Smart microfluidics: Synergy of machine learning and microfluidics in the development of medical diagnostics for chronic and emerging infectious diseases

The COVID-19 pandemic, emerging/re-emerging infections as well as other non-communicable chronic diseases, highlight the necessity of smart microfluidic point-of-care diagnostic (POC) devices and systems in developing nations as risk factors for infections, severe disease manifestations and poor clinical outcomes are highly represented in these countries. These POC devices are also becoming vital as analytical procedures executable outside of conventional laboratory settings are seen as the future of healthcare delivery. Microfluidics have grown into a revolutionary system to miniaturize chemical and biological experimentation, including disease detection and diagnosis utilizing muPads/paper-based microfluidic devices, polymer-based microfluidic devices and 3-dimensional printed microfluidic devices. Through the development of droplet digital PCR, single-cell RNA sequencing, and next-generation sequencing, microfluidics in their analogous forms have been the leading contributor to the technical advancements in medicine. Microfluidics and machine-learning-based algorithms complement each other with the possibility of scientific exploration, induced by the framework's robustness, as preliminary studies have documented significant achievements in biomedicine, such as sorting, microencapsulation, and automated detection. Despite these milestones and potential applications, the complexity of microfluidic system design, fabrication, and operation has prevented widespread adoption. As previous studies focused on microfluidic devices that can handle molecular diagnostic procedures, researchers must integrate these components with other microsystem processes like data acquisition, data processing, power supply, fluid control, and sample pretreatment to overcome the barriers to smart microfluidic commercialization.Copyright © 2023 by Begell House, Inc.

Pericytes may facilitate SARS-CoV-2 entry into the nervous system

Objective: Although the neurological and olfactory symptoms of coronavirus disease 2019 have been identified, the neurotropic properties of the causative virus, severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), remain unknown. We sought to identify the susceptible cell types and potential routes of SARS-CoV-2 entry into the central nervous system, olfactory system, and respiratory system. Method(s): We collected single-cell RNA data from normal brain and nasal epithelium specimens, along with bronchial, tracheal, and lung specimens in public datasets. The susceptible cell types that express SARS-CoV-2 entry genes were identified using single-cell RNA sequencing and the expression of the key genes at protein levels was verified by immunohistochemistry. We compared the coexpression patterns of the entry receptor angiotensin-converting enzyme 2 (ACE2) and the spike protein priming enzyme transmembrane serine protease (TMPRSS)/cathepsin L among the specimens. Result(s): The SARS-CoV-2 entry receptor ACE2 and the spike protein priming enzyme TMPRSS/cathepsin L were coexpressed by pericytes in brain tissue;this coexpression was confirmed by immunohistochemistry. In the nasal epithelium, ciliated cells and sustentacular cells exhibited strong coexpression of ACE2 and TMPRSS. Neurons and glia in the brain and nasal epithelium did not exhibit coexpression of ACE2 and TMPRSS. However, coexpression was present in ciliated cells, vascular smooth muscle cells, and fibroblasts in tracheal tissue;ciliated cells and goblet cells in bronchial tissue;and alveolar epithelium type 1 cells, AT2 cells, and ciliated cells in lung tissue. Conclusion(s): Neurological symptoms in patients with coronavirus disease 2019 could be associated with SARS-CoV-2 invasion across the blood-brain barrier via pericytes. Additionally, SARS-CoV-2-induced olfactory disorders could be the result of localized cell damage in the nasal epithelium.Copyright © Wolters Kluwer Health, Inc. All rights reserved.

Cellular and Molecular Atlas of Peripheral Blood Mononuclear Cells from a Pregnant Woman after Recovery from COVID-19

Objective This study aimed to investigate the immune response of a pregnant woman who recovered from the coronavirus disease 2019 (COVID_RS) by using single-cell transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) and to analyze the properties of different immune cell subsets. Methods PBMCs were collected from the COVID_RS patient at 28 weeks of gestation, before a cesarean section. The PBMCs were then analyzed using single-cell RNA sequencing. The transcriptional profiles of myeloid, T, and natural killer (NK) cell subsets were systematically analyzed and compared with those of healthy pregnant controls from a published single-cell RNA sequencing data set. Results We identified major cell types such as T cells, B cells, NK cells, and myeloid cells in the PBMCs of our COVID_RS patient. The increase of myeloid and B cells and decrease of T cells and NK cells in the PBMCs in this patient were quite distinct compared with that in the control subjects. After reclustering and Augur analysis, we found that CD16 monocytes and mucosal-Associated invariant T (MAIT) cells were mostly affected within different myeloid, T, and NK cell subtypes in our COVID_RS patient. The proportion of CD16 monocytes in the total myeloid population was increased, and the frequency of MAIT cells in the total T and NK cells was significantly decreased in the COVID-RS patient. We also observed significant enrichment of gene sets related to antigen processing and presentation, T-cell activation, T-cell differentiation, and tumor necrosis factor superfamily cytokine production in CD16 monocytes, and enrichment of gene sets related to antigen processing and presentation, response to type II interferon, and response to virus in MAIT cells. Conclusion Our study provides a single-cell resolution atlas of the immune gene expression patterns in PBMCs from a COVID_RS patient. Our findings suggest that CD16-positive monocytes and MAIT cells likely play crucial roles in the maternal immune response against severe acute respiratory syndrome coronavirus 2 infection. These results contribute to a better understanding of the maternal immune response to severe acute respiratory syndrome coronavirus 2 infection and may have implications for the development of effective treatments and preventive strategies for the coronavirus disease 2019 in pregnant women.Copyright © Wolters Kluwer Health, Inc. All rights reserved.

Enhancer deregulation inTET2-mutant clonal hematopoiesis is associated with increased COVID-19 severity and mortality

Background: COVID-19 causes significant morbidity and mortality, albeit with considerable heterogeneity among affected individuals. It remains unclear which host factors determine disease severity and survival. Given the propensity of clonal hematopoiesis (CH) to promote inflammation in healthy individuals, we investigated its effect on COVID-19 outcomes. Method(s): We performed a multi-omics interrogation of the genome, epigenome, transcriptome, and proteome of peripheral blood mononuclear cells from COVID-19 patients (n=227). We obtained clinical data, laboratory studies, and survival outcomes. We determined CH status and TET2-related DNA methylation. We performed single-cell proteogenomics to understand clonal composition in relation to cell phenotype. We interrogated single-cell gene expression in isolation and in conjunction with DNA accessibility. We integrated these multi-omics data to understand the effect of CH on clonal composition, gene expression, methylation of cis-regulatory elements, and lineage commitment in COVID-19 patients. We performed shRNA knockdowns to validate the effect of one candidate transcription factor in myeloid cell lines. Result(s): The presence of CH was strongly associated with COVID-19 severity and all-cause mortality, independent of age (HR 3.48, 95% CI 1.45-8.36, p=0.005). Differential methylation of promoters and enhancers was prevalent in TET2-mutant, but not DNMT3A-mutant CH. TET2- mutant CH was associated with enhanced classical/intermediate monocytosis and single-cell proteogenomics confirmed an enrichment of TET2 mutations in these cell types. We identified celltype specific gene expression changes associated with TET2 mutations in 102,072 single cells (n=34). Single-cell RNA-seq confirmed the skewing of hematopoiesis towards classical and intermediate monocytes and demonstrated the downregulation of EGR1 (a transcription factor important for monocyte differentiation) along with up-regulation of the lncRNA MALAT1 in monocytes. Combined scRNA-/scATAC-seq in 43,160 single cells (n=18) confirmed the skewing of hematopoiesis and up-regulation of MALAT1 in monocytes along with decreased accessibility of EGR1 motifs in known cis-regulatory elements. Using myeloid cell lines for functional validation, shRNA knockdowns of EGR1 confirmed the up-regulation of MALAT1 (in comparison to wildtype controls). Conclusion(s): CH is an independent prognostic factor in COVID-19 and skews hematopoiesis towards monocytosis. TET2-mutant CH is characterized by differential methylation and accessibility of enhancers binding myeloid transcriptions factors including EGR1. The ensuing loss of EGR1 expression in monocytes causes MALAT1 overexpression, a factor known to promote monocyte differentiation and inflammation. These data provide a mechanistic insight to the adverse prognostic impact of CH in COVID-19.

Multi-omics blood atlas reveals unique features of immune and platelet responses to SARS-CoV-2 Omicron breakthrough infection.

Although host responses to the ancestral SARS-CoV-2 strain are well described, those to the new Omicron variants are less resolved. We profiled the clinical phenomes, transcriptomes, proteomes, metabolomes, and immune repertoires of >1,000 blood cell or plasma specimens from SARS-CoV-2 Omicron patients. Using in-depth integrated multi-omics, we dissected the host response dynamics during multiple disease phases to reveal the molecular and cellular landscapes in the blood. Specifically, we detected enhanced interferon-mediated antiviral signatures of platelets in Omicron-infected patients, and platelets preferentially formed widespread aggregates with leukocytes to modulate immune cell functions. In addition, patients who were re-tested positive for viral RNA showed marked reductions in B cell receptor clones, antibody generation, and neutralizing capacity against Omicron. Finally, we developed a machine learning model that accurately predicted the probability of re-positivity in Omicron patients. Our study may inspire a paradigm shift in studying systemic diseases and emerging public health concerns.

Classification of tuberculosis-related programmed cell death-related patient subgroups and associated immune cell profiling.

Background: Tuberculosis (TB) is the deadliest communicable disease in the world with the exception of the ongoing COVID-19 pandemic. Programmed cell death (PCD) patterns play key roles in the development and progression of many disease states such that they may offer value as effective biomarkers or therapeutic targets that can aid in identifying and treating TB patients. Materials and methods: The Gene Expression Omnibus (GEO) was used to gather TB-related datasets after which immune cell profiles in these data were analyzed to examine the potential TB-related loss of immune homeostasis. Profiling of differentially expressed PCD-related genes was performed, after which candidate hub PCD-associated genes were selected via a machine learning approach. TB patients were then stratified into two subsets based on the expression of PCD-related genes via consensus clustering. The potential roles of these PCD-associated genes in other TB-related diseases were further examined. Results: In total, 14 PCD-related differentially expressed genes (DEGs) were identified and highly expressed in TB patient samples and significantly correlated with the abundance of many immune cell types. Machine learning algorithms enabled the selection of seven hub PCD-related genes that were used to establish PCD-associated patient subgroups, followed by the validation of these subgroups in independent datasets. These findings, together with GSVA results, indicated that immune-related pathways were significantly enriched in TB patients exhibiting high levels of PCD-related gene expression, whereas metabolic pathways were significantly enriched in the other patient group. Single cell RNA-seq (scRNA-seq) further highlighted significant differences in the immune status of these different TB patient samples. Furthermore, we used CMap to predict five potential drugs for TB-related diseases. Conclusion: These results highlight clear enrichment of PCD-related gene expression in TB patients and suggest that this PCD activity is closely associated with immune cell abundance. This thus indicates that PCD may play a role in TB progression through the induction or dysregulation of an immune response. These findings provide a foundation for further research aimed at clarifying the molecular drivers of TB, the selection of appropriate diagnostic biomarkers, and the design of novel therapeutic interventions aimed at treating this deadly infectious disease.

Single-Cell Analysis Reveals Characteristics of Myeloid Cells in Pulmonary Pasc

Background: Although our understanding of immunopathology in the risk and severity of COVID-19 disease is evolving, a detail of immune response in long-term consequences of COVID-19 infection remains unclear. Recently, few studies have detailed the immune and cytokine profiles associated with PASC. However, dysregulation of immune system driving pulmonary PASC is still largely unknown. Method(s): To characterize the immunological features of PPASC, we performed droplet-based scRNA-sequencing using 10X genomics to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from participants naive to SARS-CoV-2 (NP, n=2) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC, n=2). Result(s): Analysis of more than 34,000 PBMCs by integrating our dataset with previously reported control datasets generated cell distribution and identified 11 immune cell types based on canonical gene expression. The proportion of myeloid-lineage cells (CD14+monocyte, CD16+monocyte, and dendritic cells) and platelets were increased in PPASC compared with those of NP. Specifically, PPASC displayed up-regulation of VEGFA and transcription factors, such as ATF2, ELK, and SMAD in myeloid-lineage cells. Also, TGF-beta and WNT signaling pathways were up-regulated in these cell population. Cell-cell interaction analysis identified that myeloid-lineage cells in PPASC participated in regulation of fibrosis and immune response, such as VEGFA (increased) and MIF (decreased) interactions. Conclusion(s): Together, this study provides high-resolution insights into immune landscape in PPASC. Our results emphasize differences in myeloid lineage-mediated fibrosis and immunity between PPASC and NP, suggesting they could act as potential pathological drivers of PPASC. (Figure Presented).

CBD Treatment Improves Fatal Inflammatory Response Exhibited in SEB-Induced ARDS

The novel SARS-CoV-2 virus known to cause the COVID-19 outbreak has resulted in a global healthcare crisis that has persisted the past 3 years. Thus, understanding the mechanisms underlying this disease are vital at this time. While there are issues of research infrastructure to handle the virus and because of the refractoriness of rodents to this disease, the availability of these tools is still limited. The cytokine storm and fatality presented in patients with severe COVID-19 can be mimicked with Staphylococcal enterotoxin B (SEB)-induced Acute Respiratory Distress Syndrome (ARDS). Within ~7 days, the survival rate drops to 0% for C3H/HeJ mice exposed to a dual dose of SEB. In this study, we administered cannabidiol (CBD) intraperitoneally for 3 days pre- and post-SEB dosing and found that the clinical outcomes improved significantly. Initial evaluation of scRNASeq data from lungs comparing naive to SEB-induced ARDS mice illustrated an increase in infiltrating immune cells, and a loss in pulmonary epithelial cells in the latter group. When evaluating the effect of CBD treatment on SEB-induced ARDS, we were able to demonstrate that CBD reduced the macrophage population. To characterize the mechanism by which CBD treatment ameliorated the inflammatory response, we found that CBD treated mice had significant reduction in infiltrating immune cells and alveolar thickening. This same histology and infiltration is presented in ARDS. MicroRNA expression analysis showed a significant increase in the expression mmu-miR-298-5p and mmu-miR- 566 with CBD treatment. Ingenuity Pathway Analysis (IPA) indicated that the dysregulated miRNAs were also implicated in pathways associated with macrophage activation, respiratory disease and inflammation, interferon stimulated genes, as well as genes which have been upregulated in the disease state of this model. These targets include but are not limited to Cebpb, Efhd2, Stat3, Socs3, Cxcl5, Gbp2, and Birc3. This finding offers insights for the development of preventive and therapeutic strategies in the treatment of ARDS, including that induced in COVID-19. Supported by NIH grants P01AT003961, P20GM103641, R01ES003961, R01AI129788, R01AI123947, R01AI160896 to MN and PSN and K99GM147910 to KW.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Topic: AS04-MDS Biology and Pathogenesis/AS04a-Normal, MDS, and leukemic stem cells: CALPROTECTIN (S100A8/A9) EFFECTS ON HEALTHY CD34+ HEMATOPOIETIC STEM AND PROGENITOR CELLS ARE AMPLIFIED IN CHRONIC MYELOID MALIGNANCIES

Background And Aims: S100A8 and S100A9 alarmins and their heterodimer calprotectin are diversely involved in myeloid neoplasm pathophysiology as well as infectious and inflammatory diseases. In the context of COVID-19, circulating calprotectin was identified as a powerful biomarker of disease severity. Calprotectin impact on CD34+ hematopoietic stem and progenitor cells remains poorly understood. Method(s): Calprotectin effects on healthy donor and chronic myeloid neoplasm-derived CD34-positive hematopoietic stem and progenitor cells were tested in liquid culture for up to 7 days. The pro-inflammatory cytokine IL-6 was used as a control. Cytokine effects alone or in combination were explored by the use of bulk and single cell RNA sequencing, Assay for Transposase-Accessible Chromatin with high-throughput sequencing, cytokine secretion analyses and semi-solid cultures. Result(s): CD34+ cells exposed to IL-6 generate monocytic cells that overproduce calprotectin. Calprotectin inhibits erythroid differentiation of healthy CD34+ cells, possibly through CD36 receptor. Chronic myeloid neoplasm CD34+ cells over-react to calprotectin, with large transcriptomic rewiring of erythro-megakarocytic and granulo-monocytic populations. Calprotectin-induced inhibition of erythroid progenitor proliferation correlates with increased synthesis of ribosomal subunits and p53 pathway activation, while the cytokine impact on granulo-monocytic cells indicates an autocrine or paracrine amplification loop. Conclusion(s): Calprotectin secreted by monocytes generated by CD34+ cells upon IL-6 stimulation may be a pathophysiological component of inflammatory anemia, a role that is amplified in the context of myeloid neoplasms in which calprotectin effects extend to the granulo-monocytic lineage.Copyright © 2023 Elsevier Ltd. All rights reserved.

Surfactant Protein B Reduces Respiratory Viral Infectivity in Human Lungs

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.

Glioblastoma Susceptibility to SARS-CoV-2: Analysis of Cancer Stem Cells and Immune Tumor Microenvironment on Human Brain, Patient-Derived Tumors, and Organoid Models

INTRODUCTION: Glioblastoma (GBM) is the most common and deadliest primary brain tumor, characterized by chemoradiation resistance and an immunosuppressive tumor microenvironment (TME). SARS-CoV-2, the COVID-19 virus, produces a significant proinflammatory response and a spectrum of clinical presentations after central nervous system infection. METHOD(S): Patient-derived GBM tissue, primary cell lines, and organoids were analyzed with immunohistochemistry and pixel-line intensity quantification. Data from tumor-bulk and single-cell transcriptomics served to describe the cell-specific expression of SARS-CoV-2 receptors in GBM and its association with the immune TME phenotype. Normal brain and iPSC-derived organoids served as controls. RESULT(S): We demonstrate that patient-derivedGBMtissue and cell cultures express SARS-CoV2 entry factors such as ACE2, TMPRSS2, and NRP1. NRP1 expression was higher in GBM than in normal brains (p<0.05), where it plays a crucial role in SARS-CoV-2 infection. NRP1 was expressed in a cell-type and phenotype-specific manner and correlated with TME infiltration of immunosuppressive cells: M2 macrophages (r = 0.229), regulatory T cells (r = 0.459), NK cells (r = -0.346), and endothelial cells (r = 0.288) (p < 0.05). Furthermore, gene ontology enrichment analysis showed that leukocyte migration and chemotaxis are among the top 5 biological functions mediated by NRP1 (p < 0.05). We found our GBM organoids recapitulate tumoral expression of SARSCoV- 2 entry factors, which varies based on distance from surface as surrogate of TME oxygenation (p < 0.05). CONCLUSION(S): GBM cancer cells and immune TME cells express SARS-CoV-2 entry factors. Glioblastoma organoids recapitulate this expression and allow for currently undergoing studies analyzing the effect of SARS-CoV-2 infection in GBM. Our findings suggest that SARSCoV- 2 could potentially target GBM, opening the door to future studies evaluating SARS-CoV-2-driven immune modulation.

ANALYSIS OF EPITOPE-SPECIFIC T CELLS IN SARS-CoV-2 INFECTION AND VACCINATION

Background: T cells play a critical role in the adaptive immune response to SARS-CoV-2 in both infection and vaccination. Identifying T cell epitopes and understanding how T cells recognize these epitopes can help inform future vaccine design and provide insight into T cell recognition of newly emerging variants. Here, we identified SARS-CoV-2 specific T cell epitopes, analyzed epitope-specific T cell repertoires, and characterized the potency and cross-reactivity of T cell clones across different common human coronaviruses (HCoVs). Method(s): SARS-CoV-2-specific T cell epitopes were determined by IFNgamma ELISpot using PBMC from convalescent individuals with mild/moderate disease (n=25 for Spike (S), Nucleocapsid (N) and Membrane (M)), and in vaccinated individuals (n=27 for S). Epitope-specific T cells were isolated based on activation markers following a 6-hour peptide stimulation, and scRNAseq was performed for TCR repertoire analysis. T cell lines were generated by expressing recombinant TCRs in Jurkat cells and activation was measured by CD69 upregulation. Result(s): We identified multiple immunodominant T cell epitopes across S, N and M proteins in convalescent individuals. In vaccinated individuals, we detected many of the same dominant S-specific epitopes at similar frequencies as compared to convalescent individuals. T cell responses to peptide S205 (amino acids 817-831) were observed in 56% and 59% of individuals following infection and vaccination, respectively, while 20% and 19% of individuals responded to S302 (a.a. 1205-1219) following infection and vaccination, respectively. For S205, a CD4+ T cell response, we confirmed 8 unique TCRs and determined the minimal epitope to be a 9mer (IEDLLFNKV). While TCR genes TRAV8-6*01 and TRBV30*01 were commonly utilized across the TCRs, we did identify TCRs with unique immunogenetic properties with different potencies of cross-reactivity to other HCoVs. For S302, a CD8+ T cell response, we identified two unique TCRs with different immunogenetic properties that recognized the same 9mer (YIKWPWYIW) and cross-reacted with different HCoV peptides (Figure 1). Conclusion(s): These data identify immunodominant T cell epitopes following SARS-CoV-2 infection and vaccination and provide a detailed analysis of epitope-specific TCR repertoires. The prospect of developing a vaccine that broadly protects against multiple human coronaviruses is bolstered by the identification of conserved immunodominant SARS-CoV-2 T cell epitopes that cross react with multiple other HCoVs.

FYN, SARS-CoV-2, and IFITM3 in the neurobiology of Alzheimer's disease

Introduction: (IFITM3) is an innate immune protein that has been identified as a novel gamma-secretase (gammas) modulator. FYN is a kinase that stabilizes IFITM3 on the membrane, primes APP for amyloidogenic gammas processing and mediates tau oligomerization. The purpose of this study is to explore the role of FYN and IFITM3 in AD and COVID-19, expanding on previous research from our group. Method(s): A 520 gene signature containing FYN and IFITM3 (termed Ia) was extracted from a previously published meta-analysis of Alzheimer's disease (AD) bulk- and single nuclei sequencing data. Exploratory analyses involved meta-analysis of bulk and single cell RNA data for IFITM3 and FYN differential expression per CNS site and cellular type. Confirmatory analyses, gene set enrichment analysis (GSEA) on Ia was performed to detect overlapping enriched biological networks between COVID-19 with AD. Result(s): Bulk RNA data analysis revealed that IFITM3 and FYN were overexpressed in two CNS regions in AD vs. Controls: the temporal cortex Wilcoxon p-value=1.3e-6) and the parahippocampal cortex Wilcoxon p-value=0.012). Correspondingly, single cell RNA analysis of IFITM3 and FYN revealed that it was differentially expressed in neurons, glial and endothelial cells donated b AD patients, when compared to controls. Discussion(s): IFITM3 and FYN were found as interactors within biological networks overlapping between AD and SARS-CoV-2 infection. Within the context of SARS-CoV-2 induced tau aggregation and interactions between tau and Ab1-42, the FYN - IFITM3 regulome may outline an important innate immunity element responsive to viral infection and IFN-I signaling in both AD and COVID-19.Copyright © 2021 The Authors

Single cell mRNA sequencing of nasal swabs indicate an impaired ciliated cell function in COVID-19 convalescents with persisting dyspnea

COVID-19 convalescents often experience persistent symptoms such as fatigue, neurologic complications or dyspnea, often referred to as "long COVID". To elucidate molecular mechanisms underlying ongoing dyspnea in COVID-19 convalescents, we analyzed single-cell RNA sequencing data from nasal swabs collected during acute infection, and three, six and twelve months post infection together with matching healthy controls. Patients with and without persisting symptoms and with varying severity during the acute phase were included. Post infection, we observed a time-dependent decrease in immune cells. Transcriptional analysis of nasal epithelial cells provided evidence of an impaired cilia assembly, organization and function in COVID-19 convalescents with dyspnea compared to healthy controls and convalescents without ongoing respiratory symptoms. Moreover, differences in the differentiation trajectories of ciliated cells were evident between patients with and without dyspnea, with less diverse differentiation endpoints in the dyspnea patients than in healthy controls or convalescents without respiratory impairment. Overall, our analyses revealed a potential deficiency of ciliated cells in COVID-19 convalescents with dyspnea compared to convalescents without ongoing respiratory symptoms or compared with healthy controls. Ciliated cells clear the lung from particles and mucus. If these cells are functionally impaired, pathogens remain in the airways, causing respiratory problems and infections. Thus, it is reasonable to assume, that impaired ciliated cell function contributes to the persistent respiratory symptoms seen in COVID-19 convalescents.

Single-cell RNA-seq analysis identifies distinct myeloid cells in a case with encephalitis temporally associated with COVID-19 vaccination.

Recently accumulating evidence has highlighted the rare occurrence of COVID-19 vaccination-induced inflammation in the central nervous system. However, the precise information on immune dysregulation related to the COVID-19 vaccination-associated autoimmunity remains elusive. Here we report a case of encephalitis temporally associated with COVID-19 vaccination, where single-cell RNA sequencing (scRNA-seq) analysis was applied to elucidate the distinct immune signature in the peripheral immune system. Peripheral blood mononuclear cells (PBMCs) were analyzed using scRNA-seq to clarify the cellular components of the patients in the acute and remission phases of the disease. The data obtained were compared to those acquired from a healthy cohort. The scRNA-seq analysis identified a distinct myeloid cell population in PBMCs during the acute phase of encephalitis. This specific myeloid population was detected neither in the remission phase of the disease nor in the healthy cohort. Our findings illustrate induction of a unique myeloid subset in encephalitis temporally associated with COVID-19 vaccination. Further research into the dysregulated immune signature of COVID-19 vaccination-associated autoimmunity including the cerebrospinal fluid (CSF) cells of central nervous system (CNS) is warranted to clarify the pathogenic role of the myeloid subset observed in our study.

Avaliacao Dos Niveis Soluveis De Podoplanina E Clec-2 Em Pacientes Com Covid-19

A imunotrombose e definida pela ativacao da hemostasia resultante de uma resposta inflamatoria e/ou infecciosa. Recentemente, a via PDPN/CLEC-2 tem se destacado em processos que medeiam a ativacao plaquetaria e a formacao trombotica. Alem disso, alguns estudos em modelos animais sugerem que a via pode desempenhar um papel importante no processo de protecao pulmonar na Lesao Pulmonar Aguda (LPA). Embora o papel da via PDPN/CLEC-2 nao seja totalmente compreendido, evidencias sugerem que ela pode se encaixar como uma nova via no conceito de imunotrombose. O objetivo do estudo foi avaliar os niveis circulantes de PDPN e CLEC-2 em pacientes com COVID-19 e explorar sua relacao com a gravidade da doenca e a ativacao da hemostasia. Foram incluidos 30 pacientes diagnosticados com COVID-19, alem de 30 controles saudaveis, com aprovacao etica (CAAE 36528420.3.0000.5404 e 30227920.9.0000.5404). As medidas de PDPN foram realizadas com kits de ELISA nos momentos D0 e D4 (4degree dia apos a admissao);A dosagem de CLEC-2 tambem foi realizada por ELISA apenas no tempo D0. Os dados clinicos e laboratoriais foram obtidos de prontuarios eletronicos. Para avaliar a expressao de PDPN em celulas pulmonares foi realizada uma analise "in silico" de scRNAseq. O tempo medio de permanencia hospitalar (LOS) foi de 12,9+/-9,8 dias, doze pacientes (40%) necessitaram de UTI com tempo medio de UTI de 6,1+/-9,7 dias. O D-dimero medio foi de 3,609+/-14,440 ng/mL. A dosagem de PDPN foi menor em pacientes com COVID-19 quando comparada a individuos saudaveis (p<0,0001), e a dosagem de CLEC-2 nao mostrou diferenca entre os grupos. Houve correlacao da PDPN com hemostasia e marcadores de gravidade, tais como: D-dimero (R= -0,529), RNL (R= -0,481), fibrinogenio (R= -0,401) e vWF (R= -0,513). Em relacao aos dados clinicos, os niveis de PDPN estavam mais diminuidos no grupo que necessitou de UTI (p=0,04). Alem disso, dados "in silico" de scRNAseq mostram uma diminuicao de PDPN no tecido pulmonar em pacientes COVID-19 quando equiparados a individuos saudaveis. Recentemente, estudos "in vivo" realizados em modelos animais destacam a via PDPN/CLEC-2 como importante no processo de protecao pulmonar frente a LPA, que ocorre devido a ligacao da CLEC-2 oriunda do complexo plaqueta-neutrofilo a PDPN expressa por macrofagos alveolares, esta ligacao promove a inibicao de citocinas/quimiocinas locais, reduzindo o estado inflamatorio local. Atualmente, nenhum trabalho avaliou a via PDPN/CLEC-2 na COVID-19, sendo um modelo interessante onde ha ativacao da hemostasia concomitante a lesao pulmonar, assim nossos resultados supoem que a via possa estar envolvida no processo de protecao pulmonar, uma vez que a diminuicao da PDPN na COVID-19 foi correlacionada com hemostasia e marcadores de gravidade. Copyright © 2022

Engineering a Pseudotyped Lentiviral Platform to Enable Lineage-Specific Transduction of Immune Cells

Background Cell-specific transduction remains one of the next frontiers for virally-delivered gene therapy. Efforts to achieve cell-specific transduction have largely been limited to borrowing of preexisting viral glycoproteins and pseudotyping viral surface envelopes to result in altered tropism. VSVG is derived from vesicular stomatitis virus (VSV) and is one of the most commonly used lentiviral (LV) pseudotype glycoproteins as its cognate receptor (LDLR) is present on nearly all dividing and non-dividing cells, enabling broad tropism of VSVG-pseudotyped LVs. Methods Our lab recently developed a receptor-blinded version of VSVG, in which point mutations (K47Q, R354A) of this glycoprotein results in a mutated VSVG with inability to bind and infect LDLR-expressing cells. This mutant viral glycoprotein, which we call VSVGmut, thereby loses its broad tropism, but critically retains its fusogen ability, enabling codisplay of a new LV pseudotype ligand to drive LV tropism. Results Initial experiments displaying high-affinity anti-CD19 scFvs alongside VSVGmut on the LV surface demonstrated infection of CD19+, but not CD19- cells. Subsequent work using endogenous ligands (CD80), Fabs (a-CD3e), cytokines (IL-13), viral glycoproteins (SARS-CoV-2 RBD), and peptide MHCs (pMHCs) revealed the modularity of this platform for achieving potent transduction of on-target cells, with minimal infection of bystander cells, across a range of affinities (pM to uM) and at frequencies as low as 1 in 100,000. This technology allowed for library on library screening of 96 viral pMHC-displaying LVs against a repertoire of >450,000 TCRs in pool, which accurately uncovered EBV- and Flu-specific TCRs through scRNA sequencing. Conclusions The VSVGmut platform has resulted in our lab's ability to pair pMHCs with cognate TCRs and viral surface antigens with cognate BCRs, in addition to achieving lineagespecific transduction of T and B cell subsets, setting the stage for cell-specific gene therapy.

Integrated Analysis of Bulk RNA-Seq and Single-Cell RNA-Seq Unravels the Influences of SARS-CoV-2 Infections to Cancer Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious and pathogenic coronavirus that emerged in late 2019 and caused a pandemic of respiratory illness termed as coronavirus disease 2019 (COVID-19). Cancer patients are more susceptible to SARS-CoV-2 infection. The treatment of cancer patients infected with SARS-CoV-2 is more complicated, and the patients are at risk of poor prognosis compared to other populations. Patients infected with SARS-CoV-2 are prone to rapid development of acute respiratory distress syndrome (ARDS) of which pulmonary fibrosis (PF) is considered a sequelae. Both ARDS and PF are factors that contribute to poor prognosis in COVID-19 patients. However, the molecular mechanisms among COVID-19, ARDS and PF in COVID-19 patients with cancer are not well-understood. In this study, the common differentially expressed genes (DEGs) between COVID-19 patients with and without cancer were identified. Based on the common DEGs, a series of analyses were performed, including Gene Ontology (GO) and pathway analysis, protein-protein interaction (PPI) network construction and hub gene extraction, transcription factor (TF)-DEG regulatory network construction, TF-DEG-miRNA coregulatory network construction and drug molecule identification. The candidate drug molecules (e.g., Tamibarotene CTD 00002527) obtained by this study might be helpful for effective therapeutic targets in COVID-19 patients with cancer. In addition, the common DEGs among ARDS, PF and COVID-19 patients with and without cancer are TNFSF10 and IFITM2. These two genes may serve as potential therapeutic targets in the treatment of COVID-19 patients with cancer. Changes in the expression levels of TNFSF10 and IFITM2 in CD14+/CD16+ monocytes may affect the immune response of COVID-19 patients. Specifically, changes in the expression level of TNFSF10 in monocytes can be considered as an immune signature in COVID-19 patients with hematologic cancer. Targeting N6-methyladenosine (m6A) pathways (e.g., METTL3/SERPINA1 axis) to restrict SARS-CoV-2 reproduction has therapeutic potential for COVID-19 patients.

Characterizing Macrophages Diversity in COVID-19 Patients Using Deep Learning.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent responsible for coronavirus disease 2019 (COVID-19), has affected the lives of billions and killed millions of infected people. This virus has been demonstrated to have different outcomes among individuals, with some of them presenting a mild infection, while others present severe symptoms or even death. The identification of the molecular states related to the severity of a COVID-19 infection has become of the utmost importance to understanding the differences in critical immune response. In this study, we computationally processed a set of publicly available single-cell RNA-Seq (scRNA-Seq) data of 12 Bronchoalveolar Lavage Fluid (BALF) samples diagnosed as having a mild, severe, or no infection, and generated a high-quality dataset that consists of 63,734 cells, each with 23,916 genes. We extended the cell-type and sub-type composition identification and our analysis showed significant differences in cell-type composition in mild and severe groups compared to the normal. Importantly, inflammatory responses were dramatically elevated in the severe group, which was evidenced by the significant increase in macrophages, from 10.56% in the normal group to 20.97% in the mild group and 34.15% in the severe group. As an indicator of immune defense, populations of T cells accounted for 24.76% in the mild group and decreased to 7.35% in the severe group. To verify these findings, we developed several artificial neural networks (ANNs) and graph convolutional neural network (GCNN) models. We showed that the GCNN models reach a prediction accuracy of the infection of 91.16% using data from subtypes of macrophages. Overall, our study indicates significant differences in the gene expression profiles of inflammatory response and immune cells of severely infected patients.