Humoral and cellular immune memory response 12 years following single dose vaccination against hepatitis A in Argentinian children.

Infants' universal hepatitis A virus (HAV) single-dose vaccination has been highly effective for controlling HAV infection in Argentina, and in other Latin-American countries that adopted that strategy. Although antibodies wane over time, this has not been associated with HAV outbreaks or breakthrough infections, suggesting a relevant role for memory immunity. This study assessed long term humoral and cellular immune memory response after an average of 12 years follow-up of HAV single-dose vaccination. We selected 81 HAV-single dose vaccinated individuals from a 2015 study, including 54 with unprotective (UAL) and 27 with protective antibody levels (PAL) against HAV. Humoral memory response was assessed by measuring anti-HAV antibody titers at admission in both groups, and 30 days after a booster dose in the UAL group. Flow cytometry analysis of peripheral blood mononuclear cell samples stimulated with HAV antigen was performed in 47/81 individuals (21 with PAL, 26 with UAL) to identify activated CD4 + memory T cells or CD8 + memory T cells. The results showed that 48/52 (92%) individuals from UAL group who completed follow up reached protective levels after booster dose. In the PAL group, anti-HAV Abs waned in 2/27 (7%) individuals lacking seroprotection, while in 25/27 (93%) Abs remained >10 mUI/mL. HAV-specific memory CD4 + T cells were detected in 25/47 (53.2%) subjects while HAV-specific memory CD8 + T cells were observed in 16/47 (34.04%) individuals. HAV-specific memory CD4+ and CD8+ T cell responses were detected in 11/21 (52.4%) and in 9/21 (42.9%) subjects with PAL and in 14/26 (53.8%) and in 7/26 (26.9%) individuals with UAL, showing that the presence of memory T-cells was independent of the level or presence of anti-HAV antibodies. Long-term immunity demonstrated in the present work, including or not antibody persistence, suggests that individuals with waned Ab titers may still be protected and supports the single-dose HAV strategy.

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.

Intra-host evolution of multiple genotypes of hepatitis C virus in a chronically infected patient with HIV along a 13-year follow-up period.

The intra-host evolutionary process of hepatitis C virus (HCV) was analyzed by phylogenetic and coalescent methodologies in a patient co-infected with HCV-1a, HCV-2a, HCV-3a and human immunodeficiency virus (HIV) along a 13-year period. Direct sequence analysis of the E2 and NS5A regions showed diverse evolutionary dynamics, in agreement with different relationships between these regions and the host factors. The Bayesian Skyline Plot analyses of the E2 sequences (cloned) yielded different intra-host evolutionary patterns for each genotype: a steady state of a "consensus" sequence for HCV-1a; a pattern of lineage splitting and extinction for HCV-2a; and a two-phase (drift/diversification) process for HCV-3a. Each genotype evolving in the same patient and at the same time presents a different pattern apparently modulated by the immune pressure of the host. This study provides useful information for the management of co-infected patients and provides insights into the mechanisms behind the intra-host evolution of HCV.

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

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.

Oral cavity and extra-oral plasmablastic lymphomas in AIDS patients: report of five cases and review of the literature.

Plasmablastic lymphoma (PBL) is a distinct disease entity of the diffuse large B-cell lymphoma, which often occurs in HIV-positive patients. The immunophenotype of this lymphoid neoplasm is characterized by the presence of plasma cell-associated markers VS38c and CD138 antigens and the absence of B-cell markers such as CD20 and CD45. The most frequent site of involvement is the oral cavity and the jaw, while several reports describe the development of PBL in extra-oral sites including the lymph nodes, the anal canal, the soft tissue, the skin and the gastrointestinal tract as less frequent. Epstein-Barr virus is often associated with PBL pathogenesis and the neoplastic cells contain this virus genome. Here we review the epidemiological, clinical, immunological, histopathological and virological characteristics and their prognosis and outcome in a series of five patients with diagnoses of HIV/AIDS and PBL.

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

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.

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

UNLABELLED: 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.

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

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 +/- 117.8 cells microL(-1)) than those with HCV-positive viraemia (819.1 +/- 40.72 cells microL(-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.

Reservoirs of HIV replication after successful combined antiretroviral treatment.

Sustained reduction of viral replication can be achieved in HIV infected patients after treatment with combinations of drugs (HAART) that inhibit the viral reverse transcriptase, and protease enzymes. However, replication competent virus can still be recovered from latently infected resting memory CD4+ T-cell lymphocytes. Moreover, "covert" virus replication has been demonstrated in patients who experienced reductions in plasma viremia to levels below the limit of detection of the most sensitive PCR assays. In most studies, preferential attention has been given to latent resting CD4+ T-lymphocytes as a source of HIV persistence. However, insufficient suppression of HIV replication could also lead to viral re-emergence after HAART interruption. In addition to CD4+ T- lymphocytes, other host cells such as long-lived resident macrophages or recently infected blood monocytes could also contribute to maintain persistent HIV replication after HAART. Establishing the origin of re-emerging HIV in patients under HAART upon treatment interruption is important to design optimal treatment schemes. Therapeutic strategies aimed at reducing the number of latently infected cells involve immune activation with IL-2, or other stimulatory factors, in the presence of antiretroviral drugs. Elimination of replication-competent virus would require intensification of HAART, or the use of antiretroviral drugs achieving an effective concentration at the site of HIV replication. In this review the mechanisms of HIV persistence and the methods that can be used to distinguish latent from covert HIV replication in different cell types will be discussed.

Immortalized Epstein-Barr virus-positive B-cell lines obtained by prolonged culture of peripheral blood mononuclear cells from human immunodeficiency virus type 1-positive patients.

OBJECTIVES: To study the factors that determine malignant B cell growth in human immunodeficiency virus type 1 (HIV-1)-infected patients. STUDY DESIGN: B-cell lines (lymphocyte cell lines [LCL]) were developed after nonstimulated culture of peripheral blood mononuclear cells (PBMC) from HIV-1-positive (HIV-1(+)) patients. Human immunodeficiency virus type 1 replication in culture, Epstein-Barr virus (EBV) latent oncogene expression, and cell-to-cell interaction were studied after nonstimulated culture of HIV-1(+) PBMC, analyzing their contribution to LCL appearance. METHODS: Nonstimulated PBMC cultures of HIV-1(+) PBMC and controls (N-PBMC) were established. Lymphocyte cell lines were characterized. Epstein-Barr virus latent membrane protein 1 (LMP-1) and Epstein-Barr nuclear antigen 2 were detected by polymerase chain reaction (PCR). Clonality of LCL was determined by light chain restriction (flow cytometry) and immunoglobulin H chain rearrangement (semi-nested PCR). Peripheral blood mononuclear cell phenotypes were studied at different intervals of culture. RESULTS: Lymphocyte cell lines were obtained in 73% of HIV-1(+) PBMC cultures, compared with 6% in N-PBMC. All LCL were EBV-positive (EBV(+)). B-cell lineage was established, and up to 12 different B-cell clones were expanded from the same individual. Occurrence of LCL was more frequent in cultures with HIV-1 replication, high LMP-1 expression in viable B cells, and high CD4:CD8 ratio. Human immunodeficiency virus type 1 replication persisted in 53% of the LCL. CONCLUSIONS: In vitro HIV-1 replication and persistence of viable EBV(+) lymphoblasts favor spontaneous in vitro outgrowth of LCL in HIV-1(+) patients.

Monocyte differentiation and HIV replication after prolonged culture of peripheral blood mononuclear cells from HIV-infected individuals.

Primary cultures of peripheral blood mononuclear cells (PBMC) from 51 HIV+ hemophiliac patients (HIV+ PBMC) were set up, allowing undisturbed cellular interaction in the absence of any exogenous stimuli. The optimum time for p24 detection was between 12 and 25 days. Infective virus was recovered from the culture supernatants (HIV+ SN) and the amount of p24 released ranged from 25 to 5300 pg/ml. Cells of the monocyte/macrophage (M/M) lineage were the main source of HIV in the HIV+ SN, as judged by intracellular staining of permeabilized cells with anti-p24 (KC57 monoclonal antibody) and flow cytometry analysis. M/M activation, differentiation, and proliferation occurred along the culture before the peak of in vitro HIV replication. Release of HIV p24 was highest in patients with >200 CD4+ T lymphocytes/mm3 who did not receive highly active antiretroviral therapy (HAART), but it was still detectable in 60-90% of patients who had responded to 1-2 years of HAART, reducing their plasma viral load to undetectable levels. It is proposed that this simple experimental system can be used to assess ongoing HIV infection of M/M with the patient's own viral variants.

[HIV infected macrophages isolated from HIV+ patients with undetectable viral load undergoing combined antiretroviral treatment]. / Macrófagos infectados por HIV aislados de pacientes HIV+ con carga viral indetectable luego del tratamiento antirretroviral combinado.

A new method of culture of peripheral blood leukocytes (PBMC) from HIV+ patients, in the absence of exogenous stimuli (allogeneic cells or cytokines) (PBMC w/s) was used for the detection of persistent viral infection in HIV patients who had undergone successful highly active antiretroviral therapy (HAART) lowering their viral burden to undetectable levels (< 50 RNA copies/ml). Infected cells were always of the monocyte/macrophage lineage (M). No infection could be detected in these patients using the classical system (co-culture with HIV-CMP activated with PHA and IL-2). Differences in the class of target cells (higher proportion of proliferating M and CCR5 expressing cells in the PBMC w/s system than in PBMC-PHA cultures) may determine the relative sensitivity of each technique to achieve successful isolation of HIV from different patients.

Prescribing rates for psychotropic medication amongst east London general practices: low rates where Asian populations are greatest.

OBJECTIVES: The aim of this study was to examine the contribution of Asian ethnicity to the variation in rates of practice prescribing for antidepressant and anxiolytic medication, taking into account other population and practice organizational factors. METHODS: A practice-based cross-sectional survey was carried out of the prescribing of antidepressants and anxiolytics (daily defined dosages) in 164 general practices. The study was set in East London and the City Health Authority, which includes the multiethnic inner London boroughs of Hackney, Tower Hamlets, Newham and the City of London. The main outcome measures were the annual prescribing rates for each group of drugs, calculated as the total annual daily defined dosages divided by the practice population, and the ratio of antidepressant/ anxiolytic annual prescribing rates. RESULTS: Prescribing rates for antidepressants showed a 25-fold variation between practices; this was greater for anxiolytics. The median annual prescribing rate for all antidepressants combined was 4.13 (interquartile range 2.50-5.88). For all anxiolytics and hypnotics combined the median annual prescribing rate was 3.55 (interquartile range 1.71-6.36). Univariate analysis showed that Asian ethnicity alone accounted for 28% of the variation in antidepressant prescribing and 20.5% of the variation in the anxiolytic prescribing. A backwards multiple regression model using 10 explanatory practice and population variables accounted for 47.7% of the variance in antidepressant prescribing and 34% of the variance in the anxiolytic prescribing. CONCLUSION: In practices where the proportion of Asian patients is high, both antidepressant and anxiolytic prescribing is low. This is important for understanding interpractice prescribing variation and for setting levels of drug budgets. This study confirms that the low rates of non-psychotic disorders presented by Asian populations is not a selective feature of access to secondary care, but is evident in the prescribing behaviour of GPs. Uncertainty remains as to how much this is due to a lower prevalence rate, "culture-bound syndromes" or practical difficulties in diagnosis and management within the general practice setting.

[Cerebrospinal fluid viral load in HIV-1 positive hemophilic patients treated with HAART]. / Carga viral en líquido cefalorraquídeo de pacientes hemofilicos HIV-1 positivos bajo tratamiento con HAART.

As HIV seropositive patients with undetectable CSF viral load have a lower likelihood of developing neurologic disease, the determination of CSF viral load levels may be useful to evaluate the efficacy of HAART. We compared plasma viral load levels with HIV-1 RNA CSF levels in 18 hemophilic patients without neurocognitive involvement under HAART. We detected a significant correlation between plasma viral load levels and CSF viral load levels. Fourteen patients with undetectable plasma viral load had undetectable RNA HIV-1 CSF levels as well. Four patients with detectable plasma viral load had detectable HIV-RNA in CSF, but the latter were significantly lower. Viral load is usually lower in non-blood fluids and HAART decreases the viral load in CSF as well as in blood.

Carga viral en líquido cefalorraquídeo de pacientes hemofilicos HIV-1 positivos bajo tratamiento con HAART. / [Cerebrospinal fluid viral load in HIV-1 positive hemophilic patients treated with HAART]

As HIV seropositive patients with undetectable CSF viral load have a lower likelihood of developing neurologic disease, the determination of CSF viral load levels may be useful to evaluate the efficacy of HAART. We compared plasma viral load levels with HIV-1 RNA CSF levels in 18 hemophilic patients without neurocognitive involvement under HAART. We detected a significant correlation between plasma viral load levels and CSF viral load levels. Fourteen patients with undetectable plasma viral load had undetectable RNA HIV-1 CSF levels as well. Four patients with detectable plasma viral load had detectable HIV-RNA in CSF, but the latter were significantly lower. Viral load is usually lower in non-blood fluids and HAART decreases the viral load in CSF as well as in blood.

Macrófagos infectados por HIV aislados de pacientes HIV+ con carga viral indetectable luego del tratamiento antirretroviral combinado. / [HIV infected macrophages isolated from HIV+ patients with undetectable viral load undergoing combined antiretroviral treatment]

A new method of culture of peripheral blood leukocytes (PBMC) from HIV+ patients, in the absence of exogenous stimuli (allogeneic cells or cytokines) (PBMC w/s) was used for the detection of persistent viral infection in HIV patients who had undergone successful highly active antiretroviral therapy (HAART) lowering their viral burden to undetectable levels (< 50 RNA copies/ml). Infected cells were always of the monocyte/macrophage lineage (M). No infection could be detected in these patients using the classical system (co-culture with HIV-CMP activated with PHA and IL-2). Differences in the class of target cells (higher proportion of proliferating M and CCR5 expressing cells in the PBMC w/s system than in PBMC-PHA cultures) may determine the relative sensitivity of each technique to achieve successful isolation of HIV from different patients.

Nonhepatosplenic gamma delta T-cell lymphoma with initial testicular compromise.

We report here a case of nonhepatosplenic gammadelta T-cell lymphoma with undescribed initial localization in testis, without hepatosplenomegaly or adenopathies, and subsequent development in the maxillary sinus. The maxillar mass biopsy revealed a T-cell infiltration, and its immunologic characterization by flow cytometry showed a gammadelta T-cell phenotype (CD45+, CD3+, CD2+, TCR gammadelta+), without expression of CD7, CD5, CD1a, TdT, CD4, CD8, TCR alphabeta, or NK antigens (CD16, CD56, and CD57). Clonal gamma-chain gene rearrangement by polymerase chain reaction (PCR) was detected in testicular and maxillar biopsies. Epstein-Barr virus type 1 (EBV) sequences were detected by molecular biology in the biopsy material, suggesting that this oncogenic virus may play a role in the genesis of the clonal expansion of gammadelta T-cells. The patient was initially treated with standard chemotherapeutic protocols, with poor response and aggressive course.

Prevalence of anti-hepatitis A antibodies in an urban middle class area of Argentina: some associated factors.

OBJECTIVE: This study evaluated the seroprevalence of hepatitis A virus (HAV) antibodies in 360 middle-class subjects from Buenos Aires City and its outskirts. METHODS: The study population included 360 individuals between 10 and 89 years of age, from the socioeconomic middle class in Buenos Aires City and some suburban areas of Buenos Aires province. Antibodies to hepatitis A virus were determined by enzyme immunoassay test kits. RESULTS: The overall prevalence of HAV antibodies was 42.2%. The highest percentage of seronegativity was found in the subgroup of younger people without a history of symptomatic hepatitis and living in houses with more than one bathroom (86.9%). In the subgroup aged 21 to 60 years, the highest rates of seronegativity were found in individuals with higher level of education living in houses with tap water (66.6%). In both groups, seronegativity may be correlated with a higher socioeconomic status. CONCLUSIONS: In the middle-class community studied, more than 50% of people under 30 years of age were unprotected against HAV. Thus, the use of a vaccine against hepatitis A has to be considered for the prevention of symptomatic hepatitis, especially in adults at risk of infection, such as those who travel to areas with poor sanitation, taking into consideration that the severity of the disease increases with age.

[Apoptosis and human immunodeficiency virus infection (HIV]. / Apoptosis e infección por el virus de la inmunodeficiencia humana (HIV).

Generalized activation of the immune system after HIV infection leads to an exacerbation of all apoptotic mechanisms. CD4+, CD8+ T lymphocytes and B lymphocytes are sensitized to apoptotic stimuli. Macrophages are important in the removal of apoptotic cells, they prevent apoptotic cell accumulation during in vitro culture and they may lead to enhanced CD4+ T lymphocyte cell death through indirect mechanisms. A simple procedure for prolonged culture of peripheral blood mononuclear cells from HIV+ patients is discussed, in relation to its convenience to evaluate apoptosis, cell to cell interaction and HIV replication in the absence of exogenously added stimuli or co-culture of allogeneic cells. In this system, HIV replication takes place primarily in cells of macrophage lineage that may be activated into differentiation through removal of apoptotic debris during the culture.

Apoptosis e infección por el virus de la inmunodeficiencia humana (HIV). / [Apoptosis and human immunodeficiency virus infection (HIV]

Generalized activation of the immune system after HIV infection leads to an exacerbation of all apoptotic mechanisms. CD4+, CD8+ T lymphocytes and B lymphocytes are sensitized to apoptotic stimuli. Macrophages are important in the removal of apoptotic cells, they prevent apoptotic cell accumulation during in vitro culture and they may lead to enhanced CD4+ T lymphocyte cell death through indirect mechanisms. A simple procedure for prolonged culture of peripheral blood mononuclear cells from HIV+ patients is discussed, in relation to its convenience to evaluate apoptosis, cell to cell interaction and HIV replication in the absence of exogenously added stimuli or co-culture of allogeneic cells. In this system, HIV replication takes place primarily in cells of macrophage lineage that may be activated into differentiation through removal of apoptotic debris during the culture.