Multicenter flow cytometry proficiency testing of canine blood and lymph node samples

Background: Flow cytometry (FC) is used increasingly in veterinary medicine for further characterization of hematolymphoid cells. Guidelines for optimizing assay performance and interpretation of results are limited, and concordance of results across laboratories is unknown. Objectives: This study aimed to determine inter-investigator agreement on the interpretation of FC results from split samples analyzed in different laboratories using various protocols, cytometers, and software; and on the interpretation of archived FC standard (FCS) data files contributed by the different investigators. Methods: This was a multicenter observational cross-sectional study. Anticoagulated blood or lymph node aspirate samples from nine client-owned dogs were aliquoted and shipped to participating laboratories. Samples were analyzed with individual laboratory-developed protocols. In addition, FCS files from a set of separate samples from 11 client-owned dogs were analyzed by participating investigators. A person not associated with the study tabulated the results and interpretations. Agreement of interpretations was assessed with Fleiss\u2019 kappa statistic. Results: Prolonged transit times affected sample quality for some laboratories. Overall agreement among investigators regarding the FC sample interpretation was strong (\u3ba = 0.86 \ub1 0.19, P <.001), and for specific categories, ranged from moderate to perfect. Agreement of the lymphoproliferation or other leukocyte sample category from the analysis of the FCS files was weak (\u3ba = 0.58 \ub1 0.05, P <.001). Conclusions: Lymphoproliferations were readily identified by FC, but identification of the categories of hematolymphoid neoplasia in fresh samples or archived files was variable. There is a need for a more standardized approach to maximize the enormous potential of FC in veterinary medicine

Gallbladder microbiota in healthy dogs and dogs with mucocele formation

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Caveolae in ventricular myocytes are required for stretch-dependent conduction slowing

Mechanical stretch of cardiac muscle modulates action potential propagation velocity, causing potentially arrhythmogenic conduction slowing. The mechanisms by which stretch alters cardiac conduction remain unknown, but previous studies suggest that stretch can affect the conformation of caveolae in myocytes and other cell types. We tested the hypothesis that slowing of action potential conduction due to cardiac myocyte stretch is dependent on caveolae. Cardiac action potential propagation velocities, measured by optical mapping in isolated mouse hearts and in micropatterned mouse cardiomyocyte cultures, decreased reversibly with volume loading or stretch, respectively (by 19±5% and 26±4%). Stretch-dependent conduction slowing was not altered by stretch-activated channel blockade with gadolinium or by GsMTx-4 peptide, but was inhibited when caveolae were disrupted via genetic deletion of caveolin-3 (Cav3 KO) or membrane cholesterol depletion by methyl-β-cyclodextrin. In wild-type mouse hearts, stretch coincided with recruitment of caveolae to the sarcolemma, as observed by electron microscopy. In myocytes from wild-type but not Cav3 KO mice, stretch significantly increased cell membrane capacitance (by 98±64%), electrical time constant (by 285±149%), and lipid recruitment to the bilayer (by 84±39%). Recruitment of caveolae to the sarcolemma during physiologic cardiomyocyte stretch slows ventricular action potential propagation by increasing cell membrane capacitance