RESUMEN
In inflammatory bowel disease (IBD), inflammation is sustained by an exaggerated response of lymphocytes. This results from enhanced expression of anti-apoptotic B cell lymphoma (BCL-2) and BCL-XL associated with a diminished turnover. Azathioprine (AZA) directly targets BCL-2 family-mediated apoptosis. We investigated whether the BCL-2 family expression pattern could be used to predict treatment response to AZA and determined whether BCL-2 inhibitor A-1211212 effectively diminishes lymphocytes and ameliorates inflammation in a model of colitis. BCL-2 family expression pattern was determined by next-generation sequencing (NGS). BCL-2 inhibitor was administered orally to Il10-/- mice. Haematological analyses were performed with an ADVIA 2120 and changes in immune cells were investigated using quantitative polymerase chain reaction (qPCR) and fluorescence activated cell sorter (FACS). We determined similar expression levels of BCL-2 family members in patients with remission and patients refractory to treatment, showing that BCL-2 family expression can not predict AZA treatment response. Expression was not correlated with the modified Truelove and Witts activity index (MTWAI). BCL-2 inhibitor initiated cell death in T cells from patients refractory to AZA and reduced lymphocyte count in Il10-/- mice. FACS revealed diminished CD8+ T cells upon BCL-2 inhibitor in Il10-/- mice without influencing platelets. Tnf, Il1ß, IfnÆ and Mcp-1 were decreased upon BCL-2 inhibitor. A-1211212 positively altered the colonic mucosa and ameliorated inflammation in mice. Pro-apoptotic BCL-2 inhibitor A-1211212 diminishes lymphocytes and ameliorates colitis in Il10-/- mice without inducing thrombocytopenia. BCL-2 inhibition could be a new therapy option for patients refractory to AZA.
Asunto(s)
Azatioprina/uso terapéutico , Colitis/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Linfocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Apoptosis , Células Cultivadas , Colitis/diagnóstico , Colitis/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/genética , Interleucina-10/genética , Linfocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Resultado del TratamientoRESUMEN
Objective The anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) may contribute to the pathogenesis of systemic lupus erythematosus. The safety, tolerability, and pharmacodynamics of the selective Bcl-2 inhibitor venetoclax (ABT-199) were assessed in women with systemic lupus erythematosus. Methods A phase 1, double-blind, randomized, placebo controlled study evaluated single ascending doses (10, 30, 90, 180, 300, and 500 mg) and multiple ascending doses (2 cycles; 30, 60, 120, 240, 400, and 600 mg for 1 week, and then 3 weeks off per cycle) of orally administered venetoclax. Eligible participants were aged 18-65 years with a diagnosis of systemic lupus erythematosus for 6 months or more receiving stable therapy for systemic lupus erythematosus (which could have included corticosteroids and/or stable antimalarials). Results All patients (48/48) completed the single ascending dose, 25 continued into the multiple ascending dose, and 44/50 completed the multiple ascending dose; two of the withdrawals (venetoclax 60 mg and 600 mg cohorts) were due to adverse events. Adverse event incidences were slightly higher in the venetoclax groups compared with the placebo groups, with no dose dependence. There were no serious adverse events with venetoclax. The most common adverse events were headache, nausea, and fatigue. Venetoclax 600 mg multiple ascending dose treatment depleted total lymphocytes and B cells by approximately 50% and 80%, respectively. Naive, switched memory, and memory B-cell subsets enriched in autoreactive B cells exhibited dose-dependent reduction of up to approximately 80%. There were no consistent or marked changes in neutrophils, natural killer cells, hemoglobin, or platelets. Conclusions Venetoclax was generally well tolerated in women with systemic lupus erythematosus and reduced total lymphocytes and disease-relevant subsets of antigen-experienced B cells. Registration ClinicalTrials.gov: NCT01686555.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Relación Dosis-Respuesta a Droga , Lupus Eritematoso Sistémico/tratamiento farmacológico , Sulfonamidas/farmacocinética , Administración Oral , Adolescente , Adulto , Anciano , Anticuerpos Antinucleares/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Subgrupos de Linfocitos B/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/efectos adversos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Método Doble Ciego , Femenino , Humanos , Lupus Eritematoso Sistémico/epidemiología , Persona de Mediana Edad , Sulfonamidas/administración & dosificación , Sulfonamidas/efectos adversos , Sulfonamidas/farmacología , Resultado del Tratamiento , Adulto JovenRESUMEN
B-1 cells constitute a distinct B cell subset with characteristic phenotypic and functional features. B-1 cells are highly represented among peritoneal lymphocytes; substantial numbers of B-1 cells are also located within splenic tissue. Here a number of differences in transcription factor and gene expression were identified that separate peritoneal B-1 and splenic B-2 cells, and then splenic B-1 cells obtained from immunoglobulin transgenic mice were tested for these parameters. Splenic B-1 cells resembled splenic B-2 cells rather than peritoneal B-1 cells in terms of nuclear expression of DNA-binding STAT3, CREB, and PU.1, with respect to transcriptional activation of IL-10, and in the failure to enter cell cycle in response to PMA. Splenic B-1 cells (B-1S) appear to constitute a unique population of B-1 cells, which, while sharing with peritoneal B-1 cells (B-1P) certain phenotypic features, differ from them in transcription factor and gene expression and in signaling for cell cycle progression.
Asunto(s)
Subgrupos de Linfocitos B/clasificación , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Interleucina-10/biosíntesis , Peritoneo/citología , Proteínas Proto-Oncogénicas/biosíntesis , Bazo/citología , Transactivadores/biosíntesis , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/metabolismo , División Celular , Separación Celular , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos BALB C , Mitógenos/farmacología , Factor de Transcripción STAT3 , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
OBJECTIVE: To determine if the Trillium Biopassive Surface (Medtronic Cardiopulmonary, Minneapolis, MN) coating added to the cardiopulmonary bypass oxygenator reduces inflammatory mediators, blood loss, and transfusion requirements. DESIGN: Prospective, randomized, and blinded human trial. SETTING: Tertiary care academic medical center. PARTICIPANTS: Thirty adult patients undergoing elective coronary artery bypass graft surgery. INTERVENTIONS: Patients received visually identical coated or uncoated oxygenators. MEASUREMENTS AND MAIN RESULTS: Hemoglobin, hematocrit, leukocyte count, platelet count, terminal complement complex, complement activation, myeloperoxidase, beta-thromboglobulin, prothrombin fragment 1.2, plasmin-antiplasmin, heparin concentration, activated coagulation time, and fibrinogen concentration were measured. Blood loss and blood product usage were recorded. In both groups, there were significant inflammatory alterations with the initiation of cardiopulmonary bypass. In the postprotamine samples, the coated oxygenator group had small but significant increases in hemoglobin, hematocrit, and leukocyte count. There were no differences in inflammatory mediators, blood loss, or transfusion requirements between the coated and uncoated groups. CONCLUSION: This human trial of Trillium Biopassive Surface-coated oxygenators did not show clinical benefits or clinically important biochemical results.
Asunto(s)
Puente Cardiopulmonar , Materiales Biocompatibles Revestidos , Oxigenadores , Adulto , Anciano , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
Interleukin-2-deficient (IL-2-/-) mice develop a spontaneous, progressive, CD4+ T-cell-mediated colitis with an age-related decrease in the number of B lymphocytes. The aim of this study was to determine the mechanisms of B-cell loss in IL-2-/- mice. Serum immunoglobulin G1 (IgG1) levels in 8-week-old IL-2-/- mice were above normal but then decreased dramatically with advancing age. Between 8 and 11 weeks of age, the number of B-cell progenitors (B220+ IgM-) in the bone marrow of IL-2-/- mice was less than half of those in IL-2+/+ littermates. By 22 weeks of age, very few progenitor cells remained in the bone marrow of most mice, and spleens were almost devoid of B cells. Likewise, B1 cells were not present in the peritoneal cavity of aged IL-2-/- mice. Flow cytometry analysis of B-cell differentiation in the bone marrow suggested a progressive loss of B cells from the most mature to the least mature stages, which was not dependent on IL-2 receptor-alpha (IL-2Ralpha) expression. B cells transferred from normal animals had similar survival rates in IL-2-/- and wild-type mice. We conclude that conventional B cells in older IL-2-/- mice are lost by attrition owing to a derangement in B-cell development. Because B1 cells are less dependent on the bone marrow, a separate mechanism for their loss is suggested.
Asunto(s)
Linfocitos B/inmunología , Interleucina-2/inmunología , Animales , Líquido Ascítico/inmunología , Linfocitos B/patología , Células de la Médula Ósea/inmunología , Diferenciación Celular/inmunología , Supervivencia Celular/inmunología , Citometría de Flujo , Células Madre Hematopoyéticas/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Interleucina-2/deficiencia , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
STUDY OBJECTIVE: To develop, validate, and assess compliance with a heparin titration nomogram. DESIGN: Prospective, open-label trial. SETTING: University teaching hospital. SUBJECTS: Patients admitted with heart failure who required therapy with intravenous unfractionated heparin. Intervention. An in vitro concentration-response was determined by measuring activated partial thromboplastin times (aPTTs) on normal pooled plasma containing known concentrations of heparin. The therapeutic aPTT range was determined from the concentration-response by using the therapeutic heparin concentration range of 0.2-0.4 U/ml (protamine neutralization). Patients were consecutively enrolled, and therapy was managed by using the heparin titration nomogram. Paired aPTT-heparin concentrations were obtained, and nomogram validation was performed by comparing the in vitro and the ex vivo concentration-responses with use of linear regression. Nomogram compliance also was assessed. MEASUREMENTS AND MAIN RESULTS: The therapeutic aPTT ranges based on in vitro and ex vivo data were determined to be 45-72 seconds and 47-61 seconds, respectively. The ranges were significantly different (p<0.001). Overall compliance with the nomogram was 88%. CONCLUSION: These results confirm that, even in a relatively homogeneous disease-state patient population, in vitro data do not accurately predict ex vivo data. If in vitro data are used to develop an institution-specific nomogram, a validation procedure should be used to ensure accuracy. Although 100% compliance to a nomogram may not be attainable, it should be expected. Therefore, a compliance rate of 88% is concerning and suggests a need for increased nursing and physician education.
Asunto(s)
Anticoagulantes/administración & dosificación , Anticoagulantes/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Heparina/administración & dosificación , Heparina/uso terapéutico , Anticoagulantes/análisis , Femenino , Heparina/análisis , Hospitales Universitarios , Humanos , Masculino , Errores de Medicación/prevención & control , Persona de Mediana Edad , Tiempo de Tromboplastina Parcial , Valor Predictivo de las Pruebas , Estudios Prospectivos , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
V(H)12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine. Selection begins with the V(H) third complementarity-determining region (CDR3) at the pre-B cell stage, in which most V(H)12 pre-B cells are selectively eliminated, enriching for those with V(H)CDR3s of 10 aa and a fourth position Gly (designated 10/G4). To understand this selection, we compared B cell differentiation in mice of two V(H)12 transgenic lines, one with the favored 10/G4 V(H)CDR3 and one with a non-10/G4 V(H)CDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that V(H)12 H chains have evolved such that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/patología , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/biosíntesis , Región Variable de Inmunoglobulina/genética , Células Madre/inmunología , Células Madre/patología , Alelos , Animales , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , División Celular/genética , División Celular/inmunología , Línea Celular , Femenino , Reordenamiento Génico de Cadena Ligera de Linfocito B , Cadenas Pesadas de Inmunoglobulina/fisiología , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Células Precursoras de Linfocitos B , Receptores de Antígenos de Linfocitos B , Células Madre/metabolismo , TransfecciónRESUMEN
The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.
Asunto(s)
Neoplasias de la Mama/enzimología , Gelatinasas/metabolismo , Oxígeno/farmacología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Trofoblastos/enzimología , Anciano , Neoplasias de la Mama/patología , Movimiento Celular , Femenino , Gelatinasas/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , Embarazo , Inhibidor Tisular de Metaloproteinasa-1/genética , Trofoblastos/citologíaRESUMEN
To determine the regulation of B cells specific for the ribonucleoprotein Sm, a target of the immune system in human and mouse lupus, we have generated mice carrying an anti-Sm H chain transgene (2-12H). Anti-Sm B cells in nonautoimmune 2-12H-transgenic (Tg) mice are functional, but, in the absence of immunization, circulating anti-Sm Ab levels are not different from those of non-Tg mice. In this report, we compare the regulation of anti-Sm B cells in nonautoimmune and autoimmune MRL/Mp-lpr/lpr (MRL/lpr) and bcl-2-22-Tg mice. Activation markers are elevated on splenic and peritoneal anti-Sm B cells of both nonautoimmune and autoimmune genetic backgrounds indicating Ag encounter. Although tolerance to Sm is maintained in 2-12H/bcl-2-22-Tg mice, it is lost in 2-12H-Tg MRL/lpr mice, as the transgene accelerates and increases the prevalence of the anti-Sm response. The 2-12H-Tg MRL/lpr mice have transitional anti-Sm B cells in the spleen similar to nonautoimmune mice. However, in contrast to nonautoimmune mice, there are few if any peritoneal anti-Sm B-1 cells. These data suggest that a defect in B-1 differentiation may be a factor in the loss of tolerance to Sm and provide insight into the low prevalence of the anti-Sm response in lupus.
Asunto(s)
Autoantígenos/inmunología , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Activación de Linfocitos/genética , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Envejecimiento/genética , Envejecimiento/inmunología , Animales , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/genética , Subgrupos de Linfocitos B/metabolismo , Biomarcadores , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Relación Dosis-Respuesta Inmunológica , Regulación de la Expresión Génica/inmunología , Genes bcl-2/inmunología , Inmunofenotipificación , Ratones , Ratones Endogámicos MRL lpr , Ratones Transgénicos , Peritoneo/citología , Peritoneo/inmunología , Peritoneo/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Transgenes/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , Proteínas Nucleares snRNPRESUMEN
Systemic lupus erythematosus is an autoimmune disease characterized by the presence of autoantibodies. One of the unique targets of the immune system in systemic lupus erythematosus is Sm, a ribonucleoprotein present in all cells. To understand the regulation of B cells specific to the Sm Ag in normal mice, we have generated an Ig H chain transgenic mouse (2-12H Tg). 2-12H Tg mice produce B cells specific for the Sm that remain tolerant due to ignorance. We demonstrate here that anti-Sm B cells of 2-12H Tg mice can differentiate into Sm-specific peritoneal B-1 cells that remain tolerant. Differentiation to B-1 and tolerance are governed by the strength of B cell receptor signaling, since manipulations of the B cell receptor coreceptors CD19 and CD22 affect anti-Sm B cell differentiation and autoantibody production. These results suggest a differentiation scheme in which peripheral ignorance to Sm is maintained in mice by the differentiation of anti-Sm B cells to B-1 cells that have increased activation thresholds.
Asunto(s)
Autoantígenos/inmunología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Moléculas de Adhesión Celular , Tolerancia Inmunológica , Lectinas , Lupus Eritematoso Sistémico/inmunología , Receptores de Antígenos de Linfocitos B/fisiología , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Transducción de Señal/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos CD19/biosíntesis , Antígenos CD19/genética , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Antígenos de Diferenciación de Linfocitos B/genética , Autoanticuerpos/sangre , Autoanticuerpos/fisiología , Subgrupos de Linfocitos B/patología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Tolerancia Inmunológica/genética , Ratones , Ratones Endogámicos MRL lpr , Ratones Noqueados , Ratones Transgénicos , Peritoneo/citología , Peritoneo/inmunología , Receptores de Antígenos de Linfocitos B/biosíntesis , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Transducción de Señal/genética , Bazo/citología , Bazo/inmunología , Proteínas Nucleares snRNPAsunto(s)
Subgrupos de Linfocitos B/fisiología , Quinasas Ciclina-Dependientes/fisiología , Ciclinas/biosíntesis , Ciclinas/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas , Receptores de Quimiocina/biosíntesis , Transactivadores/metabolismo , Animales , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/enzimología , Antígenos CD5/análisis , Linaje de la Célula , Ciclina D2 , Ciclina D3 , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Ciclinas/genética , Inducción Enzimática , Humanos , Activación de Linfocitos/efectos de los fármacos , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/patología , Ratones , Ratones Transgénicos , Especificidad de Órganos , Cavidad Peritoneal/citología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores CCR2 , Receptores CCR3 , Receptores de Quimiocina/genética , Factor de Transcripción STAT3 , Transducción de Señal , Bazo/citología , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
PtC-specific B-1 cells originate from conventional B-2 (B-0) cells as a result of antigen activation. VH12 B cells specific for PtC are enriched at two developmental checkpoints in the bone marrow; first at the pre-BI to pre-BII transition where VH12 pre-B cells with anti-PtC VHCDR3 are enriched, and second at the pre-BII to immature B cell transition where L chain diversity is restricted. This restriction is due to the inability of most L chains to associate with VH12 H chains. We present evidence here of a third developmental checkpoint that enriches for PtC-specific B cells, at the transitional to mature B-2 (B-0) cell stage. Most VH12 transitional B cells do not differentiate to a mature B-2 cell and, of those that do, most have undergone receptor editing. The V kappa 4/5H L chain appears to be one of the few L chains that can support differentiation to the mature B-2 cell stage, providing an explanation for its dominance among VH12 B cells in the spleen. Once cells reach this stage, those that bind PtC are induced to differentiate to B-1. Thus, through selection at multiple differentiative stages and the induction of extensive secondary V kappa rearrangement and receptor editing, VH12 B cell differentiation is focused toward specificity for PtC and selection to the B-1 subset.
Asunto(s)
Anticuerpos Antifosfolípidos/inmunología , Subgrupos de Linfocitos B/citología , Reordenamiento Génico de Cadena Ligera de Linfocito B , Cadenas kappa de Inmunoglobulina/genética , Fosfatidilcolinas/inmunología , Bazo/citología , Animales , Anticuerpos Antifosfolípidos/genética , Especificidad de Anticuerpos , Subgrupos de Linfocitos B/inmunología , Diferenciación Celular , Linaje de la Célula , Separación Celular , Supresión Clonal , Citometría de Flujo , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Liposomas/química , Liposomas/inmunología , Activación de Linfocitos , Ratones , Ratones Transgénicos , Modelos Inmunológicos , Bazo/inmunologíaRESUMEN
Anti-neutrophil cytoplasmic Abs, directed primarily toward myeloperoxidase (MPO) and proteinase 3, are detected in the majority of patients with distinct forms of small vessel vasculitides and pauci-immune necrotizing glomerulonephritis. However, the origin of these autoantibodies remains unknown. We studied the V region gene use in murine anti-MPO Abs derived from Spontaneous Crescentic Glomerulonephritis/Kinjoh mice. A total of 13 anti-MPO-producing hybridomas were generated from four unimmunized mice. Ten of the 13 hybridomas (corresponding to 3 of 4 clones) expressed Vkappa1C but differed in their use of VH genes. The remaining three hybridomas expressed a Vkappa5 gene. Anti-MPO hybridomas from individual mice were derived from single clones as deduced by sequence similarity and splice-site identity. We found a statistically significant bias of amino acid replacement mutations to the complementarity-determining regions (CDR) in the Vkappa1C-expressing hybridomas. Intriguingly, all 10 Vkappa1C hybridomas share a lysine to glutamate mutation in the CDR1. To determine the effects of somatic V gene mutations on binding to MPO, we generated an anti-MPO Ab with an unmutated Vkappa1C L chain and compared its ability to bind MPO with its mutated counterpart. The mutated hybridoma-derived Ab has a 4.75-fold higher avidity for MPO than the unmutated Ab. These results suggest that: 1) the L chain plays a dominant role in determining Ab specificity to MPO, 2) the anti-MPO Ab response is oligoclonal, consistent with Ag selection, and 3) MPO is a driving Ag in the murine anti-MPO Ab response.
Asunto(s)
Autoanticuerpos/biosíntesis , Autoantígenos/inmunología , Reordenamiento Génico/genética , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Peroxidasa/inmunología , Secuencia de Aminoácidos , Animales , Autoanticuerpos/genética , Autoantígenos/metabolismo , Sitios de Unión de Anticuerpos/genética , Mutación de Línea Germinal/inmunología , Humanos , Hibridomas/inmunología , Hibridomas/metabolismo , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/aislamiento & purificación , Cadenas kappa de Inmunoglobulina/biosíntesis , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Peroxidasa/metabolismoRESUMEN
The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vkappa4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vkappa4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Ciclosporina/farmacología , Inhibidores de Crecimiento/farmacología , Fosfatidilcolinas/inmunología , Bazo/citología , Células Madre/citología , Células Madre/inmunología , Animales , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/trasplante , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Epítopos de Linfocito B/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Bazo/inmunología , Bazo/trasplante , Células Madre/efectos de los fármacosRESUMEN
Phosphatidyl choline (PtC)-specific B cells segregate to the B-1 subset, where they comprise up to 10% of the B-1 repertoire. About half express V(H)12 and Vkappa4/5H and are restricted in V(H)CDR3. We have previously reported that anti-PtC V(H)CDR3 is enriched among V(H)12-expressing cells by selective elimination of pre-B cells. We report here a bias for Vkappa4/5H expression among V(H)12-expressing B cells, even among those that do not bind PtC and are not B-1. This is due in part to an inability of V(H)12 to associate with many light (L) chains but must also be due to a selective advantage in survival or clonal expansion in the periphery for Vkappa4/5H-expressing cells. Thus, the bias for Vkappa4/5H expression is independent of PtC binding, and, as segregation to B-1 occurs after Ig gene expression, it precedes segregation to the B-1 subset. In 6-1 mice, splenic B-1 cells reside in follicles but segregate to follicles distinct from those that contain B-2 cells. These data indicate that selection at multiple developmental checkpoints ensures the co-expression of an anti-PtC V(H)CDR3 and L chain in a high frequency of V(H)12 B cells. This focus toward specificity for PtC facilitates the development of a large anti-PtC B-1 repertoire.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Regiones Determinantes de Complementariedad , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Subgrupos de Linfocitos B/citología , Diferenciación Celular , Supervivencia Celular , Cruzamientos Genéticos , Citometría de Flujo , Reordenamiento Génico de Cadena Pesada de Linfocito B , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Fosfatidilcolinas/inmunología , Bazo/inmunologíaRESUMEN
Autoantibodies directed at a diverse group of proteins of the U1/Sm ribonucleoprotein (snRNP) are characteristic of systemic lupus erythematosus and are found in the MRL murine model of this disease. This study examines the role of transgenic B lymphocytes in the regulation of autoreactive T cells to the snRNP autoantigen. Transgenic mice were developed bearing an Ig heavy chain gene specific for the D protein component of murine snRNP. B lymphocytes in these mice are neither deleted nor anergic and are of an immature (heat-stable Aghigh) phenotype. T lymphocytes from anti-snRNP transgenic mice were examined using a recombinant form of the D protein of the murine snRNP complex. Our results revealed that transgenic anti-snRNP B cell APCs stimulated CD4 T cells from wild-type C57BL/6 and MRL lpr/lpr mice, while nonspecific APCs failed to stimulate CD4 T cells. This study demonstrates that autoreactive T cells are not deleted from wild-type mice, although their activation is facilitated by autoantigen-specific APCs. The snRNP-reactive T cells in C57BL/6 transgenic mice are tolerized, in contrast to those T cells from MRL lpr/lpr transgenic mice. These studies implicate a role for autoreactive B lymphocytes in the in vivo activation and/or diversification of autoreactive T cells.
Asunto(s)
Genes de Inmunoglobulinas/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Autoantígenos/inmunología , Autoantígenos/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Transgénicos , Péptidos/inmunología , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Linfocitos T/metabolismoRESUMEN
Murine phosphatidyl choline (PtC)-specific B cells in normal mice belong exclusively to the B-1 subset. Analysis of anti-PtC (VH12 and VH12/Vkappa4) transgenic (Tg) mice indicates that exclusion from B-0 (also known as B-2) occurs after immunoglobulin gene rearrangement. This predicts that PtC-specific B-0 cells are generated, but subsequently eliminated by either apoptosis or differentiation to B-1. To investigate the mechanism of exclusion, PtC-specific B cell differentiation was examined in mice expressing the X-linked immunodeficiency (xid) mutation. xid mice lack functional Bruton's tyrosine kinase (Btk), a component of the B cell receptor signal transduction pathway, and are deficient in B-1 cell development. We find in C57BL/ 6.xid mice that VH12 pre-BII cell selection is normal and that PtC-specific B cells undergo modest clonal expansion. However, the majority of splenic PtC-specific B cells in anti-PtC Tg/xid mice are B-0, rather than B-1 as in their non-xid counterparts. These data indicate that PtC-specific B-0 cell generation precedes segregation as predicted, and that Btk function is required for efficient segregation to B-1. Since xid mice exhibit defective B cell differentiation, not programmed cell death, these data are most consistent with an inability of PtC-specific B-0 cells to convert to B-1 and a single B cell lineage.
Asunto(s)
Autoantígenos/inmunología , Subgrupos de Linfocitos B/inmunología , Reordenamiento Génico de Linfocito B , Células Madre Hematopoyéticas/inmunología , Inmunoglobulina M , Fosfatidilcolinas/inmunología , Agammaglobulinemia Tirosina Quinasa , Animales , Subgrupos de Linfocitos B/citología , Médula Ósea/inmunología , Diferenciación Celular , Linaje de la Célula , Ligamiento Genético , Células Madre Hematopoyéticas/citología , Enfermedades del Sistema Inmune/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Peritoneo/inmunología , Proteínas Tirosina Quinasas/genética , Aberraciones Cromosómicas Sexuales , Bazo/citología , Bazo/inmunología , Cromosoma XRESUMEN
Anti-Sm and anti-ssDNA transgenic (Tg) mice were generated using the VH-D-JH rearrangement of an anti-Sm hybridoma of MRL/Mp-lpr/lpr origin. B cells of each specificity account for 15%-35% of the splenic repertoire, but no circulating anti-Sm or anti-ssDNA antibodies are detected. Most autoreactive cells exhibit an immature B cell phenotype and have short half-lives equivalent to those of non-Tg immature B cells. However, at least some anti-Sm B cells are functional, because immunization with murine snRNPs induces anti-Sm secretion. We propose that anti-Sm and anti-ssDNA are eliminated during the transition to mature B cells and that this late stage of tolerance induction is consequential to their spontaneous activation in murine lupus.
Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Lupus Eritematoso Sistémico/inmunología , Animales , Autoantígenos/genética , Médula Ósea/inmunología , Diferenciación Celular , ADN de Cadena Simple/inmunología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Reordenamiento Génico de Cadena Pesada de Linfocito B , Células Madre Hematopoyéticas , Hibridomas , Tolerancia Inmunológica , Lupus Eritematoso Sistémico/etiología , Activación de Linfocitos , Ratones , Ratones Endogámicos MRL lpr , Ratones Transgénicos , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Bazo/inmunología , Proteínas Nucleares snRNPRESUMEN
We report the case of a 17-year-old boy with a significant history of drug and alcohol abuse, which included smoking marijuana mixed with brodifacoum. As a consequence, the patient developed a prolonged coagulopathy that persisted for more than 1 year. To our knowledge, this is the first case reported in the literature in which super-warfarin intoxication has been associated with marijuana smoking. This report should increase the awareness of pathologists and clinicians when examining a patient with a history of drug abuse who exhibits persistent vitamin K1-dependent coagulopathy.
Asunto(s)
4-Hidroxicumarinas/envenenamiento , Coagulación Sanguínea/efectos de los fármacos , Deficiencia del Factor X/inducido químicamente , Fumar Marihuana , Rodenticidas/envenenamiento , Adolescente , Factores de Coagulación Sanguínea/análisis , Deficiencia del Factor X/diagnóstico , Deficiencia del Factor X/terapia , Humanos , Masculino , Tiempo de Protrombina , Vitamina K 1/administración & dosificación , Vitamina K 1/antagonistas & inhibidoresRESUMEN
Anti-DNA induced in BALB/c mice by immunization with bacterial (Escherichia coli) DNA resemble spontaneous anti-DNA from lupus mice in V gene use and cross-reactivity with other nuclear Ags, but lack the high V(H) CDR3 arginine content seen in anti-DNA from lupus mice. Moreover, the induced anti-DNA bind bacterial and mammalian single-stranded (ss) DNA and bacterial double-stranded (ds) DNA, but do not bind mammalian dsDNA. This reactivity profile is in contrast to that of the spontaneously arising anti-DNA of lupus mice, among which mammalian dsDNA reactive Abs are prominent. In this study we demonstrate that the addition of arginine to V(H) CDR3 of an induced anti-DNA confers the mammalian dsDNA binding characteristic of anti-DNA from lupus mice. The ability to confer mammalian dsDNA binding is dependent on both the position of the arginine in V(H) CDR3 and the light chain with which the heavy chain is paired, suggesting the light chain plays a more substantial role in DNA binding by this Ab than has previously been reported for other anti-DNA. Our data support the argument that V(H) CDR3 arginines tend to confer antimammalian dsDNA reactivity, leading to censure of B cells expressing these Abs and provides an explanation for the absence of arginine-rich V(H) CDR3 in the bacterial DNA-induced response.