A DIO2 missense mutation and its impact on fetal response to PRRSV infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection. Our objectives were to identify mutation(s) within the porcine DIO2 gene and to determine if they were associated with fetal outcomes after PRRSV-2 challenge. Sequencing of the DIO2, genotyping identified variants, and association of DIO2 genotypes with fetal phenotypes including DIO2 mRNA levels, viability, survival, viral loads, cortisol and thyroid hormone levels, and growth measurements were conducted. RESULTS: A missense variant (p.Asn91Ser) was identified in the parental populations from two independent PRRSV-2 challenge trials. This variant was further genotyped to determine association with fetal PRRS outcomes. DIO2 mRNA levels in fetal heart and kidney differed by the genotypes of Asn91Ser substitution with significantly greater DIO2 mRNA expression in heterozygotes compared with wild-type homozygotes (P < 0.001 for heart, P = 0.002 for kidney). While Asn91Ser did not significantly alter fetal viability and growth measurements, interaction effects of the variant with fetal sex or trial were identified for fetal viability or crown rump length, respectively. However, this mutation was not related to dysregulation of the hypothalamic-pituitary-adrenal and thyroid axis, indicated by no differences in circulating cortisol, T4, and T3 levels in fetuses of the opposing genotypes following PRRSV-2 infection. CONCLUSIONS: The present study suggests that a complex relationship among DIO2 genotype, DIO2 expression, fetal sex, and fetal viability may exist during the course of fetal PRRSV infection. Our study also proposes the increase in cortisol levels, indicative of fetal stress response, may lead to fetal complications, such as fetal compromise, fetal death, or premature farrowing, during PRRSV infection.

Late gestation fetal hypothyroidism alters cell cycle regulation across multiple organ systems.

BACKGROUND: Hypothyroidism is a common endocrine disruption observed in utero that adversely affects fetal growth and maturation leading to long-term impacts on health; however, the exact molecular mechanisms by which these deleterious effects occur are unknown. We hypothesize that fetal hypothyroidism during late gestation will disrupt cell cycle regulation in a tissue-specific manner. To evaluate this, eight pregnant gilts were dosed with either methimazole or an equivalent negative control during days 85-106 out of 114 days of gestation (n = 4/group). Following treatment, the gilts were humanely euthanized, and tissue samples of fetal heart, ileum, kidney, lung, liver, muscle, spleen, and thymus taken from two male and two female fetuses (n = 32) from each gilt. RESULTS: The relative expression of three cell cycle promoters (CDK1, CDK2, and CDK4), and one cell cycle inhibitor (CDKN1A) was compared in each tissue to determine the effect of hypothyroidism on the developing fetus. All of the eight tissues examined experienced at least one significant up- or downregulation in the expression of the aforementioned genes as a result of treatment with methimazole. Substantial changes were observed in the liver and muscle, with the latter experiencing significant downregulations of CDK1, CDK2, and CDK4 as a result of treatment. In addition, all tissues were examined for changes in protein content, which further elucidated the impact of hypothyroidism on the fetal liver by the observation of a marked increase in protein content in the methimazole-treated group. Finally, the heart and liver were histologically examined for evidence of cellular hyperplasia and hypertrophy by measuring average nuclei density and size in each tissue, with the results showing a significant decrease in average nuclei size in the liver of hypothyroid fetuses. CONCLUSIONS: Collectively, these findings indicate the occurrence of organ-specific disruptions in cell cycle progression as a result of in utero hypothyroidism, which may explain the long term and widespread effects of hypothyroidism on fetal development.

Impact of methimazole-induced hypothyroidism on postnatal swine.

Thyroid hormones regulate metabolic rate, nutrient utilization, growth, and development. Swine are susceptible to thyroid suppression in response to disease or environmental conditions, but the physiological impact of such disruption has not been established. The objective of this study was to evaluate the impact of hypothyroidism induced with the antithyroid medication methimazole (MMI). 10 mg/kg MMI significantly decreased circulating triiodothyronine (T3) for the duration of treatment but had only a transient effect on circulating thyroxine (T4). Thyroid tissue weight was significantly increased by more than 3.5-fold in response to MMI treatment. Histologically, the eosinophilic colloid was largely absent from the thyroid follicle which displayed a disorganized columnar epithelium consistent with goiter. MMI induced hypothyroidism has no effect on growth rate over 28 days. Hepatic expression of genes associated with thyroid metabolism (DIO1, DIO2, and DIO3), lipid utilization (CD36, FASN, and ACACA), apoptosis (TP53, PERP, SIVA1, and SFN) and proliferation (CDK1, CDK2, CDK4, and CDKN1A) were unaffected by treatment. Collectively these results demonstrate that MMI induces mild systemic hypothyroidism and pronounced goiter, indicating a strong homeostatic central regulation within the hypothalamic pituitary thyroid axis. This combined with limited peripheral effects, indicates resilience to hypothyroidism in modern swine.

PRRSV-2 viral load in critical non-lymphoid tissues is associated with late gestation fetal compromise.

The impact of late gestation PRRSV-2 infection is highly variable within a litter, with a subset of fetuses displaying varying degrees of compromise following infection while others remain viable despite significant systemic viral load. To understand the underlying cause of this variation, we examined the susceptibility, distribution and impact of viral infection within non-lymphoid tissues. Samples of brain, heart, kidney, liver, lung, and skeletal muscle were obtained from fetuses of pregnant gilts at gestation day 86, and the presence and distribution of CD163+ cells within each tissue evaluated via immunohistofluorescence. Equivalent samples were collected from phenotypic extremes representing resistant, resilient and susceptible fetuses at 21 days following infection of pregnant gilts with PRRSV-2 at day 86 of gestation. Viral load and its impact in each tissue was evaluated by a combination of qPCR, in vitro viral recovery, and local expression of IFNG and CD163. Resting populations of CD163+ cells were observed in all six non-lymphoid tissues from healthy day 86 fetuses, though the apparent density and the morphology of positive cells varied between tissue. Viral RNA was detected in all six tissues derived from fetuses previously classified as highly infected, and infectious viral particles successfully recovered. Significantly more viral RNA was detected in heart, brain, lung and skeletal muscle of susceptible fetuses, relative to their viable counterparts. Infection was associated with an increase in the expression of CD163 in brain, kidney and lung. In addition, the presence of virus in each tissue coincided with a significant upregulation in the expression of IFNG, but the scale of this response was not associated with fetal susceptibility. Thus, PRRSV-2 is widely distributed across these susceptible non-lymphoid fetal tissues, and fetal outcome is associated with local viral load in critical fetal organs.

18F-fluorocholine PET/MRI versus ultrasound and sestamibi for the localization of parathyroid adenomas.

PURPOSE: Accurate preoperative localization is imperative to facilitate a minimally invasive parathyroidectomy (MIP) in primary hyperparathyroidism (pHPT). This study aims to compare the diagnostic value of standard-of-care localization techniques (ultrasound [US] and 99mTechnetium (99mTc) -sestamibi scintigraphy) to [F-18]-fluorocholine positron emission tomography/magnetic resonance imaging (FCH-PET/MRI) to determine the additional clinical usefulness of PET/MRI in a Canadian cohort. METHODS: We conducted a prospective, appropriately powered, study to compare the diagnostic value of -FCH PET/MRI to that of the US and 99mTc-sestamibi scintigraphy for localization of parathyroid adenomas in a patient with pHPT. The primary outcome was the per-lesion sensitivity and positive predictive value (PPV) of FCH-PET/MRI, US, and 99mTc-sestamibi scintigraphy. Intraoperative surgeon localization, parathormone levels, and histopathological findings were used as reference standards. RESULTS: Forty-one patients underwent FCH-PET/MRI of which 36 patients had parathyroidectomy. In these 36 patients, 41 parathyroid lesions were histologically confirmed as adenomas or hyperplastic glands. Per-lesion sensitivity of FCH-PET/MRI was 82.9% and of US and 99mTc-sestamibi scintigraphy combined at 50.0%, respectively. The sensitivity of FCH-PET/MRI was superior to that of US and 99mTc-sestamibi scintigraphy (p = 0.002). In the 19 patients in whom both US and 99mTc-sestamibi scintigraphy were negative, PET/MRI correctly identified the parathyroid adenoma in 13 patients (68%). CONCLUSIONS: FCH-PET/MRI is a highly accurate imaging modality for localization of parathyroid adenomas in a tertiary center in North America. It is a superior functional imaging modality to 99mTc-sestamibi scintigraphy alone and more sensitive for localization of parathyroid lesions than US and 99mTc-sestamibi scintigraphy combined. This imaging modality could become the most valuable preoperative localization study given its superior performance in localizing parathyroid adenomas.

Back to the future: breast surgery with tumescent local anesthesia (TLA)?

PURPOSE: Breast surgery is usually performed under general anesthesia. Tumescent local anesthesia (TLA) offers the possibility to anesthetize large areas with highly diluted local anesthetic. METHODS: In this paper, the implementation, and experiences with TLA in the field of breast surgery are discussed. CONCLUSION: For carefully selected indications, breast surgery in TLA represents an alternative to ITN.

Compensatory mechanisms in response to induced hypothyroidism in the late gestation pig fetus†.

To understand the effect of fetal thyroid gland disruption on development in swine, we evaluated thyroid hormone levels, growth and developmental characteristics, and gene expression associated with thyroid hormone metabolism in late gestation fetuses exposed to methimazole (MMI). Pregnant gilts were given either oral MMI or equivalent sham from gestation day 85-106 (n = 4/group), followed by intensive phenotyping of all fetuses (n = 120). Samples of liver (LVR), kidney (KID), fetal placenta (PLC), and the corresponding maternal endometrium (END) were collected from a subset of fetuses (n = 32). Fetuses exposed to MMI in utero were confirmed hypothyroid, with a significant increase in thyroid gland size, goitrous thyroid histology, and dramatically suppressed thyroid hormone in serum. In dams, no differences in temporal measurements of average daily gain, thyroid hormone, or rectal temperatures relative to controls suggests that MMI had little effect on maternal physiology. However, fetuses from MMI-treated gilts exhibited significant increases in body mass, girth, and vital organ weights, but no differences in crown-rump length or bone measurements suggesting non-allometric growth. The PLC and END showed a compensatory decrease in expression of inactivating deiodinase (DIO3). Similar compensatory gene expression was observed in fetal KID and LVR with a downregulation of all deiodinases (DIO1, DIO2, DIO3). Minor alterations in the expression of thyroid hormone transporters (SLC16A2 and SLC16A10) were observed in PLC, KID, and LVR. Collectively, MMI crosses the PLC of the late gestation pig, resulting in congenital hypothyroidism, alterations in fetal growth, and compensatory responses within the maternal fetal interface.

Silicosis after short-term exposure.

BACKGROUND: Silicosis develops after inhalation of dust containing respirable crystalline silica (RCS) and is recognized as an occupational disease. Workers also develop accelerated and acute silicosis after shorter exposure to respirable silica dust at high concentrations. AIMS: The objective of this study is to investigate and identify the occupational groups at the highest risk of silicosis due to short-term RCS exposure. METHODS: All confirmed cases of silicosis reported to the Central Register of Occupational Diseases in Poland between 2000 and 2019 were included. Data analysis covered: gender, age at the time of occupational disease diagnosis, exposure duration to RCS and sector of the national economy. RESULTS: A total of 2066 confirmed cases of silicosis were analysed. Thirty-two cases occurred after RCS exposure shorter than 5 years. Median age was 50. Seventy-five per cent (n = 24) of these cases were diagnosed in industrial processing workers who were mainly employed in manufacturing of non-metallic mineral products (44%, n = 14) and metal production (19%, n = 6). 16% (n = 5) of cases were associated with employment in mining and quarrying, 6% (n = 2) in conservation of monuments and 3% (n = 1) in construction. CONCLUSIONS: The findings identify occupational groups at risk of silicosis due to short-term silica exposure. Medical professionals should be aware of early silicosis symptoms, and occupational health professionals and employers should improve protective and preventive measures in silica related industries.

Effects of porcine reproductive and respiratory syndrome virus (PRRSV) on thyroid hormone metabolism in the late gestation fetus.

Porcine reproductive and respiratory syndrome virus (PRRSV) in late gestation causes a profound suppression of circulating maternal and fetal thyroid hormone during a critical window of development. To understand this relationship, we evaluated thyroid hormone metabolism at the maternal-fetal interface and within fetal tissues, along with hormone metabolite levels in serum. Fetuses were classified using an established model based on viral load in serum and thymus, and preservation status, including uninfected (UNIF), high-viral load viable (HV-VIA), and high-viral load meconium-stained (HV-MEC), with additional controls from sham-inoculated gilts (CON). Expression of three iodothyronine deiodinases, five sulfotransferases, sulfatase, and two solute carriers known to transport thyroid hormone were evaluated in maternal endometrium and fetal placenta, liver, and kidney. Serum thyroxin (T4), reverse triiodothyronine (rT3), and diiodothyronine (T2) were evaluated via liquid chromatography tandem mass spectrometry. Significant changes in gene expression were observed in all four tissues, with the liver being the most severely impacted. We observed local and fetal specific regulation of maternal tissues through significant upregulation of DIO2 and DIO3 expression in the endometrium corresponding to infected but viable fetuses relative to uninfected and control fetuses. Expression levels of DIO2 and DIO3 were significantly higher in the resilient (HV-VIA) fetuses relative to the susceptible (HV-MEC) fetuses. A substantial decrease in serum T4 was confirmed, with no corresponding increase in rT3 or T2. Collectively, these results show that thyroid hormone metabolism is altered at the maternal-fetal interface and within the PRRSV infected fetus and is associated with fetal viability.

Detection of PRRSV-2 alone and co-localized with CD163 positive macrophages in porcine placental areolae.

The porcine epitheliochorial placenta creates a barrier for the transplacental transfer of some nutrients from the dam to the fetus, as well as feto-lethal viruses such as porcine reproductive and respiratory syndrome virus 2 (PRRSV-2). Areolae are specialized structures within the porcine placenta with a high absorptive and substance transport capacity that facilitate embryonic development. The overarching aim of this study was to characterize the localization of PRRSV-2 in and adjacent to areolae to provide insight into whether transplacental transmission might occur through placental areolae. Control (CON) plus three phenotypic fetal groups were selected based on levels of virus in fetal placenta, sera and thymus, to determine if fetal resilience was related to differences in PRRSV-2 localization, alone or co-localized with CD163+ macrophages. These fetal groups represented a range of susceptibility: uninfected (UNINF) being resistant, infected in placenta only (PLCO) being resilient, and high viral load viable (HVL-VIA) being most susceptible. Finally, potential factors related to PRRSV-2 localization, including the severity of inflammation in endometrium and placenta, and intrauterine growth restriction, known resilience factors, were assessed. Thirty-one pregnant gilts were inoculated with PRRSV-2 at gestation day 86 ± 0.4. Seven pregnant gilts were sham-inoculated. Gilts were euthanized at 12 days post-infection. Presence of PRRSV and CD163+ macrophages were determined using immunofluorescence in cryosections of maternal-fetal interface (MFI) with and without areolae. In the maternal, fetal and cavity of areolar region PRRSV particles were found both independently and co-localized with CD163+ macrophages. Similarly, individual, and co-localized particles were observed in the maternal and fetal sides of the MFI region of infected fetuses. Weak positive correlations were observed between the counts of CD163+ macrophages and some inflammation scores in endometrial and placental tissues, but no correlations with PRRSV-2 localization. There were no differences across the four fetal groups evaluated. These results suggest that transplacental transmission of PRRSV may occur through the areolae, either as non-cell associated or in association with infected CD163 macrophages.

Effect of porcine reproductive and respiratory syndrome virus 2 on angiogenesis and cell proliferation at the maternal-fetal interface.

Angiogenesis and cell proliferation in reproductive tissues are essential events for the maintenance of pregnancy, and alterations can lead to compromised fetal development and survival. Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) induces reproductive disease with negative financial and production impact on the swine industry. PRRSV-2 infection alters placental physiology through inflammatory and apoptotic pathways, yet fetal susceptibility varies. This study aimed to evaluate angiogenesis and cell proliferation in the porcine maternal-fetal interface (MFI) and determine if these physiological processes were altered by PRRSV-2 infection. Thirty-one pregnant gilts were inoculated with PRRSV-2 at gestation day 86 ± 0.4 (mean ± SD). Seven control gilts were sham-inoculated. All gilts were euthanized at 12 days postinoculation. Angiogenesis and cell proliferation were determined through the detection of vascular endothelial growth factor (VEGF) and Ki-67, respectively, using immunofluorescence of the MFI from 4 fetal resilience groups: uninfected (UNIF), high viral load-viable (HVL-VIA), and HVL-meconium-stained (MEC) from PRRSV-infected gilts, as well from sham-inoculated (CON) gilts. VEGF immunolabeling in the uterine submucosa was significantly lower in MEC compared with UNIF and HVL-VIA groups. Significantly greater Ki67 immunolabeling was detected in the trophoblasts of CON fetuses versus all other groups, and in uterine epithelium of CON and UNIF fetuses versus HVL-VIA and MEC. These results suggest that fetal resilience may be related to greater cell proliferation in uterine epithelium, and fetal compromise with reduced uterine submucosal angiogenesis, except fetuses with intrauterine growth restriction, in which inherently lower submucosal angiogenesis may be protective against PRRSV infection.

Multiple endocrine neoplasia 2: an overview.

This review article discusses the diagnosis and treatment of patients with multiple endocrine neoplasia type 2 (MEN2). The most common tumors associated with MEN2 are those of the parathyroid, thyroid, and adrenal glands. Additional manifestations include characteristic clinical phenotypes or features as described in the article. This review provides an overview of clinical manifestations, screening, diagnosis, treatment, and surveillance of patients with MEN2.

Effect of porcine reproductive and respiratory syndrome virus 2 on tight junction gene expression at the maternal-fetal interface.

Understanding why intrauterine growth restricted (IUGR) fetuses are more resilient to transplacental porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) infection compared to normal fetuses may lead to alternative approaches to control PRRS. Our objective was to compare gene expression of a subset of tight junction proteins in the endometrium (END) and placenta (PLC) of i) IUGR vs N-IUGR fetuses, and ii) across disease progression phenotypes following PRRSV-2 infection. In experiment 1, snap frozen END and PLC from fetuses of non-infected control dams (CTRL) and from high viral load viable (HVL-VIA) fetuses, with both groups further classified as either IUGR or non(N)-IUGR based on brain: liver weight ratio were strategically selected from a large challenge trial. In experiment 2, similar tissues were randomly selected from CTRL and from uninfected thymus (UNIF), (HVL-VIA) and HVL meconium-stained in the body (HVL-MEC-B) of PRRSV-infected dams. The expression of claudin (CLDN) 1, 3, 4, 5, 6, 7, 10, tight junction protein 1 (TJP1) and occludin (OCLN) genes were evaluated by PCR. There were no significant group differences between IUGR and N-IUGR groups, regardless of infection status, that explained the resilience of IUGR fetuses. Regarding disease progression, elevated CLDN3 was observed in END of UNIF, CLDN6 expression was lower in PLC when the fetus became infected (HVL-VIA), and CLDN10 elevated in PLC in fetuses showing evidence of compromise (HVL-MEC-B). Lastly, OCLN gene expression was higher in the END and PLC following maternal infection. In conclusion, differences in TJ integrity were mainly observed following PRRSV-2 infection with stepwise changes corresponding with disease progression.

Porcine reproductive and respiratory virus 2 infection of the fetus results in multi-organ cell cycle suppression.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection during late gestation negatively affects fetal development. The objective of this study was to identify the fetal organs most severely impacted following infection, and evaluate the relationship between this response and fetal phenotypes. RNA was extracted from fetal heart, liver, lung, thymus, kidney, spleen, and loin muscle, collected following late gestation viral challenge of pregnant gilts. Initially, gene expression for three cell cycle promoters (CDK1, CDK2, CDK4) and one inhibitor (CDKN1A) were evaluated in biologically extreme phenotypic subsets including gestational age-matched controls (CON), uninfected (UNIF), high-viral load viable (HV-VIA), and high-viral load meconium-stained (HV-MEC) fetuses. There were no differences between CON and UNIF groups for any gene, indicating no impact of maternal infection alone. Relative to CON, high-viral load (HV-VIA, HV-MEC) fetuses showed significant downregulation of at least one CDK gene in all tissues except liver, while CDKN1A was upregulated in all tissues except muscle, with the heart and kidney most severely impacted. Subsequent evaluation of additional genes known to be upregulated following activation of P53 or TGFb/SMAD signaling cascades indicated neither pathway was responsible for the observed increase in CDKN1A. Finally, analysis of heart and kidney from a larger unselected population of infected fetuses from the same animal study showed that serum thyroxin and viral load were highly correlated with the expression of CDKN1A in both tissues. Collectively these results demonstrate the widespread suppression in cell division across all tissues in PRRSV infected fetuses and indicate a non-canonical regulatory mechanism.

Thyroid hormone suppression in feeder pigs following polymicrobial or porcine reproductive and respiratory syndrome virus-2 challenge.

Thyroid hormones are powerful regulators of growth, development, and basal metabolic rate and can be dysregulated under conditions of severe stress or illness. To understand the role of these hormones in porcine disease response, serum samples were obtained from three batches of nursery-aged pigs (n = 208) exposed to a natural polymicrobial disease challenge with an array of bacterial and viral pathogens. Levels of total thyroxin (T4) and triiodothyronine (T3) assessed in sera by radioimmunoassay, decreased significantly by 14 days post-exposure (DPE). Levels of T3 partially rebounded by 48 DPE, while T4 levels remain depressed. Post-exposure T3 and T4 levels were positively correlated with acute and long-term average daily gain (ADG). Cross-sectional sampling of animals maintained at the high health source farms, showed no equivalent change in either hormone when managed under standard industrial conditions. To further elucidate the effect of porcine reproductive and respiratory syndrome virus (PRRSV)-infection on thyroid hormone levels, archived sera over 42 days post inoculation (DPI) from nursery pigs (N = 190) challenged with one of two PRRSV2 strains by the PRRS Host Genetics Consortium were similarly assessed, with animals selected in a two-by-two design, to investigate biological extremes in ADG and viral load (VL). All animals showed a similar decrease in both thyroid hormones reaching a minimum at 7 DPI and returning to near pre-challenge levels by 42 DPI. Post-challenge T3 and T4 levels were significantly greater in high ADG groups, with no significant association with VL or strain. The results of this study demonstrate porcine susceptibility to thyroid disruption in response to disease challenge and demonstrate a relationship between this response and growth performance.

Fetal hypoxia and apoptosis following maternal porcine reproductive and respiratory syndrome virus (PRRSV) infection.

BACKGROUND: Mechanisms of fetal death following maternal PRRSV2 infection remain uncharacterized, although hypoxia from umbilical cord lesions and/or placental detachment due to apoptosis are hypothesized. We performed two experiments examining hypoxia and apoptosis in PRRSV-infected and non-infected, third-trimester fetuses to elucidate possible associations with fetal death. Fetuses were selected based on four phenotypic infection groups: fetuses from non-challenged control gilts (CTRL); low viral load fetuses (LVL; Exp 1) or uninfected fetuses (UNINF; Exp 2) from inoculated gilts; viable high viral load fetuses (HVL-VIA); and HVL meconium-stained fetuses (HVL-MEC). RESULTS: In experiment 1, paraffin embedded fetal tissues collected 21 days post maternal infection (DPI) were examined for DNA fragmentation associated with apoptosis. Positively stained foci were larger and more numerous (P < 0.05) in heart, liver, and thymus of HVL-VIA and HVL-MEC compared to CTRL and LVL fetuses. In experiment 2, group differences in gene expression within the hypoxia (HIF1a, IDO1, VEGFa, LDHA, NOS2, NOX1) and apoptosis (CASP3, CASP7, CASP8, CASP9, RIPK1, RIPK3) pathways were assessed by RT-qPCR in fetal tissues collected at 12 DPI. High viral load fetuses showed differential expression relative to the CTRL and UNINF (P < 0.05 for all). Brain tissue from HVL-VIA and HVL-MEC fetuses presented increased expression of CASP7, CASP8, RIPK3, HIF1a and IDO1. Fetal heart showed increased expression of CASP8, HIF1a, IDO and NOX1 and a decrease in NOS2 expression in infected groups. CASP7, CASP9, RIPK1 and RIPK3 were only increased in the heart of HVL-VIA while VEGFa was only increased for HVL-MEC fetuses. Thymus from HVL-MEC had decreased expression of CASP9 and there was increased IDO1 in all infected fetuses. CONCLUSIONS: There is strong evidence of apoptosis occurring in the heart, liver and thymus of highly viral load fetuses at 21 DPI. Furthermore, there was clear upregulation of apoptotic genes in the heart of high viral load infected fetuses and less prominent upregulation in the brain of PRRSV-infected fetuses, whereas thymus appears to be spared at 12 DPI. There was no strong evidence of hypoxia at 12 DPI in brain and thymus but some indication of hypoxia occurring in fetal heart.

In vivo synthesis of bacterial amyloid curli contributes to joint inflammation during S. Typhimurium infection.

Reactive arthritis, an autoimmune disorder, occurs following gastrointestinal infection with invasive enteric pathogens, such as Salmonella enterica. Curli, an extracellular, bacterial amyloid with cross beta-sheet structure can trigger inflammatory responses by stimulating pattern recognition receptors. Here we show that S. Typhimurium produces curli amyloids in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. Production of curli was associated with an increase in anti-dsDNA autoantibodies and joint inflammation in infected mice. The negative impacts on the host appeared to be dependent on invasive systemic exposure of curli to immune cells. We hypothesize that in vivo synthesis of curli contributes to known complications of enteric infections and suggest that cross-seeding interactions can occur between pathogen-produced amyloids and amyloidogenic proteins of the host.

Assessment of Immunological Response and Impacts on Fertility Following Intrauterine Vaccination Delivered to Swine in an Artificial Insemination Dose.

To protect the health of sows and gilts, significant investments are directed toward the development of vaccines against infectious agents that impact reproduction. We developed an intrauterine vaccine that can be delivered with semen during artificial insemination to induce mucosal immunity in the reproductive tract. An in vitro culture of uterine epithelial cells was used to select an adjuvant combination capable of recruiting antigen-presenting cells into the uterus. Adjuvant polyinosinic:polycytidylic acid (poly I:C), alone or in combination, induced expression of interferon gamma, tumor necrosis factor alpha, and select chemokines. A combination adjuvant consisting of poly I:C, host defense peptide and polyphosphazene (Triple Adjuvant; TriAdj), which previously was shown to induce robust mucosal and systemic humoral immunity when administered to the uterus in rabbits, was combined with boar semen to evaluate changes in localized gene expression and cellular recruitment, in vivo. Sows bred with semen plus TriAdj had decreased γδ T cells and monocytes in blood, however, no corresponding increase in the number of monocytes and macrophages was detected in the endometrium. Compared to sows bred with semen alone, sows bred with semen plus TriAdj showed increased CCL2 gene expression in the epithelial layer. These data suggest that the adjuvants may further augment a local immune response and, therefore, may be suitable for use in an intrauterine vaccine. When inactivated porcine parvovirus (PPV) formulated with the TriAdj was administered to the pig uterus during estrus along with semen, we observed induction of PPV antibodies in serum but only when the pigs were already primed with parenteral PPV vaccines. Recombinant protein vaccines and inactivated PPV vaccines administered to the pig uterus during breeding as a primary vaccine alone failed to induce significant humoral immunity. More trials need to be performed to clarify whether repeated intrauterine vaccination can trigger strong humoral immunity or whether the primary vaccine needs to be administered via a systemic route to promote a mucosal and systemic immune response.

Maternal and fetal thyroid dysfunction following porcine reproductive and respiratory syndrome virus2 infection.

To better understand the host response to porcine reproductive and respiratory virus-2 (PRRSV2) we evaluated circulating thyroid hormone and associated gene expression in a late gestation challenge model. Pregnant gilts were inoculated at gestation day 85 and fetal samples collected at either 12 or 21 days post-infection (dpi). A subset of fetuses was selected for analysis based on viability and viral load categorized as either uninfected-viable (UNIF), high viral load viable (HV-VIA) or high viral load meconium stained (HV-MEC) and were compared with gestational age matched controls (CON). In dams, circulating levels of total T3 and T4 decreased in the acute period following infection and rebounded by 21 dpi. A similar effect was observed in fetuses, but was largely restricted to HV-VIA and HV-MEC, with minimal decrease noted in UNIF relative to CON at 21 dpi. Gene expression in fetal heart at 12 dpi showed significant decompensatory transcription of thyroid hormone transporters (SLC16A2) and deiodinases (DIO2, DIO3), which was not observed in brain. Correspondingly, genes associated with cell cycle progression (CDK1,2,4) were downregulated in only the heart of highly infected fetuses, while expression of their inhibitor (CDKN1A) was upregulated in both tissues. Finally, expression of genes associated with cardiac stress including CAMKD and AGT were upregulated in the hearts of highly infected fetuses, and a shift in expression of MYH6 to MYH7 was observed in HV-MEC fetuses specifically. Collectively, the results suggest PRRSV2 infection causes a hypothyroid state that disproportionally impacts the fetal heart over the brain.