RESUMO
BACKGROUND: Asthma is a biologically heterogeneous disease and development of novel therapeutics requires understanding of pathophysiologic phenotypes. There is uncertainty regarding the stability of clinical characteristics and biomarkers in asthma over time. This report presents the longitudinal stability over 12 months of clinical characteristics and clinically accessible biomarkers from ADEPT. METHODS: Mild, moderate, and severe asthma subjects were assessed at 5 visits over 12 months. Assessments included patient questionnaires, spirometry, bronchodilator reversibility, fractional exhaled nitric oxide (FENO), and biomarkers measured in induced sputum. RESULTS: Mild (n = 52), moderate (n = 55), and severe (n = 51) asthma cohorts were enrolled from North America and Western Europe. For all clinical characteristics and biomarkers, group mean data showed no significant change from visit to visit. However, individual data showed considerable variability. FEV1/FVC ratio showed excellent reproducibility while pre-bronchodilator FEV1 and FVC were only moderately reproducible. Of note bronchodilator FEV1 reversibility showed low reproducibility, with the nonreversible phenotype much more reproducible than the reversible phenotype. The 7-item asthma control questionnaire (ACQ7) demonstrated moderate reproducibility for the combined asthma cohorts, but the uncontrolled asthma phenotype (ACQ7 > 1.5) was inconstant in mild and moderate asthma but stable in severe asthma. FENO demonstrated good reproducibility, with the FENO-low phenotype (FENO < 35 ppb) more stable than the FENO-high phenotype (FENO ≥ 35 ppb). Induced sputum inflammatory phenotypes showed marked variability across the 3 sputum samples taken over 6 months. CONCLUSIONS: The ADEPT cohort showed group stability, individual stability in some parameters e.g. low FEV1/FVC ratio, and low FENO, but marked individual variability in other clinical characteristics and biomarkers e.g. type-2 biomarkers over 12 months. This variability is possibly related to seasonal variations in climate and allergen exposure, medication changes and acute exacerbations. The implications for patient selection strategies based on clinical biomarkers may be considerable.
Assuntos
Asma/tratamento farmacológico , Testes de Função Respiratória/estatística & dados numéricos , Escarro/citologia , Adulto , Asma/epidemiologia , Biomarcadores , Broncodilatadores/uso terapêutico , Europa (Continente)/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , América do Norte/epidemiologia , Prevalência , Reprodutibilidade dos Testes , Fatores de Risco , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
BACKGROUND: Asthma is a heterogeneous disease and development of novel therapeutics requires an understanding of pathophysiologic phenotypes. The purpose of the ADEPT study was to correlate clinical features and biomarkers with molecular characteristics, by profiling asthma (NCT01274507). This report presents for the first time the study design, and characteristics of the recruited subjects. METHODS: Patients with a range of asthma severity and healthy non-atopic controls were enrolled. The asthmatic subjects were followed for 12 months. Assessments included history, patient questionnaires, spirometry, airway hyper-responsiveness to methacholine, fractional exhaled nitric oxide (FENO), and biomarkers measured in induced sputum, blood, and bronchoscopy samples. All subjects underwent sputum induction and 30 subjects/cohort had bronchoscopy. RESULTS: Mild (n = 52), moderate (n = 55), severe (n = 51) asthma cohorts and 30 healthy controls were enrolled from North America and Western Europe. Airflow obstruction, bronchodilator response and airways hyperresponsiveness increased with asthma severity, and severe asthma subjects had reduced forced vital capacity. Asthma control questionnaire-7 (ACQ7) scores worsened with asthma severity. In the asthmatics, mean values for all clinical and biomarker characteristics were stable over 12 months although individual variability was evident. FENO and blood eosinophils did not differ by asthma severity. Induced sputum eosinophils but not neutrophils were lower in mild compared to the moderate and severe asthma cohorts. CONCLUSIONS: The ADEPT study successfully enrolled asthmatics across a spectrum of severity and non-atopic controls. Clinical characteristics were related to asthma severity and in general asthma characteristics e.g. lung function, were stable over 12 months. Use of the ADEPT data should prove useful in defining biological phenotypes to facilitate personalized therapeutic approaches.
Assuntos
Antiasmáticos/uso terapêutico , Asma/diagnóstico , Asma/tratamento farmacológico , Broncodilatadores/uso terapêutico , Pulmão/efeitos dos fármacos , Medicina de Precisão , Adolescente , Adulto , Idoso , Asma/epidemiologia , Asma/metabolismo , Asma/fisiopatologia , Biomarcadores/metabolismo , Broncoconstrição/efeitos dos fármacos , Canadá/epidemiologia , Estudos de Casos e Controles , Europa (Continente)/epidemiologia , Feminino , Humanos , Estudos Longitudinais , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Fenótipo , Valor Preditivo dos Testes , Prevalência , Projetos de Pesquisa , Testes de Função Respiratória , Fatores de Risco , Índice de Gravidade de Doença , Escarro/metabolismo , Inquéritos e Questionários , Fatores de Tempo , Resultado do Tratamento , Estados Unidos/epidemiologia , Adulto JovemRESUMO
OBJECTIVE: To compare femorotibial cartilage thickness changes over a 2- vs a 1-year observation period in knees with radiographic knee osteoarthritis (OA). METHODS: One knee of 346 Osteoarthritis Initiative (OAI) participants was studied at three time points [baseline (BL), year-1 (Y1), year-2 (Y2) follow-up]: 239 using coronal fast low angle shot (FLASH) and 107 using sagittal double echo at steady state (DESS) MR imaging. Changes in cartilage thickness were assessed in femorotibial cartilage plates and subregions, after manual segmentation with blinding to time-point. RESULTS: The standardized response mean (SRM) of total joint cartilage thickness over 2 years was modestly higher than over 1 year (FLASH: -0.44 vs -0.32/-0.28 [first/second year]; DESS: -0.42 vs -0.39/-0.18). For the subregion showing the largest change per knee (OV1), the 2-year SRM was similar or lower (FLASH: -1.20 vs -1.22/-1.61; DESS: -1.38 vs -1.64/-1.51) than the 1-year SRM. The changes in total joint cartilage thickness were not significantly different in the first and second year (FLASH: -0.8% vs -0.7%; DESS: -1.3% vs -0.8%) and were negatively correlated. Analysis of smallest detectable changes (SDCs) revealed that only few participants displayed significant progression in both consecutive periods. The location of the subregion contributing to OV1 in each knee was highly inconsistent between the first and second year observation period. CONCLUSIONS: The SRM of region-based cartilage thickness change in OA is modestly larger following a 2-year vs a 1-year observation period, while it is relatively similar when an OV-approach is chosen. Structural progression displays strong temporal and spatial heterogeneity at an individual knee level that should be considered when planning clinical trials.
Assuntos
Cartilagem Articular/patologia , Osteoartrite do Joelho/patologia , Idoso , Estudos de Coortes , Progressão da Doença , Feminino , Fêmur/patologia , Humanos , Articulação do Joelho/patologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Tíbia/patologia , Fatores de TempoRESUMO
OBJECTIVE: To establish sex-specific (subregional) reference values of cartilage thickness and potential maximal Z-scores in the femorotibial joint. METHODS: The mean cartilage thickness (ThCtAB.Me) in femorotibial compartments, plates and subregions was determined on coronal magnetic resonance imaging (MRI) from a population-based sample (Framingham) and from a healthy reference sample of the Osteoarthritis Initiative (OAI). RESULTS: 686 Framingham participants (309 men, 377 women, age 62 ± 8 years) had no radiographic femorotibial osteoarthritis (OA) ("normals") and 376 (156 men, 220 women) additionally had no MRI features of cartilage lesions ("supernormals"). The Framingham "normals" had thinner cartilage in the medial (3.59 mm) than in the lateral femorotibial compartment (3.86 mm). Medially, the femur displayed thicker cartilage (1.86 mm) than the tibia (1.73 mm), and laterally the tibia thicker cartilage (2.09 mm) than the femur (1.77 mm). The thickest cartilage was observed in central, and the thinnest in external femorotibial subregions. Potential maximal Z-scores ranged from 5.6 to 9.8 throughout the subregions; men displayed thicker cartilage but similar potential maximal Z-scores as women. Mean values and potential maximal Z-scores in Framingham "supernormals" and non-exposed OAI reference participants (112 participants without symptoms or risk factors of knee OA) were similar to Framingham "normals". CONCLUSIONS: We provide reference values and potential maximal Z-scores of cartilage thickness in middle aged to elderly non-diseased populations without radiographic OA. Results were similar for "supernormal" participants without MRI features of cartilage lesions, and in a cohort without OA symptoms or risk factors. A cartilage thickness loss of around 27% is required for attaining a Z-score of -2.
Assuntos
Cartilagem Articular/patologia , Articulação do Joelho/patologia , Osteoartrite do Joelho/patologia , Idoso , Idoso de 80 Anos ou mais , Antropometria/métodos , Estudos de Coortes , Feminino , Fêmur/patologia , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Fatores Sexuais , Tíbia/patologiaRESUMO
OBJECTIVE: To assess the presence, location, type and size of denuded areas of subchondral bone (dAB) in the femorotibial joint, measured quantitatively with 3T MRI, in a large subset of OAI participants. METHODS: One knee of 633 subjects (250 men, 383 women, aged 61.7+/-9.6 y) were studied, spanning all radiographic osteoarthritis (OA) stages. dABs were determined quantitatively using segmentations of coronal FLASHwe images, representing areas where the subchondral bone was not covered by cartilage. Post hoc visual examination of segmented images determined whether dABs represented full thickness cartilage loss or internal osteophyte. RESULTS: 7% Of the knees were Kellgren & Lawrence (KL) grade 0, 6% grade 1, 41% grade 2, 41% grade 3, and 5% grade 4. 39% Of the participants (48% of the men and 33% of the women) displayed dABs; 61% of the dABs represented internal osteophytes. 1/47 Participants with KL grade 0 displayed 'any' dAB whereas 29/32 of the KL grade 4 knees were affected. Even as early as KL grade 1, 29% of the participants showed dABs. There were significant relationships of dAB with increasing KL grades (P<0.001) and with ipsi-compartimental JSN (P< or =0.001). Internal osteophytes were more frequent laterally (mainly posterior tibia and internal femur) whereas full thickness cartilage loss was more frequent medially (mainly external tibia and femur). CONCLUSIONS: dABs occur already at earliest stages of radiographic OA (KL grades 1 and 2) and become more common (and larger) with increasing disease severity. Almost all KL grade 4 knees exhibited dABs, with cartilage loss being more frequent than internal osteophytes.
Assuntos
Cartilagem Articular/patologia , Articulação do Joelho/patologia , Osteoartrite do Joelho/patologia , Idoso , Estudos de Coortes , Estudos Transversais , Feminino , Humanos , Articulação do Joelho/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/complicações , Osteoartrite do Joelho/diagnóstico por imagem , RadiografiaRESUMO
BACKGROUND AND AIMS: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor alpha (anti-TNFalpha) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. METHODS: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. RESULTS: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. CONCLUSION: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Fármacos Gastrointestinais/uso terapêutico , Mucosa Intestinal/metabolismo , Adulto , Estudos de Coortes , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Colo/metabolismo , Resistência a Medicamentos/genética , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Infliximab , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Prognóstico , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Resultado do Tratamento , Fator de Necrose Tumoral alfa/imunologia , Adulto JovemRESUMO
OBJECTIVE: Resistin is a secreted factor that is elevated in rheumatoid arthritis (RA) and believed to drive joint inflammation in vivo. This study was undertaken to determine if resistin is present in the joint following joint injury and to elucidate the role of resistin in cartilage degradation. METHODS: The level of resistin was measured in paired synovial fluid (SF) and serum samples from patients following joint injury (anterior cruciate ligament, ACL or meniscus tear). Localization of resistin was visualized by immunohistochemistry of synovial tissue and cartilage from healthy and OA donors. Mouse and human cartilage cultures were used to assess the effect of resistin on cartilage metabolism. RESULTS: In trauma patients, resistin levels declined with increasing time post injury. The resistin levels were highest in samples collected up to 1 week following traumatic injury (SF: 2980 pg/ml, serum: 7901 pg/ml) and lowest in samples collected 6-26 years post injury (SF: 686 pg/ml, serum: 5682 pg/ml). Resistin was shown to be expressed in macrophage-like cells in both healthy and OA synovial tissue. Treatment of mouse cartilage cultures with recombinant resistin led to a dose dependent loss of proteoglycan and induction of inflammatory cytokine and PGE(2) production. Recombinant resistin inhibited proteoglycan synthesis in human cartilage explants. CONCLUSION: Resistin is elevated both systemically and locally in the weeks immediately following joint injury and has a direct effect on cartilage matrix turnover and cytokine production. Resistin may play a role in the early stages of trauma-induced OA and may represent a new therapeutic target to slow joint destruction in OA.
Assuntos
Artrite Reumatoide/metabolismo , Cartilagem Articular/metabolismo , Articulações/metabolismo , Resistina/metabolismo , Líquido Sinovial/metabolismo , Adolescente , Adulto , Idoso , Animais , Cartilagem Articular/lesões , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Articulações/lesões , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto JovemRESUMO
ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Oligopeptídeos/uso terapêutico , Receptores CXCR4/antagonistas & inibidores , Adulto , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacocinética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular , Qualidade de Produtos para o Consumidor , Feminino , Infecções por HIV/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Oligopeptídeos/administração & dosagem , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores CXCR4/fisiologiaRESUMO
Receptor binding largely governs viral tropism, since the presence of CD4 and an appropriate coreceptor is a prerequisite for membrane fusion and virus infection. Env-receptor interactions are conformationally complex, involving multiple regions in both gp120 as well as in the receptors. As a result, differences in receptor conformation, posttranslational processing, and surface density all have the potential to influence viral infectivity and therefore tropism and pathogenesis. This review gives an overview of the research that led to the discovery of chemokine receptors as coreceptors for HIV-1, describes the repertoire of coreceptors described to date and addresses their in vivo relevance. We will discuss very recent studies that indicate that while the presence of CD4 and coreceptor are necessary for virus infection, their mere presence is not sufficient.
Assuntos
Infecções por HIV/imunologia , Receptores de Quimiocinas/química , Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Conformação Proteica , Processamento de Proteína Pós-Traducional , Receptores de Quimiocinas/genética , Receptores de HIV/química , Receptores de HIV/genéticaRESUMO
We have used a V3-specific heteroduplex tracking assay (V3-HTA) with probes from two different human immunodeficiency virus type 1 (HIV-1) subtypes to examine the extent and pace of HIV-1 evolution late in infection. Twenty-four subjects with advanced HIV-1 infection (CD4(+) T-cell count, <100/microl) and stable viral loads were studied using blood plasma samples collected over a study period of approximately 9 months, during which time most of the subjects were treated with reverse transcriptase inhibitors. The V3-HTA patterns from the first and last time points were evaluated initially to determine the amounts of change in V3 sequence populations, which were primarily changes in abundance in preexisting sequence populations. Three of the 24 subjects had major changes (greater than 50% total change in the relative abundance of the sequence populations), 11 subjects had intermediate changes (10 to 50% total change), and 10 subjects had minimal changes (less than 10% total change). The average total amount of change was between two- and threefold greater in subjects with X4-like variants, although there was no correlation between average viral load and the presence of X4-like variants. V3-HTA patterns in monthly samples from 11 of the subjects were also compared. In two subjects, the amount of change exceeded 40% in a 1-month period. Overall, the pace of change in V3 populations varied between subjects and was not constant within a subject over time. Sequence analysis of the V3 variants showed that R5-like variants (not containing any X4-associated substitutions) continued to be maintained in three subjects in the presence of X4-like variants, indicating that X4 variants do not always outgrow R5 variants. The coreceptor usage of the V3 sequences from two subjects was determined using a cell fusion assay. One subject had an X4 variant that was maintained at a low level for at least 9 months, during which time the predominant variants were R5X4 (dualtropic), while in the second subject the reverse situation was observed. One of the dualtropic variants had a novel sequence motif in V3, suggesting another evolutionary pathway to altered tropism. These studies begin to probe the complexities and pace of V3 evolution in vivo, revealing dynamic patterns of change among multiple V3 sequence variants in a subset of subjects.
Assuntos
Variação Genética , Proteína gp160 do Envelope de HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Sequência de Bases , Contagem de Linfócito CD4 , Humanos , Dados de Sequência Molecular , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Fatores de Tempo , Carga ViralRESUMO
B cells can either differentiate in germinal centers or in extrafollicular compartments of secondary lymphoid organs. Here we show the migration properties of B cells after differentiation in murine peripheral lymph node infected with mouse mammary tumor virus. Naive B cells become activated, infected, and carry integrated retroviral DNA sequences. After production of a retroviral superantigen, the infected B cells receive cognate T cell help and differentiate along the two main differentiation pathways analogous to classical Ag responses. The extrafollicular differentiation peaks on day 6 of mouse mammary tumor virus infection, and the follicular one becomes detectable after day 10. B cells participating in this immune response carry a retroviral DNA marker that can be detected by using semiquantitative PCR. We determined the migration patterns of B cells having taken part in the T cell-B cell interaction from the draining lymph node to different tissues. Waves of immigration and retention of infected cells in secondary lymphoid organs, mammary gland, salivary gland, skin, lung, and liver were observed correlating with the two peaks of B cell differentiation in the draining lymph node. Other organs revealed immigration of infected cells at later time points. The migration properties were correlated with a strong up-regulation of alpha(4)beta(1) integrin expression. These results show the migration properties of B cells during an immune response and demonstrate that a large proportion of extrafolliculary differentiating plasmablasts can escape local cell death and carry the retroviral infection to peripheral organs.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfonodos/imunologia , Neoplasias Mamárias Experimentais/imunologia , Vírus do Tumor Mamário do Camundongo/imunologia , Plasmócitos/imunologia , Infecções por Retroviridae/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Antígenos CD/biossíntese , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Subpopulações de Linfócitos B/virologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Feminino , Membro Posterior , Integrina alfa4 , Integrina alfa4beta1 , Integrina beta1/biossíntese , Integrinas/biossíntese , Linfonodos/patologia , Linfonodos/virologia , Ativação Linfocitária , Tecido Linfoide/imunologia , Tecido Linfoide/patologia , Tecido Linfoide/virologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/virologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmócitos/metabolismo , Plasmócitos/patologia , Plasmócitos/virologia , Receptores de Retorno de Linfócitos/biossíntese , Infecções por Retroviridae/patologia , Infecções Tumorais por Vírus/patologiaRESUMO
Mouse mammary tumor virus (MMTV) is a retrovirus encoding a superantigen that is recognized in association with major histocompatibility complex class II by the variable region of the beta chain (V(beta)) of the T-cell receptor. The C-terminal 30 to 40 amino acids of the superantigen of different MMTVs display high sequence variability that correlates with the recognition of particular T-cell receptor V(beta) chains. Interestingly, MMTV(SIM) and mtv-8 superantigens are highly homologous but have nonoverlapping T-cell receptor V(beta) specificities. To determine the importance of these few differences for specific V(beta) interaction, we studied superantigen responses in mice to chimeric and mutant MMTV(SIM) and mtv-8 superantigens expressed by recombinant vaccinia viruses. We show that only a few changes (two to six residues) within the C terminus are necessary to modify superantigen recognition by specific V(beta)s. Thus, the introduction of the MMTV(SIM) residues 314-315 into the mtv-8 superantigen greatly decreased its V(beta)12 reactivity without gain of MMTV(SIM)-specific function. The introduction of MMTV(SIM)-specific residues 289 to 295, however, induced a recognition pattern that was a mixture of MMTV(SIM)- and mtv-8-specific V(beta) reactivities: both weak MMTV(SIM)-specific V(beta)4 and full mtv-8-specific V(beta)11 recognition were observed while V(beta)12 interaction was lost. The combination of the two MMTV(SIM)-specific regions in the mtv-8 superantigen established normal MMTV(SIM)-specific V(beta)4 reactivity and completely abolished mtv-8-specific V(beta)5, -11, and -12 interactions. These new functional superantigens with mixed V(beta) recognition patterns allowed us to precisely delineate sites relevant for molecular interactions between the SIM or mtv-8 superantigen and the T-cell receptor V(beta) domain within the 30 C-terminal residues of the viral superantigen.
Assuntos
Vírus do Tumor Mamário do Camundongo/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Superantígenos/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Aminoácidos , Animais , Apresentação de Antígeno , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Alinhamento de Sequência , Superantígenos/genéticaRESUMO
After mouse mammary tumor virus (MMTV) infection, B lymphocytes present a superantigen (Sag) and receive help from the unlimited number of CD4(+) T cells expressing Sag-specific T-cell receptor Vbeta elements. The infected B cells divide and differentiate, similarly to what occurs in classical B-cell responses. The amplification of Sag-reactive T cells can be considered a primary immune response. Since B cells are usually not efficient in the activation of naive T cells, we addressed the question of whether professional antigen-presenting cells such as dendritic cells (DCs) are responsible for T-cell priming. We show here, using MMTV(SIM), a viral isolate which requires major histocompatibility complex class II I-E expression to induce a strong Sag response in vivo, that transgenic mice expressing I-E exclusively on DCs (I-EalphaDC tg) reveal a strong Sag response. This Sag response was dependent on the presence of B cells, as indicated by the absence of stimulation in I-EalphaDC tg mice lacking B cells (I-EalphaDC tg muMT(-/-)), even if these B cells lack I-E expression. Furthermore, the involvement of either residual transgene expression by B cells or transfer of I-E from DCs to B cells was excluded by the use of mixed bone marrow chimeras. Our results indicate that after priming by DCs in the context of I-E, the MMTV(SIM) Sag can be recognized on the surface of B cells in the context of I-A. The most likely physiological relevance of the lowering of the antigen threshold required for T-cell/B-cell collaboration after DC priming is to allow B cells with a low affinity for antigen to receive T-cell help in a primary immune response.
Assuntos
Células Dendríticas/imunologia , Vírus do Tumor Mamário do Camundongo/imunologia , Infecções por Retroviridae/imunologia , Superantígenos/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Apresentação de Antígeno , Antígenos Virais/imunologia , Feminino , Imunidade Celular , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mouse mammary tumor virus (MMTV) has been shown to preferentially infect B lymphocytes in vivo. We have used recombinant envelope-coated fluospheres and highly purified MMTV particles to study the distribution of the viral receptors on fresh mouse lymphocytes. A preferential dose-dependent binding to B lymphocytes was observed which could be competed with neutralizing antibodies. In contrast, T-lymphocyte binding remained at background levels. These results strongly suggest a higher density of viral receptor molecules on B lymphocytes than on T lymphocytes and correlate with the preferential initial infection of B lymphocytes observed in vivo.
Assuntos
Antígenos Virais de Tumores/metabolismo , Linfócitos B/virologia , Vírus do Tumor Mamário do Camundongo/metabolismo , Animais , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/isolamento & purificação , Linfócitos B/citologia , Linfócitos B/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Baço/citologia , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
The C-type lectins DC-SIGN and DC-SIGNR capture and transfer human immunodeficiency virus (HIV) to susceptible cells, although the underlying mechanism is unclear. Here we show that DC-SIGN/DC-SIGNR-mediated HIV transmission involves dissociable binding and transfer steps, indicating that efficient virus transmission is not simply due to tethering of virus to the cell surface.
Assuntos
Moléculas de Adesão Celular , Infecções por HIV/transmissão , HIV/fisiologia , Lectinas Tipo C , Lectinas/fisiologia , Receptores de Superfície Celular/fisiologia , Antígenos CD4/análise , Proteína do Núcleo p24 do HIV/análise , HumanosRESUMO
DC-SIGN, a C-type lectin expressed on the surface of dendritic cells (DCs), efficiently binds and transmits HIVs and simian immunodeficiency viruses to susceptible cells in trans. A DC-SIGN homologue, termed DC-SIGNR, has recently been described. Herein we show that DC-SIGNR, like DC-SIGN, can bind to multiple strains of HIV-1, HIV-2, and simian immunodeficiency virus and transmit these viruses to both T cell lines and human peripheral blood mononuclear cells. Binding of virus to DC-SIGNR was dependent on carbohydrate recognition. Immunostaining with a DC-SIGNR-specific antiserum showed that DC-SIGNR was expressed on sinusoidal endothelial cells in the liver and on endothelial cells in lymph node sinuses and placental villi. The presence of this efficient virus attachment factor on multiple endothelial cell types indicates that DC-SIGNR could play a role in the vertical transmission of primate lentiviruses, in the enabling of HIV to traverse the capillary endothelium in some organs, and in the presentation of virus to CD4-positive cells in multiple locations including lymph nodes.
Assuntos
Moléculas de Adesão Celular , Endotélio/metabolismo , HIV-1/metabolismo , HIV-2/metabolismo , Lectinas Tipo C , Lectinas/metabolismo , Receptores de Superfície Celular/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Endotélio/citologia , Humanos , Lectinas/genética , Fígado/citologia , Fígado/metabolismo , Fígado/virologia , Linfonodos/citologia , Linfonodos/metabolismo , Linfonodos/virologia , Dados de Sequência Molecular , Monócitos/virologia , Placenta/citologia , Placenta/metabolismo , Placenta/virologia , Ligação Proteica , Receptores de Superfície Celular/genética , Linfócitos T/virologiaRESUMO
The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1 alpha and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Receptores CXCR4/imunologia , Sequência de Aminoácidos , Epitopos/química , Epitopos/imunologia , Humanos , Dados de Sequência Molecular , Conformação Proteica , Receptores CXCR4/químicaRESUMO
Dendritic cells (DCs) efficiently bind and transmit human immunodeficiency virus (HIV) to cocultured T cells and so may play an important role in HIV transmission. DC-SIGN, a novel C-type lectin that is expressed in DCs, has recently been shown to bind R5 HIV type 1 (HIV-1) strains and a laboratory-adapted X4 strain. To characterize the interaction of DC-SIGN with primate lentiviruses, we investigated the structural determinants of DC-SIGN required for virus binding and transmission to permissive cells. We constructed a panel of DC-SIGN mutants and established conditions which allowed comparable cell surface expression of all mutants. We found that R5, X4, and R5X4 HIV-1 isolates as well as simian immunodeficiency and HIV-2 strains bound to DC-SIGN and could be transmitted to CD4/coreceptor-positive cell types. DC-SIGN contains a single N-linked carbohydrate chain that is important for efficient cell surface expression but is not required for DC-SIGN-mediated virus binding and transmission. In contrast, C-terminal deletions removing either the lectin binding domain or the repeat region abrogated DC-SIGN function. Trypsin-EDTA treatment inhibited DC-SIGN mediated infection, indicating that virus was maintained at the surface of the DC-SIGN-expressing cells used in this study. Finally, quantitative fluorescence-activated cell sorting analysis of AU1-tagged DC-SIGN revealed that the efficiency of virus transmission was strongly affected by variations in DC-SIGN expression levels. Thus, variations in DC-SIGN expression levels on DCs could greatly affect the susceptibility of human individuals to HIV infection.
Assuntos
Moléculas de Adesão Celular , HIV-1/metabolismo , HIV-2/metabolismo , Lectinas Tipo C , Lectinas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Membrana Celular/metabolismo , Ácido Edético , Expressão Gênica , Glicosilação , Humanos , Lectinas/genética , Dados de Sequência Molecular , Mutagênese , Receptores de Superfície Celular/genética , Receptores Virais/genética , TripsinaRESUMO
A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.
Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Proteínas Sanguíneas/farmacologia , Antígenos CD4/genética , Fusão Celular , Linhagem Celular , Epitopos/metabolismo , Glicosilação , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/genética , HIV-1/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Testes de Neutralização , Conformação Proteica , Codorniz , Receptores CXCR4/metabolismo , Transfecção , Proteínas Virais de Fusão/efeitos dos fármacosRESUMO
APJ is a seven transmembrane domain G-protein-coupled receptor that functions as a coreceptor for some primate immunodeficiency virus strains. The in vivo significance of APJ coreceptor function remains to be elucidated, however, due to the lack of an antibody that can be used to assess APJ expression, and because of the absence of an antibody or ligand that can block APJ coreceptor activity. Therefore, we produced a specific monoclonal antibody (MAb 856) to APJ and found that it detected this receptor in FACS, immunofluorescence, and immunohistochemistry studies. MAb 856 also recognized APJ by Western blot, enabling us to determine that APJ is N-glycosylated. Using this antibody, we correlated APJ expression with coreceptor activity and found that APJ had coreceptor function even at low levels of expression. However, we found that APJ could not be detected by FACS analysis on cell lines commonly used to propagate primate lentiviruses, nor was it expressed on human PBMC cultured under a variety of conditions. We also found that some viral envelope proteins could mediate fusion with APJ-positive, CD4-negative cells, provided that CD4 was added in trans. These findings indicate that in some situations APJ use could render primary cell types susceptible to virus infection, although we have not found any evidence that this occurs. Finally, the peptide ligand for APJ, apelin-13, efficiently blocked APJ coreceptor activity.