Interferon-γ and IL-10 Release Assay for Patients with Ocular Toxoplasmosis.

Peripheral blood mononuclear cells (PBMC) from patients with ocular toxoplasmosis were challenged with total antigens from Toxoplasma gondii lysate (TATL) in a cytokine release assay (CRA), run during the inactive period of the disease. Increased interferon gamma (IFN-γ) levels were detected after PBMC stimulation with either ME49 reference strain (P = 0.0015) or local TgCkAr-11-9 isolate (P = 0.0012), as compared with those recorded under basal conditions. TATL from TgCkAr11-9 isolate induced a higher release of IFN-γ than ME49 strain in CRA from all tested patients (P = 0.02). The median value of IFN-γ release on TgCkAr-11-9 stimulation (26.03 pg/mL) allowed the classification of patients into high- or low-/non-IFN-γ releasers. Clinical correlations were established with both groups. The results obtained in this study suggest the need to include local strains when performing CRA with TATL.

F127 poloxamer effect on cytotoxicity induction of tumour cell cultures treated with doxorubicin.

INTRODUCTION: Hepatocellular carcinoma is the most common liver malignancy and the third leading cause of cancer death worldwide. One crucial limitation in the pharmacotherapy for this tumour is its chemotherapy-resistant nature produced by the overexpression of several members of the ATP-binding cassette protein family that efflux drugs out of cells, as observed with the breast cancer resistant protein (BCRP). OBJECTIVES: This study aimed to assess the ability of Pluronic® F127 to reverse the multidrug resistance phenotype in two human hepatocellular cell lines. METHODS: PLC/PRF/5 and SKHep1 cells were exposed to Pluronic® F127 at several concentrations. The effect of F127 on BCRP expression (mRNA and protein), mitochondrial transmembrane potential and cell hypodiploidy was assessed. Finally, the effect of this copolymer on cytotoxicity of doxorubicin in both hepatoma cell lines was investigated, as expressed by its reverse resistance index. KEY FINDINGS: It was demonstrated that F127 in both cell lines contributes to chemosensitization, as shown by BCRP down-regulation, an altered mitochondrial transmembrane potential and hypodiploidy and reverse resistance index values. A remarkable dependence of these effects significantly correlated with the copolymer concentration. CONCLUSIONS: These findings further uncover the potential usefulness of this copolymer as multidrug resistance reversal agent, increasing the efficacy of cancer therapies.

Hepatitis C virus strategies to evade the specific-T cell response: a possible mission favoring its persistence.

Hepatitis C virus (HCV) is a small, enveloped RNA virus. The number of HCV-infected individuals worldwide is estimated to be approximately 200 million. The vast majority of HCV infections persist, with up to 80% of all cases leading to chronic hepatitis associated with liver fibrosis, cirrhosis, and hepatocellular carcinoma. The interaction between HCV and the host have a pivotal role in viral fitness, persistence, pathogenicity, and disease progression. The control of HCV infection requires both effective innate and adaptive immune responses. The HCV clearance during acute infection is associated with an early induction of the innate and a delayed initiation of the adaptive immune responses. However, in the vast majority of acute HCV infections, these responses are overcome and the virus persistence almost inexorably occurs. Recently, several host- and virus-related mechanisms responsible for the failure of both the innate and the adaptive immune responses have been recognized. Among the latter, the wide range of escape mutations to evade the specific-T-and B-cell responses as well as the T cell anergy and the CD8+ T cell exhaustion together with the interference with its function after prolonged virus exposure hold a pivotal role. Other HCV strategies include the modification or manipulation of molecules playing key roles in the induction of the interferon response and its induced effector proteins. In this review, we attempt to gain insights on the main T cell immune evasion strategies used by the virus in order to favor its persistence.

Effect of several PEO-PPO amphiphiles on bax, bcl-2, and hTERT mRNAs: An insight into apoptosis and cell immortalization induced in hepatoma cells by these polymeric excipients.

UNLABELLED: Recent data have shown that synthetic polymers and nanomaterials display phenotypic effects in cells and signal transduction mechanisms involved in inflammation, differentiation, proliferation, and apoptosis. AIM: This article aims to investigate the effect of poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) block copolymers with a wide range of biomedical and pharmaceutical applications on apoptosis and/or cell immortalization, by flow cytometry and multiplex RT-PCR for bax, bcl-2, and human telomerase reverse transcriptase (hTERT). RESULTS: PEO-PPO amphiphiles upregulated bax and hTERT and induced apoptosis of two human hepatoma cell lines. CONCLUSIONS: PEO-PPO block copolymers-considered safe for human use-can drastically alter gene expression profiles of genes related to apoptosis/cell proliferation.

Human pegivirus molecular epidemiology in Argentina: potential contribution of Latin American migration to genotype 3 circulation.

In order to determine the human pegivirus (HPgV) genotypic diversity in Argentina taking into account the potential contribution of human migration from neighboring countries, samples from 130 Argentine injecting drug users, 116 Argentine- and 50 immigrant-pregnant women were analyzed. HPgV RNA prevalence among human immunodeficiency virus (HIV)-positive injecting drug users was similar to HIV-positive pregnant women, as was the case when comparing HIV-negative injecting drug users and HIV-negative pregnant women (P > 0.05). HPgV genotype 2 (HPgV/2) was prevalent among both Argentine injecting drug users and pregnant women, in contrast to HPgV/3 observed among pregnant women from Latin American countries with predominant indigenous populations and who had experienced their initial sexual intercourses--and possibly their source of infection--in those countries (P < 0.01). In addition, HPgV vertical and horizontal transmission was proven by molecular analysis of E2 gene and construction of identity matrixes with epidemiologically non-related isolates. This study shows that human migration from neighboring Latin American countries with predominant indigenous populations might contribute to HPgV/3 circulation in Argentina.

Hepatitis B virus and hepatitis D virus in blood donors from Argentina: circulation of HBsAg and reverse transcriptase mutants.

In Argentina, current procedures to ensure the safety of the blood supply for transfusion include the serologic detection of specific blood-borne infections. The aim of this study was to evaluate the prevalence and the genetic diversity of hepatitis B virus (HBV) and hepatitis D virus (HDV) in blood donor populations from two distantly located Argentine regions. Data from 56,983 blood donations from the Favaloro Foundation, in the city of Buenos Aires (Central Region), and the Central Blood Bank of Misiones Province (Northeast Region) were analyzed. Samples that were reactive for HBsAg were analyzed for HBV-DNA characterization and HDV serological and molecular analysis. The HBV prevalence was 0.12 % for HBsAg and 1.68 % for anti-HBc antibodies in Buenos Aires, and 0.73 % and 8.55 %, respectively, in Misiones. Seventy-seven HBsAg-reactive samples were analyzed by polymerase chain reaction for HBV-DNA. Subgenotypes A2, B2, C2, F1b and F4 (Buenos Aires) and F1b and D3 (Misiones) were detected. Several mutations within the major hydrophilic region of HBsAg, the reverse transcriptase, the basal core promoter, and the precore/core were detected. HDV genotype 1 was identified in Buenos Aires. This study confirms the circulation of several HBV subgenotypes, as well as known and newly identified variants, and the presence of HDV1 in this population. A thorough investigation has to be carried out to evaluate the clinical importance of some of the documented mutations as well as those detected in the HDV1 case.

Molecular epidemiology and putative origin of hepatitis C virus in random volunteers from Argentina.

AIM: To study the subtype prevalence and the phylogenetic relatedness of hepatitis C virus (HCV) sequences obtained from the Argentine general population, a large cohort of individuals was analyzed. METHODS: Healthy Argentinian volunteers (n = 6251) from 12 provinces representing all geographical regions of the country were studied. All parents or legal guardians of individuals younger than 18 years provided informed written consent for participation. The corresponding written permission from all municipal authorities was obtained from each city or town where subjects were to be included. HCV RNA reverse transcription-polymerase chain reaction products were sequenced and phylogenetically analyzed. The 5' untranslated region (5'UTR) was used for RNA detection and initial genotype classification. The NS5B polymerase region, encompassing nt 8262-8610, was used for subtyping. RESULTS: An unexpectedly low prevalence of HCV infection in the general population (0.32%) was observed. Our data contrasted with previous studies that reported rates ranging from 1.5% to 2.5%, mainly performed in selected populations of blood donors or vulnerable groups. The latter values are in keeping with the prevalence reported by the 2007 Argentinian HCV Consensus (approximately 2%). HCV subtypes were distributed as follows: 1a (25%), 1b (25%), 2c (25%), 3a (5%), and 2j (5%). Two isolates ascribed either to genotype 1 (5%) or to genotype 3 (5%) by 5'UTR phylogenetic analysis could not be subtyped. Subtype 1a sequences comprised a highly homogeneous population and clustered with United States sequences. Genotype 1b sequences represented a heterogeneous population, suggesting that this genotype might have been introduced from different sources. Most subtype 2c sequences clustered close to the 2c reported from Italy and Southern France. CONCLUSION: HCV has a low prevalence of 0.32% in the studied general population of Argentina. The pattern of HCV introduction and transmission in Argentina appears to be a consequence of multiple events and different for each subtype.

GB virus C quasispecies detected in plasma and lymphocyte subsets in a natural human infection.

Genomic heterogeneity and quasispecies composition of GB virus C (GBV-C) within plasma and lymphocyte subsets in a naturally infected blood donor were investigated. For this purpose, fragments from the 5' untranslated region (5' UTR) and the E2 gene recovered from plasma, B and T lymphocytes, were cloned and sequenced. A total of 63 clones was analysed: 95.2 % of them (n=60) - obtained from plasma and cells - were assigned to genotype 2b, while only three derived from plasma corresponded to genotyope 3. The G215A transition within this region was present in 90.9 % of the clones from B lymphocytes, but absent in the remaining cell compartments (P<0.01). Apparently, most of the circulating GBV-C quasispecies in this blood donor were related to the viral population infecting CD8(+) T cells, and B cells to a lesser extent. This is the first report showing the quasispecies nature of GBV-C in lymphocyte subsets within peripheral blood mononuclear cells.

Intrahost evolution of HBe antigen-negative hepatitis B virus genomes ascribed to the F genotype: a longitudinal 3 year retrospective study.

The intrahost hepatitis B virus (HBV) genomic evolution process of an HBe antigen (HBeAg)-negative chronic HBV patient (designated RI) was studied. Two nearly full-length direct sequences obtained in 1995 (RI95) and 1998 (RI98) showed: (a) a mutation rate of 2.7x10(-3) nucleotides per site per year; (b) nucleotide changes mainly located at single coding regions (P=0.002); (c) mixed populations; and (d) a predominance of non-synonymous substitutions (P=0.0036). Population heterogeneity was assessed by cloning and sequencing of a fragment spanning nearly half the genome. Two-thirds of the analysed clones exhibited long nucleotide deletions. Pairwise genetic diversity revealed that diversity was higher for RI95 than for RI98 cloned sequences. In conclusion, a highly heterogeneous genomic population circulated within patient RI, which might support the persistence of HBV. Finally, the structure of the deletant genomes suggests that they might serve as intermediates for integration to the host-cell genome.

Design, development and evaluation of a competitive RT-PCR for quantitation of GBV-C RNA.

Although GB virus C (GBV-C) hepatocyte pathogenicity is still controversial, it appears that at least some strains of this virus are lymphotropic. During the past few years, several reports have documented an apparently beneficial role played by GBV-C in the course of HIV-1 infection. At present, a commercial kit for GBV-C RNA quantitation is not available. In this study, a competitive RT-PCR method for GBV-C in serum samples is described. The sensitivity of the assay proved to be 10(4) and 10(3) genomic equivalents for positive and negative sense RNAs, respectively. This method will discriminate specifically between positive and negative strand RNAs with a discrimination index of at least five log10. Out of 60 samples from different hematological disorders (n = 49), HIV-1 positive patients (n = 7), and blood donors (n = 4), 10 proved to be GBV-C RNA positive. Viral load ranged from 1.1 x 10(7) to 2.34 x 10(8) genomic equivalents/ml. Such values correlated linearly (r = 0.986) with those obtained by a 10-fold serial dilution method. In studies exploring the GBV-C pathogenicity, the measurement of viral load may contribute to understand the possible mechanisms involved.

Antígenos inactivados de virus Junín / Inactivated Junín virus antigens

El objetivo de este trabajo fue obtener un antígeno inactivado de virus Junín capaz de inducir protección en cobayos contra el desafío con la cepa XJ prototipo. Se utilizó como antígeno, la cepa XJ-Clon 3 replicada en cerebro de ratón y se ensayaron tres métodos de inactivación: formaldehido, acetona y calor. Con formaldehido se emplearon 3 dosis: concentración final de 0,05% durante 24 h, 0,2% durante 2 n y 0,05% durante 3 h. Las curvas de inactivación demostraron que en el primer caso, el virus se inactiva a la h de exposición mientras que en el segundo se logra ya a los 30 min. Aunque el formaldehido demostró ser un inactivante eficaz, ninguno de los antígenos preparados protegió a los cobayos contra el desafío XJ. No se observó retraso significativo en la fecha de muerte ni modificación en las curvas de peso con respecto a los controles desafiados y sin inmunizar. Todos los animales murieron con el cuadro hemorrágico típico de FHA, excepto el grupo que recibió la dosis máxima de antígeno inactivado con formaldehido en el que se observó mortalidad en ausencia de cuadro hemorrágico. Los antígenos inactivados con calor (37-C 48 h) o con acetona tampoco resultaron eficaces en la protección de cobayos. Solamente los antígenos inactivados con formaldehido desencadenaron una respuesta inmune no protectora, evidenciada por los bajos títulos de anticuerpos inmunofluorescentes y fijadores de complemento; por el contrario, no se detectaron anticuerpos neutralizantes. Se discuten las posibles causas de la no protección así como los riesgos de vacunas a virus vivos y atenuados versus vacunas a virus inactivados para Fiebre Hemorrágica Argentina

Falta de persistencia del virus en dos Cebus sp / Lack of viral persistence in 2 Cebus sp

Dos Cebus sp sobrevivientes a la infección por vía intramuscular e intracerebral con las cepas XJ y XJ Clon 3 virus Junín, respectivamente, no exhibieron persistencia viral. Aunque ambos primates fueron sometidos a tratamiento con drogas inmunosupresoras, so se aisló virus Junín de sangre ni de órganos, aún luego de realizarse pasajes ciegos en ráton para aquélla y cocultivos con células Vero para los últimos. Luego del tratamiento tampoco se detectaron antígenos de virus Junín por inmunofluorescencia en células BHK/21 inoculadas con sangre. Estos hallazgos coinciden con lo observado en el ser humano en quien no se ha descripto viremia más allá del período agudo de la Fiebre Hemorrágica Argentina