Functional inactivation but not structural mutation of p53 causes liver cancer.

Structural mutations in the p53 gene are seen in virtually every form of human cancer. To determine whether such mutations are important for initiating tumorigenesis, we have been studying hepatocellular carcinoma, in which most cases are associated with chronic hepatitis B virus infections. Using a transgenic mouse model where expression of a single HBV gene product, the HBx protein, induces progressive changes in the liver, we show that tumour development correlates precisely with p53 binding to HBx in the cytoplasm and complete blockage of p53 entry into the nucleus. Analysis of tumour cell DNA shows no evidence for p53 mutation, except in advanced tumours where a small proportion of cells may have acquired specific base substitutions. Our results suggest that genetic changes in p53 are late events which may contribute to tumour progression.

Contactless determination of nuclear compressibility using 3D image- and model-based analysis of drug-induced cellular deformation.

Mechanical properties of the chromatin-bearing nucleus in normal and pathological cells are of general interest for epigenetics and medicine. Conventional techniques for quantitative measurements of material properties of cellular matter are based on application of controlled forces onto the cellular or nuclear boundary and do not allow probing intracellular structures that are not directly accessible for physical contact inside the living cell. In this work, we present a novel approach for contactless determination of the nuclear compressibility (i.e. the Poisson's ratio ν) in living cells by means of image- and model-based analysis of drug-induced cell deformation. The Poisson's ratio of the HeLa cell nucleus is determined from time-series of 3D images as a parameter of constitutive model that minimizes the dissimilarity between the numerically predicted and experimentally observed images.

Clinical experience of patients referred to a multidisciplinary cardio-oncology clinic: an observational cohort study.

Introduction: Cardiovascular disease is the 2nd leading cause of long-term morbidity and mortality in cancer survivors. Cardio-oncology clinics (cocs) have emerged to address the issue; however, there is a paucity of data about the demographics and clinical outcomes of patients seen in the coc setting. Methods: Cancer patients referred to The Ottawa Hospital coc were included in this retrospective observational study. Data collected were patient demographics, cancer type and stage, reason for referral, cardiac risk factors, cardiac assessments and treatment, and clinical outcomes. Results: Between 2008 and 2015, 779 patients (516 women, 66%; 263 men, 34%) were referred to the coc. Median age of the patients at cancer diagnosis was 60 years (range: 18-90 years). The most frequent reasons for referral were decreased left ventricular ejection fraction (33%), pre-chemotherapy assessment (14%), and arrhythmia (14%). Treatment with cardiac medication was given in 322 patients (41%), 181 (56%) of whom received more than 2 cardiac medications, with 57 (18%) receiving an angiotensin-converting enzyme inhibitor (acei), 46 (14%) receiving an acei and a beta-blocker, and 38 (12%) receiving a beta-blocker. Of 163 breast cancer patients, 129 (79%) were able to complete targeted therapy with coc co-management. Most of the 779 patients (n = 643, 83%) were alive at the time of the last data collection. Conclusions: This cohort study is one of the largest to report characteristics and clinical outcomes of patients referred to a coc. Collaboration between oncologists and cardiologists resulted in completion of cancer therapy in most patients. Ongoing analysis of referral patterns, management plans, and patient outcomes will help to guide the cardiac care of oncology patients, ultimately optimizing cancer and cardiac outcomes alike.

Marine spatial planning makes room for offshore aquaculture in crowded coastal waters.

Marine spatial planning (MSP) seeks to reduce conflicts and environmental impacts, and promote sustainable use of marine ecosystems. Existing MSP approaches have successfully determined how to achieve target levels of ocean area for particular uses while minimizing costs and impacts, but they do not provide a framework that derives analytical solutions in order to co-ordinate siting of multiple uses while balancing the effects of planning on each sector in the system. We develop such a framework for guiding offshore aquaculture (bivalve, finfish, and kelp farming) development in relation to existing sectors and environmental concerns (wild-capture fisheries, viewshed quality, benthic pollution, and disease spread) in California, USA. We identify > 250,000 MSP solutions that generate significant seafood supply and billions of dollars in revenue with minimal impacts (often < 1%) on existing sectors and the environment. We filter solutions to identify candidate locations for high-value, low-impact aquaculture development. Finally, we confirm the expectation of substantial value of our framework over conventional planning focused on maximizing individual objectives.

Human osteosarcoma cell lines are dependent on insulin-like growth factor I for in vitro growth.

Osteogenic sarcoma is the most common bone tumor of childhood and typically occurs during the adolescent growth spurt when growth hormone and insulin-like growth factor I (IGF-I) may be at their lifetime highest levels. Since IGF-I is involved in normal bone growth and differentiation, we have evaluated the possible role of IGF-I signaling in the growth of human osteogenic sarcoma cell lines. In this study, we demonstrate that in vitro survival of cells is dependent on exogenously supplied IGF-I. Furthermore, we show that these cells display functional IGF-I receptors on their surface and that in vitro growth is inhibited by blocking these receptors either by monoclonal antibodies or by antisense oligonucleotides. These data demonstrate that human osteogenic sarcoma cell lines are dependent on signaling through the IGF-I receptor for in vitro survival and proliferation. Furthermore, they suggest that modulation of the growth hormone/IGF-I axis may affect the growth of these tumors in vivo.

Frequency and diversity of p53 mutations in childhood rhabdomyosarcoma.

The p53 gene was examined in primary or metastatic tumors from six patients with rhabdomyosarcoma (RMS) and in five RMS cell lines by screening methods including single-strand conformation polymorphism analysis, the RNase protection assay, sequencing of complementary DNA subclones, and Southern blotting. Six original tumors were of embryonal histology, four alveolar, and one mixed. p53 mutations were identified in four of the six tumors or cell lines derived from tumors with embryonal histology and in one of the four with alveolar histology. Consistent with p53 allele loss, each mutation was found in the homo- or hemizygous state. One tumor showed a G to C transversion at p53 codon 213 (arginine to proline), and another showed deletion of the entire gene. The p53 mutations in cell lines included a codon 248 C to T transition (arginine to tryptophan) in RD and a codon 280 A to T transversion (arginine to serine) in RH30. The cell line CTR contained a 4-base pair deletion at codons 219/220 in exon 6 with resultant frame shift and premature termination in exon 7. These data support the role of diverse types of p53 mutations in the pathogenesis and/or progression of a significant proportion of cases of childhood RMS.

Developmental regulation of annexin II (Lipocortin 2) in human brain and expression in high grade glioma.

In experiments to identify molecules that might be important in the pathogenesis of glioblastoma multiforme, the most common malignant brain tumor, we found that annexin II (Lipocortin 2, p36), a likely second messenger in several different mitogenic pathways, was highly expressed in tumor tissue of glioblastoma multiforme (9 of 9) and highly anaplastic astrocytoma (2 of 6), but not in astrocytomas of lower pathological grade (0 of 6). We also detected high levels of annexin II expression in fetal brain during the period when radial glia proliferate, although annexin II expression was not detected in normal adult brain. These data demonstrate that annexin II expression is developmentally regulated in the human central nervous system and suggest that the early progenitor radial glia share important characteristics with highly malignant glial tumors.

Regulating gene expression in transgenic animals.

Regardless of the field of application, the raison d'etre of transgenic animals is to study gene regulation and function. With increasing frequency, mammalian genes are being isolated with no concomitant knowledge of their function. The human genome mapping initiative will undoubtedly produce a cornucopia of such genes. While the merit of taking a transgenic route to study genes of unknown function is axiomatic, the choices of strategies for gene regulation in vivo may not be fully appreciated. This review will address two main points: first, the targeted and regulated expression of genes, and second, the structural and functional ablation of genes.

1 alpha, 25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531) modulation of insulin-like growth factor-binding protein-3 and induction of differentiation and growth arrest in a human osteosarcoma cell line.

1 alpha,25-Dihydroxycholecalciferol [1,25-(OH)2D3] is a potent differentiating agent in a variety of tumor cell lines. However, the induction of severe hypercalcemia has limited its clinical use. Several analogs have been synthesized that retain the antiproliferative differentiating effects of 1,25-(OH)2D3, but do not have the calcitropic effect of the parent compound. One such analog, 1 alpha,25(OH)2-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531), can induce differentiation in HL-60 cells and does not induce hypercalcemia in animal models. We, therefore, evaluated the effect of Ro24-5531 on a human osteosarcoma cell line, MG-63. Compared with 1,25-(OH)2D3, the analog Ro24-5531 is 10-100 times more potent as an inhibitor of MG-63 cell proliferation, as determined by [3H]thymidine incorporation and/or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The inhibition in cell growth is accompanied by a decrease in the expression of p34cdc2 (> 4-fold), a protein critically involved in cell cycle regulation. Ro24-5531 treatment of MG-63, at a concentration of 10(-8) mol/L, induced expression of the bone differentiation markers biglycan and osteocalcin, as determined by Northern analysis. These data suggest that Ro24-5531 treatment induces growth arrest coupled with differentiation. To begin to evaluate the mechanisms by which Ro24-5531 may exert an effect, we evaluated the effect of Ro24-5531 on components of the insulin-like growth factor I (IGF-I) signaling pathway, an important regulator of normal bone growth and differentiation. The expression of IGF-binding protein (IGFBP), IGFBP-3 messenger ribonucleic acid, and protein levels are increased 20-fold after 72 h of treatment with Ro24-5531 and are associated with a marked increase in detectable binding of ligand to binding protein, as measured by RRA. These data suggest an association between Ro24-5531-induced growth arrest and increased expression of IGFBP-3.

Influence of sex on susceptibility in the Theiler's murine encephalomyelitis virus model for multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease in humans which affects females more frequently than males. Theiler's murine encephalomyelitis virus (TMEV) induces demyelination similar to human MS in susceptible strains of mice after intracerebral inoculation. In this paper we report that sex influences susceptibility to TMEV-induced demyelination in certain genotypes derived from C57L/J and SJL/J or SWR/J mice. This is the first report of an animal model for MS that shows an association between sex and susceptibility, and this information may facilitate the investigation of human MS.

Postnatal or in utero ethanol exposure reduction of the volume of the sexually dimorphic nucleus of the preoptic area in male rats.

Adult male rats exposed to alcohol in utero results in reduction of the volume of the sexually dimorphic nucleus of the preoptic area (SND-POA). Reduction of the volume of this nucleus is also significantly reduced in rats exposed to ethanol postnatally compared to the volume in animals not exposed to ethanol (P less than 0.05). The results indicate that ethanol exposure during the postnatal period of brain development in rats is equally effective as in utero ethanol exposure in development of the SDN-POA volume. This report substantiates others demonstrating the deleterious effects of ethanol consumption during the 'critical' period of brain development.

Type 2 diabetes: update on therapy.

Type 2 diabetes mellitus is a common disorder, affecting an estimated 12 million individuals in the United States. The primary care provider should be aware of rapidly expanding treatment options, including diet, exercise, oral therapy, and insulin.

Effects of hepatitis C virus on cardiovascular risk in infected patients: a comparative study.

The role of hepatitis C virus (HCV) in the pathogenesis of atherosclerosis and cardiovascular events is unclear. The aim of this study was to evaluate the direct effect of HCV on cardiovascular risk and correlate it with pro and anti-inflammatory cytokines in patients with HCV. HCV monoinfected patients, genotype 1, naive, non-obese (BMI<30) and non-diabetics were included and compared to controls (blood donors). Patients with prior diagnosis of cardiovascular diseases, hypertension, chronic renal failure, cancer and chronic use of lipid-lowering drugs or immunosuppressants were excluded. Age, BMI, systolic blood pressure (SBP) and diastolic (DBP), fasting glucose and lipid levels were determined. Serum cytokines (IL-6, IL-10 and TNF-α) and Framingham score were also evaluated. 62 HCV patients, 34 (54.8%) were males and none of them was smoking. The Framingham scores (median and 25th and 75th percentiles) were 12% (6.5-14%), showing an intermediate cardiovascular risk in patients with HCV. There was significant direct correlation between Framingham and total cholesterol (p=0.043) and DBP (p=0.007). HDL-C (p=0.002) was inversely correlated with the Framingham score. HCV patients had higher levels of proinflammatory cytokines (IL-6 and TNF-α) compared to controls (p<0.0001) and the relation of proinflammatory/anti-inflammatory TNF-α/IL10 and IL-6/IL10 were higher in HCV patients (p<0.01). The Framingham score was directly correlated to IL-6 and TNF-α, but differences were not statistically significant. Patients with HCV monoinfected, nonobese, naïve and non diabetic have an intermediate cardiovascular risk, as measured by the Framingham score and high levels of proinflammatory cytokines (IL-6 and TNF).

Successful control of methicillin-resistant Staphylococcus aureus in a spinal cord injury center: a 10-year prospective study including molecular typing.

STUDY DESIGN: Prospective cohort study with medical record review. OBJECTIVE: To evaluate the clinical utility of an infection control program in a patient cohort at high risk for methicillin-resistant Staphylococcus aureus (MRSA) infection and to identify risk factors interfering with successful decolonization of MRSA. SETTING: All spinal cord injured (SCI) patients hospitalized at the Swiss Paraplegic Center (SPC) Nottwil from April 1991 to April 2001. METHODS: Patients whose medical records indicated laboratory-confirmed MRSA colonization or infection were included. Incidence of MRSA colonization or infection was classified as community acquired, nosocomial or transferred based on standardized criteria. Risk factors for community-acquired MRSA colonization in SCI patients were determined. MRSA subtyping and identification of nosocomial spread was performed through pulse-field gel electrophoresis (PFGE). RESULTS: Of 5992 admissions, 100 episodes of MRSA (colonization 22 cases, infection 78 cases) were identified among 76 patients. Overall incidence (1991-2001) per 1000 patient days was 0.26 cases on admission compared to 0.08 at discharge (P<0.001). Community-acquired MRSA was most frequent (56%) followed by nosocomial acquisition (34%). PFGE subtyping identified two nosocomial clusters with six and three cases, respectively. Most of community-acquired MRSA isolates were genetically unrelated and also distinct from epidemic strains identified in Switzerland during the study period. Decolonization was successful in 60 of 76 (78.9%) MRSA-positive patients. CONCLUSION: In the largest European SCI center, MRSA controlling is feasible if infection control policies are vigorously applied.

Evolving concepts in molecular pathology.

During the last decade, an understanding of the causes of many human diseases has progressed rapidly, in large measure because of the development of technologies that allow us to identify the genes that are involved. Identification of a gene that is suspected to play an important role in a particular disease opens up a whole new dimension of research to understand the molecular events that underlie the cause of that disorder. A crucial step in this process is often the development of an animal model of the disease. Again, the last decade has seen rapid advances in our ability to create such models, particularly in mice. Technologies that allow for the addition, alteration, or elimination of individual genes from the genome to create a transgenic mouse are now routine. The advantages of having a transgenic mouse model of a human disease are many. These animals often provide the first unequivocal proof that a particular gene is responsible for causing the pathological changes that occur with disease. They also can provide a system to carefully dissect the successive events that lead to the disease state, and can provide a custom-designed whole animal system to test potential therapies to treat and eventually cure the disease. Most important, new concepts relating to gene expression and gene function in disease often emerge from such transgenic studies. This review will illustrate several examples in which transgenic animals have contributed significantly to the evolution of concepts of the underlying mechanisms of human disease.

Reduction of rat pineal N-acetyltransferase activity by fetal alcohol exposure.

Pineal N-acetyltransferase (NAT) is activated by increased sympathetic activity or by administration of isoproterenol. These experiments examined the effects of fetal alcohol exposure on the pharmacological induction of NAT activity in rats. Fetal alcohol exposure resulted in a significant reduction of NAT activity compared to that in rats whose mothers received a nutritionally controlled liquid diet or a laboratory chow diet during gestation. Stimulation of NAT activity in vitro by forskolin, which directly stimulates adenylate cyclase, also was significantly less in pineal glands from fetal alcohol exposed rats when compared with pineal NAT activity from rats exposed to the control diets during gestation. The results suggest that the effects of fetal alcohol exposure is not limited to the beta-adrenergic receptor at the cell membrane, but that exposure to alcohol in utero may result in more general effects on intracellular mechanisms involved in pineal NAT enzyme synthesis or activity.

Hierarchy of effects of the MHC and T cell receptor beta-chain genes in susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible mice with Theiler's murine encephalomyelitis virus induces a demyelinating disease that is similar to human multiple sclerosis. This murine model for human multiple sclerosis is apparently immune-mediated and the genes involved in the immune response influence the outcome of disease susceptibility as observed with human multiple sclerosis. These genes include the MHC and TCR genes. However, the functional relationships among these genes on the disease susceptibility has not yet been studied. In this study, we demonstrate that the effect of the H-2s genotype from susceptible SJL/J mice overrides the resistant effect of the BALB/c TCR beta-chain gene in CXJ recombinant-inbred and BALB.S congenic mice. These results strongly suggest the presence of a hierarchy of genes involved in the immune response in Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Association between susceptibility to Theiler's virus-induced demyelination and T-cell receptor Jbeta1-Cbeta1 polymorphism rather than Vbeta deletion.

Theiler's murine encephalomyelitis virus (TMEV) induces demyelinating disease in susceptible mouse strains after intracerebral inoculation. The clinical symptoms and histopathology of the central nervous system appear to be similar to those of human multiple sclerosis (MS), and thus, this system provides an excellent infectious animal model for studying MS. The virus-induced demyelination is immune mediated, and the genes involved in the immune response such as those for the T-cell receptor beta-chain and major histocompatibility complex (MHC) haplotypes are known to influence disease susceptibility. To define whether the T-cell receptor Jbeta-Cbeta or Vbeta genes are associated with susceptibility, we have analyzed F2 mice from crosses of susceptible SJL/J (Vbeta(a)-JCbeta(b)) mice and resistant C57L (Vbeta(a)-JCbeta(a)) mice. Our results indicate that susceptibility to TMEV-induced demyelination is associated with restriction fragment length polymorphism reflecting the T-cell receptor Jbeta1-Cbeta1 region rather than the Vbeta polymorphism. This association becomes stronger when the MHC haplotype is considered in the linkage analysis. However, differences in the T-cell receptor alpha-chain haplotype have no significant influence on the pathogenesis of TMEV-induced demyelination.

[Enteral feeding--an alternative to parenteral feeding in the treatment of Crohn disease?]. / Enterale Ernährung--eine Alternative zur parenteralen Ernährung in der Behandlung des Morbus Crohn?

Efficiency of parenteral nutrition and tube feeding were compared in two groups of 25 patients each who suffered from active Crohn's disease. Indication for treatment in these patient groups was an acute phase of the disease with an activity index of higher than 150 and/or body weight below 80% of optimum weight. During the trial no other therapy was given. Target parameters were the Crohn index, body weight, serum albium, hemoglobin, and in a part of the patients the creatinine index. In both groups signs and symptoms of activity were reduced significantly and nutritional status was improved; however, there were now significant differences between groups. From these results the conclusion can be drawn, that enteral nutrition is essentially equally effective as parenteral nutrition in treatment of the acute phase of Crohn's disease. For this reason only patients where there is an absolute contraindication against tube feeding should be supplied with parenteral nutrition.

Evidence for human meiotic recombination interference obtained through construction of a short tandem repeat-polymorphism linkage map of chromosome 19.

An improved linkage map for human chromosome 19 containing 35 short tandem repeat polymorphisms (STRPs) and one VNTR (D19S20) was constructed. The map included 12 new (GATA)n tetranucleotide STRPs. Although total lengths of the male (114 cM) and female (128 cM) maps were similar, at both ends of the chromosome male recombination exceeded female recombination, while in the interior portion of the map female recombination was in excess. Cosmid clones containing the STRP sequences were identified and were positioned along the chromosome by fluorescent in situ hybridization. Four rounds of careful checking and removal of genotyping errors allowed biologically relevant conclusions to be made concerning the numbers and distributions of recombination events on chromosome 19. The average numbers of recombinations per chromosome matched closely the lengths of the genetic maps computed by using the program CRIMAP. Significant numbers of chromosomes with zero, one, two, or three recombinations were detected as products of both female and male meioses. On the basis of the total number of observed pairs of recombination events in which only a single informative marker was situated between the two recombinations, a maximal estimate for the rate of meiotic STRP "gene" conversion without recombination was calculated as 3 x 10(-4)/meiosis. For distances up to 30 cM between recombinations, many fewer chromosomes which had undergone exactly two recombinations were observed than were expected on the basis of the assumption of independent recombination locations. This strong new evidence for human meiotic interference will help to improve the accuracy of interpretation of clinical DNA test results involving polymorphisms flanking a genetic abnormality.