Use of equine embryo -derived mesenchymal stromal cells and their extracellular vesicles as a treatment for persistent breeding-induced endometritis in susceptible mares.

Persistent breeding induced endometritis (PBIE) is a significant cause of infertility in mares. The development of a safe, universal, readily available therapeutic to manage PBIE and facilitate an optimal uterine environment for embryo development may improve pregnancy rates in susceptible mares. Mesenchymal stromal cells (MSCs) are being used increasingly as a therapeutic mediator for inflammatory conditions such as endometritis, and early gestational tissue provides a unique source of multipotent stem cells for creating MSCs. Extracellular vesicles (EVs) are mediators of cell communication produced by many different cell types. This study utilized embryo-derived mesenchymal stromal cells (EDMSCs) and their EVs as a potential therapeutic modality for PBIE in two groups: a) PBIE-susceptible mares challenged with pooled dead sperm (n=5); and b) client-owned mares diagnosed as susceptible to PBIE (n=37 mares and 40 estrous cycles). Mares pre-treated with intrauterine EDMSCs or their EVs resulted in a significant reduction in the accumulation of intrauterine fluid post-breeding. Nine of 19 (47 %) mares treated with EDMSCs prior to natural breeding and 13 of 20 (65 %) mares treated with EDMSC derived EVs were pregnant after the first cycle and 12 of 18 (67 %) mares treated with EDMSCs, and 15 of 19 (79 %) mares treated with EVs conceived by the end of the breeding season. These preliminary clinical studies are the first reports of the use of EDMSCs or their EVs as a potential intrauterine therapy for the management of PBIE susceptible mares.

Development of a novel five dye insertion/deletion (INDEL) panel for ancestry determination.

The use of genetic markers, specifically Short Tandem Repeats (STRs), has been a valuable tool for identifying persons of interest. However, the ability to analyze additional markers including Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletion (INDELs) polymorphisms allows laboratories to explore other investigative leads. INDELs were chosen in this study because large panels can be differentiated by size, allowing them to be genotyped by capillary electrophoresis. Moreover, these markers do not produce stutter and are smaller in size than STRs, facilitating the recovery of genetic information from degraded samples. The INDEL Ancestry Informative Markers (AIMs) in this study were selected from the 1000 Genomes Project based on a fixation index (FST) greater than 0.50, high allele frequency divergence, and genetic distance. A total of 25 INDEL-AIMs were optimized and validated according to SWGDAM guidelines in a five-dye multiplex. To validate the panel, genotyping was performed on 155 unrelated individuals from four ancestral groups (Caucasian, African, Hispanic, and East Asian). Bayesian clustering and principal component analysis (PCA) were performed revealing clear separation among three groups, with some observed overlap within the Hispanic group. Additionally, the PCA results were compared against a training set of 793 samples from the 1000 Genomes Project, demonstrating consistent results. Validation studies showed the assay to be reproducible, tolerant to common inhibitors, robust with challenging casework type samples, and sensitive down to 125 pg. In conclusion, our results demonstrated the robustness and effectiveness of a 25 loci INDEL system for ancestry inference of four ancestries commonly found in the United States.

Development of a novel five-dye panel for human identification insertion/deletion (INDEL) polymorphisms.

DNA analysis of forensic case samples relies on short tandem repeats (STRs), a key component of the combined DNA index system (CODIS) used to identify individuals. However, limitations arise when dealing with challenging samples, prompting the exploration of alternative markers such as single nucleotide polymorphisms (SNPs) and insertion/deletion (INDELs) polymorphisms. Unlike SNPs, INDELs can be differentiated easily by size, making them compatible with electrophoresis methods. It is possible to design small INDEL amplicons (<200 bp) to enhance recovery from degraded samples. To this end, a set of INDEL Human Identification Markers (HID) was curated from the 1000 Genomes Project, employing criteria including a fixation index (FST) ≤ 0.06, minor allele frequency (MAF) >0.2, and high allele frequency divergence. A panel of 33 INDEL-HIDs was optimized and validated following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, utilizing a five-dye multiplex electrophoresis system. A small sample set (n = 79 unrelated individuals) was genotyped to assess the assay's performance. The validation studies exhibited reproducibility, inhibition tolerance, ability to detect a two-person mixture from a 4:1 to 1:6 ratio, robustness with challenging samples, and sensitivity down to 125 pg of DNA. In summary, the 33-loci INDEL-HID panel exhibited robust recovery with low-template and degraded samples and proved effective for individualization within a small sample set.

Regulation of the heparan sulfate proteoglycan, perlecan, by injury and interleukin-1alpha.

Perlecan is a specific proteoglycan that binds to amyloid precursor protein and beta-amyloid peptide, is present within amyloid deposits, and has been implicated in plaque formation. Because plaque formation is associated with local inflammation, we hypothesized that the mechanisms involved in brain inflammatory responses could influence perlecan biosynthesis. To test this hypothesis, we first studied perlecan regulation in mice after inflammation induced by a brain stab wound. Perlecan mRNA and immunoreactivity were both increased 3 days after injury. Interleukin-1alpha (IL-1alpha) is a cytokine induced after injury and plays an important role in inflammation. As such, IL-1alpha may be one of the factors participating in regulating perlecan synthesis. We thus studied perlecan regulation by IL-1alpha, in vivo. Regulation of perlecan mRNA by this cytokine was area-specific, showing up-regulation in hippocampus, whereas in striatum, perlecan mRNA was unchanged. To support this differential regulation of perlecan mRNA by IL-1alpha, basic fibroblast growth factor (bFGF), a growth factor also present in plaques, was studied in parallel. bFGF mRNA did not show any regional difference, being up-regulated in both hippocampus and striatum in vivo. In vitro, both astrocyte and microglia were immunoreactive for perlecan. Moreover, perlecan mRNA was increased in hippocampal glial cultures after IL-1alpha but not in striatal glia. These results show an increase in perlecan biosynthesis after injury and suggest a specific regulation of perlecan mRNA by IL-1alpha, which depends on brain area. Such regulation may have important implications in the understanding of regional brain variations in amyloid plaque formation.