Evaluation of alpha-synuclein immunohistochemical methods for the detection of Lewy-type synucleinopathy in gastrointestinal biopsies

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Optimizing Western Blots for the Detection of Endogenous α-Synuclein in the Enteric Nervous System

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Biomarkers of human gastrointestinal tract regions.

Dysregulation of intestinal epithelial cell performance is associated with an array of pathologies whose onset mechanisms are incompletely understood. While whole-genomics approaches have been valuable for studying the molecular basis of several intestinal diseases, a thorough analysis of gene expression along the healthy gastrointestinal tract is still lacking. The aim of this study was to map gene expression in gastrointestinal regions of healthy human adults and to implement a procedure for microarray data analysis that would allow its use as a reference when screening for pathological deviations. We analyzed the gene expression signature of antrum, duodenum, jejunum, ileum, and transverse colon biopsies using a biostatistical method based on a multivariate and univariate approach to identify region-selective genes. One hundred sixty-six genes were found responsible for distinguishing the five regions considered. Nineteen had never been described in the GI tract, including a semaphorin probably implicated in pathogen invasion and six novel genes. Moreover, by crossing these genes with those retrieved from an existing data set of gene expression in the intestine of ulcerative colitis and Crohn's disease patients, we identified genes that might be biomarkers of Crohn's and/or ulcerative colitis in ileum and/or colon. These include CLCA4 and SLC26A2, both implicated in ion transport. This study furnishes the first map of gene expression along the healthy human gastrointestinal tract. Furthermore, the approach implemented here, and validated by retrieving known gene profiles, allowed the identification of promising new leads in both healthy and disease states.

Over-expression of cyclooxygenase-2 in endoscopic biopsies of ectopic gastric mucosa

Ectopic gastric mucosa (EGM) is considered to be a congenital condition. Rare cases of adenocarcinoma have been described. There are no data justifying regular biopsies or follow-up. Cyclooxygenase-2 (COX-2) is a protein involved in gastrointestinal tumor development by inhibiting apoptosis and regulating angiogenesis. The aim of this prospective study was to evaluate COX-2 expression in EGM and compare it with normal tissue and Barrett's esophagus. We evaluated 1327 patients. Biopsies were taken from the inlet patch for histological evaluation and from the gastric antrum to assess Helicobacter pylori infection. Biopsies taken from normal esophageal, gastric antrum and body mucosa and Barrett's esophagus were retrieved from a tissue bank. EGM biopsies were evaluated with respect to type of epithelium, presence of H. pylori, and inflammation. COX-2 was detected by immunohistochemistry using the avidin-biotin complex. EGM islets were found in 14 patients (1.1%). Histological examination revealed fundic type epithelium in 58.3% of cases, H. pylori was present in 50% and chronic inflammation in 66.7%. Expression of COX-2 was negative in normal distal esophagus, normal gastric antrum and normal gastric body specimens (10 each). In contrast, EGM presented over-expression of COX-2 in 41.7% of cases and Barrett's esophagus in 90% of cases (P = 0.04 and 0.03, respectively). COX-2 immunoexpression in EGM was not related to gender, age, epithelium type, presence of inflammation or intestinal metaplasia, H. pylori infection, or any endoscopic finding. Our results demonstrate up-regulation of COX-2 in EGM, suggesting a possible malignant potential of this so-called harmless mucosa.

Mitochondrial Alterations in Nonalcoholic Fatty Liver Disease. Pediatric Case Description of Three Submitted Sequential Biopsies

Nonalcoholic fatty liver disease (NAFLD) is a clinical-pathological syndrome that encompasses a wide spectrum of morphologic alterations, ranging from simple hepatic steatosis to a more severe stage, known as nonalcoholic steatohepatitis (NASH). The purpose of this clinical report was to contribute to the understanding of mitochondrial alterations in NAFLD. The child (13-month-old) underwent initial biopsy in the year 2000 and was diagnosed with diffuse macro and microvesicular steatosis. Two additional biopsies were performed in 2001 and 2004. A high percentage of microvesicular steatosis was observed in the biopsies performed in 2000 and 2001. Mitochondrial size was slightly increased in the biopsy performed in the year 2000, significantly increased in 2001 and decreased in 2004. The presence of "mitochondrial hypertrophy" in the hepatocytes of an asymptomatic pediatric patient whose disease presentation was typical of NAFLD, excluding other pathological processes, allowed us to suspect that such a defect was considered the primary mitochondrial disorder.

Effect of age on the gastrointestinal-associated mucosal immune response of humans

Age-related changes in gastrointestinal-associated mucosal immune response have not been well studied. Thus, we investigated the effect of age on this response and compared these responses to those of peripheral immune cells. Saliva, blood, and intestinal biopsies were collected from young and old healthy subjects to determine immunoglobulin (Ig) levels and to isolate peripheral blood mononuclear cells, intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs). Although subject age did not influence the level of total IgA found in saliva, IgA levels in serum increased (p < .05) with age. Older subjects' peripheral blood mononuclear cell proliferation and IL-2 production were significantly lower than those of young subjects. LPLs from older subjects produced significantly less IL-2 in response to all stimuli than did that from the young. IEL's ability to proliferate and produce IL-2 was not affected by subject age. Thus, LPL but not IEL demonstrated an age-related decline in immune function similar to that seen in peripheral lymphocytes.

Endoscopic band ligation without resection in selected patients for small and superficial upper gastrointestinal tract lesions

Background and aim: The aim of this study was to evaluate the efficacy of endoscopic band ligation (EBL) in carefully selected patients who would benefit from this method of resection. Methods: Patients with early upper gastrointestinal and small (< 15 mm) lesions treated with EBL (Duette® Multi-Band Mucosectomy) were prospectively recruited and retrospectively analyzed between 2010 and 2015. All cases were discussed in a multidisciplinary cancer committee and it was concluded that, owing to patient conditions, surgery was not possible and that not conducting histology would not change the clinical management. A first endoscopic control with biopsies was planned at 4-8 weeks. If there was no persistence of the lesion, new controls were programmed at 6 and 12 months. Results: The group (n = 12) included 5 esophagus lesions (adenosquamous carcinoma, n = 1; carcinoma squamous, n = 2; adenocarcinoma, n = 2); 4 gastric lesions (high grade dysplasia, n = 1; adenocarcinoma, n = 2; neuroendocrine tumor [NET], n = 1), and 3 duodenal lesions (NETs) (n = 3). The mean tumor diameter was 9.6 ± 2.8 mm (range 4-15). Only one minor adverse event was described. At first follow-up (4-8 weeks), there was 91.6% and 75% of endoscopic and histological remission, respectively. At 6-month follow-up there was 70% of both endoscopic remission and negative biopsies. And at 12 months, there was 100% and 75% of endoscopic and histological remission, respectively. Persisting lesions were T1 cancers. The median follow-up was 30.6 months. Conclusion: EBL without resection is an easy and safe technique that should be considered in patients with multiple morbidities and small superficial UGI lesions.

Gastrointestinal Involvement In Lipoid Proteinosis: A Ten-year Follow-up Of A Brazilian Female Patient.

Lipoid proteinosis is a rare autosomal recessive disease characterized by the deposition of hyaline material in the skin and internal organs. The main clinical features are hoarseness and typical skin lesions. In this report we describe the endoscopic and radiologic findings in a Brazilian female patient presenting extensive gastrointestinal involvement and the evolution of the detected lesions in ten years of follow-up. Initial upper endoscopy and colonoscopy showed a similar pattern of multiple yellowish nodules throughout the esophagus, stomach, duodenum, and colons. Histological analysis confirmed the diagnosis of lipoid proteinosis. In addition, small bowel follow through demonstrated numerous well defined, round, small filling defects throughout the jejunum. Ten years later, the esophageal lesions remained the same, but none of the previous alterations were detected in the stomach, duodenum, and colons. In conclusion, lipoid proteinosis may affect all gastrointestinal organs with the same pattern of macroscopic and microscopic lesions. Some lesions may regress with increasing age.

Human Herpesvirus-6 And Cytomegalovirus Dna In Liver Donor Biopsies And Their Correlation With Hla Matches And Acute Cellular Rejection.

Herpesvirus reactivation is common after liver transplantation. Analyze the presence of cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) DNA in liver donor biopsies, seeking to better understand issues involving human donor leukocyte antigens (HLA)-A, B and DR, as well as correlations with acute cellular rejection. Fifty-nine liver transplantation patients were investigated for the presence of HCMV and HHV-6 DNA in liver donor biopsies, using the Nested-PCR technique. The clinical donor information and HLA matches were obtained from the São Paulo State Transplant System. The recipients' records regarding acute cellular rejection were studied. Seven (11.8%) biopsies were positive for HCMV DNA and 29 (49%) were positive for HHV-6 DNA. In 14 donors with HLA-DR 15 nine had HHV-6 DNA positive liver biopsy with a tendency for significant association (p=0.09), 22 recipients developed acute cellular rejection and 9/22 were positive for HLA-DR 15 (p=0.03; χ(2)=4.51), which was statistically significant in univariate analysis and showed a tendency after multivariate analysis (p=0.08). HHV-6 DNA was prevalent in liver donors studied as well as HLA-DR 15. These findings suggest that patients with HLA-DR 15 in liver donor biopsies develop more rejection after liver transplantation.

Evaluation of gastrointestinal motility in awake rats: a learning exercise for undergraduate biomedical students

Souza MA, Souza MH, Palheta RC Jr, Cruz PR, Medeiros BA, Rola FH, Magalhaes PJ, Troncon LE, Santos AA. Evaluation of gastrointestinal motility in awake rats: a learning exercise for undergraduate biomedical students. Adv Physiol Educ 33: 343-348, 2009; doi: 10.1152/advan.90176.2008.-Current medical curricula devote scarce time for practical activities on digestive physiology, despite frequent misconceptions about dyspepsia and dysmotility phenomena. Thus, we designed a hands-on activity followed by a small-group discussion on gut motility. Male awake rats were randomly submitted to insulin, control, or hypertonic protocols. Insulin and control rats were gavage fed with 5% glucose solution, whereas hypertonic-fed rats were gavage fed with 50% glucose solution. Insulin treatment was performed 30 min before a meal. All meals (1.5 ml) contained an equal mass of phenol red dye. After 10, 15, or 20 min of meal gavage, rats were euthanized. Each subset consisted of six to eight rats. Dye recovery in the stomach and proximal, middle, and distal small intestine was measured by spectrophotometry, a safe and reliable method that can be performed by minimally trained students. In a separate group of rats, we used the same protocols except that the test meal contained (99m)Tc as a marker. Compared with control, the hypertonic meal delayed gastric emptying and gastrointestinal transit, whereas insulinic hypoglycemia accelerated them. The session helped engage our undergraduate students in observing and analyzing gut motor behavior. In conclusion, the fractional dye retention test can be used as a teaching tool to strengthen the understanding of basic physiopathological features of gastrointestinal motility.

Esophageal Ulcer in Brazilian Patients with HIV: Prevalence and Comparative Analysis Among Diagnostic Methods

Esophageal ulcer (EU) represents an important comorbidity in AIDS. We evaluated the prevalence of EU, the accuracy of the endoscopic and histologic methods used to investigate viral EU in HIV-positive Brazilian patients and the numerical relevance of tissue sampling. A total of 399 HIV-positive patients underwent upper gastrointestinal (UGI) endoscopy. HIV-positive patients with EU determined by UGI endoscopy followed by biopsies were analyzed by the hematoxylin-eosin (HE) and immunohistochemical (IH) methods. EU was detected in 41 patients (mean age, 39.2 years; 23 males), with a prevalence of 10.27%. The median CD4 count was 49 cells/mm(3) (range, 1-361 cells/mm(3)) and the viral load was 58,869 copies per milliliter (range, 50-77,3290 copies per milliliter). UGI endoscopy detected 29 of 41 EU suggestive of cytomegalovirus (CMV) infection and 7 of 41 indicating herpes simplex virus (HSV) infection. HE histology confirmed 4 of 29 ulcers induced by CMV, 2 of 7 induced by HSV, and 1 of 7 induced by HSV plus CMV. IH for CMV and HSV confirmed the HE findings and detected one additional CMV-induced case. UGI endoscopy showed 100% sensitivity and 15% specificity for the diagnosis of EU due to CMV or HSV compared to HE and IH. HE proved to be an adequate method for etiologic evaluation, with 87% sensitivity and 100% specificity compared to IH. The number of samples did not influence the etiologic evaluation. The data support the importance of IH as a complementary method for HE in the diagnosis of EU of viral etiology.

Resistance of Santa Ines and crossbred ewes to naturally acquired gastrointestinal nematode infections

This trial was carried out in Piracicaba, Sao Paulo State. Brazil. to comparatively evaluate the degree of resistance to naturally acquired gastrointestinal nematode infections in sheep of the following genetic groups purebred Santa Ines (SI), SI crossbred with Dorper (DO x SI), lie de France (IF x SI), Suffolk (SU x SI), and Texel (TE < SI) Fifteen ewes from each group were raised indoors until 12 months of age. At this age, they were moved to pasture that was naturally contaminated by nematode infective larvae and were evaluated from December to May. 2007. Rainfall ranged from 267 mm in January to 37 mm in April Maximum and minimum mean temperatures ranged from 32 5 degrees C to 19 0 degrees C in March and from 25.9 degrees C to 12.8 degrees C in May. There was an increase in the mean number of eggs per gram of feces (EPG) after animals were placed on pasture with significant difference between the SI (80 EPG) and IF x SI (347 EPG) groups in January: and the DO x SI (386 EPG) and TE x SI (258 EPG) groups in May. The highest mean fecal egg count (FEC), 2073 EPG, was recorded for the TE x SI group in February. All groups showed a progressive reduction in body weight throughout the experiment of 12.0% (TE x SI) to 15.9% (SU x SI). In general. the animals with the highest FEC presented the lowest packed cell volumes (PCV): the highest correlation coefficient between FEC x PCV occurred in the SU x SI sheep in January (r = -0.70; P < 0.01). Similarly, there was an inverse relationship between FEC and blood eosinophil Values, with the highest correlation coefficient in the TE x SI sheep in February (r = -0.64; P < 0.05). Immunoglobulin G (IgG) levels against Haemonchus contortus antigens increased in all groups as a result of the exposure to parasites and remained relatively constant until the end of the study, with the exceptions of SU x SI and TE x SI, which showed a rise in IgG levels during the last sampling that coincided with a reduction in mean FEC. In conclusion. crossbreeding Santa Ines sheep with any of the breeds evaluated can result in a production increase and the maintenance of a satisfactory degree of infection resistance, especially against H. contortus and Trichostrongylus colubriformis. the major nematodes detected in this flock. (c) 2009 Elsevier B.V. All rights reserved.

Viability of Lactobacillus acidophilus in synbiotic guava mousses and its survival under in vitro simulated gastrointestinal conditions

The effects of refrigeration, freezing and substitution of milk fat by inulin and whey protein concentrate (WPC) on Lactobacillus acidophilus La-5 viability and resistance to gastric and enteric simulated conditions in synbiotic guava mousses effects were investigated. Refrigerated mousses supplemented with WPC presented the best probiotic viability. ranging from 7.77 to 6.24 log cfu/g during 28 days of storage. The highest probiotic populations, above 7.45 log cfu/g, were observed for all frozen mousses during 112 days of storage. Decreased L acidophilus survival during the in vitro gastrointestinal simulation was observed both for refrigerated and frozen mousses. Nonetheless, for the refrigerated mousses, the addition of inulin enhanced the probiotic survival during the in vitro assays in the first week of storage. L acidophilus survival in simulated gastrointestinal fluids was also improved through freezing. The frozen storage may be used to provide increased shelf-life for synbiotic guava mousses. Even though the protective effect of inulin and WPC on the probiotic microorganism tested was shown to be more specific for the refrigerated products, the partial replacement of milk fat by these ingredients may also help, as it improves the nutritional value of mousses in both storage conditions. (C) 2009 Elsevier B.V. All rights reserved.

Evaluation of the role of environmental factors in the human gastrointestinal tract on the behaviour of probiotic cultures of Lactobacillus casei Shirota and Lactobacillus casei LC01 by the use of a semi-dynamic in vitro model

This study evaluated the influence of gastrointestinal environmental factors (pH, digestive enzymes, food components, medicaments) on the survival of Lactobacillus casei Shirota and Lactobacillus casei LC01, using a semi-dynamic in vitro model that simulates the transit of microorganisms through the human GIT. The strains were first exposed to different simulated gastric juices for different periods of time (0, 30, 60 and 120 min), and then to simulated intestinal fluids for zero, 120, 180 and 240 min, in a step-wise format. The number of viable cells was determined after each step. The influence of food residues (skim milk) in the fluids and resistance to medicaments commonly used for varied therapeutic purposes (analgesics, antiarrhythmics, antibiotics, antihistaminics, proton pump inhibitors, etc.) were also evaluated. Results indicated that survival of both cultures was pH and time dependent, and digestive enzymes had little influence. Milk components presented a protective effect, and medicaments, especially anti-inflammatory drugs, influenced markedly the viability of the probiotic cultures, indicating that the beneficial effects of the two probiotic cultures to health are dependent of environmental factors encountered in the human gastrointestinal tract.