National population prevalence of antibodies to SARS-CoV-2 among pregnant women in Scotland during the second wave of the COVID-19 pandemic: a prospective national serosurvey.

OBJECTIVES: This study aimed to determine SARS-CoV-2 seroprevalence among pregnant women in the Scottish population during the second wave of the COVID-19 pandemic. STUDY DESIGN: Prospective national serosurvey. METHODS: We tested 13,428 residual samples retrieved from pregnant women participating in the first trimester combined ultrasound and biochemical screening for fetal trisomy across Scotland for SARS-CoV-2 antibodies over a 6-month period from November 2020 to April 2021. Seroprevalence estimates were adjusted for the sensitivity and specificity of the assays and weighted to reference populations. RESULTS: Seroprevalence rates in the antenatal samples significantly increased from 5.5% (95% confidence interval [CI] 4.7%-6.5%) in the 5-week period up to and including International Organization for Standardization (ISO) Week 51 (w/b Monday 14 December 2020) to 11.3% (95% CI 10.1%-12.6%) in the 5-week period up to and including ISO Week 14 (w/b Monday 5 April 2021). Increasing seroprevalence trends across the second wave were observed among all age groups. CONCLUSIONS: By the end of the second wave of the COVID-19 pandemic, approximately one in 10 women tested around the end of the first trimester of pregnancy had antibodies to SARS-CoV-2, suggesting that the vast majority were still susceptible to COVID-19 as they progressed to the later stages of pregnancy, when risks from infection are elevated for both mother and baby.

Primary sensory neurons regulate Toll-like receptor-4-dependent activity of glial cells in dorsal root ganglia.

Toll-like receptor-4 (TLR4) has been identified in primary sensory neurons, both in vivo and in vitro, but is reportedly absent from satellite glial cells (SGCs). Herein we reveal that, in rat dorsal root ganglia (DRG), SGCs do express TLR4 but this expression is inhibited by direct contact with neurons. Thus, TLR4 mRNA and protein is strongly up-regulated in isolated DRG glial cells in the absence of neurons. Lipopolysaccharide (LPS) increased cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNFα) mRNA expression with greater efficacy in DRG glial cell cultures than in mixed DRG cell cultures containing TLR4-positive neurons. Using an insert co-culture system, we have shown that neuronal inhibition of glial cell TLR4 is likely to be dependent on cell-cell contact rather than diffusible factors from neurons. LPS stimulated prostaglandin E2 (PGE2) production from DRG glial cells in a TLR4- and COX-2-dependent manner. In addition, exogenous PGE2 potentiated LPS-stimulated COX-2 mRNA while inhibiting TNFα mRNA expression by DRG cells, suggestive of a complex regulatory system to control inflammation within the DRG. In addition to LPS, conditioned medium from heat-shocked DRG neurons also increased COX-2 mRNA expression in DRG glial cells in a partially TLR4-dependent manner. We therefore hypothesize that neuronal suppression of glial TLR4 activity is a protective mechanism to prevent uncontrolled inflammation within the DRG. Under conditions where DRG neuronal viability is compromised, DRG glial cells become responsive to PAMPs (pathogen-associated molecular patterns) and DAMPs (danger-associated molecular patterns) and generate a range of classical inflammatory responses.

Lipopolysaccharide differentially modulates expression of cytokines and cyclooxygenases in dorsal root ganglion cells via Toll-like receptor-4 dependent pathways.

We have examined the functional expression of Toll-like receptor 4 (TLR4) in adult male rat dorsal root ganglion (DRG) cells in culture by studying changes in pro-inflammatory cytokines and cyclooxygenase (COX)-dependent prostanoid production. In the mixed population of DRG neurons and glial cells, only DRG neurons expressed cell surface TLR4 along with MD-2 and CD14. This classical TLR4 signaling complex on DRG neurons responded to lipopolysaccharide (LPS) with a TLR4-dependent and time-dependent increase in interleukin-1ß and tumor necrosis factor-α mRNA expression which was entirely dependent on NF-κB activity. In contrast, after 2-h incubation with DRG cells, LPS-stimulated COX-2 was regulated by both NF-κB and transactivation of epidermal growth factor receptor (EGFR) with potential downstream activation of ERK1/2 and p38 kinase. In contrast to this evidence for myeloid differentiation primary response gene-88 (MyD88)-dependent signaling, no evidence was obtained for TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent signaling from TLR4 in DRG neurons. LPS surprisingly produced a time-dependent decrease in COX-1 protein which likely facilitates the COX-2-dependent production of prostaglandin E2 and prostacyclin. Our study is the first to demonstrate the activation of TLR4-dependent production of prostaglandin E2 and prostacyclin in DRG cell cultures. Our findings support the concept that the activation of TLR4 on primary sensory neurons by endogenous ligands may underlie neuropathic and inflammatory pain states.

Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells.

BACKGROUND AND PURPOSE: Hyper-nociceptive PGE(2) EP(4) receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other G(s) -protein coupled hyper-nociceptive receptor systems: ß-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone-glial cell interactions in regulating adenylyl cyclase (AC) activity. EXPERIMENTAL APPROACH: Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. KEY RESULTS: Pharmacological analysis showed the presence of G(s) -coupled ß(2) -adrenoceptors and CGRP receptors, but not ß(1) -adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell-cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. CONCLUSIONS AND IMPLICATIONS: G(s) -coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP(4) and IP receptors, but not ß(2) -adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses.

Ribosomal frameshifting used in influenza A virus expression occurs within the sequence UCC_UUU_CGU and is in the +1 direction.

Programmed ribosomal frameshifting is used in the expression of many virus genes and some cellular genes. In eukaryotic systems, the most well-characterized mechanism involves -1 tandem tRNA slippage on an X_XXY_YYZ motif. By contrast, the mechanisms involved in programmed +1 (or -2) slippage are more varied and often poorly characterized. Recently, a novel gene, PA-X, was discovered in influenza A virus and found to be expressed via a shift to the +1 reading frame. Here, we identify, by mass spectrometric analysis, both the site (UCC_UUU_CGU) and direction (+1) of the frameshifting that is involved in PA-X expression. Related sites are identified in other virus genes that have previously been proposed to be expressed via +1 frameshifting. As these viruses infect insects (chronic bee paralysis virus), plants (fijiviruses and amalgamaviruses) and vertebrates (influenza A virus), such motifs may form a new class of +1 frameshift-inducing sequences that are active in diverse eukaryotes.

An overlapping protein-coding region in influenza A virus segment 3 modulates the host response.

Influenza A virus (IAV) infection leads to variable and imperfectly understood pathogenicity. We report that segment 3 of the virus contains a second open reading frame ("X-ORF"), accessed via ribosomal frameshifting. The frameshift product, termed PA-X, comprises the endonuclease domain of the viral PA protein with a C-terminal domain encoded by the X-ORF and functions to repress cellular gene expression. PA-X also modulates IAV virulence in a mouse infection model, acting to decrease pathogenicity. Loss of PA-X expression leads to changes in the kinetics of the global host response, which notably includes increases in inflammatory, apoptotic, and T lymphocyte-signaling pathways. Thus, we have identified a previously unknown IAV protein that modulates the host response to infection, a finding with important implications for understanding IAV pathogenesis.

Reciprocal modulation of anti-IgE induced histamine release from human mast cells by A1 and A(2B) adenosine receptors.

BACKGROUND AND PURPOSE: Adenosine is believed to participate in the pathological development of asthma through a mast cell-dependent mechanism. Our study aimed to pharmacologically characterize the functions of adenosine receptor (AR) subtypes (A1, A(2A) , A(2B) and A3) in primary human cultured mast cells (HCMC). EXPERIMENTAL APPROACH: HCMC were derived from progenitor stem cells in buffy coat and the effects of adenosine receptor ligands on basal and IgE-dependent histamine release were evaluated. KEY RESULTS: Adenosine and analogues alone did not induce HCMC degranulation. When HCMC were activated by anti-IgE after 10 min pre-incubation with adenosine, a biphasic effect on histamine release was observed with enhancement of HCMC activation at low concentrations of adenosine (10⁻9-10⁻7 mol·L⁻¹) and inhibition at higher concentrations (10⁻6-10⁻4 mol·L⁻¹). The potentiating action was mimicked by A1 AR agonists CCPA and 2'MeCCPA, and inhibited by the A1 AR antagonist PSB36. In contrast, the inhibitory action of adenosine was mimicked by the non-specific A2 AR agonist CV1808 and attenuated by A(2B) AR antagonists PSB1115 and MRS1760. The non-selective AR antagonist CGS15943 attenuated both the potentiating and inhibitory actions. CONCLUSIONS AND IMPLICATIONS: We have defined for the first time the contribution of A1 and A(2B) ARs, respectively, to the potentiating and inhibitory action of adenosine on human mast cell activation. With reference to the current trend of developing novel anti-asthmatic agents from AR ligands, our results suggest that inhibition of human mast cell activation would be a mechanism for A1 AR antagonists, but not A(2B) AR antagonists.

Nerve growth factor-induced differentiation of PC12 cells is accompanied by elevated adenylyl cyclase activity.

Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G(s)-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gα(s). These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation.

Gi-coupled prostanoid receptors are the likely targets for COX-1-generated prostanoids in rat pheochromocytoma (PC12) cells.

Cyclooxygenase-1 (COX-1) behaves as a delayed response gene in rat pheochromocytoma (PC12) cells exposed to nerve growth factor (NGF). To investigate the possible targets for COX-1 generated prostanoids in the early stages of neuronal differentiation, we have examined the expression of prostanoid receptors by PC12 cells using functional assays. Prostanoid receptor-specific agonists failed to activate adenylyl cyclase in undifferentiated and NGF-treated PC12 cells; neither did they stimulate phospholipase C activity. EP3 receptor agonists and PGF(2alpha) were the only active ligands, able to inhibit forskolin-stimulated adenylyl cyclase activity. PC12 cells expressed EP3 and FP receptor mRNA, but only the responses to EP3 receptor agonists were inhibited by the EP3 receptor antagonist ONO-AE3-240. The functional role of NGF-stimulated COX-1 remains to be determined since we found no strong evidence of a role for EP3 receptors in the morphological changes induced by NGF during the early stages of differentiation of PC12 cells.

Investigation of the prostacyclin (IP) receptor antagonist RO1138452 on isolated blood vessel and platelet preparations.

BACKGROUND AND PURPOSE: The current study examined the utility of the recently described prostacyclin (prostanoid IP) receptor antagonist RO1138452 (2-(4-(4-isopropoxybenzyl)-phenylamino) imidazoline) as a tool for classifying prostanoid receptors. EXPERIMENTAL APPROACH: pA(2) values were determined on isolated smooth muscle and platelet preparations. KEY RESULTS: RO1138452 antagonized relaxation of human pulmonary artery, guinea-pig aorta and rabbit mesenteric artery induced by the selective IP agonist cicaprost. Schild plots had slopes close to unity, generating pA(2) values of 8.20, 8.39 and 8.12 respectively. Non-surmountable antagonism was sometimes found with the higher concentrations of RO1138452, attributable to the EP(3) contractile action of cicaprost. RO1138452 did not block relaxation of guinea-pig trachea induced by the EP(2)-selective agonist butaprost. In contrast, there was a modest inhibition of butaprost-induced relaxation of human pulmonary artery by RO1138452, implying activation of both EP(2) and IP receptors by butaprost. RO1138452 did not affect relaxation induced by PGE(2) (EP(4) agonist) and substance P (NK(1)/endothelium-dependent agonist) in rabbit mesenteric artery. In human and rat platelet-rich plasmas, RO1138452 antagonized cicaprost-induced inhibition of platelet aggregation in a surmountable manner; pA(2) values may have been affected by binding of RO1138452 to plasma protein. RO1138452 did not affect the inhibitory actions of PGD(2) (DP(1) agonist) and NECA (adenosine A(2A) agonist) in human platelets. CONCLUSIONS AND IMPLICATIONS: The data indicate that RO1138452 is a potent and selective IP receptor antagonist. RO1138452 represents an important addition to our armoury of prostanoid receptor antagonists and a potential clinical agent in situations where prostacyclin has a pathophysiological function.

Conventional and toxicogenomic assessment of the acute pulmonary damage induced by the instillation of Cardiff PM10 into the rat lung.

There is strong epidemiological evidence of association between PM10 (particulate matter with an aerodynamic diameter less than or equal to 10 microm) and adverse health outcomes including death and increased hospital admissions for cardiopulmonary conditions. Ambient PM10 surrogates such as diesel exhaust particles (DEP), a common component of UK PM10 have been shown to induce lung inflammation in both humans and rodents. To date, few studies have reported on the toxicological response of UK PM10 in experimental animals. This study examines the pulmonary toxicological responses in male Sprague Dawley rats following the intratracheal instillation of Cardiff urban PM10. A mild but significant change in lung permeability was observed in the lung post-instillation of a high (10 mg) dose of the whole PM10 as adjudged by increases in lung to body weight ratio and total acellular lavage protein. Such effects were less marked following instillation of a water-soluble fraction (80% of the total mass) but histological examination showed that lung capillaries were swollen in size with this treatment. In conclusion, conventional toxicological, histological and toxicogenomic studies have indicated that Cardiff PM10 exhibits low bioreactivity in the form of mild permeability changes. Differential gene expression was observed when the lung was treated with whole PM10, containing durable particles, in comparison with the water-soluble fraction of PM10 that was devoid of particles. Such changes were linked to different histopathological events within the lung.

Agonists can discriminate between cloned human and mouse prostacyclin receptors.

The ability of prostacyclin analogues to stimulate adenylyl cyclase (AC) and phospholipase C (PLC) in Chinese hamster ovary (CHO) cells expressing cloned human (hIP) or cloned mouse (mIP) prostacyclin receptors has been compared. For hIP, the order of potency (pEC(50)) for stimulating AC and PLC pathways was similar: AFP-07 (9.3, 8.4)>cicaprost (8.3, 6.9), iloprost (7.9, 6.8)>taprostene (7.4, 6.8)>carbacyclin (6.9, 6.6), PGE(1) (6.6, 5.1). Although the standard IP agonists cicaprost and iloprost behaved similarly in both hIP and mIP receptor-expressing cells, carbacyclin and PGE(1) showed significantly higher potency at the mIP receptor, suggesting that the agonist recognition sites on hIP and mIP receptors are not identical. A further distinction between hIP and mIP receptors was found with taprostene, which had greater efficacy at hIP receptors (AC 94%, PLC 14%) than at mIP receptors (AC 77%, PLC 0%) (cicaprost=100% in each assay).

Prostanoid signal integration and cross talk.

The enzymatic machinery for the production of prostanoids and the receptors responsible for detecting their presence are widely distributed in the body. One pair of prostanoids, prostacyclin and thromboxane A(2), are particularly important in the control of haemodynamics and haemostasis. Prostacyclin achieves its antiplatelet effect by acting as a physiological antagonist, but displays some selectivity towards thromboxane A(2)-mediated platelet activation, possibly by virtue of the inability of thromboxane A(2) receptors to couple directly to G(i) proteins, and because platelet-derived endoperoxides can act as substrates for prostacyclin synthesis in endothelial cells. At low concentrations, prostaglandin E(2) can synergize with thromboxane A(2) by acting on the EP(3) subtype of prostaglandin E(2) receptor, resulting in opposition to the protective function of prostacyclin. In contrast, high concentrations of prostaglandin E(2) act on the prostacyclin receptor, and possibly the prostaglandin D(2) receptor, to turn off platelet activation. Integration of prostanoid signalling in the vascular system is similarly complex, and interpretation of data is further complicated by the regional distribution of prostanoid receptors in different vascular beds, and the poor selectivity of agonists and antagonists.

Prostacyclin receptor-independent inhibition of phospholipase C activity by non-prostanoid prostacyclin mimetics.

1. Chinese hamster ovary (CHO) cells were transiently transfected with the mouse prostacyclin (mIP) receptor to examine IP agonist-mediated stimulation of [(3)H]-cyclic AMP and [(3)H]-inositol phosphate production. 2. The prostacyclin analogues, cicaprost, iloprost, carbacyclin and prostaglandin E(1), stimulated adenylyl cyclase activity with EC(50) values of 5, 6, 25 and 95 nM, respectively. These IP agonists also stimulated the phospholipase C pathway with 10 - 40 fold lower potency than stimulation of adenylyl cyclase. 3. The non-prostanoid prostacyclin mimetics, octimibate, BMY 42393 and BMY 45778, also stimulated adenylyl cyclase activity, with EC(50) values of 219, 166 and 398 nM, respectively, but failed to stimulate [(3)H]-inositol phosphate production. 4. Octimibate, BMY 42393 and BMY 45778 inhibited iloprost-stimulated [(3)H]-inositol phosphate production in a non-competitive manner. 5. Activation of the endogenously-expressed P(2) purinergic receptor by ATP led to an increase in [(3)H]-inositol phosphate production which was inhibited by the non-prostanoid prostacyclin mimetics in non-transfected CHO cells. Prostacyclin analogues and other prostanoid receptor ligands failed to inhibit ATP-stimulated [(3)H]-inositol phosphate production. 6. A comparison between the IP receptor-specific non-prostanoid ONO-1310 and the structurally-related EP(3) receptor-specific agonist ONO-AP-324, indicated that the inhibitory effect of non-prostanoids was specific for those compounds known to activate IP receptors. 7. The non-prostanoid prostacyclin mimetics also inhibited phospholipase C activity when stimulated by constitutively-active mutant Galpha(q)RC, Galpha(14)RC and Galpha(16)QL transiently expressed in CHO cells. These drugs did not inhibit adenylyl cyclase activity when stimulated by the constitutively-active mutant Galpha(s)QL. 8. These results suggest that non-prostanoid prostacyclin mimetics can specifically inhibit [(3)H]-inositol phosphate production by targeting G(q/11) and/or phospholipase C in CHO cells, and that this effect is independent of IP receptors.

Factors affecting prostacyclin receptor agonist efficacy in different cell types.

Octimibate and related nonprostanoid prostacyclin mimetics are partial agonists displaying highly tissue-specific responses. Octimibate demonstrated considerably greater efficacy for stimulation of adenylyl cyclase activity in Chinese hamster ovary cells transiently expressing mouse prostacyclin receptors (mIP-CHO cells) when compared to human SK-N-SH neuroblastoma cells, which endogenously express prostacyclin (IP) receptors. Pretreatment of both cell types with pertussis toxin (PTx) failed to influence IP agonist efficacy or potency, indicating a lack of involvement of an agonist-stimulated inhibitory G(i)-coupled pathway. Although stimulation of mIP-CHO cells with the full agonist cicaprost increased both [3H]cyclic AMP and [3H]inositol phosphate ([3H]IP) accumulation (pEC(50) values of 8.35 and 6.82, respectively), IP receptor signalling through G(q) in SK-N-SH cells was absent. Inhibition of protein kinase C (PKC) in mIP-CHO cells increased [3H]IP accumulation but had no effect on [3H]cyclic AMP accumulation. Therefore, the poor coupling of the IP receptor in SK-N-SH cells to G(q) is unlikely to explain the relatively low efficacy of octimibate for stimulating adenylyl cyclase in these cells. Furthermore, protein kinase A (PKA) inhibition appears to enhance IP receptor signalling through both G(s) and G(q) in mIP-CHO cells.

Proportional reduction of urine supersaturation during nephrolithiasis treatment.

PURPOSE: During metabolic stone therapy, urine supersaturation decreases in proportion to pretreatment levels. We gauge the quantitative contribution of regression to the mean for reducing urine supersaturation from high pretreatment to lower values during therapy. MATERIALS AND METHODS: The 24-hour urine supersaturations for calcium oxalate, calcium phosphate and uric acid were measured on 2 pretreatment and at least 1 treatment 24-hour collection for each of the 2,667 patients in 2 networks and at a university based specialty clinic. Changes in supersaturation between the first and second pretreatment collections were an estimate of random change and compared to therapeutic changes. RESULTS: Supersaturations decreased between the first and second pretreatment collections, proportional to the supersaturation in the first collection. However, the magnitude of this effect was minor compared to therapeutic changes. Also, mean change between pretreatment collections was 0, whereas mean change with therapy was greater than 0 for all 3 supersaturations. CONCLUSIONS: Although regression to the mean can be detected, it cannot be responsible for the decrease in urine supersaturation with therapy or the fact that the decrease is proportional to pretreatment mean supersaturation. The mechanisms responsible for proportional reduction remain to be clarified.

Differences in urine volume and supersaturation in 2 physician networks.

PURPOSE: We determined whether a network of practices devoted to a broad range of urological care would achieve a decrease in metabolic stone risk comparable to that achieved by a network of similar practices that emphasized kidney stone management as a distinct specialized interest, provided that each was given equivalent access to high level urine testing and software support. MATERIALS AND METHODS: Pretreatment and treatment 24-hour urine samples were obtained from patients in a large network of practices related by the shared use of lithotripsy facilities and instruments (group 2) and a contrasting network of practices that emphasize stone treatment over other concerns (group 1). All known urine risk factors, including supersaturation, were measured and calculated. RESULTS: Treatment supersaturation values in group 2 exceeded those in group 1. The reason was unpredicted and unexplained but highly consistent lower urine volume in group 2 patients that was present before and persisted during treatment. Group 2 physicians mostly achieved changes in urine volume and stone risk factors equivalent to those of group 1 physicians but began with higher supersaturation due to lower urine volume. CONCLUSIONS: A network of physicians not specialized for stone care may achieve a decreased risk equivalent to that of more specialized physicians. Initial patient characteristics may vary significantly in the groups for reasons that are unknown to date, greatly affecting treatment outcome.