ABSTRACT
BACKGROUND: Tumour necrosis factor-alpha (TNF-α) and its inhibitors are involved in both defence against tuberculosis (TB) and damage to the host by TB. Notably, the change in receptor expression on cell density is a key mechanism in regulation of the biological properties of cytokines. OBJECTIVE: To study the differences in TNF-α receptor (TNFR) expression in patients with active pulmonary tuberculosis (aPTB) in correlation with the parameters of disease severity. METHODS: TNFR1/2 levels on peripheral blood mononuclear cells (PBMCs) from 45 patients with aPTB and 150 healthy controls were analysed by flow cytometry using monoclonal antibodies and QuantiBRITE beads. Soluble TNFR1/2 and TNF-α in serum were measured using an enzyme-linked immunosorbent assay. RESULTS: TNFR1 expression in aPTB patients was increased in the main populations of immune cells. Patients who were Mycobacterium tuberculosis culture-positive on bronchoscopy had higher levels of the soluble forms of TNFR1 (sTNFR1) than M. tuberculosis-negative patients. CONCLUSION: Active TB was shown to cause activation of different immune cell types by increasing TNFR1 expression on cells and reducing sTNFR1 expression compared with healthy controls. M. tuberculosis-positive patients with disseminated infection had the highest sTNFR1 serum level compared with other patients, but did not differ in receptor expression on PBMCs.
Subject(s)
Leukocytes, Mononuclear/immunology , Receptors, Tumor Necrosis Factor, Type II/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tuberculosis, Pulmonary/immunology , Adult , Female , Humans , Male , Mycobacterium tuberculosis/isolation & purification , Russia , Severity of Illness Index , Tuberculosis, Pulmonary/blood , Tuberculosis, Pulmonary/diagnostic imaging , Tuberculosis, Pulmonary/microbiology , Young AdultABSTRACT
Autoantibodies to cytokines are important biological effector molecules that can regulate cytokine activities. The aim of the study was to develop a protocol to purify autoantibodies to tumor necrosis factor from human serum, for use as a calibration material to determine the absolute content of autoantibodies to tumor necrosis factor by enzyme-linked immunosorbent assay. The proposed protocol includes a set of affinity chromatography methods, namely, Bio-Gel P6DG sorbent to remove albumin from serum, Protein G Sepharose 4 Fast Flow to obtain a total immunoglobulin G fraction of serum immunoglobulins, and Affi-Gel 15 to obtain specifically antibodies to tumor necrosis factor. The addition of a magnetic separation procedure to the protocol eliminated contaminant tumor necrosis factor from the fraction of autoantibodies to tumor necrosis factor. The protocol generated a pure fraction of autoantibodies to tumor necrosis factor, and enabled us to determine the absolute concentrations of different subclasses of immunoglobulin G autoantibodies to tumor necrosis factor in apparently healthy donors.
