Immunotherapy of a human papillomavirus (HPV) type 16 E7-expressing tumour by administration of fusion protein comprising Mycobacterium bovis bacille Calmette–Guérin (BCG) hsp65 and HPV16 E7

Human papillomavirus type 16 (HPV16) infection has been linked to the development of cervical and anal dysplasia and cancer. One hallmark of persistent infection is the synthesis of the viral E7 protein in cervical epithelial cells. The expression of E7 in dysplastic and transformed cells and its recognition by the immune system as a foreign antigen make it an ideal target for immunotherapy. Utilizing the E7-expressing murine tumour cell line, TC-1, as a model of cervical carcinoma, an immunotherapy based on the administration of an adjuvant-free fusion protein comprising Mycobacterium bovis BCG heat shock protein (hsp)65 linked to HPV16 E7 (hspE7) has been developed. The data show that prophylactic immunization with hspE7 protects mice against challenge with TC-1 cells and that these tumour-free animals are also protected against re-challenge with TC-1 cells. In addition, therapeutic immunization with hspE7 induces regression of palpable tumours, confers protection against tumour re-challenge and is associated with long-term survival (> 253 days). In vitro analyses indicated that immunization with hspE7 leads to the induction of a Th1-like cell-mediated immune response based on the pattern of secreted cytokines and the presence of cytolytic activity following antigenic recall. In vivo studies using mice with targeted mutations in CD8 or MHC class II or depleted of CD8 or CD4 lymphocyte subsets demonstrate that tumour regression following therapeutic hspE7 immunization is CD8-dependent and CD4-independent. These studies extend previous observations on the induction of cytotoxic T lymphocytes by hsp fusion proteins and are consistent with the clinical application of hspE7 as an immunotherapy for human cervical and anal dysplasia and cancer

A new method for assaying propantheline and its degradation product, xanthene-9- carboxylic acid using high performance liquid chromatography

A rapid, specific, and precise high-performance liquid chromatographic method is described for the simultaneous analysis of propantheline bromide and its hydrolysis product, xanthene-9-carboxylic acid. Reversed-phase chromatography was conducted using a mobile phase of 40:60, acetonitrile-0.05 M phosphate buffer (pH 2.5) delivered at 2 ml/min. Detection was at 254 nm. Methantheline bromide (internal standard), propantheline bromide, and xanthene-9-carboxylic acid gave retention times of 4.1, 5.4, and 8.3 min, respectively. Within-day, between-day, and total precision (CV) for assay of 15 mg/10 ml propantheline bromide are 1.2, 1.1, and 1.6%, respectively (n = 20). Similar precision was obtained for xanthene-9-carboxylic acid. The limit of detection was 2 ng. The assay is useful for routine quality assurance of propantheline in dosage forms and for stability and kinetic studies