Comparison of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide tube method with the conventional method and real-time polymerase chain reaction for the detection of rifampicin resistance in Mycobacterium tuberculosis

Colorimetric methods are cheap, reproducible, and rapid methods of detecting drug resistance in Mycobacterium tuberculosis. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) method is one such technique that has been established in our laboratory to detect rifampicin resistance. The present study compared the results of the MTT method with those of the proportion method and real-time polymerase chain reaction (RTPCR) in order to establish sensitivity and specificity of MTT. The mutations for rifampicin resistance occur in rpoB gene, and the commonest reported are in codons 526 and 531. Therefore, RTPCR was targeted at these two codons. The concordance of MTT with the proportion method and RTPCR was 94 and 72.77%, respectively, and that of RTPCR with the proportion method was 77.77%. While the study confirmed that the MTT method is a good method for detecting rifampicin resistance, it also brought out the fact that RTPCR when targeted for limited mutations is not a good tool. Either the genotypic method used should target the total 81-bp rpoB genome or methods such as DNA sequencing should be used. For resource-constraint laboratories, the MTT method can be considered as a better choice.     

T cell receptor (TCR) repertoire and function of human epidermal T cells: restricted TCR Vα-Vβ genes are utilized by T cells residing in the lesional epidermis in fixed drug eruption

Human epidermis contains a phenotypically heterogeneous population of T cells. No information, however, is available regarding the TCR repertoire of these T cells and their relevant physiologic and pathologic functions in vivo. To this end, T cells were prepared from the lesional epidermis in two patients with fixed drug eruption (FDE) and their phenotype, function and TCR repertoire were examined in parallel. Both epidermal T cells, termed FDE-1 and -2 cells, respectively, expressed αβ TCR, but displayed some phenotypic heterogeneity. These T cells were induced to display cytolytic activity by ligation of the CD3/TCR-αβ complex. Comparative analyses of TCR Vα and Vβ expression in the epidermal T cells and the paired peripheral blood lymphocytes (PBL) by quantitative polymerase chain reaction (PCR) demonstrated that the epidermal T cells, but not the paired PBL, utilized a very limited range of Vα and Vβ genes. These results indicate that some expansion or preferential migration of epidermal T cells that recognize a restricted set of antigens expressed within the epidermis could occur in situ following ingestion of the causative drug. The persistence of these epidermal T cells in FDE lesions suggests their pathologic role in a drug-induced flare.     

Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation.

The pattern of somatic mutations of shark and frog Ig is distinct from somatic hypermutation of Ig in mammals in that there is a bias to mutate GC base pairs and a low frequency of mutations. Previous analysis of the new antigen receptor gene in nurse sharks (NAR), however, revealed no bias to mutate GC base pairs and the frequency of mutation was comparable to that of mammalian IgG. Here, we analyzed 1023 mutations in NAR and found no targeting of the mechanism to any particular nucleotide but did obtain strong evidence for a transition bias and for strand polarity. As seen for all species studied to date, the serine codon AGC/T in NAR was a mutational hotspot. The NAR mutational pattern is most similar to that of mammalian IgG and furthermore both are strikingly akin to mutations acquired during the neutral evolution of nuclear pseudogenes, suggesting that a similar mechanism is at work for both processes. In yeast, most spontaneous mutations are introduced by the translesion synthesis DNA polymerase zeta (REV3) and in various DNA repair-deficient backgrounds transitions were more often REV3-dependent than were transversions. Therefore, we propose a model of somatic hypermutation where DNA polymerase zeta is recruited to the Ig locus. An excess of DNA glycosylases in germinal center reactions may further enhance the mutation frequency by a REV3-dependent mutagenic process known as imbalanced base excision repair.     

Defining the anatomical localisation of subsets of the murine mononuclear phagocyte system using integrin alpha X (Itgax, CD11c) and colony stimulating factor 1 receptor (Csf1r, CD115) expression fails to discriminate dendritic cells from macrophages

The murine mononuclear phagocyte (MNP) system comprises a diverse population of cells, including monocytes, dendritic cells (DC) and macrophages. Derived from the myeloid haematopoietic lineage, this group of cells express a variety of well characterized surface markers. Expression of the integrin alpha X (Itgax, CD11c) is commonly used to identify classical DC, and similarly expression of colony stimulating factor 1 receptor (Csf1r, CD115) to identify macrophages. We have characterized the expression of these markers using a variety of transgenic mouse models. We confirmed previous observations of Itgax expression in anatomically defined subsets of MNPs in secondary lymphoid organs, including all MNPs identified within the germinal centres. The majority of MNPs in the intestinal lamina propria and lung express Itgax. All mucosal Itgax expressing cells also express Csf1r suggesting Csf1-dependent haematopoietic derivation. This double-positive population included germinal centre MNPs. These data reveal that Itgax expression alone does not specifically define classical DC. These results suggest more cautious interpretation of Itgax-dependent experimentation and direct equation with uniquely DC-mediated activities, particularly in the functioning of non-lymphoid MNPs within the intestinal lamina propria.     

An emerging role of neutrophils and NETosis in chronic inflammation and fibrosis in systemic lupus erythematosus (SLE) and ANCA-associated vasculitides (AAV): Implications for the pathogenesis and treatment

Neutrophils derive from hematopoietic stem cells (HSCs) with systemic inflammation driving their activation and differentiation to myeloid progenitors to ensure enhanced myelopoiesis. Epigenetic reprograming and re-education of these HSCs produces neutrophils primed towards elimination of pathogens and increased inflammatory response. Neutrophils -an important component of acute inflammation- are not present in chronic inflammatory tissues leading to the false assumption that they may not be as important for the latter. Activated neutrophils may release Neutrophil Extracellular Traps (NETs) during a distinct form of cell death, named NETosis; NETs are rich in bioactive molecules that promote thrombosis (including atherothrombosis), inflammation and fibrosis. Thus, although neutrophils may not be present in chronic inflammatory lesions, their remnants may amplify the inflammatory response beyond their short life-span in the tissues. Herein, we review current evidence supporting a role of neutrophils and NETosis in tissue injury and dysfunction in systemic autoimmunity using as disease paradigms Systemic Lupus Erythematosus (SLE) and the ANCA-associated vasculitides (AAV). We also discuss the mechanisms involved and their potential as targets for novel therapy and drug repositioning.     

Identification and immunoregulatory function of neuromedin U (Nmu) in the Japanese pufferfish Takifugu rubripes

In this study, immunoregulatory function of neuromedin U (Nmu) in the teleost fish Fugu (Takifugu rubripes) was characterized. Three splicing variants of nmu mRNA encoding preproNMUs consisting of 164 (Nmu1), 139 (Nmu2), and 129 (Nmu3) amino acid residues were found in Fugu.The biologically active C-terminal region of Fugu Nmu showed high homology among fish and other vertebrate NMUs. The genomic organization of Fugu nmu differed from those of zebrafish and mammals. However, in phylogenetic analysis, Fugu Nmu formed a cluster with NMUs of other vertebrates, in addition to neuromedin S. The splicing variants of mRNA were identified in various tissues. Nmu-21 and Nmu-9 were purified as endogenous peptides from Fugu intestine. The synthetic Nmu-21 peptide activated phagocytic cells, and elevated the expression of cytokine mRNA in peripheral blood leukocytes.