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Methanogenic archaea are an important component of the human and animal intestinal microbiota, and yet their presence is rarely reported in publications describing the subject. One of the methods of quantifying the prevalence of methanogens is quantitative real-time PCR (qPCR) of the methanogen-specific mcrA gene, and one of the possible reasons for detection failure is usually a methodology bias. Here, we refined the existing protocol by changing one of the primers and improving the conditions of the qPCR reaction. As a result, at the expense of a slightly lower yet acceptable PCR efficiency, the new assay was characterized by increased specificity and sensitivity and a wider linear detection range of 7 orders of magnitude. The lowest copy number of mcrA quantified at a frequency of 100% was 21 copies per reaction. The other validation parameters tested, such as reproducibility and linearity, also gave satisfactory results. Overall, we were able to minimize the negative impacts of primer dimerization and other cross-reactions on qPCR and increase the number of not only detectable but also quantifiable stool samples—or in this case, chicken droppings.

Autoantibodies occur in healthy subjects as well as in children with Wilson’s disease (WD), but their prevalence and significance are unknown. Thus, we aimed to assess the prevalence of autoantibodies and autoimmune markers, and their relationship to liver injury in WD children. The study included 74 WD and 75 healthy children as a control group. Patients with WD underwent transient elastography (TE) examinations, as well as determination of liver function tests, copper metabolism markers, and serum immunoglobulins (Ig). In the sera of the WD patients and controls, anti-nuclear (ANA), anti-smooth muscle, anti-mitochondrial, anti-parietal cell, anti-liver/kidney microsomal, anti-neutrophil cytoplasmic autoantibodies, and specific celiac antibodies were determined. Among the autoantibodies, only the prevalence of ANA in children with WD was higher than in the controls. There was no significant relationship between the presence of autoantibodies and liver steatosis or stiffness after TE. However, advanced liver stiffness (E > 8.2 kPa) was related to IgA, IgG, and gamma globulin production. The type of treatment did not influence the prevalence of autoantibodies. Our results suggest that autoimmune disturbances in WD might not be directly related to liver damage as expressed by steatosis and/or liver stiffness after TE.

Disruption of epithelial junctional complex (EJC), especially tight junctions (TJ), resulting in increased intestinal permeability, is supposed to activate the enhanced immune response to gluten and to induce the development of celiac disease (CD). This study is aimed to present the role of EJC in CD pathogenesis. To analyze differentially expressed genes the next-generation mRNA sequencing data from CD326+ epithelial cells isolated from non-celiac and celiac patients were involved. Ultrastructural studies with morphometry of EJC were done in potential CD, newly recognized active CD, and non-celiac controls. The transcriptional analysis suggested disturbances of epithelium and the most significant gene ontology enriched terms in epithelial cells from CD patients related to the plasma membrane, extracellular exome, extracellular region, and extracellular space. Ultrastructural analyses showed significantly tighter TJ, anomalies in desmosomes, dilatations of intercellular space, and shorter microvilli in potential and active CD compared to controls. Enterocytes of fetal-like type and significantly wider adherence junctions were observed only in active CD. In conclusion, the results do not support the hypothesis that an increased passage of gluten peptides by unsealing TJ precedes CD development. However, increased intestinal permeability due to abnormality of epithelium might play a role in CD onset.