RESUMO
Adaptation to human leukocyte antigen (HLA)-associated immune pressure represents a major driver of human immunodeficiency virus (HIV) evolution at both the individual and population level. To date, there has been limited exploration of the impact of the initial cellular immune response in driving viral adaptation, the dynamics of these changes during infection and their effect on circulating transmitting viruses at the population level. Capturing detailed virological and immunological data from acute and early HIV infection is challenging as this commonly precedes the diagnosis of HIV infection, potentially by many years. In addition, rapid initiation of antiretroviral treatment following a diagnosis is the standard of care, and central to global efforts towards HIV elimination. Yet, acute untreated infection is the critical period in which the diversity of proviral reservoirs is first established within individuals, and associated with greater risk of onward transmissions in a population. Characterizing the viral adaptations evident in the earliest phases of infection, coinciding with the initial cellular immune responses is therefore relevant to understanding which changes are of greatest impact to HIV evolution at the population level. In this study, we utilized three separate cohorts to examine the initial CD8+ T cell immune response to HIV (cross-sectional acute infection cohort), track HIV evolution in response to CD8+ T cell-mediated immunity over time (longitudinal chronic infection cohort) and translate the impact of HLA-driven HIV evolution to the population level (cross-sectional HIV sequence data spanning 30 years). Using next generation viral sequencing and enzyme-linked immunospot interferon-gamma recall responses to peptides representing HLA class I-specific HIV T cell targets, we observed that CD8+ T cell responses can select viral adaptations prior to full antibody seroconversion. Using the longitudinal cohort, we uncover that viral adaptations have the propensity to be retained over time in a non-selective immune environment, which reflects the increasing proportion of pre-adapted HIV strains within the Western Australian population over an approximate 30-year period.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Estudos Transversais , Austrália , Antígenos de Histocompatibilidade Classe I , Antígenos HLA , Antígenos de Histocompatibilidade Classe II , Linfócitos T CD8-PositivosRESUMO
Abacavir hypersensitivity syndrome (ABC HSS) is strongly associated with carriage of human leukocyte antigen (HLA)-B*57:01, which has a 100% negative predictive value for the development of ABC HSS. However, 45% of individuals who carry HLA-B*57:01 can tolerate ABC. We investigated immune and non-immune related genes in ABC HSS (n = 95) and ABC tolerant (n = 43) HLA-B*57:01 + patients to determine other factors required for the development of ABC HSS. Assignment of phenotype showed that ABC HSS subjects were significantly less likely than tolerants to carry only ERAP1 hypoactive trimming allotypes (p = 0.02). An altered self-peptide repertoire model by which abacavir activates T cells is in keeping with observation that endoplasmic reticulum aminopeptidase 1 (ERAP1) allotypes that favour efficient peptide trimming are more common in ABC HSS patients compared to patients who tolerate ABC. Independently, non-specific immune activation via soluble cluster of differentiation antigen 14 (sCD14) may also influence susceptibility to ABC HSS.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Didesoxinucleosídeos/uso terapêutico , Síndrome de Hipersensibilidade a Medicamentos/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Antígenos HLA-B/genética , Alérgenos/imunologia , Aminopeptidases/genética , Fármacos Anti-HIV/imunologia , Didesoxinucleosídeos/efeitos adversos , Didesoxinucleosídeos/imunologia , Síndrome de Hipersensibilidade a Medicamentos/etiologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Infecções por HIV/complicações , Humanos , Receptores de Lipopolissacarídeos/genética , Masculino , Antígenos de Histocompatibilidade Menor/genética , Fenótipo , Estudos RetrospectivosRESUMO
Abacavir administration is associated with drug-induced hypersensitivity reactions in HIV+ individuals expressing the HLA-B*57:01 allele. However, the immunological effects of abacavir administration in an HLA-B57 mismatched transplantation setting have not been studied. We hypothesized that abacavir exposure could induce de novo HLA-B57-specific allorecognition. HIV-specific CD8 T cell clones were generated from HIV+ individuals, using single cell sorting based on HIV peptide/HLA tetramer staining. The T cell clones were assayed for alloreactivity against a panel of single HLA-expressing cell lines, in the presence or absence of abacavir. Cytokine assay, CD137 upregulation, and cytotoxicity were used as readout. Abacavir exposure can induce de novo HLA-B57 allorecognition by HIV-specific T cells. A HIV Gag RK9/HLA-A3-specific T cell did exhibit interferon-γ production, CD137 upregulation, and cytolytic effector function against allogeneic HLA-B57, but only in the presence of abacavir. Allorecognition was specific to the virus specificity, HLA restriction, and T cell receptor TRBV use of the T cell. We provide proof-of-principle evidence that administration of a drug could induce specific allorecognition of mismatched HLA molecules in the transplant setting. We suggest that HIV-seropositive recipients of an HLA-B57 mismatched graft should not receive abacavir until further studies are completed.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas/imunologia , Infecções por HIV/sangue , Antígenos HLA-B/imunologia , Alelos , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Infecções por HIV/complicações , Infecções por HIV/imunologia , Soropositividade para HIV/imunologia , Teste de Histocompatibilidade , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoAssuntos
Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas/imunologia , Infecções por HIV , HIV-1/imunologia , Memória Imunológica/efeitos dos fármacos , Didesoxinucleosídeos/administração & dosagem , Hipersensibilidade a Drogas/patologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/patologia , HumanosRESUMO
We hypothesized that HIV-specific CD8 T cell clonotypes can be stimulated by allogeneic HLA molecules. Multiple HIV-specific CD8 T cell clones were derived from 12 individuals with chronic HIV infection, specific for 13 different HIV Gag antigens and restricted to 7 different HLA molecules. The generated T cell clones were assayed for alloreactivity against a panel of single HLA class I expressing cell lines (SALs). HIV-specific T cells recognising at least one allogeneic HLA molecule could be identified from 7 of 12 patients tested. Allorecognition was associated with IFNγ cytokine production, CD137 upregulation and cytotoxicity, suggesting high avidity allo-stimulation. Allo-HLA recognition by HIV-specific T cells was specific to the HIV target peptide/HLA restriction and TCR TRBV usage of the T cells. HIV-specific T cells do crossreact against allogeneic HLA molecules in an epitope and TRBV specific manner. Therefore allo-HLA stimulation could be exploited to induce or augment HIV-specific T cell responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos HIV/imunologia , Infecções por HIV/imunologia , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Linfócitos T CD8-Positivos/patologia , Células Cultivadas , Células Clonais/imunologia , Estudos de Coortes , Reações Cruzadas , Infecções por HIV/patologia , Humanos , Memória Imunológica , Receptores de Antígenos de Linfócitos T/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
OBJECTIVE: To define the relative frequencies of different mechanisms of viral escape. DESIGN: A population-based approach to examine the distribution of HIV polymorphism associated with diverse population human leucocyte antigens (HLAs) at sites within and flanking CD8 T-cell epitopes as a correlate of likely mechanisms of viral escape. METHODS: Sequence windows surrounding 874 HLA allele-specific polymorphisms across the full HIV-1 proteomic consensus sequence were scanned by an epitope-prediction programme. Either already known or probable CD8 T-cell epitopes with HLA restriction matching that of the proximal HLA association were identified and synthesized. These peptides were used as stimulating antigens in automated enzyme-linked immunospot (ELISpot) assays. Peptide arrays were customized to each individual based on their HLA genotype. RESULTS: Among HLA-associated HIV polymorphisms detected in the viral sequences of a cohort of 800 individuals with chronic subtype B HIV infection, those which were likely to affect HLA peptide binding were significantly more common than polymorphisms at nonanchor HLA binding sites. HIV epitopes with such polymorphisms were associated with reduced IFNγ responses in ELISpot assays. HIV escape at sites affecting T-cell receptor (TCR) engagement and epitope processing were also evident. CONCLUSION: HIV escape from HLA-peptide binding predominates as an effective viral evasion strategy and therefore has implications for inclusion of HLA-adapted epitopes in vaccine immunogens.
Assuntos
Epitopos de Linfócito T/imunologia , HIV-1/imunologia , Evasão da Resposta Imune , Mutação de Sentido Incorreto , Fosfoproteínas/imunologia , Sítios de Ligação , Epitopos de Linfócito T/genética , HIV-1/genética , HIV-1/metabolismo , Humanos , Fosfoproteínas/metabolismo , Polimorfismo Genético , Ligação ProteicaRESUMO
HIV-1 mutations, which reduce or abolish CTL responses against virus-infected cells, are frequently selected in acute and chronic HIV infection. Among population HIV-1 sequences, immune selection is evident as human leukocyte antigen (HLA) allele-associated substitutions of amino acids within or near CD8 T-cell epitopes. In these cases, the non-adapted epitope is susceptible to immune recognition until an escape mutation renders the epitope less immunogenic. However, several population-based studies have independently identified HLA-associated viral changes, which lead to the formation of a new T-cell epitope, suggesting that the immune responses that these variants or 'neo-epitopes' elicit provide an evolutionary advantage to the virus rather than the host. Here, we examined the functional characteristics of eight CD8 T-cell responses that result from viral adaptation in 125 HLA-genotyped individuals with chronic HIV-1 infection. Neo-epitopes included well-characterized immunodominant epitopes restricted by common HLA alleles, and in most cases the T-cell responses against the neo-epitope showed significantly greater functional avidity and higher IFNγ production than T cells for non-adapted epitopes, but were not more cytotoxic. Neo-epitope formation and emergence of cognate T-cell response coincident with a rise in viral load was then observed in vivo in an acutely infected individual. These findings show that HIV-1 adaptation not only abrogates the immune recognition of early targeted epitopes, but may also increase immune recognition to other epitopes, which elicit immunodominant but non-protective T-cell responses. These data have implications for immunodominance associated with polyvalent vaccines based on the diversity of chronic HIV-1 sequences.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Interferon gama/metabolismo , Epitopos de Linfócito T/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Ativação Linfocitária , Linfócitos T Citotóxicos/imunologia , Carga ViralRESUMO
Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic hypotheses arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFN-γ responses in 290 individuals from the same cohort, which gave rise to 874 HLA-HIV associations in genetic analyses, taking into account autologous viral sequences and individual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this reverse-genomics approach. Many HLA-adapted epitopes elicited equivalent or higher-magnitude IFN-γ responses than did the nonadapted epitopes, particularly in Nef. At a population level, inclusion of all of the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef, and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We concluded that HLA-HIV associations mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations, as well as to test, train, and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA-adapted variant responses may have negative effects on natural and vaccine immunity against HIV and, therefore, has implications for diversity coverage approaches in HIV vaccine design.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Infecções por HIV/genética , HIV-1/genética , Antígenos HLA/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Estudos de Coortes , Genótipo , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Evasão da Resposta Imune/genética , Interferon gama/biossíntese , Interferon gama/imunologia , Polimorfismo Genético , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Since HLA-restricted cytotoxic T-cell responses select specific polymorphisms in HIV-1 sequences and HLA diversity is relatively static in human populations, we investigated the use of peptide epitopes based on sites of HLA-associated adaptation in HIV-1 sequences to stimulate and detect T-cell responses ex vivo. These "HLA-optimised" peptides captured more HIV-1 Nef-specific responses compared with overlapping peptides of a single consensus sequence, in interferon-gamma enzyme linked immunospot assays. Sites of immune selection can reveal more immunogenic epitopes in HLA-diverse populations and offer insights into the nature of HLA-epitope targeting, which could be applied in vaccine design.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Antígenos HLA-A/imunologia , Sequência Consenso , Feminino , HIV-1/genética , Antígenos HLA-A/genética , Humanos , Interferon gama/imunologia , Masculino , Peptídeos/imunologia , Polimorfismo Genético , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologiaRESUMO
The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, p<0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Interferon gama/imunologia , Robótica/métodos , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Humanos , Interferon gama/biossíntese , Peptídeos/imunologiaRESUMO
The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas/imunologia , Antígenos HLA-B/imunologia , Ativação Linfocitária , Inibidores da Transcriptase Reversa/efeitos adversos , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/imunologia , Fármacos Anti-HIV/metabolismo , Apresentação de Antígeno , Didesoxinucleosídeos/imunologia , Didesoxinucleosídeos/metabolismo , Hipersensibilidade a Drogas/metabolismo , Antígenos HLA-B/química , Antígenos HLA-B/metabolismo , Humanos , Inibidores da Transcriptase Reversa/imunologia , Inibidores da Transcriptase Reversa/metabolismoRESUMO
BACKGROUND: Abacavir hypersensitivity in genetically susceptible individuals implicates an abacavir-specific T-cell response to either the parent drug or a metabolite generated in vivo. We have analysed the cytokine profile in antigen-presenting cells and the T-lymphocytes that are involved in the pathological immune response to abacavir. METHODS: In this study, we compared abacavir-specific cytokine responses in cultured peripheral blood mononuclear cells (PBMCs) from HIV-infected abacavir hypersensitive, tolerant and naive individuals. Cells were cultured in the presence or absence of abacavir. Cytokine expression was determined by microarray analysis, enzyme-linked immunosorbent assays and flow cytometry. RESULTS: We demonstrated using in vitro models of immune activation that the production of interferon-gamma was specifically induced by abacavir treatment in PBMCs obtained from hypersensitive patients carrying the HLA-B*5701 allele (median 123.86 compared with -30.83 for tolerant controls, P=0.001). CONCLUSION: These results provide further insight into the immunological and metabolic basis of abacavir hypersensitivity syndrome. In vitro assays could assist in the identification of susceptible loci by providing a surrogate marker for the hypersensitivity reaction. Such a marker could be studied in unexposed individuals to shed further light on the immunopathogenesis of the abacavir hypersensitivity syndrome.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Citocinas/metabolismo , Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas , Inibidores da Transcriptase Reversa/efeitos adversos , Células Cultivadas , Hipersensibilidade a Drogas/etiologia , Hipersensibilidade a Drogas/genética , Hipersensibilidade a Drogas/imunologia , Hipersensibilidade a Drogas/fisiopatologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Antígenos HLA-B/genética , Humanos , Interferon gama/biossíntese , Leucócitos Mononucleares/imunologiaRESUMO
OBJECTIVES: HLA-B*5701 strongly predicts abacavir hypersensitivity (HSR), but implementation of effective routine screening into clinical practice requires testing be practical and accurate. We tested the proficiency of HLA-B*5701 typing among laboratories using sequence-specific primer PCR. DESIGN AND METHODS: DNA panels (1 and 2) were distributed to seven laboratories (A to G) for blinded typing of the HLA-B*5701 allele. Panel 1 (n = 10 samples; n = 7 laboratories) included 3 positives and other closely related B17 subtypes (B*5702, B*5703, B*5704 and B*5801). Panel 2 (n = 96 samples; n = 4 laboratories) included 36 positives among a broad spectrum of other B alleles. Two laboratories (A and B) also submitted 96 routine samples, typed by the same methodology, to the reference centre for additional analysis by sequence-based typing. RESULTS: All laboratories correctly typed panel 1 for HLA-B*5701 carriage. Laboratories A, B and C identified HLA-B*5701 alleles in panel 2 with 100% sensitivity and 100% specificity. Laboratory D reported one false negative, reportedly due to a sampling error. The results obtained for routine samples typed by laboratories A and B and those generated by the reference laboratory using sequencing were fully concordant. CONCLUSIONS: Detection of HLA-B*5701 alleles among laboratories was 100% specific and 99.4% sensitive, indicating that participating HIV testing laboratories were currently offering effective primary screening to identify individuals at high risk of abacavir HSR. Accurate reporting of HLA-B*5701 status is critical for the safe administration of this drug and participation in quality assurance programmes by all sites who report HLA-B*5701 status should be promoted.
Assuntos
Didesoxinucleosídeos/efeitos adversos , Testes Genéticos/normas , Antígenos HLA-B/genética , Alelos , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/uso terapêutico , Primers do DNA , Sondas de DNA de HLA , Didesoxinucleosídeos/uso terapêutico , Hipersensibilidade a Drogas/genética , Humanos , Reação em Cadeia da Polimerase , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: A potentially life-threatening hypersensitive reaction accompanies the use of HIV nucleoside analogue abacavir (ABC) in 4-8% of Caucasian individuals. HLA-B*5701 and Hsp70 493T alleles have been shown to predict susceptibility to this hypersensitivity. DESIGN AND METHODS: This study was undertaken to provide a mechanistic understanding of the highly significant genetic association of HLA Class I and Hsp70 alleles with ABC hypersensitivity. RESULTS: In this study an ABC-induced localization of intracellular HSP70 to endosomal vesicles of antigen-presenting cells was demonstrated. This ABC-stimulated redistribution of endogenous HSP70 was substantially higher in the genetically homogenous HLA-B*5701, Hsp70 493T ABC-hypersensitive individuals and ABC-naive individuals in comparison with the heterogeneous tolerant patients (P = 0.023). Increased expression of HSP70 was also detected in the hypersensitive group as measured by flow cytometry (P = 0.032). Blocking of HSP70 and HSP70 cell surface receptors CD14 and TLR2 abrogated ABC-stimulated HSP70 redistribution in sensitized individuals to basal levels (P < 0.004). In addition, the use of TcRalphabeta and HLA-B57/58 antibodies also ablated the expression of HSP70. Cells expressing the activation markers CD40 were increased after ABC stimulation in the hypersensitive patients (P = 0.006). ABC-stimulated interferon-gamma levels were higher in hypersensitive patients in comparison with ABC-tolerant individuals with a mean of 123.54 versus 0 pg/ml (P = 0.001). CONCLUSION: The present data indicates that ABC stimulates an innate immune response and activates antigen-presenting cells via the endogenous HSP70-mediated Toll-like receptor pathway in genetically susceptible individuals potentially initiating the immuno-pathological hypersensitive response.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Didesoxinucleosídeos/imunologia , Hipersensibilidade a Drogas/imunologia , Inibidores da Transcriptase Reversa/imunologia , Antígenos CD40/análise , Antígenos CD40/imunologia , Células Cultivadas , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Endossomos/genética , Endossomos/imunologia , Imunofluorescência/métodos , Soropositividade para HIV/tratamento farmacológico , Soropositividade para HIV/imunologia , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunofenotipagem/métodos , Interferon gama/análise , Interferon gama/imunologia , Microscopia Confocal/métodos , Monócitos/imunologia , Receptores de Superfície Celular/imunologiaRESUMO
BACKGROUND: Vitamin E isomers may protect against atherosclerosis. The aim of this study was to compare the effects of supplementation with either alpha-tocopherol (alphaT) or mixed tocopherols rich in gamma-tocopherol (gammaT) on markers of oxidative stress and inflammation in patients with type 2 diabetes. METHODS: In a double-blind, placebo-controlled trial, 55 patients with type 2 diabetes were randomly assigned to receive (500 mg/day) (a) alphaT, (b) mixed tocopherols, or (c) placebo for 6 weeks. Cellular tocopherols, plasma and urine F(2)-isoprostanes, erythrocyte antioxidant enzyme activities, plasma inflammatory markers, and ex vivo assessment of eicosanoid synthesis were analyzed pre- and postsupplementation. RESULTS: Neutrophil alphaT and gammaT increased (both P <0.001) with mixed tocopherol supplementation, whereas alphaT (P <0.001) increased and gammaT decreased (P <0.005) after alphaT supplementation. Both alphaT and mixed tocopherol supplementation resulted in reduced plasma F(2)-isoprostanes (P <0.001 and P = 0.001, respectively) but did not affect 24-h urinary F(2)-isoprostanes or erythrocyte antioxidant enzyme activities. Neither alphaT nor mixed tocopherol supplementation affected plasma C-reactive protein, interleukin 6, tumor necrosis factor-alpha, or monocyte chemoattractant protein-1. Stimulated neutrophil leukotriene B(4) production decreased significantly in the mixed tocopherol group (P = 0.02) but not in the alphaT group (P = 0.15). CONCLUSIONS: The ability of tocopherols to reduce systemic oxidative stress suggests potential benefits of vitamin E supplementation in patients with type 2 diabetes. In populations with well-controlled type 2 diabetes, supplementation with either alphaT or mixed tocopherols rich in gammaT is unlikely to confer further benefits in reducing inflammation.
Assuntos
Diabetes Mellitus Tipo 2/dietoterapia , Suplementos Nutricionais , Inflamação/dietoterapia , Estresse Oxidativo , Tocoferóis/uso terapêutico , Vitaminas/uso terapêutico , Biomarcadores/análise , Diabetes Mellitus Tipo 2/metabolismo , Método Duplo-Cego , Feminino , Humanos , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Tocoferóis/análise , Vitaminas/análise , alfa-Tocoferol/análise , alfa-Tocoferol/uso terapêuticoRESUMO
BACKGROUND: Abacavir hypersensitivity reaction (ABC HSR) is a potentially life-threatening adverse reaction that affects approximately 8% of patients that initiate this antiretroviral drug. Independent groups have shown a strong predictive association between ABC HSR and HLA-B*5701, indicating that exclusion of HLA-B*5701 positive individuals from abacavir treatment would largely prevent ABC HSR. However, the limited availability and relatively high cost of human leukocyte antigen (HLA) typing represent barriers to the widespread implementation of this pharmacogenetic approach to abacavir prescribing. To facilitate routine screening, we have developed a rapid flow cytometry method for HLA-B57 phenotyping using commercially available B17 monoclonal antibodies. METHODS: Whole blood samples from 84 human immunodeficiency virus (HIV) patients were examined by standard flow cytometry methods, using a two-colour B17-specific immunofluorescence assay in the CD45 lymphocyte population. RESULTS: All eight HLA-B57 individuals examined tested positive, while HLA-B57/58 negative individuals (n=74) tested negative for this flow cytometry test. Two non-HLA-B57 individuals showed weak cross-reactivity. CONCLUSION: In our predominantly Caucasian population, B17/CD45 dual staining was sufficient to identify individuals carrying B17 cell surface antigens. This approach, utilizing flow cytometry methods that are widely available in HIV laboratories, therefore offers a sensitive, rapid and cost-effective screening assay prior to abacavir prescription. Following risk stratification with this assay, it would be anticipated that identification of HLA-B*5701 using molecular HLA typing methods would be required in <10% of the screened population.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas/prevenção & controle , Testes Genéticos , Teste de Histocompatibilidade/métodos , Alelos , Fármacos Anti-HIV/uso terapêutico , Didesoxinucleosídeos/uso terapêutico , Hipersensibilidade a Drogas/genética , Citometria de Fluxo , Soropositividade para HIV , Antígenos HLA-B/genética , Humanos , Sensibilidade e EspecificidadeRESUMO
There is a growing discussion surrounding the issue of personalized approaches to drug prescription based on an individual's genetic makeup. This field of investigation has focused primarily on identifying genetic factors that influence drug metabolism and cellular disposition, thereby contributing to dose-dependent toxicities and/or variable drug efficacy. However, pharmacogenetic approaches have also proved valuable in predicting drug hypersensitivity reactions in selected patient populations, including HIV-infected patients receiving long-term antiretroviral therapy. In this instance, susceptibility has been strongly linked to genetic loci involved in antigen recognition and presentation to the immune system--most notably within the major histocompatibility complex (MHC) region--consistent with the notion that hypersensitivity reactions represent drug-specific immune responses that are largely dose independent. Here the authors describe their experiences with the development of pharmacogenetic approaches to hypersensitivity reactions associated with abacavir and nevirapine, two commonly prescribed antiretroviral drugs. It is demonstrated that prospective screening tests to identify and exclude individuals with a certain genetic makeup may be largely successful in decreasing or eliminating incidence of these adverse drug reactions in certain populations. This review also explores the broader implications of these findings.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas/fisiopatologia , Nevirapina/efeitos adversos , Animais , Biomarcadores , Hipersensibilidade a Drogas/genética , HIV-1/genética , HIV-1/imunologia , HumanosRESUMO
Susceptibility to a clinically significant drug hypersensitivity syndrome associated with abacavir use seems to have a strong genetic component. We have previously shown that the presence of HLA-B*5701 strongly predicts abacavir hypersensitivity and have identified a potential susceptibility locus within a 300-kb region between the MEGT1 and C4A6 loci in the central MHC. We now report the results of fine recombinant genetic mapping in an expanded patient population of 248 consecutive, fully ascertained, abacavir-exposed individuals in the Western Australian HIV Cohort Study, in which 18 cases of definite abacavir hypersensitivity (7.3%) and 230 tolerant controls were identified. Haplotype mapping within patients with allelic markers of the 57.1 ancestral haplotype suggests a susceptibility locus within the 14-kb Hsp70 gene cluster. HLA-B*5701 was present in 94.4% of hypersensitive cases compared with 1.7% of controls (odds ratio, 960; P < 0.00001). A haplotypic nonsynonymous polymorphism of Hsp70-Hom (HspA1L, resulting from the substitution of residue M493T in the peptide-binding subunit) was found in combination with HLA-B*5701 in 94.4% of hypersensitive cases and 0.4% of controls (odds ratio, 3,893; P < 0.00001). Individuals with abacavir hypersensitivity demonstrated increased monocyte tumor necrosis factor expression in response to ex vivo abacavir stimulation, which was abrogated with CD8(+) T cell depletion. These data indicate that the concurrence of HLA-B*5701 and Hsp70-Hom M493T alleles is necessary for the development of abacavir hypersensitivity, which is likely to be mediated by an HLA-B*5701-restricted immune response to abacavir.
Assuntos
Didesoxinucleosídeos/imunologia , Hipersensibilidade a Drogas/imunologia , Antígenos HLA-B/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Mapeamento Cromossômico , Hipersensibilidade a Drogas/epidemiologia , Hipersensibilidade a Drogas/genética , Marcadores Genéticos , Antígenos HLA-B/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Haplótipos/imunologia , Humanos , Complexo Principal de Histocompatibilidade/genética , Dados de Sequência Molecular , Prevalência , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
IL-10 inhibits the production of many pro-inflammatory cytokines. Polymorphisms in the IL10 gene promoter at positions -1082G-->A, -819C-->T and -592C-->A occur as three haplotypes, ATA, GCC and ACC. These influence several infectious and inflammatory diseases including community-acquired pneumonia, where a role for IL-10 is suggested by fluctuations in plasma levels of the cytokine. However, the effects of the haplotypes on IL-10 production are unclear. We stimulated peripheral blood mononuclear cells (PBMC) from at least five individuals homozygous for each of the three haplotypes with lipopolysaccharide (LPS, 10 microg/ml) or heat-killed Streptococcus pneumoniae (10(7)cfu/ml) and measured IL-10 mRNA by RT-PCR. Following S. pneumoniae stimulation, PBMC with the ATA haplotype had higher IL-10 mRNA levels than those with the GCC haplotype at 4 h (independent t-test; P=0.024), or the ACC haplotype at 4 h ( P<0.0001) and 8 h ( P=0.007). Following LPS stimulation, IL-10 mRNA levels were not significantly influenced by the IL10 haplotype, but similar trends were observed, consistent with the variable outcome of published studies. The results suggest that the -819 and/or -592 alleles affect transcription.
Assuntos
Interleucina-10/genética , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Streptococcus pneumoniae/patogenicidade , Transcrição Gênica , Adulto , Alelos , Feminino , Haplótipos , Homozigoto , Humanos , Interleucina-10/sangue , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-IdadeRESUMO
Immune activation associated with HIV infection declines after highly active antiretroviral therapy (HAART), but may persist or recur in some patients. It is not clear whether this reflects a resurgence of HIV replication or another cause of immune activation, such as inflammatory reactions to opportunistic pathogens (immune restoration disease [IRD]). Here, we studied plasma and cellular immune activation markers in adult HIV-1 patients who had received HAART for >12 months and maintained plasma HIV RNA levels of <400 copies/ml for >6 months. Plasma interleukin 1 receptor antagonist and tumor necrosis factor receptor I levels were similar in patients and HIV-negative control subjects, but the highest levels occurred mainly in patients with a history of IRD. In contrast, expression of HLA-DR and CD38 on monocytes and of HLA-DR on CD8(+) T cells was higher in patients than in control subjects. Thus, cellular markers of immune activation are abnormal in some patients with a good virological response to HAART, and abnormalities of plasma immune activation markers correlate with a history of IRD.