Treated Whipple disease with erythema nodosum leprosum-like lesions: cutaneous PAS-positive macrophages slowly decrease with time and are associated with lymphangiectases: a case report.

Pathologically, Whipple disease (WD) is characterized by the accumulation of myriad macrophages parasitized by Tropheryma whipplei (TW) bacilli denoted by periodic acid-Schiff (PAS) positivity. These PAS+ macrophages are typically found in the duodenum associated with lymphangiectasia. Recently, we reported the presence of PAS+ macrophages and free TW in erythema nodosum leprosum (ENL)-like lesions and normal skin in a patient with WD who suffered from the immune reconstitution inflammatory syndrome (IRIS). We extend that report by describing the clinical and pathologic findings over 5 years of follow-up. First, the IRIS gradually diminished and abated over 18-month time. Second, at no point did WD recur, and all duodenal and skin biopsies tested by polymerase chain reaction were negative for TW DNA. Third, PAS+ macrophages were identified in 26 of 27 skin biopsies (96%) and decreased along with free TW over time. Fourth, ENL-like lesions had significantly greater numbers of PAS+ macrophages than normal skin. Moreover, normal abdominal skin (region of ENL-like lesions) had greater PAS+ counts than arm skin (not a site of IRIS). Last, lymphangiectases, a histologic sign of lymphostasis, was found in all skin biopsies. Overall, these findings implicate bacillary burden as a factor in the immune tolerance to live TW in active WD and the initiation of ENL-like nodules against dead/nonreplicative TW in treated WD. In addition, poor lymphatic drainage is likely responsible for the gradual clearance of TW from the skin and the impaired delayed-type hypersensitivity reaction (absence of activated macrophages) against TW found in WD, presumptively due to reduced/absent immune cell trafficking necessary for lymphocyte-macrophage interactions and induction of adaptive immunity.

Vibrio cholerae cytolysin is essential for high enterotoxicity and apoptosis induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-O139 strain.

Cholera toxin (CT) gene-negative Vibrio cholerae non-O1, non-O139 strains may cause severe diarrhea though their pathogenic mechanism remains unclear. V. cholerae cytolysin (VCC) is a pore-forming exotoxin encoded in the hlyA gene of V. cholerae whose contribution to the pathogenesis is not fully understood. In this work, the virulence properties of a CT gene-negative V. cholerae non-O1, non-O139 strain causing a cholera-like syndrome were analyzed. Inoculation of rabbit ileal loops with the wild type strain induced extensive fluid accumulation, accompanied by severe histopathological damage characterized by villus shortening, lymphangiectasia and focal areas of necrosis. These pathogenic effects were abrogated by mutation of the hlyA gene thus pointing out the main role of VCC in the virulence of the strain. Interestingly, this toxin was capable of triggering apoptosis in human intestinal cell lines due to its anion channel activity. Moreover, the wild type strain also induced increased apoptosis of the intestinal epithelium cells which was not observed upon inoculation of the VCC null mutant strain, indicating that VCC may trigger apoptotic cell death during infection in vivo. Altogether, these results support a main role of VCC in the pathogenesis of the CT gene-negative V. cholerae non-O1, non-O139 strain and identify apoptosis as a previously unrecognized cell death pathway triggered by VCC.

Validation of 16S rDNA sequencing in microdissected bowel biopsies from Crohn's disease patients to assess bacterial flora diversity.

The bowel flora is implicated in Crohn's disease (CD) pathogenesis but its precise role is still unclear. Several non-mutually exclusive hypotheses have been proposed: an unidentified persistent pathogen; excessive bacterial translocation; an immune system abnormality in response to normal bacteria; or a breakdown in the balance between protective and harmful bacteria. These hypotheses can be tested by identifying bacteria in specific microscopic bowel structures or lesions. The present paper describes a novel technique to assess bacterial flora diversity in bowel biopsies, by combining laser capture microdissection with broad-range 16S rDNA sequencing. Fifty-four samples comprising histologically normal and pathological mucosa, MALT, ulcers, submucosal lymphangiectasias, epithelioid granulomas, and lymph nodes were microdissected out of 30 bowel biopsies from five CD patients. Bacterial 16S rDNA was successfully amplified by PCR in all samples, and PCR products from 15 samples were selected for cloning and sequence analysis. A total of 729 bacterial DNA sequences were analysed, which could be attributed to six different phyla (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria, and Planctomycetes). DNA from typical bowel bacteria (Enterobacteriaceae, Clostridiales, Bacteroidetes, Fusobacteria) was detected in all microdissected areas. It was thus convincingly demonstrated that 16S rDNA sequencing can be combined with microdissection to study the bowel flora. However, no specific persistent pathogen causal for CD was identified. The results suggest that Enterobacteriaceae may initiate or colonize ulcers in CD. Translocation of bacteria through established mucosal lesions or as a result of increased permeability may be involved in the evolution towards chronic inflammation and in the establishment of persistent lesions. Further study is needed to confirm these preliminary findings.