Pregnancy-induced differential expression of SARS-CoV-2 and influenza A viral entry factors in the lower respiratory tract

Despite differences in the clinical presentation of coronavirus disease-19 and pandemic influenza in pregnancy, fundamental mechanistic insights are currently lacking because of the difficulty in recruiting critically ill pregnant subjects for research studies. Therefore, to better understand host-pathogen interaction during pregnancy, we performed a series of foundational experiments in pregnant rats at term gestation to assess the expression of host entry factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) and genes associated with innate immune response in the lower respiratory tract. We report that pregnancy is characterized by a decrease in host factors mediating SARS-CoV-2 entry and an increase in host factors mediating IAV entry. Furthermore, using flow cytometric assessment of immune cell populations and immune provocation studies, we show an increased prevalence of plasmacytoid dendritic cells and a Type I interferon-biased environment in the lower respiratory tract of pregnancy, contrary to the expected immunological indolence. Our findings, therefore, suggest that the dissimilar clinical presentation of COVID-19 and pandemic influenza A in pregnancy could partly be due to differences in the extent of innate immune activation from altered viral tropism and indicate the need for comparative mechanistic investigations with live virus studies

Role of SIRT1 in isoflurane conditioning-induced neurovascular protection against delayed cerebral ischemia secondary to subarachnoid hemorrhage

We recently reported that isoflurane conditioning provided multifaceted protection against subarachnoid hemorrhage (SAH)-induced delayed cerebral ischemia (DCI), and this protection was through the upregulation of endothelial nitric oxide synthase (eNOS). SIRT1, an NAD-dependent deacetylase, was shown to be one of the critical regulators of eNOS. The aim of our current study is to examine the role of SIRT1 in isoflurane conditioning-induced neurovascular protection against SAH-induced DCI. Mice were divided into four groups: sham, SAH, or SAH with isoflurane conditioning (with and without EX-527). Experimental SAH via endovascular perforation was performed. Anesthetic conditioning was performed with isoflurane 2% for 1 h, 1 h after SAH. EX-527, a selective SIRT1 inhibitor, 10 mg/kg was injected intraperitoneally immediately after SAH in the EX-527 group. SIRT1 mRNA expression and activity levels were measured. Vasospasm, microvessel thrombosis, and neurological outcome were assessed. SIRT1 mRNA expression was downregulated, and no difference in SIRT1 activity was noted after isoflurane exposure. Isoflurane conditioning with and without EX-527 attenuated vasospasm, microvessel thrombosis and improved neurological outcomes. Our data validate our previous findings that isoflurane conditioning provides strong protection against both the macro and micro vascular deficits induced by SAH, but this protection is likely not mediated through the SIRT1 pathway

Upregulated influenza A viral entry factors and enhanced interferon-alpha response in the nasal epithelium of pregnant rats

Despite the increased severity of influenza A infection in pregnancy, knowledge about the expression of cell entry factors for influenza A virus (IAV) and the innate immune response in the nasal epithelium, the primary portal of viral entry, is limited. Here, we compared the expression of IAV cell entry factors and the status of the innate immune response in the nasal epithelium of pregnan

Role of endothelial nitric oxide synthase in isoflurane conditioning-induced neurovascular protection in subarachnoid hemorrhage

Background Delayed cerebral ischemia remains a common and profound risk factor for poor outcome after subarachnoid hemorrhage (SAH). The aim of our current study is to define the role of endothelial nitric oxide synthase (eNOS) in isoflurane conditioning-induced neurovascular protection after SAH. Methods and Results Ten- to 14-week-old male wild-type mice (C57BL/6) as controls and eNOS knockout male mice (strain # 002684) were obtained for the study. Animals underwent either sham surgery, SAH surgery, or SAH with isoflurane conditioning. Anesthetic post conditioning was performed with isoflurane 2% for 1 hour, 1 hour after SAH. Normothermia was maintained with the homeothermic blanket. In a separate cohort, nitric oxide synthase was inhibited by a pan nitric oxide synthase inhibitor, L-nitroarginine methyl ester. Vasospasm measurement was assessed 72 hours after SAH and neurological function was assessed daily. Isoflurane-induced changes in the eNOS protein expression were measured. eNOS protein expression was significantly increased by isoflurane conditioning in naïve mice as well as mice subjected to SAH. Vasospasm of the middle cerebral artery and neurological deficits were evident following SAH versus sham surgery, both in wild-type mice and eNOS knockout mice. Isoflurane conditioning attenuated vasospasm and neurological deficits in wild-type mice. This delayed cerebral ischemia protection was lost in L-nitroarginine methyl ester -administered mice and eNOS knockout mice. Conclusions Our data indicate isoflurane conditioning provides robust protection against SAH-induced vasospasm and neurological deficits, and that this delayed cerebral ischemia protection is critically mediated via isoflurane-induced augmentation of eNOS

Labor induction with oxytocin in pregnant rats is not associated with oxidative stress in the fetal brain

Despite the widespread use of oxytocin for induction of labor, mechanistic insights into fetal/neonatal wellbeing are lacking because of the absence of an animal model that recapitulates modern obstetric practice. Here, we create and validate a hi-fidelity pregnant rat model that mirrors labor induction with oxytocin in laboring women. The model consists of an implantable preprogrammed microprocessor-controlled infusion pump that delivers a gradually escalating dose of intravenous oxytocin to induce birth at term gestation. We validated the model with molecular biological experiments on the uterine myometrium and telemetry-supported assessment of changes in intrauterine pressure. Finally, we applied this model to test the hypothesis that labor induction with oxytocin would be associated with oxidative stress in the newborn brain. Analysis of biomarkers of oxidative stress and changes in the expression of associated genes were no different between oxytocin-exposed and saline-treated pups, suggesting that oxytocin-induced labor was not associated with oxidative stress in the developing brain. Collectively, we provide a viable and realistic animal model for labor induction and augmentation with oxytocin that would enable new lines of investigation related to the impact of perinatal oxytocin exposure on the mother-infant dyad

In utero exposure to transient ischemia-hypoxemia promotes long-term neurodevelopmental abnormalities in male rat offspring

The impact of transient ischemic-hypoxemic insults on the developing fetal brain is poorly understood despite evidence suggesting an association with neurodevelopmental disorders such as schizophrenia and autism. To address this, we designed an aberrant uterine hypercontractility paradigm with oxytocin to better assess the consequences of acute, but transient, placental ischemia-hypoxemia in term pregnant rats. Using MRI, we confirmed that oxytocin-induced aberrant uterine hypercontractility substantially compromised uteroplacental perfusion. This was supported by the observation of oxidative stress and increased lactate concentration in the fetal brain. Genes related to oxidative stress pathways were significantly upregulated in male, but not female, offspring 1 hour after oxytocin-induced placental ischemia-hypoxemia. Persistent upregulation of select mitochondrial electron transport chain complex proteins in the anterior cingulate cortex of adolescent male offspring suggested that this sex-specific effect was enduring. Functionally, offspring exposed to oxytocin-induced uterine hypercontractility showed male-specific abnormalities in social behavior with associated region-specific changes in gene expression and functional cortical connectivity. Our findings, therefore, indicate that even transient but severe placental ischemia-hypoxemia could be detrimental to the developing brain and point to a possible mitochondrial link between intrauterine asphyxia and neurodevelopmental disorders

Structural pathology and functional analysis of vitamin K-dependent protein S

Protein S deficiency is an autosomal dominant trait affecting around 10% of families thrombophilic families. The high affinity interaction of approximately 60% of protein S with C4b -binding protein (C4BP) has raised a complicated situation for the diagnosis of deficiency states. Because only free protein S functions as cofactor in the protein C anticoagulant, it is important to measure free protein S level precisely. In this study a free protein S assay was developed using its natural ligand C4BP to capture free protein S from plasma for routine clinical purpose. Although interference by C4BP is so far considered as the major hindrance for developing ELISA, the new approach has utilized this phenomenon of high affinity interaction between protein S and C4BP, thereby reversing the adverse effect of C4BP to one of good use. (1) Mutations in a variety of human genes are now known to predispose to venous thrombosis. Characterization of the wide spectrum of gene mutations causing thrombosis may allow us to relate specific gene lesions to the probability of thromboembolism as well as to the severity of thrombotic episodes, beside providing the molecular mechanism of deficiency states. To establish the relationship between genetic abnormalities of protein S and their phenotypic expressions, four naturally occurring missense mutations were chosen to analyze thier in vitro secretion profiles and functional characteristics, which illustrated the importance of in vitro experimental characterization in each and individual cases of naturally occurring missense mutations before marking them as the underlying genetic defect. (2) Since early nineties, the suggestion about the synergistic effect between two thrombotic risk factors when associated in one patient has been lacking a biochemical basis. The now observed deficient APC-cofactor activity of protein S Heerlen in the degradation of FVa Leiden suggests a possible synergistic pathogenic mechanism between these two genetic traits resulting in increased risk of thrombosis. (3) In addition, using two monoclonal antibodies as probes the structure-function relationship studies on protein S and APC interaction were carried out. R49, Q52 in TSR was found to be part of the epitope of monoclonal HPS 67 and K97, T103 in EGF1 for monoclonal HPS 54. These data implicated indirectly as the key amino acids for APC interaction, based on the fact that these two antibodies could completely block the APC-cofactor activity of protein S. The observation that HPS 67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface, suggesting that TSR is free to interact with membrane-bound APC. (4) Also, evaluation of the importance of amino acid residues 447-460 in protein S for binding to C4BP was performed. One amino acid Y456 was found to be important residue for C4BP interaction in this region. However, blocking this region by monoclonal antibody HPS 34 was not sufficient enough to inhibit protein S-C4BP interaction completely, suggesting that the interaction site constitute a fairly large binding surface

Placental dermatan sulfate: isolation, anticoagulant activity, and association with heparin cofactor II

Pregnancy is associated with hemostatic challenges that may lead to thrombosis. Heparin cofactor II (HCII) is a glycosaminoglycan-dependent thrombin inhibitor present in both maternal and fetal plasma. HCII activity increases during pregnancy, and HCII levels are significantly decreased in women with severe pre-eclampsia. Dermatan sulfate (DS) specifically activates HCII and is abundant in the placenta, but the locations of DS and HCII in the placenta have not been determined. We present evidence that DS is the major anticoagulant glycosaminoglycan in the human placenta at term. DS isolated from human placenta contains disaccharides implicated in activation of HCII and has anticoagulant activity similar to that of mucosal DS. Immunohistochemical studies revealed that DS is associated with fetal blood vessels and stromal regions of placental villi but is notably absent from the syncytiotrophoblast cells in contact with the maternal circulation. HCII colocalizes with DS in the walls of fetal blood vessels and is also present in syncytiotrophoblast cells. Our data suggest that DS is in a position to activate HCII in the fetal blood vessels or in the stroma of placental villi after injury to the syncytiotrophoblast layer and thereby inhibit fibrin generation in the placenta

Role of SIRT1 in Isoflurane Conditioning-Induced Neurovascular Protection against Delayed Cerebral Ischemia Secondary to Subarachnoid Hemorrhage

We recently reported that isoflurane conditioning provided multifaceted protection against subarachnoid hemorrhage (SAH)-induced delayed cerebral ischemia (DCI), and this protection was through the upregulation of endothelial nitric oxide synthase (eNOS). SIRT1, an NAD-dependent deacetylase, was shown to be one of the critical regulators of eNOS. The aim of our current study is to examine the role of SIRT1 in isoflurane conditioning-induced neurovascular protection against SAH-induced DCI. Mice were divided into four groups: sham, SAH, or SAH with isoflurane conditioning (with and without EX-527). Experimental SAH via endovascular perforation was performed. Anesthetic conditioning was performed with isoflurane 2% for 1 h, 1 h after SAH. EX-527, a selective SIRT1 inhibitor, 10 mg/kg was injected intraperitoneally immediately after SAH in the EX-527 group. SIRT1 mRNA expression and activity levels were measured. Vasospasm, microvessel thrombosis, and neurological outcome were assessed. SIRT1 mRNA expression was downregulated, and no difference in SIRT1 activity was noted after isoflurane exposure. Isoflurane conditioning with and without EX-527 attenuated vasospasm, microvessel thrombosis and improved neurological outcomes. Our data validate our previous findings that isoflurane conditioning provides strong protection against both the macro and micro vascular deficits induced by SAH, but this protection is likely not mediated through the SIRT1 pathway