Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA

The mild phenotype associated with targeted disruption of the mouse OGG1 and NTH1 genes has been attributed to the existence of back-up activities and/or alternative pathways for the removal of oxidised DNA bases. We have characterised two new genes in human cells that encode DNA glycosylases, homologous to the bacterial Fpg (MutM)/Nei class of enzymes, capable of removing lesions that are substrates for both hOGG1 and hNTH1. One gene, designated HFPG1, showed ubiquitous expression in all tissues examined whereas the second gene, HFPG2, was only expressed at detectable levels in the thymus and testis. Transient transfections of HeLa cells with fusions of the cDNAs to EGFP revealed intracellular sorting to the nucleus with accumulation in the nucleoli for hFPG1, while hFPG2 co-localised with the 30 kDa subunit of RPA. hFPG1 was purified and shown to act on DNA substrates containing 8-oxoguanine, 5-hydroxycytosine and abasic sites. Removal of 8-oxoguanine, but not cleavage at abasic sites, was opposite base-dependent, with 8-oxoG:C being the preferred substrate and negligible activity towards 8-oxoG:A. It thus appears that hFPG1 has properties similar to mammalian OGG1 in preventing mutations arising from misincorporation of A across 8-oxoG and could function as a back-up repair activity for OGG1 in ogg1(-/-) mice.     

MHC class II polymorphism is associated with a canine SLE-related disease complex

Nova Scotia duck tolling retrievers are predisposed to a SLE-related disease complex including immune-mediated rheumatic disease (IMRD) and steroid-responsive meningitis–arteritis (SRMA). IMRD involves symptoms that resemble those seen in systemic autoimmune rheumatic diseases, such as systemic lupus erythematosus, SLE, or SLE-related diseases, in humans. This disease complex involves persistent lameness, stiffness, mainly after resting, and palpable pain from several joints of extremities. The majority of affected dogs display antinuclear autoantibody (ANA)-reactivity. SRMA is manifested in young dogs with high fever and neck stiffness and can be treated with corticosteroids. We have investigated the possible role of MHC class II as a genetic risk factor in IMRD and SRMA etiology. We performed sequence-based typing of the DLA-DRB1, -DQA1, and -DQB1 class II loci in a total of 176 dogs including 51 IMRD (33 ANA-positive), 49 SRMA cases, and 78 healthy controls (two dogs were both IMRD- and SRMA-affected). Homozygosity for the risk haplotype DRB1*00601/DQA1*005011/DQB1*02001 increased the risk for IMRD (OR?=?4.9; ANA-positive IMRD: OR?=?7.2) compared with all other genotypes. There was a general heterozygote advantage, homozygotes had OR?=?4.4 (ANA-positive IMRD: OR?=?8.9) compared with all heterozygotes. The risk haplotype contains the five amino acid epitope RARAA, known as the shared epitope for rheumatoid arthritis. No association was observed for SRMA. We conclude that DLA class II is a highly significant genetic risk factor for ANA-positive IMRD. The results indicate narrow diversity of DLA II haplotypes and identify an IMRD-related risk haplotype, which becomes highly significant in homozygous dogs.     

Thrombin inhibitor reduces myocardial infarction in apoE-/- x LDLR-/- mice

We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE(-/-)) x LDL receptor-deficient (LDLR(-/-)) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE(-/-) x LDLR(-/-) mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 micromol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol x kg x (-1)min(-1)) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.     

Derivation of Human Induced Pluripotent Stem Cells from Patients with Maturity Onset Diabetes of the Young

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.     

Polyclonal hypergammaglobulinemia and autoantibody production induced by vaccination in farmed Atlantic salmon

The introduction of oil-adjuvanted vaccines in salmon aquaculture made large-scale production feasible by reducing the impact of infections. Vaccines given intraperitoneally (ip) contain oil adjuvant such as mineral oil. However, in rodents, a single ip injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome. We have recently reported that autoimmune disease in farmed salmon, characterized by production of various autoantibodies, immune complex glomerulonephritis, liver thrombosis, and spinal deformity, are previously unrecognized side effects of vaccination. In the present study, we examined whether vaccination-induced autoantibody production in farmed Atlantic salmon is a mere result of polyclonal B-cell activation. Sera were collected from 205 vaccinated and unvaccinated Atlantic salmon (experimental, 7 farms) and wild salmon. Total IgM levels and autoantibodies to salmon blood cell (SBC) extract in sera were measured by ELISA and the relationship between hypergammaglobulinemia and autoantibody production was analyzed. Comparison of endpoint titers vs levels/units using a single dilution of sera in detection of autoantibodies to SBC showed near perfect correlation, justifying the use of the latter for screening. Both total IgM and anti-SBC antibodies are increased in vaccinated salmon compared with unvaccinated controls, however, they do not always correlate well when compared between groups or between individuals, suggesting the involvement of antigen-specific mechanisms in the production of anti-SBC autoantibodies. The primary considerations of successful vaccine for aquaculture are cost-effectiveness and safety. Vaccination-induced autoimmunity in farmed Atlantic salmon may have consequences on future vaccine development and salmon farming strategy. Evaluation for polyclonal hypergamamglobulinemia and autoimmunity should be included as an important trait when vaccine efficacy and safety are evaluated in future.