The reconstitution of the thymus in immunosuppressed individuals restores CD4-specific cellular and humoral immune responses.

Infection with HIV-1 frequently results in the loss of specific cellular immune responses and an associated lack of antibodies. Recombinant growth hormone (rGH) administration reconstitutes thymic tissue and boosts the levels of peripheral T cells, so rGH therapy may be an effective adjuvant through promoting the recovery of lost cellular and T-cell-dependent humoral immune responses in immunosuppressed individuals. To test this concept, we administered rGH to a clinically defined group of HIV-1-infected subjects with defective cellular and serological immune responses to at least one of three commonly employed vaccines (hepatitis A, hepatitis B or tetanus toxoid). Of the original 278 HIV-1-infected patients entering the trial, only 20 conformed to these immunological criteria and were randomized into three groups: Group A (n = 8) receiving rGH and challenged with the same vaccine to which they were unresponsive and Groups B (n = 5) and C (n = 7) who received either rGH or vaccination alone, respectively. Of the eight subjects in Group A, five recovered CD4 cellular responses to vaccine antigen and four of these produced the corresponding antibodies. In the controls, three of the five in group B recovered cellular responses with two producing antibodies, whereas three of the seven in Group C recovered CD4 responses, with only two producing antibodies. Significantly, whereas seven of ten patients receiving rGH treatment in Group A (six patients) and B (one patient) recovered T-cell responses to HIVp24, only two of six in Group C responded similarly. In conclusion, reconstitution of the thymus in immunosuppressed adults through rGH hormone treatment restored both specific antibody and CD4 T-cell responses.

Human papillomavirus 16 integration and risk factors associated in anal samples of HIV-1 infected men.

BACKGROUND: The integration of HPV-16 DNA into the host genome is considered an important event in the progression of premalignant cervical lesions to cervical cancer. The aim of our study was to assess the prevalence of HPV-16 integration in anal cytologic specimens of HIV-1 infected men and its association with risk factors. PATIENTS METHODS: This cross-sectional study included 269 HIV-infected males. Detection and typing of HPV-infection was done by multiplex PCR, and integration of HPV-16 by real-time PCR. RESULTS: The overall anal HPV-infection prevalence was 78% (209/269), 29% (77/269) for HPV-16 infection, and 9% (25/269) for HPV-16 integration. In HPV-16 infected group, the integration prevalence represented 32% (25/77). The only risk factor associated with HPV-16 integration was the time since HIV diagnosis (OR = 1.2, 95% CI: 1.0-1.3; P = 0.010). The risk factors associated with abnormal cytology results were: HPV infection (OR = 17.8, 95% CI: 6.8-46.6), HPV-16 infection (OR = 4.6, 95% CI: 2.5-8.4), and presence of HPV-16 integrated forms (OR = 11.7, 95% CI: 1.5-93.5). Moreover, in the multivariate analysis, the HPV-16 integration continued representing the most important risk factor (OR = 20, 95% CI: 1.6-226) for anal cytologic abnormalities. CONCLUSION: HPV-16 infection and its integration in anal cells were highly prevalent in HIV-infected men. The assessment of HPV-16 integration rather than HPV-infection could be a good biomarker for predicting anal precancerous lesions in HIV-positive men.

Secretion of interferon-gamma by human macrophages demonstrated at the single-cell level after costimulation with interleukin (IL)-12 plus IL-18.

The interferon (IFN)-gamma component of the immune response plays an essential role in combating infectious and non-infectious diseases. Induction of IFN-gamma secretion by human T and natural killer (NK) cells through synergistic costimulation with interleukin (IL)-12 and IL-18 in the adaptive immune responses against pathogens is well established, but induction of similar activity in macrophages is still controversial, with doubts largely focusing on contamination of macrophages with NK or T cells in the relevant experiments. The possible contribution of macrophages to the IFN response is, however, an important factor relevant to the pathogenesis of many diseases. To resolve this issue, we analysed the production of IFN-gamma at the single-cell level by immunohistochemistry and by enzyme-linked immunosorbent spot (ELISPOT) analysis and unequivocally demonstrated that human macrophages derived from monocytes in vitro through stimulation with a combination of IL-12 and IL-18 or with macrophage colony-stimulating factor (M-CSF) were able to produce IFN-gamma when further stimulated with a combination of IL-12 and IL-18. In addition, naturally activated alveolar macrophages immediately secreted IFN-gamma upon treatment with IL-12 and IL-18. Therefore, human macrophages in addition to lymphoid cells contribute to the IFN-gamma response, providing another link between the innate and acquired immune responses.

HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4.

BACKGROUND: Cell-to-cell HIV transmission requires cellular contacts that may be in part mediated by the integrin leukocyte function antigen (LFA)-1 and its ligands intercellular adhesion molecule (ICAM)-1, -2 and -3. The role of these molecules in free virus infection of CD4 T cells or in transinfection mediated by dendritic cells (DC) has been previously described. Here, we evaluate their role in viral transmission between different HIV producing cells and primary CD4 T cells. RESULTS: The formation of cellular conjugates and subsequent HIV transmission between productively infected MOLT cell lines and primary CD4 T cells was not inhibited by a panel of blocking antibodies against ICAM-1, ICAM-3 and alpha and beta chains of LFA-1. Complete abrogation of HIV transmission and formation of cellular conjugates was only observed when gp120/CD4 interactions were blocked. The dispensable role of LFA-1 in HIV transmission was confirmed using non-lymphoid 293T cells, lacking the expression of adhesion molecules, as HIV producing cells. Moreover, HIV transmission between infected and uninfected primary CD4 T cells was abrogated by inhibitors of gp120 binding to CD4 but was not inhibited by blocking LFA-1 binding to ICAM-1 or ICAM-3. Rather, LFA-1 and ICAM-3 mAbs enhanced HIV transfer. All HIV producing cells (including 293T cells) transferred HIV particles more efficiently to memory than to naive CD4 T cells. CONCLUSION: In contrast to other mechanisms of viral spread, HIV transmission between infected and uninfected T cells efficiently occurs in the absence of adhesion molecules. Thus, gp120/CD4 interactions are the main driving force of the formation of cellular contacts between infected and uninfected CD4 T cells whereby HIV transmission occurs.

Expression and functionality of anti-human immunodeficiency virus and anticancer drug uptake transporters in immune cells.

Almost all drugs used in anti-human immunodeficiency virus (HIV)-1 and anticancer therapies require membrane proteins to get into the cell to develop their proper activity. Nevertheless, little is known regarding the expression and activity of specific carriers involved in the uptake of these drugs in immune cells. Here, we assessed the mRNA levels, protein expression profile, and activity of the gene families SLC28 (coding for concentrative nucleoside transporters, hCNT1-3), SLC29 (equilibrative nucleoside transporters, hENT1-2), and SLC22 (organic cation transporters, hOCT1-3 and hOCTN1-2). Both hENTs and hCNT2 were abundant in primary lymphocytes, with a preferential activity of hENT1. A significant up-regulation in hENTs expression (100-fold) and activity (30-fold) was seen under stimulation of primary T lymphocytes. In contrast, monocytes, monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells predominantly expressed hCNT3, a functional transporter in MDMs. Finally, in immune cells, hOCTs showed a more heterogeneous expression profile and a lower activity than human nucleoside transporters (hNTs), although up-regulation of hOCTs also occurred upon lymphocyte activation. Overall, the expression and activity of most of the studied transporters emphasize their relevance in relation to anti-HIV and anticancer therapies. The identification of the transporter involved in each specific drug uptake in immune cells could help to optimize pharmacological therapeutic responses.

Skewed expression and up-regulation of the IL-12 and IL-18 receptors in resting and activated CD4 T cells from HIV-1-infected patients.

IL-12 and IL-18 synergistically induce the production of IFN-gamma by resting and activated T cells. To evaluate whether this induction was affected in HIV-1-infected patients, PBMC or isolated CD4 T cells were cultured with IL-12 plus IL-18, anti-CD3 plus anti-CD28, or PHA for 72 h. Cell samples were labeled daily to assess the levels of IL-12 receptor beta1 (IL-12Rbeta1), IL-12Rbeta2, and IL-18Ralpha. Culture supernatants were analyzed for the presence of Th1- and Th2-related cytokines by ELISA or cytometric bead array and analyzed by flow cytometry. A twofold increase in the percentage of CD4-resting T cells expressing IL-12Rbeta1 and IL-18Ralpha from HIV-1-infected patients was observed when compared with cells from HIV-1-negative donors. Higher IL-12Rbeta1 and IL-18Ralpha expression correlated (r=0.87; P<0.007) to increased production of IFN-gamma by isolated CD4 T cells in the presence of IL-12 and IL-18. Moreover, exogenous IL-12 and IL-18 induced the up-regulation of IL-12Rbeta2 to twice higher in CD4 T cells from HIV-1-positive individuals compared with controls. Conversely, upon activation with anti-CD3 and anti-CD28 antibodies, only 25% of the CD4+ T cells from HIV-1 patients showed an increase in the IL-12beta2 when compared with 50% in healthy controls. Furthermore, the percentage of IL-12Rbeta1-positive cells correlated inversely with the CD4 nadir of patients, suggesting that deregulation of the IL-12 and IL-18 pathways may play a role in the immunopathogenesis of HIV-1 infection.

Genetic evolution of gp41 reveals a highly exclusive relationship between codons 36, 38 and 43 in gp41 under long-term enfuvirtide-containing salvage regimen.

OBJECTIVE: To analyse the genetic changes in the gp41 protein in HIV-infected patients with detectable plasma viraemia receiving a long-term salvage enfuvirtide regimen. METHODS: We studied 13 heavily antiretroviral-experienced patients receiving a salvage regimen containing enfuvirtide. Substitutions in gp41 were analysed by population-based sequencing at baseline and longitudinally after the initiation of enfuvirtide treatment. To investigate sequence evolution we also analysed multiple gp41 clones from four selected patients. A Fisher's two-tailed test was used to assess the distribution of resistance-associated mutations in the clonal sequences. RESULTS: Mutations at positions 36 and 38 in gp41 (HR1) emerged rapidly (median emerging time 10 weeks), but disappeared at subsequent timepoints in most of the patients. Amino acid changes did not accumulate over time, with no patient having more than two mutations in HR1 after 6 months of treatment. The mutation N43D was not observed together with changes at positions 36 or 38 in any patient. Clonal analysis showed that the three main gp41 resistance mutations were highly mutually exclusive (P < 0.001), being present in individual clones and constituting independent populations. CONCLUSION: Substitutions at positions 36 and 38 are rapidly selected but disappear thereafter in HIV-1-infected patients failing an enfuvirtide-containing salvage therapy. We found a highly exclusive relationship between the three main enfuvirtide resistance-associated mutations (amino acids 36, 38 and 43), suggesting that the genetic evolution of HIV-1 gp41 protein is a dynamic and much more complex process than previously though.

Naive CD4(+) T cells and recent thymic emigrant levels in treated individuals with HIV: clinical relevance.

An increase in the levels of naive T cells after the administration of HAART is an indicator of the quality of immune reconstitution. We investigated whether levels of naive CD4 T cells (CD4(+)/CD45RA(+)) and of recent thymic emigrants (RTEs; CD4(+)/CD45RA(+)/CD31(+)) achieved in chronically treated HIV-infected patients could predict the length of time patients could interrupt antiretroviral treatment before their CD4 counts reached values < or =350 cells/mm(3) or HIV-1 RNA levels increased to > or =100,000 copies/ml (Tibet cohort). Serial measurements revealed that the level of naive CD4 T cells among patients was extremely variable (5-95%), but the values for each patient remained stable throughout the study. We then focused on those patients who showed percentages of naive CD4 T cells above 60% or below 30%. The levels of naive T cells, RTEs, mononuclear cells containing T cell receptor excision circles (TRECs), and the strength of CD4 helper responses to HIV p24 antigen during treatment failed to predict the duration of treatment interruptions. In contrast, the median survival time of patients with a CD4 nadir > or =350 cells/mm(3) was 2-fold higher than that of patients with a CD4 nadir <350. However, the probability of restarting therapy in these two groups of patients was independent of the levels of naive T cells, RTEs, or TRECs.

Inhibition of human immunodeficiency virus type 1 infection in macrophages by an alpha-v integrin blocking antibody.

Macrophages are key cells for HIV infection and HIV spreading inside the organism. Macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (M-CSF). In the monocyte to macrophage differentiation process with M-CSF, alphav-integrins are upregulated concomitantly with the capacity of HIV to generate a productive virus infection. In the present study we show that an anti-alphav antibody, 17E6, inhibited HIV-1 infection of primary macrophages. The effect of 17E6 on HIV-1 BaL replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (EC50) of 17+/-2 microg/ml in the absence of cytotoxicity. Similarly, a monoclonal antibody targeting the alphavbeta6 integrin (14D9.F8) also inhibited HIV-1 BaL infection in this cell type. 17E6 reduced the detection of HIV-1 BaL proviral DNA in acutely infected macrophages, but was completely ineffective against HIV-1 BaL production in chronically infected macrophages, suggesting that 17E6 inhibited HIV infection at an early stage of the virus cycle. Finally, a small molecular weight antagonist of the alphavbeta6 integrin, EMD 409849, reduced HIV replication at subtoxic concentrations. Therefore, our results suggest that alphav-containing integrins could play a role in HIV replication in macrophages and suggest that small-molecular-weight compounds might interfere with HIV replication in macrophages through the interaction with alphav integrins.

The role of macrophages in angiogenesis. Comparison between HIV+ and HIV- populations with anal dysplasia and anal cancer.

BACKGROUND: While macrophages (CD68+) have been associated with angiogenesis in some inflammatory and neoplastic processes by increasing the release of vascular endothelial growth factor (VEGF), their role in anal intraepithelial neoplasia (AIN) and anal squamous cell carcinoma has not been established. This study records macrophage infiltration in anal pre-invasive and invasive lesions in HIV+ and HIV- populations, and determines their relationship with angiogenesis. MATERIALS AND METHODS: Sixty patients (31 HIV+) with AIN and anal SCC were studied. Paraffin sections were stained for CD68, VEGF and von Willebrand factor. The density of CD68 cells, the expression of VEGF and angiogenesis were quantified, and compared amongst groups and between HIV+ and HIV- populations. RESULTS: All three parameters increased linearly as the lesions became more dysplastic, in HIV+ and HIV- groups. The CD68 count was statistically lower in HIV+ (p<0.005) compared with HIV- groups, while the differences in VEGF expression and in angiogenesis were not significant between HIV+ and HIV- populations. CONCLUSION: There was a significant decrease of macrophage infiltrate in the HIV+ group. The relative increase in VEGF expression and angiogenesis in the face of lower macrophage infiltration in HIV+ patients may be explained either by a greater release of angiogenic factors by macrophages, or by VEGF expression not being solely dependent on macrophage activation.

Global gene expression profiles in fibroblasts from synovial, skin and lymphoid tissue reveals distinct cytokine and chemokine expression patterns.

We investigated the extent to which fibroblasts isolated from diverse tissues differ in their capacity to modulate inflammation by comparing the global gene expression profiles of cultured human fibroblasts from skin, acute and chronically inflamed synovium, lymph node and tonsil. The responses of these fibroblasts to TNF-alpha, IFN-gamma and IL-4 stimulation were markedly different, as revealed by hierarchical cluster analysis and principal component analysis. In the absence of exogenous cytokine, synovial and skin fibroblasts exhibited similar patterns of gene expression. However their transcriptional profiles diverged upon treatment with TNF-alpha. This proved to be biologically relevant, as TNF-alpha induced the secretion of different patterns and amounts of IL-6, IL-8 and CCL2 (MCP-1) in the two fibroblast types. Co-culture of skin or synovial fibroblasts with synovial fluid-derived mononuclear cells provided further evidence that these transcriptional differences were functionally significant in an ex vivo setting. Interestingly, the transcriptional response of skin fibroblasts to IL-4 converged with that of TNF-alpha-treated synovial fibroblasts, suggesting resident tissue fibroblasts and their blood-borne precursors may be imprinted by inflammatory cytokines that are characteristic of different tissues. Our data supports the concept that fibroblasts are heterogeneous, and that they contribute to the tissue-specificity of inflammatory reactions. Fibroblasts are therefore likely to play an active role in the persistence of chronic inflammatory reactions.

Neoplastic transformation of T lymphocytes through transgenic expression of a virus host modification protein.

Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4(+)CD8(+)CD69(-) lymphoma. The CD4(+)CD8(+)CD69(-) cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRß-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4(+)CD8(+)CD69(-) thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4(+)CD8(+) CD69(-) thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-)oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas.

Susceptibility of human lymphoid tissue cultured ex vivo to xenotropic murine leukemia virus-related virus (XMRV) infection.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) was generated after a recombination event between two endogenous murine leukemia viruses during the production of a prostate cancer cell line. Although the associations of the XMRV infection with human diseases appear unlikely, the XMRV is a retrovirus of undefined pathogenic potential, able to replicate in human cells in vitro. Since recent studies using animal models for infection have yielded conflicting results, we set out an ex vivo model for XMRV infection of human tonsillar tissue to determine whether XMRV produced by 22Rv1 cells is able to replicate in human lymphoid organs. Tonsil blocks were infected and infection kinetics and its pathogenic effects were monitored RESULTS: XMRV, though restricted by APOBEC, enters and integrates into the tissue cells. The infection did not result in changes of T or B-cells, immune activation, nor inflammatory chemokines. Infectious viruses could be recovered from supernatants of infected tonsils by reinfecting DERSE XMRV indicator cell line, although these supernatants could not establish a new infection in fresh tonsil culture, indicating that in our model, the viral replication is controlled by innate antiviral restriction factors. CONCLUSIONS: Overall, the replication-competent retrovirus XMRV, present in a high number of laboratories, is able to infect human lymphoid tissue and produce infectious viruses, even though they were unable to establish a new infection in fresh tonsillar tissue. Hereby, laboratories working with cell lines producing XMRV should have knowledge and understanding of the potential biological biohazardous risks of this virus.

A cell-to-cell HIV transfer assay identifies humoral responses with broad neutralization activity.

BACKGROUND: Cell-to-cell HIV spread through virological synapses proceeds in two steps, first HIV particles are rapidly transferred to target cells in a CD4-dependent manner and then coreceptor-dependent events allow for infection or death of single target cells and cell-to-cell fusion. METHODS: 293T or MOLT cells producing HIV particles were cocultured with primary CD4 T-cells or reporter cell lines. The extent of HIV transfer, cell fusion and target cell death was assessed. Inhibition by sera from 19 HIV-infected patients was evaluated and compared with cell-free HIV neutralization using different envelopes from clades A, B, C and E. RESULTS: Sera showed different abilities to protect CD4 T-cells from cell-to-cell transfer, fusion or death when cocultured with HIV producing 293T cells. Some sera were able to block all parameters (a property of IgGb12), while other showed lower activity against HIV transfer despite being able to block fusion and death (a property of antibodies blocking post-CD4 binding steps). Neutralization of cell-to-cell HIV transfer strongly correlated with IgG binding to native Env. Interestingly, sera that efficiently blocked HIV transfer showed broader neutralizing response, as they neutralized a higher percentage of the viruses tested compared with sera showing low CD4 binding site responses (P=0.01). Similar results were observed in a model of T cell-T cell HIV transmission, although this experimental model showed lower capacity to discriminate broadly neutralizing responses. CONCLUSION: Cell-to-cell HIV transfer assays identify sera with broadly neutralizing capacity and may help to characterize anti-HIV humoral responses.

Changes in T-cell subsets in HIV-HCV-coinfected patients during pegylated interferon-alpha2a plus ribavirin treatment.

BACKGROUND: We evaluated the effect of different doses of pegylated interferon (PEG-IFN)-alpha2a/ribavirin (RBV) on several T-cell activation markers in HIV-HCV-coinfected patients and their relationship with changes in plasma HCV RNA. METHODS: Frozen peripheral blood mononuclear cells (PBMCs) from 22 patients receiving two different PEG-IFN-alpha2a schedules were analysed by six-colour flow cytometry. Cell-surface expression of CD38 was quantified. HIV and HCV viral loads, as well as absolute CD4+ and CD8+ T-cell counts, were recorded during the follow up (72 weeks). RESULTS: PEG-IFN-alpha2a/RBV treatment decreased the absolute numbers of CD8+ and CD4+ T-cells. The decrease in CD8+ T-cells was more pronounced, resulting in increased percentages of CD4+ T-cells. Percentages of naive/memory CD4+ T-cell subsets remained unchanged, although the percentage of CD38+CD45RO+ cells significantly increased. By contrast, the CD8+ T-cell compartment significantly reduced the percentage of CD45RO+ cells and HLA-DR+ cells, whereas the percentage of CD38 expressing cells was increased because of a significant increase in cell-surface CD38 expression. Changes in CD8+ T-cells were similar for both PEG-IFN-alpha2a/RBV doses, but high doses induced more severe perturbations in CD4+ T-cells. All changes returned to baseline levels after treatment cessation and, except for the loss of naive CD4+ T-cells, were not associated with virological response. CONCLUSIONS: Transient lymphopaenia induced by PEG-IFN-alpha2a/RBV differentially affects T-cell subsets. Activated HLA-DR+ and CD45RO+ cells were selectively reduced in peripheral blood, whereas CD38 expression was up-regulated mainly in memory cells. Increasing PEG-IFN-alpha2a/RBV doses mainly affect CD4+ T-cells but failed to modify clinical outcome.

Genotypic and phenotypic evolution of HIV type-1 protease during in vitro sequential or concomitant combination of atazanavir and amprenavir.

BACKGROUND: The pathways of resistance to atazanavir (ATV) and amprenavir (APV) converge at position 50 of HIV protease. The determinants of cross-resistance were analysed during in vitro sequential or concomitant combination pressure with both drugs. METHODS: Recombinant viruses containing in vitro and in vivo selected I50L and I50V proteases were constructed and cultured in increasing concentrations of APV or ATV, respectively. In addition, in vitro resistance to ATV plus APV was performed. All the resistant viruses obtained were genotyped and phenotyped. RESULTS: ATV or APV alone selected I50L- or I50V-containing variants. Subsequent addition of ATV to I50V-containing recombinant viruses led to the reversion of this change and the later selection of I50L. By contrast, addition of APV to I50L-containing recombinant viruses was not associated with reversion. The combined pressure with ATV plus APV selected several changes but not at position 50. Phenotypically, both sequential and concomitant ATV-APV pressure yielded viruses resistant to all the drugs tested, although the emergence of I50L by ATV pressure on APV-resistant variants was associated with a reduced resistance to APV and darunavir. CONCLUSIONS: All drug combinations led to APV plus ATV cross-resistance. The different pathways select for isoleucine or leucine at position 50, whereas the I50V mutation was excluded. Sequential pressure with ATV might have an advantage in terms of modulating the sensitivity of HIV to other protease inhibitors.

CD4 T-cell hyperactivation and susceptibility to cell death determine poor CD4 T-cell recovery during suppressive HAART.

BACKGROUND: The failure to increase CD4 T-cell counts in some HAART-treated HIV-infected patients with satisfactory virological responses has been related to low CD4 T-cell production, high turnover and death. However, the relative contribution of these factors is still unclear, strongly limiting the definition of appropriate therapeutic strategies for these patients. METHODS: A cross-sectional study was designed to evaluate the contribution of thymic activity, microbial translocation, cellular activation and death to CD4 T-cell recovery. We included 230 HIV-infected individuals on suppressive HAART (>2 years); 95 of them were considered 'discordant' (CD4 T-cell count <350 cells/mul) and 135 were considered 'concordant'. Comparative and logistic regression analyses were performed. RESULTS: Discordant patients showed higher levels of activated [human leukocyte antigen (HLA)-DRCD95 and CD38CD45RA] cells in both the CD8 and CD4 T-cell compartments. Notably, the most significant differences were observed in CD4 T cells. Discordant patients showed lower naive CD4 T-cell production (CD45RACD31 cells), higher spontaneous ex-vivo CD4 T-cell death and higher plasma levels of soluble CD14. Multivariate analysis showed that activation and death of CD4 T cells, along with nadir CD4 T-cell counts, were the only predictive factors for poor immune recovery. Moreover, the low correlations found between CD4 T-cell activation or death with thymic output and bacterial translocation suggest that additional factors modulate cellular activation and death and, in turn, CD4 T-cell recovery. CONCLUSION: CD4 T-cell repopulation during HAART is determined by CD4 T-cell activation and death. Therefore, strategies aimed to reduce these parameters should be envisaged to treat discordant patients.

Antigp41 antibodies fail to block early events of virological synapses but inhibit HIV spread between T cells.

OBJECTIVE: Compared with cell-free viral infection, virological synapses increase HIV capture by target cells, viral infectivity and cytopathicity, and are believed to be less sensitive to antibody neutralization. We have evaluated the impact of antibodies against HIV envelope glycoproteins (gp120 and gp41) on cell-to-cell HIV transmission. METHODS: We analyzed the role of trogocytosis in cell-to-cell HIV transmission and the inhibitory mechanisms of antigp120 antibody IgGb12 and antigp41 antibodies 4E10 and 2F5 using cocultures of NL4-3 or BaL-infected MOLT/CCR5 cells with primary CD4 T cells. RESULTS: Analysis of early steps of HIV transmission in these cocultures showed that IgGb12, but not 4E10 and 2F5, inhibited the formation of virological synapses. Consequently, IgGb12 but not antigp41 antibodies blocked the transfer of HIV particles from infected to target cells and the trogocytic transfer of CD4 molecules from target to infected cells. Interestingly, trogocytic transfer of membranes was not detected in the HIV transmission direction. Furthermore, analysis of late events of HIV transmission showed that all neutralizing antibodies blocked productive infection of target cells, suggesting that HIV infection between T cells is transmitted by a neutralization-sensitive mechanism involving HIV budding from infected cells and capture by target cells. CONCLUSION: Despite mechanistic differences, antigp120 and antigp41 antibodies block infectious cell-to-cell HIV transmission. Our data suggest that eliciting high titers of neutralizing antibodies in vivo should be maintained as a main end of HIV vaccine design.

Epidemiological data of different human papillomavirus genotypes in cervical specimens of HIV-1-infected women without history of cervical pathology.

AIM: To study the epidemiology of different human papillomavirus (HPV) genotypes in cervical samples of HIV-1-infected women with normal Papanicolau smears. DESIGN: : Retrospective analysis of a prospective cohort. PATIENTS AND METHODS: We selected HIV-1-infected women with 2 consecutive normal Papanicolau smears at baseline and at least 1 baseline and 1 follow-up cervical sample. HPV infection was assessed by second-generation hybrid capture (HC-2) and multiplex polymerase chain reaction (mPCR). HPV genotypes were determined by mPCR. RESULTS: From a cohort of 139 women followed up to 4 years, 93 women meeting the inclusion criteria were analyzed. The mean period between samples was 20 months (range, 6-44 months). HPV baseline prevalence was 63% [59/93; 95% confidence interval (CI), 53% to 73%] using polymerase chain reaction and 41% (38/93; 95% CI, 31% to 51%) using HC-2, P = 0.007 (kappa, 0.45; P = 0.001). The most prevalent high oncogenic risk genotypes (HR-HPV) were HPV-16 (28%), HPV-33 (18%), HPV-52 (12%), HPV-58 (11%), and HPV-39 (11%). Infection with multiple HPV genotypes was detected in >40% of women. HPV infection persisted at follow-up in 86% (51/59; 95% CI, 77% to 95%) by polymerase chain reaction and 76% (29/38; 95% CI, 62% to 90%) by HC-2. HPV infection persisted in 55% of women with samples available beyond 3 years. The actuarial probabilities of clearance and incidence of HPV infection at 36 months were 16% and 45%, respectively. CONCLUSIONS: HPV infection is highly prevalent and persistent among HIV-1-infected women with normal Papanicolau smears. HR-HPV genotypes other than HPV-16 (HPV-33, HPV-52) are frequently detected in HIV-infected women. mPCR provides better surveillance of HPV infection than HC-2 methods.

HIV endocytosis after dendritic cell to T cell viral transfer leads to productive virus infection.

Contacts between HIV-producing T cells and primary CD4+ T cells may induce the uptake of HIV by target cells that are endocytosed into trypsin-resistant compartments. We have now compared the mechanism of virus transmission from T cell-to-T cell versus infected dendritic cells (DCs)-to-T cell. In cocultures of HIV-1-infected DCs with primary CD4+ T cells, virus transmission to the target cells was resistant to trypsin treatment and could only be prevented by the anti-SUgp120 antibody IgGb12 but not by TAK-779, C34 or AZT. Importantly, upon stimulation of purified HIV-1-loaded CD4+ T cells with PHA/IL-2, cells became productively infected as measured by intracellular CAp24 staining and antigen determination in the cell supernatant. These results suggest that the viral endocytic transfer may represent a escape mechanism in the presence of drugs targeting HIV-1 entry or the host immune system.