RESUMO
OBJECTIVE: To study antibody-independent contributions of B cells to inflammatory disease activity, and the immune consequences of B-cell depletion with rituximab, in patients with multiple sclerosis (MS). METHODS: B-Cell effector-cytokine responses were compared between MS patients and matched controls using a 3-signal model of activation. The effects of B-cell depletion on Th1/Th17 CD4 and CD8 T-cell responses in MS patients were assessed both ex vivo and in vivo, together with pharmacokinetic/pharmacodynamic studies as part of 2 rituximab clinical trials in relapsing-remitting MS. RESULTS: B Cells of MS patients exhibited aberrant proinflammatory cytokine responses, including increased lymphotoxin (LT):interleukin-10 ratios and exaggerated LT and tumor necrosis factor (TNF)-alpha secretion, when activated in the context of the pathogen-associated TLR9-ligand CpG-DNA, or the Th1 cytokine interferon-gamma, respectively. B-Cell depletion, both ex vivo and in vivo, resulted in significantly diminished proinflammatory (Th1 and Th17) responses of both CD4 and CD8 T cells. Soluble products from activated B cells of untreated MS patients reconstituted the diminished T-cell responses observed following in vivo B-cell depletion in the same patients, and this effect appeared to be largely mediated by B-cell LT and TNFalpha. INTERPRETATION: We propose that episodic triggering of abnormal B-cell cytokine responses mediates 'bystander activation' of disease-relevant proinflammatory T cells, resulting in new relapsing MS disease activity. Our findings point to a plausible mechanism for the long-recognized association between infections and new MS relapses, and provide novel insights into B-cell roles in both health and disease, and into mechanisms contributing to therapeutic effects of B-cell depletion in human autoimmune diseases, including MS.
Assuntos
Linfócitos B/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Citocinas/metabolismo , Esclerose Múltipla/patologia , Linfócitos T/fisiologia , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proliferação de Células/efeitos dos fármacos , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , Acetato de Glatiramer , Humanos , Imunossupressores/farmacologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Linfotoxina-alfa , Masculino , Pessoa de Meia-Idade , Mitógenos/farmacologia , Esclerose Múltipla/sangue , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Muromonab-CD3/farmacologia , Peptídeos/farmacologia , Fito-Hemaglutininas/farmacologia , Rituximab , Linfócitos T/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfaRESUMO
Adenocarcinoma (AdC) of the lung represents a common histologic subtype of non-small cell lung cancer. While there is a rising incidence of AdC in nonsmoking women, information on the cytogenetic changes involved has been minimal to date. In the present study, spectral karyotyping analysis uncovered the genome-wide chromosomal aberrations in two AdC tumors derived from women who were lifelong nonsmokers. Simple and complex structural rearrangements were indicated. A ploidy status of hypertetraploidy was suggested in both cases, with recurring derivative translocations involving chromosome arms 3q, 8q, 12q, 15q, 22q, and Xq.
Assuntos
Adenocarcinoma/genética , Aberrações Cromossômicas , Neoplasias Pulmonares/genética , Cariotipagem Espectral , Adenocarcinoma/patologia , Idoso , Aneuploidia , Transformação Celular Neoplásica/genética , Feminino , Humanos , Neoplasias Pulmonares/patologia , Pessoa de Meia-Idade , Translocação Genética , Células Tumorais Cultivadas/ultraestruturaRESUMO
Nasopharyngeal carcinoma (NPC) cell lines and xenografts represent valuable models for functional and therapeutic studies on this common malignancy in Southeast Asia. The karyotypic information in most NPC cell lines and xenografts, however, remains largely unclear to date. We have characterized the chromosomal aberrations in six commonly used human NPC cell lines and xenografts using the molecular cytogenetic technique of comparative genomic hybridization (CGH). Genomic imbalances identified in cell lines were further correlated with structural abnormalities indicated from spectral karyotyping (SKY) analysis. CGH revealed consistent overrepresentations of 8q (six out of six cases) with a smallest overlapping region identified on 8q21.1 approximately q22. Other common gains included 7p (4/6 cases), 7q (4/6 cases), 12q (4/6), and 20q (4/6 cases), where minimal overlapping regions were suggested on 7p15 approximately p14, 7q11.2 approximately q21, and 12q22 approximately q24.1. Common losses were detected on 3p12 approximately p21 (4/6 cases) and 11q14 approximately qter (4/6 cases). Although SKY analysis on cell lines revealed predominantly unbalanced rearrangements, reciprocal translocations that involved chromosome 2 [i.e., t(1;2), t(2;3), and t(2;4)] were suggested. Furthermore, SKY examination illustrated additional breakpoints on a number of apparently balanced chromosomes. These breakpoints included 3p21, 3q26, 5q31, 6p21.1 approximately p25, 7p14 approximately p22, and 8q22. Our finding of regional gains and losses and breakpoints represents information that may contribute to NPC studies in vitro.
Assuntos
Aneuploidia , Carcinoma/genética , Aberrações Cromossômicas , Neoplasias Nasofaríngeas/genética , Animais , Carcinoma/patologia , Coloração Cromossômica , Cromossomos Humanos/genética , Cromossomos Humanos/ultraestrutura , Feminino , Humanos , Cariotipagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas/patologia , Hibridização de Ácido Nucleico , Translocação Genética , Transplante Heterólogo , Células Tumorais Cultivadas/patologia , Células Tumorais Cultivadas/transplanteRESUMO
B cells have paradoxical roles in autoimmunity, exerting both pathogenic and protective effects. Pathogenesis may be antibody independent, as B cell depletion therapy (BCDT) leads to amelioration of disease irrespective of autoantibody ablation. However, the mechanisms of pathogenesis are poorly understood. We demonstrate that BCDT alleviates central nervous system autoimmunity through ablation of IL-6-secreting pathogenic B cells. B cells from mice with experimental autoimmune encephalomyelitis (EAE) secreted elevated levels of IL-6 compared with B cells from naive controls, and mice with a B cell-specific IL-6 deficiency showed less severe disease than mice with wild-type B cells. Moreover, BCDT ameliorated EAE only in mice with IL-6-sufficient B cells. This mechanism of pathogenesis may also operate in multiple sclerosis (MS) because B cells from MS patients produced more IL-6 than B cells from healthy controls, and this abnormality was normalized with B cell reconstitution after Rituximab treatment. This suggests that BCDT improved disease progression, at least partly, by eliminating IL-6-producing B cells in MS patients. Taking these data together, we conclude that IL-6 secretion is a major mechanism of B cell-driven pathogenesis in T cell-mediated autoimmune disease such as EAE and MS.
Assuntos
Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Interleucina-6/metabolismo , Depleção Linfocítica/métodos , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/terapia , Transferência Adotiva , Análise de Variância , Animais , Anticorpos Monoclonais Murinos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Primers do DNA/genética , Encefalomielite Autoimune Experimental/fisiopatologia , Citometria de Fluxo , Humanos , Interleucina-6/deficiência , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rituximab , Organismos Livres de Patógenos Específicos , Estatísticas não ParamétricasRESUMO
B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0-24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35-74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-ß1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Morte Celular/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/patologia , Oligodendroglia/imunologia , Oligodendroglia/patologia , Animais , Animais Recém-Nascidos , Subpopulações de Linfócitos B/patologia , Sistema Livre de Células/imunologia , Sistema Livre de Células/metabolismo , Sistema Livre de Células/patologia , Células Cultivadas , Humanos , Microglia/imunologia , Microglia/patologia , Esclerose Múltipla Recidivante-Remitente/metabolismo , Oligodendroglia/citologia , RatosRESUMO
OBJECTIVE: Hyperhomocysteinemia adversely affects the endothelium, although the exact mechanism is unclear. Systemic lupus erythematosus (SLE) is an inflammatory disease with a high atherothrombotic tendency. We examined whether acute hyperhomocysteinemia exacerbates endothelial and platelet dysfunction in patients with SLE. METHODS: Twelve SLE patients and 15 controls were recruited. Oral methionine was used to achieve acute hyperhomocysteinemia. Endothelial function was assessed by flow-mediated dilatation (FMD) of the brachial artery; also assessed were the levels of von Willebrand factor (vWF) and plasminogen activator inhibitor type 1 (PAI-1). Platelet activation was assessed by the levels of beta-thromboglobulin (beta-TG), fibrinogen binding, and P-selectin expression using flow cytometry. RESULTS: After oral methionine loading, vWF levels increased significantly, whereas FMD remained unchanged in both groups. PAI-1 increased significantly only in controls. Fibrinogen binding to platelets increased significantly only in SLE patients. Beta-TG remained unchanged in SLE patients but increased significantly in controls. Platelet P-selectin expression did not change in either group. CONCLUSION: These results suggest that the prothrombotic tendency after acute hyperhomocysteinemia is mediated by endothelial dysfunction and platelet activation in patients with SLE and healthy controls.