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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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Related in: MedlinePlus

Structure and production of RVFP/IL-1ra by S. gordonii. A. Structure of RVFP/IL-1ra produced by S. gordonii. By using the streptococcal M6 protein as the fusion partner, a recombinant protein composed of the M6 signal peptide (42 amino acids; hatched), the first four amino acids of M6 (RVFP; black), and the mature human IL-1ra (152 amino acids; white) was produced. After cleavage of the M6 leader peptide, RVFP/IL-1ra (156 amino acids) is secreted into the culture medium. A schematic representation of the different parts of the fusion protein is shown in the upper part of the figure. B. Immunoblot analysis of total proteins present in the culture supernatant of S. gordonii. Culture supernatants of the control GP204 strain (lane 1) and of the RVFP/IL-1ra-producing GP1300 strain (lane 2) were precipitated with TCA, separated by 15% SDS-PAGE, and reacted with a polyclonal antibody to human IL-1ra. Positions of molecular mass standards are indicated to the left.
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Figure 1: Structure and production of RVFP/IL-1ra by S. gordonii. A. Structure of RVFP/IL-1ra produced by S. gordonii. By using the streptococcal M6 protein as the fusion partner, a recombinant protein composed of the M6 signal peptide (42 amino acids; hatched), the first four amino acids of M6 (RVFP; black), and the mature human IL-1ra (152 amino acids; white) was produced. After cleavage of the M6 leader peptide, RVFP/IL-1ra (156 amino acids) is secreted into the culture medium. A schematic representation of the different parts of the fusion protein is shown in the upper part of the figure. B. Immunoblot analysis of total proteins present in the culture supernatant of S. gordonii. Culture supernatants of the control GP204 strain (lane 1) and of the RVFP/IL-1ra-producing GP1300 strain (lane 2) were precipitated with TCA, separated by 15% SDS-PAGE, and reacted with a polyclonal antibody to human IL-1ra. Positions of molecular mass standards are indicated to the left.

Mentions: By using the streptococcal M6 protein as the fusion partner [31], an S. gordonii strain secreting human IL-1ra into the culture medium was constructed and denominated GP1300. The presence of the emm6 signal sequence at the N-terminus and of two stop codons at the C-terminus of the fusion allowed translocation and secretion of the protein, respectively. After cleavage of the M6 leader peptide (42 amino acids), the recombinant IL-1ra consisted in the first four amino acid residues of M6 (RVFP) followed by 152 amino acids of the mature human IL-1ra. The resulting fusion protein (156 amino acids) was thus denominated RVFP/IL-1ra (Figure 1A). Production of RVFP/IL-1ra by GP1300 was assessed by Western blot analysis of trichloroacetic acid (TCA)-precipitated proteins released into the culture medium using IL-1ra-specific polyclonal antibodies. A protein band of approximately 19 kDa (the expected molecular mass of the fusion protein is 17.2 kDa) was detected in the GP1300 sample, while no reactivity was observed in the control sample (Figure 1B). The amount of recombinant IL-1ra secreted by S. gordonii was 0.1 mg per litre, as estimated by ELISA. These results indicate that S. gordonii is a suitable system to produce recombinant human IL-1ra.


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)

Structure and production of RVFP/IL-1ra by S. gordonii. A. Structure of RVFP/IL-1ra produced by S. gordonii. By using the streptococcal M6 protein as the fusion partner, a recombinant protein composed of the M6 signal peptide (42 amino acids; hatched), the first four amino acids of M6 (RVFP; black), and the mature human IL-1ra (152 amino acids; white) was produced. After cleavage of the M6 leader peptide, RVFP/IL-1ra (156 amino acids) is secreted into the culture medium. A schematic representation of the different parts of the fusion protein is shown in the upper part of the figure. B. Immunoblot analysis of total proteins present in the culture supernatant of S. gordonii. Culture supernatants of the control GP204 strain (lane 1) and of the RVFP/IL-1ra-producing GP1300 strain (lane 2) were precipitated with TCA, separated by 15% SDS-PAGE, and reacted with a polyclonal antibody to human IL-1ra. Positions of molecular mass standards are indicated to the left.
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Figure 1: Structure and production of RVFP/IL-1ra by S. gordonii. A. Structure of RVFP/IL-1ra produced by S. gordonii. By using the streptococcal M6 protein as the fusion partner, a recombinant protein composed of the M6 signal peptide (42 amino acids; hatched), the first four amino acids of M6 (RVFP; black), and the mature human IL-1ra (152 amino acids; white) was produced. After cleavage of the M6 leader peptide, RVFP/IL-1ra (156 amino acids) is secreted into the culture medium. A schematic representation of the different parts of the fusion protein is shown in the upper part of the figure. B. Immunoblot analysis of total proteins present in the culture supernatant of S. gordonii. Culture supernatants of the control GP204 strain (lane 1) and of the RVFP/IL-1ra-producing GP1300 strain (lane 2) were precipitated with TCA, separated by 15% SDS-PAGE, and reacted with a polyclonal antibody to human IL-1ra. Positions of molecular mass standards are indicated to the left.
Mentions: By using the streptococcal M6 protein as the fusion partner [31], an S. gordonii strain secreting human IL-1ra into the culture medium was constructed and denominated GP1300. The presence of the emm6 signal sequence at the N-terminus and of two stop codons at the C-terminus of the fusion allowed translocation and secretion of the protein, respectively. After cleavage of the M6 leader peptide (42 amino acids), the recombinant IL-1ra consisted in the first four amino acid residues of M6 (RVFP) followed by 152 amino acids of the mature human IL-1ra. The resulting fusion protein (156 amino acids) was thus denominated RVFP/IL-1ra (Figure 1A). Production of RVFP/IL-1ra by GP1300 was assessed by Western blot analysis of trichloroacetic acid (TCA)-precipitated proteins released into the culture medium using IL-1ra-specific polyclonal antibodies. A protein band of approximately 19 kDa (the expected molecular mass of the fusion protein is 17.2 kDa) was detected in the GP1300 sample, while no reactivity was observed in the control sample (Figure 1B). The amount of recombinant IL-1ra secreted by S. gordonii was 0.1 mg per litre, as estimated by ELISA. These results indicate that S. gordonii is a suitable system to produce recombinant human IL-1ra.

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