Correlation between levels of anti-cytokine antibodies, recombination activity of the mutant RAG proteins and clinical phenotype in patients with RAG deficiency

RAG mutations cause various phenotypes: SCID, Omenn syndrome (OS), leaky SCID (LS) and combined immunodeficiency (CID). We had previously reported autoantibodies targeting IFN-alpha, IFN-omega in patients with RAG deficiency. However, how the presence of such antibodies correlated with the severity of the clinical phenotype and with the recombination activity of the mutant proteins was unknown. To address this, we have studied anti-cytokine antibodies in 118 patients with RAG defects (SCID, n = 28;OS, n = 29;LS, n = 29;CID, n = 32), and in 42 controls (protocols NCT03394053 and NCT03610802). RAG mutant proteins associated with CID and LS retained 35.6 +/- 4.3 (mean +/- SE) and 29.8 +/- 5.1% recombination activity respectively, compared to wildtype protein, which was significantly higher than the recombination activity of the mutant RAG proteins associated with OS (4.1 +/- 1.5%) and SCID (5.7 +/- 2.1%) (p < 0.0001). Among 32 CID patients, 24 tested positive for anti-IFN-alpha and 21 for anti-IFN-omega antibodies. Among 29 LS patients, 15 had high levels of anti-IFN-alpha and 13 of anti-IFN-omega antibodies. A minority of the CID and LS patients had also high levels of anti-IFN-beta and anti-IL-22 antibodies. By contrast, none of the OS patients tested positive for anti-cytokine antibodies. High levels of anti-IFN-alpha and anti-IFN-omega antibodies correlated with their neutralizing activity as demonstrated in vitro by analysis of STAT1 phosphorylation upon stimulation of healthy donor monocytes in the presence of the appropriate cytokine and patient's or control plasma. Severe viral infections were recorded in 26/41 patients with CID and LS who tested positive and in 7/20 who tested negative for anti-IFN-alpha and/or anti-IFN-omega antibodies (p <0.05). Among those with anti-IFN antibodies, EBV (n = 8), CMV (n = 6), HSV (n = 5), VZV (n = 4) and adenovirus (n = 4) infections were more common. Two patients had COVID-19, which was fatal in one. Presence of the rubella virus was documented in 5 patients with anti-type I IFN antibodies. These results demonstrate that high levels of neutralizing anti-IFN-alpha and anti-IFN-omega antibodies are common in patients with RAG mutations manifesting as CID and LS, but not in those with OS, and that their presence is associated with a high risk of serious viral infections.Copyright © 2023 Elsevier Inc.

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.

Journal of Population Therapeutics & Clinical Pharmacology

The SARS-CoV-2 virus causes a contagious disease known as Coronavirus Disease 2019 (COVID-19). It began spreading globally in 2019 and is still producing pandemics today. Different COVID-19 vaccinations offer protection against this illness. Pfizer-BioNTech and Sinopharm were the two vaccine manufacturers with the highest usage in Iraq. Both vaccines use a different method to activate the immune system. This study seeks to compare the IL-22, IL-37, and IL-38 levels in those who received either the Sinopharm or the Pfizer-BioNTech COVID-19 vaccination. IL-22, IL-37, IL-38 levels have been shown to be upregulated in COVID-19 patients. In this study, IL-22, IL-37, and IL -38 levels were tested in 80 healthy controls and 100 COVID-19 patients 14-21 days after recovery. Additionally, people who received the Sinopharm or Pfizer-BioNTech vaccine (50 each) were monitored 21 days after the first dosage and 21 days after the second dose. In comparison to controls, serum levels were noticeably higher in recovered patients. Except for the first dosage of Pfizer BioNTech, the first and second doses of Sinopharm and Pfizer BioNTech were linked to considerably higher levels of IL-22, IL-37, and IL-38 compared to controls or recovered patients. where IL-22, IL -37, and IL-38 levels did not show significant differences compared to recovered patients. In conclusion, lower IL-37 and IL-38 molecule levels were linked to recovery from COVID-19, although these levels remained considerably greater in recovered patients compared to uninfected controls. Vaccination with Sinopharm or Pfizer-BioNTech confirmed the up-regulating effects of SARS-CoV-2 on IL-22, IL-37, and IL-38 levels.

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.

LOSS of TOLERANCE and ANERGY in HUMAN PERIPHERAL B CELLS during SARS-CoV-2 INFECTION

Background: Severe infection with SARS-CoV-2 induces systemic autoreactive antibodies with specificity to Type I IFN, phospholipids, nuclear or tissue specific targets. The wide breadth of targets suggests a system-wide defect in B cell tolerance during viral infection and that the source of autoreactive antibodies is likely a heterogenous subset of B cells. BND cells are mature naïve B cells that do not express IgM but do express IgD and are enriched in autoreactive specificities. BND cells are held in an anergic state in healthy humans as a mechanism of peripheral tolerance, although in vitro evidence suggests anergy can be broken with strong inflammation. We hypothesized that robust inflammation associated with viral infection from SARS-CoV2 may relax peripheral tolerance and promote breakage of BND cell anergy. Methods: Plasma and PBMCs were collected from healthy controls (N=10), subjects immunized with Pfizer BNT162b2-mRNA/Moderna mRNA-1273 (N=10), subjects with mild (N=11) or severe SARS-CoV-2 infection (N=14). BND cells were examined ex vivo for markers of activation by flow cytometry. Phosphorylation of signaling proteins downstream of the BCR were measured in vitro with or without BCR crosslinking. Inflammatory cytokines were measured in plasma by multiplex. For statistical analysis, unpaired t test between populations or paired t test between unstimulated and BCR stimulated conditions were performed. Results: BND cells from severe SARS-CoV-2 infection have lower expression of CD21, associated with loss of anergy, higher expression of activation markers CD68 and CD86 with lower expression of inhibitory receptors CD22 and CD72 when compared to BND cells from other subjects, suggesting a phenotypical breach of anergy. Upon BCR crosslinking, BND cells have higher levels of downstream signaling components of the BCR (pPLCγ2, pBlnk, and pSyk) when compared to healthy controls and immunized subjects, suggesting a functional breach in anergy with infection. Examination of plasma from severe SARS-CoV-2 infection showed higher levels of inflammatory cytokines (IFNγ, TNFα, IL-6 and CRP) where TNFα and CRP correlated with enhanced BCR signaling in BND cells. Conclusion: We demonstrate that SARS-CoV-2 viral infection relaxes peripheral tolerance of BND cells, likely through strong systemic inflammation produced during infection. These autoreactive cells overcome anergy and become activated with increased BCR signaling. Thus BND cells could be a source of autoreactive antibodies during viral infection.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.