Basal Tumor Cell Isolation and Patient-Derived Xenograft Engraftment Identify High-Risk Clinical Bladder Cancers.

Strategies to identify tumors at highest risk for treatment failure are currently under investigation for patients with bladder cancer. We demonstrate that flow cytometric detection of poorly differentiated basal tumor cells (BTCs), as defined by the co-expression of CD90, CD44 and CD49f, directly from patients with early stage tumors (T1-T2 and N0) and patient-derived xenograft (PDX) engraftment in locally advanced tumors (T3-T4 or N+) predict poor prognosis in patients with bladder cancer. Comparative transcriptomic analysis of bladder tumor cells isolated from PDXs indicates unique patterns of gene expression during bladder tumor cell differentiation. We found cell division cycle 25C (CDC25C) overexpression in poorly differentiated BTCs and determined that CDC25C expression predicts adverse survival independent of standard clinical and pathologic features in bladder cancer patients. Taken together, our findings support the utility of BTCs and bladder cancer PDX models in the discovery of novel molecular targets and predictive biomarkers for personalizing oncology care for patients.

The transmembrane heparan sulphate proteoglycan syndecan-4 is involved in establishment of the lamellar structure of the annulus fibrosus of the intervertebral disc.

The annulus fibrosus of the intervertebral disc unites adjacent vertebral bodies along the length of the spine and provides tensile resistance towards compressive, twisting and bending movements arising through gait. It consists of a nested series of oriented collagenous lamellae, arranged in cross-ply circumferentially around the nucleus pulposus. The organisation of oriented collagen in the annulus is established during foetal development by an identical arrangement of oriented fibroblasts that are precisely organised into cell sheets, or laminae. These provide a template for ordered deposition of extracellular matrix material on cell surfaces, by means of a poorly understood mechanism involving the actin cytoskeleton. In this study, we investigate the role of two cell surface heparan sulphate proteoglycans (HSPGs), glypican-6 and syndecan-4, in the matrix assembly process in the developmental rat intervertebral disc. We compare their expression patterns with those of heparan sulphate and the interactive, cell-surface adhesive glycoprotein, fibronectin, and relate these to the stage-specific collagenous architectures present within the annulus at both light and electron microscopic levels. We show that both proteoglycans are strongly associated with the development, growth and aging of the intervertebral disc. Furthermore, the immunohistochemical labelling patterns suggest that syndecan-4, in particular, plays a potentially-significant role in annulus formation. We propose that this HSPG mediates interaction between the actin cytoskeleton and nascent extracellular matrix in the lamellar organisation of annulus tissue. These data add considerably towards an understanding of how cells organise and maintain complex, oriented extracellular matrices and has particular clinical relevance to the fields of tissue engineering and repair.

Translational strategies exploiting TNF-alpha that sensitize tumors to radiation therapy.

TNFerade is a radioinducible adenoviral vector expressing tumor necrosis factor-alpha (TNF-alpha) (Ad.Egr-TNF) currently in a phase III trial for inoperable pancreatic cancer. We studied B16-F1 melanoma tumors in TNF receptor wild-type (C57BL/6) and deficient (TNFR1,2-/- and TNFR1-/-) mice. Ad.Egr-TNF+IR inhibited tumor growth compared with IR in C57BL/6 but not in receptor-deficient mice. Tumors resistant to TNF-alpha were also sensitive to Ad.Egr-TNF+IR in C57BL/6 mice. Ad.Egr-TNF+IR produced an increase in tumor-associated endothelial cell apoptosis not observed in receptor-deficient animals. Also, B16-F1 tumors in mice with germline deletions of TNFR1,2, TNFR1 or TNF-alpha, or in mice receiving anti-TNF-alpha exhibited radiosensitivity. These results show that tumor-associated endothelium is the principal target for Ad.Egr-TNF radiosensitization and implicate TNF-alpha signaling in tumor radiosensitivity.

The new consultant survey 2005.

BACKGROUND: Consultants in emergency medicine have to deal with a wide range of problems, many of which they will not have encountered during their training. One way to assess the adequacy of specialist training is to ask recently appointed consultants whether or not they feel adequately prepared for their role. METHODS: A questionnaire was sent out to 60 newly appointed consultants in emergency medicine in January and February 2005 and the results analysed. RESULTS: Many respondents feel that there should be greater emphasis on acquiring clinical skills, partly by greater consultant supervision and partly by providing more experience of anaesthetics and intensive care. New consultants also feel inadequately prepared for their management responsibilities, and this is a source of great stress. CONCLUSIONS: Specialist training in emergency medicine needs to pay more attention to the acquisition of clinical skills and to preparation for management responsibility.

On-line gel permeation chromatography/nuclear magnetic resonance of complex polymer formulations.

Separation of synthetic polymer mixtures by gel permeation chromatography (GPC), followed by on-line detection using a 500 MHz nuclear magnetic resonance (NMR) spectrometer, has been demonstrated using three different polymer formulations as examples. The mobile phase used in all cases was deutero-chloroform, and an inexpensive commercially available flow cell was used as a link between the separation and detection stages of the experiment. Using this technique it is possible to derive chemical information relating to specific molecular sizes of polymer mixtures, and not just the size-averaged information that would be obtained from standard NMR experiments. This provides an invaluable tool for the deformulation of complex mixtures such as those found in the surfactants and adhesives industries.

Iron release from corroded iron pipes in drinking water distribution systems: effect of dissolved oxygen.

Iron release from corroded iron pipes is the principal cause of "colored water" problems in drinking water distribution systems. The corrosion scales present in corroded iron pipes restrict the flow of water, and can also deteriorate the water quality. This research was focused on understanding the effect of dissolved oxygen (DO), a key water quality parameter, on iron release from the old corroded iron pipes. Corrosion scales from 70-year-old galvanized iron pipe were characterized as porous deposits of Fe(III) phases (goethite (alpha-FeOOH), magnetite (Fe(3)O(4)), and maghemite (alpha-Fe(2)O(3))) with a shell-like, dense layer near the top of the scales. High concentrations of readily soluble Fe(II) content was present inside the scales. Iron release from these corroded pipes was investigated for both flow and stagnant water conditions. Our studies confirmed that iron was released to bulk water primarily in the ferrous form. When DO was present in water, higher amounts of iron release was observed during stagnation in comparison to flowing water conditions. Additionally, it was found that increasing the DO concentration in water during stagnation reduced the amount of iron release. Our studies substantiate that increasing the concentration of oxidants in water and maintaining flowing conditions can reduce the amount of iron release from corroded iron pipes. Based on our studies, it is proposed that iron is released from corroded iron pipes by dissolution of corrosion scales, and that the microstructure and composition of corrosion scales are important parameters that can influence the amount of iron released from such systems.

Value of serum prolactin in the management of syncope.

OBJECTIVE: A meta-analysis of studies of the usefulness of raised serum prolactin in diagnosing generalised tonic-clonic seizures (GTCS) in patients presenting to the accident and emergency (A&E) department after a single episode of syncope. METHODS: A three part question was defined. Medline, EMBASE, PubMed, the Cochrane Library were searched to identify relevant studies. Studies were evaluated for eligibility and quality and data extracted to calculate sensitivity (SN), specificity (SP), and likelihood ratios (LR). RESULTS: Of 13 relevant studies only three met the criteria for evaluation. If a serum prolactin concentration is greater than three times the baseline when taken within one hour of syncope, then in the absence of test "modifiers": (1) the patient is nine times more likely to have suffered a GTCS as compared with a pseudoseizure positive LR = 8.92 (95% CI (1.31 to 60.91)), SN = 0.62 (95% CI (0.40 to 0.83)), SP = 0.89 (95% CI (0.60 to 0.98)) and (2) five times more likely to have suffered a GTCS as compared with non-convulsive syncope positive LR 4.60 (95% CI (1.25 to 16.90)), SN = 0.71 (95% CI (0.49 to 0.87)), SP = 0.85 (95% CI (0.55 to 0.98)). CONCLUSION: A positive test result is highly predictive of a GTCS, however a negative test result does not necessarily exclude a seizure. Serum prolactin should be measured in patients presenting to the A&E department within an hour of a syncopal episode, unless the cause is immediately obvious.

The significance of a defect on DMSA scan in children with renal transplants.

Since December 1995, pediatric renal transplant recipients in our unit have received a DMSA scan as soon as possible post-transplant in order to provide a baseline for comparison in the event of subsequent complications. We retrospectively reviewed the case notes and DMSA scans of the 45 patients who underwent a scan within 9 wk of their transplant to see if pre or peri-transplant factors or post-transplant complications were associated with defects on scanning. Forty percentage of scans had defects. The presence of defects was not associated with potential predisposing factors such as patient or donor age, cadaveric or live donation, cold ischemia time, multiple donor vessels, the use of non-heart beating donors, the mean time to scan, the serum creatinine, or the presence of structural renal tract anomalies predisposing to UTI. However, 87% of patients had complications before the scan, including UTI, rejection, acute tubular necrosis, transplant biopsy and drug toxicity. Children with no clinical complications had a significantly reduced risk of a defect (p = 0.035), while biopsy was associated with the presence of defects (p = 0.0034). Twenty patients had one or more follow up DMSA scans: one patient developed a new focal defect. In conclusion, renal transplant defects are frequently found on DMSA scanning even early after transplantation and are non-specifically associated with many different complications.

Recovery from pH 6.38: lactic acidosis complicated by hypothermia.

Survival after extreme arterial acidosis is uncommon. A case of metformin induced lactic acidosis is described where the presenting pH was 6.38 exacerbated by hypothermia (29 degrees C). Increased anion gap acidosis, its varied aetiology, potential reversibility, and the role of hypothermia are discussed. Early liaison with a medical toxicology unit is recommended when this rare condition is suspected.

Outcome of infants born to hepatitis C infected women.

BACKGROUND: Hepatitis C virus (HCV) can be transmitted vertically from mother to infant, either late in pregnancy or at delivery. AIMS: To determine the outcome of infants born to HCV infected women, to characterise epidemiology and to design an appropriate infant monitoring schedule. METHODS: Three hundred and fourteen infants, born to 296 HCV positive women between 1994 and 1999 were monitored for a median of 18 months (range 1-52). RESULTS: Forty per cent of infants were small for age and 46% had neonatal abstinence syndrome (NAS). Of 173 infants of defined status, 11 were infected (vertical transmission rate [VTR] 6.4%, 95% CI 2.8-10). Infected infants were diagnosed at a median of three months (range 0.5-10). Liver transaminases elevation was documented in 8% of uninfected infants. A negative HCV PCR test before one month of age did not exclude infection but all infected patients had detectable HCV RNA when next tested (range 2-10 months). CONCLUSIONS: 94% of infants born to HCV antibody positive women are not HIV infected. Liver transaminase elevation in exposed infants is not always indicative of infection. A minimum monitoring schedule of testing (PCR and antibody) at six to eight weeks, six and 18 months allows early diagnosis while detecting late seroconversions.

Antenatal hepatitis B screening - is there a need for a national policy?

Routine antenatal testing for hepatitis B carriage with maternal consent was introduced at the Rotunda in January 1998. The uptake of testing has been excellent; 99.98% of women presenting for antenatal care accepted hepatitis B (HBV) screening in the 30-months from January 1998 through June 2000. The prevalence of HBV carriage was 0.35% (58 pregnancies of 16,222 tested) increasing from 0.25% in 1998 (16 of 6227) to 0.45% in the first six months of 2000 (16 of 3484). Fifty-five women had 58 pregnancies (three women had two pregnancies). Two of these were e-antigen positive. HBV carrier status was previously unknown in 48 (87%). Two additional women had acute HBV infection in pregnancy. Forty-five infants have been born to mothers included in this screening programme. Audit of infant outcome reveals excellent compliance with immunisation and follow-up: 29 (64%) have completed the 3 dose HBV vaccination schedule to date. Thirteen infants (31%) are still attending; three are lost to follow-up including one whose family has emigrated. Routine antenatal screening for hepatitis B carriage is cost-effective and should be considered a standard of care in maternity practice.

Angiostatin effects on endothelial cells mediated by ceramide and RhoA.

Angiostatin is a cleavage product of plasminogen that has anti-angiogenic properties. We investigated whether the effects of angiostatin on endothelial cells are mediated by ceramide, a lipid implicated in endothelial cell signaling. Our results demonstrate that angiostatin produces a transient increase in ceramide that correlates with actin stress fiber reorganization, detachment and death. DNA array expression analysis performed on ceramide-treated human endothelial cells demonstrated induction of certain genes involved in cytoskeleton organization. Specifically, we report that treatment with angiostatin or ceramide results in the activation of RhoA, an important effector of cytoskeletal structure. We also show that treatment of endothelial cells with the antioxidant N-acetylcysteine abrogates morphological changes and cytotoxic effects of treatment with angiostatin or ceramide. These findings support a model in which angiostatin induces a transient rise in ceramide, RhoA activation and free radical production.

Improving the delivery of analgesia to children in pain.

OBJECTIVES: To improve the time taken for children arriving to the accident and emergency (A&E) department in pain to receive analgesia. Delivery within 30 minutes of triage was taken as an achievable goal. METHODS: 262 children who had received analgesia in the "minor injuries" area of West Middlesex University Hospital A&E department were studied over a four month period. Current practice was indicated over the first two months by retrospectively looking at data from 129 children's A&E cards. A Paediatric Pain Protocol was then introduced and another 133 children's cards studied to see if this had made an improvement. The protocol for those children aged over 4 years differed to that for children aged 4 years and under. RESULTS: For children aged 4 years and over, the introduction of the protocol significantly increased the number that received analgesia within 30 minutes of triage: 55.3% (n=54) post-protocol versus 34.0% (n=33) pre-protocol (p=0.003). However, for children aged 4 years and under there was no change in the proportion that received analgesia within 30 minutes of triage: 56.7% (n=17) postprotocol versus 59.4% (n=19) pre-protocol (p=0.829). CONCLUSIONS: The introduction of a simple Paediatric Pain Protocol has improved the time taken to deliver analgesia to children arriving in this A&E department.

Diffusion of ideas about personal hygiene and contamination in poor countries: evidence from Guatemala.

In this paper, we explore the diffusion of beliefs pertaining to the causes of childhood diarrhea in rural Guatemala. The analysis focuses on the importance of interpersonal and impersonal contacts as conduits for information and norms related to hygiene and contamination. Estimates from multivariate models reveal that there is evidence of a diffusion process through social contacts, primarily through interpersonal ones. The analysis also identifies striking differences between (1) the diffusion process related to hygiene (e.g. dirtiness) and that related to contamination (e.g. pathogens); and (2) beliefs about the causes of diarrheal illness among children in general and those among respondents' own children.

Antitumor interaction of short-course endostatin and ionizing radiation.

PURPOSE: The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment of collagen XVIII, enhances the antitumor effects of ionizing radiation (IR). Endostatin was injected to coincide with fractionated radiotherapy. METHODS: Xenografts of radioresistant SQ-20B tumor cells were established in athymic nude mice. Lewis lung carcinoma cells were injected into C57BI/6 mice. Mice bearing SQ-20B xenografts were injected intraperitoneally with 2.5 mg/kg/day of murine recombinant endostatin 5 times per week for 2 weeks 3 hours before IR treatment (50 Gy total dose). Mice bearing Lewis lung carcinoma tumors were injected intraperitoneally with endostatin (2.5 mg/kg/day) four times; the first injection was given 24 hours before the first IR dose (15 Gy) and then 3 hours before IR (15 Gy/day) for 3 consecutive days. Microvascular density was assessed on tumor tissue sections by use of CD31 immunohistochemistry and light microscopy. Endothelial cell survival analyses were employed to evaluate endostatin effects on human aortic endothelial cells and human umbilical vein endothelial cells. Endothelial cell apoptosis was examined by use of FACS analysis and DAPI microscopy. RESULTS: In SQ-20B xenografts, combined treatment with endostatin and IR produced tumor growth inhibition that was most pronounced at the nadir of regression (day 21). By day 35, tumors receiving combined treatment with endostatin and IR were 47% smaller than tumors treated with endostatin alone. Interactive cytotoxic treatment effects between endostatin and IR were also demonstrated in mice bearing Lewis lung carcinoma tumors. Significant tumor growth inhibition was observed in the endostatin/IR group at days 11 and 13 compared with IR alone. Histologic analyses demonstrated a reduction in microvascular density after combined treatment with endostatin and IR compared with endostatin treatment alone. Survival analyses confirmed interactive cytotoxicity between endostatin and IR in both human aortic endothelial cells and human umbilical vein endothelial cells but not in SQ-20B tumor cells. Combined treatment with endostatin and IR produced an increase in cow pulmonary artery endothelial apoptosis compared with either treatment alone. DISCUSSION: The tumor regression observed after combined treatment with endostatin and IR suggests additive antitumor effects in both human and murine tumors. Importantly, the concentrations of endostatin employed produced little tumor regression when endostatin was employed as a single agent. The results from the clonogenic and apoptosis assays support the hypothesis that the endothelial compartment is the target for the endostatin/IR interaction.

Angiostatin induces mitotic cell death of proliferating endothelial cells.

Angiostatin is an inhibitor of tumor angiogenesis that induces regression of experimental tumors and enhances the antitumor effects of radiation therapy. We report that the cytotoxic effects of angiostatin are restricted to the proliferating endothelial cell population. In addition, angiostatin and ionizing radiation (IR) interact by inducing death of dividing endothelial cells. We also show that angiostatin and IR interact to inhibit endothelial cell migration. These findings demonstrate that angiostatin targets the proliferating tumor vasculature and provide a mechanistic basis for the cytotoxic interaction of angiostatin and IR.

Surfactant sealing of membranes permeabilized by ionizing radiation.

Acute tissue injury and subsequent inflammation, including tissue edema and erythema, can be caused by sufficiently high levels of exposure to gamma radiation. The mechanism of this tissue injury is related to the generation of reactive oxygen intermediates (ROI) which chemically alter biological molecules and cell physiology. Cell membrane lipids are vulnerable to ROI-mediated lipid peroxidation that then leads to many of the acute tissue effects. We hypothesize that increased cell membrane permeability leading to osmotic swelling and vascular transudation is one of these effects. Thus we used adult postmitotic rhabdomyocytes in culture and microscopic fluorescence techniques to quantify radiation-induced changes in cell membrane permeability. Based on time-resolved dye flux measurements, a characteristic lag time of 34 +/- 3 min was determined between exposure to 160 Gy of gamma radiation and the decrease in membrane permeability. Administration of 0.1 mM nonionic surfactant Poloxamer 188 added to the cell medium after irradiation completely inhibited the dye loss over the time course of 2 h. Thus a reproducible model was developed for studying the mechanism of acute radiation injury and the efficacy of membrane-sealing agents. As only supportive measures now exist for treating the acute, nonlethal injuries from high-dose radiation exposure, agents that can restore cell membrane function after radiation damage may offer an important tool for therapy.

Generation of a baculovirus recombinant prostate-specific membrane antigen and its use in the development of a novel protein biochip quantitative immunoassay.

Prostate-specific membrane antigen (PSMA) is a 100-kDa transmembrane glycoprotein identified by the monoclonal antibody 7E11-C5.3 from the human prostate tumor cell line LNCaP. Because of its significant upregulation in androgen refractory and metastatic prostate cancers, PSMA may be a useful prognostic biomarker and a target for developing novel therapeutic strategies. However, the lack of abundant pure PSMA protein and the low efficacy in immunoaffinity isolation from LNCaP cells have hampered the development of clinical assays. In order to obtain a renewable and reliable source of pure antigen, we used the baculovirus/insect cell system to express and purify a recombinant PSMA. A recombinant baculovirus containing a 6x histidine-tagged PSMA gene was generated, from which rPSMA was expressed and purified using cobalt-chelating affinity chromatography. The purity and correct molecular size of rPSMA were demonstrated by gel electrophoresis and mass spectrometry. Glycosidase digestions showed that the oligosaccharides on rPSMA are primarily N-linked high-mannose type. Although the glycosylation is different from the native PSMA, it did not affect the immunoreactivity of rPSMA to antibodies specific for either the intra- or the extracellular domains of PSMA. Finally, the purified rPSMA was successfully used to develop a quantitative PSMA immunoassay using the novel ProteinChip surface-enhanced laser desorption/ionization mass spectrometry technology.

Converging health inequalities in later life--an artifact of mortality selection.

An emergent issue in the health inequalities debate is how socioeconomic status (SES) and physical health relate over the life course. Many studies indicate that the SES-health relationship diminishes in later life. The present research tests the hypothesis that this convergence in health inequalities is an artifact of mortality selection, which biases downwards the "true" association between SES and health in later life. By including respondents who had subsequently died or were loss-to-followup into the analysis, I assess the sensitivity of the age-specific association between education and health to sample selection processes. I study U.S. adults followed for approximately ten years using the NHANES I Epidemiologic Followup Study. Results based on the surviving sample are robust to the inclusion of people selected out of the sample due to mortality or attrition. Sample selection biases do not appear to explain the convergence in health inequalities in late life.