Non-Lethal Dose-Response Models Replace Lethal Bioassays for Predicting the Hazard of Para-Aminopropiophenone to Australian Wildlife.

Para-aminopropiophenone (PAPP) is a potent methaemoglobin (MetHb) forming agent used for the lethal control of exotic carnivores and mustelids. To assess the sensitivity of Australian wildlife to PAPP we developed an in vivo assay that did not use death as an endpoint. Sub-lethal dose-response data were modelled to predict PAPP doses required to achieve an endpoint set at 80% MetHb (MetHb80). The comparative sensitivity of non-target mammals referenced to this endpoint was found to be highly variable, with southern brown bandicoots (Isoodon obesulus) the most sensitive species (MetHb80 = 6.3 mg kg-1) and bush rats (Rattus fuscipes) the most tolerant (MetHb80 = 1035 mg kg-1). Published LD50 estimates were highly correlated with PAPP doses modelled to achieve the MetHb80 endpoint (r2 = 0.99, p < 0.001). Most dose-response data for native mammals were collected in the field or in semi-natural enclosures, permitting PAPP and placebo dosed animals to be fitted with tracking transmitters and transponders and released at their point of capture. A protracted morbidity and mortality was observed only in Australian ravens (Corvus coronoides). The combination of sub-lethal dose-response assay and survival data collected in the field provided more relevant information about the actual hazard of pest control agents to non-target wildlife species than laboratory-based lethal-dose bioassays. We discuss the need to replace lethal-dose data with biologically meaningful insights able to define a continuum of toxicological hazards that better serve the needs of conservation and veterinary scientists and wildlife managers.

Helicobacter pylori-infected human neutrophils exhibit impaired chemotaxis and a uropod retraction defect.

Helicobacter pylori is a major human pathogen that colonizes the gastric mucosa and plays a causative role in development of peptic ulcers and gastric cancer. Neutrophils are heavily infected with this organism in vivo and play a prominent role in tissue destruction and disease. Recently, we demonstrated that H. pylori exploits neutrophil plasticity as part of its virulence strategy eliciting N1-like subtype differentiation that is notable for profound nuclear hypersegmentation. We undertook this study to test the hypothesis that hypersegmentation may enhance neutrophil migratory capacity. However, EZ-TAXIScan™ video imaging revealed a previously unappreciated and progressive chemotaxis defect that was apparent prior to hypersegmentation onset. Cell speed and directionality were significantly impaired to fMLF as well as C5a and IL-8. Infected cells oriented normally in chemotactic gradients, but speed and direction were impaired because of a uropod retraction defect that led to cell elongation, nuclear lobe trapping in the contracted rear and progressive narrowing of the leading edge. In contrast, chemotactic receptor abundance, adhesion, phagocytosis and other aspects of cell function were unchanged. At the molecular level, H. pylori phenocopied the effects of Blebbistatin as indicated by aberrant accumulation of F-actin and actin spikes at the uropod together with enhanced ROCKII-mediated phosphorylation of myosin IIA regulatory light chains at S19. At the same time, RhoA and ROCKII disappeared from the cell rear and accumulated at the leading edge whereas myosin IIA was enriched at both cell poles. These data suggest that H. pylori inhibits the dynamic changes in myosin IIA contractility and front-to-back polarity that are essential for chemotaxis. Taken together, our data advance understanding of PMN plasticity and H. pylori pathogenesis.

Dose assessment of melatonin in sepsis (DAMSEL2) study: Pharmacokinetics of two doses of oral melatonin in patients with sepsis.

Sepsis is defined as a dysregulated host response to infection, and high-dose melatonin has been proposed as a treatment due to its antioxidant and anti-inflammatory properties. However, there are no data describing the pharmacokinetics of high-dose oral melatonin in critically ill patients. We undertook an open-label trial to determine the tolerance of melatonin administration in these patients and pharmacokinetic analysis, to inform a planned randomised controlled trial. Two cohorts of critically ill patients with sepsis due to community-acquired pneumonia received either 20 or 50 mg oral melatonin liquid as a single dose. Blood samples and clinical measures were analysed over the next 24 h. Melatonin was well tolerated and there were no adverse events. Pharmacokinetic modelling showed that a semiphysiological model, which incorporates saturable first-pass hepatic extraction, was a good fit for our data. Maximum levels of melatonin were extremely high in patients receiving the 50 mg dose and levels of the major metabolite were much lower than expected and not different from those seen after 20 mg, suggesting saturation at the higher dose. We conclude that 20 mg seems a suitable dose of liquid melatonin in patients with sepsis.

Neutrophil Survival Signaling During Francisella tularensis Infection.

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.

Metabolic Reprogramming Mediates Delayed Apoptosis of Human Neutrophils Infected With Francisella tularensis.

Neutrophils (polymorphonuclear leukocytes, PMNs) have a distinctively short lifespan, and tight regulation of cell survival and death is imperative for their normal function. We demonstrated previously that Francisella tularensis extends human neutrophil lifespan, which elicits an impaired immune response characterized by neutrophil dysfunction. Herein, we extended these studies, including our transcriptional profiling data, and employed Seahorse extracellular flux analysis, gas chromatography-mass spectrometry metabolite analysis, flow cytometry and several other biochemical approaches to demonstrate that the delayed apoptosis observed in F. tularensis-infected neutrophils is mediated, in part, by metabolic reprogramming. Specifically, we show that F. tularensis-infected neutrophils exhibited a unique metabolic signature characterized by increased glycolysis, glycolytic flux and glucose uptake, downregulation of the pentose phosphate pathway, and complex glycogen dynamics. Glucose uptake and glycolysis were essential for cell longevity, although glucose-6-phosphate translocation into the endoplasmic reticulum was not, and we identify depletion of glycogen as a potential trigger of apoptosis onset. In keeping with this, we also demonstrate that ablation of apoptosis with the pan-caspase inhibitor Q-VD-OPh was sufficient to profoundly increase glycolysis and glycogen stores in the absence of infection. Taken together, our data significantly advance understanding of neutrophil immunometabolism and its capacity to regulate cell lifespan.

Results from Phase I Extension Study Assessing Pexidartinib Treatment in Six Cohorts with Solid Tumors including TGCT, and Abnormal CSF1 Transcripts in TGCT.

PURPOSE: To assess the response to pexidartinib treatment in six cohorts of adult patients with advanced, incurable solid tumors associated with colony-stimulating factor 1 receptor (CSF1R) and/or KIT proto-oncogene receptor tyrosine kinase activity. PATIENTS AND METHODS: From this two-part phase I, multicenter study, pexidartinib, a small-molecule tyrosine kinase inhibitor that targets CSF1R, KIT, and FMS-like tyrosine kinase 3 (FLT3), was evaluated in six adult patient cohorts (part 2, extension) with advanced solid tumors associated with dysregulated CSF1R. Adverse events, pharmacokinetics, and tumor responses were assessed for all patients; patients with tenosynovial giant cell tumor (TGCT) were also evaluated for tumor volume score (TVS) and patient-reported outcomes (PRO). CSF1 transcripts and gene expression were explored in TGCT biopsies. RESULTS: Ninety-one patients were treated: TGCT patients (n = 39) had a median treatment duration of 511 days, while other solid tumor patients (n = 52) had a median treatment duration of 56 days. TGCT patients had response rates of 62% (RECIST 1.1) and 56% (TVS) for the full analysis set. PRO assessments for pain showed improvement in patient symptoms, and 76% (19/25) of TGCT tissue biopsy specimens showed evidence of abnormal CSF1 transcripts. Pexidartinib treatment of TGCT resulted in tumor regression and symptomatic benefit in most patients. Pexidartinib toxicity was manageable over the entire study. CONCLUSIONS: These results offer insight into outcome patterns in cancers whose biology suggests use of a CSF1R inhibitor. Pexidartinib results in tumor regression in TGCT patients, providing prolonged control with an acceptable safety profile.

Non-Randomized Trial of Dornase Alfa for Acute Respiratory Distress Syndrome Secondary to Covid-19.

Background: The most severe cases of Coronavirus-Disease-2019 (COVID-19) develop into Acute Respiratory Distress Syndrome (ARDS). It has been proposed that oxygenation may be inhibited by extracellular deoxyribonucleic acid (DNA) in the form of neutrophil extracellular traps (NETs). Dornase alfa (Pulmozyme, Genentech) is recombinant human deoxyribonuclease I that acts as a mucolytic by cleaving and degrading extracellular DNA. We performed a pilot study to evaluate the effects of dornase alfa in patients with ARDS secondary to COVID-19. Methods: We performed a pilot, non-randomized, case-controlled clinical trial of inhaled dornase for patients who developed ARDS secondary to COVID-19 pneumonia. Results: Improvement in arterial oxygen saturation to inhaled fraction of oxygen ratio (PaO2/FiO2) was noted in the treatment group compared to control at day 2 (95% CI, 2.96 to 95.66, P-value = 0.038), as well as in static lung compliance at days 3 through 5 (95% CI, 4.8 to 19.1 mL/cmH2O, 2.7 to 16.5 mL/cmH2O, and 5.3 to 19.2 mL/cmH2O, respectively). These effects were not sustained at 14 days. A reduction in bronchoalveolar lavage fluid (BALF) myeloperoxidase-DNA (DNA : MPO) complexes (95% CI, -14.7 to -1.32, P-value = 0.01) was observed after therapy with dornase alfa. Conclusion: Treatment with dornase alfa was associated with improved oxygenation and decreased DNA : MPO complexes in BALF. The positive effects, however, were limited to the time of drug delivery. These data suggest that degradation of extracellular DNA associated with NETs or other structures by inhaled dornase alfa can be beneficial. We propose a more extensive clinical trial is warranted. Clinical Trial Registration: ClinicalTrials.gov, Identifier: NCT04402970.

Terrestrial mesopredators did not increase after top-predator removal in a large-scale experimental test of mesopredator release theory.

Removal or loss of top-predators has been predicted to cause cascading negative effects for ecosystems, including mesopredator release. However, reliable evidence for these processes in terrestrial systems has been mixed and equivocal due, in large part, to the systemic and continued use of low-inference study designs to investigate this issue. Even previous large-scale manipulative experiments of strong inferential value have been limited by experimental design features (i.e. failure to prevent migration between treatments) that constrain possible inferences about the presence or absence of mesopredator release effects. Here, we build on these previous strong-inference experiments and report the outcomes of additional large-scale manipulative experiments to eradicate Australian dingoes from two fenced areas where dingo migration was restricted and where theory would predict an increase in extant European red foxes, feral cats and goannas. We demonstrate the removal and suppression of dingoes to undetectable levels over 4-5 years with no corresponding increases in mesopredator relative abundances, which remained low and stable throughout the experiment at both sites. We further demonstrate widespread absence of negative relationships between predators, indicating that the mechanism underpinning predicted mesopredator releases was not present. Our results are consistent with all previous large-scale manipulative experiments and long-term mensurative studies which collectively demonstrate that (1) dingoes do not suppress red foxes, feral cats or goannas at the population level, (2) repeated, temporary suppression of dingoes in open systems does not create mesopredator release effects, and (3) removal and sustained suppression of dingoes to undetectable levels in closed systems does not create mesopredator release effects either. Our experiments add to similar reports from North America, Asia, Europe and southern Africa which indicate that not only is there a widespread absence of reliable evidence for these processes, but there is also a large and continually growing body of experimental evidence of absence for these processes in many terrestrial systems. We conclude that although sympatric predators may interact negatively with each other on smaller spatiotemporal scales, that these negative interactions do not always scale-up to the population level, nor are they always strong enough to create mesopredator suppression or release effects.

Lenvatinib plus pembrolizumab in patients with either treatment-naive or previously treated metastatic renal cell carcinoma (Study 111/KEYNOTE-146): a phase 1b/2 study.

BACKGROUND: Despite advances in the first-line treatment of metastatic renal cell carcinoma (RCC), there is an unmet need for options to address disease progression during or after treatment with immune checkpoint inhibitors (ICIs). Pembrolizumab and lenvatinib are active as monotherapies in RCC; thus, we aimed to evaluate the combination of lenvatinib plus pembrolizumab in these patients. METHODS: We report results of the metastatic RCC cohort from an open-label phase 1b/2 study of lenvatinib plus pembrolizumab in patients aged at least 18 years with selected solid tumours and an Eastern Cooperative Oncology Group performance status of 0-1. Oral lenvatinib at 20 mg was given once daily along with intravenous pembrolizumab at 200 mg once every 3 weeks. Patients remained on study drug treatment until disease progression, development of unacceptable toxicity, or withdrawal of consent. Efficacy was analysed in patients with clear cell metastatic RCC receiving study drug by previous therapy grouping: treatment naive, previously treated ICI naive (previously treated with at least one line of therapy but not with an anti-PD-1 or anti-PD-L1 ICI), and ICI pretreated (ie, anti-PD-1 or anti-PD-L1) patients. Safety was analysed in all enrolled and treated patients. The primary endpoint was the objective response rate at week 24 per immune-related Response Evaluation Criteria In Solid Tumors (irRECIST) by investigator assessment. This trial is registered with ClinicalTrials.gov (NCT02501096) and with the EU Clinical Trials Register (EudraCT2017-000300-26), and is closed to new participants. FINDINGS: Between July 21, 2015, and Oct 16, 2019, 145 patients were enrolled in the study. Two patients had non-clear cell RCC and were excluded from the efficacy analysis (one in the treatment-naive group and one in the ICI-pretreated group); thus, the population evaluated for efficacy comprised 143 patients (n=22 in the treatment-naive group, n=17 in the previously treated ICI-naive group, and n=104 in the ICI-pretreated group). All 145 enrolled patients were included in the safety analysis. The median follow-up was 19·8 months (IQR 14·3-28·4). The number of patients with an objective response at week 24 by irRECIST was 16 (72·7%, 95% CI 49·8-89·3) of 22 treatment-naive patients, seven (41·2%, 18·4-67·1) of 17 previously treated ICI-naive patients, and 58 (55·8%, 45·7-65·5) of 104 ICI-pretreated patients. Of 145 patients, 82 (57%) had grade 3 treatment-related adverse events and ten (7%) had grade 4 treatment-related adverse events. The most common grade 3 treatment-related adverse event was hypertension (30 [21%] of 145 patients). Treatment-related serious adverse events occurred in 36 (25%) patients, and there were three treatment-related deaths (upper gastrointestinal haemorrhage, sudden death, and pneumonia). INTERPRETATION: Lenvatinib plus pembrolizumab showed encouraging antitumour activity and a manageable safety profile and might be an option for post-ICI treatment of metastatic RCC. FUNDING: Eisai and Merck Sharp & Dohme.

Mechanisms Driving Neutrophil-Induced T-cell Immunoparalysis in Ovarian Cancer.

T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.See related Spotlight by Cassatella, p. 725.

Microtubules and Dynein Regulate Human Neutrophil Nuclear Volume and Hypersegmentation During H. pylori Infection.

Neutrophils (also called polymorphonuclear leukocytes, PMNs) are heterogeneous and can exhibit considerable phenotypic and functional plasticity. In keeping with this, we discovered previously that Helicobacter pylori infection induces N1-like subtype differentiation of human PMNs that is notable for profound nuclear hypersegmentation. Herein, we utilized biochemical approaches and confocal and super-resolution microscopy to gain insight into the underlying molecular mechanisms. Sensitivity to inhibition by nocodazole and taxol indicated that microtubule dynamics were required to induce and sustain hypersegmentation, and super-resolution Stimulated Emission Depletion (STED) imaging demonstrated that microtubules were significantly more abundant and longer in hypersegmented cells. Dynein activity was also required, and enrichment of this motor protein at the nuclear periphery was enhanced following H. pylori infection. In contrast, centrosome splitting did not occur, and lamin B receptor abundance and ER morphology were unchanged. Finally, analysis of STED image stacks using Imaris software revealed that nuclear volume increased markedly prior to the onset of hypersegmentation and that nuclear size was differentially modulated by nocodazole and taxol in the presence and absence of infection. Taken together, our data define a new mechanism of hypersegmentation that is mediated by microtubules and dynein and as such advance understanding of processes that regulate nuclear morphology.

IL-5 mediates monocyte phenotype and pain outcomes in fibromyalgia.

ABSTRACT: Fibromyalgia (FM) is characterized by widespread chronic pain, fatigue, and somatic symptoms. The influence of phenotypic changes in monocytes on symptoms associated with FM is not fully understood. The primary aim of this study was to take a comprehensive whole-body to molecular approach in characterizing relationships between monocyte phenotype and FM symptoms in relevant clinical populations. Lipopolysaccharide-evoked and spontaneous secretion of IL-5 and other select cytokines from circulating monocytes was higher in women with FM compared to women without pain. In addition, greater secretion of IL-5 was significantly associated with pain and other clinically relevant psychological and somatic symptoms of FM. Furthermore, higher levels of pain and pain-related symptoms were associated with a lower percentage of intermediate monocytes (CD14++/CD16+) and a greater percentage of nonclassical monocytes (CD14+/CD16++) in women with FM. Based on findings from individuals with FM, we examined the role of IL-5, an atypical cytokine secreted from monocytes, in an animal model of widespread muscle pain. Results from the animal model show that IL-5 produces analgesia and polarizes monocytes toward an anti-inflammatory phenotype (CD206+). Taken together, our data suggest that monocyte phenotype and their cytokine profiles are associated with pain-related symptoms in individuals with FM. Furthermore, our data show that IL-5 has a potential role in analgesia in an animal model of FM. Thus, targeting anti-inflammatory cytokines such as IL-5 secreted by circulating leukocytes could serve as a promising intervention to control pain and other somatic symptoms associated with FM.

Use of focused intensive care echo in the diagnosis of primary cardiac angiosarcoma.

Focused Intensive Care Echo can aid diagnosis in complex patients where initial investigations are non-diagnostic. FICE is an essential skill for critical care physicians, and bedside echocardiography should be a standard investigation for all critical care patients.

Melatonin Is a Feasible, Safe, and Acceptable Intervention in Doctors and Nurses Working Nightshifts: The MIDNIGHT Trial.

Nightshift working is associated with sleep deprivation, fatigue and attention/concentration deficits which, in healthcare workers, may impact on patient safety. Clinical staff in the UK routinely work several 12 h nightshifts in a row at about 1-3 month intervals. We investigated the feasibility and acceptability of a crossover trial of melatonin administration in clinical staff working nightshifts with an exploration of effects on sleep measures and attention/concentration tasks. This was a pilot, double-blinded, randomized, placebo-controlled crossover feasibility trial in doctors and nurses working 3 consecutive nightshifts at a tertiary referral hospital in the UK. Twenty five male and female subjects were randomized to receive either 6mg Circadin™ slow release melatonin or placebo before sleep after each consecutive nightshift, followed by a washout period, before crossing over to the other experimental arm. We used actigraphy for objective assessment of sleep parameters. The trial design was feasible and acceptable to participants with negligible side effects, but elevated melatonin levels were prolonged during the active arm (P=0.016). Double digit addition testing, a concentration/attention task, improved with melatonin treatment (P<0.0001). Lapses of vigilance or judgement while doctors or nurses are working nightshifts could impact on patient safety and melatonin may be a useful intervention. This study supports the conclusion that a larger definitive trial of this design is both feasible and safe. Clinical Trial Registration: identifier ISRCTN15529655. https://www.isrctn.com/.

Avatrombopag, an Alternate Treatment Option to Reduce Platelet Transfusions in Patients with Thrombocytopenia and Chronic Liver Disease-Integrated Analyses of 2 Phase 3 Studies.

AIMS: Thrombocytopenia complicates the management of patients with chronic liver disease (CLD) undergoing invasive procedures with a bleeding risk. Until recently, prophylactic platelet transfusion was the only treatment option, but has significant safety and efficacy limitations. Phase 3 data demonstrated the superiority of avatrombopag to placebo in reducing platelet transfusions for bleeding, supporting its recent approval. METHODS: Integrated analyses of pooled data (N = 435) from two randomized, double-blind, placebo-controlled, phase 3 studies assessed the original efficacy endpoints. Additional analyses included subgroup analyses, alternate Baseline platelet count definitions, and another efficacy endpoint. RESULTS: Avatrombopag was superior to placebo in increasing patients not requiring a platelet transfusion or rescue procedure, those achieving a platelet count ≥50 × 109/L on Procedure Day, and the change in platelet counts from Baseline. The avatrombopag treatment effect was consistently positive across clinically important disease and Baseline clinical characteristic subgroups, and using alternate Baseline platelet count cohort definitions. Similarly, more avatrombopag-treated patients achieved ≥50 × 109/L platelets with an increase of ≥20 × 109/L from Baseline. The incidence and severity of adverse events were similar between avatrombopag and placebo. Further, safety data demonstrated a low risk for thromboembolic events and hepatotoxicity. CONCLUSION: These integrated analyses confirmed the superiority of avatrombopag to placebo in reducing platelet transfusions or rescue procedures for bleeding in patients with thrombocytopenia and CLD scheduled to undergo an invasive procedure, and its tolerable safety profile. Importantly, these data warrant reconsideration of clinical decision making regarding the need to treat thrombocytopenia in patients with CLD. This trial was registered with NCT01972529 and NCT01976104.

P2X4 Receptors on Muscle Macrophages Are Required for Development of Hyperalgesia in an Animal Model of Activity-Induced Muscle Pain.

Activity-induced pain is common in those with chronic musculoskeletal pain and limits participation in daily activities and exercise. Our laboratory developed a model of activity-induced pain and shows that depletion of muscle macrophages prevents development of hyperalgesia. Adenosine triphosphate (ATP) is released from fatiguing muscle and activates purinergic receptors (P2X), and P2X4 receptors are expressed on macrophages. We hypothesized that exercise releases ATP to activate P2X4 receptors on muscle macrophages, which subsequently release interleukin-1ß (IL-1ß) to produce hyperalgesia. In an animal model of activity-induced pain, using male and female C57BL6/J mice, we show increased expression of P2X4 on muscle macrophages, and blockade of P2X4 receptors in muscle prevented development of hyperalgesia. Using a lentivirus expressing an artificial micro-RNA to P2X4 under the control of a CD68 promoter, we decreased expression of P2X4 mRNA in cultured macrophages, decreased expression of P2X4 protein in muscle macrophages in vivo, and prevented development of activity-induced hyperalgesia. We further show that macrophages primed with LPS differentially released IL-1ß when treated with ATP in neutral or acidic pH. Lastly, blockade of IL-1ß in muscle prevented development of hyperalgesia in this model. Thus, our data suggest that P2X4 receptors could be a valid pharmacological target to control activity-induced muscle pain experienced by patients with chronic musculoskeletal pain.

Phagocytosis and neutrophil extracellular traps.

Neutrophils are recruited rapidly to sites of infection in response to host- and/or microbe-derived proinflammatory molecules. At such sites, neutrophils phagocytose microbes and are activated to produce superoxide and other reactive oxygen species (ROS). In addition, neutrophils contain stores of antimicrobial peptides and enzymes that work in concert with ROS to kill ingested microbes. Neutrophils can also release chromosomal DNA bound with antimicrobial peptides and enzymes to form web-like structures known as extracellular traps. Neutrophil extracellular traps (NETs) have been reported to ensnare and kill microbes and are commonly considered to be an important component of innate host defense. Notably, the formation of NETs is most often reported as a cytolytic process. Whereas intraphagosomal killing of microbes sequesters cytotoxic antimicrobial molecules that would otherwise damage host tissues, the formation of NETs and associated extracellular release of these molecules can contribute to host tissue destruction and disease. Here we compare and contrast phagocytosis and NETs in host defense, with emphasis on recent studies of NETs that ultimately underscore the importance of phagocytosis as the primary means by which neutrophils eliminate microbes.

Relationship Between Maternal Serum Bile Acid Levels and Fetal Cardiac Troponin-I Levels in Asymptomatic Pregnant Patients at Term: A Cross-sectional Observational Study.

Objective The objective of our study was to determine if a correlation exists between maternal total bile acid levels, degree of maternal pruritus, and fetal cardiac troponin-I levels in asymptomatic patients without a diagnosis of intrahepatic cholestasis of pregnancy. Study design In this cross-sectional observational study, patients were enrolled at the time of the scheduled term cesarean section. Maternal blood was drawn for fasting total bile acid levels and cord blood was collected for fetal cardiac troponin-I levels. Pruritus during pregnancy was quantified by the patient on a visual analog scale (VAS). Correlation coefficients between these variables were calculated. Results There was not a positive correlation between any of the primary variables studied (pruritis, total bile acid, cardiac troponin I). Pearson's R between total bile acid and cardiac troponin I was -0.058 (weak correlation in the opposite direction), and between total bile acid and pruritus severity, it was 0.031. Conclusion In patients without intrahepatic cholestasis of pregnancy, higher levels of maternal total bile acids did not correlate with increased cardiac troponin-I (fetal cardiomyocyte damage) or increased pruritus. This supports the current theory that the adverse outcomes associated with intrahepatic cholestasis of pregnancy require a threshold value of total bile acids, one high enough to cause clinically significant maternal pruritis.