A murine model of diarrhea, growth impairment and metabolic disturbances with Shigella flexneri infection and the role of zinc deficiency.

Shigella is one of the major enteric pathogens worldwide. We present a murine model of S. flexneri infection and investigate the role of zinc deficiency (ZD). C57BL/6 mice fed either standard chow (HC) or ZD diets were pretreated with an antibiotic cocktail and received S. flexneri strain 2457T orally. Antibiotic pre-treated ZD mice showed higher S. flexneri colonization than non-treated mice. ZD mice showed persistent colonization for at least 50 days post-infection (pi). S. flexneri-infected mice showed significant weight loss, diarrhea and increased levels of fecal MPO and LCN in both HC and ZD fed mice. S. flexneri preferentially colonized the colon, caused epithelial disruption and inflammatory cell infiltrate, and promoted cytokine production which correlated with weight loss and histopathological changes. Infection with S. flexneri ΔmxiG (critical for type 3 secretion system) did not cause weight loss or diarrhea, and had decreased stool shedding duration and tissue burden. Several biochemical changes related to energy, inflammation and gut-microbial metabolism were observed. Zinc supplementation increased weight gains and reduced intestinal inflammation and stool shedding in ZD infected mice. In conclusion, young antibiotic-treated mice provide a new model of oral S. flexneri infection, with ZD promoting prolonged infection outcomes.

Modulation of Intestinal Immune and Barrier Functions by Vitamin A: Implications for Current Understanding of Malnutrition and Enteric Infections in Children.

The micronutrient vitamin A refers to a group of compounds with pleiotropic effects on human health. These molecules can modulate biological functions, including development, vision, and regulation of the intestinal barrier. The consequences of vitamin A deficiency and supplementation in children from developing countries have been explored for several years. These children live in an environment that is highly contaminated by enteropathogens, which can, in turn, influence vitamin A status. Vitamin A has been described to modulate gene expression, differentiation and function of diverse immune cells; however, the underlying mechanisms are not fully elucidated. This review aims to summarize the most updated advances on elucidating the vitamin A effects targeting intestinal immune and barrier functions, which may help in further understanding the burdens of malnutrition and enteric infections in children. Specifically, by covering both clinical and in vivo/in vitro data, we describe the effects of vitamin A related to gut immune tolerance/homeostasis, intestinal barrier integrity, and responses to enteropathogens in the context of the environmental enteric dysfunction. Some of the gaps in the literature that require further research are also highlighted.

Docosanoids Promote Neurogenesis and Angiogenesis, Blood-Brain Barrier Integrity, Penumbra Protection, and Neurobehavioral Recovery After Experimental Ischemic Stroke.

Docosahexaenoic acid (DHA) and neuroprotectin D1 (NPD1) are neuroprotective after experimental ischemic stroke. To explore underlying mechanisms, SD rats underwent 2 h of middle cerebral artery occlusion (MCAo) and treated with DHA (5 mg/kg, IV) or NPD1 (5 µg/per rat, ICV) and vehicles 1 h after. Neuro-behavioral assessments was conducted on days 1, 2, and 3, and on week 1, 2, 3, or 4. BrdU was injected on days 4, 5, and 6, immunohistochemistry was performed on week 2 or 4, MRI on day 7, and lipidomic analysis at 4 and 5 h after onset of stroke. DHA improved short- and long-term behavioral functions and reduced cortical, subcortical, and total infarct volumes (by 42, 47, and 31%, respectively) after 2 weeks and reduced tissue loss by 50% after 4 weeks. DHA increased the number of BrdU+/Ki-67+, BrdU+/DCX+, and BrdU+/NeuN+ cells in the cortex, subventricular zone, and dentate gyrus and potentiated NPD1 synthesis in the penumbra at 5 h after MCAo. NPD1 improved behavior, reduced lesion volumes, protected ischemic penumbra, increased NeuN, GFAP, SMI-71-positive cells and vessels, axonal regeneration in the penumbra, and attenuated blood-brain barrier (BBB) after MCAo. We conclude that docosanoid administration increases neurogenesis and angiogenesis, activates NPD1 synthesis in the penumbra, and diminishes BBB permeability, which correlates to long-term neurobehavioral recovery after experimental ischemic stroke.

Zinc treatment ameliorates diarrhea and intestinal inflammation in undernourished rats.

BACKGROUND: WHO guidelines recommend zinc supplementation as a key adjunct therapy for childhood diarrhea in developing countries, however zinc's anti-diarrheal effects remain only partially understood. Recently, it has been recognized that low-grade inflammation may influence stunting. In this study, we examined whether oral zinc supplementation could improve weight, intestinal inflammation, and diarrhea in undernourished weanling rats. METHODS: Rats were undernourished using a northeastern Brazil regional diet (RBD) for two weeks, followed by oral gavage with a saturated lactose solution (30 g/kg) in the last 7 days to induce osmotic diarrhea. Animals were checked for diarrhea daily after lactose intake. Blood was drawn in order to measure serum zinc levels by atomic absorption spectroscopy. Rats were euthanized to harvest jejunal tissue for histology and cytokine profiles by ELISA. In a subset of animals, spleen samples were harvested under aseptic conditions to quantify bacterial translocation. RESULTS: Oral zinc supplementation increased serum zinc levels following lactose-induced osmotic diarrhea. In undernourished rats, zinc improved weight gain following osmotic diarrhea and significantly reduced diarrheal scores by the third day of lactose intake (p < 0.05), with improved jejunum histology (p < 0.0001). Zinc supplementation diminished bacterial translocation only in lactose-challenged undernourished rats (p = 0.03) compared with the untreated challenged controls and reduced intestinal IL-1ß and TNF-α cytokines to control levels. CONCLUSION: Altogether our findings provide novel mechanisms of zinc action in the setting of diarrhea and undernutrition and support the use of zinc to prevent the vicious cycle of malnutrition and diarrhea.

Effects of glutamine alone or in combination with zinc and vitamin A on growth, intestinal barrier function, stress and satiety-related hormones in Brazilian shantytown children.

OBJECTIVE: To determine the impact of supplemental zinc, vitamin A, and glutamine alone or in combination on growth, intestinal barrier function, stress and satiety-related hormones among Brazilian shantytown children with low median height-for-age z-scores. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in children aged two months to nine years from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for testing (a total of 120 children) were as follows: (1) glutamine alone, n = 38; (2) glutamine plus vitamin A plus zinc, n = 37; and a placebo (zinc plus vitamin A vehicle) plus glycine (isonitrogenous to glutamine) control treatment, n = 38. Leptin, adiponectin, insulin-like growth factor (IGF-1), and plasma levels of cortisol were measured with immune-enzymatic assays; urinary lactulose/mannitol and serum amino acids were measured with high-performance liquid chromatography. ClinicalTrials.gov: NCT00133406. RESULTS: Glutamine treatment significantly improved weight-for-height z-scores compared to the placebo-glycine control treatment. Either glutamine alone or all nutrients combined prevented disruption of the intestinal barrier function, as measured by the percentage of lactulose urinary excretion and the lactulose:mannitol absorption ratio. Plasma leptin was negatively correlated with plasma glutamine (p = 0.002) and arginine (p = 0.001) levels at baseline. After glutamine treatment, leptin was correlated with weight-for-age (WAZ) and weight-for-height z-scores (WHZ) (p≤0.002) at a 4-month follow-up. In addition, glutamine and all combined nutrients (glutamine, vitamin A, and zinc) improved the intestinal barrier function in these children. CONCLUSION: Taken together, these findings reveal the benefits of glutamine alone or in combination with other gut-trophic nutrients in growing children via interactions with leptin.

Effects of glutamine alone or in combination with zinc and vitamin A on growth, intestinal barrier function, stress and satiety-related hormones in Brazilian shantytown children

OBJECTIVE: To determine the impact of supplemental zinc, vitamin A, and glutamine alone or in combination on growth, intestinal barrier function, stress and satiety-related hormones among Brazilian shantytown children with low median height-for-age z-scores. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in children aged two months to nine years from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for testing (a total of 120 children) were as follows: (1) glutamine alone, n = 38; (2) glutamine plus vitamin A plus zinc, n = 37; and a placebo (zinc plus vitamin A vehicle) plus glycine (isonitrogenous to glutamine) control treatment, n = 38. Leptin, adiponectin, insulin-like growth factor (IGF-1), and plasma levels of cortisol were measured with immune-enzymatic assays; urinary lactulose/mannitol and serum amino acids were measured with high-performance liquid chromatography. ClinicalTrials.gov: NCT00133406. RESULTS: Glutamine treatment significantly improved weight-for-height z-scores compared to the placebo-glycine control treatment. Either glutamine alone or all nutrients combined prevented disruption of the intestinal barrier function, as measured by the percentage of lactulose urinary excretion and the lactulose:mannitol absorption ratio. Plasma leptin was negatively correlated with plasma glutamine (p = 0.002) and arginine (p = 0.001) levels at baseline. After glutamine treatment, leptin was correlated with weight-for-age (WAZ) and weight-for-height z-scores (WHZ) (p≤0.002) at a 4-month follow-up. In addition, glutamine and all combined nutrients (glutamine, vitamin A, and zinc) improved the intestinal barrier function in these children. CONCLUSION: Taken together, these findings reveal the benefits of glutamine alone or in combination ...

Zinc, vitamin A, and glutamine supplementation in Brazilian shantytown children at risk for diarrhea results in sex-specific improvements in verbal learning.

OBJECTIVE: To identify the impact of supplemental zinc, vitamin A, and glutamine, alone or in combination, on long-term cognitive outcomes among Brazilian shantytown children with low median height-for-age z-scores. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in children aged three months to nine years old from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for cognitive testing (total of 167 children) were: (1) placebo, n = 25; (2) glutamine, n = 23; (3) zinc, n = 18; (4) vitamin A, n = 19; (5) glutamine+zinc, n = 20; (6) glutamine+vitamin A, n = 21; (7) zinc+vitamin A, n = 23; and (8) glutamine+zinc+vitamin A, n = 18. Neuropsychological tests were administered for the cognitive domains of non-verbal intelligence and abstraction, psychomotor speed, verbal memory and recall ability, and semantic and phonetic verbal fluency. Statistical analyses were performed using SPSS, version 16.0. ClinicalTrials.gov: NCT00133406. RESULTS: Girls receiving a combination of glutamine, zinc, and vitamin A had higher mean age-adjusted verbal learning scores than girls receiving only placebo (9.5 versus 6.4, p = 0.007) and girls receiving zinc+vitamin A (9.5 versus 6.5, p = 0.006). Similar group differences were not found between male study children. CONCLUSIONS: The findings suggest that combination therapy offers a sex-specific advantage on tests of verbal learning, similar to that seen among female patients following traumatic brain injury.

Zinc, vitamin A, and glutamine supplementation in Brazilian shantytown children at risk for diarrhea results in sex-specific improvements in verbal learning

OBJECTIVE: To identify the impact of supplemental zinc, vitamin A, and glutamine, alone or in combination, on long-term cognitive outcomes among Brazilian shantytown children with low median height-for-age z-scores. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in children aged three months to nine years old from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for cognitive testing (total of 167 children) were: (1) placebo, n = 25; (2) glutamine, n = 23; (3) zinc, n = 18; (4) vitamin A, n = 19; (5) glutamine+zinc, n = 20; (6) glutamine+vitamin A, n = 21; (7) zinc+vitamin A, n = 23; and (8) glutamine+zinc+vitamin A, n = 18. Neuropsychological tests were administered for the cognitive domains of non-verbal intelligence and abstraction, psychomotor speed, verbal memory and recall ability, and semantic and phonetic verbal fluency. Statistical analyses were performed using SPSS, version 16.0. ClinicalTrials.gov: NCT00133406. RESULTS: Girls receiving a combination of glutamine, zinc, and vitamin A had higher mean age-adjusted verbal learning scores than girls receiving only placebo (9.5 versus 6.4, p = 0.007) and girls receiving zinc+vitamin A (9.5 versus 6.5, p = 0.006). Similar group differences were not found between male study children. CONCLUSIONS: The findings suggest that combination therapy offers a sex-specific advantage on tests of verbal learning, similar to that seen among female patients following traumatic brain injury.

Apolipoprotein E4 influences growth and cognitive responses to micronutrient supplementation in shantytown children from northeast Brazil.

OBJECTIVE: Apolipoprotein E4 may benefit children during early periods of life when the body is challenged by infection and nutritional decline. We examined whether apolipoprotein E4 affects intestinal barrier function, improving short-term growth and long-term cognitive outcomes in Brazilian shantytown children. METHODS: A total of 213 Brazilian shantytown children with below-median height-for-age z-scores (HAZ) received 200,000 IU of retinol (every four months), zinc (40 mg twice weekly), or both for one year, with half of each group receiving glutamine supplementation for 10 days. Height-for-age z-scores, weight-for-age z-scores, weight-for-height z-scores, and lactulose:mannitol ratios were assessed during the initial four months of treatment. An average of four years (range 1.4-6.6) later, the children underwent cognitive testing to evaluate non-verbal intelligence, coding, verbal fluency, verbal learning, and delayed verbal learning. Apolipoprotein E4 carriage was determined by PCR analysis for 144 children. RESULTS: Thirty-seven children were apolipoprotein E4(+), with an allele frequency of 13.9%. Significant associations were found for vitamin A and glutamine with intestinal barrier function. Apolipoprotein E4(+) children receiving glutamine presented significant positive Pearson correlations between the change in height-for-age z-scores over four months and delayed verbal learning, along with correlated changes over the same period in weight-for-age z-scores and weight-for-height z-scores associated with non-verbal intelligence quotients. There was a significant correlation between vitamin A supplementation of apolipoprotein E4(+) children and improved delta lactulose/mannitol. Apolipoprotein E4(-) children, regardless of intervention, exhibited negative Pearson correlations between the change in lactulose-to-mannitol ratio over four months and verbal learning and non-verbal intelligence. CONCLUSIONS: During development, apolipoprotein E4 may function concomitantly with gut-tropic nutrients to benefit immediate nutritional status, which can translate into better long-term cognitive outcomes.

Arginine decreases Cryptosporidium parvum infection in undernourished suckling mice involving nitric oxide synthase and arginase.

OBJECTIVE: This study investigated the role of L-arginine supplementation to undernourished and Cryptosporidium parvum-infected suckling mice. METHODS: The following regimens were initiated on the fourth day of life and injected subcutaneously daily. The C. parvum-infected controls received L-arginine (200 mmol/L) or phosphate buffered saline. The L-arginine-treated mice were grouped to receive NG-nitro-arginine methyl ester (L-NAME) (20 mmol/L) or phosphate buffered saline. The infected mice received orally 10(6) excysted C. parvum oocysts on day 6 and were euthanized on day 14 at the infection peak. RESULTS: L-arginine improved weight gain compared with the untreated infected controls. L-NAME profoundly impaired body weight gain compared with all other groups. Cryptosporidiosis was associated with ileal crypt hyperplasia, villus blunting, and inflammation. L-arginine improved mucosal histology after the infection. L-NAME abrogated these arginine-induced improvements. The infected control mice showed an intense arginase expression, which was even greater with L-NAME. L-arginine decreased the parasite burden, an effect that was reversed by L-NAME. Cryptosporidium parvum infection increased urine NO(3)(-)/NO(2)(-) concentrations compared with the uninfected controls, which was increased by L-arginine supplementation, an effect that was also reversed by L-NAME. CONCLUSION: These findings show a protective role of L-arginine during C. parvum infection in undernourished mice, with involvement of arginase I and nitric oxide synthase enzymatic actions.

Apolipoprotein E4 influences growth and cognitive responses to micronutrient supplementation in shantytown children from northeast Brazil

OBJECTIVE: Apolipoprotein E4 may benefit children during early periods of life when the body is challenged by infection and nutritional decline. We examined whether apolipoprotein E4 affects intestinal barrier function, improving short-term growth and long-term cognitive outcomes in Brazilian shantytown children. METHODS: A total of 213 Brazilian shantytown children with below-median height-for-age z-scores (HAZ) received 200,000 IU of retinol (every four months), zinc (40 mg twice weekly), or both for one year, with half of each group receiving glutamine supplementation for 10 days. Height-for-age z-scores, weight-for-age z-scores, weight-forheight z-scores, and lactulose:mannitol ratios were assessed during the initial four months of treatment. An average of four years (range 1.4-6.6) later, the children underwent cognitive testing to evaluate non-verbal intelligence, coding, verbal fluency, verbal learning, and delayed verbal learning. Apolipoprotein E4 carriage was determined by PCR analysis for 144 children. RESULTS: Thirty-seven children were apolipoprotein E4(+), with an allele frequency of 13.9 percent. Significant associations were found for vitamin A and glutamine with intestinal barrier function. Apolipoprotein E4(+) children receiving glutamine presented significant positive Pearson correlations between the change in height-for-age z-scores over four months and delayed verbal learning, along with correlated changes over the same period in weight-for-age z-scores and weight-for-height z-scores associated with non-verbal intelligence quotients. There was a significant correlation between vitamin A supplementation of apolipoprotein E4(+) children and improved delta lactulose/mannitol. Apolipoprotein E4(-) children, regardless of intervention, exhibited negative Pearson correlations between the change in lactulose-to-mannitol ratio over four months and verbal learning and non-verbal intelligence. CONCLUSIONS: During development, apolipoprotein E4 may function concomitantly with gut-tropic nutrients to benefit immediate nutritional status, which can translate into better long-term cognitive outcomes.

Alanyl-glutamine promotes intestinal epithelial cell homeostasis in vitro and in a murine model of weanling undernutrition.

Alanyl-glutamine (Ala-Gln) has recently been shown to enhance catch-up growth and gut integrity in undernourished children from Northeast Brazil. We hypothesized that the intestinal epithelial effects of Ala-Gln in malnourished weanling mice and mouse small intestinal epithelial (MSIE) cells would include modulation of barrier function, proliferation, and apoptosis. Dams of 10-day-old suckling C57BL/6 pups were randomized to a standard diet or an isocaloric Northeast Brazil "regional basic diet," moderately deficient in protein, fat, and minerals. Upon weaning to their dam's diet on day of life 21, pups were randomized to Ala-Gln solution or water. At 6 wk of age, mice were killed, and jejunal tissue was collected for morphology, immunohistochemistry, and Ussing chamber analysis of transmucosal resistance and permeability. Proliferation of MSIE cells in the presence or absence of Ala-Gln was measured by MTS and bromodeoxyuridine assays. MSIE apoptosis was assessed by annexin and 7-amino-actinomycin D staining. Pups of regional basic diet-fed dams exhibited failure to thrive. Jejunal specimens from undernourished weanlings showed decreased villous height and crypt depth, decreased transmucosal resistance, increased permeability to FITC-dextran, increased claudin-3 expression, and decreased epithelial proliferation and increased epithelial apoptosis (as measured by bromodeoxyuridine and cleaved caspase-3 staining, respectively). Undernourished weanlings supplemented with Ala-Gln showed improvements in weight velocity, villous height, crypt depth, transmucosal resistance, and epithelial proliferation/apoptosis compared with unsupplemented controls. Similarly, Ala-Gln increased proliferation and reduced apoptosis in MSIE cells. In summary, Ala-Gln promotes intestinal epithelial homeostasis in a mouse model of malnutrition-associated enteropathy, mimicking key features of the human disease.

Zinc and glutamine improve brain development in suckling mice subjected to early postnatal malnutrition.

OBJECTIVE: The effect of zinc and glutamine on brain development was investigated during the lactation period in Swiss mice. METHODS: Malnutrition was induced by clustering the litter size from 6-7 pups/dam (nourished control) to 12-14 pups/dam (undernourished control) following birth. Undernourished groups received daily supplementation with glutamine by subcutaneous injections starting at day 2 and continuing until day 14. Glutamine (100 mM, 40-80 microL) was used for morphological and behavioral studies. Zinc acetate was added in the drinking water (500 mg/L) to the lactating dams. Synaptophysin and myelin basic protein brain expressions were evaluated by immunoblot. Zinc serum and brain levels and hippocampal neurotransmitters were also evaluated. RESULTS: Zinc with or without glutamine improved weight gain as compared to untreated, undernourished controls. In addition, zinc supplementation improved cliff avoidance and head position during swim behaviors especially on days 9 and 10. Using design-based stereological methods, we found a significant increase in the volume of CA1 neuronal cells in undernourished control mice, which was not seen in mice receiving zinc or glutamine alone or in combination. Undernourished mice given glutamine showed increased CA1 layer volume as compared with the other groups, consistent with the trend toward increased number of neurons. Brain zinc levels were increased in the nourished and undernourished-glutamine treated mice as compared to the undernourished controls on day 7. Undernourished glutamine-treated mice showed increased hippocampal gamma-aminobutyric acid and synaptophysin levels on day 14. CONCLUSION: We conclude that glutamine or zinc protects against malnutrition-induced brain developmental impairments.

Alanyl-glutamine and glutamine supplementation improves 5-fluorouracil-induced intestinal epithelium damage in vitro.

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.

Role of retinol in protecting epithelial cell damage induced by Clostridium difficile toxin A.

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.