A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease.

OBJECTIVE: Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but identifying disease-relevant autoantigens has been a difficult challenge. In this study we aimed to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, in which infection-induced autoimmunity is thought to play an important role. METHODS: Using tandem mass spectrometry, naturally presented HLA-DR self peptides from a patient's synovium were identified, synthesized, and reacted with his peripheral blood mononuclear cells (PBMCs). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patient cells or sera. RESULTS: Of 120 HLA-DR-presented self peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMCs to proliferate. T and B cell responses to ECGF occurred systemically in ∼10-30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. Among non-antibiotic-treated historical patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis. CONCLUSION: T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence of autoimmune T and B cell responses in this illness.

Saccharomyces boulardii inhibits EGF receptor signaling and intestinal tumor growth in Apc(min) mice.

BACKGROUND & AIMS: Saccharomyces boulardii (Sb) is a probiotic yeast with anti-inflammatory and anti-microbial activities and has been used for decades in the prevention and treatment of a variety of human gastrointestinal disorders. We reported previously that Sb modulates host inflammatory responses through down-regulation of extracellular signal-regulated kinase (Erk)1/2 activities both in vitro and in vivo. The aim of this study was to identify upstream mediators responsible for extracellular signal-regulated kinase (Erk)1/2 inactivation and to examine the effects of Sb on tumor development in Apc(Min) mice. METHODS: Signaling studies of colon cancer cells were done by western blot. Cell proliferation was measured by MTS and BrdU assay. Apoptosis was examined by flow cytometry, tunel assay and caspase assay. Apc(Min) mice were orally given Sb for 9 weeks before sacrifice for tumor analysis. RESULTS: We found that the epidermal growth factor receptor (EGFR) was deactivated upon exposure to Sb, leading to inactivation of both the EGFR-Erk and EGFR-Akt pathways. In human colonic cancer cells, Sb prevented EGF-induced proliferation, reduced cell colony formation, and promoted apoptosis. HER-2, HER-3, and insulin-like growth factor-1 receptor were also found to be inactivated by Sb. Oral intake of Sb reduced intestinal tumor growth and dysplasia in C57BL/6J Min/+ (Apc(Min)) mice. CONCLUSIONS: Thus, in addition to its anti-inflammatory effects, Sb inhibits EGFR and other receptor tyrosine kinase signaling and thereby may also serve a novel therapeutic or prophylactic role in intestinal neoplasia.

Prospective derivation and validation of a clinical prediction rule for recurrent Clostridium difficile infection.

BACKGROUND & AIMS: Prevention of recurrent Clostridium difficile infection (CDI) is a substantial therapeutic challenge. A previous prospective study of 63 patients with CDI identified risk factors associated with recurrence. This study aimed to develop a prediction rule for recurrent CDI using the above derivation cohort and prospectively evaluate the performance of this rule in an independent validation cohort. METHODS: The clinical prediction rule was developed by multivariate logistic regression analysis and included the following variables: age>65 years, severe or fulminant illness (by the Horn index), and additional antibiotic use after CDI therapy. A second rule combined data on serum concentrations of immunoglobulin G (IgG) against toxin A with the clinical predictors. Both rules were then evaluated prospectively in an independent cohort of 89 patients with CDI. RESULTS: The clinical prediction rule discriminated between patients with and without recurrent CDI, with an area under the curve of the receiver-operating-characteristic curve of 0.83 (95% confidence interval [CI]: 0.70-0.95) in the derivation cohort and 0.80 (95% CI: 0.67-0.92) in the validation cohort. The rule correctly classified 77.3% (95% CI: 62.2%-88.5%) and 71.9% (95% CI: 59.2%-82.4%) of patients in the derivation and validation cohorts, respectively. The combined rule performed well in the derivation cohort but not in the validation cohort (area under the curve of the receiver-operating-characteristic curve, 0.89 vs 0.62; diagnostic accuracy, 93.8% vs 69.2%, respectively). CONCLUSIONS: We prospectively derived and validated a clinical prediction rule for recurrent CDI that is simple, reliable, and accurate and can be used to identify high-risk patients most likely to benefit from measures to prevent recurrence.

A prospective study of risk factors and historical trends in metronidazole failure for Clostridium difficile infection.

BACKGROUND & AIMS: Recent studies of Clostridium difficile infection (CDI) have indicated a dramatic increase in metronidazole failure. The aims of this study were to compare current and historical rates of metronidazole failure and to identify risk factors for metronidazole failure. METHODS: Eighty-nine patients with CDI in 2004 to 2006 were followed for 60 days and were compared with a historical cohort of 63 CDI patients studied prospectively in 1998. Metronidazole failure was defined as persistent diarrhea after 10 days of therapy or a change of therapy to vancomycin. Stool samples were analyzed for the presence of the North American pulsed-field gel electrophoresis type-1 (NAP-1) strain. RESULTS: Metronidazole failure rates were 35% in both cohorts. There was no difference in the median time to resolution of diarrhea (8 vs 5 d; P = .52) or the proportion with >10 days of diarrhea (35% vs 29%; P = .51). Risk factors for metronidazole failure included recent cephalosporin use (odds ratio [OR], 32; 95% confidence interval [CI], 5-219), CDI on admission (OR, 23; 95% CI, 3-156), and transfer from another hospital (OR, 11; 95% CI, 2-72). The frequency of NAP-1 infection in patients with and without metronidazole failure was similar (26% vs 21%; P = .67). CONCLUSIONS: We found no difference in metronidazole failure rates in 1998 and 2004 to 2006. Patients with recent cephalosporin use, CDI on admission, and transfer from another hospital were more likely to metronidazole failure. Infection with the epidemic NAP-1 strain was not associated with metronidazole failure in endemic CDI.

A mouse model of Clostridium difficile-associated disease.

BACKGROUND & AIMS: Infection with Clostridium difficile causes nosocomial antibiotic-associated diarrhea and colitis. Hamsters historically have been used to investigate disease pathogenesis and treatment, but are not ideal models because of the lack of hamster-specific reagents and genetically modified animals, and because they develop fulminant disease. The aim of this study was to establish a mouse model of antibiotic-induced C. difficile-associated disease (CDAD) that more closely resembles human disease. METHODS: C57BL/6 mice were exposed to a mixture of antibiotics (kanamycin, gentamicin, colistin, metronidazole, and vancomycin) for 3 days. Two days later, they were given injections of clindamycin and then challenged 1 day later with different doses of C. difficile. RESULTS: Mice that were exposed to antibiotics and then challenged with C. difficile developed diarrhea and lost weight. Disease severity varied from fulminant to minimal in accordance with the challenge dose. Typical histologic features of CDAD were evident. Oral vancomycin prevented CDAD in all mice, but 68% died from colitis after treatment was discontinued. All animals that survived an initial episode of CDAD showed no evidence of diarrhea or colitis after subsequent rechallenge with C. difficile. Different strains of C. difficile tested in the model showed different levels of virulence in mice. CONCLUSIONS: We have developed a mouse model of CDAD that closely represents the human disease. In light of the recent substantial increases in CDAD incidence and severity, this model will be valuable in testing new treatments, examining disease pathogenesis, and elucidating mechanisms of protective immunity.

Helicobacter pylori induces up-regulation of the epidermal growth factor receptor in AGS gastric epithelial cells.

BACKGROUND: Helicobacter pylori infection increases the risk of gastric carcinogenesis. The aim of the present study was to determine whether H. pylori could up-regulate the expression of the epidermal growth factor receptor (EGFR), a critical gene in the carcinogenic process. METHODS: AGS gastric epithelial cells were infected with cag(+) toxigenic or cag(-) nontoxigenic strains of H. pylori or isogenic mutants. EGFR protein expression was determined by Western blotting and immunofluorescence. EGFR mRNA levels were evaluated using real-time polymerase chain reaction. The signaling pathways leading to EGFR up-regulation were examined using the ERK1/2 inhibitor PD98059, the Src inhibitor pp2, the nuclear factor- kappa B inhibitor caffeic acid phenethyl ester, EGFR neutralizing antibodies, and the EGFR kinase inhibitor AG1478. RESULTS: Infection of AGS cells by H. pylori significantly increased EGFR mRNA and protein levels. We found that this effect was limited to cag(+) H. pylori strains and that mutants with a defective type IV secretion system were unable to cause EGFR up-regulation. Increased EGFR expression was found to be dependent on EGFR receptor transactivation, ERK1/2 phosphorylation, and Src activation. CONCLUSION: Infection of gastric epithelial cells by H. pylori triggers an autocrine loop whereby EGFR transactivation leads to the up-regulation of EGFR expression. This, in turn, may contribute to unrestrained epithelial cell proliferation and carcinogenesis.

Association between IgG2 and IgG3 subclass responses to toxin A and recurrent Clostridium difficile-associated disease.

BACKGROUND & AIMS: Individuals who mount a significant serum immunoglobulin (Ig)G response to toxin A are protected against recurrent Clostridium difficile-associated disease (CDAD). We investigated whether humoral immune deficiencies and/or specific IgG subclass responses are associated with recurrent CDAD. METHODS: We compared the clinical characteristics and humoral immune responses of 13 patients with recurrent CDAD with 13 matched controls with a single CDAD episode. We measured the serum IgG titers to tetanus and diphtheria toxoids, as well as total and toxin A- and toxin B-specific serum IgG, IgA, and IgG subclass concentrations. RESULTS: There were no differences between the single and recurrent CDAD subjects in terms of age, sex, ethnicity, or other potential confounding variables. The total duration of diarrhea in patients with recurrent CDAD was greater (median, 62 vs 17 days; P = .005). IgG titers to tetanus and diphtheria toxoids, total IgG, and IgG subclass levels were similar in both groups. The total IgA was somewhat lower in those with recurrent CDAD (204 vs 254 mg/dL; P = .05). IgA, IgG, IgG1, and IgG4 anti-toxin A and anti-toxin B levels were similar in both groups. However, IgG2 and IgG3 anti-toxin A levels were significantly lower in the recurrent group (P = .01 and .001, respectively). CONCLUSIONS: Subjects with recurrent CDAD did not show evidence of widespread humoral immune deficiency or of IgG subclass deficiency. Their low serum IgG anti-toxin A concentrations reflected selectively reduced IgG2 and IgG3 subclass responses. Measurement of specific IgG2/3 anti-toxin A may be useful in selecting patients for treatment with agents to prevent recurrent CDAD.

MIP-3alpha neutralizing monoclonal antibody protects against TNBS-induced colonic injury and inflammation in mice.

A characteristic feature of human inflammatory bowel disease, particularly Crohn's disease, is the presence of activated CD4(+) T cells. Recently, we have shown that colonic epithelial cell production of macrophage inflammatory protein (MIP)-3alpha, a CD4 T cell-directed chemokine, is elevated in inflammatory bowel disease. However, the functional relevance of MIP-3alpha production during intestinal inflammation is poorly understood. The aim of this study was to determine whether MIP-3alpha production is increased during murine 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and to examine the effect of anti-MIP-3alpha neutralizing monoclonal antibody administration in this model. We found that the administration of TNBS significantly increased colonic MIP-3alpha protein levels in Balb/c mice. Consistent with this, a marked increase in the number of CCR6-bearing lamina propria CD4(+) and CD8(+) T cells was also observed in TNBS-treated animals. Treatment of mice with an anti-MIP-3alpha neutralizing monoclonal antibody significantly reduced TNBS-mediated increases in colonic weight-to-length ratio, mucosal ulceration, histological damage, and myeloperoxidase activity. TNBS-mediated increases in the number of CCR6-bearing lamina propria T cells were also substantially reduced by anti-MIP-3alpha neutralizing monoclonal antibody treatment. Taken together, our findings indicate that blockade of MIP-3alpha bioactivity can significantly reduce TNBS-mediated colonic injury and T cell recruitment, suggesting a role for this chemokine in the pathophysiology of intestinal inflammation.

T-lymphocyte activity in HLA-DR17 positive patients with active and clinically recovered sarcoidosis.

BACKGROUND AND AIMS: CD4+ T-lymphocytes are accumulated at the sites of inflammation in sarcoidosis and assumed to play a key role in directing the immune events. By analysing their expression of activation molecules and comparing it between sarcoidosis patients at disease onset and patients at clinical resolution, our aims were to increase the knowledge of their role and find markers of disease activity. METHODS: Thirty-two sarcoidosis patients, 23 at disease onset and 9 after clinical resolution, and 36 healthy controls were included. All patients were HLA-DR17 positive, a group sharing several clinical and immunological features. CD4+ T-lymphocytes from bronchoalveolar lavage fluid (BALF) and peripheral blood were analysed for the expression of CD25, HLA-DR and CD69 by flow cytometry. RESULTS: The CD25 and HLA-DR expression were increased on CD4+ lymphocytes from BALF and peripheral blood at disease onset compared to patients with clinically resolved disease and controls. Interestingly, the percentage of T regulatory (Treg) cells, defined as CD4+ CD25(bright) lymphocytes, was increased in both BALF and blood from patients with active disease. The CD69 expression did not differ between the groups in either compartment. CONCLUSIONS: CD4+ BALF and blood lymphocytes from DR17 positive patients with active sarcoidosis reveal an increased expression of activity markers compared to patients after clinical resolution. An increased number of Treg cells in active sarcoidosis may be involved in the characteristic down-regulation of cell-mediated immune responses in this disease. The results indicate that analysis of these activation markers could be useful as markers of disease activity in sarcoidosis.