bluuebunny/crossref_metadata_2025 huggingface dataset:
Action control of autonomous agents in continuous valued space using RFCN
Authors: Shirakawa Shinichi , Nagao Tomoharu
Published: September 2008

Researchers on action control of autonomous agents and multiple agents have attracted increasing attention in recent years. The general methods using action control of agents are neural network, genetic programming, and reinforcement learning. In this study, we use neural network for action control of autonomous agents. Our method determines the structure and parameter of neural network in evolution. We proposed Flexibly Connected Neural Network (FCN) previously as a method of constructing arbitrary neural networks with optimized structures and parameters to solve unknown problems. FCN was applied to action control of an autonomous agent and showed experimentally that it is effective for perceptual aliasing problems. All of the experiments of FCN, however, are only in grid space. In this paper, we propose a new method based on FCN which can decide correction action in real and continuous valued space. The proposed method, called Real‐valued FCN (RFCN), optimizes input–output functions of each unit, parameters of the input–output functions and speed of each unit. In order to examine its effectiveness, we applied the proposed method to action control of an autonomous agent to solve continuous‐valued maze problems. © 2008 Wiley Periodicals, Inc. Electron Comm Jpn, 91(2): 31–39, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/eej.10032
Evaluation of glycosylation for quality assurance of antibody pharmaceuticals by capillary electrophoresis
Authors: Kamoda Satoru , Kakehi Kazuaki
Published: September 2008

The development of monoclonal antibody (mAb) pharmaceuticals has rapidly generated in this decade, and there are currently 23 FDA‐approved mAb pharmaceuticals. Carbohydrate chains in mAb pharmaceuticals play important roles for the expression of their biological activities such as antibody‐dependent cellular cytotoxicity, whereas the oligosaccharide profile is easily changed depending on the manufacturing conditions. Therefore, the evaluation of carbohydrate chains in detail is quite important for quality control in the development of mAb from early phases, but oligosaccharides in mAb have high heterogeneity and full characterization of oligosaccharide moiety in mAb has been a challenging target. Capillary electrophoresis (CE) is a powerful tool for the evaluation of glycan occupancy and oligosaccharide profile. This review focuses mainly on the application of CE to the analytical studies on glycosylation in mAb pharmaceuticals developed in our laboratory. The related techniques including the full‐image microchip isoelectric focusing and the newly developed mass spectrometry technologies are also shown. The combination of the techniques described in this review will be a good guide for the evaluation of glycosylation for quality assurance of antibody pharmaceuticals by CE from the early stage of the development to the marketing stage.
Electrophoresis studies on the contaminating glycosaminoglycans in commercially available hyaluronic acid products
Authors: Matsuno Yu‐Ki , Kakoi Naotaka , Kinoshita Mitsuhiro , Matsuzaki Yuji , Kumada Jun‐Ichi , Kakehi Kazuaki
Published: September 2008

Hyaluronic acid (HA) samples showing inhibition effect on digestion with testicular hyaluronidase (HAase) were found from 16 commercially available HA products, which were supplied from 11 different manufacturers. Most of these HA samples (six samples) were derived from the rooster comb, and one sample was derived from the human umbilical cord. HA oligosaccharides produced by exhaustive digestion of these HA samples with testicular HAases were monitored by capillary electrophoresis, and we found that a few HA samples gave no oligosaccharide products. Detailed analysis of HA samples by cellulose acetate membrane electrophoresis revealed that the HA samples were not digested with HAase because of the presence of a small amount of dermatan sulfate (DS). Analysis of disaccharide units of these HA samples produced by digestion with chondroitinase ABC supported the observations. And the content of DS in the sample was estimated to be ca. 8%. In contrast, these HA samples were easily digested with bacterial hyaluronate lyases from Streptomyces hyalurolyticus and Streptococcus dysgalactiae and gave endproducts of unsaturated disaccharide or unsaturated tetra‐ or hexasaccharides. The results suggested that the inhibitory effect of DS on HAase is specific to endo‐type hydrolase (i.e. testicular HAase). In addition, pharmaceutical preparations of HA derived from rooster comb were easily digested with testicular HAase. These findings will be useful information for clinical or cosmetic use of HA preparations in terms of their half‐life.
A highly sensitive CE‐UV method with dynamic coating of silica‐fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions
Authors: Iqbal Jamshed , Lévesque Sébastien A. , Sévigny Jean , Müller Christa E.
Published: September 2008

A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on‐line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5′‐triphosphate to adenosine 5′‐monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field‐enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica‐fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on‐line sensitivity enhancement technique, a more than 70‐fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused‐silica capillary (30 cm effective length×75 μm), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of −60 μA, UV detection at 210 nm, uridine 5′‐monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.
Analysis of the molecular size distribution of polyethylene glycols by CE‐UV versus HPLC with evaporative light scattering detection
Authors: Brinz Daniela , Holzgrabe Ulrike
Published: September 2008

A simple capillary zone electrophoresis method has been developed for the separation of polyethylene glycols (PEGs) consisting of differing amounts of polymers with different monomer numbers up to an average molecular mass of 1500. To provide both charge and detectability, the analytes were derivatized with phthalic anhydride according to a conventional method and a newly developed microwave‐assisted method. While obtaining nearly equal amounts of single and double esterificated PEGs with the conventional method, the fraction of single derivatized PEGs is considerably reduced by microwave‐assisted reaction. In order to compare the capillary zone electrophoresis results, an HPLC method with evaporative light scattering detection has been established. This method was successfully applied to the analysis of underivatized PEG 400, 1000 and 1500.
Fast assay of glucosamine in pharmaceuticals and nutraceuticals by capillary zone electrophoresis with contactless conductivity detection
Authors: Jáč Pavel , Los Petr , Spáčil Zdeněk , Pospíšilová Marie , Polášek Miroslav
Published: September 2008

A novel capillary electrophoresis (CE) method with contactless conductivity detection suitable for the determination of glucosamine (GlAm) and K+ in pharmaceuticals was devised. Under the optimum conditions (aqueous 30 mM acetate buffer of pH 5.2 as the background electrolyte; voltage 30 kV; 25°C), GlAm (migrating as glucosaminium cation) was well separated from K+ that could occur in the dosage forms as excipient. The CE analysis was performed in fused‐silica capillaries (50 µm i.d., 75 cm total length, 27 cm to detector) and the separation took <3 min. The calibration graphs were linear for both GlAm (100–300 µg/mL; r2=0.997) and K+ (15–75 µg/mL; r2=0.997) when using ethanolamine (100 µg/mL) as the internal standard. The LOD values (S/N=3) were 9.3 µg/mL for GlAm and 2.9 µg/mL for K+. The method was applied to the assay of GlAm content in various dosage forms. Intermediate precision evaluated by determining the content of GlAm in a single formulation on 3 consecutive days was characterized by RSD 2.35% (n=15). Acceptable accuracy of the CE method was confirmed by the added/found GlAm recovery experiments (recoveries 94.6–103.3%) and by statistical comparison of the results attained by the proposed CE and a reference HPLC method.
Cyclodextrin‐based nonaqueous electrokinetic chromatography with UV and mass spectrometric detection: Application to the impurity profiling of amiodarone
Authors: Mol Roelof , de Jong Gerhardus J. , Somsen Govert W.
Published: September 2008

The potential of nonaqueous electrokinetic chromatography (NAEKC) using cyclodextrins (CD) for the analysis of basic drugs and related compounds was evaluated. Both UV absorbance and mass spectrometric (MS) detection were employed. Addition of neutral CD to the NA background electrolyte did not significantly enhance the separation of a test mixture of basic drugs, and no change in selectivity was observed. In contrast, anionic single‐isomer‐sulfated CD strongly added to the selectivity of the NAEKC system inducing an improved resolution among the test compounds and increasing the migration time window. The applicability of the NAEKC system using anionic CD is demonstrated by the profiling of a sample of the drug amiodarone that had been stored for 1 year at room temperature. Amiodarone is poorly soluble in water. NAEKC‐UV analysis indicated the presence of at least seven impurities in the amiodarone sample. In order to identify these compounds, the NAEKC system was coupled directly to electrospray ionization (ESI) ion‐trap MS. The total of detected impurities increased to 12 due to the added sensitivity and selectivity of MS detection. Based on the acquired MS/MS data, three sample constituents could be identified as ‘known’ impurities (British Pharmacopoeia), whereas for three unknown impurities molecular structures could be proposed. Estimated limits of detection for amiodarone using the NAEKC method were 1 μg/mL with UV detection and 15 ng/mL with ESI‐MS detection (full‐scan). Based on relative responses, the impurity content of the stored drug substance was estimated to be 0.33 and 0.47% using NAEKC‐UV and NAEKC‐ESI‐MS, respectively.
Strategies in method development to quantify enantiomeric impurities using CE
Authors: El Deeb Sami , Hasemann Phillip , Wätzig Hermann
Published: September 2008

The growing number of chiral new drug substances requires increasing efforts in developing enantioselective methods. According to International conference on Harmonization guidelines, one should quantify the enantiomeric impurity of 0.1% relative to the major constituent. Capillary electrophoresis has evolved into an important tool for the separation of chiral drugs. The common strategies consist of two steps: firstly, initial separation conditions are evaluated. This screening usually focuses on the selection of the appropriate chiral selector. In our study 22 neutral, anionic or cationic cyclodextrins were dissolved in phosphate buffer (pH 2.5, 50 mM, CD conc.: 2.0%). Then they were investigated for the separation of 14 chiral compounds. Secondly, the obtained initial conditions for the enantiomeric separation were optimized in terms of resolution and analysis time. In our approach, important optimized factors including the concentration of the chiral selector (1–10%), the pH of the buffer (2.0–9.0), and the percentage of organic modifier (0–15%) were studied.

This common strategy was completed by elaborating final requirements for the quantification of the enantiomeric impurity. A resolution between 3 and 4 was found to be necessary for the racemic mixture during the screening and optimization steps, in order to later allow for peak overloading and thus to sufficiently increase the signal‐to‐noise ratio. The complete strategy was conducted for atenolol, isoprenaline, verapamil and mandelic acid.
A simple micellar electrokinetic capillary chromatographic method for the quantitative analysis of organic expectorants
Authors: Lin Yu‐Ting , Kou Hwang‐Shang , Wu Hsin‐Lung
Published: September 2008

A simple and rapid micellar electrokinetic capillary chromatographic method is described for the separation and quantification of five expectorant drugs, including ambroxol (AMB), bromhexine (BRM), carbocysteine, guaiacol and guaifenesin. The drugs were separated in a mixed solution of phosphate buffer (35 mM; pH 3.90) and acetonitrile (75:25, v/v) with sodium dodecylsulfate (120 mM) as the micellar source, and the separated drugs were directly monitored with UV detector (200 nm). Several key parameters affecting the separation and analysis of the drugs were studied and optimized. Based on the corrected peak‐area ratios of the drugs to an internal standard (7‐hydroxy‐4‐methyl‐coumarin) versus the concentration of the drugs, the established method is applicable to quantify AMB and BRM each over 20–150 µM, carbocysteine over 100–1500 µM, guaiacol and guaifenesin each over 10–150 µM. The detection limits (S/N=3; 0.5 psi, 5 s injection) of the method for the analytes are in the range of 3.0–5.0 µM (except that of carbocysteine at 30 µM). The precision (relative standard deviation) and accuracy (relative error) of the method for the intra‐day (n=3) and inter‐day (n=5) analyses of the analytes at three levels are all below 4%. The method is speedy with a run time of about 6 min for the analysis of the five analytes. Application of this method to the analysis of AMB and BRM in pharmaceutical preparations or AMB in the urine of a dosed subject proved simple and effective.
Sensitive analysis of donepezil in plasma by capillary electrophoresis combining on‐column field‐amplified sample stacking and its application in Alzheimer's disease
Authors: Yeh Hsin‐Hua , Yang Yuan‐Han , Ko Ju‐Yun , Chen Su‐Hwei
Published: September 2008

Field‐amplified sample stacking (FASS) in capillary electrophoresis (CE) was used to determine the concentration of donepezil, an acetylcholinesterase inhibitor, in human plasma. A sample pretreatment by liquid–liquid extraction with isopropanol/n‐hexane (v/v 3:97) and subsequent quantification by FASS‐CE was used. Before sample loading, a water plug (0.5 psi, 6 s) was injected to permit FASS. Electrokinetic injection (7 kV, 90 s) was used to introduce sample cations. The separation condition for donepezil was performed in electrolyte solutions containing Tris buffer (60 mM, pH 4.0) with sodium octanesulfonate 40 mM and 0.01% polyvinyl alcohol as a dynamic coating to reduce analytes' interaction with capillary wall. The separation was performed at 28 kV and detected at 200 nm. Using atenolol as an internal standard, the linear ranges of the method for the determination of donepezil in human plasma were over a range of 1–50 ng/mL. The limit of detection was 0.1 ng/mL (S/N=3, sampling 90 s at 7 kV). One female volunteer (54 years old) was orally administered a single dose of 10 mg donepezil (Aricept®, Eisai), and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied successfully to monitor donepezil in sixteen Alzheimer's disease patients' plasmas.
Hybrid simulation of a zipper‐braced steel frame under earthquake excitation
Authors: Yang T. Y. , Stojadinovic B. , Moehle J.
Published: September 2008

Hybrid simulation is a testing methodology that combines laboratory and analytical simulation to evaluate seismic response of complex structural framing systems. One or more portions of the structure, which may be difficult to model numerically or have properties that have not been examined before, are tested in one or more laboratories, whereas the remainder of the structure is modeled in software using one or more computers. These separate portions are assembled such that combined dynamic response of the hybrid model to excitation is computed using a time‐stepping procedure.

A hybrid simulation conducted to examine the seismic response of a type of steel concentrically braced frame, the suspended‐zipper‐braced frame, is presented. The hybrid simulation testing architecture, hybrid model, test setup, solution algorithm, and the seismic response of the suspended‐zipper‐braced frame hybrid model are discussed. Accuracy of this hybrid simulation is examined by comparing hybrid and computer‐only simulations and the errors are quantified using an energy‐based approach. This comparison indicates that the deployed hybrid simulation method can be used to accurately model the seismic response of a complex structural system such as the zipper‐braced frame. Copyright © 2008 John Wiley & Sons, Ltd.
Capn4 overexpression underlies tumor invasion and metastasis after liver transplantation for hepatocellular carcinoma #
Authors: Bai Dou-Sheng , Dai Zhi , Zhou Jian , Liu Yin-Kun , Qiu Shuang-Jian , Tan Chang-Jun , Shi Ying-Hong , Huang Cheng , Wang Zheng , He Yi-Feng , Fan Jia
Published: September 2008

Liver transplantation (LT) is one of the best therapeutic options for nonresectable hepatocellular carcinoma (HCC). Unfortunately, some HCC patients succumb to the disease after LT, which reduces long- and medium-term survival. To identify the proteins associated with HCC invasion and metastasis, HCC patients undergoing LT with complete follow-up data were included in this study and were categorized into recurrence and nonrecurrence groups. We extracted the total protein from the acquired homogeneous tumor cells and applied a cleavable isotope-coded affinity tag technology to quantitate relative changes in protein levels between the two groups. We identified a total of 149 proteins with two-dimensional liquid chromatography coupled with tandem mass spectrometry, including 52 differentially expressed proteins by at least two-fold. Among them, calpain small subunit 1 (Capn4), a protein with relevant interactions with many migration–invasion-related proteins, has attracted more attention. First, Capn4 overexpression in the recurrence group was confirmed via real-time polymerase chain reaction and western blotting in another cohort of 40 HCC patients undergoing LT. Second, Capn4 was associated with enhanced invasiveness in vitro. The small interfering RNA–mediated knockdown expression of Capn4 in HCC cell lines significantly inhibited its mobile and invasive ability. Tissue microarray in a further 192 cases revealed that Capn4 significantly correlated with invasive phenotype of HCC, and univariate and multivariate analyses indicated that Capn4 is an independent prognostic factor for recurrence and survival of HCC patients. Conclusion: Our study revealed that Capn4 overexpression underlies invasion and metastasis after LT for HCC and might be a candidate biomarker for future diagnosis and a target for therapy. (Hepatology 2008.)
Relationship of SNCG, S100A4, S100A9 and LCN2 gene expression and DNA methylation in bladder cancer
Authors: Dokun Olusola Y. , Florl Andrea R. , Seifert Hans‐Helge , Wolff Ingmar , Schulz Wolfgang A.
Published: September 2008

Microarray analysis of paired cultures of normal and cancerous urothelial cells revealed differences in cytokeratin and adhesion gene expression. Normal cells expressed autocrine growth factor genes more strongly whereas carcinoma cells were distinguished by concomitant expression of urothelial and epidermal differentiation markers. Expression of SNCG, S100A9 and LCN2 was also enhanced. In other cancers, overexpression of SNCG, LCN2 and S100A4 has been ascribed to DNA hypomethylation. We therefore investigated expression and methylation of SNCG, S100A4, S100A9 and LCN2 in urothelial cancer cell lines and tissues. SNCG and S100A4 were overexpressed in some cancer tissues and cell lines, but downregulated in others, whereas LCN2 and S100A9 were upregulated in few cancer cell lines, but regularly in tissues. Normal and cancerous urothelial cells expressing SNCG lacked promoter methylation. SNCG downregulation was associated with hypermethylation and could be reversed by the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine. S100A4 methylation at regulatory intronic sites and in the promoter region was lowest in leukocytes and fibroblasts, and denser in urothelial cells. Gene expression responded to 5‐aza‐2′‐deoxycytidine. LCN2 promoter methylation was variable and even less consistently related to expression. The S100A9 promoter was partially methylated in nonexpressing cells, but 5‐aza‐2′‐deoxycytidine had no effect. Our data indicate that SNCG methylation is cell type‐specific and the gene is hypermethylated in some urothelial cancers. S100A4, S100A9 and LCN2 are genes with moderate CpG‐density that show a less stringent relationship between DNA methylation and gene expression. Therefore, changes in methylation of these genes in cancer should be interpreted cautiously. © 2008 Wiley‐Liss, Inc.
Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to gefitinib
Authors: Servidei Tiziana , Riccardi Anna , Mozzetti Simona , Ferlini Cristiano , Riccardi Riccardo
Published: September 2008

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2‐ to 3‐fold more sensitive to gefitinib growth‐inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF‐triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF‐induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3β with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85‐PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA‐mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF‐triggered pathway and a constitutive HER3‐mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues. © 2008 Wiley‐Liss, Inc.
Endoglin (CD105) as a urinary and serum marker of prostate cancer
Authors: Fujita Kazutoshi , Ewing Charles M. , Chan David Y.S. , Mangold Leslie A. , Partin Alan W. , Isaacs William B. , Pavlovich Christian P.
Published: September 2008

We have previously shown that endoglin (CD105) is upregulated in prostatic fluid of men with large volume prostate cancer. We chose to assess endoglin levels in urine and serum from men with prostate cancer or at increased risk for the disease: Urine samples were collected after digital rectal examination (DRE) from 99 men whose cancer status was confirmed by biopsy, and serum samples were collected from 20 men without prostate cancer at low risk for the disease and from 69 men diagnosed with prostate cancer that subsequently underwent radical prostatectomy (30 pT2, 39 pT3). Endoglin levels were assessed by ELISA. Urinary endoglin was elevated in men with biopsy‐positive prostate cancer compared to biopsy‐negative men (p = 0.0014). Urinary endoglin levels in men with prostate cancer correlated with radical prostatectomy tumor volume. The area under the receiver operating characteristic (ROC) curve was 0.72 for urinary endoglin and 0.50 for serum prostate‐specific antigen (PSA; sensitivity for cancer detection 73%, specificity 63%). There were no differences in serum endoglin between normal and cancer cases, but there were increases in serum endoglin in non‐organ confined (NOC, pT3+) versus organ‐confined (OC, pT2) cases (p = 0.0004). The area under the ROC curve was 0.75 for serum endoglin and 0.63 for PSA for predicting NOC status, with a sensitivity of 67% and a specificity of 80%. In conclusion, elevations in post‐DRE urinary endoglin suggest there may be value in further studying endoglin as a urinary biomarker of prostate cancer. Endoglin levels in both urine and serum may aid in prostate cancer detection and prognostication. © 2008 Wiley‐Liss, Inc.
Novel genetic variants in microRNA genes and familial breast cancer
Authors: Shen Jie , Ambrosone Christine B. , Zhao Hua
Published: September 2008

MicroRNA (miRNA) plays an important role in tumorigenesis, but whether miRNA is a cancer predisposition factor or not is still unknown. Considering the fact that miRNA regulates a number of tumor suppressor genes (TSGs) and oncogenes, genetic variations in miRNA genes could affect the levels of expression of TSGs or oncogenes and, thereby, cancer risk. To test this hypothesis, we screened genetic variants in 17 selected miRNA genes, which are predicted to regulate key breast cancer genes, in 42 patients with familial breast cancer. Seven novel genetic variants were observed in 7 primary or precursor miRNA genes. Among them, 1 rare variant in the precursor of miR‐30c‐1 and 1 rare variant in the primary precursor of miR‐17 were only observed in noncarriers of BRCA1/2 mutations. In functional assays, these 2 variants resulted in conformational changes in the predicted secondary structures, and consequently altered the expression of mature miR‐30c‐1 and miR‐17. In the target in vitro assay, we observed that miR‐17 could bind to the 3′UTR of BRCA1 mRNAs, which is predicted to be a target for miR‐17. Our findings suggest that functional genetic variants in miRNA genes can potentially alter the regulation of key breast cancer genes. Whether they confer genetic susceptibility to breast cancer remains to be determined. © 2008 Wiley‐Liss, Inc.
Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older
Authors: Meijer Chris J.L.M. , Berkhof Johannes , Castle Philip E. , Hesselink Albertus T. , Franco Eduardo L. , Ronco Guglielmo , Arbyn Marc , Bosch F. Xavier , Cuzick Jack , Dillner Joakim , Heideman Daniëlle A.M. , Snijders Peter J.F.
Published: September 2008

Given the strong etiologic link between high‐risk HPV infection and cervical cancer high‐risk HPV testing is now being considered as an alternative for cytology‐based cervical cancer screening. Many test systems have been developed that can detect the broad spectrum of hrHPV types in one assay. However, for screening purposes the detection of high‐risk HPV is not inherently useful unless it is informative for the presence of high‐grade cervical intraepithelial neoplasia (CIN 2/3) or cancer. Candidate high‐risk HPV tests to be used for screening should reach an optimal balance between clinical sensitivity and specificity for detection of high‐grade CIN and cervical cancer to minimize redundant or excessive follow‐up procedures for high‐risk HPV positive women without cervical lesions. Data from various large screening studies have shown that high‐risk HPV testing by hybrid capture 2 and GP5+/6+‐PCR yields considerably better results in the detection of CIN 2/3 than cytology. The data from these studies can be used to guide the translation of high‐risk HPV testing into clinical practice by setting standards of test performance and characteristics. On the basis of these data we have developed guidelines for high‐risk HPV test requirements for primary cervical screening and validation guidelines for candidate HPV assays. © 2008 Wiley‐Liss, Inc.
Population differences in immune marker profiles associated with human T‐lymphotropic virus type I infection in Japan and Jamaica
Authors: Birmann Brenda M. , Breen Elizabeth C. , Stuver Sherri , Cranston Beverly , Martínez‐Maza Otoniel , Falk Kerstin I. , Okayama Akihiko , Hanchard Barrie , Mueller Nancy , Hisada Michie
Published: September 2008

The natural history of human T‐lymphotropic virus type I (HTLV‐I) has been shown to differ markedly by geographic area. The differences include contrasting patterns of risk of adult T‐cell lymphoma (ATL) and HTLV‐I‐associated myelopathy/tropical spastic paraparesis (HAM/TSP), which may be due in part to differences in host immune response to infection. To characterize variations in host immunity across populations, we compared serologic immune marker patterns in HTLV‐I‐endemic populations in Japan and Jamaica. We matched 204 participants with archived blood from the Miyazaki Cohort Study (Japan) and the Food Handlers Study (Jamaica)—i.e., 51 HTLV‐I‐positive (“carriers”) and 51 HTLV‐I‐negative individuals (“noncarriers”) from each population—by age, sex and blood collection year. We compared plasma concentrations of markers of T‐cell‐mediated (antigen‐specific) and nonspecific immunity using regression models and correlation coefficients. Compared to Jamaican HTLV‐I noncarriers, Japanese noncarriers had higher covariate‐adjusted mean levels of T‐cell activation markers, including antibody to Epstein‐Barr virus nuclear antigen‐1 (reciprocal titer 27 vs. 71, respectively, p = 0.005), soluble interleukin‐2 receptor‐α (477 vs. 623 pg/mL, p = 0.0008) and soluble CD30 (34 vs. 46 U/mL, p = 0.0001) and lower levels of C‐reactive protein (1.1 vs. 0.43 μg/mL, p = 0.0004). HTLV‐I infection was associated with activated T‐cell immunity in Jamaicans but with diminished T‐cell immunity in Japanese persons. The observed population differences in background and HTLV‐I‐related host immunity correspond closely to the divergent natural histories of infection observed among HTLV‐I carriers in Japan and Jamaica and corroborate a role for host immune status in the contrasting patterns of ATL and HAM/TSP risk. © 2008 Wiley‐Liss, Inc.
TGFB1 and TGFBR1 polymorphic variants in relationship to bladder cancer risk and prognosis
Authors: Castillejo Adela , Rothman Nathaniel , Murta‐Nascimento Cristiane , Malats Núria , García‐Closas Montserrat , Gómez‐Martínez Angeles , Lloreta Josep , Tardón Adonina , Serra Consol , García‐Closas Reina , Chanock Stephen , Silverman Debra T. , Dosemeci Mustafa , Kogevinas Manolis , Carrato Alfredo , Soto José Luis , Real Francisco X.
Published: September 2008

The transforming growth factor‐beta (TGF‐β) signalling pathway plays an important role in tumor development and progression. We aimed at analyzing whether 7 different common variants in genes coding for 2 key members of the TGF‐β signalling pathway (TGFB1 and TGFBR1) are associated with bladder cancer risk and prognosis. A total of 1,157 cases with urothelial cell carcinoma of the bladder and 1,157 matched controls where genotyped for 3 single nucleotide polymorphisms (SNPs) in TGFB1 (rs1982073, rs1800472, rs1800471) and an additional 3 SNPs and 1 indel polymorphism in TGFBR1 (rs868, rs928180, rs334358 and rs11466445, respectively). In the case‐control study, we estimated odds ratios and 95% confidence intervals for each individual genetic variant using unconditional logistic regression adjusting for age, gender, study area and smoking status. Survival analysis was performed using the Kaplan‐Meier method and Cox models. The endpoints of interest were tumor relapse, progression and death from bladder cancer. All the SNPs analyzed showed a similar distribution among cases and controls. The distribution of the TGFBR1*6A allele (rs11466445) was also similar among cases and controls, indicating no association with bladder cancer risk. Similarly, none of the haplotypes was significantly associated with bladder cancer risk. Among patients with muscle‐invasive tumors, we found a significant association between TGFBR1‐rs868 and disease‐specific mortality with an allele dosage effect (p‐trend = 0.003). In conclusion, the genetic variants analyzed were not associated with an increased risk of bladder cancer. The association of TGFBR1‐rs868 with outcome should be validated in independent patient series. © 2008 Wiley‐Liss, Inc.
Comparative evaluation of FASTPlaque assay with PCR and other conventional in vitro diagnostic methods for the early detection of pulmonary tuberculosis
Authors: Singh Sarman , Saluja Taran Prit , Kaur Manjot , Khilnani G.C.
Published: September 2008

Rapid and accurate diagnosis of symptomatic patients of pulmonary tuberculosis (TB) is highly desirable to minimize the spread of the disease in the society. We, therefore, compared the usefulness of various conventional diagnostic methods, the in‐house polymerase chain reaction (PCR), and the FASTPlaque assay in this study. Laboratory data of 150 patients with clinical diagnosis of pulmonary TB and 50 controls were included in this study. The sputa from all these 200 individuals were subjected to acid‐fast staining, culture on Lowenstein–Jensen (L‐J) slants, automated BACTEC‐MGIT‐960 culture methods, and a mycobacteriophage assay. A mycobacterium genus andMycobacterium tuberculosisspecies‐specific PCRs were also done and samples positive on both PCRs were considered as standard for comparison. Of the 5 in vitro diagnostic tests, PCR method was found to be the most rapid, sensitive, and specific, detecting all the 150 cases of pulmonary TB without any false‐positive and negative result. In comparison with PCR the sensitivity of MGIT‐960 was 90%, followed by FASTPlaque assay (76.7%), L‐J culture method (73.3%), and microscopy (60%). The mean detection time for smear‐positive and smear‐negative samples was 12.5 and 14 days in MGIT‐960 and 18 and 25 days for L‐J method, respectively. The FASTPlaque failed to detect mycobacteria from the paucibacillary samples. The contamination rates for MGIT‐960, L‐J, and FASTPlaque assays were 4, 8 and 10%, respectively. The best correlation with mycobacterial load in the specimen was observed in BACTEC‐MGIT‐960 showing 66.6% detection rate in paucibacillary, 83.3% in 1+ samples, and 100% in 2+ and 3+ samples. Out of the 150 patients, 140 (93.3%) could be diagnosed by one or more nonmolecular methods. Therefore, it could be concluded that combination of three or more in vitro diagnostic methods will have acceptable detection level. J. Clin. Lab. Anal. 22:367–374, 2008. © 2008 Wiley‐Liss, Inc.
Normal blood cells reference intervals of healthy adults at the Gaza Strip—Palestine
Authors: Sirdah Mahmoud M. , Tarazi Issa S. , Jeadi Hesham El , Al Haddad Rami M.
Published: September 2008

Hematological parameters are affected by different factors that include age, sex, smoking, ethnicity, and environmental altitude. It has been justified that each population must establish its own normal reference intervals to be used in clinical assessments and interpretations. Hematological reference intervals for adults from the Gaza Strip—Palestine have never been addressed. Therefore, this study was designed and aimed at the establishment of normal blood cells reference intervals for healthy adults at the Gaza Strip—Palestine. This study involved 89,491 apparently healthy individuals (from both sexes and from the different governorates of the Gaza Strip) who were referred to the Thalassemia Central Laboratory during the period from September 2000 until February 2008. Complete blood counts were performed. Subjects were categorized into subgroups according to gender, smoking habit, and age (15–18, 19–45, and >45 years old). For each subgroup, descriptive and comparative statistical analysis was performed for hematological parameters. The results showed substantial differences between males and females, between smokers and nonsmokers, and between the different age groups. Moreover, reference intervals derived from our population are markedly shifted downward as compared with Western European and American populations. It was concluded that separate and region‐specific reference intervals based on gender, smoking, and age for the Palestinian population at the Gaza Strip should be generalized for clinical laboratories and clinical practitioners, which could help in interpreting laboratory hematological tests more specifically, and potentially develop the quality of medical care provided to patients. J. Clin. Lab. Anal. 22:353–361, 2008. © 2008 Wiley‐Liss, Inc.
Serum adipocyte‐fatty acid binding protein discriminates patients with permanent and temporary body weight loss
Authors: Stejskal D. , Karpisek M. , Bronsky J.
Published: September 2008

Adipocyte‐fatty acid binding protein (A‐FABP) is a biomarker of adiposity and metabolic syndrome. The aim of our work was to investigate the effect of weight reduction on serum A‐FABP value. In the study, we analyzed a group of 189 probands suffering from obesity (102 women and 87 men; aged 57.3±12 years) initially, after a 3‐month low‐fat diet and once again 3 months after the termination of the diet for serum A‐FABP, insulin, glucose, total cholesterol, HDL‐cholesterol, LDL‐cholesterol, and triglycerides. Basal biomarker concentrations were typical of the metabolic syndrome, and moreover A‐FABP correlated with Quicki and BMI. We observed a reduction in BMI in 145 subjects who were divided into two subgroups: A—with persistent BMI reduction even after 6 months, B—with BMI reduction after 3 months and its regress after 6 months. Individuals with rise or no BMI difference were signed as subgroup C. In subgroup A, A‐FABP level increased and returned to the earlier level (42.3 vs 68.3 vs 37.1 µg/l) and correlated with the markers of the metabolic syndrome. In subgroup B, A‐FABP level increased less significantly, however elevated A‐FABP level persisted for 6 months (41.9 vs 53.6 vs 50.7 µg/l). Subgroup C (n=54) showed no difference in A‐FABP after 3‐month diet and after next 3 months. The A‐FABP value correlated with the some components of the metabolic syndrome. In conclusion, we describe that serum A‐FABP might be a prognostic marker of body weight loss suggesting a preventive therapeutic intervention. J. Clin. Lab. Anal. 22:380–382, 2008. © 2008 Wiley‐Liss, Inc.
Evaluation of a new commercial enzyme immunoassay for the detection of IgM antibodies to West Nile virus using a ratio method to eliminate nonspecific reactivity
Authors: Welch Ryan J. , Anderson Brian L. , Litwin Christine M.
Published: September 2008

As West Nile virus (WNV) has become endemic in the United States, following the first reported cases in New York during the summer of 1999, the demand for specific serology has increased. Several IgM capture ELISA assays for the detection of WNV‐specific IgM have been approved by the Food and Drug Administration for in vitro diagnostic testing, including kits from Focus Diagnostics and InBios International, Inc. The Focus Diagnostics IgM capture ELISA has a background subtraction protocol and the InBios IgM capture ELISA implements a ratio method to detect nonspecific reactivity due to rheumatoid factor, heterophile antibodies, and other interfering substances. We compared the InBios IgM capture ELISA with the Focus Diagnostics capture ELISA. Agreement, sensitivity, and specificity of the InBios IgM capture ELISA were 99, 98, and 100%, respectively. Samples that originally tested positive on the Focus Diagnostics IgM capture ELISA without the subtraction protocol and were then determined negative following the subtraction protocol agreed 100% with the InBios IgM capture ELISA. We conclude that a method to eliminate background reactivity is a necessary portion of any anti‐WNV IgM assay in order to eliminate false‐positive results. J. Clin. Lab. Anal. 22:362–366, 2008. © 2008 Wiley‐Liss, Inc.
Use of vaginal pH in diagnosis of infections and its association with reproductive manifestations
Authors: Mania‐Pramanik Jayanti , Kerkar S.C. , Mehta P.B. , Potdar S. , Salvi V.S.
Published: September 2008

Increase in vaginal secretion pH is an indicator of bacterial vaginosis (BV), but is yet to be in use as a diagnostic tool by clinicians. Similarly, no reports are available on the effect of cervical chlamydia infection and different reproductive manifestations on vaginal secretion pH. This study evaluated the use of vaginal pH for screening of BV, the effect of Chlamydia trachomatis (C. trachomatis) infection, and different reproductive manifestations on vaginal pH of women attending the gynecology outpatient department of a general hospital. Vaginal pH was recorded while diagnosing infections in 358 women, among which 45 were with repeated spontaneous abortion, 79 with infertility, 185 had sign and symptoms of lower genital tract infection, and 49 had no history or symptom of any complications or infections. Normal vaginal pH, BV, and C. trachomatis infection were observed in 72.6, 21.5, and 10.1% of women, respectively. BV and C. trachomatis were observed in 78.6 and 4.1% of women, respectively, with high vaginal pH; 12.3% of women with normal vaginal pH had C. trachomatis infection. C. trachomatis infection or different reproductive manifestations do not lead to change in vaginal pH but high vaginal pH correlated with BV and should be used as a simple tool for its diagnosis. J. Clin. Lab. Anal. 22:375–379, 2008. © 2008 Wiley‐Liss, Inc.
In vitro differentiation of human calvarial suture derived cells with and without dexamethasone does not induce in vivo‐like expression
Authors: Coussens Anna K. , Hughes Ian P. , Morris C. Phillip , Powell Barry C. , Anderson Peter J.
Published: September 2008

Osteogenic supplements are a requirement for osteoblastic cell differentiation during in vitro culture of human calvarial suture‐derived cell populations. We investigated the ability of ascorbic acid and β‐glycerophosphate with and without the addition of dexamethasone to stimulate in vivo‐like osteoblastic differentiation. Cells were isolated from unfused and prematurely fused suture tissue from patients with syndromic and non‐syndromic craniosynostosis and cultured in each osteogenic medium for varying lengths of time. The effect of media supplementation was investigated with respect to the ability of cells to form mineralised bone nodules and the expression of five osteodifferentiation marker genes (COL1A1, ALP, BSP, OC and RUNX2), and five genes that are differentially expressed during human premature suture fusion (GPC3, RBP4, C1QTNF3, WIF1 and FGF2). Cells from unfused sutures responded more slowly to osteogenic media but formed comparable bone nodules to fused suture‐derived cells after 16 days of culture in either osteogenic media. However, gene expression differed between unfused and fused suture‐derived cells, as did expression in each osteogenic medium. When compared to expression in the explant tissue of origin, neither medium induced a level or profile of gene expression similar to that seen in vivo. Overall, our results demonstrate that cells from the same suture that are isolated during different stages of morphogenesis in vivo, despite being de‐differentiated to a similar level in vitro, respond uniquely and differently to each osteogenic medium. Further, we suggest that neither cell culture medium recapitulates differentiation via activation of the same genetic cascades as occurs in vivo. J. Cell. Physiol. 218: 183–191, 2009. © 2008 Wiley‐Liss, Inc.
Increased bone mass, altered trabecular architecture and modified growth plate organization in the growing skeleton of SOCS2 deficient mice
Authors: MacRae V.E. , Horvat S. , Pells S.C. , Dale H. , Collinson R.S. , Pitsillides A.A. , Ahmed S.F. , Farquharson C.
Published: September 2008

Suppressor of cytokine signalling‐2 (SOCS2) negatively regulates the signal transduction of several cytokines. Socs2−/− mice show increased longitudinal skeletal growth associated with deregulated GH/IGF‐1 signalling. The present study examined the role of SOCS2 in endochondral ossification and trabecular and cortical bone formation, and investigated whether pro‐inflammatory cytokines associated with pediatric chronic inflammatory disorders mediate their effects through SOCS2. Seven‐week‐old Socs2−/− mice were heavier (27%; P < 0.001) and longer (6%; P < 0.001) than wild‐type mice. Socs2−/− tibiae were longer (8%; P < 0.001) and broader (18%; P < 0.001) than that of wild‐type mice, and the Socs2−/− mice had wider growth plates (24%; P < 0.001) with wider proliferative and hypertrophic zones (10% (P < 0.05) and 14% (P < 0.001) respectively). Socs2−/− mice showed increased total cross‐sectional bone area (16%: P < 0.001), coupled to increased total tissue area (17%; P < 0.05) compared to tibia from wild‐type mice. Socs2−/− mice showed increased percent bone volume (101%; P < 0.001), trabecular number (82%; P < 0.001) and trabecular thickness (11%; P < 0.001), with associated decreases in trabecular separation (19%; P < 0.001). TNFα exposure to growth plate chondrocytes for 48 h increased SOCS2 protein expression. Growth of metatarsals from 1‐day‐old Socs2−/− and Socs2+/+ mice, as well as expression of Aggrecan, Collagen Type II and Collagen Type X, were inhibited by TNFα, with no effect of genotype. Our data indicate that physiological levels of SOCS2 negatively regulate bone formation and endochondral growth. Our results further suggest that pro‐inflammatory cytokines mediate their inhibitory effects on longitudinal bone growth through a mechanism that is independent of SOCS2. J. Cell. Physiol. 218: 276–284, 2009. © 2008 Wiley‐Liss, Inc.