CEFYBO   02669
CENTRO DE ESTUDIOS FARMACOLOGICOS Y BOTANICOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
PF-MC: a new human anti-inflammatory fusion protein engineered to target an inflammatory site
Autor/es:
MAFFIA, PAULO; BOGADO, BETIANA; TATEOSIAN, NANCY; GUERRIERI, DIEGO; AMIANO, NICOLAS; SANCHEZ, MERCEDES; CHULUYAN, HECTOR
Lugar:
Buenos Aires
Reunión:
Congreso; First French-Argentine Immunology Congress. LVIII Reunión Anual de la Sociedad Argentina de INmunología; 2010
Institución organizadora:
Sociedad Argentina de Inmunología
Resumen:
&lt;!-- /* Font Definitions */ @font-face {font-family:"Arial Narrow"; panose-1:2 11 5 6 2 2 2 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:647 0 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --&gt; Secretory leukocyte protease inhibitor (SLPI) is a protein with anti-microbial, anti-inflammatory and wound healing activity. In vivo, SLPI is susceptible to proteolytic degradation. In order to improve the therapeutic capacity of SLPI increasing the concentration at the inflammatory site, we constructed a fusion protein comprising an anchoring peptide domain named cementoin and human SLPI. Cementoin is a substrate of the enzyme transglutaminase (TG). TG is upregulated at the inflammatory site, crosslinking proteins on the cell membrane and the extrecellular matrix. PF-MC, was cloned and expressed in E. coli. The anti proteinase activity of PF-MC was 35±5 % higher than SLPI (p<0.001). The in vitro binding of PF-MC on TNFalpha treated-A549 cell surface was analyzed by ELISA and fluorescence microscopy. The binding of PF-MC to A549 cells was higher compared to SLPI (p<0.05). In vivo, binding of the PF-MC was examined by using an inflammatory air pouch model on mice. PF-MC or SLPI (52 µg) were inoculated into the air pouch; inflammatory recruited cells were recovered after 2 h of treatment and PF-MC was detected on the inflammatory cell surface by flow cytometry. We observed PF-MC on lymphocytes and monocytes, but not on neutrophils. The binding was higher for PF-MC than SLPI (9±3 vs 4±1%; 31±7 vs 10±4% of  lymphocytes and monocytes, respectively). A higher binding of PF-MC on lymphocytes was confirmed by analysing the cells of the popliteal lymph node that were injected with LPS (in the foot pad) and PF-MC (i.p.). The biological activities of PF-MC were similar or improved than SLPI: the wound healing activity was augmented, since the diameter of the wound, measure at day 2 post-injury, was 35±8 % smaller in mice injected with PF-MC vs. SLPI. These results demonstrate that PF-MC binds to inflammatory cells both in vitro and in vivo, retains the biological activity  of SLPI and, in some cases, improve it. Therefore, PF-MC may be a new tool to treat inflammatory diseases