Browsing by Author "Kalbfleisch, Theodore S."

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    Transcriptional and Histochemical Signatures of Bone Marrow Mononuclear Cell-Mediated Resolution of Synovitis
    Menarim, Bruno C.; Ali, Hossam El-Sheikh; Loux, Shavahn C.; Scoggin, Kirsten E.; Kalbfleisch, Theodore S.; MacLeod, James N.; Dahlgren, Linda A. (Frontiers, 2021-12-08)
    Osteoarthritis (OA) may result from impaired ability of synovial macrophages to resolve joint inflammation. Increasing macrophage counts in inflamed joints through injection with bone marrow mononuclear cells (BMNC) induces lasting resolution of synovial inflammation. To uncover mechanisms by which BMNC may affect resolution, in this study, differential transcriptional signatures of BMNC in response to normal (SF) and inflamed synovial fluid (ISF) were analyzed. We demonstrate the temporal behavior of coexpressed gene networks associated with traits from related in vivo and in vitro studies. We also identified activated and inhibited signaling pathways and upstream regulators, further determining their protein expression in the synovium of inflamed joints treated with BMNC or DPBS controls. BMNC responded to ISF with an early pro-inflammatory response characterized by a short spike in the expression of a NF-ƙB- and mitogenrelated gene network. This response was associated with sustained increased expression of two gene networks comprising known drivers of resolution (IL-10, IGF-1, PPARG, isoprenoid biosynthesis). These networks were common to SF and ISF, but more highly expressed in ISF. Most highly activated pathways in ISF included the mevalonate pathway and PPAR-𝛄 signaling, with pro-resolving functional annotations that improve mitochondrial metabolism and deactivate NF-ƙB signaling. Lower expression of mevalonate kinase and phospho-PPAR-𝛄 in synovium from inflamed joints treated with BMNC, and equivalent IL-1β staining between BMNC- and DPBS-treated joints, associates with accomplished resolution in BMNC-treated joints and emphasize the intricate balance of pro- and anti-inflammatory mechanisms required for resolution. Combined, our data suggest that BMNC-mediated resolution is characterized by constitutively expressed homeostatic mechanisms, whose expression are enhanced following inflammatory stimulus. These mechanisms translate into macrophage proliferation optimizing their capacity to counteract inflammatory damage and improving their general and mitochondrial metabolism to endure oxidative stress while driving tissue repair. Such effect is largely achieved through the synthesis of several lipids that mediate recovery of homeostasis. Our study reveals candidate mechanisms by which BMNC provide lasting improvement in patients with OA and suggests further investigation on the effects of PPAR-𝛄 signaling enhancement for the treatment of arthritic conditions.
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    Using whole genome sequence to compare variant callers and breed differences of US sheep
    Stegemiller, Morgan R.; Redden, Reid R.; Notter, David R.; Taylor, Todd; Taylor, J. Bret; Cockett, Noelle E.; Heaton, Michael P.; Kalbfleisch, Theodore S.; Murdoch, Brenda M. (Frontiers, 2023-01-04)
    As whole genome sequence (WGS) data sets have become abundant and widely available, so has the need for variant detection and scoring. The aim of this study was to compare the accuracy of commonly used variant calling programs, Freebayes and GATK HaplotypeCaller (GATK-HC), and to use U.S. sheep WGS data sets to identify novel breed-associated SNPs. Sequence data from 145 sheep consisting of 14 U.S. breeds were filtered and biallelic single nucleotide polymorphisms (SNPs) were retained for genotyping analyses. Genotypes from both programs were compared to each other and to genotypes from bead arrays. The SNPs from WGS were compared to the bead array data with breed heterozygosity, principal component analysis and identifying breed associated SNPs to analyze genetic diversity. The average sequence read depth was 2.78 reads greater with 6.11% more SNPs being identified in Freebayes compared to GATK-HC. The genotype concordance of the variant callers to bead array data was 96.0% and 95.5% for Freebayes and GATK-HC, respectively. Genotyping with WGS identified 10.5 million SNPs from all 145 sheep. This resulted in an 8% increase in measured heterozygosity and greater breed separation in the principal component analysis compared to the bead array analysis. There were 1,849 SNPs identified in only the Romanov sheep where all 10 rams were homozygous for one allele and the remaining 135 sheep from 13 breeds were homozygous for the opposite allele. Both variant calling programs had greater than 95% concordance of SNPs with bead array data, and either was suitably accurate for ovine WGS data sets. The use of WGS SNPs improved the resolution of PCA analysis and was critical for identifying Romanov breed-associated SNPs. Subsets of such SNPs could be used to estimate germplasm composition in animals without pedigree information.