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Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells.

Pereira AM, de Pinheiro CG, Dos Santos LR, Teixeira NC, Chang YF, Pontes-de-Carvalho LC, de Sá Oliveira GG - BMC Res Notes (2014)

Bottom Line: Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC.However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells.The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Patologia e Bio-Intervenção, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão, No, 121, Candeal, Salvador, Bahia, Brazil. ggileno@bahia.fiocruz.br.

ABSTRACT

Background: Very few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs.

Results: Recombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells.

Conclusions: The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs.

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Interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) stimulated with recombinant canine IL-2 or IL-12 produced by COS-7 cells. PBMC of 8 adult mongrel dogs were cultured for 48 h with: complete RPMI 1640 medium alone (M), concanavalin A at 5 mg/mL, supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (SN IL-2) at 25% or supernatant of COS-7 cells transfected with pcDNA3.1-sccaIL-12 (SN IL-12) at 0.025, 0.5 or 10%. IFN-γ was assessed in the PBMC supernatants by capture ELISA. Data represent average of duplicates and bars the means obtained for the eight animals. *p < 0.01, Dunnett’s test (p < 0.0001, repeated measures ANOVA).
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Figure 4: Interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) stimulated with recombinant canine IL-2 or IL-12 produced by COS-7 cells. PBMC of 8 adult mongrel dogs were cultured for 48 h with: complete RPMI 1640 medium alone (M), concanavalin A at 5 mg/mL, supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (SN IL-2) at 25% or supernatant of COS-7 cells transfected with pcDNA3.1-sccaIL-12 (SN IL-12) at 0.025, 0.5 or 10%. IFN-γ was assessed in the PBMC supernatants by capture ELISA. Data represent average of duplicates and bars the means obtained for the eight animals. *p < 0.01, Dunnett’s test (p < 0.0001, repeated measures ANOVA).

Mentions: To evaluate the ability of the recombinant IL-12 and/or IL-2 to stimulate the production of IFN-γ by canine cells, PBMCs from 8 healthy dogs were cultured with different concentrations of COS-7 sccaIL-12 SN combined or not with various concentrations of COS-7 caIL-2 SN for 48 h. The concentration of IFN-γ (mean ± standard deviation) in the supernatant of PMBC cultures carried out with complete RPMI 1640 alone or with the addition of 5 μg/mL Con A was 0.19 ± 0.26 and 30.08 ± 22.56 ng/mL, respectively. PBMC stimulated with only COS-7 sccaIL-12 SN at 0.025% (1.56 ± 2.31 ng/mL), 0.5% (2.36 ± 3.74 ng/mL), 10% (2.14 ± 2.50 ng/mL) or COS-7 IL-2 SN at 25% (0.52 ± 0.93 ng/mL) produced a greater amount of IFN-γ compared with the cells cultivated with complete RPMI 1640 alone. However, the differences were not significant, probably because only PBMC of 3 or 4 out of 8 dogs and only 1 out of 8 dogs responded to the stimulus of IL-12 and IL-2, respectively (Figure 4). Nevertheless, when PBMC were stimulated with a combination of COS-7 sccaIL-12 SN at either 0.025% (11.34 ± 11.09 ng/mL), 0.5 (13.69 ± 12.62 ng/mL) or 10% (12.73 ± 13.57) and COS-7 caIL-2 SN at 25% or with a combination of COS-7 sccaIL-12 SN at either 0.025 (9,22 ± 10,70 ng/mL) or 0.5% (16,33 ± 16,69 ng/mL) and COS-7 caIL-2 SN at 5%, a significant higher production of IFN-γ was detected (p < 0.05, Dunnett’s test; Figure 5). The combined incitation of IL-12 and IL-2 resulted in the response of 6 or 7 out of 8 dogs.


Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells.

Pereira AM, de Pinheiro CG, Dos Santos LR, Teixeira NC, Chang YF, Pontes-de-Carvalho LC, de Sá Oliveira GG - BMC Res Notes (2014)

Interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) stimulated with recombinant canine IL-2 or IL-12 produced by COS-7 cells. PBMC of 8 adult mongrel dogs were cultured for 48 h with: complete RPMI 1640 medium alone (M), concanavalin A at 5 mg/mL, supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (SN IL-2) at 25% or supernatant of COS-7 cells transfected with pcDNA3.1-sccaIL-12 (SN IL-12) at 0.025, 0.5 or 10%. IFN-γ was assessed in the PBMC supernatants by capture ELISA. Data represent average of duplicates and bars the means obtained for the eight animals. *p < 0.01, Dunnett’s test (p < 0.0001, repeated measures ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) stimulated with recombinant canine IL-2 or IL-12 produced by COS-7 cells. PBMC of 8 adult mongrel dogs were cultured for 48 h with: complete RPMI 1640 medium alone (M), concanavalin A at 5 mg/mL, supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (SN IL-2) at 25% or supernatant of COS-7 cells transfected with pcDNA3.1-sccaIL-12 (SN IL-12) at 0.025, 0.5 or 10%. IFN-γ was assessed in the PBMC supernatants by capture ELISA. Data represent average of duplicates and bars the means obtained for the eight animals. *p < 0.01, Dunnett’s test (p < 0.0001, repeated measures ANOVA).
Mentions: To evaluate the ability of the recombinant IL-12 and/or IL-2 to stimulate the production of IFN-γ by canine cells, PBMCs from 8 healthy dogs were cultured with different concentrations of COS-7 sccaIL-12 SN combined or not with various concentrations of COS-7 caIL-2 SN for 48 h. The concentration of IFN-γ (mean ± standard deviation) in the supernatant of PMBC cultures carried out with complete RPMI 1640 alone or with the addition of 5 μg/mL Con A was 0.19 ± 0.26 and 30.08 ± 22.56 ng/mL, respectively. PBMC stimulated with only COS-7 sccaIL-12 SN at 0.025% (1.56 ± 2.31 ng/mL), 0.5% (2.36 ± 3.74 ng/mL), 10% (2.14 ± 2.50 ng/mL) or COS-7 IL-2 SN at 25% (0.52 ± 0.93 ng/mL) produced a greater amount of IFN-γ compared with the cells cultivated with complete RPMI 1640 alone. However, the differences were not significant, probably because only PBMC of 3 or 4 out of 8 dogs and only 1 out of 8 dogs responded to the stimulus of IL-12 and IL-2, respectively (Figure 4). Nevertheless, when PBMC were stimulated with a combination of COS-7 sccaIL-12 SN at either 0.025% (11.34 ± 11.09 ng/mL), 0.5 (13.69 ± 12.62 ng/mL) or 10% (12.73 ± 13.57) and COS-7 caIL-2 SN at 25% or with a combination of COS-7 sccaIL-12 SN at either 0.025 (9,22 ± 10,70 ng/mL) or 0.5% (16,33 ± 16,69 ng/mL) and COS-7 caIL-2 SN at 5%, a significant higher production of IFN-γ was detected (p < 0.05, Dunnett’s test; Figure 5). The combined incitation of IL-12 and IL-2 resulted in the response of 6 or 7 out of 8 dogs.

Bottom Line: Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC.However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells.The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Patologia e Bio-Intervenção, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão, No, 121, Candeal, Salvador, Bahia, Brazil. ggileno@bahia.fiocruz.br.

ABSTRACT

Background: Very few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs.

Results: Recombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells.

Conclusions: The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs.

Show MeSH
Related in: MedlinePlus