Galectins (Gals) are a family of animal lectins that bind β-galactosides through a carbohydrate recognition domain. Galectin-8 is a tandem-repeat galectin, secreted intracellularly and extracellularly. It is associated with neutrophil migration and has been studied as a possible therapeutic to combat inflammation. The objective of this study was to evaluate the translational and the transcriptional effects of recombinant Galectin-8 (rGal-8) on cow neutrophils. Blood was collected aseptically from Holstein-Friesian cows (n=10) from the North Carolina A&T State University Dairy Unit. Neutrophils isolated were treated with rGal-8 (2μg), or PBS (control) and were incubated at 37°C, 5% CO 2 for 1 hour. Supernatant from treated neutrophils was evaluated for total protein concentration, and galectin-8 secretion using bovine Galectin-8 Enzyme Linked-Immuno-Sorbent Assay (ELISA) kit. Total RNA was extracted, reverse transcribed, and RT-qPCR was performed using the RT² Profiler Cow Innate & Adaptive Immune Responses Array with 84 genes. The Livak method was used to calculate transcript abundance and fold change (FC>2 considered significant). Total protein concentration increased (P=0.0361) after rGal-8 treatment compared to the untreated control. Galectin-8 secretion was not significantly different in control compared to treated group (P=0.5819). Out of the 84 genes, 81 genes were differentially expressed in response to rGal-8; 14 up-regulated, 5 down-regulated, 61 genes remained unchanged. Treatment with rGal8 induced the expression of IRF7. The top five up-regulated genes include FAS, CD40, CD86, IFNGR1, STAT1; down-regulated genes were TLR9, CD14, CCR6, TICAM1, and TLR1. Selected genes were probed to validate fold change; the levels of gene expression were comparable to data from RT 2 array. Exposure of bovine neutrophils to rGal-8 modified expression of immune response genes. The functional significance of the change needs further studies.

Galectins (GAL) are β-galactoside binding proteins that can modulate both pregnancy and the response to pathogens. Increased susceptibility to infection by pathogens is linked to periparturient immune suppression and a periparturient rise (PPR) in parasite eggs on pasture. The possible role of GAL in periparturient immune relaxation and PRR needs definition.  The objective of this study was to evaluate galectin secretion and its relationship to measures of the immune relaxation and PRR in periparturient sheep. Samples were collected from pregnant St. Croix sheep (n=6) weekly at days -21 to + 21 relative to lambing. Fecal samples were collected and evaluated for strongyle and coccidia parasite eggs. The concentration of IgA and IgE coproantibodies, total microbial DNA, Bifidobacteria and Lactobacillus levels in fecal samples were used as indicators of gut health.  Blood samples were collected by jugular venipuncture and assessed for Packed Cell Volume (PCV), total and differential white blood cell counts. Total protein concentrations and protein profile were evaluated in serum. Secretion of GAL 1, 3, 9 and 14 were evaluated using ELISAs. Data were analyzed by one-way ANOVA and statistical significance was declared at P <0.05. Galectins tested were secreted in sheep blood. Differential modulation of GAL secretion and correlation with periparturient immune suppression and parasite infection was observed. Galectin secretion was modulated by the periparturient period, type and status of parasite infection. This first insight into a possible role of secreted galectins in periparturient immune relaxation and PRR improves the understanding of the immune response, informs development of management programs and therapeutics and presents Galectin profiles as biomarkers with diagnostic potential.

Galectins (GAL) are β-galactoside binding proteins. The objective of this study was to assess the effect of PMA on the regulation of galectin gene expression in bovine blood and neutrophils. Blood was collected from 3 clinically healthy Holstein cows. Neutrophils were isolated by differential centrifugation. Blood and neutrophils were treated with 10ng/ml of PMA or maintained in phosphate-buffered saline for 30 minutes at 370 C. Pooled total RNA were transcribed to cDNA for real-time PCR. Specific primers for bovine LGALS -1, -2, -3, -4, -7, -8, -9, -11, -12, and their binding proteins (bp) LGALS3bp, T cell immunoglobulin and mucin domain 3 (TIM-3) were used. Housekeeping genes RPLP0 and UCHL5 served as internal controls. Fold changes were calculated using the Livak method. Total protein concentration was assessed using the Bicinchoninic acid assay. Secretion of GAL -1, -2, -3, -4, -8, -9 was assessed using bovine GAL specific ELISA. All tested LGALS were detected. Activation with PMA differentially modulated expression and secretion of GAL in blood and neutrophils. Expression levels of LGALS -1 and LGALS -3 was not affected. Expression of LGALS -7, -8, -9, -11, -12, and TIM-3 was differentially modulated. The secretion of GAL -3 was significantly increased in response to PMA. The most pronounced effect of PMA was observed on LGALS-9, TIM-3 expression, and the secretion of GAL -3. Thus, GAL may serve as biomarkers of cell activation with functional consequences and warrant further study.

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