RESUMEN
AIMS/HYPOTHESIS: Hypoxia in the initial days after islet transplantation leads to considerable loss of islet mass and contributes to disappointing outcomes in the clinical setting. The aim of the present study was to investigate whether co-transplantation of human non-endothelial bone marrow-derived multipotent adult progenitor cells (MAPCs), which are non-immunogenic and can secrete angiogenic growth factors during the initial days after implantation, could improve islet engraftment and survival. METHODS: Islets (150) were co-transplanted, with or without human MAPCs (2.5 × 105) as separate or composite pellets, under the kidney capsule of syngeneic alloxan-induced diabetic C57BL/6 mice. Blood glucose levels were frequently monitored and IPGTTs were carried out. Grafts and serum were harvested at 2 and 5 weeks after transplantation to assess outcome. RESULTS: Human MAPCs produced high amounts of angiogenic growth factors, including vascular endothelial growth factor, in vitro and in vivo, as demonstrated by the induction of neo-angiogenesis in the chorioallantoic membrane assay. Islet-human MAPC co-transplantation as a composite pellet significantly improved the outcome of islet transplantation as measured by the initial glycaemic control, diabetes reversal rate, glucose tolerance and serum C-peptide concentration compared with the outcome following transplantation of islets alone. Histologically, a higher blood vessel area and density in addition to a higher vessel/islet ratio were detected in recipients of islet-human MAPC composites. CONCLUSIONS/INTERPRETATION: The present data suggest that co-transplantation of mouse pancreatic islets with human MAPCs, which secrete high amounts of angiogenic growth factors, enhance islet graft revascularisation and subsequently improve islet graft function.
Asunto(s)
Diabetes Mellitus Tipo 1/terapia , Trasplante de Células Madre/métodos , Células Madre/citología , Adulto , Animales , Glucemia/fisiología , Células Cultivadas , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/fisiopatología , Modelos Animales de Enfermedad , Femenino , Humanos , Islotes Pancreáticos/metabolismo , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neovascularización Fisiológica/fisiología , Células Madre/fisiologíaRESUMEN
AIMS/HYPOTHESIS: Cytotoxic T cells and macrophages contribute to beta cell destruction in type 1 diabetes at least in part through the production of cytokines such as IL-1ß, IFN-γ and TNF-α. We have recently shown the IL-17 pathway to be activated in circulating T cells and pancreatic islets of type 1 diabetes patients. Here, we studied whether IL-17A upregulates the production of chemokines by human pancreatic islets, thus contributing to the build-up of insulitis. METHODS: Human islets (from 18 donors), INS-1E cells and islets from wild-type and Stat1 knockout mice were studied. Dispersed islet cells were left untreated, or were treated with IL-17A alone or together with IL-1ß+IFN-γ or TNF-α+IFN-γ. RNA interference was used to knock down signal transducer and activator of transcription 1 (STAT1). Chemokine expression was assessed by quantitative RT-PCR, ELISA and histology. Cell viability was evaluated with nuclear dyes. RESULTS: IL-17A augmented IL-1ß+IFN-γ- and TNF-α+IFN-γ-induced chemokine mRNA and protein expression, and apoptosis in human islets. Beta cells were at least in part the source of chemokine production. Knockdown of STAT1 in human islets prevented cytokine- or IL-17A+cytokine-induced apoptosis and the expression of particular chemokines, e.g. chemokine (C-X-C motif) ligands 9 and 10. Similar observations were made in islets isolated from Stat1 knockout mice. CONCLUSIONS/INTERPRETATION: Our findings indicate that IL-17A exacerbates proinflammatory chemokine expression and secretion by human islets exposed to cytokines. This suggests that IL-17A contributes to the pathogenesis of type 1 diabetes by two mechanisms, namely the exacerbation of beta cell apoptosis and increased local production of chemokines, thus potentially aggravating insulitis.
Asunto(s)
Apoptosis , Diabetes Mellitus Tipo 1/metabolismo , Inflamación/metabolismo , Interleucina-17/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Apoptosis/inmunología , Western Blotting , Diabetes Mellitus Tipo 1/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Inflamación/inmunología , Islotes Pancreáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We previously showed that the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptor VPAC1 are negative regulators of megakaryopoiesis and platelet function, but their downstream signaling pathway that inhibits this process still remained unknown. A combined proteomic, transcriptomic, and bioinformatic approach was here used to elucidate the molecular mechanisms underlying PACAP signaling via VPAC1 in megakaryocytes. Two-dimensional difference gel electrophoresis and tandem MS were applied to detect differentially expressed proteins in megakaryocytic CHRF cells stimulated with PACAP. The majority of the 120 proteins modulated by PACAP belong to the class of "cell cycle and apoptosis" proteins. The up- or down-regulated expression of some proteins was confirmed by immunoblot and immunohistochemical analysis. A meta-analysis of our data and 12 other published studies was performed to evaluate signaling pathways involved in different cellular models of PACAP response. From 2384 differentially expressed genes/proteins, 83 were modulated by PACAP in at least three independent studies and Ingenuity Pathway Analysis further identified apoptosis as the highest scored network with NF-κB as a key-player. PACAP inhibited serum depletion-induced apoptosis of CHRF cells via VPAC1 stimulation. In addition, PACAP switched on NF-κB dependent gene expression since higher nuclear levels of the active NF-κB p50/p65 heterodimer were found in CHRF cells treated with PACAP. Finally, a quantitative real time PCR apoptosis array was used to study RNA from in vitro differentiated megakaryocytes from a PACAP overexpressing patient, leading to the identification of 15 apoptotic genes with a 4-fold change in expression and Ingenuity Pathway Analysis again revealed NF-κB as the central player. In conclusion, our findings suggest that PACAP interferes with the regulation of apoptosis in megakaryocytes, probably via stimulation of the NF-κB pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Megacariocitos/efectos de los fármacos , FN-kappa B/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Adulto , Línea Celular , AMP Cíclico/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Megacariocitos/metabolismo , Proteómica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Exposure of insulin-secreting ß-cells to inflammatory cytokines or high concentrations of free fatty acids, factors involved in the pathogenesis of type 1 and type 2 diabetes, leads to endoplasmic reticulum (ER) stress, ß-cell dysfunction, and eventually apoptotic ß-cell death. The aim of this study was to investigate the impact of ER stress on ß-cells at the protein level to evaluate the contribution of post-transcriptional and post-translational changes in ER stress-induced ß-cell damage. INS-1E cells were exposed in vitro to the ER-stress inducer cyclopiazonic acid (CPA) at two concentrations, and protein changes were evaluated using 2D-DIGE. CPA, 25 µM, led to massive apoptosis, accompanied by a near complete protein translation shut-down. CPA, 6.25 µM, led to adaptation of the ß-cells to ER stress. Identification of the differentially expressed proteins in the two conditions led to the discovery of a clear pattern of defense pathways, with post-translational modifications playing a crucial role. Key alterations included inhibition of insulin translation and post-translational modifications in ER chaperones HYOU1 and HSPA5. Also, a central role for 14-3-3 proteins is suggested. In conclusion, INS-1E cells are highly sensitive to ER stress, leading to important post-transcriptional and post-translational modifications that may contribute to ß-cell dysfunction and death.
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Retículo Endoplásmico/metabolismo , Células Secretoras de Insulina/metabolismo , Proteoma/análisis , Proteómica/métodos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Indoles/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Insulinoma/metabolismo , Insulinoma/patología , Unión Proteica/efectos de los fármacos , Proteoma/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
To investigate whether vaccination could induce lethal shock and which mechanisms are involved in this phenomenon, we tested a panel of autoantigens or diabetes-irrelevant peptides or proteins in nonobese diabetic (NOD), Balb/c, and C57Bl/6 mice. Of the antigens tested, only nondiabetogenic hen egg white lysozyme induced a severe form of shock exclusively in NOD mice. The mechanism involved is suggestive of a Th(2)-mediated anaphylactic reaction possibly connected to activation of PAF and triggering of DIC.
Asunto(s)
Muramidasa/administración & dosificación , Choque/inducido químicamente , Vacunas/efectos adversos , Animales , Pollos , Clara de Huevo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NODRESUMEN
Anaplastic thyroid cancer represents one of the most aggressive cancers. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to have antiproliferative and/or redifferentiating properties in several malignancies, including thyroid cancer. The objective of this study was to investigate the effects of 1,25(OH)(2)D(3) and the superagonistic analog CD578 in anaplastic thyroid cancer, alone or in combination with paclitaxel, a taxane, and suberoylanilide hydroxamic acid (SAHA), a potent histone deacetylase inhibitor with promising effects in undifferentiated thyroid cancer. Four human thyroid cancer cell lines (FTC-133, C643, 8505C and HTh74) were treated with 1,25(OH)(2)D(3) or CD578, alone or in combination with paclitaxel or SAHA. Effects on cell growth and differentiation were evaluated. Clear effects on growth arrest were observed in a clonogenic assay, and absolute cell counts demonstrated a 24-36% reduction in all cell lines after 72h treatment with 1,25(OH)(2)D(3) (10(-6)M) and a 60% inhibition after 120h in the most sensitive cell line HTh74. A similar growth inhibition was shown after treatment with a 1000-fold lower concentration of analog CD578. This growth arrest was explained by antiproliferative effects, further supported by an increased % of cells in the G(0)-G(1) phase of the cell cycle and by a decreased transcription factor E2F1 mRNA expression. Combination treatments of 1,25(OH)(2)D(3) or CD578 with paclitaxel or SAHA resulted in an additive and in some conditions a synergistic effect on the inhibition of proliferation. Redifferentiation analysis revealed only a modest increase in sodium iodide symporter and thyroglobulin mRNA expression after treatment with 1,25(OH)(2)D(3), without additive effect after combination treatment. No effects were observed on TSH-receptor or thyroid peroxidase mRNA expression. Our in vitro findings demonstrate that the superagonistic vitamin D analog CD578 holds promise as adjuvant antiproliferative therapy of anaplastic thyroid cancer, especially in combination with other drugs such as paclitaxel or SAHA.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Calcitriol/administración & dosificación , Calcitriol/análogos & derivados , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Humanos , Ácidos Hidroxámicos/administración & dosificación , Paclitaxel/administración & dosificación , ARN Neoplásico/química , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , VorinostatRESUMEN
IFNgamma (interferon gamma), a cytokine typically secreted by infiltrating immune cells in insulitis in Type 1 diabetes, is by itself not detrimental to beta-cells, but, together with other cytokines, such as IL-1beta (interleukin 1beta) and TNFalpha (tumour necrosis factor alpha), or dsRNA (double-stranded RNA), it induces beta-cell apoptosis. The complex gene and protein networks that are altered by the combination of cytokines clearly point towards synergisms between these agents. IFNgamma acts mostly via JAK (Janus kinase) activation, with the transcription factors STAT-1 (signal transducer and activator of transcription-1) and IRF-1 (IFNgamma regulatory factor-1) playing a central role in the downstream pathway. The study of mice with a disruption of these transcription factors has revealed a possible dual role for IFNgamma in beta-cell destruction by cytokines or dsRNA. We demonstrated that the absence of STAT-1 from beta-cells completely protects against IFNgamma+IL-1beta- and IFNgamma+dsRNA-mediated beta-cell death in vitro, whereas absence of IRF-1 does not prevent cytokine-induced beta-cell apoptosis. In vivo, a lack of the IRF-1 gene in pancreatic islets even promotes low-dose streptozotocin-induced diabetes, whereas lack of STAT-1 confers resistance against beta-cell death following low-dose streptozotocin-induced diabetes. Additionally, IRF-1(-/-) islets are more sensitive to PNF (primary islet non-function) after transplantation in spontaneously diabetic NOD (non-obese diabetic) mice, whereas STAT-1(-/-) islets are fully protected. Moreover, proteomic analysis of beta-cells exposed to IFNgamma or IFNgamma+IL-1beta confirms that very different pathways are activated by IFNgamma alone compared with the combination. We conclude that IFNgamma may play a dual role in immune-induced beta-cell destruction. Transcription factors drive this dual role, with STAT-1 driving beta-cell destruction and IRF-1 possibly playing a role in up-regulation of protective pathways induced by IFNgamma.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Interferón gamma/metabolismo , Transducción de Señal , Animales , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Factor 1 Regulador del Interferón/antagonistas & inhibidores , Interferón gamma/farmacología , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: To examine messenger (m) RNA expression of aromatase, cytokines, and adhesion factors in women with and without endometriosis. DESIGN: Patients with endometriosis were compared with control patients. SETTING: University Hospital Gasthuisberg, Leuven, Belgium. PATIENT(S): A total of 35 patients who had laparoscopic surgery during the luteal phase (n = 20) or the menstrual phase (n = 15) were selected for this study based on cycle phase and presence/absence of endometriosis. INTERVENTION(S): Tissues of endometrium and macroscopically normal peritoneum were collected during hysteroscopy and laparoscopic surgery, respectively, from 24 women with revised American Society for Reproductive Medicine stage (rASRM) stages I-II (n = 12) and III-IV (n = 12) endometriosis and 11 control patients with normal pelvic. Tissue samples were selected from a tissue bank, based on the phase of the cycle (menstrual or luteal) and the presence/absence of endometriosis. MAIN OUTCOME MEASURE(S): The mRNA levels of aromatase, vimentin, vascular cell adhesion molecule 1 (VCAM-1), alpha(V) and beta(3) integrins, interleukin (IL)-1 beta, regulated on activation normal T-cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1) were evaluated using real-time reverse transcriptase polymerase chain reaction. RESULT(S): During menstrual phase, increased endometrial mRNA levels of alpha(V) integrin, combined alpha(V)beta(3) integrins, and increased peritoneal IL-1 beta mRNA levels--but decreased peritoneal MCP-1 mRNA levels--were observed in women with endometriosis compared with control subjects. During luteal phase, endometrial mRNA levels of IL-1 beta and RANTES were increased in women with endometriosis compared with control subjects. Endometrial aromatase mRNA expression was higher in women with endometriosis than in control subjects in combined phases. Women with endometriosis had increased peritoneal mRNA expression of RANTES and VCAM-1 during menstrual compared with luteal phase. CONCLUSION(S): Aberrant mRNA expression of aromatase, cytokines, and adhesion factors in endometrium and peritoneum suggests that both tissues are involved in the pathogenesis of endometriosis.
Asunto(s)
Aromatasa/genética , Moléculas de Adhesión Celular/genética , Citocinas/genética , Endometriosis/genética , Endometrio/metabolismo , Peritoneo/metabolismo , Enfermedades Uterinas/genética , Adulto , Aromatasa/metabolismo , Moléculas de Adhesión Celular/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Citocinas/metabolismo , Endometriosis/metabolismo , Femenino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Fase Luteínica/genética , Fase Luteínica/metabolismo , Menstruación/genética , Menstruación/metabolismo , ARN Mensajero/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Enfermedades Uterinas/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMEN
OBJECTIVE: Vitamin D deficiency increases risk for type 1 diabetes in genetically predisposed individuals, while high doses of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] prevent insulitis and diabetes in NOD mice. RESEARCH DESIGN AND METHODS: Since 1,25(OH)(2)D(3) regulates gene transcription through the vitamin D receptor (VDR), we investigated the role of VDR in diabetes development by creating NOD mice without functional VDR. RESULTS: VDR(-/-) NOD mice are rachitic and have lower numbers of putative regulator cells [TCR-alpha/beta(+)CD4(-)CD8(-) (natural killer T-cells) and CD4(+)CD25(+) T-cells [in central and peripheral immune organs compared with VDR(+/+) NOD littermates. Lipopolysaccharide-stimulated VDR(-/-) NOD macrophages expressed lower interleukin (IL)-1, IL-6, and CC chemokine ligand 2 mRNA, correlating with less nuclear translocation of p65 nuclear factor-kappaB compared with VDR(+/+) NOD macrophages. Thymic and lymph node dendritic cells from VDR(-/-) NOD mice displayed an even less mature CD11c(+)CD86(+) phenotype than VDR(+/+) NOD mice. Despite this immune phenotype linked to diabetes in NOD mice, VDR(-/-) NOD mice developed insulitis and diabetes at the same rate and incidence as VDR(+/+) NOD littermates. CONCLUSIONS: Despite aggravating known immune abnormalities in NOD mice, disruption of VDR does not alter disease presentation in NOD mice in contrast to the more aggressive diabetes presentation in vitamin D-deficient NOD mice.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Receptores de Calcitriol/deficiencia , Animales , Cruzamientos Genéticos , Femenino , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Fenotipo , Receptores de Calcitriol/genética , Linfocitos T/fisiologíaRESUMEN
OBJECTIVE: To evaluate the mechanism of action of recombinant human tumor necrosis factor (TNF)-binding protein-1 by assessing differential expression of messenger RNA (mRNA) for cytokines, matrix metalloproteinases, and growth and adhesion factors in baboons. DESIGN: Analysis of gene expression in a prospective randomized study. SETTING: University Fertility Center. ANIMAL(S): In the in vivo study, 14 baboons were randomly and subcutaneously (SC) treated with either phosphate-buffered saline (PBS), GnRH antagonist, or recombinant human TNF-binding protein-1 at the time of induction. In the ex vivo study, 4 baboons were treated by menstrual endometrium that had been incubated randomly with either PBS or recombinant human TNF-binding protein-1 before intrapelvic injection. INTERVENTION(S): In the in vivo study, analysis of 11 endometrial and 10 endometriosis biopsies included either PBS (n = 5), GnRH antagonist (n = 8), or recombinant human TNF-binding protein-1 (n = 8). In the ex vivo study, 2 endometrial and 4 endometriosis biopsies were analyzed from 4 baboons. MAIN OUTCOME MEASURE(S): The mRNA expression of TNF-alpha, IL-8, IL-6, transforming growth factor-beta (TGF-beta), vascular endothelial growth factor, intercellular adhesion molecule-1, matrix metalloproteinase-1, and regulated on activation, normal T-cell expressed and secreted were investigated using real-time reverse transcriptase-polymer chain reaction (PCR). RESULT(S): TGF-beta mRNA expression was decreased in endometriotic lesions from baboons treated with recombinant human TNF-binding protein-1 when compared with the placebo group. CONCLUSION(S): Except TGF-beta, mRNA expression of inflammatory cytokines and adhesion/growth factors is not affected in endometrial and endometriosis biopsies from baboons after induction of endometriosis combined with either systemic injection of recombinant human TNF-binding or GnRH antagonist or ex vivo treatment with recombinant human TNF-binding protein-1. Further studies are needed to elucidate the mode of action on how inhibition of TNF-alpha activity prevents the development of endometriosis.
Asunto(s)
Proteínas Portadoras/farmacología , Moléculas de Adhesión Celular/genética , Citocinas/genética , Endometriosis/genética , Endometriosis/veterinaria , Endometrio/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Péptidos y Proteínas de Señalización Intercelular/genética , Papio/genética , Animales , Biopsia , Moléculas de Adhesión Celular/metabolismo , Citocinas/metabolismo , Endometriosis/metabolismo , Endometriosis/patología , Endometrio/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Humanos , Inflamación/genética , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Oligopéptidos/farmacología , Placebos , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Free fatty acids (FFA) cause apoptosis of pancreatic beta-cells and might contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary beta-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca(2+) stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor beta-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary beta-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced beta-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca(2+) depletion. The IRE1 and resulting JNK activation contribute to beta-cell apoptosis. PERK activation by palmitate also contributes to beta-cell apoptosis via CHOP.
Asunto(s)
Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/patología , Ácidos Grasos no Esterificados/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 6/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Retículo Endoplásmico/enzimología , Ácidos Grasos no Esterificados/metabolismo , Glucosa/toxicidad , Humanos , Células Secretoras de Insulina/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
OBJECTIVE: Exposure of beta-cells to inflammatory cytokines leads to apoptotic cell death through the activation of gene networks under the control of specific transcription factors, such as interferon-gamma-induced signal transducer and activator of transcription (STAT)-1. We previously demonstrated that beta-cells lacking STAT-1 are resistant to cytokine-induced cell death in vitro. The aim of this study was to investigate the effect of STAT-1 elimination on immune-mediated beta-cell destruction in vivo. RESEARCH DESIGN AND METHODS: Multiple low-dose streptozotocin (STZ) was given to C57BL/6 mice after syngeneic STAT-1(-/-) or wild-type islet transplantation. STAT-1(-/-) and wild-type islets were also transplanted in alloxan-diabetic BALB/c and spontaneously diabetic nonobese diabetic (NOD) mice. Additionally, mice were treated with interleukin (IL)-1 blockade (IL-1 receptor antagonist [IL-1ra]) and low-dose T-cell suppression (cyclosporine A [CsA]). RESULTS: When exposed to multiple low-dose STZ in an immune-competent host, STAT-1(-/-) islets were more resistant to destruction than wild-type islets (28 vs. 100% diabetes incidence, P < or = 0.05). STAT-1 deletion also protected allogeneic islet grafts against primary nonfunction in autoimmune NOD mice (0 vs. 17% using wild-type islets). However, no difference in survival time was observed. Additionally, treating recipients with IL-1ra and CsA prolonged graft survival in chemically diabetic BALB/c mice, whereas no difference was seen between STAT-1(-/-) and C57BL/6 grafts. CONCLUSIONS: These data indicate that STAT-1 is a key player in immune-mediated early beta-cell dysfunction and death. When considering the many effector mechanisms contributing to beta-cell death following islet transplantation, multiple combined interventions will be needed for prolongation of beta-cell survival in the autoimmune context of type 1 diabetes.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Rechazo de Injerto/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/metabolismo , Aloxano/farmacología , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/inmunología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Rechazo de Injerto/prevención & control , Interleucina-1beta/metabolismo , Ratones , Ratones Noqueados , Factor de Transcripción STAT1/genética , Transducción de Señal/efectos de los fármacos , Estreptozocina/farmacología , Tasa de Supervivencia , Factores de TiempoRESUMEN
The keratin-14 IL-4 transgenic (Tg) mouse model of atopic dermatitis (AD) is characterized by skin infiltration of T cells, early up-regulation of T(h)2 cytokines and late surge of T(h)1 cytokines. In the present study, we investigated the role of CCL27, a T cell skin-homing chemokine known to be elevated in sera of human AD patients, in disease development in our animal model of AD. The results showed that the mRNA and protein levels of CCL27 in the skin and serum were significantly increased in IL-4 Tg mice. The percentage of T cells expressing CCR10 in skin draining lymph nodes of IL-4 Tg mice was increased, consistent with the findings of >80% of skin-infiltrating T cells in Tg mice expressing CCR10. Chemotaxis transmigration assay demonstrated that CCL27 promotes a greater degree of migration of T cells in diseased Tg mice. Subcutaneous injection of neutralizing anti-CCL27 to IL-4 Tg mice with early skin lesions resulted in reduced clinical progression of inflammation, accompanied with decreased T cell and mast cell infiltration in the skin, and down-regulation of inflammatory cytokines. In conclusion, CCL27 and CCR10 interaction is important for the development of skin inflammation in our AD model.
Asunto(s)
Quimiocinas CC/inmunología , Dermatitis Atópica/inmunología , Interleucina-4/genética , Queratinas/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/inmunología , Quimiocina CCL27 , Quimiocinas CC/biosíntesis , Quimiocinas CC/sangre , Quimiocinas CC/metabolismo , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Interleucina-4/inmunología , Queratina-14 , Queratinocitos/citología , Queratinocitos/inmunología , Queratinocitos/metabolismo , Queratinas/metabolismo , Activación de Linfocitos , Ratones , Ratones Transgénicos , Receptores CCR10 , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/inmunología , Piel/inmunología , Regulación hacia ArribaRESUMEN
Following myocardial infarction (MI), contractile dysfunction develops not only in the infarct zone but also in noninfarcted regions of the left ventricle remote from the infarct zone. Inflammatory activation secondary to MI stimulates inducible nitric oxide synthase (iNOS) induction with excess production of nitric oxide. We hypothesized that the anti-inflammatory effects of selective A(2A)-adenosine receptor (A(2A)AR) stimulation would suppress inflammation and preserve cardiac function in the remote zone early after MI. A total of 53 mice underwent 60 min of coronary occlusion followed by 24 h of reperfusion. The A(2A)AR agonist (ATL146e, 2.4 microg/kg) was administered intraperitoneally 1, 3, and 6 h postreperfusion. Because of the 1-h delay in treatment after MI, ATL146e had no effect on infarct size, as demonstrated by contrast-enhanced cardiac MRI (n = 18) performed 24 h post-MI. ATL146e did however preserve global cardiac function at that time by limiting contractile dysfunction in remote regions [left ventricle wall thickening: 51 +/- 4% in treated (n = 9) vs. 29 +/- 3% in nontreated groups (n = 9), P < 0.01]. RT-PCR, immunohistochemistry, and Western blot analysis indicated that iNOS mRNA and protein expression were significantly reduced by ATL146e treatment in both infarcted and noninfarcted zones. Similarly, elevations in plasma nitrate-nitrite after MI were substantially blunted by ATL146e (P < 0.01). Finally, treatment with ATL146e reduced NF-kappaB activation in the myocardium by over 50%, not only in the infarct zone but also in noninfarcted regions (P < 0.05). In conclusion, A(2A)AR stimulation after MI suppresses inflammatory activation and preserves cardiac function, suggesting the potential utility of A(2A)AR agonists against acute heart failure in the immediate post-MI period.
Asunto(s)
Citocinas/inmunología , Contracción Miocárdica/inmunología , Daño por Reperfusión Miocárdica/inmunología , Óxido Nítrico Sintasa de Tipo II/inmunología , Receptor de Adenosina A2A/inmunología , Disfunción Ventricular Izquierda/inmunología , Animales , Ratones , Daño por Reperfusión Miocárdica/complicaciones , Distribución Tisular , Disfunción Ventricular Izquierda/etiologíaRESUMEN
Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.
Asunto(s)
Antígenos CD40/metabolismo , Regulación hacia Abajo/inmunología , Hipersensibilidad/inmunología , Inmunización , Interleucina-10/fisiología , Interleucina-12/fisiología , Animales , Antígenos CD40/inmunología , Citocinas/biosíntesis , Inmunidad , Inflamación , Ratones , Ratones Noqueados , Transducción de Señal/inmunología , Células Th2/inmunologíaRESUMEN
OBJECTIVE: To examine differential messenger RNA (mRNA) expression of relevant cytokines, metalloproteases, growth and adhesion factors in endometrium and peritoneum from women with endometriosis when compared with women without the disease during menstrual and luteal phases of the cycle. DESIGN: Patients with endometriosis were compared with control patients. SETTING: University hospital. PATIENT(S): A total of 35 patients (20 patients during the luteal phase and 15 patients during the menstrual phase) were selected for this study on the basis of cycle phase and presence or absence of endometriosis. INTERVENTION(S): In this study, endometriosis was laparoscopically and histologically confirmed in 24 women with endometriosis of revised American Society for Reproductive Medicine (ASRM) stage I-II (n = 12) and revised ASRM stage III-IV (n = 12), and the presence of a normal pelvis was documented by laparoscopy in 11 control patients. The macroscopically normal peritoneum tissues were collected from lateral wall left or right, near the colon ascendens or descendens. MAIN OUTCOME MEASURE(S): The expression levels were determined as ratios between the target molecules and beta-actin as housekeeping gene. RESULT(S): In women with endometriosis, peritoneal mRNA levels of matrix metalloproteinase (MMP)-3, transforming growth factor-beta, interleukin (IL)-6, and intercellular adhesion molecule-1 and endometrial mRNA levels of MMP-3, tumor necrosis factor (TNF)-alpha, and IL-8 were significantly higher during the menstrual phase when compared with luteal phase. During the menstrual phase of the cycle, both endometrial expression of TNF-alpha, IL-8, and MMP-3 mRNA levels and peritoneal expression of transforming growth factor-beta, IL-6, and intercellular adhesion molecule-1 mRNA levels were significantly higher in women with endometriosis when compared with controls. Immunohistochemical staining confirmed the presence of TNF-alpha in peritoneum and endometrium in both women with endometriosis and controls. CONCLUSION(S): Increased endometrial and peritoneal cytokine mRNA expression during menstruation may contribute to a pelvic inflammatory microenvironment favoring the development of endometriosis.
Asunto(s)
Citocinas/metabolismo , Endometriosis/metabolismo , Endometrio/metabolismo , Ciclo Menstrual/metabolismo , Peritoneo/metabolismo , Biomarcadores , Femenino , Expresión Génica , HumanosRESUMEN
CTLA-4 (CD152) expression is restricted to subsets of activated T lymphocytes and shares homology with CD28. CTLA-4 and CD28 molecules both bind to B7 molecules on antigen-presenting cells. Whereas CD28-B7 interaction enhances T cell activation, cytokine production and survival, CTLA-4 signaling down-regulates T cell responses. Here, we studied the involvement of CTLA-4 triggering in the pathogenesis of allergen-induced airway inflammation in mice. Anti-CTLA-4 mAb were injected during i.p. sensitization with ovalbumin (OVA). This treatment favored OVA-specific IgE production and augmented blood eosinophilia in BALB/c mice. In BALB/c mice, enhanced Th2 sensitization after anti-CTLA-4 mAb injections resulted in more severe airway inflammation, and increased airway hyperresponsiveness to metacholine, bronchial eosinophilia and IL-4 and IL-5 levels in broncho-alveolar lavage (BAL) fluid following repeated allergen inhalations. Importantly, aggravation of airway inflammation and enhancement of Th2 responses were accompanied by a significant reduction of pulmonary TGF-beta levels at protein level in BAL fluid as well as on mRNA level in inflamed lung tissue. In contrast to BALB/c mice, blockade of CTLA-4 did not alter IgE production nor the phenotype of airway inflammation or TGF-beta production in C57BL/6 mice. Our data suggest that CTLA-4 triggering represents an important regulatory mechanism for Th2 sensitization in genetically predisposed mice by modulating TGF-beta production.
Asunto(s)
Antígenos de Diferenciación/inmunología , Hiperreactividad Bronquial/etiología , Eosinofilia/etiología , Inmunoconjugados , Abatacept , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD , Asma/etiología , Asma/inmunología , Hiperreactividad Bronquial/inmunología , Antígeno CTLA-4 , Modelos Animales de Enfermedad , Eosinofilia/inmunología , Inmunización , Inmunoglobulina E/biosíntesis , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Especificidad de la Especie , Linfocitos T/inmunología , Células Th2/inmunología , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Interleukin (IL)-17 is produced by activated memory CD4(+) cells and induces cytokines and chemokines that stimulate neutrophil generation and recruitment. Here, we investigated the involvement of IL-17 in the bronchial influx of neutrophils in experimental allergic asthma. Inhalation of nebulized ovalbumin (OVA) by sensitized mice with bronchial eosinophilic inflammation resulting from chronic OVA exposure induced early IL-17 mRNA expression in inflamed lung tissue, concomitant with a prominent bronchial neutrophilic influx. Anti-IL-17 monoclonal antibodies (mAb) injected before allergen inhalation strongly reduced bronchial neutrophilic influx, in a manner equally as potent as the anti-inflammatory dexamethasone. Remarkably, anti-IL-17 mAb significantly enhanced IL-5 levels in both BAL fluid and serum, and aggravated allergen-induced bronchial eosinophilia. In another series of experiments, anti-IL-17 mAb were given repeatedly during the inhalatory challenge phase with OVA of sensitized mice. This treatment regimen abated bronchial neutrophilia in parallel with reduction of bone marrow and blood neutrophilia. In addition, anti-IL-17 mAb treatment elevated eosinophil counts in the bone marrow and bronchial IL-5 production, without alteration of allergen-induced bronchial hyperresponsiveness. In summary, our results demonstrate that IL-17 expression in airways is upregulated upon allergen inhalation, and constitutes the link between allergen-induced T cell activation and neutrophilic influx. Because neutrophils may be important in airway remodeling in chronic severe asthma, targeting IL-17 may hold therapeutic potential in human asthma.
Asunto(s)
Alérgenos/administración & dosificación , Bronquios/patología , Movimiento Celular/fisiología , Granulocitos/citología , Interleucina-17/fisiología , Administración por Inhalación , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Médula Ósea/inmunología , Bronquios/inmunología , Líquido del Lavado Bronquioalveolar , Citocinas/biosíntesis , Cartilla de ADN , Eosinofilia/inmunología , Eosinofilia/fisiopatología , Interleucina-17/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos AnimalesRESUMEN
Activation-induced cell death (AICD) represents a major means of peripheral tolerance induction, eliminating effector cells. NOD mice, a widely used model for autoimmune diabetes, are characterized by high levels of circulating T lymphocytes and by resistance to several apoptosis-inducing signals. The aim of this study was to analyse AICD in peripheral NOD T lymphocytes. First, we demonstrated in an in vitro AICD model that NOD T lymphocytes are more resistant to AICD (64+/-2%) compared to non-autoimmune C57BL/6 T lymphocytes (73+/-2%), but also diabetes-resistant NOR T lymphocytes (76+/-3%, P<0.05). Moreover, both CD4(+)and CD8(+)subsets were affected. Analysis of the cellular and molecular pathways revealed lower caspase 8 levels, a central caspase proximally involved in the AICD-pathway (fluorescence of 258+/-47 in NOD vs. 441+/-16 in NOR and 414+/-61 in C57BL/6 T lymphocytes, P<0.05). Gene expression analysis using real-time RT-PCR additionally revealed low expression of Fas and FasL, the death receptor system activating caspase 8 and contributing to AICD. Additionally, low IL-2 levels, together with high TGFbeta and Bclx-L levels, confirm the presence of a NOD-specific AICD-resistance profile. In conclusion, we present cellular and molecular evidence for disturbed AICD mechanisms in NOD T lymphocytes. This resistance in AICD may contribute to defective tolerance induction to autoantigens in NOD mice.