ABSTRACT
In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.
Subject(s)
Animals , Male , Mice , B-Lymphocytes/immunology , Heat-Shock Proteins/immunology , Immunomodulation/genetics , /genetics , RNA, Messenger/immunology , T-Lymphocyte Subsets/immunology , B-Lymphocytes/metabolism , Flow Cytometry , Gene Expression/genetics , Heat-Shock Proteins/therapeutic use , Immunologic Memory/physiology , Immunophenotyping/classification , Inflammation Mediators/analysis , Interferon-gamma/analysis , /immunology , /analysis , Mice, Knockout , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , Spleen/cytology , Spleen/immunology , T-Lymphocyte Subsets/classification , Vaccines, DNA/immunology , Vaccines, DNA/therapeutic useABSTRACT
The number [amount] of Argyrophilic proteins [AgNORs] were estimated in bone marrow smears of thirty-six patients with acute leukemia at initial diagnosis: 24 children [10 males 14 females] and 12 adults [6 males and 6 females] as well as 12 matched controls. The purpose of the present work was to study the possible relation of AgNORS to immunophenotypic markers and clinical outcome. Silver staining of AgNORS was applied to bone marrow smears, and immunophenotyping was done by FACS flow cytometry. Statistically significant difference in mean AgNORS count/nucleus was observed in patients versus control [p<0.05], and a different staining pattern was observed in acute lymphoblastic leukemia [ALL] [fine dots] versus acute myeloid leukemia [AML] patients [coarse dots]. Positive correlation was found between AgNORS and bone marrow blast percent, while negative correlation was found with age, hemoglobin concentration and platelet count. Strong AgNORS staining was associated with immaturity markers and poor response to treatment. Eighty percent [80%] of AML and 70% of ALL patients with mean AgNORS<2.5 achieved complete remission. Determination of AgNORS has a major predictive value and adds an additional tool to differentiate ALL from AML smear Hence, it should be included in the diagnostic and prognostic workup of patients with acute leukemia