Reinaldo B. Oriá

Malnutrition as an enteric infectious disease with long-term effects on child development

Malnutrition is a major contributor to mortality and is increasingly recognized as a cause of potentially lifelong functional disability. Yet, a rate-limiting step in achieving normal nutrition may be impaired absorptive function due to multiple repeated enteric infections. This is especially problematic in children whose diets are marginal. In malnourished individuals, the infections are even more devastating. This review documents the evidence that intestinal infections lead to malnutrition and that malnutrition worsens intestinal infections. The clinical data presented here derive largely from long-term cohort studies that are supported by controlled animal studies. Also reviewed are the mechanisms by which enteric infections lead to undernutrition and by which malnutrition worsens enteric infections, with implications for potential novel interventions. Further intervention studies are needed to document the relevance of these mechanisms and, most importantly, to interrupt the vicious diarrhea-malnutrition cycle so children may develop their full potential.

Prolonged Episodes of Acute Diarrhea Reduce Growth and Increase Risk of Persistent Diarrhea in Children

BACKGROUND & AIMS Prolonged episodes of acute diarrhea (ProD; duration 7-13 days) or persistent diarrhea (PD; duration ≥14 days) are important causes of undernutrition, yet the epidemiology and nutritional impact of ProD are poorly understood. METHODS We conducted a 10-year cohort study of 414 children from a Brazilian shantytown who were followed from birth; data were collected on diarrhea, enteric pathogens, and anthropometry. RESULTS During 1276 child-years of observation, we recorded 3257 diarrheal episodes. ProD was twice as common as PD (12% and 5% of episodes, respectively); ProD and PD together accounted for 50% of all days with diarrhea. ProD was more common in infants whose mothers had not completed primary school (relative risk [RR], 2.1; 95% confidence interval: 1.02-2.78). Early weaning was associated with earlier onset of ProD (Spearman ρ = 0.309; P = .005). Infants with ProD were twice as likely to develop PD in later childhood (log rank, P = .002) compared with infants with only acute diarrhea (AD; duration <7 days), even after controlling for confounders. Childrens growth was more severely stunted before their first episode of ProD, compared with AD (mean height-for-age Z score (HAZ) -0.81 vs -0.51, respectively, P < .05, unpaired t test). Following ProD, HAZ (ΔHAZ = -0.232) and weight-for-age (ΔWAZ = -0.26) significantly decreased (P < .005 in paired t tests). ProD was associated with Cryptosporidium and Shigella infections. CONCLUSIONS ProD accounts for significant morbidity and identifies children at risk of a vicious cycle of diarrhea and malnutrition. Further studies are needed to address the recognition and control of ProD and its consequences in resource-limited settings and assess its role in PD pathogenesis.

APOE4 Protects the Cognitive Development in Children with Heavy Diarrhea Burdens in Northeast Brazil

Polymorphisms in the apolipoprotein E (APOE) have constituted the major rationale to identify potential risk groups for developing late-onset Alzheimers disease and help to predict recovery of cognitive function after brain injury. However, the APOE impact on cognitive development in children living in poor areas of the developing world, where we have discovered profound significant associations of early childhood diarrhea (at 0–2 y) with lasting impairments of growth, cognition, and school performance, is not known. Therefore, we conducted APOE genotyping in 72 Brazilian shantytown children under active surveillance since birth, using purified DNA extracted from buccal cell samples. We found a high frequency of APOE4 alleles (18% versus 9–11% expected) in children with lower diarrhea burdens. When we examined the children who experienced the heavier diarrhea burdens (greater than or equal to the median of seven illnesses in the first 2 y of life), those with APOE4 did significantly better in the coding subtest (p = 0.01) when compared with APOE4-negative children with similar diarrhea burdens. Positive correlations between the APOE4 occurrence and coding scores remained, even after adjusting for family income, maternal education, and breast-feeding. Moreover, the APOE4-positive group, under heavy burdens of diarrhea, had preserved semantic fluency and the mean difference in fluency scores, p = 0.025, a standardized coefficient for disproportional verbal fluency impairment. Our findings show that APOE4 is relatively common in favela children and suggest a protective role of the APOE4 allele in children with a history of heavy burdens of diarrhea in their first 2 y of life.

Caspase and Bid Involvement in Clostridium difficile Toxin A-Induced Apoptosis and Modulation of Toxin A Effects by Glutamine and Alanyl-Glutamine In Vivo and In Vitro

ABSTRACT Clostridium difficile is the leading cause of nosocomial bacterial diarrhea. Glutamine and its stable and highly soluble derivative alanyl-glutamine, have been beneficial in models of intestinal injury. In this study, we extend our work on the mechanisms of Clostridium difficile toxin A (TxA)-induced apoptosis in human intestinal epithelial T84 cells and evaluate the effects of glutamine and alanyl-glutamine on TxA-induced apoptosis in vitro and disruption of ileal mucosa in vivo. T84 cells were incubated with TxA (100 ng/ml) in medium with or without glutamine or alanyl-glutamine (3 to 100 mM). Apoptosis was evaluated by DNA fragmentation in vitro and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method in vivo. Caspase and Bid involvement were investigated by Western blotting. Ligated rabbit ileal loops were used for the evaluation of intestinal secretion, mucosal disruption, and apoptosis. TxA induced caspases 6, 8, and 9 prior to caspase 3 activation in T84 cells and induced Bid cleavage by a caspase-independent mechanism. Glutamine or alanyl-glutamine significantly reduced TxA-induced apoptosis of T84 cells by 47% and inhibited activation of caspase 8. Both glutamine and alanyl-glutamine reduced TxA-induced ileal mucosal disruption and secretion. Altogether, we further delineated the apoptosis-signaling cascade induced by TxA in T84 cells and demonstrated the protective effects of glutamine and alanyl-glutamine. Glutamine and alanyl-glutamine inhibited the apoptosis of T84 cells by preventing caspase 8 activation and reduced TxA-induced intestinal secretion and disruption.

Cryptosporidium Infection Causes Undernutrition and, Conversely, Weanling Undernutrition Intensifies Infection

Abstract Cryptosporidium parvum is a leading pathogen in children in developing countries. To investigate whether early postnatal malnutrition leads to heavier C. parvum infections, we assessed intestinal adaptation and parasite load in suckling mice during the first 2 wk of life, analogous to the first postnatal yr in humans. Undernutrition was induced by daily C57BL6J pup separation from lactating dams. Half of the pups were separated daily, for 4 hr on day 4, 8 hr on day 5, and for 12 hr from day 6 until day 14. On day 6, each pup received an oral inoculum of 105 to 107 parasites in 10–25 μl of PBS. Littermate controls received PBS alone. Stools were assessed from days 8, 11, and 14 for oocyst counts. Mice were killed on day 14, 8 days postinoculation, at the peak of the infection. Ileal and colon segments were obtained for histology, real-time and reverse transcriptase PCR, and immunoassays. Villus and crypt lengths and cross-sectional areas were also measured. Undernourished and nourished mice infected with excysted 106 or 107 oocysts exhibited the poorest growth outcomes compared with their uninfected controls. Nourished 106-infected mice had comparable weight decrements to uninfected undernourished mice. Body weight and villi were additively affected by malnutrition and cryptosporidiosis. Hyperplastic crypts and heavier inflammatory responses were found in the ilea of infected malnourished mice. Undernourished infected mice exhibited greater oocyst shedding, TNF-α and IFN-γ intestinal levels, and mRNA expression compared to nourished mice infected with either 105 or 106 oocysts. Taken together, these findings show that Cryptosporidium infection can cause undernutrition and, conversely, that weanling undernutrition intensifies infection and mucosal damage.

Cryptosporidium-Malnutrition Interactions: Mucosal Disruption, Cytokines, and TLR Signaling In A Weaned Murine Model

Abstract Cryptosporidiosis is a leading cause of persistent diarrhea in children in impoverished and developing countries and has both a short- and long-term impact on the growth and development of affected children. An animal model of cryptosporidial infection that mirrors closely the complex interaction between nutritional status and infection in children, particularly in vulnerable settings such as post-weaning and malnourishment, is needed to permit exploration of the pathogenic mechanisms involved. Weaned C57BL/6 mice received a protein-deficient (2%) diet for 3–12 days, then were infected with 5 × 107 excysted C. parvum oocyts, and followed for rate of growth, parasite stool shedding, and intestinal invasion/morphometry. Mice had about 20% reduction in weight gain over 12 days of malnutrition and an additional 20% weight loss after C. parvum challenge. Further, a significantly higher fecal C. parvum shedding was detected in malnourished infected mice compared to the nourished infected mice. Also, higher oocyst counts were found in ileum and colon tissue samples from malnourished infected mice, as well as a significant reduction in the villous height–crypt depth ratio in the ileum. Tissue Th1 cytokine concentrations in the ileum were significantly diminished by malnutrition and infection. mRNA for toll-like receptors 2 and 4 were diminished in malnourished infected mice. Treatment with nitazoxanide did not prevent weight loss or parasite stool shedding. These findings indicate that, in the weaned animal, malnutrition intensifies cryptosporidial infection, while cryptosporidial infection further impairs normal growth. Depressed TLR2 and 4 signaling and Th1 cytokine response may be important in the mechanisms underlying the vicious cycle of malnutrition and enteric infection.

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 Alzheimers 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.

Novel In Vitro and In Vivo Models and Potential New Therapeutics to Break the Vicious Cycle of Cryptosporidium Infection and Malnutrition

BACKGROUND Although several animal models of cryptosporidiosis have been reported, most involve genetically or pharmacologically immune-suppressed hosts. METHODS We report challenge with excysted (in vitro and in vivo) and unexcysted (in vivo) Cryptosporidium parvum oocysts in human colonic adenocarcinoma (HCT-8) cells and weaned nourished and malnourished C57BL/6 mice, following outcomes of growth rate, stool shedding, and tissue burden. We tested treatment with an oligodeoxynucleotide containing unmethylated CpG motif (CpG-ODN) and alanyl-glutamine in vivo and in vitro. RESULTS C. parvum-challenged mice showed prolonged weight loss (>10% over 4 days), robust stool shedding (>3 logs/d over 7 days), and epithelial infection in the ileum, cecum, and colon. Of 2 potential therapeutic compounds evaluated in the model, CpG-ODN reduced body weight loss (to <6% on days 3-7 after challenge), reduced shedding of organisms (by 25% on days 1 and 3 after challenge), and decreased the burden of parasites in the ileum. Alanyl-glutamine showed similar benefits. In vitro findings suggested that effects on the epithelial component of the mucosa probably likely responsible for beneficial effects seen in vivo. CONCLUSIONS Weaned mice provide a convenient and reproducible model of cryptosporidial disease, including its vicious cycle with body weight loss and heavier infection with malnutrition, and this model may be useful in exploring innovative therapeutic solutions for this challenging infectious disease.

Clostridium difficile Toxin A Induces Intestinal Epithelial Cell Apoptosis and Damage: Role of Gln and Ala-Gln in Toxin A Effects

The aim of this study was to investigate the effect of Clostridium difficile toxin A (TxA) on intestinal epithelial cell migration, apoptosis, and transepithelial resistance and to evaluate the effect of glutamine (Gln) and its stable derivative, alanyl-glutamine (Ala-Gln), on TxA-induced damage. Migration was measured in rat intestinal epithelial cells (IEC-6) 6 and 24 hr after a razor scrape of the cell monolayer. Cell proliferation was indirectly measured utilizing the tetrazolium salt WST-1. The cells were incubated with TxA (1–100 ng/ml) in medium without Gln or medium containing Gln or Ala-Gln (1–30 mM). Apoptosis was quantified in IEC-6 cells using annexin V assay. Transepithelial resistance was measured using an epithelial voltohmmeter across T84 cells seeded on a transwell filter. TxA-induced a dose-dependent reduction of migration and also caused dose and time-dependent apoptosis in IEC-6 cells. Gln and Aln-Gln significantly enhanced IEC-6 cell migration and proliferation. Gln and Ala-Gln also prevented the inhibition of migration, apoptosis, and the initial drop in transepithelial resistance induced by TxA. In conclusion, both peptides reduced toxin-induced epithelial damage and thus might play an adjunctive role in C. difficile-induced colitis therapy.

Role of inducible nitric oxide synthase pathway on methotrexate-induced intestinal mucositis in rodents

BackgroundMethotrexate treatment has been associated to intestinal epithelial damage. Studies have suggested an important role of nitric oxide in such injury. The aim of this study was to investigate the role of nitric oxide (NO), specifically iNOS on the pathogenesis of methotrexate (MTX)-induced intestinal mucositis.MethodsIntestinal mucositis was carried out by three subcutaneous MTX injections (2.5 mg/kg) in Wistar rats and in inducible nitric oxide synthase knock-out (iNOS-/-) and wild-type (iNOS+/+) mice. Rats were treated intraperitoneally with the NOS inhibitors aminoguanidine (AG; 10 mg/Kg) or L-NAME (20 mg/Kg), one hour before MTX injection and daily until sacrifice, on the fifth day. The jejunum was harvested to investigate the expression of Ki67, iNOS and nitrotyrosine by immunohistochemistry and cell death by TUNEL. The neutrophil activity by myeloperoxidase (MPO) assay was performed in the three small intestine segments.ResultsAG and L-NAME significantly reduced villus and crypt damages, inflammatory alterations, cell death, MPO activity, and nitrotyrosine immunostaining due to MTX challenge. The treatment with AG, but not L-NAME, prevented the inhibitory effect of MTX on cell proliferation. MTX induced increased expression of iNOS detected by immunohistochemistry. MTX did not cause significant inflammation in the iNOS-/- mice.ConclusionThese results suggest an important role of NO, via activation of iNOS, in the pathogenesis of intestinal mucositis.