Background & Aims: Despite advances in surgical care and chemotherapeutic regimens, the five-year survival rate for Stage IV Hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. YAP1 and β-Catenin co-activation occurs in 80% of children’s HB; however, a lack of conditional genetic models precludes exploration of tumor maintenance and therapeutic targets. Thus, the clinical need for a targeted therapy remains unmet. Given the predominance of YAP1 and β-catenin activation in children’s tumors, I sought to evaluate YAP1 as a therapeutic target in HB. Approach & Results: Herein, I engineered the first conditional murine model of HB using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A, constitutive β-CateninDelN90, and a luciferase reporter to murine liver. Tumor regression was evaluated using in vivo bioluminescent imaging, and tumor landscape characterized using RNA sequencing, ATAC sequencing and DNA foot-printing. Here I show that YAP1 withdrawal in mice mediates >90% tumor regression with survival for 230+ days. Mechanistically, YAP1 withdrawal promotes apoptosis in a subset of tumor cells and in remaining cells induces a cell fate switch driving therapeutic differentiation of HB tumors into Ki-67 negative “hbHep cells.” hbHep cells have hepatocyte-like morphology and partially restored mature hepatocyte gene expression. YAP1 withdrawal drives formation of hbHeps by modulating liver differentiation transcription factor (TF) occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and can rescue liver damage in mice. Conclusions: YAP1 withdrawal, without modulation of oncogenic β-Catenin, significantly regresses hepatoblastoma, providing the first in vivo data to support YAP1 as a therapeutic target for HB. Modulating YAP1 expression alone is sufficient to drive long-term regression in hepatoblastoma because it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.

INTRODUÇÃO: A secreção do hormônio de crescimento (GH) está diminuída em obesos. Existem controvérsias se esta diminuição é conseqüência ou um dos fatores causais da obesidade. Perda de peso leva a alguma recuperação da secreção de GH. Não há estudos publicados sobre o efeito da derivação gástrica (gastrojejunal) com anastomose em Y-de-Roux (BPG) sobre o perfil de secreção de 24 h de GH. Por outro lado, a ghrelina é um peptídeo secretagogo de GH produzido no estômago, orexigênico, lipogênico e adipogênico, cujos níveis oscilam ao longo do dia e estão diminuídos na obesidade. As variações circadianas de ghrelina têm papel no controle da homeostase energética e secreção de GH. O nível de ghrelina eleva-se com perda de peso induzida por dieta, mas os dados são controversos sobre mudanças desses níveis após cirurgias bariátricas. Este estudo tem por objetivo caracterizar os perfis de secreção de GH e ghrelina em mulheres com obesidade grau III antes e após BPG e suas correlações com variáveis metabólicas. MÉTODOS: Coletas de sangue a cada 20 minutos por 24 horas foram realizadas em obesas mórbidas não diabéticas na pré-menopausa antes e seis meses após BPG. O procedimento foi realizado em balanço calórico neutro por quatro dias. Foram dosados glicose e insulina; GH em todas as amostras e ghrelina às 08:00h, 10:00h, 12:00h, 19:00h e 02:00h. A taxa metabólica de repouso (TMR) foi avaliada por calorimetria indireta e as massas adiposa (MA) e magra (MM) foram medidas por DEXA. RESULTADOS: Houve uma redução de 27% do peso corporal e IMC (de 55,9 ± 6,2 kg/m2 para 40,7 ± 5,8 kg/m2, p<0,001) com elevação de vários parâmetros de secreção de GH (GH basal, GH médio, p<0,05; área, amplitude e número de picos, p<0,001); redução de glicemia (p = 0,03), insulinemia de jejum (p = 0,005) e HOMA (p = 0,004). Não houve diferença nos níveis de ghrelina basal, pós-prandial e médio. O GH médio apresentou correlação negativa com as mudanças no peso (p = 0,003; r = -0,631), IMC (p <0,001; r = -0,731), MA (p = 0,003; r = -0,635), MM (p = 0,02; r = -0,507), circunferência abdominal (p = 0,01; r = -0,555), TMR (p = 0,01; p = -0,539), insulina de jejum (p = 0,014, r = -0,538) e HOMA (p = 0,01; r = -0,560), mas não com a glicemia de jejum (p = 0,13; r = -0,354) e a ghrelina (p = 0,6; r = 0,118). O melhor determinante da secreção de GH foi o IMC sendo responsável por 54% da variação do GH médio (r2 = 0,54). CONCLUSÕES: Há uma recuperação parcial da secreção de GH, reduzida no pré-operatório em obesas mórbidas, após perda de peso induzida seis meses após a cirurgia, indicando que a secreção reduzida não é um fator primário ou causal da obesidade, mas sim uma conseqüência da obesidade e essa recuperação é independente do perfil de secreção de ghrelina / INTRODUCTION: Growth hormone (GH) concentration is decreased in obesity. It is not clear if reduced GH secretion is consequence or cause of the obese state. GH secretion is partially restored by weight loss. There are no published studies about the effect of Roux-en-Y gastric bypass (RYGBP) on GH secretory profile. Ghrelin is a GH releasing peptide produced by stomach, with orexigenic, lipogenic and adipogenic actions. Ghrelin levels oscillate throughout the day and are low in obesity. Circadian changes in ghrelin levels have a role both in energy homeostasis control and GH secretion. Ghrelin levels rise after diet-induced weight loss, but results are controverse in relation to changes in ghrelin levels after bariatric surgeries. In this study, we analyzed GH and ghrelin concentrations in morbidly obese women before and after RYGBP and its relationships with metabolic parameters. METHODS: Blood was sampled at 20-minute intervals during 24 hours in non diabetic pre-menopausal morbid obese women before and six months after RYGBP. The study was done after four days in neutral caloric balance. Fasting glucose and insulin were determined in basal samples. GH concentrations were measured in all samples and ghrelin in serum collected at 08:00h, 10:00h, 12:00h, 19:00h e 02:00h. Resting metabolic rate (RMR) was evaluated by indirect calorimetry and fat mass (FM) and free-fat mass (FFM) were measured by DEXA. RESULTS: A 27% drop in body weight and BMI (55.9 ± 6.2 kg/m2 to 40.7 ± 5.8 kg/m2, p<0.001), augmentation of spontaneous GH secretory episodes (basal and mean levels, p <0.05; area, amplitude and peak frequency, p <0.001); and reduction of fasting glucose (p = 0.03), insulinemia (p = 0.005) and HOMA (p = 0.004) were observed. Neither basal, post-prandial or mean ghrelin were changed. A negative correlation was found between mean GH levels and weight changes (p = 0.003, r = -0.631), BMI (p <0.001, r = -0.731), FM (p = 0.003, r = -0.635), FFM (p = 0.02, r = -0.507), waist (p = 0.01, r = -0.555), RMR (p = 0.01, p = -0.539), fasting insulin (p = 0.014, r = -0.538), as well as HOMA (p = 0.01, r = -0.560), but not between mean GH levels and glucose (p = 0.13, r = -0.354) or ghrelin (p = 0.6, r = 0.118). BMI accounted for 54% of the mean GH variation (r2 = 0.54). CONCLUSIONS: There is a partial recovery of GH secretion after weight loss induced by RYGBP, suggesting that a blunted secretion is not a primary or causal factor of obesity, but a consequence of the obese state. This recovery is independent of ghrelin secretory profile

O volvo colônico de sigmóide, juntamente com o fecaloma e a perfuração de cólon, representam as principais complicações do megacólon chagásico. O volvo do cólon sigmóide é definido como a torção axial da alça sobre seu meso, num ângulo maior que 180 graus, e leva a um quadro clínico de obstrução intestinal clássico que pode ainda ser complicado pela associação de fecaloma, perfuração e necrose. Estudou-se retrospectivamente os prontuários de 130 doentes, 55 mulheres e 75 homens com idade média de 58,1 anos com diagnóstico de volvo de cólon sigmóide, os quais foram tratados cirurgicamente na urgência. Os doentes foram selecionados em dois grupos, sendo o grupo I submetido a tratamento derivativo na urgência, compreendendo subgrupos: grupo IA: Destorção e colostomia em alça; grupo IB: Procedimento de Hartmann; grupo IC: Colectomia total. No grupo II foram avaliados os doentes submetidos a tratamento definitivo na urgência - operação de Duhamel-Haddad. Foram analisados dados referentes aos diagnósticos clínico, radiológico, etiológico, às complicações pós-operatórias e à mortalidade. A taxa de morbidade geral foi de 17,7%; a mortalidade na urgência foi de 6,2%. A morbi-mortalidade do tratamento cirúrgico de urgência foi: grupo IA = 7,8%; grupo IB = 20,8%; grupo IC = 22,2% e grupo II = 27,3%. A permanência hospitalar média foi de 5,6 dias, sendo a maior no grupo II, com média de 10 dias. Quando se analisa os doentes que foram submetidos eletivamente à operação de Duhamel-Haddad para reconstrução do trânsito intestinal, obtém-se uma taxa de morbi-mortalidade de 38,8% e permanência hospitalar de 9,9 dias (14,1 dias no total) no grupo submetido à destorção com colostomia em alça (grupo IA). No grupo que sofreu cirurgia de Hartmann (grupo IB) de 46,2% e a permanência hospitalar foi de 11,1 dias (17,2 dias no total). Concluiu-se que: 1) o ideal é associar o tratamento definitivo do megacólon ao do volvo colônico na urgência, quando as condições clínicas o permitirem, por implicar em menor tempo de internação e menor morbi-mortalidade total; 2) a operação de Duhamel-Haddad realizada ulteriormente associou-se a maior número de complicações do que quando feita na urgência / Acute sigmoid volvulus, fecal impaction and colonic perforation are the major complications of chagasic megacolon. Sigmoid volvulus is defined as the torsion of the sigmoid colon over its mesenteric axis more than 180 degrees. Clinically, patients present signs and symptoms of bowel obstruction that may be worsen if occurs isquemia, necrosis and perforation. Files of 130 cases admitted at emergency unit and diagnosed as having sigmoid volvulus was reviewed: 55 women and 75 men with age ranging from 26 to 89 years. Patients were divided into two groups: Group I: submitted to derivative colostomy; Group IA - volvulus detorsion and loop colostomy; Group IB - Hartmann\'s procedure and Group IC - total colectomy. Group II: submitted to Duhamel-Haddad procedure, i.e., simultaneous treatment of volvulus and its cause, the megacolon. Data concerning to clinical and radiological diagnosis, etiologic factors, surgical treatment and outcomes were analyzed. Overall morbidity rate were 17.7% and mortality were 6.2%. Morbi-mortality of the surgical treatment in urgency was: Group IA= 7.8%; Group IB = 20.8%; Group IC = 22.2% and Group II = 27.3%. Median hospital stay was 5.6 and 10 days for groups I and II, respectively. Elective intestinal transit reconstruction in groups IA and IB showed: morbidity of 38% and 46.2%, median hospital stay of 9.9 and 11.1 days, respectively. Conclusions: Duhamel-Haddad procedure as a surgical option for the treatment of sigmoid volvulus is associated with short hospital stay and less outcomes; Duhamel- Haddad procedure, as an emergency operation, was associated with fewer complications than programmed ones

Esôfago de Barrett é definido como a substituição do epitélio escamoso normal por epitélio colunar com metaplasia intestinal especializada (MIE), tendo como causa a persistência do refluxo gastro-esofágico. Seu diagnóstico é baseado na identificação endoscópica e na confirmação histológica da presença de MIE. Esôfago de Barrett é a principal causa do desenvolvimento do adenocarcinoma esofágico. Aos pacientes com esôfago de Barrett é recomendada vigilância endoscópica com biópsias seriadas tentando-se diagnosticar, precocemente, lesões precursoras ou o adenocarcinoma em estágio precoce e factível de resposta à terapia. O aumento da incidência do adenocarcinoma tem contribuído para o estudo de novas técnicas endoscópicas visando melhorar a detecção destas lesões. Este estudo foi realizado objetivando-se avaliar a eficácia da cromoscopia com azul de metileno, associada a magnificação de imagem com ácido acético, na detecção de MIE, displasia e adenocarcinoma. Prospectivamente, 35 pacientes com diagnóstico de esôfago de Barrett em acompanhamento ambulatorial, com extensão superior a 2,0 cm, realizaram dois exames de endoscopia digestiva alta, sendo um convencional com biópsias seriadas e um segundo com aplicação de azul de metileno, seguida do ácido acético, magnificação de imagem e biópsias. Realizaram-se biópsias adicionais de qualquer alteração do relevo mucoso. A freqüência diagnóstica da metaplasia intestinal especializada foi de 71,4% e 77,1% para biópsias orientadas pelo método convencional e pelo método da cromoscopia/magnificação de imagem, respectivamente (p=0,41). Freqüência de displasia ou adenocarcinoma foi de 9% para as biópsias orientadas pelo método convencional e 6% para biópsias orientadas pela cromoscopia/magnifcação de imagem. Tanto os pacientes com displasia de alto grau quanto aqueles com adenocarcinoma apresentaram alterações em sua superfície mucosa visíveis em ambos os métodos endoscópicos. A sensibilidade e a especificidade da cromoscopia, quando avaliamos as áreas coradas em detectar MIE foi de 88% e 50%, respectivamente. A sensibilidade e a especificidade das áreas não coradas em detectar displasia e/ou adenocarcinoma foi de 75% e 100%, respectivamente. A sensibilidade e a especificidade da magnificação de imagem, quando avaliamos as áreas com padrão viliforme em detectar MIE foi 88% e 50%, respectivamente. Tanto a sensibilidade quanto a especificidade das áreas com padrão amorfo em detectar displasia e/ou adenocarcinoma foi de 100%. A sensibilidade e a especificidade da cromoscopia/magnificação de imagem, para padrão corado e viliforme, em detectar MIE foi de 83% e 50%, respectivamente. Já a sensibilidade e a especificidade das áreas não coradas e com padrão amorfo em detectar displasia e/ou adenocarcinoma teve seu cálculo prejudicado pela pequena amostra estudada. Na comparação dos dois métodos empregados, verificaram-se resultados similares na detecção de metaplasia intestinal, displasia e câncer. A realização de cromoscopia/magnificação de imagem proporcionou: alta sensibilidade e baixa especificidade na detecção da metaplasia intestinal especializada e baixa sensibilidade e alta especificidade na detecção de displasia ou adenocarcinoma. Alterações da superfície mucosa corresponderam as áreas neoplásicas. / Barrett\'s esophagus is defined as the replacement of the normal squamous epithelium by columnar lined esophagus. The diagnosis requires endoscopically visible columnar lined esophagus and histologic identification of characteristic specialized intestinal-type metaplasia (SIM). Gastroesophageal reflux has been proposed as a risk factor for Barrett`s esophagus and this disease has been shown to be the main cause of esophageal adenocarcinoma. After the diagnosis of Barrett`s esophagus, endoscopy surveillance is recommended with multiple biopsies of the columnar lined esophagus at quadrants of 2 cm intervals to determine epithelial dysplasia or adenocarcinoma in early and curable stage. Due to the increase in the incidence of esophageal adenocarcinoma new techniques of endoscopic surveillance have been proposed. The aim of this study was to evaluate the efficacy of magnification chromoendoscopy with methylene blue and acetic acid for the detection of intestinal metaplasia, dysplasia and cancer. Prospectively, 35 patients with Barrett\'s esophagus extending for more than 2,0 cm, underwent two upper digestive endoscopy procedures, including one with conventional biopsies and other with chromoendoscopy using methylene blue and acetic acid instillation, magnification and biopsies. Biopsies were also taken from any suspicious mucosal area. The incidence of MIE were 71,4% e 77,1% from conventional biopsies and chromoendoscopy/magnification, respectively. Dysplasia and adenocarcinoma were diagnosed in 9% and 6% throught conventional biopsies and chromoendoscopy/magnification, respectively. Patients with high grade dysplasia or adenocarcinoma revealed mucosal alterations. The sensitivity and specificity rates for chromoendoscopy for stained areas for MIE were 88% and 50%, respectively. The sensitivity and specificity rates for non stained areas for dysplasia and cancer were 75% and 100%, respectively. The sensitivity and specificity rates for magnification for villous areas for MIE were 88% and 50%, respectively. The sensitivity and specificity rates for distorted areas for dysplasia and cancer were 100%. The sensitivity and specificity rates for chromoendoscopy/magnification (for stained and villous areas) for MIE were 83% and 50%, respectively. The sensitivity and specificity rates for non stained and distorted areas couldn?t be evaluated due to the small number of patients. In conclusion, results of the two methods were similar in detecting intestinal metaplasia, dysplasia and cancer. The chromoendoscopy/magnification method procedure provides high sensitivity and low specificity rates in detecting MIE and low sensitivity and high specificity rates in detecting dysplasia and adenocarcinoma. Alterations in the mucosa corresponded to cancer and dysplasia.

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. 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The endoplasmic reticulum (ER) is a multi-functional cellular compartment that functions in protein folding, lipid biosynthesis, and calcium homeostasis. Perturbations to ER function lead to the dysregulation of ER homeostasis, causing the accumulation of unfolded and misfolded proteins in the cell. This is a state of ER stress. ER stress elicits a cytoprotective, adaptive signaling cascade to mitigate stress, the Unfolded Protein Response (UPR). As long as the UPR can moderate stress, cells can produce the proper amount of proteins and maintain a state of homeostasis. If the UPR, however, is dysfunctional and fails to achieve this, cells will undergo apoptosis. Diabetes mellitus is a group of metabolic disorders characterized by persistent high blood glucose levels. The pathogenesis of this disease involves pancreatic β-cell dysfunction: an abnormality in the primary function of the β-cell, insulin production and secretion. Activation of the UPR is critical to pancreatic β-cell survival, where a disruption in ER stress signaling can lead to cell death and consequently diabetes. There are several models of ER stress leading to diabetes. Wolcott-Rallison syndrome, for example, occurs when there is a mutation in the gene encoding one of the master regulators of the UPR, PKR-like ER kinase (PERK). In this dissertation, we show that Wolfram Syndrome 1 (WFS1), an ER transmembrane protein, is a component of the UPR and is a downstream target of two of the master regulators of the UPR, Inositol Requiring 1 (IRE1) and PERK. WFS1 mutations lead to Wolfram syndrome, a non-autoimmune form of type 1 diabetes accompanied by optical atrophy and other neurological disorders. It has been shown that patients develop diabetes due to the selective loss of their pancreatic β-cells. Here we define the underlying molecular mechanism of β-cell loss in Wolfram syndrome, and link this cell loss to ER stress and a dysfunction in a component of the UPR, WFS1. We show that WFS1 expression is localized to the β-cell of the pancreas, it is upregulated during insulin secretion and ER stress, and its inactivation leads to chronic ER stress and apoptosis. This dissertation also reveals the previously unknown function of WFS1 in the UPR. Positive regulation of the UPR has been extensively studied, however, the precise mechanisms of negative regulation of this signaling pathway have not. Here we report that WFS1 regulates a key transcription factor of the UPR, activating transcription factor 6 (ATF6), through the ubiquitin-proteasome pathway. WFS1 expression decreases expression levels of ATF6 target genes and represses ATF6-mediated activation of the ER stress response (ERSE) promoter. WFS1 recruits and stabilizes an E3 ubiquitin ligase, HMG-CoA reductase degradation protein 1 (HRD1), on the ER membrane. The WFS1-HRD1 complex recruits ATF6 to the proteasome and enhances its ubiquitination and proteasome-mediated degradation, leading to suppression of the UPR under non-stress conditions. In response to ER stress, ATF6 is released from WFS1 and activates the UPR to mitigate ER stress. This body of work reveals a novel role for WFS1 in the UPR, and a novel mechanism for regulating ER stress signaling. These findings also indicate that hyperactivation of the UPR can lead to cellular dysfunction and death. This supports the notion that tight regulation of ER stress signaling is crucial to cell survival. This unanticipated role of WFS1 for a feedback loop of the UPR is relevant to diseases caused by chronic hyperactivation of ER stress signaling network such as pancreatic β-cell death in diabetes and neurodegeneration.

INTRODUÇÃO: A secreção do hormônio de crescimento (GH) está diminuída em obesos. Existem controvérsias se esta diminuição é conseqüência ou um dos fatores causais da obesidade. Perda de peso leva a alguma recuperação da secreção de GH. Não há estudos publicados sobre o efeito da derivação gástrica (gastrojejunal) com anastomose em Y-de-Roux (BPG) sobre o perfil de secreção de 24 h de GH. Por outro lado, a ghrelina é um peptídeo secretagogo de GH produzido no estômago, orexigênico, lipogênico e adipogênico, cujos níveis oscilam ao longo do dia e estão diminuídos na obesidade. As variações circadianas de ghrelina têm papel no controle da homeostase energética e secreção de GH. O nível de ghrelina eleva-se com perda de peso induzida por dieta, mas os dados são controversos sobre mudanças desses níveis após cirurgias bariátricas. Este estudo tem por objetivo caracterizar os perfis de secreção de GH e ghrelina em mulheres com obesidade grau III antes e após BPG e suas correlações com variáveis metabólicas. MÉTODOS: Coletas de sangue a cada 20 minutos por 24 horas foram realizadas em obesas mórbidas não diabéticas na pré-menopausa antes e seis meses após BPG. O procedimento foi realizado em balanço calórico neutro por quatro dias. Foram dosados glicose e insulina; GH em todas as amostras e ghrelina às 08:00h, 10:00h, 12:00h, 19:00h e 02:00h. A taxa metabólica de repouso (TMR) foi avaliada por calorimetria indireta e as massas adiposa (MA) e magra (MM) foram medidas por DEXA. RESULTADOS: Houve uma redução de 27% do peso corporal e IMC (de 55,9 ± 6,2 kg/m2 para 40,7 ± 5,8 kg/m2, p<0,001) com elevação de vários parâmetros de secreção de GH (GH basal, GH médio, p<0,05; área, amplitude e número de picos, p<0,001); redução de glicemia (p = 0,03), insulinemia de jejum (p = 0,005) e HOMA (p = 0,004). Não houve diferença nos níveis de ghrelina basal, pós-prandial e médio. O GH médio apresentou correlação negativa com as mudanças no peso (p = 0,003; r = -0,631), IMC (p <0,001; r = -0,731), MA (p = 0,003; r = -0,635), MM (p = 0,02; r = -0,507), circunferência abdominal (p = 0,01; r = -0,555), TMR (p = 0,01; p = -0,539), insulina de jejum (p = 0,014, r = -0,538) e HOMA (p = 0,01; r = -0,560), mas não com a glicemia de jejum (p = 0,13; r = -0,354) e a ghrelina (p = 0,6; r = 0,118). O melhor determinante da secreção de GH foi o IMC sendo responsável por 54% da variação do GH médio (r2 = 0,54). CONCLUSÕES: Há uma recuperação parcial da secreção de GH, reduzida no pré-operatório em obesas mórbidas, após perda de peso induzida seis meses após a cirurgia, indicando que a secreção reduzida não é um fator primário ou causal da obesidade, mas sim uma conseqüência da obesidade e essa recuperação é independente do perfil de secreção de ghrelina / INTRODUCTION: Growth hormone (GH) concentration is decreased in obesity. It is not clear if reduced GH secretion is consequence or cause of the obese state. GH secretion is partially restored by weight loss. There are no published studies about the effect of Roux-en-Y gastric bypass (RYGBP) on GH secretory profile. Ghrelin is a GH releasing peptide produced by stomach, with orexigenic, lipogenic and adipogenic actions. Ghrelin levels oscillate throughout the day and are low in obesity. Circadian changes in ghrelin levels have a role both in energy homeostasis control and GH secretion. Ghrelin levels rise after diet-induced weight loss, but results are controverse in relation to changes in ghrelin levels after bariatric surgeries. In this study, we analyzed GH and ghrelin concentrations in morbidly obese women before and after RYGBP and its relationships with metabolic parameters. METHODS: Blood was sampled at 20-minute intervals during 24 hours in non diabetic pre-menopausal morbid obese women before and six months after RYGBP. The study was done after four days in neutral caloric balance. Fasting glucose and insulin were determined in basal samples. GH concentrations were measured in all samples and ghrelin in serum collected at 08:00h, 10:00h, 12:00h, 19:00h e 02:00h. Resting metabolic rate (RMR) was evaluated by indirect calorimetry and fat mass (FM) and free-fat mass (FFM) were measured by DEXA. RESULTS: A 27% drop in body weight and BMI (55.9 ± 6.2 kg/m2 to 40.7 ± 5.8 kg/m2, p<0.001), augmentation of spontaneous GH secretory episodes (basal and mean levels, p <0.05; area, amplitude and peak frequency, p <0.001); and reduction of fasting glucose (p = 0.03), insulinemia (p = 0.005) and HOMA (p = 0.004) were observed. Neither basal, post-prandial or mean ghrelin were changed. A negative correlation was found between mean GH levels and weight changes (p = 0.003, r = -0.631), BMI (p <0.001, r = -0.731), FM (p = 0.003, r = -0.635), FFM (p = 0.02, r = -0.507), waist (p = 0.01, r = -0.555), RMR (p = 0.01, p = -0.539), fasting insulin (p = 0.014, r = -0.538), as well as HOMA (p = 0.01, r = -0.560), but not between mean GH levels and glucose (p = 0.13, r = -0.354) or ghrelin (p = 0.6, r = 0.118). BMI accounted for 54% of the mean GH variation (r2 = 0.54). CONCLUSIONS: There is a partial recovery of GH secretion after weight loss induced by RYGBP, suggesting that a blunted secretion is not a primary or causal factor of obesity, but a consequence of the obese state. This recovery is independent of ghrelin secretory profile

Esôfago de Barrett é definido como a substituição do epitélio escamoso normal por epitélio colunar com metaplasia intestinal especializada (MIE), tendo como causa a persistência do refluxo gastro-esofágico. Seu diagnóstico é baseado na identificação endoscópica e na confirmação histológica da presença de MIE. Esôfago de Barrett é a principal causa do desenvolvimento do adenocarcinoma esofágico. Aos pacientes com esôfago de Barrett é recomendada vigilância endoscópica com biópsias seriadas tentando-se diagnosticar, precocemente, lesões precursoras ou o adenocarcinoma em estágio precoce e factível de resposta à terapia. O aumento da incidência do adenocarcinoma tem contribuído para o estudo de novas técnicas endoscópicas visando melhorar a detecção destas lesões. Este estudo foi realizado objetivando-se avaliar a eficácia da cromoscopia com azul de metileno, associada a magnificação de imagem com ácido acético, na detecção de MIE, displasia e adenocarcinoma. Prospectivamente, 35 pacientes com diagnóstico de esôfago de Barrett em acompanhamento ambulatorial, com extensão superior a 2,0 cm, realizaram dois exames de endoscopia digestiva alta, sendo um convencional com biópsias seriadas e um segundo com aplicação de azul de metileno, seguida do ácido acético, magnificação de imagem e biópsias. Realizaram-se biópsias adicionais de qualquer alteração do relevo mucoso. A freqüência diagnóstica da metaplasia intestinal especializada foi de 71,4% e 77,1% para biópsias orientadas pelo método convencional e pelo método da cromoscopia/magnificação de imagem, respectivamente (p=0,41). Freqüência de displasia ou adenocarcinoma foi de 9% para as biópsias orientadas pelo método convencional e 6% para biópsias orientadas pela cromoscopia/magnifcação de imagem. Tanto os pacientes com displasia de alto grau quanto aqueles com adenocarcinoma apresentaram alterações em sua superfície mucosa visíveis em ambos os métodos endoscópicos. A sensibilidade e a especificidade da cromoscopia, quando avaliamos as áreas coradas em detectar MIE foi de 88% e 50%, respectivamente. A sensibilidade e a especificidade das áreas não coradas em detectar displasia e/ou adenocarcinoma foi de 75% e 100%, respectivamente. A sensibilidade e a especificidade da magnificação de imagem, quando avaliamos as áreas com padrão viliforme em detectar MIE foi 88% e 50%, respectivamente. Tanto a sensibilidade quanto a especificidade das áreas com padrão amorfo em detectar displasia e/ou adenocarcinoma foi de 100%. A sensibilidade e a especificidade da cromoscopia/magnificação de imagem, para padrão corado e viliforme, em detectar MIE foi de 83% e 50%, respectivamente. Já a sensibilidade e a especificidade das áreas não coradas e com padrão amorfo em detectar displasia e/ou adenocarcinoma teve seu cálculo prejudicado pela pequena amostra estudada. Na comparação dos dois métodos empregados, verificaram-se resultados similares na detecção de metaplasia intestinal, displasia e câncer. A realização de cromoscopia/magnificação de imagem proporcionou: alta sensibilidade e baixa especificidade na detecção da metaplasia intestinal especializada e baixa sensibilidade e alta especificidade na detecção de displasia ou adenocarcinoma. Alterações da superfície mucosa corresponderam as áreas neoplásicas. / Barrett\'s esophagus is defined as the replacement of the normal squamous epithelium by columnar lined esophagus. The diagnosis requires endoscopically visible columnar lined esophagus and histologic identification of characteristic specialized intestinal-type metaplasia (SIM). Gastroesophageal reflux has been proposed as a risk factor for Barrett`s esophagus and this disease has been shown to be the main cause of esophageal adenocarcinoma. After the diagnosis of Barrett`s esophagus, endoscopy surveillance is recommended with multiple biopsies of the columnar lined esophagus at quadrants of 2 cm intervals to determine epithelial dysplasia or adenocarcinoma in early and curable stage. Due to the increase in the incidence of esophageal adenocarcinoma new techniques of endoscopic surveillance have been proposed. The aim of this study was to evaluate the efficacy of magnification chromoendoscopy with methylene blue and acetic acid for the detection of intestinal metaplasia, dysplasia and cancer. Prospectively, 35 patients with Barrett\'s esophagus extending for more than 2,0 cm, underwent two upper digestive endoscopy procedures, including one with conventional biopsies and other with chromoendoscopy using methylene blue and acetic acid instillation, magnification and biopsies. Biopsies were also taken from any suspicious mucosal area. The incidence of MIE were 71,4% e 77,1% from conventional biopsies and chromoendoscopy/magnification, respectively. Dysplasia and adenocarcinoma were diagnosed in 9% and 6% throught conventional biopsies and chromoendoscopy/magnification, respectively. Patients with high grade dysplasia or adenocarcinoma revealed mucosal alterations. The sensitivity and specificity rates for chromoendoscopy for stained areas for MIE were 88% and 50%, respectively. The sensitivity and specificity rates for non stained areas for dysplasia and cancer were 75% and 100%, respectively. The sensitivity and specificity rates for magnification for villous areas for MIE were 88% and 50%, respectively. The sensitivity and specificity rates for distorted areas for dysplasia and cancer were 100%. The sensitivity and specificity rates for chromoendoscopy/magnification (for stained and villous areas) for MIE were 83% and 50%, respectively. The sensitivity and specificity rates for non stained and distorted areas couldn?t be evaluated due to the small number of patients. In conclusion, results of the two methods were similar in detecting intestinal metaplasia, dysplasia and cancer. The chromoendoscopy/magnification method procedure provides high sensitivity and low specificity rates in detecting MIE and low sensitivity and high specificity rates in detecting dysplasia and adenocarcinoma. Alterations in the mucosa corresponded to cancer and dysplasia.

O volvo colônico de sigmóide, juntamente com o fecaloma e a perfuração de cólon, representam as principais complicações do megacólon chagásico. O volvo do cólon sigmóide é definido como a torção axial da alça sobre seu meso, num ângulo maior que 180 graus, e leva a um quadro clínico de obstrução intestinal clássico que pode ainda ser complicado pela associação de fecaloma, perfuração e necrose. Estudou-se retrospectivamente os prontuários de 130 doentes, 55 mulheres e 75 homens com idade média de 58,1 anos com diagnóstico de volvo de cólon sigmóide, os quais foram tratados cirurgicamente na urgência. Os doentes foram selecionados em dois grupos, sendo o grupo I submetido a tratamento derivativo na urgência, compreendendo subgrupos: grupo IA: Destorção e colostomia em alça; grupo IB: Procedimento de Hartmann; grupo IC: Colectomia total. No grupo II foram avaliados os doentes submetidos a tratamento definitivo na urgência - operação de Duhamel-Haddad. Foram analisados dados referentes aos diagnósticos clínico, radiológico, etiológico, às complicações pós-operatórias e à mortalidade. A taxa de morbidade geral foi de 17,7%; a mortalidade na urgência foi de 6,2%. A morbi-mortalidade do tratamento cirúrgico de urgência foi: grupo IA = 7,8%; grupo IB = 20,8%; grupo IC = 22,2% e grupo II = 27,3%. A permanência hospitalar média foi de 5,6 dias, sendo a maior no grupo II, com média de 10 dias. Quando se analisa os doentes que foram submetidos eletivamente à operação de Duhamel-Haddad para reconstrução do trânsito intestinal, obtém-se uma taxa de morbi-mortalidade de 38,8% e permanência hospitalar de 9,9 dias (14,1 dias no total) no grupo submetido à destorção com colostomia em alça (grupo IA). No grupo que sofreu cirurgia de Hartmann (grupo IB) de 46,2% e a permanência hospitalar foi de 11,1 dias (17,2 dias no total). Concluiu-se que: 1) o ideal é associar o tratamento definitivo do megacólon ao do volvo colônico na urgência, quando as condições clínicas o permitirem, por implicar em menor tempo de internação e menor morbi-mortalidade total; 2) a operação de Duhamel-Haddad realizada ulteriormente associou-se a maior número de complicações do que quando feita na urgência / Acute sigmoid volvulus, fecal impaction and colonic perforation are the major complications of chagasic megacolon. Sigmoid volvulus is defined as the torsion of the sigmoid colon over its mesenteric axis more than 180 degrees. Clinically, patients present signs and symptoms of bowel obstruction that may be worsen if occurs isquemia, necrosis and perforation. Files of 130 cases admitted at emergency unit and diagnosed as having sigmoid volvulus was reviewed: 55 women and 75 men with age ranging from 26 to 89 years. Patients were divided into two groups: Group I: submitted to derivative colostomy; Group IA - volvulus detorsion and loop colostomy; Group IB - Hartmann\'s procedure and Group IC - total colectomy. Group II: submitted to Duhamel-Haddad procedure, i.e., simultaneous treatment of volvulus and its cause, the megacolon. Data concerning to clinical and radiological diagnosis, etiologic factors, surgical treatment and outcomes were analyzed. Overall morbidity rate were 17.7% and mortality were 6.2%. Morbi-mortality of the surgical treatment in urgency was: Group IA= 7.8%; Group IB = 20.8%; Group IC = 22.2% and Group II = 27.3%. Median hospital stay was 5.6 and 10 days for groups I and II, respectively. Elective intestinal transit reconstruction in groups IA and IB showed: morbidity of 38% and 46.2%, median hospital stay of 9.9 and 11.1 days, respectively. Conclusions: Duhamel-Haddad procedure as a surgical option for the treatment of sigmoid volvulus is associated with short hospital stay and less outcomes; Duhamel- Haddad procedure, as an emergency operation, was associated with fewer complications than programmed ones

Background: Cystic fibrosis (CF) remains the most common fatal monogenic disease in the US, affecting 1 in 3,300 live births. CF is the result of mutations in CFTR, a chloride channel and regulator of other ion channels. The mechanisms by which CFTR mutations cause chronic lung disease in CF are not fully defined, but may include the combined effects of altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways. We have shown that Cftr-/- mice mount an exaggerated IgE response towards Aspergillus fumigatus (Af) when compared to Cftr+/+ mice. Along with the increased IgE levels, the Cftr-/- mice had higher levels of IL-13 and IL-4, mimicking both the Th-2 biased immune responses and predilection to mounting Af-specifc IgE seen in CF patients. Herein we hypothesize that these immune aberrations are primarily due to the lack of Cftr expression in lymphocytes rather than with Cftr deficiency in the epithelium. Results: Our results indicate that adoptive transfer experiments with Cf splenocytes confer higher IgE response to Af in host mice as compared to hosts receiving wild-type splenocytes. The predilection of Cftr-deficient lymphocytes to mount Th2 responses was confirmed by in vitro antigen recall experiments, where higher levels of IL-13 and IL-4 where seen only in the presence of Cftr-deficient lymphocytes. Conclusive data on this phenomenon were obtained with conditional Cftr knockout mice, where mice lacking Cftr in T-cell lineages developed the higher IgE titers as compared to their wild-type littermate controls. Further analysis of Cftr-deficient lymphocytes revealed an enhanced intracellular Ca 2+ flux in response to T cell receptor activation as compared to normal lymphocytes. This was accompanied by a significant increase in nuclear localization of the calcium-sensitive transcription factor NFAT, which could contribute to the enhanced secretion of IL-13 and other cytokines. Conclusions: In summary, our data identified that CFTR dysfunction in T cells can lead directly to aberrant immune responses. This is the first instance that a CF related phenotype has been entirely modeled in vivo by selectively knocking out CFTR in the immune system. Specifically, Cftr deficient lymphocytes directed skewed responses to Aspergillus fumigatus , leading to a higher than normal IgE response. These findings implicate the lymphocyte population as a potentially important target for therapeutics directed to the treatment of CF lung disease.