La sécrétion d'insuline est un phénomène physiologique majeur déclenché par l'entrée du glucose dans la cellule β-pancréatique. Elle est naturellement modulée par de nombreux facteurs comme des métabolites, des neurotransmetteurs et des hormones. Parmi les modulateurs hormonaux, les incrétines, dont le chef de file est le Glucagon Like Peptide (GLP-1), sont des amplificateurs gluco-dépendants de la sécrétion d'insuline. Ils activent la voie AMPc-PKA et leurs effets sur la glycémie sont à la base de stratégies pharmacologiques antidiabétiques. Cependant, si les actions physiologiques des incrétines sont bien connues, la compréhension des mécanismes moléculaires mis en jeu demeure incomplète. Pour ce travail, nous sommes partis de l'hypothèse suivant laquelle la PKA pourrait potentialiser la sécrétion d'insuline, par phosphorylation de cibles moléculaires spécifiques, grâce à son adressage par les A Kinase Anchoring Proteins (AKAPs) au niveau de la machinerie de l'exocytose. Cette étude s'appuie sur des techniques moléculaires d'invalidation génique (RNAi), de peptides inhibiteurs et de fractionnement subcellulaire, ainsi que sur des analyses en microscopie électronique et confocale. Durant ce travail, nous avons mis en évidence la distribution de la sous-unité RIIα de la PKA à la surface des granules d'insuline matures. Nous avons également montré que la suppression de l'expression de la sous-unité RIIα de la PKA, comme la dissociation des complexes de type PKARII-AKAPs provoquent une réduction importante de l'effet amplificateur du GLP-1 sur la sécrétion d'insuline. Ainsi, cette étude nous a permis d'identifier la sous-unité RIIα de la PKA comme l'isoforme indispensable à la potentialisation AMPc-dépendante de la sécrétion d'insuline induite par le GLP-1, et surtout de placer le granule d'insuline mature au centre des mécanismes liés à l'effet « incrétine ». Ce travail constitue une première étape vers l'identification des différents partenaires du complexe protéique impliqué dans la potentialisation de l'exocytose d'insuline par la voie AMPc-PKA. / Insulin secretion is a major physiological function triggered in the body upon glucose entry into pancreatic β cells. This process is naturally subject to modulation by numerous metabolites, neurotransmitters or hormones. Among the modulatory hormones, incretins, mainly Glucagon Like Peptide (GLP1), act on β cells as physiological amplifiers of glucose-dependent insulin secretion. Incretins are known to recruit the cAMP signaling transduction pathway and their effects on glucose homeostasis represent a current basis for promising anti-diabetic approaches. However, while physiological actions of incretins are well known, the molecular mechanisms underlying these actions are not fully understood.In this thesis, we based our work on the hypothesis that the PKA (cAMP-dependent protein kinase) might potentiate glucose-induced insulin secretion via direct phosphorylation of PKA targets at the level of the exocytosis components, through specific anchoring of the kinase to this microdomain by A-Kinase Anchoring Proteins (AKAPs).The present work involving specific molecular approaches, such as siRNA, cell-permeable peptide competitors, subcellular fractionations as well as confocal and ultrastructural analysis of β cells, culminated to provide compelling evidence that the RIIα PKA isotype is physically associated to mature insulin granules. We demonstrate that in pancreatic β cells, either suppression of RIIαPKA expression or endogenous disruption of RIIαPKA from AKAPs results in almost complete loss of GLP1-induced amplification of insulin secretion.In conclusion, the present work allowed the identification of RIIα as a crucial effector of the cAMP/PKA axis for the amplification by incretins of insulin secretion. This finding represents a first and important step towards the identification of the molecular partners involved in the GLP1-induced PKA-dependent potentiation of insulin exocytosis.

Orientador: Lício Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T15:03:00Z (GMT). No. of bitstreams: 1 Silva_CarinaSolon_D.pdf: 1769268 bytes, checksum: b8a449a504bd2aa54bb737ba42860093 (MD5) Previous issue date: 2015 / Resumo: / Resumo: A interleucina-17 (IL17) está envolvida na resposta imune contra agentes patogénicos intestinais, e a sua expressão anómala no intestino pode ocorrer em condições tais como diabetes do tipo 1 (DM1), encefalomielite auto-imune e doença de Crohn. Fatores dietéticos podem alterar a microbiota intestinal desencadeando doenças metabólicas. Nossa hipótese é de que IL17 poderia ser diretamente modulada por nutrientes e pode desempenhar um papel na obesidade e diabetes tipo 2 (DM2). Aqui, nós demonstramos que as gorduras da dieta induzem a expressão IL17, predominantemente no íleo. In vivo, ilhotas pancreáticas isoladas estimuladas com IL17 apresentaram um aumento na secreção de insulina quando comparado a ilhotas não estimuladas, enquanto que a sua inibição sistémica resultou em intolerância à glicose. Animais knockout para o receptor de IL17 (IL17RA) eram intolerantes à glucose devido ao desenvolvimento embrionário anómalo das ilhotas pancreáticos, que eram menores e foram depletados de células produtoras de insulina. Nos seres humanos, os níveis circulantes de IL17 aumentaram após uma refeição. Este aumento foi significativamente maior nos indivíduos obesos normoglicêmicos do que em indivíduos obesos com diabetes. Semelhantes aos roedores, as ilhotas humanos também foram estimulados a secretarem insulina na presença de IL17. Assim nós identificamos a IL17 como um sensor intestinal de gorduras alimentares, que exerceram um efeito semelhante a hormônios incretínicos. Além disso, a presença IL17RA é importante para o desenvolvimento normal das ilhotas pancreáticas. / Abstract: / Abstract: Interleukin-17 (IL17) is involved in the immune response against intestinal pathogens, and its anomalous expression in the gut can occur in conditions such as type 1 diabetes (T1D) 1, autoimmune encephalomyelitis 2 and Crohn¿sdisease 3. Because dietary factors can change the gut microbiota, impacting metabolic diseases 4, we hypothesized that IL17 could be directly modulated by nutrients and might play a role in obesity and type 2 diabetes (T2D). Here, we show that dietary fats induced IL17 expression, predominantly in the ileum. Both in vivo and in isolated pancreatic islets, IL17 stimulated insulin secretion, while its systemic inhibition resulted in glucose intolerance. Mice KO for the main IL17 receptor (IL17RA) were glucose intolerant due to anomalous embryonic development of the pancreatic islets, which were smaller and were depleted of insulin-producing cells. In humans, blood IL17 increased following a meal. This increase was significantly higher in obese normoglycemic individuals than in obese subjects with diabetes. Similar to those of rodents, human islets were also stimulated to secrete insulin in the presence of IL17. Thus, we identified IL17 as a gut sensor of dietary fats, which exerted an incretin-like effect. In addition, the presence IL17RA was important for normal development of the pancreatic islets / Doutorado / Fisiopatologia Médica / Doutora em Ciências

Made available in DSpace on 2016-01-26T18:55:37Z (GMT). No. of bitstreams: 1 gisele_golghetto.pdf: 1021487 bytes, checksum: 2143f9bc567e25ae13a963bafe3cc100 (MD5) Previous issue date: 2013-12-17 / Introduction: Cadmium is a heavy metal, widely used in industry. It may give rise to the appearance of tumors in multiple organs. Objective: To evaluate the effect of the pH of the water in the genesis of cancer caused by cadmium. Material and methods: We used 98 Wistar rats were divided into seven groups: A - 15 rats that received solution of cadmium chloride (CdCl2 - 400mg / L) in drinking water with a neutral pH (pH 7.0), B - 15 rats that received CdCl2 (400 mg / L) in the drinking water at acidic pH (pH 5.0), C - 15 rats that received CdCl2 (400 mg / L) in drinking water with basic pH (pH 8.0), D - 15 rats that received water at acidic pH (pH 5.0), E - 15 rats that received water with basic pH (pH 8.0), F - 15 rats that received water at neutral pH (pH 7.0 ); G - 8 rats that received cyclophosphamide as a single dose (50mg/kg) on the first day of the experiment (positive control for micronucleus test). Rats of groups A-F were euthanized after 6 months and the group G, 24 hours after the start of the experiment. We removed liver, kidney, pancreas, prostate, seminal vesicles, testes, and bone marrow. Results: There were no pre-neoplastic or neoplastic in liver, kidney, pancreas, seminal vesicles and testes in all groups. Only animals exposed to cadmium showed prostatic intraepithelial neoplasia grade one, being more prevalent in group B (p<0.05), although there was no increase in micronuclei (p>0,05). Conclusion: The acidic pH contributed to the formation of precancerous lesions in the prostate gland of animals exposed to cadmium. / Introdução: O cádmio é um metal pesado, muito utilizado na indústria. Ele poder originar o aparecimento de tumores em múltiplos órgãos. Objetivo: Avaliar o efeito do pH da água ingerida na gênese do câncer provocado por cádmio. Material e métodos: Utilizou-se 98 ratos Wistar, divididos em 7 grupos: A 15 ratos que receberam solução de cloreto de cádmio (CdCl2 - 400mg/L) na água de beber com pH neutro (pH 7,0); B 15 ratos que receberam CdCl2 (400mg/L) na água de beber com pH ácido (pH 5,0); C 15 ratos, que receberam CdCl2 (400mg/L) na água de beber com pH básico (pH 8,0). D 15 ratos que receberam água com pH ácido (pH 5,0); E 15 ratos que receberam água com pH básico (pH 8,0); F 15 ratos, que receberam água com pH neutro (pH 7,0); G 8 ratos que receberam ciclofosfamida em dose única (50mg/kg) no primeiro dia do experimento (controle positivo do teste do micronúcleo). Ratos dos grupos A-F foram eutanasiados após 6 meses e os do grupo G, 24 horas após o início do experimento. Retirou-se fígado, rim, pâncreas, próstata, vesícula seminal, testículos e medula óssea. Resultados: Não foram observadas lesões pré-neoplásicas nem neoplásicas no fígado, rim, pâncreas, vesícula seminal e testículos em todos os grupos. Somente animais expostos ao cádmio apresentaram neoplasia intraepitelial prostática de grau um, sendo mais prevalente no grupo B (p<0,05), porém não houve aumento de micronúcleos (p>0,05). Conclusão: O pH ácido contribuiu para formação de lesões pré-neoplásicas na próstata dos animais expostos ao cádmio.

Made available in DSpace on 2016-07-18T17:53:11Z (GMT). No. of bitstreams: 1 gisele_golghetto.pdf: 1021487 bytes, checksum: 2143f9bc567e25ae13a963bafe3cc100 (MD5) Previous issue date: 2013-12-17 / Introduction: Cadmium is a heavy metal, widely used in industry. It may give rise to the appearance of tumors in multiple organs. Objective: To evaluate the effect of the pH of the water in the genesis of cancer caused by cadmium. Material and methods: We used 98 Wistar rats were divided into seven groups: A - 15 rats that received solution of cadmium chloride (CdCl2 - 400mg / L) in drinking water with a neutral pH (pH 7.0), B - 15 rats that received CdCl2 (400 mg / L) in the drinking water at acidic pH (pH 5.0), C - 15 rats that received CdCl2 (400 mg / L) in drinking water with basic pH (pH 8.0), D - 15 rats that received water at acidic pH (pH 5.0), E - 15 rats that received water with basic pH (pH 8.0), F - 15 rats that received water at neutral pH (pH 7.0 ); G - 8 rats that received cyclophosphamide as a single dose (50mg/kg) on the first day of the experiment (positive control for micronucleus test). Rats of groups A-F were euthanized after 6 months and the group G, 24 hours after the start of the experiment. We removed liver, kidney, pancreas, prostate, seminal vesicles, testes, and bone marrow. Results: There were no pre-neoplastic or neoplastic in liver, kidney, pancreas, seminal vesicles and testes in all groups. Only animals exposed to cadmium showed prostatic intraepithelial neoplasia grade one, being more prevalent in group B (p<0.05), although there was no increase in micronuclei (p>0,05). Conclusion: The acidic pH contributed to the formation of precancerous lesions in the prostate gland of animals exposed to cadmium. / Introdução: O cádmio é um metal pesado, muito utilizado na indústria. Ele poder originar o aparecimento de tumores em múltiplos órgãos. Objetivo: Avaliar o efeito do pH da água ingerida na gênese do câncer provocado por cádmio. Material e métodos: Utilizou-se 98 ratos Wistar, divididos em 7 grupos: A 15 ratos que receberam solução de cloreto de cádmio (CdCl2 - 400mg/L) na água de beber com pH neutro (pH 7,0); B 15 ratos que receberam CdCl2 (400mg/L) na água de beber com pH ácido (pH 5,0); C 15 ratos, que receberam CdCl2 (400mg/L) na água de beber com pH básico (pH 8,0). D 15 ratos que receberam água com pH ácido (pH 5,0); E 15 ratos que receberam água com pH básico (pH 8,0); F 15 ratos, que receberam água com pH neutro (pH 7,0); G 8 ratos que receberam ciclofosfamida em dose única (50mg/kg) no primeiro dia do experimento (controle positivo do teste do micronúcleo). Ratos dos grupos A-F foram eutanasiados após 6 meses e os do grupo G, 24 horas após o início do experimento. Retirou-se fígado, rim, pâncreas, próstata, vesícula seminal, testículos e medula óssea. Resultados: Não foram observadas lesões pré-neoplásicas nem neoplásicas no fígado, rim, pâncreas, vesícula seminal e testículos em todos os grupos. Somente animais expostos ao cádmio apresentaram neoplasia intraepitelial prostática de grau um, sendo mais prevalente no grupo B (p<0,05), porém não houve aumento de micronúcleos (p>0,05). Conclusão: O pH ácido contribuiu para formação de lesões pré-neoplásicas na próstata dos animais expostos ao cádmio.

Introduction: The aim of this study was to evaluate the role of genetic and lifestyle factors in the risk of onset and progression of colorectal and pancreatic cancer. The first part deals with the etiological factors and the importance of polymorphisms in biotransformation enzymes and genetic alterations in the gene CHEK2 in the origin of these malignancies. In the second part, the ABC transporter genes were analyzed as potential prognostic and predictive markers of a treatment's outcome. Materials and methods: The polymorphisms and other genetic alterations were detected using real-time PCR, allelespecific PCR and PCR-RFLP methods in DNA which was extracted from the blood of patients. The frequency of polymorphisms was evaluated and their importance was assessed with regard to the available epidemiological data. Gene expressions were determined by qPCR in paired samples of tumor tissue and adjacent non-tumorous parenchyma. Results: A majority of the observed polymorphisms failed to show a relationship between their presence and the risk of any of these malignancies. CYP2A13 variant allele*7 coding inactive enzyme was found in 7 of 265 controls and in none of 235 pancreatic carcinoma patients. In contrast, GSTP1-codon 105 Val variant allele and GSTT1-null genotype were associated with an elevated...

Die Studie des menschlichen T1D (Type 1 Diabetes), als eine Autoimmunerkrankung der Bauchspeicheldrüse, könnte man mit der Entdeckung von Insulin unter Verwendung von pankreatectomisierten Tieren argumentieren. Es folgten Studien zur Verfeinerung des Insulinersatzes, Verbesserung der Technologien und zur Untersuchung von Stoffwechsel- und Autoimmunphänomenen in Blutproben. Während Tiermodelle eine Endorgananalyse erlaubten, war die Untersuchung der menschlichen Bauchspeicheldrüse im Vergleich zu Studien mit peripherem Blut stark eingeschränkt. Anhand der so genannten peripheren Blutstudien, die nPOD (Network for Pancreatic Organ Donors with Diabetes), konnte eine menschliche Bauchspeicheldrüsenbank mit Proben von Personen zusammenzustellt werden, die durch die Analyse peripherer Blut-Autoantikörper (Wasserfall, et al., 2016) für T1D als risikoreich eingestuft wurde [CW 1]. Das Hauptziel meiner Dissertation bestand darin, mit diesen noch immer ansteigenden Proben, einige grundlegende Fragen der menschlichen T1D-Biologie zu beantworten. Zuerst konnten meine Kollegen und ich Insulitis bei Autoantikörper-positiven Probanden und bei Patienten mit T1D-Diagnose (Campbell-Thompson, et al., 2016a) studieren [CW 2]. Dabei fanden wir in 2 von 5 Pankreasproben Insulitis von Autoantikörper-positiven Probanden, beide mit der Kombination GADA und IA-2A. In Pankreasproben von T1D-Spendern mit weniger als einem Jahr Krankheitsdauer hatten 100% der Patienten eine Insulitis. Während, in denen mit mehr als einem Jahr Krankheitsdauer, 23% der Insulitis aufzeigten. Dies impliziert, dass die Insulitis vorwiegend zum Krankheitsbeginn auftritt und danach abnimmt. Das steht im extremen Gegensatz zum NOD-Mausmodell, bei dem Insulitis sehr früh auftritt und bei 100% der Inzuchtmäuse, unabhängig davon, ob sie T1D entwickeln oder nicht. Auch im Gegensatz zum NOD, während β-Zellmasse in T1D-Pankreasproben reduziert wurde, fehlte es nicht ganz. Wir bestätigten ebenfalls die Ergebnisse einer reduzierten Gesamtmasse menschlicher T1D-Pankreasproben, die nicht durch fehlende β-Zellen erklärt werden, da die nur einen kleinen Teil des Organs ausmachen. Dies impliziert den Verlust von exokrinem Gewebe in einer Art und Weise, die bis heute ungeklärt bleibt. Weiterhin haben wir getestet, ob Komplement eine potenzielle Rolle in T1D spielt, indem die C4d-Deposition als Marker für (Auto) Antikörpervermittelte Ereignisse verwendet wurde (Rowe, et al., 2013) [CW 3]. Unerwarteterweise fanden wir heraus, dass es erhöhte C4d Antikörperfärbung in T1D Pankreasproben gab. Dies implizierte die Verteilung potentieller exokriner Entzündungen und nicht unbedingt Insel-spezifische Ereignisse. Ob dies mit den Befunden der kleineren Bauchspeicheldrüse in T1D zusammenhängt, ist eine naheliegende Frage für zukünftige Studien. Bei der Zusammenstellung dieser Begriffe verwendete ich eine Technik, um Proteine aus gefrorenen Abschnitten der Bauchspeicheldrüse zu extrahieren und fand die Persistenz von Proinsulin, während die Insulin- und C-Peptid-Konzentrationen niedrig bis nicht nachweisbar durch ELISA in T1D-Proben waren (Wasserfall, et al., 2017) [CW 4]. Weiterhin konnten wir zeigen, dass INS mRNA durch ISH und RT-qPCR zusammen mit Proinsulin und Insulin durch Immunhistochemie anwesend war. Wir waren ebenfalls in der Lage, die Progression von T1D semi-quantifizieren, durch den Verlust von Insulin in Inselchen, sondern die Persistenz von Insulin-positiven Einzelzellen mit längerer Krankheitsdauer. Das Enzym PCSK1 wurde außerdem in T1D-Proben durch RT-qPCR reduziert, was auf einen möglichen Mechanismus für die unvollständige Verarbeitung von Proinsulin zu reifem Insulin und CPeptid hindeutet. Die Aufklärung dieses Prozesses und die Frage, ob die einzelnen insulinpositiven Zellen als therapeutische Option gerettet werden können oder ob sie die Zellen von β dedifferenzieren, werden in Zukunft weiterverfolgt.:TABLE OF CONTENTS II LIST OF ABBREVIATIONS IV LIST OF FIGURES V SUMMARY 1 1 MOTIVATION 1 2 STATE OF THE ART 6 2.1 Type 1 diabetes is an autoimmune disease 6 2.2 Models of type 1 diabetes 6 2.3 Humoral immune responses in type 1 diabetes 7 2.4 Genetics and Cell mediated immunity in type 1 diabetes 8 2.5 The lesion in type 1 diabetes 11 2.6 Prediction of type 1 diabetes in the general human population 14 3 OBJECTIVES 17 4 OWN RESEARCH RESULTS 19 4.1 Screening organ donors for type 1 diabetes autoantibodies is feasible 19 4.2 The insulitis lesion in humans reveals the heterogeneity of type 1 diabetes 20 4.3 Complement deposition in the human pancreas is not specific to islet blood vessels 22 4.4 The pancreas of individuals with type 1 diabetes shows progressive loss of insulin, but proinsulin persists 22 5 DISCUSSION 26 5.1 Even with a low prevalence disease such as T1D it is possible to screen organ donors for the presence of disease specific autoantibodies 26 5.2 Insulitis in the natural history of T1D 26 5.3 Complement deposition in the natural history of T1D 28 5.4 Presence of endocrine hormones in the natural history of T1D 29 6 RESUME 31 SUMMARY GERMAN 33 BIBLIOGRAPHY 35 LIST OF PUBLICATIONS 53 [CW1] Validation of a rapid type 1 diabetes screening assay for community-based screening of organ donors to identify subjects at increased risk for the disease 54 [CW2] Insulitis and β-Cell Mass in the Natural History of Type 1 Diabetes 66 [CW3] Increased Complement Activation in Human Type 1 Diabetes Pancreata 82 [CW4] Persistence of Pancreatic Insulin mRNA Expression and Proinsulin Protein in Type 1 Diabetes Pancreata 87 CURRICULUM VITAE 101 ACKNOWLEDGEMENTS 121 SELBSTSTÄNDIGKEITSERKLÄRUNG 124 ANLAGE 1 125 ANLAGE 2 127

BACKGROUND. The cytologic differentiation between neoplastic and reactive/reparative processes in the endoscopic ultrasound-guided fine-needle aspirations (EUS-FNA) of the pancreas can be difficult. Malignant transformation of the pancreatic ductal epithelium changes the expression of apomucins. The goal of the current study was to determine an optimal immunohistochemical panel of mucin (MUC) antibodies that would allow the cytomorphologic distinction of pancreatic ductal adenocarcinoma and its differentiation from reactive/reparative processes and inadvertently sampled gastric and duodenal mucosa. METHODS. Pancreatic EUS-FNA specimens performed on 351 patients were reviewed. Expression profiles of MUC1, 2, 5AC, and 6 were examined on 56 cell block sections and 26 follow-up pancreatectomy specimens. RESULTS. MUC1 and 6 expression was found in nonneoplastic pancreatic samples, whereas there was an absence of expression of MUC2 and 5AC. MUC2 was detected in mucosal goblets cells of the duodenum, MUC6 in Brunner glands, and MUC5AC in gastric foveolar cells. MUC5AC expression in differentiating ductal adenocarcinomas from benign conditions demonstrated better operating characteristics than either MUC1 or MUC6. The apomucin expression pattern both in cytology and follow-up surgical pathology specimens was similar. In surgical pathology specimens, the panel of 3 antibodies, MUC1+/MUC2-/MUC5AC+, was noted in 15 of 17 ductal carcinomas (88.2%). In nonneoplastic pancreatic tissue, the expression panel MUC1+/MUC2-/MUC5AC- was observed in 14 of 17 (82.4%) cases. In cytology specimens, the combination of MUC1+/MUC2-/MUC5AC+ was noted in 21 of 30 ductal carcinoma cases (70.0%), 3 of 6 atypical cases (50%), and 1 of 1 suspicious for malignancy cases (100%). The combination MUC1+/MUC2-/MUC5AC+ was not observed in any of the negative for malignancy or reactive cases (0 of 6). CONCLUSIONS. The most optimal panel for the diagnosis of ductal adenocarcinoma in both the EUS-FNA specimens is a panel including MUC1/MUC2/MUC5AC, whereas a panel of all 4 antibodies (MUC1, 2, 5AC, and 6) will in addition aid in differentiating inadvertently sampled normal/reactive duodenal and gastric epithelium from neoplastic pancreatic tissue.

The purpose of these studies was to investigate the impact of high-fat diet (HFD) exposure during pregnancy and the early post-natal period on fetal and post-natal development of the endocrine pancreas of the Japanese macaque. Specifically I hypothesized that the HFD would alter islet morphology and lead to disturbances in glucose homeostasis in these animals. Adult female Japanese macaques were placed on either a control (CTR) or HFD diet for 4 years. Fetuses were collected at gestational day 130 (G130), while other offspring from the CTR and HFD mothers were carried to term. After birth, infant animals were maintained with their mothers on the same diet then weaned onto either the CTR or HFD diet for five months. Animals were studied up to 13 months of age, yielding 4 postnatal groups: CTR/CTR, CTR/HFD, HFD/CTR and HFD/HFD. Pancreata were collected from these offspring for gene expression and immunohistochemical analysis. Physiological measurements, including body weight, body fat percentage, fasting glucose, insulin, glucagon and response to intravenous glucose tolerance tests (IVGTTs) and an intravenous insulin tolerance test (IVITT) were collected from the post-natal offspring. Total fetal islet mass and β cell mass were not changed, but α cell mass was significantly decreased in HFD fetuses, leading to a significant increase in the β cell to α cell ratio in HFD fetal offspring. The HFD offspring displayed a significant change from CTR offspring in expression of genes involved in glucose homeostasis and islet neogenesis, including PDX1, NeuroD, Glucokinase and Glut2. Postnatal HFD animals were significantly heavier than CTR offspring and had increased adiposity by 6-7 months of age. There was no significant effect on fasting or stimulated insulin secretion at this time point, but HFD offspring were significantly insulin resistant just prior to weaning. At 13 months of age, basal and glucose-stimulated insulin secretion were elevated in HFD/HFD animals and the CTR/HFD group displayed moderate insulin resistance. There was also a significant sex effect, with males from the HFD/CTR and HFD/HFD group having increased body weight and elevated fasting glucose. Although pancreata from both the HFD/HFD and CTR/HFD animals displayed significant changes in expression of genes involved in glucose homeostasis, the pattern was distinct for the two groups. Islet mass was also elevated in both of these groups; yet, HFD/HFD only displayed an increase in β cell area, while CTR/HFD had a concomitant increase in α cell area, which served to normalize the β cell to α cell ratio to control levels. In contrast, the HFD/HFD group exhibited a 40% increase in the β cell to α cell ratio. These studies demonstrate that in-utero exposure to a HFD leads to decreased α cell plasticity in response to chronic post-natal HFD consumption. Animals exposed to the HFD during pregnancy and the early post-natal period become insulin resistant, but remain normoglycemic. HFD consumption during the post-weaning period causes similar complications in glucose homeostasis and islet mass in both the CTR/HFD and HFD/HFD animals. However, there are distinct differences in the molecular and cellular adaptive response between these two groups.

No description available.

Pancreatic cancer is the 10th most common cancer diagnosis and 4th most common cause of cancer mortality in the United States, highlighting a disparity between disease prevalence and outcome. Ineffective drug delivery to these tumors contributes to the poor prognosis for this disease, as intravenous drug delivery is hampered by poor vascularity within these tumors. Bacterial therapy, or the use of bacterial components to treat disease, is thought to be able to overcome such drug delivery challenges; through a combination of tumor homing and long-term colonization, bacteria can be utilized to produce anti-cancer molecules directly within the cores of tumors. As such, here, we interrogate the feasibility of bacterial cancer therapy for pancreatic ductal adenocarcinoma (PDAC). Before delving too deeply into bacterial therapy design, it was important to first address one major limitation in therapeutic screening models. As a therapeutic should be effective against the entirety of the tumor, without a specific emphasis on the malignant epithelia, we developed and characterized a novel protocol for culturing ex vivo (explant) murine PDAC tissue with a corresponding protocol for human PDAC tissue. We demonstrated that these tumor slice explants retain the complex cellular architecture and population complexity throughout culture, making them a useful resource for not only therapeutic screens, but also paracrine interactions, which are infeasible to explore with in vitro and in vivo models. Use of these murine and human PDAC explant models assisted in the selection of a potent, bacterial-derived cytotoxin, theta toxin, as a potential therapeutic candidate for PDAC, in both bacteria lysate and live bacteria contexts. Ultimately, we employed a strain of a probiotic bacteria, E. coli Nissle 1917, as a ‘living drug’ to selectively produce theta toxin within the confines of a PDAC tumor in a mouse model of pancreatic cancer. In in vivo studies, we demonstrated that live bacteria preferentially colonize tumor tissue following a single, direct, intratumoral injection into the primary PDAC tumor. We found that not only did the bacteria colonize the injected tumor, but also translocated to distant regions of metastasis and secondary tumors such as anogenital papillomas. However, the long-term efficacy of this strategy is in question, as bacterial colonization and therapeutic capability waned after several weeks. Despite the limited time scale of the bacterial colonization, treatment with a single dose of live, theta toxin-producing bacteria provided a nearly 3-fold improvement in overall survival compared to vehicle and standard of care chemotherapy (gemcitabine) treatment arms. Preliminary evidence suggests that this improvement is due to a combination of the direct cytotoxic effect of the theta toxin and an inherently immunostimulatory capacity of these bacteria, resulting in an influx of anti-tumor immune cells and an overall reduction in immunosuppression phenotype markers. These findings suggest that bacterial therapy could be a useful tool for the treatment of pancreatic cancer, not solely due to the direct cytotoxic effect on the tumor, but with the potential for a combination treatment with immunotherapies.