Sheri T. Dorsam

Genetic thrombophilia variants and risk for preeclampsia among American Indians.

Objective: To determine the prevalence of thrombophilic genetic variants in an American Indian population and determine if they are associated with preeclampsia. Methods: A total of 87 cases, 165 controls and an additional 75 population-based controls were genotyped for two thrombophilic polymorphisms. Results: The allelic prevalence of the factor V Leiden and 20210 G/A prothrombin variants in this population was 2.1% and 0.5% respectively. No statistically significant associations between these genetic variants and preeclampsia were found. Conclusion: The prevalence of thrombophilic variants is of possible public health significance for other morbidity; but perhaps not in relation to preeclampsia.

Identification of the Early VIP-Regulated Transcriptome and its Associated Interactome in Resting and Activated Murine CD4 T Cells

More than 40 years after the discovery of vasoactive intestinal peptide (VIP), its transcriptome in the immune system has still not been completely elucidated. In an attempt to understand the biological role of this neuropeptide in immunity, we chose CD4 T cells as a cellular system. Agilent Mouse Whole Genome microarrays were hybridized with fluorescently labeled total RNA isolated from resting CD4 T cells cultured +/-10(-7)M VIP for 5h or PMA/ionomycin activated CD4 T cells cultured +/-10(-7)M VIP for 5h. These VIP-regulated transcriptomes were analyzed by Significance Analysis of Microarrays (SAM) and Ingenuity Pathway Analysis (IPA) software to identify relevant signaling pathways modulated by VIP in the absence and presence of T cell activation. In resting CD4 T cells, VIP-modulated 368 genes, ranging from 3.49 to -4.78-fold. In the PMA/ionomycin activated CD4 T cells, 326 gene expression levels were changed by VIP, ranging from 2.94 to -1.66-fold. IPA analysis revealed that VIP exposure alters cellular function through EGFR signaling in resting CD4 T cells, and modulates immediate early genes, Fos and CREM/ICER, in activated CD4 T cells. These gene expression changes are suggested to explain at a molecular level how VIP can regulate T cell homing to the gut and induce regulatory T cell generation.

TCR signaling and environment affect vasoactive intestinal peptide receptor-1 (VPAC-1) expression in primary mouse CD4 T cells

Strict regulation of T cell function is imperative to control adaptive immunity, and dysregulation of T cell activation can contribute to infectious and autoimmune diseases. Vasoactive intestinal peptide receptor-1 (VPAC-1), an anti-inflammatory G-protein coupled receptor, has been reported to be downregulated during T cell activation. However, the regulatory mechanisms controlling the expression of VPAC-1 in T cells are not well understood. Therefore, mouse splenic CD4 T cells were treated in complete media+/-anti-CD3 for 24h, total RNA isolated and VPAC-1 levels measured by qPCR. Surprisingly, we discovered that T cells incubated in complete media steadily upregulated VPAC-1 mRNA levels over time (24h). Importantly, CD4 T cells isolated from blood also showed elevated VPAC-1 expression compared to splenic T cells. Collectively, these data support that the vascular environment positively influences VPAC-1 mRNA expression that is negatively regulated by TCR signaling. This research was supported by a national service award (1KO1 DK064828) to G.D., the Center for Protease Research (2P20RR015566), and INBRE (P20 RR016741).

Effect of melatonin or maternal nutrient restriction on vascularity and cell proliferation in the ovine placenta

Previously we reported increased umbilical artery blood flow in ewes supplemented with melatonin from mid- to late-pregnancy, while maternal nutrient restriction decreased uterine artery blood flow. To further unravel these responses, this study was designed to assess placental cell proliferation and vascularity following supplementation with melatonin or maternal nutrient restriction. For the first experiment, 31 primiparous ewes were supplemented with 5mg of melatonin per day (MEL) or no melatonin (CON) and allocated to receive 100% (adequate fed; ADQ) or 60% (restricted; RES) of their nutrient requirements from day 50 to 130 of gestation. To examine melatonin receptor dependent effects, a second experiment was designed utilizing 14 primiparous ewes infused with vehicle, melatonin, or luzindole (melatonin receptor 1 and 2 antagonist) from day 62 to 90 of gestation. For experiment 1, caruncle concentrations of RNA were increased in MEL-RES compared to CON-RES. Caruncle capillary area density and average capillary cross-sectional area were decreased in MEL-RES compared to CON-RES. Cotyledon vascularity was not different across dietary treatments. For experiment 2, placental cellular proliferation and vascularity were not affected by infusion treatment. In summary, melatonin interacted with nutrient restriction to alter caruncle vascularity and RNA concentrations during late pregnancy. Although melatonin receptor antagonism alters feto-placental blood flow, these receptor dependent responses were not observed in placental vascularity. Moreover, placental vascularity measures do not fully explain the alterations in uteroplacental blood flow.

Gene expression profiling and network analysis of peripheral blood monocytes in a chronic model of allergic asthma.

The Aspergillus fumigatus mouse model of asthma mimics the characteristics of human fungal asthma, including local and systemic inflammation. Monocyte/macrophage lineage cells direct innate immune responses and guide adaptive responses. To identify gene expression changes in peripheral blood monocytes in the context of fungal allergy, mice were exposed to systemic and intranasal inoculations of fungal antigen (sensitized), and naïve and sensitized animals were challenged intratracheally with live A. fumigatus conidia. Microarray analysis of blood monocytes from allergic versus non‐allergic mice showed ≥ twofold modulation of 45 genes. Ingenuity pathway analysis revealed a network of these genes involved in antigen presentation, inflammation, and immune cell trafficking. These data show that allergen sensitization and challenge affects gene expression in peripheral monocytes.

Effects of maternal nutrient restriction followed by realimentation during early and mid-gestation in beef cows. II. Placental development, umbilical blood flow, and uterine blood flow responses to diet alterations

The objectives were to examine the effects of maternal nutrient restriction followed by realimentation during early to mid-gestation on placental development and uterine and umbilical hemodynamics in the beef cow. On day 30 of pregnancy, multiparous, non-lactating beef cows (620.5 ± 11.3 kg) were assigned to 1 of 2 dietary treatments: control (C; 100% National Research Council [NRC] recommendations; n = 18) and restricted (R; 60% NRC; n = 30). On day 85, cows were slaughtered (C, n = 6; R, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12), or were realimented to control (RC; n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5), or were realimented to control (RRC, n = 6). On day 254, all remaining cows were slaughtered. Heart rate and umbilical and uterine hemodynamics [blood flow, resistance index (RI), and pulsatility index (PI)] were determined via Doppler ultrasonography. As expected umbilical blood flow increased and fetal heart rate decreased as gestation advanced. Umbilical PI in RRC cows was less (P = 0.01) compared to RCC and CCC. During late gestation, RCC cows had greater (P = 0.02) ipsilateral and total uterine blood flow vs. CCC and RRC. There was an increase in the number and weight of placentomes from R cows (P ≤ 0.02) compared to C cows (i.e. day 85). There were more placentomes (P = 0.03) in RR vs. CC and RC cows, but placentome weight was not affected (P = 0.18) by maternal dietary treatment at day 140. Maternal nutrient restriction during early to mid-gestation increased the weight (by day 85) and number (day 85 and 140) of placentomes, and did not reduce fetal weight compared to control cows. A longer realimentation period may enhance uterine blood flow and individual placentome size during later gestation, which may compensate for reduced nutrients experienced early in gestation.

Angiopoietin expression in ovine corpora lutea during the luteal phase: Effects of nutrition, arginine and follicle stimulating hormone

The aim of this study was to evaluate angiopoietin (ANGPT) 1 and 2, and tyrosine-protein kinase receptor 2 (TIE2) expression in the corpora lutea (CL) of FSH-treated, or non-treated sheep administered arginine (Arg) or vehicle (saline, Sal), and fed a control (C), excess (O) or restricted (U) diet. Ewes from each dietary group were treated with Arg or Sal (experiment 1), and with FSH (experiment 2). Luteal tissues were collected at the early-, mid- and/or late-luteal phases of the estrous cycle. Protein and mRNA expression was determined using immunohistochemistry followed by image analysis, and quantitative RT-PCR, respectively. The results demonstrated that ANGPT1 and TIE2 proteins were localized to luteal capillaries and endothelial cells of larger blood vessels, and ANGPT2 was localized to tunica media of larger blood vessels. TIE2 protein was also present in luteal cells. In experiment 1, ANGPT1 protein expression was greater in O than C during early- and mid-luteal phases, and was greatest during late-luteal phase, less at the mid- and least at the early-luteal phase; 2) TIE2 protein expression was greatest at the mid-, less at the early- and least at the late-luteal phase; 3) ANGPT1 and 2 mRNA expression was greater at the mid- and late- than the early-luteal phase, and TIE2 mRNA expression was greatest at the late-, less at the mid- and least at the early-luteal phase. The ANGPT1/2 ratio was less at the early- than mid- or late-luteal phases. In experiment 2, ANGPT1 protein expression was greater in O during the mid-luteal phase than in other groups, and was greater at the mid- than early-luteal phase. TIE2 protein expression was highest at the mid-, less at the early- and least during the late-luteal phase. ANGPT1 and 2, and TIE2 mRNA expression was higher at the mid- than the early-luteal phase. During mid-luteal phase, ANGPT1 mRNA expression was greater in C than O and U, ANGPT2 was greatest in C, less in O and least in U, and TIE2 mRNA expression was greater in C than O and U. The ANGPT1/2 ratio was higher in U than in any other group. Comparison of FSH vs. Sal treatment effects (experiment 2 vs. experiment 1) demonstrated that FSH affected ANGPT1 and/or -2, and TIE2 protein and mRNA expression depending on luteal phase and/or diet. Thus, expression of ANGPTs and TIE2 in the CL changes during the luteal lifespan, indicating their involvement in luteal vascular formation, stabilization and degradation. Moreover, this study has demonstrated that plane of nutrition and/or FSH treatment affect the ANGPT system, and may alter luteal vascularity and luteal function in sheep.

Follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy: An immunohistochemical study.

Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep.