We have previously reported the potent in vitro HIV-1 anti-reverse transcriptase activity of a 35-mer of 4-thio-deoxyuridylate [(s4dU)35]. In efforts to define its activity in a more physiological system, studies were carried out to determine the stage of viral infection that this compound mediates its anti-viral effect. Results of the studies reported herein show that (s4dU)35 is nontoxic and is capable of inhibiting both single and multi-drug resistant HIV strains (IC50: 0.8–25.4 μg/ml) in vitro. Besides its previously reported anti-RT activity, (s4dU)35 mediated its antiviral action by preventing virus attachment (IC50: 0.002–0.003 μg/ml), and was stable in vitro and slowly degraded by DNAses. Competition studies and fluorescence resonance energy transfer (FRET) experiments indicated that (s4dU)35 preferentially binds to CD4 receptors, but not to CD48. Confocal laser scanning microscopy (CLSM) studies showed that (s4dU)35 did not penetrate into the cells and colocalized with cell surface thioredoxin. Our studies identify (s4dU)35 as a potential novel HIV entry inhibitor that may have utility as either a systemic antiretroviral or as a preventing agent for HIV transmission.

Enhancement of in vitro infection by immune antibodies was described for several viral diseases, first for the dengue and respiratory syncytial virus infections. Immune enhancement of viral infection can be connected with vaccine failures since vaccinated individuals who develop enhancing antibodies may have an exaggerated disease upon exposure to the virus. A part of the antibody-dependent enhancement is mediated by complement activation; this enhancement is mediated by antibodies against the immunodominant part of the transmembrane protein (gp41) of HIV. Binding of the enhancing antibodies results in deposition of complement proteins (C1q) and activation products of C4 and C3 on virus particles; interaction of these proteins with the type 2 complement receptor (CR2) and the C1q receptor facilitates attachment of the HIV virions to the target cells and leads to increased virus production in vitro and probable in vivo as well. Complement mediated antibody dependent enhancement (C-ADE) was demonstrated to be clinically relevant in HIV infection by the authors of the present chapter. C-ADE was found to develop shortly after HIV-infection concomitantly with seroconversion, and strongly correlate to viral load in the course of HIV disease. C-ADE can be detected more frequently in advanced stages of HIV infection and their appearance may herald progression of disease. Combination treatment by antiviral drugs (HAART) leads to the disappearance of C-ADE; sera of the HAART-treated patients may neutralize HIV even in the presence of complement, which can be infrequently detected in untreated patients. These findings indicate that study of C-ADE may have of crucial importance at development of HIV vaccines.

The role of A/G polymorphism at nucleotide -1082 in the interleukin-10 (IL-10) promoter was assessed by following the disease course of 253 patients who had had a routine diagnostic Hybrid Capture human papillomavirus (HPV) test because of cytologic or colposcopic abnormalities of the uterine cervix. At baseline, 97 (78%) of the 125 high-risk HPV-positive and 83 (65%) of the 128 HPV-negative patients had equivocal cytologic atypia classified as P3 by the Papanicolaou classification, and the rest of the patients had mild colposcopic atypia with cytologic results of no oncogenic significance. In the high-risk HPV-infected patients, the frequency distribution of the nt -1082 genotypes (A/A: 28%; A/G: 52%; G/G: 20%) did not differ significantly from that in the controls (A/A: 25%; A/G: 51%; G/G: 24%; p = 0.70). On the other hand, the nt -1082 G allele tended to decrease susceptibility to equivocal cytologic atypia unrelated to HPV infection (A/G: OR = 0.56 [95% CI: 0.31-1.02], G/G: OR = 0.27 [95% CI: 0.11-0.63], p for trend = 0.05). With respect to the development of high-grade cervical intraepithelial neoplasia (CIN), the established risk factors, such as high-risk HPV infection (RR = 104.6, 95% CI: 14.2-769.9) and cytologic atypia (RR = 9.6, 95% CI: 2.34-39.7) but not the various nt -1082 genotypes (A/A: reference; A/G: RR = 1.11 [95% CI: 0.59-2.08]; G/G: RR = 0.62 [95% CI: 0.25-1.50]) were found to increase the risk for high-grade CIN. In conclusion, the nt -1082 polymorphism had no influence on the early phase of cervical carcinogenesis but may determine different susceptibilities to cervical abnormalities unrelated to HPV infection.

Replication of human cytomegalovirus (HCMV) was investigated in various T-cell lines expressing the tax gene product of human T-cell leukemia-lymphoma virus type I (HTLV-I). Differential patterns of HCMV replication were found in HTLV-I-carrying cell lines. HCMV gene expression was restricted to the immediate-early genes in MT-2 and MT-4 cells, whereas full replication cycle of the virus was observed in C8166-45 cells. Productive HCMV infection induced a cytopathic effect resulting in the lysis of infected cells. The results of electrophoretic mobility shift assay (EMSA) showed high levels of NF-kappaB-, CREB/ATF-1-, and SRF-specific DNA binding activity in all Tax-positive cell lines. In contrast, SP1 activity could be detected only in C8166-45 cells. Using an inducible system (Jurkat cell line JPX-9), a dramatic increase in NF-kappaB, CREB/ATF-1, SRF, and SP1 binding activity, as well as productive HCMV infection, were observed upon Tax expression. Overexpression of SP1 in MT-2 and MT-4 cells converted HCMV infection from an abortive to a productive one. These data suggest that the stimulatory effect of Tax protein on HCMV in T cells is accomplished through at least five host-related transcription factor pathways. The results of this study provide possible mechanisms whereby HCMV infections might imply suppression of adult T-cell leukemia.

The frequency and mechanism of p16(INK4A) and p14(ARF) gene alterations were studied in cell samples from 30 patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukaemia (CML), both at diagnosis and at the onset of the accelerated phase (AP) of the disease. No alterations in the p16(INK4A) or p14(ARF) genes were found in any of the chronic phase (CP) samples. DNA sequencing analyses detected p16(INK4A) or p14(ARF) mutations in 17 AP samples. All mutations were heterozygous without loss of the other allele. Aberrant methylation of the p16(INK4A) or p14(ARF) promoters was found in 14 of 30 AP samples. The most common situation was the simultaneous methylation of both promoters. Our data indicate that p16(INK4A) and p14(ARF) are primary targets for inactivation by promoter methylation in the acceleration of CML. Transcriptional silencing of the p16(INK4A) and p14(ARF) genes may be important in the conversion of CML from the CP to the AP.

Authors studied the effect of highly active antiretroviral therapy (HAART) on balance of the antibodies that enhance or neutralize growth of HIV-1IIIB strain in MT-4 cells in the presence or absence of human complement. Sequential serum samples were collected from 28 patients in advanced stage of HIV disease before and during HAART. The balance of the enhancing and neutralizing antibodies was expressed by an index value (E/N I). Samples with an E/N I of <0.5 (twofold decrease in virus production) were considered as neutralizing, whereas samples with an E/N I>2.0 (twofold increase in virus production) were considered as enhancing. At the beginning of HAART serum samples from eight patients enhanced, and samples from only two patients neutralized the virus in the presence of complement, median (25th–75th percentile) value of E/N I was 1.32 (0.79–2.29). E/N I significantly (P<0.0001) dropped to 0.37 (0.19–0.57) during the follow-up period of 18.5 (10.5–23.5) months under HAART. Similar changes were detected when serum samples were tested with no complement added. The E/N I values were also markedly decreased when cultures inoculated with mixtures of HIV and purified IgG prepared from serum pools taken before and during HAART, respectively, were compared. In the last samples of 20/28 patients, neutralization was measured even in the presence of complement while enhancement was found with none of these samples. These findings suggest that HAART results in disappearance of enhancing antibodies and switches the E/N I toward neutralization.

Human herpesvirus 6 (HHV-6) and human immunodeficiency virus type 1 (HIV-1) may interact during transplacental transmission of HIV-1. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. Patterns of replication of HHV-6 variant A (HHV-6A) and HIV-1 were analyzed in singly and dually infected human term syncytiotrophoblast cells cultured in vitro. For this purpose, the GS strain of HHV-6A and the Ba-L and IIIB strains of HIV-1 were used. HHV-6A replication was restricted at the level of early gene products in singly infected syncytiotrophoblasts, whereas no viral protein expression was found in cells infected with HIV-1 alone. Coinfection of syncytiotrophoblast cells with HHV-6A and HIV-1 resulted in production of infectious HIV-1. In contrast, no enhancement of HHV-6A expression was observed in cell cultures infected with both viruses. Uninfected syncytiotrophoblast cells were found to express CXCR4 and CCR3 but not CD4 or CCR5 receptors. Infection of syncytiotrophoblasts with HHV-6A did not induce CD4 expression and had no influence on chemokine receptor expression. Activation of HIV-1 from latency in coinfected cells was mediated by the immediate-early (IE)-A and IE-B gene products of HHV-6A. Open reading frames U86 and U89 of the IE-A region were able to activate HIV-1 replication in a synergistic manner. The data suggest that in vivo double infection of syncytiotrophoblast cells with HHV-6A and HIV-1 could contribute to the transplacental transmission of HIV-1 but not HHV-6A.

The phenotypic mixing between human immunodeficiency virus type 1 (HIV‐1) and vesicular stomatitis virus (VSV) has been exploited to assay the susceptibility of human term syncytiotrophoblast cells to penetration by various strains of HIV‐1. VSV (HIV‐1IIIB) and VSV (HIV‐1Ba‐L) pseudotypes were found to enter syncytiotrophoblasts. Infection of syncytiotrophoblasts was mediated by envelope glycoproteins of IIIB and Ba‐L strains of HIV‐1. Although certain strains of HIV‐1 could enter syncytiotrophoblasts, the cells did not exhibit permissiveness for HIV‐1. The next studies tested the possibility that placental macrophages might induce replication of HIV‐1 carried in syncytiotrophoblast cells and that infected syncytiotrophoblasts would be capable of transmitting virus into neighbouring macrophages. For this purpose, the macrophage‐tropic Ba‐L strain of HIV‐1 was used. Interactions between syncytiotrophoblasts and macrophages activated HIV‐1 from latency in syncytiotrophoblast cells, which delivered HIV‐1 to cocultured macrophages. The stimulatory effect of coculture on HIV‐1 gene expression was mediated by marked tumor necrosis factor‐α and interleukin‐6 release from macrophages, an effect caused by contact between the different placental cells. Results suggest an interactive role for the syncytiotrophoblast layer and placental macrophages in the dissemination of HIV‐1 among placental tissue.

C1q and the outer envelope protein of HIV, gp120, have several structural and functional similarities. Therefore, it is plausible to assume that proteins that are able to interact with C1q may also interact with isolated gp120 as well as the whole HIV-1 virus. Based on this hypothesis, we studied the potential ability of the recombinant form of the 33-kDa protein, which binds to the globular “heads” of C1q (gC1q-R/p33), to inhibit the growth of different HIV-1 strains in cell cultures. gC1q-R/p33 was found to effectively and dose-dependently inhibit the production of one T-lymphotropic (X4) and one macrophage-tropic (R5) strain in human T cell lines (MT-4 and H9) and human monocyte-derived macrophage cultures, respectively. At a concentration range of 5–25 μg/ml, gC1q-R caused a marked and prolonged suppression of virus production. The extent of inhibition was enhanced when gC1q-R was first incubated with and then removed from the target cell cultures before virus infection, compared to that when the cells were infected with gC1q-R–HIV mixtures. The extent of inhibition was comparable to that of the Leu3a anti-CD4 antibody. Addition of gC1q-R to the cell cultures on day 1 or 2 after infection induced markedly less inhibition of HIV-1 growth than pretreatment of the cells just before or together with the infective HIV strains. In ELISA experiments, gC1q-R did not bind to a solid-phase recombinant gp120 while strong and dose-dependent binding of gC1q-R to solid-phase CD4 was observed. Our present findings indicate that gC1q-R is an effective inhibitor of HIV-1 infection, which prevents viral entry by blocking the interaction between CD4 and gp120. Since gC1q-R is a human protein, it is most probably not antigenic in humans. It would seem logical, therefore, to consider gC1q-R or its fragments involved in the CD4 binding as potential therapeutic agents.

Intrauterine infection of the fetus is clearly an important mode of vertical transmission of human immunodeficiency virus type 1 (HIV-1). The syncytiotrophoblast layer of the human placenta must be traversed by HIV-1 in order to reach underlying cells and fetal capillaries. Although HIV-1 has been detected in the syncytiotrophoblast layer in situ, there is conflicting evidence regarding infection of syncytiotrophoblast cells with cell-free virus. The phenotypic mixing between HIV-1 and vesicular stomatitis virus (VSV) has been exploited to assay the susceptibility of human term syncytiotrophoblast cells to penetration by various strains of HIV-1. VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) pseudotypes were found to enter syncytiotrophoblast cells. In contrast, VSV pseudotyped with envelope glycoproteins of RF, MN, or Ada-M strains of HIV-1 did not infect syncytiotrophoblasts. Plating efficiency of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) was 10-fold lower on syncytiotrophoblasts than on T-cells and macrophages, respectively. Incubation of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) viruses with appropriate HIV-1 neutralizing sera before infection strongly inhibited entry of pseudotyped VSV into syncytiotrophoblast cells. These findings demonstrated that infection of syncytiotrophoblasts with VSV(HIV-1) pseudotypes was mediated by Env from IIIB and Ba-L strains of HIV-1. Monoclonal antibodies (MAb) to CD4, CXCR4, CCR5, and CCR3 were tested for their ability to block VSV(HIV-1) infection of syncytiotrophoblast cells. Neither the anti-CD4 nor the anti-CXCR4, anti-CCR5, and anti-CCR3 MAb had any inhibitory effect on infection of syncytiotrophoblast cells with VSV(HIV-1) pseudotypes. Results from this study suggest that cell-free HIV-1 can enter syncytiotrophoblasts and the susceptibility of these cells to penetration by the virus is strain dependent. Pseudotype infection merely demonstrates that the first steps in HIV-1 replication are possible in syncytiotrophoblast cells.

The syncytiotrophoblast (ST) layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Although certain strains of human immunodeficiency virus type 1 (HIV-1) may enter ST cells, the trophoblast does not exhibit permissiveness for HIV-1. The present study tested the possibility that placental macrophages might induce replication of HIV-1 carried in ST cells and, further, that infected ST cells would be capable of transmitting virus into neighboring macrophages. For this purpose, we investigated HIV-1 replication in ST cells grown alone or cocultured with uninfected placental macrophages. The macrophage-tropic Ba-L strain of HIV-1, capable of entering ST cells, was used throughout our studies. We demonstrated that interactions between ST cells and macrophages activated HIV-1 from latency and induced its replication in ST cells. After having become permissive for viral replication, ST cells delivered HIV-1 to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HIV-1 gene expression in ST cells was mediated by marked tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) release from macrophages, an effect caused by contact between the different placental cells. Results of this study suggest an interactive role for the ST layer and placental macrophages in the dissemination of HIV-1 among placental tissue. Data reported here may also explain why macrophage-tropic HIV-1 strains are transmitted preferentially during pregnancy.

Anticholesterol antibodies (ACHA) are natural antibodies against the 3 beta -OH group of cholesterol. Since lipid disorders are common in HIV infection and HAART may further enhance dislipidaemia, we determined by using an ELISA method serum ACHA concentrations in HIV patients and healthy HIV-seronegative controls.ACHA levels were almost 4 times higher in the sera of 46 patients than in 110 controls. No difference in the specificity of ACHA was found between HIV-seropositive and HIV-sero negative sera. Binding of ACI-IA to cholesterol-coated plates from a HIV-seropositive serum was dose-dependently inhibited by preincubation with HIV-1(BA-L) preparation. Serum concentration of ACHA was significantly higher in the patients with low serum cholesterol levels than in those with normal cholesterol levels. HAART induced a marked drop of ACHA concentration. We found a significant negative correlation between the length of HAART and the ACHA levels. By contrast, HAART did not significantly influence total IgG concentration and titers of antibodies against 60 kD heat shock protein.Our findings indicate that high levels of ACHA in HIV-infection may contribute to the development, of hypocholesterolaemia frequently observed in this disease.

Sensitive detection methods, such as DNA PCR and RNA PCR suggest that vertical transmission of human immunodeficiency virus (HIV) occurs at three major time periods; in utero, around the time of birth, and postpartum as a result of breastfeeding (Fig. 1). Detection of proviral DNA in infant's blood at birth suggests that transmission occurred prior to delivery. A working definition for time of infection is that HIV detection by DNA PCR in the first 48 h of life indicates in utero transmission, while peripartum transmission is considered if DNA PCR is negative the first 48 h, but then it is positive 7 or more days later [1]. Generally, in the breastfeeding population, breast milk transmission is thought to occur if virus is not detected by PCR at 3-5 months of life but is detected thereafter within the breastfeeding period [2]. Using these definitions and guidelines, studies has suggested that in developed countries the majority, or two thirds of vertical transmission occur peripartum, and one-third in utero [3-6]. The low rate of breastfeeding transmission is due to the practice of advising known HIV-positive mothers not to feed breast milk. However, since the implementation of antiretroviral treatment in prophylaxis of HIV-positive mothers, some studies have suggested that in utero infection accounts for a larger percentage of vertical transmissions [7]. In developing countries, although the majority of infections occurs also peripartum, a significant percentage, 10-17%, is thought to be due to breastfeeding [2, 8, 9].

Using the single-strand conformation polymorphism and heteroduplex analyses, the P53 and RB genes were analyzed in cell samples from twenty-eight patients with chronic myeloid leukemia (CML) both at diagnosis and at the onset of accelerated phase (AP) of the disease. No alterations of the P53 or RB genes were found in any of the chronic phase (CP) samples. Structural abnormalities of the P53 gene were observed in ten of twenty-eight AP samples within exons 4, 5, 7 and 9. Of the ten cases of AP disease with altered P53 genes, five patients also suffered from the deletion of the other allele. Alterations of the RB gene could be detected in six AP samples, and aberrant band patterns were found in the analysis of exons 2, 3, 4, 6, 7, 13, 14, 17, 21 and 26. Among the six AP samples with structural abnormalities of the RB gene, two showed the loss of the other allele. It is of note that alterations of both P53 and RB genes were observed in two AP samples. Our data strongly suggest that abnormalities of the P53 and RB genes and acceleration of CML are linked events in some cases of AP.

OBJECTIVE:We have previously demonstrated that complement-mediated antibody-dependent enhancement (C-ADE) of HIV-1 infection correlates with accelerated immunosuppression and disease progression in HIV-1-infected individuals. In the present work the relationship between C-ADE and plasma HIV-1 RNA concentrations was studied to determine the effect of C-ADE on viral replication.METHODS:Three studies were performed: (a) C-ADE and HIV-1 RNA concentrations were determined in the serum and plasma aliquots taken at the same time from 98 HIV patients, mostly in the advanced stage of the disease; (b) the above two parameters as well as HIV enzyme-linked immunosorbent assay (ELISA)-reactive antibodies (Abbott HIV 1/2 test), and p24 antigen levels (Abbott antigen test; Abbott, Delkenheim, Germany) were determined in four seroconversion panels purchased from the Boston Biomedica firm; (c) changes of HIV-1 RNA concentration and C-ADE during a 17 month follow-up period were determined in 18 HIV-infected patients. C-ADE was measured by the method previously established in our laboratories. The results were expressed by an enhancement/neutralization index (E/NI). HIV-1 RNA levels were determined with the Amplicor monitor kit (Roche, Basel, Switzerland), and in some experiments with the nucleic acid sequence based amplification (Organon Teknika, Turnhout, Belgium) kits.RESULTS:(a) We found a highly significant (P<0.0001) positive correlation between E/NI values reflecting the extent of HIV-1 infection enhancement and plasma HIV-1 RNA levels. Both E/NI and HIV-1 RNA levels negatively correlated to the CD4 cell counts. (b) C-ADE was first detected just before, or concomitantly with, seroconversion in 4/4 seroconversion panels. (c) Both E/NI values and HIV-1 RNA levels significantly (P<0.001) increased during a 17 month observation period in 18 HIV-infected patients.CONCLUSION:We found strong association between the extent of the complement-mediated antibody-dependent enhancement of HIV-1 infection and the plasma viral load in HIV patients. On the basis of these findings, C-ADE correlates with HIV replication in vivo, and potentially contributes to the progression of HIV disease.

Antibodies to solid phase C1q (C1qAb) were determined in 295 serum samples from 132 HIV-infected subjects and in sera from 140 HIV-seronegative healthy individuals as control. An ELISA method applied for the determination of C1qAb in other diseases was used. In part of these sera, other autoantibodies (antibodies reacting with 60-kDa human heat shock protein (hsp60) or mycobacterial hsp65; IgA and IgG class antibodies against the Fab and F(ab′)2 moieties of IgG) as well as complement-mediated antibody-dependent enhancement/neutralization (C′-ADE) were also determined. Increased amount of C1qAb was found in HIV-infected subjects as compared with HIV-seronegative controls (P= 0.0138). In 17 of 132 (13.0%) seropositive individuals but only in 7/140 (5.0%) samples from the controls, the amount of C1qAb exceeded the upper limit (95th percentile) of the normal values (P= 0.031). The amount of C1qAb significantly decreased during a follow-up period of 65 months. C1qAb levels were found to strongly correlate to hsp60/65 autoantibodies but did not correlate or only weakly correlated to the amount of anti-Fab or anti-F(ab′)2autoantibodies measured in the same serum samples. Anti-C1q antibodies recognized the solid phase hsp60/65. Three predicted epitope regions ofM. paratuberculosishsp65 were able to bind efficiently C1q antibodies. An inverse correlation was found between C1qAb and C′-ADE, neutralization was more frequent in the sera with detectable C1qAb, whereas sera without C1qAb more likely enhanced HIV infectionin vitro.

P033 The most common viral pathogen in chronic HTLV-I infection is the human cytomegalovirus (HCMV). We studied the interactions between HCMV and HTLV-I in macrophages cultured in vitro. Coinfection of macrophages with HCMV and HTLV-I significantly enhanced HCMV replication, whereas replication of HTLV-I was inhibited in dually infected cells. Reciprocal interactions between HCMV and HTLV-I were mediated by their trans-acting proteins. Double infection of macrophages with HCMV and HTLV-I induced rapid production of substantial amounts of interleukin 8 (IL-8) and transforming growth factor β1 (TGF-β1). Results of transfection studies showed that the Tax protein was able to induce secretion of IL-8 and TGF-β1 and to interact with HCMV synergistically in cytokine induction. Treatment of dually infected macrophage cultures with neutralizing antibodies to IL-8 and TGF-β1 led to a nearly 1000-fold decrease in release of infectious HCMV from coinfected cells. Our data suggest that in vivo double infection of macrophages with HCMV and HTLV-I may contribute to the dissemination of HCMV infection and that the stimulatory effect of HTLV-I Tax protein on HCMV replication is largely mediated by high levels of IL-8 and TGF-β1 production.