An Interpretable Classification Model Using Gluten-Specific TCR Sequences Shows Diagnostic Potential in Coeliac Disease.

Coeliac disease (CeD) is a T-cell mediated enteropathy triggered by dietary gluten which remains substantially under-diagnosed around the world. The diagnostic gold-standard requires histological assessment of intestinal biopsies taken at endoscopy while consuming a gluten-containing diet. However, there is a lack of concordance between pathologists in histological assessment, and both endoscopy and gluten challenge are burdensome and unpleasant for patients. Identification of gluten-specific T-cell receptors (TCRs) in the TCR repertoire could provide a less subjective diagnostic test, and potentially remove the need to consume gluten. We review published gluten-specific TCR sequences, and develop an interpretable machine learning model to investigate their diagnostic potential. To investigate this, we sequenced the TCR repertoires of mucosal CD4+ T cells from 20 patients with and without CeD. These data were used as a training dataset to develop the model, then an independently published dataset of 20 patients was used as the testing dataset. We determined that this model has a training accuracy of 100% and testing accuracy of 80% for the diagnosis of CeD, including in patients on a gluten-free diet (GFD). We identified 20 CD4+ TCR sequences with the highest diagnostic potential for CeD. The sequences identified here have the potential to provide an objective diagnostic test for CeD, which does not require the consumption of gluten.

Utility of Bulk T-Cell Receptor Repertoire Sequencing Analysis in Understanding Immune Responses to COVID-19.

Measuring immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), can rely on antibodies, reactive T cells and other factors, with T-cell-mediated responses appearing to have greater sensitivity and longevity. Because each T cell carries an essentially unique nucleic acid sequence for its T-cell receptor (TCR), we can interrogate sequence data derived from DNA or RNA to assess aspects of the immune response. This review deals with the utility of bulk, rather than single-cell, sequencing of TCR repertoires, considering the importance of study design, in terms of cohort selection, laboratory methods and analysis. The advances in understanding SARS-CoV-2 immunity that have resulted from bulk TCR repertoire sequencing are also be discussed. The complexity of sequencing data obtained by bulk repertoire sequencing makes analysis challenging, but simple descriptive analyses, clonal analysis, searches for specific sequences associated with immune responses to SARS-CoV-2, motif-based analyses, and machine learning approaches have all been applied. TCR repertoire sequencing has demonstrated early expansion followed by contraction of SARS-CoV-2-specific clonotypes, during active infection. Maintenance of TCR repertoire diversity, including the maintenance of diversity of anti-SARS-CoV-2 response, predicts a favourable outcome. TCR repertoire narrowing in severe COVID-19 is most likely a consequence of COVID-19-associated lymphopenia. It has been possible to follow clonotypic sequences longitudinally, which has been particularly valuable for clonotypes known to be associated with SARS-CoV-2 peptide/MHC tetramer binding or with SARS-CoV-2 peptide-induced cytokine responses. Closely related clonotypes to these previously identified sequences have been shown to respond with similar kinetics during infection. A possible superantigen-like effect of the SARS-CoV-2 spike protein has been identified, by means of observing V-segment skewing in patients with severe COVID-19, together with structural modelling. Such a superantigen-like activity, which is apparently absent from other coronaviruses, may be the basis of multisystem inflammatory syndrome and cytokine storms in COVID-19. Bulk TCR repertoire sequencing has proven to be a useful and cost-effective approach to understanding interactions between SARS-CoV-2 and the human host, with the potential to inform the design of therapeutics and vaccines, as well as to provide invaluable pathogenetic and epidemiological insights.

High Prevalence of Pre-Existing Liver Abnormalities Identified Via Autopsies in COVID-19: Identification of a New Silent Risk Factor?

A high prevalence of hepatic pathology (in 17 of 19 cases) was reported in post-mortem (PM) examinations of COVID-19 patients, undertaken between March 2020 and February 2021 by a single autopsy pathologist in two English Coronial jurisdictions. The patients in our cohort demonstrated high levels of recognised COVID-19 risk factors, including hypertension (8/16, 50%), type 2 diabetes mellitus (8/16, 50%) and evidence of arteriopathy 6/16 (38%). Hepatic abnormalities included steatosis (12/19; 63%), moderate to severe venous congestion (5/19; 26%) and cirrhosis (4/19; 21%). A subsequent literature review indicated a significantly increased prevalence of steatosis (49%), venous congestion (34%) and cirrhosis (9.3%) in COVID-19 PM cases, compared with a pre-pandemic PM cohort (33%, 16%, and 2.6%, respectively), likely reflecting an increased mortality risk in SARS-CoV-2 infection for patients with pre-existing liver disease. To corroborate this observation, we retrospectively analysed the admission liver function test (LFT) results of 276 consecutive, anonymised COVID-19 hospital patients in our centre, for whom outcome data were available. Of these patients, 236 (85.5%) had significantly reduced albumin levels at the time of admission to hospital, which was likely indicative of pre-existing chronic liver or renal disease. There was a strong correlation between patient outcome (length of hospital admission or death) and abnormal albumin at the time of hospital admission (p = 0.000012). We discuss potential mechanisms by which our observations of hepatic dysfunction are linked to a risk of COVID-19 mortality, speculating on the importance of recently identified anti-interferon antibodies.

The process of transitioning for the transgender individual and the nursing imperative: A narrative review.

AIMS: The objectives of this study were to bring the experience of the transitioning process for the transgender population to the nursing profession and address the lack of knowledge to promote improved patient outcomes. DESIGN: This study used a narrative review using the literature matrix method. Because of the dearth of trans specific literature, editorials and monologues were included. DATA SOURCE: A broad search was undertaken across all databases including CINAHL, PubMed, PsycINFO, Ovid MEDLINE, ProQuest Nursing & Allied Health and Google Scholar. Literature from June 1994 to May 2020 was appraised. Non-peer reviewed literature and published texts were procured via Google Alerts. REVIEW METHODS: Selection for inclusion was based on credibility and relevance from a variety of social science disciplines. A narrative analysis was used to identify common themes, incongruencies in schools of thought and perspectives that require consideration. RESULTS: Analysis of the literature revealed the following themes: (a) literature and terminology evolution, (b) transitioning as a process, (c) medicalization of transitioning, (d) generational views on transitioning and (e) needs during transition. CONCLUSION: This review highlights key issues about the transitioning process imperative to nursing when meeting the needs of the transgender population. IMPACT: This review addresses the lack of trans specific literature and lack of consistency in the literature about the understanding of the transitioning process for the transgender population. Main findings? Terminology to explain the transitioning process is ever evolving. Future studies about transitioning need to go beyond the medical lens. Generational views differ in the approach to transitioning, and there are needs unique to this population required during the process. Where and whom will the research impact? The review has significant implications for change in health delivery, nursing policy and formulating nursing practice and education to improve trans competent care.

Use of machine learning to identify a T cell response to SARS-CoV-2.

The identification of SARS-CoV-2-specific T cell receptor (TCR) sequences is critical for understanding T cell responses to SARS-CoV-2. Accordingly, we reanalyze publicly available data from SARS-CoV-2-recovered patients who had low-severity disease (n = 17) and SARS-CoV-2 infection-naive (control) individuals (n = 39). Applying a machine learning approach to TCR beta (TRB) repertoire data, we can classify patient/control samples with a training sensitivity, specificity, and accuracy of 88.2%, 100%, and 96.4% and a testing sensitivity, specificity, and accuracy of 82.4%, 97.4%, and 92.9%, respectively. Interestingly, the same machine learning approach cannot separate SARS-CoV-2 recovered from SARS-CoV-2 infection-naive individual samples on the basis of B cell receptor (immunoglobulin heavy chain; IGH) repertoire data, suggesting that the T cell response to SARS-CoV-2 may be more stereotyped and longer lived. Following validation in larger cohorts, our method may be useful in detecting protective immunity acquired through natural infection or in determining the longevity of vaccine-induced immunity.

Classification of intestinal T-cell receptor repertoires using machine learning methods can identify patients with coeliac disease regardless of dietary gluten status.

In coeliac disease (CeD), immune-mediated small intestinal damage is precipitated by gluten, leading to variable symptoms and complications, occasionally including aggressive T-cell lymphoma. Diagnosis, based primarily on histopathological examination of duodenal biopsies, is confounded by poor concordance between pathologists and minimal histological abnormality if insufficient gluten is consumed. CeD pathogenesis involves both CD4+ T-cell-mediated gluten recognition and CD8+ and γδ T-cell-mediated inflammation, with a previous study demonstrating a permanent change in γδ T-cell populations in CeD. We leveraged this understanding and explored the diagnostic utility of bulk T-cell receptor (TCR) sequencing in assessing duodenal biopsies in CeD. Genomic DNA extracted from duodenal biopsies underwent sequencing for TCR-δ (TRD) (CeD, n = 11; non-CeD, n = 11) and TCR-γ (TRG) (CeD, n = 33; non-CeD, n = 21). We developed a novel machine learning-based analysis of the TCR repertoire, clustering samples by diagnosis. Leave-one-out cross-validation (LOOCV) was performed to validate the classification algorithm. Using TRD repertoire, 100% (22/22) of duodenal biopsies were correctly classified, with a LOOCV accuracy of 91%. Using TCR-γ (TRG) repertoire, 94.4% (51/54) of duodenal biopsies were correctly classified, with LOOCV of 87%. Duodenal biopsy TRG repertoire analysis permitted accurate classification of biopsies from patients with CeD following a strict gluten-free diet for at least 6 months, who would be misclassified by current tests. This result reflects permanent changes to the duodenal γδ TCR repertoire in CeD, even in the absence of gluten consumption. Our method could complement or replace histopathological diagnosis in CeD and might have particular clinical utility in the diagnostic testing of patients unable to tolerate dietary gluten, and for assessing duodenal biopsies with equivocal features. This approach is generalisable to any TCR/BCR locus and any sequencing platform, with potential to predict diagnosis or prognosis in conditions mediated or modulated by the adaptive immune response. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Appropriation of GPIbα from platelet-derived extracellular vesicles supports monocyte recruitment in systemic inflammation.

Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-ß1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-ß1-stimulated cremaster muscle, while in the ApoE-/- model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic.

Prolonged systemic inflammation and damage to the vascular endothelium following intratracheal instillation of air pollution nanoparticles in rats.

BACKGROUND: Exposure to air pollution is associated with cardiovascular disease, including increased morbidity and mortality rates. OBJECTIVE: The aim of this investigation was to assess the effect, in rats, of intratracheal instillation of particulate air pollution on biomarkers of leucocyte activation and vascular endothelial damage. METHODS: Air pollution particles (PM10) were instilled into rats, and blood samples were taken three days and six weeks post instillation. Plasma neutrophil elastase and Von Willebrand factor were measured by ELISA. RESULTS: Plasma neutrophil elastase increased from 175±44 ng/ml at baseline to 288±26 ng/ml 3 days post instillation (p = 0.038). vWF increased from 0.160±0.015 IU/ml at baseline to 0.224±0.015 IU/ml at 3 days post and 0.208±0.01 IU/ml at 6 weeks post (p = 0.006, ANOVA). sICAM-1 increased from 17.75±0.70 ng/ml at baseline to 19.03±0.33 ng/ml at 3 days post and 21.72±1.16 ng/ml at 6 weeks post (p = 0.009, ANOVA). CONCLUSION: Instillation caused prolonged systemic inflammation, activation of blood leucocytes and damage to the vascular endothelium.

Heterogeneous responses of personalised high intensity interval training on type 2 diabetes mellitus and cardiovascular disease risk in young healthy adults.

Hypertension, decreased glucose tolerance, adverse lipid profiles and low physical activity levels are associated with increased type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) risk. High intensity interval training (HIIT), a low volume, reduced time, high intensity programme, may be a useful alternative to current government guidelines which specify a minimum of 150 minutes of physical activity per week. We describe a personalised programme of high intensity exercise which provides significant improvements in CVD risk markers. Healthy volunteers undertook 6 weeks of HIIT. T2DM and CVD risk predictors including glucose tolerance, VO2max, blood pressure (BP), and lipids were measured before and after HIIT. HIIT training was associated with beneficial changes in a range of predictors of blood flow and cardiovascular risk. There was a heterogeneous response to HIIT, with some subjects responding with favourable changes and others being non-responders to HIIT. In responders, HIIT was associated with a statistically significant (p = 0.023) increase in VO2max, from 45.4 (38.4,52.5) to 56.9 (51.2,65.7) (median (interquartile range)(ml/min/kg)). In responders HIIT resulted in a decrease in systolic BP from 127 (126,129) to 116 (106,122) (mmHg) with p = 0.026 and a decrease is diastolic blood pressure from 72 (69,74) to 57 (56,66) with p = 0.026. There was also some evidence of a beneficial change in blood lipid and glucose concentrations with HIIT. In conclusion, personalised HIIT has potential as an intervention to improve blood flow and cardiovascular health.

Increased monocyte actin polymerization in rat blood after intratracheal instillation of air pollution particles.

BACKGROUND: Exposure to particulate air pollution is associated with an increased risk of cardiovascular disease. The mechanism by which exposure increases risk is poorly understood but could involve changes in the flow properties of blood. OBJECTIVE: The aim of this investigation was to assess the effect, in rats, of intratracheal instillation of particulate air pollution on leukocyte flow properties by measurement of polymorphonucleocyte (PMN) and monocyte actin polymerisation. METHODS: Rats were exposed to particulate air pollution by intratracheal instillation of PM10. Blood was collected from test and control animals at 3 days (n=10) and 6 weeks (n=10) after dust instillation. Partial differential leukocyte counts were performed. The intracellular F-actin content of blood PMNs and monocytes was determined by staining with FITC-phalloidin and flow cytometric determination of mean florescence intensity (MFI). RESULTS: There were no significant changes in PMN MFI (p=0.369, ANOVA) or cell counts (p=0.753, ANOVA). There was a significant increase in monocyte MFI (p=0.004, ANOVA) and a decrease in monocyte cell count (p=0.003, ANOVA) in instilled rats. CONCLUSIONS: Intratracheal instillation of air pollution particles resulted in an increase in blood monocyte actin polymerisation, which may cause trapping of monocytes. This could be a mechanism by which exposure to air pollution increases the risk of cardiovascular disease.

The effect of physical activity on haematological predictors of cardiovascular risk: evidence of a dose response.

Cardiovascular disease is a major cause of morbidity and mortality in the developed world. Large epidemiological studies have reported a strong association between increases in haematological factors and increased cardiovascular risk. Haematological risk factors predicted cardiovascular disease at least as strongly as traditional risk factors such as blood lipid concentrations. Lifestyle factors such as physical activity level could significantly reduce risk. The aim of this study was to determine the effect of physical activity level on haematological predictors of cardiovascular risk. Healthy subjects (156) were recruited. Physical activity in subjects was assessed by IPAQ physical activity questionnaire. Blood was collected and blood cell counts were determined by automated cell counter; neutrophil elastase was determined by ELISA. Increased levels of physical activity were associated with reduced red cell (p = 0.001), white cell (p = 0.002) and platelet counts (p = 0.001) and with reduced plasma neutrophil elastase concentration (p = 0.001). There was a continuous linear relationship between increase in physical activity and decrease in haematological risk factors. Hence, the authors conclude that increased levels of physical activity improve the flow properties of blood and thus reduce the risk of developing cardiovascular disease. Even small increases in activity result in some reduction in cardiovascular risk.

Level and pattern of overstory retention interact to shape long-term responses of understories to timber harvest.

In many regions of the world, variable retention has replaced clear-cutlogging as the principal method of regeneration harvest. Partial retention of the overstory is thought to ensure greater continuity of the species and ecological processes that characterize older forests. Level (amount) and spatial pattern of overstory retention are two basic elements of forest structure that can be manipulated to achieve specific ecological or silvicultural objectives. However, experiments that elucidate the relative importance of retention level and pattern (or their interaction) are rare. Here we assess long-term (> 10 yr) responses of forest understories to experimental harvests of mature coniferous forests replicated at five sites in the Pacific Northwest (PNW). Treatments contrast both the level of retention (40% vs. 15% of original basal area) and its spatial distribution (dispersed vs. aggregated in 1-ha patches). For most vascular plant groups (early seral, forest generalist, and late seral), postharvest changes in cover and richness were reduced at higher levels of retention and in dispersed relative to aggregated treatments. Although retained forest patches were stable, changes in adjacent harvested (cleared) areas were significantly greater than in dispersed treatments. Late-seral herbs were highly sensitive to level and pattern of retention, with extirpations most frequent in the cleared areas of aggregated treatments and at low levels of dispersed retention. In contrast, early-seral species were most abundant in these environments. Forest-floor bryophytes exhibited large and persistent declines regardless of treatment, suggesting that threshold levels of disturbance or stress were exceeded. Our results indicate that 15% retention (the minimum standard on federal forestlands in the PNW) is insufficient to retain the abundance or diversity of species characteristic of late-seral forests. Although 1-ha aggregates provide refugia, they are susceptible to edge effects or stochastic processes; thus, smaller aggregates are unlikely to serve this function. The ability to achieve multiple ecological or silvicultural objectives with variable retention will require the spatial partitioning of habitats to include dispersed retention and larger undisturbed aggregates along with cleared areas.

Inflammation, edema, and peripheral blood changes in lung-compromised rats after instillation with combustion-derived and manufactured nanoparticles.

Increased exposure to pollution has been implicated in cardiovascular malfunction, and although studies show a relationship between PM10 and mortality, the exact biological causes are unclear. This study investigated how compromised lungs respond to instillation of nanoparticles, and the links between exposure to nanoparticles and the subsequent effects on the blood. Instillation of diesel exhaust particles and Cabosil caused significant permeability and inflammatory changes in both bleomycin-treated and control lungs, as shown by increased lung surface protein and lung:body weight ratio. This was true in edematous and maximally repairing lungs, but without significant hematological alterations. Plasma viscosity, a renowned marker for cardiovascular disease, correlated strongly statistically with free cell numbers, type I cell marker rT140, and lung acellular protein. These correlations are a new and novel insight into the mechanisms linking air pollution to cardiovascular mortality.

The PPAR-gamma activator, Rosiglitazone, inhibits actin polymerisation in monocytes: involvement of Akt and intracellular calcium.

Monocyte hyperactivation as seen in diabetes results in increased cytoskeletal rigidity and reduced cell deformability leading to microchannel occlusions and microvascular complications. The thiazolidinediones (TZDs) are PPAR-gamma agonists that have been reported to exert beneficial non-metabolic effects on the vasculature. This study demonstrates that the TZD, Rosiglitazone, significantly reduces f-MLP-induced actin polymerisation in human monocytic cells (p < 0.05). Two of the key signalling processes known to be involved in the regulation of cytoskeletal remodelling were investigated: PI(3)K-dependent Akt phosphorylation and intracellular calcium concentration [Ca(2+)](i). The PI(3)K inhibitor, Wortmannin, ameliorated f-MLP-induced actin polymerisation (p < 0.05), while the Ca(2+) sequestration inhibitor, thapsigargin, induced actin depolymerisation (p < 0.05), confirming the involvement of both processes in cytoskeletal remodelling. Rosiglitazone significantly reduced f-MLP activation of Akt (p < 0.05), and significantly increased [Ca(2+)](i) in both resting and f-MLP-stimulated cells (p < 0.05). Therefore, Rosiglitazone interacts with signalling events downstream of occupancy of the f-MLP receptor, to modulate cytoskeletal remodelling in a PPAR-gamma-independent manner. To our knowledge, these results are the first to present evidence that a PPAR-gamma agonist can modulate actin remodelling in monocytes, and may therefore be protective against microvascular damage in diabetes.

A randomized, controlled trial of panic disorder treatment initiation in an emergency department chest pain center.

STUDY OBJECTIVE: We examine the effect of a protocol for panic disorder recognition and treatment initiation on medication initiation rates and medication continuation rates at 1- and 3-month follow-up. METHODS: Enrolled participants, all at low to moderate risk for acute coronary syndrome, completed a 6-hour emergency department chest pain evaluation and panic disorder screen. Participants who had results positive for panic disorder completed the Panic Disorder Module of the Structured Clinical Interview (SCID) for the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) by the research psychiatrist and the treating emergency physician, each blinded to the other's rating. Participants with panic disorder were randomized to either paroxetine initiation at 20 mg/d for 1 month or usual care with telephone follow-up at 3 months. RESULTS: Fifty (32%) of 156 enrolled participants met criteria for panic disorder. All 25 (100%) participants in the paroxetine group initiated treatment, compared with 6 (24%) in the usual care group initiating any form of treatment (P =.006, relative risk [RR] 2.58, 95% confidence interval [CI] 1.20 to 5.58). Fourteen (56%) participants continued paroxetine treatment for at least 1 month, and at 3 months, 9 (36%) continued on some antipanic medication, compared with 2 (8%) for the usual care group (P =.05, RR 3.57, 95% CI 0.84 to 15.8). CONCLUSION: Panic disorder is common and severe in this sample of patients with chest pain. A screening measure and a brief (5-minute) structured interview allowed emergency physicians with no extra training to reliably diagnose panic disorder and initiate pharmacologic treatment.