ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites.

Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.

Inflammatory Pain Reduces C Fiber Activity-Dependent Slowing in a Sex-Dependent Manner, Amplifying Nociceptive Input to the Spinal Cord.

C fibers display activity-dependent slowing (ADS), whereby repetitive stimulation (≥1 Hz) results in a progressive slowing of action potential conduction velocity, which manifests as a progressive increase in response latency. However, the impact of ADS on spinal pain processing has not been explored, nor whether ADS is altered in inflammatory pain conditions. To investigate, compound action potentials were made, from dorsal roots isolated from rats with or without complete Freund's adjuvant (CFA) hindpaw inflammation, in response to electrical stimulus trains. CFA inflammation significantly reduced C fiber ADS at 1 and 2 Hz stimulation rates. Whole-cell patch-clamp recordings in the spinal cord slice preparation with attached dorsal roots also demonstrated that CFA inflammation reduced ADS in the monosynaptic C fiber input to lamina I neurokinin 1 receptor-expressing neurons (1-10 Hz stimulus trains) without altering the incidence of synaptic response failures. When analyzed by sex, it was revealed that females display a more pronounced ADS that is reduced by CFA inflammation to a level comparable with males. Cumulative ventral root potentials evoked by long and short dorsal root stimulation lengths, to maximize and minimize the impact of ADS, respectively, demonstrated that reducing ADS facilitates spinal summation, and this was also sex dependent. This finding correlated with the behavioral observation of increased noxious thermal thresholds and enhanced inflammatory thermal hypersensitivity in females. We propose that sex/inflammation-dependent regulation of C fiber ADS can, by controlling the temporal relay of nociceptive inputs, influence the spinal summation of nociceptive signals contributing to sex/inflammation-dependent differences in pain sensitivity.SIGNIFICANCE STATEMENT The intensity of a noxious stimulus is encoded by the frequency of action potentials relayed by nociceptive C fibers to the spinal cord. C fibers conduct successive action potentials at progressively slower speeds, but the impact of this activity-dependent slowing (ADS) is unknown. Here we demonstrate that ADS is more prevalent in females than males and is reduced in an inflammatory pain model in females only. We also demonstrate a progressive delay of C fiber monosynaptic transmission to the spinal cord that is similarly sex and inflammation dependent. Experimentally manipulating ADS strongly influences spinal summation consistent with sex differences in behavioral pain thresholds. This suggests that ADS provides a peripheral mechanism that can regulate spinal nociceptive processing and pain sensation.

Small GTPase-dependent regulation of leukocyte-endothelial interactions in inflammation.

Inflammation is a complex biological response that serves to protect the body's tissues following harmful stimuli such as infection, irritation or injury and initiates tissue repair. At the start of an inflammatory response, pro-inflammatory mediators induce changes in the endothelial lining of the blood vessels and in leukocytes. This results in increased vascular permeability and increased expression of adhesion proteins, and promotes adhesion of leukocytes, especially neutrophils to the endothelium. Adhesion is a prerequisite for neutrophil extravasation and chemoattractant-stimulated recruitment to inflammatory sites, where neutrophils phagocytose and kill microbes, release inflammatory mediators and cross-talk with other immune cells to co-ordinate the immune response in preparation for tissue repair. Many signalling proteins are critically involved in the complex signalling processes that underpin the inflammatory response and cross-talk between endothelium and leukocytes. As key regulators of cell-cell and cell-substratum adhesion, small GTPases (guanosine triphosphatases) act as important controls of neutrophil-endothelial cell interactions as well as neutrophil recruitment to sites of inflammation. Here, we summarise key processes that are dependent upon small GTPases in leukocytes during these early inflammatory events. We place a particular focus on the regulation of integrin-dependent events and their control by Rho and Rap family GTPases as well as their regulators during neutrophil adhesion, chemotaxis and recruitment.

Melatonin limits paclitaxel-induced mitochondrial dysfunction in vitro and protects against paclitaxel-induced neuropathic pain in the rat.

Chemotherapy-induced neuropathic pain is a debilitating and common side effect of cancer treatment. Mitochondrial dysfunction associated with oxidative stress in peripheral nerves has been implicated in the underlying mechanism. We investigated the potential of melatonin, a potent antioxidant that preferentially acts within mitochondria, to reduce mitochondrial damage and neuropathic pain resulting from the chemotherapeutic drug paclitaxel. In vitro, paclitaxel caused a 50% reduction in mitochondrial membrane potential and metabolic rate, independent of concentration (20-100 µmol/L). Mitochondrial volume was increased dose-dependently by paclitaxel (200% increase at 100 µmol/L). These effects were prevented by co-treatment with 1 µmol/L melatonin. Paclitaxel cytotoxicity against cancer cells was not affected by co-exposure to 1 µmol/L melatonin of either the breast cancer cell line MCF-7 or the ovarian carcinoma cell line A2780. In a rat model of paclitaxel-induced painful peripheral neuropathy, pretreatment with oral melatonin (5/10/50 mg/kg), given as a daily bolus dose, was protective, dose-dependently limiting development of mechanical hypersensitivity (19/43/47% difference from paclitaxel control, respectively). Melatonin (10 mg/kg/day) was similarly effective when administered continuously in drinking water (39% difference). Melatonin also reduced paclitaxel-induced elevated 8-isoprostane F2 α levels in peripheral nerves (by 22% in sciatic; 41% in saphenous) and limited paclitaxel-induced reduction in C-fibre activity-dependent slowing (by 64%). Notably, melatonin limited the development of mechanical hypersensitivity in both male and female animals (by 50/41%, respectively), and an additive effect was found when melatonin was given with the current treatment, duloxetine (75/62% difference, respectively). Melatonin is therefore a potential treatment to limit the development of painful neuropathy resulting from chemotherapy treatment.

Does unemployment worsen babies' health? A tale of siblings, maternal behaviour, and selection.

We study in-utero exposure to economic fluctuations on birth outcomes by exploiting geographical variation in the unemployment rate across local areas in England, and by comparing siblings born to the same mother. Using rich individual data from hospital administrative records for 2003-2012, babies' health is found to be strongly pro-cyclical. This overall result masks marked differences between babies born in the most affluent areas whose health at birth improves in a recession, and babies born in the average-to-lowest income deprived areas whose health deteriorates. Maternal alcohol consumption, smoking, and delay in the first antenatal care assessment - combined with parental income loss, are found to drive the results. While differences in maternal risky behaviours can explain the heterogenous effects.

Will policy to constrain GP referrals damage health? Evidence using practice level NHS emergency admissions administrative data.

Attempts to control hospital expenditure by managing down General Practitioner (GP) referrals are reoccurring features of UK health policy. However, despite the best efforts of GPs to benchmark referral criteria, patient health may be damaged and other costs created by constraining referrals to targets. This paper adopts an indirect method to indicate whether rationing practice referrals may damage population health by distorting the use of health resources away from patients' interests. We utilise a comprehensive database at practice level that allows us to explore the relationship between referrals and emergency admissions, using a panel fixed effects model of admissions that allows for the endogeneity of referrals. We find that practice referrals are positively and partially correlated with emergency admissions, which is consistent with time-varying practice-level sickness shocks driving the relationship between referrals and emergency care, rather than shocks to the practice willingness to refer, or to system reforms. In this environment, government policy to constrain referrals may make the elective care less responsive to practice-level variations in illness, and thereby lower health.

Are more GPs associated with a reduction in emergency hospital admissions? A quantitative study on GP referral in England.

BACKGROUND: Recent studies have found an association between access to primary care and accident and emergency attendances, with better access associated with fewer attendances. Analyses of an association with emergency admissions, however, have produced conflicting findings. AIM: This study investigated whether emergency admission rates in an area are associated with 1) the number of GPs, and 2) mean size of GP practice. DESIGN AND SETTING: Analysis was conducted utilising Hospital Episode Statistics, the numbers of GPs and GP practices, Office for National Statistics population data, Quality and Outcomes Framework prevalence data, and Index of Multiple Deprivation data, from 2004/2005 to 2011/2012, for all practices in England. METHOD: Regression analysis of panel data with fixed effects to address 1) a potential two-way relationship between the numbers of GPs and emergency admissions, and 2) unobservable characteristics of GP practices. RESULTS: There is not a statistically significant relationship between the number of GPs in a primary care trust area and the number of emergency admissions, when analysing all areas. In deprived areas, however, a higher number of GPs is associated with lower emergency admissions. There is also a lower emergency admission rate in areas in which practices are on average larger, holding GP supply constant. CONCLUSION: An increase in GPs was found to reduce emergency admissions in deprived areas, but not elsewhere. Areas in which GPs are concentrated into larger practices showed reduced levels of emergency admissions, all else being equal.

The 5-Phosphatase SHIP2 Promotes Neutrophil Chemotaxis and Recruitment.

Neutrophils, the most abundant circulating leukocytes in humans have key roles in host defense and in the inflammatory response. Agonist-activated phosphoinositide 3-kinases (PI3Ks) are important regulators of many facets of neutrophil biology. PIP3 is subject to dephosphorylation by several 5' phosphatases, including SHIP family phosphatases, which convert the PI3K product and lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) into PI(3,4)P2, a lipid second messenger in its own right. In addition to the leukocyte restricted SHIP1, neutrophils express the ubiquitous SHIP2. This study analyzed mice and isolated neutrophils carrying a catalytically inactive SHIP2, identifying an important regulatory function in neutrophil chemotaxis and directionality in vitro and in neutrophil recruitment to sites of sterile inflammation in vivo, in the absence of major defects of any other neutrophil functions analyzed, including, phagocytosis and the formation of reactive oxygen species. Mechanistically, this is explained by a subtle effect on global 3-phosphorylated phosphoinositide species. This work identifies a non-redundant role for the hitherto overlooked SHIP2 in the regulation of neutrophils, and specifically, neutrophil chemotaxis/trafficking. It completes an emerging wider understanding of the complexity of PI3K signaling in the neutrophil, and the roles played by individual kinases and phosphatases within.

Cross-talk between Rho GTPases and PI3K in the neutrophil.

Neutrophils are short-lived, abundant peripheral blood leukocytes that provide a first line of defense against bacterial and fungal infections while also being a key part of the inflammatory response. Chemokines induce neutrophil recruitment to inflammatory sites, where neutrophils perform several diverse functions that are aimed at fighting infections. Neutrophil effector functions are tightly regulated processes that are governed by an array of intracellular signaling pathways and initiated by receptor-ligand binding events. Dysregulated neutrophil activation can result in excessive inflammation and host damage, as is evident in several autoimmune diseases. Rho family small GTPases and agonist-activated phosphoinositide 3-kinases (PI3Ks) represent 2 classes of key regulators of the highly specialized neutrophil. Here we review cross-talk between these important signaling intermediates in the context of neutrophil functions. We include PI3K-dependent activation of Rho family small GTPases and of their guanine nucleotide exchange factors and GTPase activating proteins, as well as Rho GTPase-dependent regulation of PI3K.

HoxB8 neutrophils replicate Fcγ receptor and integrin-induced neutrophil signaling and functions.

Neutrophils are short-lived, terminally differentiated leukocytes that form an essential part of host immunity and play a key role in acute and chronic inflammation. The analysis of these important cells is hindered by the fact that neutrophils are not amenable to culture, transfection, or transduction. Conditionally HoxB8-immortalized mouse hematopoietic progenitors are suitable for in vitro differentiation of a range of myeloid cells, including neutrophils. Integrins and FcγRs are cell surface receptors, the ligation of which is required for a range of neutrophil functions that are important in health and disease. We show here that HoxB8 neutrophils express major neutrophil integrins and FcγRs. They respond to FcγR and integrin stimulation in a manner that is comparable with primary neutrophils, in terms of intracellular signaling. HoxB8 neutrophils also perform a range of FcγR/integrin-dependent neutrophil functions, including, generation of reactive oxygen species, degranulation, and chemotaxis. Our findings suggest that HoxB8 neutrophils represent a faithful experimental model system for the analysis of Fc and integrin receptor-dependent neutrophil functions.

Comparative morbidities and the share of emergencies in hospital admissions in deprived areas: a method and evidence from English administrative data.

OBJECTIVE: Various studies find that the share of emergencies in hospital admissions is higher in deprived areas, but both the explanation and policy implications are unclear. We estimate the extent to which this finding is due to a different disease mix in deprived areas, rather than other explanations such as patient behaviour and general practitioner effectiveness. DESIGN: Secondary analysis using English Hospital Episode Statistics data, with disease for elective and emergency admissions in 2008/2009 coded at 186 blocks or 1230 categories and aggregated to lower layer super output area of residence. It is then linked to an appropriate measure of deprivation. OUTCOME MEASURES: The difference in the share of emergencies in hospital admissions between communities in the highest and lowest deciles of deprivation; and the percentage of this difference that is explained if areas in the least deprived decile have the same disease mix as those in the most deprived decile. RESULTS: Using the finest disease classification scheme (1230 categories), 71% of the higher share of admissions that were emergencies in decile 1 areas relative to decile 10, is explained by the "adverse" case mix (CM) in deprived areas. The remainder reflects the higher relative use of emergency care in deprived areas for the same conditions. Higher incidence of respiratory and circulatory diseases in deprived areas explains about 30% of the CM contribution. Diseases of the digestive system and abdomen have a high relative use of emergency care in deprived areas. CONCLUSIONS: The higher use of emergency care in deprived areas is primarily a symptom of the higher prevalence of diseases which have high national rates of emergency to elective care-especially respiratory diseases-rather than an indication of less effective primary care. Nevertheless, there is a higher share of emergency care in admissions in deprived areas for several diseases, most notably of the digestive system.

The ageing society and emergency hospital admissions.

There is strong policy interest, in England as elsewhere, in slowing the growth in emergency hospital admissions, which for older people increased by 3.3% annually between 2001/2 and 2012/3. Resource constrains have increased the importance of understanding rising emergency admissions, which in policy discourse is often explained by population aging. This study examines how far the rise in emergency admissions of people over 65 was due to population ageing, how far to the changing likelihood of entering hospital at each age, and how far to other factors which might be more amenable to policy measures. It shows that: admission rates rose with age from age 40 upward but each successive birth cohort experienced lower emergency admission rates after standardising for age and other effects. This downward cohort effect largely offset the consequences of an older and larger population aged over 65. Other factors which could explain increasing admissions, such as new technologies or rising expectations, appear more important than the changing size and age structure of the population as drivers of rising emergency admissions in old age. These findings suggest that stemming the rate of increase in emergency admissions of older people may be feasible, if challenging, despite population ageing.

Hospital care and costs for homeless people.

The national picture of the comparative costs and diagnoses of hospitalised homeless patients are examined using the 'no fixed abode' flag in English hospital statistics. Comparable studies sample patients in single cities, eg New York and Toronto. The most common diagnosis is substance misuse; the share of homeless NHS patients with this diagnosis is rising, and now equals that found in North American cities. About half of the cost of homeless patients relates to diagnoses of mental illness, although these comprise a much smaller share of homeless patients than in North America. Hospital costs for homeless patients - both total and per admission - have fallen significantly in recent years, primarily because of fewer admissions and shorter lengths of stay for mentally ill patients. Aims to reduce NHS costs at the level of individual institutions have often shaped policy. Broader policy to prevent and reduce homelessness offers substantial long-term reductions in the cost of chronic care.

Randomised controlled trial of GP-led in-hospital management of homeless people ('Pathway').

Homeless people have complex problems. GP enhanced care (Pathway) has shown benefits. We performed a randomised, -parallel arm trial at two large inner city hospitals. Inpatient homeless adults were randomly allocated to either standard care (all management by the hospital-based clinical team) or enhanced care with input from a homeless care team. The hospital data system provided healthcare usage information, and we used questionnaires to assess quality of life. 206 patients were allocated to enhanced care and 204 to usual care. Length of stay (up to 90 days after admission) did not differ between groups (standard care 14.0 days, enhanced care 13.3 days). Average reattendance at the emergency department within a year was 5.8 visits in the standard care group and 4.8 visits with enhanced care, but this decrease was not significant. -Quality of life scores after discharge (in 108 patients) improved with enhanced care (EQ-5D-5L score increased by 0.12 [95% CI 0.032 to 0.22] compared wtih 0.03 [-0.1 to 0.15; p=0.076] with standard care). The proportion of people sleeping on the streets after discharge was 14.6% in the standard care arm and 3.8% in the enhanced care arm (p=0.034). The quality-of-life cost per quality-adjusted life-year was £26,000. The Pathway approach doesn't alter length of stay but improves quality of life and reduces street -homelessness.

Use of vocalic information in the identification of /s/ and /sh/ by children with cochlear implants.

OBJECTIVE: When a syllable such as "sea" or "she" is spoken, listeners with normal hearing extract evidence of the fricative consonant from both the fricative noise and the following vocalic segment. If the fricative noise is made ambiguous, listeners may still perceive "s" or "sh" categorically, depending on information in the vocalic segment. Do children whose auditory experience comes from electrical stimulation also display this effect, in which a subsequent segment of speech disambiguates an earlier segment? DESIGN: Unambiguous vowels were appended to ambiguous fricative noises to form tokens of the words "she," "sea," "shoe," and "Sue." A four-choice identification test was undertaken by children with normal hearing (N = 29), prelingually deaf children with the Nucleus Spectra-22 implant system using the SPEAK coding strategy (N = 13), postlingually deafened adults with the same implant system (N = 26), and adults with normal hearing (N = 10). The last group undertook the test before and after the stimuli were processed to simulate the transformations introduced by the SPEAK coding strategy. RESULTS: All four groups made use of vocalic information. Simulated processing reduced the use made by normal-hearing adults. Implanted subjects made less use than the other groups, with no significant difference between implanted children and implanted adults. The highest levels of use by implanted subjects were within one standard deviation of the mean level displayed when normal-hearing adults listened to processed stimuli. Analyses showed that the SPEAK strategy distorted formant contours in the vocalic segments of the stimuli in ways that are compatible with the errors of identification made by implanted subjects. CONCLUSIONS: Some children with implants can extract information from a following vowel to disambiguate a preceding fricative noise. The upper limit on this ability may be set by distortions introduced by the implant processor, rather than by the auditory experience of the child.