Aim: There is little information in the literature regarding assays for measuring CDH17 in tissues. Numerous studies indicate overexpression of CDH17 in a variety of diseases including hepatocellular carcinoma, colorectal and gastric cancer. Here we present an immunoaffinity enrichment LC–MS/MS approach for analysis of CDH17 in human tissues, plasma and serum as well as preclinical models. Results: CDH17 levels were measured in colon and ileum tissues from healthy donors and inflamed tissues from patients with Ulcerative Colitus or Crohn’s disease. Applicability of the immunocapture LC–MS/MS approach is demonstrated for colon tissues from non-diseased mouse and cynomolgus monkey. Conclusion: The analytical approaches discussed here are suitable for quantitation of CDH17 in various tissues to enable both preclinical and clinical assessment.
Tremorgenic mycotoxicosis can arise from dietary exposure to secondary metabolite products of various fungal species, particularly those from the Penicillium genus. Although general toxin screens often rely on gas chromatography-mass spectrometry (GC/MS) and well-developed mass spectral library databases, two principal representative Penicillium mycotoxins, roquefortine and penitrem A, are unamenable to GC/MS owing to high molecular weights, low volatilities and/or high thermal instabilities. Reliance on GC/MS screens alone could therefore inadvertently result in failure to collect evidence of exposure to such tremorgenic mycotoxins. In this report we describe a newly discovered tremorgenic marker compound (TMC), the presence of which correlates highly with conclusive exposure to Penicillium toxins in explanation of clinical manifestations of tremorgenic mycotoxicosis. According to detailed mass spectral deconvolution, the compound is 210.0892 molecular weight, and amenable to GC/MS whether chemically underivatized or derivatized by methylation or trimethylsilylation. 1D and 2D NMR (nuclear magnetic resonance) studies on the isolated compound determined the TMC to be the Penicillium product terrestric acid, C11H14O4, which matches the molecular formula determined by high resolution mass spectrometry and thus provides an excellent target for assessment of mycotoxicosis by GC/MS.
Free intracellular magnesium (Mg2+) plays a critical role in diverse immune cell functions. Human individuals with a genetic deficiency in the magnesium transporter MagT1 present with XMEN disease, a X-linked immunodeficiency characterized by uncontrolled Epstein-Barr virus infection and neoplasia, uncovering a critical role for Mg2+ in the regulation of NK and CD8T cell function. To interrogate the role of MagT1 in the immune response, we generated conditional knockout mice utilizing a tamoxifen inducible (Cre-ERT2) system to genetically delete Magt1. Here, we characterized the requirement for MagT1 in immune cell function following in vivo genetic deletion. MagT1 is not required for lymphocyte homeostasis following tamoxifen deletion, as T and B cell populations were unperturbed in the spleen, lymph node and thymus following short and long-term tamoxifan treatment. Magt1-deficient CD4+ T cells were defective in response to antigenic challenge following Ova immunization in vivo, as the numbers of BRDU+ CD4+ T cells were reduced. This observation was not due to a defect in proliferation, as the percentage of BRDU+ CD4+ T cells was similar between wild-type and Magt1-deficient mice. In support of this finding, Magt1-deficient CD4+ T proliferation is normal in multiple in vitro proliferation assays. While this observation is inconsistent with human findings from X-MEN patients, differences in human and mouse physiology might contribute to this discrepancy. Future interrogation of MagT1 function in NK and CD8+ T cells could provide mechanistic insight into the manifestations observed in X-MEN disease.
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