Defective Apoptosis In Intestinal And Mesenteric Adipose Tissue Of Crohn's Disease Patients.

Crohn's disease (CD) is associated with complex pathogenic pathways involving defects in apoptosis mechanisms. Recently, mesenteric adipose tissue (MAT) has been associated with CD ethiopathology, since adipose thickening is detected close to the affected intestinal area. However, the potential role of altered apoptosis in MAT of CD has not been addressed. To evaluate apoptosis in the intestinal mucosa and MAT of patients with CD. Samples of intestinal mucosa and MAT from patients with ileocecal CD and from non-inflammatory bowel diseases patients (controls) were studied. Apoptosis was assessed by TUNEL assay and correlated with the adipocytes histological morphometric analysis. The transcriptional and protein analysis of selected genes and proteins related to apoptosis were determined. TUNEL assay showed fewer apoptotic cells in CD, when compared to the control groups, both in the intestinal mucosa and in MAT. In addition, the number of apoptotic cells (TUNEL) correlated significantly with the area and perimeter of the adipose cells in MAT. Transcriptomic and proteomic analysis reveal a significantly lower transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the controls; low protein levels of Bax were found localized in the lamina propria and not in the epithelium of this tissue. Furthermore, higher level of Bcl-2 and low level of Caspase 3 were seen in the MAT of CD patients. The defective apoptosis in MAT may explain the singular morphological characteristics of this tissue in CD, which may be implicated in the pathophysiology of the disease.9e9854

Apoptosis evaluation in mucosa intestinal and mesenteric adipose tissue of Crohn disease patients

Orientadores: Raquel Franco Leal, Luciana Rodrigues de MeirellesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A doença de Crohn (DC) está associada com as vias patogênicas complexas envolvendo alterações nos mecanismos de apoptose. Recentemente, o tecido adiposo mesenterial (TAM) tem sido associado com a etiopatogenia DC, uma vez que é verificado um aumento da espessura do tecido adiposo mesenterial e envolvimento circunferencial da alça intestinal próximo à área intestinal afetada. Entretanto, não há estudos do mecanismo de apoptose no TAM na DC. Desta forma, o objetivo desse estudo foi avaliar apoptose na mucosa intestinal e no TAM de pacientes com DC, além de correlacionar estes achados com o estudo morfométrico dos adipócitos neste tecido. Casuística e Métodos: Foram estudadas amostras de mucosa intestinal e TAM de 10 pacientes com DC ileocecal e de 8 pacientes sem doenças inflamatórias intestinais (controles). A apoptose foi avaliada pelo ensaio de TUNEL e correlacionada com a análise histológica e morfométrica dos adipócitos. Determinou-se a análise transcripcional e proteica de uma seleção de genes e proteínas relacionadas com o mecanismo de apoptose. Resultados: O ensaio de TUNEL mostrou menor número de células em apoptose na DC, quando comparado com os grupos controles, tanto na mucosa intestinal quanto no TAM. Além disso, o número de adipócitos em apoptose (TUNEL) correlacionou significativamente com a área e perímetro destas células. A análise transcripcional e proteica revelaram níveis de transcritos e de proteína Bax significativamente mais baixos na mucosa intestinal de DC, em comparação com os respectivos controles; sendo que os baixos níveis de Bax foram encontrados na lâmina própria e não no epitélio intestinal; não houve diferença na expressão de Bax no TAM. Além disso, maior nível de Bcl-2 e baixo nível de Caspase 3 foram vistos no TAM de pacientes com DC. Conclusão: A alteração da apoptose no TAM pode explicar as características morfológicas singulares deste tecido na DC, que podem estar implicadas na fisiopatologia da doençaAbstract: Crohn¿s disease (CD) is associated with complex pathogenic pathways involving defects in apoptosis mechanisms. Recently, mesenteric adipose tissue (MAT) has been associated with CD ethiopathology, since adipose thickening is detected close to the affected intestinal area. However, there are no studies concern apoptosis pathways in MAT of CD. Therefore, the aim of this study was to evaluate apoptosis in the intestinal mucosa and MAT of patients with CD, besides to correlate these findings with the morphometricstudy of the adipocytes from MAT.Patients and Methods: Samples of intestinal mucosa and MAT from 10 patients with ileocecal CD and from 8 non-inflammatory bowel diseases patients (controls) were studied. Apoptosis was assessed by TUNEL assay and correlated with the adipocytes histological morphometric analysis. The transcriptional and protein analysis of selected genes and proteins related to apoptosis were determined. Results: TUNEL assay showed fewer apoptotic cells in CD, when compared to the control groups, both in the intestinal mucosa and in MAT. In addition, the number of apoptotic cells (TUNEL) correlated significantly with the area and perimeter of the adipose cells in MAT. Transcriptomic and proteomic analysis reveal a significantly lower transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the controls; low protein levels of Bax were found localized in the lamina propria and not in the epithelium of this tissue. Furthermore, higher level of Bcl-2 and low level of Caspase 3 were seen in the MAT of CD patients. Conclusion: The defective apoptosis in MAT may explain the singular morphological characteristics of this tissue in CD, which may be implicated in the pathophysiology of the diseaseDoutoradoFisiopatologia CirúrgicaDoutora em Ciência

Study of swallowing function, food intake and nutritional profile of patients after stroke

Orientador: Lucia Figueiredo MourãoDissertação (mestrado profissional) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: O acidente vascular cerebral (AVC) é uma doença cérebro-vascular, sendo uma das maiores causas de mortalidade em todo o mundo. Os principais fatores de risco para as doenças cardiocerebrovasculares são: hipertensão, a obesidade e o sobrepeso, o diabetes, dietas aterogênicas. Dentre as seqüelas mais encontradas estão: alteração motora global, complicações respiratórias, nutricionais e na deglutição. Este trabalho tem como objetivo, avaliar aspectos clínicos, nutricionais e da deglutição em sujeitos após AVC acompanhados em um Hospital Público Terciário da cidade de Campinas/ SP. Estudo de corte transversal, os sujeitos da pesquisa realizaram avaliação da deglutição e nutricional. Foi realizada o exame endoscópico da deglutição (FEES) com base no procedimento realizada a classificação da severidade da disfagia (SD) e a funcionalidade da ingestão oral (Functional Oral Intake Scale - FOIS). Para traçar o perfil nutricional desta população, utilizaram-se métodos antropométricos, anamnese e recordatório alimentar de 24 horas. Os sujeitos foram avaliados no ambulatório de Otorrinolaringologia/disfagia de um hospital terciário do interior do estado de São Paulo. Foram incluídos sujeitos que tiveram AVC (até 1 ano). Realizou-se análise estatística dos dados e adotou-se como nível de significância p<0,05.Resultados: População constituiu-se de 38 sujeitos, sendo 21 do gênero masculino, com idade de média 60 anos e 17 do gênero feminino com idade média 59 anos. Destes 34 eram hipertensos e 19 diabéticos. O número de AVC se único ou múltiplo para mulheres e homens foram respectivamente: 9 e 8; 14 e 7. As médias dos dados bioquímicos encontram-se dentro do padrão de recomendação. Houve correlação positiva de modo estatisticamente significante entre as variáveis: idade com circunferência abdominal; IMC com as circunferências abdominal, da cintura, do braço, peso corporal. Além do percentual de gordura com a circunferência abdominal. Na avaliação da deglutição os sujeitos apresentaram classificação para FOIS de nível 5,6,7, respectivamente 73%, 8%, 19%; a severidade da disfagia 23% normal, 58% leve, 15% moderado, 4% severa. A média dos dados antropométricos foram: IMC 25,76kg/m2; circunferência da cintura 92,3cm; circunferência abdominal 99,44cm. A ingestão alimentar apresentou-se acima das recomendações diárias para carboidratos, proteínas e lipídios. Ao correlacionar diabetes, hipertensão, número de AVC com a SD e a FOIS, verificou-se que não houve correlação estatística significativa entre SD e hipertensão (r= 0,196), e diabetes (r= 1,177), nem entre o número de AVC e funcionalidade (r=0,29) ou SD. No entanto, identificou-se correlação moderada entre FOIS e diabetes (r= 0,0496) e hipertensão (r= 0,0376). Conclui-se que os sujeitos desta pesquisa possuem hábitos alimentares inadequados, alto consumo de nutrientes calóricos, baixa ingestão de alguns nutrientes não calóricos. O peso, IMC e circunferências da cintura e abdominais estavam acima do recomendado. Entretanto, a manutenção destes fatores parecem não apresentar correlação com a recorrência de AVC. O Diabete favorece o AVC recorrente. Foram encontradas correlações positivas de modo estatisticamente significantes entre FOIS com diabetes e hipertensão, e das variáveis: idade com Circunferência abdominal; IMC com a circunferência abdominal, da cintura e do braço, peso corporal. Além do percentual de gordura com a circunferência abdominal. A média dos exames bioquímicos encontra-se, em sua maioria, dentro dos valores de referênciaAbstract: Stroke is a cerebrovascular disease, being a major cause of mortality worldwide. The main risk factors for diseases cardiocerebrovasculares are hypertension, obesity and overweight, diabetes, atherogenic diets. Among the most frequent sequel are: global motor impairment, respiratory complications, nutritional and swallowing. This study has a purpose to analyze clinical aspects, nutritional and swallowing of the stroke subjects in the tertiary hospital of the Campinas city. Cross-sectional study, the subjects were submitted to nutritional and swallowing evaluation. To swallowing evaluation were realized the Fiber endoscopic evaluation swallowing (FEES) and degreed the dysphagia severity (DS) and Functional Oral Intake Scale (FOIS). To trace the nutritional profile of this population was used anthropometric methods, history and food record of 24 hours. We included all subjects after a recent stroke event (up to 1 year). Was conducted statistical analysis and adopted the significance level of p <0.05. Results: Population composed of 38 subjects, 21 males, mean age 60 years and 17 female, mean age 59. Of these 34 are hypertensive and 19 diabetic subjects. The number of single or multiple strokes if women and men are respectively: 9:8, 14:7. The means of the biochemical test were within the recommended standards. A positive correlation was a statistically significant between the variables: age with waist circumference, BMI and waist circumferences, waist, arms, body weight. Besides the percentage of fat with waist circumference. In the evaluation of swallowing the subjects showed the following classification for FOIS level 5,6,7, respectively 73%, 8%, 19%, the severity of dysphagia by 23% normal FEES, 58% mild, 15% moderate, 4% severe. Mean anthropometric data: BMI 25.76 kg/m2, waist circumference 92.3 cm, waist circumference 99.44 cm. The food intake presented above daily recommendations for carbohydrates, proteins and lipids. By correlating diabetes, hypertension, number of strokes with the FOIS and DS was realized that there was no statistical correlation between DS and hypertension (r = 0.196) and diabetes (r = 1.177), nor between the number of strokes and functionality (r = 0.29) or DS. However, we identified a moderate correlation between FOIS and diabetes (r = 0.0496) and hypertension (r = 0.0376). It is concluded that the subjects in this study have poor dietary habits, high caloric intake of nutrients, low intake of some nutrients, non-caloric. Subjects maintain weight, BMI and waist circumference and abdominal higher than recommended. Although the maintenance of these factors seem not present correlation with the recurrence of stroke. The Diabetes favors recurrent stroke. Positive correlations were found between a statistically significant FOIS with diabetes and hypertension, and the variables of age with waist circumference, BMI and waist circumference, waist and arm, body weight. Besides the percentage of fat with waist circumference. The average biochemical lies mostly within the reference valuesMestradoInterdisciplinaridade e ReabilitaçãoMestre em Saúde, Interdisciplinaridade e Reabilitaçã

Transcriptional and Molecular Pathways Activated in Mesenteric Adipose Tissue and Intestinal Mucosa of Crohn’s Disease Patients

Crohn’s disease (CD) is a chronic inflammatory disorder, characterized by cytokine imbalance and transcription signaling pathways activation. In addition, the increase of mesenteric adipose tissue (MAT) near the affected intestinal area is a hallmark of CD. Therefore, we evaluated the transcription signaling pathways and cytokines expression in intestinal mucosa and MAT of active CD patients. Ten patients with ileocecal CD and eight with noninflammatory diseases were studied. The biopsies of intestinal mucosa and MAT were snap-frozen and protein expression was determined by immunoblotting. RNA levels were measured by qPCR. The pIkB/IkB ratio and TNFα level were significantly higher in intestinal mucosa of CD when compared to controls. However, STAT1 expression was similar between intestinal mucosa of CD and controls. Considering the MAT, the pIkB/IkB ratio was significantly lower and the anti-inflammatory cytokine IL10 was significantly higher in CD when compared to controls. Finally, the protein content of pSTAT1 was higher in MAT of CD compared to controls. These findings reinforce the predominance of the proinflammatory NF-kB pathway in CD intestinal mucosa. For the first time, we showed the activation of STAT1 pathway in MAT of CD patients, which may help to understand the physiopathology of this immune mediated disease

Transcriptional and molecular pathways activated in mesenteric adipose tissue and intestinal mucosa of Crohn's disease patients

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCrohn’s disease (CD) is a chronic inflammatory disorder, characterized by cytokine imbalance and transcription signaling pathways activation. In addition, the increase of mesenteric adipose tissue (MAT) near the affected intestinal area is a hallmark of C20171110FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçã

Immunofluorescence staining of Caspase 3 in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD) compared to the respective control biopsy samples (AC).

<p>(A) Representative staining of fixed paraffin-embedded mesenteric adipose tissue from the AC and ACD groups, showing a higher number of positive cells for FITC (green-fluorescent) in the cytosol, co-labeled with DAPI (nuclear staining: blue-fluorescent) in the ACD group, compared to the control (AC). The arrows show the positive cells. Images were obtained using a 40x objective. (B) Quantitative analysis of immunofluorescence staining for Caspase 3 of ACD group compared to the respective control group (AC). For ACD, n = 10; for AC, n = 8, *p<0.05 vs control.</p

Bax and Bcl2 gene expressions, as determined by RT-PCR; low transcript levels of Bax and Bcl2 are observed in the intestinal mucosa of the Crohn’s disease group (ICD), compared to the respective control (IC).

<p>Low transcript levels of Bcl2 are also seen in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD), compared to the respective control (AC), while no differences in Bax transcripts were found in the MAT groups. For ICD, n = 10; for ACD, n = 10; for IC, n = 8; and, for AC, n = 8, *p<0.05 vs control.</p

Immunohistochemical staining of Bax in the intestinal tissue (epithelium and <i>lamina propria</i>) of the Crohn’s disease group (ICD) and in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD), compared to the respective control biopsy samples (IC and AC).

<p>(A) Representative staining of fixed paraffin-embedded tissue of terminal ileum from IC and ICD groups showing fewer positive cells (brown) in the <i>lamina propria</i> of ICD, compared to the IC group. (B) Representative staining of fixed paraffin-embedded mesenteric adipose tissue in the AC and ACD groups; no differences were found among the groups. Images were obtained using a 40x objective. The positive control was from tissue section of prostatic cancer. For ACD, n = 10; and for AC, n = 8. (C) Quantitative analysis of immunohistochemical staining for Bax in the intestinal mucosa of the Crohn’s disease group (ICD), compared to the respective control (IC). The graphs show the quantitative analysis for the epithelium and <i>lamina propria</i> immunostainings separately. For ICD, n = 10; for IC, n = 8, *p<0.05 vs control.</p

TUNEL assay shows different patterns in the intestinal mucosa (ICD) and in the mesenteric adipose tissue (MAT) of Crohn’s disease (ACD), compared to the respective control biopsy samples (IC and AC).

<p>(A) Enterocyte and <i>lamina propria</i> cell apoptosis are shown by immunofluorescence staining (overlay image); TUNEL+ cells are showed in orange (co-labeled by PI and FITC). Low numbers of TUNEL+ enterocytes and <i>lamina propria</i> cells were detected in the ICD group compared to IC. (C) Adipocyte apoptosis, shown by immunofluorescence staining (overlay image); TUNEL+ cells are showed in orange (co-labeled by PI and FITC). Low numbers of TUNEL+ adipocytes were detected in the ACD group compared to AC. Note the high density of TUNEL+ adipocytes, in orange, in the control (AC). Images were obtained using a 40x objective. (B) and (D) Quantitative analysis of TUNEL staining in the ICD and ACD groups, compared to the respective controls (IC and AC). The graphs of intestinal tissue show the quantitative analysis for the epithelium and <i>lamina propria</i> TUNEL staining, separately. For ICD, n = 10; for ACD, n = 10; for IC, n = 8; and, for AC, n = 8, *p<0.05 vs control. (E) Representative hematoxylin-eosin (H&E) staining of fixed paraffin-embedded MAT from AC and ACD groups shows lower area and perimeter of the adipocytes in the ACD group, compared to the control (AC). Images were obtained using a 40x objective. (F) Quantitative morphometric histological analysis in the mesenteric adipose tissue (MAT) of Crohn’s disease (ACD), compared to the respective control group (AC). The graphs show the decreased perimeter (µm) and area (µm²) of the adipocytes from the MAT of Crohn’s disease, compared to the control biopsy samples. For ACD, n = 10; for AC, n = 8, *p<0.05 vs control. (G) The graphs dispersion show a significant correlation between the perimeter (µm) and the number of apoptotic cells (TUNEL+), (r = 0.89, p<0.05) and also between the area (µm²) and the number of apoptotic cells (TUNEL+), (r = 0.92, p<0.05). (H) Immunohistochemical staining of Ki67 in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD), compared to the control biopsy samples (AC); no evidence of proliferation were found in all samples (ACD, n = 10; AC, n = 8). Images were obtained using a 40x objective. The positive control was from tissue section of intestinal mucosa.</p