J. Thomas Lamont

Clostridium difficile Infection

This article reviews the pathogenesis, epidemiology, diagnosis, and treatment of this nosocomial and potentially fatal infectious diarrhea, as well as the associated risk factors. New treatments include fecal microbiota transplantation for disease that is resistant to vancomycin.

Treatment with intravenously administered gamma globulin of chronic relapsing colitis induced by Clostridium difficile toxin

We tested the hypothesis that children with chronic relapsing colitis induced by Clostridium difficile toxin have defective antibody responses to C. difficile toxins as a cause of their underlying illness. Six such children were tested for serum IgG and IgA antibody to C. difficile toxin A. These six children had lower IgG anti-toxin A levels than 24 healthy children (p = 0.026) and 18 healthy adults (p = 0.0008). Five patients treated with 400 mg intravenously administered gamma-globulin per kilogram every 3 weeks had significant increases in IgG (p = 0.01) but not IgA anti-toxin A (p = 0.406) levels, and all five had clinical resolution of their gastrointestinal symptoms as well as clearing of C. difficile cytotoxin B from their stools. These observations suggest that a deficiency of IgG anti-toxin A may predispose children to the development of chronic relapsing C. difficile-induced colitis. In such cases, intravenous gamma-globulin therapy may be effective in producing clinical remission.

p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis

Clostridium difficile toxin A causes acute neutrophil infiltration and intestinal mucosal injury. In cultured cells, toxin A inactivates Rho proteins by monoglucosylation. In monocytes, toxin A induces IL-8 production and necrosis by unknown mechanisms. We investigated the role of mitogen-activated protein (MAP) kinases in these events. In THP-1 monocytic cells, toxin A activated the 3 main MAP kinase cascades within 1 to 2 minutes. Activation of p38 was sustained, whereas stimulation of extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinase was transient. Rho glucosylation became evident after 15 minutes. IL-8 gene expression was reduced by 70% by the MEK inhibitor PD98059 and abrogated by the p38 inhibitor SB203580 or by overexpression of dominant-negative mutants of the p38-activating kinases MKK3 and MKK6. SB203580 also blocked monocyte necrosis and IL-1beta release caused by toxin A but not by other toxins. Finally, in mouse ileum, SB203580 prevented toxin A-induced neutrophil recruitment by 92% and villous destruction by 90%. Thus, in monocytes exposed to toxin A, MAP kinase activation appears to precede Rho glucosylation and is required for IL-8 transcription and cell necrosis. p38 MAP kinase also mediates intestinal inflammation and mucosal damage induced by toxin A.

Clostridium difficile toxin B disrupts the barrier function of T84 monolayers

The contribution of toxin B to Clostridium difficile-associated infection is undefined. Toxin B induces dramatic phenotypic alterations (cytotoxic effects) in cultured mesenchymal and nonintestinal epithelial cells, yet its effects on intestinal epithelial cells are not clearly understood. The alterations induced by toxin B in nonintestinal cells appear to be secondary to toxin-induced redistribution of filamentous actin. It has not been determined whether toxin B exerts similar effects on cultured intestinal epithelial cells or whether such phenotypic alterations are of any physiological consequence. To address these questions, we examined the effect of C. difficile toxin B on the phenotype and barrier function of T84 cell monolayers. Our studies show that the cytotoxic effects of toxin B, i.e., cell rounding, do extend to cultured intestinal epithelial cells (T84). In addition, toxin B dramatically reduces the barrier function of T84 monolayers grown on collagen-coated filters. Toxin B-induced redistribution of filamentous actin appears to be responsible for the alterations in both intestinal epithelial cell phenotype and barrier function. Specifically, filamentous actin comprising the perijunctional actomyosin ring, known to be important in regulating tight junction permeability, is condensed into discrete plaques. Flux studies confirm that the permeability defect is at the level of the tight junction. We conclude that toxin-induced changes in actin distribution perturb intercellular junctional contacts and thereby ablate epithelial barrier function. There was no evidence of cell death as determined by lactate dehydrogenase release assays.

Saccharomyces boulardii stimulates intestinal immunoglobulin A immune response to Clostridium difficile toxin A in mice.

ABSTRACT Saccharomyces boulardii is a nonpathogenic yeast that protects against antibiotic-associated diarrhea and recurrentClostridium difficile colitis. The administration ofC. difficile toxoid A by gavage to S. boulardii-fed BALB/c mice caused a 1.8-fold increase in total small intestinal immunoglobulin A levels (P = 0.003) and a 4.4-fold increase in specific intestinal anti-toxin A levels (P < 0.001). Enhancing host intestinal immune responses may be an important mechanism for S. boulardii-mediated protection against diarrheal illnesses.

pH-dependent conformational change of gastric mucin leads to sol-gel transition.

We present dynamic light scattering (DLS) and hydrophobic dye-binding data in an effort to elucidate a molecular mechanism for the ability of gastric mucin to form a gel at low pH, which is crucial to the barrier function of gastric mucus. DLS measurements of dilute mucin solutions were not indicative of intermolecular association, yet there was a steady fall in the measured diffusion coefficient with decreasing pH, suggesting an apparent increase in size. Taken together with the observed rise in depolarized scattering ratio with decreasing pH, these results suggest that gastric mucin undergoes a conformational change from a random coil at pH >/= 4 to an anisotropic, extended conformation at pH < 4. The increased binding of mucin to hydrophobic fluorescent with decreasing pH indicates that the change to an extended conformation is accompanied by exposure of hydrophobic binding sites. In concentrated mucin solutions, the structure factor S(q, t) derived from DLS measurements changed from a stretched exponential decay at pH 7 to a power-law decay at pH 2, which is characteristic of a sol-gel transition. We propose that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated.

Microbial factors in inflammatory bowel disease

An unsolved puzzle in IBD research is whether germs, genes, or a combination of the two with excessive immune responses to gut-associated bacteria explains the pathogenesis of UC and CD. Whatever the answer, there is little doubt that microbial factors are involved intimately in IBD pathogenesis. Although a long search has failed to confirm a direct pathogenic role for a specific infectious agent, compelling evidence suggests that commensal enteric bacteria and their products provide a local environmental trigger that initiates and perpetuates IBD, reactivates quiescent disease, results in the frequent septic complications of CD, and contributes to the development of several extraintesinal manifestations. The most compelling evidence for involvement of the enteric flora in the pathogenesis of IBD has been generated from studies of animal models, which collectively support the view that IBD is due to genetically determined dysregulation of the mucosal immune response to luminal antigens derived from the normal bacterial flora. Although removing or dampening the dominant antigenic stimuli with antibiotics or probiotics is conceptually superior to the current array of immunosuppressive and anti-inflammatory agents that nonspecifically block the inflammatory cascade, more definitive, rigorously designed, controlled trials of treatments directed at the microflora are needed. Future research investigating mechanisms of tolerance to luminal bacteria and an understanding of how probiotics can manipulate the intestinal flora beneficially will bring clinicians closer to identifying potential therapeutic targets and unraveling the bacterial connection to IBD pathogenesis.

Treatment of Clostridium difficile-associated disease.

Clostridium difficile infection is an increasing burden to the health care system, totaling more than

IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes

1 billion/year in the United States. Treatment of patients with C difficile infection with metronidazole or vancomycin reduces morbidity and mortality, although the number of patients that do not respond to metronidazole is increasing. Despite initial response rates of greater than 90%, 15%-30% of patients have a relapse in symptoms after successful initial therapy, usually in the first few weeks after treatment is discontinued. Failure to develop specific antibody response has recently been identified as a critical factor in recurrence. The review discusses the different management strategies for initial and recurrent symptomatic C difficile infections.

Clostridium difficile Toxin A Regulates Inducible Cyclooxygenase-2 and Prostaglandin E2 Synthesis in Colonocytes via Reactive Oxygen Species and Activation of p38 MAPK

Neutrophil infiltration is central to the pathogenesis of Clostridium difficile toxin A-induced enterocolitis. This study examines whether monocyte activation by C. difficile toxins is instrumental in initiating neutrophil activation and recruitment. Human monocytes were exposed to low concentrations of highly purified C. difficile toxins, and the conditioned media were harvested for cytokine and functional assays. Monocytes exposed to C. difficile toxin A (10(-10) M) or toxin B (10(-12) M) released 100 and 20 times basal levels, respectively, of the neutrophil chemoattractant interleukin-8 (IL-8). Reverse transcriptase-polymerase chain reaction demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h after toxin exposure. Conditioned media from toxin A- and toxin B-treated monocytes stimulated neutrophil migration (324 and 245% of control, respectively). This effect was completely blocked by IL-8 antiserum. These media also upregulated neutrophil CD11b/CD18 and endothelial cell intercellular adhesion molecule-1 expression. C. difficile toxins, at low concentrations, potently activate monocytes to release factors, including IL-8, that facilitate neutrophil extravasation and tissue infiltration. Our findings indicate a major role for toxin-mediated monocyte and macrophage activation in C. difficile colitis.