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Background/Objectives: COVID-19 still represents a lifethreatening disease in individuals with a specific genetic background. We successfully applied a new Machine Learning method on WES data to extract a set of coding variants relevant for COVID- 19 severity. We aim to identify personalized add-on therapy. Method(s): A subset of identified variants, "actionable" by repurposed drugs, were functionally tested by in vitro and in vivo experiments. Result(s): Males with either rare loss of function variants in the TLR7 gene or L412F polymorphism in the TLR3 gene benefit from IFN-gamma, which is specifically defective in activated PBMCs, restoring innate immunity. Females heterozygous for rare variants in the ADAMTS13 gene and males with D603N homozygous polymorphism in the SELP gene benefit from Caplacizumab, which reduces vWF aggregation and thrombus formation. Males with either the low-frequency gain of function variant T201M in CYP19A1 gene or with poly-Q repeats >=23 in the AR gene benefit from Letrozole, an aromatase inhibitor, which restores normal testosterone levels, reducing inflammation and which rescues male golden hamsters from severe COVID-19. Conclusion(s): By adding these commonly used drugs to standard of care of selected patients, the rate of intubation is expected to decrease consistently, especially in patients with high penetrance rare genetic markers, mitigating the effect of the pandemic with a significant impact on the healthcare system.
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Background/Objectives: The severity of the symptoms of coronavirus disease 2019 (COVID-19) has been associated to age, comorbidity, and male sex. Besides virus characteristics, host genetic factors influence the infection outcome. Different genome-wide association studies and meta-analyses investigated the contribution of common variants, whereas the role of rare variants just started to be elucidated. Our goal is to determine the contribution of rare variants to the development of severe COVID-19 in the Italian population. Method(s): We compared the genetic background of 215 severe COVID-19 patients with 1764 individuals from the general population. Rare variants (minor allele frequency <1%) from wholeexome sequencing data were retrieved using a bioinformatics variant discovery pipeline. We tested the impact of rare variants (classified according to their predicted effect on the gene product) both using a burden test design, and an iterative machine learning (ML) approach. Result(s): We identified a total of 690,000 rare variants in the entire examined population. Among them, 250 were associated with COVID-19 severity at a nominal P < 0.05. Gene-based burden test revealed a gene with an excess of loss-of-function mutations at P < 0.05. Finally, the ML approach, analysing all the 690,000 rare variants, identified the best combination of variants that is able to predict COVID-19 severity in our cohort. Conclusion(s): Our work provides new insights on the genetic signature of COVID-19 in the Italian population. The most informative rare variants could be exploited to define individuals' risk profiles to COVID-19 severity for the Italian population.
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Background/Objectives: We previously demonstrated that carrying a single pathogenic CFTR allele increases the risk for COVID-19 severity and mortality rate. We now aim to clarify the role of several uncharacterized rare alleles, including complex (cis) alleles, and in trans combinations. Method(s): LASSO logistic regression was used for the association of sets of variants, stratified by MAF, with severity. Immortalized cystic fibrosis bronchial epithelial cell lines and Fischer Rat Thyroid cells were transfected by plasmid carrying specific CFTR mutations. YFP-based assays were used to measure CFTR activity. Result(s): Here we functionally demonstrate that the rare (MAF=0.007) complex G576V/R668C allelemitigates the disease by a gain of function mechanism. Several novel CFTR ultra-rare (MAF <0.001) alleles were proved to have a reduced function;they are associated with disease severity either alone (single or complex alleles) or with another hypomorphic allele in the second chromosome, with a global reduction of CFTR activity between 40 to 72%. Conclusion(s): CFTR is a bidirectional modulator of COVID-19 outcome. At-risk subjects do not have open cystic fibrosis before viral infection and therefore are not easily recognisable in the general population unless a genetic analysis is performed. As the CFTR activity is partially retained, CFTR potentiator drugs could be an option as add-on therapy for at-risk patients.
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RNA viruses are the major class of human pathogens responsible for many global health crises, including the COVID-19 pandemic. However, the current repertoire of U.S. Food and Drug Administration (FDA)-approved antivirals is limited to only nine out of the known 214 human-infecting RNAviruses, and almost all these antivirals target viral proteins. Traditional antiviral development generally proceeds in a virus-centric fashion, and successful therapies tend to be only marginally effective as monotherapies, due to dose-limiting toxicity and the rapid emergence of drug resistance. Host-based antivirals have potential to alleviate these shortcomings, but do not typically discriminate between infected and uninfected cells, thus eliciting unintended effects. In infected cells where host proteins are repurposed by a virus, normal host protein functions are compromised;a situation analogous to a loss-of-function mutation, and cells harboring the hypomorph have unique vulnerabilities. As well-established in model systems and in cancer therapeutics, these uniquely vulnerable cells can be selectively killed by a drug that inhibits a functionally redundant protein. This is the foundation of synthetic lethality (SL). To test if viral induced vulnerabilities can be exploited for viral therapeutics, we selectively targeted synthetic lethal partners of GBF1, a Golgi membrane protein and a critical host factor for many RNA viruses including poliovirus, Coxsackievirus, Dengue, Hepatitis C and E virus, and Ebola virus. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. A genome-wide chemogenomic CRISPR screen identified synthetic lethal partners of GBF1 and revealed ARF1 as the top hit. Disruption of ARF1, selectively killed cells that synthesize poliovirus 3A alone or in the context of a poliovirus replicon. Combining 3A expression with sub-lethal amounts of GCA - a specific inhibitor of GBF1 further exacerbated the GBF1-ARF1 SL effect. Together our data demonstrate proof of concept for host-based SL targeting of viral infection. We are currently testing all druggable synthetic lethal partners of GBF1 from our chemogenomic CRISPR-screen, in the context of dengue virus infection for their abilities to selectively kill infected cells and inhibit viral replication and infection. Importantly, these SL gene partners of viral-induced hypomorphs only become essential in infected cells and in principle, targeting them will have minimal effects on uninfected cells. Our strategy to target SL interactions of the viral-induced hypomorph has the potential to change the current paradigm for host-based therapeutics that can lead to broad-spectrum antivirals and can be applied to other intracellular pathogens. This work is supported by National Institutes of Health grants R01 GM112108 and P41 GM109824, R21 AI151344 and foundation grant FDN-167277 from the Canadian Institutes of Health Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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Purpose of Study: COVID pneumonia caused by SARS-CoV-2 can result in a depletion of surfactant & lung injury, which resembles neonatal respiratory distress syndrome. Exogenous surfactant has shown promise as a therapeutic option in intubated hospitalized patients. Our preliminary data in human lung organoids (LOs) with a deficiency of surfactant protein B (SP-B) showed an increased viral load compared to normal LOs. Single cell RNA sequencing (scRNAseq) revealed that SP-B-deficient cells showed increased viral entry genes (ACE2 receptor) & dysregulated inflammatory markers emanating from the lung cells themselves. Our objective was to determine: (1) cell-specific transcriptional differences between normal & SP-B deficient human lung cells after infection with SARS-CoV-2 and (2) a therapeutic role of SP-B protein & surfactant in COVID-19 pneumonia. Methods Used: We used normal and SP-B mutant (homozygous, frameshift, loss of function mutation p.Pro133GlnfsTer95, previously known as 121ins2) human induced pluripotent stem cells (hiPSC) and differentiated them into 3D proximal lung organoids. The organoids were infected with the delta variant of SARS-CoV-2 for 24 hours at an MOI of 1. Infected and uninfected organoids were fixed in trizol in triplicate and underwent processing for bulk RNA sequencing. We tested for differentially expressed genes using the program DEseq. We also plated normal iPSC derived lung organoids as a monolayer and pre-treated them with 1mg/ml of Poractant alfa or 5 uM of recombinant SP-B protein. The delta strain of SARS-CoV-2 was added to the 96 wells at an MOI of 0.1 for one hour with shaking, then an overlay with DMEM/CMC/FBS was added and left on for 23 hours. The plate was fixed and stained for nucleocapsid (NC) protein. Summary of Results: Bioinformatic analysis of the bulk RNA sequencing data showed an increase in the multiple cytokines and chemokines in the SP-B mutant LOs compared to control. We also saw differential gene expression patterns in the SP-B mutant LOs including a reduction in SFTPC, FOXA2, and NKX2-1 and an increase in IL1A, VEGFA, PPARG and SMAD3. In the exogenous surfactant experiments, there was a decrease in total expression of viral NC in the Poractant alfa & rSP-B-treated cells compared to SARS-CoV-2 infection alone (p<0.001). Conclusion(s): Surfactant modulates the viral load of SARS-CoV-2 infection in the human lung. Deficiency in SP-B results in the dysregulation of the lung epithelial inflammatory signaling pathways resulting in worsening infections.
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Background: The pathophysiology of viral-infections is highly complex and involves host immunocompetence, host genetics, and gene-environment interactions. We hypothesized that polymorphic variants in host genes, blood group and previous vaccination status against H1N1 may affect the clinical course of covid-19 infection. Method(s): A total of 202 subjects who were RT-PCR negative after Covid-19 infection were recruited. We investigated association between Covid-19 infection (Severity and recovery period) and multiple factors including ABO and Rh blood groups, H1N1 vaccination, polymorphism in Viral susceptibility genes (ACE2 G8790A), and polymorphism in host response genes (ACE I/D rs4646994, IL6- 174G/C, GSTT1/GSTM1 I/D and GSTP1 Ile 105 Val). Result(s): B-ve and O-ve ABO and Rh blood groups had significantly higher Covid-19 recovery period applied on one-vs.-all in a nonparametric t-test (p<0.05). Subjects who had vaccinated themselves against H1N1 presented with a lower recovery-period (p<0.05). Both variables (blood group and H1N1 vaccination) were not however associated with Covid-19 severity. Out of the studied polymorphisms, ACE2 G8790A and GSTT1/GSTM1 were significantly associated with covid-19 infection. Our results indicated that G/G genotype of ACE2 G8790A (OR 3.52, P 0.007) and GSTT1/ GSTM1 null (M1 - / - OR = 3.98, P = 0.0004;T1 - / - OR 3.84, P = 0.004) and double null (M1 - / - /T1 - / - OR = 9.66, P = 0.001) are likely to be associated with an increased risk for severe-critical outcomes in individuals with COVID-19. Other polymorphisms analyzed in this study were found to have no significant association with Covid-19 outcome. Conclusion(s): This study suggests that outcome of Covid-19 infection is affected by both clinical and genetic factors. Thus it seems plausible to utilize these factors as prediction and susceptibility markers in the prognosis of COVID-19, which may help to personalize the treatment.
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Background: Speckle tracking echocardiography provides quantification of myocardial deformation and is useful in the assessment of myocardial function. Right ventricular (RV) strain has been suggested as a sensitive tool for diagnosing cardiomyopathies and assessing long term patient outcomes for patients with pulmonary hypertension, severe tricuspid regurgitation and COVID-19 infection. Recent advances in deep learning (DL) have made promising advances in automating the labour-intensive delineation of regions of interest (ROIs). However, compared to echocardiograms with left ventricular (LV) strain, RV strain data is scarce, making DL models difficult to train. Purpose(s): To investigate whether annotated LV strain data could be beneficial in training a DL model for automatic RV strain when using a limited RV dataset. Method(s): The dataset consisted of anonymized still frames from 141 echocardiograms of the RV in the RV-focused 4 chamber view with corresponding cardiologist-defined ROI. Exams included healthy subjects and patients with heart failure, valvular disease, and conduction abnormalities. ROIs and still images were extracted at the mid-systole, and then quality assessed by an experienced cardiologist as high, medium, or low. The dataset was randomly split into 68%/17%/15% sets for training, validation, and testing. A convolutional neural network for image segmentation (UNet) with a residual neural network (ResNet50) encoder was used, with a combination of binary cross entropy and Dice loss functions. Augmentation, predefined ImageNet weights and pre-training were also employed. For pre-training, 715 still images in the apical 4 chamber view with LV defined ROIs were used, both in their original and horizontally flipped view. Predicted ROIs were reintroduced into commercially available echocardiogram analysis software to automatically calculate longitudinal strain (LS) values. Result(s): The model pre-trained with the flipped LV images achieved the highest performance with a mean absolute difference of 1.26 percentage points (95% confidence interval (CI): 0.62-1.89 percentage points) between manually measured and DL-assisted LS. Median absolute LS difference was 0.85 (95% CI: 0.28-1.57) percentage points. A Bland-Altman plot revealed two outliers and no obvious trends. In comparison, the mean and median absolute LS differences for the model without pre-training were 1.87 (95% CI: 0.73-3.00) and 1.09 (95% CI: 0.56-1.63) percentage points, respectively. Conclusion(s): The current study demonstrates that DL-assisted, automated RV strain measurement is feasible even with a small dataset, and that performance can be increased by using images annotated for LV strain. While the majority of the predicted RV strain results were within the typical range of intra- and interobserver variability, a few outliers were observed. These outliers could possibly be avoided with the use of larger datasets.
ABSTRACT
Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4+ T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2-specific Th cells could be detected as early as days 2-4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8+ T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells - basophiles and eosinophils - were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased "regulatory" Tfh1 cell and increased "pro-inflammatory" Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS-CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of "naive" and memory B cell subsets, as well as increased level of CD27hiCD38hiCD24- plasma cell precursors and atypical CD21low B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.
ABSTRACT
Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4+ T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2-specific Th cells could be detected as early as days 2–4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8+ T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells — basophiles and eosinophils — were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased "regulatory” Tfh1 cell and increased "pro-inflammatory” Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS-CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of "naïve” and memory B cell subsets, as well as increased level of CD27hiCD38hiCD24– plasma cell precursors and atypical CD21low B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.
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Background & Aim: From SARS-CoV-1 outbreak in 2002 to the most recent SARS-CoV-2 pandemic (COVID-19), emergence of viral diseases has repeatedly threatened humanity over the recent decades. These viral diseases mainly cause respiratory symptoms, which can even lead to death when appropriate measures are not taken. In this study, we investigated whether adipose tissue-derived mesenchymal stem cell EVs (ASC-EVs) can attenuate acute lung injury (ALI) induced by H1N1 influenza A virus and SARS-CoV-2 and by what mechanism the ant-viral effect may occurs. Methods, Results & Conclusion: EVs were isolated from ASC or HEK293T conditioned media by tangential flow filtration, and were characterized according to MISEV recommendation. Influenza A/ Puerto Rico/08/1934 (H1N1) and SARS-CoV-2 (NCCP43326) were used to model highly pathogenic human influenza A and SARS-CoV-2 virus infection, respectively, in mice and Syrian hamsters respectively. Treatment of ASC-EVs, from 0.15 x 109 to 5.0 x 109 particles/mL, showed inhibitory activities on cytopathic effects and replication of H1N1 and SARS-CoV-2 in MDCK cells and Vero E6 cells, respectively. In the mouse H1N1 influenza A virus induced acute lung injury (ALI) model, total of 4 daily injections of 1 x 1010 particles of ASC-EVs administration resulted in significantly increased survival rate by 30 – 40%, recovery of body weight, and improved clinical disease score from 9 dpi. In addition, ASC-EV treatment downregulated various inflammatory cytokines such as IL-1β, IL-6 and TNFα in lung tissue by up to 77%. In the Syrian hamster SARS-CoV-2 induced ALI model, total of 4 daily injections of ASC-EVs at a dose of 3 x 1010 or 1 x 1010 particles resulted recovery of body weights from 5 dpi, in a dose-dependent manner, by 9.7% - 12.75%. Further, ASC-EV treatment resulted in significant downregulation of viral genes and IL-1 beat in lung tissue. To elucidate the molecular mechanisms of the observed anti-viral effects of ASC-EVs, the role of multiple miRNAs and proteins present in the ASC-EVs were assessed in vitro. We identified one specific protein that conveyed anti-viral efficacy against the two studied viruses including SARS-CoV-2. Loss and gain of function studies revealed that this protein may be involved in the anti-viral efficacy of the ASC-EVs. Our findings support the concept that that ASC-EVs have anti-viral effects against virus induced ALI, which may have implications for the treatment of not only treatment COVID-19, but also future ALI-inducing virus diseases.
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Background: TGF-Beta plays an important role in immune evasion in oncology. Similarly, SARSCov-2, the causal agent of the COVID-19 pandemic, also has an immune evasion function. This is mediated by ORF-8 through its interaction with multiple immune regulatory elements, including TGF-beta. This is a mutational analysis of ORF-8. Methods: We took advantage of the database of millions of SARS-CoV-2 genomes are archived and organized in phylogenetic relationships to show the evolution of ORF-8. Site numbering and genome structure use Wuhan-Hu-1/2019 as reference. The phylogeny is rooted relative to early samples from Wuhan. Temporal resolution assumes a nucleotide substitution rate of 8 × 10-4 subs per site per year. ( https://nextstrain.org/). The epidemiological data provided at https://ourworldindata.org/coronavirus was used to determine the property of the variants using mortality and infectivity data at the site. Results: Scan of ORF-8 revealed a high rate of mutation at aa119 and aa120. More importantly, the mutation at 120 or 119 that resulted in null ORF8 clearly delineates the pre-Delta and Delta SARSCov-2. In fact, all the delta lineages exhibited the null mutation at 119/120. This region is important for the dimerization of ORF-8 and possibly its interaction with host TGF-beta. All other variants, including the alpha variants, are wild type (aa120 = F). Monitoring the mutations over the last several months indicated that the delta variants have now picked up the wild type F at aa120 (Faa120) in Egypt or the L at aa 120 (Laa120) in India. The epidemiology of Egypt and India indicates that the Faa120 is more immune evasive and suggestive that more infectious but not more lethal. Conclusions: This is an opportunity to monitor in real-time the evolution of ORF-8 and how it is interacting with the host immune system. Additionally, since our current clinical trial on TGF-beta inhibitors is in India and Latin America, it is an opportunity to correlate clinical findings to molecular and epidemiological data for these variants. If we are correct, the Faa120 will emerge as the dominant variant in the next wave of COVID-19.
ABSTRACT
Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4+ T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2-specific Th cells could be detected as early as days 2–4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8+ T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells — basophiles and eosinophils — were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased “regulatory” Tfh1 cell and increased “pro-inflammatory” Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS-CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of “naïve” and memory B cell subsets, as well as increased level of CD27hiCD38hiCD24– plasma cell precursors and atypical CD21low B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.
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The interferon (IFN) response is a critical arm of the innate immune response and a major host defense mechanism against viral infections. Numerous genes that contribute to this antiviral state remain to be identified and characterized. Using large-scale loss-of-function strategies, we screened siRNAs or gRNAs libraries targeting hundreds of IFNstimulated genes (ISGs) in IFN-treated cells infected with human RNA viruses, including SARS-CoV-2, Zika virus or tick-borne encephalitis virus. We recovered previously unrecognized human genes able to modulate the replication of these RNA viruses in an IFN-dependent manner. For instance, we identified the chromatin remodeling protein MTA2 as a potent flavivirus-specific antiviral factor. Mechanistic studies to decipher the molecular mechanisms by which these novel antiviral genes are functioning are on-going. We are also expanding our studies to the identification and characterization of ISGs in animal species that serve as viral reservoirs, such as bats. Our work should open new perspectives to target weakness points in the life cycle of these emerging RNA viruses.
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Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions. We are currently investigating the molecular mechanisms behind IFN-dependent flaviviral restriction of MTA2. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent way, opening new perspectives to target weakness points in the life cycle of these viruses.
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Background: The clinical spectrum of coronavirus disease 2019 (COVID19) is wide. While some individuals have severe disease, themajority of individuals are either asymptomatic or have mildsymptoms with minimal hypoxia. There is emerging evidence thatrare genetic variation can contribute to risk for more severe COVID19infection. The goal of this study was to investigate if rare geneticvariation was contributing to severe disease presentation in a familywith varying clinical responses to COVID19 infection.Methods: This case series describes clinical, laboratory and radiographicfeatures in a three generation family of seven individualswithout previous known immunodeficiency that were all directlyexposed to COVID19. Four individuals developed COVID19 infection:three individuals had critical disease, and one had mild symptoms.Three exposed family members were asymptomatic and did not haveclinical evidence of COVID19 infection. All family members werepreviously healthy and did not have a history ofmajor chronic diseaseincluding respiratory disease, known immunodeficiency, or any othergenetic disorder. Exome sequencing analysis was completed toinvestigate monogenic risk factors segregating with severe diseasein this family.Results: Seven family members spanning three generations wereincluded in final analysis. Individuals with severe COVID19 diseasewere male, had a mean age of 71 years old (range 61–87), and a meanbody mass index (BMI) of 27 (range 28–32). All three severely affectedmales were intubated and died within 33 days of presentation (mean25 days, range 16–33 days). One female family member with COVID19infection and a milder clinical coursewas 68 years old on presentationand had a BMI of 34. She did not require intubation but was mildlyhypoxic on room air requiring nasal cannula for oxygenation. All fourfamily members with symptomatic COVID19 infection receivedRemdesivir antiviral therapy and systemic steroids as part of thetreatment course. Unaffected family members (n = 3) had a mean ageof 35 years old (range 30–58). All were exposed to affected familymembers and all remained clinically asymptomatic. Whole exomesequencing and segregation analysis of this family identified amissense alteration of SPDEF that segregated with family memberswith severe COVID19 infection and was not detected in the mildlyaffected and unaffected family members. SPDEF is a transcriptionfactor that is highly intolerant to loss of function (pLI 0.97). Thealteration detected in this family (c.830G>A;p.Gly277Asp) is withinthe functional DNA binding domain of the protein product, and ispredicted to be damaging by in-silico models.Conclusions: Here we report exome findings from a family withvariable clinical response to COVID19 infection and describe a raremissense alteration in SPDEF segregating with severe COVID19infection. SPDEF is essential for goblet cell differentiation andmucociliary clearance within respiratory epithelial cells and has arole in mediating innate immune response. This report demonstratesthat studying large families with variable clinical outcomes can be auseful approach for identifying rare genetic variation associated withincreased risk for severe COVID19 infection. Moreover, our findingsprovide insight into the putative link between the altered inflammatoryresponse and respiratory comprise observed in some individualswith severe COVID19 infection
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Introduction: Head and neck cancer (HNC) is a heterogeneous group of subsites, with differing natural histories. Its management is complex and it may have a long-term effect on patients’ quality of life (QOL). The purpose of this survey was to assess patients’ preferences using a validated 12 point ‘Priority Assessment Tool’ developed by Sharp et al [1]. Materials and Methods: Sixty consecutive HNC patients being treated with radiotherapy at our centre were approached. Patients were asked to rank their priorities from 1 to 12 (1 = very important, 12 = least important). Results: 45 patients (75%) completed the survey. The most important, overwhelming priority for the cohort as a whole, was ‘being cured of my cancer’ which was first priority in 73% patients and within first three priorities for 96% of patients. Similarly, ‘living as long as possible’ was within the first three priorities for 67% of patients although for 13% patients, living longer was, surprisingly the least priority. For more than two third of patients (69%), ‘having no pain’ was within the first four important priorities. There is next, a group of six QOL priorities (voice, speech, chewing, swallowing, taste/smell, moist mouth), which have a much wider range and vary considerably in importance to individual patients, being very important to some and much less important to others. There then remains a group of three priorities that have a relatively low importance to patients, not absolutely, but in relation to their other priorities, with ‘keeping appearance unchanged’, being the lowest priority chosen by 40% patients. The other two least priorities were ‘returning to activities as soon as possible’ and ‘having a normal amount of energy’. Conclusions: Our survey has shown that most HNC patients markedly prioritise survival over function and other QOL measures. Having such information is crucial, to being able to make a valid decision with regard to the treatment options and the trade-offs between increased probability of cure or extending survival and loss of function, and late morbidity. This makes shared decision-making even more important in the present COVID-19 era, so that added information about the risks of infection and potential changes in risk benefit ratio may alter patients’ priorities and preferred outcomes.
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Objective: Previous studies provided the first evidence that the importance of olfaction decreases with the duration of smell loss. It is currently unknown whether the olfactoryrelated quality of life (QoL) also differs between patients with new-onset and persistent smell loss (longer than four weeks) during the coronavirus-19 (COVID-19) pandemic and patients with persistent postinfectious smell loss (PIOD) that were recruited before the pandemic. Methods: This was a retrospective study that included 149 patients with self-reported olfactory dysfunction (OD). The olfactory-related QoL was measured using the Questionnaire of Olfactory Dysfunction (QOD). The QOD measures the degree to which patients (i) adjust and cope with smell loss (QOD-PS) and (ii) suffer from OD in general (QOD-NS). Self-perceived chemosensory function, demographics, olfactory function, and duration of smell loss were evaluated. Analyses of variance were used to depict differences in QoLoutcomes between different OD groups. Results: All patients included during the COVID-19 pandemic reported an extensive loss of chemosensory functions smell, taste, and flavor perception. Retronasal testing revealed olfactory impairments in more than half of these patients. One-way analysis of variance and posthoc tests revealed that the QOD-NS was significantly higher in the new-onset OD group than the PIOD group. At the same time, the QOD-PS score was significantly higher in the PIOD and the persistent COVID-19 OD group compared to the new-onset OD group. Conclusions: We showed that patients with persistent OD experienced better olfactory-related adjustment and lower QoL-impairment than those with recent-onset OD, suggesting that the olfactory-related QoL might change as a function of time after symptom onset.
ABSTRACT
Clinical manifestations of infection with the novel SARS-CoV-2 in humans are widely varied, ranging from asymptomatic to COVID-19 respiratory failure and multiorgan damage. Profound inflammation is the hallmark of severe COVID-19 disease, and commonly does not occur until the second week of infection. Although risk factors for this late hyperinflammatory disease have been identified, most notably age and pre-existing co-morbidities, even within high-risk groups the specific factors leading to severe COVID-19 illness remain elusive. Acquired somatic mutations in hematopoietic stem and progenitor cells (HSPCs), termed clonal hematopoiesis (CH), are associated with advanced age, and loss of function (LOF) mutations in certain genes, most commonly DNMT3A and TET2, have been linked to a marked hyperinflammatory phenotype as well as clonal expansion of mutant HSPCs. Given the similar age range of frequent CH and severe COVID-19 disease, the presence of CH could impact the risk of severe COVID-19. Several human cohort studies have suggested this relationship may exist, but results to date are conflicting. Rhesus macaques (RM) have been established as a model for SARS-CoV infection and are being utilized to test therapies and vaccine development, but up to now, macaques have not been reported to develop late hyperinflammatory COVID-19 disease. We have created a robust RM model of CH by introducing LOF TET2 mutations into young adult HSPC via CRISPR/Cas9 followed by autologous transplantation, recapitulating the clonal expansion and hyperinflammatory phenotype. Thus, we hypothesized that macaques with CH could develop severe late COVID-19 disease and be utilized as a model to study disease pathophysiology or test therapeutic approaches. Macaques with either engineered (n=2) or natural CH (n=1) along with age-matched transplanted controls (n=3) were inoculated with SARS-CoV-2 and monitored clinically and via laboratory studies until 12 days post-inoculation (dpi). Macaques normally clear infection and symptoms within 3-5 days of infection. No significant differences in clinical symptoms and blood counts were noted, however, an aged animal with natural DNMT3A CH died on 10 dpi. IL-6 levels were somewhat higher in sera of the CH animals until 12 dpi, and in BAL, mean concentrations of MCP-1, IL-6, IL-8 and MIP-1b were consistently higher in CH macaques compared to controls. Interestingly, we found the median copy number of subgenomic SARS-CoV-2 RNA was higher at every timepoint in the CH group as compared with the control group, in both upper and lower respiratory samples. Lung sections from euthanasia at 10 or 12 dpi showed evidence of mild inflammation in all animals. However, in the immunohistochemical analysis, the viral antigen was detected in the lung tissues of all three animals in the CH group even at the time of autopsy, whereas only one animal of three controls had detectable viral antigen. Although the striking inflammation and serious disease have not been observed, data so far provide evidence of potential pathophysiological differences with or without CH upon SARS-CoV-2 infection. We continue to expand sample size and conduct further analyses to draw a solid conclusion, but we believe this model may be of benefit to understand the relationship between COVID-19 disease and CH. Disclosures: No relevant conflicts of interest to declare.
ABSTRACT
Background: COVID-19 is a pandemic that had already infected more than forty-six million people and caused more than a million deaths by 1st of November 2020. The virus pandemic appears to have had a catastrophic effect on the global population's safety. Therefore, efficient detection of infected patients is a key phase in the battle against COVID-19. One of the main screening methods is radiological testing. The goal of this study is using chest X-ray images to detect COVID-19 pneumonia patients while optimizing detection efficiency. Methods: As shown in Figure 1, we combined three methods to detect COVID-19 namely: convolutional neural network, transfer learning, and the focal loss1 function which are used for unbalanced classes, to build three binary classifiers which are COVID-19 versus normal, COVID-19 versus pneumonia, and COVID-19 versus normal pneumonia (normal and pneumonia). The database used2 includes a mixture of 400 COVID-19, 1,340 viral pneumonia, 2,560 bacterial pneumonia, and 1,340 normal chest X-ray images for training, validation, and testing of four pre-trained deep convolutional neural networks. Then, the pre-trained model that gives the best results was chosen to improve its performances by two enhancement techniques which are image augmentation, allowing us to reach approximately 2,500 images per class, and the adjustment of focal loss hyperparameters. Results: A comparative study was conducted of our proposed classifiers with well-known classifiers and obtained much better results in terms of accuracy, specificity, sensitivity and precision, as illustrated in Table 1. Conclusion: The high performance of this computer-aided diagnostic technique may greatly increase the screening speed and reliability of COVID-19 diagnostic cases. Particularly, at the crowded emergency services, it will be particularly helpful in this pandemic when the risk of infection and the necessity for prevention initiatives run contrary to the available resources.
ABSTRACT
Introduction: It was recently observed and described an association between a pediatric hyperinflammatory state and the infection by SARS-CoV-2 which was named Multisystem inflammatory syndrome in children related to COVID-19 (PIMS- TS) or Multisystem inflammatory syndrome (in children) MIS-C. Objectives: We aimed our study at describing the clinical features, epidemiologic characteristics, management, and prognosis. Methods: This is a retrospective, descriptive and observational study performed at the Hospital Infantil de Mexico Federico Gomez, a thirdlevel children's hospital in Mexico City. The study includes all the cases that met criteria for PIMS-TS/MIS-C of the RCPCH, WHO, and/or CDC, that were diagnosed and treated between March 2020 and March 2021. We identified a total of 41 cases. Results: The depicted table describe the demographics of our studied population. The highest incidence was seen in previously healthy, school-aged children. No differences were noted based on sex. In 50% of the cases, there was history of exposure to COVID-19. 7.3% of patients had an associated comorbidity. The SARS-CoV2 was isolated from CSF in one patient with PIMS. There were no documented cases of macrophage activation syndrome (MAS). While coagulopathy was observed, there were no cases of disseminated intravascular coagulation (DIC). These results are consistent with the results reported by Hoste L et al. Eur J Pediatr. 2021. Conclusion: The present study depicts the experience of our institution with the new nosological entity named PIMS. We highlight the null mortality, the effectiveness of steroids and gammaglobulin in lieu of biologic therapy as part of the management, and the predominance of previously healthy patients without significant comorbidities. We consider this a broad evaluation, as our sample size consisted of 41 patients from the same location.