Role of CD14+ monocyte-derived oxidised mitochondrial DNA in the inflammatory interferon type 1 signature in juvenile dermatomyositis

OBJECTIVES: To define the host mechanisms contributing to the pathological interferon (IFN) type 1 signature in Juvenile dermatomyositis (JDM). METHODS: RNA-sequencing was performed on CD4+, CD8+, CD14+ and CD19+ cells sorted from pretreatment and on-treatment JDM (pretreatment n=10, on-treatment n=11) and age/sex-matched child healthy-control (CHC n=4) peripheral blood mononuclear cell (PBMC). Mitochondrial morphology and superoxide were assessed by fluorescence microscopy, cellular metabolism by 13C glucose uptake assays, and oxidised mitochondrial DNA (oxmtDNA) content by dot-blot. Healthy-control PBMC and JDM pretreatment PBMC were cultured with IFN-α, oxmtDNA, cGAS-inhibitor, TLR-9 antagonist and/or n-acetyl cysteine (NAC). IFN-stimulated gene (ISGs) expression was measured by qPCR. Total numbers of patient and controls for functional experiments, JDM n=82, total CHC n=35. RESULTS: Dysregulated mitochondrial-associated gene expression correlated with increased ISG expression in JDM CD14+ monocytes. Altered mitochondrial-associated gene expression was paralleled by altered mitochondrial biology, including 'megamitochondria', cellular metabolism and a decrease in gene expression of superoxide dismutase (SOD)1. This was associated with enhanced production of oxidised mitochondrial (oxmt)DNA. OxmtDNA induced ISG expression in healthy PBMC, which was blocked by targeting oxidative stress and intracellular nucleic acid sensing pathways. Complementary experiments showed that, under in vitro experimental conditions, targeting these pathways via the antioxidant drug NAC, TLR9 antagonist and to a lesser extent cGAS-inhibitor, suppressed ISG expression in pretreatment JDM PBMC. CONCLUSIONS: These results describe a novel pathway where altered mitochondrial biology in JDM CD14+ monocytes lead to oxmtDNA production and stimulates ISG expression. Targeting this pathway has therapeutical potential in JDM and other IFN type 1-driven autoimmune diseases

Prediction of thioguanine-induced cytotoxicity by dual-parameter flow cytometric analysis

A method is presented for the quantitative analysis of delayed cytokinetic effects resulting from the treatment of L1210 cells with 6-thioguanine (TG). By using dual-parameter (DNA/protein) flow cytometry, we could observe the accumulation of late S/G2/M cells with abnormally high green fluorescence (i.e., protein content), indicative of unbalanced growth. The use of mitotic cells from a pseudotetraploid line (HT29) as external markers for both red and green fluorescence facilitated highly reproducible measurement of the mean green fluorescence (GFL mean ) of the arrested late S/G2/M population. We found that the dose dependence of the observed GFL mean values followed the same unusual biphasic pattern as did cytotoxicity in this cell line, indicating that this parameter might be a suitable means of predicting TG-induced toxicity in vivo. We propose that the low background expected for this kind of measurement would make it particularly appropriate for the analysis of clinical specimens (e.g., mononuclear bone marrow cells) from leukemic patients receiving thiopurines, to monitor (and, hopefully, predict) their response to treatment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46920/1/280_2004_Article_BF00304760.pd

Male × Female Interaction for a Pre-Copulatory Trait, but Not a Post-Copulatory Trait, among Cosmopolitan Populations of Drosophila melanogaster

Sexual coevolution occurs when changes in the phenotype of one sex select for changes in the other sex. We can identify the “footprint” of this coevolution by mating males and females from different populations and testing for a male-female genotype interaction for a trait associated with male (or female) performance. Here we mated male Drosophila melanogaster from five different continents with females from their own and different continents to test for a male-female interaction for mating speed, a pre-copulatory trait, and female reproductive investment, a post-copulatory trait. We found a strong male-female interaction for mating speed, consistent with previous studies using different populations, suggesting that the potential for sexual coevolution for this trait is present in this species. In contrast, we did not detect a male-female interaction for female reproductive investment. Although a male-female interaction for mating speed is compatible with the hypothesis of ongoing sexual coevolution, the nature of our experimental design is unable to exclude alternate explanations. Thus, the evolutionary mechanisms promoting male-female genotype interactions for pre-copulatory mating traits in D. melanogaster warrant further investigation

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies

An HR-MAS MR Metabolomics Study on Breast Tissues Obtained with Core Needle Biopsy

BACKGROUND: Much research has been devoted to the development of new breast cancer diagnostic measures, including those involving high-resolution magic angle spinning (HR-MAS) magnetic resonance (MR) spectroscopic techniques. Previous HR-MAS MR results have been obtained from post-surgery samples, which limits their direct clinical applicability. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we performed HR-MAS MR spectroscopic studies on 31 breast tissue samples (13 cancer and 18 non-cancer) obtained by percutaneous core needle biopsy. We showed that cancer and non-cancer samples can be discriminated very well with Orthogonal Projections to Latent Structure-Discriminant Analysis (OPLS-DA) multivariate model on the MR spectra. A subsequent blind test showed 69% sensitivity and 94% specificity in the prediction of the cancer status. A spectral analysis showed that in cancer cells, taurine- and choline-containing compounds are elevated. Our approach, additionally, could predict the progesterone receptor statuses of the cancer patients. CONCLUSIONS/SIGNIFICANCE: HR-MAS MR metabolomics on intact breast tissues obtained by core needle biopsy may have a potential to be used as a complement to the current diagnostic and prognostic measures for breast cancers

Effects of Hydrogen Peroxide on Wound Healing in Mice in Relation to Oxidative Damage

10.1371/journal.pone.0049215PLoS ONE711

Alcohol Facilitates CD1d Loading, Subsequent Activation of NKT Cells, and Reduces the Incidence of Diabetes in NOD Mice

Background: Ethanol ('alcohol') is a partly hydrophobic detergent that may affect the accessibility of glycolipids thereby influencing immunological effects of these molecules. Methods: The study included cellular in vitro tests using α-galactosylceramide (αGalCer), and in vivo NOD mice experiments detecting diabetes incidence and performing behavioural and bacterial analyses. Results: Alcohol in concentrations from 0.6% to 2.5% increased IL-2 production from NKT cells stimulated with αGalCer by 60% (p<0.05). CD1d expressed on HeLa cells contained significantly increasing amounts of αGalCer with increasing concentrations of alcohol, suggesting that alcohol facilitated the passive loading of αGalCer to CD1d. NOD mice were found to tolerate 5% ethanol in their drinking water without signs of impairment in liver function. Giving this treatment, the diabetes incidence declined significantly. Higher numbers of CD3+CD49b+ NKT cells were found in spleen and liver of the alcohol treated compared to the control mice (p<0.05), whereas the amount of CD4+Foxp3+ regulator T cells did not differ. Increased concentrations of IFN-γ were detected in 24-hour blood samples of alcohol treated mice. Behavioural studies showed no change in attitude of the ethanol-consuming mice, and bacterial composition of caecum samples was not affected by alcohol, disqualifying these as protective mechanisms. Conclusion: Alcohol facilitates the uptake of glycolipids and the stimulation of NKT cells, which are known to counteract Type 1 diabetes development. We propose that this is the acting mechanism by which treatment with alcohol reduces the incidence of diabetes in NOD mice. This is corroborated by epidemiology showing beneficial effect of alcohol to reduce the severity of atherosclerosis and related diseases

Type 2 diabetes increases and metformin reduces total, colorectal, liver and pancreatic cancer incidences in Taiwanese: a representative population prospective cohort study of 800,000 individuals

<p>Abstract</p> <p>Background</p> <p>Metformin protection against cancer risk in Orientals is uncertain. We examined the possible metformin effect on total, esophageal, gastric, colorectal (CRC), hepatocellular (HCC) and pancreatic cancers in a Taiwanese cohort.</p> <p>Methods</p> <p>A representative sample of 800,000 was drawn from the Taiwanese National Health Insurance data of 2000. A cohort of 480,984 participants 20 years or older, diabetes-cancer-free on 1st January 2000 was formed and categorized as four groups by DM and metformin usage status. Eligible incident cancer events had to occur one year after the index date until the end of 2007. The Cox proportional-hazards model evaluated relative risk of cancer for treated DM patients with or without metformin. The covariates included age, gender, other oral anti-hyperglycemic medication, Charlson comorbidity index (CCI) score and metformin exposure dosage and duration.</p> <p>Results</p> <p>With diabetes but no anti-hyperglycemic medication, cancer incidence density increased at least 2-fold for total, CRC and HCC. On metformin, total, CRC and HCC incidences decreased to near non-diabetic levels but to varying degrees depending on gender and cancer type (CRC in women, liver in men). Adjustment for other oral anti-hyperglycemic agents usage and CCI made the benefit of metformin more evident [hazard ratios (95% confidence intervals): total 0.12 (0.08-0.19), CRC 0.36 (0.13-0.98), liver 0.06 (0.02-0.16), pancreas 0.15 (0.03-0.79)]. There was a significant gender interaction with metformin in CRC which favored women. Metformin dosage for a significant decrease in cancer incidence was ≤500 mg/day.</p> <p>Conclusions</p> <p>Metformin can reduce the incidences of several gastroenterological cancers in treated diabetes.</p