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.

Immunotherapy of a human papillomavirus type 16 E7-expressing tumor by administration of fusion protein comprised of Mycobacterium bovis 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 tumor cell line, TC-1, as a model of cervical carcinoma, an immunotherapy based on the administration of an adjuvant-free fusion protein comprised of Mycobacterium bovis BCG Hsp65 linked to HPV16 E7 (HspE7) has been developed. Initial in vitro analyses indicate that immunization with HspE7 results in the induction of a type 1 immune response based on the pattern of secreted cytokines and the presence of cytolytic activity following antigenic recall. It has been previously shown that prophylactic immunization with HspE7 protected mice against challenge with TC-1 cells and that these tumor-free animals are also protected against rechallenge with TC-1 cells. The present report shows that a single therapeutic immunization with HspE7 induces regression of palpable tumors, confers protection against tumor rechallenge, and is associated with long-term survival (>253 days). In vivo studies using mice with targeted mutations in CD8 or MHC class II or depleted of CD8 or CD4 lymphocyte subsets demonstrate that tumor regression following therapeutic HspE7 immunization is CD8 dependent and CD4 independent. These studies extend previous observations on the induction of CTL by Hsp fusion proteins and are consistent with the clinical application of HspE7 as an immunotherapy for human cervical and anal dysplasia and cancer.

Priming of CD8+ CTL effector cells in mice by immunization with a stress protein-influenza virus nucleoprotein fusion molecule.

Literature is accumulating which suggests the potential for stress proteins to form the basis of a novel vaccine technology. Immunization with mammalian tumor-derived stress proteins and their associated peptides promote anti-tumor immunity. Vaccination with HIV-1 p24 antigen fused to mycobacterial heat shock protein (Hsp) Hsp71 enhances p24-specific immunity, as measured by p24-specific antibody production and in vitro cell proliferation and cytokine induction. An ovalbumin-Hsp71 fusion protein primes ovalbumin-specific CTL activity and resistance to challenge with an ovalbumin-expressing tumor. We have extended these observations by using a mycobacterial Hsp65 fusion molecule to prime CTL specific for a viral antigen. Gene fusion constructs were generated from DNA encoding Mycobacterium bovis strain BCG Hsp65 and individual fragments of influenza virus nucleoprotein (NP) encompassing H-2Kd- and H-2Db-restricted CTL epitopes. The ability of these purified recombinant fusion proteins to prime NP-specific CTL was assessed in mice of appropriate H-2 haplotypes. We observed that adjuvant-free immunization with either fusion protein elicited significant CTL activity when administered at doses of 10-100 micrograms per mouse. An NP fusion protein made with glutathione-S-transferase failed to elicit NP-specific CTL, indicating that the phenomenon requires Hsp65 sequences. A single immunization with the Hsp65-NP fusion protein elicited CTL activity which persisted for a minimum of 4 months post-immunization, at which time it could be boosted by a second immunization. To our knowledge, this is the first report of a member of the Hsp60 family priming for antigen-specific CTL activity when employed as a fusion protein partner.

Gene replacement in Bordetella pertussis by transformation with linear DNA.

We replaced the wild-type TOX operon of Bordetella pertussis with in vitro mutated, detoxified alleles by electroporetic transformation using unmarked linear DNA. Uptake of DNA was selected by transient ampicillin resistance and two simultaneous recombination events resulted in gene-replacement at the natural locus with no integration of heterologous DNA. TOX alleles were stable without selection and recombinant strains secreted non-toxic, fully assembled, protective pertussis toxin (PT) analogues with kinetics similar to the parental vaccine strain under production-scale fermentation conditions. Strains generated in this way are suitable for the production of recombinant whole-cell or component whooping cough vaccines that require no chemical modification of PT.

Base-catalyzed decomposition of N-nitrosobis(2-oxopropyl)amine.

N-Nitrosobis(2-oxopropyl)amine, a potent carcinogen for the pancreas in Syrian hamsters, undergoes a facile, base-catalyzed, intramolecular aldol condensation to yield N-nitroso-5-hydroxy-5-methyl-3-piperidone. This cyclic nitrosamine then decomposes to yield 3-hydroxy-5-methylpyridine.

The activation and DNA binding of 7-methylbenz[c]-acridine catalysed by mouse liver microsomes.

The metabolism of the carcinogen, 7-methylbenz[c]acridine (7MBAC), in liver microsomes prepared from untreated, and phenobarbital sodium (PB) and 3-methylcholanthrene (MC) induced male C57BL/6 mice was examined by high pressure liquid chromatography (HPLC). The abilities of control and MC induced liver microsomes to catalyse covalent binding of the carcinogen to DNA were comparable (60 pmol 7MBAC bound/mg DNA), although this maximum binding level observed was achieved at different 7MBAC concentrations for control (100 microM) and MC induced (25 microM) preparations. In vivo binding of 7MBAC to liver DNA was greater than that observed for lung DNA of the same animals but over 21 h bound 7MBAC levels decreased to a greater extent in liver than in lung. One fraction of a digest of in vitro alkylated DNA gave a fluorescence emission spectrum very similar to that of 7-methyl-1,2,3,4-tetrahydrobenz[c]acridine. This result suggests that diol epoxides functionalised at the 1,2,3,4-positions may be ultimate carcinogens derived from 7MBAC.

The metabolism of the carcinogen 7-methylbenz[c]acridine by hepatocytes isolated from untreated and induced rats.

The metabolic fate of the carcinogenic aza-aromatic hydrocarbon 7-methyl[7-(14)C]benz[c]acridine (14C-7MBAC) was studied in hepatocytes freshly isolated from untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated rats. 14C-7MBAC (4-200 microM) was metabolized in a concentration-dependent manner; Michaelis-Menten kinetics were not followed. Using 100 microM 14C-7MBAC, the bulk of the ethyl acetate-extractable metabolites were found in the incubation medium; about 50% of the total metabolites were not extractable into ethyl acetate. The nature of the water-soluble metabolites was examined by enzyme hydrolysis of glucuronides and sulphates, and by glutathione-depletion experiments. Organo-extractable metabolites were examined by reverse-phase h.p.l.c. and quantified by co-chromatography with standards. pretreatment of the rats with mixed-function oxidase inducers, phenobarbital and 3-methylcholanthrene resulted in 2.85- and 5.70-fold increases, respectively, in total metabolism of 14C-7MBAC. Major metabolites for all three hepatocyte preparations co-chromatographed with 7-hydroxymethylbenz[c]acridine, trans-5,6-dihydro-5,6-dihydroxy-7-methylbenz[c]acridine and trans-8,9-dihydro-8,9-dihydroxy-7-methylbenz[c]acridine.

Metabolism of 7-methylbenz[c]acridine: comparison of rat liver and lung microsomal preparations and identification of some minor metabolites.

The metabolism of the carcinogenic polycyclic aza-aromatic compound, 7-methylbenz[c]acridine, has been studied in lung and liver microsomal preparations obtained from control and induced rats. Minor metabolites not previously identified included, trans-10,11-dihydro-10,11-dihydroxy-7-methylbenz[c]acridine, trans-1,2-dihydro-1,2-dihydroxy-7-methylbenz[c]acridine, 7-methylbenz[c]acridine-5,6-oxide and 7-hydroxymethylbenz[c]acridine-5,6-oxide. Metabolite profiles from liver microsomes showed 7-hydroxymethylbenz[c]acridine, trans-8,9-diydro-8,9-dihydroxy-7-methylbenz[c]acridine, 7-methylbenz[c]acridine-5,6-oxide and phenols to be major products. Metabolite distributions obtained with lung microsomes were very similar although activities were much lower than those of liver microsomes prepared from the same animals.

Enzymatic reduction of beta-ketonitrosamines.

The reduction of N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-oxopropyl)propylamine (NOPPA) by hepatic and pancreatic cytosol and microsomes from Syrian golden hamsters and Sprague-Dawley rats has been examined. All hepatic fractions reduced both substrates, although the activity depended on the fraction tested and the cofactor employed (NADH or NADPH). Generally, hamster hepatic fractions contained higher activity than the rat hepatic fractions and BOP was a better substrate than NOPPA. Of the pancreatic fractions, only cytosol exhibited reductase activity. The hamster cytosol was able to utilise both cofactors, but the rat fraction exhibited activity only when NADPH was present. BOP was the better substrate for the pancreatic enzymes and in the presence of NADPH, the rat and hamster activities were about equal. These results suggest that the pancreatic reduction of BOP to HPOP is unlikely to be a significant factor in the species-specific induction of pancreatic cancer by BOP.

Thin-layer chromatographic and high-performance liquid chromatographic separation of metabolites of the weak carcinogen, 7-methylbenz[c]acridine.

The metabolism of [14C]7-methylbenz[c]acridine, a weakly carcinogenic polycyclic aza aromatic hydrocarbon, was studied in rates in vivo, and in rat hepatocytes and hepatic microsomes. Evidence for oxidation of the methyl group and excretion of 7-hydroxymethylbenz[c]acridine and benz[c]acridine-7-carboxylic acid in rat bile was obtained by thin-layer chromatography. A reversed-phase high-performance liquid chromatographic separation of metabolites was developed and chromatographic profiles of metabolites found in vivo and formed in vitro are presented. Several synthetic potential oxidation products were used to characterise the chromatographic profiles and evidence for the in vitro formation of 7-hydroxymethylbenz[c]acridine and 5,6-dihydro-5,6-dihydroxy-7-methylbenz[c]acridine was obtained.

Anabolic steroids. Part 2: the disposition of ethylestrenol in the rat.

1. The absorption, distribution, metabolism and excretion of [3H]ethylestrenol were studied in the rat. 2. Approximately one third of an intragastric dose was absorbed; 17% of the dose was excreted in urine and 83% in faeces within 10 days. 3. The dose is distributed throughout the rat, and kidney and liver were found to contain respectively 2.5-3 and 5-7 times the average specific activity of all other tissues. 4. Unchanged ethylestrenol was the only component detected in urine. Ethylestrenol was also found in faeces, along with two different dihydroxylated dihydro derivatives and one trihydroxylated dihydro derivative.

Anabolic steroids. Part 3: metabolism of ethylestrenol in rat liver preparations in vitro.

1. Ethylestrenol incubated with a post-mitochondrial supernatant fraction of rat liver plus co-factors gives norethandrolone as the major metabolite. 2. A second (minor) metabolite was tentatively identified as 17 alpha-ethyl-5 epsilon-estrane-3 epsilon,17 beta-diol. 3. A pathway is suggested for the metabolism of ethylestrenol in the rat.