4.5. Clinical Experiment Measures
TNF-α
LYSING, EVENTS, APC, FRANKLIN
Consecutive patients in the study were randomly assigned to one of two groups using a computer-generated list of numbers. The first group, referred to as the intervention group, received 200 mg of potassium canrenoate (Aldactone) dissolved in 100 mL of 0.9% sodium chloride intravenously twice daily for a duration of 7 days. The second group, known as the placebo group, received 100 mL of 0.9% sodium chloride intravenously twice daily for the same 7-day period.For the above-mentioned groups of patients, a detailed immunophenotype analysis of blood cell cytometry on day 1 and day 7 of the intervention was performed, including parameters such as T CD4+ lymphocytes, T CD3+ lymphocytes, IL-1ß on lymphocytes, total count of IL-1ß and IL-2 on lymphocytes, and total count of IL-2 and TNF-α on lymphocytes vs. total count of TNF-α. Immunophenotyping results were compared with those from the control group. For flow cytometrical analysis, peripheral blood samples for frequency analysis were collected in ethylenediaminetetraacetic acid-containing tubes. The cells were examined based on unstained control, FMO control, and stained cells with monoclonal antibodies conjugated with fluorescent dyes using 3-color labeling with the following surface antibodies: mouse fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 (clone SK3)/mouse peridinin chlorophyll protein (PerCP)-conjugated anti-CD3 (clone SK7) to determine the proportion of the CD4 + CD3+ T lymphocytes (Becton Dickinson, East Rutherford, NJ, USA), anti-human IL-1β (PE) (BD FastImmune, San Jose, CA, USA), anti-human TNF-α (PE) (FastImmune, San Jose, CA, USA) and anti-human IL-2 (APC) (FastImmune, San Jose, CA, USA). Cells were incubated with an Fc receptor (FcR) blocking reagent (Miltenyi Biotec, Auburn, CA, USA) for 10 min at room temperature to block specific FcR-mediated binding of antibodies. Next, the cells were incubated for 20 min at room temperature with 20 μL of each mAb per sample for 30 min at 4 °C in the dark. After staining of samples with antibodies, cells were treated with lysing solution (Becton Dickinson) and incubated for 15 min at 4 °C in the dark. The cells were then washed twice with phosphate-buffered saline. Cell subsets were detected using cell labeling and gating methods, which start by removing doublets (FSC-A vs. FSC-H), followed by a dot plot, in which lymphocyte populations were defined (FSC vs. SSC). The data were collected on an eight-colour FACSCantoII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Diva software (BD Biosciences, Franklin Lakes, NJ, USA) was used for data analysis, and the percentage of positive cells was recorded. At least 10,000 total events in the physical parameters were acquired for each sample. The percentage of IL-1, IL-2 and TNF-positive cells was calculated from the total number of lymphocytes. Levels of IL-6 have been routinely assessed by hospital laboratory using ELISA as part of the local treatment protocol.