Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Phenotypic and functional characterization of NK cells in human immune response against the dimorphic fungus Paracoccidioides brasiliensis.

doi: 10.4049/jimmunol.1102563

Figure Lengend Snippet: FIGURE 4. Expression of cytotoxic granules’ components (mRNA and protein) by NK cells. (A–D) Analysis by qRT-PCR of the mRNA relative ex- pression for granzyme A (A), granzyme B (B), perforin (C), and granulysin (D) in NK cells isolated from peripheral blood of controls (n = 9) unstimulated (US), stimulated with IL-15 (5 ng/ml) or with P. brasiliensis yeast cells (strains Pb18 or Pb265; yeast cell/NK cell ratio 1:100) for 24 h. Results are expressed as mean 6 SEM of the relative expression determined as described in Materials and Methods. Statistical analysis: ANOVA for repeated measures with Bonferroni posttest. *p # 0.05 (in relation to unstimulated cells), #p # 0.05 (in relation to IL-15–stimulated cells). (E–H) Flow cytometry analysis of the expression of granzyme A (E), granzyme B (F), perforin (G), and granulysin (H) in NK cells isolated from peripheral blood of controls (n = 10) and PCM patients (n = 7) unstimulated (US), stimulated with IL-15 (5 ng/ml) or with P. brasiliensis yeast cells (strains Pb18 or Pb265; yeast cell/NK cell ratio 1:100) for 24 h. Results are expressed as mean 6 SEM of the percentage of positive cells for each parameter. Statistical analysis: ANOVA for repeated measures with Bonferroni posttest. #p # 0.05 (in relation to unstimulated cells), *p # 0.05 (in relation to NK cells from healthy individuals). (I) Dosage of granulysin in supernatants of culture of NK cells from controls (C) or PCM patients supplemented with recombinant IL-15 (5 ng/ml). The values are expressed as micromolars of granulysin. Values represent the mean 6 SEM. Statistical analysis: paired t test. p values are shown in the graphics. (J) Analysis of the effect of recombinant granulysin on P. brasiliensis yeast cells (Pb18 or Pb265 strain). Yeast cells were incubated with the indicated concentrations of granulysin at 37˚C for 48 h. After incubation, the samples were diluted 1:100 with distilled water and spread on BHI agar plates, and after 5–10 d the number of CFUs was determined. The results shown are represented as mean 6 SEM of three independent experiments. (K) Representative image of Western blotting analysis of supernatants from IL-15–stimulated NK cells untreated (Ctrl) or depleted of granulysin by immunoprecipitation (GNL-D). Membranes were labeled with specific Abs against granulysin, perforin, granzyme B, and actin. (L) Number of CFU/ml of P. brasiliensis (strain Pb18) cultured for 48 h with supernatants from IL-15–stimulated NK cells untreated (Ctrl) or depleted of granulysin by immunoprecipitation (GNL-D) or RPMI. Results expressed as mean 6 SEM of the number of CFU/ml. Statistical analysis: ANOVA test with Bonferroni posttest. *p # 0.05 (in relation to the GNL-D supernatants or RPMI). (M–P) Immunohistochemical analysis of granulysin+ cells (M), granzyme B+ cells (N), and perforin+ cells (O) (brown-stained cells) in a representative biopsy lesion from a patient with PCM. Cells were stained using specific mAbs as described in the Materials and Methods. Note that positive cells were found surrounding P. brasiliensis yeast cells (arrows). (P) Representative result of a control slide (without the primary Ab). Original magnification (M–P) 31000.

Article Snippet: The dosage of granulysin in supernatants of NK cells cultured with P. brasiliensis yeast cells stimulated or not with rhIL-15 was performed by ELISA as previously described (18), and the results are expressed as micromolars of granulysin determined by comparison with a standard curve made of recombinant human granulysin (R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Recombinant, Incubation, Western Blot, Immunoprecipitation, Labeling, Cell Culture, Immunohistochemical staining, Staining, Control