The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Monitoring of Bip promoter activation during cancer cell growth by bioluminescence imaging technique at single cell level

Cancer cells require the regulation of organelle-specific unfolded protein responses, such as endoplasmic reticulum (ER) stress, because of their increased metabolic activity during rapid proliferation and cell growth, which are executed through the activation of diverse signaling pathways. In this study, we focused on the dynamic regulation of ER stress in accordance with cancer cellular demand, and we performed real-time monitoring of the activation of the binding immunoglobulin protein (Bip) promoter, which is one of the most responsive genes to ER stress during cancer cell growth, in two and three dimensional (2D and 3D) cell culture using bioluminescence imaging at the single-cell level. Bioluminescence images were obtained from living single cancer cells after transient transfection of the reporter gene, and we observed Bip promoter activation during cell growth. Bip promoter activation was also observed in 2D and 3D culture using stably transfected glioblastoma cancer cells with the reporter gene. The Bip promoter was activated especially in dividing cells during cell growth. We then performed real-time monitoring of Bip promoter activation by bioluminescence imaging in tissue slices obtained from U251/pBipPro-Luc tumors. Luminescence intensity was not constant and was different in individual regions of the tumor slices, and the Bip promoter was activated in several regions during monitoring in vitro. These results show that real-time monitoring by bioluminescence imaging at the single-cell level is a suitable tool for not only gene analysis of signal transduction and regulation of the dynamics of the unfolded protein response in cancer cells but also for the evaluation of the efficacy of anti-cancer agents, and could provide additional information that has been difficult to obtain using conventional assays

Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω‑Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid

Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid that plays various beneficial roles in organisms from bacteria to humans. Although its beneficial physiological functions are well-recognized, a molecular probe that enables the monitoring of its in vivo behavior without abolishing its native functions has not yet been developed. Here, we designed and synthesized an ω-ethynyl EPA analog (eEPA) as a tool for analyzing the in vivo behavior and function of EPA. eEPA has an ω-ethynyl group tag in place of the ω-methyl group of EPA. An ethynyl group has a characteristic Raman signal and can be visualized by Raman scattering microscopy. Moreover, this group can specifically react in situ with azide compounds, such as those with fluorescent group, via click chemistry. In this study, we first synthesized eEPA efficiently based on the following well-known strategies. To introduce four C–C double bonds, a coupling reaction between terminal acetylene and propargylic halide or tosylate was employed, and then, by simultaneous and stereoselective partial hydrogenation with P-2 nickel, the triple bonds were converted to <i>cis</i> double bonds. One double bond and an ω-terminal C–C triple bond were introduced by Wittig reaction with a phosphonium salt harboring an ethynyl group. Then, we evaluated the in vivo function of the resulting probe by using an EPA-producing bacterium, <i>Shewanella livingstonensis</i> Ac10. This cold-adapted bacterium inducibly produces EPA at low temperatures, and the EPA-deficient mutant (ΔEPA) shows growth retardation and abnormal morphology at low temperatures. When eEPA was exogenously supplemented to ΔEPA, eEPA was incorporated into the membrane phospholipids as an acyl chain, and the amount of eEPA was about 5% of the total fatty acids in the membrane, which is comparable to the amount of EPA in the membrane of the parent strain. Notably, by supplementation with eEPA, the growth retardation and abnormal morphology of ΔEPA were almost completely suppressed. These results indicated that eEPA mimics EPA well and is useful for analyzing the in vivo behavior of EPA

Serum IgG titers against periodontal pathogens are associated with cerebral hemorrhage growth and 3-month outcome.

To assess the influence of periodontal disease on cerebral hemorrhage and its clinical course, we examined the association of the serum IgG titer of periodontal pathogens with hemorrhage growth and 3-month outcome. We consecutively enrolled 115 patients with acute cerebral hemorrhage (44 females, aged 71.3 ± 13.1 years) and used ELISA to evaluate the serum IgG titers of 9 periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter (A.) actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Fusobacterium (F.) nucleatum, Treponema denticola, Tannerella forsythensis, Campylobacter rectus, and Eikenella corrodens. Significant hematoma growth was defined as an increase in the volume of >33% or an absolute increase in the volume of >12.5 mL. A poor outcome was defined as a 3 or higher on the modified Rankin Scale. We observed hemorrhage growth in 13 patients (11.3%). Multivariate analysis revealed that increased IgG titers of A. actinomycetemcomitans independently predicted the elevated hemorrhage growth (odds ratio 5.26, 95% confidence interval 1.52-18.25, p = 0.01). Notably, augmented IgG titers of F. nucleatum but not A. actinomycetemcomitans led to a poorer 3-month outcome (odds ratio 7.86, 95% confidence interval 1.08-57.08, p = 0.04). Thus, we demonstrate that elevated serum IgG titers of A. actinomycetemcomitans are an independent factor for predicting cerebral hemorrhage growth and that high serum IgG titers of F. nucleatum may predict a poor outcome in patients with this disease. Together, these novel data reveal how systemic periodontal pathogens may affect stroke patients, and, should, therefore, be taken into consideration in the management and treatment of these individuals

Serum IgG titers to periodontal pathogens predict 3-month outcome in ischemic stroke patients.

Several cohort studies have shown that periodontal disease is associated with an increased risk for stroke. However, it remains unclear whether serum antibody titers for a specific periodontal pathogen are associated with outcome after ischemic stroke, and which kinds of pathogens are associated with ischemic stroke. We examined the relationship between serum IgG titers to periodontal pathogens and outcome in ischemic stroke patients. A total of 445 patients with acute ischemic stroke (194 female [44.0%], mean age 71.9±12.3 years) were registered in this study. Serum IgG titers to 9 periodontal pathogens (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum, Treponema denticola, Tannerella forsythensis, Campylobacter rectus, Eikenella corrodens) were evaluated using the enzyme-linked immunosorbent assay (ELISA) method. An unfavorable outcome was defined as a 3 or higher on the modified Rankin Scale. The proportion of patients with unfavorable outcome was 25.4% (113 patients). Based on multivariate logistic regression analysis, numbers of IgG antibodies positive for periodontal pathogens (odds ratio 1.20, 95% CI 1.02-1.41, p = 0.03) were independent predictors of unfavorable outcome in ischemic stroke patients

Association between periodontal disease due to Campylobacter rectus and cerebral microbleeds in acute stroke patients.

Oral health conditions and cerebral small vessel disease, such as white matter lesions or cerebral microbleeds (CMBs), are associated with the incidence of stroke. The purpose of this study was to examine the associations between oral health conditions (serum IgG titers of periodontal pathogens) with the presence or severity of CMBs in acute stroke patients. From January 2013 to April 2016, acute stroke patients were registered in two hospitals. Serum samples were evaluated for antibody titers against 9 periodontal pathogens using the ELISA method. The cut-off points for reactivity (the positive decision point) to each antigen were defined as more than a mean ELISA unit + 1 standard deviation (after logarithmic transformation) in all subjects. CMBs were evaluated on T2*-weighted MRI. In all, 639 patients were evaluated (ischemic, n = 533 and hemorrhagic, n = 106; 73.1 ± 12.9 years old). Among these patients, 627 were available for CMB evaluation. Among the 9 evaluated periodontal pathogens, only Campylobacter rectus (C. rectus) was associated with the presence of CMBs. the prevalence of positive serum antibody titers against C. rectus was higher among patients with CMBs than among those without CMBs (14.6% vs. 8.7%, P = 0.025). In addition, positive serum antibody titers against C. rectus remained one of the factors associated with the presence of CMBs in multivariate logistic analysis (odds ratio 2.03, 95% confidence interval 1.19-3.47, P = 0.010). A positive serum antibody titer against C. rectus was associated with the presence of CMBs in acute stroke patients

Short-term inhalation of sargramostim with concomitant high-dose steroids does not hasten recovery in moderate COVID-19 pneumonia: a double-blind, randomised, placebo-controlled trial

Granulocyte-macrophage colony stimulating factor (GM-CSF) inhalation may alleviate pulmonary inflammation caused by viral pneumonia. To investigate this, we evaluated its efficacy on COVID-19 pneumonia. This double-blind, randomised, placebo-controlled study (ClinicalTrials.gov: NCT04642950) evaluated patients in the first half of 2021 at seven Japanese hospitals. Hospitalised patients with COVID-19 pneumonia with moderate hypoxaemia inhaled sargramostim or placebo for 5 days. The primary endpoint was days to achieve a ≥ 2-category improvement from baseline on a modified 7-category ordinal scale. Secondary endpoints included degree of oxygenation, defined by amount of oxygen supply, and serum CCL17 level. Seventy-five patients were randomly assigned in a 2:1 ratio to receive sargramostim or placebo, of which 47 and 23 were analysed, respectively. No difference was observed between groups regarding the primary endpoint (8.0 and 7.0 days for sargramostim and placebo, respectively) or in the secondary endpoints, except for CCL17. A post hoc sub-analysis indicated that endpoint assessments were influenced by concomitant corticosteroid therapy. When the cumulative corticosteroid dose was ≤500 mg during Days 1–5, recovery and oxygenation were faster in the sargramostim group than for placebo. Bolus dose corticosteroids were associated with temporarily impaired oxygenation and delayed clinical recovery. The increase in serum CCL17, a candidate prognostic factor, reflected improvement with sargramostim inhalation. The number of adverse events was similar between groups. Two serious adverse events were observed in the sargramostim group without causal relation. Inhaled sargramostim was likely to be effective for COVID-19 pneumonia unless the concomitant corticosteroid dose was high.</p