IIhotas pancreáticas humanas viáveis para o transplante através do aumento da massa de células e do imunoisolamento com microcápsulas biocompatíveis / Obtention of human pancreatic islets for transplantation through an increase in cell mass and an immunoisolation with biocompatible microcapsules

O transplante de ilhotas pancreáticas humanas representa uma estratégia promissora para a cura do diabetes mellitus tipo 1 (DM1) mas a aplicação a todos os pacientes diabéticos ainda é impraticável devido à limitada disponibilidade de ilhotas ou células β e à necessidade de utilização de drogas imunossupressoras pelo paciente transplantado. O tratamento com imunossupressores após o transplante de ilhotas pode ser abolido quando se realiza o microencapsulamento das ilhotas pancreáticas. Neste trabalho investigou-se um novo biomaterial, Biodritina (alginato/sulfato de condroitina) adequado ao microencapsulamento que gelifica na presença de íons de cálcio ou bário. A biocompatibilidade das microcápsulas tem sido avaliada segundo o grau de pureza do alginato utilizado na sua confecção. Amostras de alginato comercial purificado foram analisadas, comprovando-se a presença de impurezas (polifenóis, endotoxinas, proteínas) em níveis elevados, que impedem sua aplicação clínica. Optou-se, portanto pela utilização do alginato comercial ultrapurificado nos experimentos descritos neste trabalho. Das formulações de biomateriais avaliadas, as microcápsulas de bário-Biodritina apresentaram o melhor desempenho em testes de estabilidade físico-química...

[Effect of oral vanadyl sulphate on ultrastructure of pancreatic islets beta cells in streptozotocin-induced diabetic rats]

Considering insulin like effects of vanadium salts, these compounds have been evaluated as a therapeutic agent for treatment of diabetes mellitus in the experimental models of the disease in animals. This study was performed to study the ultrastructrure of islet beta cells in streptozotocin [STZ]- induced diabetes in rats after treatment with vanadyl sulfate [VS]. diabetes was induced in male Wistar rats by intravenous injection of 40 mg/kg STZ. Equal volume of normal saline was injected via lateral tail vein in sham animals. Seven days after injection animals in both groups were divided into treated and control groups. VS was added to the drinking water of the diabetic treated [DT] and Sham treated [NT] animals with a concentration of 0.5 mg/ml for one week and 1mg/ml up to three months. Untreated diabetic [DC] and sham rats [NC] received tap water during this period. Two months later all animals were killed. Langerhans islets were isolated from exocrine parts by use of collagen digestion, and were fixed in glutaraldehyde. Ultrastructure of islet beta cells were studied by means of transmission electron microscope. VS treatment led to amelioration of the symptoms of diabetes including hyperglycemia and polydepsia in DT rats. DC rats remained diabetic during the period of study. No significant changes were observed in plasma glucose and fluid intake of NT animals. Ultrastructural studies of islet beta cells of DT rats showed normal cells with normal chromatin distribution, well-developed rough endoplasmic reticulum, increased cytoplasmic granules and no clear sign of cell injury. Lymphocytic infiltration was not detected in langerhans islets of DT group. Nuclear pyknosis, cytoplasmic vacuolization, lymphocytic infiltration and signs of cell death such as cell necrosis were found in the islets of beta cells of DC rats. Cytoplasm of islets beta cells of NT rats were more granular in comparison to NC rats. Considering the results of this study we concluded that amelioration of diabetes signs in VS treated STZ induced diabetic rats are accompanied by preservation of islets beta cells ultrastructure

Modificaciones dinámicas en los islotes de Langerhans de dos Líneas de ratas espontaneamente diabeticas / Dynamic modifications in islets of Langerhans in two lines of spontaneously diabetic rats

La rata eSMT derivó del cruzamiento de eSS y b, líneas de la cepa IIM. eSS es un modelo de diabetes tipo 2 sin sobrepeso; b desarrolla obesidad moderada e intolerancia tardía a la glucosa. Fueron comparados características metabólicas y hallazgos histopatológicos del páncreas endocrino entre eSS y eSMT. Cotejados con eSS, los animales eSMT jóvenes son más corpulentos y desarrollan hiperglucemia de ayuno e intolerancia a la glucosa más precoces e intensas. En los machos eSMT de 6 y 9 meses existen islotes de formas alteradas y con fibrosis, detectándose esporádicas imágenes de apoptosis. En los de un año se tornan más pequeños y escasos, remedando la histoarquitectura de los machos eSS en el segundo año de vida; posteriormente los islotes van disgregándose, a la vez que muestran ocasionales mitosis y se observa nesidioblastosis. Se sugiere que estas modificaciones dinámicas constituyen una respuesta a la hiperglucemia. Las hembras eSS conservan por más tiempo la estructura insular y tienen menores alteraciones de la glucemia. El dimorfismo sexual del síndrome diabético de eSMT es atenuado respecto de eSS. La construcción de una tipología de individuos mediante el análisis multivariado separó tres clusters, evidenciando diferencias genéticas, etáreas y de sexo.

The eSMT rat is derived from the crossing of eSS and b, both lines belonging to the IIM strain, while eSS is a model of type 2 diabetes without overweight and b develops moderate obesity and late glucose intolerance. Metabolic characteristics and histopathological findings in endocrine pancreas of eSS and eSMT were compared. Young eSMT animals are more robust than eSS and develop more intense fasting hyperglycemia and glucose intolerance at an earlier age. eSMT males of 6 and 9 months show islets with altered shapes and fibrosis, as well as sporadic images of apoptosis. At 12 months of age, islets are reduced in number and size, resembling the histoarchitecture of eSS males during their second year of life; eventually islets undergo disruption and, at the same time, occasional mitoses and nesidioblastosis are seen. These dynamic modifications may be expressing a response to hyperglycemia. eSS females preserve their insular structure for a longer time and have less glycemic alterations. Sexual dimorphism of the diabetic syndrome of eSMT is attenuated when compared with eSS. The construction of a typology of individuals through multivariate analysis separated three clusters, evidencing genetic, age and sex differences.

Avaliação morfológica e dos mecanismos de mobilização de Ca2+ pela glicose e acetilcolina em células pancreáticas humanas / Morphologic evaluation and Ca2+ mobilization by glicose and acetylcholine in human pancreatic cells

OBJETIVOS: Avaliar a morfologia das organelas e do citoesqueleto em células pancreáticas humanas cultivadas e a mobilização de Ca2+ em resposta à glicose e ACh por medidas fluorimétricas. MATERIAL E MÉTODOS: As células foram semeadas em lamínulas, fixadas e marcadas com uma combinação de fluoróforos: o núcleo foi corado com DAPI e as mitocôndrias, com Mytotracker Red. Foram utilizados faloidina e anticorpos secundários conjugados com Alexa Fluor verde e vermelho fluorescentes (488 e 594) para identificar proteína actina F e receptor muscarínico tipo M3, respectivamente. Para estudar a mobilização de Ca2+, as células foram incubadas com fura-2/AM. RESULTADOS: As células pancreáticas humanas apresentaram morfologia preservada com grande quantidade de mitocôndrias. Na região de maior densidade celular, evidenciou-se as pseudo-ilhotas e os receptores muscarínicos M3. Por meio da elevação da [Ca2+]c, devido à ação da glicose e ACh, mostrou-se preservação da capacidade responsiva a esses estímulos e foi dependente de concentração desses agonistas. A glicose promoveu uma resposta sustentada e a ACh induziu uma resposta bifásica. CONCLUSÃO: As células pancreáticas humanas cultivadas conservaram sua morfologia. A mobilização de Ca2+ em resposta à glicose e a ACh confirma a sua funcionalidade. Os receptores muscarínicos M3 estão presentes nessas células.

AIMS: The proposal of this study was to analyze morphology of the organelles and cytoskeleton in human pancreatic cells cultured and the mobilization of the cytosolic calcium ([Ca2+]c) in response to glucose and ACh by fluorimetry method. MATERIAL AND METHODS: The cells were plated on glass coverslips, fixed and stained with a combination of fluorophores: the nuclei were stained with DAPI and mitochondria with Mytotracker Red. It was used phalloidin and the secondary antibodies Alexa Fluor conjugated green and red-fluorescent (488 and 594) to identify the protein cell actin F and type M3 muscarinic receptor respectively. The cells also were loaded with fura-2/AM to study Ca2+ mobilization. RESULTS: The human pancreatic cells show characteristics morphologically preserved with great amount of mitochondria. In region major cell density was evidenced pseudo-islets and type M3 muscarinic receptors. Through increase of [Ca2+]c due to action of glucose and ACh were shown that the cellsÆ capacity to respond to these stimuli were conserved. The elevation of the [Ca2+]c depended on concentration by glucose-induced promoting sustained phase and ACh-induced a biphasic response. CONCLUSION: The morphologic characteristics of human pancreatic cells cultured were preserved. The Ca2+ mobilization in response to glucose and ACh confirmed its functionality. The expression of the M3 muscarinic receptors in human pancreatic cell cultured was demonstrated.

Indução in vitro da trans-diferenciação de células ovais hepáticas em estruturas semelhantes às ilhotas pancreáticas / In vitro induction of hepatic oval cell of hepatic transdifferentiation into structures similar to pancreatic islets

Embora as células-tronco possuam grande capacidade de diferenciação em diversos tipos celulares no organismo, tanto "in vivo" como "in vitro", a produção de células endócrinas pancreáticas e de células produtoras de insulina, a partir destes tipos celulares em animais adultos, não está completamente demonstrada. A proliferação das células ovais hepáticas (COH) pode ser induzidas pela administração / Although stem cells have the ability to differentiale "in vivo" and "in vitro" into several types tof cells in the body, the production of endocrine pancreatic cells and insulin producing cells from adult stem cells is not completely elucidated. Hepatic oval cell (Hoc) proliferation can be induced by 2-AAF administration. Studies have showed that HOC can transdifferentiate 'in vitro' into endocrine pancreatic cells, when cultured for long periods and with high glucose concentrations. The aim of this study was to induce rat HOC transfifferentiation in shorter...

Development of fetal rat pancreatic islet A cells. A quantitative and immunocytochemical study

To perform a detailed quantitative immunocytochemical study of the development of fetal rat pancreatic islet A cells. Pancreases were obtained from 19 and 21-day- old fetal rats. Ten rats were used per each group. Non-fasting blood glucose levels were measured to confirm that the animals were normoglycemic. The pregnant rats were anesthetized by ether inhalation, and the fetuses were removed from their uteruses. They were fixed in buffered neutral formalin, dehydrated and embedded in paraplast and serially sectioned [5 um]. We examined 32-48 islets [8-12 per section] for each fetus. Sections were stained by avidin biotin complex technique. A quantitative study was performed on the pancreatic islet A cells. Carl Zeiss software from Zeiss was used in this study. This study was carried out at the Department of Anatomy, King Abdul-Aziz University, Jeddah, Kingdom of Saudi Arabia during the period January to December 2005. The volume density and the number of A cells showed a significant increase during the last days of gestation. All other parameters showed a significant increase during the last days of gestation. The A cell nuclear diameter and volume did not increase significantly during the last days of pregnancy. The A cells were well stained and occupied the peripheral part of the islets. The present study represented a detailed quantitative immunohistochemical study and demonstrated that the size of the endocrine tissue and the islet A cells increased significantly during the last days of gestation

Estructura y ultraestructura del pancreas endocrino de ratones transgenicos diabeticos / Structure and ultrastructure of the endocrine pancreas in diabetic transgenic mice

El objetivo del trabajo fue corroborar los cambios estructurales y determinar las alteraciones ultraesctructurales ocurridas en el páncreas endocrino de ratones con un síndrome inducido de diabetes mellitus insulinodependiente (DMID). Para ello se utilizaron ratones transgénicos (OVE 27) que sobreexpresan el gen de calmodulina en las células beta del páncreas endocrino; en estos animales, el aumento de calmodulina disminuye los niveles de calcio citosólico de las células beta, produciendo alteraciones morfológicas y funcionales que desencadenan una DMID de curso severo. Para nuestro estudio se obtuvieron y procesaron porciones del páncreas (cola) de 4 ratones transgénicos machos diabéticos de 5 semanas de edad (glucemia: 376 + 2 mg/del) y de 4 controles no transgénicos de la misma cepa, sexo y edad (glucemia: 113 + 13 mg/dl). El estudio inmunohistoqu­mico con microscop­a de luz confirmó que los ratones transgénicos presentan una disminución del número y tama o de los islotes con distorsión de su arquitectura, sin respuesta inflamatoria asociada. Los estudios ultraestructurales demostraron diversos grados de da o en las células Beta, tales como la presencia de interdigitaciones de membrana y alteraciones de sus organelas y de sus gránulos de secreción. Estos hallazgos coinciden con el deterioro cuantitativo y funcional de las células beta y la conservación de las problaciones celulares no-beta de los islotes. Estos cambios ultraestructurales en las células beta del páncreas del modelo experimental estudiado, sumados a las alteraciones inmunohistoquímicas previamente descriptas, contribuyen a explicar la manifestación de la diabetes.

Respuesta Metabólica al trauma en el Páncreas Endocrino

Objetivo: Revisar el tema de la respuesta endocrinometabólica del páncreas ante un trauma. Fuente de datos: Trauma experimental de páncreas, trasplante de páncreas y de islotes y manejo de pacientes con adenomas insulares de células beta de páncreas. Síntesis de lo hallado: las células beta pancreáticas son heterogéneas, aún en su capacidad de regeneración. Aceptando los conceptos de regeneración y glucotoxicidad pancreática, se requiere una pérdida del 90 por ciento de la masa de células beta para inducir un estado diabético. Pérdidas menores causan alteración en la tolerancia a los carbohidratos, sólo si hay exposición a cargas prolongadas de glucosa. El número de células beta por páncreas probablemente varie con la especie que se estudie y con su estado de desarrollo, en humanos adultos se ha calculado en 600.000 islotes. La capacidad de respuesta secretoria de las células beta o el efecto de ella en el metabolismo de los carbohidratos es mejor evaluado por medio de la prueba con arginina en presencia de hiperglicemia. Sujetos sanos que han donado parcialmente su páncreas, tienen al año un deterioro en la secreción de insulina y en la tolerancia a la glucosa. Los trasplantes de páncreas, cursan con un hiperinsulinismo periférico y con una adecuada tolerancia a los carbphidratos. Se ha demostrado que la función de las células beta se mantienen cuando se trasplantan más de 200.000 islotes. Conclusiones: La información sobre el tema es escasa, es necesario establecer un protocolo de manejo temprano y seguimiento de los pacientes con trauma pancreático

Islet cells-tight junctions: changes in its number induced by glucose

El número de uniones estrechas intercelulares fue cuantificado en islotes aislados de rata, incubados durante diferentes lapsos en un medio conteniendo distintas concentraciones de glucosa. El número de uniones estrechas aumentó en función de la duración de la incubación y de la concentración de glucosa en el medio. Estos resultados sugerirían la participación de las uniones estrechas en la regulación de la secreción de insulina en respuesta a la glucosa

Quantitative ultrastructural changes induced by glucose in pancreatic B cells

En el presente trabajo se estudian los cambios cuantitativos inducidos por la glucosa en la ultraestructura de las células B provenientes de páncreas de rata perfundidos con 3.3 y 16.6 mmol/1 de glucosa. En presencia de 16.6 mmol/l de glucosa se observó un incremento significativo en el volumen de RER, microtúbulos, mitocondrias, lisosomas y gránulos B, hallándose cambios significativos en el volumen total de la célula B o en tamaño del núcleo, citoplasma y complejo de Golgi. En los páncreas perfundidos con 16.6 mmol/l de glucosa, el número de gránulos secretorios está marcadamente reducido, y su diámetro está aumentado significativamente. Las células B de estos páncreas también presentan un incremento en el número de granos pálidos y de las imágenes de exocitosis. Estos datos sugieren que el incremento en la concentración de la glucosa extracelular produce no solamente la típica curva bifásica de secreción de insulina, sino también cambios cuantitativos significativos en el volumen de las distintas organelas de la célula B. Estas modificaciones ultraestructurales se correlacionan con el efecto de la glucosa sobre el proceso de síntesis y secreción de insulina

Hipoglicemia asociada con hiperplasia difusa de las células de los Islotes y bajo nivel de insulina

Un hombre de 87 años presentó hipoglicemia severa espontánea y su evaluación inicial no fue diagnosticá para exceso de insulina. Una extensa investigación no reveló tumor extrapancreatico. Después de ayuno prolongado, se encontró una elevación inapropiada de la insulina sérica inmunorreactiva (IIR). En la autopsia se confirmó la hiperplasia dufusa de células de los Islotes característica sugestiva de nesidioblastosis