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Background: A novel single-use, analyser-free, molecular point-of-care test for SARS-CoV-2 (Veros COVID-19 test, Sherlock Biosciences) could reduce time to results and improve patient care and flow in the emergency department (ED), but its performance in this setting is unknown., Methods: Adults aged ≥18 years presenting to Southampton General Hospital (UK) with suspected COVID-19 were tested with the Veros COVID-19 test in addition to standard of care near-patient PCR. Measures of diagnostic accuracy were calculated for the Veros COVID-19 test stratified by Ct value. Discrepant results underwent viral culture., Findings: Between Jan 16 and May 2, 2023, 400 patients were enrolled with a median (IQR) age of 60 (34-77) and 141 (35·3%) were SARS-CoV-2 positive by PCR. The Veros test gave valid results on the first test in 384 (96·0%), and sensitivity and specificity were 127/141 (90·1%, 95%CI 83·9-94·5) and 258/259 (99·6%, 95%CI 97·9-100) overall. For those with high or moderate viral load (Ct ≤30), sensitivity was 125/129 (96·9%, 95%CI 92·3-99·2). One (7·1%) of 14 PCR positive/Veros test negative samples was culture positive. Median (IQR) time from sample collection to result was 19 (18-20) mins with the Veros test versus 73 (59-92) mins with PCR (p < 0·0001)., Interpretation: The Veros COVID-19 test generated results in near real-time, around 1 h sooner than rapid, near-patient, analyser-based PCR, and accuracy was excellent for samples with moderate and high viral loads. The Veros test represents a step-change in molecular diagnostics for infection and could significantly reduce time to results and improve patient management in EDs and other settings., Competing Interests: Declaration of Competing Interest The author Professor Tristan W Clark is an Associate Editor for Journal of Infection and was not involved in the editorial review or the decision to publish this article. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TWC has received speaker fees, honoraria, travel reimbursement, and equipment and consumables at discount or free of charge for the purposes independent of research, outside of this submitted study, from BioFire diagnostics, BioMerieux and QIAGEN. He has received consultancy fees from Cepheid, Synairgen research, Roche, Janssen, Biofire diagnostic and BioMerieux. He has received honoraria for participation in advisory boards from Cepheid, Roche, Janssen, Shionogi, GSK, Seqirus and Sanofi. He is a member of an independent data monitoring committee for a trial sponsored by Roche. He has acted as the UK chief investigator for a study sponsored by Janssen. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: FebriDx is a CE-marked, FDA-approved point-of-care test that detects the antiviral host response protein Myxovirus Resistance Protein A (MxA), in addition to C-reactive protein, using finger-prick blood. FebriDx MxA detection had a high negative predictive value for COVID-19 in symptomatic adults presenting to hospital in the first waves of the pandemic and was used subsequently as a 'rule out' triage tool in Emergency departments. The diagnostic accuracy of FebriDx MxA in the current context of co-circulation of influenza, SARS-CoV-2, and Respiratory Syncytial Virus (RSV), and in the era of COVID-19 vaccination, is unknown., Methods: We retrospectively evaluated the diagnostic performance of FebriDx MxA in adults with acute respiratory symptoms presenting to the Emergency Department (ED) of a large UK teaching hospital using Reverse Transcription Polymerase Chain Reaction (RT-PCR) as the reference standard (Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV)., Results: Between March 9th 2022 and March 8th 2023, 5426 patients had both FebriDx and RT-PCR testing with valid results. 999 (18.4%) of patients had influenza detected, 520 (9.6%) SARS-CoV-2, and 190 (3.5%) RSV. Negative Predictive Value (NPV) of MxA detection by FebriDx was 97.5% (96.9-98.0) for influenza, 97.1% (96.4-97.7) for SARS-CoV-2, 98.1% (97.5-98.6) for RSV, and 92.8% (91.8-93.7) for all viruses combined., Conclusions: In symptomatic adults, FebriDx MxA had a high NPV for influenza and RSV, and retained a high NPV for SARS-CoV-2, in the context of virus co-circulation and widespread COVID-19 vaccination. FebriDx continues to be a useful 'rule out' triage tool in the ED and could potentially be scaled to provide a national triage solution for future viral pandemics., Competing Interests: Declaration of Competing Interest TWC has received equipment and consumables at discount or free of charge for the purposes independent of research, outside of this submitted study, from BioFire diagnostics, BioMerieux, QIAGEN, and SenseBio. He has received travel re-imbursement and speaker fees from BioFire diagnostics, BioMerieux, and QIAGEN. He has received consultancy fees from Cepheid, Synairgen research, Roche, Sanofi, BioMerieux, and Biofire diagnostics. He has received honoraria for participation in advisory boards from Cepheid, Roche, Janssen, GSK, Shionogi, Seqirus and Sanofi. He is a member of an independent data monitoring committee for a trial sponsored by Roche. He has acted as the UK chief investigator for a study sponsored by Janssen. He owns shares in Synairgen research. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19) 1 . The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod 2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 infection is spreading at an alarming rate and has created an unprecedented health emergency around the globe. There is no effective vaccine or approved drug treatment against COVID-19 and other pathogenic coronaviruses. The development of antiviral agents is an urgent priority. Biochemical events critical to the coronavirus replication cycle provided a number of attractive targets for drug development. These include, spike protein for binding to host cell-surface receptors, proteolytic enzymes that are essential for processing polyproteins into mature viruses, and RNA-dependent RNA polymerase for RNA replication. There has been a lot of ground work for drug discovery and development against these targets. Also, high-throughput screening efforts have led to the identification of diverse lead structures, including natural product-derived molecules. This review highlights past and present drug discovery and medicinal-chemistry approaches against SARS-CoV, MERS-CoV and COVID-19 targets. The review hopes to stimulate further research and will be a useful guide to the development of effective therapies against COVID-19 and other pathogenic coronaviruses., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction ( P  < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis. IMPORTANCE Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses., (Copyright © 2020 American Society for Microbiology.)

Conventional indicator dilution techniques for measuring body fluid volume are laborious, expensive, and highly invasive. Bioimpedance spectroscopy (BIS) may be a useful alternative due to being rapid, minimally invasive, and allowing repeated measurements. BIS has not been reported in mice; hence we examined how well BIS estimates body fluid volume in mice. Using C57/Bl6 mice, the BIS system demonstrated <5% intermouse variation in total body water (TBW) and extracellular (ECFV) and intracellular fluid volume (ICFV) between animals of similar body weight. TBW, ECFV, and ICFV differed between heavier male and lighter female mice; however, the ratio of TBW, ECFV, and ICFV to body weight did not differ between mice and corresponded closely to values in the literature. Furthermore, repeat measurements over 1 wk demonstrated <5% intramouse variation. Default resistance coefficients used by the BIS system, defined for rats, produced body composition values for TBW that exceeded body weight in mice. Therefore, body composition was measured in mice using a range of resistance coefficients. Resistance values at 10% of those defined for rats provided TBW, ECFV, and ICFV ratios to body weight that were similar to those obtained by conventional isotope dilution. Further evaluation of the sensitivity of the BIS system was determined by its ability to detect volume changes after saline infusion; saline provided the predicted changes in compartmental fluid volumes. In summary, BIS is a noninvasive and accurate method for the estimation of body composition in mice. The ability to perform serial measurements will be a useful tool for future studies.

There has been extensive interest in the role of serotonin (5-hydoxytryptamine, 5-HT) in the pathogenesis of pulmonary hypertension because episodes of pulmonary arterial hypertension in humans have been linked to serotoninergic appetite-suppressant drugs. In this study, we investigated the role of serotonin in the development of pulmonary hypertension induced by intravenously injecting bacterial lipopolysaccharide (LPS, endotoxin) and cellulose microparticles. In experiment 1, we used a 5-HT ELISA kit for the in vitro quantitative determination of 5-HT in plasma during the development of pulmonary hypertension induced by injecting LPS and cellulose microparticles i.v. in broilers. In experiment 2, broilers were either chronically infused with 5-HT via surgically implanted osmotic pumps or received sham surgery as a control. After a period of 10 d, the pulmonary arterial pressure was recorded during challenge with injected LPS or microparticles. Microparticles elicited 5-HT plasma levels more than 2-fold greater than those elicited by LPS from 15 to 45 min postinjection. This indicates that 5-HT is an important mediator in the pulmonary hypertensive response of broilers to microparticles, but may not play a prominent role in the pulmonary hypertensive response to LPS. Furthermore, chronic 5-HT infusion via osmotic pumps caused an increase in the duration of the pulmonary hypertensive response of broilers to microparticles, indicating that the infused 5-HT was sequestered by circulating thrombocytes and then released upon microparticle-mediated thrombocyte activation. Serotonin appears to play a less prominent role in the pulmonary hypertensive response of broilers to LPS, indicating that other mediators within the innate response to inflammatory stimuli may also be involved. These results are consistent with our hypothesis that pulmonary arterial hypertension ensues when vasoconstrictors such as 5-HT overwhelm the dilatory affects of vasodilators such as nitric oxide, thereby effectively reducing the pulmonary vascular capacity of pulmonary arterial hypertension-susceptible broilers.

Nitric oxide is a potent vasodilator synthesized from l-Arg by NO synthase (NOS). Constitutive NOS in endothelial cells (eNOS) produces transient bursts of NO in low but physiologically effective levels. Activated monocytes and macrophages express inducible NOS (iNOS), which produces copious quantities of NO. Previous studies showed that NO attenuates pulmonary hypertensive responses induced by i.v. injections of lipopolysaccharide (LPS) or cellulose microparticles (MP). The present study determined whether changes in plasma NO concentrations could be used to assess the time course of NO production in response to LPS or MP injections. Broilers were injected i.v. with 1 mL of PBS (control), 1 mL of LPS (1 mg/mL), or 0.4 mL of MP (0.02 g/mL). Plasma samples were collected from 10 different broilers per group at 15, 30, 45, and 60 min and at 2, 3, 4, 5, 6, 8, 10, and 12 h postinjection. Total plasma NO concentrations were analyzed by nitrate + nitrite assay. After PBS or MP injection, plasma NO levels did not change throughout the 12-h period. Nitric oxide measured in the plasma increased in LPS-injected broilers from 4.8 +/- 0.8 microM at 15 min to 46.6 +/- 5.7 microM by 4 h postinjection, reached peak levels of 85.1 +/- 10.6 microM at 5 h, and returned to baseline levels similar to PBS-injected broilers by 12 h postinjection. We conclude that LPS triggered widespread iNOS expression by circulating monocytes and macrophages, resulting in copious NO production as reflected by significant increases in total plasma NO. Proportionally few monocytes and macrophages responded to MP entrapped in pulmonary arterioles. Consequently, NO produced by iNOS in activated leukocytes or by eNOS in the pulmonary vasculature had a minimal impact on total plasma NO. Total plasma NO from broilers did reflect the time course of massive iNOS activation in response to LPS, but biologically relevant quantities of NO produced by iNOS and eNOS activated during the local inflammatory response to entrapped MPs were too low to affect total plasma NO concentrations.

Broilers are susceptible to pulmonary hypertension syndrome (PHS; ascites syndrome) when their pulmonary vascular capacity is anatomically or functionally inadequate to accommodate the requisite cardiac output without an excessive elevation in pulmonary arterial pressure. The consequences of an inadequate pulmonary vascular capacity have been demonstrated experimentally and include elevated pulmonary vascular resistance (PVR) attributable to noncompliant, fully engorged vascular channels; sustained pulmonary arterial hypertension (PAH); systemic hypoxemia and hypercapnia; specific right ventricular hypertrophy, and right atrioventricular valve failure (regurgitation), leading to central venous hypertension and hepatic cirrhosis. Pulmonary vascular capacity is broadly defined to encompass anatomical constraints related to the compliance and effective volume of blood vessels, as well as functional limitations related to the tone (degree of constriction) maintained by the primary resistance vessels (arterioles) within the lungs. Surgical occlusion of 1 pulmonary artery halves the anatomical pulmonary vascular capacity, doubles the PVR, triggers PAH, eliminates PHS-susceptible broilers, and reveals PHS-resistant survivors whose lungs are innately capable of handling sustained increases in pulmonary arterial pressure and cardiac output. We currently are using i.v. microparticle injections to increase the PVR and trigger PAH sufficient in magnitude to eliminate PHS-susceptible individuals while allowing PHS-resistant individuals to survive as progenitors of robust broiler lines. The microparticles obstruct pulmonary arterioles and cause local tissues and responding leukocytes to release vasoactive substances, including the vasodilator NO and the highly effective vasoconstrictors thromboxane A(2) and serotonin [5-hydroxytryptamine (5-HT)]. Nitric oxide is the principal vasodilator responsible for modulating (attenuating) the PAH response and ensuing mortality triggered by i.v. microparticle injections, whereas microparticle-induced increases in PVR can be attributed principally to 5-HT. Our observations support the hypothesis that susceptibility to PHS is a consequence of anatomically inadequate pulmonary vascular capacity combined with the functional predominance of the vasoconstrictor 5-HT over the vasodilator NO. The contribution of TxA(2) remains to be determined. Selecting broiler lines for resistance to PHS depends upon improving both anatomical and functional components of pulmonary vascular capacity.

There has been considerable interest in the role of serotonin (5-hydroxytryptamine, 5-HT) in the pathogenesis of pulmonary hypertension due to episodes of primary pulmonary hypertension in humans linked to serotoninergic appetite-suppressant drugs. In this study, we investigated the effect of 5-HT on the development of pulmonary hypertension induced by injecting bacterial lipopolysaccharide (LPS; endotoxin) and cellulose microparticles intravenously, using the nonselective 5-HT(1/2)receptor, antagonist methiothepin. In Experiment 1, broilers selected for ascites susceptibility or resistance under conditions of hypobaric hypoxia were treated with methiothepin or saline, followed by injection of LPS, while recording pulmonary arterial pressure (PAP). In Experiment 2 ascites-susceptible broilers were treated with methiothepin or saline, followed by injection of cellulose microparticles, while recording PAP. In Experiment 3, an i.v. microparticle injection dose shown to cause 50% mortality was injected into ascites-susceptible and ascites-resistant broilers after methiothepin or saline treatment. Injecting methiothepin reduced PAP below baseline values in ascites-susceptible and ascites-resistant broilers, suggesting a role for 5-HT in maintaining the basal tone of the pulmonary vasculature in broilers. Injecting microparticles into the wing vein had no affect on the PAP in the broilers treated with methiothepin, suggesting that 5-HT is an important mediator in the pulmonary hypertensive response of broilers to microparticles. Furthermore, injecting an 50% lethal dose of microparticles into ascites-susceptible and ascites-resistant broilers pretreated with methiothepin resulted in reduced mortality. Serotonin appears to play a less prominent role in the pulmonary hypertensive response of broilers to intravenously injected LPS, indicating that other mediators within the innate response to inflammatory stimuli may also be involved. These results are consistent with our hypothesis that pulmonary hypertension syndrome ensues when vasoconstrictors, such as 5-HT, overwhelm the dilatory effects of vasodilators, such as NO, thereby effectively reducing the pulmonary vascular capacity of pulmonary hypertension syndrome-susceptible broilers.

The pathogenesis of pulmonary hypertension remains incompletely understood. Many factors have been implicated; however, there has been great interest in the potent pulmonary vasoconstrictor serotonin (5-HT) due to episodes of primary pulmonary hypertension in humans triggered by serotoninergic appetite-suppressant drugs. Pulmonary hypertensive patients have elevated blood 5-HT levels and pulmonary vasoconstriction induced by 5-HT is believed to be mediated through 5-HT1B/1D and 5-HT2A receptors that are expressed by pulmonary smooth muscle cells. The vascular remodeling associated with pulmonary hypertension also appears to require the serotonin transporter. We investigated the roles of 5-HT receptor blockers on the development of pulmonary hypertension induced by infusing 5-HT i.v. in broilers. For this purpose, we treated broilers with the selective 5-HT2A receptor antagonist ketanserin (5 mg/ kg of BW) or with the nonselective 5-HT1/2 receptor antagonist methiothepin (3 mg/kg of BW). Receptor blockade was followed by infusion of 5-HT while recording pulmonary arterial pressure and pulmonary arterial blood flow. The results demonstrate that methiothepin, but not ketanserin, eliminated the 5-HT-induced pulmonary hypertensive responses in broilers. The 5-HT2A receptor does not, therefore, appear to play a role in the 5-HT-induced pulmonary hypertensive responses in broilers. Methiothepin did not inhibit pulmonary vascular contractility per se, because the pulmonary hypertensive response to the thromboxane A2 mimetic U44069 remained intact in methiothepin-treated broilers. Methiothepin will be a useful tool for evaluating the role of 5-HT in the pathogenesis of pulmonary hypertension syndrome (ascites) as well as the onset of pulmonary hypertension triggered by inflammatory stimuli such as bacterial lipolysaccharide.

The pulmonary hypertensive response to pulmonary vascular obstruction caused by intravenously injected microparticles is amplified by pretreatment with N(omega)nitro-L-arginine methyl ester (L-NAME). The L-NAME prevents the synthesis of the potent vasodilator nitric oxide (NO) by inhibiting both the constitutive [endothelial NO synthase (eNOS or NOS-3)] and inducible [inducible NO synthase (iNOS or NOS-2)] forms of NO synthase. In the present study we used the selective iNOS inhibitor aminoguanidine (AG) to evaluate the role of iNOS in modulating the pulmonary hypertension (PH) triggered by microparticle injections. Experiment 1 was conducted to confirm the ability of AG to inhibit NO synthesis by iNOS in broiler peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide (LPS, endotoxin). Mononuclear leukocytes treated with LPS produced 10-fold more NO than untreated (control) cells. The LPS-stimulated production of NO was partially inhibited by L-NAME and was fully inhibited by AG, thereby confirming that AG inhibits LPS-mediated iNOS activation in broilers. In Experiment 2 we evaluated the responses of male progeny from a base population (MP Base) and from a derivative line selected for one generation from the survivors of an LD50 microparticle injection (MP Select). The pulmonary arterial pressure (PAP) was lower in MP Select than in MP Base broilers. Both lines exhibited similar percentage increases in PAP after microparticles were injected, and AG modestly amplified the PH triggered by microparticles in both lines. In Experiment 3 we evaluated the responses of male progeny from a second base population (PAC Base) and from a derivative line selected for 3 generations using the unilateral pulmonary artery clamp technique (PAC Select). The PAP was lower in PAC Select than in PAC Base broilers, and both lines exhibited similar percentage increases in PAP in response to the microparticles. The PH triggered by microparticles was not amplified by AG but was doubled by L-NAME. These experiments demonstrate that during the 30 min following pulmonary vascular entrapment of microparticles, iNOS modulated the PH elicited in broilers derived from the MP pedigree line, but not in broilers from the PAC pedigree line. Different NOS-mediated responses among broiler populations may affect pulmonary hemodynamic characteristics of broiler lines selected using i.v. microparticle injections.

Nitric oxide (NO) is a potent vasodilator that is synthesized by constitutive and inducible isoforms of the enzyme NO synthase (eNOS and iNOS, respectively). The half-life of NO averages only 3 to 4 s in biological fluids, where it is rapidly converted to the stable oxidation products nitrite (NO2-) and nitrate (NO3-). Our objectives were to use 2 commercial kits to measure total plasma NO, as NO2- + NO3-, and to assess plasma NO values during experimental protocols designed to influence NO accumulation in the plasma. One kit employed copper-coated cadmium as a catalyst for reducing NO3- to NO2-; the second kit employed the enzyme NO3- reductase for the same purpose. Both then employed Griess reagent for the colorimetric determination of NO2- as a measure of total plasma NO. Broilers in Experiment 1 were infused i.v. with solutions containing increasing concentrations of sodium NO2-. Broilers in Experiment 2 were injected with 1 mg of lipopolysaccharide (LPS), which is known to stimulate iNOS activity. Both commercial kits successfully detected increases in total plasma NO attributable to ongoing i.v. NO2- infusion or to increased iNOS expression at 5 h after the LPS injection. In Experiment 3, we compared the total plasma NO responses to LPS in the presence and absence of aminoguanidine (AG), a selective inhibitor of iNOS. The AG significantly attenuated the LPS-mediated increase in total plasma NO at 5 h post-injection. In Experiment 4, broilers were infused with sodium nitroprusside (SNP), an exogenous NO donor molecule that previously had been shown to lower the pulmonary arterial pressure in broilers. The SNP infusion did substantially reduce the pulmonary arterial pressure, but an increase in total plasma NO was not detected during the SNP infusion. Overall, NO accumulation in the plasma was successfully detected after sustained infusion of NaNO2 and administration of LPS for 5 h, but biologically effective levels of NO released from SNP were not detected. Therefore, total plasma NO concentrations (assayed as NO2- + NO3-) qualitatively reflect whole-body NO synthesis, but biologically relevant quantities of NO may be produced at levels that cannot be detected by colorimetric assays.

The acquisition of immunity to Eimeria maxima by chicks infected 18 hr after hatch with a single dose of 100 oocysts was investigated. In the first experiment, birds were moved each day to clean cages in order to prevent the possibility of secondary infection resulting from ingestion of oocysts passed in their feces. Immunity was measured at 4 wk of age by calculation of oocyst production following challenge with 500 oocysts or weight gain following challenge with 100,000 oocysts. Large numbers of oocysts were produced by infected birds following challenge, although numbers were significantly less than those from birds that had been reared in the absence of infection (susceptible controls). The weight gain of infected birds following challenge was significantly greater than that of susceptible controls but less than that of unchallenged controls. Thus, only partial protection had been acquired, whether parasite replication or body weight gain was used to assess the extent of immunity development. In a second experiment, acquisition of immunity at 4 wk by chicks infected 18 hr after hatch with 100 oocysts of E. maxima and reared in floor pens in contact with their droppings was investigated. Infected birds produced no oocysts following challenge, and weight gains were not significantly different from the unchallenged controls, which indicates that full immunity had developed by 4 wk. It is concluded that if oocysts of Eimeria species are used to vaccinate day-old chicks, reinfection by oocysts present in the litter is necessary for the establishment of protective immunity.

We tested the hypothesis that microparticles entrapped within the pulmonary vasculature elicit the production of nitric oxide (NO) in quantities sufficient to modulate the combined impact of physical occlusion plus contemporaneously released vasoconstrictors. In experiment 1, male broilers were given an injection of the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME), followed by an intravenous injection of cellulose microparticles while the pulmonary arterial pressure (PAP) and cardiac output (CO) were recorded. When L-NAME was used to block NO synthesis induced by the microparticles, an early peak of pulmonary hypertension was revealed that rarely developed in the absence of L-NAME. The subsequent more prolonged increases in PAP and pulmonary vascular resistance (PVR) were greater in amplitude and duration in broilers pretreated with L-NAME than in broilers in the control group. These amplified responses occurred in spite of a simultaneous reduction in CO, thereby conclusively demonstrating that inhibiting NOS permitted the development of a much more profound increase in the PVR. In experiment 2 the mortality triggered within 48 h after injecting microparticles was evaluated in the presence and absence of L-NAME. The 48 h postinjection mortality more than doubled when L-NAME was combined with microparticle injection doses that otherwise caused relatively low mortality in the absence of L-NAME. Experiment 3 was conducted to determine whether NO contributes to the systemic hypoxemia that develops after microparticles are injected. L-NAME administration had no impact on the magnitude and duration of the microparticle induced decline in the percentage saturation of hemoglobin with oxygen (%HbO2). Evidently hypoxemia per se contributes relatively little to the amplified pulmonary vasoconstriction and 48 h postinjection mortality triggered by microparticle injections in broilers pretreated with L-NAME. These observations indicate that NO modulates the responses to vasoconstrictors released when microparticles become entrapped in the pulmonary vasculature. Inhibition of NOS by L-NAME exposed a more dramatic increase in PVR and pulmonary hypertension leading to enhanced mortality in response to microparticle injections.

Previous studies demonstrated that bacterial lipopolysaccharide (LPS, endotoxin) triggers pulmonary vasoconstriction leading to pulmonary hypertension (PHS, ascites) in broilers. The lungs of broilers are constantly challenged with LPS that can trigger pulmonary vasoconstriction. Among broilers from a single genetic line, some individuals respond to LPS with large increases in pulmonary arterial pressure (PAP), whereas others fail to exhibit any response to the same supramaximal dose of LPS. In the present study we evaluated the impact of a variety of factors on the magnitude of the PAP response of male broilers to LPS, including: (1) the role of the initial PAP (low vs. high initial PAP); (2) the source of the LPS (Salmonella typhimurium vs. Escherichia coli); (3) the dose of LPS (0.02, 0.1, and 0.5 mg/kg of BW); and (4) the role of micro-particle selection for improved pulmonary vascular capacity (cellulose survivors vs. saline-injected controls). Broilers in the low initial PAP group (21 +/- 0.34 mmHg, mean +/- SEM) did not differ in their pulmonary hypertensive response to LPS compared with broilers in the high initial PAP group (29 +/- 0.55 mmHg, mean +/- SEM). Lipopolysaccharide from S. typhimurium elicited pulmonary hypertensive responses qualitatively similar to those elicited by E. coli LPS. A detailed evaluation revealed that an LPS dose of 0.1 mg/kg of BW elicits a maximal pulmonary hypertensive response in male broilers, and broilers selected by micro-particle injection for a robust pulmonary vascular capacity did not differ in their pulmonary hypertensive response to LPS compared with unselected broilers. This research confirms that the variable pulmonary hypertensive responses among broilers cannot be attributed to the source or dosage of LPS, or to differences in the baseline pulmonary arterial pressure or micro-particle selection before injecting LPS. These findings are consistent with the hypothesis that innate rather than acquired variability may influence the profile of chemical mediators released during the inflammatory cascade.

The eicosanoid vasodilator prostacyclin (PGI2) reduces resistance to pulmonary blood flow and attenuates pulmonary hypertension in mammals. However, sparse information is available regarding the responsiveness of the avian pulmonary vasculature to PGI2. Accordingly, in 3 experiments we evaluated the pulmonary vascular responses to PGI2 in male broilers. In experiment 1, infusing PGI2 (10 microg/min) into clinically healthy broilers did not reduce their pulmonary vascular resistance (PVR) but did reduce their pulmonary arterial pressure (PAP) by lowering their cardiac output. Within 4 min after stopping the PGI2 infusion, the cardiac output and PAP returned to preinfusion levels. In experiment 2, the responses to PGI2 were evaluated after arachidonic acid (AA) had been infused to preconstrict the pulmonary vasculature. The AA infusion (400 microg/min) consistently triggered dramatic, sustained pulmonary vasoconstriction (increased PVR) and pulmonary hypertension (increased PAP). Concurrent PGI2 infusions did not reduce PVR but did reduce PAP by lowering cardiac output. Within 4 min after stopping the PGI2 infusion, PAP and cardiac output returned to their previous (hypertensive) levels attributable to the ongoing AA infusion. In experiment 3, PGI2 was infused (10 microg/min) into clinically healthy (PAP < or = 24 mmHg) or subclinically hypertensive (PAP > or = 27 mmHg) broilers. Throughout this experiment broilers in the hypertensive group had higher PAP values than broilers in the healthy group. The PGI2 infusion reduced PAP in both groups but did not reduce PVR. Instead, the pulmonary hypotensive response to PGI2 infusion was associated with a reduction in cardiac output in both groups. In all 3 experiments PGI2 reduced PAP by reducing cardiac output rather than by reducing PVR. There was no evidence that PGI2 acts as an effective pulmonary vasodilator in broilers regardless of whether their pulmonary vasculature was apparently normal (clinically healthy), had been pharmacologically preconstricted (AA infusion), or initially exhibited the vasoconstriction that is typical of the pathogenesis of pulmonary hypertension syndrome in broilers (PAP > or = 27 mmHg). The consistent failure of PGI2 to elicit pulmonary vasodilation in this study suggests fundamental differences in AA metabolism or the etiology of pulmonary hypertension may exist when broilers are compared with mammals.

The effects of diclazuril and monensin, when included in the feed of turkeys from 0 to 10 wk, upon performance and development of immunity to Eimeria species was investigated. Birds were initially inoculated with a low dose of oocysts of three species of Eimeria at 3, 5, 7, and 9 days of age in order to simulate a natural infection. Weight gain and feed intake from 0 to 6 wk of age was significantly greater in medicated birds compared with those that received no anticoccidial medication. Weight gain and feed intake from 6 to 10 wk was greater in birds that received diclazuril than in unmedicated birds. No differences in performance were evident after drug withdrawal from 10 to 16 wk. Immunity to Eimeria species developed by 10 wk in birds that received no anticoccidial medication but did not develop in those given diclazuril or monensin.

The lungs of broilers are constantly challenged with lipopolysaccharide (LPS, endotoxin) that can activate leukocytes and trigger thromboxane A2 (TxA2)- and serotonin (5HT)-mediated pulmonary vasoconstriction leading to pulmonary hypertension. Among broilers from a single genetic line, some individuals respond to LPS with large increases in pulmonary arterial pressure, whereas others fail to exhibit any response to the same supramaximal dose of LPS. This extreme variability in the pulmonary hypertensive response to LPS appears to reflect variability in the types or proportions of chemical mediators released by leukocytes. Our research has confirmed that TxA2 and 5HT are potent pulmonary vasoconstrictors in broilers and that broilers hatched and reared together consistently exhibit pulmonary hypertension after i.v. injections of TxA2 or 5HT. Previous in vitro studies conducted using macrophages from different lines of chickens demonstrated innate variability in the LPS-stimulated induction of nitric oxide synthase (iNOS) followed by the onset of an LPS-refractory state. The NOS enzyme converts arginine to citrulline and nitric oxide (NO). It is known that NO produced by endothelial NOS serves as a key modulator of flow-dependent pulmonary vasodilation, and it is likely that NO generated by iNOS also contributes to the pulmonary vasodilator response. Accordingly, it is our hypothesis that the pulmonary hypertensive response to LPS in broilers is minimal when more vasodilators (NO, prostacyclin) than vasoconstrictors (TxA2, 5HT) are generated during an LPS challenge. Indeed, inhibiting NO production through pharmacological blockade of NOS with the inhibitor Nomega-nitro-L-arginine methyl ester modestly increased the baseline pulmonary arterial pressure and dramatically increased the pulmonary hypertensive response to LPS in all broilers evaluated. Innate differences in the effect of LPS on the pulmonary vasculature may contribute to differences in susceptibility of broilers to pulmonary hypertension syndrome (ascites).

The pulmonary hypertensive response to bacterial lipopolysaccharide (LPS, endotoxin) varies widely among individual broilers, leading to the suggestion that innate variability may exist in the proportions or profiles of chemical mediators released during the ensuing inflammatory cascade. LPS induces the expression of nitric oxide synthase (iNOS), which produces the vasodilator nitric oxide (NO) to modulate the responses to concurrently produced vasoconstrictors. In experiment 1, broilers were given the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), followed by a supra-maximal dose of LPS while the pulmonary arterial pressure was recorded. In experiment 2 the cardiac output also was recorded before and following the i.v. injection of L-NAME. In both experiments, injection with L-NAME modestly increased the pulmonary arterial pressure when compared with control values, confirming previous reports that tonic/basal NO synthesis is required to promote flow-dependent pulmonary vasodilation in chickens. This response to L-NAME occurred in spite of a tendency for cardiac output and stroke volume to decline and, therefore, can be attributed to pulmonary vasoconstriction (an increase in the pulmonary vascular resistance) rather than an increase in pulmonary blood flow. When L-NAME was used to block NO synthesis induced by LPS, an early peak of pulmonary hypertension was revealed that rarely develops in broilers in the absence of L-NAME, and that has been correlated with the release of platelet activating factor and thromboxane A2 in mammals. The control group responded to LPS with a delayed-onset pulmonary hypertension that was typical in timing, amplitude, and duration of the responses previously observed in broilers and that has been attributed to endothelin-mediated thromboxane A2 synthesis in mammals. This delayed-onset pulmonary hypertensive response to LPS was longer in duration and higher in amplitude in the L-NAME group when compared with the control group. These observations are consistent with the hypothesis that NO modulates the responses to vasoconstrictors released concurrently during the LPS-mediated inflammatory cascade. Inhibition of NOS by L-NAME apparently reduced the modulatory influence of NO and exposed a more dramatic pulmonary hypertensive response to LPS.

Serotonin is a potent pulmonary vasoconstrictor actively accumulated by mammalian platelets and avian thrombocytes and released into the plasma during platelet or thrombocyte aggregation. Serotonin has been implicated in the mechanisms responsible for pulmonary hypertension in several human and animal studies. However, the role of serotonin in pulmonary hypertension syndrome (PHS, ascites) in broilers previously had not been evaluated. In the present study we evaluated the pulmonary hemodynamic responses of broilers to intravenous infusions of serotonin dissolved in 2.5% (wt/vol) mannitol solution (carrier vehicle). Carrier vehicle infusion alone had no influence on any of the hemodynamic variables. Serotonin infusion triggered rapid increases in pulmonary arterial pressure to approximately 50% above pre-infusion baseline values, accompanied by decreases in mean systemic arterial pressure and cardiac output. The peak pulmonary arterial pressure response occurred within approximately 70 s after the start of serotonin infusion and remained elevated above baseline values over the course of a 10-min infusion period. Pulmonary arterial pressure and cardiac output returned to pre-infusion baseline values upon cessation of serotonin infusion, whereas mean systemic arterial pressure returned toward pre-infusion base-line values. Pulmonary hypertensive responses were associated with increased pulmonary vascular resistance (pulmonary vasoconstriction). The peak pulmonary arterial pressure attainable was inadequate to propel the normal cardiac output through the elevated pulmonary vascular resistance. Consequently, the impeded venous return to the left ventricle caused dependent reductions in stroke volume, cardiac output, and mean systemic arterial pressure. Reductions in cardiac output were associated with reductions in stroke volume but not heart rate. Any factor that reduces the pulmonary vascular capacity or increases the pulmonary vascular resistance theoretically can increase the incidence of PHS. The present study provides direct evidence that serotonin can trigger pulmonary vasoconstriction and pulmonary hypertension in broilers.

Bacterial endotoxins stimulate endothelin-mediated, thromboxane-dependent increases in pulmonary vascular resistance in mammals, and thromboxane has been shown to cause an immediate but transient pulmonary vasoconstriction in broiler chickens. In the present study, i.v. injections of 1 mg endotoxin into anesthetized male broilers caused a pulmonary vasoconstrictive response that was delayed in onset by 15 min and that elevated the pulmonary arterial pressure by 10 mm Hg within 25 min postinjection. Thereafter, pulmonary hemodynamic variables gradually (> or = 15 min) returned toward pre-injection levels, and supplemental injections of 4 mg endotoxin during this recovery period failed to reinitiate pulmonary hypertension. In contrast, injecting the thromboxane A2 mimetic U44069 during the endotoxin recovery period triggered pulmonary vasoconstriction and pulmonary hypertension similar in magnitude to the responses triggered by U44069 before endotoxin had been administered. The time course and magnitude of the pulmonary hemodynamic responses to endotoxin were highly variable among individual broilers, whereas the individual responses to U44069 were more consistent. Unanesthetized broilers resembled anesthetized broilers in the time course, magnitude, and variability of their pulmonary hemodynamic responses to endotoxin. Overall, these observations are consistent with the hypothesis that endotoxin initiates a biochemical cascade, culminating in the delayed onset of pulmonary vasoconstriction and pulmonary hypertension within 20 min postinjection. Subsequently, the pulmonary vasculature remains responsive to large bolus injections of exogenous thromboxane mimetic; however depletion of endogenous vasoconstrictive components of the endotoxin-mediated cascade, a compensatory increase in endogenous vasodilators, or the induction of a transient cellular tolerance to endotoxin prevented fourfold higher doses of endotoxin from reversing the return toward a normal pulmonary vascular tone. Individual differences among broilers in their susceptibility to pulmonary hypertension syndrome (ascites) may be related to innate or acquired variability in their pulmonary vascular responsiveness to vasoactive mediators.

High retrograde pressure through the pulmonary venous system caused by failure of the left ventricle or left atrio-ventricular valve may result in the elevated pulmonary arterial pressure and right ventricular hypertrophy associated with pulmonary hypertension syndrome (PHS; ascites) in broiler chickens. In the present study, unanaesthetized male broilers from an ascites-resistant line, the base population from which the resistant line was derived, and a separate unselected line were used to determine whether changes in wedge pressure (thought to be similar to left atrial pressure) are predictive of differences in the pulmonary arterial pressure of clinically healthy and pre-ascitic broilers. Venous, right atrial, right ventricular, pulmonary arterial, and wedge pressures were obtained by inserting a catheter into a wing vein and progressively advancing the catheter into a pulmonary branch artery until the catheter tip became wedged in and occluded the flow through a terminal artery. Mean right ventricular and pulmonary arterial pressures were lower in the resistant line than in the base population, but wedge pressures did not differ between the resistant, base, and unselected lines. Right:total ventricular weight ratios (RV:TV) and the percentage saturation of hemoglobin with oxygen in arterial blood ranged in value from 0.18 to 0.44 and 65 to 96%, respectively. Wedge pressure, however, remained similar when pre-ascitic broilers with high RV:TV values and low oximetry values were compared with clinically healthy broilers. In all birds, whether healthy or showing pre-ascitic characteristics, the wedge pressure was slightly higher than the right atrial pressure but substantially lower than pulmonary arterial pressure. These observations provide definitive proof that pulmonary hypertension is initiated as a consequence of excessive pulmonary arterial or arteriole resistance. Pulmonary venous pressure is estimated by measuring the pulmonary arterial wedge pressure, and high wedge pressures would be evident if pulmonary hypertension was caused by the elevated downstream resistances associated with left-sided heart failure.

The performance of broilers reared in floor pens and given monensin in the feed at 121 ppm was compared with that of birds given no drug. Feed intake and BW gain of medicated birds was significantly lower than that of unmedicated birds from 0 to 22 d of age. Feed intake and feed conversion of medicated birds was significantly reduced, compared with unmedicated birds, from 22 to 53 and 0 to 60 d of age. Total mortality, and mortality due to leg abnormalities from 22 to 53 and 0 to 60 d, was significantly lower in birds given monensin. There was no difference in the incidence of tibial dyschondroplasia (TD) by 60 d. No differences in mortality due to pulmonary hypertension syndrome (PHS) were observed for any age period. Birds removed from pens at 28 d that had received monensin had lower hematocrit and percentage saturation of hemoglobin with oxygen in the blood than unmedicated birds. No differences in these variables were found at 54 d. There were no differences in the right ventricle weight: total ventricular weight ratios or electrocardiogram lead II values at 28 or 54 d. The results indicate that PHS does not occur more frequently in broilers medicated with monensin.

A prospective analysis of the incidence of paranasal sinus opacification in 100 patients referred for cranial computerized tomography (CT) for non-sinus related problems is described. The findings were correlated with symptomatic assessment. Twenty-seven per cent of asymptomatic patients had sinus opacification. The study illustrates the importance of careful clinical correlation when interpreting CT scans of the paranasal sinuses.

A postal survey of British and Irish interventional radiologists was carried out in 1991 in order to assess current practice with respect to sedation and monitoring of patients during angiography and interventional procedures. The response rate was 65%. 49% of patients are fasted prior to angiography and 68% prior to interventional procedures. Radiologists participate in obtaining consent in 60% of cases. Patients are often (50%) sedated for angiography and usually (62-94% depending on the procedure) sedated for interventional procedures. Nurses are present for most procedures, but are given the task of monitoring the patient's vital signs in only 49% of cases. Anaesthetists are present for less than 10% of interventional procedures. Pulse oximetry is used routinely in 20% of departments, and automatic blood pressure monitors in 16%. 28% of radiologists never administer oxygen to patients under sedation, whereas 4% always do. 43% of departments have a staffed recovery area. Most vascular/interventional suites are stocked with emergency drugs and 80% with a defibrillator. 28% of departments report at least one death during or shortly after a procedure during the last 10 years. 18% of interventional radiologists have taken a refresher course in cardiopulmonary resuscitation in the past year. These findings indicate a wide variation in practice and a need to standardize practice at a uniform high level.

Closed osteotomy and nailing were performed on 37 patients for leg-length inequality or rotational deformities. Shortening operations were performed in 31 patients, derotation in six. Preoperatively, the leg-length discrepancy ranged from 2 to 6.6 cm. All femoral shortenings resulted in correction to less than 1 cm. Preoperatively, rotational deformities averaged 58 degrees; all were corrected to within 5 degrees of normal. Follow-up observation averaged 3.3 years. There were no nonunions or infections. All patients regained preoperative joint range of motion (ROM). Thirteen patients were Cybex tested one year or more postoperatively; all had quadriceps and hamstring strength equal to or greater than the contralateral leg, except for two patients who had suffered additional trauma to the shortened femur. Closed femoral osteotomy is an effective, safe, and reproducible means to obtain lower limb length correction in patients with leg-length inequality or rotational abnormality.

A case of florid thyroid acropachy affecting a single bone is described. Its potential to mimic malignancy is illustrated.

The sources of noradrenergic (NA) innervation to the hypoglossal nucleus (nXII) in the rat were investigated with double-labeling histochemical/immunocytochemical and lesion/degeneration techniques. Following injection of wheat germ-agglutinin conjugated to horseradish peroxidase into nXII, brain stem sections were reacted with tetramethylbenzidine, stabilized, and incubated in antiserum to tyrosine hydroxylase (TH). Double-labeled neurons were observed in three pontine sites bilaterally, although mainly ipsilaterally, that included the nucleus subceruleus (nSC; 68.75%) and the A7 (21.09%) and A5 (10.15%) cell groups. Confirmation of the above results and identification of the course taken by descending NA-nXII projections was accomplished by lesioning the rostral pons, the nSC, or the medullary catecholamine bundle (MB), the suspected route by which NA afferents reach nXII. Quantitative estimates of the reduction of TH immunoreactivity on the lesioned compared to nonlesioned side of nXII were made densitometrically. In each case, TH immunostaining was significantly decreased (75%) in the ipsilateral caudoventromedial district of nXII, the predominant target area of NA input. The results from this study establish that multiple NA sources in the pons project to nXII in the rat, the majority of NA-nXII afferents are derived from the nSC, and descending NA-nXII projections course in the MB. These data are discussed relative to tongue control.

Seventeen patients with malignant disease underwent percutaneous nephrostomy to relieve renal failure secondary to ureteric obstruction. Renal function improved in 88%. The median survival time was 18 weeks and 58% left the primary hospital to return home or to a terminal care hospice. Minor complications occurred in 58%. The use of bilateral nephrostomy tubes conferred no greater benefit than a unilateral tube. Intervention in malignant ureteric obstruction may confer some benefit and should not necessarily be viewed with pessimism.