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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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Related in: MedlinePlus

Evaluation of proliferation of canine peripheral blood mononuclear cells (PBMC) stimulated with recombinant canine IL-2 produced by E. coli or COS-7 cells. PBMC of 6 adult mongrel dogs were cultured for 4 to 14 days with: (A) affinity purified rcaIL-2-F43L produced in E. coli at 50 ng/mL (squares) or excipient (circles); (B, C, and D) supernatant of COS-7 cells transfected with either pcDNA3.1 without insert (circles) or pcDNA3.1-caIL-2 (squares) at 0.5% (B), 5% (C) or 50% (D). The cells were pulsed with 3H-thymidine for 18 h. The data represent the median of the CPM obtained for the six animals and the bars the 25 and 75 percentiles. *p < 0.05, **p < 0.01; Dunn’s post-test (p < 0.001, Friedman’s test).
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Figure 3: Evaluation of proliferation of canine peripheral blood mononuclear cells (PBMC) stimulated with recombinant canine IL-2 produced by E. coli or COS-7 cells. PBMC of 6 adult mongrel dogs were cultured for 4 to 14 days with: (A) affinity purified rcaIL-2-F43L produced in E. coli at 50 ng/mL (squares) or excipient (circles); (B, C, and D) supernatant of COS-7 cells transfected with either pcDNA3.1 without insert (circles) or pcDNA3.1-caIL-2 (squares) at 0.5% (B), 5% (C) or 50% (D). The cells were pulsed with 3H-thymidine for 18 h. The data represent the median of the CPM obtained for the six animals and the bars the 25 and 75 percentiles. *p < 0.05, **p < 0.01; Dunn’s post-test (p < 0.001, Friedman’s test).

Mentions: To determine if the rcaIL-2 produced in E. coli or the COS-7 caIL-2 SN would promote proliferation of resting lymphocytes, PBMC from six healthy dogs were cultured for 4 to 14 days with rcaIL-2-F43L at 50 ng/mL or COS-7 caIL-2 SN at 0.5, 5 or 50% and thymidine incorporation was measured. Whereas PBMC cultured in complete RPMI medium alone, used to determine the basal proliferation, presented low incorporation of thymidine, with CPM values ranging from M = 9 to M = 31 along the time points assessed, cells cultured with rcaIL-2-F43L displayed significantly higher CPM values at days 8 (M = 3,542), 10 (M = 7,854) and 12 (M = 11,928), corresponding to an increase of up to 918 times above the basal levels (p < 0.05, Dunn’s test; Figure 3A). Moreover, the PBMC incubated with COS-7 caIL-2 SN showed significantly higher thymidine incorporations when tested at 0.5% for 8 days (M = 237 vs 14 for the control) and 12 days (262 vs 10) , 5% for 6 days (388 vs 22), 8 days (2,161 vs 26) and 10 days (2,375 vs 28), and 50% for 8 days (780 vs 26) and 10 days (735 vs 32), reaching up to an 87-fold increase, as compared with the COS-7 SN-negative control (Dunn’s test p <0.05, Figure 3B, C and D).


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 proliferation of canine peripheral blood mononuclear cells (PBMC) stimulated with recombinant canine IL-2 produced by E. coli or COS-7 cells. PBMC of 6 adult mongrel dogs were cultured for 4 to 14 days with: (A) affinity purified rcaIL-2-F43L produced in E. coli at 50 ng/mL (squares) or excipient (circles); (B, C, and D) supernatant of COS-7 cells transfected with either pcDNA3.1 without insert (circles) or pcDNA3.1-caIL-2 (squares) at 0.5% (B), 5% (C) or 50% (D). The cells were pulsed with 3H-thymidine for 18 h. The data represent the median of the CPM obtained for the six animals and the bars the 25 and 75 percentiles. *p < 0.05, **p < 0.01; Dunn’s post-test (p < 0.001, Friedman’s test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Figure 3: Evaluation of proliferation of canine peripheral blood mononuclear cells (PBMC) stimulated with recombinant canine IL-2 produced by E. coli or COS-7 cells. PBMC of 6 adult mongrel dogs were cultured for 4 to 14 days with: (A) affinity purified rcaIL-2-F43L produced in E. coli at 50 ng/mL (squares) or excipient (circles); (B, C, and D) supernatant of COS-7 cells transfected with either pcDNA3.1 without insert (circles) or pcDNA3.1-caIL-2 (squares) at 0.5% (B), 5% (C) or 50% (D). The cells were pulsed with 3H-thymidine for 18 h. The data represent the median of the CPM obtained for the six animals and the bars the 25 and 75 percentiles. *p < 0.05, **p < 0.01; Dunn’s post-test (p < 0.001, Friedman’s test).
Mentions: To determine if the rcaIL-2 produced in E. coli or the COS-7 caIL-2 SN would promote proliferation of resting lymphocytes, PBMC from six healthy dogs were cultured for 4 to 14 days with rcaIL-2-F43L at 50 ng/mL or COS-7 caIL-2 SN at 0.5, 5 or 50% and thymidine incorporation was measured. Whereas PBMC cultured in complete RPMI medium alone, used to determine the basal proliferation, presented low incorporation of thymidine, with CPM values ranging from M = 9 to M = 31 along the time points assessed, cells cultured with rcaIL-2-F43L displayed significantly higher CPM values at days 8 (M = 3,542), 10 (M = 7,854) and 12 (M = 11,928), corresponding to an increase of up to 918 times above the basal levels (p < 0.05, Dunn’s test; Figure 3A). Moreover, the PBMC incubated with COS-7 caIL-2 SN showed significantly higher thymidine incorporations when tested at 0.5% for 8 days (M = 237 vs 14 for the control) and 12 days (262 vs 10) , 5% for 6 days (388 vs 22), 8 days (2,161 vs 26) and 10 days (2,375 vs 28), and 50% for 8 days (780 vs 26) and 10 days (735 vs 32), reaching up to an 87-fold increase, as compared with the COS-7 SN-negative control (Dunn’s test p <0.05, Figure 3B, C and D).

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