A precise identification and phenotypic characterization of human B-cell subsets is of crucial importance in both basic research and medicine. In the literature, flow cytometry studies for the ...phenotypic characterization of B-lymphocytes are mainly focused on the description of a particular cell stage, or of specific cell stages observed in a single type of sample. In the present work, we propose a backbone of 6 antibodies (CD38, CD27, CD10, CD19, CD5 and CD45) and an efficient gating strategy to identify, in a single analysis tube, a large number of B-cell subsets covering the whole B-cell differentiation from precursors to memory and plasma cells. Furthermore, by adding two antibodies in an 8-color combination, our approach allows the analysis of the modulation of any cell surface marker of interest along B-cell differentiation. We thus developed a panel of seven 8-colour antibody combinations to phenotypically characterize B-cell subpopulations in bone marrow, peripheral blood, lymph node and cord blood samples. Beyond qualitative information provided by biparametric representations, we also quantified antigen expression on each of the identified B-cell subsets and we proposed a series of informative curves showing the modulation of seventeen cell surface markers along B-cell differentiation. Our approach by flow cytometry provides an efficient tool to obtain quantitative data on B-cell surface markers expression with a relative easy-to-handle technique that can be applied in routine explorations.
Background
The SARS-CoV-2 infection triggers excessive immune response resulting in increased levels of pro-inflammatory cytokines, endothelial injury, and intravascular coagulopathy. The complement ...system (CS) activation participates to this hyperinflammatory response. However, it is still unclear which activation pathways (classical, alternative, or lectin pathway) pilots the effector mechanisms that contribute to critical illness. To better understand the immune correlates of disease severity, we performed an analysis of CS activation pathways and components in samples collected from COVID-19 patients hospitalized in Grenoble Alpes University Hospital between 1 and 30 April 2020 and of their relationship with the clinical outcomes.
Methods
We conducted a retrospective, single-center study cohort in 74 hospitalized patients with RT-PCR-proven COVID-19. The functional activities of classical, alternative, and mannose-binding lectin (MBL) pathways and the antigenic levels of the individual components C1q, C4, C3, C5, Factor B, and MBL were measured in patients’ samples during hospital admission. Hierarchical clustering with the Ward method was performed in order to identify clusters of patients with similar characteristics of complement markers. Age was included in the model. Then, the clusters were compared with the patient clinical features: rate of intensive care unit (ICU) admission, corticoid treatment, oxygen requirement, and mortality.
Results
Four clusters were identified according to complement parameters. Among them, two clusters revealed remarkable profiles: in one cluster (n = 15), patients exhibited activation of alternative and lectin pathways and low antigenic levels of MBL, C4, C3, Factor B, and C5 compared to all the other clusters; this cluster had the higher proportion of patients who died (27%) and required oxygen support (80%) or ICU care (53%). In contrast, the second cluster (n = 19) presented inflammatory profile with high classical pathway activity and antigenic levels of complement components; a low proportion of patients required ICU care (26%) and no patient died in this group.
Conclusion
These findings argue in favor of prominent activation of the alternative and MBL complement pathways in severe COVID-19, but the spectrum of complement involvement seems to be heterogeneous requiring larger studies.
Background/Aims The fate of intrahepatic NK cell subsets in the course of HCV and HBV infections is not clearly understood. Methods Blood and intrahepatic CD56+ NK cell subsets (expressing NKG2A, ...CD158a,h or CD158b,j receptors) from HCV or HBV patients were quantified by flow cytometry and localized by immunohistochemistry in liver biopsies. Results A significant reduction in NK cell frequency and a quantitative imbalance between CD56bright and CD56dim subsets were observed in chronic HCV patients as compared to HBV patients, underlining that the inflammatory environment is not the only cause of these phenomena. The proportions of intrahepatic NK cells expressing either NKG2A, and/or CD158a,h, CD158b,j differed significantly between HCV and HBV patients. A higher frequency of perforin among intrahepatic CD56+ CD3− cells was observed in HCV compared to HBV patients. Double immunohistochemical staining showed that CD56+ CD3− cells were localized within necrotic areas. Immune monitoring of circulating CD56 subsets revealed that CD3− CD56bright NKG2A+ and CD3− CD56dim NKG2A+ cells were positively correlated with the necroinflammatory score and inversely correlated with viral load, respectively, in HCV patients. Conclusions HCV and HBV affect NK cell subsets according to the status of the diseases, especially CD3− CD56dim NKG2A+ and CD3− CD56bright NKG2A+ cells, may be of interest for disease monitoring.
Inaugural and fatal anaphylaxis to wasp venom Chatain, Catharina; Sedillot, Nicholas; Pernollet, Martine ...
The World Allergy Organization journal,
August 2020, 2020-08-00, 2020-08-01, Letnik:
13, Številka:
8
Journal Article
Primary C3 deficiency, a rare autosomal inherited disease (OMIM 120700), was identified in a 2-year-old male suffering from recurrent pyogenic infections from early infancy with undetectable total ...complement hemolytic activity (CH50) and C3 values. The nonconsanguineous parents and the two patients' two siblings had 50% normal serum C3 concentration. The molecular abnormality associated a paternal allele coding C3 with the missense mutation p.Ser(550)Pro and an apparently null maternal allele, with production of a defective protein that could no longer be secreted. Vaccination of the child did not induce a long-term Ab response. Accordingly, switched memory IgD(-)CD27(+) B cells were barely detected, amounting to only 2.3% of peripheral blood CD19(+) cells. Cells were significantly defective in stimulating alloreactive responses. The in vitro development of immature dendritic cells and their maturation capacity were greatly impaired, with decreased CD1a expression and IL-12p70 secretion ability. These cells were unable to induce autologous B cell proliferation and Ig secretion in the presence of CD40L and C3. Finally, the regulatory T cell development ability of CD4(+) T cells after CD3 and CD46 activation in the presence of IL-2 was significantly impaired. Thus, the association of important functional defects of dendritic cells, acquisition of B cell memory, and regulatory T cells with human C3 deficiency strongly supports a major role for C3 in bridging innate and adaptive immunity in humans.
We present a case of documented acute hepatitis C that occurred in a health care worker who sustained a needlestick injury while caring for an individual who was infected with both hepatitis C virus ...(HCV) and human immunodeficiency virus (HIV). According to the findings of third-generation serological assays performed during a follow-up of >1 year, the health care worker, who was treated with interferon-α (during weeks 2-6) and ribavirin (during weeks 5-9), did not develop antibodies against HCV, in spite of documentation of an HCV-specific T cell response.
Background & Aims In chronic hepatitis C (CHC), HCV-specific T-cell responses are often dysfunctionnal. In vitro data point out that regulatory T cells (Treg) are able to suppress HCV-specific ...lymphocyte proliferation and cytokine secretion but their implication in this pathology is still debated. Methods Three complementary approaches were performed to investigate phenotype, frequency or localization of intra-hepatic Treg in treatment naïve CHC patients. Double immunohistochemical analysis was performed in 20 formalin-fixed biopsies with CD8/FoxP3 and CD4/FoxP3 antibodies. Cellular markers and cytokines were investigated by quantitative RT-PCR in 27 additional frozen biopsies. Eight other fresh liver biopsies were selected for complementary analysis of immunophenotyping and frequency of intra-hepatic Treg. Results Immunohistochemical analyses showed the presence of intra-hepatic CD4+ FoxP3+ T cells while CD8+ FoxP3+ T cells were very scarce. CD4+ FoxP3+ T cells were located in necro-inflammatory areas in contact with CD8+ T cells, suggesting that Treg-mediated inhibition of CD8+ T cell proliferation may occur by cell–cell contact. RT-PCR analyses showed strong correlations between CD8, FoxP3, and IL-10 with emergence of four distinct gene clusters, CD8-FoxP3, CD8-IL-10, TGF-β−IL-10, and TNF-α-TGF-β. No correlation was found between serum viral load and any immune markers. Interestingly, the FoxP3+ /CD8+ cells ratio significantly decreased in severe fibrosis ( F >3) due to the dramatic decline of FoxP3 cells. Conclusions This study provides new insights into the histological localization of Treg within HCV-infected liver, with a special accumulation of CD4+ FoxP3+ Treg cells in necro-inflammatory areas, in contact with CD8+ T cells. Our results suggest a link between Treg, CD8, and IL-10 which altogether could balance immune responses against the virus to avoid immunopathogenesis.
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