Retroviral-mediated transfer and amplification of a functional human factor VIII gene

Hemophilia A results from a deficiency in factor VII (FVIII), a cofactor in the intrinsic pathway of blood coagulation. As an approach toward genetic therapy of this disease, we constructed a retroviral vector encoding human FVIII and a selectable and amplifiable genetic marker, human adenosine deaminase (Ada). A retrovirus packaging line was transfected with this vector and stable transformants were selected for Ada expression. Isolated transformants produced both FVIII activity in the conditioned medium and retrovirus capable of transferring the Ada selectable marker and FVIII expression to the mouse 3T3 fibroblasts. Selection of virus-producer cell lines for increasing levels of Ada expression yielded a 20-fold increase in both FVIII expression and viral titer. Similarly, selection of infected 3T3 fibroblasts for Ada gene amplification yielded a 20-fold increase in FVIII expression. The results demonstrate the feasibility of retrovirus- mediated transfer of human FVIII, and also the utility of selection for gene amplification to increase retrovirus titers in producer cell lines as well as expression levels in infected cells.     

The inter‐rater reliability between nurse‐assessors clinically assessing infection of chronic wounds using the WUWHS criteria

The aim of this study was to determine the inter‐rater reliability between one expert‐nurse and four clinical‐nurses who were asked to clinically assess infection of chronic wounds by using the World Union of Wound Healing Societies (WUWHS) criteria. A quasi‐experimental design was used to collect the data. In comparison to phase 1 in which ‘open questions’ were asked, in phase 2 a pre‐printed form (checklist) was introduced. In both phases, 55 chronic wounds were clinically assessed. For each WUWHS criterion the inter‐rater reliability of signs and symptoms was expressed by Cohens Kappa (κ). A substantial agreement (κ ≥ 0·6) was considered as adequate. In both phases pocketing (p < 0·02), and erythema (p < 0·004) scored statistically significant results. Phase 2 showed higher inter‐rater agreements compared with phase 1 (three substantial agreements (easily bleeding/friable granulation tissue, delayed healing, increasing exudate), an almost perfect‐ and a perfect agreement for malodour and pain, respectively. According to the results it can be concluded that the clinical assessment of infection of chronic wounds may be better supported by a pre‐printed form than making use of an ‘open questions’ form. To provide this with a higher level of evidence, there is need for more well conducted studies.