Expression of cellular adhesion molecule 'OPCML' is down-regulated in gliomas and other brain tumours.

The four GPI-anchored cell adhesion molecules that exemplify the IgLON family are most highly expressed in the nervous system and associate to form up to six different heterodimeric 'Diglons' that can modify cell adhesion and inhibit axon migration. Recently, two members, OPCML and LSAMP, were identified as putative tumour suppressor genes in ovarian and renal carcinomas respectively. In this study, we investigated OPCML expression in nonneoplastic brain tissue and 35 brain tumours (18 glioblastoma multiformes, five anaplastic gliomas, five meningiomas, six metastases and one medulloblastoma) and four glioma cell lines using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). OPCML was highly expressed in cerebellum, less so in cerebral cortex, frontal lobe and meninges and was significantly reduced or absent in 83% of brain tumours and all cell lines compared with nonneoplastic whole brain. Two OPCML splice variants have been identified in humans, termed alpha1 and alpha2, but the latter has not been demonstrated in human neural tissues. Using PCR with specific primers, nonneoplastic brain and 3/6 of tested brain tumours expressed both splice variants, whereas the remaining brain tumours only expressed the alpha2 variant. Hypermethylation of the alpha1 OPCML promoter, associated with down-regulation of expression in ovarian tumours, did not correlate with expression levels in the subset of brain tumours tested, implying transcription of OPCML from an alternative promoter or a different mechanism of down-regulation. This study demonstrates that OPCML down-regulation occurs in the majority of brain tumours tested, warranting further investigation of OPCML and other IgLONs in the development and progression of brain tumours.

An open-label study of darbepoetin alfa administered once monthly for the maintenance of haemoglobin concentrations in patients with chronic kidney disease not receiving dialysis.

OBJECTIVE: To demonstrate the efficacy and safety of once-monthly (QM) darbepoetin alfa administration in maintaining haemoglobin (Hb) 11.0-13.0 g dL(-1) in subjects with chronic kidney disease (CKD) not receiving dialysis and previously treated with darbepoetin alfa every other week (Q2W). SUBJECTS: This open-label study enrolled subjects > or =18 years of age who had glomerular filtration rate > or =15 and < or =60 mL min(-1)/1.73 m(2), had Hb 11.0-13.0 g dL(-1), and were receiving Q2W darbepoetin alfa. DESIGN: Subjects were switched to QM darbepoetin alfa therapy for 28 weeks; the QM dose was titrated to maintain Hb levels. Primary end-point: proportion of subjects maintaining Hb > or =11.0 g dL(-1) during the final 8 weeks of the study (evaluation phase). Secondary end-points: Hb concentration during evaluation, darbepoetin alfa dose during the study, adverse events, laboratory parameters, and blood pressure. RESULTS: The study enrolled 152 subjects (female 52%, white 64%). Mean Hb > or =11.0 g dL(-1) during evaluation was achieved by 76% of the 150 subjects who received at least one dose of darbepoetin alfa [95% confidence interval (CI): 68%, 83%]. Mean (SD) Hb during evaluation was 11.71 (0.92) g dL(-1). Eighty-five per cent of 129 subjects who completed the study (95% CI: 78%, 91%) had Hb > or =11.0 g dL(-1) during evaluation. The dose of darbepoetin alfa over the study period was median (95% CI) 124.4 mug (106.2, 140.0). Darbepoetin alpha administered QM was well tolerated in study subjects. CONCLUSION: Darbepoetin alpha administered QM maintained Hb in study subjects with CKD not receiving dialysis.

Expression of ADAMTS-8, a secreted protease with antiangiogenic properties, is downregulated in brain tumours.

Angiogenesis and extracellular matrix degradation are key events in tumour progression, and factors regulating stromal-epithelial interactions and matrix composition are potential targets for the development of novel anti-invasive/antiangiogenic therapies. Here, we examine the expression of ADAMTS-8, a secreted protease with antiangiogenic properties, in brain tissues. Using quantitative RT-polymerase chain reaction (PCR), high, equivalent expression of ADAMTS-8 was found in normal whole brain, cerebral cortex, frontal lobe, cerebellum and meninges. ADAMTS-8 expression in 34 brain tumours (including 22 high-grade gliomas) and four glioma cell lines indicated at least two-fold reduction in mRNA compared to normal whole brain in all neoplastic tissues, and no detectable expression in 14 out of 34 (41%) tumours or four out of four (100%) cell lines. In contrast, differential expression of TSP1 and VEGF was seen in nine out of 15 (60%) and seven out of 13 (54%) tumours, with no relationship in the expression of these genes. Immunohistochemistry and Western analysis indicated downregulation of ADAMTS-8 protein in >77% tumours. Methylation-specific PCR analysis of ADAMTS-8 indicated promoter hypermethylation in one out of 24 brain tumours (a metastasis) and three out of four glioma cell lines suggesting an alternative mechanism of downregulation. These data suggest a role for ADAMTS-8 in brain tumorigenesis, warranting further investigation into its role in regulation of tumour angiogenesis and local invasion.

High-level expression of recombinant Fc chimeric proteins in suspension cultures of stably transfected J558L cells.

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.

Feasibility of planar virtual pathology: a new paradigm in volume-rendered CT colonography.

Planar virtual pathology (PVP) is an isometric rendering method for examining the CT colonography dataset, which renders the colon in discrete colonic segments. Ten patients with 36 polyps were evaluated using traditional 2D axial, 2D multiplanar reformatted, and 3D endoluminal images as well as PVP. PVP displayed 13 of 17 (76%) polyps of >1 cm, whereas 11 of 17 (65%) were detected using traditional rendering methods. PVP may be a useful adjunct in detecting additional polyps at CT colonography.

New heterocyclic precursors for thermal generation of reactive, electron-rich 1,2-diaza-1,3-butadienes.

[reaction--see text] [corrected] The preparation and thermolysis of new stable heterocyclic precursors of 1,2-diaza-1,3-butadienes is described. The resulting reactive diazadienes are trapped in situ with N-phenylmaleimide [corrected]. The effect of precursor structure on the temperature at which the diazadienes are generated is discussed.

A novel route to 2,3-pyrazol-1(5H)-ones via palladium-catalyzed carbonylation of 1,2-diaza-1,3-butadienes.

[reaction--see text] A novel Pd(0)-catalyzed carbonylation of both isolable 1,2-diaza-1,3-butadienes and those generated in situ by extrusion of SO(2) and CO(2) from heterocyclic precursors is described. The reaction proceeds at room temperature to 110 degrees C under 1-2 atm of CO to afford 2,3-pyrazol-1(5H)-ones in good to excellent yields. The effect of catalyst structure and stability on the carbonylation reaction is evaluated.

CT colonography without cathartic preparation: feasibility study.

PURPOSE: To evaluate methods for contrast material labeling of stool in the unprepared colon for computed tomographic (CT) colonography and to determine their sensitivity for polyp detection. MATERIALS AND METHODS: Fifty-six patients with suspected or known polyps were assigned to five groups. Two to seven doses of 225 mL of dilute contrast material were orally administered during 24 or 48 hours. Transverse CT images were assessed for effectiveness of stool labeling. Colonoscopy was performed in all patients and was the standard. Two radiologists blinded to prior imaging and colonoscopic results assessed polyp detection. RESULTS: For each group, average stool labeling scores and ranges were as follows: 24 hour two dose, 16% and 8%-21%; 24 hour five dose, 53% and 27%-66%; 48 hour four dose, 38% and 22%-48%; 48 hour six dose, 68% and 54%-77%; and 48 hour seven dose, 88% and 75%-98%. Sensitivity for the two radiologists for the identification of patients with polyps 1 cm or larger for each group was as follows: 24 hour two dose, 50% and 67%; 24 hour five dose, 100% and 100%; 48 hour four dose, 58% and 75%; 48 hour six dose, 56% and 67%; and 48 hour seven dose, 100% and 80%. CONCLUSION: Ingestion of contrast material adequately labels stool for lesion identification; a 48-hour lead time and multiple doses of contrast material are required. Sensitivity for polyp detection in patients with adequate stool labeling approaches the sensitivity for polyp detection in prepared colons.

Automated polyp detection at CT colonography: feasibility assessment in a human population.

PURPOSE: To test the feasibility of and improve a computer algorithm to automatically detect colonic polyps in real human computed tomographic (CT) colonographic data sets. MATERIALS AND METHODS: Twenty patients with known polyps underwent CT colonography in the supine position. CT colonographic data were processed by using a shape-based algorithm that depicts masses that protrude into the lumen. We studied nine shape criteria and three isosurface threshold settings. Results were compared with those of conventional colonoscopy performed the same day. RESULTS: There were 50 polyps (28 were > or =10 mm in size; 12, 5-9 mm; 10, <5 mm). The sensitivity with optimal settings for detecting polyps 10 mm or greater was 64% (18 of 28). Sensitivity improved to 71% (10 of 14) for polyps 10 mm or greater in well-distended colonic segments. Performance decreased for polyps less than 10 mm, poorly distended colonic segments, and other shape algorithms. There was a mean of six false-positive lesion sites per colon. These sites were reduced 39% to 3.5 per colon by sampling CT attenuation at the lesion site and discarding sites having attenuation less than a threshold. CONCLUSION: Automated detection of colonic polyps, especially clinically important large polyps, is feasible. Colonic distention is an important determinant of sensitivity. Further increases in sensitivity may be achieved by adding prone CT colonography.

Optimization of CT colonography technique: prospective trial in 180 patients.

PURPOSE: To assess the added benefits of prone positioning in addition to supine positioning and oral iodinated contrast medium for help in the detection of colonic polyps at computed tomographic (CT) colonography. MATERIALS AND METHODS: CT colonography was performed in prone and supine positions in 180 patients with polyps or risk factors for colonic neoplasia. Patients were randomly assigned to receive a standard bowel preparation or a standard preparation plus oral iodinated contrast medium. One radiologist interpreted supine images alone, and another analyzed supine and prone images. All patients subsequently underwent colonoscopy. RESULTS: At colonoscopy, 121 large (> or =1-cm-diameter) polyps and 142 smaller (0.5-0.9-cm) polyps were identified. Prone positioning resulted in increased sensitivity for identification of patients with large (> or =1-cm) polyps (increase from 70% to 85%, P: =.004) and of patients with polyps 0.5 cm or larger (increase from 75% to 88%, P: <.005), with no change in specificity. Use of oral contrast medium did not significantly improve polyp detection even in the subset of patients in whom colonic fluid attenuation was markedly increased. CONCLUSION: Acquisition and review of supine and prone CT colonographic images significantly improves the ability to identify patients with polyps 0.5 cm in diameter or larger. Administration of oral iodinated contrast medium does not significantly improve polyp detection.

Comparison of two different software systems for electron-beam CT-derived quantification of coronary calcification.

OBJECTIVE: The growing interest in coronary calcium quantification by electron-beam CT (EBCT) has led to the development of various software systems for the analysis of EBCT raw data, but it is unknown whether these software systems yield comparable results. METHODS: Two sets of EBCT scans were obtained in 73 asymptomatic patients less than 15 minutes apart. Both scans of each patient were analyzed using two different software systems, the Mayo Clinic software and the AccuImage Scoring System. The authors compared the calcium quantities yielded by the two different software systems, analyzed the interscan variability, and calculated the interobserver variability. Finally, they investigated the influence of the CT density factor inherent in the widely used Agatston score for the quantification of coronary calcium on reproducibility. RESULTS: The mean score determined by the Mayo Clinic software was 14% greater than that determined by the AccuImage system. The mean difference between the two systems was 14% +/- 25%, and the median difference was 3%. The relative mean and the median difference between the two scans of one patient were 15.3% and 6% determined by the AccuImage system and 17% and 6.5% determined by the Mayo Clinic software. The interobserver reliability calculated by the Mayo Clinic software was better than that of the AccuImage system. There was a trend for better reproducibility using calcium area rather than the Agatson score. CONCLUSIONS: Two different scoring systems do not necessarily yield the same result. Calcium quantities were systematically determined to be greater by one system than the other, and there were significant differences with regard to interobserver reliability. Hence, software should be tested with regard to reproducibility data, and the interpretation of calcium quantities should acknowledge which type of software was used.

An algorithm for noninvasive identification of angiographic three-vessel and/or left main coronary artery disease in symptomatic patients on the basis of cardiac risk and electron-beam computed tomographic calcium scores.

OBJECTIVES: We sought to model an algorithm for noninvasive identification of angiographically obstructive three-vessel and/or left main disease based on conventional cardiac risk assessment and site and extent of coronary calcium determined by electron-beam computed tomography (EBCT). BACKGROUND: Such an algorithm would greatly facilitate clinical triage in symptomatic patients with no previous diagnosis of coronary artery disease (CAD). METHODS: We examined 291 patients with suspected, but not previously diagnosed, CAD who underwent coronary angiography for clinical indications. Cardiac risk factors were determined as defined by the National Cholesterol Education Program. An EBCT scan was performed in all patients, and a coronary calcium score (Agatston method) was computed. Total per-patient calcium scores and separate scores for the major coronary arteries were generated. These scores were also analyzed for localization of coronary calcium in the more distal versus proximal tomographic sections. These parameters and the risk factors were considered for the model described in the following section. RESULTS: Sixty-eight patients (23%) had angiographic three-vessel and/or left main CAD. Multiple logistic regression analysis determined male sex, presence of diabetes and left anterior descending (LAD) and circumflex (LCx) coronary calcium scores, independent from more distal calcium localization, as independent predictors for identification of three-vessel and/or left main CAD. Based on this four variable model, a simple noninvasive index (NI) was constructed as the following: loge(LAD score) + log(e)(LCx score) + 2[if diabetic] + 3[if male]. Receiver operating characteristic curve analysis for this NI yielded an area under the curve of 0.88+/-0.03 (p < 0.0001) for separating patients with, versus without, angiographic three-vessel and/or left main CAD. Various NI cutpoints demonstrated sensitivities from 87-97% and specificities from 46-74%. The NI values >14 increased the probability of angiographic three-vessel and/or left main CAD from 23% (pretest) to 65-100% (posttest), and NI values <10 increased the probability of no three-vessel and/or left main CAD from 77% (pretest) to 95-100% (posttest). CONCLUSIONS: On the basis of a simple algorithm ("noninvasive index"), EBCT calcium scanning in conjunction with risk factor analysis can rule in or rule out angiographically severe disease, i.e., three-vessel and/or left main CAD, in symptomatic patients.

Pituitary desensitization to gonadotropin-releasing hormone increases abdominal adiposity in hyperandrogenic anovulatory women.

OBJECTIVE: To determine whether hyperandrogenism in anovulatory women affects body fat distribution. DESIGN: Prospective nonrandomized study. SETTING: An academic research environment. PATIENT(S): Ten hyperandrogenic anovulatory patients and 10 healthy women matched by body mass index. INTERVENTION(S): Regional body fat analysis was performed before and after 3 months of GnRH analogue (GnRH-a) therapy. MAIN OUTCOME MEASURE(S): Body fat distribution was measured by waist-to-hip circumference ratio, single-slice computed tomography imaging (L2-3 interspace), and total body dual-energy x-ray absorptiometry. RESULT(S): Weight, body mass index, waist-to-hip circumference ratio, total body and leg fat mass, and subcutaneous adipose area were unaffected by the presence of hyperandrogenism or the use of GnRH-a therapy. Basal abdominal fat mass, abdomen-to-leg fat mass ratio, visceral adipose area, and total visceral adipose volume were comparable in both study groups. The abdominal fat mass increased in both groups during GnRH-a therapy, whereas the abdomen-to-leg fat mass ratio rose significantly only in the hyperandrogenic patients. During GnRH-a therapy, the hyperandrogenic patients demonstrated a significant increase in visceral adipose area compared with the healthy women so that total visceral adipose volume increased significantly in the former but not the latter. CONCLUSION(S): Three months of GnRH-a administration preferentially increased abdominal fat, as measured by single-slice computed tomography imaging and total body dual-energy x-ray absorptiometry, in hyperandrogenic anovulatory women.

Colorectal lesions: evaluation with CT colography.

Computed tomographic (CT) colography is a promising technique for differentiating malignant or premalignant colorectal disease from benign lesions. In this technique, helical CT data are used to produce reformated two-dimensional (2D) CT images and simulated endoscopic images of the colon. Adenomatous polyps 0.7 cm in diameter or larger are easily detected on CT colographic images: A pedunculated polyp is identified by means of its stalk, whereas a sessile polyp appears as a polypoid soft-tissue mass projecting into the air-filled lumen of the colon. However, flat adenomas (lesions raised less than 2 mm from the surface of the colon) are difficult to detect with CT colography. In cases of colorectal cancer, both intraluminal and extraluminal disease can be evaluated with CT colography. Although CT colography does not allow differentiation between hyperplastic and adenomatous polyps, lipomas can be confidently diagnosed because of their fatty attenuation on reformatted 2D images. Pseudolesions that can produce false-positive findings at CT colography include the ileocecal valve, retained stool, retained barium, respiratory artifacts, and a stool-filled diverticulum.

Detection of colorectal polyps with CT colography: initial assessment of sensitivity and specificity.

PURPOSE: To estimate the sensitivity and specificity of computed tomographic (CT) colography in detection of colorectal polyps and to compare these findings with those at axial CT. MATERIALS AND METHODS: In 70 consecutive patients, CT colography and colonoscopy were performed. Helical axial CT images and CT colographic images (multiplanar two- and three-dimensional endoluminal images) were evaluated separately by two radiologists blinded to results from colonoscopy and other imaging studies. Findings were compared with those at colonoscopy, which was the standard. RESULTS: The sensitivity and specificity for the two observers with CT colography averaged 75% and 90% in patients with adenomas 10 mm in diameter or larger, 66% and 63% in patients with adenomas 5 mm in diameter or greater, and 45% and 80% for patients with adenomas less than 5 mm in diameter, respectively. Sensitivity and specificity with axial CT were lower than those with CT colography (58% and 74%, respectively) in patients with adenomas 10 mm in diameter or larger. CONCLUSION: Compared with axial CT, CT colography appears to have superior sensitivity and specificity in detection of clinically important colorectal adenomas. Early performance of CT colography seems promising for detection of colorectal polyps 5 mm and larger.

Computed tomographic colonography (Virtual colonoscopy): a new method for detecting colorectal neoplasms.

Computed tomographic (CT) colonography is an exciting new technique that uses volumetric CT data combined with advanced imaging software to create two-dimensional and three-dimensional images of the colon. The technique uses both three-dimensional images that simulate the endoluminal perspective of the colonoscope, as well as axial and reformatted two-dimensional images. The two-dimensional and three-dimensional images are complementary, and in combination offer the most robust performance for the detection of colorectal polyps. Currently, CT colonographic examinations are performed in the fully cleansed and air-inflated colon using a slice thickness of 5 mm, a reconstruction interval of 3 mm, a pitch of 1.3, and 70 mA. In a blinded, prospective study of 70 patients (half with a known lesion, and half from a surveillance population with a low disease prevalence) the sensitivity for the detection of polyps of 1 cm or more is 75%, and the specificity is 90%. The most commonly encountered problems include retained colonic fluid and stool, suboptimally distended colonic segments, and long interpretation times. Many of these problems can be solved using both supine and prone imaging. It is expected that the performance of this examination will improve, and that a new era of colorectal screening will begin.

Automatic segmentation, tissue characterization, and rapid diagnosis enhancements to the computed tomographic colonography analysis workstation.

An image processing system developed to support examination of computed tomographic colonoscopy (CTC) was developed in 1995. The clinical viability of CTC is enhanced by the solution of several technical problems. These problems include the limited detectability of sessile polyps and difficulties in discrimination between polypoid masses and retained stool. CTC is also made more feasible by simplifying the required colon preparation and reducing the time required to analyze scan results. Each of these challenges have been addressed by enhancements to the CTC analysis workstation software. Endoluminal volume rendering has been enhanced by the addition of automatic segmentation to facilitate analysis of colon segments, which contain tagged liquid stool. By automating this function, the system is able to process scans that are acquired following a wide variety of colon preparation protocols. Similar approaches have been used to identify retained stool. Automatic tissue characterization has also been incorporated into the volume rendering routines to help identify and diagnose polypoid masses. These enhancements have improved the quality of CTC interpretation, while reducing the time required to perform the analysis. This time reduction was necessary to reduce the cost of CTC enough to make it viable for asymptotic population screening. To date, over 150 patient examinations have been performed using this new technique. A recent blinded, prospective study reporting the results from two independent observers has been presented. The technique is feasible, reliable, and has been implemented clinically with results reported within 1 hour of the examination.