Limits...
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.

Show MeSH

Related in: MedlinePlus

Evaluation of CTLL-2 proliferation following stimulation with recombinant canine IL-2 produced by E. coli or COS-7 cells. CTLL-2 cells were cultured for 48 h with: (A) different concentrations of affinity purified rcaIL-2-F43L produced in E. coli or (B) different concentrations of supernatant of COS-7 cells transfected with pcDNA3.1 without insert (circles) or pcDNA3.1-caIL-2 (squares). The cells were pulsed with 3H-thymidine for 24 h. The data represent the median of CPM of triplicates and the bars the 25 and 75 percentiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Evaluation of CTLL-2 proliferation following stimulation with recombinant canine IL-2 produced by E. coli or COS-7 cells. CTLL-2 cells were cultured for 48 h with: (A) different concentrations of affinity purified rcaIL-2-F43L produced in E. coli or (B) different concentrations of supernatant of COS-7 cells transfected with pcDNA3.1 without insert (circles) or pcDNA3.1-caIL-2 (squares). The cells were pulsed with 3H-thymidine for 24 h. The data represent the median of CPM of triplicates and the bars the 25 and 75 percentiles.

Mentions: To assess whether the canine rcaIL-2-F43L subjected to refolding or the COS-7 caIL-2 SN would present biological activity, the CTLL-2 cell proliferation assay was performed, and median (M) values of CPM, that are related to thymidine incorporation, were used to analyze the results. CTLL-2 cells cultured in supplemented RPMI 1640 medium alone (negative control) or stimulated with 10% T-STIM (positive control) showed no (M = 14.0) or high (M = 73,166) proliferation activity, respectively. CTLL-2 cells cultured with rcaIL-2-F43L in concentrations ranging from 0.22 ng/mL (M = 413) to 700 ng/mL (M = 3,239) displayed higher proliferative response than cells incubated with supplemented RPMI 1640 alone (Figure 2A). However, the proliferation peak was reached when rcaIL-2-F43L was used at 140 ng/mL (M = 20,380) (Figure 2A).


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 CTLL-2 proliferation following stimulation with recombinant canine IL-2 produced by E. coli or COS-7 cells. CTLL-2 cells were cultured for 48 h with: (A) different concentrations of affinity purified rcaIL-2-F43L produced in E. coli or (B) different concentrations of supernatant of COS-7 cells transfected with pcDNA3.1 without insert (circles) or pcDNA3.1-caIL-2 (squares). The cells were pulsed with 3H-thymidine for 24 h. The data represent the median of CPM of triplicates and the bars the 25 and 75 percentiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Evaluation of CTLL-2 proliferation following stimulation with recombinant canine IL-2 produced by E. coli or COS-7 cells. CTLL-2 cells were cultured for 48 h with: (A) different concentrations of affinity purified rcaIL-2-F43L produced in E. coli or (B) different concentrations of supernatant of COS-7 cells transfected with pcDNA3.1 without insert (circles) or pcDNA3.1-caIL-2 (squares). The cells were pulsed with 3H-thymidine for 24 h. The data represent the median of CPM of triplicates and the bars the 25 and 75 percentiles.
Mentions: To assess whether the canine rcaIL-2-F43L subjected to refolding or the COS-7 caIL-2 SN would present biological activity, the CTLL-2 cell proliferation assay was performed, and median (M) values of CPM, that are related to thymidine incorporation, were used to analyze the results. CTLL-2 cells cultured in supplemented RPMI 1640 medium alone (negative control) or stimulated with 10% T-STIM (positive control) showed no (M = 14.0) or high (M = 73,166) proliferation activity, respectively. CTLL-2 cells cultured with rcaIL-2-F43L in concentrations ranging from 0.22 ng/mL (M = 413) to 700 ng/mL (M = 3,239) displayed higher proliferative response than cells incubated with supplemented RPMI 1640 alone (Figure 2A). However, the proliferation peak was reached when rcaIL-2-F43L was used at 140 ng/mL (M = 20,380) (Figure 2A).

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