Effect of α-difluoromethylornithine on the polyamine levels and proliferation in two transplantable tumours

Summary The effect of inhibition of polyamine biosynthesis by-difluoromethylornithine (DFMO) on the growth of two murine transplantable tumours was studied. Female CBA mice were implanted with either the sarcoma F (SaF) or an anaplastic mammary carcinoma (CaNT), and 3% DFMO in the drinking water was provided once the tumours were established. Over a 10-day period control SaF tumours increased exponentially from 20 mm³ to over 800 mm³, whereas DFMO-treated SaF reached only 300 mm³. CaNT grew more slowly, requiring 22 days to achieve a similar volume increase, and DFMO was as effective in retarding growth as it had been in SaF. DFMO depleted tumour tissues of putrescine and spermidine, but did not reduce spermine levels. Metaphase arrest experiments with vincristine demonstrated that DFMO could substantially reduce the rates of tumour cell production, but there was no indication the DFMO accelerated the rate of cell loss from the tumours. Despite reduced rates of cell production, labelling studies with bromodeoxyuridine failed to detect differences between control and treated tumours: an increase in transit time through the S-phase was suspected. The number of nuclear organizer regions, detected by the argyrophilia of their associated proteins, was less in DFMO-treated tumours, and within a tumour the degree of silver deposition unequivocally reflected the proliferative heterogeneity. Ultrastructural studies revealed no differences between DFMO-treated and untreated tumours.     

Therapeutic Vaccines for Cervical Cancer

Human papillomavirus (HPV) infection is associated with transformation and clonal expansion of infected epithelial cells, resulting in the production of a benign growth, i.e. a wart. Recently, however, HPV has emerged as the primary causative agent of cervical carcinoma, malignancy being associated with the presence of the viral genome (predominantly genotypes 16 and 18) in cancerous cells. The only HPV proteins reliably expressed in neoplastic lesions are the 'oncogenic' E6 and E7 proteins, that serve both as tumour-specific markers and potential targets for immunotherapeutic intervention. As intracellular (nuclear) proteins, the E6 and E7 gene products may be hidden from the humoral immune response. Attention has thus focused on the generation of a vaccine capable of inducing or stimulating a cellular immune response to HPV 16 and HPV 18 E6 and E7. Vaccine development has been constrained by the absence of an appropriate animal model, the oncogenic nature of E6 and E7 and technical difficulties associated with detection of cytotoxic T cell responses to these antigens. Despite these difficulties, vaccine strategies have now been devised based on immunisation with synthetic peptide, whole protein and a vaccinia virus recombinant. Phase I/II human clinical trials have been initiated, and preliminary results have demonstrated the induction of specific cellular immune responses after immunisation. The HPV-associated neoplasia in cervical cancer represents an excellent target for therapeutic intervention because the tumour-associated antigens are so clearly defined. As such, it provides an appropriate model for establishing the general principles of cancer immunotherapy in humans.     

Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection

Cattle are important reservoirs of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin(O157)) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of < or =100 were vaccinated twice with purified intimin(O157) or sham-vaccinated with sterile buffer. Intimin(O157)-specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin(O157). Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10(6) CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin(O157)-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin(O157) as a viable candidate for an EHEC O157:H7 antitransmission vaccine.     

An introduction to stem cells

1998 saw the publication of two papers describing the growth in vitro of human embryonic stem (ES) cells derived either from the inner cell mass (ICM) of the early blastocyst or the primitive gonadal regions of early aborted fetuses. Work on murine ES cells over many years had already established the amazing flexibility of ES cells, essentially able to differentiate into almost all cells that arise from the three germ layers. The realization of such pluripotentiality (see below) has, of course, resulted in the field of stem cell research going into overdrive, the establishment of many new biotechnology companies (http://www.stemcellresearchnew.com/catalog1677.html), and a genuine belief that stem cell research will deliver a revolution in terms of how we treat cardiovascular disease, neurodegenerative disease, cancer, diabetes, and the like. However, many people believe that early human embryos should be accorded the same status as any sentient being and thus their 'harvesting' for stem cells is morally unjustifiable. With this in mind, other sources of malleable stem cells have been sought. In the adult, organ formation and regeneration was thought to occur through the action of organ- or tissue-restricted stem cells (i.e. haematopoietic stem cells giving rise to all the cells of the blood, neural stem cells making neurons, astrocytes, and oligodendrocytes). However, it is now believed that stem cells from one organ system, for example the haematopoietic compartment can develop into the differentiated cells within another organ system, such as the liver, brain or kidney. Thus, certain adult stem cells may turn out be as malleable as ES cells and so also be useful in regenerative medicine. This brief overview summarizes the important attributes of tissue-based stem cells and clarifies the terms used.     

Multiple sites of oxygen sensing and their contributions to hypoxic pulmonary vasoconstriction

Oxygen sensing by the pulmonary vasculature is important for the regulation of vessel tone and the matching of lung perfusion to ventilation. Airways hypoxia is a major stimulus for vasoconstriction, which diverts blood from hypoxic alveoli to better ventilated areas of the lung. Several hypotheses have emerged to explain how pulmonary arteries sense a decrease in oxygen and mediate hypoxic pulmonary vasoconstriction (HPV). They differ mainly in where they place the main site of HPV: in the endothelial or smooth muscle cells of the artery wall. HPV probably results from synergistic actions on both cell types, but it can proceed in the absence of endothelium, suggesting that the primary oxygen sensor is the smooth muscle cell and endothelium-derived agents modulate the muscle response. Several oxygen-sensing targets have been identified in smooth muscle, including potassium channels, Ca(2+) stores in the sarcoplasmic reticulum (SR) and the Ca(2+) sensitivity of the contractile proteins. The evidence for different oxygen-sensing mechanisms in pulmonary vessels is discussed.     

Angiotensin-1-converting enzyme (ACE) plasma concentration is influenced by multiple ACE-linked quantitative trait nucleotides

Circulating angiotensin-1-converting enzyme (ACE) is a highly heritable trait, and a major component of the genetic variance maps to the region of the ACE gene. The strong effect of the locus, and the interest in ACE as a candidate gene for cardiovascular disorders, has led to extensive investigation of its relationship to the ACE phenotype, providing one of the most complete examples of quantitative trait locus (QTL) analysis in humans. Resequencing of ACE followed by haplotype analysis in families of British and French origin has shown that the genetic variants that are primarily associated with the ACE trait map to an 18 kb interval flanked by two intragenic, ancestral recombination breakpoints. This critical interval contains dozens of ACE-associated variants in Caucasians, but identification of which of these directly influence ACE concentration is ambiguous because of the almost complete linkage disequilibrium in European populations. In a complementary sequencing and genotyping study of individuals from West African families, we show that this population has much greater haplotype diversity across the gene. Through analysis of the contrasting relationships of the trait phenotype with haplotypes that carry different allelic combinations from those observed in Caucasians, we demonstrate that (at least) two major intragenic sites within the critical interval and (at least) one minor promoter site are associated with the ACE quantitative trait through additive effects. These results point to the importance of analysing diverse populations with different gene genealogies in gene-association studies.     

Delineation of an estimated 6.7 MB candidate interval for an anophthalmia gene at 3q26.33-q28 and description of the syndrome associated with visible chromosome deletions of this region

Anophthalmia or microphthalmia occur in approximately one in 10 children who have severe visual impairment. These eye malformations are often of unknown aetiology, but can be inherited in autosomal dominant, recessive or X-linked forms, and can also occur in association with specific chromosome abnormalities. Four children are described in the medical literature with microphthalmia or anophthalmia in association with chromosome rearrangements involving distal 3q, suggesting the presence of a micro/anophthalmia gene in this region. We have identified two further patients with micro/anophthalmia and chromosome rearrangements involving 3q26-->3q27 and identified a 6.7 MB common deleted region. Patient 1 had multiple abnormalities including bilateral anophthalmia, abnormalities of the first and second cranial nerves and partial absence of the corpus callosum. His karyotype was 46,XY,del(3)(q26.33q28). Patient 2 had right anophthalmia and left extreme microphthalmia. Her karyotype was 46,XX,del(3)(q26.33q28)t(3;7)(q28;q21.1). Both patients had intrauterine growth retardation (IUGR) and strikingly similar dysmorphic facies consisting of bossed forehead, downward-slanting palpebral fissures, grooved bridge of the nose, prominent low-set ears, small down-turned mouth and small mandible. We identified BAC clones mapping to distal 3q from the ENSEMBL and NCBI Entrez databases. These BAC clones were used as fluorescence in situ hybridisation (FISH) probes to identify the minimum deleted region common to both patients. This interval, between clones RPC11-134F2 and RPC11-132N15, was estimated to be 6.7 MB. We conclude that there is an anophthalmia locus within this interval. Candidate genes mapping to this region include Chordin and DVL3, a homologue of the Drosophila Dishevelled gene.     

The behaviour of strains of the virus of foot-and-mouth disease in pig,calf, ox and lamb kidney tissue cultures

Summary Strains of the virus of foot-and-mouth disease obtained from different hosts and tissue cultures were tested in tissue cultures of pig, calf, ox and lamb kidneys for their ability to multiply and produce cytopathogenic effects. It was found that whereas cattle and kidney strains of the virus multiplied well in the cultures with cytopathogenic effect, mouse and egg adapted strains did not multiply or show cytopathogenic effect to the same extent especially in the ox and calf kidneys, and this could be correlated with their behaviour in cattle, pigs and cattle tongue epithelium tissue cultures. With all the strains used it was found possible to produce plaques on pig kidney monolayers, but the size and shape of the plaque varied as well as the relation of plaque titre to the titre in mice. The plaque size and plaque population from different sources were compared, and it was found that the relative number of the different plaque sizes varied with the source of the virus and changed in passage in the different systems. The possible significance of these findings in relation to vaccine preparation and adaptation of the virus is discussed.Part of the work described represents the kidney tissue culture side of experiments on attenuated strains of the virus of foot-and-mouth disease. We are grateful to our colleagues at Pirbright for the supply of virus strains and the results of many cattle, egg and mouse titrations.We would also like to thank MissP. Tremayne-Smitli, Mr.W. Chapman and Mr.D. Maskell for their excellent assistance in this work.