Determination of intracellular cytokines produced by Th1 and Th2 cells using flow cytometry in patients with brucellosis.
ABSTRACT The aim of the study was to evaluate intracellular interferon-gamma (IFN-gamma), and interleukin-4 (IL-4) levels in pre- and post-treatment periods of brucellosis patients and to determine the relationship between these parameters and patients' clinical findings. Twenty-five patients diagnosed as brucellosis and 11 aged-matched healthy volunteers were included in the study. CD3+CD4+ T lymphocytes levels were significantly lower in patients with brucellosis as compared to the control group. CD3+CD8+ T lymphocytes and CD3+IFN-gamma+ levels were increased in brucellosis patients compared with the control group. CD4+IFN-gamma+ and CD4+IL-4+ levels were no different between patients and healthy individuals. CD3+IL-4+ levels decreased in patients compared with healthy controls. Pre-treatment CD3+IFN-gamma+ levels dramatically increased in patients responsive to management compared with the unresponsive ones. In responsive cases, CD3+IFN-gamma+ levels decreased statistically after the treatment while in unresponsive cases no meaningful change was observed with respect to treatment. Adding IFN-gamma to the treatment for improving the depleted levels of IFN-gamma can be beneficial in patients with brucellosis who shows a tendency to chronicity or patients who do not respond to the treatment.
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ABSTRACT: Both CD4(+) and CD8(+) T lymphocytes play key roles in immunity to Brucella, in part because they secrete interferon (IFN)-γ and activate the bactericidal functions in macrophages. Therefore, use of markers, which can evaluate macrophage activation, may have diagnostic and prognostic significance. High mobility group-box 1 protein (HMGB1) is a late-onset pro-inflammatory cytokine secreted from activated macrophages. Soluble hemoglobin scavenger receptor (sCD163) is a specific marker of anti-inflammatory macrophages. The aim of this study was to investigate associations of HMGB1 and sCD163 levels with Brucellosis. Serum HMGB1 and sCD163 levels in 49 Brucellosis patients were compared with 52 healthy control subjects. Both serum HMGB1 and sCD163 levels were significantly higher in Brucellosis patients compared those of with healthy controls (p < 0.001). There was no statistically significant difference in serum levels of HMGB1 and sCD163 among acute, subacute and chronic cases with Brucellosis. Additionally, serum HMGB1 levels were positively correlated with sCD163 levels, whilst neither HMGB1 nor sCD163 levels were correlated with CRP, WBC and sedimentation values. Our study demonstrated that the levels of HMGB1 and sCD163 may be diagnostic markers for Brucellosis, but none of them can be used for differentiating three different forms of disease (acute, subacute, chronic).Microbiology and Immunology 12/2012; · 1.55 Impact Factor
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ABSTRACT: Introduction. Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes have ever been delineated that might provide new approaches to classifying and prognosticating B. melitensis infection.Methods. Twenty-seven pools of 500 MHC-II restricted peptides were created by computational prediction of promiscuous MHC-II CD4(+) T cell derived from the top 50 proteins recognized by IgG in human sera on a genome-level B. melitensis protein microarray. Interferon-γ (IFN-γ) and Interleukin-5 (IL-5) ELISPOT analysis were used to quantify Th1 vs Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (N=9) vs. patients who relapsed (N=5).Results. Four peptide epitopes derived from 3 B. melitensis proteins (BMEI1330, DegP/HtrA protease; BMEII0029, Type IV secretion system component VirB5; BMEII0691, predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant.Conclusions. These experiments are the first to systematically identify B. melitensis MHC class II-restricted CD4(+) T cell epitopes recognized by human immune response, with the potential for new approaches to brucellosis diagnostics and understanding immunopathogenesis related to this intracellular pathogen.Infection and immunity 10/2013; · 4.21 Impact Factor
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ABSTRACT: Brucella spp., are Gram negative bacteria that cause disease by growing within monocyte/macrophage lineage cells. Clinical manifestations of brucellosis are immune mediated, not due to bacterial virulence factors. Acquired immunity to brucellosis has been studied through observations of naturally infected hosts (cattle, goats), mouse models (mice), and human infection. Even though Brucella spp. are known for producing mechanisms that evade the immune system, cell-mediated immune responses drive the clinical manifestations of human disease after exposure to Brucella species, as high antibody responses are not associated with protective immunity. The precise mechanisms by which cell-mediated immune responses confer protection or lead to disease manifestations remain undefined. Descriptive studies of immune responses in human brucellosis show that TH(1) (interferon-γ-producing T cells) are associated with dominant immune responses, findings consistent with animal studies. Whether these T cell responses are protective, or determine the different clinical responses associated with brucellosis is unknown, especially with regard to undulant fever manifestations, relapsing disease, or are associated with responses to distinct sets of Brucella spp. antigens are unknown. Few data regarding T cell responses in terms of specific recognition of Brucella spp. protein antigens and peptidic epitopes, either by CD4+ or CD8+ T cells, have been identified in human brucellosis patients. Additionally because current attenuated Brucella vaccines used in animals cause human disease, there is a true need for a recombinant protein subunit vaccine for human brucellosis, as well as for improved diagnostics in terms of prognosis and identification of unusual forms of brucellosis. This review will focus on current understandings of antigen-specific immune responses induced Brucella peptidic epitopes that has promise for yielding new insights into vaccine and diagnostics development, and for understanding pathogenetic mechanisms of human brucellosis.Frontiers in Cellular and Infection Microbiology 01/2012; 2:1.