The macrophage resistance gene NRAMP1 regulates priming/activation of macrophages for enhanced TNFa, ILIl, and MHC class II expression. Since all of these functions are of potential importance in the induction or maintenance or both of autoimmune disease, samples from the Arthritis and Rheumatism Council's repository of multicase rheumatoid arthritis families were typed for a dinucleotide repeat in the NRAMP1 promoter region and four other 2q34 (TNP1) or 2q35 (IL8R, VILl, DES) marker genes.Identity by descent (IBD) sib pair analysis using a three locus haplotype NRAMP1-IL8RB-VIL1, or NRAMP1 alone, provided preliminary evidence (maximum lod score = 1.01, p = 0.024) for a gene in this region contributing to susceptibility to rheumatoid arthritis. Candidacy for NRAMP1 as the disease susceptibility gene was supported by a significant bias (p=0.048) towards transmission of the NRAMP1 promoter region allele 3 in affected offspring.

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Leprosy type 1 reactions (T1R) are due to increased cell-mediated immunity and result in localized tissue damage. The anti-inflammatory drug prednisolone is used for treatment, but there is little good in vivo data on the molecular actions of prednisolone. We investigated the effect of prednisolone treatment on tumor necrosis factor alpha (TNF-␣), interleukin-1␤ (IL-1␤), IL-10, and transforming growth factor ␤1 (TGF-␤1) mRNA and protein expression in blood and skin biopsies from 30 patients with T1R in India. After 1 month of prednisolone treatment the sizes of the skin granulomas were reduced, as were the grades of cells positive for TNF-␣ and IL-10 in skin lesions. Increased production of TGF-␤1 was seen in skin lesions after 6 months of prednisolone treatment. Expression of mRNA for TNF-␣, IL-1␤, and TGF-␤1 was reduced, whereas no change in IL-10 mRNA expression was detected during treatment. The circulating cytokine profiles were similar in patients with and without T1R, and prednisolone treatment had no detectable effects on cytokine expression in the blood. The data emphasize the compartmentalization of pathology in T1R and the importance of the immune response in the skin. Clinical improvement and cytokine expression were compared. Surprisingly, patients with improved skin and nerve function and patients with nonimproved skin and nerve function had similar cytokine profiles, suggesting that clinical improvement is not directly mediated by the cytokines studied here. This in vivo well-controlled study of the immunosuppressive effects of prednisolone showed that the drug does not switch off cytokine responses effectively.

Gamma interferon (IFN-γ)-secreting CD4+ T cells have long been established as an essential component of the protective immune response against Mycobacterium tuberculosis. It is now becoming evident from studies with the murine model of tuberculosis that an important role also exists for major histocompatibility complex (MHC) class I-restricted CD8+ T cells. These cells are capable of acting as both IFN-γ secretors and cytotoxic T lymphocyte (CTL) effectors; however, their exact role in immunity against tuberculosis remains unclear. This study demonstrates the presence ofMycobacterium bovis BCG-reactive CD8+ T cells in healthy BCG-vaccinated donors and that these CD8+ T cells are potent cytokine producers as well as cytotoxic effector cells. Using FACScan analysis, we have shown that restimulation with live M. bovis BCG induced more CD8+-T-cell activation than the soluble antigen purified protein derivative and that these cells are actively producing the type 1 cytokines IFN-γ and tumor necrosis factor alpha (TNF-α). These CD8+ T cells also contain the cytolytic granule perforin and are capable of acting as potent CTLs against M. bovis BCG-infected macrophages. The mycobacterial antigens 85A and B (Ag85A and Ag85B, respectively), and to a lesser extent the 19- and 38-kDa proteins, are major antigenic targets for these mycobacterium-specific CD8+ T cells, while whole-M. bovis BCG activated effector cells from these BCG-vaccinated donors, as expected, failed to recognize the 6-kDa ESAT-6 protein. The use of metabolic inhibitors and blocking antibodies revealed that the CD8+ T cells recognize antigen processed and presented via the classical MHC class I pathway. These data suggest that CD8+ T cells may play a critical role in the human immune response to tuberculosis infection.

The mycobacterial diseases leprosy and tuberculosis (TB) and the leishmaniases are characterized by a wide spectrum of disease phenotypes, and by the fact that the majority of individuals exposed to the causative organisms Mycobacterium leprae, M. tuberculosis and Leishmania sp. become infected but do not present with clinical disease. In order to determine whether a human homologue to the murine macrophage resistance gene Lsh/Ity/Bcg influences susceptibility to human disease, multicase families for all three diseases have been collected, and linkage analysis performed using a panel of markers in the region of human chromosome 2q33-q37 known to be conserved with the Lsh/Ity/Bcg-containing region of murine chromosome 1. Because of the paucity of available polymorphic markers/linkage information for 2q33-q37, data from 35 multicase leprosy, TB and visceral leishmaniasis families (310 individuals) were first pooled to produce a detailed RFLP map of the region. Peak LOD scores well in excess of 3 were observed for linkage between adjacent pairs of a more proximal (2q33-q35) set of markers CRYGP1, MAP2, FN1, TNP1, VIL1 and DES, and between adjacent pairs of a more distal (2q35-q37) set COL6A3, D2S55 and D2S3. These peak LOD scores and the corresponding values for theta were used in the MAP92 program to generate a multiple two-point map with gene order/map intervals (cM) of: CRYGP1-4.65-MAP2-3.45-FN1-5.95-TNP1-3.41-VIL1-3. 01- DES-20.14-COL6A-10.91-D2S55-3.67-D2S3. Although local support for the placement of loci in this order was weak (LOD < 2, except for DES-COL6A3 where LOD = 6.02), the map is consistent with the gene order for those loci (Cryg, Fn-1, Tp-1, Vil, Des, Col6a3) previously mapped in the mouse. Data from 17 multicase leprosy families (149 individuals) were further analysed for linkage between a putative disease susceptibility locus (DSL) controlling susceptibility to leprosy per se and each of the marker loci. Assuming 100% penetrance for the susceptibility allele, no positive LOD score was obtained for linkage between the DSL and any of the marker genes. Instead, the data provide convincing evidence (LOD scores < -2) that a DSL does not fall within 10-20 cM of CRYGP1, MAP2, TNP1, VIL1, DES or D2S55, or within 5-10 cM of FN1, COL6A3 or D2S3. This effectively excludes a putative DSL controlling susceptibility to leprosy per se from the entire region 2q33-q37.(ABSTRACT TRUNCATED AT 400 WORDS)

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This is the first study to examine the expression of TLR2 and TLR4 in vivo in individuals experiencing leprosy Type 1 reactions. The data support the possibility of an important role for TLR2 and TLR4 in the pathogenesis of this important complication of leprosy.

Abstract Objectives Management of colorectal cancer warrants mutational analysis of KRAS/NRAS when considering anti–epidermal growth factor receptor therapy and BRAF testing for prognostic stratification. In this multicenter study, we compared a fully integrated, cartridge-based system to standard-of-care assays used by participating laboratories. Methods Twenty laboratories enrolled 874 colorectal cancer cases between November 2017 and December 2018. Testing was performed on the Idylla automated system (Biocartis) using the KRAS and NRAS-BRAF cartridges (research use only) and results compared with in-house standard-of-care testing methods. Results There were sufficient data on 780 cases to measure turnaround time compared with standard assays. In-house polymerase chain reaction (PCR) had an average testing turnaround time of 5.6 days, send-out PCR of 22.5 days, in-house Sanger sequencing of 14.7 days, send-out Sanger of 17.8 days, in-house next-generation sequencing (NGS) of 12.5 days, and send-out NGS of 20.0 days. Standard testing had an average turnaround time of 11 days. Idylla average time to results was 4.9 days with a range of 0.4 to 13.5 days. Conclusions The described cartridge-based system offers rapid and reliable testing of clinically actionable mutation in colorectal cancer specimens directly from formalin-fixed, paraffin-embedded tissue sections. Its simplicity and ease of use compared with other molecular techniques make it suitable for routine clinical laboratory testing.