Journal:

Article Title: Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

doi: 10.1046/j.1365-2567.2002.01363.x

Figure Lengend Snippet: Concentration and activity of IL-10 secreted by IL-10-transfected B16-melanoma cell lines. The B16-melanoma cell line was transfected with IL-10 cDNA and the production of IL-10 was quantified from cell culture supernatants by ELISA. Three clones that secreted high (B16-10), medium (B16-13) and low (B16-5) amounts of IL-10 were selected. Results are expressed in pg/ml of IL-10/2 × 106 cells/48 hr. The biological activity of the secreted IL-10 was determined by measuring the nitrite (NO2) concentrations after macrophages were incubated with cell line supernatants and then activated by LPS/IFN-γ (see Material and Methods). Labels at the left of the slash indicate the cell-line supernatant and the sign ‘−’ indicates not incubated with cell-line supernatant; ‘+’ or ‘−’ signals at the right of the slash indicate activation or not activation of macrophages. The bar representing the nitrite basal production by macrophages is indicated as ‘−/−’ (no cell line supernatant/not activated) and total nitrite production after stimulation is indicated as −/+ (no cell line supernatant/activated). The mean of triplicates is presented and error bars represent the standard errors of means.

Article Snippet: Mouse monoclonal antibodies were detected with a biotinylated anti-mouse made in rabbit, rat monoclonal antibodies with a biotinylated anti-rat made in mouse and polyclonal antibodies were detected with biotinylated horse anti-goat (IL-10) and goat anti-rabbit (IL-10R; Vector Laboratories), using streptavidin–peroxidase and 3,3′-diaminobenzidine (Sigma Chemical Co.) or aminoethyl carbazole (Zymed Laboratories, Inc.) as chromogens.

Techniques: Concentration Assay, Activity Assay, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Clone Assay, Incubation, Activation Assay

Journal:

Article Title: Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

doi: 10.1046/j.1365-2567.2002.01363.x

Figure Lengend Snippet: Growth and IL-10 production of tumours generated with IL-10-transfected and non-transfected B16-melanoma cells. (a) Groups of 14 mice were injected subcutaneously with 3 × 105 cells from clones that secreted high (B16-10), medium (B16-13) and low (B16-5) amount of IL-10 or from non-transfected B16-melanoma cell line, and the tumour growth was measured by 18 days in six animals per group. IL-10 was determined by ELISA assay in tumour samples obtained from two mice of each group, killed at days 13, 14, 16, 17 and 18. Results are expressed in pg/ml of IL-10/0·3 g of tumour tissue (see Materials and Methods). Error bars represent the standard error of the mean in one representative experiment.

Article Snippet: Mouse monoclonal antibodies were detected with a biotinylated anti-mouse made in rabbit, rat monoclonal antibodies with a biotinylated anti-rat made in mouse and polyclonal antibodies were detected with biotinylated horse anti-goat (IL-10) and goat anti-rabbit (IL-10R; Vector Laboratories), using streptavidin–peroxidase and 3,3′-diaminobenzidine (Sigma Chemical Co.) or aminoethyl carbazole (Zymed Laboratories, Inc.) as chromogens.

Techniques: Generated, Transfection, Injection, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

doi: 10.1046/j.1365-2567.2002.01363.x

Figure Lengend Snippet: Tumour growth of IL-10-transfected B16-melanoma cells in mice treated with anti-IL-10 antibody. Three groups of six C57BL/6 mice were used in this experiment, two of them were inoculated with B16-10 cells and the other was inoculated with B16-0 cells. At day 11 post-inoculation, 2 days before tumour development started in B16-10 groups, one of these groups was treated with 3 µg of neutralizing antibody (anti-IL-10). The other B16-10 group and the group inoculated with B16-0 cells were treated only with PBS. All groups of mice were treated (with antibody or PBS) for 7 days, tumour growth was measured every day and mice were killed at day 18. Error bars represent the standard error of the mean of one representative experiment.

Article Snippet: Mouse monoclonal antibodies were detected with a biotinylated anti-mouse made in rabbit, rat monoclonal antibodies with a biotinylated anti-rat made in mouse and polyclonal antibodies were detected with biotinylated horse anti-goat (IL-10) and goat anti-rabbit (IL-10R; Vector Laboratories), using streptavidin–peroxidase and 3,3′-diaminobenzidine (Sigma Chemical Co.) or aminoethyl carbazole (Zymed Laboratories, Inc.) as chromogens.

Techniques: Transfection

Journal:

Article Title: Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

doi: 10.1046/j.1365-2567.2002.01363.x

Figure Lengend Snippet: Histological and immunohistochemical comparisons in tumours produced after 18 days of melanoma cell inoculation. (a) Non-transfected tumours have extensive areas of ischaemic necrosis, characterized by picnotic nuclei (arrow) surrounded by deep acidophilic cytoplasm. (b) In contrast, IL-10-transfected tumours do not have necrotic areas. (c) Necrotic areas are produced when the activity of IL-10 is suppressed using blocking antibodies in animals bearing transfected tumours. (d) There is not IL-10 immunoreactivity in tumoral non-transfected cells. (e) In contrast, the majority of neoplastic transfected cells show strong IL-10 immunostaining. (f) Many transfected neoplastic cells have cytoplasmic IL-10 immunoreactivity after administration of IL-10-blocking antibodies. (g) The immunohistochemical detection of Von Willebrand factor as a marker of endothelial cells, shows fewer blood vessels in non-transfected than in IL-10-transfected tumours (h). (i) When mice bearing IL-10-transfected tumours are treated with blocking anti-IL-10 antibodies, the number of blood vessels is similar to that in non-transfected tumours. (All micrographs ×100, except panels d–f, ×200).

Article Snippet: Mouse monoclonal antibodies were detected with a biotinylated anti-mouse made in rabbit, rat monoclonal antibodies with a biotinylated anti-rat made in mouse and polyclonal antibodies were detected with biotinylated horse anti-goat (IL-10) and goat anti-rabbit (IL-10R; Vector Laboratories), using streptavidin–peroxidase and 3,3′-diaminobenzidine (Sigma Chemical Co.) or aminoethyl carbazole (Zymed Laboratories, Inc.) as chromogens.

Techniques: Immunohistochemical staining, Produced, Transfection, Activity Assay, Blocking Assay, Immunostaining, Marker

Journal:

Article Title: Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

doi: 10.1046/j.1365-2567.2002.01363.x

Figure Lengend Snippet: Cell proliferation of IL-10-transfected and non-transfected B16-melanoma cells. Cell line proliferation was measured by the incorporation of BrdU with a colorimetric method. Cell lines (1440 tumour cells/well) were cultured in triplicates for 24 hr, then treated only with culture medium or 3 µg/ml of IL-10-neutralizing antibody for 48 hr. Cells were then labelled by the addition of BrdU for 5 hr and incorporation of BrdU was quantified by a colorimetric reaction measuring the absorbance at 370 or 492 nm (see Materials and methods). The mean OD of triplicates is presented and bars represent the standard error of means.

Article Snippet: Mouse monoclonal antibodies were detected with a biotinylated anti-mouse made in rabbit, rat monoclonal antibodies with a biotinylated anti-rat made in mouse and polyclonal antibodies were detected with biotinylated horse anti-goat (IL-10) and goat anti-rabbit (IL-10R; Vector Laboratories), using streptavidin–peroxidase and 3,3′-diaminobenzidine (Sigma Chemical Co.) or aminoethyl carbazole (Zymed Laboratories, Inc.) as chromogens.

Techniques: Transfection, Cell Culture

Journal:

Article Title: Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

doi: 10.1046/j.1365-2567.2002.01363.x

Figure Lengend Snippet: Representative immunohistochemical features in tumours produced after 18 days of melanoma cell inoculation. (a) Non-transfected tumours have occasional cells with nuclear immunoreactivity to PCNA. (b) In contrast, IL-10-transfected tumours have numerous PCNA-positive cells denoting a higher proliferative activity. (c) The administration of IL-10-blocking antibodies in transfected tumour-bearing mice significantly reduced the number of PCNA-positive cells. (d) Occasional neoplastic cells have immunoreactivity to IL-10R. (e) In comparison, numerous cells are IL-10R-positive in transfected tumours. (f) Animals bearing IL-10-transfected tumours treated with blocking antibodies have scarce IL-10R-positive cells. (g) Non-transfected tumours have many infiltrating macrophages (× 100). (h) Macrophages are usually settled in the periphery of the IL-10-transfected tumours (× 100). (i) A similar number and distribution of macrophages as in non-transfected tumours is observed in IL-10-transfected tumours treated with blocking antibodies (× 100). (j) The majority of neoplastic cells have MHC-I molecules in non-transfected tumours.(k) In contrast, occasional cells are MHC-I positive in IL-10-transfected tumours. (l) Many neoplastic cells express MHC-I molecules in IL-10-transfected tumours after the administration of blocking antibodies. (All micrographs ×200, except panels g–i).

Article Snippet: Mouse monoclonal antibodies were detected with a biotinylated anti-mouse made in rabbit, rat monoclonal antibodies with a biotinylated anti-rat made in mouse and polyclonal antibodies were detected with biotinylated horse anti-goat (IL-10) and goat anti-rabbit (IL-10R; Vector Laboratories), using streptavidin–peroxidase and 3,3′-diaminobenzidine (Sigma Chemical Co.) or aminoethyl carbazole (Zymed Laboratories, Inc.) as chromogens.

Techniques: Immunohistochemical staining, Produced, Transfection, Activity Assay, Blocking Assay