Evidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here, we describe the generation of ...HepG2-derived transmitochondrial cybrids, to investigate the impact of mtDNA variation on mitochondrial (dys)function and susceptibility to iDILI. This study created 10 cybrid cell lines, each containing distinct mitochondrial genotypes of haplogroup H or haplogroup J backgrounds.
HepG2 cells were depleted of mtDNA to make rho zero cells, before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide, and tolcapone, and their less toxic counterparts bicalutamide and entacapone utilizing ATP assays and extracellular flux analysis.
Whilst only slight variations in basal mitochondrial function were observed between haplogroups H and J, haplogroup-specific responses were observed to the mitotoxic drugs. Haplogroup J showed increased susceptibility to inhibition by flutamide, 2-hydroxyflutamide, and tolcapone, via effects on selected mitochondrial complexes (I and II), and an uncoupling of the respiratory chain.
This study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest. This provides a practical and reproducible system to investigate the cellular consequences of variation in the mitochondrial genome, against a constant nuclear background. Additionally, the results show that inter-individual variation in mitochondrial haplogroup may be a factor in determining sensitivity to mitochondrial toxicants.
This work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).
The postinfectious irritable bowel syndrome (PI-IBS) suggests that impaired resolution of inflammation could cause IBS symptoms. The authors hypothesised that polymorphisms in genes whose expression ...were altered by gastroenteritis might be linked to IBS with diarrhoea (IBS-D) which closely resembles PI-IBS.
Part 1: 25 healthy volunteers (HVs), 21 patients 6 months after Campylobacter jejuni infection, 37 IBS-D and 19 IBS with constipation (IBS-C) underwent rectal biopsy for gene expression analysis and peripheral blood mononuclear cell cytokine production assessment. Part 2: Polymorphisms in genes whose expression was altered in Part 1 were assessed in 179 HV, 179 IBS-D, 122 IBS-C and 41 PI-IBS.
Part 1: Mucosal expression of seven genes was altered in IBS: CCL11, CCL13, Calpain 8 and TNFSF15 increased while NR1D1, GPR161 and GABRE decreased with similar patterns after infection with C jejuni. Part 2: The authors assessed 21 known single nucleotide polymorphisms (SNPs) in these seven genes and one SNP in each of the TNFα and IL-10 genes. Three out of five TNFSF15 SNPs (rs6478108, rs6478109 and rs7848647) showed reduced minor allele frequency (MAF) (0.28, 0.27 and 0.27) in subjects with IBS-D compared with HV (0.38, 0.36 and 0.37; p=0.007, 0.015 and 0.007, respectively) confirming others recent findings. The authors also replicated the previously reported association of the TNFα SNP rs1800629 with PI-IBS which showed an increase in the MAF at 0.30 versus 0.19 for HV (p=0.04).
IBS-D and PI-IBS patients are associated with TNFSF15 and TNFα genetic polymorphisms which also predispose to Crohn's disease suggesting possible common underlying pathogenesis.
The anterior nares are regarded as the primary site for Staphylococcus aureus colonization, although studies have highlighted the potential importance of colonization at extra-nasal sites, including ...the oropharynx. Accordingly, the aims of this study were to assess the prevalence, persistence and molecular epidemiology of S. aureus colonization in the nares and oropharynx of Māori and Pacific children, a population with strikingly high rates of S. aureus infection.
A cross-sectional study of predominantly Māori and Pacific school-aged children in Auckland, New Zealand was performed in October 2013, and swabs were taken from the nares and oropharynx. Sampling was repeated from the same schools in October 2014. All S. aureus isolates underwent antimicrobial susceptibility testing and spa typing.
Overall, 506/893 (56.7%) children were colonized with S. aureus, and the colonization prevalence was significantly higher in the oropharynx than nares (41.1% vs. 31.5%; P < 0.001). Longitudinal colonization was significantly higher in the oropharynx than the nares, and children with longitudinal oropharyngeal colonization were more likely to be colonized with the same spa type than those colonized in the nares (67.6% vs. 37.0%; P = 0.01). Approximately 40% of children had discordant spa types at the nares and oropharynx.
Oropharyngeal S. aureus colonization represents a significant reservoir of S. aureus and it is possible that the oropharynx may represent a protected anatomical niche, enabling persistent colonization with the same S. aureus strain. Future study should attempt to better understand the determinants of oropharyngeal carriage.
We analyzed all cases of community-onset Staphylococcus aureus skin and soft tissue infection in children presenting to our hospital between 2007 and 2010. A total of 1860 children were included. ...There was significant sociodemographic disparity, with the incidence disproportionately higher in Maori and Pacific Island children residing in deprived areas.
To determine whether basal serum or plasma cortisol concentration can be used as a screening test to rule out hypoadrenocorticism in dogs.
Retrospective case-control study.
110 dogs with nonadrenal ...gland illnesses and 13 dogs with hypoadrenocorticism.
Sensitivity and specificity of basal serum or plasma cortisol concentrations of either <or= 1 microg/dL or <or= 2 microg/dL to detect dogs with hypoadrenocorticism were estimated by use of the ACTH stimulation test as the gold standard.
Basal cortisol concentrations of <or= 1 microg/dL had excellent sensitivity (100%) and specificity (98.2%) for detecting dogs with hypoadrenocorticism. For basal cortisol concentrations of <or= 2 microg/dL, sensitivity was 100% but specificity was 78.2%.
On the basis of sensitivity and specificity, basal serum or plasma cortisol concentrations had high negative predictive values over a wide range of prevalence rates and can be used to rule out a diagnosis of hypoadrenocorticism. Dogs with basal cortisol concentrations > 2 microg/dL that are not receiving corticosteroids, mitotane, or ketoconazole are highly unlikely to have hypoadrenocorticism. However, if the basal cortisol concentration is <or= 2 microg/dL, little to no information regarding adrenal gland function can be obtained and an ACTH stimulation test should be performed.
The relative contributions of superoxide anion (O2−) and peroxynitrite (PN) were evaluated in the pathogenesis of intestinal microvascular damage caused by the intravenous injection of E. coli ...lipopolysaccharide (LPS) in rats. The superoxide dismutase mimetic (SODm) SC‐55858 and the active peroxynitrite decomposition catalysts 5,10,15,20‐tetrakis(2,4,6‐trimethyl‐3,5‐disulphonatophenyl)‐porphyrinato iron (III) and 5,10,15,20‐tetrakis(N‐methyl‐4′‐pyridyl)‐porphyrinato iron (III) (FeTMPS, FeTMPyP respectively) were used to assess the roles of O2− and PN respectively.
The intravenous injection of LPS elicited an inflammatory response that was characterized by a time‐dependent infiltration of neutrophils, lipid peroxidation, microvascular leakage (indicative of microvascular damage), and epithelial cell injury in both the duodenum and jejunum.
Administration of the SODm SC‐55858, FeTMPS or FeTMPyP at 3 h post LPS reduced the subsequent increase in microvascular leakage, lipid peroxidation and epithelial cell injury. Inactive peroxynitrite decomposition catalysts exhibited no protective effects. Only, SC‐55858 inhibited neutrophil infiltration.
Our results suggest that O2− and peroxynitrite play a significant role in the pathogenesis of duodenal and intestinal injury during endotoxaemia and that their removal by SODm and peroxynitrite decomposition catalysts offers a novel approach to the treatment of septic shock or clinical conditions of gastrointestinal inflammation. Furthermore, the remarkable protection of the intestinal epithelium by these agents suggests their use during chemo‐ and radiation therapy, cancer treatments characterized by gastrointestinal damage. Potential mechanisms through which these radicals evoke damage are discussed.
British Journal of Pharmacology (1999) 127, 685–692; doi:10.1038/sj.bjp.0702604
The aim of our study was to determine the clinical course of children with idiopathic childhood nephrotic syndrome (ICNS) who received intravenous methylprednisolone (ivMP) following failure to ...achieve remission with standard oral prednisolone therapy. This study was designed as a retrospective case record review from 1993 to 2007. Sixteen children received ivMP over the 15-year study period, of whom ten responded, achieving clinical remission. The remaining six children with steroid resistant nephrotic syndrome (SRNS) underwent biopsy four focal segmental glomerulosclerosis (FSGS), two minimal change disease (MCD). Three responders developed late secondary steroid resistance (two FSGS, one MCD). At the latest follow-up (mean 6.7 years), three of the ten ivMP responders and none (0/6) of the children with SRNS had heavy proteinuria and chronic kidney disease (CKD) stage 3-5. The remaining 13 children demonstrated significant steroid dependency but had achieved stable remission following cyclophosphamide and/or ciclosporin therapy. The majority of children with ICNS who do not respond to 4 weeks of daily prednisolone therapy will enter remission following three to five doses of ivMP, thus avoiding a renal biopsy at initial presentation. These children are likely to develop steroid dependency, and the majority will require treatment with alkylating agents and/or ciclosporin to maintain remission. The requirement for ivMP in this setting appears to be associated with a risk of developing CKD in the longer term.
T cell activation in response to antigenic stimulation is a complex process, involving changes in the expression level of a large number of genes. We have used cDNA array technology to characterize ...the differences in gene expression between human CD4+ and CD8+ T cells. PBMC from six healthy donors were stimulated with live Mycobacterium tuberculosis, and the gene expression profiles of each donor's CD4+ and CD8+ T cells were analyzed separately. ANOVA revealed 518 genes that were consistently differentially expressed between CD4+ and CD8+ T cells. These differentially expressed genes include a combination of well-known, previously characterized genes with a range of biological functions and unknown in silico predicted hypothetical genes. Where possible, the novel genes have been characterized using bioinformatics, and putative transcription factors, signaling molecules, transmembrane, and secreted factors have been identified. A subset of these differentially expressed genes could be exploited as markers of CD4+ and CD8+ T cell activation for use in vaccine trials. These observed differences in the gene expression profile of CD4+ and CD8+ T cells following activation by a human pathogen contribute to an increased understanding of T cell activation and differentiation and the roles these T cell subsets may play in immunity to infection.
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