Liliana Belmonte

The intestinal mucosa as a reservoir of HIV-1 infection after successful HAART.

The presence of HIV-1 RNA in distal duodenal mucosa was evaluated in 44 HIV-1-positive patients. HIV-1 RNA was detected in gut tissue in antiretroviral-naive patients with high plasma viral loads, as well as in patients on HAART with plasma viral loads below the limit of detection and in patients on HAART with virological failure. The intestinal mucosa seems to serve as a reservoir poorly influenced by levels of plasma viral load or HAART.

Macrophage HIV-1 infection in duodenal tissue of patients on long term HAART.

Mucosal surfaces play a major role in human immunodeficiency virus type 1 (HIV-1) transmission and pathogenesis. Since the role of intestinal macrophages as viral reservoirs during chronic HIV-1 infection has not been elucidated, we investigated the effects of successful therapy on intestinal HIV-1 persistence. Intestinal macrophage infection was demonstrated by the expression of p24 antigen by flow cytometry and by the presence of proviral DNA, assessed by PCR. Proviral DNA was detected in duodenal mucosa of HIV-infected patients under treatment with undetectable plasma viral load. These findings confirm that intestinal macrophages can act as viral reservoirs and permit HIV-1 production even after viral suppression following antiretroviral therapy.

Expression of Signaling Lymphocytic Activation Molecule- Associated Protein Interrupts IFN-γ Production in Human Tuberculosis

Production of the Th1 cytokine IFN-γ by T cells is considered crucial for immunity against Mycobacterium tuberculosis infection. We evaluated IFN-γ production in tuberculosis in the context of signaling molecules known to regulate Th1 cytokines. Two populations of patients who have active tuberculosis were identified, based on their T cell responses to the bacterium. High responder tuberculosis patients displayed significant M. tuberculosis-dependent T cell proliferation and IFN-γ production, whereas low responder tuberculosis patients displayed weak or no T cell responses to M. tuberculosis. The expression of the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) on cells from tuberculosis patients was inversely correlated with IFN-γ production in those individuals. Moreover, patients with a nonfunctional SAP gene displayed immune responses to M. tuberculosis similar to those of high responder tuberculosis patients. In contrast to SAP, T cell expression of SLAM was directly correlated with responsiveness to M. tuberculosis Ag. Our data suggest that expression of SAP interferes with Th1 responses whereas SLAM expression contributes to Th1 cytokine responses in tuberculosis. The study further suggests that SAP and SLAM might be focal points for therapeutic modulation of T cell cytokine responses in tuberculosis.

HCV recovery from peripheral blood mononuclear cell culture supernatants derived from HCV–HIV co-infected haemophilic patients with undetectable HCV viraemia

Summary.  Hepatitis C viraemia, in 38 human immunodeficiency virus positive (HIV+)/hepatitis C virus positive (HCV+) patients, was determined in haemophilic patients during the 4 years since initiation of highly active antiretroviral therapy (HAART). Six of 38 patients had persistently HCV‐negative viraemia for more than 2 years. No correlation between HCV‐negative viraemia and CD4+ T‐cell counts, HIV viral load, age, type or severity of haemophilia could be established. Reduced levels of HIV viral load and the immune reconstitution that follows the initiation of HAART were not enough to explain the disappearance of HCV from plasma. Individuals who cleared plasma HCV had significantly higher CD8+ T‐cell counts (P = 0.0013) (mean ± SE: 1153 ± 117.8cells μL−1) than those with HCV‐positive viraemia (819.1 ± 40.72 cells μL−1). Because HCV could maintain a low replication level in peripheral blood mononuclear cells (PBMC), we cultured PBMC of five of six patients with undetectable HCV viraemia. We found four of five HCV RNA‐positive cultures. The presence of HCV RNA in our cultures proved that these cells may be an important viral reservoir that could contribute to HCV recurrence in plasma even after long periods of negative viraemia. In summary, our results indicate that in spite of prolonged HCV‐negative plasma viraemia, HCV patients that are co‐infected with HIV may harbour replication‐competent HCV in their PBMC. Therefore, true clearance of HCV infection is difficult to achieve in these patients.

HIV-1 Infection and Chemokine Receptor Modulation

In addition to the CD4 molecule that binds to the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp120, productive HIV-1 infection requires interaction with cellular receptors for alpha- or beta- chemokines (CXCR4 and CCR5 respectively). Isolates of HIV-1 exhibit different tropism depending on the chemokine receptor type that they use to infect their cellular targets. HIV-1 strains that use preferentially CCR5 are known as R5 strains. They are more frequently found in asymptomatic individuals during the initial stages of the disease and are involved in the transmision of infection from mother to child. HIV-1 species using CXCR4 (X4 strains) are observed mainly in patients with advanced disease. While X4 isolates are associated with syncitium formation, in general R5 strains are not. Interaction of X4 and R5 with their specific receptors is necessary to establish productive HIV-1 infection and trigger a series of intracellular signals. Modulation of CXCR4 and CCR5 expression after HIV-1 infection is one of the results of such interaction and may have important consequences on the course of the infection. Down regulation of CCR5 and CXCR4 after HIV-1 infection could be the result of indirect events linked to HIV-1 infection, such as the induction of alpha- or beta-chemokines competing with the virions for receptor binding. They could also reflect direct effects of HIV-1 on chemokine-receptor turnover. In this review, the mechanisms of modulation of CXCR4 and CCR5 expression after HIV-1 infection will be discussed.

Impact of Human Immune Deficiency Virus Infection on Hepatitis C Virus Infection and Replication

Human immune deficiency virus (HIV) and human hepatitis C virus (HCV) infection are frequent in patients who have been exposed to blood or blood-derived products. It has been suggested that HIV infection increases HCV replication altering the course of HCV-related disease. However, it is not known if HIV directly enhances HCV replication or if its effect is the consequence of HIV infection of other cell types that control HCV replication (lymphocytes, macrophages). While the main cell targets for HIV infection are mononuclear leukocytes bearing CD4 and the chemokine receptors CCR5 and CXCR4, HCV was originally thought to be strictly hepatotropic, but it is now known that HCV can also replicate in peripheral blood mononuclear cells (PBMC). Therefore, in co-infected individuals, these two different viruses could share cell targets and interact either directly or indirectly. Some membrane receptors can be used by both HCV and HIV for entry into target cells, but the intracellular mechanisms shared by these viruses are not known. Lack of experimental systems providing suitable methods for the study of HCV replication in the presence or absence of HIV co-infection has hampered advances in this research area, but recent investigations are currently going on in order to answer these questions. This is an important issue, as knowledge of HIV/HCV interactions is required for the design of effective antiviral therapies.

Decreased recovery of replication-competent HIV-1 from peripheral blood mononuclear cell-derived monocyte/macrophages of HIV-positive patients after 3 years on highly active antiretroviral therapy.

We studied the release of p24 antigen from peripheral blood mononuclear cell-derived monocyte/macrophages obtained from 13 HIV-positive patients receiving highly active antiretroviral therapy (HAART). Although HIV-infected monocyte/macrophages were detected in 80% of patients after 36 months of continuous treatment, additional exposure to HAART reduced the chance of detecting HIV-releasing monocyte/macrophages. Therefore, after more than 3 years of HAART, recently infected monocytes may play a less important role as a source of emerging HIV-1 upon HAART interruption.

Factors involved in the generation of memory CD8+ T cells in patients with X-linked lymphoproliferative disease (XLP)

We have analysed the phenotype of T lymphocytes in two X‐linked lymphoproliferative disease (XLP) patients with the same SH2D1A mutation differing in initial exposure to Epstein–Barr virus (EBV) and treatment. While memory T lymphocytes (with low CCR7 and CD62L expression) prevailed in both XLP patients, in patient 9, who developed acute infectious mononucleosis (AIM) and received B cell ablative treatment, the predominant phenotype was that of late effector CD8 T cells (CD27–, CD28–, CCR7–, CD62L–, CD45 RA+, perforin+), while in patient 4 (who did not suffer AIM) the prevalent phenotype of CD8 T lymphocytes was similar to that of normal controls (N) or to that of adult individuals who recovered from AIM: CD27+ , CD28+, CCR7–, CD62L–, CD45 RO+ and perforin–. CD57 expression (related to senescence) was also higher in CD8 T cells from patient 9 than in patient 4, AIM or N. Persistently high EBV viral load was observed in patient 9. The results obtained from this limited number of XLP patients suggest that events related to the initial EBV encounter (antigen load, treatment, cytokine environment) may have more weight than lack of SH2D1A in determining the long‐term differentiation pattern of CD8 memory T cells.

Increased lymphocyte viability after non-stimulated peripheral blood mononuclear cell (PBMC) culture in patients with X-linked lymphoproliferative disease (XLP)

Survival of lymphocytes after prolonged culture was studied in two asymptomatic XLP patients. Viability of XLP PBMC after 30 days of non-stimulated culture was higher than that of normal controls (N), mainly due to the persistence of CD8 memory lymphocytes. IFNgamma high CD8 T lymphocytes remained higher in XLP than in N after 30 days. The number of perforin+ CD8 lymphocytes was markedly reduced after 30 days in XLP and in N. Increased viability was not related to CD127, PD-1, CD27, or CD62L expression. Concerning B lymphocytes, memory CD27+ CD19+ cells prevailed over CD27- cells after 30 days in both XLP and N, with far more surviving cells in XLP. In N, few CD19+ B lymphocytes were viable after prolonged culture. In XLP, these cells were also IgD+, IgM+ and EBNA2+. These results demonstrate that IFNgamma-positive memory CD8 T cells persist in XLP after prolonged culture in association with a subset of viable memory CD27+ B cells expressing latent EBV antigens. The survival advantage of XLP cells might be related to increased frequency of extranodal lymphoma in XLP patients.