Early and mid-gestation Zika virus (ZIKV) infection in the olive baboon (Papio anubis) leads to fetal CNS pathology by term gestation.

Zika virus (ZIKV) infection in pregnancy can produce catastrophic teratogenic damage to the developing fetus including microcephaly and congenital Zika syndrome (CZS). We previously described fetal CNS pathology occurring by three weeks post-ZIKV inoculation in Olive baboons at mid-gestation, including neuroinflammation, loss of radial glia (RG), RG fibers, neuroprogenitor cells (NPCs) resulting in disrupted NPC migration. In the present study, we explored fetal brain pathologies at term gestation resulting from ZIKV exposure during either first or second trimester in the Olive baboon. In all dams, vRNA in whole blood resolved after 7 days post inoculation (dpi). One first trimester infected dam aborted at 5 dpi. All dams developed IgM and IgG response to ZIKV with ZIKV IgG detected in fetal serum. Placental pathology and inflammation were observed including disruption of syncytiotrophoblast layers, delayed villous maturation, partially or fully thrombosed vessels, calcium mineralization and fibrin deposits. In the uterus, ZIKV was detected in ¾ first trimester but not in second trimester infected dams. While ZIKV was not detected in any fetal tissue at term, all fetuses exhibited varying degrees of neuropathology. Fetal brains from ZIKV inoculated dams exhibited a range of gross brain pathologies including irregularities of the major gyri and sulci of the cerebral cortex and cerebellar pathology. Frontal cortices of ZIKV fetuses showed a general disorganization of the six-layered cortex with degree of disorganization varying among the fetuses from the two groups. Frontal cortices from ZIKV inoculation in the first but not second trimester exhibited increased microglia, and in both trimester ZIKV inoculation, increased astrocyte numbers (white matter). In the cerebellum, increased microglia were observed in fetuses from both first and second trimester inoculation. In first trimester ZIKV inoculation, decreased oligodendrocyte precursor cell populations were observed in fetal cerebellar white matter. In general, our observations are in accordance with those described in human ZIKV infected fetuses.

Maternal Zika Virus (ZIKV) Infection following Vaginal Inoculation with ZIKV-Infected Semen in Timed-Pregnant Olive Baboons.

Zika virus (ZIKV) infection is now firmly linked to congenital Zika syndrome (CZS), including fetal microcephaly. While Aedes species of mosquito are the primary vector for ZIKV, sexual transmission of ZIKV is a significant route of infection. ZIKV has been documented in human, mouse, and nonhuman primate (NHP) semen. It is critical to establish NHP models of the vertical transfer of ZIKV that recapitulate human pathogenesis. We hypothesized that vaginal deposition of ZIKV-infected baboon semen would lead to maternal infection and vertical transfer in the olive baboon (Papio anubis). Epidemiological studies suggest an increased rate of CZS in the Americas compared to the original link to CZS in French Polynesia; therefore, we also compared the French Polynesian (FP) ZIKV isolate to the Puerto Rican (PR) isolate. Timed-pregnant baboons (n = 6) were inoculated via vaginal deposition of baboon semen containing 106 focus-forming units (FFU) of ZIKV (n = 3 for FP isolate H/PF/2013; n = 3 for PR isolate PRVABC59) at midgestation (86 to 95 days of gestation [dG]; term, 183 dG) on day 0 (all dams) and then at 7-day intervals through 3 weeks. Maternal blood, saliva, and cervicovaginal wash (CVW) samples were obtained. Animals were euthanized at 28 days (n = 5) or 39 days (n = 1) after the initial inoculation, and maternal/fetal tissues were collected. Viremia was achieved in 3/3 FP ZIKV-infected dams and 2/3 PR ZIKV-infected dams. ZIKV RNA was detected in CVW samples of 5/6 dams. ZIKV RNA was detected in lymph nodes but not the ovaries, uterus, cervix, or vagina in FP isolate-infected dams. ZIKV RNA was detected in lymph nodes (3/3), uterus (2/3), and vagina (2/3) in PR isolate-infected dams. Placenta, amniotic fluid, and fetal tissues were ZIKV RNA negative in the FP isolate-infected dams, whereas 2/3 PR isolate-infected dam placentas were ZIKV RNA positive. We conclude that ZIKV-infected semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate appeared more capable of widespread dissemination to tissues, including reproductive tissues and placenta, than the FP isolate.IMPORTANCE Zika virus remains a worldwide health threat, with outbreaks still occurring in the Americas. While mosquitos are the primary vector for the spread of the virus, sexual transmission of Zika virus is also a significant means of infection, especially in terms of passage from an infected to an uninfected partner. While sexual transmission has been documented in humans, and male-to-female transmission has been reported in mice, ours is the first study in nonhuman primates to demonstrate infection via vaginal deposition of Zika virus-infected semen. The latter is important since a recent publication indicated that human semen inhibited, in a laboratory setting, Zika virus infection of reproductive tissues. We also found that compared to the French Polynesian isolate, the Puerto Rican Zika virus isolate led to greater spread throughout the body, particularly in reproductive tissues. The American isolates of Zika virus appear to have acquired mutations that increase their efficacy.

Zika virus infection at mid-gestation results in fetal cerebral cortical injury and fetal death in the olive baboon.

Zika virus (ZIKV) infection during pregnancy in humans is associated with an increased incidence of congenital anomalies including microcephaly as well as fetal death and miscarriage and collectively has been referred to as Congenital Zika Syndrome (CZS). Animal models for ZIKV infection in pregnancy have been developed including mice and non-human primates (NHPs). In macaques, fetal CZS outcomes from maternal ZIKV infection range from none to significant. In the present study we develop the olive baboon (Papio anubis), as a model for vertical transfer of ZIKV during pregnancy. Four mid-gestation, timed-pregnant baboons were inoculated with the French Polynesian ZIKV isolate (104 ffu). This study specifically focused on the acute phase of vertical transfer. Dams were terminated at 7 days post infection (dpi; n = 1), 14 dpi (n = 2) and 21 dpi (n = 1). All dams exhibited mild to moderate rash and conjunctivitis. Viremia peaked at 5-7 dpi with only one of three dams remaining mildly viremic at 14 dpi. An anti-ZIKV IgM response was observed by 14 dpi in all three dams studied to this stage, and two dams developed a neutralizing IgG response by either 14 dpi or 21 dpi, the latter included transfer of the IgG to the fetus (cord blood). A systemic inflammatory response (increased IL2, IL6, IL7, IL15, IL16) was observed in three of four dams. Vertical transfer of ZIKV to the placenta was observed in three pregnancies (n = 2 at 14 dpi and n = 1 at 21 dpi) and ZIKV was detected in fetal tissues in two pregnancies: one associated with fetal death at ~14 dpi, and the other in a viable fetus at 21 dpi. ZIKV RNA was detected in the fetal cerebral cortex and other tissues of both of these fetuses. In the fetus studied at 21 dpi with vertical transfer of virus to the CNS, the frontal cerebral cortex exhibited notable defects in radial glia, radial glial fibers, disorganized migration of immature neurons to the cortical layers, and signs of pathology in immature oligodendrocytes. In addition, indices of pronounced neuroinflammation were observed including astrogliosis, increased microglia and IL6 expression. Of interest, in one fetus examined at 14 dpi without detection of ZIKV RNA in brain and other fetal tissues, increased neuroinflammation (IL6 and microglia) was observed in the cortex. Although the placenta of the 14 dpi dam with fetal death showed considerable pathology, only minor pathology was noted in the other three placentas. ZIKV was detected immunohistochemically in two placentas (14 dpi) and one placenta at 21 dpi but not at 7 dpi. This is the first study to examine the early events of vertical transfer of ZIKV in a NHP infected at mid-gestation. The baboon thus represents an additional NHP as a model for ZIKV induced brain pathologies to contrast and compare to humans as well as other NHPs.

Domain Analysis and Motif Matcher (DAMM): A Program to Predict Selectivity Determinants in Monosiga brevicollis PDZ Domains Using Human PDZ Data.

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 µM affinity, a value commonly considered the threshold for cellular PDZ-peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.

Zika Virus Infection, Reproductive Organ Targeting, and Semen Transmission in the Male Olive Baboon.

Zika virus (ZIKV) infection in pregnant women is a serious threat to the development and viability of the fetus. The primary mode of ZIKV transmission to humans is through mosquito bites, but sexual transmission has also been well documented in humans. However, little is known of the short- and long-term effects of ZIKV infection on the human male reproductive system. This study examines the effects of ZIKV infection on the male reproductive organs and semen and the immune response of the olive baboon (Papio anubis). Nine mature male baboons were infected with ZIKV (French Polynesian strain) subcutaneously. Six animals were euthanized at 41 days, while three animals were euthanized at 10 or 11 days postinfection (dpi). Viremia and clinical evidence of infection were present in all nine baboons. ZIKV RNA was present in the semen of five of nine baboons. ZIKV was present in the testes of two of three males euthanized at 10 or 11 dpi, but in none of six males at 41 dpi. Immunofluorescence of testes suggested the presence of ZIKV in sperm progenitor cells, macrophage penetration of seminiferous tubules, and increased tumor necrosis factor alpha (TNF-α), particularly in vascular walls. These data demonstrate that male olive baboons approximate the male human ZIKV response, including viremia, the adaptive immune response, and persistent ZIKV in semen. Although gross testicular pathology was not seen, the demonstrated breach of the testes-blood barrier and targeting of spermatogenic precursors suggest possible long-term implications in ZIKV-infected primates.IMPORTANCE Zika virus (ZIKV) is an emerging flavivirus spread through mosquitoes and sexual contact. ZIKV infection during pregnancy can lead to severe fetal outcomes, including miscarriage, fetal death, preterm birth, intrauterine growth restriction, and fetal microcephaly, collectively known as congenital Zika syndrome. Therefore, it is important to understand how this virus spreads, as well as the resulting pathogenesis in translational animal models that faithfully mimic ZIKV infection in humans. Such models will contribute to the future development of efficient therapeutics and prevention mechanisms. Through our previous work in olive baboons, we developed a nonhuman primate model that is permissive to ZIKV infection and transfers the virus vertically from mother to fetus, modeling human observations. The present study contributes to understanding of ZIKV infection in male baboon reproductive tissues and begins to elucidate how this may affect fertility, reproductive capacity, and sexual transmission of the virus.

PETRA: Drug Engineering via Rigidity Analysis.

Rational drug design aims to develop pharmaceutical agents that impart maximal therapeutic benefits via their interaction with their intended biological targets. In the past several decades, advances in computational tools that inform wet-lab techniques have aided the development of a wide variety of new medicines with high efficacies. Nonetheless, drug development remains a time and cost intensive process. In this work, we have developed a computational pipeline for assessing how individual atoms contribute to a ligand's effect on the structural stability of a biological target. Our approach takes as input a protein-ligand resolved PDB structure file and systematically generates all possible ligand variants. We assess how the atomic-level edits to the ligand alter the drug's effect via a graph theoretic rigidity analysis approach. We demonstrate, via four case studies of common drugs, the utility of our pipeline and corroborate our analyses with known biophysical properties of the medicines, as reported in the literature.

Translational Model of Zika Virus Disease in Baboons.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus with devastating outcomes seen recently in the Americas due to the association of maternal ZIKV infection with fetal microcephaly and other fetal malformations not previously associated with flavivirus infections. Here, we have developed the olive baboon (Papio anubis) as a nonhuman primate (NHP) translational model for the study of ZIKV pathogenesis and associated disease outcomes to contrast and compare with humans and other major NHPs, such as macaques. Following subcutaneous inoculation of adult male and nonpregnant female baboons, viremia was detected at 3 and 4 days postinfection (dpi) with the concordant presentation of a visible rash and conjunctivitis, similar to human ZIKV infection. Furthermore, virus was detected in the mucosa and cerebrospinal fluid. A robust ZIKV-specific IgM and IgG antibody response was also observed in all the animals. These data show striking similarity between humans and the olive baboon following infection with ZIKV, suggesting our model is a suitable translational NHP model to study ZIKV pathogenesis and potential therapeutics.IMPORTANCE ZIKV was first identified in 1947 in a sentinel rhesus monkey in Uganda and subsequently spread to Southeast Asia. Until 2007, only a small number of cases were reported, and ZIKV infection was relatively minor until the South Pacific and Brazilian outbreaks, where more severe outcomes were reported. Here, we present the baboon as a nonhuman primate model for contrast and comparison with other published animal models of ZIKV, such as the mouse and macaque species. Baboons breed year round and are not currently a primary nonhuman primate species used in biomedical research, making them more readily available for studies other than human immunodeficiency virus studies, which many macaque species are designated for. This, taken together with the similarities baboons have with humans, such as immunology, reproduction, genetics, and size, makes the baboon an attractive NHP model for ZIKV studies in comparison to other nonhuman primates.

The role of prostaglandins E1 and E2, dinoprostone, and misoprostol in cervical ripening and the induction of labor: a mechanistic approach.

PURPOSE: Prostaglandins play a critical role in cervical ripening by increasing inflammatory mediators in the cervix and inducing cervical remodeling. Prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) exert different effects on these processes and on myometrial contractility. These mechanistic differences may affect outcomes in women treated with dinoprostone, a formulation identical to endogenous PGE2, compared with misoprostol, a PGE1 analog. The objective of this review is to evaluate existing evidence regarding mechanistic differences between PGE1 and PGE2, and consider the clinical implications of these differences in patients requiring cervical ripening for labor induction. METHODS: We conducted a critical narrative review of peer-reviewed articles identified using PubMed and other online databases. RESULTS: While both dinoprostone and misoprostol are effective in cervical ripening and labor induction, they differ in their clinical and pharmacological profiles. PGE2 has been shown to stimulate interleukin-8, an inflammatory cytokine that promotes the influx of neutrophils and induces remodeling of the cervical extracellular matrix, and to induce functional progesterone withdrawal. Misoprostol has been shown to elicit a dose-dependent effect on myometrial contractility, which may affect rates of uterine tachysystole in clinical practice. CONCLUSIONS: Differences in the mechanism of action between misoprostol and PGE2 may contribute to their variable effects in the cervix and myometrium, and should be considered to optimize outcomes.

Fetal endocrine and metabolic adaptations to hypoxia: the role of the hypothalamic-pituitary-adrenal axis.

In utero, hypoxia is a significant yet common stress that perturbs homeostasis and can occur due to preeclampsia, preterm labor, maternal smoking, heart or lung disease, obesity, and high altitude. The fetus has the extraordinary capacity to respond to stress during development. This is mediated in part by the hypothalamic-pituitary-adrenal (HPA) axis and more recently explored changes in perirenal adipose tissue (PAT) in response to hypoxia. Obvious ethical considerations limit studies of the human fetus, and fetal studies in the rodent model are limited due to size considerations and major differences in developmental landmarks. The sheep is a common model that has been used extensively to study the effects of both acute and chronic hypoxia on fetal development. In response to high-altitude-induced, moderate long-term hypoxia (LTH), both the HPA axis and PAT adapt to preserve normal fetal growth and development while allowing for responses to acute stress. Although these adaptations appear beneficial during fetal development, they may become deleterious postnatally and into adulthood. The goal of this review is to examine the role of the HPA axis in the convergence of endocrine and metabolic adaptive responses to hypoxia in the fetus.

Altitude, attitude and adaptation.

The fetus has the extraordinary capacity to respond to stress during development, which, in a large part, is mediated by the hypothalamo- pituitary-adrenal (HPA) axis. Hypoxia represents a significant risk to fetal homeostasis and can occur in a wide range of settings including maternal smoking, preeclampsia, preterm labor and high altitude. To study fetal adaptation to chronic, gestational hypoxia, we developed a model of high-altitude, long-term hypoxia (LTH) in pregnant sheep. We discuss the role of LTH on the HPA axis and potential programming of adaptive responses. LTH causes significant activation of the hypothalamic paraventricular nucleus (PVN) and anterior pituitary. In marked contrast, there is an adaptive inhibition in the adrenal, thus balancing the potentially maladaptive centrally mediated responses to LTH. Additionally, we discuss effects of LTH on adipose tissue development. LTH enhances leptin production, which in turn has a regulatory role on the adrenal cortex. Importantly, LTH also has a significant impact on programming of adipose tissue function. Together, our studies show that LTH induces a number of adaptive responses in the ovine fetus. Although they may be beneficial during fetal life, these adaptations could prove to be deleterious in the postnatal period and adulthood.

Extracellular signal-regulated kinases (ERK1/2) signaling pathway plays a role in cortisol secretion in the long-term hypoxic ovine fetal adrenal near term.

This study assessed the role of the extracellular signal-regulated kinase (ERK) signaling pathway on the previously observed enhanced cortisol secretion in response to adrenocorticotropic hormone (ACTH) treatment in fetal adrenocortical cells (FACs) from long-term hypoxic (LTH) ovine fetuses. Ewes were maintained at high altitude (3,820 m) from ~40 to 138-141 days gestation when FACs were collected and challenged with either ACTH (10 nM) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP, 10 mM) in the presence or absence of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MEK)/ERK inhibitor UO126 (10 µM). FACs from age-matched normoxic fetuses served as controls. Media and FACs were collected at selected time intervals after ACTH or 8-bromo-cAMP stimulation for cortisol measurement and Western analysis of ERK1/2 and phospho-ERK1 and -2 (pERK1/2). After ACTH or 8-bromo-cAMP treatment, cortisol production was greater in the LTH group compared with control (P < 0.05). UO126 reduced ACTH and 8-bromo-cAMP-mediated cortisol output in both groups (P < 0.01 vs. ACTH or 8-bromo-cAMP alone). Under basal conditions, ERK1/2 and pERK1/2 were not different between LTH and normoxic fetuses. In response to ACTH or 8-bromo-cAMP treatment, ERK1/2 were not different between groups; however, pERK1/2 were elevated in the LTH FACs compared with normoxic control FACs. ERK1/2 phosphorylation declined following ACTH treatment in the control group, but UO126 had no effect on ERK1/2 compared with untreated levels. Both ACTH and 8-bromo-cAMP treatment resulted in a decline of protein levels. UO126 pretreatment virtually eliminated pERK1/2 expression. We conclude that basal ERK signaling in FACs is necessary for normal cortisol production and sustained pERK in LTH adrenals enhances cortisol production.

Leptin receptor antagonist treatment ameliorates the effects of long-term maternal hypoxia on adrenal expression of key steroidogenic genes in the ovine fetus.

We previously reported elevated adipose leptin expression, plasma leptin concentrations, and adrenocortical leptin receptor expression in the long-term hypoxic (LTH) ovine fetus. This study addressed whether leptin antagonist (LA) administration to LTH fetal sheep altered expression of key genes governing cortisol synthesis. Ewes were maintained at high altitude (3,820 meters) from 40 to 130 days gestation (dG), returned to Loma Linda University, and implanted with a maternal tracheal catheter. Reduced Po2 was maintained by nitrogen infusion. On 132 dG, LTH (n = 11) and age-matched, normoxic control (n = 11) fetuses underwent vascular catheter implantation. At 138 dG, fetuses were continuously infused with either saline or the LA (1.5 mg·kg(-1)·day(-1)) for 4 days and samples collected for blood gases, ACTH, and cortisol. Fetal adrenal cortex was collected for determination of steriodogenic acute regulatory protein (StAR), ACTH, and leptin receptor, cholesterol side-chain cleavage (CYP11A1), cytochrome P-450 11ß-hydroxylase (CYP11B1), 17α-hydroxylase (CYP17), 21-hydroxylase (CYP21), signal transducer and activator of transcription 3 (STAT3), pSTAT3, and 17ß-hydroxysteroid dehydrogenase (HSD3B) expression. In the saline-infused LTH fetuses, StAR, ACTH receptor, CYP11A1, and CYP17 expression was significantly lower compared with control (P < 0.05), whereas levels of CYP11B1, CYP21, and HSD3B mRNA were similar between groups. LA infusion restored expression of StAR, pSTAT3, CYP11A1, and CYP17, but not ACTH receptor, to normal ontogenic levels in the LTH group while having no effect on control fetuses. Neither fetal plasma ACTH nor cortisol concentrations were altered by LA infusion. We speculate that while leptin plays a role in governing expression of key enzymes and StAR in response to LTH, other factors play a role in modulating cortisol synthesis in these fetuses.

The Isolation and In Vitro Differentiation of Primary Fetal Baboon Tracheal Epithelial Cells for the Study of SARS-CoV-2 Host-Virus Interactions.

The mucociliary airway epithelium lines the human airways and is the primary site of host-environmental interactions in the lung. Following virus infection, airway epithelial cells initiate an innate immune response to suppress virus replication. Therefore, defining the virus-host interactions of the mucociliary airway epithelium is critical for understanding the mechanisms that regulate virus infection, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Non-human primates (NHP) are closely related to humans and provide a model to study human disease. However, ethical considerations and high costs can restrict the use of in vivo NHP models. Therefore, there is a need to develop in vitro NHP models of human respiratory virus infection that would allow for rapidly characterizing virus tropism and the suitability of specific NHP species to model human infection. Using the olive baboon (Papio anubis), we have developed methodologies for the isolation, in vitro expansion, cryopreservation, and mucociliary differentiation of primary fetal baboon tracheal epithelial cells (FBTECs). Furthermore, we demonstrate that in vitro differentiated FBTECs are permissive to SARS-CoV-2 infection and produce a potent host innate-immune response. In summary, we have developed an in vitro NHP model that provides a platform for the study of SARS-CoV-2 infection and other human respiratory viruses.

Western diet-induced shifts in the maternal microbiome are associated with altered microRNA expression in baboon placenta and fetal liver.

Maternal consumption of a high-fat, Western-style diet (WD) disrupts the maternal/infant microbiome and contributes to developmental programming of the immune system and nonalcoholic fatty liver disease (NAFLD) in the offspring. Epigenetic changes, including non-coding miRNAs in the fetus and/or placenta may also underlie this risk. We previously showed that obese nonhuman primates fed a WD during pregnancy results in the loss of beneficial maternal gut microbes and dysregulation of cellular metabolism and mitochondrial dysfunction in the fetal liver, leading to a perturbed postnatal immune response with accelerated NAFLD in juvenile offspring. Here, we investigated associations between WD-induced maternal metabolic and microbiome changes, in the absence of obesity, and miRNA and gene expression changes in the placenta and fetal liver. After ~8-11 months of WD feeding, dams were similar in body weight but exhibited mild, systemic inflammation (elevated CRP and neutrophil count) and dyslipidemia (increased triglycerides and cholesterol) compared with dams fed a control diet. The maternal gut microbiome was mainly comprised of Lactobacillales and Clostridiales, with significantly decreased alpha diversity (P = 0.0163) in WD-fed dams but no community-wide differences (P = 0.26). At 0.9 gestation, mRNA expression of IL6 and TNF in maternal WD (mWD) exposed placentas trended higher, while increased triglycerides, expression of pro-inflammatory CCR2, and histological evidence for fibrosis were found in mWD-exposed fetal livers. In the mWD-exposed fetus, hepatic expression levels of miR-204-5p and miR-145-3p were significantly downregulated, whereas in mWD-exposed placentas, miR-182-5p and miR-183-5p were significantly decreased. Notably, miR-1285-3p expression in the liver and miR-183-5p in the placenta were significantly associated with inflammation and lipid synthesis pathway genes, respectively. Blautia and Ruminococcus were significantly associated with miR-122-5p in liver, while Coriobacteriaceae and Prevotellaceae were strongly associated with miR-1285-3p in the placenta; both miRNAs are implicated in pathways mediating postnatal growth and obesity. Our findings demonstrate that mWD shifts the maternal microbiome, lipid metabolism, and inflammation prior to obesity and are associated with epigenetic changes in the placenta and fetal liver. These changes may underlie inflammation, oxidative stress, and fibrosis patterns that drive NAFLD and metabolic disease risk in the next generation.

Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air-liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.

Noninvasive tissue oxygen saturation measurements identify supply dependency.

BACKGROUND: Hemorrhagic shock can lead to multiple organ failure and death. We have previously shown that noninvasive measurement of tissue oxygen saturation (StO(2)) has predictive value for outcomes in patients suffering hemorrhagic shock. Our study objectives were twofold: (1) to compare invasive and noninvasive measurements of local and systemic tissue hemoglobin oxygenation and (2) to compare the effects of various physiologic conditions seen in patients in hemorrhagic shock on tissue hemoglobin oxygenation. MATERIALS AND METHODS: We studied pigs in controlled conditions mimicking shock induced by one of the following: hypothermia, isovolemic hemodilution, or manipulations of vascular tone. We obtained both invasive and noninvasive measurements in a hind limb of StO(2), tissue hemoglobin index, femoral artery and venous flows, blood pressures, temperature, pH, pO(2), pCO(2), oxygen saturation, lactate, hemoglobin, and base excess. In all cases, we measured baseline values in both experimental and control hind limbs. RESULTS: We found that tissue hemoglobin oxygenation did not vary significantly over relevant physiologic temperatures. Under all physiologic conditions tested, we found supply-dependent oxygen consumption at oxygen levels less than 7 mL O(2)/min/kg. Similarly, we found that local oxygen delivery in animals subjected to varying degrees of isovolemic hemodilution or altered vascular tone was correlated with supply-dependent oxygen consumption, as measured by local noninvasive StO(2). CONCLUSIONS: Noninvasive StO(2) measurements are valid and durable over a wide range of physiologic conditions and correlate with invasively-measured oxygen delivery.

Long term hypoxia during gestation alters perirenal adipose tissue gene expression in the lamb.

We previously reported that following long-term hypoxia (LTH), the ovine foetus exhibits enhanced expression of brown/beige adipose genes. This study was designed to determine if these changes are preserved after birth. Pregnant ewes were divided among three groups, 1) Control, sea level, 2) LTH, high altitude (3,820 m, LTH-HA) from ~ day 40 of gestation through ~14 days post-delivery and 3) LTH from ⁓ day 40 through day 137 of gestation then returned to the laboratory where atory reduced maternal PO2 was maintained by nitrogen infusion. Following delivery, lambs remained at sea level (LTH-SL). Perirenal adipose tissue was collected at ~day 14, and qRT-PCR was used to quantify mRNA. Uncoupling protein 1 (UCP-1), PPAR gamma coactivator 1 (PGC1α), and deiodinase-2 (DIO2) mRNA levels were significantly lower in both LTH groups while PR domain containing 16 (PRDM16) levels did not differ. Peroxisome proliferator-activated receptor (PPARγ) was maintained in the LTH-HA group and significantly increased in the LTH-SL group, compared to control. Unlike our previous LTH foetal studies, the brown/beige fat phenotype was rapidly lost by day 14 postpartum compared to control, while PPARγ was maintained. This loss of the brown fat phenotype may promote obesity due to decreased energy expenditure, favouring fat deposition.

Long-term hypoxia enhances ACTH response to arginine vasopressin but not corticotropin-releasing hormone in the near-term ovine fetus.

This study tested the hypothesis that long-term hypoxia (LTH) results in enhanced fetal corticotrope sensitivity to the ACTH secretagogues, corticotropin-releasing hormone (CRH), and AVP. Ewes were maintained at high altitude (3,820 m) from 40 to 130-131 days of gestation. Upon return to the laboratory, hypoxia was maintained by maternal nitrogen infusion. Vascular catheters were placed in both LTH (n = 4) and normoxic controls (n = 4). Each fetus received a 15-min infusion of either saline, 100 ng/kg of ovine CRH, or 20 ng/kg of AVP/min over 3 consecutive days in a randomized order. Fetal blood samples were collected at 0, 15, 30, 60, and 90 min after the start of infusion and analyzed for ACTH(1-39), ACTH precursors, and cortisol. Anterior pituitaries were collected from additional noninstrumented fetuses for analysis of vasopressin receptor 1b (V1b) mRNA and protein. Basal plasma concentrations of both ACTH(1-39) and ACTH precursors were higher in LTH fetuses and were not altered by saline infusion. In response to CRH, ACTH(1-39) increased in both groups and was higher in the LTH group compared with control (P < 0.05). When analyzed as sum of ACTH(1-39) released (Delta0-90 min) above basal, CRH released equal amounts of ACTH(1-39) in both groups. In LTH fetuses, AVP evoked a greater ACTH(1-39) release (P < 0.05) when analyzed as an increased sum of ACTH(1-39) (Delta0-90 min) above basal. Both CRH and AVP elicited a release of ACTH precursors with no differences observed between LTH and control. AVP and CRH elicited significant increases in cortisol, which were higher in response to AVP than CRH. V1b mRNA and protein were elevated in the anterior pituitary of LTH fetuses compared with control. LTH significantly increases pituitary sensitivity to AVP. This enhanced sensitivity may be a mechanism of our previously observed enhanced corticotrope function.

Tissue hemoglobin index: a non-invasive optical measure of total tissue hemoglobin.

INTRODUCTION: The tissue hemoglobin index (THI) is a hemoglobin signal strength metric provided on the InSpectra StO2 Tissue Oxygenation Monitor, Model 650. There is growing interest regarding the physiologic meaning of THI and whether a clinically useful correlation between THI and blood hemoglobin concentration exists. A series of in vitro and in vivo experiments was performed to evaluate whether THI has potential utility beyond its primary purpose of helping InSpectra device users optimally position a StO2 sensor over muscle tissue. METHODS: The THI and tissue hemoglobin oxygen saturation (StO2) were measured using the InSpectra StO2 Tissue Oxygenation Monitor, Model 650, with a 15 mm optical sensor. A THI normal reference range was established in the thenar eminence (hand) for 434 nonhospitalized human volunteers. In 30 subjects, the thenar THI was also evaluated during 5-minute arterial and venous blood flow occlusions, and with blood volume exsanguination in the hand induced with an Esmarch bandage. In addition, correlation of the THI to blood total hemoglobin concentration (Hbt) was studied in five pigs whose Hbt was isovolumetrically diluted from 13 to 4 g/dl systemically and 0.5 g/dl locally in the hind limb. The sensitivity and specificity of the THI to measure tissue hemoglobin concentration (THC) were characterized in vitro using isolated blood tissue phantoms. RESULTS: In human thenar tissue, the average THI was 14.1 +/- 1.6 (mean +/- standard deviation). The THI extrapolated to 100% blood volume exsanguination was 3.7 +/- 2.0 units presumably from myoglobin. On average, the THI increased 1.5 +/- 1.0 units with venous occlusion and decreased 4.0 +/- 2.0 units with arterial occlusion. In porcine hind limbs, the THI weakly correlated with Hbt (r2 = 0.26) while DeltaTHI during venous occlusion had a stronger correlation (r2 = 0.62). In vitro tests indicated that THI strongly correlated (r2 > 0.99) to phantom THC and was insensitive to StO2 changes. CONCLUSIONS: Steady-state THI values do not reliably indicate Hbt. The THI is a reproducible quantitative index for THC, and THI trends can discriminate between arterial or venous blood flow occlusions. The THI magnitude permits the estimation of myoglobin's contribution to StO2.

Assessment of tissue oxygen saturation during a vascular occlusion test using near-infrared spectroscopy: the role of probe spacing and measurement site studied in healthy volunteers.

INTRODUCTION: To assess potential metabolic and microcirculatory alterations in critically ill patients, near-infrared spectroscopy (NIRS) has been used, in combination with a vascular occlusion test (VOT), for the non-invasive measurement of tissue oxygen saturation (StO2), oxygen consumption, and microvascular reperfusion and reactivity. The methodologies for assessing StO2 during a VOT, however, are very inconsistent in the literature and, consequently, results vary from study to study, making data comparison difficult and potentially inadequate. Two major aspects concerning the inconsistent methodology are measurement site and probe spacing. To address these issues, we investigated the effects of probe spacing and measurement site using 15 mm and 25 mm probe spacings on the thenar and the forearm in healthy volunteers and quantified baseline, ischemic, reperfusion, and hyperemic VOT-derived StO2 variables. METHODS: StO2 was non-invasively measured in the forearm and thenar in eight healthy volunteers during 3-minute VOTs using two InSpectra tissue spectrometers equipped with a 15 mm probe or a 25 mm probe. VOT-derived StO2 traces were analyzed for base-line, ischemic, reperfusion, and hyperemic parameters. Data were categorized into four groups: 15 mm probe on the forearm (F15 mm), 25 mm probe on the forearm (F25 mm), 15 mm probe on the thenar (T15 mm), and 25 mm probe on the thenar (T25 mm). RESULTS: Although not apparent at baseline, probe spacing and measurement site significantly influenced VOT-derived StO2 variables. For F15 mm, F25 mm, T15 mm, and T25 mm, StO2 ownslope was -6.4 +/- 1.7%/minute, -10.0 +/- 3.2%/minute, -12.5 +/- 3.0%/minute, and -36.7 +/- 4.6%/minute, respectively. StO2 upslope was 105 +/- 34%/minute, 158 +/- 55%/minute, 226 +/- 41%/minute, and 713 +/- 101%/minute, and the area under the hyperemic curve was 7.4 +/- 3.8%.minute, 10.1 +/- 4.9%.minute, 12.6 +/- 4.4%.minute, and 21.2 +/- 2.7%.minute in these groups, respectively. Furthermore, the StO2 parameters of the hyperemic phase of the VOT, such as the area under the curve, significantly correlated to the minimum StO2 during ischemia. CONCLUSIONS: NIRS measurements in combination with a VOT are measurement site-dependent and probe-dependent. Whether this dependence is anatomy-, physiology-, or perhaps technology-related remains to be elucidated. Our study also indicated that reactive hyperemia depends on the extent of ischemic insult.