Etiology and severity of diarrheal diseases in infants at the semiarid region of Brazil: A case-control study.

BACKGROUND: Diarrheal diseases are an important cause of morbidity and mortality among children in developing countries. We aimed to study the etiology and severity of diarrhea in children living in the low-income semiarid region of Brazil. METHODOLOGY: This is a cross-sectional, age-matched case-control study of diarrhea in children aged 2-36 months from six cities in Brazil's semiarid region. Clinical, epidemiological, and anthropometric data were matched with fecal samples collected for the identification of enteropathogens. RESULTS: We enrolled 1,200 children, 596 cases and 604 controls. By univariate analysis, eight enteropathogens were associated with diarrhea: Norovirus GII (OR 5.08, 95% CI 2.10, 12.30), Adenovirus (OR 3.79, 95% CI 1.41, 10.23), typical enteropathogenic Escherichia coli (tEPEC), (OR 3.28, 95% CI 1.39, 7.73), enterotoxigenic E. coli (ETEC LT and ST producing toxins), (OR 2.58, 95% CI 0.99, 6.69), rotavirus (OR 1.91, 95% CI 1.20, 3.02), shiga toxin-producing E. coli (STEC; OR 1.77, 95% CI 1.16, 2.69), enteroaggregative E. coli (EAEC), (OR 1.45, 95% CI 1.16, 1.83) and Giardia spp. (OR 1.39, 95% CI 1.05, 1.84). By logistic regression of all enteropathogens, the best predictors of diarrhea were norovirus, adenovirus, rotavirus, STEC, Giardia spp. and EAEC. A high diarrhea severity score was associated with EAEC. CONCLUSIONS: Six enteropathogens: Norovirus, Adenovirus, Rotavirus, STEC, Giardia spp., and EAEC were associated with diarrhea in children from Brazil's semiarid region. EAEC was associated with increased diarrhea severity.

Milk with and without lactoferrin can influence intestinal damage in a pig model of malnutrition.

Malnutrition remains a leading contributor to the morbidity and mortality of children under the age of five worldwide. However, the underlying mechanisms are not well understood necessitating an appropriate animal model to answer fundamental questions and conduct translational research into optimal interventions. One potential intervention is milk from livestock that more closely mimics human milk by increased levels of bioactive components that can promote a healthy intestinal epithelium. We tested the ability of cow milk and milk from transgenic cows expressing human lactoferrin at levels found in human milk (hLF milk) to mitigate the effects of malnutrition at the level of the intestine in a pig model of malnutrition. Weaned pigs (3 weeks old) were fed a protein and calorie restricted diet for five weeks, receiving cow, hLF or no milk supplementation daily from weeks 3-5. After three weeks, the restricted diet induced changes in growth, blood chemistry and intestinal structure including villous atrophy, increased ex vivo permeability and decreased expression of tight junction proteins. Addition of both cow and hLF milk to the diet increased growth rate and calcium and glucose levels while promoting growth of the intestinal epithelium. In the jejunum hLF milk restored intestinal morphology, reduced permeability and increased expression of anti-inflammatory IL-10. Overall, this pig model of malnutrition mimics salient aspects of the human condition and demonstrates that cow milk can stimulate the repair of damage to the intestinal epithelium caused by protein and calorie restriction with hLF milk improving this recovery to a greater extent.

Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. METHODS: Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 µg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). RESULTS: Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. CONCLUSIONS: These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

Increased gene targeting in Ku70 and Xrcc4 transiently deficient human somatic cells.

The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell's genome is important to advance strategies for biotechnology and genetic medicine.

Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway.

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.

Determination of genetic status in a closed colony of rhesus monkeys (Macaca mulatta).

The long-term management of breeding colonies requires some measure of genetic diversity in the animal population. For the maintenance of breeding colonies of monkeys used for biomedical research, known pedigrees supply precise data to determine the genetic status of colonies. We present data of genetic analyses in an old closed colony of rhesus macaques (Macaca mulatta) that was established in 1932 with 100 animals. For more than 40 years, the animals were kept on an isolated island and, in 1980, single-male breeding groups were established. A total of 333 DNA samples of these animals were typed to 20 microsatellite markers using multiplex PCR in order to verify inbreeding coefficient (alpha) and level of heterozygosity. We found an average heterozygosity of 64% and obtained alpha=-0.03293 (+/-0.00573). Our results indicate that the reproductive strategy used was effective because consanguineous breeding was avoided. A continuous genetic program must be carried out in order to obtain better quality primates for biomedical research.

Growth, development, and gene expression by in vivo- and in vitro-produced day 7 and 16 bovine embryos.

The effects of the embryo production system on growth and transcription rate of day 7 and 16 bovine embryos were investigated. In vivo- (controls) and in vitro-produced (IVP) embryos were transferred to female recipients on day 7 of development, and were allowed to develop in a synchronous uterine environment to day 16. Embryonic transcripts for insulin-like growth factors-1 and -2 (IGF-1 and -2), their receptors (IGF-1r and -2r), facilitative glucose transporters-1 and -3 (Glut-1 and -3), and interferon-tau (IFN-tau) were determined by real-time quantitative PCR (TaqMan); gender diagnosis was performed on day 16 concepti only. On day 7, IVP embryos presented lower mRNA levels than controls (P < 0.05), but these differences were generally reduced on day 16. No IGF-1 transcripts were detected on day 7, but a low IGF-1 mRNA level was observed in day 16 embryos. In the IVP group, IFN-tau mRNA levels were lower on day 7 (P < 0.05), but higher than controls on day 16 (P < 0.05). Control embryos showed a temporal decrease in the relative transcription from day 7 to 16 (P < 0.05), except IGF-1 mRNA. On day 16, IVP concepti were shorter and displayed smaller embryonic discs (P < 0.05). Female concepti were generally smaller than males, and IGF-2r mRNA and growth were negatively correlated. The in vitro production of bovine embryos negatively affected the amount of gene expression on day 7 and the rate of development on day 16. Physical traits and transcriptional activity on day 16 were associated with one another, which appeared to be significant for growth and development.