Study Subjects
Participants were recruited for this study under protocols approved by the ethics committee of Central India Institute of Medical sciences (CIIMS), Nagpur, India and enrolled after obtaining informed consents. All the subjects aged 18-45 years were recruited having no history of pulmonary illness, tuberculosis, seronegative for HIV and HBV and had been vaccinated with BCG.
PBMC model
PBMCs were separated from whole blood of healthy volunteers (n = 15) included in this study by density gradient centrifugation using the Ficoll Histopaque method. BCG vaccine (Moscow Strain) was obtained from Serum Institute of India, Pune and stored at 4-8°C. Prior to use, vaccine was reconstituted in sterile saline. After counting, the cells were cultured in RPMI-1640 medium keeping the concentration at 2 × 105 cells/well and were induced with the BCG vaccine (10 μl/ml or 104 CFU/ml). The cells not induced with BCG vaccine were taken as controls. Induced cells were then incubated for 0, 4, 24, 48, 72, 96, and 120 hrs in a CO2 incubator. Booster doses of BCG (10 μl) were given after 24 and 72 hrs. The cells were then taken out from the incubator and were centrifuged for 10 mins at 1000 rpm. Supernatant was separated and was analyzed for anti-BCG IgG titer, adenosine deaminase activity, and interferon γ levels. The pellet was suspended in phosphate buffer saline (pH 7.2) and was used for flow cytometry analysis to determine CD 4 and CD 8 T cell count. The detailed experimental sketch is given in figure 1.
Anti-BCG (IgG) estimation
An in-house developed Indirect ELISA method was employed using a BCG vaccine. (Bacillus Calmette Guerin strain, Serum Institute Of India Ltd, Pune, India) to estimate the Anti-BCG (IgG) titer/level. Briefly the 96-well microtiter plate (MaxisorpImmunoplate, Nalge Nunc International, Naperville, III.) were coated with 10 ng of BCG (diluted in sterile saline). After 3 hours of incubation at 37°C, the plates were washed and blocked with 0.25% BSA in Phosphate buffered saline (PBS) pH 7.40. After 60 minutes of incubation, plates were washed once and kept overnight at 4°C. Next day the plates were incubated with supernatant (1:400 diluted) in PBS. After 45 minutes of incubation plates were washed and incubated with rabbit anti-mouse IgG, HRP conjugate (1:10,000) for 45 minutes. For color development substrate Tetramethyl benzidine in hydrogen peroxide (TMB/H2O2) was added and incubated for 10 min. The reaction was stopped by adding 2.5N sulphuric acid and the optical density of plates was read at 450 nm.
Interferon γ (gamma)
IFN-γ was measured by an enzyme linked immunosorbant assay (ELISA) according to the manufacturer's instructions (Bender Med System, Austria). In brief, anti IFN-γ monoclonal coating antibody was adsorbed onto the microwells. After two hours of incubation at room temperature, the wells were washed and blocked with 0.5% BSA in phosphate buffer. After one hour of incubation at room temperature, supernatant followed by biotin-conjugated anti-cytokine antibody was added to the coated wells. After another two hours of incubation, streptavidin-HRP (horseradish peroxidase) was added to the wells. After one hour of incubation, streptavidin-HRP was removed by washing and substrate solution reactive with HRP was added to the wells. A colored product was formed in proportion to the amount of cytokine present in the sample. The reaction was terminated by the addition of 4 N sulphuric acid and the absorbance was measured at 450 nm.
Adenosine deaminase (ADA)
ADA activity in the supernatant was determined at 37°C according to the method of Guisti and Galanti [14] based on the Berthlot reaction, in which there is the formation of colored indophenol complex from ammonia liberated from adenosine and quantified spectrophotometrically (U.V. Visible spectrophotometer, Systronic-Model). One unit of ADA is defined as the amount of enzyme required to release 1 m mol of ammonia/min from adenosine in standard assay conditions. Results were expressed as units per litre per minute (U/L/min). The assays were performed in triplicate and blind to the diagnosis.
Flow cytometric analysis
After performing experiments, the PBMCS obtained were suspended in PBS and subjected to flow cytometry analysis to determine CD 4 and CD 8 T cell level/cell count. Flow cytometry was performed on a fluorescence activated cell sorter (FACS-SCAN) instrument (Becton Dickinson Biosciences) Briefly the cells were centrifuged and 50 ul of sediment was stained using 5 ul antibodies attached to specific flourochromes against CD8-PER CP, CD4-PE and CD-3 -FITC (all from Becton Dickinson Biosciences) and incubated for 30 minutes at room temperature. The cells were again washed and resuspended in 1 ml saline and subjected to flowcytometry. Cells collected using flow cytometry on a FACS were analyzed using FlowJo software by gating on the lymphocyte population in forward scatter (FSC) and side scatter (SSC). The gate was set around the lymphocytes to exclude other cells from analysis. Routinely 10,000 cells per tube were counted.
