Comparison of 2 simulation models for teaching obstetric anal sphincter repair.

OBJECTIVE: The purpose of this study was to compare the utility of 2 surgical models for obstetric anal sphincter repair for improving resident knowledge, confidence level, and repair technique. STUDY DESIGN: Third- and fourth-year obstetrics and gynecology residents at LAC + USC Medical Center performed simulated anal sphincter repair on both sponge and beef tongue surgical models. Resident knowledge, confidence, technical skill, and preference were assessed after each model was performed. RESULTS: Both models significantly improved resident confidence (sponge, P = 0.017; tongue, P = 0.016) and knowledge (60% correct before intervention vs. 92% correct after intervention, P < 0.001). There was no difference detected between the 2 models. All participants preferred the tongue model to the sponge model. When performing both models, performing the tongue model after the sponge model resulted in a further increase in confidence (P = 0.008) CONCLUSIONS: Both models are excellent tools to aid in resident teaching of obstetric anal sphincter repair and significantly increase residents' knowledge and confidence.

Improved yield and stability of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation, by protein engineering.

The L49 single-chain Fv fused to beta-lactamase (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen. However, large-scale production of L49-sFv-bL from refolded E. coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein. Sequence analysis of the L49-sFv framework regions revealed three residues in the framework regions at positions L2, H82B, and H91, which are not conserved for their position, occurring in <1% of sequences in Fv sequence databases. One further unusual residue, found in <3% of variable sequences, was observed at position H39. Each unusual residue was mutated to a conserved residue for its position and tested for refolding yield from inclusion bodies following expression in E. coli. The three V(H) single mutants showed improvement in the yield of active protein and were combined to form double and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein. In an attempt to further improve yield, the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resulting in a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein. The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations. Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts, respectively. These results show that a rational protein-engineering approach improved the yield, stability, and refolding characteristics of L49-sFv-bL while maintaining binding affinity and therapeutic efficacy.

The anti-CD30 monoclonal antibody SGN-30 promotes growth arrest and DNA fragmentation in vitro and affects antitumor activity in models of Hodgkin's disease.

The leukocyte activation marker CD30 is highly expressed on the Reed Sternberg cells of Hodgkin's disease (HD). On normal tissues, CD30 has a restricted expression profile limited to activated T cells, activated B cells, and activated natural killer cells. This expression profile makes CD30 an ideal target for monoclonal antibody (mAb)-based therapies of Hodgkin's disease. CD30 mAbs have been shown to be effective in in vitro and in vivo models of hematologic malignancies such as anaplastic large cell lymphoma, yet these mAb have not been efficacious in HD models. We have found that a mAb against CD30, AC10, was able to inhibit the growth of HD cell lines in vitro. To generate a more clinically relevant molecule, the variable regions from AC10 were cloned into an expression construct containing the human gamma1 heavy chain and kappa light chain constant regions. The resulting chimeric antibody, designated SGN-30, retained the binding and in vitro growth-inhibitory activities of the parental antibody. Treatment of HD cell lines with SGN-30 in vitro resulted in growth arrest in the G(1) phase of the cell cycle and DNA fragmentation consistent with apoptosis in the HD line L540cy. Severe combined immunodeficient mouse xenograft models of disseminated HD treated with SGN-30 produced significant increases in survival. Similarly, xenograft models of localized HD demonstrated dose-dependent reduction in tumor mass in response to SGN-30 therapy. SGN-30 is being developed for the treatment of patients who have HD that is refractory to initial treatment or who have relapsed and have limited therapeutic options.