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In vivo mucosal delivery of bioactive human interleukin 1 receptor antagonist produced by Streptococcus gordonii.

Ricci S, Macchia G, Ruggiero P, Maggi T, Bossù P, Xu L, Medaglini D, Tagliabue A, Hammarström L, Pozzi G, Boraschi D - BMC Biotechnol. (2003)

Bottom Line: RFVP/IL-1ra displayed full biological activity in vitro in assays of inhibition of IL-1beta-induced lymphocyte proliferation and was released by recombinant S. gordonii in vivo both at the vaginal and the gastrointestinal mucosa of mice.RFVP/IL-1ra appeared beneficial in the model of ulcerative colitis represented by IL-2-/- mice (knock-out for the interleukin-2 gene), as shown by the body weight increase of IL-2-/- mice locally treated with S. gordonii producing RFVP/IL-1ra.These results indicate that recombinant S. gordonii can be successfully used as a delivery system for the selective targeting of mucosal surfaces with therapeutic proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Microbiology and Biotechnology, Department of Molecular Biology, University of Siena, Policlinico Le Scotte, Viale Bracci, 53100 Siena, Italy. riccisus@unisi.it

ABSTRACT

Background: Interleukin-1 (IL-1) is a cytokine involved in the initiation and amplification of the defence response in infectious and inflammatory diseases. IL-1 receptor antagonist (IL-1ra) is an inactive member of the IL-1 family and represents one of the most potent mechanisms for controlling IL-1-dependent inflammation. IL-1ra has proven effective in the therapy of acute and chronic inflammatory diseases in experimental animal models and also in preliminary clinical trials. However, optimisation of therapeutic schedules is still needed. For instance, the use of drug delivery systems targeting specific mucosal sites may be useful to improve topical bioavailability and avoid side effects associated with systemic administration.

Results: In order to develop systems for the delivery of IL-1ra to mucosal target sites, a Streptococcus gordonii strain secreting human IL-1ra was constructed. The recombinant IL-1ra produced by S. gordonii was composed of the four amino acid residues RVFP of the fusion partner at the N-terminus, followed by the mature human IL-1ra protein. RFVP/IL-1ra displayed full biological activity in vitro in assays of inhibition of IL-1beta-induced lymphocyte proliferation and was released by recombinant S. gordonii in vivo both at the vaginal and the gastrointestinal mucosa of mice. RFVP/IL-1ra appeared beneficial in the model of ulcerative colitis represented by IL-2-/- mice (knock-out for the interleukin-2 gene), as shown by the body weight increase of IL-2-/- mice locally treated with S. gordonii producing RFVP/IL-1ra.

Conclusions: These results indicate that recombinant S. gordonii can be successfully used as a delivery system for the selective targeting of mucosal surfaces with therapeutic proteins.

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Inhibition of IL-1-mediated thymocyte proliferation. A. Thymocyte proliferation induced by 0.3 ng/ml of IL-1β (β; grey bar) could be inhibited in a dose-dependent fashion by a standard preparation of wild type human IL-1ra expressed in E. coli (open triangles). The IL-1 inhibitory activity of the wild type IL-1ra reference standard was compared to that of the culture supernatant of recombinant S. gordonii GP1300 (solid triangles), which contained 0.1 mg/litre RVFP/IL-1ra (as assessed by ELISA). B. Thymocyte proliferation to 0.3 ng/ml IL-1β (β; grey bar) was inhibited in a dose-dependent fashion by both the wild type IL-1ra reference standard (open triangles) and the recombinant RVFP/IL-1ra expressed in E. coli (solid squares). Results are the mean ± SEM of 3–9 replicate determinations within single experiments, representative of five performed. SEM lower than 10 % are not shown. Background thymocyte proliferation in culture medium without IL-1β (m, open bar) is also reported.
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Figure 2: Inhibition of IL-1-mediated thymocyte proliferation. A. Thymocyte proliferation induced by 0.3 ng/ml of IL-1β (β; grey bar) could be inhibited in a dose-dependent fashion by a standard preparation of wild type human IL-1ra expressed in E. coli (open triangles). The IL-1 inhibitory activity of the wild type IL-1ra reference standard was compared to that of the culture supernatant of recombinant S. gordonii GP1300 (solid triangles), which contained 0.1 mg/litre RVFP/IL-1ra (as assessed by ELISA). B. Thymocyte proliferation to 0.3 ng/ml IL-1β (β; grey bar) was inhibited in a dose-dependent fashion by both the wild type IL-1ra reference standard (open triangles) and the recombinant RVFP/IL-1ra expressed in E. coli (solid squares). Results are the mean ± SEM of 3–9 replicate determinations within single experiments, representative of five performed. SEM lower than 10 % are not shown. Background thymocyte proliferation in culture medium without IL-1β (m, open bar) is also reported.

Mentions: In order to verify that RVFP/IL-1ra produced by the GP1300 strain retains IL-1ra-like biological activity, its ability to inhibit IL-1-mediated proliferation of murine thymocytes was assessed. As a control, a reference standard of wild type human IL-1ra expressed in Escherichia coli was used. IL-1β-induced proliferation could be inhibited in a dose-dependent fashion by both wild type IL-1ra and the culture medium of S. gordonii GP1300 (Figure 2A). Specific inhibitory activities of the GP1300 culture medium (containing 0.1 mg/litre of RVFP/IL-1ra) and of wild type IL-1ra were 1.25 × 106 Antagonist Units (AU) per mg and 1.0 × 106 AU/mg, respectively. Culture supernatant of the control S. gordonii GP204 strain did not contain measurable levels of IL-1 inhibitory activity (data not shown). In order to verify that the presence of the RVFP residues did not affect the biological activity of IL-1ra produced by S. gordonii, RVFP/IL-1ra was also expressed in E. coli, purified, and assayed for IL-1 inhibitory activity as described above. Also in this case, the dose-dependent inhibition of IL-1β-induced thymocyte proliferation by RVFP/IL-1ra was fully comparable to that of the reference standard wild type IL-1ra, with a specific inhibitory activity of approximately 1.25 × 106 AU/mg in both cases (Figure 2B). These results show that RVFP/IL-1ra is capable of inhibiting IL-1 to an extent fully comparable to that of wild type IL-1ra, indicating that the presence of RVFP at the N-terminal does not affect the biological activity of IL-1ra produced by S. gordonii.


In vivo mucosal delivery of bioactive human interleukin 1 receptor antagonist produced by Streptococcus gordonii.

Ricci S, Macchia G, Ruggiero P, Maggi T, Bossù P, Xu L, Medaglini D, Tagliabue A, Hammarström L, Pozzi G, Boraschi D - BMC Biotechnol. (2003)

Inhibition of IL-1-mediated thymocyte proliferation. A. Thymocyte proliferation induced by 0.3 ng/ml of IL-1β (β; grey bar) could be inhibited in a dose-dependent fashion by a standard preparation of wild type human IL-1ra expressed in E. coli (open triangles). The IL-1 inhibitory activity of the wild type IL-1ra reference standard was compared to that of the culture supernatant of recombinant S. gordonii GP1300 (solid triangles), which contained 0.1 mg/litre RVFP/IL-1ra (as assessed by ELISA). B. Thymocyte proliferation to 0.3 ng/ml IL-1β (β; grey bar) was inhibited in a dose-dependent fashion by both the wild type IL-1ra reference standard (open triangles) and the recombinant RVFP/IL-1ra expressed in E. coli (solid squares). Results are the mean ± SEM of 3–9 replicate determinations within single experiments, representative of five performed. SEM lower than 10 % are not shown. Background thymocyte proliferation in culture medium without IL-1β (m, open bar) is also reported.
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Figure 2: Inhibition of IL-1-mediated thymocyte proliferation. A. Thymocyte proliferation induced by 0.3 ng/ml of IL-1β (β; grey bar) could be inhibited in a dose-dependent fashion by a standard preparation of wild type human IL-1ra expressed in E. coli (open triangles). The IL-1 inhibitory activity of the wild type IL-1ra reference standard was compared to that of the culture supernatant of recombinant S. gordonii GP1300 (solid triangles), which contained 0.1 mg/litre RVFP/IL-1ra (as assessed by ELISA). B. Thymocyte proliferation to 0.3 ng/ml IL-1β (β; grey bar) was inhibited in a dose-dependent fashion by both the wild type IL-1ra reference standard (open triangles) and the recombinant RVFP/IL-1ra expressed in E. coli (solid squares). Results are the mean ± SEM of 3–9 replicate determinations within single experiments, representative of five performed. SEM lower than 10 % are not shown. Background thymocyte proliferation in culture medium without IL-1β (m, open bar) is also reported.
Mentions: In order to verify that RVFP/IL-1ra produced by the GP1300 strain retains IL-1ra-like biological activity, its ability to inhibit IL-1-mediated proliferation of murine thymocytes was assessed. As a control, a reference standard of wild type human IL-1ra expressed in Escherichia coli was used. IL-1β-induced proliferation could be inhibited in a dose-dependent fashion by both wild type IL-1ra and the culture medium of S. gordonii GP1300 (Figure 2A). Specific inhibitory activities of the GP1300 culture medium (containing 0.1 mg/litre of RVFP/IL-1ra) and of wild type IL-1ra were 1.25 × 106 Antagonist Units (AU) per mg and 1.0 × 106 AU/mg, respectively. Culture supernatant of the control S. gordonii GP204 strain did not contain measurable levels of IL-1 inhibitory activity (data not shown). In order to verify that the presence of the RVFP residues did not affect the biological activity of IL-1ra produced by S. gordonii, RVFP/IL-1ra was also expressed in E. coli, purified, and assayed for IL-1 inhibitory activity as described above. Also in this case, the dose-dependent inhibition of IL-1β-induced thymocyte proliferation by RVFP/IL-1ra was fully comparable to that of the reference standard wild type IL-1ra, with a specific inhibitory activity of approximately 1.25 × 106 AU/mg in both cases (Figure 2B). These results show that RVFP/IL-1ra is capable of inhibiting IL-1 to an extent fully comparable to that of wild type IL-1ra, indicating that the presence of RVFP at the N-terminal does not affect the biological activity of IL-1ra produced by S. gordonii.

Bottom Line: RFVP/IL-1ra displayed full biological activity in vitro in assays of inhibition of IL-1beta-induced lymphocyte proliferation and was released by recombinant S. gordonii in vivo both at the vaginal and the gastrointestinal mucosa of mice.RFVP/IL-1ra appeared beneficial in the model of ulcerative colitis represented by IL-2-/- mice (knock-out for the interleukin-2 gene), as shown by the body weight increase of IL-2-/- mice locally treated with S. gordonii producing RFVP/IL-1ra.These results indicate that recombinant S. gordonii can be successfully used as a delivery system for the selective targeting of mucosal surfaces with therapeutic proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Microbiology and Biotechnology, Department of Molecular Biology, University of Siena, Policlinico Le Scotte, Viale Bracci, 53100 Siena, Italy. riccisus@unisi.it

ABSTRACT

Background: Interleukin-1 (IL-1) is a cytokine involved in the initiation and amplification of the defence response in infectious and inflammatory diseases. IL-1 receptor antagonist (IL-1ra) is an inactive member of the IL-1 family and represents one of the most potent mechanisms for controlling IL-1-dependent inflammation. IL-1ra has proven effective in the therapy of acute and chronic inflammatory diseases in experimental animal models and also in preliminary clinical trials. However, optimisation of therapeutic schedules is still needed. For instance, the use of drug delivery systems targeting specific mucosal sites may be useful to improve topical bioavailability and avoid side effects associated with systemic administration.

Results: In order to develop systems for the delivery of IL-1ra to mucosal target sites, a Streptococcus gordonii strain secreting human IL-1ra was constructed. The recombinant IL-1ra produced by S. gordonii was composed of the four amino acid residues RVFP of the fusion partner at the N-terminus, followed by the mature human IL-1ra protein. RFVP/IL-1ra displayed full biological activity in vitro in assays of inhibition of IL-1beta-induced lymphocyte proliferation and was released by recombinant S. gordonii in vivo both at the vaginal and the gastrointestinal mucosa of mice. RFVP/IL-1ra appeared beneficial in the model of ulcerative colitis represented by IL-2-/- mice (knock-out for the interleukin-2 gene), as shown by the body weight increase of IL-2-/- mice locally treated with S. gordonii producing RFVP/IL-1ra.

Conclusions: These results indicate that recombinant S. gordonii can be successfully used as a delivery system for the selective targeting of mucosal surfaces with therapeutic proteins.

Show MeSH
Related in: MedlinePlus