Single-Cell Analysis Reveals Characteristics of Myeloid Cells in Pulmonary Pasc

Background: Although our understanding of immunopathology in the risk and severity of COVID-19 disease is evolving, a detail of immune response in long-term consequences of COVID-19 infection remains unclear. Recently, few studies have detailed the immune and cytokine profiles associated with PASC. However, dysregulation of immune system driving pulmonary PASC is still largely unknown. Method(s): To characterize the immunological features of PPASC, we performed droplet-based scRNA-sequencing using 10X genomics to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from participants naive to SARS-CoV-2 (NP, n=2) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC, n=2). Result(s): Analysis of more than 34,000 PBMCs by integrating our dataset with previously reported control datasets generated cell distribution and identified 11 immune cell types based on canonical gene expression. The proportion of myeloid-lineage cells (CD14+monocyte, CD16+monocyte, and dendritic cells) and platelets were increased in PPASC compared with those of NP. Specifically, PPASC displayed up-regulation of VEGFA and transcription factors, such as ATF2, ELK, and SMAD in myeloid-lineage cells. Also, TGF-beta and WNT signaling pathways were up-regulated in these cell population. Cell-cell interaction analysis identified that myeloid-lineage cells in PPASC participated in regulation of fibrosis and immune response, such as VEGFA (increased) and MIF (decreased) interactions. Conclusion(s): Together, this study provides high-resolution insights into immune landscape in PPASC. Our results emphasize differences in myeloid lineage-mediated fibrosis and immunity between PPASC and NP, suggesting they could act as potential pathological drivers of PPASC. (Figure Presented).

Pbmc Immunophenotyping and Plasma Inflammatory Profile of Children with Long Covid

Background: Long COVID can be developed by individuals after an infection with SARS-CoV-2 as described by the WHO. Although this condition is more commonly described in adults, it can occur in children and adolescents with a wide range of estimated prevalence of 1-25%. Little is known about the role of the immune system in long COVID. However, one of the main hypotheses about the underlying mechanism in long COVID is that there is an immune and inflammatory dysregulation that persists after the acute infection. The objective of this study is to compare immune cells populations, and inflammatory biomarkers in paediatric populations with and without long COVID. Method(s): We analyzed 55 blood samples from the pediaCOVID cohort (Hospital Germans Trias i Pujol), which includes more than 130 children diagnosed with long COVID and 23 controls. We measured different immune cell populations using spectral cytometry with a panel of 37 cellular markers, and 42 inflammatory markers using Luminex or ELISA. EdgeR was used for statistical analysis of the spectral data;p-values of inflammatory markers were calculated using the likelihood ratio test and they were corrected for multiple comparisons. Result(s): The study cohort had a median age of 14.3 (IQR, 12.5-15.2) and 69.1% female. Patients had at least 3 symptoms associated with long COVID (median [IQR];10 [7-16]). The most common symptom was asthenia/fatigue (98.2%). Compared to the control cohort, children with long COVID had increased numbers of CD4+CD8+ T cells, IgA+CD21+CD27+ memory B cells, and IgA+CD21-CD27- memory B cells, while CD4+ TEMRA cells (CD45RA+, CCR7-), intermediate monocytes (CD14+, CD16+) and classical monocytes (CD14+, CD16-) were decreased (all p< 0.05;q=n.s.). None of the 42 inflammatory biomarkers showed significant differences between children with and without long COVID. Conclusion(s): The results of this study suggest that specific populations of peripheral blood immune cells might be involved in the mechanisms underlying prolonged COVID in children and adolescents. The increase in both IgA+CD21-CD27- and IgA+CD21+CD27+ memory B cells could be associated with the persistence of viral antigen in the gut and/or gut dysbiosis. Moreover, the decrease in CD4+ TEMRA cells could be related to autoantibodies against G-protein coupled receptors (GPCRs), since this cell population can express GPR56, and autoantibodies against GPCRs were previously reported to be elevated in adults with long COVID.

Leukocyte Cell Population Data as Potential Markers of Covid-19 Disease Characterization

Background: The usefulness of leukocyte cell population data (CPD) is currently being investigated. In COVID-19 pandemic several reports showed the clinical importance of hematological parameters. Our study aimed to assess CPDs in Sars CoV-2 patients as new disease markers. Method(s): From February to April 2020 (1st wave) 540 and from September to December 2020 (2nd wave) 2821 patients respectively were enrolled. SARS CoV-2 infection diagnosis was carried out by Multiplex rRT-PCR from nasopharyngeal swabs. CPDs were detected by XN 2000 hematology analyzer (Sysmex Corporation). A comparison between two disease waves was performed. Additionally, C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assayed. Result(s): CPDs were classified into: cell complextity, DNA/RNA content and abnormal sized cells. We detected parameters increased from the reference population for all cell types for both 1st and 2nd wave (p<0.05). However, in the 2nd vs 1st wave 5 CPDs vs 9 CPDs were found. In addition we observed higher CPD values of the 1st compared to 2nd wave: (NE-SFL) (p<0.001), (LY-Y) (p<0.0001), (LY-Z) (p<0.0001), (MO-X) (p<0.0001), (MO-Y) (p<0.0001). These findings were confirmed by the higher concentrations of CRP and LDH in the 1st vs 2nd wave: 17.3 mg/L (8.5-59.3) vs 6.3 mg/L (2.3-17.6) (p<0.001) and 241.5 IU/L (201-345) vs 195 IU/L (174-228) (p< 0.001) (median, interquartile range) respectively. Conclusion(s): CPDs showed increased cell activation in 1st wave patients confirmed by clinical and biochemical data, associated with worse clinical conditions. Results highlighted the CPDs as disease characterization markers or useful for a risk model.Copyright © 2023 Sciendo. All rights reserved.

Evaluation of lung cell populations in severe COVID-19 pneumonia

Introduction/Aim: Coronavirus disease 2019 (COVID-19) is a novel viral infection that can cause severe pneumonia and acute respiratory failure;however, the mechanism of disease progression is still unclear. The aim of this study is to evaluate inflammatory cells in the lung by analysing cell populations of bronchial aspirates of COVID-19 pneumonia. Method(s): Eligible cases were diagnosed as COVID-19, confirmed by SARS-CoV-2 PCR. All cases had developed severe COVID-19 pneumonia and undergone invasive positive pressure ventilation for the treatment of respiratory failure. Bronchial aspirates were collected during endotracheal intubation, and SARS-CoV-2 PCR was done. The populations of obtained cells from bronchial aspirates were examined by Giemsa staining and immunohistochemical staining of CD3, CD4, CD8, CD20 and CD68 antigens. Bronchial aspirates were cultured to confirm respiratory bacterial co-infections. Result(s): A total of 14 cases (median age 70;eleven male and three female) were enrolled in this study. Their bronchial aspirates were all positive for SARS-CoV-2 PCR. Bacterial co-infections were developed in 10 cases, including 6 cases of pneumonia/respiratory tract infection, 2 cases of sepsis, and 2 cases of urinary tract infection. Cell populations of bronchial aspirates with or without bacterial co-infections were as follows: neutrophils 33.0% vs. 21.5%;CD3+ mononuclear cells (MNCs) 2.5% vs. 5.8%;CD4+ MNCs 4.6% vs. 3.4%;CD8+ MNCs 3.5% vs. 5.2%;CD20+ MNCs 0.2% vs. 0.1%;CD68+ MNCs 39.7% vs. 38.8%, respectively. Conclusion(s): CD68 antigen is mainly expressed in monocytes/macrophages. CD68+ MNCs were dominant in bronchial aspirates of the cases with severe COVID-19 pneumonia. Our data suggests that CD68+ MNCs, presumably macrophages, would play an essential role during the innate immune response to acute SARS-CoV-2 infection in the lung.

Changes in Juvenile Dermatomyositis after the Covid-19 Pandemic

Background. Prior research has shown that viruses may trigger JDM, although the degree to which COVID-19 may serve as a trigger for JDM remains unknown. We present two case reports of JDM occurring after COVID-19 infection. We also provide case numbers of new JDM diagnoses pre-and post-COVID-19 as well as an analysis of JDM population characteristics pre-and post-COVID-19. A 5year-old female developed upper respiratory infection (URI) symptoms and was diagnosed with COVID-19 in December of 2020. She developed Gottron's sign, heliotrope rash, and weakness resulting in admission in February of 2021. She had elevated CK, AST, ALT, LDH, and aldolase. Her CMAS (childhood myositis assessment scale) was 24. An MRI showed diffuse myositis. Myositis specific antibody (MSA) testing revealed a positive MJ antibody. She was diagnosed with JDM and started on steroids, methotrexate, hydroxychloroquine, and IVIG with improvement. The second patient was a 4year-old female who was diagnosed with COVID-19 in October 2020. In January 2021, she developed heliotrope rash and Gottron's papules. She developed decreased exercise tolerance in May 2021 found to have elevated Aldolase and LDH. Her CMAS was 34. An MRI showed diffuse myositis. MSA testing was significant for a positive P155/140 antibody. She was started on hydroxychloroquine, steroids, IVIG and methotrexate with improvement. Due to the aforementioned cases a retrospective analysis was performed assessing the characteristics of JDM pre-and post-COVID-19 at Lurie Children's Hospital. Methods. The Cure JM biorepository houses clinical data, laboratory data, and patient samples obtained at the onset of JDM. The following information was obtained from newly diagnosed JDM patients: MSA, DAS (disease activity score), flow cytometry results, vWF antigen, neopterin, CMAS, capillary end row loop(ERL), LDH, Aldolase, ESR, CRP, IgG, complements, ANA, and age at diagnosis. We identified 10 patients with a diagnosis of JDM from January 1st 2020 -July 1st 2021 who were designated as the post-COVID-19 group. This population was compared to a total of 51 patients diagnosed with JDM between Jan 1st 2010 and December 31st 2019 who were designated as the pre-COVID-19 group. Data analysis was performed using Welch T-testing. Research enrollment was impacted due to the COVID-19 pandemic. To better assess JDM rates, chart review and EMR reports were obtained to determine the total number of JDM diagnoses. Results. T-testing showed no significant change in DAS, ERL count, T or B cell flow cytometry, vWF antigen, CK, CMAS, CRP, Aldolase, LDH, IgG, complements or ANA titer between the pre-and post-COVID-19 JDM groups. The analysis showed a significant change in NK cell population with a decrease in the absolute NK cell number (pre 163, post 90.75. P value 0.03), and NK cell percentage (pre 6.6%, post 3.625%, P value 0.008). Both of the patients presented in this case report showed a low NK cell number (1% and 3% respectively). The total number of new JDM cases rose from an average of 6.3 cases per year to an average 9 cases per year from January 1st 2020 to December 31st 2021. Conclusion. This study provides two case reports of COVID-19 likely triggering JDM. This study also shows a modest increase in the number of new JDM cases since the onset of the pandemic. Interestingly, the NK cell population in the post-COVID-19 JDM patients were significantly decreased. NK cells have multiple roles in not only immune regulation, but also the immune response to viruses. This study suggests that NK cells play a role in the development of in virally mediated JDM, specifically in cases triggered by COVID-19. Future studies will be important to further delineate the function of NK cells in these patients. Markers of JDM disease severity, including DAS, Neopterin, CK, and CMAS, did not significantly change in our institution's JDM population after the onset of the COVID-19 pandemic.

Immune response and allergenic components of COVID-19 vaccines induced delayed cutaneous reactions

Background: Delayed-type cutaneous adverse reactions (DCARs) are potential adverse reactions induced by COVID-19 vaccinations. Objective(s): To investigate the immune pathomechanism of COVID-19 vaccination-related DCARs. Method(s): We conducted a prospective observational study on patients with COVID-19 vaccine-DCARs and tolerant subjects. Serum immune molecules and high-parameter blood cell analysis were analyzed.In vitro lymphocyte activation test (LAT) was performed to evaluate the causative allergens of COVID-19 vaccines for DCARs. Result(s): We enrolled 103 patients with COVID-19 vaccine-DCARs. Patients suffered from DCARs mainly after the first vaccination dose (75.7%). Compared to the tolerant controls, patients with DCARs showed significantly higher serum levels of IL-4, IL-6, IL-8, IL-17A, IL-18, IFN-gamma, IP-10, MIG, granulysin, PARC and TARC(P=3.40x10-5-0.028). High-parameter flow cytometric analysis revealed significant increased CD4+Th2, CD4+Th17, CD4+Th22, CD4+LAG-3+, CD4+CD103+Trm, Tfr, CD8+CXCR3+, CD8+Tc2, CD8+Tc17 and CD8+CTLA4+cell populations relative to total DCARs and specific phenotypes(P=0.001-0.042). In vitro LAT assays measuring IFN-gamma, granulysin, and granzyme B showed that patients with AZD1222-DCARs were significantly reactive to polysorbate 80 and spike protein;BNT162b2-DCARs were significantly reactive to polyethene glycol(PEG) 2000, and spike protein;while mRNA-1273-DCARs were significantly reactive to PEG 2000, tris and spike protein(P<0.05). Conclusion(s): We demonstrated a distinct immune response related to variable clinical phenotypes involved in the immune mechanism of COVID-19 vaccines-DCARs. In vivo LAT assays showed that COVID-19 vaccines excipients and spike protein were potential major components related to the COVID-19 vaccines-induced DCARs.Copyright © 2023

SUBPOPULATION COMPOSITION OF PERIPHERAL BLOOD LYMPHOCYTES IN COVID-19.

The pathogenesis of severe coronavirus infection COVID-19 is associated with activation of immune system, cytokine storm, impaired blood clotting, microvascular thrombosis, organ ischemia and multiple organ dysfunction syndrome. The role of various lymphocyte subpopulations in COVID-19 is still debated. The aim of our study was to analyze the subpopulational profile of peripheral blood lymphocytes in COVID-19 patients as compared with healthy donors. The study included 20 COVID-19 patients (11 males and 9 females,) and 26 healthy donors. Average age of the patients was 52 and 56 years, respectively. Clinical examinations were performed by standard laboratory methods. Peripheral blood lymphocytes were isolated in the Ficoll gradient. The cells were stained with antibodies to specific antigens of main lymphocyte populations, endothelial cells, and apoptotic cell markers. The analysis was performed by flow cytometry. The results showed that all patients had elevated C-reactive protein (14- to 35-fold), ferritin (1.2- to 13-fold), D-dimers (1.2- to 90-fold). 55% of men had a decrease in the absolute number of lymphocytes, in women this index was at the low normal limit. Cytometric analysis showed that, among peripheral blood lymphocytes, the proportion of functional cells expressing the CD45 marker ranged from 2 to 12% in 70% of patients, as compared with 80-99% among the donors. The proportion of CD45+ lymphocytes significantly correlated with the level of hemoglobin, but not with the levels of inflammatory biochemical markers. Among the functional lymphocytes of patients, there was a decrease in the proportion of CD3+, CD4+, CD8+T cells, increased proportion of natural killer CD56+ and the apoptotic (AnnexinV+) cell contents, but the proportion of CD19 and HLA-DR+B cells was not changed. Analysis of the lymphocyte (LC) subpopulations that did not express CD45 marker showed that this fraction contained different lymphocyte subsets with reduced expression of CD4, CD8, CD19, CD56 etc. in the blood of patients and donors. Higher percentage of endothelial cells expressing CD62P marker made the difference between patients and donors. Laboratory determination of lymphocyte subsets in blood samples of COVID-19 patients does not reflect the real severity pattern of the disease, thus requiring studies of the CD45-expressing functional cell populations.Copyright © Svirshchevskaya E.V. et al., 2023 The article can be used under the Creative Commons Attribution 4.0 License.

FEATURES OF POST-VACCINATION HUMORAL IMMUNE RESPONSE IN THE PERSONS WHO UNDERWENT COVID-19 OF VARIOUS SEVERITY.

The studies on humoral immune response in the individuals who have undergone COVID-19 and vaccinated with anti-COVID vaccines allows us to assess the development of "hybrid" immunity, which contributes to understanding the mechanisms of its formation from the effector phase to the step of immunological memory. We assessed the relative and absolute contents of B cell populations and subpopulations, development of humoral immunity in the patients who suffered with COVID-19 of varying severity being thereafter vaccinated with "KoviVak" and "Sputnik V". The study involved volunteers (age 47.3+/-14.5 years) who beared COVID-19 asymptomatically (n = 32), at moderate severity (n = 21), or had severe form of the disease (n = 12), then being vaccinated with "KoviVak" and "Sputnik V" 6-9 months after their recovery. The groups of vaccinated persons consisted of those who beared severe disease being vaccinated with "KoviVak" (n = 6) or "Sputnik V" (n = 6);moderate cases, vaccinated with "KoviVak" (n = 10) and "Sputnik V" (n = 11);asymptomatic cases vaccinated with "KoviVak" (n = 10) and "Sputnik V" (n = 22). We have determined relative and absolute numbers of B lymphocytes (CD45+CD19+), B1 lymphocytes (CD45+CD5+CD19-CD27-), B2 lymphocytes (CD45+CD19+CD5-CD27-), total population of memory B cells (CD45+CD19+CD5-CD27+), non-switched (CD45+CD19+IgD+CD27+), and switched (CD45+CD19+IgD-CD27+) memory B cells;mature naive B lymphocytes (CD45+CD19+CD27-IgD+), plasmoblasts (CD45+CD19+ CD38+++IgD-CD27+), as well as presence of IgG to S(RBD)-SARS-CoV-2 protein. We have found that the humoral immunity among survivors of COVID-19 of varying severity is expressed for up to nine months. The largest number of volunteers who raised antibodies to SARS-CoV-2 S-protein was registered in the group of seriously ill patients. As soon as 1 month after "Sputnik V" vaccination and until the end of the observation, all the examined subjects in this group became seropositive. 4-5 months after injection of this vaccine, specific immunoglobulins were present in all patients who had asymptomatic or average-severity infection. All volunteers who received "KoviVak" had antibodies to the COVID-19 viral S protein from the beginning to the end of the study. Vaccination, especially with "KoviVak", contributed to the highest increase, both in relative and absolute numbers of memory B lymphocytes in asymptomatic patients. Less pronounced changes in the content of B lymphocytes in COVID-19 patients who had severe and moderate clinical course may be associated with higher levels of these cells prior to injection of the vaccines. A positive correlation was found between the number of memory B cells and presence of immunoglobulins to the S protein SARS-CoV-2 in all examined patients.Copyright © 2023 Russian Association of Allergologists and Clinical Immunologists, St. Petersburg Regional Branch (SPb RAACI). All rights reserved.

Immunological aspects of the lethal HIV/COVID-19 coinfected cohort.

SARS-CoV-2 pandemic is now a global medical and social problem. Little is known about its impact on some vulnerable subgroups, such as immunocompromised patients. Therefore, there is still a strong interest in exploring the impact of SARS- CoV-2 infection among HIV-positive individuals worldwide. Aim of the study: to analyze immunological aspects of the deceased, patients with HIV/COVID-19 coinfection. Materials and. methods. We provided retrospective analysis of 258 patient's electronic medical records. All patients were admitted, to the Infectious diseases hospital N2 with HIV/COVID-19 coinfection and died in May 2020 - February 2022. Standard, immunological parameters were analyzed, like CD4+, CD8+ counts and. immunoregulatory index for different patient's subgroups. Statistical data processing was provided by SPSS 17 version (allowable error E=5%). Results and. discussion. The study demonstrated. CD4+ and CD8+ reduction in HIV-infected with COVID-19. Late HIV-presenters didn't display such phenomenon probably because of immune system, exhaustion. COVID-19 itself in some cases could, lead, to immunodeficiency worsening due to depletion of T cell populations in HIV-patients on effective antiretroviral therapy. Conclusion. Comprehension, of different immunological characteristics in HIV/COVID-19 coinfected patients could, improve therapeutic approaches for this challenging cohort.Copyright © 2022 Interregional public organization Association of infectious disease specialists of Saint-Petersburg and Leningrad region (IPO AIDSSPbR). All rights reserved.

34th Annual Meeting of the Japanese Society for Neuroimmunology (JSNI)

The proceedings contain 14 papers. The topics discussed include: MOG-positive anti-NMDA receptor encephalitis with no demyelinating lesions: two case reports;safety and tolerability of rozanolixizumab in the randomized phase 3 MycarinG study;Outcomes from RAISE: A randomized, phase 3 trial of zilucoplan in generalized myasthenia gravis;efficacy and safety of zilucoplan in myasthenia gravis: responder analysis from the randomized Phase 3 RAISE trial;distinct effects among calcium-binding proteins for microglia to produce chemokines associated with the clinical severity of ALS;astroglial connexin 43 is a novel therapeutic target for a chronic multiple sclerosis model;targeting lymphocytes in SPMS: Th cell populations as a biomarker to predict the efficacy of Siponimod;CSF lysophospholipids as a novel biomarker in relapsing-remitting multiple sclerosis;the immune response to SARS-COV-2 MRNA vaccines in siponimod-treated patients with secondary progressive multiple sclerosis;patient characteristics of siponimod-treated SPMS patients in Japan: interim results from post-marketing surveillance;and efficacy of ravulizumab across sex and age subgroups of patients with generalized myasthenia gravis: a post hoc analysis of the CHAMPION MG study.

Increased CD14+ monocytes and OPN+ positive airway macrophages in the BAL are associated with Post-COVID19 induced altered respiratory function

The long term consequences of severe COVID-19 in the lungs remain speculative, however interstitial abnormalities in these patients may arise. In this study previously identified fibrotic markers from the BAL were evaluated in PostCOVID-19 patients. 26 patients were referred for evaluation of respiratory symptoms and/or abnormalities on HRCT, on average 5.3 months from the acute disease phase, to the Post COVID-19 clinic. 20 patients showed persistent radiological findings with fibrotic changes and/or altered respiratory function. Bronchoalveolar lavage cellular composition was determined by MGG staining and CD45, CD14, CD11c, CD163 and Osteopontin staining. Airway monocytes were identified by SSClo/CD45+ parameters and surface expression of CD11c, CD14 and CD16 by flow cytometry. FVC% and DLCO% were used as measures of disease severity. Collectively, monocyte percentages in the BAL were associated with lower FVC% (Rs=-0.53,p=0.02). Importantly, patients with DLCO% below 60 showed higher monocyte infiltration (p=0.015). CD14 positivity on monocytes was more pronounced in patients with DLCO% below 60, while CD16 and CD11c were not associated with DLCO. Increased Osteopontin expression in airway macrophages was also linked with lower DLCO% levels (Rs=-0.661, p=0.019), in contrast to CD163 macrophage expression which tended to be higher in patients with higher DLCO%. Neutrophils were negatively associated with DLCO% in Post-COVID-19 patients (Rs=-0.62,p=0.01). Airway immune cell populations from BAL were associated with Post-COVID-19 induced altered respiratory function.

An integrative systems biology view of host-pathogen interactions: The regulation of immunity and homeostasis is concomitant, flexible, and smart.

The systemic bio-organization of humans and other mammals is essentially "preprogrammed", and the basic interacting units, the cells, can be crudely mapped into discrete sets of developmental lineages and maturation states. Over several decades, however, and focusing on the immune system, we and others invoked evidence - now overwhelming - suggesting dynamic acquisition of cellular properties and functions, through tuning, re-networking, chromatin remodeling, and adaptive differentiation. The genetically encoded "algorithms" that govern the integration of signals and the computation of new states are not fully understood but are believed to be "smart", designed to enable the cells and the system to discriminate meaningful perturbations from each other and from "noise". Cellular sensory and response properties are shaped in part by recurring temporal patterns, or features, of the signaling environment. We compared this phenomenon to associative brain learning. We proposed that interactive cell learning is subject to selective pressures geared to performance, allowing the response of immune cells to injury or infection to be progressively coordinated with that of other cell types across tissues and organs. This in turn is comparable to supervised brain learning. Guided by feedback from both the tissue itself and the neural system, resident or recruited antigen-specific and innate immune cells can eradicate a pathogen while simultaneously sustaining functional homeostasis. As informative memories of immune responses are imprinted both systemically and within the targeted tissues, it is desirable to enhance tissue preparedness by incorporating attenuated-pathogen vaccines and informed choice of tissue-centered immunomodulators in vaccination schemes. Fortunately, much of the "training" that a living system requires to survive and function in the face of disturbances from outside or within is already incorporated into its design, so it does not need to deep-learn how to face a new challenge each time from scratch. Instead, the system learns from experience how to efficiently select a built-in strategy, or a combination of those, and can then use tuning to refine its organization and responses. Efforts to identify and therapeutically augment such strategies can take advantage of existing integrative modeling approaches. One recently explored strategy is boosting the flux of uninfected cells into and throughout an infected tissue to rinse and replace the infected cells.

Leukocyte differential and cell population data in the evaluation of SARS-CoV-2 and other infections

Background: Cell population data (CPD) are reported as part of leukocyte differentials by Mindray BC 6800Plus analyzer, give information of the size (Neu Z, Lym Z, Mon Z) nucleic acid content (Neu Y, Lym Y, Mon Y) and internal structure (Neu X, Lym X, Mon X) of leukocytes. We evaluated the potential utility of the leukocyte differential and CPD as laboratory indicators for the detection of coronavirus disease 2019 (COVID-19) in patients when admitted to the Emergency Department. Methods: A total of 672 patients with suspected infection were recruited at admission to the Hospital. The study group included 237 (151 COVID-19, 33 other virus, 53 bacterial infections). We applied the unsupervised K-means clustering method and principal component analysis (PCA). A validation group of 435 patients, 268 COVID-19 and 167 non-COVID-19 was used to verify the reliability of our model. Results: The COVID-19 cases presented the typical neutrophilia and lymphopenia, high Mon Z, and intermediate values of Neu X and Neu Y. The study group was classified into two clusters. This model was applied to the validation set;PCA analysis showed that almost 45.71% of the data variability could be explained by the two clusters. Cluster 1 had higher neutrophil counts, Neutrophil Lymphocyte ratio, Neu X, Neu Y and Mon Z, and Cluster 2, higher lymphocyte counts (P<0.05). Cluster 1 which included 91.4% of the COVID-19 patients and 60.5% of non-COVID-19 patients were assigned to Cluster 2, notably 100% of other viral infections. Conclusions: Leukocyte count (WBC) differential and CPD seem reliable parameters for the initial evaluation of patients with fever of unknown origin. By means of K analysis, the patients can be classified into distinct groups according to the etiology of the infection. © Journal of Laboratory and Precision Medicine. All rights reserved.

Interest of the Combination of Cell Population Data of the Complete Blood Count and Clinical Data to Identify, among Patients Hospitalized for Covid-19, Those at High Risk of Transfer to Intensive Care Unit

Introduction: COVID-19 is characterized by a maladaptive cytokine release, requiring hospitalization in intensive care unit (ICU) for the more severe forms, or in conventional wards for less aggressive forms. However, in a recent meta-analysis, the prevalence of complications during hospitalization in patients with COVID-19 was as high as 36%, requiring in some cases transfer into ICU, particularly in patients with comorbidities such as diabetes, hypertension or obesity. Cell Population Data (@CPD), provided as part of CBC-Diff analysis by the DxH 800 analyzers (Beckman Coulter) were previously shown to be affected by SARS-CoV-2 infection. Thus, the aim of this study was to analyze whether it was possible, from conventional biological tests collected on admission, and comorbidities, to identify patients with a moderate initial form but at high risk of worsening, who will require subsequent ICU admission. Method(s): The study included 357 consecutive hospitalised COVID-19 patients, from which 40 patients required ICU after staying in the ward. CBC-Diff results including @ CPD from DxH 800, C-reactive protein (CRP) and clinical information (hypertension, diabetes, Body Mass Index (BMI)) were used for the statistical analysis with MedCalc software, version 20.106 (MedCalc Software Ltd, Ostend, Belgium). Result(s): Using logistic regression we obtained ICU factor, which allowed to identify those patients who will require transfer to ICU. ICU factor, based on several @CPD, platelet count, BMI and diabetes, demonstrated AUC 0.843, sensitivity 40% and specificity 97.5% at cut-off 0.39. This approach would allow us correct identification of 16 out of 40 (40%) patients who require ICU hospitalization, with only 8 (2.5%) incorrectly classified patients from hospitalization only group. Adding CRP did not improve the performance of the model. Conclusion(s): ICU factor can constitute an easy and inexpensive tool for screening patients with a moderate form of COVID-19, but at high risk of worsening. This also makes it possible to anticipate the management of intensive care beds, a limiting factor when patients arrive in large numbers at the hospital in the event of a new aggressive variant.

Leukocyte Cpd by in the Detection of Sarscov- 2 Pneumonia

Introduction: Leukocyte differential, present certain features in SARS-CoV2 infected patients neutrophilia, lymphopenia and morphology alterations, which could be useful for screening. Cell population data (CPD) are reported as part of leukocyte differentials by Sysmex XN analyze;they are morphometric parameters that characterize neutrophils, lymphocytes and monocytes and classify them according to their volume, granularity and their content in nucleic acids. CPD reflects in numbers the changes in morphology and activation status triggered by infections. We aimed to evaluate the predictive power of CPDs for the differential diagnosis of COVID19 versus non-COVID19 pneumonia. Method(s): The prospective, observational, multicenter study was conducted in 3 hospitals, including patients > 18 years admitted with the diagnosis community-acquired pneumonia in the period November 2019 - October 2020. Complete blood count were analyzed using Sysmex XN counters. Diagnosis of SARS-CoV-2 infection was done using real-time reverse transcriptionpolymerase chain reaction. Patients were divided into two groups: (1) referral cohort in a hospital for the development of the model (2) the sample of two other hospitals for its validation. Multivariate logistic regression model has been developed for the detection of COVID patients. Robustness of the model has been evaluated by means of the area under the ROC curve and the calibration of the model. Statisticalsignificance p < 0.05. Result(s): 598 patients were recruited, 322 in the referral cohort and 276 in the validation group. The average age was 67.0 years (Standard Deviation 14.59 years ) and 61.49 % male. Neutrophil lymphocyte ratio, NE-WZ, LYY, LY-Z, LY-WX, LY- WY, MO- WY, MO-Z were included in the multivariate analysis, and presented a significant association for the differential diagnosis of SARS-CoV-2 infection with AUC of 0.84 (95% CI 0.82-0.92) in the referral cohort and 0.77 (0.69-0.85) in the validation cohort Conclusion(s): Leukocyte differential and CPDs could be very useful in the differential diagnosis of SARS-CoV-2 pneumonia and lead to a cheap and early diagnosis of the disease.

Term placenta transcriptomic atlas identifies cells with discrete transcription programs implicated in gestational diabetes subtypes

Objective: Single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics have identified novel cell subtypes and microenvironments which compartmentalize diverse functions at the maternal-fetal interface. We aimed to combine these high-resolution technologies with a rigorous classification of transcription alterations associated with diabetes subtypes in pregnancy. We hypothesized characteristic transcriptome profiles in specific cell populations would be linked to these classifications. Study Design: We clinically validated gestational diabetes mellitus type 1 (GDMA1), GDMA2, and type 2 diabetes (T2DM) classes within a cohort of placentae and compared them to healthy controls by bulk RNA-seq (N=53). We then integrated our non-diabetic term placentae spatial transcriptomics data (N=12) with 273,944 publicly available transcriptomes from term placenta scRNA-seq or single-nuclei RNA-seq (snRNA-seq) datasets (accessions phs001886, GSE173193, EGAS00001002449) with control, GDM, or SARS-CoV-2 positive subjects to create a placental transcriptomic catalog. Result(s): In bulk, we identified 104 significantly differentially expressed transcripts (-2< log2fold-change >2,p< 0.05) in our GDMA1 samples, 102 with GDMA2, and 121 with T2DM (Fig. 1a). Comparisons revealed 88 transcripts uniquely marking GDMA1, 68 for GDMA2, and 85 for T2DM, while FGA and CYP1A1 perturbations were shared across diabetes classes (Fig. 1b). We then compared these bulk GDM subtype markers with the 5,211 significantly differentially expressed transcripts associated with 22 cell-type clusters in our term placenta atlas (Fig. 2), and 20 unique GDM bulk markers aligned with extravillous trophoblast, stromal, endometrial epithelia, endothelial, NK, and dendritic single-cell placental markers. Conclusion(s): Together, these results detail the gene expression profiles and the cell types in the maternal-fetal milieu of pregnancies affected by diabetes. Consistent with their distinct clinical outcomes, GDM and T2DM have unique cellular transcriptomes and would thus be targets for new therapeutics. [Formula presented] [Formula presented] Copyright © 2022

First person - Sarantis Korniotis

First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Sarantis Korniotis is first author on 'GM-CSF-activated human dendritic cells promote type 1 T follicular helper cell polarization in a CD40-dependent manner', published in JCS. Sarantis conducted the research described in this article while a postdoctoral researcher in Professor Vassili Soumelis's lab at Saint-Louis Hospital, Universite de Paris, France. He is now a Senior Scientific Associate in Immunology at Intelligencia AI, Athens, Greece, investigating new signals and factors that make cells acquire regulatory or immune-suppressive properties. Copyright © 2022 Company of Biologists Ltd. All rights reserved.

Identification and Characterization of an Unconventional NK Subset in COVID-19

Background. Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is associated with dysregulation in the innate immune response including NK cells. NK cells are integral in the innate immune response against viral infections. Canonical NK cells are classified as CD56dim CD16+ and CD56bright CD16-. An unconventional subset of CD56dim CD16 - NK cells has previously been identified in COVID-19 that is not present in other viral infections. Here we characterize phenotypic changes in the NK cells of patients with severe COVID-19 as work towards determining the functional status of this unconventional subset. Methods. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from healthy donors (n=5) and patients with severe COVID-19 on Extra Corporeal Membrane Oxygenation (ECMO) (n =15). Primary NK cells were stimulated in vitro with plasma from patients with severe COVID-19 or healthy donors. Flow cytometry was used to phenotype the NK cells. A separate cohort of PBMC samples (n =7) from patients requiring hospitalization for COVID-19 underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) analysis. Results. The CD56bright CD16 - NK subset was expanded in PBMCs from patients with severe COVID-19 as compared to healthy controls. CITE-Seq demonstrated that NK cells without surface CD16 clustered separately based on transcriptional profiling and did express FCGR3A at the translational level. Stimulation with COVID-19 plasma recapitulated the loss of CD16 from primary human NK cells and led to increased activity of Caspase 3/7. a) Representative gating of NK cell subsets by Flow Cytometry in healthy and COVID-19 patient peripheral blood mononuclear cells (PBMCs). b) Percentage of total NK cells belonging to a particular cell subset compared between healthy donor samples (n=4) and COVID-19 patient samples (n =8). Data points represent an individual patient sample. Error bars represent the standard deviation of the mean. Differences between groups was analyzed using a two tailed t-test. *: p< 0.05, ns: not significant Figure 2. NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset after stimulation with COVID-19 plasma in vitro a) Representative gating of NK cell subsets by Flow Cytometry analysis in healthy donor NK cells stimulated by healthy plasma and COVID-19 patient plasma. b)Relative change in percentage of total NK cells belonging to a particular cell subset compared between healthy donor plasma (n=6)and COVID-19 patient plasma (n=15) stimulation conditions. Error bars represent the standard deviation of the mean and the difference between groups was analyzed using a two-tailed T-test. * *: p< 0.01, * * *: p< 0.001, ns: not significant. Conclusion. We demonstrate and characterize a nonclassical population of CD56dim CD16 - NK cells that are present in patients with severe COVID-19 and replicate this phenotype in vitro. Reproduction of this in vivo phenotype in an in vitro system will allow for additional studies on the functional state of NK cell subsets in COVID-19. The presence of this NK cell population may reflect a dysregulated innate immune response and immunopathogenesis of COVID-19.

Myeloid-derived suppressor cells as a potential biomarker and therapeutic target in rhino-orbital mucormycosis patients

Background: Mucormycosis is a deadly fungal infection that emerges in patients affected with COVID-19. All fungal illnesses are caused by dysregulated adaptive immunity, but Myeloid-derived suppressor cells (MDSC) have added a new di-mension to the chronic inflammatory response. Objective(s): We attempted to enumerate the MDSC immune response in rhino-orbital mucormycosis patients before and after treatment and compared the data with healthy control. Method(s): A total of 3 ml of blood samples were taken in an EDTA vial from 20 patients with mucormycosis and 20 age-matched healthy control. A second blood sample was collected to examine the immune system post three months of treatment. Mycological identification was performed on nasal crust retrieved aftersurgery using KOH/culture.The expression of the MDSC marker was analyzed by immunostaining with the antibodies against CD14, HLA-DR, CD11b, CD33, CD66 (Biolegend). Flu-orescence profiles were recorded by Flow Cytometer (BD FACSAria TM III) and analyzed by Flow Jo s oftware (BD Biosciences). The percentage of positive cells is used to express the results.The GraphPad Prism (version 8, GraphPad s oftware, LaJolla, CA, USA) was used to analyze the data. All of the results were considered significant when P <.05. Result(s): All of the patients tested positive for Rhizopus arrhizus, which was confirmed by the culture. The percentages of Monocytic-MDSC (mMDSC: CD14 + HLA-DR-/low) cells were significantly high in patients compared to healthy control. In post-3-month treatment, the percentages of mMDSC were found significantly low and comparable with healthy control. Granulocytic MDSC (gMDSC: HLA-DR-/low CD33 + CD11b + CD66 +) cell population was higher in patients compared with healthy control and patients with post-3-month treatment. Conclusion(s): MDSC regulates T cells and other immune cells with a different mode of action. The findings in this study imminently indicatethe mechanism of immunedysregulation involvingMDSCpathways inmucormycosis andprovide evidence that restoration of immune balance causes reduction of MDSCcells may be considered a therapeutic option for long-term benefit.

Blood cytometry parameters in Sars Cov-2 patients: potential markers of disease characterization

IntroductionThe usefulness of leukocyte cell population data (CPD) derived from optical signals of new hematology analyzers is currenctly being investigated. In Covid-19 pandemic several reports showed the clinical importance of functional and quantitative blood parameters. Our study aimed to assess CPDs in positive Sars Cov-2 patients as potential disease markers.MethodsFrom February to April 2020 (1st wave), 540 patients (490 negative and 50 SARS CoV-2 positive) were enrolled in this retrospective study, as well as 2821 patients from September to December 2020 (2nd wave) (2762 negative and 59 SARS CoV-2 positive). SARS CoV-2 infection diagnosis was carried out by Multiplex rRT-PCR from nasopharyngeal swabs and clinical information collected in cardiology emergency department (ED). CPDs were detected by XN 2000 hematology analyzer (Sysmex Corporation) considering a single determination on whole blood. Comparisons between disease waves and SARS CoV-2 negative and positive patients were performed. Additionally, C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assayed. Statistical analysis using the univariate and multivariate general linear regressions were made.ResultsLeukocyte CPDs were classified into: cell complextity (NE, LY, MO X-axis), DNA/RNA content (NE, LY, MO Y-axis) and abnormal sized cells (NE, LY, MO Z-axis). We detected cytometric parameters increased from the reference population for all cell types for both 1st and 2nd wave (p<= 0.03). However, smaller quantitative alterations were found in the 2nd vs 1st wave: 5 CPDs vs 9 CPDs. In addition we found higher CPD values of the 1st compared to 2nd wave: (NESFL) (p=00004), (LY-Y) (p<=0.0001), (LY-Z) (p<=0.0001), (MO-X) (p<=0.0001), (MO-Y) (p<=0.0001). These findings were confirmed by the higher concentrations of CRP and LDH in the 1st vs 2nd wave: 17.3mgdL (8.5-59.3) vs 6.3 mg/dL (2.3-17.6) (p=0.0003) and 241.5 U/L (201-345) vs 195 U/L (174-228) (p=0.0005) (median, interquartile range) respectively.Conclusions Leukocyte CPDs showed increased cell activation in patients of 1st wave confirmed by biochemical data, correlated with worse clinical conditions of hospitalized patients. Our results highlighted the CPDs as disease characterization markers or useful for a predictive risk model.