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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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Evaluation of rcaIL-2 expression and affinity purification by SDS-PAGE and Western blot. A. Samples analysed in 15% polyacrilamide gel stained with Commassie blue. Lane 1, Molecular weight markers; Lane 2, E. coli BL21(DE3)pLysS-pRSET without insert; Lane 3, BL21(DE3)pLysS-pRSET-caIL-2; Lane 4, affinity purified rcaIL-2-F43L. B. Samples evaluated by Western blot developed with an anti-His tag monoclonal antibody conjugated with alkaline phosphatase. Lane 1, E. coli BL21(DE3)pLysS-pRSET without insert; Lane 2, BL21(DE3)pLysS-pRSET-caIL-2; Lane 3, affinity purified rcaIL-2-F43L; and Lane 4, affinity purified His-tagged enhanced green fluorescent protein (EGFP, positive control). Arrows and arrow head indicate rcaIL-2F43L and EGFP bands, respectively.
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Figure 1: Evaluation of rcaIL-2 expression and affinity purification by SDS-PAGE and Western blot. A. Samples analysed in 15% polyacrilamide gel stained with Commassie blue. Lane 1, Molecular weight markers; Lane 2, E. coli BL21(DE3)pLysS-pRSET without insert; Lane 3, BL21(DE3)pLysS-pRSET-caIL-2; Lane 4, affinity purified rcaIL-2-F43L. B. Samples evaluated by Western blot developed with an anti-His tag monoclonal antibody conjugated with alkaline phosphatase. Lane 1, E. coli BL21(DE3)pLysS-pRSET without insert; Lane 2, BL21(DE3)pLysS-pRSET-caIL-2; Lane 3, affinity purified rcaIL-2-F43L; and Lane 4, affinity purified His-tagged enhanced green fluorescent protein (EGFP, positive control). Arrows and arrow head indicate rcaIL-2F43L and EGFP bands, respectively.

Mentions: Canine rIL-2-F43L with an N-terminus histidine tag, produced in E. coli and purified by affinity chromatography, displayed a single band with a molecular weight of 19.3 kDa in SDS-PAGE and Western blot analysis (Figure 1).


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)

Evaluation of rcaIL-2 expression and affinity purification by SDS-PAGE and Western blot. A. Samples analysed in 15% polyacrilamide gel stained with Commassie blue. Lane 1, Molecular weight markers; Lane 2, E. coli BL21(DE3)pLysS-pRSET without insert; Lane 3, BL21(DE3)pLysS-pRSET-caIL-2; Lane 4, affinity purified rcaIL-2-F43L. B. Samples evaluated by Western blot developed with an anti-His tag monoclonal antibody conjugated with alkaline phosphatase. Lane 1, E. coli BL21(DE3)pLysS-pRSET without insert; Lane 2, BL21(DE3)pLysS-pRSET-caIL-2; Lane 3, affinity purified rcaIL-2-F43L; and Lane 4, affinity purified His-tagged enhanced green fluorescent protein (EGFP, positive control). Arrows and arrow head indicate rcaIL-2F43L and EGFP bands, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109786&req=5

Figure 1: Evaluation of rcaIL-2 expression and affinity purification by SDS-PAGE and Western blot. A. Samples analysed in 15% polyacrilamide gel stained with Commassie blue. Lane 1, Molecular weight markers; Lane 2, E. coli BL21(DE3)pLysS-pRSET without insert; Lane 3, BL21(DE3)pLysS-pRSET-caIL-2; Lane 4, affinity purified rcaIL-2-F43L. B. Samples evaluated by Western blot developed with an anti-His tag monoclonal antibody conjugated with alkaline phosphatase. Lane 1, E. coli BL21(DE3)pLysS-pRSET without insert; Lane 2, BL21(DE3)pLysS-pRSET-caIL-2; Lane 3, affinity purified rcaIL-2-F43L; and Lane 4, affinity purified His-tagged enhanced green fluorescent protein (EGFP, positive control). Arrows and arrow head indicate rcaIL-2F43L and EGFP bands, respectively.
Mentions: Canine rIL-2-F43L with an N-terminus histidine tag, produced in E. coli and purified by affinity chromatography, displayed a single band with a molecular weight of 19.3 kDa in SDS-PAGE and Western blot analysis (Figure 1).

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