Pathogenesis of Salmonella-induced enteritis

Infections with Salmonella serotypes are a major cause of food-borne diseases worldwide. Animal models other than the mouse have been employed for the study of nontyphoidal Salmonella infections because the murine model is not suitable for the study of Salmonella-induced diarrhea. The microbe has developed mechanisms to exploit the host cell machinery to its own purpose. Bacterial proteins delivered directly into the host cell cytosol cause cytoskeletal changes and interfere with host cell signaling pathways, which ultimately enhance disease manifestation. Recently, marked advances have been made in our understanding of the molecular interactions between Salmonella serotypes and their hosts. Here, we discuss the molecular basis of the pathogenesis of Salmonella-induced enteritis.

Effect of hydroxyurea on G gamma chain fetal hemoglobin synthesis by sickle-cell disease patients

Hydroxyurea is used for sickle-cell disease patients in order to increase fetal hemoglobin synthesis and consequently decrease the severity of pain episodes. Fetal hemoglobin, which is formed by gamma-globin chains A and G, is present in a constant composition throughout fetal development: about 75% of Ggamma and 25% of Agamma. In contrast, adult red cells contain about 40% of Ggamma and 60% of Agamma. In the present study, we analyzed the effect of hydroxyurea induction on the gamma chain composition of fetal hemoglobin in 31 sickle-cell disease patients treated with hydroxyurea. The control group was composed of 30 sickle-cell disease patients not treated with hydroxyurea in clinical steady state. The patients were older than 13 years and were not matched for age. All patients were seen at Hemocentro/UNICAMP and Boldrini Infantile Center, Campinas, SP, Brazil. The levels of total hemoglobin were significantly higher in patients treated with hydroxyurea (mean ± SD, 9.6 ± 2.16 g/dl) than in untreated patients (8.07 ± 0.91 g/dl). Fetal hemoglobin levels were also higher in treated patients (14.16 ± 8.31%) than in untreated patients (8.8 ± 4.09%), as was the Ggamma/Agamma ratio (1.45 ± 0.78 vs 0.98 ± 0.4, P < 0.005). The increase in the Ggamma/Agamma ratio in patients treated with hydroxyurea suggests the prevalence of a pattern of fetal hemoglobin synthesis, whereas patients not treated with hydroxyurea maintain the adult pattern of fetal hemoglobin synthesis. Because no correlation was observed between the Ggamma/Agamma ratio and total hemoglobin or fetal hemoglobin levels, the increase in Ggamma chain synthesis may not imply a higher production of hemoglobin.

Thiol-independent activity of a cholesterol-binding enterohemolysin produced by enteropathogenic Escherichia coli

Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 µg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 µg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

Etiology of diarrheal infections in children of Porto Velho (Rondonia, Western Amazon region, Brazil)

In the present study, 470 children less than 72 months of age and presenting acute diarrhea were examined to identify associated enteropathogenic agents. Viruses were the pathogens most frequently found in stools of infants with diarrhea, including 111 cases of rotavirus (23.6% of the total diarrhea cases) and 30 cases of adenovirus (6.3%). The second group was diarrheogenic Escherichia coli (86 cases, 18.2%), followed by Salmonella sp (44 cases, 9.3%) and Shigella sp (24 cases, 5.1%). Using the PCR technique to differentiate the pathogenic categories of E. coli, it was possible to identify 29 cases (6.1%) of enteropathogenic E. coli (EPEC). Of these, 10 (2.1%) were typical EPEC and 19 (4.0%) atypical EPEC. In addition, there were 26 cases (5.5%) of enteroaggregative E. coli, 21 cases (4.4%) of enterotoxigenic E. coli, 7 cases (1.4%) of enteroinvasive E. coli (EIEC), and 3 cases (0.6%) of enterohemorrhagic E. coli. When comparing the frequencies of diarrheogenic E. coli, EPEC was the only category for which significant differences were found between diarrhea and control groups. A low frequency of EIEC was found, thus EIEC cannot be considered to be a potential etiology agent of diarrhea. Simultaneous infections with two pathogens were found in 39 diarrhea cases but not in controls, suggesting associations among potential enteropathogens in the etiology of diarrhea. The frequent association of diarrheogenic E. coli strains was significantly higher than the probability of their random association, suggesting the presence of facilitating factor(s).

Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8% identity for nucleotides and 96.8% identity for amino acids) and more similar to the BCoV-ENT strain (98.7% for nucleotides and 98.7% for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

ApoE polymorphisms and diarrheal outcomes in Brazilian shanty town children

A series of studies have shown that the heavy burdens of diarrheal diseases in the first 2 formative years of life in children living in urban shanty towns have negative effects on physical and cognitive development lasting into later childhood. We have shown that APOE4 is relatively common in shanty town children living in Brazil (13.4%) and suggest that APOE4 has a protective role in cognitive development as well as weight-for-height in children with heavy burdens of diarrhea in early childhood (64/123; 52%), despite being a marker for cognitive decline with Alzheimer’s and cardiovascular diseases later in life. APOE2 frequency was higher among children with heaviest diarrhea burdens during the first 2 years of life, as detected by PCR using the restriction fragment length polymorphism method, raising the possibility that ApoE-cholesterol balance might be critical for growth and cognitive development under the stress of heavy diarrhea burdens and when an enriched fat diet is insufficient. These findings provide a potential explanation for the survival advantage in evolution of genes, which might raise cholesterol levels during heavy stress of diarrhea burdens and malnutrition early in life.

Evaluation of piroxicam-β-cyclodextrin as a preemptive analgesic in functional endoscopic sinus surgery

The preemptive analgesic efficacy and adverse effects of preoperatively administered piroxicam-β-cyclodextrin for post-endoscopic sinus surgery pain was determined in a prospective, double-blind, randomized, clinical study. Seventy-five American Society of Anesthesiologists status I-II patients, aged 18-65 years, were divided into three groups with similar demographic characteristics: group 1 received 20 mg piroxicam-β-cyclodextrin, group 2 received 40 mg piroxicam-β-cyclodextrin and group 3 received placebo orally before induction of general anesthesia. A blinded observer recorded the incidence and severity of pain at admission to the post-anesthesia care unit (PACU), at 15, 30, and 45 min in the PACU, and 1, 2, 4, 6, and 24 h postoperatively. All patients received patient-controlled morphine analgesia during the postoperative period and consumption was recorded for 24 h. During the PACU period, mean visual analogue scale values were significantly lower in groups 1 and 2 compared to group 3 (P < 0.05). During the postoperative period, morphine consumption was 3.03 ± 2.54, 2.7 ± 2.8, and 5.56 ± 3.12 mg for each group, respectively (P < 0.05). As a side effect, bleeding was observed in groups 1 and 3, nausea and vomiting in all groups, and edema only in group 3. However, no significant differences were detected in any of the parameters analyzed, which also included epigastric pain, constipation/diarrhea and headache. Similar hematological test results were obtained for all groups. Preemptive administration of piroxicam-β-cyclodextrin effectively reduced analgesic consumption, and 40 mg of the drug was more effective than 20 mg piroxicam-β-cyclodextrin without side effects during the postoperative period.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

Probiotics and oral health: In vitro and clinical studies

Aino Toiviainen Probiotics and oral health: in vitro and clinical studies University of Turku, Faculty of Medicine, Institute of Dentistry, Periodontology, Finnish Doctoral Program in Oral Sciences (FINDOS-Turku), Turku, Finland Annales Universitatis Turkuensis, Sarja – Ser. D, Medica-Odontologica. Painosalama Oy, Turku, Finland, 2015 Probiotics are used, for example, to prevent and treat diarrhea, allergies and respiratory infections, and there is an increasing interest to use probiotics also for oral health purposes. The most commonly used probiotic bacteria are lactobacilli and bifidobacteria, which are acidogenic and aciduric. From the oral point of view, use of these probiotics may, at least in theory, mean an increased risk of caries. In this thesis, the effects of probiotics on oral microbial composition, acid production of dental plaque and gingival health were studied through in vitro studies and two clinical studies. In a randomized, double-blind and crossover study, 13 healthy adults were allocated into two groups. Half of the subjects first consumed Lactobacillus rhamnosus GG tablets twice a day for two weeks, and after the washout period, L. reuteri tablets twice a day for two weeks. The other half of the subjects used the tablets in reverse order. In another controlled, randomized and double-blind study, 62 healthy adults were allocated into two groups. One group used the test tablets containing L. rhamnosus GG and B. lactis BB-12 and the other group used control tablets without probiotics. The recommendation for the use of the tablets was 4 per day for 4 weeks. Probiotic lactobacilli interfered with S. mutans biofilm formation and the adhesion of S. mutans to saliva-coated hydroxyapatite in vitro. No effect was found in S. mutans levels in the three-species biofilms. In clinical studies, the studied probiotics had no effect on the acid production of plaque. The counts of mutans streptococci and the oral microbial composition remained the same. Tablets containing L. rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12 did decrease the amount of plaque and gingival bleeding. According to our results, it seems that probiotics have beneficial effects on gingival health. The present results confirmed that probiotics are safe and have beneficial effects on oral health. Since the consumption of probiotics by the general population is steadily increasing, an understanding of the functions of probiotics in the oral cavity has become more important. Keywords: lactobacilli, bifidobacteria, caries, periodontal disease, mutans streptococci, probiotics

Chemical composition of tempeh from soybean cultivars specially developed for human consumption

Tempeh is a food obtained by fermentation of soybean grains by the fungus Rizophus oligosporus. It is a traditional Indonesian food that presents benefits for human health protecting against diarrhea and chronic diseases. Tempeh processing includes dehulling, cooking, inoculation, and fermentation. In this study, chemical characteristics of tempeh prepared with soybean cultivars specially developed for human consumption (BRS 216, BRS 232, BRS 257, and BRS 267) were investigated. Soybean grains and tempeh obtained from these cultivars were analyzed for oil, protein, antinutrional factors, and isoflavone content. Cultivar BRS 216 presented the highest protein content in the grains (36.81%) and in tempeh (51.99%). On average, the protein content in tempeh increased 16% in relation to that of soybean grains. Isoflavone content was higher in the grains than in tempeh with significant differences among the cultivars. However, the aglycones content increased about 50% in tempeh (49.00 mg.100 g-1 on average) compared to that of raw material (soybean grains - 21.49 mg.100 g-1, on average). The content of Kunitz trypsin inhibitor (KSTI) reduced 83% in tempeh, on average, as compared to the value found in the grains. Phytic acid content was similar in both tempeh and the grains.

Lactose intolerance and cow's milk protein allergy

Abstract Adverse reactions to food intake have very diverse etiology and symptomatology. Regarding milk, its food allergy is presented as lactose intolerance, the sugar in milk, or allergy to milk protein. Despite having different symptomatology, confusions among allergic conditions to dairy and its mediators are common. Milk protein allergy originates from protein components present in milk, causing reactions to either the protein fractions in emulsion (caseins) or in whey (milk albumin). The allergic reaction is type IV mediated by T lymphocytes. The allergic reaction produces severe cellular damage and it triggers physical, mental and emotional symptomatology that may vary in time, intensity and severity. Lactose intolerance is originated by total or partial absence of the enzyme that digests this disaccharide. Lactose intolerance can be primary or congenital and secondary; the former being more rare and severe, the latter being more common. Lactase deficiency can be diagnosed by symptoms associated with cramping and diarrhea. Thus, the objective of this study was to conduct a review of available literature on cow’s milk protein allergy and lactose intolerance.

Roles of keratins in intestinal health and disease

Intermediate filament keratins (K) play a pivotal role in protein targeting and epithelialcytoprotection from stress as evidenced by keratin mutations predisposing to human liver and skin diseases and possibly inflammatory bowel disease (IBD). The K8-null (K8-/-) mice exhibit colonic phenotype similar to IBD and marked spontaneous colitis, epithelial hyperproliferation, decreased apoptosis, mistargeting of proteins leading to defective ion transport and diarrhea. The K8-heterozygote (K8+/-) mouse colon appears normal but displays a defective sodium (Na+) and chloride (Cl-) transport similar to, but milder than K8-/-. Characterization of K8+/- colon revealed ~50% less keratins (K7, K8, K19, K20) compared to K8 wild type (K8+/+). A similar ~50% decrease was seen in K8+/- mRNA levels as compared to K8+/+, while the mRNA levels for the other keratins were unaltered. K8+/- keratins were arranged in a normal colonic crypt expression pattern, except K7 which was expressed at the top of crypts in contrast to K8+/+. The K8+/- colon showed mild hyperplasia but no signs of inflammation and no resistance to apoptosis. Experimental colitis induced by using different concentrations of dextran sulphate sodium (DSS) showed that K8+/- mice are slightly more sensitive to induced colitis and showed a delayed recovery compared to K8+/+. Hence, the K8+/- mouse with less keratins and without inflammation, provided a novel model to study direct molecular mechanisms of keratins in intestinal homeostasis and ion transport. Different candidate ion transporters for a possible role in altered ion transport seen in the K8-/- and K8+/- mouse colon were evaluated. Besides normal levels of CFTR, PAT-1 and NHE-3, DRA mRNA levels were decreased 3-4-fold and DRA protein nearly entirely lost in K8-/- caecum, distal and proximal colon compared to K8+/+. In K8+/- mice, DRA mRNA levels were unaltered while decreased DRA protein level and patchy distribution was detected particularly in the proximal colon and as compared to K8+/+. DRA was similarly decreased when K8 was knocked-down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammation-independent manner. The dramatic loss of DRA in colon and caecum of K8-/- mice was responsible for the chloride transport defect. The milder ion transport in K8+/- colon might be related to DRA suggesting a role for K8 in regulation of DRA expression and targeting. The current study demonstrates the importance of keratins in stress protection and cell signaling. Furthermore, we have also successfully developed a novel, simple, fast, cost effective, non-invasive in vivo imaging method for the early diagnosis of murine colitis with specificity for both genetic and experimental colitis. The said modality provides continuous measurements of reactive oxygen and nitrogen species (RONS) and minimizes the use of an increased number of experimental animals by using a luminal derivative chemiluminescent probe, L-012 which provides a cost-effective tool to study the level and longitudinal progression of colitis.

薛院判醫案二十四種Xie yuan pan yi an er shi si zhong.Archives médicales du médecin impérial Xie.Nei ke.Médecine interne.

Contient : I十四經絡發揮Shi si jing luo fa hui.Examen des quatorze vaisseaux ; II難經本義Nan jing ben yi.Sens du Nan jing ; III本草發揮Ben cao fa hui.Traité de matière médicale ; IV平治會萃Ping zhi hui zui.Traité sur diverses maladies ; V家居醫錄內科摘要Jia ju yi lu nei ke zhe yao.Éléments de médecine interne ; VI明醫雜著Ming yi za zhu.Traités divers de médecins célèbres ; VII傷寒鈐法Shang han qian fa.Étude du traité de la fièvre typhoïde ; VIII敖氏外傷金鏡錄圖Ao shi wai shang jin jing lu tu.Traité de la langue dans la fièvre typhoïde ; IX原機啟微Yuan ji qi wei.Traité sur les maladies et les médicaments.You ke.Médecine infantile ; X保嬰撮要Bao ying tshoo yao.Traité général de médecine infantile ; XI錢氏小兒直（眞）訣Qian shi xiao er zhi (zhen) jue.Traité de médecine infantile ; XII陳氏小兒痘疹方論Chen shi xiao er dou zhen fang lun.Formules et traité pour la petite vérole chez les enfants ; XIII保嬰金鏡錄Bao ying jin jing lu.Traité de médecine infantile.Nü ke.Gynécologie ; XIV婦人良方Fu ren liang fang.Formules pour les maladies des femmes ; XV女科撮要Nü ke tshoo yao.Principes de gynécologie.Wai ke.Médecine externe ; XVI立齋外科發揮Li zhai wai ke fa hui.Examen de la médecine externe ; XVII外科心法Wai ke xin fa.Préceptes de médecine externe ; XVIII外科樞要Wai ke chu yao.Principes de médecine externe ; XIX外科精要Wai ke jing yao.Éléments de médecine externe ; XX癰疽神秘驗方Yong ju shen bi yan fang.Formules pour les furoncles ; XXI外科經驗方Wai ke jing yan fang.Formules éprouvées de médecine externe ; XXII正體類要Zheng ti lei yao.Exposé méthodique du corps humain ; XXIII口齒類要Kou chi lei yao.Exposé méthodique de la bouche et des dents ; XXIV癘瘍機要Li yang ji yao.Traité des ulcères