Adenocarcinoma pancreático é um tumor maligno com mau prognóstico, e por isso há necessidade de aperfeiçoamento ou criação de novas estratégias terapêuticas. A vacinação baseada em DCs é uma das abordagens mais promissoras, uma vez que as DCs são as células apresentadoras de antígenos (APCs) mais potentes e centrais para a indução e manutenção de uma resposta imune. Entretanto, em pacientes com câncer, a geração e a função de DCs podem ser deficientes, impondo um obstáculo para o sucesso de seu uso. Neste trabalho, nós descrevemos a geração in vitro de DCs a partir de células mononucleares do sangue periférico (PBMC) obtidas de pacientes ictéricos com câncer de pâncreas e, também, o efeito do plasma ictérico (PI) sobre as culturas de DCs de doadores saudáveis. PBMCs foram separadas do sangue obtido de 22 pacientes e 22 doadores saudáveis. Células aderentes foram cultivadas com GM-CSF e IL-4 (50ng/mL) por 7 dias. No 5o dia, TNF-<font face=\"symbol\">a (50ng/mL) foi adicionado para a maturação das DCs. Culturas foram realizadas em 10% PI ou plasma normal (PN). Células não aderentes foram coletadas no 7o dia, marcadas com anticorpos monoclonais anti-CD86, CD11c, CD14 e HLA-DR e analisados por citometria de fluxo. Células de pacientes, cultivadas em 10% de PI, quando comparadas com células de doadores saudáveis, cultivadas em 10% PN, apresentaram expressão reduzida (p<0,05) de CD86 e HLADR. É interessante observar que células geradas de PBMCs de pacientes não expressaram CD11c, diferente das células derivadas de doadores saudáveis. A presença de PI nas culturas dos doadores saudáveis causou uma significante diminuição na porcentagem de expressão de células HLA-DR+, CD11c+, CD86. Finalmente, quando PBMCs de pacientes foram cultivadas em PN, houve um aumento na expressão de HLA-DR e CD86 (p<0,05). Os ensaios de proliferação demonstraram também que as células de pacientes ictéricos tiveram capacidade aloestimuladora de linfócitos reduzida, quando comparada a de células de doadores saudáveis. Estes dados indicam uma alteração importante na capacidade das células de pacientes se diferenciarem em DCs in vitro, um fenômeno que parece depender tanto de fatores solúveis presentes no plasma e sobre as próprias células. / Pancreatic adenocarcinoma (PAdc) is an aggressive malignancy with poor prognosis, urging for improved or new therapeutic strategies. DC-based vaccination is one of such promising approaches. DC are the most potent antigen-presenting cells and central to the induction and maintenance of an immune response. However, in cancer patients DC generation and function may be deficient, imposing an obstacle to the success of their use. Here, we describe the in vitro generation of DC from peripheral blood mononuclear cells (PBMC) obtained from jaundiced patients with PAdc and, also, the effect of jaundiced plasma (JP) in the phenomenon. PBMC were separated from blood obtained from 10 patients and 10 healthy controls over a density gradient. Adherent cells were cultured with GM-CSF and IL-4 (50ng/mL) for 7 days. On the 5th day, TNF-<font face=\"symbol\">a (50ng/mL) was added for DC activation. Cultures were performed in 10% JP or normal plasma (NP). Non-adherent cells were harvested at day 7, labeled with FITC- or PE-conjugated monoclonal antibodies against CD86, CD11c, CD14, HLA-DR and analyzed by flow cytometry. Patients\' cells, cultured in 10% JP, compared to healthy donors\' cells, cultured in 10% NP, had a significantly (p<0.05) lower expression of CD86 and HLA-DR. It is noteworthy that cells generated from patients\' PBMC did not express CD11c, while from those derived from healthy donors\' cells did so. The presence of JP in healthy donors\' cells cultures caused a significant decrease in the percentage of HLA-DR+, CD11c+ and CD86+ cells. Finally, when patients\' PBMC were cultured in NP, a significant increase in HLA-DR and in CD86 expression occurred. MLR assays also demonstrated that cells from jaundice patients had decreased capacity to stimulate alloestimulation of lymphocytes when compared to healthy donors. These data indicate a significant alteration in the patients\' PBMC ability to differentiate into DC in vitro, a phenomenon that seems to depend both on soluble factors present in plasma and on the cells, themselves.

Submitted by MARCOS LEANDRO TEIXEIRA DE OLIVEIRA (marcosteixeira@ufv.br) on 2018-11-09T11:48:24Z No. of bitstreams: 1 texto completo.pdf: 3413556 bytes, checksum: e09637dfbfae1e8cfbcf4cf3d3485622 (MD5) / Made available in DSpace on 2018-11-09T11:48:24Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3413556 bytes, checksum: e09637dfbfae1e8cfbcf4cf3d3485622 (MD5) Previous issue date: 2018-06-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A dieta sangue é explorada somente por três espécies de morcegos vampiros entre os mamíferos. Considerando seu alto conteúdo proteico, várias adaptações fisiológicas são reportadas no morcego comum vampiro, Desmodus rotundus. O objetivo deste trabalho foi analisar a histomorfologia do pâncreas e rins de morcegos Desmodus rotundus durante o seu desenvolvimento, desde a fase intra-uterina, passando pela lactação, até a fase adulta, período pelo qual ocorre a transição de dietas, a fim de investigar o surgimento de mecanismos de adaptações associados à plasticidade das estruturas endócrinas pancreáticas e renais. O IPS foi maior em lactentes em relação aos fetos e em adultos, em relação aos outros dois grupos. A área proporcional das ilhotas, no entanto, foi maior em lactentes em relação aos fetos, porém menor em adultos quando comparada aos lactentes. Da mesma forma, a densidade volumétrica de ilhotas foi maior em lactente e menor em adultos. No tecido renal os resultados reportaram um aumento AG, como também uma maior V vglomérulos e túbulos uriníferos corticais, V v túbulos corticais encontrados nos animais adultos em relação aos lactentes e fetos . Estes resultados justificam a hipertrofia renal e o maior IRS encontrados nos animais adultos também em relação aos lactentes e fetos. Além disso, foram analisados índices renais relacionados com a capacidade máxima de concentração urinária como o MI/C e o MT/C mostrando uma medula interna mais espessa em morcegos vampiros adultos em relação aos morcegos vampiros lactentes e fetos. Os resultados pancreáticos indicam um menor investimento em massa pancreática em adultos quando comparados a lactentes, o que pode refletir uma menor participação da regulação hormonal pancreática sobre o controle glicêmico, sendo que estas adaptações parecem ocorrer entre as fases de lactação e a fase adulta, quando a dieta de sangue torna-se exclusiva. Os resultados no tecido renal traduzem uma maior habilidade de concentração urinária em adultos em relação aos lactentes. A maior espessura da medula nos animais adultos reflete uma importante adaptação no tecido renal para suportar o ambiente azotêmico em que vive. Observamos também uma maior densidade e rearranjos de podócitos na cápsula glomerular nos animais adultos comparados aos lactentes. Trata-se de uma adaptação morfológica provocada pelo aumento da força mecânica de distensão e cisalhamento na MBG, em consequência de um grande aumento de volume extracelular e, portanto um grande fluxo de filtração glomerular após a alimentação com uma dieta hiperproteica. Assim, nós concluímos que as alterações morfológicas reportadas nos morcegos vampiros após a transição da dieta sangue pode constituir uma programação fetal culminando nas adaptações em adultos para que este animal sobrevivesse a uma dieta de sangue. / The blood diet is exploited only by three species of vampire bats among mammals. Considering its high protein content, several physiological adaptations are reported in the common vampire bat, Desmodus rotundus. The objective of this work was to analyze the histomorphology of the pancreas and kidneys of bats Desmodus rotundus during their development, from the intrauterine phase, through lactation, to the adult stage, period through which the transition of diets occurs, in order to investigate the appearance of adaptation mechanisms associated with the plasticity of the pancreatic and renal endocrine structures. The PSI was higher in suckling vampire compared to fetuses and in adults, in relation to the other two groups. The proportionate area of the islets, however, was higher in suckling than in fetuses, but lower in adults when compared to suckling. Likewise, volumetric density of islets was higher in suckling and lower in adults. In renal tissue the results reported a GA increase, as well as a larger Vv glomerules and cortical urinary tubules, and cortical tubules found in adult animals in relation to suckling vampire and fetuses. These results justify renal hypertrophy and the highest RSI found in adult animals also in relation to infants and fetuses. In addition, renal indices related to maximal urinary concentration capacity, such as IM/C and TM/C, was analyzed showing a thicker internal medulla in adult vampire bats in relation to suckling vampire bats. Pancreatic results indicate a lower pancreatic mass investment in adults when compared to suckling vampire, which may reflect a lower participation of pancreatic hormonal regulation on glycemic control, and these adaptations seem to occur between the lactation and adult phases, when the blood diet becomes exclusive. The results in the renal tissue translate a greater ability of urinary concentration in adults in relation to suckling vampire. The increased thickness of the marrow in adult animals reflects an important adaptation in renal tissue to support the azotemic environment in which it lives. We also observed a higher density and rearrangements of podocytes in the glomerular capsule in adult animals compared to suckling. It is a morphological adaptation provoked by the increase of the mechanical force of distension and shear in the GBM, as a consequence of a large increase in extracellular volume and, therefore, a large flow of glomerular filtration after feeding with a hyperproteic diet. Thus, we conclude that the morphological changes reported in vampire bats after the transition from the blood diet may constitute a fetal schedule culminating in adult adaptations for this animal to survive a blood diet.

Orientadores: Mario Mantovani, Gustavo Pereira Fraga / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T17:58:25Z (GMT). No. of bitstreams: 1 Silveira_HenriqueJoseVirgili_D.pdf: 1825654 bytes, checksum: 21235a597528aaba03e13e883055a983 (MD5) Previous issue date: 2008 / Resumo: Embora incomuns, lesões traumáticas do pâncreas estão associadas a significativos níveis de complicações e mortalidade. O objetivo deste estudo foi definir os fatores preditivos de morbidade e mortalidade em pacientes vítimas de trauma pancreático. Nesta casuística, foram estudados 131 pacientes atendidos pela Disciplina de Cirurgia do Trauma no Hospital de Clínicas da UNICAMP no período entre janeiro de 1994 a dezembro de 2007, com seus parâmetros epidemiológicos, fisiológicos e anatômicos registrados em protocolo específico prospectivamente e, comparados e analisados aos fatores preditivos de evolução, com estudo estatístico. Trauma penetrante, com predomínio de ferimentos por projétil de arma de fogo ocorreu em 64% dos casos. A maioria, 91,6% era do sexo masculino e a idade média de 29,8 anos. A morbidade global foi de 64,9%, com 29% de complicações diretamente relacionadas ao pâncreas, como fístulas e sangramento. A mortalidade foi de 27,5%, principalmente em decorrência de choque hipovolêmico e falência de múltiplos órgãos e sistemas. Houve maior morbidade e mortalidade em pacientes com lesões complexas (graus IV e V) do pâncreas quando comparados com lesões menos graves (graus I e II), porém a morbidade e mortalidade neste grupo não foi desprezível, devido a valores alterados de RTS, valores elevados de ISS e ATI. Pacientes com valores alterados de RTS, pressão arterial sistólica menor que 90 mmHg, valor de ISS maior ou igual a 25 e valor de ATI maior ou igual a 25 apresentaram maior incidência de complicações mórbidas e maior mortalidade. Pacientes com valores elevados de TRISS ainda apresentaram complicações mórbidas e óbito. O presente estudo conclui que lesões complexas do pâncreas, pressão aterial sistólica inferior a 90 mm Hg, RTS com valor alterado, ISS maior ou igual a 25 e ATI maior ou igual a 25 são preditivos de maior morbidade e mortalidade em pacientes traumatizados com lesão pancreática / Abstract: Although uncommon, traumatic pancreatic injury is associated with significative index of morbidity and mortality. The objective of the study was to define the predictors' factors of increase in the morbidity and mortality in patients with pancreatic trauma. In this casuistic 131 patients were studied, since January 1994 through December 2007, with theirs epidemiological, physiological and anatomic parameters compared and the analysis of the predictive values for the occurrence of bad evolution, with an appropriate statistical study. Prospective coorte study. Penetrating trauma occurred in 64% and blunt trauma in 36%, and 91,6% was male. The mean age was 29,8 years.The global morbidity in this series was 64,9% with 29% prevalence of pancreas related complications, such as pancreatic fistula and bleeding occurrence. The overall mortality was 27,5% and occurred by hemorrhagic shock and multiple organs and system failed. Higher morbidity and mortality was related with complex injuries of the pancreas (grade IV and V), but morbidity and mortality in the group of injuries grade I and II are not minimal in patients with changed values of RTS and high values of ISS and ATI. Systolic blood pressure lower 90 mmHg, changed values of RTS index, values of ISS higher 25 and values of ATI higher 25 are predictive factors of morbidity and mortality. This study conclude complex injuries of pancreas, systolic blood pressure lower 90 mmHg, changed values of RTS, values of ISS and ATI higher 25 are predictive factors of elevation morbidity and mortality / Doutorado / Cirurgia / Cirurgia

Objetivamos identificar os efeitos celulares pelo qual o DHEA melhora a função das ilhotas pancreáticas. Ratos wistar com 12-14 meses de idade receberam uma única injeção subcutânea de DHEA (10mg.kg-1) ou veículo. Após uma semana, ilhotas pancreáticas foram isoladas para realização dos testes: secreção de insulina, respiração mitocondrial, oxidação de glicose, atividade da citrato sintase, fragmentação de DNA, expressão gênica e dosagem plasmáticas de hormônios esteróides sexuais. DHEA apresentou melhoria significativa na secreção de insulina. Notou-se um aumento na oxidação da glicose, respiração mitocondrial, atividade da citrato sintase, expressão do PCNA mRNA e reduzida apoptose. O uso de inibidores de receptores de andrógeno e estrógeno inibiu a secreção de insulina estimulada por glicose. Concluímos que o DHEA é capaz de melhorar a funcionalidade metabólica e reduzir o índice de apoptose da ilhota pancreática, melhorando a capacidade secretora, sendo este papel modulado, provavelmente, pela ligação do DHEA aos receptores de andrógenos e estrógenos. / We sought to identify the cellular effects by which DHEA improves pancreatic islets function. Male Wistar rats, 12-14 month-old, receive one subcutaneous injection of DHEA (10mg.kg-1) or vehicle. After a week, pancreatic islets from these animals were isolated and submitted to the following tests: insulin secretion, mitochondrial respiration, glucose oxidation, citrate synthase activity, DNA fragmentation, gene expression and the dosage of plasmatic steroid hormones. DHEA treatment resulted in significant improvement in insulin secretion. We observed an increase in glucose oxidation, mitochondrial respiration, citrate synthase activity, PCNA mRNA expression, and reduced apoptosis. Glucose-stimulated insulin secretion was inhibited by androgen and estrogen receptor inhibitors. We conclude that DHEA is capable of improving metabolic functionality and reduces apoptosis index in pancreatic islets, improving the secretory capacity stimulated by different secretagogues, with this being probably modulated by the binding of DHEA to androgen and estrogen receptors.

Foram avaliados, com o auxílio de um aparelho dinâmico, bidimensional e transdutores eletrônicos multifreqüênciais (convexo de 4 a 7 MHz e linear de 7 a 12 MHz), os pâncreas de 33 cães da raça Poodle Toy, 17 fêmeas e 16 machos, com idade entre seis e 168 meses, peso corpóreo entre 1,85 a 6,15 quilogramas, sem processos patológicos aparentes. Estudaram-se as características anátomo sonográficas e suas correlações com as variáveis: sexo, idade e peso. A avaliação ultra-sonográfica do pâncreas permitiu identificar pelo menos uma região (lobo direito) e uma estrutura interna pancreática (veia pancreaticoduodenal), em todos os animais. O sexo, a idade e o peso não influenciaram na visibilização da glândula. Os valores métricos, referentes à espessura média e o desvio padrão das diferentes regiões e estruturas internas pancreáticas foram: lobo direito, tanto no plano longitudinal, como no transversal 0,72 &plusmn; 0,10 cm, lobo esquerdo 0,77 &plusmn; 0,11 cm, corpo 0,66 ± 0,09 cm, veia pancreaticoduodenal, em ambos planos, 0,18 &plusmn; 0,03 cm, artéria pancreaticoduodenal, no plano longitudinal 0,13 &plusmn; 0,01 cm, e no plano transversal 0,13 &plusmn; 0,02 cm, ducto pancreático, no plano longitudinal 0,08 &plusmn; 0,01 cm e no plano transversal 0,08 &plusmn; 0,02 cm. Observaram-se correlações positivas entre a espessura do lobo direito e a idade (p = 0,00), e entre essa espessura e o peso (p = 0,00). Correlações positivas foram observadas entre a espessura do lobo esquerdo e a idade (p = 0,04), e entre essa espessura o peso (p = 0,04). O estabelecimento dos padrões anátomo ultra-sonográficos permitiram verificar que a topologia e a arquitetura foram semelhantes em todos os animais. Os contornos foram classificados como pouco definidos ou definidos e não variaram com o sexo, o peso e a idade. A ecogenicidade pancreática, quando comparada ao baço, foi predominantemente hipoecogênica, independente de sexo, idade ou peso; a ecogenicidade em relação ao fígado, apresentou-se isoecogênica ou discretamente hiperecogênica; e em relação à gordura mesentérica foi hipoecogênica ou isoecogênica. A ecotextura mostrou-se homogênea a difusamente heterogênea. Observaram-se correlações positivas entre a ecogenicidade em relação ao fígado e a idade (p = 0,00) e o peso (p = 0,00), e entre a ecogenicidade em relação à gordura mesentérica, e a idade (p = 0,01) e ao peso (p = 0,02). Quanto à ecotextura pancreática, também foram observadas correlações positivas em relação à idade (p = 0,001) e ao peso (p = 0,002). / Pancreas of 33 healthy Toy Poodles, 16 male and 17 bitches, ranging from 6 to 168 months of age and weighing from 1,85 to 6,15 kg where evaluated with a dynamic bidimensional equipament and multifrequency ultrasound (curvilinear array 4-7 MHz and linear array 7-12 MHz) scanner. Ultrasonographic anatomy and correlation with gender, age and weight were studied. Ultrasonography allowed visualization of at least one region (right lobe) and one internal structure (pancreaticoduodenal vein) in all animals. Gender, age and weight had no influence on visualization. Measures of thickness where 0,72 &plusmn; 0,10 cm for the right lobe in longitudinal and transversal cut, 0,77 &plusmn; 0,11 cm for the left lobe and 0,66 &plusmn; 0,09 cm for the body. Diameter of the pancreaticoduodenal vein was 0,18 &plusmn; 0,03 cm in both cuts, diameter of the pancreaticoduodenal artery measured 0,13 &plusmn; 0,01 cm in the longitudinal cut and 0,13 &plusmn; 0,02 cm in the transversal; values obtained for the pancreatic duct where 0,08 &plusmn; 0,01 cm (longitudinal) and 08 &plusmn; 0,02 cm (transversal). Right lobe thickness was positively correlated to age (p = 0,00) and weight (p = 0,00). Left lobe thickness was positively correlated to age (p = 0,04) and weight (p = 0,04). Topology and architecture, similar in all animals, allowed recognition of a pattern. Outline was classified as poorly defined or defined and did not change with gender, weight and age. Echogenicity showed to be predominately hypoechoic compared to the spleen, iso or slightly hyperechoic when compared to liver and iso or hypoechoic when compared to mesenteric fat. Echotexture varied from homogeneous to diffusely heterogeneous. Pancreatic ecogenicity compared to the liver ecogenicity changed positively with age (p = 0,00) and weight (p = 0,00). Echogenicity compared to the mesenteric fat changed positively with age (p = 0,01) and weight (p = 0,02). Pancreatic echotexture showed a positive correlation with age (p = 0,001) and weight (p = 0,002).

Advanced pancreatic cancer (APC) has a poor prognosis due to the high degree of resistance after systemic chemotherapy. Celastrol (CSL), a quinone methyl triterpenoid monomer extracted from Tripterygium wilfordii Hook F, exhibits superior antitumor activity on pancreatic cancer (PC) both in vitro and in vivo. In addition, CLS counteracts multiple mechanisms involved in multi-drug resistance (MDR) of PC cells. However, CSL induced toxicity to normal tissues (e.g. liver) is the major impediment to its clinical application. Thus, it is desirable to seek strategy to facilitate CSL selectively targeting PC tissues and simultaneously reducing its exposure to healthy tissues (e.g. liver). Aptamers are single-stranded oligonucleotides which specifically recognize and bind to targets by distinct secondary or tertiary structures. Nucleolin, a protein overexpressed on the plasma membrane of PC cells than that of normal cells (e.g. liver cell), which shuttle between cell surface, cytoplasm and nucleus, work as a cell surface receptor. Nucleolin aptamer is an anti-proliferative G-rich oligonucleotide with high affinity and specificity to nucleolin, which has been proved to be safe in clinical research. Then, nucleolin aptamer, as a target moiety, provide an approach to facilitate CLS selectively targeting PC cells. Taken together, our hypothesis is that the nucleolin aptamer modification could facilitate the conjugated CSL selectively targeting pancreatic cancer cells to achieve higher antitumor activity and less liver toxicity. In our study, CSL was conjugated to nucleolin aptamer to form Nucleolin Aptamer-Celastrol Conjugate (NACC). A CRO Aptamer-Celastrol conjugate (CACC) was also synthesized as a control for comparison. The water solubility of NACC was significantly higher than that of CSL. Then, the molecular weight of NACC was detected by ESI mass sepectrum (MS). The anti-proliferative efficacy of NACC was higher than CSL in vitro. NACC could selectively bind to PANC-1 cells over normal liver cells. The cellular uptake of NACC by PANC-1 cell was stronger than CSL. Moreover, NACC could be taken up by PANC-1 cells mainly via macropinocytosis. Tissue distribution study revealed that NACC could selectively accumulate in pancreatic tumor tissue and reduce the distribution in liver in vivo. In addition, NACC demonstrated higher antitumor activity and less liver toxicity in vivo, compared with CSL and CACC. The above results revealed that the nucleolin aptamer modification could facilitate the conjugated CSL selectively targeting PC cells to achieve higher antitumor activity and less liver toxicity.

Au-delà de l’intérêt cognitif de la démarche, la compréhension des mécanismes qui régissent le développement pancréatique humain reste la clé pour décrypter les acteurs physiopathologiques des maladies du pancréas et pour développer des approches thérapeutiques innovantes. En outre, alors que la cellule bêta de rongeurs et la cellule bêta humaine partagent un grand nombre de similitudes, certaines données indiquent également des différences marquées entre les espèces. L'absence de systèmes expérimentaux robustes , à partir de matériel humain, n'a pas permis un examen détaillé du développement pancréatique humain jusqu’à présent. Dans le laboratoire, il a été précédemment validé un modèle de xénogreffe de pancréas immature humain sous la capsule rénale de souris immuno-incompétentes SCID. Il a été démontré que celui-ci permettait de récapituler l’ensemble des étapes du développement endocrine humain. Néanmoins le site de greffe limitait les possibilités de modifier ce développement, notamment par infection virale. Dans ce travail, nous avons développé et validé un nouveau site de greffe dans le muscle squelettique, plus simple et plus superficiel. En outre, nous démontrons qu’il est possible de créer un pancréas humain partiellement transgénique in vivo en réalisant du transfert de gènes médié par des lentivirus, après injection directe de la solution virale dans le greffon. Ce modèle de greffe dans le muscle est une nouvelle approche permettant d’envisager des études longitudinales, dans lesquelles il serait possible d’étudier la régulation de certains gènes ou le devenir de certaines lignées marquées par des gènes rapporteurs apportés par le virus à différents stades de développement. / Whilst sporadic human genetic studies have permitted some comparisons between rodent and human pancreatic development, the lack of a robust experimental system has not permitted detailed examination of human pancreatic development. We previously developed a xenograft model of immature human fetal pancreas grafted under the kidney capsule of immune-incompetent mice, which allowed the development of human pancreatic beta cells. Here, we compared the development of human and murine fetal pancreatic grafts either under skeletal muscle epimysium or under the renal capsule. We demonstrated that human pancreatic beta cell development occurs) both by differentiation of pancreatic progenitors and by proliferation of developing beta cells. The superficial location of the skeletal muscle graft and its easier access permitted in vivo lentivirus-mediated gene transfer which targeted specific cells. This model of engraftment under the skeletal muscle epimysium is a new approach for longitudinal studies, which allows localized manipulation to determine the regulation of human pancreatic development.

Type 1 diabetes (T1D) is an autoimmune disorder, targeting the β-cells of the pancreas. Processes occurring in the gut and pancreas are inferred to be involved. The pre-diabetic expression signature in these tissues is largely uncharacterized. HYPOTHESIS: Spontaneous models of T1D, the LEW.1AR1/Ztm-iddm rat (LEW-DP) and BioBreeding diabetes-prone rat (BBdp) exhibit a distinct transcriptional signature prior to T1D onset. Transcriptional profiling was used to elucidate the expression signatures of the LEW-DP gut and BBdp pancreas. The LEW-DP gut displayed decreased expression of markers of anti-inflammatory M2 macrophages. The LEW-DP rats showed an upregulation of markers of pro-inflammatory signaling when fed a diabetes-promoting cereal diet compared with LEW-DP rats fed a protective hydrolyzed casein diet. Prospective pancreatectomy was used to analyze T1D development in the BBdp rat. Significant upregulation of β-cell markers Reg3α, Reg3β, and Trim26 was observed in pre-diabetic rats. Thus, it was shown that environment modifies the transcriptional program and the transcriptional profile is programmed early to affect T1D development.

Total parenteral nutrition (TPN) provides an experimental situation where adequate nutrition is provided intravenously, bypassing the gastrointestinal tract. Under these conditions the importance of orally ingested nutrients in the control of gastric and pancreatic endocrine secretion can be assessed. The objectives of this thesis were two-fold. First, to examine the effects of TPN on the enteroinsular axis component of insulin secretion. Second, to study the importance of orally ingested nutrients in the regulation of gastric hormone secretion using the TPN rat model. In order to carry out these objectives, techniques for TPN and enteral feeding (TEN) of the rat were first developed. A dietary regimen for use in TPN and TEN rats was formulated from commercially-available, human TPN components. Under most circumstances, the TPN/TEN regimen met or exceeded the nutritional requirements for growing rats, as determined by the National Research Council (1978). Hematological analysis revealed few side effects of — intravenous or intragastric feeding. Parenterally and enterally-fed animals demonstrated comparable weight gain to that of a control group (ORAL) fed a rat chow (#5012, Ralston Purina) diet ad libitum. In addition, both TPN and TEN animals appeared healthy after the 7-day infusion period. These studies indicated that the infusion formulation was suitable for chronic intravenous and intragastric feeding. In the first series of experiments, the effects of TPN and TEN on the hormonal component of the enteroinsular axis were studied. TPN animals exhibited hyperinsulinemia and mild hyperglycemia. Conversely, TEN animals exhibited normal plasma glucose and immunoreactive insulin (IRI) concentrations. These data suggested that enterally delivered nutrients were assimilated with greater efficiency than intravenously administered nutrients. It was hypothesized that gut factors normally released by oral food intake facilitated the disposal of nutrients by hepatic and/or peripheral tissues. During the infusion period, TPN animals exhibited chronically depressed circulating IR-gastric inhibitory polypeptide (GIP) levels, in contrast to TEN animals where IR-GIP was elevated. Seven days of TPN or TEN resulted in no change in fasting plasma IRI or IR-GIP levels. However, an exaggerated insulin response to an oral glucose challenge (OGC) occurred after TPN, while the glucose response was reduced. The insulin response from the perfused pancreata of TPN animals to a GIP gradient was 20% and 40% greater than from ORAL and TEN pancreata respectively. Shorter periods of TPN (3 and 5-day periods) indicated that the hypersensitivity of the pancreas to GIP was a progressive condition, increasing with longer periods of infusion. Immunocytochemical and morphometric analysis revealed no differences in the jejunal GLP-cell population after chronic (7-day) intravenous or intragastric feeding. In addition, these routes of feeding had no effect on pancreatic islet area or endocrine cell composition of the islets. Based on these results, it was hypothesized that the increased B-cell sensitivity to GIP may have been causally related to the exposure of the pancreas to chronically low plasma GIP levels during the infusion period. To further test this hypothesis, chronically depressed plasma GIP levels, observed during TPN, were elevated by exogenous GIP infusion to levels seen in TEN rats. Chronic GIP —treatment in TPN animals (TPN-GIP) resulted in normalization of the insulin response to an OGC and in the in vitro insulin response of the isolated pancreas to GIP. These data were taken as further evidence that B-cell sensitivity to GIP was affected by ambient plasma GIP levels, and it was hypothesized that changes in sensitivity may be mediated by alteration at the receptor or post-receptor level. The effect of TPN on nutrient and neuronally mediated insulin release was also investigated. During TPN, metabolites and neuronal elements provided the main stimulus for insulin release, since hormonal components of the enteroinsular axis remained inactive. The present experiments indicated that the B-cell was hypersensitive to glucose, vagal stimulation and the cholinergic agonist methacholine, but normally sensitive to vasoactive intestinal polypeptide (VIP) and the insulinotropic amino acid arginine. These results indicated that TPN was associated with an increased B-cell sensitivity to specific hormonal, nutritive and neuronal stimuli. It was hypothesized that an increased B-cell sensitivity to these specific stimuli contributed to hyperinsulinemia observed in TPN animals during the infusion period, and to the exaggerated insulin response observed after an oral glucose challenge. Total parenteral nutrition also provided an experimental situation in which to study the importance of gastric nutrients in the regulation of Gl-hormone secretion. TPN resulted in a rapid and progressive depletion of circulating gastrin levels. G-cell secretory activity in vivo under basal and stimulatory conditions was also reduced by TPN. This condition persisted in vitro in the isolated stomach. The antral G-cell population was shown to decrease progressively with longer TPN periods, but G-cell hypoplasia and reductions in antral gastrin content were less dramatic than reductions in G-cell secretory activity. It was hypothesized that reductions in G-cell secretory activity were in part causally related to antral G-cell hypoplasia. The present data further suggested, however, that mechanisms which control synthesis and/or secretion within G-cells may have also been impaired, since various stimulants of gastrin release could not reverse gastrin hyposecretion observed during basal periods. Gastrin hyposecretion also could not be reversed by chronic bombesin administration, but was reversed by a 6-day period of oral — refeeding, indicating that the presence of nutrients in the gastric lumen was the primary regulator of tissue gastrin levels and G-cell secretory activity. The gastric D-cell was much less affected by the absence of nutrients in the gastric lumen than was the G-cell, and antral somatostatin hypersecretion may have contributed to G-cell hyposecretion. The experiments presented in this thesis indicated that total parenteral nutrition had marked effects on both B- and G-cell secretory activity. These studies clearly demonstrated the importance of enteral feeding in the maintenance of normal pancreatic and gastrointestinal endocrine secretion. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13418号 / 論医博第2226号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 長船 健二, 教授 伊藤 貴浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM