RESUMO
PURPOSE OF THE STUDY: In ABO-incompatible bone marrow transplantation, an efficient depletion of red blood cells (RBC) within the graft is mandatory to avoid adverse events in transplanted patients. Using non therapeutic products, we evaluated the substitution of the standard density gradient-based separation (DGBS) over Ficoll-Paque with the use of an automated procedure intended for buffy coat only (SmartRedux software) introducing modifications within the settings to achieve a drastic reduction of the initial volume of the product. Both methods were conducted on the Sepax-2 device. SAMPLES AND METHODS: RBC depletion rates and CD34+ cells recoveries from eight procedures with SmartRedux software using "in-house" settings (method A) were compared to those obtained from four procedures using NeatCell software, an automated DGBS over Ficoll-Paque (method B). RESULTS: Median erythrocyte depletion of 95,4% (92,7%-99,0%) and 99,8% (99,0%-99,9%) were observed using methods A and B, respectively. Median residual RBC volumes in the final product were 19 mL (4,4 mL-31,2 mL) and 0,7 mL (0,4 mL-4,7 mL), respectively (p = 0,014). CD34+ cells recoveries of 90,9% (62,7%-102,1%) and 78,4% (64,1%-86,2%) were achieved for methods A and B. Median platelet depletion was 16,6% (10%-42,7%) and 89,8% (88,5%-92,4%) using methods A and B, respectively (p = 0,004). Processing duration was shorter using method A (168 ± 29 min) than method B (295 ± 21 min) (p = 0,004). CONCLUSION: Both methods achieved satisfactory erythrocyte depletion and CD34+ recovery. The use of Sepax-2 device in association with SmartRedux software could be extended to efficiently deplete RBC from large-volume BM in a raw instead of DGBS.
Assuntos
Sistema ABO de Grupos Sanguíneos , Incompatibilidade de Grupos Sanguíneos , Transplante de Medula Óssea/métodos , Separação Celular/instrumentação , Separação Celular/métodos , Eritrócitos/citologia , Reação Transfusional/prevenção & controle , Sistema ABO de Grupos Sanguíneos/sangue , Sistema ABO de Grupos Sanguíneos/imunologia , Adulto , Incompatibilidade de Grupos Sanguíneos/sangue , Incompatibilidade de Grupos Sanguíneos/terapia , Células da Medula Óssea/citologia , Transplante de Medula Óssea/efeitos adversos , Equipamentos e Provisões , Volume de Eritrócitos , Estudos de Viabilidade , Feminino , Ficoll/química , Humanos , Masculino , Reação Transfusional/sangueRESUMO
AIMS: Candida albicans biofilms are commonly associated with severe oral infections. We previously discovered that a crude extract from the Solidago virgaurea plant (SV extract) was a potent inhibitor of C. albicans biofilm formation. Here, we further investigate the mechanisms underlying C. albicans biofilm inhibition by the SV extract. METHODS AND RESULTS: The SV extract was shown to inhibit laboratory and clinical C. albicans isolates adherence and hyphal transition on inert support and epithelial human cells, without affecting viability and growth of planktonic yeasts. Interestingly, RT-PCR-based experiments demonstrated that some key genes involved in adhesion and hyphal morphological switch (e.g. Hwp1p, Ece1p, Als3p) were strongly down-regulated by the SV extract. Moreover, antimicrobial synergy testing (checkerboard assay) demonstrated that antifungal effects of miconazole, nystatin or a common antiseptic mouthwash were synergistically improved when used in combination with the SV extract. CONCLUSIONS: The SV extract prevents C. albicans biofilm formation through direct inhibition of key adherence and hyphae-associated genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm is considered as a key virulence factor of C. albicans infection. Our discovery of an inhibitor specifically acting on genes involved in biofilm formation paves the way for the future development of a new class of antifungal product.
Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Extratos Vegetais/farmacologia , Solidago/química , Antifúngicos/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Expressão Gênica/efeitos dos fármacos , Humanos , Hifas/efeitos dos fármacos , Miconazol/farmacologia , Nistatina/farmacologia , Extratos Vegetais/químicaRESUMO
Epstein-Barr virus (EBV), in addition to its transforming properties, contributes to the pathogenesis of several inflammatory diseases. Here, we investigated its involvement in oral lichen planus (OLP), a common autoimmune-like disease of unknown etiopathogenesis that can display a malignant potential. EBV-infected cells (EBV+ cells) were sought in a large series of clinically representative OLPs ( n = 99) through in situ hybridization to detect small noncoding EBV-encoded RNAs. Overall, our results demonstrated that EBV was commonly found in OLP (74%), with significantly higher frequency (83%) in the erosive form than in the reticular/keratinized type mild form (58%). Strikingly, many erosive OLPs were massively infiltrated by large numbers of EBV+ cells, which could represent a large part of the inflammatory infiltrate. Moreover, the number of EBV+ cells in each OLP section significantly correlated with local inflammatory parameters (OLP activity, infiltrate depth, infiltrate density), suggesting a direct relationship between EBV infection and inflammatory status. Finally, we characterized the nature of the infiltrated EBV+ cells by performing detailed immunohistochemistry profiles ( n = 21). Surprisingly, nearly all EBV+ cells detected in OLP lesions were CD138+ plasma cells (PCs) and more rarely CD20+ B cells. The presence of EBV+ PCs in erosive OLP was associated with profound changes in cytokine expression profile; notably, the expression of key inflammatory factors, such as IL1-ß and IL8, were specifically increased in OLP heavily infiltrated with EBV+ PCs. Moreover, electron microscopy-based experiments showed that EBV+ PCs actively produced EBV viral particles, suggesting possible amplification of EBV infection within the lesion. Our study thus brings conclusive evidence showing that OLP is commonly infiltrated with EBV+ PCs, adding a further puzzling element to OLP pathogenesis, given that PCs are now considered to be major regulatory immune cells involved in several autoimmune diseases (ClinicalTrials.gov NCT02276573).
Assuntos
Herpesvirus Humano 4 , Líquen Plano Bucal/virologia , Plasmócitos/virologia , Adulto , Biópsia , Estudos de Casos e Controles , Citocinas/metabolismo , Feminino , França , Interações Hospedeiro-Patógeno , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Masculino , Microscopia Eletrônica , Estudos RetrospectivosRESUMO
Radiotherapy is advocated in the treatment of cancer of over 50 % of patients. It has long been considered as a focal treatment only. However, the observation of effects, such as fatigue and lymphopenia, suggests that systemic effects may also occur. The description of bystander and abscopal effects suggests that irradiated cells may exert an action on nearby or distant unirradiated cells, respectively. A third type of effect that involves feedback interactions between irradiated cells was more recently described (cohort effect). This new field of radiation therapy is yet poorly understood and the definitions suffer from a lack of reproducibility in part due to the variety of experimental models. The bystander effect might induce genomic instability in non-irradiated cells and is thus extensively studied for a potential risk of radiation-induced cancer. From a therapeutic perspective, reproducing an abscopal effect by using a synergy between ionizing radiation and immunomodulatory agents to elicit or boost anticancer immune responses is an interesting area of research. Many applications are being developed in particular in the field of hypofractionated stereotactic irradiation of metastatic disease.
Assuntos
Efeito Espectador/efeitos da radiação , Neoplasias/radioterapia , Humanos , Radioterapia/métodos , Dosagem RadioterapêuticaRESUMO
Adipose tissue of HIV-1-infected patients shows severe abnormalities such as profound changes in adipose tissue morphology and metabolism. Does HIV-1 infect the adipose cell remains an unsolved question since previous attempts showed that HIV-1 poorly infects human adipocytes in vitro. In the present study, preadipose cells from human subcutaneous fat pads were differentiated in vitro, checked for HIV receptor expression, then infected with R5 and X4 HIV1 strains. Using a sensitive RT-PCR assay, we showed that HIV-1 tat and rev early viral transcripts were expressed in infected adipocytes giving a clear evidence of HIV-1 transcriptional activity in these cells. However, at the same time, no sign of productive infection was demonstrated since infected adipocytes did not efficiently produce Gag p24 antigen. We hypothesized that such a limitation could result from the lack of activation of adipocyte-signaling pathways able to stimulate HIV-1 gene expression in quiescent adipocytes. Indeed, a significant increase in Gag p24 production was observed after stimulation of infected adipocytes with pro-inflammatory cytokines, such as tumor necrosis factor alpha or interleukin-1-beta. Taken together, these results demonstrate that HIV-1 does infect human adipose cells in vitro and suggest that the initial limited infection can be overcome upon pro-inflammatory cytokine treatment.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/virologia , HIV-1/efeitos dos fármacos , Síndrome de Lipodistrofia Associada ao HIV/virologia , Fator de Necrose Tumoral alfa/farmacologia , Replicação Viral/efeitos dos fármacos , Adipócitos/metabolismo , Células Cultivadas , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/fisiologia , Produtos do Gene rev/genética , Produtos do Gene rev/metabolismo , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/fisiologia , Síndrome de Lipodistrofia Associada ao HIV/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Replicação Viral/fisiologia , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We have previously shown that secreted phospholipases A2 (sPLA2) from bee and snake venoms have potent anti-human immunodeficiency virus (HIV) activity. These sPLA2s block HIV-1 entry into host cells through a mechanism linked to sPLA2 binding to cells. In this study, 12 synthetic peptides derived from bee venom sPLA2 (bvPLA2) have been tested for inhibition of HIV-1 infection. The p3bv peptide (amino acids 21 to 35 of bvPLA2) was found to inhibit the replication of T-lymphotropic (T-tropic) HIV-1 isolates (ID(50) = 2 microM) but was without effect on monocytotropic (M-tropic) HIV-1 isolates. p3bv was also found capable of preventing the cell-cell fusion process mediated by T-tropic HIV-1 envelope. Finally, p3bv can inhibit the binding of radiolabeled stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and the binding of 12G5, an anti-CXCR4 monoclonal antibody. Taken together, these results indicate that p3bv blocks the replication of T-tropic HIV-1 strains by interacting with CXCR4. Its mechanism of action however appears distinct from that of bvPLA2 because the latter inhibits replication of both T-tropic and M-tropic isolates and does not compete with SDF-1alpha and 12G5 binding to CXCR4.
Assuntos
Fármacos Anti-HIV/farmacologia , Venenos de Abelha/enzimologia , HIV-1/efeitos dos fármacos , Fosfolipases A/farmacologia , Receptores CXCR4/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Comunicação Celular/efeitos dos fármacos , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , HIV-1/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Fosfolipases A2 , Receptores CXCR4/efeitos dos fármacos , Linfócitos T/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Determining the cis-acting elements controlling nuclear export of RNA is critical, because they specify which RNA will be selected for transport. We have characterized the nuclear export motif of the adenoviral VA1 RNA, a small cytoplasmic RNA transcribed by RNA polymerase III. Using a large panel of VA1 mutants in both transfected COS cells and injected Xenopus oocytes, we showed that the terminal stem of VA1 is necessary and sufficient for its export. Surprisingly, we found that the nucleotide sequence within the terminal stem is not important. Rather, the salient features of this motif are its length and its relative position within the RNA. Such stems thus define a novel and degenerate cytoplasmic localization motif that we termed the minihelix. This motif is found in a variety of polymerase III transcripts, and cross-competition analysis in Xenopus oocytes revealed that export of one such RNA, like hY1 RNA, is specifically competed by VA1 or artificial minihelix. Taken together these results show that the minihelix defines a new cis-acting export element and that this motif could be exported via a novel and specific nuclear export pathway.
Assuntos
RNA Polimerase III/química , RNA/química , Animais , Sequência de Bases , Transporte Biológico , Células COS , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA/genética , RNA/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Especificidade por SubstratoAssuntos
Antivirais/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Lamivudina/uso terapêutico , Fosfato de Vidarabina/uso terapêutico , Adulto , Resistência Microbiana a Medicamentos , Feminino , Hepatite B Crônica/imunologia , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Humanos , Assistência de Longa Duração , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Resultado do TratamentoRESUMO
Mammalian and venom secreted phospholipases A(2) (sPLA(2)s) have been associated with a variety of biological effects. Here we show that several sPLA(2)s protect human primary blood leukocytes from the replication of various macrophage and T cell-tropic HIV-1 strains. Inhibition by sPLA(2)s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA(2)s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA(2)s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA(2) have no effect on HIV-1 infection, suggesting that sPLA(2) catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA(2)s to host cells. Indeed, of 11 sPLA(2)s from venom and mammalian tissues assayed, 4 venom sPLA(2)s were found to be very potent HIV-1 inhibitors (ID(50) < 1 nM) and also to bind specifically to host cells with high affinities (K(0.5) < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA(2)s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA(2)s are being characterized in humans.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Fosfolipases A/farmacologia , Animais , Venenos de Abelha/enzimologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Células Gigantes/efeitos dos fármacos , Fosfolipases A2 do Grupo II , Proteína do Núcleo p24 do HIV/biossíntese , Proteína gp120 do Envelope de HIV/metabolismo , Células HeLa , Humanos , Macrófagos/virologia , Mamíferos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Provírus/efeitos dos fármacos , Provírus/genética , Receptores CCR5/metabolismo , Venenos de Serpentes/enzimologia , Peçonhas/enzimologia , Replicação Viral/efeitos dos fármacosRESUMO
The first and second generations of the Cobas Amplicor HCV assay were compared among patients at risk of hepatitis C virus (HCV) infection. The second-generation test was found to be of greater sensitivity and of good specificity among clinical specimens containing HCV RNA of different genotypes. Finally, this new test is shown to predict the outcome of interferon therapy better.
Assuntos
Hepacivirus/genética , RNA Viral/análise , Hepatite C/terapia , Humanos , Interferons/uso terapêutico , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The purposes of this study were to analyse the prevalence and histologic impact of hepatitis G virus (HGV), a newly discovered virus, in alcoholic patients, a population known to be at risk for viral hepatitis. METHODS: One hundred and thirty-nine consecutive alcoholics admitted to our liver unit (106 men and 33 women; mean age, 47.1 +/- 10.9 years) were included in the study. All patients had consumed more than 60 g of ethanol per day for at least 1 year. One hundred healthy blood donors constituted a control group. Antibodies to HGV E2 protein and HGV-RNA testing by reverse transcription-polymerase chain reaction (RT-PCR) with primers derived from the NS5 coding region were performed in all serum samples. RESULTS: A significantly higher seroprevalence of anti-E2 antibodies was observed in alcoholic patients than in healthy blood donors (41 (29.5%) versus 8 (8%); P < 0.0001). Moreover, the prevalence of HGV-RNA was significantly higher in alcoholic patients (13 (9.3%) versus 1 (1%); P = 0.01). HGV-RNA and anti-HGV antibodies were never detected simultaneously. HGV viraemia was not associated with an increased risk of cirrhosis or hepatocarcinoma in alcoholic subjects. CONCLUSIONS: Our study reports a high prevalence of HGV in alcoholic patients. HGV infection does not modify or aggravate the course of alcoholic liver disease.
Assuntos
Flaviviridae/isolamento & purificação , Hepatite Viral Humana/virologia , Hepatopatias Alcoólicas/virologia , Estudos de Casos e Controles , Feminino , Flaviviridae/imunologia , Hepatite Viral Humana/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estudos Soroepidemiológicos , Viremia/epidemiologia , Viremia/virologiaRESUMO
Polymerase III (pol III)-dependent genes, like the adenoviral VA1 gene, are of particular interest for expressing small therapeutic RNAs into cells. A new VA1 RNA carrier molecule was generated through the deletion of the VA1 RNA central domain to give rise to the VAdeltaIV RNA vector that was devoid of undesirable physiologic activity (i.e., inhibition of the interferon-induced protein kinase, PKR). This vector was used to express in human cells hammerhead ribozymes targeted against the human immunodeficiency virus (HIV). Eight anti-HIV ribozymes were inserted at the 3'-end of this vector immediately before the four T-residues that serve as a transcription termination signal. Although the constructs were active in vitro, they failed to inhibit HIV replication in transient assays. Analysis of the intracellular ribozyme expression in cells revealed several anomalies. First, using mutant derivatives, we showed that the presence of two or three consecutive T-residues in the ribozyme portion was sufficient to promote the release of anomalous short transcripts. Second, when the ribozyme did not contain T-rich sequence, full-length transcripts were produced, but these transcripts were very unstable and were retained in the cell nucleus. In contrast, insertion of the ribozyme in place of the central domain of VA1 RNA led to production of full-length transcripts that were stable and located in the cytoplasm but that were not found to be active in vitro. Taken together, these results have important consequences for the future design of active intracellular ribozymes based on the use of pol III-transcribed genes.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Adenoviridae/genética , RNA Catalítico/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Regiões 3' não Traduzidas/fisiologia , Sequência de Bases , Células Cultivadas , Regulação Viral da Expressão Gênica/fisiologia , Genes Virais/genética , Humanos , Hibridização In Situ , Rim/química , Rim/embriologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Catalítico/análise , RNA Catalítico/biossíntese , RNA Catalítico/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Alinhamento de SequênciaRESUMO
There is an increasing demand for the genotyping of hepatitis C virus (HCV), since it has been shown that different HCV genotypes are associated with distinct profiles of pathogenicity and responses to antiviral treatment. Hence, there is a need for a simple and precise genotyping assay for routine diagnosis of HCV types and subtypes. Here we show that direct sequencing, considered as the reference method, can provide an accurate and rapid method for large-scale screening of HCV genotypes. PCR-amplified cDNAs of the HCV 5' non-coding region (5' NCR) were obtained from the widespread "Amplicor" HCV detection system. Semi-purified PCR products were directly cycle-sequenced in a single tube using multicolour dye terminator chemistry. Sample loading, electrophoresis and sequence analysis were automatically achieved by a capillary electrophoresis-based genetic analyser. Out of a total of 500 samples, HCV subtype 1b accounted for the majority of the infections (41%), followed by HCV 3 (31%) and HCV 1a (22%). This procedure failed to identify a genotype in only 3 samples. In addition, several cases of mixed HCV infection were also documented. The combination of direct cycle sequencing of PCR products with capillary electrophoresis provides a simple and rapid method convenient for routine HCV genotyping analysis.
Assuntos
Eletroforese Capilar/métodos , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Automação , DNA Complementar/sangue , DNA Complementar/genética , Feminino , Genótipo , Humanos , Masculino , RNA Viral/sangue , RNA Viral/genética , Kit de Reagentes para Diagnóstico , Análise de Sequência de DNARESUMO
The aim of this work was to specify the time course of response to interferon (IFN) of hepatitis G virus (HGV) and hepatitis C virus (HCV) in coinfected individuals. A group of 33 patients, undergoing 12 months of IFN therapy for chronic hepatitis C, was screened for the presence of both HGV and HCV RNAs to select seven coinfected patients. Spontaneous recovery from HGV infection was excluded through the detection of antibodies to the envelope-2 protein of HGV and HCV isolates were genotyped. Within three months of treatment, we found that HGV RNA was transiently cleared in 6/7 patients, but the rate of long-term favorable response was very low (1/7). In addition, considering the same individuals separately, it was shown that HGV and HCV responded to IFN with different kinetics in 5/7 patients. Taken together, these results underscore the importance of the virological basis of the resistance to IFN treatment.
Assuntos
Antivirais/uso terapêutico , Flaviviridae , Hepatite C Crônica/terapia , Hepatite Viral Humana/terapia , Interferon-alfa/uso terapêutico , Flaviviridae/genética , Genótipo , Hepacivirus/genética , Hepatite C Crônica/complicações , Hepatite Viral Humana/virologia , Humanos , Interferon alfa-2 , Reação em Cadeia da Polimerase , RNA Viral/análise , Proteínas Recombinantes , Fatores de Tempo , Resultado do TratamentoRESUMO
Among HIV viral proteins, envelope glycoproteins and Nef have been both suggested to participate in CD4 downregulation during the course of HIV infection. In a previous study, we provided evidence that a mutant form of CD4 that does not bind gp120 was never downregulated in chronically HIV-1- and HIV-2-infected CEM cells. To further investigate the relative effects of Nef or glycoproteins in CD4 downregulation, recombinant vaccinia virus (VV) vectors were used to express high levels of HIV-1 viral proteins in cells expressing both wild-type and mutant CD4. It was demonstrated that during HIV infection, overexpression of Nef, achieved through the VV expression system, was necessary to induce CD4 downregulation in the mutant CD4-expressing cell model. These results are consistent with the hypothesis that Nef-mediated CD4 downregulation depends on the cellular levels of Nef expression. We concluded that during the late stage of viral replication, CD4 downregulation is mostly due to gp120 and not to Nef because of a low level of Nef expression.
Assuntos
Antígenos CD4/metabolismo , Regulação para Baixo , Produtos do Gene nef/análise , HIV/crescimento & desenvolvimento , Linfócitos T/virologia , Antígenos CD4/genética , Linhagem Celular , HIV-1/crescimento & desenvolvimento , HIV-2/crescimento & desenvolvimento , Mutação , Linfócitos T/citologia , Acetato de Tetradecanoilforbol/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaAssuntos
Doenças Autoimunes/complicações , Hepatite C/complicações , Imunossupressores/uso terapêutico , Idoso , Autoanticorpos/análise , Doenças Autoimunes/tratamento farmacológico , Azatioprina/uso terapêutico , Doença Crônica , Feminino , Hepatite C/tratamento farmacológico , Hepatite C/imunologia , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/imunologia , Masculino , Prednisona/uso terapêuticoRESUMO
The host immune responses have been suggested to play a role in liver injury occurring in patients with chronic hepatitis C. In order to explore the relationship between the relative proportions of intrahepatic and peripheral blood lymphocytes (IHL, PBL), the levels of viremia, and the histological hepatitis activity score, three-color fluorescence-activated cytometric analysis was performed for 36 patients with chronic hepatitis C and six control subjects without chronic hepatitis. The liver biopsy was performed before any antiviral therapy. Each liver specimen was divided into two parts: one for histological examination and one for immunological analysis. Tricolor CD45 was used to improve "lymphogating." Fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies with specificity for CD3, CD4, CD8, and CD20 (lymphocyte subpopulations), for CD69 (activated lymphocytes), and for CD16/56 (natural killer cells) were used. The livers of patients with chronic hepatitis C contained a greater proportion of CD4+ lymphocytes that exhibited marked expression of CD69 than in control subjects (20.7 +/- 7.3% vs 10.2 +/- 4.6%, P = 0.027). Moreover, in patients with chronic hepatitis C, the proportion of CD4+ IHL correlated with the histological hepatitis activity evaluated by the Knodell score (r = 0.48, P = 0.004). No correlation was found between the percentage of CD4+ IHL and the level of viremia or transaminase activities. Our findings clearly indicate that a cellular immune response does take place in HCV-infected livers and could thus contribute to the outcome of hepatitis C virus infection.
Assuntos
Hepatite C Crônica/imunologia , Subpopulações de Linfócitos/imunologia , Adulto , Idoso , Complexo CD3/análise , Antígenos CD4/análise , Antígeno CD56/análise , Antígenos CD8/análise , Feminino , Citometria de Fluxo , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Humanos , Células Matadoras Naturais , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores de IgG/análise , Transaminases/sangueRESUMO
CD44 is known to interfere in HIV replication and to participate in many physiological processes such as lymphocyte binding to high endothelial venules of lymphoid tissue, lymph nodes, and mucosal endothelium. The T cell lines MOLT-4 and CEM, and CEM subclones were infected with the HIV-1 LAI strain and monitored for the expression of CD44 during the course of chronic virus production until the infected cells were at the stage of latent infection. The levels of CD44 protein expression were quantified using cell surface immunostaining and biotinylation. The maturation of CD44 molecules was evaluated by metabolic sulforadiolabeling and CD44 mRNA was visualized by Northern blot analysis. We show a downmodulation of CD44 expression in infected T cell lines and subclones. This phenomenon was most evident at the stage of latent infection. Then, CD44 molecules were undetectable at both the protein and mRNA levels in latently infected CEM cells and CEM subclones. In addition, the 97-kDa standard CD44 isoform showed a shift upward, while detectable during the stage of chronic virus production. In latently infected MOLT-4 cells, the CD44 protein levels were dramatically decreased; CD44 mRNA was detected, but the sizes differed from the mRNA in uninfected cells. Since CD44 is known to regulate in part lymphocyte homing and HIV replication, the alterations that were observed in the expression of this molecule could interfere with the particular homing of HIV-infected cells and/or viral latency.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , HIV-1/fisiologia , Receptores de Hialuronatos/genética , Linfócitos T/imunologia , Biotina , Linhagem Celular , Membrana Celular/imunologia , Humanos , Receptores de Hialuronatos/imunologia , RNA Mensageiro , Solubilidade , Sulfatos , Linfócitos T/citologia , Células Tumorais CultivadasRESUMO
Autoantibodies to lymphocytes have been detected in sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals, and several autoantigens have been described. Among them, hyposialylated CD43 has been shown to be a target for autoantibodies in up to 47% of HIV+ individuals. However, the corresponding autoantigen (ie, the incompletely sialylated CD43) has not been isolated from blood cells of HIV-1-infected individuals. Recently, we have observed in vitro that HIV-1 productively or latently infected CEM cells (CEMLAI/NP) express CD43 molecules with modified glycosylation (mogly CD43). Using CEMLAI/NP cells, which do not express any structural viral antigen, we show now that all of the tested HIV+ sera from asymptomatic individuals, and up to 86% of those from subjects at the acquired immunodeficiency syndrome stage contain antibodies (mainly IgM and, to a lesser degree, IgG) that recognize the surface of CEMLAI/NP cells, and precipitate mogly CD43 molecules from the cells lysates. Taken together with our previous demonstration of altered glycosylation of CD43 from HIV-1-infected CEM cells in vitro, the constant antimogly CD43 autoimmune response observed from asymptomatic HIV-1+ subjects is likely to illustrate the occurrence of an altered glycosylation in vivo of the major lymphocyte surface CD43 glycoprotein, associated with HIV-1 infection.