Enhanced activity of meprin-?, a pro-migratory and pro-angiogenic protease, in colorectal cancer

Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, protein and proteolytic activity in primary tumors at progressive stages and in liver metastases of patients with colorectal cancer, as well as inhibitory activity towards meprin-α in sera of cancer patient as compared to healthy controls. We found that the hepatocyte growth factor (HGF)-induced migratory response of meprin-transfected epithelial cells was increased compared to wild-type cells in the presence of plasminogen, and that the angiogenic response in organ-cultured rat aortic explants was enhanced in the presence of exogenous human meprin-α. In patients, meprin-α mRNA was expressed in colonic adenomas, primary tumors UICC (International Union Against Cancer) stage I, II, III and IV, as well as in liver metastases. In contrast, the corresponding protein accumulated only in primary tumors and liver metastases, but not in adenomas. However, liver metastases lacked meprin-α activity despite increased expression of the corresponding protein, which correlated with inefficient zymogen activation. Sera from cancer patients exhibited reduced meprin-α inhibition compared to healthy controls. In conclusion, meprin-α activity is regulated differently in primary tumors and metastases, leading to high proteolytic activity in primary tumors and low activity in liver metastases. By virtue of its pro-migratory and pro-angiogenic activity, meprin-α may promote tumor progression in colorectal cancer.

Low rate of methicillin-resistant coagulase-positive staphylococcal colonization of veterinary personnel in Hong Kong

Elevated rates of methicillin-resistant Staphylococcus aureus (MRSA) carriage have been reported in veterinary personnel, suggesting an occupational colonization risk. Hong Kong veterinary personnel (n = 150) were sampled for coagulase-positive staphylococci (CPS) nasal colonization. Risk factors for colonization were assessed by questionnaire. Isolates were identified and antibiotic susceptibility determined. All CPS isolates were investigated for mecA carriage, SCCmec type and PVL genes. Two subjects were colonized with methicillin-resistant CPS: one with MRSA (spa type t002 (CC5), SCCmec type II) and one with methicillin-resistant Staphylococcus pseudintermedius (MRSP) (MLST type ST71, SCCmec type II-III). MLST type ST71 S. pseudintermedius strain is the predominant MRSP clone circulating in dogs in Europe and in Hong Kong. The low MR-CPS colonization rate may be associated with low levels of large animal exposure or low rates of MRSA colonization of companion animals in Hong Kong. Colonization with non-aureus CPS, which may cause human infection, must also be considered in veterinary personnel.

Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study

OBJECTIVES: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. METHODS: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. RESULTS: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). CONCLUSIONS: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

Transient rhythmic network activity in the somatosensory cortex evoked by distributed input in vitro

The initiation and maintenance of physiological and pathophysiological oscillatory activity depends on the synaptic interactions within neuronal networks. We studied the mechanisms underlying evoked transient network oscillation in acute slices of the adolescent rat somatosensory cortex and modeled its underpinning mechanisms. Oscillations were evoked by brief spatially distributed noisy extracellular stimulation, delivered via bipolar electrodes. Evoked transient network oscillation was detected with multi-neuron patch-clamp recordings under different pharmacological conditions. The observed oscillations are in the frequency range of 2-5 Hz and consist of 4-12 mV large, 40-150 ms wide compound synaptic events with rare overlying action potentials. This evoked transient network oscillation is only weakly expressed in the somatosensory cortex and requires increased [K+]o of 6.25 mM and decreased [Ca2+]o of 1.5 mM and [Mg2+]o of 0.5 mM. A peak in the cross-correlation among membrane potential in layers II/III, IV and V neurons reflects the underlying network-driven basis of the evoked transient network oscillation. The initiation of the evoked transient network oscillation is accompanied by an increased [K+]o and can be prevented by the K+ channel blocker quinidine. In addition, a shift of the chloride reversal potential takes place during stimulation, resulting in a depolarizing type A GABA (GABAA) receptor response. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), N-methyl-D-aspartate (NMDA), or GABA(A) receptors as well as gap junctions prevents evoked transient network oscillation while a reduction of AMPA or GABA(A) receptor desensitization increases its duration and amplitude. The apparent reversal potential of -27 mV of the evoked transient network oscillation, its pharmacological profile, as well as the modeling results suggest a mixed contribution of glutamatergic, excitatory GABAergic, and gap junctional conductances in initiation and maintenance of this oscillatory activity. With these properties, evoked transient network oscillation resembles epileptic afterdischarges more than any other form of physiological or pathophysiological neocortical oscillatory activity.

Cellular mechanisms of burst firing-mediated long-term depression in rat neocortical pyramidal cells

During wakefulness and sleep, neurons in the neocortex emit action potentials tonically or in rhythmic bursts, respectively. However, the role of synchronized discharge patterns is largely unknown. We have recently shown that pairings of excitatory postsynaptic potentials (EPSPs) and action potential bursts or single spikes lead to long-term depression (burst-LTD) or long-term potentiation, respectively. In this study, we elucidate the cellular mechanisms of burst-LTD and characterize its functional properties. Whole-cell patch-clamp recordings were obtained from layer V pyramidal cells in somatosensory cortex of juvenile rats in vitro and composite EPSPs and EPSCs were evoked extracellularly in layers II/III. Repetitive burst-pairings led to a long-lasting depression of EPSPs and EPSCs that was blocked by inhibitors of metabotropic glutamate group 1 receptors, phospholipase C, protein kinase C (PKC) and calcium release from the endoplasmic reticulum, and that required an intact machinery for endocytosis. Thus, burst-LTD is induced via a Ca2+- and phosphatidylinositol-dependent activation of PKC and expressed through phosphorylation-triggered endocytosis of AMPA receptors. Functionally, burst-LTD is inversely related to EPSP size and bursts dominate single spikes in determining the sign of synaptic plasticity. Thus burst-firing constitutes a signal by which coincident synaptic inputs are proportionally downsized. Overall, our data thus suggest a mechanism by which synaptic weights can be reconfigured during non-rapid eye movement sleep.

Efficiency of arthroscopic lysis and lavage for internal derangement of the temporomandibular joint correlated with Wilkes classification

OBJECTIVES: To compare the outcome of arthroscopic lysis and lavage of TMJ with internal derangement of Wilkes stages II, III, IV, and V. STUDY DESIGN: Arthroscopic lysis and lavage was performed in 45 TMJ of 39 patients with internal derangement. The cases were divided into 4 groups corresponding to Wilkes stages II, III, IV, and V. Two parameters were compared pre- and postoperatively: pain and mouth opening. Statistical significance was determined using the chi(2) test. RESULTS: Overall success rate was 86.7% (Wilkes stage II 90.9%, Wilkes stage III 92.3%, Wilkes stage IV 84.6%, Wilkes stage V 75%). There were no statistically significant differences between the success rates for Wilkes stages II, III, IV, and V. CONCLUSION: Arthroscopic lysis and lavage should be performed as a standard operation for internal derangement of the TMJ after failure of conservative treatment in all Wilkes stages.

Tenomodulin is necessary for tenocyte proliferation and tendon maturation

Tenomodulin (Tnmd) is a member of a new family of type II transmembrane glycoproteins. It is predominantly expressed in tendons, ligaments, and eyes, whereas the only other family member, chondromodulin I (ChM-I), is highly expressed in cartilage and at lower levels in the eye and thymus. The C-terminal extracellular domains of both proteins were shown to modulate endothelial-cell proliferation and tube formation in vitro and in vivo. We analyzed Tnmd function in vivo and provide evidence that Tnmd is processed in vivo and that the proteolytically cleaved C-terminal domain can be found in tendon extracts. Loss of Tnmd expression in gene targeted mice abated tenocyte proliferation and led to a reduced tenocyte density. The deposited amounts of extracellular matrix proteins, including collagen types I, II, III, and VI and decorin, lumican, aggrecan, and matrilin-2, were not affected, but the calibers of collagen fibrils varied significantly and exhibited increased maximal diameters. Tnmd-deficient mice did not have changes in tendon vessel density, and mice lacking both Tnmd and ChM-I had normal retinal vascularization and neovascularization after oxygen-induced retinopathy. These results suggest that Tnmd is a regulator of tenocyte proliferation and is involved in collagen fibril maturation but do not confirm an in vivo involvement of Tnmd in angiogenesis.

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

BACKGROUND CONTEXT Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PURPOSE To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. STUDY DESIGN Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. METHODS Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. RESULTS A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. CONCLUSIONS By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

Laparoscopic computer-navigated ablation of liver metastases

Objective In order to benefit from the obvious advantages of minimally invasive liver surgery there is a need to develop high precision tools for intraoperative anatomical orientation, navigation and safety control. In a pilot study we adapted a newly developed system for computer-assisted liver surgery (CALS) in terms of accuracy and technical feasibility to the specific requirements of laparoscopy. Here, we present practical aspects related to laparoscopic computer assisted liver surgery (LCALS). Methods Our video relates to a patient presenting with 3 colorectal liver metastases in Seg. II, III and IVa who was selected in an appropriate oncological setting for LCALS using the CAScination system combined with 3D MEVIS reconstruction. After minimal laparoscopic mobilization of the liver, a 4- landmark registration method was applied to enable navigation. Placement of microwave needles was performed using the targeting module of the navigation system and correct needle positioning was confirmed by intraoperative sonography. Ablation of each lesion was carried out by application of microwave energy at 100 Watts for 1 minute. Results To acquire an accurate (less 0.5 cm) registration, 4 registration cycles were necessary. In total, seven minutes were required to accomplish precise registration. Successful ablation with complete response in all treated areas was assessed by intraoperative sonography and confirmed by postoperative CT scan. Conclusions This teaching video demonstrates the theoretical and practical key points of LCALS with a special emphasis on preoperative planning, intraoperative registration and accuracy testing by laparoscopic methodology. In contrast to mere ultrasound-guided ablation of liver lesions, LCALS offers a more dimensional targeting and higher safety control. This is currently also in routine use to treat vanishing lesions and other difficult to target focal lesions within the liver.

Genetic Relatedness, Antimicrobial and Biocide Susceptibility Comparative Analysis of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius from Portugal

Forty methicillin-resistant and -susceptible Staphylococcus pseudintermedius (MRSP and MSSP, respectively) from colonization and infection in dogs and cats were characterized for clonality, antimicrobial, and biocide susceptibility. MSSP were genetically more diverse than MRSP by multi-locus sequence typing and pulsed-field gel electrophoresis. Three different spa types (t06, t02, t05) and two SCCmec types (II-III and V) were detected in the MRSP isolates. All MRSP and two MSSP strains were multidrug-resistant. Several antibiotic resistance genes (mecA, blaZ, tet(M), tet(K), aac(6')-Ie-aph(2')-Ia, aph(3')-III, ant(6)-Ia, sat4, erm(B), lnu(A), dfr(G), and catpC221) were identified by microarray and double mutations in the gyrA and grlA genes and a single mutation in the rpoB gene were detected by sequence analysis. No differences were detected between MSSP and MRSP in the chlorhexidine acetate (CHA) minimum inhibitory concentrations (MICs). However, two MSSP had elevated MIC to triclosan (TCL) and one to benzalkonium chloride and ethidium bromide. One MSSP isolate harboured a qacA gene, while in another a qacB gene was detected. None of the isolates harboured the sh-fabI gene. Three of the biocide products studied had high bactericidal activity (Otodine(®), Clorexyderm Spot Gel(®), Dermocanis Piocure-M(®)), while Skingel(®) failed to achieve a five log reduction in the bacterial counting. S. pseudintermedius have become a serious therapeutic challenge in particular if methicillin- resistance and/or multidrug-resistance are involved. Biocides, like CHA and TCL, seem to be clinically effective and safe topical therapeutic options.

Characterization of new staphylococcal cassette chromosome mec (SCCmec) and topoisomerase genes in fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius

Fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius isolates harbor two new staphylococcal cassette chromosome mec (SCCmec) elements that belong to class A, allotype 3 (SCCmec II-III), and to the new allotype 5 (SCCmec VII). Analysis of the complete nucleotide sequences of the topoisomerase loci gyrB/gyrA and grlB/grlA revealed mutations involved in fluoroquinolone resistance.

Strain typing and antimicrobial susceptibility of methicillin-resistant coagulase-positive staphylococcal species in dogs and people associated with dogs in Thailand

AIMS This study was to investigate and to characterize methicillin-resistant coagulase-positive staphylococci (MRCoPS) harboring in dogs and people associated with dogs in Thailand. METHODS AND RESULTS Staphylococci were collected from 100 dogs, 100 dog owners, 200 small animal veterinarians and 100 people without pet association. Species of MRCoPS were identified phenotypically and genotypically. Molecular characteristics were determined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and SCCmec typing, and antimicrobial susceptibility was assayed by broth microdilution and by microarray analysis for resistance genes. Methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSSc) and methicillin-resistant Staphylococcus aureus (MRSA) were isolated from dogs (45, 17 and 1%, respectively), veterinarians (8, 2 and 1·5%, respectively) and dog owners (3, 2 and 0%, respectively). Seventeen sequence types (STs) were identified among 83 MRSP isolates which specifically carried SCCmec V, II-III, ΨSCCmec57395 and three uncharacterized SCCmec types. MRSP ST 45, 68 and novel STs including 169, 178, 181 and 183 were shared among canine and human isolates. Most of MRSA ST398 and MRSSc carried SCCmec type V. The MRCoPS commonly displayed multiple resistances to tested antimicrobials and carried various resistance genes. CONCLUSION Variety of MRCoPS, especially new MRSP clones, distributed in dogs and people in Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY The existence of MRCoPS circulating between dogs and humans in Thailand provides indirect evidence of interspecies transmission and represents a potential public health hazard.

The Hundred Most-Cited Publications in Orthopaedic Knee Research

BACKGROUND: Despite its limitations, citation analysis remains one of the best currently available tools for quantifying the impact of articles. Bibliometric studies list the "best-sellers" in a single location, and they have been published frequently in many fields during recent years. The purpose of the present study was to report the qualities and characteristics of citation classics in orthopaedic knee research. METHODS: The database of the Institute for Scientific Information (ISI) was utilized for identification of articles published from 1945 to March 2014. All knee articles that had been published in sixty-five orthopaedic and twenty-nine rheumatology journals and that had been cited at least 200 times were identified. The top 100 were selected for further analysis of authorship, source journal, number of citations, citation rate (both since publication and in 2013), geographic origin, article type, and level of evidence. RESULTS: The publication dates of the 100 most-cited articles ranged from 1948 to 2007, with the greatest number of articles published in the 1980s. Citations per article ranged from 2640 to 287. All articles were published in eleven of the ninety-four journals. The leading countries of origin were the U.S. followed by the U.K. and Sweden. The two main focus areas were sports traumatology and degenerative disease. The number of citations per article was also greatest for articles published in the 1980s. Basic research articles were cited more quickly, but not more often, than clinical articles. Most articles represented Level-IV evidence, followed by Levels II, III, and I. CONCLUSIONS: This bibliometric study is likely to include a list of intellectual milestones in orthopaedic knee research. It is apparent that a high level of evidence is not mandatory for an article to gain a large number of citations. Bibliometric reports provide a reflection of the quality of cited research published in a specific field and should therefore provoke thinking within the scientific community.

The impact of neural invasion severity in gastrointestinal malignancies: a clinicopathological study.

OBJECTIVES Because neural invasion (NI) is still inconsistently reported and not well characterized within gastrointestinal malignancies (GIMs), our aim was to determine the exact prevalence and severity of NI and to elucidate the true impact of NI on patient's prognosis. BACKGROUND The union internationale contre le cancer (UICC) recently added NI as a novel parameter in the current TNM classification. However, there are only a few existing studies with specific focus on NI, so that the distinct role of NI in GIMs is still uncertain. MATERIALS AND METHODS NI was characterized in approximately 16,000 hematoxylin and eosin tissue sections from 2050 patients with adenocarcinoma of the esophagogastric junction (AEG)-I-III, squamous cell carcinoma (SCC) of the esophagus, gastric cancer (GC), colon cancer (CC), rectal cancer (RC), cholangiocellular cancer (CCC), hepatocellular cancer (HCC), and pancreatic cancer (PC). NI prevalence and severity was determined and related to patient's prognosis and survival. RESULTS NI prevalence largely varied between HCC/6%, CC/28%, RC/34%, AEG-I/36% and AEG-II/36%, SCC/37%, GC/38%, CCC/58%, and AEG-III/65% to PC/100%. NI severity score was uppermost in PC (24.9±1.9) and lowest in AEG-I (0.8±0.3). Multivariable analyses including age, sex, TNM stage, and grading revealed that the prevalence of NI was significantly associated with diminished survival in AEG-II/III, GC, and RC. However, increasing NI severity impaired survival in AEG-II/III and PC only. CONCLUSIONS NI prevalence and NI severity strongly vary within GIMs. Determination of NI severity in GIMs is a more precise tool than solely recording the presence of NI and revealed dismal prognostic impact on patients with AEG-II/III and PC. Evidently, NI is not a concomitant side feature in GIMs and, therefore, deserves special attention for improved patient stratification and individualized therapy after surgery.