Immunotherapy: SYNERGISTIC PERITONEAL ANTI-TUMOR EFFECT OF ALLOCETRAOTS, A CELLULAR IMMUNE-THERAPY, IN COMBINATION WITH IMMUNE CHECKPOINT INHIBITORS/CHEMOTHERAPY, THROUGH IN-VIVO REPROGRAMMING OF MACROPHAGES AND DENDRITIC CELLS

Background & Aim: Immune checkpoint inhibitors (ICI) revolutionized solid tumor treatment, however, in many tumors only partial response is achieved. Allocetra-OTS has an immune modulating effect on macrophages and dendritic cells and showed an excellent safety profile in patients including patients with sepsis and Covid-19. Here we investigated the anti-tumoral effect of Allocetra-OTS cellular therapy, in peritoneal solid tumor animal models. Methods, Results & Conclusion(s): Allocetra-OTS is manufactured from enriched mononuclear fractions and induced to undergo early apoptosis. Balb/c mice were inoculated intraperitoneally (IP) with AB12 (mesothelioma) with pLenti-PGK-V5-Luc-Neo and treated with anti- CTLA4 with or without Allocetra-OTS. Mice were monitored daily for clinical score and weekly using IVIS (Fig.1). Kaplan-Meier log rank test was done for survival. For Allocetra-OTS preparation, enriched mononuclear fractions were collected by leukapheresis from healthy eligible human donors and induced to undergo early apoptosis. Anti- CTLA4 standalone therapy significantly improved survival (Fig.2) from mean 34+/-9 to 44.9 +/-20 days. However, OTS standalone therapy was non-inferior and improved survival to 52.3 +/-20 days. Anti-CTLA4 + Allocetra-OTS combination therapy, ameliorated survival to 86.7+/-20 days with complete cancer remission in 60-100% of mice. Similar anti- tumoral effects of Allocetra-OTS were seen in mesothelioma model in a combination therapy with either anti-PD1 or cisplatin and using anti-PD1 in ID8 ovary cancer model. Based on single cell analysis confirmed by flow cytometry and pathology, the mechanism of action seems to be related or at least associated with an increase in f/480high peritoneal macrophages and a decrease in recruited macrophages, and to f/480high infiltration of the tumor. However, further studies are needed to confirm these observations. During IP tumor progression, Allocetra-OTS as a standalone therapy or in combination with ICI, or cisplatin, significantly reduced tumor size and resulted in complete remission in up to 100% treated mice. Similar results were obtained in ID8 ovary cancer. Based on excellent safety profile in > 50 patients treated in prior clinical trials for sepsis and Covid-19, Phase I/II clinical trial of Allocetra-OTS plus chemotherapy has started and three patient already recruited. A second phase I/II clinical trial of Allocetra- OTS plus anti-PD1, as a second- and third-line therapy in various cancers, was initiated in Q1 2023. [Figure presented]Copyright © 2023 International Society for Cell & Gene Therapy

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.

Identification of 3 key genes as novel diagnostic and therapeutic targets for OA and COVID-19.

Background: Corona Virus Disease 2019 (COVID-19) and Osteoarthritis (OA) are diseases that seriously affect the physical and mental health and life quality of patients, particularly elderly patients. However, the association between COVID-19 and osteoarthritis at the genetic level has not been investigated. This study is intended to analyze the pathogenesis shared by OA and COVID-19 and to identify drugs that could be used to treat SARS-CoV-2-infected OA patients. Methods: The four datasets of OA and COVID-19 (GSE114007, GSE55235, GSE147507, and GSE17111) used for the analysis in this paper were obtained from the GEO database. Common genes of OA and COVID-19 were identified through Weighted Gene Co-Expression Network Analysis (WGCNA) and differential gene expression analysis. The least absolute shrinkage and selection operator (LASSO) algorithm was used to screen key genes, which were analyzed for expression patterns by single-cell analysis. Finally, drug prediction and molecular docking were carried out using the Drug Signatures Database (DSigDB) and AutoDockTools. Results: Firstly, WGCNA identified a total of 26 genes common between OA and COVID-19, and functional analysis of the common genes revealed the common pathological processes and molecular changes between OA and COVID-19 are mainly related to immune dysfunction. In addition, we screened 3 key genes, DDIT3, MAFF, and PNRC1, and uncovered that key genes are possibly involved in the pathogenesis of OA and COVID-19 through high expression in neutrophils. Finally, we established a regulatory network of common genes between OA and COVID-19, and the free energy of binding estimation was used to identify suitable medicines for the treatment of OA patients infected with SARS-CoV-2. Conclusion: In the present study, we succeeded in identifying 3 key genes, DDIT3, MAFF, and PNRC1, which are possibly involved in the development of both OA and COVID-19 and have high diagnostic value for OA and COVID-19. In addition, niclosamide, ciclopirox, and ticlopidine were found to be potentially useful for the treatment of OA patients infected with SARS-CoV-2.

COVID-19 ACE2 related to hepatocellular carcinoma in single cell analyses and immuno-cancer microenvironment infiltrated with macrophage

Background: Hepatocellular carcinoma (HCC) is the second leading cause of malignancy-related mortality and the fifth most common worldwide. Immuno-cancer microenvironment (ICME) was highlighted recently because scientists want to unlock the detailed mechanism in carcinogenesis pathway and find the novel interactions in ICME. Besides, single cell analysis could mitigate the interrupted signals between cells and tissues. On the other hand, COVID-19 angiotensin I converting enzyme (ACE) previously was reported associated with cancer. However, the robust association between COVID-19 and HCC ICME is still unaddressed. Aim(s): We plan to investigate the COVID-19 ACE relevant genes to HCC ICME regarding survival. Method(s): We used Reactome for COVID-19 ACE gene pathway mapping and explored the positive relevant gene expression. DISCO website was applied for single cell analyses using the above-collected genes from Reactome. Finally, we implanted the biomedical informatics into TIMER 2.0 for ICME survival analyses. Result(s): In Fig. 1, the gene-gene interaction mapping was shown. We collected 13 genes (CPB2, ACE2, AGT, MME, ANPEP, CPA3, ENPEP, GZMH, CTSZ, CTSD, CES1, ATP6AP2, and AOPEP) for further single cell relevant analyses, in Table 1, with detailed expression level (TPM). Among the above 13 genes, AGT, GZMH, CTSZ, CTSD, CES1, and ATP6AP2 were strongly expressed in liver tissue. We then applied the initial 13 genes to TIMER 2.0 for HCC ICME 2-year survival analyses. CPA3 and GZMH low expressions with high macrophage infiltration in HCC ICME showed significantly worse 2-year cumulative survival [hazard ratio (HR):CPA3 2.21, p-value 0.018;GZMH 2.07, p-value 0.0341]. ACE2, CPB2, AGT, MME, ANPEP, ENPEP, CTSZ, CTSD, CES1, and ATP6AP2 high expressions with high macrophage infiltration in HCC ICME revealed significantly worse 2-year cumulative survival. Conclusion(s): We demonstrate that ACE2 was strongly associated with HCC clinical survival with macrophage infiltration. However, the bidirectional translational roles about ACE2 relevant genes in HCC should be documented.

Cell-Specific Mechanisms in the Heart of COVID-19 Patients.

From the onset of the pandemic, evidence of cardiac involvement in acute COVID-19 abounded. Cardiac presentations ranged from arrhythmias to ischemia, myopericarditis/myocarditis, ventricular dysfunction to acute heart failure, and even cardiogenic shock. Elevated serum cardiac troponin levels were prevalent among hospitalized patients with COVID-19; the higher the magnitude of troponin elevation, the greater the COVID-19 illness severity and in-hospital death risk. Whether these consequences were due to direct SARS-CoV-2 infection of cardiac cells or secondary to inflammatory responses steered early cardiac autopsy studies. SARS-CoV-2 was reportedly detected in endothelial cells, cardiac myocytes, and within the extracellular space. However, findings were inconsistent and different methodologies had their limitations. Initial autopsy reports suggested that SARS-CoV-2 myocarditis was common, setting off studies to find and phenotype inflammatory infiltrates in the heart. Nonetheless, subsequent studies rarely detected myocarditis. Microthrombi, cardiomyocyte necrosis, and inflammatory infiltrates without cardiomyocyte damage were much more common. In vitro and ex vivo experimental platforms have assessed the cellular tropism of SARS-CoV-2 and elucidated mechanisms of viral entry into and replication within cardiac cells. Data point to pericytes as the primary target of SARS-CoV-2 in the heart. Infection of pericytes can account for the observed pericyte and endothelial cell death, innate immune response, and immunothrombosis commonly observed in COVID-19 hearts. These processes are bidirectional and synergistic, rendering a definitive order of events elusive. Single-cell/nucleus analyses of COVID-19 myocardial tissue and isolated cardiac cells have provided granular data about the cellular composition and cell type-specific transcriptomic signatures of COVID-19 and microthrombi-positive COVID-19 hearts. Still, much remains unknown and more in vivo studies are needed. This review seeks to provide an overview of the current understanding of COVID-19 cardiac pathophysiology. Cell type-specific mechanisms and the studies that provided such insights will be highlighted. Given the unprecedented pace of COVID-19 research, more mechanistic details are sure to emerge since the writing of this review. Importantly, our current knowledge offers significant clues about the cardiac pathophysiology of long COVID-19, the increased postrecovery risk of cardiac events, and thus, the future landscape of cardiovascular disease.

Nanokit coupled electrospray ionization mass spectrometry for analysis of angiotensin converting enzyme 2 activity in single living cell.

Angiotensin-converting enzyme 2 (ACE2) is not only an enzyme but also a functional receptor on cell membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, the activity of ACE2 in single living cell is firstly determined using a nanokit coupled electrospray ionization mass spectrometry (nanokit-ESI-MS). Upon the insertion of a micro-capillary into the living hACE2-CHO cell and the electrochemical sorting of the cytosol, the target ACE2 enzyme hydrolyses angiotensin II inside the capillary to generate angiotensin 1-7. After the electrospray of the mixture at the tip of the capillary, the product is differentiated from the substrate in molecular weight to achieve the detection of ACE2 activity in single cells. The further measurement illustrates that the inflammatory state of cells does not lead to the significant change of ACE2 catalytic activity, which elucidates the relationship between intracellular ACE2 activity and inflammation at single cell level. The established strategy will provide a specific analytical method for further studying the role of ACE2 in the process of virus infection, and extend the application of nanokit based single cell analysis.

Increased mTOR Signaling and Impaired Autophagic Flux Are Hallmarks of SARS-CoV-2 Infection.

The COVID-19 (Coronavirus Disease 2019), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), severely affects mainly individuals with pre-existing comorbidities. Here our aim was to correlate the mTOR (mammalian/mechanistic Target of Rapamycin) and autophagy pathways with the disease severity. Through western blotting and RNA analysis, we found increased mTOR signaling and suppression of genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2 as well as in transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients. Immunofluorescence co-localization assays also indicated that SARS-CoV-2 colocalizes within autophagosomes but not with a lysosomal marker. Our findings indicate that SARS-CoV-2 can benefit from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling.

FLUIDOT: A Modular Microfluidic Platform for Single-Cell Study and Retrieval, with Applications in Drug Tolerance Screening and Antibody Mining.

Advancements in lab-on-a-chip technologies have revolutionized the single-cell analysis field. However, an accessible platform for in-depth screening and specific retrieval of single cells, which moreover enables studying diverse cell types and performing various downstream analyses, is still lacking. As a solution, FLUIDOT is introduced, a versatile microfluidic platform incorporating customizable microwells, optical tweezers and an interchangeable cell-retrieval system. Thanks to its smart microfluidic design, FLUIDOT is straightforward to fabricate and operate, rendering the technology widely accessible. The performance of FLUIDOT is validated and its versatility is subsequently demonstrated in two applications. First, drug tolerance in yeast cells is studied, resulting in the discovery of two treatment-tolerant populations. Second, B cells from convalescent COVID-19 patients are screened, leading to the discovery of highly affine, in vitro neutralizing monoclonal antibodies against SARS-CoV-2. Owing to its performance, flexibility, and accessibility, it is foreseen that FLUIDOT will enable phenotypic and genotypic analysis of diverse cell samples and thus elucidate unexplored biological questions.

Single-cell analytical technologies: uncovering the mechanisms behind variations in immune responses.

The immune landscape varies among individuals. It determines the immune response and results in surprisingly diverse symptoms, even in response to similar external stimuli. However, the detailed mechanisms underlying such diverse immune responses have remained mostly elusive. The utilization of recently developed single-cell multimodal analysis platforms has started to answer this question. Emerging studies have elucidated several molecular networks that may explain diversity with respect to age or other factors. An elaborate interplay between inherent physical conditions and environmental conditions has been demonstrated. Furthermore, the importance of modifications by the epigenome resulting in transcriptome variation among individuals is gradually being revealed. Accordingly, epigenomes and transcriptomes are direct indicators of the medical history and dynamic interactions with environmental factors. Coronavirus disease 2019 (COVID-19) has recently become one of the most remarkable examples of the necessity of in-depth analyses of diverse responses with respect to various factors to improve treatment in severe cases and to prevent viral transmission from asymptomatic carriers. In fact, determining why some patients develop serious symptoms is still a pressing issue. Here, we review the current "state of the art" in single-cell analytical technologies and their broad applications to healthy individuals and representative diseases, including COVID-19.

In-vivo reprogramming macrophages and dendritic cells with Allocetra-OTS: Successful mono- and combination-antitumor therapy

Background: Chimeric antigen receptor (CAR) T cells can activate an immune response to a cancer-specific antigen but is less effective in solid tumors. Immune check point inhibitors (ICI) revolutionized the treatment of solid tumors, however, in many tumors only partial response is achieved. Here we questioned the role of synergistic effect of Allocetra-OTS (cellular therapy for in-vivo reprogramming macrophages and dendritic cells, Enlivex Therap.) on solid tumor progression. Methods: To follow tumor growth in vivo, HeLa-CD19 cells were stably transduced with pLenti-PGK-V5-Luc-Neo. For CAR preparation, fresh mononuclear cells (MNC) were transfected with CD19-CAR plasmids. For the intraperitoneal solid tumor model, SCID-Bg mice were injected intraperitoneally (IP) with human HeLa- CD19 or HeLa-CD19-luciferase cells, 10×106 allocetra-OTS or vehicle, and 10×106 CD19-CAR T cells or mock T cells. In an immune-competent model, Balb/c mice were treated IP with AB12 (mesothelioma) with pLenti-PGK-V5-Luc-Neo and treated with anti-CTLA4 with or without Allocetra-OTS. Mice were monitored daily for clinical signs and peritoneal fluid accumulation and weekly for tumor growth. Kaplan-Meier log rank test was done for survival. Peritoneal cells were evaluated using single cell analysis and flow cytometry. Tumors were examined for bacterial presence by immunohistochemistry staining with antilipoteichoic acid (LTA) and antilipopolysaccharide (LPS). For allocetra-OTS preparation, enriched mononuclear fractions were collected by leukapheresis from healthy eligible human donors and induced to undergo early apoptosis. Results: SCID mice survived 30±5 days (range 27-37) and were sacrificed or died from solid tumor in the peritoneal cavity after accumulation of bloody peritoneal fluid and clinical deterioration. Results were verified using IVIS of intraperitoneal HeLaCD19- Luc cells. CAR T cell therapy significantly ameliorated survival to 55±11 days (p < 0.05 vs MOCK) but Alloctra-OTS further ameliorated survival to 75±10 (p < 0.001) with 20-40% complete remission. In AB12 model, anti CTLA4 therapy significantly ameliorated survival from 26±5 to 38 ±9 days (p < 0.05). However, Allocetra-OTS monotherapy ameliorated survival to 45 ±12 days (p < 0.02) and combinational therapy to 75±9 days (p < 0.0001) with complete remission in 60-75% of mice. Single cell analysis revealed that restoration of large peritoneal macrophages (LPM), were associated with antitumor activity. Conclusions: During intraperitoneal tumor progression, allocetra-OTS as monotherapy or combinational therapy with CAR or anti-CTLA4 significantly reduced tumor size and enable complete remission in up to 75% treated mice. Based on excellent safety profile in > 30 patients treated for sepsis and Covid19, human phase I/II of allocetra-OTS plus ICI, for peritoneal metastases, is planned for 2022.

From COVID to fibrosis: lessons from single-cell analyses of the human lung.

The increased resolution of single-cell RNA-sequencing technologies has led to major breakthroughs and improved our understanding of the normal and pathologic conditions of multiple tissues and organs. In the study of parenchymal lung disease, single-cell RNA-sequencing has better delineated known cell populations and identified novel cells and changes in cellular phenotypes and gene expression patterns associated with disease. In this review, we aim to highlight the advances and insights that have been made possible by applying these technologies to two seemingly very different lung diseases: fibrotic interstitial lung diseases, a group of relentlessly progressive lung diseases leading to pulmonary fibrosis, and COVID-19 pneumonia, an acute viral disease with life-threatening complications, including pulmonary fibrosis. We discuss changes in cell populations and gene expression, highlighting potential common features, such as alveolar cell epithelial injury and aberrant repair and monocyte-derived macrophage populations, as well as relevance and implications to mechanisms of disease and future directions.

Coronavirus disease 2019 and the placenta: A literature review.

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has been implicated in the clinical pathology of multiple organs and organ systems. Due to the novelty of the disease, there is a need to review emerging literature to understand the profile of SARS-CoV-2 in the placenta. This review sought to evaluate the literature on the mediators, mechanism of entry, pathogenesis, detection, and pathology of SARS-CoV-2 in the placenta. Systematic literature searches found 96 eligible studies. Our review revealed that SARS-CoV-2 canonical mediators, angiotensin-converting enzyme-2 (ACE2), and transmembrane serine protease-2 (TMPRSS2) are variably expressed in various placenta compartments, including the villous cytotrophoblasts, syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs) throughout pregnancy. Placental SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs), including basigin (BSG/CD147), dipeptidyl peptidase-4 (DPP4/CD26), cathepsin B/L (CTL B/L), furin, interferon-induced transmembrane protein (IFITM1-3), and lymphocyte antigen 6E (LY6E) may increase or reduce the permissiveness of the placenta to SARS-CoV-2. EVTs express genes that code for proteins that may drive viral pathogenesis in the placenta. Viral RNA, proteins, and particles were detected primarily in the STBs by in situ hybridization, immunohistochemistry, electron microscopy, and polymerase chain reaction. Placental pathology in SARS-CoV-2-infected placentas included maternal and fetal vascular malperfusion and a generally nonspecific inflammatory-immune response. The localization of SARS-CoV-2 receptors, proteases, and genes involved in coding proteins that drive viral pathogenesis in the placenta predisposes the placenta to SARS-CoV-2 infection variably in all pregnancy trimesters, with antecedent placental pathology. There is a need for further studies to explicate the mechanism of entry and pathogenesis of SARS-CoV-2 in the placenta.

Single-cell transcriptome profiling of the immune space-time landscape reveals dendritic cell regulatory program in polymicrobial sepsis.

Rationale: Evident immunosuppression has been commonly seen among septic patients, and it is demonstrated to be a major driver of morbidity. Nevertheless, a comprehensive view of the host immune response to sepsis is lacking as the majority of studies on immunosuppression have focused on a specific type of immune cells. Methods: We applied multi-compartment, single-cell RNA sequencing (scRNA-seq) to dissect heterogeneity within immune cell subsets during sepsis progression on cecal ligation and puncture (CLP) mouse model. Flow cytometry and multiplex immunofluorescence tissue staining were adopted to identify the presence of 'mature DCs enriched in immunoregulatory molecules' (mregDC) upon septic challenge. To explore the function of mregDC, sorted mregDC were co-cultured with naïve CD4+ T cells. Intracellular signaling pathways that drove mregDC program were determined by integrating scRNA-seq and bulk-seq data, combined with inhibitory experiments. Results: ScRNA-seq analysis revealed that sepsis induction was associated with substantial alterations and heterogeneity of canonical immune cell types, including T, B, natural killer (NK), and myeloid cells, across three immune-relevant tissue sites. We found a unique subcluster of conventional dendritic cells (cDCs) that was characterized by specific expression of maturation- and migration-related genes, along with upregulation of immunoregulatory molecules, corresponding to the previously described 'mregDCs' in cancer. Flow cytometry and in stiu immunofluorescence staining confirmed the presence of sepsis-induced mregDC at protein level. Functional experiments showed that sepsis-induced mregDCs potently activated naive CD4+ T cells, while promoted CD4+ T cell conversion to regulatory T cells. Further observations indicated that the mregDC program was initiated via TNFRSF-NF-κB- and IFNGR2-JAK-STAT3-dependent pathways within 24 h of septic challenge. Additionally, we confirmed the detection of mregDC in human sepsis using publicly available data from a recently published single-cell study of COVID-19 patients. Conclusions: Our study generates a comprehensive single-cell immune landscape for polymicrobial sepsis, in which we identify the significant alterations and heterogeneity in immune cell subsets that take place during sepsis. Moreover, we find a conserved and potentially targetable immunoregulatory program within DCs that associates with hyperinflammation and organ dysfunction early following sepsis induction.

Microfluidics as a Novel Technique for Tuberculosis: From Diagnostics to Drug Discovery.

Tuberculosis (TB) remains a global healthcare crisis, with an estimated 5.8 million new cases and 1.5 million deaths in 2020. TB is caused by infection with the major human pathogen Mycobacterium tuberculosis, which is difficult to rapidly diagnose and treat. There is an urgent need for new methods of diagnosis, sufficient in vitro models that capably mimic all physiological conditions of the infection, and high-throughput drug screening platforms. Microfluidic-based techniques provide single-cell analysis which reduces experimental time and the cost of reagents, and have been extremely useful for gaining insight into monitoring microorganisms. This review outlines the field of microfluidics and discusses the use of this novel technique so far in M. tuberculosis diagnostics, research methods, and drug discovery platforms. The practices of microfluidics have promising future applications for diagnosing and treating TB.

GeneTrail: A Framework for the Analysis of High-Throughput Profiles.

Experimental high-throughput techniques, like next-generation sequencing or microarrays, are nowadays routinely applied to create detailed molecular profiles of cells. In general, these platforms generate high-dimensional and noisy data sets. For their analysis, powerful bioinformatics tools are required to gain novel insights into the biological processes under investigation. Here, we present an overview of the GeneTrail tool suite that offers rich functionality for the analysis and visualization of (epi-)genomic, transcriptomic, miRNomic, and proteomic profiles. Our framework enables the analysis of standard bulk, time-series, and single-cell measurements and includes various state-of-the-art methods to identify potentially deregulated biological processes and to detect driving factors within those deregulated processes. We highlight the capabilities of our web service with an analysis of a single-cell COVID-19 data set that demonstrates its potential for uncovering complex molecular mechanisms. GeneTrail can be accessed freely and without login requirements at http://genetrail.bioinf.uni-sb.de.

On Deep Landscape Exploration of COVID-19 Patients Cells and Severity Markers.

COVID-19 is a disease with a spectrum of clinical responses ranging from moderate to critical. To study and control its effects, a large number of researchers are focused on two substantial aims. On the one hand, the discovery of diverse biomarkers to classify and potentially anticipate the disease severity of patients. These biomarkers could serve as a medical criterion to prioritize attention to those patients with higher prone to severe responses. On the other hand, understanding how the immune system orchestrates its responses in this spectrum of disease severities is a fundamental issue required to design new and optimized therapeutic strategies. In this work, using single-cell RNAseq of bronchoalveolar lavage fluid of nine patients with COVID-19 and three healthy controls, we contribute to both aspects. First, we presented computational supervised machine-learning models with high accuracy in classifying the disease severity (moderate and severe) in patients with COVID-19 starting from single-cell data from bronchoalveolar lavage fluid. Second, we identified regulatory mechanisms from the heterogeneous cell populations in the lungs microenvironment that correlated with different clinical responses. Given the results, patients with moderate COVID-19 symptoms showed an activation/inactivation profile for their analyzed cells leading to a sequential and innocuous immune response. In comparison, severe patients might be promoting cytotoxic and pro-inflammatory responses in a systemic fashion involving epithelial and immune cells without the possibility to develop viral clearance and immune memory. Consequently, we present an in-depth landscape analysis of how transcriptional factors and pathways from these heterogeneous populations can regulate their expression to promote or restrain an effective immune response directly linked to the patients prognosis.

Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions.

COVID-19 can cause severe neurological symptoms, but the underlying pathophysiological mechanisms are unclear. Here, we interrogated the brain stems and olfactory bulbs in postmortem patients who had COVID-19 using imaging mass cytometry to understand the local immune response at a spatially resolved, high-dimensional, single-cell level and compared their immune map to non-COVID respiratory failure, multiple sclerosis, and control patients. We observed substantial immune activation in the central nervous system with pronounced neuropathology (astrocytosis, axonal damage, and blood-brain-barrier leakage) and detected viral antigen in ACE2-receptor-positive cells enriched in the vascular compartment. Microglial nodules and the perivascular compartment represented COVID-19-specific, microanatomic-immune niches with context-specific cellular interactions enriched for activated CD8+ T cells. Altered brain T-cell-microglial interactions were linked to clinical measures of systemic inflammation and disturbed hemostasis. This study identifies profound neuroinflammation with activation of innate and adaptive immune cells as correlates of COVID-19 neuropathology, with implications for potential therapeutic strategies.

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

Single Cell Transcriptomic Re-analysis of Immune Cells in Bronchoalveolar Lavage Fluids Reveals the Correlation of B Cell Characteristics and Disease Severity of Patients with SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic (severe acute respiratory syndrome coronavirus 2) is a global infectious disease with rapid spread. Some patients have severe symptoms and clinical signs caused by an excessive inflammatory response, which increases the risk of mortality. In this study, we reanalyzed scRNA-seq data of cells from bronchoalveolar lavage fluids of patients with COVID-19 with mild and severe symptoms, focusing on Ab-producing cells. In patients with severe disease, B cells seemed to be more activated and expressed more immunoglobulin genes compared with cells from patients with mild disease, and macrophages expressed higher levels of the TNF superfamily member B-cell activating factor but not of APRIL (a proliferation-inducing ligand). In addition, macrophages from patients with severe disease had increased pro-inflammatory features and pathways associated with Fc receptor-mediated signaling, compared with patients with mild disease. CCR2-positive plasma cells accumulated in patients with severe disease, probably because of increased CCL2 expression on macrophages from patients with severe disease. Together, these results support the hypothesis that different characteristics of B cells might be associated with the severity of COVID-19 infection.