C.

Polymerase chain reaction (PCR) and analysis ofHIVDNA in JEG-3

At 3 h postinfection, HIV-exposed JEG-3 cells (104) werewashed and lysed in DNA extraction buffer containing 10 mMTris buffer, pH 8.3, 50 mM KC1, 2.5 mM MgCl2, 0.05%Nonidet P-40, and 100 p.g/ml of proteinase K. After incubationat 57°C for 1 h and 15 minutes at 95°C, 10 p.1 lysate was

transferred into plastic tubes containing 90 p.1 PCR reactionmixture with 10 mM Tris buffer, pH 8.3, 50 mM KC1, 2.5 mMMgCl2, 200 u.M dATP, dGTP, dTTP, and dCTP, 2 U TaqDNA polymerase, and SK68 and SK69 env primers (Synthe-cell, Columbia, MD). PCR was performed in a Perkin-Elmer(Emeryville, CA) thermal cycler for 35 cycles of 2 minutes at94°C, 2 minutes at 55°C, and 2 minutes at 72°C as de-scribed/19' Amplified product was hybridized with [32P]ATP-labeled oligonucleotide SK70 probe (Synthecell) internal to theprimer pairs for 1 h at 55°C and was subjected to electrophoresisin 8% acrylamide gel. The hybrid was visualized by autoradiog-raphy after overnight exposure to x-ray film (X-Omat AR;Kodak, Rochester, NY) at —20°C. Every amplification in-cluded DNA from noninfected JEG-3 and H9/IIIB as negativeand positive controls, respectively.

Determination of IFN dose effect by coculturewith MT-4With the exception of monocytes and lymphocytes, the la-

tency phenomenon is characteristic of most cells and tissuesthat are permissive to HIV. Human trophoblasts infected withHIV do not produce sufficient quantity of virus that can be

directly measured by p24 or reverse transcription assays. Toassess productive HIV infection, one must rescue the virus fromlatently infected placental cells. In our assay, the protectiveeffect of IFN was measured after amplification of virus fromJEG-3 by coculture with cord blood-derived fetal MT-4 lym-phocytes/20' MT-4 (106 cells/ml) were washed twice and addedto mock-infected and infected trophoblasts at day 7 postinfec-tion. No IFN was added at this time. The lymphocytes remainedin contact with the trophoblasts for 3 h and were resuspended innew wells after washing three times. By the fourth day, MT-4cells cocultured with infected JEG-3 formed multinuclear giantcells. To prevent the bias associated with the counting ofsyncy-tia in clump-growing MT-4 cells, productive viral infection was

instead quantified by measuring the release of p24 HIV gagantigen by enzyme-linked immunosorbent assay (ELISA,Coulter, Hialeah, FL).

Colorimetric cell-cell adhesion assay usingbiotin-labeled lymphocytesThis assay was used to test the possibility that IFN may affect

the adherence of lymphocytes to trophoblasts. If increasingdoses of IFN were to prevent the adherence, then the results ofboth PCR and coculture assays could be interpreted as if IFNhad a dose-dependent effect in hindering the association of H9lymphocytes with JEG-3 cultures rather than a direct effect onthe cell-cell transmission of HIV. The details of this assay havebeen described elsewhere/2" This technique is very sensitiveand allows detection of fewer than 100 lymphocytes per well.Sulfo-NHS-biotin-labeled H9 lymphocytes (Pierce, Rockford,IL) were added at a ratio of 10:1 to 96-well plates with JEG-3monolayers. After 3 h incubation, the plate was washed andadherent lymphocytes were detected by adding avidin-conju-gated peroxidase (Sigma, St. Louis, MO) followed by o-phe-nylenediamine substrate. The difference between light absor-bance in controls without IFN and the reading of wellscontaining epithelium-adherent lymphocytes in the presence ofvarious concentrations of IFN was judged as IFN-dependentadherence.

RESULTS

Effect of IFN on cell-to-cell HIV transmission observedby monitoring the levels ofproviral DNA in JEG-3

The intensity and size of env-specific bands in virus-exposedtrophoblasts exhibit an IFN-dependent dose pattern (Fig. 1).The absence of an HIV-specific signal in lanes corresponding toIFN concentrations starting from 100 IU indicates that lowdoses of IFN present for 3 h in coculture can efficiently blockviral transfer resulting from cell-cell contact. Identical resultswere obtained when JEG-3 were exposed to concentrated prep-arations of cell-free HIV containing an equivalent of 1 p,g p24per ml.

Effect of IFN on cell-to-cell HIV transmission observedby examining the infection ofMT-4 lymphocytescocultured with JEG-3

In these experiments, we examined the degree of productiveinfection in cord blood MT-4 lymphocytes after coincubation

IFN-a INHIBITS CELL-TO-CELL HIV TRANSMISSION 505

p24 ng/ml % adherence

100

100 IU

1000 IU

JEG-3FIG. 1. HIV DNA in JEG-3 trophoblasts infected by contactwith H9/IIIB lymphocytes. At 3 h postinfection, JEG-3 cellswere washed from adherent H9/IIIB lymphocytes and lysed inDNA extraction buffer containing proteinase K. Proviral DNAformed after a single round of infection was amplified by PCRusing SK68 and SK69 env primers. HIV env-specific signalrevealed by autoradiography after hybridization with [32P]ATP-labeled oligonucleotide SK70 probe is absent in trophoblaststhat were protected with doses of IFN starting at 100 IU.

with JEG-3, which were previously infected by contact with H9in the presence of different doses of IFN. In the absence of IFN,there was an obvious cytopathic effect characterized by syncy-tium formation in MT-4 starting from day 4 and followed bylymphocyte death after 1 week. These findings indicate thatlatently infected JEG-3 can be induced to produce HIV uponphysical contact with fetal cord blood lymphocytes. The num-

ber of syncytial cells, as detected visually under the micro-scope, appeared to correlate inversely with the concentrationsof IFN present during initial coculture with H9. To quantify thisphenomenon, duplicate samples of supernatants from JEG-3-exposed MT-4 cultures were examined for productive infectionby p24 ELISA at day 4 postinfection. The levels of p24 viralantigen diminished linearly with increasing log titers of IFN.The highest dose of IFN (103 IU/ml) used in this study was ofsimilar restrictive efficiency as the lower dose of 102 IU/ml,both providing complete protection against HIV infection (Fig.2). Thus, the outcome of these experiments correlates with theresults of PCR analysis, suggesting that HIV transmission can

be blocked by low physiologic doses of IFN.

Determination of the size of viral inoculum transferredby cell-to-cell contactTo verify that the effect of low-dose IFN was not simply a

result of the insufficient production of HIV from H9 lympho-cytes, we estimated the amount of HIV transmitted within theconfined intercellular space formed between physically associ-ated cells. In such a "claustrophilic" mode of transmission, it isevident that HIV particles are not available to the direct probingof the coculture medium. By lysing H9 cells cocultured withJEG-3 for 3 h and then by measuring the total levels of p24, we

0 0.1 1 10 100 1000

IFN concentration (IU)FIG. 2. Effect of IFN on cell-to-cell HIV transmission andlymphocyte adhesion. IFN was present during 3 h of cocultureof infected H9 lymphocytes with JEG-3 monolayers, and doseeffect was measured by p24 ELISA at 7 days postinfection,after rescuing HIV by coculture with MT-4 cord blood T lym-phocytes (left y axis). The experiments were repeated threetimes. The inhibition of viral transmission from H9 to JEG-3 isobserved at 100 IU IFN. The adherence of H9 lymphocytes toJEG-3 (shown as % of control on the right ordinate) in thepresence of log dilutions of IFN is not statistically differentfrom the control wells without IFN (p < 0.01 by Student's ttest).

could estimate the concentration of released virus. The amountof HIV transferred during 3 h of cell-cell contact was equivalentto 1 |xg p24. According to our calculations of the number ofvirions in relation to the mass of gag antigen, 1 p,g p24 corre-

sponds to 1010 viral particles'22,23' of which 108 particles are

infectious as determined by end-point titration/24' On the basisof 105 JEG-3 cells in a culture well, this represents at least 1000infectious virions per trophoblast. Therefore, it is unlikely thatthe neutralizing action of low doses of IFN can be attributedsolely to a small viral inoculum.

Effect of IFN on cell-to-cell adhesionBecause the action of IFN was restricted to the period of

cell-cell contact, we used an adherence assay to determinewhether the effect of IFN was associated with the prevention oflymphocyte-trophoblast attachment (Fig. 2). In this assay,however, we were unable to demonstrate any IFN-dependenteffect, indicating that IFN influenced the process of viral trans-mission but not the association ofH9 cells with JEG-3 cultures.

DISCUSSION

We report here a dose-dependent effect of IFN-a in prevent-ing the HIV infection of trophoblasts resulting from physical

506 BOURINBAIAR ET AL.

association with virus-carrying H9 lymphocytes. A single 100IU dose of IFN present during the brief period of cell-to-cellcontact successfully prevented cell-mediated viral transmis-sion. This effect cannot be attributed to low viral output fromH9 lymphocytes because viral challenge dose via cell contactwas equivalent to an inoculum containing up to 108 free infec-tious particles. The protective effect of IFN conferred uponchallenge with a similar amount of cell-free virus in controlstudies further supported this conclusion (data not shown).Earlier in vitro studies indicated that the permissiveness of

CD4-negative trophoblasts to HIV depends on the mode ofvirus delivery. Primary cultures of placental trophoblasts or

trophoblast lines were resistant to physiologic levels of cell-freevirus"112' or when exposed to HIV-carrying cells with im-paired adhesion capacity/14' In contrast, exposure to very highdoses of free virus (<1000 HIV particles per target cell) or toinfected lymphocytes that adhere to substrate cells resulted in-variably in infection of trophoblasts/1314' Although it is notknown whether such a mode of infection may occur in vivo, it isunlikely that the low transmission rate of HIV across the pla-centa can be accounted for solely by insufficient viral burden orthe absence of CD4.

The examination of placental tissues from HIV-positivewomen has revealed that the occurrence of HIV antigens or

viral genomic material in these samples is very low/25-32'Follow-up studies by Rouzioux et al. further supported theprevailing concept that intrauterine HIV transmission is an ex-

tremely rare event, and the majority of the infants are infectedduring delivery/29' These observations imply that some intrin-sic pregnancy factors may prevent mother-to-child HIV trans-mission. During past few years we have carried out a series ofstudies to explore the possibility that pregnancy proteins andhormones may interfere with retroviral infection. In the course

of these studies, it has been established that physiologic levelsof major pregnancy hormones, human chorionic gonadotropin(hCG) and steroid female hormones, progesterone, and estro-

gen, can inhibit viral replication in already infected cells andcell-to-cell transmission to uninfected trophoblasts/20,33,34'

The present study supports the possibility that IFN is anotherpregnancy hormone that may play a role in local protectionagainst HIV transmission in utero. Because at the time of thisstudy two other types of pregnancy-associated IFNs, ß and t,were not available commercially, we have not had the chance totest them. However, based on prior data demonstrating theiranti-HIV activity in v/'/ro/9,35' it is likely they will displaysimilar activity in our system as well.

We also cannot exclude the possibility that some other stillunidentified factors may contribute to the low transmission rateof HIV. For example, we have recently described a high-molec-ular-weight trophoblast-derived factor that inhibits lymphocyte-trophoblast adhesion. This factor appears to be similar to whatwas described earlier in different mammalian species and inhumans/36' It is conceivable that this substance may contributeto the prevention of viral spread via inhibition of cell-to-cellcontact. Dextran sulfate, a polysaccharide anticoagulant, hasbeen shown to block cell-mediated HIV infection of tropho-blasts through such a mechanism/2" Adhesion assays haveshown that IFN does not affect the cell-to-cell attachment,ruling out the possibility that higher doses of IFN may havepreferentially eliminated residual H9 lymphocytes from JEG-3

cultures. In the contrary case, one can attribute the dose-depen-dent decrease in HIV yield by PCR or in coculture experimentsto the persistence ofHIV+ lymphocytes in wells with low dosesof IFN.

The mode of antiviral action of IFN, present during a briefperiod of time in our system, seems to support the model ac-cording to which IFN-a can restrict viral infection throughmodulation of the plasma membrane fluidity rather thanthrough suppression of transcriptional step in viral expres-sion/9,37,38' The reduction in amount of proviral DNA inJEG-3 and the subsequent failure of IFN-treated trophoblasts toinfect cord blood-derived fetal lymphocytes can be explainedby the local effect of IFN confined to the cell surface. Thisalternative mode of action explains the effect of IFN on the finalstages of HIV morphogenesis in host cells and/or the earlierstages of viral entry into target cell/39"43' Using pulse-chaseelectron microscopy, we showed earlier that viral sheddingfrom donor lymphocytes toward target cell initiates within fewminutes of cell-cell contact and ceases after 3 h, suggesting thatvirions released by lymphocytes were already preassembledand their expression was not dependent on IFN-inducible virus-regulating genes/14,16,17' This possibility is supported by ob-servations that unrelated biologic substances without knowneffect on transcription/translation machinery can prevent HIVspread through interference with the host membrane/21,44'The IFN blockade of HIV transmission from virus-carrying

lymphocytes in contact with placental cells can be pertinent tothe in vivo situation. Several studies have shown that, duringpregnancy, the expression of IFN-a, hCG, and steroids is inter-dependent. For example, IFN-a enhances the expression ofhCG/45' whereas steroid hormones appear to suppress IFN-aand hCG production/46,47' It has been already proposed thatsubtle variations in the levels of these hormones, includingIFN-a, can significantly affect the success of implantation andmaintenance of pregnancy/48' It is also possible that an as yetunknown dysfunction in the expression of these hormones ina relatively small proportion of HIV-positive women maybe correlated with the occurrence of virus infection in thefetus/49'50'During normal pregnancy, as much as 100 units IFN can be

found in the amniotic fluid/7,8' but the precise local concentra-tions of IFN at the placental surface are not known/51-53' Itcannot be ruled out that mothers who transmit HIV to theiroffspring, as opposed to mothers who do not infect their fe-tuses, may have lower levels of IFN. However, no one hasinvestigated whether there are alterations in the production ofIFN in seropositive mothers and what role, if any, this may playin maternofetal HIV transmission. It was recently indicated thatvitamin A deficiency in HIV-positive pregnant women is one ofthe risk factors associated with higher transmission rate ofHIV/54' Despite the fact that underlying biologic mechanismfor this phenomenon has not been established, clinical trials ofvitamin A supplement are now being planned.IFN-a has now been approved for treatment of 13 malignant

and viral disorders/55' and based on experience with the IFN-atherapy of pregnant women with leukemia, it has been provensafe for the developing embryo/56,57' Although exogenous IFNcan be recommended for antiviral prophylaxis, it needs to beestablished whether IFN-a can be indeed effective in providingprotection to the fetus. In vitro data cannot be readily extrapo-

IFN-a INHIBITS CELL-TO-CELL HIV TRANSMISSION 507

lated to an in vivo intervention strategy: the treatment of preg-nant rhesus monkeys with recombinant IFN-a has not inhibitedC-type virus budding in the placenta/58'

In conclusion, our findings indicate that low doses of IFNcorresponding to physiologic levels during normal gestationcan inhibit HIV spread in a model that represents the interactionof infected maternal lymphocytes with fetal trophoblasts.

ACKNOWLEDGMENTS

Katsuaki Hayashibara from Hayashibara Biochemical Labs,Inc., generously provided the commercial preparation of natu-ral IFN-a that was approved for clinical use in Japan and forclinical trials in the United States. It is our pleasure to thankCaroline F. Coleman for editorial assistance. Heinz Annus pro-vided photographic assistance. This work was supported in partby grants from the National Institutes of Health T32 AI07382,NIH ROI AI31343, ROI DA04791, ROI HD26613, andgrants from the Schubert Foundation and Metatron, Inc.

REFERENCES

1. BULMER, J.N., MORRISON, L., JOHNSON, P.M., and MEA-GER, A. (1990). Immunohistochemical localization of interferonsin human placental tissues in normal, ectopic, and molar preg-nancy. Am. J. Reprod. Immunol. 22, 109-116.

2. ABOAGYE-MATHIESEN, G., TOTH, F.D., JUHL, C.B.,NORSKOV-LAURITSEN, N., PETERSEN, P.M., and EBBE-SEN, P. (1991). Purification of human placental trophoblast inter-feron by two-dimensional high performance liquid chromatogra-phy. Prep. Biochem. 21, 35-51.

3. WHALEY, A.E., MEKA, C.S., HARBISON, L.A., HUNT,J.S., and IMAKAWA, K. ( 1994). Identification and cellular local-ization of unique interferon mRNA from human placenta. J. Biol.Chem. 269, 10864-10868.

4. CHARD, T. (1989). Interferon in pregnancy. J. Dev. Physiol. 11,271-276.

5. ROBERTS, R.M., CROSS, J.C., and LEAMAN, D.W. (1992).Interferons as hormones of pregnancy. Endocr. Rev. 13,432-452.

6. TOTH, F.D., NORSKOV-LAURITSEN, N, JUHL, C.B., ABO-AGYE-MATHIESEN, G., and EBBESEN, P. (1990). Interferonproduction by cultured human trophoblasts and choriocarcin-oma cell lines induced by Sendai virus. J. Gen. Virol. 71, 3067-3069.

7. TAGUCHI, F., KAJIOKA, J., and SHIMADA, N. (1985). Pres-ence of interferon and antibodies to BK virus in amniotic fluid ofnormal pregnant women. Acta Virol. (Praha) 29, 299-304.

8. YAMADA, K., OMURA, K., NAKAKUKI, M., OZAWA, N.,and YAJIMA, A. (1986). Antiviral activity of the human amnioticfluid and placenta. Acta Obstet. Gynaecol. Jpn. 38, 1535-1539.

9. LAURENCE, J. (1990). Immunology ofHIV infection. I. Biologyof interferons. AIDS Res. Hum. Retroviruses 6, 1149-1156.

10. BOURINBAIAR, A.S., and NAGORNY, R. (1993). Inhibitoryeffect of natural interferon alpha on HIV transmission from epithe-lial cells to lymphocytes in vitro. Eur. J. Pharmacol. 230, 15-22.

11. ZACHAR, V., SPIRE, B., HIRSCH, I., CHERMANN, J.C., andEBBESEN, P. (1991). Human transformed trophoblast-derivedcells lacking CD4 receptor exhibit restricted permissiveness forhuman immunodeficiency virus type 1. J. Virol. 65, 2102-2107.

12. MANO, H., and CHERMANN, J.C. (1991). Replication of hu-man immunodeficiency virus type 1 in primary cultured placentalcells. Res. Virol. 142, 95-104.

13. DOUGLAS, G.C., FRY, G.N., THIRKILL, T., HOLMES, E.,HAKIM, H., JENNINGS, M., and KING, B.F. (1991). Cell-mediated infection of human placental trophoblast with HIV invitro. AIDS Res. Hum. Retroviruses 7, 735-740.

14. BOURINBAIAR, A.S., and NAGORNY, R. (1993). Human im-munodeficiency virus type 1 infection of choriocarcinoma-derivedtrophoblasts. Acta Virol. (Praha) 37, 21-28.

15. SABIN, A.S. (1993). HIV vaccination dilemma. Nature (Lond.)362,212.

16. BOURINBAIAR, A.S., and PHILLIPS, D.M. (1991). Transmis-sion of human immunodeficiency virus from monocytes to epithe-lia. J. Acquir. Immune Defic. Syndr. 4, 56-63.

17. PHILIPPS, D.M., and BOURINBAIAR, A.S. (1992). Mecha-nism of HIV spread from lymphocytes to epithelia. Virology 186,261-273.

18. FUKUDA, S., ANDO, S., OSAMU, S., TANIAI, M., FUJII,M., MASAKI, N., NAKAMURA, K., ANDO, O., TORIGOE,K., and KURIMOTO, M. (1988). Simultaneous production ofnatural human tumor necrosis factor-a, -ß and interferon-a fromBALL-1 cells stimulated by HVJ. Lymphokine Res. 7, 175-185.

19. BORKOWSKY, W., KRASINSKI, K., POLLACK, H.,HOOVER, W., KAUL, A., and ILMET-MOORE, T. (1992).Early diagnosis of human immunodeficiency virus infection inchildren < 6 months of age: comparison of polymerase chainreaction, culture, and plasma antigen capture techniques. J. Infect.Dis. 166, 616-619.

20. BOURINBAIAR, A.S., and NAGORNY, R. (1992). Inhibitoryeffect of human chorionic gonadotropin (hCG) on HIV-1 transmis-sion from lymphocytes to trophoblasts. FEBS Lett. 309, 82-84.

21. BOURINBAIAR, A.S., and NAGORNY, R. (1992). Associationof anti-HIV-1 effect of dextran sulfate with prevention of lympho-cyte-to-trophoblast adhesion. Immunol. Infect. Dis. 2, 245-247.

22. BOURINBAIAR, A.S. (1991). HIV and gag. Nature (Lond.)349, HI.

23. BOURINBAIAR, A.S. (1992). Weight of HIV. AIDS Res. Hum.Retroviruses 8, 1545.

24. BOURINBAIAR, A.S. (1994). The ratio of defective HIV-1 parti-cles to replication-competent infectious virions. Acta Virol.(Praha) 38, 59-61.

25. HILL, W.C., BOLTON, V., and CARLSON, J.R. (1987) Isola-tion of acquired immunodeficiency syndrome virus from the pla-centa. Am. J. Obstet. Gynecol. 157, 10-11.

26. MAURY, W., POTTS, B.J., and RABSON, A.B. (1989). HIV-1infection of first-trimester and term human placental tissue: a pos-sible mode of maternal-fetal transmission. J. Infect. Dis. 160,583-588.

27. LEWIS, S.H., REYNOLDS-KOHLER, C, FOX, H.E., andNELSON, J.A. (1990). HIV-1 in trophoblastic and villous Hof-bauer cells, and haematological precursors in eight week fetuses.Lancet 335, 565-568.

28. CHANDWANI, S., GRECO, M.A., MITTAL, K., ANTOINE,C, KRASINSKI, K., and BORKOWSKY, W. (1991). Pathologyand human immunodeficiency virus expression in placentas ofseropositive women. J. Infect. Dis. 163, 1134-1138.

29. ROUZIOUX, C, COSTAGLIOLA, D., BURGARD, M.,BLANCHE, S., MAYAUX, M.J., GRISCELLI, C, VALLE-RON, J.J., and the HIV Infection in Newborns French Collabora-tive Study Group. (1993). Timing of mother-to-child HIV-1 trans-mission depends on maternal status. AIDS 7, S49-S52.

30. MATTERN, CF., MURRAY, K., JENSEN, A., FARZADE-GAN, H., PANG, J., and MODLIN, J.F. (1992). Localization ofhuman immunodeficiency virus core antigen in term human pla-centas. Pediatrics 89, 207-209.

508 BOURINBAIAR ET AL.

31. MARTIN, A.W., BRADY, K., SMITH, S.I., DECOSTE, D.,PAGE, D.V., MALPICA, A., WOLF, B., and NEIMAN, R.S.(1992). Immunohistochemical localization of human immunodefi-ciency virus p24 antigen in placental tissue. Hum. Pathol. 23,411-414.

32. BACKE, E., JIMENEZ, E., UNGER, M., SCHAFER, A., JAU-NIAUX, E., and VOGEL, M. (1992). Demonstration of HIV-1infected cells in human placenta by in situ hybridization and immu-nohistochemistry. J. Clin. Pathol. 45, 871-874.

33. BOURINBAIAR, A.S., and NAGORNY, R. (1992). Effect ofhuman chorionic gonadotropin (hCG) on reverse transcriptase ac-

tivity in HIV-1 infected lymphocytes and monocytes. FEMS Mi-crobiol. Lett. 96, 27-30.

34. BOURINBAIAR, AS., NAGORNY, R., and TAN, X. (1992).Pregnancy hormones, estrogen and progesterone, prevent HIV-1synthesis in monocytes but not in lymphocytes. FEBS Lett. 302,206-208.

35. BAZER, F.W. (1992). Mediators of maternal recognition of preg-nancy in mammals. Proc. Soc. Exp. Biol. Med. 199, 373-384.

36. BOURINBAIAR, A.S., and TAN, X. (1993). Inhibitory effect ofchoriocarcinoma-derived high molecular weight factor (HMWF)on lymphocyte proliferation and adhesion to the trophoblasts. J.Reprod. Immunol. 23, 145-154.

37. PFEFFER, L.M., and LANDSBERGER, F.R. (1990). Inter-feron-a modulates the plasma membrane-cytoskeletal complex ofhuman lymphoblastoid cells sensitive to the antiproliferative actionof interferon-a. J. Interferon Res. 10, 91—97.

38. APOSTOLOV, K., and BARKER, W. (1981). The effects ofinterferon on the fatty acids in uninfected cells. FEBS Lett. 126,261-264.

39. YASUDA, Y., MIYAKE, S., KATO, S., KITA, M., KISHIDA,T., KIMURA, T., and IKUTA, K. (1990). Interferon-et treatmentleads to accumulation of virus particles on the surface of cellspersistently infected with the human immunodeficiency virus type1. J. Acquir. Immune Defic. Syndr. 3, 1046-1051.

40. SMITH, M.S., THRESHER, R.J., and PAGANO, J.S. (1991).Inhibition of human immunodeficiency virus type 1 morphogene-sis in T cells by alpha interferon. Antimicrob. Agents Chemother.35, 62-67.

41. GENDELMAN, H.E., BACA, L., TURPIN, J.A., KALTER,D.C., HANSEN, B.D., ORENSTEIN, J.M., FRIEDMAN, R.N.,and MELTZER, M.S. (1990). Restriction of HIV replication ininfected T cells and monocytes by interferon-a. AIDS Res. Hum.Retroviruses 6, 1045-1051.

42. GENDELMAN, H.E., BACA, L., TURPIN, J, KALTER, D.C.,HANSEN, B., ORENSTEIN, J.M., DIEFENBACH, C.W.,FRIEDMAN, R.M., and MELTZER, M.S. (1990). Regulation ofHIV replication in infected monocytes by IFN-alpha. Mechanismsfor viral restriction. J. Immunol. 145, 2669-2676.

43. SHIRAZI, Y., and PITHA, P.M. (1992). Alpha interferon inhibitsearly stages of the human immunodeficiency virus type 1 replica-tion cycle. J. Virol. 66, 1321-1328.

44. BOURINBAIAR, A.S., KRASINSKI, K., and BORKOWSKY,W. (1994). Anti-HIV effect of gramicidin in vitro: potential forspermicide use. Life Sei. 54, PL5-PL9.

45. ILES, R.K., and CHARD, T. (1989). Enhancement of ectopicbeta-human chorionic gonadotrophin expression by interferon-alpha. J. Endocrinol. 123, 501-507.

46. HANING, R.V., JR, CHOI, L., CURET, L.B., HENDERSON,P.A., KIGGENS, A.J., LEIFHEIT, T.L., OLSON, R.W.,SCHWABE, M.G., SCHWARTZ, D.B., and TRAVER, M.

(1983). Interrelationships among human chorionic gonadotropin(hCG), 17ß-estradiol, progesterone and estriol in maternal serum:evidence for an inhibitory effect of the fetal adrenal on secretion ofhCG. J. Endocrinol. Metab. 56, 1188-1194.

47. ONODERA, M., OKAMURA, K., TANIGAWARA, S., WA-TANABE, T., SHINTAKU, Y., ENDOH, H., TAEGASHI, N.,TSUMURAYA, T., HAMAZAKI, Y„ and YAJIMA, A. (1989).Influence of pregnancy and steroid hormones on interferon produc-tion of lymphocytes. Acta Obstet. Gynaecol. Jpn. 41, 1736-1742.

48. JONES, K.P., WARNOCK, S.H., URRY, R.L., EDWIN, S.S.,and MITCHELL, M.D. (1992). Immunosuppressive activity andalpha interferon concentrations in human embryo culture media as

an index of potential for successful implantation. Fértil. Steril. 67,637-640.

49. BORKOWSKY, W., KRASINSKI, K., CAO, Y.Z., HO, D.D.,POLLACK, H., MOORE, T., CHEN, S.H., ALLEN, M., andTAO, P.T. (1994). Correlation of perinatal transmission of humanimmunodeficiency virus type 1 with maternal viremia and lympho-cyte phenotypes. J. Pediatr. 125, 345-351.

50. AMIRHESSAMI-AGHILI, N., and SPECTOR, S.A. (1991). Hu-man immunodeficiency virus type 1 infection of human placenta:potential route for fetal infection. J. Virol. 65, 2231-2236.

51. BOCCI, V., PAULESU, L., and RICCI, M.G. (1985). The phys-iological interferon response. IV. Production of interferon by theperfused human placenta at term. Proc. Soc. Exp. Biol. Med. 180,137-143.

52. DUC-GORAIN, P., ROBERT-GALLIOT, B., LOPEZ, J., andCHANY, C. (1985). Unusual apparently constitutive interferonsand antagonists in human placental blood. Proc. Nati. Acad. Sei.USA 82, 5010-5014.

53. CHARD, T., CRAIG, P.H., MENABAWEY, M., and LEE, C.(1986). Alpha interferon in human pregnancy. Br. J. Obstet. Gy-naecol. 93, 1145-1149.

54. SEMBA, R.D., MIOTTI, P.G., CHIPHANGWI, J.D., SAAH,A.J., CANNER, J.K., DALLABETTA, J.A., and HOOVER,D.R. (1994). Maternal vitamin A deficiency and mother-to-childtransmission of HIV-1. Lancet 343, 1593-1597.

55. GUTTERMAN, J.U. (1994). Cytokine therapeutics: lessons frominterferon a. Proc. Nati. Acad. Sei. USA 91, 1198-1205.

56. BAER, M.R., OZER, H., and FOON, K.A. (1992). Interferon-alpha therapy during pregnancy in chronic myelogenous leukaemiaand hairy cell leukaemia. Br. J. Haematol. 81, 167-169.

57. CRUMP, M., WANG, X.H., SERMER, M., and KEATING, A.(1992). Successful pregnancy and delivery during alpha-interferontherapy for chronic myeloid leukemia. Am. J. Hematol. 40,238-239.

58. FELDMAN, D., VALENTINE, T., NIEMANN, W.H., HOAR,R.M., CUKIERSKI, M., and HENDRICKX, A. (1989). C-typevirus particles in placentas of rhesus monkeys after maternal treat-ment with recombinant leukocyte A interferon. J. Exp. Pathol. 4,193-198.

Address reprint requests to:Dr. Aldar S. Bourinbaiar

Department ofBiochemistryNew York University Medical Center

550 First AvenueNew York, NY 10016

Received 5 December 1994/Accepted 11 January 1995

This article has been cited by:

1. Vladimir Zachar, Gabriella Fazio-Tirrozzo, Trine Fink, Drucilla J. Roberts, Robin L. Broadhead, Bernard Brabin, PeterEbbesen. 2000. Lack of protection against vertical transmission of HIV-1 by interferons produced during pregnancy in a cohortfrom East African Republic of Malawi. Journal of Medical Virology 61:2, 195-200. [CrossRef]

2. William A. Bennett, S. LagooDeenadayalan, N.S. Whitworth, J.A. Stopple, W.H. Barber, E. Hale, M.N. Brackin, B.D.Cowan. 1999. First-Trimester Human Chorionic Villi Express Both Immunoregulatory and Inflammatory Cytokines: A Rolefor Interleukin-10 in Regulating the Cytokine Network of Pregnancy. American Journal of Reproductive Immunology 41:1,70-78. [CrossRef]

3. W.A. Bennett, S. Lagoo-Deenadayalan, J.A. Stopple, W.H. Barber, E. Hale, M. N. Brackin, B. D. Cowan. 1998. CytokineExpression by First-Trimester Human Chorionic Villi. American Journal of Reproductive Immunology 40:5, 309-318. [CrossRef]

4. James Reuben, Bang-Ning Lee, Edwina J. Popek. 1998. HIV AND THE PLACENTA. Immunology and Allergy Clinics ofNorth America 18:2, 371-400. [CrossRef]

5. Aldar S. Bourinbaiar, William Borkowsky, Keith M. Krasinski, Eric C. Fruhstorfer. 1997. Failure of neutralizing gp120monoclonal antibodies to prevent HIV infection of choriocarcinoma-derived trophoblasts. Journal of Biomedical Science 4:4,162-168. [CrossRef]