Patricia Baré

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.

Evidence of occult HCV genotypes in haemophilic individuals with unapparent HCV mixed infections

Summary.  Individuals with haemophilia who received non heat‐treated factor concentrates were likely to undergo multiple exposures to the hepatitis C virus (HCV). Therefore, HCV mixed‐genotype infections might be more frequent in these patients than in the general population. Their prevalence is extremely variable in similar groups of patients tested by different assays due to the fact that currently available genotyping techniques are not suitable to detect multiple HCV genotypes in a viral population. As an HCV viral reservoir, the peripheral blood mononuclear cell (PBMC) might harbor viral variants distinct from the genotypes detected in plasma. We investigated the presence of HCV genotypes in a group of chronically infected haemophilic patients in the PBMC compartment using a non‐stimulated cell culture system that allows the detection of the HCV genome in culture supernatants. We compared them to the HCV genotypes found in plasma samples. Cell culture experiments performed with PBMC demonstrated the presence of additional HCV genotypes that were undetected in the corresponding plasma samples with the same genotyping technique. Although mixed infections at HCV genotype level became evident in 5.6% of the patients (16/288), the culture methodology increased the number of HCV infections with multiple genotypes to 62.5% (10/16) (P < 0.0001). Once more, the role of mononuclear cells as HCV viral reservoirs is emphasized. Considering minor strains could influence the outcome of treatment, detection of covert HCV mixed‐genotype infections might be essential for choosing the adequate therapeutic regimen.

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.

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.

American tegumentary leishmaniasis: T-cell differentiation profile of cutaneous and mucosal forms—co-infection with Trypanosoma cruzi

American tegumentary leishmaniasis displays two main clinical forms: cutaneous (CL) and mucosal (ML). ML is more resistant to treatment and displays a more severe and longer evolution. Since both forms are caused by the same Leishmania species, the immunological response of the host may be an important factor determining the evolution of the disease. Herein, we analyzed the differentiation and memory profile of peripheral CD4+ and CD8+ T lymphocytes of patients with CL and ML and their Leishmania–T. cruzi co-infected counterparts. We measured the expression of CD27, CD28, CD45RO, CD127, PD-1 and CD57, together with interferon-γ and perforin. A highly differentiated phenotype was reflected on both T subsets in ML and preferentially on CD8+ T cells in CL. A positive trend toward a higher T differentiation profile was found in T. cruzi-infected CL and ML patients as compared with Leishmania single infections. Association between CD8+ T-cell differentiation and illness duration was found within the first year of infection, with progressive increase of highly differentiated markers over time. Follow-up of patients with good response to therapy showed predominance of early differentiated CD8+ T cells and decrease of highly differentiated cells, while patients with frequent relapses presented the opposite pattern. CD8+ T cells showed the most striking changes in their phenotype during leishmaniasis. Patients with long-term infections showed the highest differentiated degree implying a relation between T differentiation and parasite persistence. Distinct patterns of CD8+ T differentiation during follow-up of different clinical outcomes suggest the usefulness of this analysis in the characterization of Leishmania-infected patients.

Hepatitis C virus long-term persistence in peripheral blood mononuclear cells in patients with haemophilia. Detection of occult genotype 1

Peripheral blood mononuclear cells (PBMC) from chronic hepatitis C virus‐infected persons can harbour viral variants that are not detected in plasma samples. We explored the presence and persistence of HCV genotypes in plasma and PBMC cultures from 25 HCV‐monoinfected and 25 HIV/HCV‐coinfected patients with haemophilia. Cell cultures were performed at different time points between 1993 and 2010‐2011, and the HCV genome was examined in culture supernatants. Sequential plasma samples were studied during the same time period. Analysing sequential plasma samples, 21% of patients had mixed‐genotype infections, while 50% had mixed infections determined from PBMC culture supernatants. HIV coinfection was significantly associated with the presence of mixed infections (OR = 4.57, P = 0.02; 95% CI = 1.38–15.1). In our previous study, genotype 1 was found in 72% of 288 patients of this cohort. Similar results were obtained with the sequential plasma samples included in this study, 69% had genotype 1. However, when taking into account plasma samples and the results from PBMC supernatants, genotype 1 was identified in 94% of the population. The PBMC‐associated variants persisted for 10 years in some subjects, emphasizing their role as long‐term reservoirs. The presence of genotype 1 in PBMC may be associated with therapeutic failure and should not be disregarded when treating haemophilic patients who have been infected by contaminated factor concentrates. The clinical implications of persistent lymphotropic HCV variants deserve further examination among multiple exposed groups of HCV‐infected patients.

Intrafamilial transmission of hepatitis C virus in patients with severe haemophilia A

Summary.  The history behind the production of clotting factor concentrates produced differences in the prevalence of Hepatitis C Virus (HCV) and other blood‐borne infections in haemophilic patients. Prevalence rates of HCV infection up to 100% were reported in patients treated with concentrates before 1985. Conversely, nowadays, viral inactivation and recombinant technologies have effectively prevented transfusion‐transmitted viral pathogens. Recently, new HCV infections in three young brothers were observed. In the absence of any other risk of transmission, their HIV/HCV coinfected uncle, who was living in the same house, was subject to study. Plasma samples of the four relatives were investigated in order to test whether the infections have a common source. A phylogenetic approach using the most variable (E2) viral sequences was carried out using samples from the four family members. The HCV sequences from the study resulted highly related, being those obtained from the uncle the most ancestral ones. Because of the chronological order in which the infections occurred and the relatedness of the sequences, an infection from the uncle to his nephews is the most likely explanation. Special cares must be applied in the case of household contact among members of a family with inherited bleeding disorders.

Evolutionary dynamics of Hepatitis C virus in a chronic HIV co-infected patient and its correlation with the immune status

The HCV evolutionary dynamics play a key role in the infection onset, maintenance of chronicity, pathogenicity, and drug resistance variants fixation, and are thought to be one of the main caveats in the development of an effective vaccine. Previous studies in HCV/HIV co-infected patients suggest that a decline in the immune status is related with increases in the HCV intra-host genetic diversity. However, these findings are based on single point sequence diversity measures or coalescence analyses in several virus-host interactions. In this work, we describe the molecular evolution of HCV-E2 region in a single HIV-co-infected patient with two clearly defined immune conditions. The phylogenetic analysis of the HCV-1a sequences from the studied patient showed that he was co-infected with three different viral lineages. These lineages were not evenly detected throughout time. The sequence diversity and coalescence analyses of these lineages suggested the action of different evolutionary patterns in different immune conditions: a slow rate, drift-like process in an immunocompromised condition (low levels of CD4+ T lymphocytes); and a fast rate, variant-switch process in an immunocompetent condition (high levels of CD4+ T lymphocytes).

Progressive reduction of circulating B lymphocytes in patients with X-linked lymphoproliferative disease (XLP).

Fil: Bare, Patricia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina