Clinical and pathological analysis of deaths from different SARS-CoV-2 coronavirus genotypes.

The genotype of the SARS-CoV-2 virus pathogen plays an important role in the epidemiological and clinical characteristics of a new coronovirus infection. There are no published data on the morphological features of lesions caused by different virus genotypes. The aim of the study was to evaluate clinical, laboratory and morphological changes depending on the genotype of the SARS-CoV-2 virus. Materials and methods. A retrospective analysis of the medical records of 39 patients with COVID-19 with a severe course of the disease, which ended in death, who were hospitalized at the St. Petersburg State Budgetary Infectious Diseases Clinical Hospital named after S.P. Botkin" in 2020-2022. Clinical and laboratory characteristics were assessed, including determination of the virus genotype, levels of leukocytes, lymphocytes, alanine aminotransferase, creatinine, ferritin, C-reactive protein, D-dimer, interleukin-6. Macro- and microscopic changes were assessed, including immunohistochemical examination of the lungs and other organs using sera to CD14 68, 163, type 1 and 3 collagen. The preparations were digitized on a Panoramic scanner, morphometric studies were carried out using the SlideViewer program, including the quantitative determination of the content of CD68+ macrophages in 12 cases. Results. In all patients, the disease was complicated by the development of pneumonia, the majority had concomitant diseases (94.6%). The average time of hospitalization was 19.0+/-1.6 days, the average time of stay in the intensive care unit was 7.7+/-1.2 days. The analysis, depending on the genotype of the SARS-CoV-2 virus, showed a statistical difference between the age of patients, the length of stay in the intensive care unit and the level of lymphocytes. Differences in the average duration of hospitalization, the level of laboratory parameters were not revealed. Histopathological picture in all examined was approximately the same. The content of CD68+ macrophages per unit area in different genotypes did not differ, but varied significantly within the same genotype. Conclusion. Thus, it was not possible to identify significant differences between the changes caused by different genotypes of the new coronavirus, which can probably be explained by the fact that mutations do not include genome regions that are relevant to virulence factors, although further research is needed.Copyright © 2022 Interregional public organization Association of infectious disease specialists of Saint-Petersburg and Leningrad region (IPO AIDSSPbR). All rights reserved.

Clinical and pathological analysis of deaths from different SARS-CoV-2 coronavirus genotypes.

The genotype of the SARS-CoV-2 virus pathogen plays an important role in the epidemiological and clinical characteristics of a new coronovirus infection. There are no published data on the morphological features of lesions caused by different virus genotypes. The aim of the study was to evaluate clinical, laboratory and morphological changes depending on the genotype of the SARS-CoV-2 virus. Materials and methods. A retrospective analysis of the medical records of 39 patients with COVID-19 with a severe course of the disease, which ended in death, who were hospitalized at the St. Petersburg State Budgetary Infectious Diseases Clinical Hospital named after S.P. Botkin" in 2020-2022. Clinical and laboratory characteristics were assessed, including determination of the virus genotype, levels of leukocytes, lymphocytes, alanine aminotransferase, creatinine, ferritin, C-reactive protein, D-dimer, interleukin-6. Macro- and microscopic changes were assessed, including immunohistochemical examination of the lungs and other organs using sera to CD14 68, 163, type 1 and 3 collagen. The preparations were digitized on a Panoramic scanner, morphometric studies were carried out using the SlideViewer program, including the quantitative determination of the content of CD68+ macrophages in 12 cases. Results. In all patients, the disease was complicated by the development of pneumonia, the majority had concomitant diseases (94.6%). The average time of hospitalization was 19.0+/-1.6 days, the average time of stay in the intensive care unit was 7.7+/-1.2 days. The analysis, depending on the genotype of the SARS-CoV-2 virus, showed a statistical difference between the age of patients, the length of stay in the intensive care unit and the level of lymphocytes. Differences in the average duration of hospitalization, the level of laboratory parameters were not revealed. Histopathological picture in all examined was approximately the same. The content of CD68+ macrophages per unit area in different genotypes did not differ, but varied significantly within the same genotype. Conclusion. Thus, it was not possible to identify significant differences between the changes caused by different genotypes of the new coronavirus, which can probably be explained by the fact that mutations do not include genome regions that are relevant to virulence factors, although further research is needed.Copyright © 2022 Interregional public organization Association of infectious disease specialists of Saint-Petersburg and Leningrad region (IPO AIDSSPbR). All rights reserved.

Clinical and pathological analysis of deaths from different SARS-CoV-2 coronavirus genotypes.

The genotype of the SARS-CoV-2 virus pathogen plays an important role in the epidemiological and clinical characteristics of a new coronovirus infection. There are no published data on the morphological features of lesions caused by different virus genotypes. The aim of the study was to evaluate clinical, laboratory and morphological changes depending on the genotype of the SARS-CoV-2 virus. Materials and methods. A retrospective analysis of the medical records of 39 patients with COVID-19 with a severe course of the disease, which ended in death, who were hospitalized at the St. Petersburg State Budgetary Infectious Diseases Clinical Hospital named after S.P. Botkin" in 2020-2022. Clinical and laboratory characteristics were assessed, including determination of the virus genotype, levels of leukocytes, lymphocytes, alanine aminotransferase, creatinine, ferritin, C-reactive protein, D-dimer, interleukin-6. Macro- and microscopic changes were assessed, including immunohistochemical examination of the lungs and other organs using sera to CD14 68, 163, type 1 and 3 collagen. The preparations were digitized on a Panoramic scanner, morphometric studies were carried out using the SlideViewer program, including the quantitative determination of the content of CD68+ macrophages in 12 cases. Results. In all patients, the disease was complicated by the development of pneumonia, the majority had concomitant diseases (94.6%). The average time of hospitalization was 19.0+/-1.6 days, the average time of stay in the intensive care unit was 7.7+/-1.2 days. The analysis, depending on the genotype of the SARS-CoV-2 virus, showed a statistical difference between the age of patients, the length of stay in the intensive care unit and the level of lymphocytes. Differences in the average duration of hospitalization, the level of laboratory parameters were not revealed. Histopathological picture in all examined was approximately the same. The content of CD68+ macrophages per unit area in different genotypes did not differ, but varied significantly within the same genotype. Conclusion. Thus, it was not possible to identify significant differences between the changes caused by different genotypes of the new coronavirus, which can probably be explained by the fact that mutations do not include genome regions that are relevant to virulence factors, although further research is needed.Copyright © 2022 Interregional public organization Association of infectious disease specialists of Saint-Petersburg and Leningrad region (IPO AIDSSPbR). All rights reserved.

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.

Development of thermosensitive hydrogel of Amphotericin-B and Lactoferrin combination-loaded PLGA-PEG-PEI nanoparticles for potential eradication of ocular fungal infections: In-vitro, ex-vivo and in-vivo studies.

The most prevalent conditions among ocular surgery and COVID-19 patients are fungal eye infections, which may cause inflammation and dry eye, and may cause ocular morbidity. Amphotericin-B eye drops are commonly used in the treatment of ocular fungal infections. Lactoferrin is an iron-binding glycoprotein with broad-spectrum antimicrobial activity and is used for the treatment of dry eye, conjunctivitis, and ocular inflammation. However, poor aqueous stability and excessive nasolacrimal duct draining impede these agens' efficiency. The aim of this study was to examine the effect of Amphotericin-B, as an antifungal against Candida albicans, Fusarium, and Aspergillus flavus, and Lactoferrin, as an anti-inflammatory and anti-dry eye, when co-loaded in triblock polymers PLGA-PEG-PEI nanoparticles embedded in P188-P407 ophthalmic thermosensitive gel. The nanoparticles were prepared by a double emulsion solvent evaporation method. The optimized formula showed particle size (177.0 ± 0.3 nm), poly-dispersity index (0.011 ± 0.01), zeta-potential (31.9 ± 0.3 mV), and entrapment% (90.9 ± 0.5) with improved ex-vivo pharmacokinetic parameters and ex-vivo trans-corneal penetrability, compared with drug solution. Confocal laser scanning revealed valuable penetration of fluoro-labeled nanoparticles. Irritation tests (Draize Test), Atomic force microscopy, cell culture and animal tests including histopathological analysis revealed superiority of the nanoparticles in reducing signs of inflammation and eradication of fungal infection in rabbits, without causing any damage to rabbit eyeballs. The nanoparticles exhibited favorable pharmacodynamic features with sustained release profile, and is neither cytotoxic nor irritating in-vitro or in-vivo. The developed formulation might provide a new and safe nanotechnology for treating eye problems, like inflammation and fungal infections.

Perspectivas Inovadoras Na Abordagem Na Doenca Do Olho Seco (Dos) E O Interfaceamento Com Bancos De Sangue De Cordao Umbilical Humano

Os bancos de sangue de cordao umbilical e placentario (BSCUP), os laboratorios de processamento de medula ossea/ sangue periferico para transplante e os centros de tecnologia celular, passaram a receber a denominacao comum de Centros de Processamento Celular - CPC. O pais tem BSCUP 14 unidades publicas e 19 de natureza privada, totalizando 33 BSCUP. Uma analise retrospectiva dos relatorios da ANVISA identifica que em 2003 foram coletadas 26 unidades, com desqualificacao de 15,38% delas, o crescimento da coleta foi exponencial e dez anos depois, em 2013, foram coletadas 13.995 unidades (5,82% de desqualificacao). Em 2020, houve uma diminuicao expressiva de coletas, reflexo da pandemia de COVID-19: 4.918 unidades (desqualificacao de 12,69%). Este tipo de produtividade compromete a viabilidade financeira destes servicos, e encontrar formas de otimizar bolsas desqualificadas por volume ou quantidade de celulas para demais finalidades e uma vertente de gestao que deve ser estabelecida. O objetivo deste projeto foi a coleta de segmentos de cordao umbilical das unidades ja previamente validadas pelos BSCUP para extracao de celulas tronco, caracterizacao imunofenotipica e producao de meio condicionado isento de soro fetal bovino. A obtencao do meio condicionado (MC) da cultura de celulas tronco. Tem crescido cada vez mais o interesse pelo uso dos fatores de crescimento, citocinas e moleculas sinalizadoras livres no MC alem das vesiculas extracelulares, que se tornaram relevantes, tanto para diagnostico como para terapeutica, inclusive para aplicacoes oftalmologicas. Neste campo, identificamos a DOS que impacta profundamente a qualidade de vida das pessoas. Ha 15 anos o Laboratorio de Biologia Celular tem desenvolvido o soro autologo, para atendimento dos pacientes refratarios aos tratamentos convencionais e farmacologicos disponiveis, em especial aqueles pacientes submetidos ao Transplante de Medula Ossea e que desenvolveram DOS2ario a doenca de enxerto versus hospedeiro. No entanto, existem pacientes com impossibilidade de acesso venoso ou com sorologias reagentes para doencas infecciosas que sao impedidos de utilizar o produto. Diante disto, optou-se por produzir o MC de celulas tronco de cordao umbilical de parturientes jovens e sem comorbidades para obtencao do secretoma das celulas para avaliacao terapeutica na DOS. Um segmento do cordao umbilical foi retirado e processado seguido de plaqueamento e expansao para posterior identificacao de adesao ao plastico, caracterizacao imunofenotipica por citometria de fluxo utilizando marcadores como CD11b, CD13, CD14, CD34, CD31, CD36, CD45,CD73, CD90, CD 105, CD106 e HLA-DR. Todas as amostras tiveram adesao ao plastico com aspecto fibroblastoides e perfil imunofenpotipico corrobora com o determinado pela SITC. Para a obtencao de MC foram semeadas CTMcup ate 70% de confluencia e foram submetidas ao wash out, recebendo meio de cultura DMEM-F12 aditivado por 48 horas. Apos isto, foi coletado 60mL do secretoma das celulas para o experimento especificos in vitro. Copyright © 2022

Leucemia Aguda De Fenotipo Misto - Apresentacao Inicial Atipica - Um Relato De Caso

Introducao: Com desafiador tratamento e de diagnostico criterioso, a leucemia aguda de fenotipo misto (LAFM) e uma entidade rara dentro do espectro das leucemias agudas. Requer a presenca imunofenotipica de marcadores de linhagem B (CD19, CD22, CD79a), T (CD3) em conjunto com a linhagem mieloide (mieloperoxidase e diferenciacao monocitica - CD11c, CD14, CD64 ou lisozima). Relato de caso: Paciente masculino, 30 anos, obeso e diabetico tipo 2, hipertrigliceridemia, inicia com febre (38C), dor abdominal em hipocondrio direito e fadiga. Com dois dias de sintomas procura atendimento sendo liberado com sintomaticos. No quarto dia de sintomas houve piora da febre (39degreeC) e da dor, surgindo maculas hiperemiadas pruriginosas pelo corpo, ictericia e coluria. Retornou ao hospital de sua cidade sendo prescrito azitromicina e liberado com suspeita de influenza. No sexto dia de sintomas notou piora da ictericia, procurando, novamente, atendimento. Encaminhado, entao ao servico de referencia da regiao. Interna inicialmente na equipe da gastroenterologia como suspeita de hepatite viral. Na chegada: Hb 14,9, leucocitos 6140 com 2793 neutrofilos, 442 monocitos e 2812 linfocitos, 53 mil plaquetas;AST 57, ALT 733, hiperbilirrubinemia as custas de bilirrubina direta. Com todos os marcadores virais negativos, prosseguiu a investigacao de hepatite. No dia em que realiza ressonancia magnetica, que indicava processo infiltrativo/inflamatorio em figado e rim esquerdo, alem de testar positivo para COVID-19, ha evolucao no hemograma: Hb 10,7, leucocitos 7450 com 1192 blastos, 60 neutrofilos, 2012 monocitos e 4187 linfocitos, 23 mil plaquetas. Com o aparecimento de blastos, piora dos niveis de bilirrubinas e das lesoes de pele, foi realizado imunofenotipagem de sangue periferico que indicava leucemia monocitica aguda. Transferido a equipe da hematologia, sendo realizada biopsia de medula e iniciado protocolo 7 + 3 com substituicao das antraciclinas em falta no mercado por doxorrubicina 45 mg/m2. No terceiro dia da inducao, foi liberado o resultado da imunofenotipagem que confirmava o diagnostico de leucemia aguda de fenotipo misto B/mieloide, marcando CD19, CD22 e CD79a, com diferenciacao monocitica (CD14 e CD64). Cariotipo nao houve crescimento e PCR BCR/ABL negativo. Optado por seguir tratamento com 7 + 3, apresentando medula no D14 aplasiada e medula no D28 com doenca residual minima (DRM) negativa. Realiza tres consolidacoes com altas doses de citarabina (3g/m2). Paciente sustenta DRM negativa, estando em remissao completa. Iniciado manutencao com vincristina, mercaptopurina, metotrexato e prednisona. Aguarda transplante de celulas tronco hematopoieticas (TCTH). Discussao: Com o diagnostico de LAFM, o tratamento requer o maior numero de quimioterapicos, sendo sugerido o uso de protocolos para leucemia linfoblastica aguda. Como ja havia sido instituido o tratamento com doxorrubicina e citarabina, foi optado por seguir protocolo e, na manutencao da remissao completa, terminar as consolidacoes e iniciar a manutencao prevista pelo protocolo HyperCVAD. Devido a ser uma leucemia de alto risco, a realizacao do TCTH e necessaria e, neste caso relatado, a manutencao sera mantida ate a realizacao do transplante. Conclusao: Contudo, por se tratar de doenca rara e com poucos estudos publicados, requer compartilhamento de conhecimentos e condutas para melhora da abordagem. Copyright © 2022

ELEVATED CIRCULATING MONOCYTES AND MONOCYTE ACTIVATION IN PULMONARY POSTACUTE SEQUELAE OF SARS-COV-2 INFECTION

SESSION TITLE: Long COVID: It Can Take Your Breath Away SESSION TYPE: Original Investigations PRESENTED ON: 10/16/2022 10:30 am - 11:30 am PURPOSE: Nearly one third of patients who recover from acute SARS-CoV2 infection will experience persistent symptoms known as Post-Acute Sequelae of SARS-CoV2 infection (PASC). Among individuals with PASC, pulmonary complications are common. Patients with severe COVID-19 have observed high systemic levels of cytokines and profound immune dysregulation. During acute SARS-CoV-2 infection, CD169, a type I interferon-inducible receptor, is overexpressed on monocytes. CD169+ macrophages are involved in hyperinflammation, viral spread, and immune regulatory function. Although monocytes/macrophage play a pivotal role in inflammation during acute SARS-CoV2 infection, less is known about how these cells contribute to lung sequelae and immunopathology in PPASC. METHODS: Cross section study conducted comparing three groups: participants with PPASC with a reduced predicted diffusing capacity for carbon monoxide (DLCOc, <80%) on pulmonary function test;participants who fully recovered (RC) from SARS-CoV-2 with no residual symptoms;and healthy participants (HC) negative for SARS-CoV-2. These groups were age and gender matched from similar community settings. Among the groups, we compared the numbers of monocyte subsets (classical, intermediate and non-classical monocytes) and monocyte activation by assessing CD169 expression using flow cytometry analysis of peripheral blood mononuclear cells. RESULTS: Ten participants enrolled in each group with median age 53 years, 38.7% males. We found that PPASC and RC had higher median levels of total circulating monocytes than in HC, 59374 (IQR: 43161-91523), 65661 (40049-89490) and 2689 (1378-28125), respectively (p<0.01, p<0.01). Regarding monocyte subsets based on CD14+CD16+ expression, we observed significant increase in the number of classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14-CD16+) monocytes in PPASC and RC, compared to HC (p<0.01, p=0.01, respectively). There was no difference in the number of monocytes and in the proportion of each subset between PPASC and RC. We observed increased CD169+ monocyte counts in PPASC and RC compared to HC 56.8 (23.0-92.5), 66.75 (4.3-968.7), and 2.095 (0-16.9), respectively (p<0.01, p<0.01). Furthermore, a rising trend of CD169 expression was observed in intermediate and non-classical monocytes from PPASC compared to HC. In addition, CD169+ non-classical monocytes were positively correlated with D-dimer levels in PPASC (ρ=0.72, p=0.03). CONCLUSIONS: This study present evidence that patients with COVID infection exhibit persistent alterations in monocytes even after the acute COVID infection period. Correlation of D-dimer level with CD169+ non-classical monocytes in patients with PPASC provides a further rational for determining if a specific monocyte subset contributes to the pathogenesis of PPASC. CLINICAL IMPLICATIONS: Further studies are required for understanding of the development and progression of PPASC. DISCLOSURES: No relevant relationships by Dominic Chow No relevant relationships by Logan Dean No relevant relationships by Gehan Devendra No relevant relationships by FRITZIE IGNO No relevant relationships by Boonyanudh Jiyarom No relevant relationships by Juwon Park No relevant relationships by Parthav Shah No relevant relationships by Cecilia Shikuma No relevant relationships by Chathura Siriwardhana

Cell specific pro- and antiinflammatory lipidmediator shift in critically ill Covid19 patients

Introduction The COVID-19 pandemic had a significant impact on morbidity and mortality in Germany challenging intensive care unit (ICU) capacities across the country. To delineate the high variability in disease severity, clinical presentation and outcome, we focused on cellular regulators of inflammation and resolution on a single cell level to gain a deeper understanding of the patient's individual inflammatory response and their impact on survival. Methods Written informed consent was obtained from all patients and healthy controls. The study was approved by the local ethical review board (Az249/20 S-EB). To characterize the peripheral immune landscape, we performed a 14 parameter flow cytometric analysis of PBMCs of 32 critically ill CoV2 patients and a targeted HPLC-MS/MS of previously sorted PBMCs. All data was analyzed and correlated to clinical parameters and patients' outcomes (Fig. 4). Results As known [1], computational analysis of flow cytometry revealed a strong decrease of B Cell and CD8+ T Cell ratios and an increase of monocytes in critically ill CoV19 patients compared to control (Fig. 1A). Interestingly, non-survivors displayed an increased ratio of CD16+ monocytes and proinflammatory IL- 1beta in monocytes, B and T cells, while HLADR receptors were downregulated correlating with clinical outcome (Fig. 1B). Not unexpectedly, we saw a major increase in proinflammatory lipidmediators, such as PGJ2, PGF2, TxB2 (Fig. 1C). Additionally, our analysis revealed that not only the amount, but also the source of those mediators was shifted from CD16 to classical CD14 monocytes, even more pronounced in non-survivors. CD16 monocytes of CoV2 patients, however, lost the ability to generate proresolving lipidmediators depending on cytochrome p450 (Cyp450) or soluble epoxide hydrolase (sEH) TxB2 (Fig. 1D). Conclusions Even though a lot of insight into CoV2 has been gained over the last 2 years, relatively little is known about the impact of immune changes in critically ill patients. With this study, we are the first to attribute lipid mediators to specific cell types. Our findings show that TxB2 in critically ill CoV2 patients, which correlates with mortality in CoV2 [2], is produced mainly in CD14 monocytes. We further report that specifically non-survivors display increased ratios of non-classical CD16 monocytes, which are impaired to generate a major class of lipidmediators depending on Cyp450. In conclusion, these data provide evidence that not only the absolute amount of pro- and anti-inflammatory mediators, but also the cellular source of these mediators remains key to fully understand their role in critically ill CoV2 patients. (Figure Presented).

OA Biology

Purpose: The field of osteoarthritis (OA) biology is rapidly evolving and brilliant progress has been made this year as well. Methods: Landmark studies of OA biology published in 2021 and early 2022 were selected through PubMed searches and classified by their molecular mechanisms, and it was largely divided into the intra-cellular mechanisms and the inter-compartment or inter-cellular interaction in OA progression. Results: The intra-cellular mechanisms involving OA progression included 1) Piezo1/TRPV4-mediated calcium signaling, 2) low grade inflammation by TLR-CD14-LBP complex and IKKβ-NFkB signaling, 3) PGRN/TNFR2/14-3-3ε/Elk-1 anabolic cascade, 4) G protein-coupled receptor (GPCR) signaling, 5) mechanical loading-cilia/Ift88-hedgehog signaling, 6) mitochondrial fission by ERK1/2-DRP1 pathway, and 7) hypoxia-DOT1L-H3K79 methylation pathway. The studies on inter-compartment or inter-cellular interaction in OA progression included the following subjects: 1) the anabolic role of Lubricin, a proteoglycan from superficial zone cells, 2) osteoclast-chondrocyte interaction via exosomal miRNA and sphingosine 1-phosphate (S1P), 3) αV integrin-mediated TGFβ activation by mechanical loading, 4) TGFβ-mediated suppression of sclerostin in osteocytes, 5) catabolic role of Flightless I as a DAMPs-triggering molecule, and 6) catabolic role of paracrine signaling from fat. Conclusions: Despite the disastrous Covid-19 pandemic situation, many outstanding studies have expanded the boundary of OA biology. They give us not only critical insight on pathophysiology, but also clue for the treatment of OA.

High Monocyte Count Associated with Human Cytomegalovirus Replication In Vivo and Glucocorticoid Therapy May Be a Hallmark of Disease.

Cytomegalovirus (CMV) syndrome and infectious disease are defined as pathogen detection with appropriate clinical symptoms, but there are not pathognomonic signs of CMV disease. Although the prodrome of acute minor viral infections leukopenia (lymphopenia and neutropenia) is noted with onset of fever, followed by monocytosis, the role of monocytosis in CMV disease has not been described. Furthermore, under influence of corticosteroid therapy, CMV reactivation and monocytosis are described, but without a strict relationship with steroids dose. In the study, the monocyte level was investigated during the CMV infectious process. Regrettably, a non-selected group of 160 patients with high CMV viremia showed high dispersion of monocyte level and comparable with the median value for healthy subjects. Therefore, we investigated monocyte level in CMV-infected patients in relation to the logarithmic phase of the infectious process. Samples from patients with active CMV replication (exponential growth of CMV viremia) were tested. Significant monocytosis (above 1200/µL) during the logarithmic phase of CMV infection (with exponent between 3.23 and 5.77) was observed. Increased count and percentage of monocytes correlated with viral replication in several clinical situations except when there was a rapid recovery without relapse. Furthermore, glucocorticoids equivalent to 10 and 20 mg of dexamethasone during a 2-3-week period caused monocytosis-significant increase (to 1604 and 2214/µL, respectively). Conclusion: In light of the logarithmic increase of viral load, high monocytosis is a hallmark of CMV replication. In the COVID-19 era, presence of high virus level, especially part of virome (CMV) in the molecular technique, is not sufficient for the definition of either proven or probable CMV replication at any site. These preliminary observations merit additional studies to establish whether this clinical response is mediated by monocyte production or by decrease of differentiation to macrophages.

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

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

Immune phenotypes correlate with the outcome of severe Covid-19 infection

Despite the tremendous impact of the Sars-CoV-2 global pandemic and becoming focus of scientific research[1], many aspects of the disease and its pathophysiology, especially concerning prognostic parameters and treatments remain uncertain. The aim of our study is to assess and link immune profiles of the dysregulated cellular immune response in patients hospitalized with severe Covid-19 to their outcomes. Therefore, we immune phenotyped severly ill Sars-CoV-2 patients on our ICU and to surviving to non-surviving patients and healthy controls. Methods Using flow cytometry (BD-Fortessa), we created a 14-parameter immunoprofile of 25 Covid-19 patients from our ICU and 11 of healthy control individuals. The analysis was based on live/dead control, CD3, CD4, CD8, CD19, CD66b, CD14, CD16, IL-10, TNF-α, IL-1β, HLA-DR and IL-6 antibodies. Both clusters (survivors, n=16;non-survivors, n=9) and healthy controls (n=11) were compared with each other by Kruskal-Wallis test with Dunns-Ls post-test correction for multiple testing (Prism V.9.0). The patients in both groups had a similar age and at the time of analysis (Fig. 1A), the treatment was insignificantly more invasive in non-survivors than in survivors (7-point WHO ordinal scale means 5.8 vs. 5.3, p = 0.43) (Fig.1 D). Similarly, the blood tests and the viral loads were comparable in both groups. Study has permission from the ethic commission (AZ-249/20 S-EB). Results The study showed that cell specific cytokine expressions are distinct in survivors compared to non-survivors even at an early stage of the critical disease. Surviving Covid patients showed increased TFNá levels throughout all cell populations, which met significance in CD4+ T (Fig.2 A) Cells and CD135+ DC. (Fig. 4 C). IL-6 levels, however, were significantly lower in CD4+ T cells of survivors (Fig. 2 B). Similarly, proinflammatory, classical CD16+ monocytes of non-survivors exhibited an increase in IL-6 and IL-1â. Moreover, dendritic cells of non-survivors seemed to be exhausted revealing less TNFá and IL-6 and IL-1â (Fig 4) Conclusion: Taken together, a disability of monocyte activation and exhaustion of dendritic cell reaction was associated with a worsened outcome of severely ill Covid-19 patients [2,3]. On the contrary a sufficient TNFá response, especially of CD4 and dendritic cells might be required to overcome the infection. Therefore, our findings suggest that measuring cell specific levels of cytokines and cell population shifts might be of high clinical relevance to predict the outcome of the disease and offer new therapeutical options for these patients.

Interactive Web-Based Lung Cell Atlases Lower Barriers to Transcriptomic Data Sharing, Mining and Dissemination

Rationale: Recent advancements in sequencing technologies have led to a substantial increase in the scale and resolution of transcriptomic data. Despite this progress, accessibility to this data, particularly among those who are coming from non-computational backgrounds is limited. To facilitate improved access and exploration of our single-cell RNA sequencing data, we generated several data sharing, mining and dissemination portals to accompany our idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and lung endothelial cells (Lung EC) cell atlases. Descriptions and links of each website can be found here: https://medicine.yale.edu/lab/kaminski/research/atlas/. Methods: Each interactive data mining website is coded in the R language using the Shiny package and is hosted by Shinyapps.io. Percell expression data for each website is stored on a MySQL database hosted by Amazon Web Services (AWS). Time-associated website engagement statistics and gene query information is collected for each website using a combination of Google Analytics and a gene search table stored on our MySQL database. User exploration of available data is facilitated through several easy-touse visualization tools available on each website. Results: Website usage statistics since the publication of each website shows that 9,772 unique users from 56 countries and five continents have accessed at least one of the three websites. At the time of writing, 300,748 total queries have been made for 15,627 unique genes across the websites. The top five searched genes for the IPF Cell Atlas are CD14, ACE2, ACTA2, IL11 and MUC5B while for the COPD Cell Atlas they are FAM13A, MIRLET7BHG, HHIP, ISM1 and DDT. Finally, the top searched genes for the Lung Endothelial Cell Atlas are BMPR2, PECAM1, EDNRB, APLNR and PROX1. Of note, interaction with the IPF Cell Atlas increased dramatically at the start of the COVID-19 pandemic, with queries for the ACE2 gene, the putative binding receptor for the SARS-CoV-2 virus, increasing substantially at the pandemic's onset in the United States. Conclusions: Usage statistics, gene query information and feedback from users, both within academia and industry, have shown broad engagement with our websites by individuals across computational and non-computational backgrounds. We envision widespread adoption of web-based portals similar to ours will facilitate novel discoveries within these complex datasets and new scientific collaborations.

The Dual-Endothelin1/VEGFsp Receptor (DEspR) - Potential Role in Chronic Respiratory Diseases

Background The translatability of the dual-endothelin1/VEGFsp receptor (DEspR) in human was first described in 2016 and its functionality is largely unknown since DEspR is not expressed in healthy human tissues except for kidney tissue and certain tumors. Recently, DEspR expression was reported on human neutrophil subsets of acute respiratory distress syndrome (ARDS) and COVID19-ADRS patients. DEspR+ neutrophil levels correlated with disease severity and mortality which may root in their delayed apoptosis and facilitated formation of neutrophil extracellular traps. Neutrophils play a major role in inflammation of chronic respiratory diseases and altered levels of DEspR ligands ET-1 and VEGF are found in COPD and asthma phenotypes. Here, we investigated the DEspR expression on human leukocyte populations of asthmatics, COPD patients and healthy smokers as well as on the human promyelocytic leucaemic cell line HL-60. Methods DEspR expression was measured on undifferentiated, promyelocytic HL-60 cells and after differentiation towards a neutrophilic phenotype using 1.25% DMSO. Expression was also measured after stimulation with 50 μg/mL poly I:C or 100 ng/mL LPS. Whole blood cells of COPD patients (step III, IV), healthy smokers and asthmatics (step III) were stained directly after blood draw or after stimulation with 50 μg/mL poly I:C or 100 ng/mL LPS. HL-60 and whole blood leukocytes were stained with Annexin V, 7AAD, DEspR (rhIgG4, clone 6g8), CD11b, CD14 and CD16a. Results Undifferentiated CD11b-, CD14- CD16a- and differentiated CD11b+, CD14-, CD16a- HL-60 cells did not express DEspR, neither with or without inflammatory stimulation. DespR was not expressed on whole blood leukocytes at baseline level (mean±SD: 0.15±0.26 to 0.91±0.60%) but poly I:C induced DEspR expression on neutrophils (34.10±18.52%), monocytes (29.16±20.00%), lymphocytes (9.67±6.11%) and eosinophils (6.14±4.39%). The distribution of DEspR+ cells upon poly I:C stimulation was not significantly altered among different disease types, however, healthy smokers showed a trend to higher DEspR levels. The median fluorescence intensity was not significantly altered among disease types but among the cell populations. Conclusion First experiments demonstrated that DEspR expression can be induced on leukocytes upon inflammatory stimulation. In contrast to previous results of us, LPS did not induce DEspR expression which might be related to differences in the age and disease severity of investigated patients. Interestingly, poly I:C induced a strong DEspR expression indicating a toll-like receptor 3 related mechanism. The sample size needs to be increased to confirm these first results and to investigate the underlying mechanism in detail.

SARS-CoV-2-INFECTED MACAQUES EXHIBIT MICROBIAL TRANSLOCATION ACROSS INTESTINAL MUCOSA

Background: Gastrointestinal symptoms and viral RNA (vRNA) in stool have been described in human SARS-CoV-2 infections. However, intestinal pathology and related inflammation have not been extensively described in humans or animal models. Here we investigate the effect of SARS-CoV-2 infection on the gut mucosa and inflammation in rhesus macaques (RM) and humans. Methods: Fourteen adult RM were infected with US/WA-1/2020 SARS-CoV-2 instilled intranasally and intratracheally. Animal clinical features (mass, temperature, etc.) and samples (nasal swabs, throat swabs, blood, stool, etc.) were collected at baseline and up to day 10 post-infection at necropsy. RNA was extracted from swab and stool samples and vRNA measured by qRT-PCR. Plasma samples were assessed for inflammatory biomarkers by ELISA. Tissues collected at necropsy were fixed and evaluated for microbial translocation through immunohistochemical (IHC) staining of bacterial products;H&E staining was also performed. Tissues were additionally collected from uninfected RM and processed in the same manner. Human plasma samples from individuals with moderate COVID-19 were collected at early infection and recovery time points and assessed for inflammatory biomarkers. Results: SARS-CoV-2 infection of RM did not induce fever nor weight loss over five percent. vRNA was detected in all animals in nasal and throat swabs. vRNA, including subgenomic RNA indicative of viral replication, was also detected in stool samples. Scores for translocating bacteria in colon sections stained by IHC for bacterial products were higher for SARS-CoV-2 infected RM than uninfected controls. Additionally, follicles made up a higher percentage of total mesenteric lymph node area in SARS-CoV-2 infected animals than control RM. Furthermore, soluble CD14 in plasma increased significantly from baseline to day 10 of SARS-CoV-2 infection (p=0.0006) and decreased significantly in humans from early infection to recovery time points (p=0.0295). Conclusion: Thus, adult RM experienced mild to moderate SARS-CoV-2 infections yet demonstrated evidence of microbial translocation. Humans similarly demonstrated evidence of microbial translocation that decreased upon recovery from COVID-19. These data suggest gut pathology in SARS-CoV-2 infection may be contributing to systemic inflammation in COVID-19.

ALTERAÇÕES NOS SUBTIPOS DE MONÓCITOS (CMO, IMO E NCMO) NA COVID-19 E SUA RELAÇÃO COM A GRAVIDADE DA DOENÇA

Objetivos: Este estudo teve como objetivo avaliar as frequências e números absolutos de monócitos e seus subtipos: clássico (cMo), intermediário (iMo) e não clássico (ncMo) em pacientes com COVID-19, a fim de esclarecer sua relação com as alterações do sistema imunológico periférico e a gravidade da doença. Material e métodos: Este estudo incluiu um total de 30 controles saudáveis (CS) e 157 pacientes com COVID-19. Os pacientes hospitalizados foram atendidos no Hospital Universitário Polydoro Ernani de São Thiago e Hospital Nereu Ramos. A gravidade da doença foi classificada como leve (n = 36), moderada (n = 30), grave (n = 32) e crítica (n = 59). Os monócitos foram subclassificados de acordo com a expressão de CD14 e CD16 em: cMo (CD14++CD16-), iMo (CD14+CD16+) e ncMo (CD14-/CD16+). Para analisar a expressão de HLA-DR nos subtipos de monócitos, a intensidade de fluorescência média (IFM) desse marcador foi avaliada. Em seguida, os valores das razões de IFM foram comparados entre os grupos. Resultados: Os pacientes com COVID-19 não apresentaram diferença significativa na contagem absoluta de monócitos quando comparados aos CS. Nos valores relativos, foi observada uma redução nos pacientes graves (p < 0,001). Já em relação aos subtipos de monócitos, foram encontradas diferenças significativas no valor percentual dentro do compartimento monocítico. Foi observado um aumento de cMo, principalmente nos casos de moderado a crítico (p < 0,001), no entanto, houve uma redução expressiva de ncMo, com redução de mais de 30× nos pacientes críticos quando comparado aos CS (p < 0,001). As frequências de iMo não apresentaram variação quando comparadas aos CS. Além disso, nos pacientes com quadro leve foi observado um aumento da expressão de HLA-DR pelos monócitos, quando comparados aos CS, que decai a partir dos pacientes moderados e reduz de forma mais expressiva nos pacientes graves e críticos (p < 0,001). Em relação aos subtipos de monócitos, esse padrão se repete, com um aumento de HLA-DR nos cMo, iMo e ncMo nos pacientes leves e uma redução significativa, principalmente nos quadros grave-crítico (p < 0,001). Essa redução ocorre principalmente nos cMo, o que levou a um aumento da razão HLA-DR iMo/cMo nos pacientes moderado-crítico (p < 0,001). Discussão: A COVID-19 é uma infecção respiratória causada pelo SARS-CoV-2, onde diversos estudos demonstram uma ligação entre a resposta imune do hospedeiro e a gravidade da doença. Nesse contexto, os monócitos circulantes possuem funções pró-inflamatórias e de resolução. Várias evidências indicam que essas células desempenham um papel importante na imunopatogênese da doença, uma vez que a resposta hiperinflamatória induzida pelo vírus parece ser a principal causa de gravidade da doença. Os resultados apresentados demonstram uma alteração no compartimento dos monócitos, com aumento de cMo e redução expressiva de ncMo, alterações que se mostram relacionadas com o aumento da gravidade da doença. Além disso, em pacientes com quadros mais severos, foi observada uma redução na expressão de HLA-DR, um MHC de classe II, o que indica um possível quadro de imunossupressão. Conclusão: Os resultados obtidos demonstram uma correlação dos subtipos de monócitos com a gravidade da doença, o que poderá servir como um possível marcador de prognóstico em pacientes com COVID-19.

DIAGNÓSTICO DE LEUCEMIA MIELOMONOCÍTICA CRÔNICA EM PACIENTE COM INFECÇÃO PELO NOVO CORONAVÍRUS: RELATO DE CASO

Mulher de 68 anos, hipotireoidea, em uso de levotiroxina, COVID confirmado em exame realizado há 3 dias;deu entrada no pronto atendimento de um hospital de grande porte devido a piora dos sintomas gripais, febre, astenia e dispneia aos esforços com piora progressiva. Apresentava palidez discreta, sem outros achados dignos de nota. À admissão, apresentou leucocitose (Gl 18900/mm³) com desvio à esquerda sem anemia (Hb 13.1 g/dL) ou monocitose, associada a trombocitopenia moderada (98000/mm³). Tomografia de tórax mostrou infiltrado difuso sugestivo de pneumonia por Covid-19. Iniciada dexametasona 6mg/dia e antibioticoterapia empírica (ceftriaxona e azitromicina). Após 3 dias de internação, manteve febre;solicitadas hemoculturas e escalonada antibioticoterapia para Piperacilina-tazobactam. Evoluiu com anemia normocítica e normocrômica discreta (Hb 11.8g/dL), trombocitopenia leve (132000/mm³) e piora de leucocitose (16600/mm³), monocitose (4628/mm³) e surgimento de blastos em hemograma. As hemoculturas fecharam negativas. Devido à piora da leucocitose (Gl 46700/mm³) e da monócitos (9807/mm³) foi solicitada avaliação hematológica;realizada hematoscopia, que evidenciou alterações displásicas nas três séries. Realizada imunofenotipagem de sangue periférico, que mostrou presença de 15,8% de blastos mielóides, CD14- e CD16-, sem basofilia, com desvio à esquerda até promielócitos. Estudo medular inicial foi inconclusivo, com hiperplasia granulocítica e megacariocítica. Após 7 dias de antibioticoterapia, optado por alta hospitalar com proposta de acompanhamento ambulatorial, uma vez que a paciente manteve estabilidade clínica. No seguimento, evoluiu com queixa de astenia;novo hemograma mostrou piora da anemia e da trombocitopenia, assim como piora da leucocitose. Novo estudo medular realizado, com pesquisa da translocação BCR-ABL, mutaçõesJAK2, CALR, MPL e pesquisa de sideroblastos em anel, todos negativos. No mielograma, encontrado hipercelularidade granulocítica com maturação normoblástica, monocitose (13,2%), aumento do blastos(15.4%) e de megacariócitos displásicos. Cariótipo 46, XX, del (20) (q13.1). Biópsia de medula óssea com hiperplasia granulocítica e ausência de fibrose. Diante do exposto, foi feito o diagnóstico de Leucemia mielomonocítica crônica (LMMC-2). Realizado tratamento com azacitidina por 5 ciclos, mantendo leucocitose. No momento, aguardando transplante de medula óssea alogênico. Discussão: A LMMC é uma neoplasia hematológica mieloide com características de ambas as síndromes mielodisplásicas (SMD) e neoplasia mieloproliferativa (NMP), com até um quinto dos casos relacionados a condições inflamatórias/auto-imunes, conforme ilustrado neste caso, onde o seguimento é necessário para o diagnóstico. Trata-se de doença rara, predominante em idosos do sexo masculino. A apresentação varia conforme características mielodisplásicas ou mieloproliferativas, podendo se apresentar como monocitose incidental em indivíduos assintomáticos. Para o diagnóstico, é necessária a exclusão das doenças mieloproliferativas crônicas e de causas reativas, obedecendo os critérios da OMS de 2016. O tratamento varia conforme estratificação de risco e características clínico-laboratoriais mieloproliferativas/mielodisplásicas, com uso de terapia citorredutora ou hipometilante, respectivamente. O transplante alogênico de medula óssea é a única alternativa curativa disponível no momento, no entanto muitos candidatos não são elegíveis devido suas comorbidades.

The natural history of covid-19 in vaccinated inflammatory bowel disease patients

Background: Patients with Inflammatory Bowel Disease (IBD), especially those on immunosuppressive (IMS) treatment should be vaccinated against SARS-CoV-2 to prevent hospitalization, mechanical ventilation, and death. However, IMS may adversely affect vaccination, raising concerns as to how vulnerable these patients are to break through COVID-19 infections. Thus, we aimed to assess the proportion of IBD patients who despite complete vaccination developed COVID-19, as well as the course of the infection. Methods: This study was an initiative of the Hellenic Group for the study of IBD which involved seven IBD referral Centers. Patients attending these Centers who reported a COVID-19 infection at least 3 weeks after vaccination completion were asked to complete an on-line anonymous questionnaire which included patient demographics and IBD clinical and therapeutic data, a detailed vaccination history, and the course and outcome of COVID-19, especially the need for hospitalization, oxygen supply, and admission to ICU. In patients with grave outcome information was sought by family members Results: On estimate, 2940 patients reported full vaccination (Pfizer vaccine) in the 7 centers. Between 1st May 2021 and 30th October 2021, 46 (1.5%) fully vaccinated IBD patients reported COVID-19 infection [25 male, 32 CD, 14 UC, mean (SD) age 40.8 (13.7) years, mean (SD) IBD duration mean, 11.2 (10.8) years]. Five patients were receiving 5-ASAs, 2 corticosteroids, 5 azathioprine/methotrexate, 23 anti-TNFs as monotherapy and 3 in combination with azathioprine/methotrexate, and 1 with corticosteroids, 3 vedolizumab and 1 each ustekinumab, tofacitinib and rizakinzumab at the time of COVID-19 diagnosis;one patient was receiving no treatment. IBD was in remission in 37/46 patients (80.4%). Comorbidities were seen in 21 patients (thyroid disease 11;diabetes mellitus 2;hypertension 2;psoriasis 1;prior breast cancer 1;spondyoartropathy 2;dyslipidemia 1;and PSC 1 patient). The mean (SD) time between last vaccination dose and infection was 3.2 (1.4) months. Overall, 40 (86.9%) patients reported mild constitutional and respiratory symptoms, 4 (8.7%) were asymptomatic and only 2 patients (4.3%) required hospitalization which was uneventful in both. None needed high flow oxygen supply or ICU admission, and none reported symptoms of long COVID. No deaths were reported by patient relatives. IBD medications were stopped in 21 patients (45.6%) during the COVID-19 infection. Conclusion: A minority of fully vaccinated IBD vaccinated patients developed COVID-19 which was relatively mild and uneventful. These results reinforce the importance of vaccination especially in vulnerable populations.