Fig 1: Antibacterial activity of BPI at lactation-relevant concentrations and in BPI-supplemented milk formula. The reference strain E. coli Seattle 1946 and (A) a clinical isolate of E. coli (B) were inoculated at 1.5 × 103 CFU/mL and treated with BPI at three concentrations (0.638 ng/mL, 0.236 μg/mL, and 1.645 μg/mL) or amikacin (1.25 mg/mL, positive control) to evaluate bacterial growth inhibition in Muller–Hinton broth over 24 h using a resazurin-based colorimetric assay). Short-term effects (8 h) of BPI (1.645 μg/mL) and amikacin on the reference strain (C) or clinical isolate of E. coli in the milk formula (D). The x-axis represents time post-inoculation (hours), and the y-axis corresponds to metabolic activity, measured as the resorufin/resazurin ratio (A570 nm/A600 nm). Resazurin was used at a final concentration of 0.02%. Data are shown as the mean ± standard deviation (SD) from four independent experiments. Statistical significance was calculated using a two-way ANOVA, denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.
Fig 2: BPI used at colostrum concentrations favors the uptake of E. coli by THP-1 cells. THP-1 monocytes interacted with the E. coli Seattle 1946 strain and a clinical isolate stained with carboxyfluorescein succinimidyl ester (CFSE) and exposed to 1.695 μg/mL BPI or human serum before being incubated with THP-1 cells for 10 min. Finally, THP-1 cells were analyzed by flow cytometry. (A) Left: uptake of E. coli Seattle 1946 by THP-1 cells; right: uptake of E. coli clinical isolate by THP-1 cells. A representative histogram shows the binding of THP-1 cells (gray) to the bacteria in question, whether untreated (orange), pretreated with BPI (green), or in human serum (blue). (B) The graphs represent the mean ± standard deviation of five independent experiments showing the THP-1 cell binding to E. coli Seattle 1946 (left) and the clinical isolate (right). Statistical significance was assessed by a one-tailed ANOVA and is indicated as ** p < 0.01, and *** p < 0.001. (C) Flow cytometry analysis to evaluate the binding of BPI protein present in the protein fraction of human colostrum samples to E. coli Seattle 1946. A significant increase in BPI-positive bacteria is observed in the sample treated with Alexa Fluor 488-labeled anti-BPI compared to isotype control. Data represent the average of six independent experiments.
Fig 3: Determination of interleukin-8 (IL-8) release by HT 29 cells in the presence of BPI and E. coli strains. HT-29 cells were seeded and exposed to different conditions for two hours. Conditions included no bacteria present, inoculated with the Seattle 1946 strain of E. coli (A) or with the clinical isolate (B), inoculated with bacteria and the antibiotic amikacin, and inoculated with bacteria plus BPI at the mean concentrations found in each lactation phase (colostrum: 1.645 µg/mL; transition: 0.236 µg/mL; and mature milk: 0.638 ng/mL). In addition, the co-presence of the antibiotic and recombinant BPI was evaluated. Graphs represent the mean ± standard deviation of IL-8 release by HT-29 cells from six replicates, and statistical significance was determined by a one-tailed exhaustive ANOVA multiple comparison test, expressed as ** p < 0.01, and *** p < 0.001. DMEM (Dulbecco’s Modified Eagle Medium), C (colostrum), T (transition milk), M (mature milk), and ABX (antibiotic; amikacin).
Fig 4: Human breast milk contains BPI. (A) Human breast milk samples were collected from 75 clinically healthy lactating women, with 25 samples per stage: colostrum (1–5 days postpartum), transitional milk (6–15 days postpartum), and mature milk (16 days to 6 months postpartum). BPI levels were measured by ELISA. The graph shows the concentration obtained in each of the milk samples per individual and the average and standard deviation per stage. Statistical analysis was performed by Tukey’s multiple comparison test, with significance indicated as follows: * p < 0.05, and **** p < 0.0001. (B) Cells were purified from colostrum samples from clinically healthy women (2 days postpartum), fixed on slides, permeabilized, and stained with a monoclonal antibody (mAb) against BPI. Nuclei were counterstained with DAPI to identify cells. Representative images are shown from one of three independent assays in which five fields were analyzed per sample. Brightfield image of colostrum cells (top left), staining with DAPI (blue) to highlight nuclei (top right), immunofluorescence staining for BPI (green) (bottom left), and the combined image showing the colocalization of BPI with DAPI (bottom right).
Fig 5: Phenotypic characterization and intracellular expression of BPI in colostrum and mature milk cells. (A) Flow cytometry analysis of cells from colostrum and mature milk shows populations expressing CD11b (a myeloid cell marker) and cytokeratins CK14, CK15, CK16, and CK19 (epithelial cell markers). The forward scatter (FSC; x-axis) plots display gated populations, indicating the percentage of positive cells. Corresponding histograms represent intracellular BPI expression in these gated populations (blue) compared to isotype controls (gray). The x-axis of the histograms denotes fluorescence intensity, while the y-axis indicates the number of cells (counts). (B–D) Quantitative data are derived from panel (A). Panel (B) compares the percentages of CD11b+ and CKs+ cells between colostrum (C) and mature milk (MM). Panel (C) shows the percentages of BPI+ cells within the CD11b+ and CKs+ populations in both conditions. Panel (D) presents the fluorescence intensity mean (MFI) of intracellular BPI expression in these cell subsets. Data are represented as mean ± SD, and statistical analysis was performed by Tukey’s multiple comparison test, with significance indicated as follows * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 from six independent assays at each stage.
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