RESUMO
AIM: Much focus of immunotherapy for type 1 diabetes (T1D) has been devoted on selectively boosting regulatory T (Treg) cells using low dose IL-2 due to their constitutive expression of IL-2Rα, CD25. However, several clinical trials using a low dose of IL-2 only showed a limited improvement of metabolic control. It can therefore be hypothesized that further decreasing IL-2 dosage may increase the selective responsiveness of Treg cells. METHODS: We induced experimental T1D using multiple low dose streptozotocin (STZ) injections and treated the mice with an ultra-low dose IL-2 (uIL-2, approximately 7-fold lower than low dose). Immune response was studied using multicolor flow cytometry. RESULTS: We found that uIL-2 did not protect STZ mice from developing hyperglycemia. It did neither increase Treg cell proportions, nor did it correct the phenotypic shift of Treg cells seen in T1D. It only partially decreased the proportion of IFN-γ+ T cells. Likewise, uIL-2 also did not protect the dysfunction of regulatory B (Breg) cells. Strikingly, when administered in combination with an anti-inflammatory cytokine IL-35, uIL-2 abrogated IL-35's protective effect. Low dose IL-2, on the other hand, protected half of the STZ mice from developing hyperglycemia. No difference was found in the Treg and Breg response, and it only tended to decrease CD80 expression in macrophages and dendritic cells. CONCLUSION: In conclusion, further decreasing IL-2 dosage may not be a suitable approach for T1D therapy, and the limited success suggests that an alternative low dose IL-2 therapy strategy or other immunotherapies should be considered.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Interleucina-2 , Linfócitos T Reguladores , Animais , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Camundongos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/imunologia , Interleucinas , Masculino , Camundongos Endogâmicos C57BL , Imunoterapia/métodosRESUMO
The anti-inflammatory role of regulatory B cells (Breg cells) has been associated with IL-35 based on studies of experimental autoimmune uveitis and encephalitis. The role of Breg cells and IL-35+ Breg cells for type 1 diabetes (T1D) remains to be investigated. We studied PBMCs from T1D subjects and healthy controls (HC) and found lowered proportions of Breg cells and IL-35+ Breg cells in T1D. To elucidate the role of Breg cells, the lymphoid organs of two mouse models of T1D were examined. Lower proportions of Breg cells and IL-35+ Breg cells were found in the animal models of T1D compared with control mice. In addition, the systemic administration of recombinant mouse IL-35 prevented hyperglycemia after multiple low dose streptozotocin (MLDSTZ) injections and increased the proportions of Breg cells and IL-35+ Breg cells. A higher proportion of IFN-γ+ cells among Breg cells were found in the PBMCs of the T1D subjects. In the MLDSTZ mice, IL-35 administration decreased the proportions of IFN-γ+ cells among the Breg cells. Our data illustrate that Breg cells may play an important role in the development of T1D and that IL-35 treatment prevents the development of hyperglycemia by maintaining the phenotype of the Breg cells under an experimental T1D condition.
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Anti-Inflamatórios/farmacologia , Linfócitos B Reguladores/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Hiperglicemia/prevenção & controle , Interleucinas/farmacologia , Adulto , Animais , Anti-Inflamatórios/sangue , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Hiperglicemia/induzido quimicamente , Interferon gama/sangue , Interleucinas/sangue , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Estreptozocina/toxicidadeRESUMO
Heparanase, the sole heparan sulfate degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, angiogenesis and metastasis. Much of the impact of heparanase on tumor progression is related to its function in mediating tumor-host crosstalk, priming the tumor microenvironment to better support tumor growth and metastasis. We have utilized mice over-expressing (Hpa-tg) heparanase to reveal the role of host heparanase in tumor initiation, growth and metastasis. While in wild type mice tumor development in response to DMBA carcinogenesis was restricted to the mammary gland, Hpa-tg mice developed tumors also in their lungs and liver, associating with reduced survival of the tumor-bearing mice. Consistently, xenograft tumors (lymphoma, melanoma, lung carcinoma, pancreatic carcinoma) transplanted in Hpa-tg mice exhibited accelerated tumor growth and shorter survival of the tumor-bearing mice compared with wild type mice. Hpa-tg mice were also more prone to the development of metastases following intravenous or subcutaneous injection of tumor cells. In some models, the growth advantage was associated with infiltration of heparanase-high host cells into the tumors. However, in other models, heparanase-high host cells were not detected in the primary tumor, implying that the growth advantage in Hpa-tg mice is due to systemic factors. Indeed, we found that plasma from Hpa-tg mice enhanced tumor cell migration and invasion attributed to increased levels of pro-tumorigenic factors (i.e., RANKL, SPARC, MIP-2) in the plasma of Hpa-Tg vs. wild type mice. Furthermore, tumor aggressiveness and short survival time were demonstrated in wild type mice transplanted with bone marrow derived from Hpa-tg but not wild type mice. These results were attributed, among other factors, to upregulation of pro-tumorigenic (i.e., IL35+) and downregulation of anti-tumorigenic (i.e., IFN-γ+) T-cell subpopulations in the spleen, lymph nodes and blood of Hpa-tg vs. wild type mice and their increased infiltration into the primary tumor. Collectively, our results emphasize the significance of host heparanase in mediating the pro-tumorigenic and pro-metastatic interactions between the tumor cells and the host tumor microenvironment, immune cells and systemic factors.
Assuntos
Glucuronidase/genética , Glucuronidase/metabolismo , Metástase Neoplásica/patologia , Neoplasias/patologia , Regulação para Cima , Animais , Antracenos/efeitos adversos , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Metástase Neoplásica/genética , Metástase Neoplásica/imunologia , Transplante de Neoplasias , Neoplasias/induzido quimicamente , Neoplasias/genética , Neoplasias/metabolismo , Piperidinas/efeitos adversos , Microambiente TumoralRESUMO
In type 1 diabetes (T1D), the insulin-producing ß cells are destructed by immune mechanisms. It has been hypothesized that the very first immune response in T1D onset comes from innate immune cells, which further activates the adaptive immune cells to attack the islets. Despite intensive research on characterization of islet-infiltrating immune cells, the kinetics of different immune cells in multiple low-dose streptozotocin (MLDSTZ)-induced T1D mouse model is still much unclear. Therefore, we investigated the proportions of innate immune cells such as neutrophils, dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), macrophages, natural killer (NK) cells, and adaptive immune cells (T and B lymphocytes) in thymi, pancreatic-draining lymph nodes, and spleens of MLDSTZ mice on days 3, 7, 10, and 21 after the first injection of STZ by flow cytometry. The proportions of DCs and B cells were increased from day 3, while the proportions of B-1a lymphocytes and interferon-γ+ cells among NK cells were increased, but NK cells were decreased on day 10 in MLDSTZ-treated mice, illustrating that the initial immune response is induced by DCs and B cells. Later, the proportions of T helper 1 and cytotoxic T cells were increased from day 7, suggesting that the innate immune cells precede adaptive immune cell response in MLDSTZ mice. Altogether, our data demonstrate a possible sequence of events regarding the involvement of DCs, pDCs, NK cells, B-1a lymphocytes, B, and T cells at the early stage of T1D development.
RESUMO
To assess mechanisms responsible for breast carcinoma metastasis, 4T1 breast carcinomas were grown orthotopically in wild type or Shb knockout mice. Tumor growth, metastasis, vascular characteristics and immune cell properties were analyzed. Absence of Shb did not affect tumor growth although it increased lung metastasis. Shb knockout mouse tumors showed decreased redness and less developed vascular plexa located at the periphery of the tumors. No difference in overall tumor vascular density, leakage or pericyte coverage was noted between the genotypes although the average vessel size was smaller in the knockout. Tumors induced an increase of CD11b+ cells in spleen, lymph node, thymus, bone marrow and blood. Numbers of Shb knockout CD11b/CD8+ cells were decreased in lymph nodes and bone marrow of tumor bearing mice. Mice with tumors had reduced numbers of CD4+ lymphocytes in blood/lymphoid organs, whereas in most of these locations the proportion of CD4+ cells co-expressing FoxP3 was increased, suggesting a relative increase in Treg cells. This finding was reinforced by increased blood interleukin-35 (IL-35) in wild type tumor bearing mice. Shb knockout blood showed in addition an increased proportion of IL-35 expressing Treg cells, supporting the notion that absence of Shb further promotes tumor evasion from immune cell recognition. This could explain the increased number of lung metastases observed under these conditions. In conclusion, 4T1 tumors alter immune cell responses that promote tumor expansion, metastasis and escape from T cell recognition in an Shb dependent manner.
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OBJECTIVE: Many patients with long-standing type 1 diabetes have remaining functional ß-cells. This study investigated immunological differences between patients with or without measurable remaining endogenous insulin production after ≥10 years duration of disease. RESEARCH DESIGN AND METHODS: Patients (n = 113; ≥18 years of age) with type 1 diabetes and with disease duration of ≥10 years were recruited at Uppsala University Hospital. Residual ß-cell function was determined with an ultrasensitive C-peptide ELISA. Circulating cytokines, including interleukin-35 (IL-35), were determined in plasma. Additional blood samples were collected from 14 of the identified C-peptide-positive patients and 12 of the C-peptide-negative patients, as well as from 15 healthy control subjects, and were used for immediate investigation of peripheral blood mononuclear cells. RESULTS: The blood concentration of the cytokine IL-35 was markedly lower in C-peptide-negative patients, and this was associated with a simultaneous decrease in the proportion of IL-35+ regulatory T cells (Tregs), IL-35+ regulatory B cells, and IL-35-producing CD8+Foxp3+ cells. IL-35 has previously been shown to maintain the phenotype of Tregs, block the differentiation of T-helper 17 cells, and thereby dampen immune assaults to ß-cells. We found that the proportions of IL-17a+ cells among the Tregs, CD4+ T cells, and CD8+ T cells were lower in the C-peptide-positive patients. CONCLUSIONS: Patients with remaining endogenous ß-cell function after >10 years duration of type 1 diabetes differ immunologically from other patients with long-standing type 1 diabetes. In particular, they have a much higher IL-35 production.
Assuntos
Peptídeo C/sangue , Diabetes Mellitus Tipo 1/sangue , Interleucinas/sangue , Adulto , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Feminino , Humanos , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Interleucina-17/sangue , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismoRESUMO
Heparanase is an endo-glucuronidase that degrades heparan sulfate chains. The enzyme is expressed at a low level in normal organs; however, elevated expression of heparanase has been detected in several inflammatory conditions, e.g. in the synovial joints of rheumatoid arthritis (RA) patients. Herein, we have applied the model of collagen-induced arthritis (CIA) to transgenic mice overexpressing human heparanase (Hpa-tg) along with wildtype (WT) mice. About 50% of the induced animals developed clinical symptoms, i.e. swelling of joints, and there were no differences between the Hpa-tg and WT mice in the incidence of disease. However, Hpa-tg mice displayed an earlier response and developed more severe symptoms. Examination of cells from thymus, spleen and lymph nodes revealed increased innate and adaptive immune responses of the Hpa-tg mice, reflected by increased proportions of macrophages, antigen presenting cells and plasmacytoid dendritic cells as well as Helios-positive CD4+ and CD8+ T cells. Furthermore, splenic lymphocytes from Hpa-tg mice showed higher proliferation activity. Our results suggest that elevated expression of heparanase augmented both the innate and adaptive immune system and propagated inflammatory reactions in the murine RA model.
Assuntos
Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Glucuronidase/metabolismo , Inflamação/patologia , Linfócitos T/imunologia , Animais , Antígenos CD/metabolismo , Artrite Experimental/imunologia , Artrite Experimental/patologia , Galinhas , Modelos Animais de Doenças , Imunidade Inata , Interferon gama/metabolismo , Interleucina-17/metabolismo , Linfonodos/patologia , Contagem de Linfócitos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Baço/patologiaRESUMO
Aim. To characterize the plasma levels of vascular endothelial growth factor (VEGF) in type 1 diabetes mellitus (T1D) and its relation to both present and historical metabolic control and microvascular complications. Methods. Plasma levels of VEGF and routine clinical parameters were analyzed in 115 patients with long-standing T1D and 45 healthy controls (HC). All patients were under clinical routine diabetes treatment at Uppsala University Hospital. Results. The plasma levels of VEGF were increased by 37% in patients with T1D when compared to HC (18.2 ± 0.8 versus 13.2 ± 1.0 pg/ml, p < 0.001). The levels of VEGF correlated to insulin needs and BMI but not to present or historical metabolic control. The levels of VEGF were similar in patients with T1D and microvascular complications (microalbuminuria and retinopathy) when compared with patients without microvascular complications. Historical HbA1c levels were found to be the best predictor for present metabolic control. Conclusion. Circulating plasma levels of VEGF do not correlate to present or historical metabolic control in long-standing T1D and the levels are not affected by the presence of microvascular complications.
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Albuminúria/etiologia , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/sangue , Nefropatias Diabéticas/etiologia , Retinopatia Diabética/etiologia , Hemoglobinas Glicadas/metabolismo , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Humanos , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , MasculinoRESUMO
The anti-inflammatory cytokine IL-35 is produced by regulatory T (Treg) cells to suppress autoimmune and inflammatory responses. The role of IL-35 in type 1 diabetes (T1D) remains to be answered. To elucidate this, we investigated the kinetics of Treg cell response in the multiple low dose streptozotocin induced (MLDSTZ) T1D model and measured the levels of IL-35 in human T1D patients. We found that Treg cells were increased in MLDSTZ mice. However, the Treg cells showed a decreased production of anti-inflammatory (IL-10, IL-35, TGF-ß) and increased pro-inflammatory (IFN-γ, IL-2, IL-17) cytokines, indicating a phenotypic shift of Treg cells under T1D condition. IL-35 administration effectively both prevented development of, and counteracted established MLDSTZ T1D, seemingly by induction of Eos expression and IL-35 production in Treg cells, thus reversing the phenotypic shift of the Treg cells. IL-35 administration reversed established hyperglycemia in NOD mouse model of T1D. Moreover, circulating IL-35 levels were decreased in human T1D patients compared to healthy controls. These findings suggest that insufficient IL-35 levels play a pivotal role in the development of T1D and that treatment with IL-35 should be investigated in treatment of T1D and other autoimmune diseases.
Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Interleucinas/administração & dosagem , Linfócitos T Reguladores/metabolismo , Animais , Citocinas/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Hiperglicemia/prevenção & controle , Interleucina-10/sangue , Interleucina-2/metabolismo , Interleucinas/sangue , Interleucinas/genética , Interleucinas/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Antígenos de Histocompatibilidade Menor , Fenótipo , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Baço/metabolismo , Estreptozocina/toxicidade , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Timo/metabolismo , Fator de Crescimento Transformador beta/sangueRESUMO
BACKGROUND: Somatostatin acts through five receptor subtypes (SSTRs 1-5). We aimed to investigate SSTRs mRNA expression and protein distribution in whole rat embryos, with special emphasis on the pancreas. MATERIAL AND METHODS: Rat embryos were collected on embryonal days 10, 11, 12, 14, 15, 17, 19, 21, and at birth. Presence of SSTRs was investigated with RT-PCR techniques and immunohistochemistry. RESULTS: There was no SSTR5 mRNA expression in the whole rat embryos. All SSTR1-5 proteins were observed at embryonal day 10, but the localization varied between the different subtypes. From day 11 to birth SSTRs protein presence increased with time in major structures such as skin and cartilage. It remained similar over time in the heart and liver. In the fetal pancreas mRNA expression of SSTR2 and 4 was detected at day 14, and there was an increase up to birth. Only SSTR1 protein co-localized to a higher extent with the islet hormones studied. SSTR2 was present in all islet endocrine cells except for ß-cells. In contrast, the immunostaining for SSTR3-4 was co-localized with insulin and PP, and, finally, SSTR5 with glucagon and pancreatic polypeptide. In mRNA isolated from whole rat embryos SSTR1-2 and SSTR4 expression showed a peak at day 14, while SSTR3 mRNA was not present until day 15. CONCLUSION: The present data suggest a role for SSTRs during the development of the rat embryo. Subsequent functional studies may elucidate regulatory roles of specific SSTRs for the growth and differentiation of the pancreas as well as other organs.
Assuntos
Embrião de Mamíferos/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Pâncreas/embriologia , Pâncreas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Somatostatina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TranscriptomaRESUMO
Regulatory T (Treg) cells are characterized by the expression of CD4, CD25 and the intracellular Foxp3. However, these markers do not indicate whether Treg cells are thymic derived Treg (tTreg) cells or peripherally induced Treg (pTreg) cells. Recently, Helios and Neuropilin-1 (Nrp1) has been reported as potential markers for tTreg cells. Herein, we used flow cytometry to examine the proportion of CD4(+)CD8(-)CD25(+) Treg cells expressing Helios, Nrp1 and Foxp3 in thymus, pancreatic draining lymph nodes (PDLNs) and spleen of CD-1 mice, and thymus of NOD and C57BL/6 mice. The frequency of Helios(+) cells was higher than that of Nrp1(+) cells in CD4(+)CD8(-)CD25(+) and CD4(+)CD8(-)CD25(+)Foxp3(+) Treg cells in thymus. Interestingly, the proportion of IL-10(+), Ebi3(+)and CTLA-4(+) cells was higher in Helios(+) than Nrp1(+) tTreg cells. The anti-apoptotic activity of Helios(+) tTreg cells was higher in thymus compared to Nrp1(+) tTreg cells. Nrp1 seems to be expressed at a later developmental stage compared to Helios and Foxp3. Furthermore, the expression of Nrp1 in CD4(+)CD25(+) T cells of younger mice did not increase after stimulating them in vitro with anti-CD3 and -CD28. Thus, under these conditions, Helios could be considered a more reliable marker for distinguishing tTreg cells from pTreg cells than Nrp1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neuropilina-1/metabolismo , Linfócitos T Reguladores/metabolismo , Timo/citologia , Fatores de Transcrição/metabolismo , Animais , Apoptose , Biomarcadores/metabolismo , Antígeno CTLA-4/metabolismo , Interleucina-10/metabolismo , Linfonodos/citologia , Contagem de Linfócitos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Antígenos de Histocompatibilidade Menor , Receptores de Citocinas/metabolismo , Baço/citologiaRESUMO
BACKGROUND: It has been proposed that the histamine 1-receptor (H1-receptor) not only promotes allergic reactions, but also modulates innate immunity and autoimmune reactions. In line with this, we have recently reported that the H1-receptor antagonist cetirizine partially counteracts cytokine-induced beta-cell signaling and destruction. Therefore, the aim of this study was to determine whether cetirizine affects diabetes in NOD mice, a model for human type 1 diabetes, and glucose intolerance in high-fat diet C57BL/6 mice, a model for human glucose intolerance. METHODS: Female NOD mice were treated with cetirizine in the drinking water (25 mg/kg body weight) from 9 until 30 weeks of age during which precipitation of diabetes was followed. Male C57BL/6 mice were given a high-fat diet from 5 weeks of age. When the mice were 12 weeks of age cetirizine was given for 2 weeks in the drinking water. The effects of cetirizine were analyzed by blood glucose determinations, glucose tolerance tests, and insulin sensitivity tests. RESULTS: Cetirizine did not affect diabetes development in NOD mice. On the other hand, cetirizine treatment for 1 week protected against high-fat diet-induced hyperglycemia. The glucose tolerance after 2 weeks of cetirizine treatment was improved in high-fat diet mice. We observed no effect of cetirizine on the insulin sensitivity of high-fat diet mice. CONCLUSION: Our results suggest a protective effect of cetirizine against high-fat diet-induced beta-cell dysfunction, but not against autoimmune beta-cell destruction.
Assuntos
Cetirizina/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Intolerância à Glucose/tratamento farmacológico , Antagonistas não Sedativos dos Receptores H1 da Histamina/farmacologia , Animais , Autoimunidade , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Dieta Hiperlipídica , Feminino , Teste de Tolerância a Glucose , Hipersensibilidade , Imunidade Inata , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Receptores Histamínicos H1/metabolismoRESUMO
AIM: To investigate colonic mucus thickness in vivo in health and during experimental inflammatory bowel disease. METHODS: Colitis was induced with 5% DSS in drinking water for 8 days prior to experiment, when the descending colonic mucosa of anesthetized rats was studied using intravital microscopy. Mucus thickness was measured with micropipettes attached to a micromanipulator. To assess the contributions of NOS and prostaglandins in the regulation of colonic mucus thickness, the non-selective NOS-inhibitor L-NNA (10 mg/kg bolus followed by 3 mg/kg/h), the selective iNOS-inhibitor L-NIL (10 mg/kg bolus followed by 3 mg/kg/h) and the non-selective COX-inhibitor diclofenac (5 mg/kg) were administered intravenously prior to experiment. To further investigate the role of iNOS in the regulation of colonic mucus thickness, iNOS -/- mice were used. RESULTS: Colitic rats had a thicker firmly adherent mucus layer following 8 days of DSS treatment than untreated rats (88±2 µm vs 76±1 µm). During induction of colitis, the thickness of the colonic mucus layer initially decreased but was from day 3 significantly thicker than in untreated rats. Diclofenac reduced the mucus thickness similarly in colitic and untreated rats (-16±5 µm vs -14±2 µm). While L-NNA had no effect on colonic mucus thickness in DSS or untreated controls (+3±2 µm vs +3±1 µm), L-NIL reduced the mucus thickness significantly more in colitic rats than in controls (-33±4 µm vs -10±3 µm). The importance of iNOS in regulating the colonic mucus thickness was confirmed in iNOS-/- mice, which had thinner colonic mucus than wild-type mice (35±3 µm vs 50±2 µm, respectively). Furthermore, immunohistochemistry revealed increased levels of iNOS in the colonic surface epithelium following DSS treatment. CONCLUSION: Both prostaglandins and nitric oxide regulate basal colonic mucus thickness. During onset of colitis, the thickness of the mucus layer is initially reduced followed by an iNOS mediated increase.
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Colite Ulcerativa/enzimologia , Colo/enzimologia , Muco/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Colite Ulcerativa/induzido quimicamente , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana , Células Caliciformes/metabolismo , Lisina/análogos & derivados , Lisina/farmacologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Nitroarginina/farmacologia , Prostaglandinas/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
We previously reported that IL-1 Trap (a hybrid molecule consisting of the extracellular domain of IL-1 receptor accessory protein and IL-1 receptor type 1 arranged inline and fused to the Fc-portion of IgG1) can protect rat pancreatic islets in vitro against noxious effects induced by IL-1ß. In this study we tested the effect of administration of a murine IL-1 Trap on the recurrence of disease (ROD) model in non-obese diabetic (NOD) mice. Spontaneously diabetic female NOD mice received implantation of a curative number (600) of syngeneic pancreatic islets beneath their left kidney capsule from young healthy NOD mouse donors. Once a day, the mice were injected subcutaneously with IL-1 Trap (30mg/kg bodyweight), or an equimolar dose Fc-control protein (8.4mg/kg bodyweight) or saline. The treatments were maintained until ROD (i.e. a blood glucose value ⩾11.1mM for 2 consecutive days) or until 5days after transplantation. 3 out of 11 mice treated with IL-1 Trap showed a significantly increased graft survival compared to all other mice, and analysis of relative cytokine mRNA levels in isolated spleen cells showed elevated IL-4 mRNA levels, but no differences in FoxP3 or iNOS staining of grafts, from mice treated with IL-1 Trap, at both endpoints, compared to both control groups. Administration of IL-1 Trap counteracts islet cell destruction in the NOD mouse model of type 1 diabetes. In part this could be due to a shift towards Th2 cytokine production seen in IL-1 Trap treated animals.
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Proteína Acessória do Receptor de Interleucina-1/metabolismo , Ilhotas Pancreáticas/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Interleucina-4/genética , Interleucina-4/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos NOD , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Baço/citologia , Baço/metabolismoRESUMO
Although several reports suggest a potentially deleterious role of macrophage migration inhibitory factor (MIF) in type 2 diabetes (T2D) pathology, it is still unclear how this pro-inflammatory cytokine acts on pancreatic beta cells. The aim of the present study was to evaluate MIF effects on murine beta cells in the in vitro settings mimicking T2D-associated conditions. Results indicate that recombinant MIF further increased apoptosis of pancreatic islets or MIN6 cells upon exposure to palmitic acid or glucose. This was accompanied by upregulation of several pro-apoptotic molecules. Furthermore, MIF potentiated nutrient-induced islet cell dysfunction, as revealed by lower glucose oxidation rate, ATP content, and depolarized mitochondrial membrane. The final outcome was potentiation of mitochondrial apoptotic pathway. The observed upregulation of nutrient-induced islet cell dysfunction and apoptosis by MIF implicates that silencing MIF may be beneficial for maintaining integrity of endocrine pancreas in obesity-associated T2D.
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Glucose/metabolismo , Células Secretoras de Insulina/citologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Ácido Palmítico/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Linhagem Celular , Citocinas/metabolismo , Fragmentação do DNA , Diabetes Mellitus Tipo 2/metabolismo , Perfilação da Expressão Gênica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sais de Tetrazólio/química , Tiazóis/químicaRESUMO
As a result of chronic exposure to high levels of free fatty acids, glucose and inflammatory mediators ß-cell apoptosis occurs at the end stage of obesity-associated type 2 diabetes (T2D). One potentially deleterious molecule for ß-cell function associated with T2D and obesity in humans is macrophage migration inhibitory factor (MIF). Therefore, the aim of this study was to explore MIF expression in vivo during development of obesity and insulin resistance in high-fat diet (HFD)-fed C57BL/6 mice and whether MIF inhibition could affect ß-cell apoptosis and dysfunction induced by palmitic acid (PA) in vitro. Indeed, increase in systemic and locally produced MIF correlated well with the weight gain, triglyceride upregulation, glucose intolerance and insulin resistance, which developed in HFD-fed mice. In in vitro settings PA dose-dependently induced MIF secretion before apoptosis development in islets. Further, mif gene deletion, mRNA silencing or protein inhibition rescued ß-cells from PA-induced apoptosis as measured by MTT assay and histone-DNA enzyme linked immuno sorbent assay. Protection from induced apoptosis was mediated by altered activation of caspase pathway and correlated with changes in the level of Bcl-2 family members. Further, MIF inhibition conveyed a significant resistance to PA-induced downregulation of insulin and PDX-1 expression and ATP content. However, ß-cell function was not entirely preserved in the absence of MIF judging by low glucose oxidation and depolarized mitochondrial membrane. In conclusion, the observed considerable preservation of ß-cells from nutrient-induced apoptosis might implicate MIF as a potential therapeutic target in the later stage of obesity-associated T2D.
Assuntos
Apoptose/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Fatores Inibidores da Migração de Macrófagos/deficiência , Ácido Palmítico/farmacologia , Animais , Glicemia/metabolismo , Caspase 9/genética , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica/efeitos adversos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Insulina/sangue , Ilhotas Pancreáticas/patologia , Isoxazóis/farmacologia , Fatores Inibidores da Migração de Macrófagos/sangue , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/sangue , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/genéticaRESUMO
Undergraduate medical education in Sweden has moved from nationally regulated, subject-based courses to programmes integrated either around organ systems or physiological and patho-physiological processes, or organised around basic medical science in conjunction with clinical specialities, with individual profiles at the seven medical schools. The national regulations are restricted to overall academic and professional outcomes. The 5½ year long university undergraduate curriculum is followed by a mandatory 18 months internship, delivered by the County Councils. While quality control and accreditation for the university curriculum is provided by the Swedish National Agency for Higher Education, no such formal control exists for the internship; undergraduate medical education is therefore in conflict with EU directives from 2005. The Government is expected to move towards 6 years long university undergraduate programmes, leading to licence, which will facilitate international mobility of both Swedish and foreign medical students and doctors. Ongoing academic development of undergraduate education is strengthened by the Bologna process. It includes outcome (competence)-based curricula, university Masters level complying with international standards, progression of competence throughout the curriculum, student directed learning, active participation and roles in practical clinical education and a national assessment model to assure professional competence. In the near future, the dimensioning of Swedish undergraduate education is likely to be decided more by international demands and aspects of quality than by national demands for doctors.
Assuntos
Currículo/normas , Educação de Pós-Graduação em Medicina/normas , Educação de Graduação em Medicina/normas , Faculdades de Medicina/normas , Currículo/tendências , Educação de Pós-Graduação em Medicina/tendências , Educação de Graduação em Medicina/tendências , Escolaridade , Política de Saúde , Humanos , Internato e Residência/normas , Internato e Residência/tendências , Modelos Educacionais , Faculdades de Medicina/tendências , Estudantes de Medicina , SuéciaRESUMO
Somatostatin acts via five receptors (sst(1-5)). We investigated if the changes in pancreatic islet sst expression in diabetic NOD mice compared to normoglycemic mice are a consequence of hyperglycemia or the ongoing immune reaction in the pancreas. Pancreatic islets were isolated from NOD mice precultured for 5 days and further cultured for 3 days at high or low glucose before examined. Islets were also isolated from NOD mice and transplanted to normal or diabetic mice in a number not sufficient to cure hyperglycemia. After three days, the transplants were removed and stained for sst(1-5) and islet hormones. Overall, changes in sst islet cell expression were more common in islets cultured in high glucose concentration in vitro as compared to the islet transplantation in vivo to diabetic mice. The beta and PP cells exhibited more frequent changes in sst expression, while the alpha and delta cells were relatively unaffected by the high glucose condition. Our findings suggest that the glucose level may alter sst expressed in islets cells; however, immune mechanisms may counteract such changes in islet sst expression.
Assuntos
Regulação da Expressão Gênica , Glucose/metabolismo , Ilhotas Pancreáticas/citologia , Receptores de Somatostatina/biossíntese , Animais , Glicemia/metabolismo , Células Cultivadas , Feminino , Células Secretoras de Glucagon/citologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Microscopia de Fluorescência/métodos , Receptores de Somatostatina/metabolismo , Células Secretoras de Somatostatina/citologia , Fatores de TempoRESUMO
OBJECTIVE: cAMP is a critical messenger for insulin and glucagon secretion from pancreatic ß- and α-cells, respectively. Dispersed ß-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown. RESEARCH DESIGN AND METHODS: The subplasma-membrane cAMP concentration ([cAMP](pm)) was recorded in α- and ß-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and ß-cells. RESULTS: In islets exposed to 3 mmol/L glucose, [cAMP](pm) was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP](pm), often with oscillations synchronized among ß-cells. Whereas glucagon also induced [cAMP](pm) oscillations in most α-cells, <20% of the α-cells responded to GLP-1. Elevation of the glucose concentration to 11-30 mmol/L in the absence of hormones induced slow [cAMP](pm) oscillations in both α- and ß-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in the ß-cells but not caused by the changes in [Ca(2+)](i). The transmembrane adenylyl cyclase (AC) inhibitor 2'5'-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP](pm) elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-ß-triol perturbed cell metabolism and lacked effect, respectively. CONCLUSIONS: Oscillatory [cAMP](pm) signaling in secretagogue-stimulated ß-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP](pm) oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.
Assuntos
AMP Cíclico/metabolismo , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/metabolismo , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Cálcio/farmacologia , Inibidores Enzimáticos/farmacologia , Epinefrina/farmacologia , Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução/efeitos dos fármacos , Oximas/farmacologiaRESUMO
Until now, there have been few assays to measure C-peptide and proinsulin in the rat. We used a well-established rat insulin ELISA and validated two novel ELISAs for rat C-peptide and rat/mouse proinsulin to examine secretion and content of insulin, proinsulin, and C-peptide from rat islets cultured for 72 h at different glucose concentrations in culture medium. To examine long-term effects in vitro rather than short-term effects of exposure to low, normal, and high glucose, the exposure time to the different glucose concentrations was set to 72 h. The measurement uncertainty of the values obtainable from the ELISAs was determined by calculation of the variation pattern from the intraassay variation generated by unknown samples, and repeatability was determined by analysis of controls. The precision study and the analysis of controls confirm that the validated ELISAs for rat C-peptide and proinsulin would be useful for further studies on the effects of preculture in different glucose concentrations. The higher the glucose concentration used during the 72-h culture period of rat islets, the higher insulin, C-peptide and proinsulin values were obtained in a subsequent short-term glucose-challenge experiment. The proportion of proinsulin to insulin secreted increased, as did islet content, with increasing glucose concentration during preculture. We also observed a nonequimolar, glucose-dependent secretion and content of rat insulin over rat C-peptide after culture at 11.1 and 28 mM glucose.