BACKGROUND: SARS-CoV-2, which causes COVID-19, has killed more than 7 million people worldwide. Understanding the development of postinfectious and postvaccination immune responses is necessary for effective treatment and the introduction of appropriate antipandemic measures. OBJECTIVES: We analysed humoral and cell-mediated anti-SARS-CoV-2 immune responses to spike (S), nucleocapsid (N), membrane (M), and open reading frame (O) proteins in individuals collected up to 1.5 years after COVID-19 onset and evaluated immune memory. METHODS: Peripheral blood mononuclear cells and serum were collected from patients after COVID-19. Sampling was performed in two rounds: 3-6 months after infection and after another year. Most of the patients were vaccinated between samplings. SARS-CoV-2-seronegative donors served as controls. ELISpot assays were used to detect SARS-CoV-2-specific T and B cells using peptide pools (S, NMO) or recombinant proteins (rS, rN), respectively. A CEF peptide pool consisting of selected viral epitopes was applied to assess the antiviral T-cell response. SARS-CoV-2-specific antibodies were detected via ELISA and a surrogate virus neutralisation assay. RESULTS: We confirmed that SARS-CoV-2 infection induces the establishment of long-term memory IgG+ B cells and memory T cells. We also found that vaccination enhanced the levels of anti-S memory B and T cells. Multivariate comparison also revealed the benefit of repeated vaccination. Interestingly, the T-cell response to CEF was lower in patients than in controls. CONCLUSION: This study supports the importance of repeated vaccination for enhancing immunity and suggests a possible long-term perturbation of the overall antiviral immune response caused by SARS-CoV-2 infection.

• MeSH
• B-Lymphocytes immunology   MeSH
• Immunity, Cellular immunology   MeSH
• COVID-19 * immunology   MeSH
• Adult MeSH
• Enzyme-Linked Immunospot Assay MeSH
• Spike Glycoprotein, Coronavirus immunology   MeSH
• Immunity, Humoral MeSH
• Immunologic Memory MeSH
• Leukocytes, Mononuclear immunology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Antibodies, Viral * blood   immunology   MeSH
• SARS-CoV-2 * immunology   MeSH
• Aged MeSH
• T-Lymphocytes immunology   MeSH
• COVID-19 Vaccines immunology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

The ELISpot assay is a sensitive technique applied to assess cytokine-producing memory/effector T cells and human leukocyte antigens (HLA)-specific IgG-producing B cells. Besides the fact that the method is laborious and is difficult to standardise between laboratories, it may provide valuable information on the immune response of recipients before and after organ transplantation. In this article, we briefly review the recent literature and discuss the clinical significance of the ELISpot assay in predicting the risk and incidence of allograft rejection and survival.

• MeSH
• Enzyme-Linked Immunospot Assay MeSH
• Interferon-gamma MeSH
• Humans MeSH
• Graft Rejection * MeSH
• T-Lymphocytes MeSH
• Kidney Transplantation * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Laboratorní diagnostika SARS-CoV-2 je v současné době založena na přímém průkazu viru pomocí metody PCR používané k potvrzení, zda je jedinec infikován SARS-CoV-2, a průkazem přítomnosti protilátek anti SARS-CoV-2, které svědčí o proběhlé infekci SARS-CoV-2. Zejména v poslední době se hromadí důkazy, které jednoznačně podporují roli T buněk v imunitní odpovědi. Zatímco jak PCR, tak průkaz protilátek mohou detekovat aktuální a postinfekční stavy, sledování T buněčné odpovědi by mohlo být důležitou součástí testování SARS-CoV-2 při terapeutickém monitorování a identifikaci imunního stavu vyšetřovaného jedince. V našem případě byla k testování T buněčné imunity použita testovací souprava T-SPOT Discovery SARS-CoV-2, která je zjednodušenou variantou metody ELISPOT. Porovnány byly výsledky pacientů z hlediska klinického průběhu onemocnění, pozitivity PCR testu na SARS-CoV-2 a přítomnosti protilátek proti SARS-CoV-2. Na základě námi dosažených dat by bylo možno říci, že tato metoda představuje účinný a technicky dostupný nástroj k monitorování T buněčné imunity.

Laboratory diagnosis of SARS-CoV-2 is currently based on direct detection of the virus using the PCR method used to confirm whether an individual is infected with SARS-CoV-2 and the presence of anti-SAR-CoV-2 antibodies, which indicate SARS–CoV-2 infection. Especially recently, evidence has been accumulating that clearly supports the role of T cells in the immune response. While both PCR and antibody detection can detect current and post-infectious conditions, monitoring the T cell response could be an important part of SARS-CoV-2 testing in therapeutic monitoring and identification of the subject’s immune status. In our case, the T-SPOT Discovery SARS-CoV-2 test kit, which is a simplified variant of the ELISPOT method, was used to test T cell immunity. The results of patients were compared both in terms of the clinical course of the disease, the positivity of the PCR test for SARS-CoV-2 and the presence of antibodies against SARS-CoV-2. Based on our data, we can say that this method is an effective and technically available tool for monitoring T cell immunity.

• MeSH
• Lymphocyte Activation MeSH
• Immunity, Cellular MeSH
• COVID-19 diagnosis   immunology   MeSH
• Enzyme-Linked Immunospot Assay methods   instrumentation   MeSH
• Humans MeSH
• T-Lymphocytes immunology   MeSH
• COVID-19 Testing * methods   instrumentation   MeSH
• Check Tag
• Humans MeSH

Chronic kidney disease stage 5 (CKD5) dialysis patients who stay long term in uremic environment often exhibit several, poorly defined, immune impairments. In this study, we assessed peripheral virus-specific effector/memory cells and subpopulations of T, B and DC cells using ELISPOT and FACS methods in 74 low-risk kidney transplant candidates without anti-HLA antibodies, prior to transplantation in pre-emptive (never experienced dialysis) and dialysis cohorts. There was difference in circulating marginal zone B cells (MZB) (IgDhighCD27high) between dialysis patients and those receiving kidney grafts pre-emptively (P = .002). Patients treated on dialysis >12 months had also 4.2-fold greater risk of increased absolute numbers of MZB (95%CI:1.6-11.2; P = .004). There were no other differences in B-, T- and DC-cell subsets. Numbers of effector/memory T cells reactive to major opportunistic virus-specific antigens (CMV, BKV and EBV) were not affected by dialysis. Non-sensitised dialysis-treated patients displayed significantly more circulating MZB compared to those CKD5 patients that had never undergone dialysis therapy.

• MeSH
• Tumor Necrosis Factor Receptor Superfamily, Member 7 metabolism   MeSH
• B-Lymphocytes immunology   MeSH
• Renal Insufficiency, Chronic immunology   therapy   MeSH
• Dendritic Cells immunology   MeSH
• Dialysis MeSH
• Adult MeSH
• Enzyme-Linked Immunospot Assay MeSH
• Immunologic Memory MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Aged MeSH
• Spleen pathology   MeSH
• T-Lymphocytes immunology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Transplantace ledviny od žijících dárců představují nejlepší metodu léčby nezvratného selhání ledvin. Výskyt akutních rejekcí je v tomto programu nyní vyšší než u kadaverosních transplantací, především v důsledku rozšíření geneticky nepříbuzných transplantací. Riziko vzniku rejekce se pomocí běžně používaných metod (stanovení panel reaktivních protilátek a crossmatch) dá odhadnout jen velmi těžko. Cílem této studie je posoudit aloreaktivitu příjemce pomocí panelu laboratorních testů a odhadnout riziko vzniku T buňkami a protilátkami zprostředkované rejekce. Předmětem výzkumu je detekce předtransplantačních a potransplantačních anti HLA protilátek spolu s určením dárcovsky specifické T a B lymfocytární aloreaktivity (ELISPOT), vyšetření periferních populací T a B lymfocytů a jejich specifických periferních transkriptů. Získaná data budou analyzována ve vztahu k potransplantačnímu průběhu a fenotypu rejekce.; Living donor kidney transplantation represents the best therapy of end stage renal disease. Recently, the acute rejection incidence has been observed to be higher than in deceased donor transplantation, mainly because of increasing number of genetically unrelated procedures. Prediction of acute rejection with recently available techniques often fails. The aim of this project is to analyze recipient alloreactivty using multilevel laboratory platforms to better predict risks of acute rejection, both T cell- and antibody mediated. In this project pre and posttransplant analyzes will be performed to detect anti HLA antibodies, T and B cell donor specific alloreactivity, specific populations of T and B cells along with their peripheral transcripts. Data will be analyzed in relation to postransplant outcome and distinct rejection phenotype.

• MeSH
• B-Lymphocytes MeSH
• Enzyme-Linked Immunospot Assay MeSH
• Monitoring, Immunologic MeSH
• Antibodies adverse effects   MeSH
• Graft Rejection diagnosis   MeSH
• T-Lymphocytes MeSH
• Kidney Transplantation adverse effects   MeSH
• Living Donors MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• transplantologie
• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

The current diagnostic algorithm for beta-lactam allergy is based on skin and provocation tests, both of which carry a certain risk of inducing hypersensitivity reactions. Thus, non-invasive in vitro tests reliable enough to replace skin and provocation tests at least in a portion of patients are desirable. We aimed to verify the utility of IFN-γ ELISPOT as a first-line test in patients with suspected non-immediate hypersensitivity reaction to amoxicillin (AMX) and penicillin (PNC). The prospective observational study included 24 patients with recent, suspected non-immediate hypersensitivity reaction to AMX or PNC and 6 recently-exposed healthy subjects. In vitro tests were performed in all patients and healthy subjects: a) IFN-γ ELISPOT with PNC, AMX and amoxicillin plus clavulanic acid (AMX-CL); b) penicillin specific IgE; c) basophil activation test (BAT). Skin and provocation tests followed only in certain patients. IFN-γ ELISPOT results with PNC and AMX stimulation did not differ from the unstimulated condition. The highest IFN-γ responses to AMX-CL were close to previously published criteria in three patients; one of which had true hypersensitivity according to drug provocation tests. Five patients with confirmed hypersensitivity by skin tests showed no response to the culprit antibiotic on IFN-γ ELISPOT assay. Our results did not support the utility of IFN-γ ELISPOT in the diagnosis of mild, non-immediate hypersensitivity to amoxicillin and penicillin.

• MeSH
• Amoxicillin adverse effects   immunology   MeSH
• Anti-Bacterial Agents adverse effects   immunology   MeSH
• Adult MeSH
• Enzyme-Linked Immunospot Assay methods   MeSH
• Immunoenzyme Techniques MeSH
• Drug Hypersensitivity diagnosis   immunology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Penicillins adverse effects   immunology   MeSH
• Prospective Studies MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Observational Study MeSH

Hodláme studovat klinický význam Interferon-gama produkujících buněk určených metodikou ELISpot s cílem hodnotit riziko vývoje celulární a protilátkami-zprostředkované rejekce po transplantaci a zlepšit tak přežití a dobrou funkci transplantovaných orgánů.; We are aiming to study the clinical significance of Interferon-gamma producing cells as detected by the ELISpot method with the goal to assess the risk of development of cellular and antibody-mediated transplant rejection and consequently to improve the optimal function and survival of the kidney allograft.

• MeSH
• Biostatistics MeSH
• Enzyme-Linked Immunospot Assay MeSH
• HLA Antigens MeSH
• Immunosuppression Therapy MeSH
• Interferon-gamma MeSH
• Graft Rejection MeSH
• Kidney Transplantation MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• alergologie a imunologie
• transplantologie
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR

BACKGROUND: The reactivation of human cytomegalovirus (HCMV) in immunosuppressed patients is associated with significant morbidity. Testing HCMV-specific T-cell responses can help determine which patients are at high risk of HCMV disease. We optimized selection of HCMV antigens for detection of T-cell response of patients after allogeneic hematopoietic stem cell transplantation (HSCT) with the aim of identifying patients with insufficient control of HCMV reactivation. METHODS: T-cell immune response to HCMV was monitored in 30 patients during the first year after HSCT. The HSCT recipients were classified according to their anti-HCMV T-cell response and the presence of HCMV DNA in the blood. RESULTS: We observed an inverse relationship between the magnitude of HCMV-specific T-cell responses against CMV lysate, phosphoprotein (pp) 65, immediate early-1 (IE-1), UL36, and UL55, but not to US3 and US29 detected by interferon-gamma (IFNγ)- ELISPOT and the level of HCMV DNA in the blood of patients during the 30 days following sampling. The study has revealed that patients who received a graft from a seronegative donor have a lower T-cell response against HCMV and increased probability of HCMV reactivation in comparison to the patients who had received their graft from a seropositive donor. CONCLUSION: The individual peptide pools and native HCMV antigens were useful for monitoring the time course of the anti-HCMV response by IFNγ-ELISPOT, which proved to have a prognostic value. Besides widely employed peptide pools of pp65 and IE-1, the use of antigens UL36 and UL55, but not US3 or US29, increased sensitivity of the test.

• MeSH
• Antigens, Viral genetics   immunology   MeSH
• Antiviral Agents therapeutic use   MeSH
• CD8-Positive T-Lymphocytes immunology   virology   MeSH
• Cytomegalovirus Infections drug therapy   immunology   virology   MeSH
• Cytomegalovirus genetics   immunology   MeSH
• Enzyme-Linked Immunospot Assay MeSH
• Phosphoproteins immunology   MeSH
• Immunocompromised Host MeSH
• Interferon-gamma blood   MeSH
• Middle Aged MeSH
• Humans MeSH
• Hematopoietic Stem Cell Transplantation adverse effects   MeSH
• Viral Proteins genetics   immunology   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

V současné době jsou pro nepřímou diagnostiku tuberkulózy k rutinnímu použití dostupné dvě komerční IGRA (Interferon Gamma Release Assay) metody – starší QuantiFERON?-TB Gold, nově na trhu dostupná verze QuantiFERON?-TB Gold Plus (čtyřzkumavková, s diferenciací aktivity CD4+ a CD8+) a T-SPOT ?.TB. Obě využívají podobný princip, ale liší se v provedení. Ve sdělení jsou porovnány výsledky vyšetření 284 pacientů, u nichž byly provedeny obě metody. Shoda ve výsledcích obou metod byla v 81,3 %, což odpovídá hodnocení indexem Cohenovým kappa 0,72. Pro správnou interpretaci IGRA je při rozporu výsledku jedné z metod s dalšími nálezy u pacienta (klinický stav, zobrazovací metody apod.) vhodné výsledek konfirmovat provedením druhé metody.

For indirect diagnosis of tuberculosis, two commercial IGRA (Interferon Gamma Release Assay) assays are available – primal QuantiFERON?-TB Gold test, new version QuantiFERON?-TB Gold Plus test (four tube, differentiation in activity CD4+ a CD8+) and T-SPOT?.TB test. Both methods are based on the same principle, but their workflows are different. In this article, both assays are compared on the collection of 284 patients. Inter-rate agreement measure showed 81.3% consistency and Cohen’s kappa index was calculated as 0.72. In case of discrepancy between IGRA and other methods (clinical aspects, X-ray diagnostic, etc.), results should be confirmed by second IGRA assay for correct interpretation.

• Keywords
• Quantiferon T-SPOT. TB,
• MeSH
• Enzyme-Linked Immunosorbent Assay methods   MeSH
• Enzyme-Linked Immunospot Assay methods   MeSH
• Interferon-gamma blood   MeSH
• Clinical Trials as Topic MeSH
• Humans MeSH
• Mycobacterium tuberculosis pathogenicity   MeSH
• Interferon-gamma Release Tests * methods   MeSH
• Tuberculosis diagnosis   MeSH
• Check Tag
• Humans MeSH

BACKGROUND: Our retrospective study included a cohort of 47 patients who underwent living donor kidney transplantation.The pre-transplant frequencies of donor-specific Interferon-gamma (IFN-γ) producing cells were define dusing enzyme-linked immunosorbent spot (ELISpot) assay and correlated with incidence of acute cellular(ACR), antibody-mediated rejection (AMR) and kidney graft survival up to one year after transplantation. RESULTS: We found a statistically significant correlation between the frequencies of IFN-γ-producing cells and the number of mismatches in HLA antigens between patients and their respective donors – for Class I – A and B (r = 0.399, p b 0.01) and for Class I and Class II antigens – A, B and DR (r = 0.409, p b 0.01). No significant relationship was observed between the numbers of IFN-γ-secreting cells and incidence of acute rejection (neither ACR, nor AMR). However, there was a trend of elevated frequencies of IFN-γ-producing cells in patients who developed ACR or AMR in comparison with kidney recipients free of rejection (91 ± 82 and 114 ± 75 vs. 72 ± 70/5 × 10(4) peripheral blood mononuclear cells respectively). Patients with concurrent acute cellular and antibody-mediated rejection had also higher numbers of IFN-γ-producing memory/effector cells compared to patients with cellular rejection only. CONCLUSION: Pre-transplant determination of the numbers of IFN-γ-producing donor-specific memory cells using the ELISpot technique may provide clinically relevant results when evaluating the risk of development of acute cellular and antibody-mediated rejection. These frequencies are influenced by the degree of HLA mismatching between patients and their respective kidney donors.

• MeSH
• Acute Disease MeSH
• Antibody-Dependent Cell Cytotoxicity MeSH
• Adult MeSH
• Enzyme-Linked Immunospot Assay MeSH
• HLA Antigens immunology   MeSH
• Interferon-gamma metabolism   MeSH
• Cohort Studies MeSH
• Cells, Cultured MeSH
• Leukocytes, Mononuclear immunology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Follow-Up Studies MeSH
• Graft Survival MeSH
• Graft Rejection diagnosis   immunology   MeSH
• Retrospective Studies MeSH
• Aged MeSH
• Kidney Transplantation * MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH