[Show abstract][Hide abstract] ABSTRACT: Clostridium difficile is a major cause of antibiotic-associated colitis and is associated with significant morbidity and mortality. Glutamine (Gln) is a major fuel for the intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. IEC-6 cells were used in the in vitro experiments. Cell morphology was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cell proliferation was assessed by WST-1 and Ki-67 and apoptosis was assessed by TUNEL. Cytoskeleton was evaluated by immunofluorescence for RhoA and F-actin. RhoA was quantified by immunoblotting. TcdA induced cell shrinkage as observed by AFM, SEM, and fluorescent microscopy. Additionally, collapse of the F-actin cytoskeleton was demonstrated by immunofluorescence. TcdA decreased cell volume and area and increased cell height by 79%, 66.2%, and 58.9%, respectively. Following TcdA treatment, Ala-Gln and Gln supplementation, significantly increased RhoA by 65.5% and 89.7%, respectively at 24 h. Ala-Gln supplementation increased cell proliferation by 137.5% at 24 h and decreased cell apoptosis by 61.4% at 24 h following TcdA treatment. In conclusion, TcdA altered intestinal cell morphology and cytoskeleton organization, decreased cell proliferation, and increased cell apoptosis. Ala-Gln and Gln supplementation reduced intestinal epithelial cell damage and increased RhoA expression.
BioMed research international. 01/2013; 2013:152052.
[Show abstract][Hide abstract] ABSTRACT: The accuracy of a nested PCR in gastric DNA obtained by string test for the diagnosis of H. pylori infection in asymptomatic children was 94.0%. The cagA-positive toxigenic vacA s1m1 strains were the most prevalent indicating that this population is early colonized by the strains associated with gastric cancer.
Journal of clinical microbiology 12/2012; · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human Immunodefiency Virus (HIV) protease inhibitors (PI) remain a crucial component of highly active therapy (HAART) and recently have been demonstrated to have potent antitumor effect on a wide variety of tumor cell lines. However, discontinuation of therapy is an important issue, which may be related to various side-effects, especially diarrhea. The aim of this study was to evaluate the effects of nelfinavir (NFV), an HIV PI, and of alanyl-glutamine (AQ) supplementation, on intestinal cell migration, proliferation, apoptosis and necrosis, using IEC-6 cells and on intestinal crypt depth, villus length, villus area, mitotic index and apoptosis in Swiss mice. Methods: Migration was evaluated at 12 and 24h after injury using a wound healing assay. Cellular proliferation was measured indirectly at 24 and 48h using tetrazolium salt WST-1. Apoptosis and necrosis were measured by flow cytometry using the Annexin V assay. Intestinal morphometry and mitotic index in vivo were assessed following a seven-day treatment with 100mg/kg of NFV, given orally. In vivo proliferation and apoptosis were evaluated by intestinal crypt mitotic index and immunohistochemistry, respectively. Results: In vitro, AQ supplementation enhanced IEC-6 cell migration and proliferation, following challenge with NFV. In vivo, AQ increased intestinal villus length, villus area, crypt depth and cell proliferation and cell migration, following treatment with NFV. AQ did not decrease cell death induced by NFV both in vivo and in vitro. Conclusions: AQ supplementation is potentially beneficial in preventing the effects of PIs, such as NFV, in the intestinal tract.
Cancer biology & therapy 09/2012; 13(14). · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the prevalence of more virulent H. pylori genotypes in relatives of gastric cancer patients and in patients without family histories of gastric cancer.
We evaluated prospectively the prevalence of the infection by more virulent H. pylori strains in 60 relatives of gastric cancer patients comparing the results with those obtained from 49 patients without family histories of gastric cancer. H. pylori status was determined by the urease test, histology and presence of H. pylori ureA. The cytotoxin associated gene (cagA), the cagA-EPIYA and vacuolating cytotoxin gene (vacA) were typed by PCR and the cagA EPIYA typing was confirmed by sequencing.
The gastric cancer relatives were significant and independently more frequently colonized by H. pylori strains with higher numbers of CagA-EPIYA-C segments (OR = 4.23, 95%CI = 1.53-11.69) and with the most virulent s1m1 vacA genotype (OR = 2.80, 95%CI = 1.04-7.51). Higher numbers of EPIYA-C segments were associated with increased gastric corpus inflammation, foveolar hyperplasia and atrophy. Infection by s1m1 vacA genotype was associated with increased antral and corpus gastritis.
We demonstrated that relatives of gastric cancer patients are more frequently colonized by the most virulent H. pylori cagA and vacA genotypes, which may contribute to increase the risk of gastric cancer.
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori causes chronic gastric inflammation and significantly increases the risk of duodenal and gastric ulcer disease and distal gastric carcinoma. In this study, we evaluated the Helicobacter pylori vacA and cagA genotypes in patients from a Brazilian region where there is a high prevalence of gastric cancer. Polymerase chain reaction (PCR) was used to investigate vacA mosaicism and cagA status in the gastric mucosa of 134 H. pylori-positive patients, including 76 with gastritis: 28 with peptic ulcer disease and 30 with gastric cancer. The s1m1 variant was the predominant vacA genotype observed, whereas the s1 allele was more frequently observed in patients with more severe diseases associated with H. pylori infection [p = 0.03, odds ratio (OR) = 5.72, 95% confidence interval (CI) = 1.15-38.60]. Furthermore, all of the s1 alleles were s1b. Mixed vacA m1/m2 strains were found more frequently in patients with gastric cancer and a cagA-positive status was significantly associated with gastric cancer (p = 0.016, OR = 10.36, 95% CI = 1.35-217.31). Patients with gastric cancer (21/21, 100%, p = 0.006) or peptic ulcers (20/21, 95%, p = 0.02) were more frequently colonised by more virulent H. pylori strains compared to gastritis patients (41/61, 67.2%). In conclusion, in the northeastern of Brazil, which is one of the regions with the highest prevalence of gastric cancer in the country, infection with the most virulent H. pylori strains, carrying the cagA gene and s1m1 vacA alleles, predominates and is correlated with more severe H. pylori-associated diseases.
Memórias do Instituto Oswaldo Cruz 06/2012; 107(4):561-3. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori infection is acquired predominantly in childhood. There is also evidence that children loss the infection. Therefore, factors that account for children remain infected need to be investigated because once established the infection persists throughout the life unless treated.
This study aimed to evaluate the H. pylori infection in children of a low-income community at baseline and 8years later to determine the predictor factors linked to the maintenance, acquisition, and loss of the infection using regression models of generalized estimating equations. H. pylori status was determined by (13) C-urea breath test.
Data from 37.7% (133/353) of the children were available. No difference between the characteristics of the included and nonincluded children was observed. The prevalence of infection increased from 53.4 to 64.7%. Thirty-nine children (29.3%) remained noninfected, 47.4% remained infected, 17.3% became infected, and 6.0% lost the infection. Factors associated with to remain infected compared with to remain noninfected included the age, increased number of children in the household, and the use of well water instead of municipal water. The acquisition of the infection was associated with the male gender.
Factors linked to remain and to gain H. pylori infection in a poor region were increased number of children in the household and the male gender. Also, the acquisition rates were higher than the loss rates, which lead to an increase in the infection prevalence with age.
[Show abstract][Hide abstract] ABSTRACT: Methotrexate treatment has been associated to intestinal epithelial damage. Studies have suggested an important role of nitric oxide in such injury. The aim of this study was to investigate the role of nitric oxide (NO), specifically iNOS on the pathogenesis of methotrexate (MTX)-induced intestinal mucositis.
Intestinal mucositis was carried out by three subcutaneous MTX injections (2.5 mg/kg) in Wistar rats and in inducible nitric oxide synthase knock-out (iNOS-/-) and wild-type (iNOS+/+) mice. Rats were treated intraperitoneally with the NOS inhibitors aminoguanidine (AG; 10 mg/Kg) or L-NAME (20 mg/Kg), one hour before MTX injection and daily until sacrifice, on the fifth day. The jejunum was harvested to investigate the expression of Ki67, iNOS and nitrotyrosine by immunohistochemistry and cell death by TUNEL. The neutrophil activity by myeloperoxidase (MPO) assay was performed in the three small intestine segments.
AG and L-NAME significantly reduced villus and crypt damages, inflammatory alterations, cell death, MPO activity, and nitrotyrosine immunostaining due to MTX challenge. The treatment with AG, but not L-NAME, prevented the inhibitory effect of MTX on cell proliferation. MTX induced increased expression of iNOS detected by immunohistochemistry. MTX did not cause significant inflammation in the iNOS-/- mice.
These results suggest an important role of NO, via activation of iNOS, in the pathogenesis of intestinal mucositis.
[Show abstract][Hide abstract] ABSTRACT: This study conducted in Northeastern Brazil, evaluated the prevalence of H. pylori infection and the presence of gastritis in HIV-infected patients.
There were included 113 HIV-positive and 141 age-matched HIV-negative patients, who underwent upper gastrointestinal endoscopy for dyspeptic symptoms. H. pylori status was evaluated by urease test and histology.
The prevalence of H. pylori infection was significantly lower (p < 0.001) in HIV-infected (37.2%) than in uninfected (75.2%) patients. There were no significant differences between H. pylori status and gender, age, HIV viral load, antiretroviral therapy and the use of antibiotics. A lower prevalence of H. pylori was observed among patients with T CD4 cell count below 200/mm3; however, it was not significant. Chronic active antral gastritis was observed in 87.6% of the HIV-infected patients and in 780.4% of the control group (p = 0.11). H. pylori infection was significantly associated with chronic active gastritis in the antrum in both groups, but it was not associated with corpus chronic active gastritis in the HIV-infected patients.
We demonstrated that the prevalence of H. pylori was significantly lower in HIV-positive patients compared with HIV-negative ones. However, corpus gastritis was frequently observed in the HIV-positive patients, pointing to different mechanisms than H. pylori infection in the genesis of the lesion.
[Show abstract][Hide abstract] ABSTRACT: In this study, we have examined the role of glutamine derivatives in reducing 5-fluorouracil (5-FU)-induced epithelial damage in an undifferentiated crypt intestinal cell line, IEC-6. In this model, we have investigated proliferation indirectly by detecting the enzyme-derived formazan dye from the tetrazolium salt WST-1 in viable cells at 24 and 48 h after 5-FU treatment. Migration was measured at 12 and 24 h after razor scraping of the cell monolayer. Cell death was measured by quantifying the percentage of apoptotic and necrotic figures by flow cytometry at 12 and 24 h following 5-FU challenge. Neither glutamine nor alanyl-glutamine prevented 5-FU-induced apoptosis and necrosis in IEC-6 cells at 12 and 24 h after 5-FU challenge. However, glutamine and alanyl-glutamine enhanced migration and proliferation when compared with 5-FU-treated controls (P < 0.05). These new findings support our earlier study on the benefit of oral glutamine in enhancing epithelial recovery after 5-FU challenge.
Digestive Diseases and Sciences 04/2008; 53(10):2687-96. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vitamin A (retinol), a fat-soluble vitamin, is an essential nutrient for the normal functioning of the visual system, epithelial cell integrity and growth, immunity, and reproduction. Our group has investigated the effect of high doses of oral vitamin A on early childhood diarrhea in our prospective community-based studies from Northeast Brazil and found a beneficial role in reducing the mean duration but not incidence of diarrheal episodes. In this study, we explored the role of retinol supplementation in intestinal cell lines following Clostridium difficile toxin A (TxA) challenge. C. difficile is the most common anaerobic pathogen borne with antibiotic-borne diarrhea and pseudomembranous colitis. Since retinol is critical for the integrity of tight junctions and to modulate the cell cycle, we have focused on changes in transepithelial electrical resistance (TEER) in Caco-2, a more differentiated intestinal cell line, and on models of cell proliferation, migration and viability in IEC-6 cells, an undifferentiated crypt cell line, following TxA injury. In this model, retinol therapy reduced apoptosis, improved cell migration and proliferation, and prevented the reduction in TEER, following C. difficile TxA challenge in a glutamine-free medium. These results suggest the role of retinol in protecting intestinal epithelial barrier function from C. difficile TxA enterotoxic damage.
[Show abstract][Hide abstract] ABSTRACT: Background: HIV protease and reverse transcriptase inhibitors are components of highly-active antiretroviral therapy (HAART). Diarrhea is a common adverse effect of HAART with unknown pathogenesis.
Methods: We investigated the effects of these HIV drugs on intestinal barrier function, morphometry, and ion and water secretion in vivo in mice and on cell proliferation and migration in vitro in IEC-6 cells.
Results: Swiss mice were treated with nelfinavir (NFV), indinavir (IND), zidovudine (AZT), didanosine (DDI) or PBS. NFV decreased villus length (VL) in duodenum by 28% and jejunum by 19% and crypt depth (CD) in ileum by 18%. IND reduced VL in duodenum by 28%, jejunum by 30%, and ileum by 12%, and increased duodenal CD by 18% and reduced ileal CD by 26%. AZT reduced VL in duodenum by 27%, jejunum by 15% and increased duodenal CD by 23% and jejunal CD by 22%. DDI reduced the VL in duodenum by 30%, jejunum by 22% and ileal CD by 25%. NFV increased urinary excretion of both mannitol (7.9% vs. 3.2%) and lactulose (5.3% vs. 1.4%). IND, DDI, and AZT did not alter intestinal permeability. NFV, IND and AZT increased intestinal secretion of Na+ (-58.5, -43.2 and -44.4 vs -21.9µEq/min/g), Cl- (-69.3, -43.2, and -43.4 vs -31.7µEq/min/g) and water (-59.0, -49.0 and -49.6 vs -23.0µL/min/g), respectively. DDI increased Na+ excretion (-32.3 vs -21.9µEq/min/g). Proliferation of IEC-6 cells decreased in response to NFV, IND, saquinavir (SQV), lopinavir (LPV), ritonavir (RTV) and efavirenz (EFV), but not to DDI or nevirapine (NVP). Migration of IEC-6 cells decreased in response to NFV, IND, SQV, LPV, RTV and EFV, but not to DDI or NVP.
Conclusion: Protease inhibitors reduced intestinal villus and crypt length in vivo, caused significant change in intestinal permeability and in ion and water secretion, and decreased cell proliferation and migration in vitro. This data suggests intestinal barrier dysfunction as a potential cause of HAART-associated diarrhea.
Infectious Diseases Society of America 2007 Annual Meeting; 10/2007