The SARS-CoV-2 virus significantly evolved with several new strains identified. Transmissibility has increased with each strain, corresponding with a decrease in mortality. We previously demonstrated novel dysfunctional neutrophil responses in alpha COVID-19 patients compared to community acquired pneumonia controls. We investigated if strain variation altered our previously observed neutrophil dysfunction. Patients with COVID-19 not requiring intensive care were recruited between January 2021 and May 2022 from the Queen Elizabeth Hospital Birmingham; 41 patients with alpha, 32 delta and 14 omicron. Neutrophils isolated from whole blood were investigated for phagocytosis of labelled Streptococcus pneumoniae, transwell migration towards interleukin-8, neutrophil extracellular (NET) formation and surface phenotype. Neutrophil phagocytosis was significantly increased in delta variant (vs alpha p=0.0012, vs omicron p=0.0209). Transwell migration was also significantly reduced in omicron patients (vs alpha p=0.0002, vs delta p=0.0129). There was a significant reduction in NET formation from omicron patients (vs alpha p=0.0031, vs delta p=0.0231). Compared to alpha patients, neutrophils from omicron patients had reduced expression of CD10 (p=0.0004), CD54 (p=0.0015), CD62L (p=0.0013) and CD11c (p<0.0001). CXCR2 expression was higher in neutrophils from omicron compared to alpha patients (p=0.0001). Neutrophil function and phenotype differ between the variants of COVID-19 infection in hospitalised patients. Neutrophil changes suggest more accurate migration in omicron patients, leading to decreased neutrophil host-mediated tissue damage. This may contribute to the overall milder clinical omicron phenotype. Ongoing research and review of therapies remains important alongside viral evolution.

Background: Smoking cessation remains the only effective strategy for slowing the progression of Chronic Obstructive Pulmonary Disease (COPD). E-cigarettes (EC) have been shown effective in smoking cessation trials, however real-world data suggests long-term usage after cessation is common. Acute exposure studies suggest signals of harm in immune cells from young healthy volunteers, yet data is lacking on the impact of vaping on COPD patient neutrophils. Hypothesis: EC exposure, despite being less harmful than tobacco smoking, impairs neutrophil functions, indicating that EC exposure may enhance the pathophysiology of COPD patients. Methods: Neutrophils were isolated from the blood of healthy older adults and COPD patients by Percoll density gradient separation, then exposed to a total of 40 EC puffs (4 seconds on, 30 seconds off), in a modified hypoxia chamber. Viability was analysed by flow cytometry; annexin V and Propidium iodide. NETosis was measured using SYTOX® green, following a 3hour phorbol myristate acetate (PMA) incubation. Results: EC exposure did not affect viability of neutrophils from healthy older adult age matched controls (AMC) or COPD patient neutrophils. Baseline and PMA stimulated NETosis was lower in in COPD patient neutrophils compared to AMC, indicating an overall lower capacity to produce ex vivo. Our preliminary data suggests nicotine containing EC exposure reduced PMA stimulated NETosis in both groups. Conclusion: Exposure to EC vapour impairs the capacity to produce NETs in response in both healthy older adult and COPD patient neutrophils. Further reduction in COPD neutrophil NETosis following exposure to EC, suggests additional harm in this group.

Background: Tissue hypoxia is implicated as a contributing factor in the progressive fibrosis seen in idiopathic pulmonary fibrosis (IPF). IPF lung tissue contains hypoxia markers and patients experience hypoxaemia. Although hypoxia drives fibroproliferation in epithelial cells and fibroblasts, the effect on the homeostatic function of macrophages is unknown. Hypothesis: Hypoxia impairs alveolar macrophage (AM) function and promotes the profibrotic phenotype seen in IPF AMs. Aim: To model the effects of hypoxia on AM function. Methods: Human AMs isolated by lavage of normal lung tissue (n=4) from non-smokers undergoing cancer resection, were cultured for 48 hours in hypoxia (1% O2, 5% CO2) or normoxia (21% O2, 5% CO2). AM viability and phagocytosis were measured by flow cytometry using Annexin V/Propidium Iodide staining and heat-killed Streptococcus pneumoniae respectively. mRNA was measured by reverse transcriptase qPCR. Results: Hypoxia impaired AM phagocytosis by 46%, whilst viability was unaffected. mRNA expression of vascular endothelial growth factor A (VEGF-A) was increased in hypoxia. However, matrix metalloproteinase (MMP)-9 and MMP-7; pro-resolution factor, transforming growth factor β; pro-inflammatory cytokines, tumour necrosis factor and interleukin 1β, were unchanged in hypoxic conditions. Conclusion: These results show that hypoxia impairs AM phagocytosis. Consequently, in hypoxic IPF lung, bacterial clearance may be reduced, increasing acute exacerbation risk. Upregulated VEGFA expression by hypoxic macrophages may also be a pro-fibrotic driver. Further work is needed to explore the mechanism of these changes, and ascertain effects on the lung environment.

Rational: Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics. Cell functions are interwoven pathways, so understanding the effect of COVID-19 across neutrophil function may identify therapeutic targets. We examined neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia (CAP) patients. Methods: Isolated neutrophils underwent ex vivo analyses for migration, phagocytosis and NETosis, and the effect of PI3K inhibition. Circulating DNAse 1 activity and levels of cfDNA were measured. Results: Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p=0.0397, A) and NETosis (p=0.0366, B), but impaired phagocytosis (p=0.0236, C) associated with impaired ROS generation (p<0.0001). COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p=0.0153) and impaired DNAse activity (p<0.0.001, D). Ex vivo inhibition of PI3K γ and δ reduced NET release by COVID-19 neutrophils (p=0.0156). Conclusion: COVID-19 is associated with neutrophil dysfunction across all main effector functions, with elevated migration, impaired antimicrobial responses and elevated NETosis. These changes represent a clear mechanism for tissue damage and highlight that targeting neutrophil function via PI3k may help modulate COVID-19 severity.

Background The global COVID-19 pandemic has significantly evolved since first identified in November 2019. Viral antigenic shift has seen the emergence of several new strains leading to spikes in infection. Transmissibility has increased with each emerging strain, corresponding with a decrease in overall mortality. We have previously demonstrated dysfunctional neutrophil responses in COVID-19 patients compared to community acquired pneumonia control.1 Here we aimed to correlate strain severity with change in underlying neutrophil function. Methods Patients were recruited between January 2021 to May 2022 from the Queen Elizabeth Hospital Birmingham. Patients were recruited if not admitted to the ITU. 33 patients presented infection with alpha COVID-19, 13 delta COVID-19 and 14 omicron COVID-19 infections. Neutrophils were isolated from whole blood. Phagocytosis of labelled Streptococcus pneumoniae, transwell migration towards IL-8, neutrophil extracellular trap (NETosis) formation and surface extracellular marker expression were analysed. Results There were no significant differences in demographics between patients recruited for different variants. Patients infected with the alpha variant had significantly raised CRP compared to omicron patients (p=0.0427). Phagocytosis was significantly increased between alpha and delta variants (figure 1A). Transwell migration was significantly reduced in omicron patients compared to the other variants (figure 1B). There was reduction in neutrophil extracellular trap in omicron patients compared to alpha variant patients (figure 1C). Compared to alpha patients, neutrophils from omicron patients had reduced expression of CD10 (p=0.0004), CD54 (p=0.0015), CD62L (p=0.0013) and CD11c (p<0.0001). CXCR2 expression was higher in neutrophils from omicron patients (p=0.0001), while there was no difference in CD11b, CXCR4, PD-L1 and CD66b expression. Discussion Our results showing changes in neutrophil "function and phenotype differ between variants of COVID-19 infection, potentially reflect viral evolution. This change in neutrophil function may contribute to the evolving clinical phenotype observed in patients. Our population of ward-based COVID-19 patients represents the majority of inpatient hospital burden where early intervention may prevent clinical deterioration. Targeting neutrophil function may be an effective way of improving infection outcome in the future. Reference Belchamber K, et al. Altered neutrophil phenotype and function in non-ICU hospitalised COVID-19 patients correlated with disease severity. medRxiv, 2021: p. 2021.06.08.21258535.

Background Internationally, researchers have called for evidence to support tackling health inequalities during the COVID19 pandemic. UK Office for National Statistics data suggests that patients in regions of most deprived overall Index of Multiple Deprivation Score (IMDS) are twice as likely to die of COVID19 than other causes. The Intensive Care National Audit and Research Centre (ICNARC) report that Black, Asian and Minority Ethnic (BAME) patients account for 34% of critically ill COVID19 patients nationally despite constituting 14% of the population. This paper is the first to explore the roles of social determinants of health, including specific IMDS sub-indices with indicators for household overcrowding deprivation (Barriers to Housing and Services subindex (BHS)), indoor housing quality deprivation and outdoor air pollution deprivation (Living Environment subindex (LE)) as modulators of presentation, Intensive Care Unit(ITU) admission and outcomes among COVID19 patients of all ethnicities. Methods An in-depth retrospective cohort study of 408 hospitalised COVID19 patients admitted to Queen Elizabeth Hospital, Birmingham was conducted. Quantitative data analyses including two-step cluster analyses were applied. Results Patients admitted from highest LE deprivation sub-indices were at increased risk of presenting with multi-lobar pneumonia and, in turn, ITU admission. Patients admitted from highest BHS deprivation sub-indices were at increased risk of ITU admission. BAME patients were more likely, than white patients, to present with multi-lobar pneumonia, be admitted to ITU and be admitted from highest BHS and LE deprivation indices. Comorbidities and frailty significantly increased the risk of death among COVID19 patients irrespective of deprivation. Conclusions Air pollution and housing quality deprivation are potential modulators of presentation with multi-lobar pneumonia. Household overcrowding deprivation and presentation with multi-lobar pneumonia are potential modulators of ITU admission. Patents of BAME ethnicity are more likely to be admitted from regions of highest air pollution, housing quality and household overcrowding deprivation; this is likely to contribute an explanation towards the higher ITU admissions reported among COVID19 BAME patients. Consideration of Charlson Comorbidity and Clinical Frailty Scores on admission supports clinicians in stratifying high risk patients. These findings have urgent implications for supporting front line clinical decisions, disseminating practical advice around applying social distancing messages at the household level and informing wider pandemic strategy. This study has been cited by several national and international public bodies including Public Health England and UK Parliament as evidence to support the COVID19 strategic response.

Background : Internationally, researchers have called for evidence to support tackling health inequalities during the Severe acute respiratory syndrome coronavirus 2(COVID19) pandemic. UK Office for National Statistics data suggests that patients in regions of most deprived overall, generic Index of Multiple Deprivation Score(IMDS) are twice as likely to die of COVID19 than other causes. The Intensive Care National Audit and Research Centre (ICNARC) report that BAME patients account for 34% of critically ill COVID19 patients nationally despite constituting 14% of the population. This paper is the first to explore the roles of social determinants of health, including specific IMDS sub-indices with indicators for household quality deprivation, household overcrowding deprivation and air pollution deprivation, as modulators of presentation, Intensive Care Unit(ITU) admission and outcomes among COVID19 patients of all ethnicities. Methods: An in-depth retrospective cohort study of 408 hospitalised COVID19 patients admitted to the Queen Elizabeth Hospital, Birmingham was conducted. Quantitative data analyses including two-step cluster analyses were applied. Results: Patients admitted from highest living environment(LE) deprivation indices were at increased risk of presenting with multi-lobar pneumonia and, in turn, ITU admission whilst patients admitted from highest Barriers to Housing and Services(BHS) deprivation indices were at increased risk of ITU admission. Admission to ITU significantly increased the risk of death. Black, Asian and Minority Ethnic(BAME) patients were more likely, than white patients, to present with multi-lobar pneumonia, be admitted to ITU and be admitted from highest BHS and LE deprivation indices. Comorbidities and frailty significantly increased the risk of death among COVID19 patients irrespective of deprivation. Conclusions: Air pollution and housing quality deprivation are potential modulators of presentation with multi-lobar pneumonia. Household overcrowding deprivation and presentation with multi-lobar pneumonia are potential modulators of ITU admission. Patents of BAME ethnicity are more likely to be admitted from regions of highest air pollution, housing quality and household overcrowding deprivation; this is likely to contribute an explanation towards the higher ITU admissions reported among COVID19 BAME patients. These findings have urgent implications for supporting front line clinical decisions, disseminating practical advice around applying social distancing messages at the household level and informing wider pandemic strategy.

Introduction Malignant pleural effusions (MPE) remain a significant problem with approximately 50% of all cancer patients developing a MPE during their disease process. Our pleural service is one of the largest in the country. This day case service has the potential to accelerate MPE diagnosis, management and thus enhance patient experience. Objectives The aim was to assess the timeline of patients referred to pleural clinic with suspected malignant (unilateral) effusion. Method Retrospective analysis of 178 consecutive patients referred to pleural clinic with suspected MPE from March 2015 to November 2016. Data was collated from electronic patient records, including route of referral, diagnosis methodology, speed of diagnosis (MDT) and procedures performed. Results 126 (70.8%) of the 178 patients had pleural effusion and underwent pleural aspiration. 61 patients (48.4%) had positive malignant fluid cytology. 26 (43%) and 35 (57%) were thoracic and extra thoracic malignancies respectively. Out of the 61 patients, 26 (43%) had systemic treatment and 35 (57%) had palliative management. These patients were diagnosed on average within 17 days from referral to clinic (SD 17.3). Mean time taken from referral to pleural clinic review was 5 days (SD 6.6) and 12.3 days (SD 16.6) elapsed from pleural clinic review to diagnosis. Average time from cytology diagnoses to treatment was 26 days. 20 (16%) patients were referred for VATS (Video Assisted Thoracoscopic Surgery). The average time from VATS diagnosis to treatment was 37 days (for further breakdown see Table 1). The remaining (64%) were benign till to date. Conclusion The data demonstrates that a dedicated pleural service has the ability to rapidly review and diagnose patients with suspected MPE (especially the cytology positive). There is a need for improvement in patient’s timeline for those referred for VATS. Perhaps a dedicated pleural multidisciplinary meeting may help to reduce the delay and improve patient care.

Introduction: cFLIP is a major anti-apoptotic protein that blocks the apoptotic pathway mediated by death receptors. It is overexpressed in many cancers resulting in chemoresistance and limiting the effectiveness of commonly used anticancer therapies. Both the long (FLIPL) and the short (FLIPS) splice forms compete with procaspase 8 for binding with the adaptor protein FADD at the death inducing signaling complex (DISC), which is formed after stimulation of death receptors by their ligands. Materials and Methods:Molecular modeling of the FLIP–FADD interaction was performed using published structures viral FLIP (MC159) and human FADD. Site directed mutagenesis of was performed to study the importance of specific residues of FLIP. Protein–protein interactions were studied by GST-pulldown assay. DISC recruitment of FLIP mutants was investigated in a DISC IP assay. Western blotting was performed to assess protein levels. Results: Modelling identified several key residues which appeared to be important for the cFLIP–FADD interaction. Site directed mutagenesis was performed against these key residues, and a panel of cFLIP and FADD mutants was generated. These mutants were then screened for their ability to interact with either FADD or cFLIP using GST-pulldown assays. A functional assay for DISC recruitment was also carried out to confirm which of these mutations resulted in impaired recruitment. This analysis established the critical amino acids which mediate DED interactions between cFLIP and FADD. Conclusions: Iterative rounds of computer modelling and mutagenesis have characterised the molecular features of the interaction between the pro-apoptotic adaptor molecule FADD and the anti-apoptotic protein FLIP.

BACKGROUND:The terrorist attacks on the World Trade Center (WTC) of September 11, 2001 resulted in the deaths of 2,823 persons. They also generated a long-lasting burden of multiple physical and mental health illnesses among the cohort of 50,000 rescue workers who responded to the attacks and in the 400,000 residents and workers in nearby areas of New York City. A comprehensive health surveillance program was developed from the first months after the accidents and was further developed in the subsequent ears. Individual exposure and health data were stored in ad hoc databases and produced epidemiological outcomes on the various exposure-related illnesses.METHODS:About 10 years of longitudinal assessment of this large cohort of WTC rescue and recovery workers, yielded data from participants in the WTC Screening, Monitoring, and Treatment Program. Police officers, firefighters, construction workers, and municipal workers were included in the cohort. Cumulative and annual incidence were estimated for various physical disorders including asthma, sinusitis, and gastroesophageal reflux disease, mental health disorders including depression, post-traumatic stress disorder [PTSD], and panic disorder. Respiratory functionality was also assessed. Exposure was characterized with qualitative parameter including working on the pile and being engulfed in the dust cloud, and quantitative parameters including the time of arrival on site and the exposure duration.RESULTS:Upper and lower respiratory conditions such as rhinosinusitis and asthma have been found in a significant number of people in WTC-exposed populations. A lack of appropriate respiratory protection may have contributed to these effects. Other commonly observed physical health conditions include gastro-esophageal reflux disease, obstructive sleep apnea and musculo-skeletal injuries. Many WTC-exposed individuals also suffer from mental health conditions, primarily post-traumatic stress disorder, depression, panic disorder, and substantial stress reaction. Recent studies suggest that WTC exposure may increase the risk of cancer and of mortality from cardiac disease.CONCLUSION:Ten years of systematic health surveillance after the 9/11 WTC attacks, show long lasting burden of physical and mental health problems. Continued monitoring and treatment of this population is needed for early diagnoses of initial clinical conditions that can be treated more effectively. The experience of September 11 offers also indications on how to approach the acute and delayed health effects of civilian catastrophes. Critical lessons are derived about the importance of having trained responders--medical and non-medical--in place in advance of disasters, and about the need to proceed with adequate exposure assessment in a timely manner.

Background King County has a large Latino immigrant population, some of whom are undocumented. Many undocumented immigrants do not receive medical care due to fear of deportation, language barriers, or lack of knowledge of health care resources. Lack of primary care in this population is associated with more emergency department visits for preventable conditions, more hospitalizations, and increased spread of infection. To address the lack of health care access in undocumented immigrants, a brochure was created describing undocumented immigrants9 right to health care and the commitment of a local community health center to provide medical services to all people. Methods The project idea emerged from a community assessment, conversations with Latino immigrants in the clinic and discussions with administrators about health care gaps in the community. The brochure was directed towards undocumented Latino immigrants because Latinos represent 70% of the undocumented immigrants in this country. These brochures, written in Spanish, will be available to the community health center, and will be given to community leaders such as priests who can provide the brochures in the community. Summary Undocumented Latino immigrants face barriers to receiving primary care for a variety of reasons. In an attempt to increase health care access for this population, an educational brochure was created. The two main objectives for the brochure were to let undocumented immigrants know that it is safe, from an immigration perspective, to receive care at a local community health center and to describe the available resources at the health center. It is hoped that the brochure will reach the target population and help increase primary health care utilization among this underserved group.