RESUMEN
Cancers are characterized by genomic instability, and the resulting intraclonal diversity is a prerequisite for tumor evolution. Therefore, metrics of tumor heterogeneity may prove to be clinically meaningful. Intraclonal heterogeneity in follicular lymphoma (FL) is apparent from studies of somatic hypermutation (SHM) caused by activation-induced deaminase (AID) in IGH. Aberrant SHM (aSHM), defined as AID activity outside of the IG loci, predominantly targets noncoding regions causing numerous "passenger" mutations, but it has the potential to generate rare significant "driver" mutations. The quantitative relationship between SHM and aSHM has not been defined. To measure SHM and aSHM, ultradeep sequencing (>20,000-fold coverage) was performed on IGH (~1650 nt) and nine other noncoding regions potentially targeted by AID (combined 9411 nt), including the 5' untranslated region of BCL2. Single-nucleotide variants (SNVs) were found in 12/12 FL specimens (median 136 SHMs and 53 aSHMs). The aSHM SNVs were associated with AID motifs (p < 0.0001). The number of SNVs at BCL2 varied widely among specimens and correlated with the number of SNVs at eight other potential aSHM sites. In contrast, SHM at IGH was not predictive of aSHM. Tumor heterogeneity is apparent from SNVs at low variant allele frequencies; the relative number of SNVs with variable allele frequency < 5% varied with clinical grade, indicating that tumor heterogeneity based on aSHM reflects a clinically meaningful parameter. These data suggest that genome-wide aSHM may be estimated from aSHM of BCL2 but not SHM of IGH. The results demonstrate a practical approach to the quantification of intratumoral genetic heterogeneity for clinical specimens.
Asunto(s)
Regiones no Traducidas 5' , Genes bcl-2/genética , Genoma Humano , Linfoma Folicular/genética , Polimorfismo de Nucleótido Simple , Hipermutación Somática de Inmunoglobulina/genética , Alelos , Linfocitos B/inmunología , Linfocitos B/patología , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 18 , Células Clonales , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Expresión Génica , Frecuencia de los Genes , Genes bcl-2/inmunología , Sitios Genéticos , Inestabilidad Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Linfoma Folicular/inmunología , Linfoma Folicular/patología , Hipermutación Somática de Inmunoglobulina/inmunología , Translocación GenéticaRESUMEN
HCV (hepatitis C virus) chronic hepatitis has become one the most expensive diseases for public health systems all over the world in the past 10-20 years, a real epidemic, the second most frequent, after hepatitis B virus infection. Due to the complex manifestations, one may consider HCV infection as a "systemic" disease. Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of HCV infection, but cryoglobulinemic vasculitis (CV) is considered to be relatively sparse although prevalence studies are needed. Presence of serum cryoglobulins is essential for MC diagnosis, but serum levels do not correlate with the disease activity or prognosis. MC can be defined as a B lymphocyte proliferation disease being characterized by polyclonal activation and antibody synthesis. Evolution to lymphoma should be considered continuous but also other infectious, environmental or genetic factors could be involved. The t (14.18) translocation and Bcl-2 activation in B lymphocytes, B cell-activating factor (BAFF), E2-CD81 interaction, immunoregulatory T CD4+CD25(high) + lymphocytes and type III IFNs might play an important role in MC and lymphoma evolution in HCV patients.
Asunto(s)
Linfocitos B/metabolismo , Crioglobulinemia/epidemiología , Crioglobulinemia/inmunología , Epidemias , Hepatitis C Crónica , Linfoma , Factor Activador de Células B/metabolismo , Linfocitos B/inmunología , Estudios Transversales , Crioglobulinemia/etiología , Crioglobulinemia/fisiopatología , Crioglobulinas/análisis , Ambiente , Genes bcl-2/inmunología , Predisposición Genética a la Enfermedad , Salud Global , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/epidemiología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/fisiopatología , Humanos , Fenómenos Inmunogenéticos , Linfoma/etiología , Linfoma/genética , Linfoma/inmunología , Monitorización InmunológicaRESUMEN
Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/inmunología , Apoptosis/inmunología , Autofagia/inmunología , Virus de la Ectromelia/fisiología , Genes bcl-2/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Beclina-1 , Línea Celular , Chlorocebus aethiops , Replicación del ADN , Ectromelia Infecciosa/inmunología , Ectromelia Infecciosa/fisiopatología , Citometría de Flujo , Ratones , Microscopía Fluorescente , ARN Interferente Pequeño/farmacología , Células Vero , Replicación ViralRESUMEN
Subcompartments of the plasma membrane are believed to be critical for lymphocyte responses, but few genetic tools are available to test their function. Here we describe a previously unknown X-linked B cell-deficiency syndrome in mice caused by mutations in Atp11c, which encodes a member of the P4 ATPase family thought to serve as 'flippases' that concentrate aminophospholipids in the cytoplasmic leaflet of cell membranes. Defective ATP11C resulted in a lower rate of phosphatidylserine translocation in pro-B cells and much lower pre-B cell and B cell numbers despite expression of pre-rearranged immunoglobulin transgenes or enforced expression of the prosurvival protein Bcl-2 to prevent apoptosis and abolished pre-B cell population expansion in response to a transgene encoding interleukin 7. The only other abnormalities we noted were anemia, hyperbilirubinemia and hepatocellular carcinoma. Our results identify an intimate connection between phospholipid transport and B lymphocyte function.
Asunto(s)
Adenosina Trifosfatasas/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Endocitosis/inmunología , Fosfoserina/inmunología , Adenosina Trifosfatasas/genética , Animales , Linfocitos B/enzimología , Secuencia de Bases , Femenino , Citometría de Flujo , Genes bcl-2/inmunología , Interleucina-7/genética , Interleucina-7/inmunología , Hígado/citología , Hígado/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis/inmunología , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Multiple evidences support the notion that cell-cycle deregulation or apoptosis alterations can lead to autoimmune syndrome (AIS). Inactivation of the cell-cycle regulator E2F2 or over-expression of the anti-apoptotic Bcl-2 protein induces spontaneously an AIS in certain mouse strains. In the present study, we have examined the contribution of the genetic background on the development of autoimmunity after E2F2 gene inactivation, and the effect that a simultaneous inactivation of the E2F2 gene and over-expression of the Bcl-2 gene in B cells has on lymphoid homeostasis and autoimmunity. We show that E2F2(- / - ) mice carrying wild-type levels of Bcl-2 do not develop AIS when they are in a non-pro-autoimmune background (C57BL/6). However, mice harboring both genetic alterations concomitantly develop late AIS characterized by the presence of serum anti-nuclear antibodies, double and single strand anti-DNA antibodies, and the development of a mild glomerulonephritis with mesangial immunoglobulins, mainly IgA, deposits. These results suggest that alterations in cell-cycle and cell survival are critical contributing factors for the development of autoimmunity.
Asunto(s)
Enfermedades Autoinmunes/genética , Factor de Transcripción E2F2/genética , Genes bcl-2/genética , Predisposición Genética a la Enfermedad/genética , Animales , Apoptosis/genética , Enfermedades Autoinmunes/inmunología , Linfocitos B/inmunología , Recuento de Células , Ciclo Celular , Separación Celular , Factor de Transcripción E2F2/inmunología , Citometría de Flujo , Genes bcl-2/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Síndrome , Regulación hacia ArribaRESUMEN
IL-7 signaling is essential for optimal CD8 T cell function, homeostasis and establishment of memory. We have previously shown decreased expression of the IL-7 receptor alpha-chain (CD127) on CD8 T cells from HIV-infected patients with active viral replication. We have also shown that soluble HIV Tat protein specifically down-regulates CD127 on the surface of CD8 T cells and impairs cell proliferation and cytolytic potential following stimulation with IL-7 in vitro. We now show that soluble HIV Tat protein and IL-7 at near physiologic concentrations act synergistically to suppress CD127 expression. While soluble HIV Tat protein and IL-7 both independently reduce CD127 expression on the surface of CD8 T cells, Tat concentrations of 10 microg ml(-1) and IL-7 concentrations of 500 pg ml(-1) are required in vitro to have an appreciable effect. However, where 0.5 microg ml(-1) of Tat has no effect on CD127 expression and 200 pg ml(-1) of IL-7 decreases CD127 by only 14%, these two together at these same concentrations induce a 35% reduction in CD127 expression after 24 h. Inhibition of Janus kinase (JAK) completely blocks IL-7's ability to down-regulate CD127 on the surface of CD8 T cells and also abolishes synergy with Tat. Interestingly, while Tat acts synergistically with IL-7 to reduce CD127 expression, it antagonizes IL-7-induced cell proliferation and Ki-67 expression and has no effect on IL-7-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation or expression of the anti-apoptotic gene Bcl-2. Thus, by affecting different IL-7 signal transduction pathways, HIV Tat protein is able to impair both CD8 T cell activation and proliferation without inducing apoptosis.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Infecciones por VIH/inmunología , VIH-1/inmunología , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Interleucina-7/metabolismo , Antígeno Ki-67/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Apoptosis/inmunología , Linfocitos T CD8-positivos/patología , Proliferación Celular , Regulación hacia Abajo , Genes bcl-2/inmunología , Infecciones por VIH/virología , Humanos , Separación Inmunomagnética , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-7/inmunología , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/inmunología , Quinasas Janus/inmunología , Antígeno Ki-67/genética , Antígeno Ki-67/inmunología , Activación de Linfocitos/inmunología , Fosforilación , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The purpose of this study was to evaluate the beneficial effect of Hochu-ekki-to (TJ-41) combined with interferon-gamma (IFN gamma) on daily activity, immunological and neurological alternation in a mouse model of chronic fatigue syndrome (CFS). CFS was induced by 6 times of repeated injection of Brucella abortus antigen every 2 weeks. Both single TJ-41 and TJ-41 combined with IFN gamma increased running activity and thymus weight of CFS mice, while thicker thymic cortex together with elevation of natural killer cell activity was only found in the combined treatment group. No significant improvement was observed in the atrophic brain and decreased expression level of brain-derived neurotrophic factor and Bcl-2 mRNA in hippocampus in both treatment groups. Our results suggest that TJ-41 combined with IFN gamma might have a protective effect on the marked reduction in the activity in a model of CFS via normalization of host immune responses, but not neuroprotection.
Asunto(s)
Medicamentos Herbarios Chinos/administración & dosificación , Síndrome de Fatiga Crónica/tratamiento farmacológico , Interferón gamma/administración & dosificación , Células Asesinas Naturales/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Actividades Cotidianas , Animales , Antígenos Bacterianos/inmunología , Factor Neurotrófico Derivado del Encéfalo/análisis , Factor Neurotrófico Derivado del Encéfalo/inmunología , Brucella abortus/inmunología , Síndrome de Fatiga Crónica/inmunología , Síndrome de Fatiga Crónica/patología , Femenino , Genes bcl-2/efectos de los fármacos , Genes bcl-2/inmunología , Hipocampo/efectos de los fármacos , Hipocampo/inmunología , Hipocampo/patología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos BALB C , Actividad Motora/inmunología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/inmunología , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/inmunología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Timo/efectos de los fármacos , Timo/inmunología , Timo/patologíaRESUMEN
UNLABELLED: Most of extranodal lymphomas are localized in gastrointestinal tract, gastric lymphoma representing more than 50% of them. The role that Helicobacter pylori (H. pylori) plays in pathogenesis of gastric lymphoma has changed the therapeutic approach. AIMS: Description of morphological features and immunohistochemical pattern of gastric lymphomas from patients admitted in University Hospital "Sf. Spiridon" Iasi. MATERIAL AND METHODS: Thirty four gastric lymphomas were investigated using routine histopathological technics and immunohistochemical staining based on a large panel of antibodies: CD3, CD5, CD20, CD79á, CD23, CD30, cyclin-D1, BCL2, BCL6, ALK1, Ki67, CK-cocktail, anti-H. pylori. RESULTS: All gastric lymphomas were localized in the antrum, most of them being solitary and large-sized tumors. Ninety-seven percent were B-cell lymphomas, 41.17% were mucosa-associated lymphatic tissue lymphomas (MALT lymphomas), and the remaining were high grade lymphomas. Only one case was classified as peripheral T-cell lymphoma. Cytokeratin cocktail immunostaining improved the detection of typical lymphoepithelial lesions, which characterized exclusively the MALT lymphomas. The sensibility for H. pylori detection in gastric lymphoma cases was increased by 22% using anti-H. pylori antibodies. CONCLUSIONS: Immunohistochemistry is a diagnostic method for gastric lymphomas, being useful in identification of lymphoepithelial lesions, detection of H. pylori infection, and is mandatory for lymphomas classification according to WHO criteria.
Asunto(s)
Biomarcadores de Tumor/análisis , Linfoma/química , Linfoma/patología , Neoplasias Gástricas/química , Neoplasias Gástricas/patología , Receptores de Activinas Tipo II/análisis , Anticuerpos Antibacterianos/análisis , Antígenos CD/análisis , Biomarcadores de Tumor/inmunología , Ciclina D , Ciclinas/análisis , Proteínas de Unión al ADN/análisis , Genes bcl-2/inmunología , Infecciones por Helicobacter/complicaciones , Helicobacter pylori/inmunología , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Linfoma/microbiología , Proteínas Proto-Oncogénicas c-bcl-6 , Estudios Retrospectivos , Neoplasias Gástricas/microbiologíaRESUMEN
OBJECTIVE: To investigate the effect of HIV Tat protein on Bcl-2 expression in human monocytes, and observe apoptosis of Tat-stimulated monocytes induced by TNF-alpha-related apoptosis-induced ligand (TRAIL). METHODS: Western blot was used to detect Bcl-2 expression in monocytes stimulated by HIV Tat protein, and Annexin V and 7-AAD staining were used to detect apoptosis of monocytes induced by TRAIL. RESULTS: HIV Tat protein increased Bcl-2 expression in human monocytes in a dose-dependent manner. Annexin V staining showed that 51.54% of monocytes underwent apoptosis after being treated with 100 ng/ml recombinant TRAIL. When monocytes were prestimulated with HIV Tat, only 15.46% of monocytes underwent apoptosis. This effect can be inhibited by polyclonal anti-Tat serum. 7-AAD staining showed similar results. CONCLUSION: HIV Tat protein increases Bcl-2 expression in monocytes which inhibited apoptosis induced by TRAIL. HIV Tat protein may play an important role in the mechanisms of HIV-persistent infection in monocytes.
Asunto(s)
Apoptosis , Regulación de la Expresión Génica , Productos del Gen tat/fisiología , Genes bcl-2/inmunología , VIH/inmunología , Monocitos/virología , Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Anexina A5/metabolismo , Western Blotting , Células Cultivadas , Dactinomicina/análogos & derivados , Dactinomicina/metabolismo , Citometría de Flujo , Humanos , Monocitos/citología , Monocitos/inmunología , Coloración y Etiquetado , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
It is now well established that the cytokine environment influences the activation, differentiation, proliferation and death of T lymphocytes during the primary response to antigen. Using an in vitro model, we investigated the influence of IL-4, added at the onset of TCR stimulation, on phenotypic and functional markers of naive CD8+ T cell activation including the up-regulation of activation markers, proliferation as well as the susceptibility to activation-induced cell death (AICD). We report that IL-4, unlike IL-2 added at the onset of repeated TCR stimulation of naive CD8+ T cells prevents AICD, in part due to its ability to maintain the level of the survival-related protein Bcl-2. Moreover, TCR-triggered activation of naive CD8+ T cells in the presence of IL-4 leads to the development of a CD8+ T cell subset that proliferates normally, but which fails to exhibit characteristic activation parameters such as the up-regulation of CD25 and Granzyme B. Taken together, these results demonstrate that exposure to IL-4 during primary activation influences CD8+ T cell differentiation by inducing the development of a sub-population of AICD-resistant, proliferation-competent cells that do not show some of the typical features of CD8+ T cell activation.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Interleucina-4/inmunología , Activación de Linfocitos/inmunología , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Genes bcl-2/inmunología , Granzimas , Humanos , Interleucina-2/inmunología , Interleucina-2/farmacología , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Interleucina-2/inmunología , Serina Endopeptidasas/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
The TLX1/HOX11 homeobox gene is frequently activated in T-cell acute lymphoblastic leukaemia (T-ALL) by the t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations or by as yet unknown transcriptional mechanisms in the absence of 10q24 cytogenetic abnormalities. Almost all TLX1(+) T-ALLs exhibit a CD4(+)CD8(+) double-positive (DP) phenotype. To investigate the role of TLX1 as an initiating oncogene in T-ALL pathogenesis, we assessed the consequences of retroviral vector-directed TLX1 expression during the differentiation of murine and human thymocytes in fetal thymic organ cultures. Interestingly, enforced expression of TLX1 disrupted the differentiation of murine fetal liver precursors and human cord blood CD34(+) stem/progenitor cells prior to the DP thymocyte stage. Although differentiation arrest was associated with an increased percentage of apoptotic thymocytes, it could only be partially bypassed by coexpression of transgenic BCL2. Mutation of the invariant asparagine residue at position 51 of the homeodomain - which is required for efficient DNA binding - released the block, consistent with the notion that TLX1 inhibits thymocyte differentiation and promotes T-cell oncogenesis by functioning as a transcription factor. The relevance of these findings is discussed in the context of activating NOTCH1 mutations and the other genetic lesions implicated in the multistep transformation process of TLX1(+) T-ALL.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de Homeodominio/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas Proto-Oncogénicas/genética , Timo/inmunología , Animales , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Genes Homeobox , Genes bcl-2/inmunología , Vectores Genéticos , Proteínas de Homeodominio/metabolismo , Humanos , Inmunofenotipificación , Leucemia-Linfoma de Células T del Adulto/inmunología , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas/metabolismo , Retroviridae/genética , Transducción GenéticaRESUMEN
OBJECTIVES: To investigate the effects of primary HIV-1 infection (PHI) and of two antiretroviral therapies [highly active antiretroviral therapy (HAART) or reverse transcriptase inhibitors (RTI)] on activation, differentiation and survival of B cells. METHODS: Naive and memory B cells from three groups [PHI (31), chronic infection (26) and healthy donors (12)] were studied for surface expression of Fas, LAIR-1, CD70, intracellular expression of Bcl-2 and spontaneous apoptosis. Fluorescence activated cell sorting (IgD+IgM+CD19+CD27+) and short-term cell culture to analyse induction of CD25 on B cells were performed in five patients with PHI. Patients with PHI were sampled at baseline, and after 1 and 6 months of therapy. Results were analysed by parametric and non-parametric tests and by mathematical modelling. RESULTS: In PHI, B cells were significantly decreased; naive and memory B lymphocytes showed a high degree of activation, manifested by hypergammaglobulinaemia, altered expression of Fas and LAIR-1, and high rate of spontaneous apoptosis. Antiretroviral treatment improved the activation/differentiation status of B cells, reduced apoptosis to levels comparable to those in healthy individuals and restored the ability of B cells to respond to T cell-dependent activation. B cells showed slightly better recovery in patients taking HAART than in those taking RTI. Decreased IgM-positive memory B cells and lower induction of CD25 expression on B cells upon T cell activation at diagnosis of PHI was shown in five patients tested. These parameters normalized after 6 months of therapy. CONCLUSION: B cell dysfunctions found in chronic HIV-1 infection appear during PHI and initiation of antiretroviral therapy early during infection may help to preserve the B cell compartment.
Asunto(s)
Linfocitos B/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Adolescente , Adulto , Anciano , Antígenos CD/inmunología , Terapia Antirretroviral Altamente Activa/métodos , Apoptosis/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Enfermedad Crónica , Femenino , Genes bcl-2/inmunología , Infecciones por VIH/tratamiento farmacológico , Humanos , Hipergammaglobulinemia/inmunología , Memoria Inmunológica/inmunología , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Fenotipo , Receptores Inmunológicos/análisis , Receptores del Factor de Necrosis Tumoral/análisis , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Linfocitos T/inmunología , Receptor fasRESUMEN
Reticuloendotheliosis viral oncogene homolog/nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (Rel/NF-kappaB) activation is a ubiquitous outcome of engaging Toll-like receptors (TLRs), yet the cell-type-specific functions of this pathway in response to particular microbial signals remain poorly defined. Here we show that NF-kappaB1 and C-Rel, Rel/NF-kappaB proteins induced in conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) by cytosine-phosphate-guanosine (CpG) DNA, a TLR-9 ligand, serve markedly different functions in these DC subsets. With the exception of impaired interleukin-12 (IL-12) production, cultured Nfkb1(-/-)C-Rel(-/-) cDCs responded relatively normally to CpG DNA. In contrast, CpG-treated Nfkb1(-/-)C-Rel(-/-) pDCs, which were still able to produce type I interferon and regulated on activation normal T-cell expressed and secreted (RANTES), but not IL-6 or IL-12, failed to acquire an activated dendritic phenotype and underwent apoptosis. Although the TLR-9-mediated death of Nfkb1(-/-)C-Rel(-/-) pDCs, which coincided with a failure to up-regulate the prosurvival proteins B-cell lymphoma apoptosis regulator xL (Bcl-x(L)) and A1, was blocked by Bcl-2 transgene expression, this inhibition of apoptosis still failed to rescue the differentiation defects. This indicated that these NF-kappaB transcription factors independently regulate TLR-9-mediated pDC morphogenesis and survival. Collectively, these findings establish that NF-kappaB1 and c-Rel, while largely dispensable for TLR-9-induced cDC activation, are critical for regulating differentiation and survival programs during pDC activation.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Subunidad p50 de NF-kappa B/inmunología , Células Plasmáticas/inmunología , Proteínas Proto-Oncogénicas c-rel/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 9/inmunología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Islas de CpG/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Células Dendríticas/citología , Genes bcl-2/genética , Genes bcl-2/inmunología , Ratones , Ratones Noqueados , Subunidad p50 de NF-kappa B/genética , Oligodesoxirribonucleótidos/inmunología , Oligodesoxirribonucleótidos/farmacología , Células Plasmáticas/citología , Proteínas Proto-Oncogénicas c-rel/genética , Virus de la Reticuloendoteliosis/inmunología , Transducción de Señal/efectos de los fármacos , Proteína bcl-X/genética , Proteína bcl-X/inmunologíaAsunto(s)
Apoptosis , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Genes bcl-2/inmunología , Infecciones por VIH/virología , Vacunas contra el SIDA/uso terapéutico , Animales , Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Diferenciación Celular/inmunología , Citocinas/inmunología , Regulación de la Expresión Génica , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos , Activación de Linfocitos , Proteínas de los Retroviridae/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Subgrupos de Linfocitos T/virología , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/virologíaRESUMEN
Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.
Asunto(s)
Apoptosis/inmunología , Complemento C3/inmunología , Complemento C9/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales de Origen Murino , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/farmacología , Inhibidores de Caspasas , Caspasas/inmunología , Inhibidores Enzimáticos/farmacología , Etopósido/metabolismo , Citometría de Flujo , Genes bcl-2/inmunología , Humanos , Células Jurkat , Meliteno/inmunología , Meliteno/farmacología , Estaurosporina/metabolismo , Estreptolisinas/inmunología , Estreptolisinas/farmacología , TransfecciónRESUMEN
Genetic vaccines encoding pancreatic beta cell antigens can prevent autoimmune (type 1) diabetes when delivered into murine model systems, but there is a need to improve their efficacy. Here, we investigated the effects of intramuscular delivery of DNA coding for the pro-apoptotic protein BAX together with an intracellular or a secreted form of the beta cell antigen glutamic acid decarboxylase (GAD) on diabetes onset and immune responses in non-obese diabetic (NOD) mice. We hypothesized that induction of apoptosis in vaccine-containing cells could lead to GAD tolerance and disease suppression. Remarkably, monitoring of spontaneous diabetes onset indicated that only delivery of DNA coding for secreted GAD and BAX resulted in significant prevention of the disease. Using GFP as a model plasmid-encoded antigen revealed that co-delivery of BAX resulted in the recruitment of GFP-containing dendritic cells (DCs) in the draining lymph nodes and spleen of NOD mice. Furthermore, data indicated that subcellular localization of GAD had an effect on both the number and function of antigen presenting cells (APCs) recruited by BAX as well as on IFN-gamma secretion, and that diabetes suppression was unlikely to be caused by increased T helper 2 (Th2)-like activity. Our results indicate that, under certain conditions, co-delivery of DNA encoding BAX can improve the efficacy of genetic vaccination for prevention of pathogenic autoimmunity via a mechanism likely to involve modulation of antigen presenting cell function. In addition, our data also suggest that properties associated with subcellular localization of an antigen in apoptotic cells can have a significant effect on induced immune responses.
Asunto(s)
Apoptosis/genética , Enfermedades Autoinmunes/prevención & control , Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos/análisis , Apoptosis/inmunología , Glucemia/metabolismo , Citocinas/biosíntesis , Citocinas/genética , ADN Complementario/genética , ADN Complementario/inmunología , Diabetes Mellitus Tipo 1/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Genes bcl-2/genética , Genes bcl-2/inmunología , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/inmunología , Humanos , Immunoblotting , Inyecciones Intramusculares , Isoenzimas/genética , Isoenzimas/inmunología , Luciferasas/biosíntesis , Luciferasas/genética , Luciferasas/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos NOD , Plásmidos/genética , Plásmidos/inmunología , Proteínas Proto-Oncogénicas/genética , Fracciones Subcelulares/metabolismo , Células TH1/inmunología , Células Th2/inmunología , Vacunas de ADN/genética , Proteína X Asociada a bcl-2RESUMEN
The responsiveness and diversity of peripheral B-cell repertoire decreases with age, possibly because of B-cell clonal expansions, as suggested by the incidence of serum monoclonal immunoglobulins and of monoclonal chronic lymphocytic leukemia (CLL)-like B lymphocytes in clinically silent adults. We phenotyped peripheral blood cells from 500 healthy subjects older than 65 years with no history or suspicion of malignancies and no evidence of lymphocytosis. In 19 cases (3.8%) a kappa/lambda ratio of more than 3:1 or less than 1:3 was found: 9 were CD5+, CD19+, CD23+, CD20low, CD79blow, sIglow (classic CLL-like phenotype); 3 were CD5+, CD19+, CD23+, CD20high, CD79blow, sIglow (atypical CLL-like), and 7 were CD5-, CD19+, CD20high, CD23-, CD79bbright, FMC7+, sIgbright (non-CLL-like). In 2 subjects, 2 phenotypically distinct unrelated clones were concomitantly evident. No cases were CD10+. Polymerase chain reaction (PCR) analysis demonstrated a monoclonal rearrangement of IgH genes in 15 of 19 cases. No bcl-1 or bcl-2 rearrangements were detected. Using a gating strategy based on CD20/CD5/CD79 expression, 13 additional CLL-like B-cell clones were identified (cumulative frequency of classic CLL-like: 5.5%). Thus, phenotypically heterogeneous monoclonal B-lymphocyte expansions are common among healthy elderly individuals and are not limited to classic CLL-like clones but may have the phenotypic features of different chronic lymphoproliferative disorders, involving also CD5- B cells.
Asunto(s)
Envejecimiento/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Antígenos CD5/inmunología , Anciano , Anciano de 80 o más Años , Envejecimiento/sangre , Células Clonales , Femenino , Reordenamiento Génico , Genes de Inmunoglobulinas , Genes bcl-1/inmunología , Genes bcl-2/inmunología , Humanos , Cadenas Ligeras de Inmunoglobulina/sangre , Cadenas Ligeras de Inmunoglobulina/genética , Inmunofenotipificación , Leucemia Linfocítica Crónica de Células B/inmunología , MasculinoRESUMEN
Cardiac glycosides are commonly used drugs in clinical medicine. We analyzed the cytotoxic effect of six steroids belonging to the bufadienolide family on malignant T lymphoblasts and normal peripheral blood mononuclear cells (PBMC). One compound was a natural bufadienolide glycoside (hellebrin) with cardiac activity. The other five compounds were chemically modified derivatives that did not contain cardioactive groups. We found that these steroids were able to cause time-dependent apoptosis in Jurkat T lymphoblasts, whereas they only minimally affected PBMC. Preferential killing of malignant cells was induced by the natural cardioactive substance hellebrin and by three of the five chemically modified non-cardioactive derivatives. The substances caused mitochondrial transmembrane potential disruption and internucleosomal DNA fragmentation in tumor cells. The cytoplasmic and nuclear events of bufadienolide-induced apoptosis were strongly inhibited in the presence of caspase 8, caspase 9, or caspase 3 inhibitors, as well as in the presence of the broad-spectrum caspase inhibitor Z-VAD-FMK. Overexpression of Bcl-2 significantly protected bufadienolide-treated cells from phosphatidylserine translocation, transmembrane potential disruption, and internucleosomal DNA fragmentation. Our results show that the analyzed bufadienolide derivatives preferentially kill malignant human lymphoblasts by initiating apoptosis via the classical caspase-dependent pathway. Apoptosis-inducing agents specific for tumor cells might be ideal anti-tumor drugs. The therapeutic use of bufadienolides has been hampered by their concomitant cardiac activity. The description of compounds without cardiac activity but with tumor-specific cytotoxicity suggests the potential of using them in cancer therapy.
Asunto(s)
Apoptosis/inmunología , Cardenólidos/efectos adversos , Muerte Celular/efectos de los fármacos , Células Jurkat , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Bufanólidos/farmacología , Cardenólidos/química , Cardenólidos/farmacología , Caspasa 3 , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Colenos/efectos adversos , Colenos/antagonistas & inhibidores , Colenos/química , Fragmentación del ADN/efectos de los fármacos , Expresión Génica , Genes bcl-2/genética , Genes bcl-2/inmunología , Corazón/efectos de los fármacos , Lesiones Cardíacas/inducido químicamente , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/patología , TransfecciónRESUMEN
Signalling through CD40 is essential for the development of immunoglobulin G (IgG) antibody responses, germinal centres and B-cell memory against T-dependent antigens. In addition, engagement of CD40 in B cells promotes cell survival by inducing the expression of anti-apoptotic members of the bcl-2 family of cell-death regulators. In the present study we analysed whether T-dependent immune responses can be developed in mice deficient in CD40 if the anti-apoptotic activity mediated by the engagement of CD40 in B cells is compensated by the constitutive over-expression of anti-apoptotic genes of the bcl-2 family. We showed that the over-expression of either hbcl-2 or hbcl-xL transgenes in B cells is not sufficient to restore IgG antibody responses and germinal centre formation in CD40-deficient mice. These results indicate that CD40 functions, other than those mediated through survival, are required for the establishment of T-dependent B-cell responses.
Asunto(s)
Linfocitos B/inmunología , Antígenos CD40/inmunología , Genes bcl-2/inmunología , Linfocitos T/inmunología , Animales , Apoptosis/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica/inmunología , Centro Germinal/inmunología , Inmunidad Celular/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteína bcl-XRESUMEN
OBJECTIVE: The aim of this study was to analyze the expression of apoptosis-related molecules in rheumatoid arthritis (RA) synovium, with special emphasis on the apoptosis accelerator Bax. METHODS: Immunohistochemical analysis of Bax, Bcl-2, and Bcl-x(L) was performed in tissue specimens of patients with RA and compared to normal synovial tissue. Expression of Bax was additionally determined by double labeling with CD68, p53, and Ki-67 (clone MIB-1). Apoptotic cells were further identified by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method. RESULTS: In RA, expression of Bax was higher than in healthy controls and occurred in CD68-positive and -negative synoviocytes. Strong Bax staining was also found in chondrocytes at sites of cartilage degradation. Bax-positive synoviocytes could be detected with p53 and also with Ki-67. Bax and Bcl-x(L) were markedly colocalized in synovium. The TUNEL method revealed only few positive synoviocytes. CONCLUSIONS: The marked colocalization of Bax and antiapoptotic Bcl-x(L) as well as the low frequency of TUNEL-positive cells in RA synovium suggest that Bax activity is not sufficient to decrease synovial hyperplasia in RA. Apoptotic mechanisms in RA chondrocytes might also be important for the pathogenesis of joint damage.