Plasma cytokine profile in patients with severe COVID-19

Background/Objectives: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease (COVID-19) enters the lung tissue through exocytosis, leading to the release of a large amount of pro-inflammatory cytokines called 'cytokine storm'. The aim was to provide more insight into relationship between plasma cytokines profile and fatal outcome of COVID-19. Method(s): Plasma cytokines (IL-17F,GM-CSF,IFNg,IL-10,CCL20/ MIP3a,IL-12P70,IL-13, IL-15,IL-17A,IL-22,IL-9,IL-1b,IL-33,IL-2,IL-21,IL-4,IL-23,IL-5,IL-6,IL-17E/IL-25,IL-27,IL-31,TNFa,TNFb,IL-28A) were detected in 30 patients with severe COVID-19 by a Luminex assay system with Milliplex Human Th17 Magnetic Premix 25 Plex Kit (HT17MG-14K-PX-25, Merk-Millipore, USA) according to the instructions. Patients were followed up for 30 days since admission to intensive care. 18 patients died and 12 patients survived during the period of observation. The control group comprised 10 individuals who had never been diagnosed with COVID-19. Result(s): IL-10 and CCL20/MIP3a plasma levels were elevated in non-survivors patients with COVID-19 compared to controls (p = 0.0027, p = 0.012, respectively). IL-15, IL-6, IL-27 plasma levels were higher in survivors with COVID-19 compared to controls (p = 0.049, p = 0.026, p = 0.00032, respectively). Interestingly, IL-15, IL-27 plasma levels were increased in non-survivors with COVID-19 compared to controls and survivors with severe COVID-19 (IL-15: p = 0.00098, p = 0.00014, respectively;IL-27: p = 0.011, p < 0.0001, respectively). Receiver operating characteristic (ROC) analysis has been conducted for IL-15 and IL-27. Cut-off value was estimated as 25.50 pg/ml for IL-15 and 1.51 pg/ml for IL-27. Conclusion(s): Our study demonstrated a more pronounced immune response in non-surviving patients with severe COVID-19. IL-15, IL-27 could be considered as a sensitive biomarker of the fatal outcome from COVID-19.

Immunotherapy: FAMILIAL CD45RA DEPLETED T CELLS TO TREAT SEVERE REFRACTORY INFECTIONS IN IMMUNOCOMPROMISED PATIENTS

Background & Aim: Immunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. If available, most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. Methods, Results & Conclusion(s): We have previously demonstrated the safety of adoptive cell therapy for COVID-19 patients with CD45RA negative cells containing SARS-CoV-2-specific T cells from a donor, chosen based on HLA compatibility and cellular response to SARS-CoV-2 peptide pools. After finishing a Phase 2 randomized multicenter clinical trial (RELEASE, NCT04578210), we concluded that the infusion is safe, effective, accelerates lymphocyte recovery and shows hallmarks of an immune response. To use adoptive cell therapy to treat COVID-19 it would be necessary to develop a biobank of living drugs. For that, we examined the immune evolution performing a longitudinal analysis from previously SARS-CoV-2 infected and infection- naive individuals covering 21 months from infection. Cellular responses were maintained over time while humoral responses increased after vaccination but were gradually lost. Therefore, the best donors would be recovered individuals and two months after vaccination. We also evaluated the effect of dexamethasone (current standard of care treatment for COVID-19 and other infections involving lymphopenia) and Interleukin-15 (cytokine involved in T-cell maintenance and survival) on CD45RA negative. Dexamethasone did not alter cell functionality, proliferation or phenotype at a clinical-practice concentration, while interleukin-15 increased the memory T-cell and T-regulatory cell activation state, and interferon gamma release. Furthermore, we applied the adoptive passive transfer of CD45RA negative cells containing pathogen-specific memory T-cells to other infectious diseases characterized by sustained lymphopenia. We infused six immunocompromised patients with Cytomegalovirus, BK virus, Aspergillus, and Epstein-Barr virus lymphoproliferative disease. Patients experienced pathogen clearance, resolution of symptoms and lymphocyte increase. Transient microchimerism was detected in three patients. The use of CD45RA negative cells containing specific memory T cells of a third-party donor for treating severe pathogenic diseases in immunocompromised patients is feasible, safe, and effective, and has an advantage over other cell therapies such as lower costs and a less complex regulatory environment.Copyright © 2023 International Society for Cell & Gene Therapy

Pathogenesis of encephalopathy and delirium: neocortical neuronal membrane potential and action potential firing are changed by coronavirus-associated cytokines

Introduction: Encephalopathy and delirium are common following coronavirus infection [1], and the associated neuroinflammation often results in long-term behavioral and cognitive impairment. Neurovirulent cytokines (NVC) are strongly implicated in the pathogenesis of coronavirus encephalopathy [2]. We hypothesized that characterizing the abnormal signaling in NVC exposed neurons will enable us to identify targets to treat encephalopathy and prevent its downstream effects. Method(s): We incubated primary mouse neocortical cultures in NVC known to be increased in coronavirus encephalopathy (TNF-alpha, IL-1beta, IL-6, IL-12 and IL-15). Using whole-cell patch clamp methods, we tested how neuronal function was impacted by 22-28-h exposure to NVC. Result(s): We found that NVC depolarized the resting membrane potential (RMP), reduced the firing threshold of neocortical neurons, and increased baseline spontaneous action potential (AP) firing. NVC altered the sensitivity (or input-output properties) of single neurons to changes in their microenvironment. Specifically, decreasing external Ca2+ and Mg2+ from physiological to low (1.1-0.2 mM) levels increased evoked AP firing in control, but not following exposure to NVC. AP firing threshold and spontaneous firing rates returned to control levels 1 h after NVC wash-out. However, the RMP and attenuated sensitivity of evoked APs to changes in the microenvironment remained persistently abnormal suggesting two distinct mechanisms were at play. Interestingly, hyperpolarizing the RMP reversed this altered response. Conclusion(s): Sustained exposure to NVC reversibly depolarizes neocortical neuronal RMP, altering excitability and the ability of neurons to respond to microenvironment changes. By characterizing the pathogenesis of the underlying changes in neuronal function in our model of coronavirus encephalopathy we will identify intervenable drug targets.

Cytokine Profile in Different Post-Covid-19 Condition Phenotypes

Background: At least 10% of SARS-CoV-2 infected patients suffer from persistent symptoms for >12 weeks, known as post-COVID-19 condition (PCC) or Long Covid. Reported symptomatology is diverse with >200 physical and neurological debilitating symptoms. Here, we analyzed pro-inflammatory cytokine levels as a potential mechanism underlying persistent symptomatology. Method(s): Clinical data and samples used belong to the KING cohort extension, which includes clinically well characterized PCC (N=358, 59 persistent symptoms evaluated), COVID-19 recovered and uninfected subjects. We used Gower distances to calculate symptom's similarity between PCC and Ward's hierarchical clustering method to identify different symptom patterns among PCC patients. Cytokine levels of randomly selected PCC, recovered and uninfected subjects (N=193) were measured on plasma samples collected >6 months after acute infection using the 30-Plex Panel for Luminex. Mann- Whitney t-test was used to compare PCC vs recovered groups and Kruskal-Wallis t-test for >2 groups comparisons (PCC vs recovered vs Uninfected and within PCC clusters). FDR correction was applied for statistical significance (p-adj). Result(s): Hierarchical clustering identified 5 different PCC clusters according to their symptomatology, where PCC3 and PCC5 clusters showed higher prevalence of women ( >80%) and more persistent symptoms, while acute COVID-19 was mild in >80% of the patients. We selected 91 PCC (belonging to each cluster), 57 recovered and 45 uninfected subjects for cytokine profiling (Table 1). 13 soluble markers were significantly elevated (IL-1beta, Eotaxin, MIP-1beta, MCP-1, IL-15, IL-5, HGF, IFN-alpha, IL-1RA, IL-7, MIG, IL-4 and IL-8) in PCC and recovered groups compared to uninfected subjects (all p-adj< 0.04). In addition, PCC subjects tended towards higher levels of IL-1RA compared to recovered group (padj= 0.071). Within PCC clusters, FGF-basic and RANTES were elevated while IL-2 and MIG were decreased in PCC3 and PCC5 compared to the other PCC clusters (all p-adj< 0.04). TNF-alpha, IP-10, G-CSF and MIP-1alpha were decreased in PCC3 and PCC5 not reaching statistical significance (all p-adj=0.07). Conclusion(s): Some cytokines remained altered in all SARS-CoV-2 infected subjects independently of persistent symptoms after 6 months from acute infection. Differences between PCC and recovered individuals are limited after correction. Importantly, PCC cytokine profiles showed differences between clusters, which suggests different PCC subsyndromes with distinct etiology. Subjects Characteristics (Table Presented).

NK CELLS ARE CRITICAL FOR IN VIVO CONTROL OF SARS-CoV-2 REPLICATION AND DISEASE

Background: Natural killer (NK) cells play a critical role in control of viral infections. However, empirical evidence thus far has been unclear on the role of NK cells in pathogenesis and control of SARS-CoV-2 infection with some research suggesting NK cell accumulation as beneficial while others indicate it as deleterious. To address this crucial deficit in understanding, we employed a non-human primate infection model with a validated experimental NK cell depletion technique. Method(s): A total of 12 experimentally naive (75% female) cynomolgus macaques (CM) of Cambodian origin were used in this study. Six CM were NK cell-depleted using an anti-IL-15 neutralizing antibody, while six controls received placebo, prior to intranasal and intratracheal challenge with the SARS-CoV-2 Delta variant at a TCID50 of 1X105. The cohort was monitored for five weeks with scheduled blood, colorectal (CR) biopsies, and lymph node (LN) collections. Total envelope and sub-genomic viral loads (VL) were measured in the nasal cavity, throat, and bronchoalveolar lavage (BAL). 23-color flow cytometry, pathology, and 27-plex inflammatory analyte Luminex analyses were conducted. Statistical tests used were Mann-Whitney U and Spearman's Correlation. Result(s): Control CM exhibited an increase in the frequency of circulating NK cells, reaching a peak at 10 days post-infection (DPI) and returning to baseline by 22DPI. Simultaneously, NK cells expressing activation and tissue retention marker, CD69, also significantly increased. Cytotoxic NK cells were positively associated with VL (r=0.66;p=0.02), suggestive of a virus-induced mobilization. Total experimental NK cell ablation was verified in blood, CR, and LN of NK celldepleted CM, which had higher VL compared to controls in all tissues evaluated, reaching significance at 10DPI (p=0.01) and demonstrated a longer duration of viremia. Although Luminex measures were similar in plasma, BAL samples from NK cell-depleted CM had universally higher concentrations of inflammatory mediators, most notably a 25-fold higher concentration of IFN-alpha compared to controls. Lung pathology scores were also higher in NK cell-depleted CM with increased evidence of fibrosis, syncytia, pneumocyte hyperplasia, and endothelialitis. Conclusion(s): Overall, we find significant and conclusive evidence for NK cell-mediated control of SARS-CoV-2 virus replication and disease pathology. These data suggest adjunct therapies for infection could largely benefit from NK cell-targeted approaches.

Trained innate immunity and diseases: Bane with the boon

Emerging research shows that innate immunity can also keep the memory of prior experiences, challenging the long-held notion that immunological memory is only the domain of the adaptive immune cells. However, the absence of immunological memory in innate immune responses has recently been brought into question. Now it is known that after a few transient activations, innate immune cells may acquire immunological memory phenotype, resulting in a stronger response to a subsequent secondary challenge. When exposed to particular microbial and/or inflammatory stimuli, trained innate immunity is characterized by the enhanced non-specific response, which is regulated by substantial metabolic alterations and epigenetic reprogramming. Trained immunity is acquired by two main reprogramming, namely, epigenetic reprogramming and metabolic adaptation/reprogramming. Epigenetic reprogramming causes changes in gene expression and cell physiology, resulting in internal cell signaling and/or accelerated and amplified cytokine release. Metabolic changes due to trained immunity induce accelerated glycolysis and glutaminolysis. As a result, trained immunity can have unfavorable outcomes, such as hyper inflammation and the development of cardiovascular diseases, autoinflammatory diseases, and neuroinflammation. In this review, the current scenario in the area of trained innate immunity, its mechanisms, and its involvement in immunological disorders are briefly outlined.Copyright © 2022

Unraveling and targeting the innate immune response in Multisystem Inflammatory Syndrome in Children (MIS-C)

OBJECTIVES/GOALS: The innate immune responses to Multisystem Inflammatory Syndrome in Children (MIS-C) are not fully known. Using samples from MIS-C, we will assess the cellular responses and develop a novel Tri-Specific Killer Engager (TRiKE) that engages innate immune cells to improve those responses. METHODS/STUDY POPULATION: We collected blood samples from 60 pediatric patients from which we isolated plasma and peripheral blood mononuclear cells. We received blood samples from 13 MIS-C, 32 severe acute COVID, 5 COVID-19 asymptomatic, and 15 COVID-19 negative patients. Using plasma, we then performed ELISAs to determine IgG antibody levels against SARS-CoV-2 and plaque reduction neutralization tests to determine neutralizing antibody functions. We isolated DNA to look at Fc receptor genetics. We also utilized utilize flow cytometry assays determine the phagocytosis and killing abilities of the innate cells from these patients. This data will be correlated with clinical outcomes. Additionally, we have developed a novel SARS-CoV-2 TRiKE which directs natural killer (NK) cell killing specifically to of COVID-19 infected cells. RESULTS/ANTICIPATED RESULTS: MIS-C patients had higher IgG antibody titers against SARS-CoV-2 compared to children with symptomatic or asymptomatic COVID. MIS-C patients also neutralized SARS-CoV-2 more effectively than children with acute symptomatic or asymptomatic COVID-19. We found natural killer cells and monocytes are dysfunctional in MIS-C patients and do not kill SARS-CoV-2 infected cells as well. Specifically, NK cells do not kill COVID-19 infected cells as well. To combat this, we have successfully generated and are now testing a Tri-Specific Killer engager (TRiKE) which binds one ends to NK cells, one end to the Spike protein on COVID-19 infected cells and contains IL-15 to improve NK cell function. We anticipate that we can improve NK cell killing of COVID-19 infected cells with this TRiKE. DISCUSSION/SIGNIFICANCE: We found that MIS-C patients have antibodies that can neutralize SARS-CoV-2 but that that innate immune cells that engage antibodies are dysfunctional. We are have successfully developed and are targeting this response with a TRiKE to improve innate immune cell functional;this may serve as an adjunctive therapeutic if proven successful.

Predictive Value of Specific Cytokines for Lethal Covid-19 Outcome

In our study, we aimed to evaluate the significance of specific cytokines in blood plasma as predictive markers of COVID-associated mortality. Materials and methods. In plasma samples of 29 patients with PCR-confirmed COVID-19 we measured the concentrations of 47 molecules. These molecules included: interleukins and selected pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A/CTLA8, IL-17-E/IL-25, IL-17F, IL-18, IL-22, IL-27, IFNalpha2, IFNgamma, TNFalpha, TNFbeta/Lymphotoxin-alpha(LTA));chemokines (CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL11/Eotaxin, CCL22/MDC, CXCL1/GROalpha, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CX3CL1/Fractalkine);anti-inflammatory cytokines (IL-1Ra, IL-10);growth factors (EGF, FGF-2/FGF-basic, Flt-3 Ligand, G-CSF, M-CSF, GM-CSF, PDGF-AA, PDGFAB/BB, TGFalpha, VEGF-A);and sCD40L. We used multiplex analysis based on xMAP technology (Luminex, USA) using Luminex MagPix. As controls, we used plasma samples of 20 healthy individuals. Based on the results, we applied Receiver Operating Characteristic (ROC) analysis and Area Under Curve (AUC) values to compare two different predictive tests and to choose the optimal division point for disease outcome (survivors/non-survivors). To find optimal biomarker combinations, we as used cytokines concentrations as dependent variables to grow a regression tree using JMP 16 Software.Results. Out of 47 studied cytokines/chemokines/growth factors, we picked four pro-inflammatory cytokines as having high significance in evaluation of COVID-19 outcome: IL-6, IL-8, IL-15, and IL-18. Based on the results received, we assume that the highest significance in terms of predicting the outcome of acute COVID-19 belongs to IL-6 and IL-18. Conclusion. Analyzing concentrations of IL-6 and IL-18 before administering treatment may prove valuable in terms of outcome prognosis. Copyright © Arsentieva N.A. et al., 2022.

of the 2nd dose of Pfizer-BioNTech vaccine administration which was given to the medical college students at Diyala University

Background: Fighting the Covid-19 pandemic is one of the global priorities now, and the most important type of pandemic control is vaccination. Pfizer-Biotech is considered one of the most important vaccines currently because of its high effectiveness in stimulating the immune system, despite limited data regarding the duration of the response and its side effects. The goal of this study is to assess the response ofSARS CoV-2 S1-RBD IgG andInterleukin-15 after 30 and 120days fromthe 2nd dose ofPfizer-BioNTech vaccine which applied on themedical college students at Diyala university. Methodology: This study began after the obtainment of the Medical College of the University of Diyala, the Medical College of Al-IraqiaUniversity, and the Iraqi Ministry of Health approvals . It continued from October 2021 until March 2022.A total of45 male and femaleparticipants from the College of Medicine( DiyalaUniversity)students who took thetwo doses of Pfizer-BioNTech and were divided into two groups: 1 month (30 days) and 4 months (120 days) after the full vaccination (two doses).A 5 ml of their blood was taken two times (30 days and 120 days after the 2nd dose of thePfizer-BioNTech vaccine) in the postgraduate laboratories inside the Diyala Medical College. A serological analysis to quantify IL-15 and SARS CoV-2 S1-RBD IgG has been done using BT LAB/ Bioassay Technology Laboratory/ Human Interleukin 15 ELISA Kit from CHINA andDiasino/ SARS CoV-2 S1-RBD IgG ELISA Kit/ CHINA respectively. All the lab work happened in the postgraduate laboratories inside the Diyala Medical College. Demographic information (Age and Gender) has been collected from the participants. These participants were split into two groups depending on the time after the 2nd of Pfizer-BioNTech vaccine dose (1 month and 4 months, respectively). STATISTICA (version 12 )and SPSS (version 26 ) were used to input, review and data analysis. Essential approaches of percentages and frequencies were used for qualitative variables, while, average and standard deviation were used for quantitative variables. For both IL-15 and SARS CoV-2 S1-RBD IgG, less than 0.05 of a P-value was considered considerable. Result(s): The ratio, according to gender, was (17.8: 82.2) while the age Average was (20.9 years old). The serum data of IL-15 and SARS-CoV-2 S1-RBD IgG levels after 1 month (30 days) and 4 months (120 days) were statistically non-parametric. Mann-Whitney test (Independent two samples), showeda considerabledrop(P<0.05) of IL-15as well as SARS CoV-2 S1-RBD IgGserum levels in the 4th-monthsgroup compared to the 1-monthgroup. Conclusion(s): Interleukin-15 and SARS CoV-2 S1-RBD IgG serum levels significantly droped after 120days of the 2nd dose of Pfizer-BioNTech vaccine. Copyright © 2022, Anka Publishers. All rights reserved.

IS PSORIATIC ARTHRITIS A RISK FACTOR for SEVERE COVID-19 INFECTION? DATA from the ARGENTINIAN REGISTRY SAR-COVID

Background: Comorbidities, particularly cardio-metabolic disorders, are highly prevalent in patients with psoriatic arthritis (PsA) and they were associated with an increased risk of atherosclerotic cardiovascular disease, which have been associated with higher morbidity and mortality. Whether PsA enhances the risk of SARS-CoV-2 infection or affects the disease outcome remains to be ascertained. Objectives: To describe the sociodemographic, clinical and treatment characteristics of patients with PsA with confrmed SARS-CoV-2 infection from the SAR-COVID registry and to identify the variables associated with poor COVID-19 outcomes, comparing them with those with rheumatoid arthritis (RA). Methods: Cross-sectional observational study including patients ≥18 years old, with diagnosis of PsA (CASPAR criteria) and RA (ACR/EULAR 2010 criteria), who had confrmed SARS-CoV-2 infection (RT-PCR or serology) from the SAR-COVID registry. Recruitment period was between August 13, 2020 and July 31, 2021. Sociodemographic variables, comorbidities, and treatments were analyzed. To assess the severity of the infection, the ordinal scale of the National Institute of Allergy and Infectious Diseases (NIAID)1 was used, and it was considered that a patient met the primary outcome, if they presented criteria of categories 5 or higher on the severity scale. For this analysis, Chi2 test, Fisher's test, Student's test or Wilcoxon test, and binomial logistic regression using NIAID>=5 as dependent variable were performed. Results: A total of 129 PsA patients and 808 with RA were included. Clinical characteristics are shown in Table 1. Regarding PsA treatment, 12.4% of PsA were receiving IL-17 inhibitors, 5.4% IL12-23 inhibitors, one patient apremilast and one abatacept. The frequency of NIAID≥5 was comparable between groups (PsA 19.5% vs RA 20.1%;p=0.976). (Figure 1). PsA patients with NIAID≥5 in comparison with NIAID<5 were older (58.6±11.4 vs 50±12.5;p=0.002), had more frequently hypertension (52.2% vs 23%;p=0.011) and dyslipidemia (39.1% vs 15%;p=0.017). In the multivariate analysis, age (OR 1.06;95% CI 1.02-1.11) was associated with a worse outcome of the COVID-19 (NIAID≥5) in patients with PsA, while those who received methotrexate (OR 0.34;95% CI 0.11-0.92) and biological DMARDs (OR 0.28;95% CI 0.09-0.78) had a better outcome. Conclusion: Although PsA patients have a higher frequency of cardiovascular and metabolic comorbidities than those with RA, the COVID-19 severity was similar. Most of the patients had mild SARS-CoV-2 infection and a low death rate.

Salivary Cytokines in Children with and Without Severe Disease Due to sars-cov-2 Infection

Background: Most children exposed to SARS-CoV-2 virus present with mild symptoms, but some may experience severe illnesses such as Multisystem inflammatory syndrome (MISC) or respiratory failure. Currently there are no established biomarkers to predict progression to severe disease. Although specific serum cytokines have been found to be higher in adults with severe COVID-19, their role as predictors of severe disease in children remains unclear. Further, the role of salivary cytokines in COVID-19 associated inflammation is unknown. Our objective was to compare cytokine levels in saliva of children with and without severe disease due to SARS-CoV-2 infection. Methods: This prospective observational study, conducted at two tertiary children's hospitals, was supported by a grant from the National Institute of Health RADx Program. Children ≤ 18 years of age with symptoms due to SARS-CoV-2 infection (positive PCR test, serology or immunological link) were enrolled after informed consent. Severe cases were defined as the occurrence of any of the following within 30 days of testing: diagnosis of MISC or Kawasaki disease, requirement for >2L oxygen, inotropes, mechanical ventilation or ECMO, or death. A saliva sample was obtained through passive drool using MicroSAL kits (Oasis Diagnostics) and a viral transport medium (VTM-C19, Biomed). Abundance levels of six cytokines (TNFR1, IL13, IL-15, CCL7, CXCL10 and CXCL9) were measured in triplicate using microfluidic immunoassays (Ella, Protein Simple). Mean concentrations for each sample were determined against a standard curve and corrected for dilution. Levels of the six cytokines were compared between those with severe or nonsevere SARS-CoV-2 symptoms using a non-parametric t-test. The relationship between salivary levels of individual cytokines was assessed among children with severe and non-severe SARS-CoV2 using a Pearson correlation analysis Results: A total of 150 children were enrolled from 03/29/2021 to 05/30/2021 (mean age of 7.1 years ± 5.7 years, 54.6% females). Of the total, 38 (25.3%) children met criteria for severe SARS-CoV-2 infection. CXCL10 displayed significantly (fold change>2, p < 0.05) elevated levels in the saliva of children with severe SARS-CoV-2 (Figure 1). The relationship between levels of CXCL9 (MIG) and CXCL10 showed greater levels association (R2 = 0.93) in children with severe SARS-CoV-2 than in peers with non-severe SARS-CoV-2 (R2 = 0.65;Figure 2). Conclusion: In this preliminary analysis of salivary cytokines among children with SARS-CoV-2 infection, we found CXCL10 displayed differential expression with severe symptoms. These findings may provide critical information about the pathophysiology of severe SARS-CoV-2. Confirmation in further studies is necessary. Saliva concentrations of CXCL10 in children with severe SARSCoV-2 symptoms. The whisker box plots display salivary concentrations of CXCL10 in children with severe (green) and non-severe (red) SARS-CoV-2 infection as measured with next generation enzyme linked immunosorbent assay. Levels of CXCL10 (p < 0.01;fold change = 3.04) were elevated in children with severe SARS-CoV-2 symptoms on Wilcoxon testing. .

COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis.

BACKGROUND: In the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options. METHODS: Using an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR. FINDINGS: COVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric), and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2. INTERPRETATION: Like IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung diseases. FUNDING: This work was supported by the National Institutes for Health grants R01- GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780).

Understanding Coronavirus-Related Delirium: Neurovirulent Cytokines and Their Impact on Neuronal Excitability

Rationale Delirium affects a majority of critically-ill patients, increasing mortality and dementia risk. The absence of effective therapy reversing neuronal changes in delirium emphasizes the need for greater understanding of delirium pathophysiology. Neuroinflammation represents a common pathway through which delirium-triggering diseases act. Glial cells sense systemic inflammation across the blood-brain barrier and become activated, releasing cytokines within the brain. In one disease model, systemic infection with neurovirulent coronaviruses caused delirium and specifically increased levels of TNF-α, IL-1β, IL-6, IL-12 and IL-15 in the mouse brain. Methods Here, we tested how neuronal function was affected in a coronavirus-induced neurovirulent cytokine (NVC) model of delirium. Using whole-cell patch clamp methods, we examined how single neuron excitability in murine primary neocortical cultures was impacted by 22-28 hour incubation in NVC. Results NVC treatment depolarized the resting membrane potential (RMP) compared to control (-65 ± 1.6 mV versus -73 ± 1 mV;P < 0.0001, n = 37 and 31 respectively) without affecting action potential characteristics. Delirium is often diagnosed due to altered responses to external stimuli. NVC exposure altered the sensitivity of neurons to changes in external Ca2+ and Mg2+ from physiological (1.1 mM, T1.1) to low (0.2 mM, T0.2) levels. The frequency of spontaneous firing was substantially increased following T0.2 application in control but not in NVC-treated neurons (p=0.026, ANOVA, control: 0.02 ± 0.01 Hz to 2.1 ± 1.2 Hz, n=16, p=0.046;NVC: 0.5 ± 0.4 Hz to 0.9 ± 0.3 Hz, n = 15, p=0.16). Consistent with this, evoked spiking following current injection was also observed in control but not NVC-treated neurons following the switch from T1.1 to T0.2 (P = 0.006, 2WRM ANOVA, Control: 3.9 ± 1.2 Hz vs. 8.5 ± 1.3 Hz n=37, p<0.0001;NVC: 4.8 ± 1.3 Hz vs. 4.5 ± 1.0 Hz, n=31, p=0.78). The attenuated excitability observed in NVC-treated neurons was reversed by hyperpolarization of the RMP. Evoked firing was substantially improved in NVC-treated cells after correcting the RMP (p =0.049, ANOVA, Control: 7.5 ± 1.8 Hz vs. 10 ± 2.5 Hz, n=15, p=0.25;NVC: 6.7 ± 2.2 Hz vs. 11.7 ± 2.8 Hz, n=16 p=0.01). Conclusion Our studies indicate that NVC-treated neurons have attenuated sensitivity to microenvironment changes. As these changes are reversible by correction of the RMP, further characterization of the underlying pathophysiological mechanism is essential to identify biologically plausible targets for delirium.

Effect of hydroxychloroquine on an overactive endometrial immune profile: A Pilot Case Study

Problem: A37 y.o, P1+1 presented with a 1.5 year Hx of secondary infertility. Initial ovulation induction with clomifene citrate was unsuccessful, followed by a single IUI attempt with gonadotropin stimulation. The couple progressed to IVF treatment with 3 failed embryo transfers. There were no endocrinological abnormalities, endometrial/ uterine anatomy was normal on transvaginal USS and saline infusion sonograpy, so an Endometrial Immune Profile(EIP) and Receptivity Array(ERA) were performed prior to further treatment. ERA was in the receptive range, and EIP demonstrated an overactive profile with high IL15:Fn14, suggestive of NK overactivation. In the absence of other pathology this was hypothesised as a potential cause for implantation failure. Immunotherapy options were discussed, including IVIG and adalumimab. Risks of these during the Covid pandemic resulted in the decision to try oral hydroxychloroquine, with cost benefits and potentially less adverse side effects. Unfortunately there is a paucity of published data and outcomes, but proposed benefits of this treatment were based on demonstration of improvemed serum TH1:TH2 cytokine ratios (reduction in TNFa and increase in IL:10), and demonstration of a reduction in miscarriage rate. Method of Treatment: Hydroxycholoroquine 200mgPOBD was commenced 6 weeks prior to commencing treatment, followed by a frozen transfer of a single blastocyst (5AA). Unfortunately this transfer after 8/52 treatment was unsuccessful. A repeat cycle was scheduled after a further 8 weeks, continuing the hydroxychloroquine for >3 months. Initial hCG 13 days post transfer was 2934, but the patient presented with sudden PV bleeding after 6 days, follow up hCG was only 3650. Transvaginal USS demonstrated a collapsed intrauterine gestation sac in keeping with a non-viable pregnancy. Onward referral to an Early Pregnancy unit for follow up confirmed a miscarriage. PGS was not incorporated into the cycles to assess for embryo aneuploidy. Results: Overall the implantation rate had increased from 0 (0/3) to 50% (1/2), but due to sample size this was not statistically significant (p = 0.81). Pregnancy rate per embryo transfer also increased form 0/3 to 1/2, but again was not significant because of low numbers. Due to the failure to achieve an ongoing pregnancy, a repeat biopsy was performed while using hydroxychloroquine to assess its effects on the endometrial immunological environment. This showed a normalisation of the IL15:Fn14 ratio (5.680 to 0.831), but with a slight elevation in the IL18:Tweak ratio (0.088 to 0.114).CD56 remained in the normal range (0.993 to 1.344). Conclusion: Although prescribed for inconsistent indications, there is little published data on hydroxychloroquine use for adverse reproductive outcome. This case report demonstrates the effect of oral hydroxychloroquine therapy on an overactive endometrial profile, leading to a major reduction in IL15:Fn4 ratio, suggesting a potential role in reducing uNK cytotoxicity. Anecdotally a 3 month course is recommended prior to transfer, which would be supported by these events. Unfortunately there is limited ability to make treatment recommendations based on a single sample, however, the findings suggest that a larger study to explore if this pattern is reproducible would have important clinical value.

Intrathecal inflammatory responses in the absence of SARS-CoV-2 nucleic acid in the CSF of COVID-19 hospitalized patients

Objective: Evaluate SARS-CoV-2 RNA and inflammatory cytokines and chemokines in the CSF of patients with acute COVID-19 and neurologic symptoms, and to compare these to controls and patients with known neurotropic pathogens. Background: Neurologic symptoms have been described in 30-60% of hospitalized patients with Coronavirus Disease 2019 (COVID-19). However, little is known about CSF profiles in these patients. Design/Methods: CSF from twenty-seven consecutive patients with COVID-19 and neurological symptoms was assayed for SARS-CoV-2 RNA using quantitative reverse transcription PCR (RT-qPCR) and unbiased metagenomic sequencing. Assays for blood brain barrier (BBB) breakdown (CSF:serum albumin ratio (Q-Alb)), and proinflammatory cytokines and chemokines (IL-6, IL-8, IL-15, IL-16, monocyte chemoattractant protein -1 (MCP-1) and monocyte inhibitory protein - 1β (MIP-1β)) were performed in 23 patients and compared to CSF from patients with HIV-1 (16 virally suppressed, 5 unsuppressed), West Nile virus (WNV) (n=4) and 16 healthy controls (HC). Results: Median CSF cell count for COVID-19 patients was 1 white blood cell/μL;two patients were infected with a second pathogen (Neisseria, Cryptococcus neoformans). No CSF samples had detectable SARS-CoV-2 RNA by either detection method. In patients with COVID-19 only, CSF IL-6, IL-8, IL-15, and MIP-1β levels were higher than HC and suppressed HIV (corrected-p < 0.05). MCP-1 and MIP-1β levels were higher, while IL-6, IL-8, IL-15 were similar in COVID-19 compared to WNV patients. Q-Alb correlated with all proinflammatory markers, with IL-6, IL-8, and MIP-1β (r≥0.6, p<0.01) demonstrating the strongest associations. Conclusions: Lack of SARS-CoV-2 RNA in CSF is consistent with pre-existing literature. Evidence of intrathecal proinflammatory markers in a subset of COVID-19 patients with BBB breakdown despite minimal CSF pleocytosis is atypical for neurotropic pathogens.

Cytokine profile in hospitalized patients with COVID-19 of different severity

Analysis of cytokine profile markers in conjunction with the clinical manifestations of coronavirus disease 2019 (COVID-19) can provide valuable information about the pathogenetic manifestations of the disease, and therefore, in the future, determine drugs that affect the cytokine storm and have an anti-inflammatory effect. Aim. To identify correlations between the parameters of the developed cytokine profile and the clinical course in hospitalized patients with COVID-19 of different severity. Material and methods. The study included 70 hospitalized patients with a confirmed diagnosis of COVID-19, with a mean age of 58 [50;69] years, including 40 men (57%) and 30 women (43%). The average lung involvement according to computed tomography (CT) at admission was CT-2 [1;3]. Peripheral venous blood was taken at admission, which averaged 7 [6;8] days from the symptom onset. Standard biochemical parameters were studied, as well as 47 cytokines and chemokines using the Multiplex system (Merck KGaA, Darmstadt, Germany). Results. Correlations was found between the lung involvement degree and the level of IL-8 (r=0,31, p<0,05), IL-15 (r=0,35, p<0,05), IL-18 (r=0,31, p<0,05), MCP-1 (r=0,36, p<0,05), MIG (r=0,50, p<0,05), TNF-α (r=0,41, p<0,05). An inverse correlation was also found in the level of blood oxygen saturation with the same indicators as follows: IL-8 (r=-0,27, p<0,05), IL-15 (r=-0,34, p<0,05), IL-18 (r=-0,31, p<0,05), MCP-1 (r=-0,40, p<0,05), MIG (r=-0,56, p<0,05), TNF-α (r=-0,45, p<0,05). IL-6 levels were significantly elevated in patients with severe COVID-19 (CT3, CT4), while no increase in IL-6 was observed in patients with moderate disease (CT1, CT2). It is noteworthy that in patients with diabetes, the highest values of IL-12, IL-9 were recorded. Conclusion. Hyperinflammatory syndrome in severe COVID-19 is manifested by high levels of IL-6, MIG, MDC, MCP-1, M-CSF, TNF-α, β, IL-8, IL-18, IL-15. With the CT-1 and CT-2, an increase in only the level of IL-18, IL-8 is noted. The identified patterns prove and make it possible to explain a number of systemic inflammatory changes that occur with COVID-19.

CD64 GENE-MODIFIED PRIMARY NK CELLS LOADED with ANTI-HIV bNAbs TRIGGER HIGH ADCC

Background: As part of a combined HIV CURE immuno-therapy strategy, we transduced primary human NK cells with the high affinity CD64 Fc receptor and pre-loaded them with HIV-specific bNAbs. We named these chimeric NK cells "NuKES" (NK Enhancement Strategy) for their augmented capacity to mediate ADCC and their potential clinical application as an autologous primary NK cell immuno-therapy against HIV. Methods: We transduced primary NK cells from control donors with a lentivirus expressing human CD64 in the presence or absence of irradiated K562 feeder cells expressing co-stimulatory molecules (CD40, 4-1BB) and/or cytokine pre-stimulation (IL-2, IL-21, IL-15). CD64 expressing NK cells were CFSE labeled and expanded ex vivo or FACS sorted at various times post transduction to high purity. CD64 expressing NK cells were then pre-loaded with HIV-specific bNAbs and tested in a functional ADCC CD107a degranulation assay against HIV-1 infected autologous CD4+ primary T cells. Results: After pre-stimulation with cytokines and/or irradiated K562 Feeder Cells, we could routinely achieve (n=5) greater than 40% CD64 expression in primary human NK cells (Day 14 post-transduction shown in Figure 1A). NK cells maintained strong proliferation potential with greater than 6 cells divisions beyond 10 days post transduction as determined by CFSE dilution (Day 10 post-transduction shown in Figure 1B). Phenotypically, CD64 transduced NK cells were similar to control NK cells and possessed strong expression of CD56, CD16, CD69 with intermediate levels of the NK maturation marker CD57. CD64 transduced NK cells could be successfully pre-loaded with HIV-specific bNAbs and possessed an enhanced capacity (GMFI of 2,014 versus 276) to retain 10-1074 for several hours as compared to control NK cells (Figure 1C). Functionally, CD64 transduced NK cells showed a significant two-fold increase in ADCC-triggered degranulation capacity against autologous HIV-1 infected CD4+ primary T cells compared to control NK cells after pre-loading with HIV-specific bNAbs (27.6% versus 13.2% CD107a). Conclusion: Primary human NK cells can be successfully transduced with CD64 and expanded ex vivo to high purity. Preparation of bNAbs specific NuKES represent a viable autologous NK immuno-therapy approach against HIV-1 with potential adaptation for added disease targets (i.e., COVID, Cancer) moving forward.

Longitudinal Plasma Cytokine Profiles Differentiating COVID-19 Severity Groups

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infection with widely varying clinical severity. Severe COVID-19 was initially proposed to be secondary to cytokine storm syndrome (CSS). However, studies since showed that patients with severe COVID-19 rarely display CSS cytokine phenotypes, and may have more limited inflammatory responses instead. Methods. Prospective cohorts, aged 0-90 years of age who tested positive by polymerase chain reaction (PCR) for SARS-CoV-2 were enrolled from inpatient hospitals and outpatient testing centers in Memphis, TN from May 2020-January 2021. Longitudinal blood samples were obtained including acute, sub-acute and convalescent timepoints. Severity scores of asymptomatic, mild, moderate, and severe COVID-19 were assigned at time of convalescent assessment. Plasma was analyzed with a quantitative human magnetic 38-plex cytokine assay. Results. : 169 participants were enrolled, including 8 asymptomatic, 117 mild, 22 moderate and 17 severe cases, and 5 children with post-COVID-19 multisystem inflammatory syndrome in children (MIS-C). All moderate and severe patients were hospitalized and received treatment (39%). Clear distinctions were seen between asymptomatic-mild cases and moderate-severe cases at acute timepoints and during disease progression for GCSF, IL-8, IL-10, IL-15, IL-1Ra, IP-10, MIP-1a, MIP-1β, and TGFα. There was a significant difference between participants who did and did not require hospitalization for acute timepoint levels of IL-10, IL-15, MIP-1 β and TGFα (p< 0.01). Only 4 participants with active COVID-19 were found to meet criteria for CSS (2%), only 3 of which were severe. MIS-C participants showed nearly universally elevated cytokine levels compared to those with active COVID-19. Conclusion. Moderate and severe acute COVID-19 has a distinct cytokine profile from asymptomatic and mild cases, as detected from acute, subacute and convalescent plasma.

Myocardial infarction versus COVID-19 blood secretome

Background: Both myocardial infarction (MI) and COVID-19 are characterized by cytokine storm in blood. Purpose: The objective of this study was to compare the concentration of 39 cytokines, chemokines, and growth factors in blood sera of patients with MI, COVID-19 (COV), and healthy donors. Methods: Patients' blood was collected within 1-2 days after hospitalization in the cardiovascular or COVID intensive care units. All COV patients were in a severe condition;all had increased C reactive protein, 86 and 95% had increased ferritin and D-dimers levels accordingly, 8-10 times decreased lymphocyte numbers. The analysis of the humoral factors in blood serum of MI (n=22), COV (n=23) and donors (n=27) was performed using a 39-plex cytometric analysis. Results: Among all factors analyzed TGFa, IL-1b, 2, 3, 5, 9, 13, 17A were almost not detectable both in patient and donor sera. The concentrations of the other 31 humoral factors in normal sera differed significantly from 0 to 22000 pg/mL. We divided them into house-keeping factors HKF ranged from 1000 to 22000 pg/mL;sentinel innate immunity factors SIF (30-200 pg/mL), and acute phase factors APF (0-30 pg/mL). HKF were detected in all samples. Among SIF and APF IL-1a, G-CSF, IFNa2, IL-7, MIP-1a, IL- 12, and IFNg were detected in 56-80% donor blood while IL-1RA, MCP-3, IL-2, 6, 10, 12, 15, FLT-3F, GM-CSF, TNF-b - only in 10-55%. At the same time all MI patients were 100% positive in all these factors showing extensive activation of blood secretome. Among low incidence APF cytokines in COV patients, percentage of IL-1RA, MCP-3, IFNa2, IL-6, 10, 15, FLT-3L negative sera decreased 3-5 times;and all sera were positive for MIP-1a and IL-12. At the same time TNF-a level decreased significantly from 0 in control to 85% of negative sera in COV patients. Summarized results are shown as the ratios of factor concentrations in MI or COV sera to normal control (Fig). Blood secretome of MI changed more significantly than of COV patients. The major factors (shown in red) in MI were IL-6, IL-12, IFNg, FLT-3L, GM-CSF, and IL-15, which increased 12, 9, 6, 6, 6, and 5 times accordingly. In COV sera IL-6, IL-10, IP-10, and MCP-3 increased by 28, 12, 10, and 9 times accordingly. Less expressed however significant increases are marked with asterisks. Conclusions: Acute MI is characterized by severe disturbances in blood secretome with an increased level of 25 out of 39 factors studied. Contrary to it, in COV patients the levels of IL-6, 10, IP-10, and MCP-3 were more enhanced while only 15 out of 31 exceeded normal levels.

Two types of humoral response in acute myocardial infraction

Background: Atherothrombosis and myocardial infarction are accompanied by the development of an inflammatory reaction. The severity of the immune reaction and its role in the acute myocardial infarction (AMI) remain contradictory to date. Purpose: The objective of this study was to analyze 39 cytokines and chemokines in the serum of patients hospitalized with AMI compared to the healthy volunteers. Methods: All patients included in the study were COVID-19 negative. Patients' blood was collected within 1-2 days after hospitalization in the cardiology department. Cytokine and chemokine detection in the serum of patients (n=20) and donors (n=20) was performed using a 39-plex set of cytometric beads. Results: Among all factors analyzed TGFa, IL-17A, IL-1b, 3, 5, 9 were not detected both in patient and donor sera. Three groups of factors were identified in the normal serum: housekeeping chemokines and vascular factors (F1) ranged from 1000 to 22000 pg/mL (Fig. a);sentinel innate immunity factors F2 (Fig. b), 30-200 pg/mL;and acute phase factors F3 (Fig. c, d), 0-30 pg/mL, detected only in 0-30% of donors but in all AMI patients. Severe imbalance was found in AMI sera at all three levels including chemokine, growth factors, and cytokines. Among AMI patients 65% (Gr1) demonstrated 2-4 times increased level (Fig. a, grey brackets) while 35% (Gr2) had a decreased level of F1 factors in a comparison with donor sera. There was not significant difference between clinical features of the patients in Gr1 and Gr2. GRO, PDGF-AA, and sCD40L levels decreased 35, 15, and 10 times accordingly. Gr1 and Gr2 also differed in F2 and some F3 concentrations: Gr1 had 3-5 times increased level of multiple factors (Fig, b), among them - IL-6, IL-8, and IL-10 were increased 5, 6, and 14 times. At the same time Gr2 had a normal level of these factors (Fig, b, blue brackets). Finally, multiple cytokines and growth factors F3 were significantly increased in both AMI groups (Fig, b, d, red brackets). Of note, IL-12, IFN-g, IL-15, GM-CSF, FLT-3T were increased 8, 6, 5, 5, 5 times accordingly in pooled Gr1+Gr2. There were no correlations found between cytokine profiles in Gr1 and Gr2 and their clinical parameters. Conclusions: Two types of humoral response in AMI patients were identified which differed in the levels of GRO, PDGF-AA, and sCD40L. IL-6 as well as TNF-a can not serve as master cytokines because their levels were increased only in Gr1 patients. These data show that Th1 cytokine increase is specific for AMI. Further studies are needed to identify groups of patients who may be exposed to new therapeutic targets.