Při obraně organismu vůči infekčním agens hrají B lymfocyty zcela nezastupitelnou úlohu. Kromě schopnosti prezentovat antigen T lymfocytům jsou především producenty protilátek. Ty jsou nezbytné pro efektivní eliminaci infekce a podílejí se též na regulaci imunitní odpovědi. Stanovení B-lymfocytárních subpopulací v periferní krvi, pomocí kterého můžeme sledovat část vývoje B lymfocytů až do stadia plasmablastů produkujících protilátky, je proto cenným laboratorním parametrem, který má význam ve studiu patogeneze některých onemocnění, stejně jako napomáhá správnému stanovení diagnózy některých onemocnění. Laboratorní stanovení B lymfocytárních subpopulací je v současné době rutinním laboratorním vyšetřením zejména díky rozvoji mnohobarevné průtokové cytometrie. Tento článek shrnuje základní poznatky, které jsou v současné době při rutinním vyšetření B lymfocytárních subpopulací v imunologické laboratoři uplatňovány.

B cells play a vital role in the defence of the body against infectious agents. Apart from their ability to present antigen to T cells, B cells are mainly producers of antibodies. These play a crucial role in the effective elimination of infection and are also involved in the regulation of the immune response. The analysis of peripheral blood B cell subpopulations that makes it possible to monitor the development of B cells to the stage of antibody producing plasmablasts provides a valuable laboratory parameter which is important for both the study of the pathogenesis and diagnosis of some diseases. Laboratory analysis of B cell subpopulations is now a routinely available laboratory option thanks to the development of multicolour flow cytometry. This article summarizes the core knowledge which is currently applied to the analysis of B cell subpopulations in immunological laboratories.

• Klíčová slova
• marginální zóna sleziny,
• MeSH
• B-lymfocyty * MeSH
• krevní obraz metody   MeSH
• lidé MeSH
• průtoková cytometrie metody   MeSH
• Check Tag
• lidé MeSH

Introduction: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients. Methods: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube, standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD). Results: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5+ CD38+/++ CD21het CD24++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5+ CD38het CD21+ CD24+ (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21-CD24- (6.1 vs 0.74 cells/µl, p<0.0001) and CD21-CD24++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD+ (21 vs 32 cells/µl, p=0.03) and IgMD- (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%). Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID.

• MeSH
• běžná variabilní imunodeficience diagnóza   imunologie   metabolismus   MeSH
• CD antigeny analýza   MeSH
• dospělí MeSH
• fenotyp MeSH
• imunofenotypizace * MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• prekurzorové B-lymfoidní buňky imunologie   metabolismus   MeSH
• průtoková cytometrie * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH

Common variable immunodeficiency disorders (CVID) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and impaired specific Ab response, resulting in recurrent infections due to dysfunctional immune response. The specific mechanisms mediating immune deficiency in CVID remain to be determined. Previous studies indicated that immune dysregulation in CVID patients is associated with chronic microbial translocation, systemic immune activation, and altered homeostasis of lymphocytic and myeloid lineages. A detailed phenotypic, functional characterization of plasma markers and immune cell populations was performed in 46 CVID patients and 44 healthy donors. CVID patients displayed significantly elevated plasma levels of a marker of neutrophil activation neutrophil gelatinase-associated lipocalin. Neutrophils from CVID patients exhibited elevated surface levels of CD11b and PD-L1 and decreased levels of CD62L, CD16, and CD80, consistent with a phenotype of activated neutrophils with suppressive properties. Neutrophils from CVID patients actively suppressed T cell activation and release of IFN-γ via the production of reactive oxygen species. Furthermore, CVID was associated with an increased frequency of low-density neutrophils (LDNs)/granulocytic myeloid-derived suppressor cells. LDN/granulocytic myeloid-derived suppressor cell frequency in CVID patients correlated with reduced T cell responsiveness. Exogenous stimulation of whole blood with bacterial LPS emulated some but not all of the phenotypic changes observed on neutrophils from CVID patients and induced neutrophil population with LDN phenotype. The presented data demonstrate that neutrophils in the blood of CVID patients acquire an activated phenotype and exert potent T cell suppressive activity. Specific targeting of myeloid cell-derived suppressor activity represents a novel potential therapeutic strategy for CVID.

• MeSH
• aktivace neutrofilů MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD274 metabolismus   MeSH
• běžná variabilní imunodeficience imunologie   MeSH
• dospělí MeSH
• granulocyty fyziologie   MeSH
• imunologická tolerance MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipokalin-2 krev   MeSH
• mladý dospělý MeSH
• myeloidní supresorové buňky fyziologie   MeSH
• neutrofily fyziologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• T-lymfocyty imunologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Neutrophils impact on processes preceding the formation of bradykinin, a major swelling mediator in hereditary angioedema (HAE), yet their potential role in HAE pathogenesis has not been sufficiently studied. We assessed the relative mRNA expression of 10 genes related to neutrophil activation using RNA extracted from the peripheral blood neutrophils of 23 HAE patients in a symptom-free period and 39 healthy donors. Increased relative mRNA expression levels of CD274, IL1B, IL1RN, IL8, MMP9, and TLR4, together with a lack in their mutual correlations detected in HAE patients compared to healthy controls, suggested a preactivated state and dysregulation of patients' neutrophils. Patients' neutrophil-alerted state was further supported by increased CD11b, decreased CD16 plasma membrane deposition, and increased relative CD274+ and CD87+ neutrophil counts, but not by increased neutrophil elastase or myeloperoxidase plasma levels. As CD274 mediates inhibitory signals to different immune cells, neutrophils were cocultured with T-cells/PBMC. The decrease in CD25+ and IFN-γ+ T-cell/PBMC ratio in patients indicated the patients' neutrophil suppressive functions. In summary, the results showed neutrophils' alerted state and dysregulation at the transcript level in patients with HAE types I and II even in a symptom-free period, which might make them more susceptible to edema formation. Neutrophils' T-cell suppressive capacity in HAE patients needs to be further investigated.

• MeSH
• antagonista receptoru pro interleukin 1 metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD274 metabolismus   MeSH
• dítě MeSH
• dospělí MeSH
• ELISA MeSH
• hereditární angioedém, typy I a II metabolismus   MeSH
• interleukin-1beta metabolismus   MeSH
• interleukin-8 metabolismus   MeSH
• kultivované buňky MeSH
• leukocyty mononukleární metabolismus   MeSH
• lidé MeSH
• matrixová metaloproteinasa 9 metabolismus   MeSH
• messenger RNA MeSH
• mladiství MeSH
• mladý dospělý MeSH
• neutrofily metabolismus   MeSH
• pankreatická elastasa krev   MeSH
• peroxidasa krev   MeSH
• průtoková cytometrie MeSH
• receptory IgG metabolismus   MeSH
• receptory urokinázového aktivátoru plazminogenu metabolismus   MeSH
• toll-like receptor 4 metabolismus   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• Publikační typ
• časopisecké články MeSH

Surface IgD is coexpressed with IgM on naive mature B cells. Still, the role of surface IgD remains enigmatic even 50 y after its initial discovery. In this study, we examined the in vivo role of surface IgD in human B cell homeostasis and Ab responses in four individuals with heterozygous nonsense mutations in IGHD All IGHD heterozygous individuals had normal numbers of B cells and serum Igs and did not show signs of immunodeficiency or immune dysregulation. IgD+ and IgD- naive mature B cells were present in equal numbers and showed similar immunophenotypes, except for decreased expression of CD79b in the IgD- subset. Furthermore, both IgD+ and IgD- naive mature B cells had normal replication histories and similar capacities to differentiate into plasma cells upon in vitro stimulation, and Ig class-switched memory B cells showed similar levels of somatic hypermutations. Thus, human B cells lacking IgD expression develop normally and generate immunological memory in vivo, suggesting that surface IgD might function more restrictedly in regulating of B cell activation to specific antigenic structures.

• MeSH
• aktivace lymfocytů MeSH
• B-lymfocyty imunologie   MeSH
• buněčná diferenciace MeSH
• haploinsuficience MeSH
• homeostáza MeSH
• imunoglobulin D genetika   metabolismus   MeSH
• imunoglobulin M metabolismus   MeSH
• imunologická paměť MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• plazmatické buňky imunologie   MeSH
• podskupiny B-lymfocytů imunologie   MeSH
• přesmyk imunoglobulinových tříd MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

Introduction: Previous studies showed that several lymphocyte abnormalities seen in the most frequent symptomatic immunoglobulin deficiency, common variable immunodeficiency (CVID), were also observed in a genetically related asymptomatic disorder - selective IgA deficiency (IgAD). In this study we searched for abnormalities in the differentiation stages of T cells as well as for similarities of these abnormalities in CVID and IgAD patients. Material and methods: Using flow cytometry in 80 patients with IgAD, 48 patients with CVID, and 80 control persons we determined T-lymphocyte subsets: both CD4 and CD8 were divided into the naïve CD45RO-CD27+, early differentiated CD45RO+CD27+, late differentiated CD45RO+CD27- and fully differentiated effector CD45RO-CD27- memory T cells, as well as Treg cells, defined as CD4+CD25highCD127low T cells. Results: An increase of CD4+ and CD8+ late differentiated memory cells was observed comparing CVID patients to controls, as well as comparing IgAD patients to controls. In CVID patients an increase of CD4+ early differentiated memory cells, a decrease of CD8+ intermediate memory cells, and CD4+ and CD8+ naïve cells were found as well. The abnormalities in IgAD patients might be explained by higher CMV seropositivity observed in our IgAD. We confirmed the repeatedly published decrease of Treg cells in CVID patients, while Treg cells in IgAD patients were increased compared to controls. Conclusions: Our results show T-cell activation not only in CVID, but also in IgAD patients. The increase in IgAD patients may be influenced by a more frequent CMV infection in our group of IgAD patients.

• Publikační typ
• časopisecké články MeSH

PURPOSE: Primary selective IgM deficiency (sIgMD) is a primary immunodeficiency with unclear pathogenesis and a low number of published cases. METHODS: We reviewed clinical and laboratory manifestations of 17 sIgMD patients. Serum IgM, IgG, and its subclasses, IgA, IgE, antibodies against tetanus toxoid, pneumococcal polysaccharides and Haemophilus influenzae type b, isohemagglutinins, and T and B lymphocyte subsets, expressions of IgM on B cells and B lymphocyte production of IgM were compared with previously reported case reports and a small series of patients, which included 81 subjects in total. RESULTS: We found that some patients in our cohort (OC) and published cases (PC) had increased IgE levels (OC 7/15; PC 21/37), decreased IgG4 levels (OC 5/14), very low titers of isohemagglutinins (OC 8/8; PC 18/21), increased transitional B cell counts (OC 8/9), decreased marginal zone B cell counts (OC 8/9), and increased 21low B cell counts (OC 7/9). Compared with the PC (20/20), only two of five OC patients showed very low or undetectable production of IgM after stimulation. A majority of the patients had normal antibody production to protein and polysaccharide antigens, basic lymphocyte subset counts, and expression of surface IgM molecules on B cells. CONCLUSIONS: Low IgM levels are associated with various immunopathological disorders; however, pathogenic mechanisms leading to decreased IgM serum level in selective IgM deficiency remain unclear. Moreover, it is difficult to elucidate how strong these associations are and if these immunopathological conditions are primary or secondary.

• MeSH
• autoimunita MeSH
• B-lymfocyty imunologie   MeSH
• dospělí MeSH
• imunoglobulin E krev   MeSH
• imunoglobulin G krev   MeSH
• imunoglobulin M nedostatek   metabolismus   MeSH
• infekce imunologie   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• senioři MeSH
• syndromy imunologické nedostatečnosti imunologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

In international cooperation with EuroFlow PID, we will introduce uniform and optimized methodology for a detailed immunophenotyping of lymphocytes using multicolor flow cytometry (8-12 parameters) in patients with immunodeficiency, which are examinated in regional centers in Prague and Brno. The project will lead to improved diagnostics and classification of immunodeficiencies, particularly the rare types of these diseases, as well as to revealing of patophysiological mechanisms and mechanisms that modify the clinical progress of the primary and secondary immunodeficiencies. The project is designed as multicentric to cover the majority of immunodeficient patients in the Czech Republic and to reach the maximal application of the results in practice. Cooperation with Institute of Molecular Genetics of Academy of Science of the Czech Republic will allow detection of patophysiological mechanisms on detailed molecular level.

U pacientů s imunodeficiencí vyšetřeným v regionálních centrech (Praha a Brno) zavedeme v mezinárodní spolupráci s EuroFlow-PID jednotnou a optimalizovanou metodiku vyšetření podrobného imunofenotypu lymfocytů metodou mnohobarevné průtokové cytometrie (8-12 parametrů). Projekt povede ke zlepšení diagnostiky a klasifikace imunodeficiencí, především u vzácných typů onemocnění. Povede také k objevení patofysiologických mechanismů a mechanismů jež modifikují klinický průběh onemocnění (u primárních i sekundárních imunodeficiencí). Projekt je koncipován jako multicentrický, což povede jednak k pokrytí většiny imunodeficientních pacientů v ČR, jednak k maximálnímu uplatnění výsledků v praxi. Spolupráce s ÚMG AV ČR umožní odhalení patofysiologických mechanismů na detailní molekulární úrovni.

• MeSH
• algoritmy MeSH
• B-lymfocyty cytologie   MeSH
• běžná variabilní imunodeficience diagnóza   klasifikace   MeSH
• imunofenotypizace MeSH
• průtoková cytometrie MeSH
• T-lymfocyty cytologie   MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• alergologie a imunologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

• Publikační typ
• abstrakt z konference MeSH