ABSTRACT
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.
ABSTRACT
Background: SARS CoV 2 infection alters the immunological profiles of natural killer (NK) cells. However, whether NK anti-viral functions (direct cytotoxicity and/or antibody-dependent cell cytotoxicity (ADCC)) are impaired during severe COVID-19 and what host factors modulate these functions remain unclear. Method(s): Using functional assays, we examined the ability of NK cells from SARS-CoV-2 negative controls (n=12), mild COVID-19 patients (n=26), and hospitalized COVID-19 patients (n=41) to elicit direct cytotoxicity and ADCC [NK degranulation by flow] against cells expressing SARS-CoV-2 antigens. SARS-CoV- 2 N antigen plasma load was measured using an ultra-sensitive Simoa assay. We also phenotypically characterized the baseline expression of NK activating (CD16 and NKG2C), maturation (CD57), and inhibitory (NKG2A and the glyco-immune negative checkpoint Siglec-9) by flow cytometry. Finally, an anti-Siglec-9 blocking antibody was used to examine the impact of Siglec-9 expression on anti-SARS-CoV-2-specific ADCC [degranulation and target cell lysis]. Result(s): NK cells from hospitalized COVID-19 patients degranulate less against SARS-CoV-2-antigen-expressing cells (in direct cytolytic and ADCC assays) than did cells from mild COVID-19 patients or negative controls (Fig. 1A). The lower NK degranulation was associated with higher plasma levels of SARS-CoV-2 N-antigen (P<=0.02). Phenotypic and functional analyses showed that NK cells expressing Siglec-9 elicited higher ADCC than Siglec-9- NK cells (P<0.05;Fig. 1B). Consistently, Siglec-9+ NK cells expressed an activated and mature phenotype with higher expression of CD16, CD57, and NKG2C, and lower expression of NKG2A, than Siglec-9- NK cells (P<=0.03). These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains NK cytotoxicity during cancer and other infections. Indeed, blocking Siglec-9 significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells (P<=0.05;Fig. 1C). Conclusion(s): These data support a model (Fig. 1D) in which the Siglec-9+ CD56dim NK subpopulation is cytotoxic even while being restrained by the inhibitory effects of Siglec-9. However, alleviating the Siglec-9-mediated restriction on NK cytotoxicity can further improve NK immune surveillance and presents an opportunity to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells. (Figure Presented).
ABSTRACT
The relationship between the incidence of COVID-19 in pregnant women who have had a coronavirus infection at different gestational ages and the health status of paired neonates is of great interest. However, no sufficient convincing data fully reflecting features of subsequent neonatal period, the state of the immune system in this category of children, affecting characteristics of postnatal period have been accumulated. Based on this, it underlies the relevance of the current study aimed at investigating parameters of clinical and immunological state of neonatal health after paired mothers recovered from COVID-19 at different gestational ages. The prospective study included 131 women and 132 children. The main group consisted of women (n = 61) who had COVID-19 during pregnancy and paired newborns (n = 62) at gestational age (GA) of 37-41 weeks, the comparison group - women without laboratory-confirmed COVID-19 during pregnancy (n = 70) and paired newborns (n = 70) of similar gestational age. While analyzing the anamnesis of the patients, no significant differences in somatic and obstetric-gynecological diseases were found. Analyzing course of pregnancy revealed that low molecular weight heparins were significantly more often applied in the main group. The term and frequency of delivery by caesarean section in pregnant women in the main group did not significant differ from that of the control group. No significant difference in the frequency of causes accounting for the severity of the condition of neonates in paired mothers with COVID-19 at different trimester of gestation was found. Investigating lymphocyte subset composition, neutrophil phagocytic activity, and IgG class antibodies specific to SARS-CoV-2 was carried out. It was found that lymphocyte subset profile in newborns from paired mothers with COVID-19 at different trimesters of gestation differed only in the level of NK cells (CD56+) in children born to mothers recovered from COVID-19 in the first trimester. In this study, in general, no severe perinatal outcomes in newborns from paired mothers with COVID-19 during pregnancy were documented. No cases of moderate or severe maternal COVID-19 were observed. Therefore, further prospective studies are needed to assess an impact of COVID-19 severity on maternal and fetal birth outcomes and clarify optimal management of pregnant women in such cases.Copyright © 2023 Saint Petersburg Pasteur Institute. All rights reserved.
ABSTRACT
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.
ABSTRACT
Background: To search for molecular biomarkers of pulmonary pathologies using non-invasive samples, such as urine, is of high clinical relevance. However, there are almost no proteomic studies using urine applied to respiratory diseases. Aim(s): To develop a biomarker discovery strategy using non-targeted proteomics in urine with applicability to different pulmonary diseases. Method(s): Urine samples were centrifuged and DTT treated to decrease uromodulin (THP). Low-THP samples were concentrated (ultrafiltration), ultracentrifugated, and exosome free urine was analysed using LC-MS/MS. GO terms/Pathway analyses were performed using STRING database. Result(s): Urine proteome (765 proteins) was enriched (FDR < 0.05) in proteins from different tissues, including respiratory system (N = 124), lung (N = 107), and immune system (N = 88). We detected an enrichment of relevant pathways for respiratory diseases, including several innate (e.g., TLR and NFkB pathways, complement system), and adaptive (e.g., interleukin signalling) immune system pathways. Some of these proteins have been previously studied in respiratory system disease (e.g., MPO, NAPSA, CHL1, FREM2, PLG), lower respiratory tract disease (e.g., NCAM1, MTOR, SERPINA1), viral infectious disease (e.g., ITIH4, CD209, CLEC4M, CD55), or specific pathologies such as coronavirus infection (e.g., ACE2, TMPRSS2), bronchiectasis (e.g., SAA1, SAA2, ELANE) or asthma (e.g., IGFALS, IGFBP7, HSPG2, DPP4, CD44, IL6R, MASP1). Conclusion(s): We have developed a protocol for the detection of proteomic biomarkers in urine. This proteome is enriched in proteins from the immune and respiratory systems, with a potential clinical and translational relevance.
ABSTRACT
Currently, as the SARS-CoV-2 pandemic evolves, there has been increasingly more attention paid to building natural and vaccine-induced immunity against SARS-CoV-2 and related disease known as COVID-19. Widespread preventive vaccination plays an important role in effectively protecting people from viral infections and can reduce national economic costs. Purpose - to study peripheral blood cell subset composition and magnitude of humoral response in vaccinated Gam-COVID-Vac subjects. The prospective study included 352 patients, of which 194 (119 women and 75 men) underwent an immunogram study and assessed level of anti-SARS-CoV-2 antibodies. In patients, the study of the lymphocyte subset composition and estimation of anti-SARS-CoV-2 antibodies was carried out at two time points - prior to vaccination and 90 days after inoculated component 1 of the Gam-COVID-Vac vaccine. In general, vaccination was well tolerated by patients, with no serious adverse events after immunization. The reaction to the vaccine (fever, malaise, headache, local reactions) was short-term (1-2 days) and more often noted after inoculated vaccine component 2. Comparatively analyzed immunogram parameters in females before and after vaccination revealed increased relative level of T-lymphocytes (CD3+), T-helper cell subset (CD3+CD4+), increased absolute and relative level of activated CD3+CD25+ T-lymphocytes, but decreased absolute and relative level of natural killer (CD3-CD56+CD16+) and natural killer T-cell (CD3+CD56+CD16+) cell subsets as well as decreased CD147 receptor expression on T-lymphocytes. Similar patterns were also found while examining the immunogram in males exepting increased level of lymphocytes and lowered CD147 expression on both T- and B-lymphocytes. No changes in the parameters of the immune T-cell arm was found. The high efficacy of the vaccine was confirmed by development of SARS-CoV-2-specific class G antiviral antibodies in 97.5% and 92.3% of vaccinated females and males, respectively. The data obtained evidence that: 1) vaccination induces a specific humoral immune response determined three months post-vaccination, and 2) it caused no serious disturbances in the immune system functioning, which could be reflected in the peripheral blood lymphocyte subset composition. Thus, the data presented allow to conclude that Gam-COVID-Vac is effective vaccine against SARS-CoV-2 infection.Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.
ABSTRACT
Currently, as the SARS-CoV-2 pandemic evolves, there has been increasingly more attention paid to building natural and vaccine-induced immunity against SARS-CoV-2 and related disease known as COVID-19. Widespread preventive vaccination plays an important role in effectively protecting people from viral infections and can reduce national economic costs. Purpose - to study peripheral blood cell subset composition and magnitude of humoral response in vaccinated Gam-COVID-Vac subjects. The prospective study included 352 patients, of which 194 (119 women and 75 men) underwent an immunogram study and assessed level of anti-SARS-CoV-2 antibodies. In patients, the study of the lymphocyte subset composition and estimation of anti-SARS-CoV-2 antibodies was carried out at two time points - prior to vaccination and 90 days after inoculated component 1 of the Gam-COVID-Vac vaccine. In general, vaccination was well tolerated by patients, with no serious adverse events after immunization. The reaction to the vaccine (fever, malaise, headache, local reactions) was short-term (1-2 days) and more often noted after inoculated vaccine component 2. Comparatively analyzed immunogram parameters in females before and after vaccination revealed increased relative level of T-lymphocytes (CD3+), T-helper cell subset (CD3+CD4+), increased absolute and relative level of activated CD3+CD25+ T-lymphocytes, but decreased absolute and relative level of natural killer (CD3-CD56+CD16+) and natural killer T-cell (CD3+CD56+CD16+) cell subsets as well as decreased CD147 receptor expression on T-lymphocytes. Similar patterns were also found while examining the immunogram in males exepting increased level of lymphocytes and lowered CD147 expression on both T- and B-lymphocytes. No changes in the parameters of the immune T-cell arm was found. The high efficacy of the vaccine was confirmed by development of SARS-CoV-2-specific class G antiviral antibodies in 97.5% and 92.3% of vaccinated females and males, respectively. The data obtained evidence that: 1) vaccination induces a specific humoral immune response determined three months post-vaccination, and 2) it caused no serious disturbances in the immune system functioning, which could be reflected in the peripheral blood lymphocyte subset composition. Thus, the data presented allow to conclude that Gam-COVID-Vac is effective vaccine against SARS-CoV-2 infection.
ABSTRACT
In the context of the COVID-19 pandemic, scientific interest is growing in studying the impact of the proposed vaccination on women's reproductive health. As is known, alterations in the state of the immune system and activation of an autoimmune response can lead to reproductive failure in women and potential complications of subsequent pregnancy. Objective(s): to evaluate the effect of the "Gam-COVID-Vac" on the immune status parameters, the relationship of their changes and the specific immune response to vaccination with the dynamics of the level of autoantibodies in women of reproductive age. The prospective study included 120 women who were vaccinated against COVID-19 with the "Gam-COVID-Vac". The criteria for inclusion in the study were: the age from 18 to 49 years, the absence of COVID-19 in the anamnesis, a negative result of a study on SARS-CoV-2 by PCR and negative results of tests for antibodies of classes G and M to SARS-CoV-2 before vaccination, the absence of pregnancy and serious somatic diseases. The patients were examined twice: immediately before vaccination and 90-100 days after the introduction of the 1st component of the vaccine. The level of IgG antibodies to SARS-CoV-2 after vaccination was assessed using ELISA. Before and after vaccination, the levels of antiphospholipid, anti-nuclear, organ-specific and antihormonal autoantibodies were determined, peripheral blood lymphocytes were immunophenotyped to determine the main subpopulations (CD3, CD4, CD8, CD19, CD5, CD16, CD56), as well as the expression of activation markers of lymphocytes (HLA-DR, CD25, CD147) using monoclonal antibodies. The effectiveness and safety of the combined vector vaccine against COVID-19 were high. Specific IgG antibodies to SARS-CoV-2 were produced in 98.3% of vaccinated women, no serious adverse reactions were observed. After vaccination, there was an increase in the level of some autoantibodies within the reference ranges, only IgM antibodies to phosphatidylethanolamine (PE) and IgG antibodies to DNA increased above the reference values. However, this increase was transient. After vaccination, the following changes in the parameters of the immunogram were observed: an increase in the content of cells with CD3+CD25+, CD19+ phenotype in peripheral blood and a decrease in the content of cells with CD56+CD16+ phenotype within the reference ranges, a decrease in CD147+/CD3+. Weak correlations were noted between these changes in immunogram parameters and the levels of some autoantibodies. The specific antiviral immune response to vaccination did not correlate with the autoimmune response. Vaccination with "Gam-COVID-Vac" is effective and safe and does not lead to disorders in the immune system. The observed increase in the level of autoantibodies to PE and DNA is transient. Changes in the parameters of the immune status within the reference ranges may be due to vaccination and the development of a specific antiviral immune response. Copyright © 2022, SPb RAACI.
ABSTRACT
In the context of the COVID-19 pandemic, scientific interest is growing in studying the impact of the proposed vaccination on women's reproductive health. As is known, alterations in the state of the immune system and activation of an autoimmune response can lead to reproductive failure in women and potential complications of subsequent pregnancy. Objective: to evaluate the effect of the "Gam-COVID-Vac” on the immune status parameters, the relationship of their changes and the specific immune response to vaccination with the dynamics of the level of autoantibodies in women of reproductive age. The prospective study included 120 women who were vaccinated against COVID-19 with the "Gam-COVID-Vac”. The criteria for inclusion in the study were: the age from 18 to 49 years, the absence of COVID-19 in the anamnesis, a negative result of a study on SARS-CoV-2 by PCR and negative results of tests for antibodies of classes G and M to SARS-CoV-2 before vaccination, the absence of pregnancy and serious somatic diseases. The patients were examined twice: immediately before vaccination and 90-100 days after the introduction of the 1st component of the vaccine. The level of IgG antibodies to SARS-CoV-2 after vaccination was assessed using ELISA. Before and after vaccination, the levels of antiphospholipid, anti-nuclear, organ-specific and antihormonal autoantibodies were determined, peripheral blood lymphocytes were immunophenotyped to determine the main subpopulations (CD3, CD4, CD8, CD19, CD5, CD16, CD56), as well as the expression of activation markers of lymphocytes (HLA-DR, CD25, CD147) using monoclonal antibodies. The effectiveness and safety of the combined vector vaccine against COVID-19 were high. Specific IgG antibodies to SARS-CoV-2 were produced in 98.3% of vaccinated women, no serious adverse reactions were observed. After vaccination, there was an increase in the level of some autoantibodies within the reference ranges, only IgM antibodies to phosphatidylethanolamine (PE) and IgG antibodies to DNA increased above the reference values. However, this increase was transient. After vaccination, the following changes in the parameters of the immunogram were observed: an increase in the content of cells with CD3+CD25+, CD19+ phenotype in peripheral blood and a decrease in the content of cells with CD56+CD16+ phenotype within the reference ranges, a decrease in CD147+/CD3+. Weak correlations were noted between these changes in immunogram parameters and the levels of some autoantibodies. The specific antiviral immune response to vaccination did not correlate with the autoimmune response. Vaccination with "Gam-COVID-Vac” is effective and safe and does not lead to disorders in the immune system. The observed increase in the level of autoantibodies to PE and DNA is transient. Changes in the parameters of the immune status within the reference ranges may be due to vaccination and the development of a specific antiviral immune response. © 2022, SPb RAACI.
ABSTRACT
Objetivos: Sabe-se que os tumores neuroectodermicos primitivos (PNETs) sao tumores raros envolvendo o sistema nervoso central, ossos ou tecidos moles, com pico de incidencia na adolescencia. O PNET e um tumor extremamente agressivo cuja sobrevida livre de doenca em 2 a 3 anos varia de 25 a 60%. Cerca de 30% dos pacientes apresentam metastase no momento do diagnostico. O prognostico varia de acordo com sitio acometido e a extensao da doenca ao diagnostico. O objetivo e relatar um caso raro de PNET, avaliado pela hematologia, com hipotese diagnostica inicial de leucemia aguda. Materiais e metodos: Coleta dos dados clinicos da paciente nas Unidades de Clinica Medica e Hematologia do HUCAM, bem como o levantamento em prontuario dos resultados de provas laboratoriais e exames especializados. Resultado: Paciente do sexo feminino, 19 anos, admitida no hospital com plaquetopenia grave. A paciente relatava mialgia, cefaleia e calafrios que iniciaram apos a terceira dose da vacinacao para Covid, evoluindo 2 dias apos com o surgimento de equimoses em membros e metrorragia. Ao exame fisico apresentava-se levemente palida, afebril, com sangramento cutaneo, sem linfonodomegalias perifericas ou visceromegalia. Os exames iniciais revelaram uma anemia normocitica (Hb-10.5g/dl), leucocitos-8200/mm3, plaquetopenia (12000/mm3), aumento de desidrogenase latica (DHL-4550), beta HCG negativo. A morfologia inicial do sangue periferico nao revelou alteracoes leucocitarias, porem o mielograma mostrou uma infiltracao intensa da medula ossea por celulas com caracteristicas imaturas, compativel com celulas blasticas. A imunofenotipagem estas celulas eram negativas para CD45, e para os marcadores de linhagens mieloide, linfoides B e T, e celulas dendriticas, e positivas para CD56. O diagnostico final foi de um tumor neuroectodermico primitivo com base na histopatologia da biopsia de medula. Discussao: Apresentamos o caso de uma paciente jovem com o diagnostico de PNET avancado, com manifestacoes hemorragicas cutaneo-mucosas de evolucao aguda, associado a plaquetopenia grave e infiltracao da medula ossea por celulas imaturas com caracteristicas blasticas, que faziam suspeitar fortemente de uma leucemia aguda, portanto, uma apresentacao atipica para um tumor solido raro e de comportamento agressivo, cujo diagnostico so foi possivel atraves de exames especializados. Conclusao: O diagnostico de certas neoplasias pode ser desafiador devido a sua rara incidencia e, por vezes, apresentacao clinica atipica, que pode simular outras doencas. O envolvimento primario do sangue periferico e da medula ossea suscita a avaliacao inicial do hematologista que, com base nos conhecimentos clinico e laboratorial, e capaz de estabelecer um raciocinio amplo e diferencial, cuja confirmacao requer o conhecimento de um especialista experiente para diagnostico assertivo e precoce. Copyright © 2022
ABSTRACT
Objectives: A 36-year-old Caucasian woman developed acute hepatitis and morbilliform eruption arising ten days after the first dose of the mRNA BNT162b2 SARS-CoV-2 vaccine. Materials and Methods: The patient was asymptomatic apart from the skin rash. Liver function tests showed predmoninantly severe transaminitis (AST 523 U/L, ALT 1550 U/L, GGT 151 U/L, ALP 128 U/L, bilirubin 12 umol/L). Only the ANA 1:160 was abnormal. Other serology for autoimmune and infectious diseases were negative. Multiphase computed tomography of the abdomen was unremarkable. The SARS-CoV-2 anti-spike IgG titre was 67.5 AU/mL (cut-off[15 AU/mL). The skin histology revealed spongiotic reaction pattern with focal interface lymphocytic inflammation. Multiple eosinophils and a few plasma cells were present. The epidermal lymphocytes were composed of CD2, CD3, C4, CD5, CD7 and CD8-positive T cells, with a CD4:CD8 ratio of 1:5. A small number stained positive with TIA1, PD1 and granzyme B. CD56 staining was negative. A liver biopsy was performed after 2 days of steroids. Liver histology showed mild steatosis and mild inflammatory portal infiltrate comprising mainly of small lymphocytes that were CD3 positive with retained staining for CD7 and CD8. Lobular architecture was preserved with inconspicuous interface hepatitis or piecemeal necrosis. Results: The patient was treated with intravenous hydrocortisone (400 mg/day) followed by prednisone (50 mg/day). There was rapid improvement in her liver function tests and cutaneous manifestations (Fig. 1). Conclusion: mRNA COVID-19 vaccine induced hepatitis is a rare phenomenon that is steroid-responsive and has associations with cutaneous eruptions. Our patient's lack of hepatic histological abnormalties is most likely due to early immunosuppression. She had epidermal lymphocytosis with predominance of CD8-positive T cells that were not of cytotoxic phenotype and we are uncertain as to their significance. There is limited guidance on the safety of SARS-CoV-2 vaccination in those who have had developed significant hepatic and cutaenous reactions. Further work is needed.
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Background: Although respiratory failure is the hallmark of severe disease, it is increasingly clear that Coronavirus Disease 2019 (COVID-19) is a multi-system disorder. The presence of gastrointestinal (GI) involvement by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been suggested by epidemiological, clinical, non-human primate, in-vitro (enteroid) and ex-vivo (human biopsy) studies. Having recently documented persistence of SARS-CoV-2 within the intestinal epithelium 7 months after infection, here we aimed to study mucosal immune cell abnormalities in individuals with prior history of COVID-19. Methods: Individuals with previous COVID-19 diagnosis (by either RT–PCR or seroconversion) and controls (without RT-PCR or serological evidence of prior COVID-19 infection) undergoing endoscopic evaluation were recruited into the study (Table 1). Colonic and small intestinal (duodenal and ileal) biopsies were analyzed by multiparameter flow cytometry for mucosal immune cell populations including myeloid cells (classical and non-classical monocytes, dendritic cell subsets), T cells (subsets and activation state), B cells (including plasma cells) and NK cells. Persistence of viral antigens was determined by immunofluorescence microscopy (n=30) using a previously published anti-nucleocapsid (NP) antibody. Results: Thirty subjects with a previous history of COVID-19 (post-COVID), median of 4 months from diagnosis (range 1-10 months), were recruited and compared with 40 normal volunteer (NV) controls. Relative to controls, post-COVID subjects displayed higher frequencies of classical (CD14+) monocytes in both, the colon and the small bowel, while significantly higher frequencies of conventional dendritic cells (cDC)1 (lin-HLA-DRhiCD14- CD11c+CD141+) and cDC2 (lin-HLA-DRhiCD14-CD11c+CD1c+) were noted in the colon. Among NK subsets, CD56bright CD16- NK cells were significantly higher in the colon of post-COVID subjects. Among T cell subsets, CD8+ tissue resident memory T cells (CD8+CD69+CD103+) were significantly increased in colon of post-COVID subjects compared to NV. Among B cell subsets, plasma cells (CD3-CD27+CD38hi) trended higher (p= 0.06), while mucosal B cells (CD3-CD19+) were significantly lower in the terminal ileum of post-COVID subjects compared to NV. Finally, with IF, we detected SARS-CoV-2 NP in 10 out of 30 (33%) of post-COVID subjects (Figure 1). Conclusion: Innate and adaptive immune cell abnormalities persist in the intestinal mucosa of post-COVID subjects for up to 10 months and may reflect viral persistence or immune cell dysregulation in the intestines. These findings have major implications for understanding the pathogenesis of long-term sequelae of COVID-19, including long-haul COVID.(Table Presented)(Figure Presented)
ABSTRACT
Problem: A37 y.o, P1+1 presented with a 1.5 year Hx of secondary infertility. Initial ovulation induction with clomifene citrate was unsuccessful, followed by a single IUI attempt with gonadotropin stimulation. The couple progressed to IVF treatment with 3 failed embryo transfers. There were no endocrinological abnormalities, endometrial/ uterine anatomy was normal on transvaginal USS and saline infusion sonograpy, so an Endometrial Immune Profile(EIP) and Receptivity Array(ERA) were performed prior to further treatment. ERA was in the receptive range, and EIP demonstrated an overactive profile with high IL15:Fn14, suggestive of NK overactivation. In the absence of other pathology this was hypothesised as a potential cause for implantation failure. Immunotherapy options were discussed, including IVIG and adalumimab. Risks of these during the Covid pandemic resulted in the decision to try oral hydroxychloroquine, with cost benefits and potentially less adverse side effects. Unfortunately there is a paucity of published data and outcomes, but proposed benefits of this treatment were based on demonstration of improvemed serum TH1:TH2 cytokine ratios (reduction in TNFa and increase in IL:10), and demonstration of a reduction in miscarriage rate. Method of Treatment: Hydroxycholoroquine 200mgPOBD was commenced 6 weeks prior to commencing treatment, followed by a frozen transfer of a single blastocyst (5AA). Unfortunately this transfer after 8/52 treatment was unsuccessful. A repeat cycle was scheduled after a further 8 weeks, continuing the hydroxychloroquine for >3 months. Initial hCG 13 days post transfer was 2934, but the patient presented with sudden PV bleeding after 6 days, follow up hCG was only 3650. Transvaginal USS demonstrated a collapsed intrauterine gestation sac in keeping with a non-viable pregnancy. Onward referral to an Early Pregnancy unit for follow up confirmed a miscarriage. PGS was not incorporated into the cycles to assess for embryo aneuploidy. Results: Overall the implantation rate had increased from 0 (0/3) to 50% (1/2), but due to sample size this was not statistically significant (p = 0.81). Pregnancy rate per embryo transfer also increased form 0/3 to 1/2, but again was not significant because of low numbers. Due to the failure to achieve an ongoing pregnancy, a repeat biopsy was performed while using hydroxychloroquine to assess its effects on the endometrial immunological environment. This showed a normalisation of the IL15:Fn14 ratio (5.680 to 0.831), but with a slight elevation in the IL18:Tweak ratio (0.088 to 0.114).CD56 remained in the normal range (0.993 to 1.344). Conclusion: Although prescribed for inconsistent indications, there is little published data on hydroxychloroquine use for adverse reproductive outcome. This case report demonstrates the effect of oral hydroxychloroquine therapy on an overactive endometrial profile, leading to a major reduction in IL15:Fn4 ratio, suggesting a potential role in reducing uNK cytotoxicity. Anecdotally a 3 month course is recommended prior to transfer, which would be supported by these events. Unfortunately there is limited ability to make treatment recommendations based on a single sample, however, the findings suggest that a larger study to explore if this pattern is reproducible would have important clinical value.
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Introduction: In January 2020, the genome of the novel coronavirus, SARS-CoV-2, was sequenced, the etiologic RNA virus for COVID- 19. Several variants from the initial strain of SARS-CoV-2 have been reported, notably the Delta variant and most recently, the Omicron variant. However, how the virus affects the cellular immune system has been elusive. This study aims to investigate peripheral blood immunophenotype, natural killer (NK) cell cytotoxicity, and T helper (Th) 1/ Th2 ratios in pregnantwomen undergoing immunotherapy with a history of recurrent pregnancy losses (RPL) and repeated implantation failures (RIF). Materials and Methods: A prospective cohort study was performed on pregnant women with a history of RIF and RPL. The study group comprised 20 pregnant women with COVID-19, of whom eight had documented vaccinations. All were undergoing personalized immunemodulation and/or anticoagulation treatment. When they were diagnosed with COVID-19, immunomodulating medications were tapered off or decreased until recovery. Peripheral blood immunophenotype, NK cell cytotoxicity (NKC) at effector to target cell (E: T) ratio at 50:1 and 25:1, and Th1/Th2 cell ratios (TNF-a/IL-10, IFN-g/IL-10 producing Th cell ratios)were measured by flow cytometry within 5weeks before and after COVID-19. Statistical analysis was performed by using the student t-test. Results: Peripheral blood immunophenotypes including % CD3 (77.38 ± 2.15 % vs. 79.98 ± 1.65 %, P = 0.34), % CD19 (13.63 ± 1.63 % vs. 12.63 ± 1.79 %, P = 0.68), % CD56 (7.61 ±1.32 % vs. 6.15± 1.08 %, P = 0.40), % CD19/CD5 (4.68 ±1.41 % vs. 4.17 ± 0.72 %, P = 0.75) were not significantly different before and after COVID- 19. NKC at E: T ratio of 50:1 (Mean ±SE), before and after COVID- 19 were 21.16 ± 0.66% and 22.21 ± 1.06 % respectively (P = 0.40). NKC at E: T cell ratios of 25:1 were 15.71 ± 0.69 % and 16.44 ± 0.93 % respectively (P = 0.52). TNF-a/IL-10 (29.61 ± 2.43 vs 28.95 ±2.06, P = 0.83) and IFN-g/IL-10 (16.36 ±2.22 vs 12.61 ± 0.94, P = 0.14) producing Th1/Th2 cell ratios were comparable before and after COVID-19. Conclusions: Our findings suggest that even though patients are affected by the COVID-19 during the Omicron phase, ©2022 The Authors. American Journal of Reproductive Immunology ©2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. there were no significant flares in cellular immune responses when patients recovered from COVID-19 in not hospitalized cases.
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Background: Neuroendocrine cancer of the prostate can present in diverse clinical pictures, potentially hampering the diagnosis and probably leading to underdiagnosis. Methods: Two cases are presented corresponding respectively to two forms of the disease: de novo neuroendocrine cancer and dedifferenciation of an adenocarcinoma of the prostate to neuroendocrine cancer under long term luteinising hormone releasing hormone (LHRH) agonist treatment. Results: Suspicion of neuroendocrine cancer may be raised in prostate cancer patients presenting either clinical or radiological metastatic progression without prostate specific antigen (PSA) rise, or relatively extended metastatic disease right at diagnosis associated to relatively low PSA, yet any non-pulmonary visceral metastases. Neuroendocrine cancer of the prostate can also turn out to be the origin of an adenocarcinoma of unknown primary. Conclusion: In case these considerations are respected the risk of missing the correct diagnosis of a neuroendocrine cancer of the prostate may be minimised.
ABSTRACT
Background: About 10% of individuals with mild infection with SARS-CoV-2 suffer from Long COVID-19, defined as signs and symptoms developed during or following COVID-19 that continue for more than twelve weeks and cannot be explained by an alternative diagnosis. In this study, we analyzed the ADCC response and the reactivation of CMV and EBV in Long COVID-19 syndrome, in comparison with patients who completely recovered from mild COVID-19 Methods: 30 patients with Long COVID-19 (Long COVID-19) and 20 individuals who suffered mild COVID-19 and were completely recovered (Recovered) were recruited for this study. Specific anti-SARS-CoV-2 IgG titers were analyzed by direct ELISA and their neutralizing capability was measured by using pseudovirus neutralization assay. Phenotype of CD4+ and CD8+ T cells, NK, NKT, and B cells in peripheral blood was analyzed by flow cytometry. ADCC activity was analyzed using rituximab-coated Raji cells as target. EBV and CMV reactivation in plasma was analyzed by qPCR. Results: 1) 86.6% and 55.50% participants were female in Long COVID-19 and Recovered cohorts, respectively. Median age at COVID-19 diagnosis was 42y(IQR 37-46) and 45y (IQR 28-57), respectively. 2) Similar levels of CD4+ T cells were observed in both groups. However, Tregs were increased 2.8-fold in Long COVID-19 participants (p=0.0007). 3) CD8+ T cells, CD8+TCRγδ and CD8+TCRγδ were increased 1.3-(p=0.0005), 2.0-(p=0.049), and 2.5-fold (p=0.005) in Long COVID-19 individuals. 4) Expression of CD56 in NK cells and CD3-CD56+CD16+ cells were increased 1.7-(p=0.0005) and 1.7-fold (p=0.032) in Long COVID-19, respectively. 5) Specific anti-SARS-CoV-2 IgG titers were increased 2.3-fold in Long COVID-19 individuals (p=0.02) and their neutralizing capacity was increased 4.2-fold (p=0.034) in this cohort. However, ADCC activity was decreased 1.4-fold (p=0.0044). 6) Resting memory B cells were increased 2.3-fold during Long COVID-19, whereas plasmablasts were reduced 3.1-fold. 7) EBV was reactivated in 33.3% of Long COVID-19 individuals (p<0.0001), whereas CMV was not reactivated in any individual. Conclusion: Despite high levels of neutralizing antibodies and cytotoxic immune populations, an impaired antibody-dependent cytotoxic activity was observed in PBMCs from individuals with Long COVID-19. This defective cytotoxic immune response may impede viral clearance, which may also contribute to EBV reactivation observed in these individuals, thereby influencing on the persistent COVID-19 symptoms.
ABSTRACT
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.
ABSTRACT
Backgroung: COVID-19 pandemic (SARS-CoV-2) has affected an increasing number of people worldwide, with death rates higher than previous viral epidemics. It is possible that NK cells, known to have great cytokine secreting potential are competent at the onset of the condition and that in some individuals, the viral load is able to exhaust them. Balance between tolerant (CD27- CD11b-), secretory (CD27+ CD11b-/ CD27+ CD11b+) and cytotoxic (CD27- CD11b+) NK cells involved in the inflammatory response and their anti-SARS-CoV-2 activity are still not well established. Strategies that can restore function of NK cells against the virus are worth investigating. Here, we aimed to characterize NK cells frequency, functional subtypes and maturation in early phase of COVID-19 patients, by Multiparametric Flow Cytometry (MFC). Methods: Peripheral blood from 15 COVID-19 patients in early stage of infection (day 1-14, confirmed by RT-PCR), categorized according comorbidities in: G1 (not oncologic;n = 6), G2 (oncologic;n = 3), G3 (hematologic neoplasms;n = 3) and G4 (without comorbidities;n = 3), and 10 healthy samples enrolled the study. Clinical and laboratorial data were collected from electronic medical records. Samples were stained with CD45, CD19, CD3, CD56, CD11b, CD27, acquired on a FACS Canto II (BD Biosciences) and data analyzed with FlowJo V10 software. Results: A lower frequency of lymphocytes was observed in the disease when compared to controls (P < 0.0001) and frequency of NK cells were similar in both groups (P = 0.6605). Although frequency of CD27- CD11b- NK cells was lower in the disease (P = 0.0109), there was a significantly higher frequency of CD27+ CD11b- NK cells in COVID-19 samples when compared to controls (P < 0.0001), featuring a mostly immature profile in the disease. On the other hand, no statistical significance was observed regarding the frequencies of CD27+ CD11b+ (P = 0.1370) and CD27- CD11b+ NK cells with a more mature profile (P = 0.3094). Amongst disease groups, no statistical significance was found regarding frequency of NK cells and G1 showed lower frequency of CD27- CD11b- NK cells (P = 0.0226), while G3 group had an increased frequency of CD27+ CD11b- NK cells (P = 0.0238) when compared to the other groups and controls. Finally, no statistical significance was found in the frequency of CD27+ CD11b+ (P = 0.6691) and CD27- CD11b+ (P = 0.6270) NK cells between disease groups and controls. Conclusion: Although the frequency of NK cells did not show a significant difference between COVID-19 patients and healthy controls, our findings showed a possible change in their maturation profile, which seems to be inversely proportional to normal, with the frequency of CD27+ CD11b- NK cells considerably higher in the disease. This phenotype is directly associated with secretory function of a more immature NK cell and is responsible for triggering inflammatory responses that could lead to severe respiratory failure, what seems to be consistent with COVID-19 profile. A high frequency of cytotoxic cells was observed, which seemed to be similar to what we found in normal heathy samples. Even though unregulated maturation might be associated to a dysfunctional mature NK cell, additional studies of cytotoxicity and activation of NK cells in COVID-19 are required to affirm whether there is functional exhaustion or hyperactivation of the cytotoxic subtypes of these cells.
ABSTRACT
As células Natural Killer (NK) têm um papel definido na imunidade inata e adaptativa. São os linfócitos que primeiro se reconstituem após o transplante de células hematopoéticas, desempenhando um papel na proteção contra a recaída precoce. Para pacientes pediátricos e adultos, o transplante de sangue do cordão umbilical (SCU) é uma opção terapêutica para uma variedade de doenças hematológicas, genéticas e metabólicas. Estudos clínicos em andamento avaliam o uso das células NK na imunoterapia contra o câncer e tratamento da Covid-19. A quantificação das células NK no sangue de cordão umbilical não é realizada de rotina como um fator de qualificação do produto. O conhecimento da curva de normalidade deste tipo celular no sangue de cordão permitirá o avanço em sua aplicação. Objetivo: O presente estudo teve como objetivo determinar o percentual de células NK presentes no SCUP para retratar a composição celular e avaliar a possível correlação entre algumas características do parto e tipos celulares presentes no sangue de cordão. Material e métodos: Estudo observacional descritivo retrospectivo, do banco de dados de centro de processamento celular de sangue de cordão umbilical e placentário privado, no período 15 meses. Foram analisadas 829 USCUP de doadoras saudáveis. O SCUP foi submetido a determinação do volume coletado, hemograma automatizado e processamento para a obtenção de células nucleadas totais. Após o processamento as células foram analisadas quanto a viabilidade celular (método de exclusão do azul de Trypan). O percentual de células mononucleares e CD34+ viáveis foram quantificadas por citometria de fluxo (ISHAGE) e pelo corante 7AAD. A quantificação das células NK foi feita por citometria de fluxo segundo o método de ISHAGE usando marcadores CD16 e CD56. Resultado: A análise das 829 USCUP mostrou que a média de volume colhido foi de 79 mL, a média de células nucleadas totais viáveis de 9,2 x 108 ±4,6;o percentual de células CD34+ viáveis de 0,28 ± 0,14;o percentual de células mononucleares 84 ± 7, enquanto a média percentual das células NK foi de 14 ± 8. O percentual de células NK apresentou uma fraca correlação com os demais parâmetros analisados: idade gestacional, tipo de parto, percentual de células mononucleares e CD34+ viáveis. Discussão: O sangue do cordão umbilical oferece vantagens únicas, muitas das quais são diretamente aplicáveis à alorreatividade dirigida pelas células NK. A imunoterapia baseada nas células NK derivadas de SCU tem se mostrado como um tratamento promissor, assim como o grande potencial para sua expansão in vitro. Alguns autores já demostraram que as células NK expandidas possuem alta atividade antitumoral tanto in vitro quanto in vivo. Além disso, SCUP contém populações de células que representam progenitores de células NK presentes em números muito pequenos no sangue periférico. O resultado da quantificação das células NK no SCUP confirma os achados anteriores. Sua concentração é uma variável independente e não correlacionada com a concentração das células CD 34+, tipo de parto ou idade gestacional. Um possível produto de terapia avançada produzido a partir de células NK derivado de SCUP, com potencial aplicação autóloga ou alogênica “pronto para uso”na imunoterapia celular anticâncer, é uma opção promissora. A quantificação destas células no SCU armazenado deve ser considerada como mais um dos controles de qualidade do material para um possível uso futuro em terapias avançadas.
ABSTRACT
Descrição do caso: Paciente masculino, 76 anos, sem comorbidades, transferido de unidade básica de saúde por choque hipovolêmico devido a lesão vascular após punção arterial. O mesmo tinha sido levado por tosse, febre e alteração do estado de consciência. Foi iniciado antibiótico, reposição volêmica, afastado Covid e angioTc de membros inferiores com extravasamento do contraste em artéria femoral direita e infiltração difusa na medula óssea. Exame físico: Taquicardia, SO2 95% com O2 2 litros/min, Glasgow 11 sem déficit focal, roncos pulmonares bilaterais e presença de hematoma em região inguinal à direita com extensão para área lombar, glútea e escrotal sem palpação de frêmito. Exames: Hb 6.2 g/dL, plaquetas 67.000/uL, leucócitos totais 17.810/uL, creatinina 4.42 mg/dL, cálcio 10.6 mg/dL, albumina 2.6 g/dL, TP 63%, TTPa 50.9s, D-dímeros 3.34 ug/mL, LDH 443 U/L, fibrinogênio 240 mg/dL. RNM do neuroeixo com infiltração difusa na medula óssea da coluna vertebral e fraturas em T3, L1 e L3. Eletroforese de proteínas com imunofixação sérica e urinária sem banda monoclonal, cadeias leves Lambda 2.66 mg/L, cadeias leves kappa 2.62 mg/L, relação Kappa/Lambda 0.98, IgG 269 mg/dL, IgM 13 mg/dL, IgA mg/dL e B2-microglobulina 6.441 ng/mL. Demais exames sem alterações significativas: perfil hepático, reumatológico, metabólico, punção lombar, tomografias crânio, tórax e abdominal. Mielograma com infiltração por plasmócitos de aspecto neoplásico (78%), imunofenotipagem 57.51% de células plasmáticas totais (57.5% compatíveis com plasmócitos neoplásicos). BMO hipercelular (80%), infiltração difusa e multifocal por plasmócitos. IHQ CD138+, CD38+, ciclina+, CD3+, CD20-, CD56-, Kappa-, Lambda-, vermelho congo negativa, sugerindo Mieloma múltiplo (MM) não secretor, não produtor - ISS III. Ante ECOG de 4 e idade foi optado por paliação. Na alta hospitalar o paciente estava com resolução da coagulopatia, melhora da anemia e normalização da função renal. Discussão: O MM é uma neoplasia de células plasmocitárias caracterizada pela produção de imunoglobulina monoclonal, representa 10 a 15% das malignidades hematológicas com predomínio no sexo masculino. Incidência aumenta com idade, sendo 90% em pacientes > 50 anos. Critérios diagnósticos são resumidos no acrônimo SLiM CRAB. A distribuição da proteína monoclonal sérica ou urinária identifica-se em 97%: 50% IgG, 20% IgA, 20% cadeias leves e < 10% entre IgD, IgE, IgM ou pico biclonal. O restante 3% corresponde à variante não secretora, e, se os plasmócitos clonais são incapazes de produzir imunoglobulina, configura-se além, como não produtor. O MM não secretor (MMNS) verdadeiro baseia-se na presença de 30% de células plasmocitárias monoclonais OU plasmocitoma confirmado por biópsia, tem sido descrita associação com t(11,14)(q13;q32). Dentro das manifestações clínicas estão a imunoparesia secundária, ausência de insuficiência renal e hipercalcemia. Na apresentação do paciente a avaliação foi prejudicada decorrente do choque hipovolêmico e insuficiência renal aguda pré-renal. O prognóstico do MMNS é melhor na vigência de tratamento que inclua imunomodulador e inibidor de proteassoma quando comparado ao do MM com proteína M detectável, embora o desfecho dos pacientes que foram a TCTH autólogo não teve diferença significativa. Conclusão: MMNS é um subtipo raro com pouca literatura disponível, sendo um desafio realizar o diagnóstico, avaliação da resposta e recaída ante a falta de quantificação do pico monoclonal.