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Influenza viruses unable to express NS1 protein (delNSl) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally as-sumed to result from the inability to counter the antiviral host response, as delNSl viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNSl virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene seg-ment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA edit-ing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing effi-ciency. In one virus, 2 amino acid substitutions in Ml additionally enhanced virus replication, possibly through changes in the Ml distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNSl virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induc-tion but rather is related to changes in Ml expression or localization. The mutations identified in this paper may be used to en-hance delNSl virus replication for vaccine production. [ABSTRACT FROM AUTHOR]

Neovascularization by endothelial progenitor cells (EPC) for the treatment of ischaemic diseases has been a topic of intense research. The CD34+ cell is often designated as EPC, because it contributes to repair of ischaemic injuries through neovascularization. However, incorporation of CD34+ cells into the neovasculature is limited, suggesting another role which could be paracrine. CD14+ cells can also differentiate into endothelial cells and contribute to neovascularization. However, the low proliferative capacity of CD14+ cell-derived endothelial cells hampers their use as therapeutic cells. We made the assumption that an interaction between CD34+ and CD14+ cells augments endothelial differentiation of the CD14+ cells. In vitro, the influence of CD34+ cells on the endothelial differentiation capacity of CD14+ cells was investigated. Endothelial differentiation was analysed by expression of endothelial cell markers CD31, CD144, von Willebrand Factor and endothelial Nitric Oxide Synthase. Furthermore, we assessed proliferative capacity and endothelial cell function of the cells in culture. In monocultures, 63% of the CD14+-derived cells adopted an endothelial cell phenotype, whereas in CD34+/CD14+ co-cultures 95% of the cells showed endothelial cell differentiation. Proliferation increased up to 12% in the CD34+/CD14+ co-cultures compared to both monocultures. CD34-conditioned medium also increased endothelial differentiation of CD14+ cells. This effect was abrogated by hepatocyte growth factor neutralizing antibodies, but not by interleukin-8 and monocyte chemoattractant protein-1 neutralizing antibodies. We show that co-culturing of CD34+ and CD14+ cells results in a proliferating population of functional endothelial cells, which may be suitable for treatment of ischaemic diseases such as myocardial infarction. [ABSTRACT FROM AUTHOR]

Although scientific evidence shows that therapy improves movement recovery following a stroke, the duration of the reimbursed therapy available to patients is decreasing. To compensate for the reduction in personal therapy self-training procedures using robotic arms have been developed for hemiplegic patients. In this Dutch pilot study, the experiences of stroke patients and their therapists with the second prototype of a robotic arm device for ACtive REhabilitation (ACRE2) are evaluated. Positive results for patients and therapists are reported and the development of a system that can be used at home (ACRE3) is recommended. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To provide insight into parents' awareness of and knowledge about urinary tract infections (UTIs) in young children. METHODS: Twenty interviews with parents who had a child recently diagnosed with a UTI were audiotaped, transcribed verbatim, and qualitatively analysed. RESULTS: Most parents knew the typical symptoms related to UTI. But, according to the parents, neither they nor all general practitioners (GPs) thought of a UTI in case of atypical symptoms. The awareness that UTI can be a serious illness usually came to parents later, partly because health care workers often did not explicitly mention this. According to the parents, health care workers should be more aware of UTIs in children. Parents felt that health education or mass screening might not be desirable because it would increase anxiety or would be perceived as not relevant. CONCLUSION: Parents could not consistently recognise UTI in their children and were most times unaware of the possible consequences of a UTI. Nevertheless, parents were sceptical about health education and mass screening. PRACTICE IMPLICATIONS: There seems little scope for health education addressed at parents or screening for UTI in young children. Instead, physicians and nurses should be alert for the possibility of UTIs in young children, and more information should be given once a UTI is diagnosed. [ABSTRACT FROM AUTHOR]

Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.Cancer Gene Therapy (2006) 13, 150–158. doi:10.1038/sj.cgt.7700882; published online 12 August 2005 [ABSTRACT FROM AUTHOR]

We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with chymosin in yeast. From this library, we obtained two VHH clones that stimulated chymosin secretion by screening colonies for the level of chymosin secreted. These VHHs bound biotinylated chymosin in an immunoblot procedure but failed to bind chymosin in ELISA, suggesting that their interaction with chymosin was of low affinity. In a second approach, chymosin-specific VHHs were first selected using phage display and then coexpressed with chymosin in yeast cells. Screening yeast cells for higher levels of chymosin secretion resulted in 11 VHHs. Sequence analysis revealed that these 11 VHHs formed four sets of related VHHs that were different from the previously isolated two VHHs. Although binding of VHHs to chymosin could not be demonstrated in ELISA using soluble VHHs, it could be unambiguously demonstrated for clones isolated by phage display, using phage-displayed VHHs. Finally, quantitative Western blot analysis of chymosin amounts demonstrated that coexpression with VHH domains can stimulate the level of secreted chymosin 1.5- to 6-fold. [ABSTRACT FROM AUTHOR]