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1.
Immunity ; 57(2): 256-270.e10, 2024 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-38354703

RESUMEN

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.


Asunto(s)
Proteínas Tirosina Fosfatasas , Transducción de Señal , Proteínas Tirosina Fosfatasas/metabolismo , Antígenos CD28 , Receptores Inmunológicos
2.
Proc Natl Acad Sci U S A ; 116(40): 19802-19804, 2019 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-31527237

RESUMEN

The gut microbiome is an established regulator of aspects of host metabolism, such as glucose handling. Despite the known impacts of the gut microbiota on host glucose homeostasis, the underlying mechanisms are unknown. The gut microbiome is also a potent mediator of gut-derived serotonin synthesis, and this peripheral source of serotonin is itself a regulator of glucose homeostasis. Here, we determined whether the gut microbiome influences glucose homeostasis through effects on gut-derived serotonin. Using both pharmacological inhibition and genetic deletion of gut-derived serotonin synthesis, we find that the improvements in host glucose handling caused by antibiotic-induced changes in microbiota composition are dependent on the synthesis of peripheral serotonin.


Asunto(s)
Microbioma Gastrointestinal , Glucosa/metabolismo , Homeostasis , Serotonina/fisiología , Animales , Antibacterianos/farmacología , Área Bajo la Curva , Glucemia/metabolismo , Eliminación de Gen , Prueba de Tolerancia a la Glucosa , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria
3.
EMBO Rep ; 19(12)2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30389725

RESUMEN

Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo , Proteínas Musculares/metabolismo , Termogénesis , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo Beige/efectos de los fármacos , Tejido Adiposo Beige/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Adrenérgicos/farmacología , Animales , Calcineurina/metabolismo , Proteínas de Unión al Calcio , Diferenciación Celular/efectos de los fármacos , Frío , Femenino , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/metabolismo , Masculino , Síndrome Metabólico/metabolismo , Metabolismo/efectos de los fármacos , Ratones , Ratones Noqueados , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Estriado/metabolismo , Obesidad/metabolismo , Obesidad/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Regiones Promotoras Genéticas/genética , Proteolípidos/genética , Proteolípidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Termogénesis/efectos de los fármacos , Proteína Desacopladora 1/metabolismo
4.
PLoS Genet ; 12(5): e1006033, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27195491

RESUMEN

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic ß-cell dysfunction. Reduced mitochondrial function is thought to be central to ß-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in ß-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D ß-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D ß-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their ß-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of ß-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D ß-cells where we had little knowledge of which changes cause ß-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to ß-cell mitochondrial dysfunction in T2D.


Asunto(s)
Diabetes Mellitus Tipo 2/genética , Síndrome de Down/genética , Insulina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Musculares/genética , Adenosina Trifosfato/metabolismo , Aneuploidia , Animales , Proteínas de Unión al Calcio , Cromosomas Humanos Par 21/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Síndrome de Down/metabolismo , Síndrome de Down/patología , Regulación de la Expresión Génica , Glucosa/metabolismo , Humanos , Hiperglucemia/genética , Hiperglucemia/metabolismo , Hiperglucemia/patología , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Mitocondrias/genética , Mitocondrias/patología , Proteínas Musculares/metabolismo , Biosíntesis de Proteínas/genética
5.
Curr Diab Rep ; 18(11): 117, 2018 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-30267158

RESUMEN

PURPOSES OF REVIEW: Scattered throughout the pancreas, the endocrine islets rely on neurovascular support for signal relay to regulate hormone secretion and for maintaining tissue homeostasis. The islet accessory cells (or components) of neurovascular tissues include the endothelial cells, pericytes, smooth muscle cells, neurons (nerve fibers), and glia. Research results derived from experimental diabetes and islet transplantation indicate that the accessory cells are reactive in islet injury and can affect islet function and homeostasis in situ or in an ectopic environment. RECENT FINDINGS: Recent advances in cell labeling and tissue imaging have enabled investigation of islet accessory cells to gain insights into their network structures, functions, and remodeling in disease. It has become clear that in diabetes, the islet neurovascular tissues are not just bystanders damaged in neuropathy and vascular complications; rather, they participate in islet remodeling in response to changes in the microenvironment. Because of the fundamental differences between humans and animal models in neuroinsular cytoarchitecture and cell proliferation, examination of islet accessory cells in clinical specimens and donor pancreases warrants further attention.


Asunto(s)
Homeostasis , Islotes Pancreáticos/citología , Animales , Células Endoteliales/citología , Humanos , Trasplante de Islotes Pancreáticos , Neuroglía/citología , Pericitos/citología
6.
J Cell Physiol ; 231(7): 1593-600, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26574734

RESUMEN

Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing ∼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles.


Asunto(s)
Células Enterocromafines/metabolismo , Vesículas Secretoras/metabolismo , Serotonina/biosíntesis , Corteza Suprarrenal/citología , Corteza Suprarrenal/metabolismo , Animales , Exocitosis/genética , Cobayas , Fusión de Membrana/genética , Ratones , Serotonina/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genética , Proteínas de Transporte Vesicular de Monoaminas/metabolismo
7.
Cancers (Basel) ; 15(8)2023 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-37190281

RESUMEN

An overabundance of desmoplasia in the tumour microenvironment (TME) is one of the defining features that influences pancreatic ductal adenocarcinoma (PDAC) development, progression, metastasis, and treatment resistance. Desmoplasia is characterised by the recruitment and activation of fibroblasts, heightened extracellular matrix deposition (ECM) and reduced blood supply, as well as increased inflammation through an influx of inflammatory cells and cytokines, creating an intrinsically immunosuppressive TME with low immunogenic potential. Herein, we review the development of PDAC, the drivers that initiate and/or sustain the progression of the disease and the complex and interwoven nature of the cellular and acellular components that come together to make PDAC one of the most aggressive and difficult to treat cancers. We review the challenges in delivering drugs into the fortress of PDAC tumours in concentrations that are therapeutic due to the presence of a highly fibrotic and immunosuppressive TME. Taken together, we present further support for continued/renewed efforts focusing on aspects of the extremely dense and complex TME of PDAC to improve the efficacy of therapy for better patient outcomes.

8.
Cell Death Dis ; 13(10): 911, 2022 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-36309486

RESUMEN

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic ß-cells. Although ß-cell targeted autoimmune processes and ß-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports ß-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing ß-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced ß-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine ß-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of ß-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes.


Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Animales , Humanos , Ratones , Supervivencia Celular , Desmogleínas/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Estreptozocina
9.
Gen Comp Endocrinol ; 174(2): 71-9, 2011 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-21821034

RESUMEN

The New world primates (NWP) Callithrix jacchus separated from man approximately 50 million years ago and is a potential alternative small non-human primate model for diabetes research. Ultrastructure, and gene expression of pancreatic islets and the recently described diabetes auto antigenic zinc transporters families in human, NWP and pig pancreas were studied. Morphologically NWP islets were larger than pig islets and similar in size to human islets. NWP islets alpha cells had high dense core surrounded by a limiting membrane, beta cells by the mixed morphology of the granule core, and delta cells by moderate opaque core. Antibody staining for insulin, glucagon, somatostatin and Glucagon-like peptide-1 (GLP-1) showed that the distribution pattern of the different cell types within islets was comparable to pig and human islets. In all three species protein expression of zinc transporter ZnT8 was detected in most of the insulin producing beta cells whereas Zip14 expression was widely expressed in alpha and beta cells. In both human and NWP little or no expression of Glut2 was observed compared to Glut1 and glucokinase at the protein level, however the messenger RNA level of Glut2 was greater than Glut1 and glucokinase. In contrast all three glucose transporters were expressed in pig islets at the protein level. The expression of Zip14 in islets is reported for the first time. In conclusion NWP pancreatic islets express comparable islet cell types and distribution to humans and pigs. Importantly, marmosets have a similar glucose transporter profile to humans, making this non-endangered primate species a useful animal model for pancreatic biology.


Asunto(s)
Callithrix/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Proteínas Portadoras/genética , Técnica del Anticuerpo Fluorescente , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Humanos , Islotes Pancreáticos/ultraestructura , Microscopía Electrónica , Reacción en Cadena en Tiempo Real de la Polimerasa
10.
Neurogastroenterol Motil ; 32(8): e13869, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32378785

RESUMEN

BACKGROUND: Enterochromaffin (EC) cells are specialized enteroendocrine cells lining the gastrointestinal (GI) tract and the source of almost all serotonin (5-hydroxytryptamine; 5-HT) in the body. Gut-derived 5-HT has a plethora of physiological roles, including regulation of gastrointestinal motility, and has been implicated as a driver of obesity and metabolic disease. This is due to 5-HT influencing key metabolic processes, such as hepatic gluconeogenesis, adipose tissue lipolysis and hindering thermogenic capacity. Increased circulating 5-HT occurs in humans with obesity and type 2 diabetes. However, despite the known metabolic roles of gut-derived 5-HT, the mechanisms underlying the cellular-level change in EC cells under obesogenic conditions remains unknown. METHODS: We use a mouse model of diet-induced obesity (DIO) to identify the regional changes that occur in primary EC cells from the duodenum and colon. Transcriptional changes in the nutrient sensing profile of primary EC cells were assessed, and responses to nutrient stimuli in culture were determined by 5-HT ELISA. KEY RESULTS: We find that obesogenic conditions affect EC cells in a region-dependent manner. Duodenal EC cells from DIO mice have impaired sugar sensing even in the presence of increased 5-HT content per cell, while colonic EC cell numbers are significantly increased, but have unaltered nutrient sensing capacity. CONCLUSIONS & INFERENCES: Our findings from this study add novel insights into the mechanisms by which functional changes to EC cells occur at a cellular level, which may contribute to the altered circulating 5-HT seen with obesity and metabolic disease, and associated gastrointestinal disorders.


Asunto(s)
Dieta , Células Enterocromafines/metabolismo , Intestino Grueso/metabolismo , Intestino Delgado/metabolismo , Obesidad/metabolismo , Serotonina/metabolismo , Animales , Glucemia/metabolismo , Masculino , Ratones
11.
Physiol Rep ; 5(6)2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28320893

RESUMEN

Enterochromaffin (EC) cells located in the gastrointestinal (GI) tract provide the vast majority of serotonin (5-HT) in the body and constitute half of all enteroendocrine cells. EC cells respond to an array of stimuli, including various ingested nutrients. Ensuing 5-HT release from these cells plays a diverse role in regulating gut motility as well as other important responses to nutrient ingestion such as glucose absorption and fluid balance. Recent data also highlight the role of peripheral 5-HT in various pathways related to metabolic control. Details related to the manner by which EC cells respond to ingested nutrients are scarce and as that the nutrient environment changes along the length of the gut, it is unknown whether the response of EC cells to nutrients is dependent on their GI location. The aim of the present study was to identify whether regional differences in nutrient sensing capability exist in mouse EC cells. We isolated mouse EC cells from duodenum and colon to demonstrate differential responses to sugars depending on location. Measurements of intracellular calcium concentration and 5-HT secretion demonstrated that colonic EC cells are more sensitive to glucose, while duodenal EC cells are more sensitive to fructose and sucrose. Short-chain fatty acids (SCFAs), which are predominantly synthesized by intestinal bacteria, have been previously associated with an increase in circulating 5-HT; however, we find that SCFAs do not acutely stimulate EC cell 5-HT release. Thus, we highlight that EC cell physiology is dictated by regional location within the GI tract, and identify differences in the regional responsiveness of EC cells to dietary sugars.


Asunto(s)
Colon/metabolismo , Duodeno/metabolismo , Células Enterocromafines/metabolismo , Serotonina/metabolismo , Animales , Calcio/metabolismo , Colon/efectos de los fármacos , Duodeno/efectos de los fármacos , Células Enterocromafines/efectos de los fármacos , Ácidos Grasos Volátiles/farmacología , Fructosa/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Sacarosa/farmacología
12.
Endocrinology ; 158(5): 1049-1063, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-28323941

RESUMEN

Serotonin (5-hydroxytryptamine or 5-HT) is a multifunctional bioamine with important signaling roles in a range of physiological pathways. Almost all of the 5-HT in our bodies is synthesized in specialized enteroendocrine cells within the gastrointestinal (GI) mucosa called enterochromaffin (EC) cells. These cells provide all of our circulating 5-HT. We have long appreciated the important contributions of 5-HT within the gut, including its role in modulating GI motility. However, evidence of the physiological and clinical significance of gut-derived 5-HT outside of the gut has recently emerged, implicating 5-HT in regulation of glucose homeostasis, lipid metabolism, bone density, and diseases associated with metabolic syndrome, such as obesity and type 2 diabetes. Although a new picture has developed in the last decade regarding the various metabolic roles of peripheral serotonin, so too has our understanding of the physiology of EC cells. Given that they are scattered throughout the lining of the GI tract within the epithelial cell layer, these cells are typically difficult to study. Advances in isolation procedures now allow the study of pure EC-cell cultures and single cells, enabling studies of EC-cell physiology to occur. EC cells are sensory cells that are capable of integrating cues from ingested nutrients, the enteric nervous system, and the gut microbiome. Thus, levels of peripheral 5-HT can be modulated by a multitude of factors, resulting in both local and systemic effects for the regulation of a raft of physiological pathways related to metabolism and obesity.


Asunto(s)
Metabolismo Energético , Serotonina/fisiología , Animales , Metabolismo Energético/efectos de los fármacos , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/metabolismo , Células Enteroendocrinas/metabolismo , Motilidad Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/metabolismo , Humanos , Serotonina/metabolismo , Serotonina/farmacología , Transducción de Señal/efectos de los fármacos
13.
Diabetes ; 66(5): 1301-1311, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28174291

RESUMEN

Pancreatic islet transplantation is a promising clinical treatment for type 1 diabetes, but success is limited by extensive ß-cell death in the immediate posttransplant period and impaired islet function in the longer term. Following transplantation, appropriate vascular remodeling is crucial to ensure the survival and function of engrafted islets. The sphingosine kinase (SK) pathway is an important regulator of vascular beds, but its role in the survival and function of transplanted islets is unknown. We observed that donor islets from mice deficient in SK1 (Sphk1 knockout) contain a reduced number of resident intraislet vascular endothelial cells. Furthermore, we demonstrate that the main product of SK1, sphingosine-1-phosphate, controls the migration of intraislet endothelial cells in vitro. We reveal in vivo that Sphk1 knockout islets have an impaired ability to cure diabetes compared with wild-type controls. Thus, SK1-deficient islets not only contain fewer resident vascular cells that participate in revascularization, but likely also a reduced ability to recruit new vessels into the transplanted islet. Together, our data suggest that SK1 is important for islet revascularization following transplantation and represents a novel clinical target for improving transplant outcomes.


Asunto(s)
Movimiento Celular/genética , Diabetes Mellitus Experimental/cirugía , Diabetes Mellitus Tipo 1/cirugía , Células Endoteliales/citología , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/irrigación sanguínea , Lisofosfolípidos/metabolismo , Neovascularización Fisiológica/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Esfingosina/análogos & derivados , Animales , Citometría de Flujo , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Esfingosina/metabolismo , Trasplantes/irrigación sanguínea
14.
Diabetes ; 65(5): 1328-1340, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26961116

RESUMEN

Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by 'conventional' therapies, memory T-cell mediated attack is a substantial challenge in islet transplantation and this will extend to application of personalized approaches using stem-cell derived replacement ß cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement ß cells. Here we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions inactivates established memory CD8+ T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8+ memory T cell-mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell-mediated gene therapy effectively terminates antigen-specific memory T-cell responses and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and importantly, the clinical application of personalized ß-cell replacement therapies using patient-derived stem cells.

15.
Invest Ophthalmol Vis Sci ; 46(5): 1675-81, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15851568

RESUMEN

PURPOSE: Allograft rejection is the leading cause of corneal graft failure. CD4(+) T cells control the allograft response and represent targets for antirejection therapy. The purpose of this study was to transfer cDNA encoding a monomeric anti-CD4 antibody fragment to donor corneal endothelium, to attempt to modulate orthotopic corneal allograft rejection in the rat. METHODS: A replication-deficient adenoviral vector (AdV) encoding anti-CD4 single-chain, variable-domain antibody fragment (scFv) and enhanced green fluorescent protein (eGFP) was constructed (AdCD4GFP). AdV encoding eGFP alone (AdGFP) was used as a control. Transgenic product was detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, flow cytometry, and fluorescence microscopy. The alloinhibitory capacity of anti-rat CD4 scFv was measured in the one-way mixed lymphocyte reaction (MLR). The survival of Wistar-Furth corneas transduced with AdV either immediately or 3 days before orthotopic transplantation in Fischer 344 recipients was examined. RESULTS: ScFv and eGFP mRNAs were detected in rat corneas transduced in vitro, and active scFv secreted in corneal supernatants peaked at days 4 to 5 after transduction at 23 +/- 4 ng of protein per cornea per day. Antibody and scFv against rat CD4 blocked alloproliferation in MLR. However, transduction of corneas with AdCD4GFP ex vivo, immediately before transplantation, or in vivo, 3 days before transplantation, did not significantly prolong corneal allograft survival (P > 0.05). CONCLUSIONS: Anti-CD4 scFvs were capable of blocking allostimulation, but their local expression within the eye did not prolong corneal allograft survival, suggesting that sensitization may still occur.


Asunto(s)
Trasplante de Córnea , Técnicas de Transferencia de Gen , Rechazo de Injerto/prevención & control , Adenoviridae/genética , Animales , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Córnea/inmunología , Citometría de Flujo , Vectores Genéticos , Rechazo de Injerto/metabolismo , Supervivencia de Injerto , Proteínas Fluorescentes Verdes , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Microscopía Fluorescente , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas WF , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única , Trasplante Homólogo
16.
Ocul Surf ; 3(4 Suppl): S165-8, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17216111

RESUMEN

More effective therapies are required to improve clinical corneal allograft outcomes. The strategies that have led to improvements in vascularized organ graft survival are not always applicable to the cornea. New treatments that modulate the afferent arm of the immune response to a corneal allograft are more likely to prove effective than are treatments that target the effector arm of the immune response. We briefly discuss two such options: the use of engineered antibody fragments to block antigen presentation, and the use of gene therapy approaches to abrogate antigen presenting-cell function.

17.
Front Mol Neurosci ; 8: 29, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26283908

RESUMEN

Chronic pain is one of the most burdensome health issues facing the planet (as costly as diabetes and cancer combined), and in desperate need for new diagnostic targets leading to better therapies. The bioactive lipid sphingosine 1-phosphate (S1P) and its receptors have recently been shown to modulate nociceptive signaling at the level of peripheral nociceptors and central neurons. However, the exact role of S1P generating enzymes, in particular sphingosine kinase 2 (Sphk2), in nociception remains unknown. We found that both sphingosine kinases, Sphk1 and Sphk2, were expressed in spinal cord (SC) with higher levels of Sphk2 mRNA compared to Sphk1. All three Sphk2 mRNA-isoforms were present with the Sphk2.1 mRNA showing the highest relative expression. Mice deficient in Sphk2 (Sphk2(-/-)) showed in contrast to mice deficient in Sphk1 (Sphk1(-/-)) substantially lower spinal S1P levels compared to wild-type C57BL/6 mice. In the formalin model of acute peripheral inflammatory pain, Sphk2(-/-) mice showed facilitation of nociceptive transmission during the late response, whereas responses to early acute pain, and the number of c-Fos immunoreactive dorsal horn neurons were not different between Sphk2(-/-) and wild-type mice. Chronic peripheral inflammation (CPI) caused a bilateral increase in mechanical sensitivity in Sphk2(-/-) mice. Additionally, CPI increased the relative mRNA expression of P2X4 receptor, brain-derived neurotrophic factor and inducible nitric oxide synthase in the ipsilateral SC of wild-type but not Sphk2(-/-) mice. Similarly, Sphk2(-/-) mice showed in contrast to wild-type no CPI-dependent increase in areas of the dorsal horn immunoreactive for the microglia marker Iba-1 and the astrocyte marker Glial fibrillary acidic protein (GFAP). Our results suggest that the tightly regulated cell signaling enzyme Sphk2 may be a key component for facilitation of nociceptive circuits in the CNS leading to central sensitization and pain memory formation.

18.
Cell Transplant ; 24(1): 37-48, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-24069942

RESUMEN

The success of pancreatic islet transplantation is limited by delayed engraftment and suboptimal function in the longer term. Endothelial progenitor cells (EPCs) represent a potential cellular therapy that may improve the engraftment of transplanted pancreatic islets. In addition, EPCs may directly affect the function of pancreatic ß-cells. The objective of this study was to examine the ability of EPCs to enhance pancreatic islet transplantation in a murine syngeneic marginal mass transplant model and to examine the mechanisms through which this occurs. We found that cotransplanted EPCs improved the cure rate and initial glycemic control of transplanted islets. Gene expression data indicate that EPCs, or their soluble products, modulate the expression of the ß-cell surface molecule connexin 36 and affect glucose-stimulated insulin release in vitro. In conclusion, EPCs are a promising candidate for improving outcomes in islet transplantation, and their mechanisms of action warrant further study.


Asunto(s)
Conexinas/biosíntesis , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Trasplante de Islotes Pancreáticos , Trasplante de Células Madre , Células Madre/metabolismo , Animales , Células Endoteliales/patología , Células Endoteliales/trasplante , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/patología , Masculino , Ratones , Células Madre/patología , Edulcorantes/farmacología , Trasplante Isogénico , Proteína delta-6 de Union Comunicante
19.
Diabetes ; 63(1): 3-11, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24357688

RESUMEN

Within the pancreatic islet, the ß-cell represents the ultimate biosensor. Its central function is to accurately sense glucose levels in the blood and consequently release appropriate amounts of insulin. As the only cell type capable of insulin production, the ß-cell must balance this crucial workload with self-preservation and, when required, regeneration. Evidence suggests that the ß-cell has an important ally in intraislet endothelial cells (ECs). As well as providing a conduit for delivery of the primary input stimulus (glucose) and dissemination of its most important effector (insulin), intraislet blood vessels deliver oxygen to these dense clusters of metabolically active cells. Furthermore, it appears that ECs directly impact insulin gene expression and secretion and ß-cell survival. This review discusses the molecules and pathways involved in the crosstalk between ß-cells and intraislet ECs. The evidence supporting the intraislet EC as an important partner for ß-cell function is examined to highlight the relevance of this axis in the context of type 1 and type 2 diabetes. Recent work that has established the potential of ECs or their progenitors to enhance the re-establishment of glycemic control following pancreatic islet transplantation in animal models is discussed.


Asunto(s)
Comunicación Celular/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Glucemia/metabolismo , Células Endoteliales/citología , Insulina/metabolismo , Células Secretoras de Insulina/citología
20.
Oxid Med Cell Longev ; 2014: 520316, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25009690

RESUMEN

Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Down's syndrome (DS) and Alzheimer's disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1(ox)) and demonstrate an increased susceptibility of neurons from these mice to oxidative stress. Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12(RCAN1)). Similar to RCAN1(ox) neurons, PC12(RCAN1) cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Proteínas Musculares/metabolismo , Estrés Oxidativo , Animales , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN , Femenino , Peróxido de Hidrógeno/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismo
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