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1.
BMC Nephrol ; 16: 212, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26666710

RESUMEN

BACKGROUND: Pyrrolidine dithiocarbamate (PDTC) reduces renal cyst growth in a rodent model of polycystic kidney disease (PKD) but the mechanism of action is not clear. Here, we investigated the hypothesis that PDTC reduces the proliferation of cystic epithelial cells in vitro in a nuclear factor (NF)-κB-dependent manner. METHODS: Immortalized autosomal dominant PKD (ADPKD) cells that are heterozygous (WT9-7) and homozygous (WT-9-12) for a truncating Pkd1 mutation, and immortalized normal human tubular cells (HK-2), were exposed to NF-κB-inducing agents with or without PDTC. Cell proliferation and apoptosis were assessed by bromodeoxyuridine assay and Annexin V flow cytometry, respectively. NF-κB activity was assessed by luciferase reporter assay and western blotting for nuclear p65, p50, and RelB subunits and cytoplasmic phosphorylated-IκBα. RESULTS: Serum-induced proliferation was similar in all cell lines over 72 h. PDTC demonstrated anti-proliferative effects that were delayed in ADPKD cells compared to HK-2. Basal NF-κB-dependent luciferase reporter activity was lower in ADPKD cells compared to normal cells. Classical NF-κB stimulants, lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α, increased NF-κB luciferase activity in HK-2, whereas in PKD cell lines, NF-κB activity was only induced by TNF-α. However, neither stimulant altered proliferation in any cell line. PDTC reduced TNF-α-stimulated NF-κB activity in HK-2 only. CONCLUSIONS: PDTC reduced proliferation in ADPKD cells but did not consistently alter NF-κB activation, suggesting that other signalling pathways are likely to be involved in its ability to attenuate renal cyst growth in vivo.


Asunto(s)
Proliferación Celular/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Riñón Poliquístico Autosómico Dominante/patología , Pirrolidinas/farmacología , Tiocarbamatos/farmacología , Células Cultivadas , Humanos
2.
J Immunol ; 182(2): 793-801, 2009 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-19124722

RESUMEN

The cytokine B cell activation factor of the TNF family (BAFF) is considered to perform a proinflammatory function. This paradigm is particularly true for B cell-dependent immune responses; however the exact role for BAFF in regulating T cell immunity is ill-defined. To directly assess the effect of BAFF upon T cells, we analyzed T cell-dependent immune responses in BAFF-transgenic (Tg) mice. We found that T cell responses in BAFF-Tg mice are profoundly compromised, as indicated by their acceptance of islet allografts and delayed skin graft rejection. However, purified BAFF-Tg effector T cells could reject islet allografts with a normal kinetic, suggesting that the altered response did not relate to a defect in T cell function per se. Rather, we found that BAFF-Tg mice harbored an increased number of peripheral CD4+Foxp3+ T cells. A large proportion of the BAFF-expanded CD4+CD25+Foxp3+ regulatory T cells (Tregs) were CD62LlowCD103high and ICAM-1high, a phenotype consistent with an ability to home to inflammatory sites and prevent T cell effector responses. Indeed, depletion of the endogenous BAFF-Tg Tregs allowed allograft rejection to proceed, demonstrating that the increased Tregs were responsible for preventing alloimmunity. The ability of BAFF to promote Treg expansion was not T cell intrinsic, as Tregs did not express high levels of BAFF receptor 3, nor did excessive BAFF trigger NF-kappaB2 processing in Tregs. In contrast, we found that BAFF engendered Treg expansion through an indirect, B cell-dependent mechanism. Thus, under certain conditions, BAFF can play a surprising anti-inflammatory role in T cell biology by promoting the expansion of Treg cells.


Asunto(s)
Factor Activador de Células B/genética , Proliferación Celular , Factores de Transcripción Forkhead/biosíntesis , Rechazo de Injerto/prevención & control , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Supervivencia de Injerto/genética , Supervivencia de Injerto/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Trasplante de Piel/inmunología , Tolerancia al Trasplante/genética
3.
iScience ; 24(4): 102379, 2021 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-33981968

RESUMEN

Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic ß-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription.

4.
Pathogens ; 9(9)2020 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-32839374

RESUMEN

Cryptococcus neoformans is a human fungal pathogen that adapts its metabolism to cope with limited oxygen availability, nutrient deprivation and host phagocytes. To gain insight into cryptococcal metabolism, we optimized a protocol for the Seahorse Analyzer, which measures extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indications of glycolytic and respiratory activities. In doing so we achieved effective immobilization of encapsulated cryptococci, established Rotenone/Antimycin A and 2-deoxyglucose as effective inhibitors of mitochondrial respiration and glycolysis, respectively, and optimized a microscopy-based method of data normalization. We applied the protocol to monitor metabolic changes in the pathogen alone and in co-culture with human blood-derived monocytes. We also compared metabolic flux in wild-type C. neoformans, its isogenic 5-PP-IP5/IP7-deficient metabolic mutant kcs1∆, the sister species of C. neoformans, Cryptococcus deuterogattii/VGII, and two other yeasts, Saccharomyces cerevisiae and Candida albicans. Our findings show that in contrast to monocytes and C. albicans, glycolysis and respiration are tightly coupled in C. neoformans and C. deuterogattii, as no compensatory increase in glycolysis occurred following inhibition of respiration. We also demonstrate that kcs1∆ has reduced metabolic activity that correlates with reduced mitochondrial function. Metabolic inflexibility in C. neoformans is therefore consistent with its obligate aerobe status and coincides with phagocyte tolerance of ingested cryptococcal cells.

5.
Diabetes ; 69(8): 1735-1748, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32381646

RESUMEN

Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell-mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5-1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-ß production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-2/farmacología , Sirolimus/farmacología , Aloinjertos , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Rechazo de Injerto , Humanos , Ratones , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo
7.
JCI Insight ; 52019 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-31361602

RESUMEN

Human islet isolation is a cost-/resource-intensive program generating islets for cell therapy in Type 1 diabetes. However, only a third of cadaveric pancreas get to clinical transplantation due to low quality/number of islets. There is a need to identify biomarker(s) that predict the quality of islets, prior to initiating their isolation. Here, we sequenced transcriptome from 18 human islet preparations stratified into three groups (Gr.1: Best quality/transplantable islets, Gr.2: Intermediary quality, Gr.3: Inferior quality/non-transplantable islets) based on routine measurements including islet purity/viability. Machine-learning algorithms involving penalized regression analyses identified 10 long-non-coding(lnc)RNAs significantly different across all group-wise comparisons (Gr1VsGr2, Gr2vsGr3, Gr1vsGr3). Two variants of Metastasis-Associated Lung Adenocarcinoma Transcript-1(MALAT1) lncRNA were common across all comparisons. We confirmed RNA-seq findings in a "validation set" of 75 human islet preparations. Finally, in 19 pancreas samples, we demonstrate that assessing the levels of MALAT1 variants alone (ROC curve AUC: 0.83) offers highest specificity in predicting post-isolation islet quality and improves the predictive potential for clinical islet transplantation when combined with Edmonton Donor Points/Body Mass Index(BMI)/North American Islet Donor Score(NAIDS). We present this resource of islet-quality-stratified lncRNA transcriptome data and identify MALAT1 as a biomarker that significantly enhances current selection methods for clinical (GMP)-grade islet isolation.


Asunto(s)
Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Biomarcadores , Índice de Masa Corporal , Modelos Animales de Enfermedad , Humanos , Ratones Desnudos , Páncreas/patología , Trasplante de Páncreas , Donantes de Tejidos , Transcriptoma , Trasplante Heterólogo
8.
Cell Rep ; 27(8): 2370-2384.e6, 2019 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-31116982

RESUMEN

The development of autoimmune disease type 1 diabetes (T1D) is determined by both genetic background and environmental factors. Environmental triggers include RNA viruses, particularly coxsackievirus (CV), but how they induce T1D is not understood. Here, we demonstrate that deletion of the transcription factor hypoxia-inducible factor-1α (HIF-1α) from ß cells increases the susceptibility of non-obese diabetic (NOD) mice to environmentally triggered T1D from coxsackieviruses and the ß cell toxin streptozotocin. Similarly, knockdown of HIF-1α in human islets leads to a poorer response to coxsackievirus infection. Studies in coxsackievirus-infected islets demonstrate that lack of HIF-1α leads to impaired viral clearance, increased viral load, inflammation, pancreatitis, and loss of ß cell mass. These findings show an important role for ß cells and, specifically, lack of ß cell HIF-1α in the development of T1D. These data suggest new strategies for the prevention of T1D.


Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Subunidad alfa del Factor 1 Inducible por Hipoxia/uso terapéutico , Animales , Apoptosis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/farmacología , Masculino , Ratones
9.
Diabetes ; 55(9): 2491-501, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16936197

RESUMEN

Apoptotic beta-cell death is central to the pathogenesis of type 1 diabetes and may be important in islet graft rejection. Despite this, genetic control of beta-cell apoptosis is only poorly understood. We report that inhibition of gene transcription sensitized beta-cells to tumor necrosis factor (TNF)-alpha-induced apoptosis, indicating the presence of a regulated antiapoptotic response. Using oligonucleotide microarrays and real-time PCR, we identified TNFAIP3/A20 as the most highly regulated antiapoptotic gene expressed in cytokine-stimulated human and mouse islets. Cytokine induction of A20 mRNA in primary islets and insulinoma cells was rapid and observed within 1 h, consistent with A20 being an immediate early response gene in beta-cells. Regulation of A20 was nuclear factor-kappaB (NF-kappaB)-dependent, two NF-kappaB sites within the A20 promoter were found to be necessary and sufficient for A20 expression in beta-cells. Activation of NF-kappaB by TNF receptor-associated factor (TRAF) 2, TRAF6, NF-kappaB-inducing kinase, or protein kinase D, which transduce signals downstream of Toll-like receptors, TNF receptors, and free radicals, respectively, were all potent activators of the A20 promoter. Moreover, A20 expression was induced in transplanted islets in vivo. Finally, A20 expression was sufficient to protect beta-cells from TNF-induced apoptosis. These data demonstrate that A20 is the cardinal antiapoptotic gene in beta-cells. Further, A20 expression is NF-kappaB dependent, thus linking islet proinflammatory gene responses with protection from apoptosis.


Asunto(s)
Apoptosis , Células Secretoras de Insulina/citología , FN-kappa B/fisiología , Proteínas/fisiología , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Cisteína Endopeptidasas , Proteínas de Unión al ADN , Perfilación de la Expresión Génica , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1 , Péptidos y Proteínas de Señalización Intracelular , Islotes Pancreáticos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Proteínas Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factor 2 Asociado a Receptor de TNF/farmacología , Factor 6 Asociado a Receptor de TNF/farmacología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Quinasa de Factor Nuclear kappa B
10.
Transplantation ; 101(7): 1587-1599, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-27653300

RESUMEN

BACKGROUND: Although islet xenotransplantation is a promising therapy for type 1 diabetes, its clinical application has been hampered by cellular rejection and the requirement for high levels of immunosuppression. The aim of this study was to determine the role of Foxp3 regulatory T (Treg) cells in costimulation blockade-induced dominant tolerance to porcine neonatal islet cell cluster (NICC) xenografts in mice. METHODS: Porcine-NICC were transplanted under the renal capsule of BALB/c or C57BL/6 recipients and given a single dose of CTLA4-Fc at the time of transplant and 4doses of anti-CD154 mAb to day 6. Depletion of Foxp3Treg cell was performed in DEpletion of REGulatory T cells mice at day 80 posttransplantation. Foxp3Treg cell from spleens of treated BALB/c mice (tolerant Treg cell), and splenocytes were cotransferred into islet transplanted nonobese diabetic background with severe combined immunodeficiency mice to assess suppressive function. RESULTS: In treated mice, increased numbers of Foxp3Treg cell were identified in the porcine-NICC xenografts, draining lymph node, and spleen. Porcine-NICC xenografts from treated mice expressed elevated levels of TGF-ß, IL-10 and IFN-γ. Porcine-NICC xenograft tolerance was abrogated after depletion of Foxp3Treg cell. Tolerant Treg cell produced high levels of IL-10 and had diverse T cell receptor Vß repertoires with an oligoclonal expansion in CDR3 of T cell receptor Vß14. These tolerant Treg cells had the capacity to transfer dominant tolerance and specifically exhibited more potent regulatory function to porcine-NICC xenografts that naive Treg cell. CONCLUSIONS: This study demonstrated that short-term costimulation blockade-induced dominant tolerance and that Foxp3Treg cell played an essential role in its maintenance. Foxp3Treg cells were activated and had more potent regulatory function in vivo than naive Treg cells.


Asunto(s)
Abatacept/farmacología , Ligando de CD40/antagonistas & inhibidores , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Inmunosupresores/farmacología , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/cirugía , Linfocitos T Reguladores/efectos de los fármacos , Traslado Adoptivo , Animales , Animales Recién Nacidos , Ligando de CD40/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Rechazo de Injerto/inmunología , Xenoinjertos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Trasplante de Islotes Pancreáticos/efectos adversos , Trasplante de Islotes Pancreáticos/inmunología , Activación de Linfocitos/efectos de los fármacos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , Sus scrofa , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/trasplante , Factores de Tiempo , Tolerancia al Trasplante/efectos de los fármacos
11.
World J Nephrol ; 5(4): 339-57, 2016 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-27458563

RESUMEN

AIM: To determine the temporal expression and pattern of Rel/nuclear factor (NF)-κB proteins in renal tissue in polycystic kidney disease (PKD). METHODS: The renal expression of Rel/NF-κB proteins was determined by immunohistochemistry, immunofluorescence and immunoblot analysis in Lewis polycystic kidney rats (LPK, a genetic ortholog of human nephronopthsis-9) from postnatal weeks 3 to 20. At each timepoint, renal disease progression and the mRNA expression of NF-κB-dependent genes (TNFα and CCL2) were determined. NF-κB was also histologically assessed in human PKD tissue. RESULTS: Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation (peaking at weeks 3 and 10 respectively), and progressive interstitial fibrosis (with α smooth muscle actin and Sirius Red deposition significantly increased compared to Lewis kidneys from weeks 3 to 6 onwards). Rel/NF-κB proteins (phosphorylated-p105, p65, p50, c-Rel and RelB) were expressed in cystic epithelial cells (CECs) of LPK kidneys as early as postnatal week 3 and sustained until late-stage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBα protein levels, and TNFα and CCL2 expression, were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human PKD. The DNA damage marker γ-H2AX was also identified in the CECs of LPK and human polycystic kidneys. CONCLUSION: Several NF-κB proteins are consistently expressed in CECs in human and experimental PKD. These data suggest that the upregulation of both the canonical and non-canonical pathways of NF-κB signaling may be a constitutive and early pathological feature of cystic renal diseases.

12.
Transplant Direct ; 2(6): e77, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-27500267

RESUMEN

INTRODUCTION: The instant blood-mediated inflammatory reaction (IBMIR) causes major loss of islets after transplantation and consequently represents the initial barrier to survival of porcine neonatal islet cell clusters (NICC) after xenotransplantation. METHODS: This study used novel assays designed to characterize the various immunologic components responsible for xenogeneic IBMIR to identify initiators and investigate processes of IBMIR-associated coagulation, complement activation and neutrophil infiltration. The IBMIR was induced in vitro by exposing NICC to platelet-poor or platelet-rich human plasma or isolated neutrophils. RESULTS: We found that xenogeneic IBMIR was characterized by rapid, platelet-independent thrombin generation, with addition of platelets both accelerating and exacerbating this response. Platelet-independent complement activation was observed as early as 30 minutes after NICC exposure to plasma. However, membrane attack complex formation was not observed in NICC histopathology sections until after 60 minutes. We demonstrated for the first time that NICC-mediated complement activation was necessary for neutrophil activation in the xenogeneic IBMIR setting. Finally, using the Seahorse extracellular flux analyzer, we identified substantial loss of islet function (up to 40%) after IBMIR with surviving NICC showing evidence of mitochondrial damage. CONCLUSIONS: This study used novel assays to describe multiple key pathways by which xenogeneic IBMIR causes islet destruction, allowing further refinement of future interventions aimed at resolving the issue of IBMIR in xenotransplantation.

13.
Cell Transplant ; 22(12): 2355-66, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23127588

RESUMEN

Islet grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB). Thus, NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically engineering islets to express the NF-κB superrepressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early immediate proinflammatory response, with the most highly upregulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10, and Ccl2; the cytokines Tnf-α and Il-6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95% in islets and MIN6 ß-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at postoperative day (POD) 10 revealed that IκBα-transduced islet allografts exhibited improved islet architecture and strong insulin-labeling with decreased Ccl2 and Il-6 mRNA levels compared to the GFP-transduced control grafts. Despite these protective effects, NF-κB-blocked islet allografts were promptly rejected in our MHC-mismatched mouse model. However, IκBα-expressing grafts did harbor localized "pockets" of Foxp3(+) mononuclear cells not evident in the control grafts. This result suggested that the effect of the NF-κB blockade might synergize with regulatory T-cell-sparing rapamycin. Indeed, combining intragraft IκBα expression with low-dose rapamycin increased the mean survival time of islet allografts from 20 to 81 days, with 20% of the grafts surviving for greater than 100 days. In conclusion, rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.


Asunto(s)
Diabetes Mellitus Experimental/cirugía , Supervivencia de Injerto/efectos de los fármacos , Quinasa I-kappa B/metabolismo , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/citología , FN-kappa B/antagonistas & inhibidores , Sirolimus/farmacología , Animales , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Diabetes Mellitus Experimental/mortalidad , Diabetes Mellitus Experimental/patología , Sinergismo Farmacológico , Quinasa I-kappa B/genética , Inmunosupresores/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Porcinos , Trasplante Homólogo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo
14.
Diabetes ; 61(9): 2269-79, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22688333

RESUMEN

Insulin is secreted as discrete insulin secretory bursts at ~5-min intervals into the hepatic portal vein, these pulses being attenuated early in the development of type 1 and type 2 diabetes mellitus (T2DM). Intraportal insulin infusions (pulsatile, constant, or reproducing that in T2DM) indicated that the pattern of pulsatile insulin secretion delivered via the portal vein is important for hepatic insulin action and, therefore, presumably for hepatic insulin signaling. To test this, we examined hepatic insulin signaling in rat livers exposed to the same three patterns of portal vein insulin delivery by use of sequential liver biopsies in anesthetized rats. Intraportal delivery of insulin in a constant versus pulsatile pattern led to delayed and impaired activation of hepatic insulin receptor substrate (IRS)-1 and IRS-2 signaling, impaired activation of downstream insulin signaling effector molecules AKT and Foxo1, and decreased expression of glucokinase (Gck). We further established that hepatic Gck expression is decreased in the HIP rat model of T2DM, a defect that correlated with a progressive defect of pulsatile insulin secretion. We conclude that the physiological pulsatile pattern of insulin delivery is important in hepatic insulin signaling and glycemic control. Hepatic insulin resistance in diabetes is likely in part due to impaired pulsatile insulin secretion.


Asunto(s)
Diabetes Mellitus Tipo 2/fisiopatología , Insulina/fisiología , Hígado/fisiología , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental , Perros , Factores de Transcripción Forkhead/metabolismo , Glucoquinasa/metabolismo , Insulina/administración & dosificación , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina/fisiología , Secreción de Insulina , Masculino , Proteínas del Tejido Nervioso/metabolismo , Vena Porta , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología
15.
Diabetes ; 58(7): 1568-77, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19336675

RESUMEN

OBJECTIVE: Tumor necrosis factor ligand family members B-cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) can exert powerful effects on B-cell activation and development, type 1 T-helper cell (Th1) immune responses, and autoimmunity. We examined the effect of blocking BAFF and APRIL on the development of autoimmune diabetes. RESEARCH DESIGN AND METHODS: Female NOD mice were administered B-cell maturation antigen (BCMA)-Fc from 9 to 15 weeks of age. Diabetes incidence, islet pathology, and T- and B-cell populations were examined. RESULTS: BCMA-Fc treatment reduced the severity of insulitis and prevented diabetes development in NOD mice. BCMA-Fc-treated mice showed reduced follicular, marginal-zone, and T2MZ B-cells. B-cell reduction was accompanied by decreased frequencies of pathogenic CD4(+)CD40(+) T-cells and reduced Th1 cytokines IL-7, IL-15, and IL-17. Thus, T-cell activation was blunted with reduced B-cells. However, BCMA-Fc-treated mice still harbored detectable diabetogenic T-cells, suggesting that regulatory mechanisms contributed to diabetes prevention. Indeed, BCMA-Fc-treated mice accumulated increased CD4(+)CD25(+) regulatory T-cells (Tregs) with age. CD4(+)CD25(+) cells were essential for maintaining euglycemia because their depletion abrogated BCMA-Fc-mediated protection. BCMA-Fc did not directly affect Treg homeostasis given that CD4(+)CD25(+)Foxp3(+) T-cells did not express TACI or BR3 receptors and that CD4(+)CD25(+)Foxp3(+) T-cell frequencies were equivalent in wild-type, BAFF(-/-), TACI(-/-), BCMA(-/-), and BR3(-/-) mice. Rather, B-cell depletion resulted in CD4(+)CD25(+) T-cell-mediated protection from diabetes because anti-CD25 monoclonal antibody treatment precipitated diabetes in both diabetes-resistant NOD.microMT(-/-) and BCMA-Fc-treated mice. CONCLUSIONS: BAFF/APRIL blockade prevents diabetes. BCMA-Fc reduces B-cells, subsequently blunting autoimmune activity and allowing endogenous regulatory mechanisms to preserve a prehyperglycemic state.


Asunto(s)
Autoinmunidad , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Subunidad alfa del Receptor de Interleucina-2/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/inmunología , Antígeno de Maduración de Linfocitos B/farmacología , Femenino , Interleucina-15/inmunología , Interleucina-17/inmunología , Interleucina-7/inmunología , Activación de Linfocitos , Depleción Linfocítica , Ratones , Ratones Endogámicos NOD
16.
Diabetes ; 57(2): 395-404, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18025414

RESUMEN

OBJECTIVE: B-cells are important for disease pathogenesis in the nonobese diabetic (NOD) mouse model of type 1 diabetes. Recent studies demonstrate that marginal-zone B-cells (MZBs), which connect innate with adaptive immune responses, are increased in NOD mice. However, beyond this, the contribution of different B-cell subsets to diabetes pathogenesis is poorly understood. RESEARCH DESIGN AND METHODS: To better understand the role of different B-cell subsets in the etiology of type 1 diabetes, we have examined the MZB compartment in NOD mice, with respect to their number, distribution, and function. RESULTS: We demonstrate that splenic MZB numbers in female NOD mice undergo a marked, approximately threefold expansion between approximately 12 and 16 weeks of age, coincident with the onset of frank diabetes. Functionally, NOD MZBs are hyperresponsive to toll-like receptor 9 ligation and CD40 ligation, as well as sphingosine-1-phosphate-dependent chemotactic cues, suggesting an increased sensitivity to selective innate- and activation-induced stimuli. Intriguingly, at 16 weeks of age, approximately 80% of female NOD mice present with MZB-like cells in the pancreatic lymph node (PLN). These MZB-like cells express major histocompatibility complex class II and high levels of CD80 and CD86, and their presence in the PLN is associated with an increased frequency of activated Vbeta4(+) CD4(+) T-cells. Significantly, we demonstrate that purified MZBs are able to present the autoantigen insulin to diabetogenic T-cells. CONCLUSIONS: These data are consistent with MZBs contributing to the pathogenesis of type 1 diabetes as antigen-presenting cells. By integrating innate-derived inflammatory signals with the activation of autoreactive T-cells, MZBs may help to direct T-cell responses against beta-cell self-constituents.


Asunto(s)
Diabetes Mellitus Tipo 1/patología , Células Secretoras de Insulina/patología , Ganglios Linfáticos/patología , Linfocitos T/inmunología , Animales , Autoantígenos , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos NOD , Linfocitos T/patología
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