TYPE I INTERFERON SCORE IS ASSOCIATED WITH THE SEVERITY AND POOR PROGNOSIS IN ANTI-MDA5 ANTIBODY-POSITIVE DERMATOMYOSITIS PATIENTS

BackgroundAnti-MDA5 antibody-positive dermatomyositis (anti-MDA5+DM) is a rare autoimmune disease associated with a high mortality rate due to rapid-progressive interstitial lung disease (RP-ILD), particularly in East Asia[1]. MDA5, acts as a cytoplasmic sensor of viral RNA, thus activating antiviral responses including the type I interferon (IFN) signaling pathway[2]. The involvement of type 1 IFN in the pathogenesis of MDA5+DM has been proposed based on the significantly elevated expression of its downstream stimulated genes(ISG) in muscle, skin, lung, and peripheral blood[3;4]. Janus kinase inhibitor, which targets the IFN pathway, combined with glucocorticoid could improve the survival of early-stage MDA5+DM-ILD patients[5]. In clinical practice, there is still an urgent demand for sensitive biomarkers to facilitate clinical risk assessment and precise treatment.ObjectivesThis study aimed to investigate the clinical significance of interferon score, especially IFN-I score, in patients with anti-MDA5+DM.MethodsDifferent subtypes of idiopathic inflammatory myopathy, including anti-MDA5+DM(n=61), anti-MDA5-DM(n=20), antisynthetase syndrome(ASS,n=22),polymyositis(PM,n=6) and immune-mediated necrotizing myopathy(IMNM,n=9), and 58 healthy controls were enrolled.. A multiplex quantitative real-time PCR(RT-qPCR) assay using four TaqMan probes was utilized to evaluate two type I ISGs (IFI44, MX1, which are used for IFN-I score), one type II ISG (IRF1), and one housekeeping gene (HRPT1). Clinical features and disease activity index were compared between high and low IFN-I score groups in 61 anti-MDA5+DM patients. The association between laboratory findings and the predictive value of baseline IFN-I score level for mortality was analyzed.ResultsThe IFN scores were significantly higher in patients with anti-MDA5+DM than in HC (Figure 1A). The IFN-I score correlated positively with serum IFN α(r = 0.335, P =0.008), ferritin (r = 0.302, P = 0.018), and Myositis Disease Activity Assessment Visual Analogue Scale (MYOACT) score(r=0.426, P=0.001). Compared with patients with low IFN-I scores, patients with high IFN-I scores showed increased MYOACT score, CRP, AST, ferritin, and the percentages of plasma cells (PC%) but decreased lymphocyte count, natural killer cell count, and monocyte count. The 3-month survival rate was significantly lower in patients with IFN-I score > 4.9 than in those with IFN-I score ≤ 4.9(72.9% vs. 100%, P=0.044)(Figure 1B).ConclusionIFN score, especially IFN-I score, detected by multiplex RT-qPCR, can be a valuable biomarker for monitoring disease activity and predicting mortality in anti-MDA5+DM patients.References[1]I.E. Lundberg, M. Fujimoto, J. Vencovsky, R. Aggarwal, M. Holmqvist, L. Christopher-Stine, A.L. Mammen, and F.W. Miller, Idiopathic inflammatory myopathies. Nat Rev Dis Primers 7 (2021) 86.[2]G. Liu, J.H. Lee, Z.M. Parker, D. Acharya, J.J. Chiang, M. van Gent, W. Riedl, M.E. Davis-Gardner, E. Wies, C. Chiang, and M.U. Gack, ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity. Nat Microbiol 6 (2021) 467-478.[3]G.M. Moneta, D. Pires Marafon, E. Marasco, S. Rosina, M. Verardo, C. Fiorillo, C. Minetti, L. Bracci-Laudiero, A. Ravelli, F. De Benedetti, and R. Nicolai, Muscle Expression of Type I and Type II Interferons Is Increased in Juvenile Dermatomyositis and Related to Clinical and Histologic Features. Arthritis Rheumatol 71 (2019) 1011-1021.[4]Y. Ye, Z. Chen, S. Jiang, F. Jia, T. Li, X. Lu, J. Xue, X. Lian, J. Ma, P. Hao, L. Lu, S. Ye, N. Shen, C. Bao, Q. Fu, and X. Zhang, Single-cell profiling reveals distinct adaptive immune hallmarks in MDA5+ dermatomyositis with therapeutic implications. Nat Commun 13 (2022) 6458.[5]Z. Chen, X. Wang, and S. Ye, Tofacitinib in Amyopathic Dermatomyositis–Associated Interstitial Lung Disease. New England Journal of Medicine 381 (2019) 291-293.AcknowledgementsThis work was supported by the National Natural Science Foundation of China [81974251], and Shanghai Hospital Develop ent Center, Joint Research of New Advanced Technology Project [SHDC12018106]Disclosure of InterestsNone Declared.

COVID-19: AFTER TWO YEARS OF IATROGENIC AND BIOLOGIC IMMUNIZATION/ VACCINATION DO WE ACTUALLY NEED FURTHER BOOSTER IMMUNIZATION/ VACCINATION?

BackgroundThe Covid19 pandemic started in late 2019 and went through different phases by spreading from China around the whole globe. During the pandemic different mutation types got predominant from original Wuhan type through Alpha, Delta and Omicron variate BA 1/2 to BA 4/5 with different infectiousity and different potential to harm people´s health status. Immunization/ vaccination program started late 2020, first booster phase started midst of 2021, second booster phase in late 2021/ beginning of 2022 and Omicron specific booster phase midst of 2022.ObjectivesIs there a need of further iatrogenic (booster) immunization/ vaccination after 2 years of immunization/ vaccination program from efficacy driven analysis and safety issues standpoint?MethodsAnalysis of Covid-19 antibody development every three months since August 2021 with comparison of infection rates and assessment of safety parameters by assessing D-Dimers as potential endothelium damage marker in 725 patients (600 female, 125 male, age mean: 62,2 years) of a German rheumatological practice to improve the medical care.ResultsIn 99 % of the patients longstanding immune memory could be shown by analyzing the antibody curves in different exemplary shown biologic and iatrogenic immunization pathways after 2 years of immunization/ vaccination program and biologic immunization, mainly by Delta variate since late 2021 and Omicron variate since beginning of 2022. In 38.5 % of the patients the safety concerns of potential endothelium damage by analysing D-Dimers every 3 months showed a side effect potential of at least 8 months after every MRNA/ Vector immunization, but not after protein based vaccination and even not after infections in that amount.ConclusionOut of the obligation "nil nocere” no further iatrogenic Covid-19 immunization/ vaccination is of need in nearly all (99 %) already immunized people. At present only adult people with very low antibody levels (at least below 64 BAU/ml) (considering the infection or iatrogenic immunization/ vaccination status and time since last spike protein contact) and not yet immunized adult people should be forseen for iatrogenic immunization/ vaccination with protein based or attenuated viral vaccines or in rare cases one Omicron specific MRNA immunization drug. In that case D-Dimer controls for up to 8 months should be obligatory to detect endothelial damage side effect of MRNA (or Vector) technique. Intense cardiovascular monitoring (small vessels) of MRNA/ Vector immunized people in the next 10 – 20 years is necessary.Figure 1.References[1] Pohl C;SAFETY AND EFFICACY ASSESSMENT OF COVID-19 IMMUNIZATIONS/ VACCINATIONS IN PATIENTS OF A GERMAN GENERAL RHEUMATOLOGICAL PRACTICE;EULAR 2022 Poster POS1213;https://doi.org/10.1136/annrheumdis-2022-eular.1389[2] McConeghy KW et al. Effectiveness of a Second COVID-19 Vaccine Booster Dose Against Infection, Hospitalization, or Death Among Nursing Home Residents - 19 States, March 29-July 25, 2022. MMWR Morb Mortal Wkly Rep. 2022 Sep 30;71(39):1235-1238. doi: 10.15585/mmwr.mm7139a2. PMID: 36173757;PMCID: PMC9533729.[3] Bowe, B. Et al. Acute and postacute sequelae associated with SARS-CoV-2 reinfection. Nat Med 28, 2398–2405 (2022). https://doi.org/10.1038/s41591-022-02051-3[4] Hui-Lee Wong et al. Surveillance of COVID-19 vaccine safety among elderly persons aged 65 years and older, Vaccine, Volume 41, Issue 2, 2023, Pages 532-539, ISSN 0264-410X, https://doi.org/10.1016/j.vaccine.2022.11.069.[5] Maher AK et al. Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19. Nat Commun. 2022 Dec 26;13(1):7947. doi: 10.1038/s41467-022-35638-y. PMID: 36572683;PMCID: PMC9791976.[6] Erich Freisleben;Sie wollten alles richtig machen – Dokumentation eines verschwiegenen Leidens – Bericht eines Hausarztes über die Nebenwirkungen der Corona Impfungen;Nov 11, 2022;Cajus Verlag[7] Positive Testrate Germany – https://www.rki.de/DE/Content/InfAZ/N/Neuartiges_Coronavirus/Testzahl.htmlAcknowledgementsThanks to my fami y, all my patients and my collegues for supporting me in my research to improve my personal patient care.Disclosure of InterestsNone Declared.

Features of the Immune Response in COVID-19

BACKGROUND This review is devoted to the analysis of the features of the immune response in COVID-19. The review indicates the clinical manifestations of COVID-19, modern data on the immunopathogenesis of the disease and its complications are considered. AIM OF STUDY To clarify some pathogenetic mechanisms of the immune response in COVID-19, which can help in creating an algorithm for examining patients for early prognosis and prevention of severe course and complications of the disease. MATERIAL AND METHODS To achieve this goal, the results of domestic and foreign scientific studies on the pathogenesis, diagnosis and treatment of COVID-19 were analyzed. The literature search was carried out in electronic search engines Scopus and PubMed. For the analysis, scientific articles published in the period from 2019 to 2021 were selected;88% of analyzed works are not older than 5 years. CONCLUSION The late production of type I IFN, an increase in the level of pro-inflammatory monocytes, a decrease in the expression of HLA-DR on monocytes, violation of the presentation of the virus and the formation of specific lymphocytes, the death of T-lymphocytes and profound immunosuppression are of greatest importance for the development of a severe form of COVID-19. © 2023 Sklifosovsky Research Institute for Emergency Medicine. All rights reserved.

IDDF2023-ABS-0045 Spatiotemporal immune landscape of Mas-driven pro-inflammatory microenvironment with hyperactivated glycolysis in acetaminophen-induced liver injury

IDDF2023-ABS-0045 Figure 1 IDDF2023-ABS-0045 Figure 2 IDDF2023-ABS-0045 Figure 3 IDDF2023-ABS-0045 Figure 4

NEUTROPHIL TO LYMPHOCYTE RATIO (NLR) PREDICTS FRACTURE IN PATIENTS ON LONG-TERM GLUCOCORTICOID

BackgroundLong-term glucocorticoid (GC) exposure leads to systemic bone loss and fracture. In addition, GC is known to increase white blood cell (WBC) amount and change the distribution of differential count (DC). Neutrophil-to-Lymphocyte ratio (NLR) has been studied as an optimal marker of subclinical inflammation, predicting the prognosis of cardiovascular diseases, cancers and even covid-19 infection. For patients under long-term GC exposure, the hemogram change might be a potential parameter to predict prognosis.ObjectivesThis pilot study aims to investigate if GC related WBC-DC change, including NLR, is associated with future fractures during 3 years follow-up.MethodsThis retrospective study is based on a registry, conducted in Kaohsiung Chang Gung Memorial Hospital, Taiwan, from September 2014 till April 2021, aimed to monitor bone mineral density (BMD) changes and fractures in patients with autoimmune diseases. All recruited patients were followed at least 3 years and took X-ray images annually to capture new fragility fracture, including morphometric vertebral fractures. We screened participants who used GC continuously at least 3 months before the index day. We recorded the complete blood count (CBC) and WBC-DC values at least twice during the period of 3 months before and after the index day, and excluded patients who were febrile, under infection status, diagnosed as cancers or cardiovascular diseases at the index day. The NLR was calculated by the absolute neutrophil count divided by absolute lymphocyte count individually.ResultsA total of 346 participants were enrolled in current study, and 101 (29.2%) suffered from new fragility fracture in 3 years. Among patients with fracture and non-fracture, conventional fracture risk factors, such as age, BMD, and previous fracture remained significantly different, while the WBC revealed no difference (Table 1). Nevertheless, the absolute neutrophil and lymphocyte count were significantly higher and lower in the fracture group, respectively, and no difference in the monocyte, eosinophil, and basophil count. We compared different WBC ratio, and NLR is significantly higher in the fracture group, providing the odds ratio of 1.24 (95% confidence interval 1.07-1.44, p=0.005). Figure 1 showed that the observed fracture risk raised as the NLR values increased.ConclusionIn patients under long-term GC, NLR might be a helpful marker to predict fracture, and higher NLR indicates higher fracture risks.Figure 1.Observed fracture rate is associated with baseline NLR[Figure omitted. See PDF]Table 1.Demographic characteristics of enrolled patients on long-term glucocorticoid.Fracture N=101No-Fracture N=245p-valueAge63.7 ± 9.056.5 ± 9.6<0.001*Sex(women)89(88.1)210(85.7)0.55BMI24.1 ± 3.923.4 ± 3.90.14Previous Fracture64(63.4)55(22.4)<0.001*Total hip BMD0.738 ± 0.1330.790 ± 0.1220.001*Femoral neck BMD0.575 ± 0.1130.626 ± 0.109<0.001*Lumbar BMD0.841 ± 0.2000.855 ± 0.1500.49WBC7.3 ± 2.16.9 ±1.70.14Hemoglobin12.8 ± 1.512.9 ± 1.40.33Platelet239.2 ± 64.7247.9 ± 71.40.30Neutrophil67.3 ± 9.764.3 ± 9.70.009*Lymphocyte24.3 ± 8.726.6 ± 9.50.04*Monocyte6.2 ± 1.86.3 ± 1.60.52Eosinophil1.8 ± 1.81.9 ± 1.30.77Basophil0.4 ± 0.20.4 ± 0.20.18NLR (Neutrophil to lymphocyte)3.3 ± 1.72.8 ± 1.40.004*NMR (Neutrophil to monocyte)11.9 ± 4.511.0 ± 3.60.04*LMR (Lymphocyte to monocyte)4.2 ± 1.74.5 ± 1.90.20AcknowledgementsThis work was supported by funding grant CMRPG8J0331 from the Chang Gung Memorial Hospital (https://www.cgmh.org.tw).Disclosure of InterestsNone Declared.

Systems analysis of human innate immunity in COVID-19.

Recent developments in sequencing technologies, the computer and data sciences, as well as increasingly high-throughput immunological measurements have made it possible to derive holistic views on pathophysiological processes of disease and treatment effects directly in humans. We and others have illustrated that incredibly predictive data for immune cell function can be generated by single cell multi-omics (SCMO) technologies and that these technologies are perfectly suited to dissect pathophysiological processes in a new disease such as COVID-19, triggered by SARS-CoV-2 infection. Systems level interrogation not only revealed the different disease endotypes, highlighted the differential dynamics in context of disease severity, and pointed towards global immune deviation across the different arms of the immune system, but was already instrumental to better define long COVID phenotypes, suggest promising biomarkers for disease and therapy outcome predictions and explains treatment responses for the widely used corticosteroids. As we identified SCMO to be the most informative technologies in the vest to better understand COVID-19, we propose to routinely include such single cell level analysis in all future clinical trials and cohorts addressing diseases with an immunological component.

Alterations in immunophenotype and metabolic profile of mononuclear cells during follow up in children with multisystem inflammatory syndrome (MIS-C).

Introduction: Although children seem to be less susceptible to COVID-19, some of them develop a rare but serious hyperinflammatory condition called multisystem inflammatory syndrome in children (MIS-C). While several studies describe the clinical conditions of acute MIS-C, the status of convalescent patients in the months after acute MIS-C is still unclear, especially the question of persistence of changes in the specific subpopulations of immune cells in the convalescent phase of the disease. Methods: We therefore analyzed peripheral blood of 14 children with MIS-C at the onset of the disease (acute phase) and 2 to 6 months after disease onset (post-acute convalescent phase) for lymphocyte subsets and antigen-presenting cell (APC) phenotype. The results were compared with six healthy age-matched controls. Results: All major lymphocyte populations (B cells, CD4 + and CD8+ T cells, and NK cells) were decreased in the acute phase and normalized in the convalescent phase. T cell activation was increased in the acute phase, followed by an increased proportion of γ/δ-double-negative T cells (γ/δ DN Ts) in the convalescent phase. B cell differentiation was impaired in the acute phase with a decreased proportion of CD21 expressing, activated/memory, and class-switched memory B cells, which normalized in the convalescent phase. The proportion of plasmacytoid dendritic cells, conventional type 2 dendritic cells, and classical monocytes were decreased, while the proportion of conventional type 1 dendritic cells was increased in the acute phase. Importantly the population of plasmacytoid dendritic cells remained decreased in the convalescent phase, while other APC populations normalized. Immunometabolic analysis of peripheral blood mononuclear cells (PBMCs) in the convalescent MIS-C showed comparable mitochondrial respiration and glycolysis rates to healthy controls. Conclusions: While both immunophenotyping and immunometabolic analyzes showed that immune cells in the convalescent MIS-C phase normalized in many parameters, we found lower percentage of plasmablasts, lower expression of T cell co-receptors (CD3, CD4, and CD8), an increased percentage of γ/δ DN Ts and increased metabolic activity of CD3/CD28-stimulated T cells. Overall, the results suggest that inflammation persists for months after the onset of MIS-C, with significant alterations in some immune system parameters, which may also impair immune defense against viral infections.

Unique molecular signatures sustained in circulating monocytes and regulatory T cells in convalescent COVID-19 patients.

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including convalescent COVID-19 and sero-negative controls. Flow cytometry analyses revealed reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells within the patients who have recovered from severe COVID-19. sc-RNA seq analysis identifies seven heterogeneous clusters of monocytes and nine Treg clusters featuring distinct molecular signatures in association with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocytes and Tregs expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters featuring S100 family genes: one monocyte cluster of S100A8 & A9 coupled with high HLA-I and another cluster of S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, as well as a unique TGF-ß high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-lived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (≥ 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.

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.

Elevated circulating monocytes and monocyte activation in COVID-19 convalescent individuals.

Background: Monocytes and macrophages play a pivotal role in inflammation during acute SARS-CoV-2 infection. However, their contribution to the development of post-acute sequelae of SARS-CoV-2 infection (PASC) are not fully elucidated. Methods: A cross-sectional study was conducted comparing plasma cytokine and monocyte levels among three groups: participants with pulmonary PASC (PPASC) with a reduced predicted diffusing capacity for carbon monoxide [DLCOc, <80%; (PG)]; fully recovered from SARS-CoV-2 with no residual symptoms (recovered group, RG); and negative for SARS-CoV-2 (negative group, NG). The expressions of cytokines were measured in plasma of study cohort by Luminex assay. The percentages and numbers of monocyte subsets (classical, intermediate, and non-classical monocytes) and monocyte activation (defined by CD169 expression) were analyzed using flow cytometry analysis of peripheral blood mononuclear cells. Results: Plasma IL-1Ra levels were elevated but FGF levels were reduced in PG compared to NG. Circulating monocytes and three subsets were significantly higher in PG and RG compared to NG. PG and RG exhibited higher levels of CD169+ monocyte counts and higher CD169 expression was detected in intermediate and non-classical monocytes from RG and PG than that found in NG. Further correlation analysis with CD169+ monocyte subsets revealed that CD169+ intermediate monocytes negatively correlated with DLCOc%, and CD169+ non-classical monocytes positively correlated with IL-1α, IL-1ß, MIP-1α, Eotaxin, and IFN-γ. Conclusion: This study present evidence that COVID convalescents exhibit monocyte alteration beyond the acute COVID-19 infection period even in convalescents with no residual symptoms. Further, the results suggest that monocyte alteration and increased activated monocyte subsets may impact pulmonary function in COVID-19 convalescents. This observation will aid in understanding the immunopathologic feature of pulmonary PASC development, resolution, and subsequent therapeutic interventions.

Deficient Phagocytosis in Circulating Monocytes from Patients with COVID-19-Associated Mucormycosis.

Cases of rhino-orbital mucormycosis in patients suffering from severe coronavirus disease 2019 (COVID-19) were reported in different parts of the world, especially in India. However, specific immune mechanisms that are linked to susceptibility to COVID-19-associated mucormycosis (CAM) remain largely unexplored. We aimed to explore whether the differential regulation of circulating cytokines in CAM patients had any potential pathogenic links with myeloid phagocyte function and susceptibility to mucormycosis. A small cohort of Indian patients suffering from CAM (N = 9) as well as COVID-19 patients with no evidence of mucormycosis (N = 5) were recruited in the study. Venous blood was collected from the patients as well as from healthy volunteers (N = 8). Peripheral blood mononuclear cells and plasma were isolated. Plasma samples were used to measure a panel of 48 cytokines. CD14+ monocytes were isolated and used for a flow cytometric phagocytosis assay as well as a global transcriptome analysis via RNA-sequencing. A multiplex cytokine analysis of the plasma samples revealed reduction in a subset of cytokines in CAM patients, which is known to potentiate the activation, migration, or phagocytic activity of myeloid cells, compared to the COVID-19 patients who did not contract mucormycosis. Compared to monocytes from healthy individuals, peripheral blood CD14+ monocytes from CAM patients were significantly deficient in phagocytic function. The monocyte transcriptome also revealed that pathways related to endocytic pathways, phagosome maturation, and the cytoskeletal regulation of phagocytosis were significantly downregulated in CAM patients. Thus, the study reports a significant deficiency in the phagocytic activity of monocytes, which is a critical effector mechanism for the antifungal host defense, in patients with CAM. This result is in concordance with results regarding the specific cytokine signature and monocyte transcriptome. IMPORTANCE A number of cases of mucormycosis, often fatal, were reported among severe COVID-19 patients from India as well as from some other parts of the world. However, specific immunocellular mechanisms that underlie susceptibility to this fungal infection in COVID-19 remain largely unexplored. Our study reports a deficiency in phagocytosis by monocytes in COVID-19 patients who are concomitantly afflicted with mucormycosis, with this deficiency being linked to a characteristic monocyte transcriptome as well as a circulating cytokine signature. The functional phenotype and cytokine signature of the monocytes may provide useful biomarkers for detecting potential susceptibility to mucormycosis in COVID-19 as well as in other viral infections.

To investigate the importance of the CBC, derived parameters, and morphology of peripheral blood cells in COVID-19 patients

Aim: To investigate the importance of the CBC, derived parameters, and morphology of peripheral blood cells in Covid-19 patients. Material and methods: According to their symptoms, patients were classified as asymptomatic, mild, or moderate-severe. This research included all paediatric and adult patients who had two CBC samples available (one at admission and another during discharge) throughout their hospital stay. Those who were already undergoing therapy for their cancer, haematological illness, liver disease, or chronic lung disease were not allowed to participate. Results: Patients' ages varied from 8 to 71. The patients' average age was 36.15+or-14.58 years. Sixty percent of research participants were male, making up a sex ratio of 1.5:1. (M: F). The average white blood cell count was 6.87+or-3.51 x109/L, the average red blood cell count was 4.61+or-0.88 x106/microL, and the average haemoglobin level was 12.80+or-2.15 g/dl upon admission. The average absolute neutrophil count was 3.81+or-3.46x109/L, the average absolute lymphocyte count was 2.31+or-1.40x109/L, the average absolute monocyte count was 0.38+or-0.31x109/L, and the average absolute eosinophil count was 0.15+or-0.18x109/L. Overall, the average number of platelets per microliter of blood was 149.21+or- 80.25. Neutrophil to lymphocyte ratio (NLR) at admission was 3.806;platelet to lymphocyte ratio (PLR) was 116.32+or-13.1;lymphocyte to monocyte ratio (LMR) was 8.91+or-5.25, and derivative neutrophil to lymphocyte ratio (d-NLR) was 2.61+or-1.36. Twenty (40%) of the patients were asymptomatic at admission, while 44% had mild symptoms, and 16% required oxygen and ventilator support due to moderate to severe symptoms. The RT-PCR test was positive for all of the patients examined. There was a noteworthy shift in both the mean WBC and mean platelet counts after the follow-up evaluation. No correlation was seen between clinical state on admission and any of the other CBC measures (p>0.05) Conclusion: The significance of CBC values and morphological inspection of the peripheral blood smear at baseline and subsequent assessment is highlighted in the research.

Critical evaluation of haematological and biomarker variation in COVID -19 patients - a prospective study in level 2 tertiary care hospital (Lucknow West)

Background: In India, the first case of COVID-19 was reported on January 30, 2020. The case reporting is based on the testing of individuals by Real-time Reverse Transcription- Polymerase Chain Reaction (RT-qPCR). The present study was conducted to evaluatedifferent parameters, Haematological and Biomarker variations in patients with SARS-CoV2 Infection to assess the prognostic significance. Material & Methods: The present prospective study was conducted among 70 patients who were diagnosed with COVID-19 infection. Relavant physical examination and clinical data of the patient and routine blood investigations including, CBC, serum biochemistry, coagulation function and measurement of inflammatory markers were performed. The results were analyzed by using a SPSS Statistics software version 25.0. Results: In the present study total patients were 70 out of which 58.6% were males and 41.4% were females. Maximum subjects belong to age group 61-80 yrs (47.1%). Mean haemoglobin was 12.89g/l, mean platelet was 9.96x103/l. Mean neutrophil were 88.21%, mean lymphocyte were 8.84%, mean eosinophil were 1.47%, mean monocyte was 1.59%, mean TLC was 12007.14/l. Mean random blood sugar was 148.09 mg/dl. Mean D-dimer was 0.56. Mean CRP levels were 65.5 mg/l. Mean LDH was 516.03 IU/L, mean IL-6 was 282.6pg/ml, and mean procalcitonin was 0.8 ng/ml. Mean SGOT was 62.36u/l, mean ALP was 171.87IU/L, mean urea levels were 57.10 mg/dl and mean INR was 1.22. Outcome mortality was present in total 14 subjects (5 were male and 9 were female) out of all 70 subjects. Conclusion: The present study concluded that Mean values of neutrophil, eosinophil, TLC, random blood sugar, IL6, SGOT, ALP, urea levels and INR were increased in patients with SARS-CoV2 Infection.

Evaluation of Inosine-Acedoben-Dimepranol as an immunomodulator in broiler chicks vaccinated with Infectious bronchitis virus vaccine

Protecting livestock against diseases by enhancing its immunity is essential and required in poultry industry. Therefore, the aim of the present study was to evaluate the possible immunoenhancing effects of Inosine-Acedoben-Dimepranol (IAD) in broiler chicks. A total of 150 chicks were used in the present study, divided into 6 groups (25 for each) and subjected to different treatments. It has been found that IAD significantly (P 0.05) increased total leukocytic count, with increased granulocyte (neutrophils, eosinophils, basophils), lymphocyte and monocyte counts compared to control chicks. IAD significantly (P 0.05) increased total protein as a result of increased globulins in plasma when compared with those of control. IAD has been found to significantly (P 0.05) increase immune response of IB vaccine in IAD + IB vaccine-treated groups compared to control measured by ELISA. IAD exhibited antiviral effect indicated by increased survival percent of chicks challenged with virulent IB virus with survival 100% in the groups received IAD large dose plus vaccine. Data of the present study may indicate that supplying chicks with IAD in drinking water is a good recommendation in poultry industry based on its immune enhancing properties.

Monocyte distribution width - new sepsis biomarker

Sepsis is a global health problem, annually over 45 million patients are diagnosed and over 11 million deaths are recorded. Activation of monocytes in sepsis by the pathogen agent or hypoxia brings about functional, morphological and phenotypic changes in these cells. Monocyte Distribution Width (MDW) is a new biomarker, defined as a measure of monocyte size heterogeneity and has been approved by the Food and Drug Administration for the early diagnosis of sepsis in the adult patient in the emergency department. In intensive care services, this biomarker can be used as a prognostic index in the follow-up of patients with sepsis. The indicator is a measure of the increased morphological variability of monocytes in response to infections, regardless of bacterial, viral or fungal etiology. This new marker also has increased values in the infection with COVID-19 and correlates positively with the severity of the disease.

The Flow Cytometry Study of Cellular Immunity in Rhesus Monkeys after Experimental Infection with SARS-CoV-2 Virus

Cellular immunity plays an important role in the pathogenesis and formation of protective immune defense against the SARS-CoV-2 virus. The aim of the work was to study the cellular immunity of rhesus monkeys applying flow cytometry after experimental infection with the SARS-CoV-2 virus. Materials and methods. Male rhesus monkeys were intranasally inoculated with the SARS-CoV-2 virus, Isolate B strain and hCoV-19/Russia/SP48-1226/2020 strain (abbreviated name U-2), at a dose of 5.0 lg PFU. Using flow cytometry, the levels of 21 populations/subpopulations of mononuclear cells in the peripheral blood of animals were determined before experimental infection with the pathogen and on day 14 after infection. SARS-CoV-2 coronavirus RNA was assessed using real-time polymerase chain reaction. Determination of the titer of virus-neutralizing antibodies to the SARS-CoV-2 virus in the blood sera of animals was conducted through neutralization test evaluating the ability to suppress negative colonies. Results and discussion. Infection with Isolate B strain culture has led to an increase in the relative content of total T-lymphocytes (p<0.2), cytotoxic T-lymphocytes (p<0.1), as well as monocytes expressing the early activation marker CD25 (p<0.2). The decrease in levels has been observed for total B-lymphocytes (p<0.2) and T-helper cells (p<0.1). Infection with the U-2 strain culture revealed an increase in the relative content of monocytes expressing the early activation marker CD25 (p<0.2). Thus, for the first time in the Russian Federation, flow cytometry was used to study the cellular immunity of rhesus monkeys before and after experimental infection with the SARS-CoV-2 virus. The obtained information can be used for studying the pathogenesis of SARS-CoV-2 infection, course, and outcome of the disease, and developing strategies for vaccination and treatment. © 2022 Russian Research Anti-Plague Institute. All rights reserved.

Prevotella differentially regulates the inflammatory response of human monocytes to SARS-CoV-2 spike glycoproteins

Patients suffering severe COVID-19 show an aggressive and excessive immune response against the SARS-CoV-2 coronavirus, known as a cytokine storm. If left untreated these patients face the risk of tissue damage, multi-organ failure and death. A high relative abundance of Prevotella copri has been reported in patients with newly diagnosed rheumatoid arthritis (RA). On the other hand, it has been observed that Prevotella histicola can modulate the inflammatory manifestations of autoimmune diseases like multiple sclerosis, and it is now being evaluated as a monoclonal microbial treatment in COVID-19. We observed that pre-treatment with P. histicola decreased NF-kB activation, while pre-treatment with P. histicola and P. copri decreased IRF activation in monocytes upon SARS-CoV-2 glycoprotein. Our findings suggest that exposure of blood immune cells, such as monocytes, to commensal species of Prevotella may reduce the inflammatory response to SARS-CoV-2 glycoprotein. Besides treatments targeting the viral infection, other treatments such as immunomodulation by bacteria aiming to reduce or regulate the inflammatory process in COVID-19 to avoid the development of related complications may be considered.

Upregulation of coronavirus (MERS-CoV) receptor dipeptidyl peptodase 4 on camel leukocytes after bacterial stimulation in vitro

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging zoonotic pathogen which causes high mortality rate in humans. Dromedary camels may play a central role in virus transmission to humans. Dipeptidyl peptidase-4 (DPP4), a transmembrane protein located on the cell surface of many epithelial and endothelial tissues was identified as the receptor for MERS-CoV. The current study investigated the possibility that bacterial stimulation of camel blood could affect the expression level of DPP4 on camel leukocyte subpopulation, which in turn may contribute to the higher susceptibility of camels with bacterial infection to MERS-CoV infection. DPP4 expression was evaluated by membrane immunofluorescence and flow cytometry. Stimulation of camel blood with the bacterial species S. aureus or E. coil resulted in the upregulation of DPPV on both monocytes and granulocytes, while S. agalactiae did not significantly modulate DPPV expression on either of the immune cells (p > 0.05). None of the bacterial species could induce a change in DPPV expression on lymphocytes from stimulated blood. Collectively, the present study showed an enhancing effect of bacterial stimulation on DPPV expression on camel monocytes and granulocytes.

Chemokines and chemokine receptors during COVID-19 infection.

Chemokines are crucial inflammatory mediators needed during an immune response to clear pathogens. However, their excessive release is the main cause of hyperinflammation. In the recent COVID-19 outbreak, chemokines may be the direct cause of acute respiratory disease syndrome, a major complication leading to death in about 40% of severe cases. Several clinical investigations revealed that chemokines are directly involved in the different stages of SARS-CoV-2 infection. Here, we review the role of chemokines and their receptors in COVID-19 pathogenesis to better understand the disease immunopathology which may aid in developing possible therapeutic targets for the infection.

Multi-omics and immune cells' profiling of COVID-19 patients for ICU admission prediction: in silico analysis and an integrated machine learning-based approach in the framework of Predictive, Preventive, and Personalized Medicine.

Background: Intensive care unit admission (ICUA) triage has been urgent need for solving the shortage of ICU beds, during the coronavirus disease 2019 (COVID-19) surge. In silico analysis and integrated machine learning (ML) approach, based on multi-omics and immune cells (ICs) profiling, might provide solutions for this issue in the framework of predictive, preventive, and personalized medicine (PPPM). Methods: Multi-omics was used to screen the synchronous differentially expressed protein-coding genes (SDEpcGs), and an integrated ML approach to develop and validate a nomogram for prediction of ICUA. Finally, the independent risk factor (IRF) with ICs profiling of the ICUA was identified. Results: Colony-stimulating factor 1 receptor (CSF1R) and peptidase inhibitor 16 (PI16) were identified as SDEpcGs, and each fold change (FCij) of CSF1R and PI16 was selected to develop and validate a nomogram to predict ICUA. The area under curve (AUC) of the nomogram was 0.872 (95% confidence interval (CI): 0.707 to 0.950) on the training set, and 0.822 (95% CI: 0.659 to 0.917) on the testing set. CSF1R was identified as an IRF of ICUA, expressed in and positively correlated with monocytes which had a lower fraction in COVID-19 ICU patients. Conclusion: The nomogram and monocytes could provide added value to ICUA prediction and targeted prevention, which are cost-effective platform for personalized medicine of COVID-19 patients. The log2fold change (log2FC) of the fraction of monocytes could be monitored simply and economically in primary care, and the nomogram offered an accurate prediction for secondary care in the framework of PPPM. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-023-00317-5.