7), IL-6 (0.9), IL-4 (4.5), IL-1ra (8.3), MIP-1α (2.9), and MCP-1 (1.9). Leukocytes subsets were characterized find more in ChL and PL using the BD Multitest IMK kit following the manufacturer’s protocol (BD Biosciences, CA, USA; Cat. No. 340504): total leukocytes/CD45+(clone 2D1-HLe-1), NK cells/CD16+ (clone B73.1), CD56+ (clone NCAM 16.2), B cells/CD19+ (clone SJ25C1), and monocytes/macrophages/CD14+ (clone HCD14), and subsets of T cells/CD3+ (clone SK7), CD8+ (clone SK1), and CD4+ (clone SK3). Leukocyte subsets were analyzed within the CD45+ gate using a FACSCalibur flow cytometer, and data analysis was performed by BD Cell Quest software (BD Biosciences, CA, USA). Gelatinase activity in culture media
was determined by SDS–PAGE containing 1% gelatin under non-denaturing conditions as described previously. Culture supernatants (0.5 μg of total protein) were loaded into each well. Enzymatic activity standards for MMP-2 and MMP-9 were included using conditioned media on the U-937 promyelocyte cell line. Specific quantification of active and total MMP-9 in culture supernatants of choriodecidual and peripheral leukocytes was carried out using the Biotrak MMP-9 Activity Assay System (General Electric Healthcare, Buckinghamshire, UK) following the protocol suggested by the manufacturer. To measure the total MMP-9 content, bound enzyme was activated with
p-aminophenylmercuric acetate. The MI-503 concentration of total and active MMP-9 in the samples is reported as nanograms (ng) of MMP-9 per μg of protein. Protein was measured by Bradford’s method. For each variable, descriptive
statistics (mean, standard deviation, standard error, median, and range) were obtained, and the data distribution was tested for normality using the Kolmogorov–Smirnoff and Shapiro–Wilk tests. Student’s t-test was Progesterone performed to compare leukocytes subsets between ChL and PL. A P value ≤0.05 was considered to be statistically significant. Two-way analysis of variance using repeated measurement model was used to compare cytokines/chemokines concentrations in the culture media from ChL and PL. Differences with P ≤ 0.05 were considered statistically significant. All statistical analyses were carried out using spss, version 20 software (IBM Corporation, Armonk, NY, USA). The two-step method, using a density gradient followed by selection by plastic adherence, yielded in 1,33,000 ± 3,500 choriodecidual leukocytes per cm of fetal membranes (n = 18). According to the flow cytometry data, this method also allowed enriching and purifying (≥80%) choriodecidual leukocytes. Flow cytometry analysis revealed that T lymphocytes and natural killer cells were the major subsets in the ChL and PL preparations (Table 1). Choriodecidual leukocytes showed a distinct secretion pattern of cytokines and chemokines when compared with intervillous placental blood leukocytes (Fig. 1).