Analysis of canine myeloid-derived suppressor cells (MDSCs) utilizing fluorescence-activated cell sorting, RNA protection mediums to yield quality RNA for single-cell RNA sequencing.

Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for the isolation of specific cell populations that can then be further analyzed by single-cell RNA sequencing (scRNA-seq). When utilizing FACS for population isolation prior to sequencing, it is essential to consider the protection of RNA from RNase activity, environmental conditions, and the sorting efficiency to ensure optimum sample quality. This study aimed to optimize a previously published MDSC flow cytometry strategy to FACS sort canine Myeloid-Derived Suppressor Cells (MDSC) with various permutations of RNAlater ™ and RiboLock™ before and after FACS sorting. Concentrations of RNAlater™ greater than 2 % applied before flow analysis affected cell survival and fluorescence, whereas concentrations ≤ 2 % and time ≤ 4 h had little to no effect on cells. To shorten the procedural time and to enhance the sorting of rare populations, we used a primary PE-conjugated CD11b antibody and magnetic column. The combination of RiboLock™ pre- and post-sorting for FACS provided the best quality RNA as determined by the RNA integrity number (RIN ≥ 7) for scRNA-seq in a normal and dog and a dog with untreated oral melanoma dog. As proof of principle, we sequenced two samples, one from a normal dog another from a dog with untreated oral melanoma. Applying scRNA-Seq analysis using the 10X Genomic platform, we identified 6 clusters in the Seurat paired analysis of MDSC sorted samples. Two clusters, with the majority of the cells coming from the melanoma sample, had genes that were upregulated (> log2); these included MMP9, MMP1, HPGD, CPA3, and GATA3 and CYBB, CSTB, COX2, ATP6, and COX 17 for cluster 5 and 6 respectively. All genes have known associations with MDSCs. Further characterization using pathway analysis tools was not attempted due to the lower number of cells sequenced in the normal sample. The benefit deriving from the results of the study helped to gain data consistency when working with cells prone to RNase activity, and the scRNA-seq provided data showing transcriptional heterogeneity in MDSC populations and potentially identifying previously unreported or rare cell populations.

Enrichment of IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets is associated with reduction of NKp44+ ILC3s in the terminal ileum of Crohn's disease patients.

Crohn's disease (CD) is a chronic inflammatory condition of the human gastrointestinal tract whose aetiology remains largely unknown. Dysregulated adaptive immune responses and defective innate immunity both contribute to this process. In this study, we demonstrated that the interleukin (IL)-17A+ interferon (IFN)-γ+ and IL-22+ IFN-γ+ T cell subsets accumulated specifically in the inflamed terminal ileum of CD patients. These cells had higher expression of Ki-67 and were active cytokine producers. In addition, their proportions within both the IL-17A-producer and IL-22-producer populations were increased significantly. These data suggest that IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets might represent the pathogenic T helper type 17 (Th17) population in the context of intestinal inflammation for CD patients. In the innate immunity compartment we detected a dramatic alteration of both phenotype and function of the intestinal innate lymphoid cells (ILCs), that play an important role in the maintenance of mucosal homeostasis. In the inflamed gut the frequency of the NKp44- CD117- ILC1s subset was increased significantly, while the frequency of NKp44+ ILC3s was reduced. Furthermore, the frequency of human leucocyte antigen D-related (HLA-DR)-expressing-NKp44+ ILC3s was also reduced significantly. Interestingly, the decrease in the NKp44+ ILC3s population was associated with an increase of pathogenic IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets in the adaptive compartment. This might suggest a potential link between NKp44+ ILC3s and the IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets in the terminal ileum of CD patients.

Sustained reduction of intraocular pressure by supraciliary delivery of brimonidine-loaded poly(lactic acid) microspheres for the treatment of glaucoma.

Although effective drugs that lower intraocular pressure (IOP) in the management of glaucoma exist, their efficacy is limited by poor patient adherence to the prescribed eye drop regimen. To replace the need for eye drops, in this study we tested the hypothesis that IOP can be reduced for one month after a single targeted injection using a microneedle for administration of a glaucoma medication (i.e., brimonidine) formulated for sustained release in the supraciliary space of the eye adjacent to the drug's site of action at the ciliary body. To test this hypothesis, brimonidine-loaded microspheres were formulated using poly(lactic acid) (PLA) to release brimonidine at a constant rate for 35 days and microneedles were designed to penetrate through the sclera, without penetrating into the choroid/retina, in order to target injection into the supraciliary space. A single administration of these microspheres using a hollow microneedle was performed in the eye of New Zealand White rabbits and was found to reduce IOP initially by 6 mmHg and then by progressively smaller amounts for more than one month. All administrations were well tolerated without significant adverse events, although histological examination showed a foreign-body reaction to the microspheres. This study demonstrates, for the first time, that the highly-targeted delivery of brimonidine-loaded microspheres into the supraciliary space using a microneedle is able to reduce IOP for one month as an alternative to daily eye drops.

Equine CRISP3 modulates interaction between spermatozoa and polymorphonuclear neutrophils.

Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1-0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.

Effects of testing context on ball skill performance in 5-year-old children with and without developmental delay.

BACKGROUND AND PURPOSE: Physical therapists often test ball-playing skills of children with disabilities using standardized testing, which may not predict performance of ball skills in games with peers. This type of testing is used by physical therapists to determine whether children have delays in ball-handling skills. The purpose of this study was to compare ball skill performance of children with and without developmental delay in a one-to-one testing situation and in a structured game with peers. SUBJECTS: Five-year-old children with developmental delay (n=20) and 5-year-old children without disabilities (n=20) participated in the study. METHODS: We used the Peabody Developmental Motor Scales receipt and propulsion scale to test children one-to-one with a therapist and during a structured game with peers. RESULTS: Subjects without developmental delay performed better than subjects with developmental delay under both testing conditions. Children with developmental delay performed better in the one-to-one testing condition than in the game with peers. The performance of children without developmental delay did not differ under the 2 conditions. Boys performed better than girls. CONCLUSION AND DISCUSSION: Physical therapists should consider the potential effect of environment on the ball-handling skills of children with disabilities.

An economic consideration of same-gender marriage and fertility.

"This paper is an extension of Gary Becker's economic theory on families and marriage with particular attention to same-gender marriage and family formation. Summary discussion of several concepts central to the economics of the family as they relate to same-gender family formation are considered.... First, this article will present a general discussion of marriage markets and decisions and rationales for cohabiting or marrying. Second, the economic gains to marriage for both homosexual and heterosexual couples will be examined. Third, fertility alternatives and demand for children by same-gender couples will be considered. The article concludes with a discussion of future outcomes and policy implications relating to gay and lesbian marriage and fertility." The geographical focus is on the United States.