High level of anticholesterol antibodies (ACHA) in HIV patients. Normalization of serum ACHA concentration after introduction of HAART.

Anticholesterol antibodies (ACHA) are natural antibodies against the 3beta-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-seronegative sera. Binding of ACHA 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.

Pseudotypes of vesicular stomatitis virus-bearing envelope antigens of certain HIV-1 strains permissively infect human syncytiotrophoblasts cultured in vitro: implications for in vivo infection of syncytiotrophoblasts by cell-free HIV-1.

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.

Soluble gC1q-R/p33, a cell protein that binds to the globular "heads" of C1q, effectively inhibits the growth of HIV-1 strains in cell cultures.

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 microg/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.

Induction of HIV-1 replication in latently infected syncytiotrophoblast cells by contact with placental macrophages: role of interleukin-6 and tumor necrosis factor-alpha.

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.

Vertical transmission of human immunodeficiency virus.

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].

Alterations of P53 and RB genes and the evolution of the accelerated phase of chronic myeloid leukemia.

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.

Differential patterns of interaction between HIV type 1 and HTLV type I in monocyte-derived macrophages cultured in vitro: implications for in vivo coinfection with HIV type 1 and HTLV type I.

The interaction between human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia-lymphoma virus type I (HTLV-I) has generated substantial interest. However, there is disagreement on the in vivo consequences of the double infection. We investigated the interactions between HIV-1 and HTLV-I in monocyte-derived macrophages cultured in vitro. For study, the T cell-tropic strain IIIB and the macrophagetropic strain Ada-M of HIV-1 were used. The HTLV-I was prepared from the supernatants of the virus-producing MT-2 cell line. We found that coinfection of macrophages with T cell-tropic HIV-1 and HTLV-I significantly enhanced HIV-1 replication, whereas double infection of the cells with macrophage-tropic HIV-1 and HTLV-I resulted in marked upregulation of HTLV-I production. Stimulatory interactions between HIV-1 and HTLV-I were mediated by their trans-acting proteins. Results of study on nuclear translocation of proviral DNA showed that the tax gene product of HTLV-I was able to facilitate the nuclear import of the reverse-transcribed HIV-1(IIIB) DNA. In contrast, the HIV-1 Tat protein did not increase the intranuclear trafficking of HTLV-I DNA, which suggests another mechanism for HTLV-I enhancement by the tat gene product. In conclusion, this study provides possible mechanisms whereby coinfection of an individual with HIV-1 and HTLV-I may influence the clinical outcome of double infection.

Placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1.

Although syncytiotrophoblast (ST) cells can be infected by human cytomegalovirus (HCMV), in vitro studies have indicated that ST cells do not support the complete viral reproductive cycle, or HCMV replication may occur in less than 3% of ST cells. The present study tested the possibility that placental macrophages might enhance activation of HCMV carried in ST cells and, further, that infected ST cells would be capable of transmitting virus to neighboring macrophages. For this purpose, we studied HCMV replication in ST cells grown alone or cocultured with uninfected placental macrophages. Our results demonstrated that HCMV gene expression in ST cells was markedly upregulated by coculture with macrophages, resulting in release of substantial amounts of infectious virus from HCMV-infected ST cells. After having become permissive for viral replication, ST cells delivered HCMV to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HCMV gene expression in ST cells was mediated by marked interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1) release from macrophages, an effect caused by contact between the different placental cells. Our findings indicate an interactive role for the ST layer and placental macrophages in the dissemination of HCMV among placental tissue. Eventually, these interactions may contribute to the transmission of HCMV from mother to the fetus.

Role of interleukin-8 and transforming growth factor-beta1 in enhancement of human cytomegalovirus replication by human T cell leukemia-lymphoma virus type I in macrophages coinfected with both viruses.

Human cytomegalovirus (HCMV) is one of the most frequent opportunistic agents causing severe illness in chronic human T cell leukemia-lymphoma virus type I (HTLV-I) infection. Our previous studies have shown that coinfection of macrophages with HCMV and HTLV-I significantly enhances HCMV replication, resulting in release of infectious HCMV from dually infected cells. We found that double infection of macrophages with HCMV and HTLV-I induced a rapid production of substantial amounts of interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1). Results of transfection studies demonstrated that the tax gene product of HTLV-I was able to induce secretion of IL-8 and TGF-beta1. In addition to its cytokine-inducing effect, the Tax protein could interact with HCMV synergistically to result in production of much higher levels of IL-8 and TGF-beta1 than expected on the basis of their separate activities. Treatment of dually infected macrophage cultures with neutralizing antibodies to IL-8 and TGF-beta1 led to a nearly 1000-fold decrease in release of infectious HCMV from coinfected cells. Similar results were obtained when anti-IL-8 and anti-TGF-beta1 treatments were combined in macrophage cultures transfected with the tax gene before HCMV infection. Our results suggest that the stimulatory effect of HTLV-I Tax protein on HCMV replication in coinfected macrophages is largely mediated by high levels of IL-8 and TGF-beta1 production.

Changes in oncogene expression implicated in evolution of chronic granulocytic leukemia from its chronic phase to acceleration.

Expression of nine oncogenes was investigated in cell samples from fifteen patients with Philadelphia chromosome (Ph)-positive chronic granulocytic leukemia (CGL) both at diagnosis and at the onset of accelerated phase (AP) of the disease. The bcr-abl fusion gene, the H-ras gene and the c-myb gene were universally expressed. In comparison with the chronic phase (CP) of the disease, an increase in the levels of bcr-abl-, c-myb- and H-ras-related transcripts was found in three, two and three AP samples, respectively. Elevation of the bcr-abl-related message was associated with duplication of the Ph chromosome and amplification of the bcr-abl fusion gene in one AP sample. No CP samples were positive for c-myc or c-sis expression. On the contrary, c-myc and c-sis were expressed in three and four AP samples, respectively. The presence of c-myc-related transcript was associated with trisomy 8 with or without amplification of the c-myc oncogene in leukemia cells of two patients with CGL in AP. No changes of oncogene expression were found in four AP samples. However, we observed deletions of chromosome 13 and 17 or i(17q) in three of them, suggesting that tumor suppressor gene alterations may also be responsible for the development of AP of CGL. Our data indicate that heterogeneous alterations in oncogenes and tumor suppressor genes accompany the evolution of CGL-CP to the AP of the disease.

Reciprocal interactions between human cytomegalovirus and human T cell leukemia-lymphoma virus type I in monocyte-derived macrophages cultured in vitro.

Infection of macrophages with human cytomegalovirus (HCMV) has been shown to be nonlytic and exclusively cell associated. Human T cell leukemia-lymphoma virus type I (HTLV-I) is capable of establishing productive infection in macrophages. We studied the interactions between HCMV and HTLV-I in monocyte-derived macrophages cultured in vitro. We found that coinfection of macrophages with HCMV and HTLV-I significantly enhanced HCMV replication, resulting in release of infectious HCMV from dually infected cells. On the other hand, HCMV inhibited HTLV-I replication in macrophages coinfected with both viruses. Reciprocal interactions between HCMV and HTLV-I were mediated by their trans-acting proteins. Results of transfection studies demonstrated that the tax gene product of HTLV-I alone was capable of upregulating HCMV production. In a transient gene expression assay the immediate-early 2 (IE2) protein of HCMV alone could inhibit HTLV-I replication, whereas the IE1 protein, which had no effect by itself, produced a synergistic inhibitory effect together with the IE2 protein. Results from this study suggest that in vivo double infection of macrophages with HCMV and HTLV-I may contribute to the dissemination of HCMV infection in patients suffering from HTLV-I-associated T cell leukemia-lymphoma.