A novel strategy for NQO1 (NAD(P)H:quinone oxidoreductase, EC 1.6.99.2) mediated therapy of bladder cancer based on the pharmacological properties of EO9.

The indolequinone EO9 demonstrated good preclinical activity but failed to show clinical efficacy against a range of tumours following intravenous drug administration. A significant factor in EO9's failure in the clinic has been attributed to its rapid pharmacokinetic elimination resulting in poor drug delivery to tumours. Intravesical administration of EO9 would circumvent the problem of drug delivery to tumours and the principal objective of this study is to determine whether or not bladder tumours have elevated levels of the enzyme NQO1 (NAD(P)H:quinone oxidoreductase) which plays a key role in activating EO9 under aerobic conditions. Elevated NQO1 levels in human bladder tumour tissue exist in a subset of patients as measured by both immunohistochemical and enzymatic assays. In a panel of human tumour cell lines, EO9 is selectively toxic towards NQO1 rich cell lines under aerobic conditions and potency can be enhanced by reducing extracellular pH. These studies suggest that a subset of bladder cancer patients exist whose tumours possess the appropriate biochemical machinery required to activate EO9. Administration of EO9 in an acidic vehicle could be employed to reduce possible systemic toxicity as any drug absorbed into the blood stream would become relatively inactive due to an increase in pH.

Progress in cancer gene therapy.

The "First International Symposium on Genetic Anticancer Agents," which took place in Amsterdam on March 8-9, 2000, served as a forum to review the results of preclinical and clinical gene therapy studies for cancer endeavored to date. Despite the fact that gene therapy was initially conceptualized as an approach for inherited genetic disease, it is currently finding its widest employ for treating neoplastic disorders. In this regard, more than 70% of patients treated to date in human clinical gene therapy protocols have been in the context of anticancer regimens. Of note, the application of gene therapy for cancer has proceeded from the same rational basis as was originally conceptualized for inherited genetic disorders. Specifically, the molecular basis of these disorders is increasingly being understood, therapeutic genes are available, and alternative therapies are often lacking. Most recently, the field of gene therapy has enjoyed the realization of the first incontrovertible evidence of clinical benefit, for hemophilia and cardiovascular disease, in its first 15 years of human application. This recent recognition of the potential power of gene therapy, and the current lack of realizing such ends for neoplastic disease, has led to a reassessment of the field. Such a critical analysis is a necessary step in defining the means to progress the technology toward achieving the potential benefits of gene therapy for cancer.

Discovery of new anti-cancer agents.

Anticancer drug discovery has changed fundamentally owing to recent progress in basic research. Historically, the discovery of potential new drugs for the treatment of cancer has largely relied on large-scale random screening. Although several useful agents have become available, sometimes after the development of chemical analogues of the original OhitO, this approach has generally been disappointing in terms of the efficacy/toxicity balance. To date, a range of cancer-specific molecular and biological drug targets are available providing opportunities for the design and discovery of specific anti-cancer agents with better tumour selectivity, and therefore less toxicity, than conventional agents. Among the many innovative approaches currently explored in pre-clinical and clinical research and development programs are inhibition of angiogenesis and metastasis, tumour vaccines/immunotherapy and gene therapy approaches. Several promising drug development projects are currently underway in these areas. The advent of innovative anticancer agents also has important consequences for the organisation of new drug development. Traditional drug development methodologies are often not appropriate for agents having completely new modes of action. The design of new drug development templates for such agents therefore has high priority in anti-cancer drug development organisations.

Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains.

Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions. There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions. Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions. For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector. Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express. This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors.

A helper T-cell epitope of the E7 protein of human papillomavirus type 16 in BALB/c mice.

The helper T-cell response to the E7 protein of human papillomavirus type 16 (HPV16) was studied using BALB/c (H-2d) mice. Twenty-two overlapping synthetic peptides spanning the HPV16 E7 protein were split into 6 groups. Mice were sensitized using mixtures of synthetic peptides corresponding to each of the groups. Lymph node cell suspensions were cultured with the corresponding mixture of synthetic peptides that was used for sensitization. Two mixtures induced a proliferative response. Analysis of the individual peptides from these mixtures showed that two (overlapping) peptides induced a proliferative response. This response was mediated by CD4+ cells. The common region of the two peptides was found to be a single epitope, and a minimal epitope was demonstrated (AHYNIVTFCCK). In conclusion, in contrast to others, we demonstrated a helper T-cell response in BALB/c mice. This may be due to the fact that we used synthetic peptides as immunizing agent. The helper T-cell epitopes in HPV16 E7 demonstrated previously are partly overlapping with the (minimal) epitope demonstrated here, underlining the 'public' nature of the epitope.

Identification of a unique group of human papillomavirus type 16 sequence variants among clinical isolates from Barbados.

The naturally occurring sequence variation of human papillomavirus type 16 (HPV-16) was analysed by direct sequence analysis of the PCR products of the long control region (LCR), the E5 and E7 open reading frames (ORFs), a segment of the L2 ORF overlapping the early viral poly(A) signal and a small segment of the L1 ORF or clinical isolates from Barbados and The Netherlands. Despite the widely different geographical and ethnic origin of the two groups of specimens, sequence analysis revealed relatively few mutational differences. Analysis of the LCR and the E5 ORF appeared to be the minimum requirement for the correct positioning of these variants in the HPV-16 phylogenetic tree. Most of the Barbadian variants appeared to be located at a unique position in the HPV-16 phylogenetic tree, at the internal branch close to the point where the European and Asian branches diverge. In contrast, most of the Dutch samples were located on the European branch.

Human papillomavirus, lichen sclerosus, and squamous cell carcinoma of the vulva: detection and prognostic significance.

Detection of HPV-DNA in squamous vulvar carcinoma, its prognostic significance, and investigation of the presence of lichen sclerosus near HPV-DNA-positive vulvar carcinomas were the objectives of this study. In 60 samples of squamous vulvar cancer, we looked for HPV-DNA by means of PCR. The same samples were examined for the presence of lichen sclerosus. The possible prognostic significance of the presence of HPV-DNA and lichen sclerosus was studied. Nineteen (32%) of the samples were HPV-DNA positive using PCR. Patients with an HPV-positive tumor had a better prognosis than those with an HPV-negative tumor (P = 0.03). Lichen sclerosus was found in 19 tumor samples, of which 7 had detectable HPV-DNA. Only a minority of vulvar cancers contain HPV-DNA. In contrast to previous statements, near some of these HPV-positive cancers, lichen sclerosus can be found.

[Progressive multifocal leukoencephalopathy in a female patient with chronic lymphatic leukemia]. / Progressieve multifocale leuko-encefalopathie bij een patiënte met chronische lymfatische leukemie.

This case report describes a 78-year old female patient with chronic lymphocytic leukaemia and neurological symptoms due to progressive multifocal leukoencephalopathy (PML). At autopsy the histopathology was characteristic, the involvement of JC virus was established. PML occurs in immunocompromised patients and is caused by infection with JC papova virus. Epidemiology, clinical course, diagnostic procedures with special reference to the use of the polymerase chain reaction, and therapy are briefly reviewed.

Analysis of Escherichia coli isolates from subjects with travellers' diarrhoea using DNA probes and serotyping.

Escherichia coli isolated from faeces of 54 healthy volunteers who visited Tunisia for eight days were examined. These volunteers participated in a randomized double-blind placebo-controlled study to establish whether ciprofloxacin could prevent travellers' diarrhoea. Escherichia coli strains isolated before travel, during episodes of travellers' diarrhoea, immediately after return and five weeks after return were serotyped and tested for the presence of virulence genes indicating diarrheogenic properties by hybridization with a set of four non-radioactively labelled DNA probes. Subjects receiving ciprofloxacin prophylactically to prevent travellers' diarrhoea were asymptomatic and no Escherichia coli could be cultured shortly after return home. Sixty-four percent of subjects (18/28) who did not receive antibiotic prophylaxis suffered from travellers' diarrhoea. Hybridization tests detected 8 enterotoxigenic Escherichia coli strains producing heat stable toxin, 13 enterotoxigenic strains producing heat labile toxin and 10 strains which produced both heat labile and stable toxin. Of the 31 probe positive strains, 29 (94%) were cultured from 11 volunteers with travellers' diarrhoea. A bacterial cause was thus determined in 61% of the volunteers who experienced travellers' diarrhoea.

Cell communication compartments in molluscan embryos.

Early embryos of Patella vulgata have been injected with Lucifer Yellow. No restriction of dye spread was found. We show that later in the development, the larval trochophore stage present evidence of compartments of cell communication. These dye compartments coincide with different presumptive regions.