Cirle A. Warren

Diagnosis and Treatment of Acute or Persistent Diarrhea

Studies of microbial pathogens and the toxins they produce are important for determining the mechanisms by which they cause disease and spread throughout a population. Some bacteria produce secretory enterotoxins (such as cholera toxin or the heat-labile or stable enterotoxins produced by Escherichia coli) that invade cells directly. Others invade cells or produce cytotoxins (such as those produced by Shigella, enteroinvasive E coli, or Clostridium difficile) that damage cells or trigger host responses that cause small or large bowel diseases (such as enteroaggregative or enteropathogenic E coli or Salmonella). Viruses (such as noroviruses and rotaviruses) and protozoa (such as Cryptosporidium, Giardia, or Entamoeba histolytica) disrupt cell functions and cause short- or long-term disease. Much epidemiologic data about these pathogens have been collected from community- and hospital-acquired settings, as well as from patients with travelers or persistent diarrhea. These studies have led to practical approaches for prevention, diagnosis, and treatment.

Pediatric Diarrhea in Southern Ghana: Etiology and Association with Intestinal Inflammation and Malnutrition

Diarrhea is a major public health problem that affects the development of children. Anthropometric data were collected from 274 children with (N = 170) and without (N = 104) diarrhea. Stool specimens were analyzed by conventional culture, polymerase chain reaction for enteroaggregative Escherichia coli (EAEC), Shigella, Cryptosporidium, Entamoeba, and Giardia species, and by enzyme-linked immunosorbent assay for fecal lactoferrin levels. About 50% of the study population was mildly to severely malnourished. Fecal lactoferrin levels were higher in children with diarrhea (P = 0.019). Children who had EAEC infection, with or without diarrhea, had high mean lactoferrin levels regardless of nutritional status. The EAEC and Cryptosporidium were associated with diarrhea (P = 0.048 and 0.011, respectively), and malnourished children who had diarrhea were often co-infected with both Cryptosporidium and EAEC. In conclusion, the use of DNA-biomarkers revealed that EAEC and Cryptosporidium were common intestinal pathogens in Accra, and that elevated lactoferrin was associated with diarrhea in this group of children.

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.

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.

Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea.

Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases.

Amixicile, a Novel Inhibitor of Pyruvate:Ferredoxin Oxidoreductase, Shows Efficacy against Clostridium difficile in a Mouse Infection Model

ABSTRACT Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-μg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.

Vancomycin Treatment's Association with Delayed Intestinal Tissue Injury, Clostridial Overgrowth, and Recurrence of Clostridium difficile Infection in Mice

ABSTRACT Antibiotic treatment, including vancomycin, for Clostridium difficile infection (CDI) has been associated with recurrence of disease in up to 25% of infected persons. This study investigated the effects of vancomycin on the clinical outcomes, intestinal histopathology, and anaerobic community during and after treatment in a murine model of CDI. C57BL/6 mice were challenged with C. difficile strain VPI 10463 after pretreatment with an antibiotic cocktail. Twenty-four hours after infection, mice were treated daily with vancomycin, nitazoxanide, fidaxomicin, or metronidazaole for 5 days. Mice were monitored for either 6 or 12 days postinfection. Clinical, diarrhea, and histopathology scores were measured. Cecal contents or stool samples were assayed for clostridial or Bacteroides DNA and C. difficile toxins A and B. Vancomycin treatment of infected mice was associated with improved clinical, diarrhea, and histopathology scores and survival during treatment. However, after discontinuation of the drug, clinical scores and histopathology were worse in treated mice than in untreated infected controls. At the end of the study, 62% of the vancomycin-treated mice succumbed to recurrence, with an overall mortality rate equivalent to that of the untreated infected control group. Fidaxomicin-treated mice had outcomes similar to those of vancomycin-treated mice. C. difficile predominated over Bacteroides in cecal contents of vancomycin-treated mice, similar to findings for untreated infected mice. Decreasing the duration of vancomycin treatment from 5 days to 1 day decreased recurrence and deaths. In conclusion, vancomycin improved clinical scores and histopathology acutely but was associated with poor outcome posttreatment in C. difficile-infected mice. Decreasing vancomycin exposure may decrease relapse and improve survival in CDI.

Apolipoprotein E Plays a Key Role against Cryptosporidial Infection in Transgenic Undernourished Mice

Apolipoliprotein E (apoE), a critical targeting protein in lipid homeostasis, has been found to have immunoinflammatory effects on murine models of infection and malnutrition. The effects of apoE in undernourished and Cryptosporidium parvum-infected mice have not been investigated. In order to study the role of apoE in a model of C. parvum infection, we used the following C57BL6J mouse genetic strains: APOE-deficient, wild-type controls, and APOE targeted replacement (TR) mice expressing human APOE genes (E3/3; E4/4). Experimental mice were orally infected with 107-unexcysted-C. parvum oocysts between post-natal days 34–35 followed by malnutrition induced with a low-protein diet. Mice were euthanized seven days after C. parvum-challenge to investigate ileal morphology, cytokines, and cationic arginine transporter (CAT-1), arginase 1, Toll-like receptor 9 (TLR9), and inducible nitric oxide synthase (iNOS) expression. In addition, we analyzed stool oocyst shedding by qRT-PCR and serum lipids. APOE4/4-TR mice had better weight gains after infection plus malnutrition compared with APOE3/3-TR and wild-type mice. APOE4/4-TR and APOE knockout mice had lower oocyst shedding, however the latter exhibited with villus blunting and higher ileal pro-inflammatory cytokines and iNOS transcripts. APOE4/4-TR mice had increased ileal CAT-1, arginase-1, and TLR9 transcripts relative to APOE knockout. Although with anti-parasitic effects, APOE deficiency exacerbates intestinal inflammatory responses and mucosal damage in undernourished and C. parvum-infected mice. In addition, the human APOE4 gene was found to be protective against the compounded insult of Cryptosporidium infection plus malnutrition, thus extending our previous findings of the protection against diarrhea in APOE4 children. Altogether our findings suggest that apoE plays a key role in the intestinal restitution and immunoinflammatory responses with Cryptosporidium infection and malnutrition.

Role of Leptin-Mediated Colonic Inflammation in Defense against Clostridium difficile Colitis

ABSTRACT The role of leptin in the mucosal immune response to Clostridium difficile colitis, a leading cause of nosocomial infection, was studied in humans and in a murine model. Previously, a mutation in the receptor for leptin (LEPR) was shown to be associated with susceptibility to infectious colitis and liver abscess due to Entamoeba histolytica as well as to bacterial peritonitis. Here we discovered that European Americans homozygous for the same LEPR Q223R mutation (rs1137101), known to result in decreased STAT3 signaling, were at increased risk of C. difficile infection (odds ratio, 3.03; P = 0.015). The mechanism of increased susceptibility was studied in a murine model. Mice lacking a functional leptin receptor (db/db) had decreased clearance of C. difficile from the gut lumen and diminished inflammation. Mutation of tyrosine 1138 in the intracellular domain of LepRb that mediates signaling through the STAT3/SOCS3 pathway also resulted in decreased mucosal chemokine and cell recruitment. Collectively, these data support a protective mucosal immune function for leptin in C. difficile colitis partially mediated by a leptin-STAT3 inflammatory pathway that is defective in the LEPR Q223R mutation. Identification of the role of leptin in protection from C. difficile offers the potential for host-directed therapy and demonstrates a connection between metabolism and immunity.