A comparative study on the immunotherapeutic efficacy of recombinant Semliki Forest virus and adenovirus vector systems in a murine model for cervical cancer.

Currently, various therapeutic strategies are being explored as a potential means to immunize against metastatic malignant cells or even primary tumours. Using recombinant viral vectors systems or protein-based immunization approaches, we are developing immunotherapeutic strategies against cervical cancer or premalignant cervical disease, as induced by high-risk type human papillomaviruses (HPVs). We previously demonstrated that immunization of mice with recombinant replication-defective Semliki Forest virus (rSFV) encoding a fusion protein of HPV16 E6 and -E7 (SFV-eE6,7) induces strong cytotoxic T-lymphocyte (CTL) activity and eradication of established HPV-transformed tumours. In this study, we compared the antitumour efficacy of SFV-eE6,7 with that of a recombinant adenovirus (rAd) type 5 vector, expressing the same antigen construct (Ad-eE6,7). Prime-boosting with SFV-eE6,7 resulted in higher precursor CTL frequencies and CTL activity compared to prime-boosting with Ad-eE6,7 and also in murine tumour treatment experiments SFV-eE6,7 was more effective than Ad-eE6,7. To elicit a therapeutic effect with Ad-eE6,7, 100/1000-fold higher doses were needed compared to SFV-eE6,7. In vivo T-cell depletion experiments demonstrated that these differences could not be explained by the induction of a different type of effector cells, since CD8+ T cells were the main effector cells involved in the protection against tumour growth in both rSFV- and rAd-immunized mice. Also comparable amounts of in vivo transgene expression were found upon immunization with rSFV and rAd encoding the reportor gene luciferase. However, anti-vector responses induced by a single injection with rAd resulted in a more than 3-log decrease in luciferase expression after a second injection of rAd. With rSFV, transgene expression was inhibited by only one to two orders of magnitude in preinjected mice. As an antigen-specific booster immunization strongly increases the level of the CTL response and is essential for efficient induction of immunological memory, it is likely that (part of) the difference in efficacy between rSFV and rAd type 5 can be ascribed to a diminished efficacy of the booster immunization in the case of rAd due to anti-vector antibody responses.

Expression of Rab small GTPases in epithelial Caco-2 cells: Rab21 is an apically located GTP-binding protein in polarised intestinal epithelial cells.

Rab proteins belong to a subfamily of small GTP-binding protein genes of the Ras superfamily and play an important role in intracellular vesicular targeting. The presence of members of this protein family was examined in Caco-2 cells by a PCR-based strategy. Twenty-five different partial cDNA sequences were isolated, including 18 Rab protein family members. Seven novel human sequences, representing Rab2B, Rab6A', Rab6B, Rab10, Rab19B, Rab21 and Rab22A, were identified. For one clone, encoding Rab21, full-length cDNA was isolated from a Caco-2 cDNA library. Northern blot analysis showed a ubiquitous expression pattern of Rab21. To study Rab21 protein expression in Caco-2 cells, polyclonal antibodies were raised against GST-Rab21 fusion protein and characterised. The antibodies recognised Rab21 as a protein of approximately 25 kDa. Interestingly, the protein shows a general ER-like staining in nonpolarised Caco-2 cells in contrast to an apically located vesicle-like staining in polarised Caco-2 cells. Furthermore, immunohistochemical staining on human jejunal tissue showed a predominant expression of Rab21 in the epithelial cell layer with high expression levels in the apical region, whereas stem cells in the crypts were negative. We therefore suggest an alternative role for Rab21 in the regulation of vesicular transport in polarised intestinal epithelial cells.

Adherence to the guidelines of a regional formulary.

BACKGROUND: Pharmacotherapeutical guidelines, called formularies, have been developed to facilitate effective, efficient and cost-conscious prescribing. Monitoring adherence to such guidelines may be a reasonable way of assessing prescribing practices. OBJECTIVE: The aim of this study was to assess how strictly the GPs participating in our department's registration network adhere to the guidelines of the regional formulary, and which indications and drugs the GPs used. METHODS: This is a descriptive study, concerning 1000 consecutive prescriptions from each of the 17 participating GPs. The third edition of the Groningen formulary (GFIII), published in 1995, was used. If the drug prescribed was advised in the formulary, we considered it to be global adherence. If the indication was mentioned in the formulary, and the drug prescribed was advised for that indication in the formulary, it was considered to be specific adherence. Both the medications prescribed and the health problems registered by the GPs, but not mentioned in the GFIII, were analysed. RESULTS: The 17 000 prescriptions chosen for analysis formed approximately 25% of all prescriptions written by the GPs in 1 year. The indications for only 24 prescriptions (0. 14%) were missing. Among the 17 GPs, the number of different drugs prescribed varied between 167 and 219 per 1000 prescriptions. The global adherence varied from 76 to 89% among the GPs, and the specific adherence varied from 55 to 71%. Of the 17 000 prescriptions, 11 457 (67%) concerned indications mentioned in the GFIII. Prescriptions for indications not mentioned in the GFIII contained 4353 (78.5%) drugs advised in the formulary. Of the 251 medications mentioned in the GFIII, only 15 (6%) were not prescribed. DISCUSSION: The GPs in our study were neither representative, nor were they chosen at random. Their patient population was comparable in age, sex and insurance status. These findings are an example of what level of adherence is obtainable. The formulary covered approximately two-thirds of the indications registered by GPs, and did not contain many unnecessary medications (6%).

[Comparison of regional formularies for general practitioners]. / Regionale formularia voor huisartsen vergeleken.

OBJECTIVE: To inventory the similarities and differences of the contents of regional general practice formularies. DESIGN: Descriptive. SETTING: Department of General Practice Medicine, University of Groningen, the Netherlands. METHOD: Through personal contacts and an appeal in the journal Medisch Contact, 14 formularies were acquired. Of these, seven recent, still valid Dutch general practice formularies were compared as to pharmacotherapy and health problems listed. RESULTS: Volume and contents of these seven formularies showed major, inexplicable differences with regard to pharmacotherapy, while the choice of the health problems listed was not argued anywhere, and not based on prevalence figures. CONCLUSION: The regional general practice formularies examined showed little similarity in the drugs and health problems listed.