Therapeutic role of rifaximin in inflammatory bowel disease: clinical implication of human pregnane X receptor activation.

Human pregnane X receptor (PXR) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a human PXR activator, is in clinical trials for treatment of IBD and has demonstrated efficacy in Crohn's disease and active ulcerative colitis. In the current study, the protective and therapeutic role of rifaximin in IBD and its respective mechanism were investigated. PXR-humanized (hPXR), wild-type, and Pxr-null mice were treated with rifaximin in the dextran sulfate sodium (DSS)-induced and trinitrobenzene sulfonic acid (TNBS)-induced IBD models to determine the protective function of human PXR activation in IBD. The therapeutic role of rifaximin was further evaluated in DSS-treated hPXR and Pxr-null mice. Results demonstrated that preadministration of rifaximin ameliorated the clinical hallmarks of colitis in DSS- and TNBS-treated hPXR mice as determined by body weight loss and assessment of diarrhea, rectal bleeding, colon length, and histology. In addition, higher survival rates and recovery from colitis symptoms were observed in hPXR mice, but not in Pxr-null mice, when rifaximin was administered after the onset of symptoms. Nuclear factor κB (NF-κB) target genes were markedly down-regulated in hPXR mice by rifaximin treatment. In vitro NF-κB reporter assays demonstrated inhibition of NF-κB activity after rifaximin treatment in colon-derived cell lines expressing hPXR. These findings demonstrated the preventive and therapeutic role of rifaximin on IBD through human PXR-mediated inhibition of the NF-κB signaling cascade, thus suggesting that human PXR may be an effective target for the treatment of IBD.

Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases.

Mycobacterium leprae, the causative agent of leprosy, is uncultivable in defined media. Development of new diagnostic tools which do not depend on growth of bacteria is needed for the early detection of M. leprae and for monitoring the effectiveness of chemotherapy. We used a real-time PCR-based assay to quantify the copy number of bacterial DNA and hsp18 mRNA from 47 leprosy patients using paraffin-embedded biopsy samples. The assay used was specific, sensitive and reproducible. The applicability of this approach in monitoring the chemotherapy of leprosy was examined. A reduction in DNA and mRNA during chemotherapy was observed and hsp18 mRNA could not be detected in patients who underwent 2 years of multidrug therapy (MDT). However, a considerable amount of M. leprae DNA could be detected even after 2 years of MDT. A significant amount of hsp18 mRNA was found in reactional cases as well. This raises important questions regarding the role of bacterial antigens in leprosy reactions and the rationale of omitting antibiotics in the treatment of reactional cases. Results in this study show that real-time PCR could be a better tool for the careful monitoring of bacillary DNA and mRNA in lesions, which will help to improve diagnosis, disease progression and the treatment regimen.

Cytokine profiles in paraffin-embedded biopsy samples of lepromatous leprosy patients: semi-quantitative measure of cytokine mRNA using RT-PCR.

A reproducible technique for fixation of tissue, RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis from paraffin-embedded leprosy biopsies, has been developed and used to study the mRNA profiles. This approach is valuable in retrospective analysis of gene expression, and the handling of infectious biopsy material is also minimized. Among the methods of RNA extraction compared, the most efficient method was found to be incubation of the tissue sections in digestion buffer followed by extraction with Trizol. The experimental conditions were optimized for first strand cDNA synthesis and PCR, and used to measure the quantity of cytokine transcripts in biopsy materials. Interleukin-10 (IL-10) mRNA was detectable in all cases examined, which correlates well with other earlier reports using frozen tissues. However, IL-5 transcripts were present in 60% of the biopsies, unlike the earlier reports which showed IL-5 mRNA in all LL cases. Transforming growth factor-beta (TGF-beta) mRNA was detected in 80% of the biopsies, and this confirmed earlier immuno-cytochemical data which showed TGF-beta protein in all cases. Tumor necrosis factor-alpha mRNA was found in about 60% of the biopsies; whereas interferon gamma mRNA was detected in 30% of the LL cases. In conclusion, the results obtained in our study confirm and extend earlier observations which examined cytokines in peripheral blood cells and dermal lesions of leprosy. The simplicity of this method would allow the examination of a large number of samples across the spectrum of leprosy.