bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery of potent and selective inhibitors of procollagen C‐proteinase for the treatment of fibrotic disorders
Authors: Butt R.P. , Huggins J.P. , Greiling D. , Hopkins B. , Gaboardi S. , Winslow D. , Ronald M. , Lewis S. , Ward S. , Levett E. , Owen J. , Burslem F. , Collis M. , Bailey S. , Fish P.V. , Whitlock G. , Billotte S. , James K. , Mcelroy A. , Blagg J.
Published: September 2009

Introduction  Fibrosis is a component of many tissue pathologies leading to loss of normal tissue function, primarily due to excessive collagen deposition. Collagen is deposited following cleavage of the C‐ and N‐ terminal peptides from the pro‐collagen molecule. The cleavage of the globular C‐peptide by PCP reduces solubility of the fibrillar collagen molecule, resulting in deposition of insoluble collagen. Increased insoluble collagen deposition is a feature of all organ fibroses, with inhibition of this process, a key potential anti‐fibrotic mechanism. The aim of this work was to discover potent and selective PCP inhibitors as experimental, topically applied, anti‐fibrotic drugs for clinical evaluation.

Materials and methods  PCP was cloned from human osteosarcoma cells and enzymatic activity demonstrated using a PCP‐specific peptide cleavage assay. Activities were confirmed by measuring cleavage of [3H]C‐peptide from type‐I pro‐collagen. A cell‐based fibroplasias model was employed to demonstrate compound efficacy using collagen deposition, liberated C‐peptide and histological endpoints. The activities of PCP inhibitors in fibroblast and epithelial in vitro cell proliferation and migration assays, and selectivity vs. a panel of MMPs were also determined.

Discussion  In summary, we have identified and characterized potent and selective inhibitors of PCP for progression to clinical studies for investigation as a treatment paradigm for fibrotic disease.

  Results  

Assay
UK‐383,367
UK‐421,045

Inhibition of peptide cleavage (Ki, nM)
33.3 ± 1.92
8.7 ± 2.9

Inhibition of pro‐collagen cleavage (IC50, nM)
27.5 ± 3.2
3.4 ± 1.0

Maximal inhibition of collagen deposition in fibroplasia model
75.5% @10 µm
76.0% @10 µm

Maximal inhibition of C‐peptide liberation
(IC50 approximately 2 µm)
(IC50 approximately 1 µm)

n.d.
72.2% @10 µm

(IC50 approximately 1 µm)

Effect on fibroblast proliferation/migration
No effect
No effect

Selectivity vs. MMPs (fold vs. PCP Ki in peptide substrate assay)
MMP‐1,‐2,‐9,13,‐14 >2273
>10,000

MMP‐3 1684
9,000

TACE >500
>5,000

All values mean ± SEM; n ≥ 3 replicate experiments. Compounds: UK‐383,367 and UK‐421,045. MMP, matrix metalloproteinase; PCP, procollagen C‐proteinase.
Novel strategies for enhancing tissue integration in cartilage repair
Authors: Davies L.C. , Caterson B. , Duance V.C.
Published: September 2009

Introduction  Articular cartilage is unable to initiate a spontaneous repair response when injured due to its avascular and aneural properties. Within adult cartilage, chondrocytes are entrapped within an extensive extracellular matrix and are unable to migrate to sights of injury to regulate tissue repair. Injury to this tissue therefore inevitably leads to degeneration of the cartilage and the development of degenerative diseases such as osteoarthritis.

The surgical technique of autologous chondrocyte transplantation (ACT) was developed for the treatment of full‐thickness cartilage defects (Brittberg et al. 1994). Implantation of chondrocytes into the defect site repairs the injury site with a mixture of fibrocartilaginous and hyaline‐like tissue that poorly integrates with the existing cartilage and frequently degenerates with time. In this current study, we have developed an in vitro model to investigate methods for enhancing this integration and the development of a more biomechanically stable repair tissue.

Materials and methods  Bovine articular cartilage explants from the metacarpalphalangeal joint were experimentally injured using a stainless steel trephine and cultured for a period of 28 days. Autologous chondrocytes in an agarose suspension were injected into the interface region at the injury site. Media was collected and analysed for proteoglycan and collagen content using the DMMB and hydroxyproline assays, respectively. Matrix metalloproteinase (MMP) expression was also analysed using zymography and an adapted collagen fibril assay.

Results  Morphological analyses indicate attempts at repair and integration within both control and experimental treatment groups, although the presence of autologous chondrocytes appeared to amplify this repair response. Although not statistically significant, considerable differences in proteoglycan release between injured explants and the intact control group were seen. Collagen release into the media was only seen at day 28 within experimental cultures. An up‐regulation of MMP‐2 and MMP‐9 was seen within the experimental cultures compared to the controls. Preliminary data also suggest up‐regulation of collagenases in the experimental group when compared to controls.

Discussion  As seen with clinical ACT treatment, the presence of autologous chondrocytes appears to enhance repair and integration attempts; however, morphologically, this repair tissue appears to be fibrocartilaginous. Further analysis will establish whether the repair tissue is true hyaline cartilage and monitor the synthesis and turnover of macromolecules within the established culture system.
Expression profiling of metalloproteinases and inhibitors in cartilage
Authors: Kevorkian Lara , Young David A. , Darrah Clare , Donell Simon T. , Shepstone Lee , Porter Sarah , Brockbank Sarah , Edwards Dylan R. , Parker Andrew E. , Clark Ian M.
Published: September 2009

Objective  To profile the expression of all known members of the matrix metalloproteinase (MMP), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and tissue inhibitor of metalloproteinases (TIMPs) gene families in normal cartilage and that from patients with osteoarthritis (OA).

Methods  Human cartilage was obtained from femoral heads at joint replacement for either osteoarthritis or following fracture to the neck of femur. Total RNA was purified and expression of genes assayed using quantitative real‐time PCR.

Results  Several members of the above gene families were regulated in OA. Genes increasing in expression in OA were: at P < 0.001, MMP‐13, MMP‐28, ADAMTS‐16; at P < 0.01, MMP‐9, MMP‐16, ADAMTS‐2, ADAMTS‐14 and at P < 0.05, MMP‐2, TIMP‐3, ADAMTS‐12. Genes decreasing in expression in OA were: at P < 0.001, MMP‐1, MMP‐3, ADAMTS‐1; at P < 0.01, MMP‐10, TIMP‐1, ADAMTS‐9 and at P < 0.05, TIMP‐4, ADAMTS‐5, ADAMTS‐15. Correlation analysis revealed that groups of genes across the gene families are co‐expressed in cartilage.

Conclusion  This is the first comprehensive expression profile of all known MMP, ADAMTS and TIMP genes in cartilage. Patterns of expression provide a foundation on which to understand mechanisms of gene regulation in OA and potentially for refining the specificity of anti‐proteolytic therapies.
TGF‐β1 induces epithelial–mesenchymal transition but not myofibroblast transdifferentiation in primary cultures of human epithelial renal tubular cells
Authors: Anglani Franca , Forino Monica , Murer Luisa , Vella Manuela Dalla , Prete Dorella Del , Ceol Monica , Gambaro Giovanni , D'Angelo Angela
Published: September 2009

Introduction  Although the origin of renal interstitial myofibroblasts is still a matter of debate (Powell et al. 1999), emerging evidences suggest that they may derive from tubular epithelial cells. Since the well‐known mesenchymal origin of human tubular epithelial cells (HUTECs), our working hypothesis is that in renal fibrogenesis, fibrogenetic cytokines could reactivate the mesenchymal program [epithelial–mesenchymal transition (EMT)], switched‐off during renal nephrogenesis, leading to differentiation into MF. TGF‐β1 is a key modulator of the myofibroblast phenotype in fibroblasts during the process of wound healing and in mesangial cells in culture. TGF‐β1 is also able to induce EMT in a variety of cell types. However, most of these studies including the recent one on SV40‐transformed HKC‐8 human tubular cells (Yang & Liu 2001) have been carried out in immortalized cells.

Materials and methods  Primary HUTEC cultures were established from histologically normal human renal cortexes, obtained from surgical biopsies performed following informed consent in paediatric patients undergoing surgery because of extrinsic pyelouretheral obstruction. HUTECs were cultured for 4 and 6 days on plastic or type‐I collagen‐coated plates with or without 1, 5, 10 and 50 ng/ml TGF‐β1. Time‐course experiments (24 h to 6 days) with 1 ng/ml TGF‐β1 were also performed on primary human dermal fibroblasts, used as control mesenchymal cells, and HUTECs. Control conditions were represented by cells maintained for 4 and 6 days in 1% serum without TGF‐β1. The EMT process was monitored by morphology and immunophenotyping for α‐SMA, cytokeratin 8–18, E‐cadherin, vimentin and Ki67. Quantitative comparative RT/PCR evaluated the expression of collagen‐III and ‐V, fibronectin, tenascin, MMP‐2, CTGF, E‐cadherin and cadherin 11 genes. TGF‐β1 regulation of α‐SMA was investigated at both transcriptional and translational (Western blot) levels.

Results and discussion  TGF‐β1‐driven EMT was documented morphologically, biochemically and molecularly. HUTEC morphology changes following TGF‐β1 exposure were already evident at 24 h and with as little as 1 ng/ml of TGF‐β1; HUTECs acquired a spindle shape with front‐end/back‐end polarity, like fibroblasts. The transition was characterized by drastic up‐regulation of all the mesenchymal markers studied, including CTGF, and down‐regulation of cytokeratin and E‐cadherin expression. These phenomena were dose‐dependent and favuored by growth on collagen‐I. TGF‐β1 treatment did not induce MF conversion, because it induces neither de novo expression of α‐SMA gene nor the myofibroblast phenotype. We demonstrate that the TGF‐β1‐driven EMT is characterized by the re‐appearance of developmental gene networks which, in incomplete or uncoordinate fashion, could contribute to the pathogenesis of renal fibrosis.
Decorin affects endothelial cells by interacting with IGF‐I and its receptor
Authors: Schönherr Elke
Published: September 2009

Introduction  Endothelial cells (ECs) undergoing angiogenesis start to synthesize decorin, a member of the family of small proteoglycans with leucine‐rich repeats. Previously, we could show that decorin synthesis in ECs leads to capillary formation and survival of ECs in an angiogenesis model (Schönherr et al. 1999). In this model, decorin enhanced the phosphorylation of protein kinase B (Akt) and subsequently induced the cyclin‐dependent kinase inhibitor p21 (Schönherr et al. 2001).

Materials and methods  Cell culture: ECs of the permanent cell line EA.hy 926 were cultured on collagen type‐I‐coated dishes in Waymouth MAB 87/3 medium containing 1% heat‐inactivated fetal calf serum and antibiotics. Decorin purified from fibroblast cultures and/or the respective inhibitors were added.

Binding studies:  Decorin and IGF‐I were labelled with 125I‐iodine. (1) Decorin was run on an SDS‐PAGE and transferred to nitrocellulose. Blots were probed with 125I‐IGF‐I and exposed to X‐ray films. (2) A Sepharose CL‐4B gel filtration column was equilibrated with or without decorin containing buffer and 125I‐IGF‐I (500.000 cpm) was applied. The elution profiles were monitored. (3) IGF‐receptor was immunoprecipitated from EA.hy 926 cells with the antibody (sc‐713) against the β‐chain of the receptor. The immune complex was incubated with labelled or unlabelled decorin or IGF‐I (as indicated). Immune complexes without receptor were used as controls.

Results  To analyse how decorin activates Akt, inhibitors of different receptor tyrosine kinases were tested. The EGF‐receptor inhibitor tyrphostin AG1478 (10 µm) had no effect on decorin‐induced Akt posphorylation, but preincubation with tyrphostin AG1024 (10 µm), an inhibitor of the insulin and the IGF‐I receptor tyrosine kinases, inhibited Akt phophorylation and p21 expression. Combined addition of IGF‐I and decorin to ECs in culture had an additive effect on Akt phosphorylation. Binding experiments with 125I‐IGF‐I to decorin immobilized on nitrocellulose revealed that both the core protein and the proteoglycan bind 125I‐IGF‐I. Binding of 125I‐IGF‐I to decorin in solution showed the dose‐dependent formation of a high molecular weight complex that eluted in the included volume of the column. Immunoprecipitated IGF‐I receptor bound 125I‐‐IGF‐I as well as 125I‐decorin. In addition, 125I‐IGF‐I bound to the receptor could be displaced by unlabelled decorin.

Discussion  These results suggest that decorin can bind to IGF‐I and to its receptor and that this interaction enhances Akt phosphorylation in ECs. The effect of decorin on the IGF‐I receptor could be mediated by direct interaction. However, a further possibility is that decorin couples trace amounts of IGF‐I from the medium or the collagen on the dishes to a larger complex, which can more effectively activate the IGF‐I receptor. These possibilities as well as the role of IGF binding proteins will need further investigation.
The isoprostane 8‐iso‐PGF2α suppresses monocyte adhesion to microvascular endothelial cells: a role in limiting inflammation and fibrosis?
Authors: Kumar Anila , Kingdon Edward , Norman Jill
Published: September 2009

Introduction  Isoprostanes, produced in vivo by nonenzymatic free radical‐induced lipid peroxidation, are recognized markers of oxidative stress with elevated serum and urine levels of 8‐iso‐PGF2α (iP) reported in a variety of fibrotic diseases including end‐stage renal disease, systemic sclerosis and atherosclerosis. It has been suggested that iP may also have pathogenic functions. Many fibrotic diseases are characterized by early perivascular inflammatory infiltrates, and inflammation has been shown to be a critical component of the fibrotic process. Adhesion of monocytes to the capillary endothelium is an initiating event in inflammation and, in line with a proposed pathological role for iP, we hypothesized that iP might stimulate monocyte adhesion and thus promote fibrosis.

Methods  Human monocytes (U937 or THP‐1) were added to confluent MECs and the number of adhered monocytes measured colorimetrically or by cell counting.

Results  In dose–response assays (10−12−10−5 M), iP > 10−7 M inhibited monocyte adhesion (by 76%P < 0.01) to quiescent or proliferating human and rat renal MECs. In contrast, iP stimulated U937 adhesion to HUVEC (as reported by Leitinger et al. FASEB J 15,1254, 2001) demonstrating diverse effects of iP on different ECs. iP had no effect on viability (trypan blue exclusion), proliferation (3H‐thymidine incorporation) or apoptosis (Annexin V) of U937 or MECs. Inhibition of adhesion was dependent on preincubation (minimum 1–2 h) of iP; simultaneous addition of monocytes and iP to HMEC or treatment of U937 had no effect; suggesting that inhibition is due to iP‐induced changes in HMEC. These changes appear to be independent of de novo protein synthesis. Furthermore, iP, added either before or after TNF‐α, blocked TNF‐α‐induced monocyte adhesion. The inhibitory effect of iP was mimicked by a thromboxane receptor (TxR) agonist (U46619, 10 µm) and blocked by a receptor antagonist (SQ29548, 10 µm) indicating a TxR‐mediated process. Experiments using signal transduction pathway inhibitors (SB203580, curcumin and PD98059) implicate p38 and JNK, but not ERK, in iP‐induced suppression of monocyte adhesion. In addition to a direct effect, conditioned medium (CM) transfer experiments suggest that iP induces a secondary mediator which also suppresses monocyte adhesion but via an alternative mechanism which is TxR‐independent and dependent on new protein synthesis. This pathway also involves activation of p38 but is only partially dependent on JNK.

Conclusion  The data show that iP can suppress the attachment of monocytes to MECs via two independent pathways indicating an unexpected, potentially protective effect of iP in the microvasculature. Characterization of the mechanisms and mediators involved in this process may provide novel targets for intervention in inflammation and subsequent fibrosis.
Investigating the PKR‐signalling pathway in the articular joint
Authors: Gilbert S.J. , Duance V.C. , Mason D.J.
Published: September 2009

Introduction  Our previous studies have shown that the protein kinase, PKR and its activator, PACT are involved in TNF‐α signalling in articular cartilage (Gilbert et al. 2002). The sphingolipid ceramide is known to play an important role in signal transduction of TNF‐α and is a potent apoptotic agent. The PKR pathway is also known to be activated by ceramide (Ruvolo et al. 2001). Recent studies have shown that ceramide stimulates proteoglycan degradation and mRNA expression of MMP‐1, MMP‐3 and MMP‐13 in rabbit cartilage suggesting a role for this second messenger in cartilage degradation (Sabatini et al. 2000). In the current study, we investigated the role of ceramide, TNF‐α and PKR in bovine articular cartilage degradation. In addition, we treated human primary synoviocytes with TNF and investigated whether PKR is activated and if NFkB, a transcription factor downstream of PKR, is translocated to the nucleus.

Materials and methods  Bovine articular cartilage explants were stimulated with C2‐ceramide (50 µm) or TNF‐α (100 ng/ml) for 24 h. To inhibit the activation of PKR, 2‐aminopurine (10 mm) was added to duplicate cultures 1 h prior to the addition of treatments. Media was collected and MMPs analysed by gelatin zymography. Proteoglycan release was measured by the DMMB assay and cell viability determined by the Cytotox® assay. Human primary synoviocytes were stimulated with TNF‐α (10 ng/ml) for 30 min, and phosphorylated PKR and the Rel A p65 subunit of NFκB were detected by immunofluorescence.

Results  C2‐ceramide treatment resulted in a significant release of both pro and active MMP‐2. Explants incubated with the PKR inhibitor, 2‐aminopurine, prior to TNF‐α or C2‐ceramide treatment resulted in a marked reduction in both MMP‐2 and MMP‐9 activation. A significant increase in proteoglycan release was observed following treatment with TNF‐α and C2‐ceramide, which was significantly inhibited by 2‐aminopurine. A significant loss of cell viability was observed when explants were treated with C2‐ceramide, which was also found to be regulated by a PKR‐dependant pathway. Studies using antisense oligonucleotides to PACT and PKR are currently being undertaken to confirm these results.

An increase in phosphorylation of PKR and NFkB translocation was observed in synoviocytes treated with TNF.

Discussion  Our data has shown that PKR potentially is an important mediator of degradative and death pathways in chondrocytes. Collectively, these results suggest a novel role for the PKR pathway in the turnover of articular cartilage and support our hypothesis that PKR and PACT are implicated in the cartilage loss that occurs in arthritic disease.
Modulation of the expression of connective tissue growth factor (CTGF) by alterations of the cytoskeleton
Authors: Ott Christian , Graness Angela , Giehl Klaudia , Goppelt‐Struebe Margarete
Published: September 2009

Introduction  In closing wounds or developing fibrotic tissue, fibroblasts are exposed to mechanical stress, which leads to alterations in cell morphology and reorganization of the cytoskeleton. The impact of morphological changes on gene expression was investigated in the present study. As a model system, we used a human renal fibroblast cell line and studied the expression of connective tissue growth factor (CTGF). CTGF is a downstream mediator of TGF‐β mediating many of the pro‐fibrotic actions of this growth factor and has also been shown to be up‐regulated by static or dynamic pressure. The molecular mechanisms regulating CTGF expression related to stress are not yet known.

Materials and methods  A human renal fibroblast cell line was kindly provided by Dr Müller, Göttingen, Germany. These cells express CTGF upon treatment with soluble stimuli (Heusinger‐Ribeiro et al. 2001; Eberlein et al. 2001). CTGF expression was detected by northern and Western blot analysis. The actin cytoskeleton was visualized by rhodamine phalloidin staining, and microtubules were detected by immunocytochemistry.

Results  Low concentrations of the microtubule disrupting agents nocodazole and colchicine strongly up‐regulated CTGF mRNA and protein expression in the human renal fibroblast cell line TK173. The up‐regulation was prevented by stabilization of the microtubules by paclitaxel (taxol). As a consequence of microtubule disruption, the small GTPase RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction: interference with RhoA signalling by simvastatin, toxin B and Y27632 prevented up‐regulation of CTGF. The important role of RhoA was supported by an increased CTGF expression upon overexpression of constitutively active RhoA. Direct disassembly of the actin cytoskeleton by latrunculin B interfered with colchicine‐mediated up‐regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D unexpectedly increased CTGF expression. This indicated that the content of F‐actin per se was not the major determinant for CTGF gene expression. It has been shown, however, that cytochalasin D sequesters G‐actin, whereas latrunculin increases the level of G‐actin. Our data are thus in accordance with an inverse correlation between G‐actin levels and CTGF expression.

Discussion  These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF. Recently, decreased levels of G‐actin were observed in vascular smooth muscle cells in response to increased vascular pressure (Cipolla et al. 2002). Our findings thus provide a molecular basis for the observation that CTGF is up‐regulated in cells exposed to mechanical stress.
Homophilic complex formation is prerequisite for MT1‐MMP to degrade type‐I collagen on the cell surface
Authors: Itoh Yoshifumi , Ito Noriko , Nagase Hideaki , Seiki Motoharu
Published: September 2009

Introduction  MT1‐MMP degrades a wide variety of extra‐cellular matrix macromolecules and some cell‐surface proteins such as CD44, transglutaminase and αv‐integrin, and activates zymogen of MMP‐2 and MMP‐13. Among these activities, the collagenolytic activity is one of the most important functions of the enzyme in biology, as the phenotypes of MT1‐MMP gene knockout mice, such as abnormal skeletal development and various connective tissue abnormalities, are thought to be attributed to the lack of cellular collagenase activity. We have previously shown that MT1‐MMP forms homophilic complex through its haemopexin (HPX) domain facilitating the activation of proMMP‐2 (Itoh et al. 2001). In this report, we present data that collagen‐degrading activity of MT1‐MMP also requires homophilic complex formation.

Methods  COS7 cells were cultured on type‐I collagen film and transfected with MT1‐MMP and its mutant gene to look at collagen degradation activity of these gene products.

Results  Co‐expression of the catalytic domain‐deleted mutant of MT1‐MMP (MT1δCat) with the wild‐type enzyme inhibited its homophilic complex formation as well as its ability to activate proMMP‐2 on the cell surface, and MT1δCat also inhibited cell‐surface collagenolytic activity of the wild‐type MT1‐MMP. A chimeric MT1‐MMP mutant (MT‐MMP13) whose catalytic domain, hinge region and HPX domain were replaced with those derived from collagenase‐3 (MMP‐13) did not degrade collagen though MT‐MMP13 was expressed and maintained its proteolytic activity against gelatin on the cell surface. When an interface for homophilic complex formation, the HPX domain derived from MT1‐MMP, was further added to the chimeric enzyme (MT‐MMP13‐HPXmt1), it degraded collagen. Co‐expression of MT1dCat and MT‐MMP13‐HPXmt1 also inhibited the collagenolytic activity.

Discussion  Homphilic complex formation is prerequisite for the expression of collagenase activity on the cell surface. Presumably, each MT1‐MMP in the complex has different roles; one unwinds triple‐helical structure of collagen locally and the other cuts peptide bond.
LAPTM4B‐35, a novel tetratransmembrane protein and its PPRP motif play critical roles in proliferation and metastatic potential of hepatocellular carcinoma cells
Authors: Liu Xinrong , Xiong Fuxia , Wei Xuanhui , Yang Hua , Zhou Rouli
Published: September 2009

Lysosomal protein transmembrane 4 beta (LAPTM4B) was originally identified as a hepatocellular carcinoma (HCC)‐associated gene. This gene and its protein product LAPTM4B‐35, are both overexpressed in a variety of human cancers. However, its specific role in cell transformation and malignancy has remained elusive. In the present study we investigated the effects of LAPTM4B‐35 overexpression on the malignant phenotypic features in the HLE cell line. Our data show that overexpression of LAPTM4B‐35 promotes cell proliferation, exogenous growth‐stimulating factor‐independent and anchorage‐independent growth, and enhances metastatic potential, including promotion of both cell migration and invasion. Study of the underlying mechanisms demonstrated alterations of molecular events involved in these processes, which included upregulation of proliferation‐promoting transcription factors such as c‐Myc, c‐Jun, and c‐Fos, and cell cycle‐promoting proteins such as cyclin D1 and cyclin E. In addition, mutagenesis study showed that the PPRP motif in the N‐terminal region of LAPTM4B‐35 plays a critical role in promoting proliferation, migration, and invasion, as well as in the upregulation of the oncoproteins noted above. These data offer insight into the mechanism by which this novel tetratransmembrane protein contributes to the pathogenesis of liver cancer, and suggest that it may be a potential target for cancer therapy. (Cancer Sci 2009)
High expression of the evolutionarily conserved α/β hydrolase domain containing 6 (ABHD6) in Ewing tumors
Authors: Max Daniela , Hesse Manuela , Volkmer Ines , Staege Martin S.
Published: September 2009

Despite improvements in the treatment of patients with Ewing family tumors (EFT), the prognosis for patients with advanced disease is still unsatisfactory. Recently, we identified lipase I as an EFT‐associated gene that might be interesting for the development of new immunological or pharmacological treatment strategies. Lipase I is a member of the large protein superfamilies of α/β hydrolases and serine hydrolases. In the present paper we describe high expression of another member of these superfamilies in EFT. By DNA microarray data base mining we found exceptional high expression of α/β hydrolase domain containing 6 (ABHD6) in EFT but not in other sarcomas. Expression of ABHD6 in EFT correlated with expression of another EFT‐associated gene, aristaless. Analysis of ABHD6‐associated GGAA microsatellites revealed shorter microsatellites in EFT with lack of ABHD6 expression. ABHD6 homologues were found in varying chordata but not in other animal species. Based on homology modeling we predicted the 3D‐structure of ABHD6, which shows high similarity with bacterial homoserine transacetylases. High expression of ABHD6 in EFT in comparison to normal tissues and other tumors suggests that ABHD6 might be an interesting new diagnostic or therapeutic target for EFT. However, knock down of ABHD6 in EFT cells did not inhibit tumor cell growth. (Cancer Sci 2009; 100: 2383–2389)
Withdrawal of corticosteroids in inflammatory bowel disease patients after dependency periods ranging from 2 to 45 years: a proposed method
Authors: MURPHY S. J. , WANG L. , ANDERSON L. A. , STEINLAUF A. , PRESENT D. H. , MECHANICK J. I.
Published: September 2009

Background  Even in the biologic era, corticosteroid dependency in IBD patients is common and causes a lot of morbidity, but methods of withdrawal are not well described.

Aim  To assess the effectiveness of a corticosteroid withdrawal method.

Methods  Twelve patients (10 men, 2 women; 6 ulcerative colitis, 6 Crohn’s disease), median age 53.5 years (range 29–75) were included. IBD patients with quiescent disease refractory to conventional weaning were transitioned to oral dexamethasone, educated about symptoms of the corticosteroid withdrawal syndrome (CWS) and weaned under the supervision of an endocrinologist. When patients failed to wean despite a slow weaning pace and their IBD remaining quiescent, low dose synthetic ACTH stimulation testing was performed to assess for adrenal insufficiency. Multivariate analysis was performed to assess predictors of a slow wean.

Results  Median durations for disease and corticosteroid dependency were 21 (range 3–45) and 14 (range 2–45) years respectively. Ten patients (83%) were successfully weaned after a median follow‐up from final wean of 38 months (range 5–73). Disease flares occurred in two patients, CWS in five and ACTH testing was performed in 10. Multivariate analysis showed that longer duration of corticosteroid use appeared to be associated with a slower wean (P = 0.056).

Conclusions  Corticosteroid withdrawal using this protocol had a high success rate and durable effect and was effective in patients with long‐standing (up to 45 years) dependency. As symptoms of CWS mimic symptoms of IBD disease flares, gastroenterologists may have difficulty distinguishing them, which may be a contributory factor to the frequency of corticosteroid dependency in IBD patients.
Trajectory analysis: concepts and applications
Authors: Helland‐Hansen W. , Hampson G. J.
Published: September 2009

Shoreline and shelf‐edge trajectories describe the migration through time of sedimentary systems, using geomorphological breaks‐in‐slope that are associated with key changes in depositional processes and products. Analysis of these trajectories provides a simple descriptive tool that complements and extends conventional sequence stratigraphic methods and models. Trajectory analysis offers four advantages over a sequence stratigraphic interpretation based on systems tracts: (1) each genetically related advance or retreat of a shoreline or shelf edge is viewed in the context of a continuously evolving depositional system, rather than as several discrete systems tracts; (2) subtle changes in depositional response (e.g. within systems tracts) can be identified and honoured; (3) trajectory analysis does not anticipate the succession of depositional events implied by systems‐tract models; and (4) the descriptive emphasis of trajectory analysis does not involve any a priori assumptions about the type or nature of the mechanisms that drive sequence development. These four points allow the level of detail in a trajectory‐based interpretation to be directly tailored to the available data, such that the interpretation may be qualitative or quantitative in two or three dimensions. Four classes of shoreline trajectory are recognized: ascending regressive, descending regressive, transgressive and stationary (i.e. nonmigratory). Ascending regressive and high‐angle (accretionary) transgressive trajectories are associated with expanded facies belt thicknesses, the absence of laterally extensive erosional surfaces, and relatively high preservation of the shoreline depositional system. In contrast, descending regressive and low‐angle (nonaccretionary) transgressive trajectories are associated with foreshortened and/or missing facies belts, the presence of laterally extensive erosional surfaces, and relatively low preservation of the shoreline depositional system. Stationary trajectories record shorelines positioned at a steeply sloping shelf edge, with accompanying bypass of sediment to the basin floor. Shelf‐edge trajectories represent larger spatial and temporal scales than shoreline trajectories, and they can be subdivided into ascending, descending and stationary (i.e. nonmigratory) classes. Ascending trajectories are associated with a relatively large number and thickness of shoreline tongues (parasequences), the absence of laterally extensive erosional surfaces on the shelf, and relatively low sediment supply to the basin floor. Descending trajectories are associated with a few, thin shoreline tongues, the presence of laterally extensive erosional surfaces on the shelf, and high sediment supply to basin‐floor fan systems. Stationary trajectories record near‐total bypass of sediment across the shelf and mass transfer to the basin floor.
A short low‐dose imatinib trial allows rapid identification of responsive patients in hypereosinophilic syndromes
Authors: Intermesoli Tamara , Delaini Federica , Acerboni Sara , Salmoiraghi Silvia , Spinelli Orietta , Guerini Vittoria , Vannucchi Alessandro M. , Mappa Silvia , Rossi Giuseppe , Rossi Valentina , Di Bona Eros , Paratore Simona , Carobbio Alessandra , Rambaldi Alessandro , Barbui Tiziano , Bassan Renato
Published: September 2009

Although imatinib may be effective in hypereosinophilic syndromes, the exact response kinetics are not known. Imatinib was administered at 100–400 mg/d each week in a 12‐week response‐oriented schedule, targeting a complete clinical and haematological remission (CR). CR was achieved in 11/23 patients (6/6 with FIP1L1‐PDGRFA rearrangement and 5/17 without, P = 0·006), most after 2 weeks of 100 mg/d imatinib. The maximum imatinib dose had no effect in early unresponsive patients. Low‐dose, short‐course imatinib may represent a rational choice for identifying responsive cases, both within and outside the pre‐defined FIP1L1 rearrangement subset.
How individuals with phenylketonuria experience their illness: an age‐related qualitative study
Authors: Vegni E. , Fiori L. , Riva E. , Giovannini M. , Moja E. A.
Published: September 2009

Background  The aim of the study was to explore the illness experience of individuals affected by phenylketonuria (PKU) and its differences in different patient age groups.

Methods  A qualitative–interpretative methodology was used through in‐depth interviews. Textual data were explored using the principles of grounded theory.

Results  Forty‐seven patients participated in the study, aged from 10 to >25 years old. The results suggested the age‐related PKU experience: (1) a paradox, either to feel normal but isolated from the social context, or to be different while participating in the convivial aspects of the social being; and (2) the need for education about the disease tailored to the individual and growing needs. Specific themes seem to characterize each age range.

Conclusions  This study constitutes a first attempt at understanding PKU from a non‐medical‐biological perspective.
Perception of vulnerability among mothers of healthy infants in a middle‐income country
Authors: Dogan D. G. , Ertem I. O. , Karaaslan T. , Forsyth B. W.
Published: September 2009

Background  Although four decades have passed since the concept of ‘vulnerable children’ has been introduced into paediatric literature, research on vulnerability is limited to high‐income, Western countries. To adapt and adopt practices that have been advised for paediatricians to prevent ‘the vulnerable child syndrome’, information is needed also on the prevalence and correlates of perceived vulnerability in children in low‐ and middle‐income (LAMI) countries.

Objective  To determine the rate and correlates of the perception of vulnerability among healthy young children in a healthy population of children in Ankara, Turkey.

Methods  In this cross‐sectional observational study, participants comprised of a ‘prescriptive sample’ of healthy, thriving children with no known health risk for vulnerability. Maternal perception of child vulnerability was assessed using the Child Vulnerability Scale (CVS). Potential risks factors for vulnerability including history of threatened abortion during pregnancy, child gender, birth order, maternal and paternal age and education were collected using a structured questionnaire.

Results  A total of 519 children – 264 boys (50.9%) and 255 girls (49.1%) – comprised the sample. The internal consistency of the CVS was 0.71. Item‐total scale corrrelations were 0.30 or above for all of the eight items. The median CVS score of the sample was 2.0 and 30 mothers (5.8%) were found to perceive their children as vulnerable. None of the socio‐demographic variables that were investigated were found to be associated with high vulnerability scores.

Conclusion  This study is the first to examine maternal perceived vulnerability of healthy children in a middle‐income country. The findings imply that a high proportion of healthy children are perceived as vulnerable by their mothers and that previously studied socio‐demographic factors do not explain perceived vulnerability. The results of this study may provide a comparison point for studies on childhood vulnerability in LAMI countries.
Disabled children in the UK: a quality assessment of quantitative data sources
Authors: Read J. , Blackburn C. , Spencer N.
Published: September 2009

Background  The limitations of reliable and accessible UK national and local quantitative data on the prevalence of childhood disability and the characteristics and circumstances of disabled children and their households have been recognized for some time. This paper reports the findings from a study that scoped and quality‐assessed existing quantitative UK national and regional data sets on disabled children and their families.

Methods  A comprehensive search of relevant data sources with information on disabled children was undertaken. Data sources were evaluated with reference to: disability definitions and questions; potential to generate nationally representative prevalence estimates of disabled children; study design; population coverage; sampling issues; social and demographic data; appropriateness for identifying childhood as opposed to adult disability.

Results  Thirty‐seven data sources with information on childhood disability were identified, of which 30 met the inclusion criteria: nine cross‐sectional surveys, nine longitudinal and panel studies, seven administrative data sets, four specific condition databases and one was another type. Definitions and questions varied across data sources. Long‐standing illness and limiting long‐standing illness were the most consistently used definitions. Repeated cross‐sectional surveys were found to be most appropriate for estimating overall prevalence but, with the exception of the Population Census, sample sizes were too small to study prevalence and characteristics by some population sub‐groups such, including age by year, minority ethnic status and socio‐economic position. Few data sources included questions appropriate for identifying childhood as opposed to adult disability or collected information from disabled children themselves.

Conclusions  Our findings summarize the currently available quantitative data sources on childhood disability and highlight the limitations. We make recommendations for the future development of more robust childhood disability data and issues requiring further research. To assist policy makers and service providers to make use of current data sources we have produced a brief online guide based on our findings.
Allelic variants of CD40 and CD40L genes interact to promote antibiotic‐induced cutaneous allergic reactions
Authors: Kim Sae‐H , Lee J‐E , Kim Sang‐H , Jee Y‐K , Kim Y‐K , Park H‐S , Min K‐U , Park H‐W ,   The Adverse Drug Reaction Research Group in Korea
Published: September 2009

Background
The danger hypothesis provides a new perspective of the mechanisms underlying drug allergy. In this study, we evaluated associations between variations in the genes involved in danger signal pathways and antibiotic‐induced cutaneous allergic reactions (AICARs).

Methods
Two hundred cases with urticaria, angio‐oedema, maculopapular rash, and erythema multiforme caused by antibiotics were extracted from the database of the Adverse Drug Reaction Research Group in Korea. All cases were confirmed by an allergy specialist. Causative antibiotics included penicillin, cephalosporin, quinolone, and others (approximately 40 different types). Ten single nucleotide polymorphisms (SNPs) in seven genes (−318C>T, +49A>G, and +6230G>A in CTLA4, IVS+17T>C in CD28, −3479T>G and I170V in CD86, −1C>T in CD40, −3458A>G in CD40LG, −308G>A in TNF, and −31T>C in IL1B) were scored for cases and for healthy subjects without a history of AICARs.

Results
Our analysis failed to reveal differences in the distribution of the 10 SNPs between cases and controls. However, we could find a gene–gene interaction between −1C>T in CD40 and −3458A>G in CD40L using multifactor dimensionality reduction analysis. Subjects with minor alleles of both SNPs showed a significant risk for developing AICARs [P=0.017, odds ratio (OR) (95% confidence interval)=2.93 (1.20–7.97)].

Conclusion
Our findings suggest that a genetic interaction between CD40 and CD40L favours the development of AICARs.
Cytology of the ‘penile’ neovagina in transsexual women
Authors: Weyers S. , Lambein K. , Sturtewagen Y. , Verstraelen H. , Gerris J. , Praet M.
Published: September 2009

S. Weyers, K. Lambein, Y. Sturtewagen, H. Verstraelen, J. Gerris and M. Praet Cytology of the ‘penile’ neovagina in transsexual women

Objective:  The primary objective was to describe the neovaginal cytology in transsexual patients (n = 50) treated with the inverted penile skin technique. Secondary objectives were to compare our cytological findings with patient characteristics including use of oestrogens, sexual orientation and penetrative intercourse.

Methods:  The medical and surgical history, sexual orientation and whether there was a current relationship were ascertained. A speculum examination was followed by microscopy of a Pap smear of the neovaginal vault.

Results:  Well‐preserved nucleated squamous cells were found in 72%. The correlation between their presence and sexual orientation was highly significant (P = 0.016), with those not sexually interested and homosexually oriented all having nucleated cells on the Pap smear. However, the correlation between these cells and penetrative intercourse failed to reach significance. Four samples showed atypical squamous cells of undetermined significance, all were negative for high‐risk human papillomavirus (HR‐HPV) types. One patient showed a low‐grade squamous intraepithelial lesion that was HR‐HPV positive. There was a significant correlation between the presence of cytological lesions and sexual orientation (P = 0.006). Four percentage of the specimens showed Döderlein bacilli. Inflammation was found in 30.6% of samples with squamous cells.

Conclusions:  The penile skin‐lined neovagina of transsexual women can reflect the cytological findings present in biological women. However ‘normal’ cervical cytology, with superficial, intermediate and parabasal cells as well as Döderlein bacilli, was found in only 4% of transsexual women. Although one patient’s Pap test showed koilocytes and was HR‐HPV positive, no high‐grade squamous intraepithelial lesions were identified.
Review paper: more than ringing in the ears: a review of tinnitus and its psychosocial impact
Authors: Holmes Susan , Padgham Nigel D
Published: September 2009

Aim and objectives.  To provide an overview of tinnitus, current management and its psychosocial impact offering strategies for managing acute and chronic tinnitus in practice.

Background.  Tinnitus, characterised by the perception of sound in the absence of external stimuli, is experienced by about 10% of the population at some time in their lives. It may be temporary/longstanding; approximately 5% adults experience severe, persistent tinnitus affecting their lifestyle. Although many adjust successfully, others are disabled by the condition. Though often unrecognised, tinnitus affects many patients regardless of their presenting illness.

Design.  A literature review including descriptive, theoretical and empirical material. Databases were searched using the keyword ‘tinnitus’ providing diverse information which was used to address the research questions.

Results.  Tinnitus represents more than ‘simple’ ringing in the ears and may be accompanied by many distressing changes. It may be acute or chronic. It is difficult to treat, care may be directed towards management rather than cure. Many patients are, however, told that ‘nothing can be done’.

Relevance to clinical practice.  Despite the high prevalence of tinnitus, there is a paucity of relevant nursing literature suggesting that there is an information deficit amongst nurses. The information provided shows that understanding the full impact of the condition and identification of patients’ needs are essential to effective care. Strategies to help affected patients are given.

Conclusions.  Tinnitus, a widespread, often intractable condition, affects millions of people; there is considerable debate about its causes. Tinnitus is distressing and may be severe enough to affect lifestyle and quality of life. Affected patients need considerable support and advice on healthcare options, encouragement to try different treatments and recognition that help and hope are available. Though patients may have to learn to live with tinnitus, the most important thing is that they recognise that help is available.
A cost–effectiveness analysis of a multimedia learning education program for stoma patients
Authors: Lo Shu‐Fen , Wang Yun‐Tung , Wu Li‐Yue , Hsu Mei‐Yu , Chang Shu‐Chuan , Hayter Mark
Published: September 2009

Study aims. The purpose of this study was to compare the costs and effectiveness of enterostomal education using a multimedia learning education program (MLEP) and a conventional education service program (CESP).

Background. Multimedia health education programs not only provide patients with useful information in the absence of health professionals, but can also augment information provided in traditional clinical practice. However, the literature on the cost–effectiveness of different approaches to stoma education is limited.

Design. This study used a randomised experimental design.

Methods. A total of 54 stoma patients were randomly assigned to MLEP or CESP nursing care with a follow‐up of one week. Effectiveness measures were knowledge of self‐care (KSC), attitude of self‐care (ASC) and behavior of self‐care (BSC). The costs measures for each patient were: health care costs, MLEP cost and family costs.

Results. Subjects in the MLEP group demonstrated significantly better outcomes in the effectiveness measures of KSC, ASC and BSC. Additionally, the total social costs for each MLEP patient and CESP patient were US$7396·90 and US$8570·54, respectively. The cost–effectiveness ratios in these two groups showed that the MLEP model was better than the CESP model after one intervention cycle. In addition, the Incremental Cost Effectiveness Ratio was −20·99.

Conclusion. This research provides useful information for those who would like to improve the self‐care capacity of stoma patients. Due to the better cost–effectiveness ratio of MLEP, hospital policy‐makers may consider these results when choosing to allocate resources and develop care and educational interventions.

Relevance to clinical practice. This study provides a cost effective way of addressing stoma care in the post‐operative period that could be usefully transferred to stoma care settings internationally.