In vitro effects of hexapeptide -arginyl- alpha- aspartyl- lysyl- valyl- tyrosyl- arginine on 2 subsets and functions of neutrophilic granulocytes in moderate COVID-19

Background: The hyperactivated subsets of neutrophilic granulocytes (NG) play a negative role on the development, course and outcome of the COVID-19. With this position, NG are interesting target for creation of new therapeutic approaches. Method(s): The study group (SG) included 31 patients with moderate COVID-19, aged 61(57;71)years. Before and after incubation of whole blood with HP (106 g/l, 60 min., 37degreeC) 2 NG subsets were tested: major -CD64- CD16+CD32+CD11b+, minor -CD64+ CD16+CD32+CD11b+ with detection of their phenotypes using MFI (FC 500, Beckman Coulter, USA). Phagocytic activity of NG against S. aureus was studied. Comparison group(CG) consisted 22 healthy volunteers 58 (57;70) years old. Result(s): In patients with COVID-19 the minor subset of NG CD64+CD16+CD32+CD11b+(% ) was significantly increased in 5-fold versus the CG (p <= 0.005) and had transformed phenotype: CD64dimCD16brightCD32midCD11bbright versus CD64midCD16dimCD32midCD11b brightin the CG (p <= 0.005). This transformed phenotype had high expression levels of receptors of activation: CD16 and CD11b, that suggested its negative hyperactivation. The % of the major subset NG did not change (p > 0.05), but an altered phenotype of CD64-CD16brightCD32midCD11bdimNG was determine against CD64-CD16dimCD32midCD11boNG in the CG. Defects of phagocytic activities of NG were found: the decrease of % an active phagocytic NG, absorbing and digesting abilities (p1 <= 0.005;p2 <= 0.005;p3 <= 0.005).The effects of HP in vitro were shown: the significant decreasing of NG minor subset (%) in comparison with it's level before HP influences (p <= 0.005) reached to the values of the CG (p > 0.05). In parallel, the phenotype of minor subset changed to CD64brightCD16dimCD32midCD11bo, with decreasing level of CD16 to normal (p > 0.05). The transformed phenotype of the major subset was changed to CD64-CD16midCD32brightCD11 bmidNG: a decrease in MFI CD16, an increase in MFI CD32 and MFI CD11b (p1 <= 0.005;p2 <= 0.005;p3 <= 0.005). The restoration of defective phagocytic function of NG was received. Conclusion(s): Immunomodulating effects of HP in vitro on NG in moderate COVID-19 were shown: positive remodeling of the phenotype of minor aggressive NG subset from hyperactivated to normal and the restoration of defective NG phagocytic function.

Kinetics of CD169, HLA-DR, and CD64 expression as predictive biomarkers of SARS-CoV2 outcome.

INTRODUCTION: Discriminating between virus-induced fever from superimposed bacterial infections is a common challenge in intensive care units. Superimposed bacterial infections can be detected in severe SARS-CoV2-infected patients, suggesting the important role of the bacteria in COVID-19 evolution. However, indicators of patients' immune status may be of help in the management of critically ill subjects. Monocyte CD169 is a type I interferon-inducible receptor that is up-regulated during viral infections, including COVID-19. Monocyte HLA-DR expression is an immunologic status marker, that decreases during immune exhaustion. This condition is an unfavorable prognostic biomarker in septic patients. Neutrophil CD64 upregulation is an established indicator of sepsis. METHODS: In this study, we evaluated by flow cytometry the expression of cellular markers monocyte CD169, neutrophil CD64, and monocyte HLA-DR in 36 hospitalized patients with severe COVID-19, as possible indicators of ongoing progression of disease and of patients' immune status. Blood testings started at ICU admission and were carried on throughout the ICU stay and extended in case of transfer to other units, when applicable. The marker expression in mean fluorescence intensity (MFI) and their kinetics with time were correlated to the clinical outcome. RESULTS: Patients with short hospital stay (≤15 days) and good outcome showed higher values of monocyte HLA-DR (median 17,478 MFI) than long hospital stay patients (>15 days, median 9590 MFI, p= 0.04) and than patients who died (median 5437 MFI, p= 0.05). In most cases, the recovery of the SARS-CoV2 infection-related signs was associated with the downregulation of monocyte CD169 within 17 days from disease onset. However in three surviving long hospital stay patients, a persistent upregulation of monocyte CD169 was observed. An increased neutrophil CD64 expression was found in two cases with a superimposed bacterial sepsis. CONCLUSION: Monocyte CD169, neutrophil CD64, and monocyte HLA-DR expression can be used as predictive biomarkers of SARS-CoV2 outcome in acutely infected patients. The combined analysis of these indicators can offer a real-time evaluation of patients' immune status and of viral disease progression versus superimposed bacterial infections. This approach allows to better define the patients' clinical status and outcome and may be useful to guide clinicians' decisions. Our study focused on the discrimination between the activity of viral and bacterial infections and on the detection of the development of anergic states that may correlate with an unfavorable prognosis.

Monocyte HLADR and Immune Dysregulation Index as Biomarkers for COVID-19 Severity and Mortality.

Immune dysregulation in COVID-19 is the major causal factor associated with disease progression and mortality. Role of monocyte HLA-DR (mHLA-DR), neutrophil CD64 (nCD64) and Immune dysregulation index (IDI) were studied in COVID-19 patients for assessing severity and outcome. Results were compared with other laboratory parameters. Antibody bound per cell for mHLA-DR, nCD64 and IDI were measured in 100 COVID-19 patients by flow cytometry within 12 h of hospital admission. Thirty healthy controls (HC) were included. Clinical and laboratory parameters like C - reactive protein (CRP), Procalcitonin (PCT), Absolute Lymphocyte count (ALC), Absolute Neutrophil count (ANC) and Neutrophil to Lymphocyte ratio (NLR) were recorded. Patients were followed up until recovery with discharge or death. Parameters from 54 mild (MCOV-19), 46 severe (SCOV-19) and 30 HC were analysed. mHLA-DR revealed significant and graded down regulation in MCOV-19 and SCOV-19 as compared to HC whereas IDI was lowest in HC with increasing values in MCOV-19 and SCOV-19. For diagnostic discrimination of MCOV-19 and SCOV-19, IDI revealed highest AUC (0.99). All three immune parameters revealed significant difference between survivors (n = 78) and non-survivors (n = 22). mHLA-DR < 7010 and IDI > 12 had significant association with mortality. Four best performing parameters to identify patients with SCOV-19 at higher risk of mortality were IDI, NLR, ALC and PCT. mHLA-DR and IDI, in addition to NLR and ALC at admission and during hospital stay can be utilized for patient triaging, monitoring, early intervention, and mortality prediction. IDI reported for the first time in this study, appears most promising. Immune monitoring of 'in hospital' cases may provide optimized treatment options. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01087-z.

IgG antispike persistence and immunophenotype in children infected by SARS-CoV-2.

AIM: The immune status of children recovering from SARS-CoV-2 infection is not completely understood. We describe IgG antispike persistence in children infected during the first two pandemic waves. In addition, we compared with healthy controls their leukocyte populations and CD64 expression. METHODS: Cross-sectional study. Carried out from October 2021 to February 2022 in nonreinfected and nonvaccinated children with SARS-CoV-2 in 2020. The presence of antispike IgG was studied using chemiluminescent immunoassay. Leukocyte populations were analysed using flow cytometry and marked for CD45, CD4, CD8 and CD64. Statistical minor than 0.05 was considered significant. RESULTS: One hundred and eighty-three control and 77 patients were included. IgG antispike determinations were performed after a median of 501 days (262-464); 52 of 77 children were positive. Cases showed significantly higher percentages of monocytes, lymphocytes, CD8+ and CD4+ . In addition, CD64 expression was higher in monocytes and neutrophils. The presence of IgG antispike was accompanied by a higher percentage of CD64+ neutrophils. CONCLUSION: In our series, the SARS-CoV-2 IgG antispike protein was usually positive beyond 1 year after infection. Furthermore, leukocyte populations from cases differ from controls, with higher CD64 expression on neutrophils and monocytes. Prospective clinical observations are required to confirm the implications of these findings.

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

Eficacia Do Tratamento Baseado Em Venetoclax Em Paciente Jovem Com Leucemia Mieloide Aguda Recidivada Candidato a Transplante De Medula Ossea Alogenico

Introducao: A Leucemia Mieloide Aguda (LMA) e uma doenca agressiva, e em geral, de prognostico reservado. O tratamento com intuito curativo e realizado com altas doses de quimioterapia, seguido ou nao por transplante de medula ossea (TMO) alogenico. Porem, a recidiva ainda representa um desafio a ser superado, levando a busca de novas opcoes terapeuticas, como o inibidor de bcl-2, Venetoclax. Objetivo: Relatar o caso de um paciente jovem com LMA recidivada com resposta ao tratamento baseado em Venetoclax, seguido por TMO alogenico. Metodo: Levantamento de dados do prontuario e revisao da literatura. Relato do caso: VSV, sexo masculino, 21 anos, diagnosticado com LMA em 18/02/2020, mielograma com 60,4% blastos, com fenotipo CD45+ intermediario;CD34+;CD13+;CD33 parcial;CD64 parcial;HLA-DR parcial;MPO parcial e cariotipo 46, XY [20]. Sem possibilidade de avaliacao molecular da doenca. O paciente recebeu tratamento de inducao padrao (D3A7) atingindo remissao completa apos um ciclo e seguindo com consolidacao com 3 ciclos de ARA-C 3g/m2 ate maio de 2020 (optou-se por nao realizar o 4ciclo devido a pandemia por COVID19). Apresentava doenca residual minima (DRM) negativa em reavaliacao medular de agosto de 2020, mantendo-se ate junho de 2021, quando apresentou recidiva com 33,8% de blastos em imunofenotipagem de reavaliacao. Internado em julho de 2021 para QT de resgate com FLAG, evoluiu com choque septico e insuficiencia respiratoria, sendo necessario suporte em unidade de terapia intensiva. Apos recuperacao clinica, obteve novamente DRM negativa, sendo encaminhado para TMO alogenico aparentado (irma "full match"). Entretanto, enquanto aguardava o transplante, apresentou nova recidiva (IF com 5,4% de blastos), sendo optado por tratamento com Venetoclax + Azacitidina, com reducao de DRM para 1,5%. Recebeu o segundo ciclo de Venetoclax combinado com citarabina, devido a indisponibilidade da Azacitidina, seguido por DRM negativa. O paciente foi submetido ao TMO alogenico em 31/03/22, com avaliacao medular no D+60 com quimerismo de 100%, mantendo DRM negativa no D+120. Discussao: O uso do inibidor de BCL-2 (Venetoclax), em combinacao com agentes hipometilantes ou doses baixas de citarabina, foi aprovado para pacientes recem-diagnosticados com LMA inelegiveis para quimioterapia intensiva, revolucionando o tratamento da doenca. Publicacoes recentes vem demonstrando novos beneficios dessa associacao, tanto previamente ao TMO (em primeira linha ou com doenca recidivada), como na terapia de resgate para recidiva pos-TMO. Tambem tendo sido evidenciado sucesso terapeutico em faixas etarias menores (incluindo criancas). Esses novos estudos motivaram o uso de terapia baseada em venetoclax nesse paciente jovem, com doenca recidivada, conseguindo atingir e manter DRM negativa. Conclusao: O caso relatado demonstra eficacia do Venetoclax (em combinacao com hipometilante ou citarabina) em promover remissao profunda da doenca, tornando o paciente habil para o transplante e aumentando a probabilidade de sobrevida a longo prazo. Copyright © 2022

The FLARE score, circulating neutrophils, and association with COVID-19 outcomes in patients with solid tumors

Background: Inflammation and neutrophils play a central role in severe Covid-19 disease. In previous data, we showed that the FLARE score, combining both tumor and Covid-19-induced proinflammatory status (proinflamstatus), predicts early mortality in cancer patients (pts) with Covid-19 infection. We aimed to assess the impact of this score in a larger cohort and characterize the immunophenotype (IF) of circulating neutrophils. Methods: Multicenter retrospective cohort (RC) of pts with cancer and Covid-19 infection across 14 international centers. Circulating inflammatory markers were collected at two timepoints: baseline (-15 to -45d before Covid-19 diagnosis) and Covid-19 diagnosis. Tumor-induced proinflam-status was defined by high dNLR (neutrophils/(leucocytes-neutrophils)> 3) at baseline. Covid-19-induced proinflam-status was defined by +100% increase of dNLR between both timepoints. We built the FLARE score combining both Tumor and Infection-induced inflammation: T+/I+ (poor), if both proinflam-status;T+/I- (T-only), if inflammation only due to tumor;T-/I+ (I-only), if inflammation only due to Covid;T-/I- (favorable), if no proinflam-status. The IF of circulating neutrophils by flow cytometry was determined in a unicenter prospective cohort (PC) of pts with cancer during Covid-19 infection and in healthy volunteers (HV). Primary endpoint was 30-day mortality. Results: 524 pts were enrolled in the RC with a median follow- up of 84d (95%CI 78-90). Median age was 69 (range 35-98), 52% were male and 78% had baseline PS <1.Thoracic cancers were the most common (26%). 70% had active disease, 51% advanced stage and 57% were under systemic therapy. dNLR was high in 25% at baseline vs 55% at Covid-19 diagnosis. The median dNLR increase between both timepoints was +70% (IQR: 0-349%);42% had +100% increase of dNLR. Pts distribution and mortality across FLARE groups is resumed in the Table. Overall mortality rate was 26%. In multivariate analysis, including gender, stage and PS, the FLARE poor group was independently associated with 30-day mortality [OR 5.27;1.37-20.3]. 44 pts were enrolled in the PC. Median circulating neutrophils were higher in pts with cancer (n=10, 56.7% [IQR: 39-78.4%]) vs HV (n=6, 35.8% [IQR: 25.6-21%]), and particularly higher in pts with cancer and severe Covid-19 infection (n=7, 88.6% [IQR: 80.9-94%] (p=0.003). A more comprehensive characterization of the IF of circulating neutrophils, including Lox1/CD62/CD64, will be presented at ASCO. Conclusions: The FLARE score, combining tumor and Covid-19-induced proinflam-status, can identify the population at higher risk for mortality. A better characterization of circulating neutrophils may help improve the prediction of Covid-19 outcomes in pts with cancer. (Table Presented).

The Low Expression of Fc-Gamma Receptor III (CD16) and High Expression of Fc-Gamma Receptor I (CD64) on Neutrophil Granulocytes Mark Severe COVID-19 Pneumonia.

Hyperinflammation through neutrophil granulocytes contributes to disease severity in COVID-19 pneumonia and promotes acute lung failure. Understanding the mechanisms of the dysregulations within the myeloid cell compartment may help to improve therapies for severe COVID-19 infection. Here, we investigated the immunopathological characteristics of circulating neutrophil granulocytes and monocytes in 16 patients with COVID-19 pneumonia by multiparameter flow cytometry in comparison to 9 patients with pulmonary infiltrates but without COVID-19. We correlated the immunophenotypes with the scores of the severity-of-disease classification system, APACHE-II. We found that the mean fluorescence intensity (MFI) of CD15, which is important for the transendothelial migration, was significantly reduced in the patients with COVID-19 (difference ± SD; 295.70 ± 117.50 MFI; p = 0.02). In addition, the granularity was significantly lower in the neutrophil granulocytes of patients with COVID-19 (difference ± SD; 1.11 ± 0.43 side-scatter ratio; p = 0.02). Moreover, the Fc-gamma receptor III (CD16) and Fc-gamma receptor I (CD64) on the neutrophil granulocytes were expressed discordantly with COVID-19 severity. CD16 correlated as inversely proportional (ρ = (-)0.72; 95% CI (-)0.92-(-)0.23; p = 0.01) and CD64 as proportional (ρ = 0.76; 95% CI 0.31-0.93; p = 0.01) with the APACHE-II scores of the patients. We conclude that the deviant expression of the Fc-gamma receptors might play role in a dysregulated antibody-mediated phagocytosis in severe cases of COVID-19 pneumonia.

A rapid, easy, and scalable whole blood monocyte CD169 assay for outpatient screening during SARS-CoV-2 outbreak, and potentially other emerging disease outbreaks.

Objective: The COVID-19 corona virus disease outbreak is globally challenging health systems and societies. Its diagnosis relies on molecular methods, with drawbacks revealed by mass screening. Upregulation of neutrophil CD64 or monocyte CD169 has been abundantly reported as markers of bacterial or acute viral infection, respectively. We evaluated the sensitivity of an easy, one-step whole blood flow cytometry assay to measure these markers within 10 min, as a potential screening test for COVID-19 patients. Methods: Patients (n = 177) with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were tested on 10 µL blood and results were compared with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Results: We observed 98% and 100% sensitivity in early-stage (n = 52) and asymptomatic patients (n = 9), respectively. Late-stage patients, who presented for a second control RT-qPCR, were negative for both assays in most cases. Conversely, neutrophil CD64 expression was unchanged in 75% of cases, without significant differences between groups. Conclusion: Monocyte CD169 evaluation was highly sensitive for detecting SARS-CoV-2 infection in first-presentation patients; and it returns to basal level upon infection clearance. The potential ease of fingerprick collection, minimal time-to-result, and low cost rank this biomarker measurement as a potential viral disease screening tool, including COVID-19. When the virus prevalence in the tested population is usually low (1%-10%), such an approach could increase the testing capacity 10 to 100-fold, with the same limited molecular testing resources, which could focus on confirmation purposes only.

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

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

RELATO DE CASO: PACIENTE COM LEUCEMIA MIELÓIDE AGUDA EXTRA MEDULAR

Introdução: Leucemia cutânea é caracterizada pela infiltração por leucócitos neoplásicos na pele. As formas mais comuns de leucemia que acometem a pele são Leucemia linfocítica crônica (LLC) e Leucemia mielóide aguda (LMA), dentre essas com morfologia monocítica ou mielomonocítica. Geralmente, o acometimento cutâneo é indicativo de doença avançada. Relato: Paciente masculino, 62 anos, sem comorbidades ou uso de medicações. Encaminhado da UPA para emergência do HCPA referindo astenia progressiva, perda ponderal de 12 kg há 2 meses com surgimento de “equimoses” em membros inferiores há 3 semanas, febre e secreção nasal recorrente. Ao exame: mau estado geral, desidratado, extremidades frias. Sinais vitais com taquicardia leve (FC 110) demais estáveis. Exames externos: Hb 5,8 g/dL, VCM 102, RDW 18,8%, leucócitos totais 11080/uL com diferencial de neutrófilos 665 /uL, monócitos 6759/uL, plaquetas 35.000/uL. Recebeu suporte transfusional após exames admissionais: Hb 3,7, plaquetas 26.000, demais sem alterações significativas. Exames de investigação: tomografia compatível com rinossinusite fúngica invasiva. Iniciado voriconazol empírico e realizada turbinectomia, etmoidectomia anterior, uncinectomia e antrostomia maxilar. Realizou medulograma com celularidade diminuída nas três séries com 4% de blastos e 60,5% de monócitos atípicos, sugestivo de Leucemia Mielomonocítica Crônica e imunofenotipagem compatível com LMA de comprometimento da linhagem monocítica (43,7% de células monocíticas com expressão de HLADR, CD64 forte, CD33 forte e coexpressão de CD56). Lesões de pele avaliadas pela dermatologia: máculas e manchas purpúricas e confluentes em coxas e pernas, com descamação. Lesões semelhantes de aspecto residual em abdome e membros superiores. Pápulas com brilho perolado em face, com telangiectasias a dermatoscopia. Biópsia de pele com punch de 4 mm teve o resultado do anatomopatológico de infiltrado mononuclear intersticial discreto com células maduras incluindo plasmócitos e células de tamanho médio e padrão imaturo e a imunohistoquímica demonstrou infiltração dérmica por células imaturas de perfil mielomonocítico, compatível com sarcoma mielóide. Devido a infecção fúngica invasiva foi optado por não iniciar quimioterapia até sua resolução. Paciente apresentou piora do padrão ventilatório no dia 05/07 tendo PCR para SARS-CoV-2 positivo, sendo transferido a UTI e evoluindo a óbito por complicações do quadro pulmonar. Discussão: A leucemia de apresentação cutânea é rara e na LMA está presente em 13% dos casos. As formas de apresentação mais comuns são pápulas e nódulos (60%) e em casos mais raros ocorre eritema, eritrodermia ou úlceras. A lesão do paciente era atípica e inicialmente foi avaliada como equimose, o que corrobora a importância de diagnósticos diferenciais de lesões cutâneas. O tratamento se dá através de protocolos guiados para a leucemia de base com quimioterápico sistêmico agressivo, que no caso do nosso paciente seria o protocolo 7+3. Em alguns casos, pode-se realizar radioterapia, porém ela é mais usada no tratamento de casos refratários ou paliativos. Leucemia cutânea geralmente tem o prognóstico desfavorável e o follow-up de dois anos dos pacientes com LMA demonstrou 6% de taxa de sobrevivência para casos com acometimento cutâneo versus 30% sem tal apresentação.

PIMS-TS immunophenotype: description and comparison with healthy children, Kawasaki disease and severe viral and bacterial infections.

BACKGROUND: A new clinical syndrome named Paediatric Inflammatory Multisystem Syndrome Temporally Associated with SARS-CoV-2 (PIMS-TS) has been described. This new disease is a leading cause of hospital and paediatric intensive care unit (PICU). It has been related to immunity dysregulation. METHODS: Prospective-retrospective observational study to describe the innate cell signature and immunophenotype of children admitted to PICU because of PIMS-TS (from March 2020 to September 2020). The immunophenotype was done through the expression analysis of these proteins of mononuclear cells: CD64, CD18, CD11a and CD11b. They were compared with previous healthy controls and children admitted to PICU because of bacterial infection, viral infection and Kawasaki disease (KD). Two hundred and forty-seven children were studied: 183 healthy controls, 25 viral infections, 20 bacterial infections, 6 KD and 13 PIMS-TS. RESULTS: PIMT-TS showed the lowest percentage of lymphocytes and monocytes with higher relative numbers of CD4+ (p = .000). Monocytes and neutrophils in PIMS-TS showed higher levels of CD64 expression (p = .000). Also, CD11a and CD11b were highly expressed (p =,000). CONCLUSION: We observed a differential cell innate signature in PIMS-TS. These findings are consistent with a proinflammatory status (CD64 elevated expression) and lymphocyte trafficking to tissues (CD11a and CD11b). More studies should be carried out to confirm our results.

Neutrophil Activation/Maturation Markers in Chronic Heart Failure with Reduced Ejection Fraction.

BACKGROUND: Neutrophils are critically involved in the immune response. Inflammatory stimuli alter the expression status of their surface molecule toolset, while inflammation-stimulated granulopoiesis might also influence their maturation status. Data on neutrophil status in heart failure with reduced ejection fraction (HFrEF) are scarce. The present study aims to evaluate the role of neutrophil CD11b, CD66b and CD64 expression in HFrEF. METHODS: A total of 135 HFrEF patients and 43 controls were recruited. Mean fluorescence intensity of the activation/maturation markers CD11b, CD66b and CD64 was measured on neutrophils by flow cytometry. CD10 (neprilysin) expression was simultaneously determined. RESULTS: Neutrophil CD64 expression was higher in HFrEF compared with controls, while CD11b/CD66b levels were similar. Neutrophil CD11b and CD66b showed a significant direct correlation to neutrophil CD10 expression (rs = 0.573, p < 0.001 and rs = 0.184, p = 0.033). Neutrophil CD11b and CD66b correlated inversely with heart failure severity reflected by NT-proBNP and NYHA class (NT-proBNP: rs = -0.243, p = 0.005 and rs = -0.250, p = 0.004; NYHA class: p = 0.032 and p = 0.055), whereas no association for CD64 could be found. Outcome analysis did not reveal a significant association between the expression of CD11b, CD66b and CD64 and all-cause mortality (p = ns). CONCLUSIONS: The results underline the potential role of neutrophils in HFrEF disease pathophysiology and risk stratification and should stimulate further research, characterizing subpopulations of neutrophils and searching for key molecules involved in the downward spiral of inflammation and heart failure.

High expression of neutrophil and monocyte CD64 with simultaneous lack of upregulation of adhesion receptors CD11b, CD162, CD15, CD65 on neutrophils in severe COVID-19.

BACKGROUND AND AIMS: The pronounced neutrophilia observed in patients with coronavirus disease 2019 (COVID-19) infections suggests a role for these leukocytes in the pathology of the disease. Monocyte and neutrophil expression of CD64 and CD11b have been reported as early biomarkers to detect infections. The aim of this study was to study the expression of receptors for IgG (CD64) and adhesion molecules (CD11b, CD15s, CD65, CD162, CD66b) on neutrophils and monocytes in patients with severe COVID-19 after admission to an intensive care unit (ICU). METHODS: The expression of receptors was analyzed using flow cytometry. EDTA blood from 23 patients with confirmed COVID-19 infection was sampled within 48 h of admission to the ICU. Leukocytes were labeled with antibodies to CD11b, CD15s, CD65s, CD162, CD64, and CD66b. Expression of receptors was reported as mean fluorescence intensity (MFI) or the percentage of cells expressing receptors. RESULTS: Results are presented as comparison of COVID-19 patients with the healthy group and the receptor expression as MFI. Neutrophil receptors CD64 (2.5 versus 0.5) and CD66b (44.5 versus 34) were increased and CD15 decreased (21.6 versus 28.3) when CD65 (6.6 versus 4.4), CD162 (21.3 versus 21.1) and CD11b (10.5 versus 12) were in the same range. Monocytes receptors CD64 (30.5 versus 16.6), CD11b (18.7 versus 9.8), and CD162 (38.6 versus 36.5) were increased and CD15 decreased (10.3 versus 17.9); CD65 were in the same range (2.3 versus 1.96). CONCLUSION: Monocytes and neutrophils are activated during severe COVID-19 infection as shown by strong upregulation of CD64. High monocyte and neutrophil CD64 can be an indicator of a severe form of COVID19. The adhesion molecules (CD11b, CD162, CD65, and CD15) are not upregulated on otherwise activated neutrophils, which might lead to relative impairment of tissue migration. Low adhesion profile of neutrophils suggests immune dysfunction of neutrophils. Monocytes maintain upregulation of some adhesion molecules (CD11b, CD162) suggesting the persistence of an increased ability to migrate into tissues, even during a severe stage of COVID-19. Future research should focus on CD64 and CD11b kinetics in the context of prognosis.

Validation of a Quick Flow Cytometry-Based Assay for Acute Infection Based on CD64 and CD169 Expression. New Tools for Early Diagnosis in COVID-19 Pandemic.

Objectives: Several parameters aid in deciphering between viral and bacterial infections; however, new tools should be investigated in order to reduce the time to results and proceed with an early target-therapy. Validation of a biomarker study, including CD64 and CD169 expression, was conducted. Material and Methods: Patients with active SARS-CoV-2 infection (ACov-2), bacterial infection (ABI), healthy controls, and antiretroviral-controlled chronic HIV infection were assessed. Whole blood was stained and, after lysing no-wash protocol, acquired by flow cytometry. The median fluorescence intensity (MFI) of CD64 and CD169 was measured in granulocytes, monocytes, and lymphocytes. The CD64 MFI ratio granulocytes to lymphocytes (CD64N) and CD169 MFI ratio monocytes to lymphocytes (CD169Mo) were evaluated as biomarkers of acute bacterial and viral infection, respectively. Results: A CD64N ratio higher than 3.3 identified patients with ABI with 83.3 and 85.9% sensitivity and specificity, with an area under the curve (AUC) of 83.5%. In contrast, other analytic or hematological parameters used in the clinic had lower AUC compared with the CD64N ratio. Moreover, a CD169Mo ratio higher than 3.3 was able to identify ACov-2 with 91.7 and 89.8 sensitivity and specificity, with the highest AUC (92.0%). Conclusion: This work confirms the previous data of CD64N and CD169Mo ratios in an independent cohort, including controlled chronic viral HIV infection patients as biomarkers of acute bacterial and viral infections, respectively. Such an approach would benefit from quick pathogen identification for a direct-therapy with a clear application in different Health Care Units, especially during this COVID pandemic.

Inflammatory Type 2 cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity to Respiratory Virus Infection.

The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs.