A New Murine Undernutrition Model Based on Complementary Feeding of Undernourished Children Causes Damage to the Morphofunctional Intestinal Epithelium Barrier.

BACKGROUND: Complementary feeding is critical in establishing undernutrition. However, experimental undernourished diets do not represent the amount of nutrients in the complementary diets of undernourished children. OBJECTIVES: To develop, validate, and evaluate the impact of a new murine model of undernutrition on the intestinal epithelium, based on the complementary diet of undernourished children from 7 countries with low-socioeconomic power belonging to the Malnutrition-Enteric Diseases (MAL-ED) cohort study. METHODS: We used the difference in the percentage of energy, macronutrients, fiber and zinc in the complementary diet of children without undernutrition compared with stunting (height-for-age Z-score < -2) for the MAL-ED diet formulation. Subsequently, C57BL/6 mice were fed a control diet (AIN-93M diet) or MAL-ED diet for 28 d. Weight was measured daily; body composition was measured every 7 d; lactulose:mannitol ratio (LM) and morphometry were evaluated on days 7 and 28; the cotransport test and analysis of intestinal transporters and tight junctions were performed on day 7. RESULTS: The MAL-ED diet presented -8.03% energy, -37.46% protein, -24.20% lipid, -10.83% zinc, +5.93% carbohydrate, and +45.17% fiber compared with the control diet. This diet rapidly reduced weight gain and compromised body growth and energy reserves during the chronic period (P < 0.05). In the intestinal epithelial barrier, this diet caused an increase in the LM (P < 0.001) and reduced (P < 0.001) the villous area associated with an increase in FAT/CD36 in the acute period and increased (P < 0.001) mannitol excretion in the chronic period. CONCLUSIONS: The MAL-ED diet induced undernutrition in mice, resulting in acute damage to the integrity of the intestinal epithelial barrier and a subsequent increase in the intestinal area during the chronic period. This study introduces the first murine model of undernutrition for the complementary feeding phase, based on data from undernourished children in 7 different countries.

Cytokines, chemokines, and cells growth factors in patients with mild to moderate SARS-CoV-2 infection: A case-control study.

Several biomarkers have been evaluated as predictors of severity or in directing the treatment of COVID-19, however there are no conclusive results. In this study, we evaluated serum levels of cytokines, chemokines, and cell growth factors in association with the pathobiology of mild to moderate SARS-CoV-2 infection. Serum levels of SARS-CoV-2 infected patients (n = 113) and flu symptoms individuals negative for SARS-CoV-2 (n = 58), tested by the RT-qPCR test-nasal swab were compared to healthy controls (n = 53). Results showed that the proinflammatory cytokines IL-1ß, MCP-3, TNF-α, and G-CSF were increased in symptomatic patients and the cytokines IL-6 and IL-10 were associated with patients positive for SARS-CoV-2 when compared to healthy controls. Symptoms associated with COVID-19 were fever, anosmia, ageusia, and myalgia. For patients without SARS-CoV-2 infection, their major symptom was sore throat. The pathobiology of mild to moderate SARS-CoV-2 infection was associated with increasing proinflammatory cytokines and a pleiotropic IL-6 and anti-inflammatory IL-10 cytokines compared to healthy controls. Thus, knowledge about the pathophysiology and the involvement of biomarkers in the mild to moderate profile of the disease should be evaluated. Monitoring these biomarkers in patients with mild to moderate disease can help establish adequate treatment and prevention strategies for long-term COVID-19.

Consumption of a multi-deficient diet causes dynamic changes in the intestinal morphofunctional barrier, body composition and impaired physical development in post-weaning mice.

Few studies have focused on nutrient-deficient diets and associated pathobiological dynamics of body composition and intestinal barrier function. This study evaluated the impact of a nutrient-deficient diet on physical development and intestinal morphofunctional barrier in mice. C57BL/6 (21 days of age) mice were fed a Northeastern Brazil regional basic diet (RBD) or a control diet for 21 d. The animals were subjected to bioimpedance analysis, lactulose test, morphometric analysis and quantitative reverse transcription-PCR to evaluate tight junctions and intestinal transporters. RBD feeding significantly reduced weight (P < 0·05) from day 5, weight gain from day 3 and tail length from day 14. The intake of RBD reduced total body water, extracellular fluid, fat mass and fat-free mass from day 7 (P < 0·05). RBD induced changes in the jejunum, with an increase in the villus:crypt ratio on day 7, followed by reduction on days 14 and 21 (P < 0·05). Lactulose:mannitol ratio increased on day 14 (P < 0·05). Changes in intestinal barrier function on day 14 were associated with reductions in claudin-1 and occludin, and on day 21, there was a reduction in the levels of claudin-2 and occludin. SGLT-1 levels decreased on day 21. RBD compromises body composition and physical development with dynamic changes in intestinal barrier morphofunctional. RBD is associated with damage to intestinal permeability, reduced levels of claudin-1 and occludin transcripts and return of bowel function in a chronic period.

Detection of SARS-CoV-2 in different human biofluids using the loop-mediated isothermal amplification assay: A prospective diagnostic study in Fortaleza, Brazil.

We adopted the reverse-transcriptase-loop-mediated isothermal amplification (RT-LAMP) to detect severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) in patient samples. Two primer sets for genes N and Orf1ab were designed to detect SARS-CoV-2, and one primer set was designed to detect the human gene Actin. We collected prospective 138 nasopharyngeal swabs, 70 oropharyngeal swabs, 69 salivae, and 68 mouth saline wash samples from patients suspected to have severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 to test the RT-LAMP in comparison with the gold standard technique reverse-transcription quantitative polymerase chain reaction  (RT-qPCR). The accuracy of diagnosis using both primers, N5 and Orf9, was evaluated. Sensitivity and specificity for diagnosis were 96% (95% confidence interval [CI]: 87-99) and 85% (95% CI: 76-91) in 138 samples, respectively. Accurate diagnosis results were obtained only in nasopharyngeal swabs processed via extraction kit. Accurate and rapid diagnosis could aid coronavirus disease 2019 (COVID-19) pandemic management by identifying, isolating, and treating patients rapidly.

Intervention and Mechanisms of Alanyl-glutamine for Inflammation, Nutrition, and Enteropathy: A Randomized Controlled Trial.

OBJECTIVE: Determine the minimum dosage of alanyl-glutamine (Ala-Gln) required to improve gut integrity and growth in children at risk of environmental enteropathy (EE). METHODS: This was a double-blinded randomized placebo-controlled dose-response trial. We enrolled 140 children residing in a low-income community in Fortaleza, Brazil. Participants were 2 to 60 months old and had weight-for-age (WAZ), height-for-age (HAZ), or weight-for-height (WHZ) z-scores less than -1. We randomized children to 10 days of nutritional supplementation: Ala-Gln at 3 g/day, Ala-Gln at 6 g/day, Ala-Gln at 12 g/day, or an isonitrogenous dose of glycine (Gly) placebo at 12.5 g/day. Our primary outcome was urinary lactulose-mannitol excretion testing. Secondary outcomes were anthropometry, fecal markers of inflammation, urine metabolic profiles, and malabsorption (spot fecal energy). RESULTS: Of 140 children, 103 completed 120 days of follow-up (24% dropout). In the group receiving the highest dose of Ala-Gln, we detected a modest improvement in urinary lactulose excretion from 0.19% on day 1 to 0.17% on day 10 (P = 0.05). We observed significant but transient improvements in WHZ at day 10 in 2 Ala-Gln groups, and in WHZ and WAZ in all Ala-Gln groups at day 30. We detected no effects on fecal inflammatory markers, diarrheal morbidity, or urine metabolic profiles; but did observe modest reductions in fecal energy and fecal lactoferrin in participants receiving Ala-Gln. CONCLUSIONS: Intermediate dose Ala-Gln promotes short-term improvement in gut integrity and ponderal growth in children at risk of EE. Lower doses produced improvements in ponderal growth in the absence of enhanced gut integrity.

Virulence-Related Genes and Coenteropathogens Associated with Clinical Outcomes of Enteropathogenic Escherichia coli Infections in Children from the Brazilian Semiarid Region: a Case-Control Study of Diarrhea.

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in children from developing countries and presents high genetic variability. We aimed to characterize the EPEC virulence-related gene (VRG) distribution and copathogens associated with diarrhea and nutrition-related outcomes in children from the low-income Brazilian semiarid region. A cross-sectional case-control study of diarrhea was conducted in 1,191 children aged 2 to 36 months from the northeast region of Brazil. Stool samples were collected and clinical, epidemiological, and anthropometric data were identified from each child. A broad molecular evaluation of enteropathogens was performed, and EPEC-positive samples were further investigated for 18 VRGs using five multiplex PCRs. EPEC was detected in 28.2% of the study population, with similar proportions among cases and controls. Typical EPEC (tEPEC) infections were more often associated with diarrhea than atypical EPEC (aEPEC) infections, while aEPEC infections presented a higher prevalence. The VRG ler, a negative regulator of the locus of enterocyte effacement, was associated with the absence of diarrhea in aEPEC-positive children; espB, a major component of the type 3 secretion system, was associated with diarrhea in tEPEC-positive children; the presence of procolonization VRGs-the combination of cesT positivity, espP negativity, and the presence of the map gene-was associated with undernutrition; and Campylobacter spp., norovirus, and enteroaggregative E. coli (EAEC) coinfections were associated with increased clinical severity in EPEC-infected children. These data identified tEPEC strains associated with diarrhea and specific VRGs of EPEC (ler, espB, cesT, and map genes) and Campylobacter spp., norovirus, and EAEC to be major contributors to diarrhea and undernutrition in children from a low-income Brazilian region.

Virulence-related genes are associated with clinical and nutritional outcomes of Shigella/Enteroinvasive Escherichia coli pathotype infection in children from Brazilian semiarid region: A community case-control study.

Shigella/Enteroinvasive Escherichia coli (EIEC) pathotype is a major enteropathogen associated with diarrhea and malnutrition in children from developing countries. This study aimed to correlate Shigella/EIEC virulence-related genes (VRGs) with clinical symptoms, nutritional status and coenteropathogens in children from the Brazilian semiarid region. We designed a case-control study of community diarrhea in six cities of the Brazil semiarid region with 1200 children aging 2-36 months. Standardized questionnaire was applied for collecting sociodemographic, nutritional status and clinical information of the children. DNA samples were extracted from stools and diagnosed for Shigella/EIEC using PCR-based approaches. Positive samples were tested for 28 VRGs using four multiplex PCRs. Intestinal inflammation was determined by measuring fecal myeloperoxidase (MPO). Shigella/EIEC pathotype was detected in 5% of the children and was significantly associated with diarrhea. The genes sen (encoding Shigella enterotoxin 2), ipgB2, ipgB1 (both encoding type 3 secretion system-T3SS effectors that modulate actin filament), and ospF (encoding a T3SS effector involved in suppression of host responses) were further associated with diarrhea in Shigella/EIEC positive children. Among children presenting diarrhea, virA gene (encoding a T3SS effector that promotes microtubule destabilization) was associated with fever, while virB (encoding a major transcriptional activator) was associated with low height-for-age z-score. In addition, these VRGs were associated with increased fecal MPO, and coinfection with Salmonella spp. was associated with increased abdominal pain. These data reinforce the impact of Shigella/EIEC on diarrhea in children from Brazilian semiarid region and highlighted the contributions of specific virulence genes for its pathobiology.

Rotavirus A Infections in Community Childhood Diarrhea in the Brazilian Semiarid Region During Postvaccination Era.

BACKGROUND: Rotavirus A (RVA) is one of the leading causes of acute gastroenteritis worldwide; however, few studies assessed RVA genetics with community surveillance. OBJECTIVES: This study aimed to investigate clinical data, genetic diversity, and coinfection patterns of RVA infections in children from 2 to 36 months old with or without community childhood diarrhea in the Brazilian semiarid region during postvaccination era. METHODS: We enrolled and collected socioeconomic/clinical information using a standardized questionnaire and fecal samples from 291 children. Viral RNA samples were extracted and analyzed using quantitative reverse transcription polymerase chain reaction to establish the diagnosis of RVA. Sequencing of VP7 and VP4 (VP8*) regions and phylogenetic analysis were performed. RESULTS: RVA-negative diagnosis was associated with children 24 to 36 months old with complete vaccination schedule. Genotype G1P[8] was the most prevalent (57%), whereas unusual genotypes including G1P[4], G2P[8], and G3P[9] were also detected. G1- and P[8]-positive samples showed high degrees of similarity with the vaccine strain. RVA coinfections were frequently observed, and enteroaggregative Escherichia coli was the most prevalent copathogen. CONCLUSIONS: These results demonstrate that genotype G1P[8] is the most prevalent strain. VP7 and/or VP8* gene segments arising from RV1 vaccine strain were documented in these children, suggesting shedding or herd vaccination. Moreover, our study indicates full vaccination is important for protection against RVA infections.

Alanyl-glutamine Protects Against Damage Induced by Enteroaggregative Escherichia coli Strains in Intestinal Cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an important pathogen causing enteric infections worldwide. This pathotype is linked to malnutrition in children from developing countries. Alanyl-glutamine (Ala-Gln) is an immune modulator nutrient that acts during intestinal damage and/or inflammation. This study investigated the effect of EAEC infection and Ala-Gln on cell viability, cell death, and inflammation of intestinal epithelium cells (IEC-6). METHODS: Cells were infected with an EAEC prototype 042 strain, an EAEC wild-type strain isolated from a Brazilian malnourished child, and a commensal E coli HS. Gene transcription and protein levels of caspases-3, -8, and -9 and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL1) were evaluated using RT-qPCR, western blot analysis, and ELISA. RESULTS: Infections with both EAEC strains decreased cell viability and induced apoptosis and necrosis after 24 hours. Ala-Gln supplementation increased cell proliferation and reduced cell death in infected cells. Likewise, EAEC strain 042 significantly increased the transcript levels of caspases-3, -8, and -9 when compared to the control group, and Ala-Gln treatment reversed this effect. Furthermore, EAEC induced CXCL1 protein levels, which were also reduced by Ala-Gln supplementation. CONCLUSION: These findings suggest that EAEC infection promotes apoptosis, necrosis, and intestinal inflammation with involvement of caspases. Supplementation of Ala-Gln inhibits cell death, increases cell proliferation, attenuates mediators associated with cell death, and inflammatory pathways in infected cells.

Epidemiology and Risk Factors for Cryptosporidiosis in Children From 8 Low-income Sites: Results From the MAL-ED Study.

Background: Cryptosporidium species are enteric protozoa that cause significant morbidity and mortality in children worldwide. We characterized the epidemiology of Cryptosporidium in children from 8 resource-limited sites in Africa, Asia, and South America. Methods: Children were enrolled within 17 days of birth and followed twice weekly for 24 months. Diarrheal and monthly surveillance stool samples were tested for Cryptosporidium by enzyme-linked immunosorbent assay. Socioeconomic data were collected by survey, and anthropometry was measured monthly. Results: Sixty-five percent (962/1486) of children had a Cryptosporidium infection and 54% (802/1486) had at least 1 Cryptosporidium-associated diarrheal episode. Cryptosporidium diarrhea was more likely to be associated with dehydration (16.5% vs 8.3%, P < .01). Rates of Cryptosporidium diarrhea were highest in the Peru (10.9%) and Pakistan (9.2%) sites. In multivariable regression analysis, overcrowding at home was a significant risk factor for infection in the Bangladesh site (odds ratio, 2.3 [95% confidence interval {CI}, 1.2-4.6]). Multiple linear regression demonstrated a decreased length-for-age z score at 24 months in Cryptosporidium-positive children in the India (ß = -.26 [95% CI, -.51 to -.01]) and Bangladesh (ß = -.20 [95% CI, -.44 to .05]) sites. Conclusions: This multicountry cohort study confirmed the association of Cryptosporidium infection with stunting in 2 South Asian sites, highlighting the significance of cryptosporidiosis as a risk factor for poor growth. We observed that the rate, age of onset, and number of repeat infections varied per site; future interventions should be targeted per region to maximize success.

Genome-Wide Analysis in Brazilians Reveals Highly Differentiated Native American Genome Regions.

Despite its population, geographic size, and emerging economic importance, disproportionately little genome-scale research exists into genetic factors that predispose Brazilians to disease, or the population genetics of risk. After identification of suitable proxy populations and careful analysis of tri-continental admixture in 1,538 North-Eastern Brazilians to estimate individual ancestry and ancestral allele frequencies, we computed 400,000 genome-wide locus-specific branch length (LSBL) Fst statistics of Brazilian Amerindian ancestry compared to European and African; and a similar set of differentiation statistics for their Amerindian component compared with the closest Asian 1000 Genomes population (surprisingly, Bengalis in Bangladesh). After ranking SNPs by these statistics, we identified the top 10 highly differentiated SNPs in five genome regions in the LSBL tests of Brazilian Amerindian ancestry compared to European and African; and the top 10 SNPs in eight regions comparing their Amerindian component to the closest Asian 1000 Genomes population. We found SNPs within or proximal to the genes CIITA (rs6498115), SMC6 (rs1834619), and KLHL29 (rs2288697) were most differentiated in the Amerindian-specific branch, while SNPs in the genes ADAMTS9 (rs7631391), DOCK2 (rs77594147), SLC28A1 (rs28649017), ARHGAP5 (rs7151991), and CIITA (rs45601437) were most highly differentiated in the Asian comparison. These genes are known to influence immune function, metabolic and anthropometry traits, and embryonic development. These analyses have identified candidate genes for selection within Amerindian ancestry, and by comparison of the two analyses, those for which the differentiation may have arisen during the migration from Asia to the Americas.

Enteroaggregative Escherichia coli subclinical and clinical infections.

PURPOSE OF REVIEW: The current review is to update the results on epidemiology, pathobiology, and genes related to virulence, clinical presentation, molecular diagnosis, antimicrobial resistance, and extraintestinal infection of enteroaggregative Escherichia coli (EAEC). RECENT FINDINGS: EAEC subclinical infection was significantly associated with reduced length at 2 years of age and EAEC and coinfections were associated with reduced delta weight-for-length and weight-for-age z-scores in the first 6 months of age in the MAL-ED birth cohort study. EAEC was associated with malnutrition in children 6-24 months of age in prospective case-control studies in Bangladesh and Brazil. Virulence gene-based studies have suggested aggregative fimbriae II may be a major contributor to disease, whereas AggR-activated regulator a marker of less severe disease. The high ability of EAEC colonization likely exacerbates effects of other microbial virulence strategies. Molecular diagnosis has been useful for understanding EAEC burden, although different criteria may relate to different pathogenic outcomes. SUMMARY: EAEC gained special interest in the past few years, especially due to association with growth decrements in children with subclinical infections and its important role as a copathogen. Understanding of EAEC pathogenesis advanced but further research is needed for elucidating both microbial and host factors influencing infection outcomes.

Campylobacter jejuni virulence genes and immune-inflammatory biomarkers association with growth impairment in children from Northeastern Brazil.

Campylobacter spp. have been associated with anthropometric Z-score decrements, but the role of specific virulence genes associated with these outcomes has not been explored. This study aimed to investigate whether specific Campylobacter jejuni virulence-related gene and immune-inflammatory biomarkers are associated with malnutrition in children from Northeastern Brazil. A case-control study was performed in Fortaleza, Brazil. Children aging 6-24 months were characterized as malnourished (cases) if weight-for-age Z-score (WAZ) = 2 and as nourished (controls) if WAZ ≥ 1. DNA samples were extracted from stools and screened for C. jejuni/coli by real-time PCR. A subsequent C. jejuni-specific PCR was employed and positive samples were evaluated for 18 C. jejuni virulence genes by using four multiplex PCRs. C. jejuni was detected in 9.71% (33/340) of the children's samples, being 63.63% (21/33) from nourished and 37.37% (12/33) from malnourished children. The cadF, iamA, cheW, and sodB genes were the most frequent genes (100%, 90.9%, 87.9%, and 75.8%, respectively), while some others (ceuE, jlpA, pldA, and pVir) showed low rates (all below 6%). Malnourished children were significantly associated with infection with C. jejuni strains lacking cdtB gene (active subunit of cytolethal distending toxin) and harboring flgE gene (flagellar hook protein). These strains were also associated with children presenting increased serum SAA and sCD-14, but decreased IgG anti-LPS. These data reinforce the impact of Campylobacter jejuni infection on children without diarrhea and highlight the contribution of a specific virulence gene profile, cdtB(-)flgE(+) and increased systemic response in malnutrition children.

Genetic Diversity of Norovirus Infections, Coinfections, and Undernutrition in Children From Brazilian Semiarid Region.

BACKGROUND AND OBJECTIVE: Norovirus (NoV) infections are known to have high-morbidity and mortality rates and are a major health problem globally. The impact of NoV on child development is, however, poorly understood. We evaluated the distribution of NoV genotypes in children from a low-income Brazilian semiarid region, in relation with their clinical symptoms, nutritional status, and co-pathogens. METHODS: The test population included children aged 2 to 36 months from 6 cities of the Brazilian semiarid region. Fecal samples were collected from each child, along with the information regarding their socioeconomic/clinical conditions using a standardized questionnaire. Detection and quantification of NoV were performed by reverse-transcription quantitative polymerase chain reaction, followed by molecular and phylogenetic analyses. RESULTS: The NoV detection rate was 45.2%. Presence of NoV was associated with lower z scores for weight-for-age (P = 0.03), weight-for-height (P = 0.03), and body mass index-for-age (P = 0.03). NoV infection was associated with more frequent respiratory illnesses (P < 0.01). GII.P7 (polymerase) and GII.3 (capsid) were the most frequent NoV genotypes. Analysis of the open reading frame (ORF)1-2 junction identified recombinant NoV strains in 80% of the sequenced samples. Enteroaggregative Escherichia coli coinfection was the major predictor for diarrhea in NoV-positive samples (P < 0.02). Moreover, Shigella spp was also associated with NoV-positive diagnosis (P = 0.02). CONCLUSIONS: This study highlights the genetic variability of NoV and, associated co-infections and undernutrition in infants from low-income Brazilian semiarid region.

Enteroaggregative Escherichia coli Subclinical Infection and Coinfections and Impaired Child Growth in the MAL-ED Cohort Study.

OBJECTIVE: We evaluated the impact of subclinical enteroaggregative Escherichia coli (EAEC) infection alone and in combination with other pathogens in the first 6 months of life on child growth. METHODS: Nondiarrheal samples from 1684 children across 8 Multisite Birth Cohort Study, Malnutrition and Enteric Diseases (MAL-ED) sites in Asia, Africa, and Latin America were tested monthly; more than 90% of children were followed-up twice weekly for the first 6 months of life. RESULTS: Children with subclinical EAEC infection did not show altered growth between enrollment and 6 months. Conversely, EAEC coinfection with any other pathogen was negatively associated with delta weight-for-length (P < 0.05) and weight-for-age (P > 0.05) z scores between 0 and 6 months. The presence of 2 or more pathogens without EAEC was not significantly associated with delta weight-for-length and weight-for-age. The most frequent EAEC coinfections included Campylobacter spp, heat-labile toxin-producing enterotoxigenic E coli, Cryptosporidium spp, and atypical enteropathogenic E coli. Myeloperoxidase levels were increased with EAEC coinfection (P < 0.05). EAEC pathogen codetection was associated with lower neopterin levels compared to those of no-pathogen control children (P < 0.05). Mothers of children with EAEC coinfections had lower levels of education, poorer hygiene and sanitation, lower socioeconomic status, and lower breast-feeding rates compared to mothers of children in whom no pathogen was detected (P < 0.05). CONCLUSIONS: These data emphasize the public health importance of subclinical EAEC infection in early infancy in association with other pathogens and the need for improved maternal and child care, hygiene, sanitation, and socioeconomic factors.

Extracellular acidosis selectively inhibits pharmacomechanical coupling induced by carbachol in strips of rat gastric fundus.

NEW FINDINGS: What is the central question of this study? Acute acidosis that results from short-term exercise is involved in delayed gastric emptying in rats and the lower responsiveness of gastric fundus strips to carbachol. Does extracellular acidosis decrease responsiveness to carbachol in tissues of sedentary rats? How? What is the main finding and its importance? Extracellular acidosis inhibits cholinergic signalling in the rat gastric fundus by selectively influencing the Gq/11 protein signalling pathway. Acute acidosis that results from short-term exercise delays gastric emptying in rats and decreases the responsiveness to carbachol in gastric fundus strips. The regulation of cytosolic Ca2+ concentrations appears to be a mechanism of action of acidosis. The present study investigated the way in which acidosis interferes with gastric smooth muscle contractions. Rat gastric fundus isolated strips at pH 6.0 presented a lower magnitude of carbachol-induced contractions compared with preparations at pH 7.4. This lower magnitude was absent in carbachol-stimulated duodenum and KCl-stimulated gastric fundus strips. In Ca2+ -free conditions, repeated contractions that were induced by carbachol progressively decreased, with no influence of extracellular pH. In fundus strips, CaCl2 -induced contractions were lower at pH 6.0 than at pH 7.4 but only when stimulated in the combined presence of carbachol and verapamil. In contrast, verapamil-sensitive contractions that were induced by CaCl2 in the presence of KCl did not change with pH acidification. In Ca2+ store-depleted preparations that were treated with thapsigargin, the contractions that were induced by extracellular Ca2+ restoration were smaller at pH 6.0 than at pH 7.4, but relaxation that was induced by SKF-96365 (an inhibitor of store-operated Ca2+ entry) was unaltered by extracellular acidification. At pH 6.0, the phospholipase C inhibitor U-73122 relaxed carbachol-induced contractions less than at pH 7.4, and this phenomenon was absent in tissue that was treated with the RhoA kinase blocker Y-27632. Thus, extracellular acidosis inhibited pharmacomechanical coupling in gastric fundus by selectively inhibiting the Gq/11 protein signalling pathway, whereas electromechanical coupling remained functionally preserved.

Amyrins from Protium heptaphyllum Reduce High-Fat Diet-Induced Obesity in Mice via Modulation of Enzymatic, Hormonal And Inflammatory Responses.

Obesity remains a global problem. In search of phytochemicals that have antiobesity potential, this study evaluated α,ß-amyrin, a triterpenoid mixture from Protium heptaphyllum, on high-fat diet-induced obesity in mice. Groups of mice (n = 8) were fed a normal diet or a high-fat diet, and were orally treated or not treated with either α,ß-amyrin (10 or 20 mg/kg) or sibutramine (10 mg/kg) for 15 weeks. Variables measured at termination were body weight, visceral fat accumulation, adipocyte surface area, peroxisome proliferator-activated receptor gamma, and lipoprotein lipase expressions in adipose tissue, the levels of plasma glucose and insulin, the satiety hormones ghrelin and leptin, the digestive enzymes amylase and lipase, and the inflammatory mediators TNF-α, interleukin-6, and MCP-1. Results showed that α,ß-amyrin treatment resulted in lower high-fat diet-induced increases in body weight, visceral fat content, adipocyte surface area, peroxisome proliferator-activated receptor gamma, and lipoprotein lipase expressions, and blood glucose and insulin levels. Additionally, the markedly elevated leptin and decreased ghrelin levels seen in the high-fat diet-fed control mice were significantly modulated by α,ß-amyrin treatment. Furthermore, α,ß-amyrin decreased serum TNF-α and MCP-1. These results suggest that α,ß-amyrin could be beneficial in reducing high-fat diet-induced obesity and associated disorders via modulation of enzymatic, hormonal, and inflammatory responses.

Group a rotavirus and norovirus genotypes circulating in the northeastern Brazil in the post-monovalent vaccination era.

Group A rotaviruses (RVA) and noroviruses (NoV) are the leading cause of acute gastroenteritis (AGE) worldwide. Childhood diarrhea deaths and hospital admissions have declined since the introduction of the monovalent (G1P[8]) vaccine (Rotarix(®) [RV1]) in the National Immunization Program in Brazil in 2006. This study aims to investigate the epidemiological profile of NoV and RVA infections from children with AGE in the Northeastern region of Brazil in the post vaccine season. Two-hundred fecal samples collected from children up to 10 years old in Fortaleza, Ceará between 2008-2009 were screened for the presence of RVA and NoV. Positive samples were genotyped and sequenced. The RVA screening revealed 12% prevalence and all RVA strains belonged to G2P[4] genotype. Phylogenetic analysis based on the 11 RVA genome segments sequenced from eight samples revealed a DS-1-like genotype constellation: I2-R2-C2-M2-A2-N2-T2-E2-H2. For NoV screening, the prevalence observed was 17% and the following genotypes were detected: GII.4 (59%), GII.12 (17%), GII.6 (9%), GII.3 (6%), and GII.? (9%). At least four different NoVs genotypes and two RVA G2P[4] variants were identified circulating in the Northeastern region of Brazil. RVA phylogenetic analysis suggests that the RVA G2P[4] strains might have originated from intragenogroup reassortment events. Whether the genetic modifications observed in these contemporary G2P[4] RVA strains may impact the long-term effectiveness of the current vaccination programs remains to be explored. These data reinforce the importance of surveillance for monitoring the emergence of new strains of RVA and NoV and their impact on cases of acute gastroenteritis.

Update on molecular epidemiology of Shigella infection.

PURPOSE OF REVIEW: Shigella spp. are important etiologic agents of diarrhea worldwide. This review summarizes the recent findings on the epidemiology, diagnosis, virulence genes, and pathobiology of Shigella infection. RECENT FINDINGS: Shigella flexneri and Shigella sonnei have been identified as the main serogroups circulating in developing and developed countries, respectively. However, a shift in the dominant species from S. flexneri to S. sonnei has been observed in countries that have experienced recent improvements in socioeconomic conditions. Despite the increasing usage of molecular methods in the diagnosis and virulence characterization of Shigella strains, researchers have been unsuccessful in finding a specific target gene for this bacillus. New research has demonstrated the role of proteins whose expressions are temperature-regulated, as well as genes involved in the processes of adhesion, invasion, dissemination, and inflammation, aiding in the clarification of the complex pathobiology of shigellosis. SUMMARY: Knowledge about the epidemiologic profile of circulating serogroups of Shigella and an understanding of its pathobiology as well as of the virulence genes is important for the development of preventive measures and interventions to reduce the worldwide spread of shigellosis.

Microbiologic methods utilized in the MAL-ED cohort study.

A central hypothesis of The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study is that enteropathogens contribute to growth faltering. To examine this question, the MAL-ED network of investigators set out to achieve 3 goals: (1) develop harmonized protocols to test for a diverse range of enteropathogens, (2) provide quality-assured and comparable results from 8 global sites, and (3) achieve maximum laboratory throughput and minimum cost. This paper describes the rationale for the microbiologic assays chosen and methodologies used to accomplish the 3 goals.