Early molecular and developmental events impacting many incurable mitochondrial disorders are not fully understood and require generation of relevant patient-and disease-specific stem cell models. In this study, we focus on the ability of a nonviral and integration-free reprogramming method for deriving clinical-grade induced pluripotent stem cells (iPSCs) specific to Leigh's syndrome (LS), a fatal neurodegenerative mitochondrial disorder of infants. The cause of fatality could be due to the presence of high abundance of mutant mitochondrial DNA (mtDNA) or decline in respiration levels, thus affecting early molecular and developmental events in energy-intensive tissues. LS patient fibroblasts (designated LS1 in this study), carrying a high percentage of mutant T8993G mtDNA, were reprogrammed using a combined mRNA-miRNA nonviral approach to generate human iPSCs (hiPSCs). The LS1-hiPSCs were evaluated for their self-renewal, embryoid body (EB) formation, and differentiation potential, using immunocytochemistry and gene expression profiling methods. Sanger sequencing and next-generation sequencing approaches were used to detect the mutation and quantify the percentage of mutant mtDNA in the LS1-hiPSCs and differentiated derivatives. Reprogrammed LS-hiPSCs expressed pluripotent stem cell markers including transcription factors OCT4, NANOG, and SOX2 and cell surface markers SSEA4, TRA-1-60, and TRA-1-81 at the RNA and protein level. LS1-hiPSCs also demonstrated the capacity for self-renewal and multilineage differentiation into all three embryonic germ layers. EB analysis demonstrated impaired differentiation potential in cells carrying high percentage of mutant mtDNA. Nextgeneration sequencing analysis confirmed the presence of high abundance of T8993G mutant mtDNA in the patient fibroblasts and their reprogrammed and differentiated derivatives. These results represent for the first time the derivation and characterization of a stable nonviral hiPSC line reprogrammed from a LS patient fibroblast carrying a high abundance of mutant mtDNA. These outcomes are important steps toward understanding disease origins and developing personalized therapies for patients suffering from mitochondrial diseases.
BackgroundGroup A streptococcus (GAS) is a common cause of skin and soft-tissue infections (SSTIs). Current diagnostic techniques are culture-based and time intensive, requiring the prescription of empiric antibiotics before results are available. New detection tools are needed to hasten the diagnosis and appropriate treatment of SSTIs. The Cobas® Liat® System is a point of care (POC), real-time PCR system developed by Roche Molecular Diagnostics and is used in the United States and Europe to detect GAS from throat swabs within 15 minutes. We evaluated the feasibility and performance characteristics of POC for the detection of GAS in non-severe SSTIs.MethodsWound swabs collected from patients presenting to the Whiteriver Indian Health Service Hospital with non-severe SSTIs requiring only outpatient treatment were eligible for inclusion. Two swabs were collected: one swab was cultured on sheep’s blood agar, and the other swab was tested using POC. Compared with culture, we determined the sensitivity (SN), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) for POC to detect GAS in wound samples. We performed chart reviews 30-days from eligibility to assess the potential impact of POC systems on antibiotic use and healthcare utilization for SSTIs.ResultsTo date, we have tested 100 (25%) of our target 400 samples (enrollment will be complete in August 2019). Of the 100 samples, 50 (50%) tested positive for GAS by POC, all of which were culture positive for GAS, 49 tested negative by POC (2 after a first invalid result), all of which tested culture negative for GAS (table), and 1 had an invalid POC result even after repeat testing (culture positive for MRSA only) and was excluded from further analysis. Among samples with a valid POC result, POC SN was 100%, SP was 100%, PPV was 100%, and NPV was 100%. The most common mono-infections were MRSA (22%), GAS (18%), and CoNS (6%). Among GAS cases, MSSA (32%) and MRSA (18%) co-infection was common.ConclusionPOC PCR is highly sensitive and specific for the detection of GAS in non-severe SSTIs. To our knowledge, this is the first prospective study to use this technology for wound samples. POC PCR methods have the potential to accelerate identification of SSTI pathogens and improve antibiotic prescribing. Disclosures All authors: No reported disclosures.
Objectives Statin-associated autoimmune myopathy is a rare condition associated with the formation of autoantibodies to 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Underlying environmental and genetic risk factors remain poorly understood. American Indians have high rates of cardiovascular disease and associated co-morbidities that require lipid-lowering therapies. We observed this autoimmune myopathy in a series of American Indian statin users in rural Arizona. Methods We reviewed the charts of six American Indian patients with statin-associated autoimmune myopathy. We provide an illustrative case in addition to summaries of clinical presentations and treatment courses. Results This is the first report of statin-associated autoimmune myopathy in American Indians. These cases were all identified at the same geographically isolated hospital that exclusively serves an American Indian population with only 1800 statin users. There is relatively low migration. Each case was consistent with the previously described classical presentations for the disease. All six of our cases had diabetes and developed myopathy on high-dose atorvastatin, often with a recent change in statin type or dose. Conclusion Providers serving American Indians need to be aware of the possibility of statin-associated autoimmune myopathy and familiar with its presentation. Larger, inclusive, population-based investigations are needed to elucidate risk factors for this condition, in particular the potential interactions between predisposing HLA alleles, diabetes and specific statin exposures. This is necessary to identify effective and safe lipid-lowering medications.
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