Inherited deficiency of the OAS-RNase L pathway in children with MIS-C

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic dsRNA-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the ssRNA-degrading RNase L. Consistent with the absence of pneumonia in these patients, epithelial cells and fibroblasts defective for this pathway restricted SARS-CoV-2 normally. This contrasted with IFNAR1-deficient cells from patients prone to hypoxemic pneumonia without MIS-C. Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNASEL deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or SARS-CoV- 2 stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-but not RNase L- deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by MAVS deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.Copyright © 2023 Elsevier Inc.

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.

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).

Fc RECEPTOR-MEDIATED INFECTION OF MYELOID CELLS BY SARS-CoV-2

Background: The role of myeloid cells in the pathogenesis of SARS-CoV-2 is well established, in particular as drivers of cytokine production and systemic inflammation characteristic of severe COVID-19. However, the potential for myeloid cells to act as bona fide targets of productive SARS-CoV-2 infection remains unclear. Method(s): Using anti-SARS-CoV-2 mAbs with a range of neutralisation potencies and binding specificities, we performed a detailed assessment of mAb-mediated infection of monocytes/macrophages. THP-1 cells were used as a model system, with results confirmed in primary macrophages. Result(s): Infection of THP-1 cells was seen via mAbs targeting the spike RBD, but not with those targeting the NTD or S2 subunit. mAbs with the most consistent potential to mediate infection targeted a conserved region of the RBD (group 1/class IV). No infection was seen with the same quantity of virus but in the absence of antibody, and pre-treating the cells with FcgammaRI and -II blocking antibodies inhibited infection. Thus, antibody-FcR interactions are able to expand the tropism of SARS-CoV-2. Time-course studies demonstrated high-level and productive infection. Studies performed in human iPSC-derived macrophages and primary monocyte-derived macrophages paralleled results seen in THP-1 cells but with lower infection levels. Up to 2% of macrophages were infected, with infected cells appearing multinucleated and syncytial. Addition of ruxolitinib, an inhibitor of JAK1/2 signalling, increased infection up to 10-fold, indicating limitation of infection through innate immune mechanisms. Sera from primary infections (n=80) mediated rare infection events, with a minority of samples (n=3) promoting significant infection. Competition assays confirmed results seen in sera, with the addition of neutralising mAbs diminishing the infection seen with infection-mediating mAbs. Thus, the presence of antibodies with potential to mediate infection is not sufficient to predict myeloid cell infection, rather, the context in which the antibodies are produced is key. Conclusion(s): We hypothesise that a nascent antibody response during peak viral replication in primary infection presents a window of opportunity for myeloid cells to become infected, while establishment of a robust polyclonal response via vaccination or prior infection reduces the likelihood of this occurring. Infection via antibody-FcR interactions could contribute to pathogenesis in primary infection, systemic virus spread or persistent infection.

Mesenchymal Stem Cells: A Potent Cell Source for COVID-19

Coronaviruses are enveloped positive-stranded RNA viruses that cause mild to acute respiratory illness. Coronaviruses can merge envelope proteins with the host cell membranes and de-liver their genetic material. Coronavirus disease 2019 (COVID-19) is the seventh coronavirus clos-est to the severe acute respiratory syndrome (SARS) in bats that infects humans. COVID-19 at-tacks the respiratory system and stimulates the host inflammatory responses, promotes the recruit-ment of immune cells, and enhances angiotensin-converting enzyme 2 (ACE2) activities. Patients with confirmed COVID-19 have experienced fever, dry cough, headache, dyspnea, acute kidney injury (AKI), acute respiratory distress syndrome (ARDS), and acute heart injury. Several strategies such as oxygen therapy, ventilation, antibiotic or antiviral therapy, and renal replacement therapy are commonly used to decrease COVID-19-associated mortality. Inflammation is a common and important factor in the pathogenesis of COVID-19. In recent years, stem cell-based therapies represent a promising therapeutic option against various diseases. Mesenchymal stem cells (MSCs) are multipotent stem cells that can self-renew and differentiate into various tissues of mesodermal ori-gin. MSCs can be derived from bone marrow, adipose tissue, and umbilical cord blood. MSCs, with their unique immunomodulatory properties, represent a promising therapeutic alternative against diseases associated with inflammation. Several previous studies have shown that MSCs with a strong safety profile can improve the treatment of patients with COVID-19. The information in this review provides a summary of the prevention and diagnosis of COVID-19. Also, we focus on the current clinical application of MSCs for treatments of patients with COVID-19.Copyright © 2021 Bentham Science Publishers.

Phase 3 RCT of C5a-Specific Vilobelimab in Severe COVID-19 Pneumonia

Background: Blocking the C5a-C5aR axis in COVID-19 patients could improve outcomes by limiting myeloid cell infiltration in damaged organs and preventing excessive lung inflammation and endothelialitis. Aims and Objectives: Vilobelimab (VILO), an anti-C5a mAb that preserves the membrane attack complex (MAC), was tested in a Phase III adaptively designed multicenter, double-blind placebo (P)-controlled study for survival in critically ill COVID-19 patients. Method(s): COVID-19 pneumonia patients (N=369;VILO n=178, P n=191) within 48 hrs of intubation were randomly assigned to receive 6, 800 mg infusions of VILO or P on top of standard of care. Primary outcome was 28-day allcause mortality. Result(s): 28-day all-cause mortality was 31.7% VILO vs 41.6% P (Kaplan-Meier estimates;Cox regression site stratified, HR 0.73;95%CI:0.50-1.06;P=0.094) with a 22.7% relative mortality reduction to Day 60. In predefined primary outcome analysis without site stratification, VILO significantly reduced 28-day mortality (HR 0.67;95%CI:0.48-0.96;P=0.027);needed to treat number, 10 to save 1. VILO significantly reduced 28-day mortality in severe patients with baseline WHO ordinal scale score of 7 (n=237, HR 0.62;95%CI:0.40-0.95;P=0.028) or severe ARDS/PaO2/FiO2<=100 mmHg (n=98, HR 0.55;95%CI:0.30-0.98;P=0.044) or eGFR<60 mL/min/1.73m2 (n=108, HR 0.55;95%CI:0.31-0.96;P=0.036). Treatment emergent AEs were 90.9% VILO vs 91.0% P. Infections were comparable;VILO (62.9%), P (59.3%). Serious AEs were 58.9% VILO, 63.5% P. Conclusion(s): VILO reduced mortality at 28 to 60 days in severe COVID-19 pneumonia patients with no increase in infections suggesting the importance of targeting C5a while preserving MAC.

Impact of Tet2-Mutant Clonal Hematopoiesis on Solid Tumor Immunology and Response to Checkpoint Blockade

Background Clonal hematopoiesis (CH) is an age-related phenomenon characterized by the overrepresentation of blood cells arising from a single, mutant clone and is detectable in 10-20% of individuals over 70.1 CH has now been implicated in a variety of non-hematological disorders, such as cardiovascular diseases and Covid-19 infections, by exacerbating the innate inflammatory response.2-4 However, the impact of CH in solid tumors and response to immune checkpoint blockade (ICB) is unknown. Methods To assess the prevalence and role of CH in patients with solid tumors, we analyzed publicly available data from the MSKCC-IMPACT study.5, 6 To mechanistically study CH in solid tumors, we established an orthotopic model of pancreatic adenocarcinoma (PDAC) in mice with Tet2+/- CH. CH and WT mice were treated with either ICB (aCTLA-4 + aPD-1) or vehicle control. Single-cell (sc-) RNAseq was performed on tumor infiltrating lymphocytes (n=3/group) while remaining mice were observed for disease progression and overall survival (n=10/group). Results Analyzing CH frequencies in a cohort of patients with solid tumors, we observed that the prevalence of CH was approximately 5 times higher in patients with cancer when compared to healthy age-matched controls. Further, patients with detectable CH clones had significantly worse overall survival (figure 1A). In vivo, sc-RNAseq data revealed that myeloid cells present within the pancreatic tumors of mice with Tet2+/- CH were significantly enriched for both type I and type II interferon (IFN) signaling (figure 1B). Further, these IFN+ myeloid cells were ablated after ICB therapy in Tet2+/+ WT mice but persisted in mice with Tet2+/- CH (figure 1C). PDAC tumors from mice with Tet2+/- CH had approximately half the total number of infiltrating CD8 T cells at baseline when compared to those from Tet2+/+ WT mice. Upon ICB treatment, CD8 effector cells only expanded in the tumors from Tet2+/+ WT mice. Functionally, this translated to more rapidly progressing tumors, resistance to ICB, and reduced overall survival in mice with Tet2+/- CH (figure 1D). Conclusions CH is present in upwards of 30% of patients with solid tumors and is associated with significantly worsened prognosis. Modeling PDAC in the presence of Tet2+/- CH in vivo revealed distinct alterations in the tumor microenvironment that ultimately influenced tumor progression and response to ICB. This proposed research bridges the fields of solid tumor immunology and clonal hematopoiesis to address novel mechanisms of immunotherapy resistance that will span cancer type and, ultimately, improve patient care.

Chronic exposure to waterpipe smoke elicits immunomodulatory and carcinogenic effects in the lung

Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector as a socially acceptable form of smoking, particularly among the youth. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using both an experimental animal model and an exploratory observational analysis of human waterpipe smokers and non-smokers. Mice exposed to increasing doses of WPS (once or five times per week for 20 weeks), through an exclusively devised exposure system for this study, were more prone to develop lung tumors compared to control-air exposed littermates. This effect was accompanied by various pro-tumor immune phenotypes, including increased IL-17A+ levels in T/B cells as well as elevated expression of the immune checkpoint PD-L1 and the pro-inflammatory cytokine IL-1β in myeloid cells. While flow cytometry analysis revealed increased CD4 and CD8 T cell infiltration in WPSexposed mice, gene signatures of cytotoxic and expanded immune response were, conversely, decreased in animals exposed to heavy WPS relative to control air. Interestingly, mice heavily exposed to WPS exhibited increased expression of Tmprss4, Cd55, and Ace2, cell receptors and mediators of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and, thus, COVID-19 pathogenesis. We also perform RNA-sequencing analysis of bronchial airway epithelial brushings of cancer-free waterpipe smokers and non-smokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe non-smokers, harbored gene programs that were associated with poor clinical outcomes in lung adenocarcinoma (LUAD) patients, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Overall, our analyses demonstrate immunomodulatory and carcinogenic effects of WPS on the murine and human lung. Our study also shows that WPS exposure leads to a field of injury that may be associated with increased risk for lung cancer. This study is unique for interrogating carcinogenic effects of chronic exposure to WPS with the longest, to our knowledge, follow-up time in vivo. Our findings accentuate the need for additional studies that can guide evidence-based policies to counteract shortfalls in public health control of waterpipe smoking.

PERSISTENT IMMUNE CELL ABNORMALITIES IN THE INTESTINAL MUCOSA OF PATIENTS AFTER RECOVERY FROM COVID-19 INFECTION

Background: Although respiratory failure is the hallmark of severe disease, it is increasingly clear that Coronavirus Disease 2019 (COVID-19) is a multi-system disorder. The presence of gastrointestinal (GI) involvement by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been suggested by epidemiological, clinical, non-human primate, in-vitro (enteroid) and ex-vivo (human biopsy) studies. Having recently documented persistence of SARS-CoV-2 within the intestinal epithelium 7 months after infection, here we aimed to study mucosal immune cell abnormalities in individuals with prior history of COVID-19. Methods: Individuals with previous COVID-19 diagnosis (by either RT–PCR or seroconversion) and controls (without RT-PCR or serological evidence of prior COVID-19 infection) undergoing endoscopic evaluation were recruited into the study (Table 1). Colonic and small intestinal (duodenal and ileal) biopsies were analyzed by multiparameter flow cytometry for mucosal immune cell populations including myeloid cells (classical and non-classical monocytes, dendritic cell subsets), T cells (subsets and activation state), B cells (including plasma cells) and NK cells. Persistence of viral antigens was determined by immunofluorescence microscopy (n=30) using a previously published anti-nucleocapsid (NP) antibody. Results: Thirty subjects with a previous history of COVID-19 (post-COVID), median of 4 months from diagnosis (range 1-10 months), were recruited and compared with 40 normal volunteer (NV) controls. Relative to controls, post-COVID subjects displayed higher frequencies of classical (CD14+) monocytes in both, the colon and the small bowel, while significantly higher frequencies of conventional dendritic cells (cDC)1 (lin-HLA-DRhiCD14- CD11c+CD141+) and cDC2 (lin-HLA-DRhiCD14-CD11c+CD1c+) were noted in the colon. Among NK subsets, CD56bright CD16- NK cells were significantly higher in the colon of post-COVID subjects. Among T cell subsets, CD8+ tissue resident memory T cells (CD8+CD69+CD103+) were significantly increased in colon of post-COVID subjects compared to NV. Among B cell subsets, plasma cells (CD3-CD27+CD38hi) trended higher (p= 0.06), while mucosal B cells (CD3-CD19+) were significantly lower in the terminal ileum of post-COVID subjects compared to NV. Finally, with IF, we detected SARS-CoV-2 NP in 10 out of 30 (33%) of post-COVID subjects (Figure 1). Conclusion: Innate and adaptive immune cell abnormalities persist in the intestinal mucosa of post-COVID subjects for up to 10 months and may reflect viral persistence or immune cell dysregulation in the intestines. These findings have major implications for understanding the pathogenesis of long-term sequelae of COVID-19, including long-haul COVID.(Table Presented)(Figure Presented)

A SINGLE-CELL SURVEY OF GASTRIC MUSCULARIS MACROPHAGES IN DIABETIC GASTROPARESIS

Background: Gastric muscularis propria immune cells play an instrumental role in homeostasis and disease. A subset of these cells, muscularis macrophages (MMs) are involved in the pathobiology of diabetic gastroparesis (DG) but are poorly understood. This study aims to survey transcriptional and functional profiling of gastric MMs in DG and diabetes. Methods: Full-thickness gastric body biopsies were obtained from patients with DG and diabetic controls. CD45+ cells were isolated from dissociated muscle tissue using magnetic beads. 10xGenomics was used for scRNA-seq library prep and cells sequenced by Illumina HiSeq4000. Bioinformatic analyses was performed using Suite and Seurat. Myeloid cells were annotated through a pseudogating strategy that identifies cells by differential expression levels of HLA-DR, CD14, CD11b, and CD11c based on flow cytometry-based gating utilized in a recent analysis of human small intestinal MMs. Canonical signaling pathways were determined using Ingenuity Pathway Analysis (IPA). Results: A total of 21,740 high-quality single-cell transcriptomes were generated from 16 subjects (DG=6, age 32±8 yr, BMI 23.7±3.9, 48.2±40.1% 4 hr gastric retention, average GCSI score 3.7±0.5;Diabetic controls= 10, age 53±13 yr, BMI 42.2±5.7). Through annotating 8,693 myeloid cells (DG 1509, Controls 7184), we characterized 1,788 as MMs (CD45+HLA-DR+) and 448 as dendritic cells (CD14-CD11c+). Utilizing a priori markers for pseudogating, the MMs were divided into four populations (Figure 1): subset 1 (CD14+CD11c+HLA-DRint, 5.6%), subset 2 (CD14+CD11c+HLA-DRhi, 36.0%), subset 3 (CD14+CD11c-CD11b-, 41.8%), and subset 4 (CD14+CD11c-CD11b+, 16.6%). The overall proportions of cells in the 4 subsets were similar to a prior approach in small bowel using gating. The expected ratio of cells from DG/diabetic control was 21% based on imputed cells. Subsets 1 and 4 were significantly decreased in DG compared to controls with ratios 15% and 14% respectively while subsets 2 and 3 were unchanged (21% and 20%). On IPA, phagosome formation and immune cell trafficking represented canonical signaling pathways of subset 1 and coronavirus phagocytosis pathway and phagosome formation of subset 4. Canonical genes of subset 1 included S100A12, A8, A9, and CSTA and subset 4 as LYVE1, MAF, MRC1 (CD206), MS4A4, and A2M. Subset 4 also had the highest expression of neuron-related genes (NPTX2, BMP2) similar to that observed in the small intestine. Conclusions: Pseudogating based on the transcriptomic expression of gastric immune cells reveal MM clusters similar in gene expression and proportions to previously characterized MMs in human small bowel using gating. The reduction of MM clusters associated with anti-inflammatory, phagocytosis, and neuronal signaling in specialized MMs subsets may suggest candidate targets in the pathophysiology of DG. Supported by NIHDK074008. (Figure Presented) Figure 1. Single-Cell RNA-Seq Profiling of Human Gastric Muscularis Macrophages in DG and Diabetes. T-distributed Stochastic Neighbor Embedding (tSNE) plot of muscularis macrophages in DG and diabetic control subjects by their differential genes from MAST (FDR < 0.05), color-coded by Status. *Mf1 and Mf2 not visualized as distinct clusters due to inadequate separation of overall gene expression in cells distinguished by HLA-DRint (Mf1) and HLA-DRhi (Mf2)

Biomarker Identification in Fatal Covid-19 and Post-Acute Sequalae of SARS-CoV-2 (PASC) Lungs

Rationale: SARS-CoV-2 has affected millions worldwide. Among those individuals infected with this coronavirus, most recover without hospitalization. However, COVID-19 is characterized by chronic lung failure and death in a significant number of hospitalized patients. Indeed, there is growing evidence that SARS-CoV-2 causes ARDS leading to lung fibrosis that shares similarities with interstitial lung diseases (ILDs) including idiopathic pulmonary fibrosis (IPF). Herein, we addressed the hypothesis that fatal COVID-19, PASC, and ILDs share key biomarkers of interest in lung fibrosis. Methods: This study included 9 fatal COVID-19 and 13 PASC cases, who received lung transplants due COVID-19 associated lung failure. Clinical characteristics such as duration of mechanical ventilation, length of hospitalization, age, sex, and BMI were evaluated in each patient. Autopsy and explanted lung samples were subjected to histopathological and/or immunohistochemical (IHC) analysis for key biomarkers of interest in lung fibrosis including bromodomain-containing protein-4(BRD4), interferon alpha 2(IFNα2), interleukin-1(IL-11), growth differentiation factor 15(GDF15), and keratin 8(KRT8). COVID-19 and PASC lung samples were also compared with lung samples from fatal ARDS due to other causes (i.e., non-COVID-19 ARDS).Results: In the fatal COVID-19 patient group, the mean age was 60.6(50-71) years-old and included 6 males and 3 females. In the transplanted-PASC patient group, the mean age was 46(31-71) years-old and included 12 males and 2 females. The average BMI was 28.3(21-35.5) for fatal COVID-19 and 25.2(19-29.5) for PASC. In fatal COVID-19, comorbidities included hypertension(22%), diabetes(44%), immunocompromised status(11%). The mean duration of mechanical ventilation was 23(8-65) days while hospitalization was 25(8-67) days. Conversely, PASC patients averaged 168(71-539) days from diagnosis to transplant date. The SARS-CoV-2 ARDS survivors that developed chronic lung failure had diffuse interstitial fibrosis frequently with organization into a non-specific interstitial fibrosis (NSIP) pattern. Key IHC findings in fatal COVID-19 and in PASC lung samples included BRD4, IFNalpha2, and IL-11 receptor alpha (IL-11RA) protein expression, which were markedly increased in several cell types most notably macrophages or myeloid cells localized in the alveolar space in COVID-19 lung samples. Although these markers were detected in non-COVID-19 ARDS the levels of each were markedly lower than that detected in the COVID-19 lung samples.Conclusions: These data suggest that key profibrotic pathways in the lung are shared among COVID-19 and chronic fibrotic ILDs. The identification of these pathways provides the impetus to further explore treatment strategies which might survival benefit to chronically ventilated COVID-19 patients and mitigate the need from lung transplantation in PASC patients.

Effects of favipiravir on hematologic parameters and bone marrow in the rats

Favipiravir, a selective RNA polymerase inhibitor agent, is an antiviral drug currently used effectively in treating pandemic diseases such as Covid-19. The present study aims to determine the effects of favipiravir use on bone marrow and blood cells. Twelve male Wistar rats were divided into two groups, namely control and favipiravir groups. Physiological saline at a dose of 1 ml/kg was administered to the rats in the control group by oral gavage for 10 days. Rats in the favipiravir group were administered favipiravir by oral gavage at a dose of 200 mg/kg for 10 days. At the end of the study, the blood tissue was collected from the heart, and bone marrow samples were collected from the femur bone of the rats sacrificed under anesthesia. The hematologic parameters in the blood samples obtained were measured using an auto-analyzer device with the help of rat compatible kits. Bone marrow cell counts were performed by examining structural changes and myeloid and erythroid cell series in the smear samples. The results obtained in the study revealed that favipiravir use caused a decrease in the counts of some hematologic parameters containing erythrocytes, lymphocytes, and monocytes. In addition, it was determined that the ratio of myeloid and erythroid cells in bone marrow smears changed significantly with the use of favipiravir. It was concluded that treatment with favipiravir caused suppression of erythrocyte and some leukocyte series. The suppressor effects were also determined in bone marrow cell series in the rats.

Research progress on the BCG-induced trained immunity:mechanism and role

The purpose of Bacillus Calmette-Guérin (BCG) vaccination is to prevent Mycobacterium tuberculosis infection, but studies have shown that BCG activates innate immunity, causes epigenetic reprogramming and metabolic changes of myeloid cells, and forms innate immune memory or trained immunity. When bone marrow-like cells are stimulated by pathogens again, they show enhanced immune response and promote the host's nonspecific defense ability. Innate immune memory is also called training immunity. In recent years, BCG-induced innate immune memory has attracted much attention, and it will guide the design of novel vaccine. This article reviews the application of BCG in prevention and treatment of corone virus disease 2019, the non-specific protection and mechanism of BCG-mediated trained immunity.

Myeloid and lymphoid cell abnormalities persist in the intestinal mucosa of patients who recover from COVID-19 infection

Background: Although respiratory failure is the hallmark of severe disease, it is increasingly clear that Coronavirus Disease 2019 (COVID-19) is a multi-system disorder. The presence of gastrointestinal (Gl) involvement by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been suggested by epidemiological, clinical, non-human primate, invitro (enteroid) and ex-vivo (human biopsy) studies. Having recently documented persistence of SAR-CoV-2 within the intestinal epithelium 7 months after infection, here we aimed to study mucosal immune cell abnormalities in individuals with prior history of COVID-19. Methods: Individuals with previous COVID-19 diagnosis (by either RT- PCR or seroconversion) and controls (without RT-PCR or serological evidence of prior COVID-19 infection) undergoing endoscopic evaluation were recruited into the study (Table 1,2). Colonic and small intestinal (duodenal and ileal) biopsies were analyzed by multiparameter flow cytometry for mucosal immune cell populations including myeloid cells (classical and non-classical monocytes, dendritic cell subsets), T cells (subsets and activation state), B cells (including plasma cells). Persistence of viral antigens was determined by immunofluorescence microscopy (n=30) using a previously published anti-nucleocapsid (NP) antibody. Results: Thirty subjects with a previous history of COVID-19 (post- COVID), median of 4 months from diagnosis (range 1-10 months), were recruited and compared with 40 normal volunteer (NV) controls. Relative to controls, post-COVID subjects displayed higher frequencies of classical (CD14+) monocytes in both, the colon and the small bowel, while significantly higher frequencies of conventional dendritic cells (cDC) 1 (lin-HLA-DRhiCD14-CD11c+CD141+) and cDC2 (lin-HLA-DRhiCD14-- CD11c+CD1c+) were noted in the colon only. Among T cell subsets, CD8+ tissue resident memory T cells (CD8+CD69+CD103+) were significantly increased in colon of post-COVID subjects compared to NV. Among B cell subsets, plasma cells (CD3-CD27+CD38hi) trended higher (p=0.06), while mucosal B cells (CD3-CD19+) were significantly lower in the terminal ileum of post-COVID subjects compared to NV. Finally, with IF, we detected SARS-CoV-2 NP in 10 out of 30 (33%) of post-COVID subjects (Figure 1). There were no significant correlations of these cell populations with either time after the infection or IF positivity. Conclusion: Innate and adaptive immune cell abnormalities persist in the intestinal mucosa of post-COVID subjects for up to 10 months and may reflect viral persistence or immune cell dysregulation in the intestines. These findings have major implications for understanding the pathogenesis of long term sequela of COVID-19, including long-haul COVID.

Vancomycin Induced Maturation Arrest with Reactive Promyelocyte Proliferation: A Diagnosticand Therapeutic Challenge

Introduction: Drug induced leucopenia complicates any clinicalsituation especially when it's associated with COVID-19 infection.Here we report a case of vancomycin induced myeloid maturationarrest in a patient with COVID-19 infection where his conditionreverted to normal after stopping the drug.Aims &Objectives: It highlights the adverse effects of Vancomycinleading to promyelocyte proliferation posing a diagnostic challenge todifferentiate it from neoplastic promyelocyte proliferation.Materials &Methods: Case report: A 47 year old male presentedwith fever, dry cough and difficulty in breathing for 12 days. He wasCOVID-19 positive with CT lung showing bilateral pneumonic consolidations for which Vancomycin was started. Hemogram showedhemoglobin-6.8 gm/dl, total leucocyte count-3200/cumm with a differential count revealing: Neutrophil-28% (with left shift and featuresof dyspoiesis), Lymphocyte-66%, Monocyte-4%, Eosinophil-2%(Fig. 1a), and platelets-90,000/cumm. Bone marrow aspiration(BMA) and biopsy was advised. BMA was hypercellular for age withmarked promyelocyte proliferation &maturation arrest with strongMPO positivity (1b, c d). Bone marrow biopsy also reflected similarfindings (Fig-1e). FISH for PML-RARA translocation turned out to benegative, differentiating it from Acute promyeloytic Leukemia(Fig. 1f). A diagnosis of drug induced leucopenia with reactivepromyelocyte proliferation was made. Considering worsening of thesehematological findings, Vancomycin was stopped and patient'shematological findings improved drastically with stabilization ofhematological parameters.Result: Discussion: Drug induced leukopenia occurs in a dosedependent or dose-independent (idiosyncratic) reaction. Vancomycindependent antibodies against neutrophils lead to an autoimmunereaction directly affecting progenitor cell growth especially of myeloid cell lineage leading to maturation arrest. Secondly cytotoxicT-cell mediated response also has damaging effects on hematopoieticcells. Infections like COVID-19 can also lead to suppression ofnormal myeloid maturation due to release of interleukins.Conclusions: Myeloid maturation arrest with marked promyelocyteproliferation poses a diagnostic dilemma especially in patients presenting with cytopenia as they are confused with Acute promyelocytic Leukemia. This case highlights the importance of detailed knowledgerelated to drug induced myeloid maturation arrest which is reversibleafter stoppage of the offending drug.

Multi-Arm Trial of Inflammatory Signal Inhibitors (MATIS) for Hospitalised Patients with Mild or Moderate COVID-19 Pneumonia: A Structured Summary of a Study Protocol for a Randomised Controlled Trial

Introduction: Severe COVID-19 pneumonia is characterised by respiratory and multi-organ failure in the context of marked systemic inflammation. This hyperinflammatory syndrome is reflected by the elevation of several inflammatory molecules, such as C-reactive protein (CRP), ferritin, IL-6, troponin, and D-dimer. In a subset of patients, early intervention with signal inhibitors may treat the Covid-19 hyperinflammatory syndrome before the development of acute lung injury and organ failure. We present a summary of a study protocol for a randomised controlled, multi-arm trial with two novel inflammatory signal inhibitors;Ruxolitinib (RUX) and Fostamatinib (FOS) for the treatment of Covid-19 pneumonia. RUX is an oral Janus Associated Kinase (JAK1/JAK2) inhibitor approved for the treatment of splenomegaly, myelofibrosis and polycythaemia vera. Inhibition of STAT3 downregulates IL-6 and IL-23, which are important for the inflammatory effects of Th17 cells. Further, JAK2 inhibition has been shown to reduce levels of TNFa and CRP, as well as reducing viral cellular entry and assembly. FOS is an oral spleen tyrosine kinase (SYK) inhibitor approved for the treatment of chronic immune thrombocytopenia. Studies of severe acute respiratory distress syndrome (ARDS) suggest that the pathogenesis relies on a series of SYK events leading to cytokine and chemokine release. FOS acts by inhibiting SYK activity, blocking the production and release of cytokines induced via C-lectin receptors and Fc receptor activation, ameliorating the cytokine storm which precedes ARDS. Primary Objective: The primary objective of MATIS is to determine the efficacy of RUX or FOS compared to standard of care (SOC) to reduce the proportion of hospitalised patients progressing from mild or moderate to severe COVID-19 pneumonia at 14 days from baseline. Secondary objectives at 7, 14 and 28 days: - Determine the efficacy of RUX or FOS to reduce mortality - Determine the efficacy of RUX or FOS to reduce the need for invasive ventilation or ECMO - Determine the efficacy of RUX or FOS to reduce the need for non-invasive ventilation - Determine the efficacy of RUX or FOS to reduce the proportion of patients suffering significant oxygen desaturation - Determine the efficacy of RUX or FOS to reduce the need for renal replacement therapy - Determine the efficacy of RUX and FOS to reduce the incidence of venous thromboembolism COVID-19 pneumonia - Determine the efficacy of RUX and FOS to reduce the severity of COVID-19 pneumonia [graded by a modified WHO Ordinal Scale] - Determine the efficacy of RUX or FOS to reduce the level of inflammatory biomarkers - Determine the efficacy of RUX or FOS to reduce the duration of hospital admission - Evaluate the safety of RUX and FOS for COVID-19 pneumonia Study Design: This is a multi-arm, two-stage, open-label, randomised (1:1:1) controlled trial. Participants will be recruited during hospitalisation for COVID-19 in multiple centres in the UK. Eligible participants (table 1) are randomised to one of the three interventions (RUX, FOS, SOC) by a central web-based randomisation service. This uses randomisation sequences with random block sizes, stratified by age (<65 and ≥65 years) and site. The treatment duration is 14 days from baseline. Patients receiving RUX will be administered 10mg BD for Day 1-7 and 5mg BD for Day 8-14. FOS will be administered as 150mg BD day 1-7 and 100mg BD day 8-14. Participants receive follow up assessments on days 7, 14 and 28 after the first dose. Outcomes: Primary endpoints will be assessed with a pairwise comparison (FOS vs SOC and RUX vs SOC) of the proportion of participants diagnosed with severe COVID-19 pneumonia within 14 days. Severe COVID-19 pneumonia is defined by a modified WHO COVID-19 Ordinal Score 5, comprising the following indicators of disease severity: - Death - Requirement for invasive ventilation - Requirement for non-invasive ventilation including CPAP or high flow oxygen - O2 saturation < 90% on 60% inspired oxygen Samples size: In stage 1 of this multi-arm study, 171 parti ipants will be randomised (57 per arm). Following an interim analysis, if either intervention shows a signal of efficacy, stage 2 will recruit a further 95 participants per arm (Fig 1). Trial Status: Recruitment is ongoing and commenced 2nd October 2020. Currently 127 patients are recruited and stage 1 is projected to be completed by 1st September 2021. The full protocol can be accessed via the trial's website. [Formula presented] Disclosures: Milojkovic: Novartis: Honoraria, Speakers Bureau;Incyte: Honoraria, Speakers Bureau;Bristol-Myers Squibb: Honoraria, Speakers Bureau;Pfizer: Honoraria, Speakers Bureau. Cooper: Principia and Sanofi: Consultancy;Sanofi and Principia: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations expenses. OffLabel Disclosure: Fostamatanib - is a tyrosine kinase inhibitor with activity against spleen tyrosine kinase (SYK). In the context of treating COVID-19, Fostamatanib acts by inhibiting SYK activist, blocking the production and release of cytokines induced via C-lectin receptors and Fc receptor activation, ameliorating the cytokine storm which precedes ARDS. Studies of severe acute respiratory syndrome induced by coronavirus, suggest that pathogenesis relies on a series of SYK events. SYK medicates ctuokine and chemokine release, induced by the activation of C-lectin receptors and immunoglobulin Fc receptors, resulting in neutrophil and monocyte lung ingress, sequential activation of neutrophil extracellular traps and activation of lung epithelium and multiple myeloid cell. This is followed by inflammation and tissue destruction that contribute to ARDS. Ruxolitinib - A JAK1/JAK2 inhibitor. JAK and STAT molecules are proteins that trance extracellular stimulation into intracellular signalling, leading to expression of host inflammatory cytokines and a variety of immune cells. In the context of MATIS, we use low dose ruxolitinib to treat COVID-19 by targeting key signalling pathways implicated in the hyper-inflammatory response of patients with COVID-19 infection. The mechanisms of Ruxolitinib to act in COVID-19 is through inhibition of STAT3 activation, down regulating IL-6 and IL-23, signalling important for the inflammatory effects of Th17 cells. Furthermore it leads to reductions of TNFa and CRP.

A Phase 2/3 Randomized, Placebo-Controlled, Open-Label, Multi-Center Trial of Lenzilumab to Improve the Safety and Efficacy of CAR-T Cell Therapy in Adults with Relapsed or Refractory Large B-Cell Lymphoma (The SHIELD Study)

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive sub-type of non-Hodgkin's lymphoma(Liu, et al. Am J Hematol 2019). All three approved CD19-directed CAR-T therapies (axicabtagene ciloleucel, tisagenlecleucel, lisocabtagene maraleucel) are associated with toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) that can be severe, resulting in non-relapse mortality, ICU admission, and significant non-drug related health resource utilization which represent barriers to access and adoption (Nabhan, et al. J Clin. Pathway 2017). Studies have shown that early elevation of granulocyte-macrophage colony-stimulating factor (GM-CSF) levels 1-day post CAR-T infusion correlates with severe ICANS (Rossi, et al. EMA Workshop 2016), which is a negative prognostic factor for overall survival (Karschnia, et al. Blood 2019). It has been proposed that upon contact with the tumor, CAR-Ts produce GM-CSF, which serves as a communication conduit between the specific immune response of CAR-T and the off-target inflammatory cascade produced by myeloid lineage cells, causing myeloid cells to expand and promote the production of other downstream proinflammatory chemokines (MCP-1, IL-8, IP-10), cytokines (IL-1, IL-6), and other markers of systemic inflammation (CRP, Ferritin) (Sterner, et al. Blood 2019). Moreover, IL-6 is predominately released by tumor cells in a contact-independent manner (Barrett et al. Blood 2016), which helps explain why the prophylactic administration of tocilizumab is not effective in reducing the overall incidence of CRS or ICANS, as this cytokine is downstream in the inflammatory cascade. Further, the prophylactic use of tocilizumab has been shown to increase the incidence of all-grades and grade >3 ICANS (Locke, et al. Blood 2017). Lenzilumab is a novel Humaneered ® monoclonal antibody that neutralizes GM-CSF and has demonstrated potential to reduce the hyper-immune mediated cytokine storm induced by SARS-CoV-2 infection and significantly improve the likelihood of survival without ventilation in hospitalized COVID-19 patients, as reported in the LIVE-AIR phase 3 study (Temesgen, et al. medRxiv 2021). Methods: Eligible patients are adults (≥ 18 y) with relapsed or refractory DLBCL or are chemorefractory. Prior therapy must have included an anti-CD20 monoclonal antibody and an anthracycline-containing regimen. Patients will undergo leukapheresis and may receive optional corticosteroid bridging therapy. Patients will then receive lymphodepleting chemotherapy on Days ‒3 to ‒5 followed by infusion of lenzilumab on Day 0, 6-hrs prior to CAR-T infusion. Approximately 40 accredited sites across the U.S. certified to administer the three commercially available CAR-Ts have been engaged to participate in this 2-part study. In Part 1, all patients will receive lenzilumab 1800mg via a single 2-hour infusion prior to CAR-T administration. The objective of Part 1 is to evaluate the optimal regimen and assess whether a second dose of lenzilumab post-CAR-T infusion is required. A translational assessment of GM-CSF axis suppression, levels of CAR-T cells in blood, other inflammatory markers and lenzilumab PK/PD will be evaluated, along with the incidence and severity of CRS and ICANS, objective response rates (ORR) and rates of complete response (CR) by Day 28 to select the optimal regimen to carry forward into Part 2. The objective of Part 2 is to confirm whether lenzilumab can improve the toxicity and tolerance of CAR-T while maintaining or improving efficacy and durability of response. Up to 250 patients will be randomized 1:1 to receive lenzilumab or placebo with CAR-T per standard of care. The primary endpoint of the study is incidence of grade >2 CRS and/or ICANS by Day 28, with a key secondary endpoint of CR at 6-months in patients without grade ≥ 2 CRS and/or ICANS at Day 28 (Toxicity-free CR). This design and sample size yields 90% power to detect a 50% reduction in the primary outcome measure. Secondary endpoints includ incidence of all grades and grade >3 CRS and/or ICANS, respectively;ORR and CR at 1, 3, 6, 12 months;durability of CR;progression-free survival, overall survival and health related quality of life using validated patient reported outcome measures. In addition, the study will explore the CRS and ICANS grading criteria that have been utilized with each of the approved CAR-Ts. Disclosures: Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding. Durrant: Humanigen, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Chappell: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Ahmed: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company. Kilcoyne: Humanigen, Inc.: Current Employment, Current equity holder in publicly-traded company.