Abstract Both immunosuppressive and nifedipine medication have been associated with drug-induced gingival overgrowth. There are several hypothetical mechanisms for drug-induced gingival overgrowth, such as the influence of genetic predisposition, alterations in gingival tissue homeostasis, especially in the function of fibroblasts, and drug-induced action on growth factors. Clinical studies have also shown that, those with poor oral hygiene status drug-induced gingival overgrowth is more prevalent and severe than those with good oral hygiene status. The working hypothesis was that immunosuppressive medication and/or nifedipine medication affects inflammatory cell profile and mitotic activity of keratinocytes in human overgrown gingiva. We studied gingival samples, collected from nifedipine-medicated cardiac outpatients and immunosuppression-medicated organ-transplant recipients. Patients were placed into four groups: 1) the immunosuppression group, patients receiving cyclosporin-A (CsA), azathioprine (AZA) and prednisolone (Pred) 2) the immunosuppression plus nifedipine group, patients receiving CsA, AZA, Pred. and nifedipine 3) the nifedipine group patients receiving only nifedipine and 4) the non-medicated control group. All of the samples related to moderate to severe degrees of gingival overgrowth, covering half to two thirds of the clinical crown. The aim of the study was to investigate the occurrence of Langerhans cells, macrophages, mast cells and mitotic activity of keratinocytes in human drug-induced overgrown gingiva, and consequently to assess their possible role in the pathogenesis of drug-induced gingival overgrowth. We found that immunosuppressive medication increased the numbers of reparative macrophages (RM3/1) and decreased the numbers of tryptase- and chymase-positive mast cells (MCTC) cells. We have also shown that immunosuppressive and nifedipine medication decreased the numbers of Langerhans cells (CD1a) and increased the numbers of 27E10-macrophages parallelly. Additionally we found increase in the mitotic activity of gingival keratinocytes and even two-fold thickening of gingival epithelium in immunosuppressive and nifedipine medication-induced gingival overgrowth as compared with healthy gingiva. Immunosuppressive medication activated gingival epithelium (27E10 expression in gingival keratinocytes) more than nifedipine medication. In conclusion, our results suggest that gingival overgrowth among immunosuppressive- and nifedipine-medicated patients is related to alteration of tissue homeostasis. First, this suggestion is supported by changes found in the numbers of cells that directly affect connective tissue turnover, e.g. reparative macrophages (RM3/1) and mast cells. Changes in the numbers of these cells could alter the cytokine- and growth factor-profile, which affects fibroblast function. Secondly, we found changes in the numbers of cells involved in regulation of inflammation, e.g. Langerhans cells and monocytes as compared with healthy controls. Immunosuppressive medication could directly activate gingival keratinocytes. We suggest that our findings mainly reflect the effects of immunosuppressive medication, but the role of inflammation cannot be excluded. The changes observed above represent differences of the pathogenesis of drug-induced gingival overgrowth between immunosuppressive and nifedipine medication. It must be however remembered that drug-induced gingival overgrowth is a result of multicausal intrinsic and extrinsic factors. Age, gender, concomitant medication with multiple drugs, plaque accumulation, and genetic disposition are additional risk factors. The abnormal distribution of specific immune system cell subpopulations does not alone prove a functional relationship to gingival overgrowth.

Orientador: Everardo Magalhães Carneiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T17:22:21Z (GMT). No. of bitstreams: 1 Goncalves_LucianaMateus_M.pdf: 1375240 bytes, checksum: fe3cbb519b479a70d1a3fcd2f6fd3b82 (MD5) Previous issue date: 2015 / Resumo: incidência do diabetes mellitus tipo 2 vem crescendo em todo o mundo, e a estimativa para os próximos anos é que continue aumentando. Dentro desse panorama, destacamos estudos relacionados com substâncias classificadas como desreguladores endócrinos, caracterizados tanto por alterar a síntese, liberação e ação de hormônios bem como atuar sobre receptores hormonais. Dentre os desreguladores endócrinos, alguns apresentam potencial obesogênico e diabetogênico, como é o caso do Bisfenol A (BPA). Estudos demonstram que o BPA, substância utilizada na manufatura do plástico policarbonato e resina epóxi, possui ação sobre receptores de estrógeno. Devido sua ampla aplicação em itens utilizados cotidianamente, estamos constantemente expostos ao BPA desde o desenvolvimento fetal. O objetivo desse estudo foi investigar o efeito da exposição de ilhotas pancreáticas de camundongos neonatos ao BPA. Foram utilizadas ilhotas de neonatos (4 a 6 dias de vida) cultivadas por 48 horas com 0,1nM de BPA para avaliar: 1) secreção de insulina; 2) expressão gênica; 3) conteúdo proteico. Os resultados foram analisados pelo teste t-Student, com nível de significância p<0,05. A secreção de insulina induzida por glicose ou solução de KCl 40 mM foi prejudicada nas ilhotas tratadas com BPA. O conteúdo total de insulina, assim como a expressão dos genes da insulina não foram alterados. Houve redução tanto da expressão gênica como do conteúdo proteico de conexina 36 nas ilhotas tratadas. O BPA reduziu a expressão gênica da subunidade beta 2 do canal de cálcio do tipo L e o conteúdo proteico das subunidades kir6.2 e sur1 do canal de potássio ATP-dependente. O conteúdo de vamp2 e sintaxina 1A também foi menor nas ilhotas tratadas. Concluímos que a exposição das ilhotas de neonatos ao BPA na concentração e tempo utilizados promove alterações dos mecanismos moleculares envolvidos na secreção de insulina / Abstract: Type 2 diabetes is increasing worldwide. Recently, studies have shown the diabetogenic effects of some chemical components used in packages of food and beverages. An obsesogenic and diabetogenic substance is the bisphenol A (BPA), used in manufacture of polycarbonate plastic and epoxy resin. Our exposure to this substance may begin during fetal development and in the first hours of life still in hospital. The aim of this study was to investigate the effects of BPA in islets from newborn mice. Neonate pancreatic islets (4-6 days) were isolate by collagenase method and were cultured with BPA (0.1 nM) for 48 hours, and then used to evaluate: 1) insulin secretion; 2) gene expression; 3) protein content. The results were analyzed by Student t-test, p<0.05. We observed a reduced insulin secretion in response to both glucose and 40 mM KCl solution. However, total insulin content and Ins1/Ins2 gene expression was not altered. On the other hand, connexin 36 gene and protein expression were decreased. Another genes involved with b-cell maturation were unchanged. Furthermore, BPA decreased gene expression of beta 2 subunit of L-type calcium channel. Protein content of K-ATP channel (kir6.2 and sur1), vamp2 and syntaxin 1A were also reduced. We concluded that 0.1 nM BPA exposure for 48 h alters molecular mechanisms involved with insulin secretion from newborn islets in culture / Mestrado / Fisiologia / Mestra em Biologia Funcional e Molecular

Vitiligo is an autoimmune disease of the skin characterized by epidermal depigmentation that results from CD8+ T cell-mediated destruction of pigment producing melanocytes. Vitiligo affects up to 1% of the population and current treatments are moderately effective at facilitating repigmentation by suppressing cutaneous autoimmune inflammation to promote melanocyte regeneration. In order to cause disease, CD8+ T cells must overwhelm the mechanisms of peripheral tolerance in the skin and if we understand the suppressive mechanisms that are compromised during vitiligo, we can potentially use this information to improve existing treatments or engineer novel interventions. Therefore, my goal is to characterize the regulatory factors in the skin that suppress depigmentation during vitiligo. Our lab has developed a mouse model of vitiligo that accurately reflects human disease and I used this model to demonstrate that regulatory T cells suppress CD8+ T cell-mediated depigmentation and interact with CD8+ T cells in the skin during vitiligo. In this model of disease, I investigated the molecules involved in regulatory T cell function and observed that the chemokine receptors CCR5 and CCR6 play different roles in regulatory T cell suppression. While CCR6 facilitates regulatory T cell migration to the skin, CCR5 is dispensable for migration but required for optimal regulatory T cell function. Additionally, I used our mouse model to demonstrate that Langerhans cells suppress the incidence of disease during vitiligo. Taken together the results from these studies provide novel insights into the mechanisms of suppression during vitiligo.

No description available.

No description available.

Le diabète de type 2 (DT2) résulte d’une résistance à l’insuline par les tissus périphériques et par un défaut de sécrétion de l’insuline par les cellules β-pancréatiques. Au fil du temps, la compensation des îlots de cellules β pour la résistance à l’insuline échoue et entraine par conséquent une baisse progressive de la fonction des cellules β. Plusieurs facteurs peuvent contribuer à la compensation de la cellule β. Toutefois, la compréhension des mécanismes cellulaires et moléculaires sous-jacents à la compensation de la masse de la cellule β reste à ce jour inconnue. Le but de ce mémoire était d’identifier précisément quel mécanisme pouvait amener à la compensation de la cellule β en réponse à un excès de nutriments et plus précisément à l’augmentation de sa prolifération et de sa masse. Ainsi, avec l’augmentation de la résistance à l’insuline et des facteurs circulants chez les rats de six mois perfusés avec du glucose et de l’intralipide, l’hypothèse a été émise et confirmée lors de notre étude que le facteur de croissance HB-EGF active le récepteur de l’EGF et des voies de signalisations subséquentes telles que mTOR et FoxM1 impliquées dans la prolifération de la cellule β-pancréatique. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la compensation de la masse de la cellule β dans un état de résistance à l’insuline et peuvent servir de nouvelles approches thérapeutiques pour prévenir ou ralentir le développement du DT2. / Type 2 diabetes (T2D) results from insulin resistance in peripheral tissues and impaired insulin secretion from the pancreatic β-cell. Over the time, compensation of the β cell islets for insulin resistance fails, and therefore leads to a gradual decline in β-cell function. Several factors may contribute to β-cell compensation. However, the cellular and molecular mechanisms underlying β-cell compensation remain unknown. The purpose of this thesis was to identify what mechanism could lead to β cell compensation in response to nutrients excess and specifically the increase in proliferation and β-cell mass. Thus, with increasing insulin resistance and circulating factors in the 6 month rats infused with glucose + intralipid, the hypothesis was made and confirmed in our study that the growth factor HB-EGF would activate the EGF receptor, and subsequent signaling pathways such as mTOR and FoxM1, both involved in the proliferation of the pancreatic beta-cell. Collectively, these results allow us to understand better the molecular mechanisms involved in the β cell compensation in the insulin resistance state and may serve as a potential new therapeutic approach to prevent or delay T2D development.

L’amyloïdose, une maladie progressive et incurable, implique une vaste panoplie de pathologies et de pathogénèses, qui est expliquée par la grande variabilité biologique et structurale des protéines responsables de la formation des dépôts d’amyloïde. L’amyline (polypeptide amyloïde des îlots pancréatiques, IAPP) est une protéine très susceptible de subir des changements de conformation impliquant les feuillets bêta et conférant aussi des propriétés physicochimiques distinctes. Cette protéine prend alors une forme fibrillaire et se dépose dans les îlots de Langerhans chez les humains atteints de diabète de type 2 ou d’insulinome. Ces dépôts d’amyloïde pancréatique (AIAPP) ont été décrits chez certaines espèces animales telles que les félins domestiques, les grands félins, le raton laveur et les primates non humains. La formation de dépôts d’amyloïde contribue à la pathogénèse du diabète de type 2, mais les mécanismes qui induisent la conversion de l’amyline (IAPP) en amyloïde (AIAPP) ne sont pas complètement compris. Les hypothèses du projet sont que certaines variations présentes dans les séquences peptidiques de l’IAPP provenant de différentes espèces animales jouent un rôle critique pour la formation de fibrilles et que plusieurs composés chimiques aromatiques/phénoliques sont capables d’abroger la formation de dépôts d’amyloïde. Le projet de recherche consiste donc à caractériser la propension des différentes isoformes animales d’IAPP à former de l’amyloïde in vitro afin d’identifier les acides aminés jouant un rôle clé dans cette transformation structurale et ultimement d’inhiber la formation d’amyloïde pancréatique. Le projet se divise en deux volets principaux. Le premier consiste à identifier les différentes séquences peptidiques de l’IAPP retrouvées chez les espèces animales. L’objectif est d’identifier les acides aminés jouant un rôle clé dans la formation d’amyloïde. Le gène de l’IAPP a été séquencé chez plus d’une quarantaine d’espèces. Le potentiel d’agrégation des séquences obtenues a été simulé à l’aide d’outils bioinformatique. Une librairie de 23 peptides a été commandée afin de procéder à des analyses physicochimiques in vitro permettant d’évaluer le potentiel amyloïdogénique (test fluorimétrique à la thioflavine T, essai de liaison au rouge Congo, dichroïsme circulaire, microscopie électronique à transmission) et cytotoxique (sur une lignée cellulaire provenant d’insulinome : INS-1). Les analyses effectuées à partir de la librairie constituée de 23 peptides ont permis d’identifier trois séquences ne formant pas d’amyloïde et qui proviennent des espèces animales suivantes : le tamarin lion doré (Leontopithecus rosalia), le grand dauphin (Tursiops truncatus) et l’alpaga (Vicugna pacos). Un site potentiellement critique est le segment 8-20 présentant le motif NFLVH qui ne forme plus d’amyloïde lorsqu’il est remplacé par le motif DFLGR ou KFLIR. Les acides aminés 29P, 14K et 18R sont également impliqués dans l’inhibition de la transformation structurale en fibrille. La dernière partie du projet consiste à inhiber la formation de l’amyloïde en utilisant des composés chimiques commercialisés (hypoglycémiants, anti-inflammatoires non stéroïdiens) ou nouvellement synthétisés dans notre laboratoire (les aryles éthyles urées). Un criblage d’une soixantaine de composés chimiques a été conduit dans cette étude. Leur efficacité a été testée sur l’IAPP humaine, qui possède un fort potentiel amyloïdogénique. Les techniques utilisées sont les mêmes que celles exploitées précédemment. L’essai de liaison croisée photo-induite ("photo-induced cross-linking of unmodified proteins", PICUP) a été réalisé afin d’étudier les formes intermédiaires (monomères, oligomères). Un total de 11 composés chimiques a démontré un potentiel à inhiber l’agrégation des fibrilles. Pour la classe des hypoglycémiants, le glyburide, le répaglinide et la troglitazone ont montré l’activité thérapeutique la plus élevée pour retarder et réduire la formation de fibrilles. Les anti-inflammatoires antiamyloïdogènes actifs incluaient le diclofenac, le méloxicam, le phénylbutazone, le sulindac et le ténoxicam. Les aryles étyles urées les plus intéressantes étaient la EU-362 et la EU-418. Tous ces composés ont conféré une protection cellulaire contre l’activité cytotoxique des fibrilles. Les molécules actives possèdent des éléments structuraux communs tels des substituants donneurs d’électrons (alcool, amine, halogène) sur un noyau benzène. En conclusion, ce projet de recherche a permis de caractériser l’IAPP chez diverses espèces animales, dont plusieurs chez lesquelles elle n’avait pas encore été décrite, de déterminer les sites jouant un rôle clé dans sa transformation en amyloïde et, ultimement, de tester le potentiel thérapeutique de nouveaux agents antiamyloïdogènes dans le diabète de type 2. Nous espérons que ce projet ouvrira ainsi la porte à de nouvelles stratégies de traitement. / Amyloidosis is a progressive and, as of now, incurable disease caused by the deposition of insoluble proteins. Amyloid research over the past decades focused on the characterization of the substantive biological variability of amyloid deposits. Amyloidosis encompasses a diversity of pathological manifestations, explained by the diversity of underlying causal proteins. In the pancreas of susceptible species, islet amyloid polypeptide (IAPP) is a precursor for an amyloid protein (AIAPP), which has a characteristic fibrillar structure and resistance to physical agents. This folded protein deposits in the islets of Langerhans of patients with type 2 diabetes or islet cell tumors (insulinoma). Amyloid deposits have also been well characterized, anatomically, in feline and non-human primate species. Amyloid fibril formation contributes to the pathogenesis of diabetes mellitus but the precise pathophysiologic factors involved in the fibrillization of IAPP as well as resultant islet injury remain to be elucidated. Further understanding of the causative factors in the fibrillogenesis of IAPP will be requisite in the development of therapeutic strategies to disrupt the amyloidosis process. This project hypothesizes that the specific variations found in IAPP peptide sequences among different animal species are critical for IAPP fibrillization. Also, some aromatic/polyphenolic compounds can abrogate fibrillization. The main objective forms the basis for development of new therapeutic tactics to impede amyloid formation and associated cellular injury. Thus this project has two specific aims. The first specific aim was to identify critical variations in IAPP amino acid sequences from different animal species and to assess their amyloidogenic potential. To accomplish this, the IAPP gene was isolated and sequenced from paraffin-embedded tissues from various animals (40 species). The aggregation potency was assessed for each sequence using in silico analysis. A library of 23 peptides was prepared from sequences that were distinctly different and their amyloidogenic potential was assessed in vitro using physicochemical analysis (thioflavin-T assay, Congo red binding assay, far-UV circular dichroism, transmission electron microscopy) and cytotoxicity assays (insulinoma cell line INS-1). Among this peptide library, three were non-amyloidogenic and corresponded to the following animal species: golden lion tamarin (Leontopithecus rosalia), commun bottlenose dolphin (Tursiops truncates) and alpaca (Vicugna pacos). Segment 8-20 of the peptide was critical for amyloid formation and the substitution of the NFLVH motif found in this region by a DFLGR or KFLIR motif impeded fibrillization. The amino acids 29P, 14K and 18R were also demonstrated to abrogate fibril formation. The second objective consisted in abrogating IAPP fibrillogenesis using conceptualized aromatic/polyphenolic structures, specifically hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl urea agents. This part of the project involved molecular screening of more than 60 compounds. Their efficacy at inhibiting amyloid formation was assessed in vitro on human IAPP, which exhibits the highest amyloidogenic potential. Techniques included the above-mentioned methods, with the addition of photo-induced cross-linking of unmodified proteins (PICUP). A total of 11 compounds showed potential in abrogating IAPP aggregation. Among the hypoglycemic agents evaluated, glyburide, repaglinide and troglitazone showed the highest potency in reducing fibril formation. The NSAIDs that displayed anti-amyloidogenic activity were diclofenac, meloxicam, phenylbutazone, sulindac and tenoxicam. EU-362 and EU-418 were the hit compounds resulting from the screening of the aryl ethyl urea (EU) class. Additionally, these anti-amyloidogenic molecules conferred a protection against fibril cytotoxicity. All of the active molecules bear a commun motif composed of benzene ring with electron donor moieties, such as alcohol, amine or halide. To conclude, this project characterized IAPP in several animal species in which it has not been previously described and improves our understanding of the amyloidogenesis process. Moreover, the therapeutic potential of hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl ureas agents as anti-amyloidogenic compounds was evaluated. It is conceivable that the additional information hereby gained on the regulation of amyloidogenesis may point towards new therapeutic strategies for diabetic patients.

A histiocitose de células de Langerhans (HCL) é uma neoplasia mieloide inflamatória comumente afetando pacientes pediátricos e apresenta frequentemente mutações ativadoras somáticas em genes da via MAPK, incluindo BRAF e MAP2K1. Vários estudos sugerem que as células lesionais da HCL podem recrutar e modular células inflamatórias e cujas citocinas parecem fornecer sinais recíprocos de sobrevivência celular. Para o presente estudo foram selecionados 15 casos de HCL (10 crianças, 5 adultos), sendo as amostras de tecido avaliadas através de imunoistoquímica utilizando marcadores para macrófagos (CD68 e CD163), células dendríticas maduras (CDm) (CD83 e CD208), linfócitos T regulatórios (LTregs) (CD4, CD25 e FOXP3) e linfócitos citotóxicos (LCs) (CD56, CD57, perforina e granzima B). Além disso, marcadores de células B (CD20), células T (CD3, CD8) e confirmatórios de HCL foram analisados. Todos os casos de HCL foram positivos para S100, CD1a, CD207 e CD4; enquanto que Bcl-2 e Ciclina D1 foram positivos em 13/15 (86,7%) casos. No microambiente imune intralesional, macrófagos M2 (CD68+/CD163+), seguidos por LTregs, foram as populações celulares mais predominantes. Em quantidade significativamente menor, foram observadas CDm, seguidas por escassos LCs. Considerando a população linfoide, linfócitos T CD3+ foram mais numerosos do que linfócitos B CD20+. Dentro dos linfócitos T, linfócitos T CD4+ foram mais numerosos do que linfócitos T CD8+ (p<0,05). Nossos resultados sugerem que a infiltração de células imunes na HCL, provavelmente através de mecanismos pró-tumorais, inflamatórios e/ou imunossupressores mediados por citocinas, pode promover o desenvolvimento e sobrevivência das células lesionais da HCL, fornecendo uma justificativa para a combinação de imunoterapia e terapia gênica (BRAF) na HCL / Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia often affecting children with constitutively somatic activating mutations in MAPK pathway genes including BRAF and MAP2K1. Several studies suggest that LCH cells can recruit and modulate inflammatory cells and whose cytokines appear provide reciprocal survival signals. For the present study, 15 cases of LCH (10 children, 5 adults) were selected, and the tissue samples were evaluated through immunohistochemistry using markers for macrophages (CD68 and CD163), mature dendritic cells (mDC) (CD83 and CD208), regulatory T-cells (Tregs) (CD4, CD25 and FOXP3) and cytotoxic lymphocytes (CLs) (CD56, CD57, perforin and granzime B). Moreover, B-cell (CD20), T-cell (CD3, CD8) and LCH markers were analyzed. All LCH cases were positive for S100, CD1a, CD207 and CD4, while Bcl-2 and Cyclin D1 were positive in 13/15 cases (86.7%). In the immune microenvironment, M2-polarized macrophages (CD68+/CD163+), followed by LTregs, were the predominant cell populations. In a significantly lower amount, mDC were observed, followed by scarce CLs. Moreover, CD3+ Tcells than CD20+ B-cells were more numerous (p>0.05), the former presenting a higher number of CD4+ than CD8+ T-cells (p<0.05). Our results suggest that immune cell infiltration in LCH, probably through cytokine-mediated pro-tumoral, inflammatory and/or immunosupressive mechanisms, can promote LCH cell development and survival, providing a rationale for combining immunotherapy and BRAF-targeted therapy in LCH

Diabetes mellitus tipo 1 resulta da produção insuficiente ou da ausência de insulina, decorrente da destruição de células &#946;, por mecanismo auto-imune. O tratamento deste tipo de diabetes consiste na administração subcutânea de insulina exógena. Recentemente, foi demonstrado que o transplante de ilhotas pancreáticas é capaz de tornar o portador de diabetes tipo 1 independente de insulina exógena. Apesar do sucesso alcançado, a necessidade permanente de imunossupressão é uma das principais barreiras para que o transplante de ilhotas possa ser realizado em número maior de pacientes. Assim, o desenvolvimento de novas metodologias que evitem a rejeição do enxerto, como o macro e o microencapsulamento de ilhotas, continua sendo crucial para o estabelecimento definitivo do transplante de ilhotas como opção terapêutica no tratamento de diabetes tipo 1. Neste trabalho, foi padronizado um modelo animal para avaliar, in vivo, a funcionalidade das ilhotas pancreáticas humanas isoladas e purificadas na Unidade de Ilhotas Pancreáticas Humanas do IQUSP. Ratos NIH nude foram tornados diabéticos através de injeção de estreptozotocina para o implante de ilhotas pancreáticas humanas nuas e microencapsuladas. As ilhotas foram microencapsuladas em Biodritina, um novo heteropolissacarídeo patenteado e cedido ao nosso laboratório, tendo sido possível padronizar a produção de microcápsulas uniformes e homogêneas, com tamanho médio entre 400&#181;m e 600 &#181;m. A reversão do diabetes ocorreu em 24% dos ratos nude transplantados com ilhotas pancreáticas humanas nuas. Por outro lado, não observamos reversão do diabetes quando ilhotas encapsuladas foram implantadas, apesar do teste de atividade funcional realizado in vitro ter demonstrado que elas continuam a secretar insulina e a responder ao estímulo com glicose após o encapsulamento. Para elucidar este efeito, cápsulas vazias foram implantadas em ratos nude e em ratos imunocompetentes, os quais desenvolveram processo inflamatório acompanhado de processo fibrótico no local do implante. Estudo imuno-histoquímico está sendo realizado para esclarecer a natureza e a intensidade destes processos. / Type 1 diabetes mellitus results from insufficient or absence of insulin production, as a consequence of destruction of pancreatic &#946; cells, by an auto-imune mechanism. Treatment for this type of diabetes consists of subcutaneous administration of exogenous insulin. Recently, it has been demonstrated that pancreatic islet cell transplantation is capable of rendering type I diabetic patients independent of exogenous insulin. However, in spite of the success achieved, permanent immunosuppression is still required, being the main barrier to expand this treatment to a large number of patients. Therefore, development of new technologies, such as islet macro and microencapsulation to avoid rejection of the tissue implanted, is still crucial for definitive establishment of islet transplantation as a therapeutic alternative for type I diabetes. In the present work, an animal model was established for in vivo evaluation of the functional ability of human pancreatic islets, which were isolated and purified at the Human Pancreatic Islet Unit of the University of São Paulo Chemistry Institute. Diabetes was induced in NIH nude rats through streptozotocin injection followed by implantation of naked or microencapsulated human pancreatic islets. Biodritin, a new and patented heteropolyssaccaride was used to microencapsulate the islets. The production of uniform and homogeneous microcapsules with diameters in the range of 400&#181;m e 600 &#181;m was successfully established. Reversion of diabetes occurred in 24% of the nude rats transplanted with human pancreatic islets. On the other hand, no reversion of diabetes was observed when encapsulated islets were implanted, although their functional activity in vitro indicated that they secreted insulin and responded to glucose stimulation upon encapsulation. In order to elucidate this effect, empty capsules were implanted in nude rat and in immunocompetent rats, both of which developed an inflammatory process accompanied by a fibrotic process in the site of the implant. Immunohistochemical studies are underway to address the nature and the intensity of these inflammatory processes.

O uso de dieta hiperlipídica com banha de porco em roedores induz obesidade, resistência à insulina e disfunção das células beta do pâncreas. Em diversos tecidos de animais tratados com dieta hiperlipídica já se observou aumento de expressão e/ou atividade da NAD(P)H oxidase, que pode estar envolvida em processos fisiopatológicos. O objetivo deste trabalho foi avaliar se a dieta hiperlipídica altera a expressão e/ou a atividade da NAD(P)H oxidase em ilhotas pancreáticas e se este fato pode estar associado às disfunções das células beta relatadas na literatura para este modelo animal. As ilhotas pancreáticas dos animais tratados com dieta apresentam maior secreção de insulina em alta glicose, maior metabolização da glicose, menos apoptose, menor expressão protéica de subunidades da enzima, menor produção de superóxido e não apresentam estresse oxidativo. O papel da enzima provavelmente se relaciona ao processo de secreção de insulina. A regulação de sua expressão e atividade deve estar relacionada à adaptação das ilhotas aos efeitos deletérios da dieta. / Feeding animals with high fat diet containing lard causes obesity, insulin resistance and dysfunction of pancreatic beta cells. High fat diet induces oxidative stress and modulates NAD(P)H oxidase expression and activity in many tissues. This enzyme may be involved in many pathophysiological processes. The objective of this study was to evaluate the action of high fat diet on NAD(P)H oxidase activity and expression and if this fact can be connected to the beta cell dysfunction reported in the literature on this animal model. In pancreatic islets of rats fed the high fat diet apoptosis was reduced, glucose metabolism increased, insulin secretion elevated at high glucose, protein expression of NAD(P)H oxidase subunits reduced and the superoxide production was diminished. There was no difference between the groups for oxidative stress markers. It is possible that the enzyme has a role in the process of insulin secretion. Probably the islets are regulating their activity and function to compensate the deleterious effect of lard.