Carboplatin/taxane-induced gastrointestinal toxicity: a pharmacogenomics study on the SCOTROC1 trial.

Carboplatin/taxane combination is first-line therapy for ovarian cancer. However, patients can encounter treatment delays, impaired quality of life, even death because of chemotherapy-induced gastrointestinal (GI) toxicity. A candidate gene study was conducted to assess potential association of genetic variants with GI toxicity in 808 patients who received carboplatin/taxane in the Scottish Randomized Trial in Ovarian Cancer 1 (SCOTROC1). Patients were randomized into discovery and validation cohorts consisting of 404 patients each. Clinical covariates and genetic variants associated with grade III/IV GI toxicity in discovery cohort were evaluated in replication cohort. Chemotherapy-induced GI toxicity was significantly associated with seven single-nucleotide polymorphisms in the ATP7B, GSR, VEGFA and SCN10A genes. Patients with risk genotypes were at 1.53 to 18.01 higher odds to develop carboplatin/taxane-induced GI toxicity (P<0.01). Variants in the VEGF gene were marginally associated with survival time. Our data provide potential targets for modulation/inhibition of GI toxicity in ovarian cancer patients.

EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury.

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have a variety of roles in the developing and adult central nervous system that require direct cell-cell interactions; including regulating axon path finding, cell proliferation, migration and synaptic plasticity. Recently, we identified a novel pro-survival role for ephrins in the adult subventricular zone, where ephrinB3 blocks Eph-mediated cell death during adult neurogenesis. Here, we examined whether EphB3 mediates cell death in the adult forebrain following traumatic brain injury and whether ephrinB3 infusion could limit this effect. We show that EphB3 co-labels with microtubule-associated protein 2-positive neurons in the adult cortex and is closely associated with ephrinB3 ligand, which is reduced following controlled cortical impact (CCI) injury. In the complete absence of EphB3 (EphB3(-/-)), we observed reduced terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), and functional improvements in motor deficits after CCI injury as compared with wild-type and ephrinB3(-/-) mice. We also demonstrated that EphB3 exhibits dependence receptor characteristics as it is cleaved by caspases and induces cell death, which is not observed in the presence of ephrinB3. Following trauma, infusion of pre-clustered ephrinB3-Fc molecules (eB3-Fc) into the contralateral ventricle reduced cortical infarct volume and TUNEL staining in the cortex, dentate gyrus and CA3 hippocampus of wild-type and ephrinB3(-/-) mice, but not EphB3(-/-) mice. Similarly, application of eB3-Fc improved motor functions after CCI injury. We conclude that EphB3 mediates cell death in the adult cortex through a novel dependence receptor-mediated cell death mechanism in the injured adult cortex and is attenuated following ephrinB3 stimulation.

The effects of intravenous tobramycin on renal tubular function in children with cystic fibrosis.

BACKGROUND: Tobramycin, used to treat respiratory exacerbations in cystic fibrosis (CF), is also a renal tubular toxin. Tubular dysfunction leads to increased urinary levels of the proximal tubular lysosomal enzyme, N-acetyl-beta-D-glucosaminidase (NAG) and the proximal tubular protein, retinol-binding protein (RBP). Hypermagnesuria and resulting hypomagnesaemia are indicative of more severe tubular damage, occasionally seen following repeated courses of intravenous tobramycin. Using these biochemical markers we studied the effect of a 2-week course of this agent on tubular function. METHODS: Twenty-two children (11 boys) with CF were studied. Median age = 10.9 years, range 3.1-16.4 years. All had a normal predicted glomerular filtration rate (pGFR). They received tobramycin 3 mg/kg/dose tds. Urinary NAG, RBP, creatinine and plasma magnesium and creatinine were assayed: a) immediately before commencing tobramycin, b) immediately following the course, c) 4 weeks after the end of the course. RESULTS: Mean log UrNAG and UrRBP rose significantly between time points a) and b) before falling to almost pre-treatment levels by time c). Using two way ANOVA analysis the results for UrNAG and UrRBP were both highly statistically significant (p<0.0001). Paired t-tests on the logged values revealed highly significant differences between all time points for UrNAG and in the case of UrRBP for all other than a) compared to c). In all patients plasma magnesium and pGFR remained within normal limits. CONCLUSIONS: Intravenous tobramycin produces acute tubular injury, which showed evidence of almost complete recovery after 4 weeks. The insult to the tubules was not sufficient to produce hypomagnesaemia in our study group. To assess cumulative tubular damage in more detail it would be necessary to repeat this study after further courses of tobramycin. We recommend monitoring plasma magnesium during courses of intravenous tobramycin.

Characterization of partially sintered ceramic powder compacts using fluorinated gas NMR imaging.

We use nuclear magnetic resonance (NMR) imaging of C2F6 gas to characterize porosity, mean pore size, and permeability of partially sintered ceramic (Y-TZP Yttria-stabilized tetragonal-zirconia polycrystal) samples. Conventional measurements of these parameters gave porosity values from 0.18 to 0.4, mean pore sizes from 10 nm to 40 nm, and permeability from 4 nm(2) to 25 nm(2). The NMR methods are based on relaxation time measurements (T(1)) and the time dependent diffusion coefficient D(Delta). The relaxation time of C2F6 gas is longer in pores than in bulk gas and it increases as the pore sizes decrease. NMR yielded accurate porosity values after correcting for surface adsorption effects. A model for T(1) dependence on pore size that accounts for collisions between gas molecules and walls as well as surface adsorption effects is proposed. The model fits the experimental data well. Finally, the long time limit of D(Delta)/D(o), where D(o) is the bulk gas diffusion coefficient is useful for measuring tortuosity, while the short time limit was not achieved experimentally and could not be used for calculating surface-area to volume (S/V) ratios.

SCF(beta-TRCP) and phosphorylation dependent ubiquitinationof I kappa B alpha catalyzed by Ubc3 and Ubc4.

NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536

Antisense targeting of E6AP elevates p53 in HPV-infected cells but not in normal cells.

Invasive cervical cancer is very highly correlated with the presence of high-risk human papillomavirus (HPV) types 16 and 18. Two viral proteins, E6 and E7, act in concert to subvert growth control of infected cells by inactivating the tumor suppressor proteins, p53 and Rb, respectively. E6 is thought to abrogate p53 function by stimulating its degradation via ubiquitin-mediated proteolysis in a reaction requiring E6AP (E6-Associated Protein). Here we evaluate the in vivo role of E6AP in p53 degradation in normal and HPV-infected cell types using antisense phosphorothioate oligodeoxynucleotides (S-ODNs). This study shows that reduction of E6AP in vivo in high-risk HPV-infected cells leads to an elevation of p53, confirming the function of E6AP predicted by in vitro experiments. Further, we demonstrate that reduction of E6AP in normal cells has no effect on p53 levels, indicative of an E6AP-indpendent mechanism for p53 degradation. These experiments show that inhibition of intermediate proteins in the ubiquitin-mediated proteolysis pathway (ubiquitin-conjugating enzymes or associated recognition proteins) can result in specific inhibition of substrate degradation. We propose that modulation of p53 levels by elimination of E6AP function may have therapeutic potential for cervical cancer.

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

Fecal isolation of Pseudomonas aeruginosa from patients with cystic fibrosis.

Fecal isolation of Pseudomonas aeruginosa was observed in 8 of 10 patients with cystic fibrosis who at the time of sampling also exhibited colonization of the respiratory tract. In contrast, P. aeruginosa cells were isolated at low frequency (9.1%) from the stools of 44 patients with cystic fibrosis with no previous history of chronic colonization. The results of this study suggest that the gastrointestinal tract is not a significant chronic reservoir of P. aeruginosa prior to pulmonary colonization.

Serum concentrations of a granulocyte-derived calcium-binding protein in cystic fibrosis patients and heterozygotes.

Serum samples from cystic fibrosis homozygotes and heterozygotes contain elevated levels of a protein, known as cystic fibrosis antigen, which is synthesized primarily in granulocytes. We have produced monoclonal antibodies against cystic fibrosis antigen and developed a sensitive two-site immunoassay. Antigen levels were evaluated in serum samples from 50 cystic fibrosis homozygotes, 34 heterozygotes, 60 healthy controls and 25 disease controls. Simultaneous measurement was made of another granulocyte-derived serum protein, lactoferrin, and also of the acute-phase reactant, C-Reactive Protein. Attempts to use the concentrations of cystic fibrosis antigen in serum to distinguish cystic fibrosis patients from heterozygotes were unsuccessful, even when these concentrations were expressed as a ratio with lactoferrin or with C-Reactive Protein. However, examination of the ratio of cystic fibrosis antigen to lactoferrin in serum samples from cystic fibrosis heterozygotes suggests that there is some specific association between this antigen and the cystic fibrosis gene.

Rational parameters for antibiotic therapy in patients with cystic fibrosis.

Data from the literature and the authors' experiences were used to review aspects of antibiotic therapy of patients with cystic fibrosis; attention was paid to in vitro antimicrobial susceptibility tests and assessment of therapy directed against mucoid Pseudomonas aeruginosa. The heterogeneity of P. aeruginosa within single sputa with respect to antibiotic susceptibility is stressed. Quantitative viable counts of bacteria based on an analysis of homogenised sputum is recommended. The mode of in vivo growth of mucoid P. aeruginosa is discussed to explain the survival of hypersusceptible P. aeruginosa in vivo, and the clinical benefit observed in the absence of a significant reduction of the pathogen. The value of ceftazidime in the treatment of exacerbations due to Haemophilus influenzae is emphasised. The social benefits from oral administration of ciprofloxacin also emphasises that the patient's quality of life must also be considered.

Mortality and morbidity caused by measles in children with malignant disease attending four major treatment centres: a retrospective review.

Measles is a major cause of mortality and morbidity in children receiving treatment for leukaemia. A review was made of all the documented cases of measles in children in first remission from acute lymphoblastic leukaemia at four major treatment centres in 1974-84. Over the 11 years reviewed 1043 children with acute lymphoblastic leukaemia were referred to these centres. Fifty one (4.9%) died while in first remission and 15 (29.4%) of these deaths were due to measles or its complications: 12 cases of pneumonia, 10 of them fatal; and six cases of encephalitis, five of them fatal and the sixth child left severely handicapped. These children would have had at least a 50% chance of long term survival. The severity of measles in the immunocompromised patient reinforces the need to improve the poor uptake of measles immunisation in Britain.

Scottish validation study of Cancer Registration data childhood leukaemia 1968-1981--I.

This study attempted to validate central registration data on all childhood leukaemia cases in Scotland between 1968 and 1981 in line with the Black Enquiry concerning West Cumbria. Missing files precluded a complete verification, but minor errors of registration were found in 44% of cases. A small number of important mistakes of omission (eight cases), wrong diagnosis (six cases) and postal code errors (nine cases) were found which might affect epidemiological studies of these relatively rare diseases. Precise and verified prospective data collection at the time of diagnosis is essential if the spatial distribution of childhood cancers is being studied.

Scottish validation study of Cancer Registration data childhood leukaemia 1968-1981. Bone marrow review--II.

Following a review of central leukaemia registration data for Scottish children 1968-1981 a retrospective assessment of leukaemia type was made by inspection of bone marrow slides. Only 57% of slides were still available and in 72% of these the review panel confirmed the initial diagnosis of leukaemia and its type. In eight cases the panel disagreed with the diagnosis or the type of leukaemia designated. Central slide review at diagnosis in childhood leukaemia is essential if subsequent epidemiological studies are to be meaningful.

Pseudomonas cepacia--fatal pulmonary infection in a patient with cystic fibrosis.

A 9-year-old girl with cystic fibrosis (CF) was admitted with an exacerbation of respiratory infection and subsequently died. At death Pseudomonas cepacia was cultured from her sputum, in large numbers, 10(7) colony forming units/ml.

Reticuloendotheliosis in breeder turkeys.

Reticuloendotheliosis virus (REV) was isolated at 21 weeks from a flock of breeder turkeys with lymphoid neoplasms. Except for about 2% mortality due to tumors mainly between 20 and 30 weeks of age, the livability and egg production of the flock were normal. Egg transmission as a source of infection seemed unlikely, because no REV antibodies or production problems were observed in two sister flocks (hatchmates) and in three progeny flocks, and because eggs obtained from the breeder flock at 45 weeks lacked REV group-specific antigen and produced poults negative for REV infection. The virus isolate, designated REV strain 339, produced an acute neoplastic disease when inoculated into young chickens.

Rotavirus antibody in chickens as measured by enzyme-linked immunosorbent blocking assay.

The antibody titer of 202 chickens to SA-11 rotavirus was determined by enzyme-linked immunosorbent blocking assay. The chickens were from 15 separate flocks on six farms. The titer was detectable (1:18 or greater dilution) in 43% of the chickens. The wide variation in infection rate between flocks on individual farms (0% to 90%) indicated there is probably not significant transmission of the virus between flocks on the farm. Significant differences in numbers of serotest-positive birds were found between separate farms, possibly reflecting different management practices.