Es in the tested groups. (*) P values less than or equal

Es in the tested groups. (*) P values less than or equal to 0.02 were considered significant.In vivo inhibition of complement activationTo evaluate the ability of MB12/22 to prevent complementmediated tissue damage by inhibiting C5 cleavage, a model of antigen-induced arthritis (AIA) obtained by injecting (mBSA) in the rat knee was used. This experimental model was selectedResults In vitro characterization of DNA encoding for CI-1011 anti-C5 neutralizing antibodyWe have efficiently produced scFv-Fc Anlotinib fusion proteins in the supernatant of transient and stable transfected HEK 293T or CHO-S cells [18,22,23,24,25]. More recently we have improve this strategy by using an optimized UCOE containing vector [20] that guarantees both a longer expression as well as an increased production yield of the recombinant protein of interest. In this study we selected the scFv TSA12/22 directed to C5 complement component. This scFv is able to neutralize the complement component C5 from man, and various animal species including rat, mouse and rabbit. The scFv was expressed as a scFv-Fc fusion protein containing the rat Fc region. In order to compare the performance of the standard pMB vector with the UCOE containing vector, CHO-S cells were transfected with both and the recombinant antibody released in the culture medium was tested for its ability to recognize C5. The results presented in Fig 1A confirm that both recombinant antibodies were able toFigure 3. Effect of the intraarticular injection of DNA in the production of MB12/22. Rats were treated with either DMRI-C + MB12/22 DNA or saline as a negative control. Three days later, the animals were euthanized and the synovial tissue was analyzed. Note that the synovial tissue exhibit essentially a normal histologic structure in both groups of rats. doi:10.1371/journal.pone.0058696.gAnti-C5 DNA Therapy for Arthritis PreventionFigure 4. Effect of DNA encoding MB12/22 on the activation of the complement system in the model of antigen-induced arthritis in rats. Immunofluorescence analysis of synovial membranes for deposition of C3 and C9 obtained from rats receiving mBSA 3 days after intraarticular injection of Saline (indicated as BSA), DMRI-C + MB12/22 DNA or DMRI-C alone. The animals were sacrificed 3 days after arthritis induction and samples of synovial tissue were analyzed for deposition of C3 and C9. Saline-treated groups represent a negative control group in order to show the absence of complement activation. Note that while C3 is detected in the synovium of mBSA + saline, mBSA + DMRI-C containing control DNA animals, we failed to reveal binding of C9 on the synovial tissue of DMRI-C + MB12/22 DNA-treated rats. The graph in the lower part of the figure shows the mean fluorescence intensity (MFI) of the images 6 SD. for each experimental group. (*): P values less than or equal to 0.02 were considered significant. Pictures were taken at original magnification 200X. doi:10.1371/journal.pone.0058696.gbecause the inflammatory process is restricted to a single joint enabling the comparison of the biochemical and structural changes with those of the unaffected controlateral joint. In addition, this model of monoarthritis is characterized by a `well defined’ time of onset, with the development of joint inflammation a few hours after intraarticular injection of mBSA [16,18,22]. Based on the finding that the production of MB12/22 reaches its peak 3 days after DNA administration (Figure 2), we decided to inject the DNA-conj.Es in the tested groups. (*) P values less than or equal to 0.02 were considered significant.In vivo inhibition of complement activationTo evaluate the ability of MB12/22 to prevent complementmediated tissue damage by inhibiting C5 cleavage, a model of antigen-induced arthritis (AIA) obtained by injecting (mBSA) in the rat knee was used. This experimental model was selectedResults In vitro characterization of DNA encoding for anti-C5 neutralizing antibodyWe have efficiently produced scFv-Fc fusion proteins in the supernatant of transient and stable transfected HEK 293T or CHO-S cells [18,22,23,24,25]. More recently we have improve this strategy by using an optimized UCOE containing vector [20] that guarantees both a longer expression as well as an increased production yield of the recombinant protein of interest. In this study we selected the scFv TSA12/22 directed to C5 complement component. This scFv is able to neutralize the complement component C5 from man, and various animal species including rat, mouse and rabbit. The scFv was expressed as a scFv-Fc fusion protein containing the rat Fc region. In order to compare the performance of the standard pMB vector with the UCOE containing vector, CHO-S cells were transfected with both and the recombinant antibody released in the culture medium was tested for its ability to recognize C5. The results presented in Fig 1A confirm that both recombinant antibodies were able toFigure 3. Effect of the intraarticular injection of DNA in the production of MB12/22. Rats were treated with either DMRI-C + MB12/22 DNA or saline as a negative control. Three days later, the animals were euthanized and the synovial tissue was analyzed. Note that the synovial tissue exhibit essentially a normal histologic structure in both groups of rats. doi:10.1371/journal.pone.0058696.gAnti-C5 DNA Therapy for Arthritis PreventionFigure 4. Effect of DNA encoding MB12/22 on the activation of the complement system in the model of antigen-induced arthritis in rats. Immunofluorescence analysis of synovial membranes for deposition of C3 and C9 obtained from rats receiving mBSA 3 days after intraarticular injection of Saline (indicated as BSA), DMRI-C + MB12/22 DNA or DMRI-C alone. The animals were sacrificed 3 days after arthritis induction and samples of synovial tissue were analyzed for deposition of C3 and C9. Saline-treated groups represent a negative control group in order to show the absence of complement activation. Note that while C3 is detected in the synovium of mBSA + saline, mBSA + DMRI-C containing control DNA animals, we failed to reveal binding of C9 on the synovial tissue of DMRI-C + MB12/22 DNA-treated rats. The graph in the lower part of the figure shows the mean fluorescence intensity (MFI) of the images 6 SD. for each experimental group. (*): P values less than or equal to 0.02 were considered significant. Pictures were taken at original magnification 200X. doi:10.1371/journal.pone.0058696.gbecause the inflammatory process is restricted to a single joint enabling the comparison of the biochemical and structural changes with those of the unaffected controlateral joint. In addition, this model of monoarthritis is characterized by a `well defined’ time of onset, with the development of joint inflammation a few hours after intraarticular injection of mBSA [16,18,22]. Based on the finding that the production of MB12/22 reaches its peak 3 days after DNA administration (Figure 2), we decided to inject the DNA-conj.