Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.

DNA hypomethylation and oxidative stress-mediated increase in genomic instability in equine sarcoid-derived fibroblasts

It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.

International online survey to assess current practice in equine anaesthesia.

REASONS FOR PERFORMING STUDY Multicentre Confidential Enquiries into Perioperative Equine Fatalities (CEPEF) have not been conducted since the initial CEPEF Phases 1-3, 20 years ago. OBJECTIVES To collect data on current practice in equine anaesthesia and to recruit participants for CEPEF-4. STUDY DESIGN Online questionnaire survey. METHODS An online questionnaire was prepared and the link distributed internationally to veterinarians possibly performing equine anaesthesia, using emails, posters, flyers and an editorial. The questionnaire included 52 closed, semiclosed and open questions divided into 8 subgroups: demographic data, anaesthetist, anaesthesia management (preoperative, technical equipment, monitoring, drugs, recovery), areas of improvements and risks and motivation for participation in CEPEF-4. Descriptive statistics and Chi-squared tests for comparison of categorical variables were performed. RESULTS A total of 199 questionnaires were completed by veterinarians from 14 different countries. Of the respondents, 43% worked in private hospitals, 36% in private practices and 21% in university teaching hospitals. In 40 institutions (23%) there was at least one diplomate of the European or American colleges of veterinary anaesthesia and analgesia on staff. Individual respondents reported routinely employ the following anaesthesia monitoring modalities: electrocardiography (80%), invasive arterial blood pressures (70%), pulse oximetry (60%), capnography (55%), arterial blood gases (47%), composition of inspired and expired gases (45%) and body temperature (35%). Drugs administered frequently or routinely as part of a standard protocol were: acepromazine (44%), xylazine (68%), butorphanol (59%), ketamine (96%), diazepam (83%), isoflurane (76%), dobutamine (46%), and, as a nonsteroidal anti-inflammatory drug, phenylbutazone (73%) or flunixin meglumine (66%). Recovery was routinely assisted by 40%. The main factors perceived by the respondents to affect outcome of equine anaesthesia were the preoperative health status of the animal and training of the anaesthetist. CONCLUSIONS Current practice in equine anaesthesia varies widely, and the study has highlighted important topics relevant for designing a future prospective multicentre cohort study (CEPEF-4). The Summary is available in Chinese - see Supporting information.

Mucosal Erosion of the Cricoid Cartilage After the Use of an i-Gel Supraglottic Airway Device in a Patient with Diffuse Idiopathic Skeletal Hyperostosis

After standard hip arthroplasty, an 82-year-old patient with previously undiagnosed diffuse idiopathic skeletal hyperostosis of the cervical spine experienced life-threatening side effects after use of a supraglottic airway device (i-gel). Extensive mucosal erosion and denudation of the cricoid cartilage caused postoperative supraglottic swelling and prolonged respiratory failure requiring tracheostomy. In this case report, we highlight the importance of evaluating risk factors for failure of supraglottic airway devices.

Development of a method for analysis of ketamine and norketamine enantiomers in equine brain and cerebrospinal fluid by capillary electrophoresis

Ketamine and norketamine are being transported across the blood brain barrier and are also entering from blood into cerebrospinal fluid (CSF). Enantioselective distributions of these compounds in brain and CSF have never been determined. The enantioselective CE based assay previously developed for equine plasma was adapted to the analysis of these compounds in equine brain via use of an acidic pre-extraction of interferences prior to liquid/liquid extraction at alkaline pH. CSF can be treated as plasma. With 100 mg of brain tissue and 0.5 mL of CSF or plasma, assay conditions for up to 30 nmol/g and 6 μM, respectively, of each enantiomer with LOQs of 0.5 nmol/g and 0.1 μM, respectively, were established and the assays were applied to equine samples. CSF and plasma samples analyzed stemmed from anesthetized patient horses and brain, CSF and plasma were obtained from anesthetized horses that were euthanized with an overdose of pentobarbital. Data obtained indicate that ketamine and norketamine enantiomers are penetrating into brain and CSF with those of ketamine being more favorably transported than norketamine, whereas metabolites of norketamine are hindered. More work is required to properly investigate possible stereoselectivities of the ketamine metabolism and transport of metabolites from blood into brain tissue and CSF.

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

The transcriptome of equine peripheral blood mononuclear cells.

Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.

Plate fixation of extra-articular fractures of the proximal phalanx: do new implants cause less problems?

BACKGROUND Limited range of finger motion is a frequent complication after plate fixation of phalangeal fractures. The purpose of this study was to evaluate the results of plate fixation of extra-articular fractures of the proximal phalanx using current low-profile mini-fragment-systems. METHODS From 2006 to 2012, 32 patients with 36 extra-articular fractures of the proximal phalanx of the triphalangeal fingers were treated with open reduction and plate fixation (ORPF) using 1.2 and 1.5 mm mini-fragment systems. Patients presenting with open fractures grade 2 and 3 or relevant laceration of adjacent structures were excluded from the study. We retrospectively evaluated the rate of mal-union or non-union after ORPF, the need for revision surgery, for plate removal, and for tenolysis. Data were analyzed for further complications with regard to infections or complex regional pain syndrome (CRPS). RESULTS No infections were noted. Five patients developed transient symptoms of CRPS. Six weeks postoperatively, total active finger motion (TAM) averaged 183°, and all 32 patients underwent formal hand therapy. At the latest follow-up or at the time of plate removal, respectively, the mean TAM improved to 213°. Extension lag of proximal interphalangeal joints was found in 67 % of all fractured fingers. Secondary surgery was necessary in 14 of 32 patients (2 corrective osteotomies, 12 plate removals including 7 procedures explicitly because of reduced mobility). CONCLUSIONS Despite of new implant designs significant problems persist. Adhesions of extensor tendons leading to limited range of finger motion are still the most frequent complications after ORPF of proximal phalangeal fractures, even in absence of significant soft-tissue damage. LEVEL OF EVIDENCE Therapeutic, Retrospective, Level IV.

An equine chromosome 3 inversion is associated with the tobiano spotting pattern in German horse breeds

The tobiano white-spotting pattern is one of several known depigmentation phenotypes in horses and is desired by many horse breeders and owners. The tobiano spotting phenotype is inherited as an autosomal dominant trait. Horses that are heterozygous or homozygous for the tobiano allele (To) are phenotypically indistinguishable. A SNP associated with To had previously been identified in intron 13 of the equine KIT gene and was used for an indirect gene test. The test was useful in several horse breeds. However, genotyping this sequence variant in the Lewitzer horse breed revealed that 14% of horses with the tobiano pattern did not show the polymorphism in intron 13 and consequently the test was not useful to identify putative homozygotes for To within this breed. Speculations were raised that an independent mutation might cause the tobiano spotting pattern in this breed. Recently, the putative causative mutation for To was described as a large chromosomal inversion on equine chromosome 3. One of the inversion breakpoints is approximately 70 kb downstream of the KIT gene and probably disrupts a regulatory element of the KIT gene. We obtained genotypes for the intron 13 SNP and the chromosomal inversion for 204 tobiano spotted horses and 24 control animals of several breeds. The genotyping data confirmed that the chromosomal inversion was perfectly associated with the To allele in all investigated horses. Therefore, the new test is suitable to discriminate heterozygous To/+ and homozygous To/To horses in the investigated breeds.

Impaired Cell Cycle Regulation in a Natural Equine Model of Asthma.

Recurrent airway obstruction (RAO) is a common and potentially debilitating lower airway disease in horses, which shares many similarities with human asthma. In susceptible horses RAO exacerbation is caused by environmental allergens and irritants present in hay dust. The objective of this study was the identification of genes and pathways involved in the pathology of RAO by global transcriptome analyses in stimulated peripheral blood mononuclear cells (PBMCs). We performed RNA-seq on PBMCs derived from 40 RAO affected and 45 control horses belonging to three cohorts of Warmblood horses: two half-sib families and one group of unrelated horses. PBMCs were stimulated with hay dust extract, lipopolysaccharides, a recombinant parasite antigen, or left unstimulated. The total dataset consisted of 561 individual samples. We detected significant differences in the expression profiles between RAO and control horses. Differential expression (DE) was most marked upon stimulation with hay dust extract. An important novel finding was a strong upregulation of CXCL13 together with many genes involved in cell cycle regulation in stimulated samples from RAO affected horses, in addition to changes in the expression of several HIF-1 transcription factor target genes. The RAO condition alters systemic changes observed as differential expression profiles of PBMCs. Those changes also depended on the cohort and stimulation of the samples and were dominated by genes involved in immune cell trafficking, development, and cell cycle regulation. Our findings indicate an important role of CXCL13, likely macrophage or Th17 derived, and the cell cycle regulator CDC20 in the immune response in RAO.

Intra-articular corticosteroid for knee osteoarthritis.

BACKGROUND Knee osteoarthritis is a leading cause of chronic pain, disability, and decreased quality of life. Despite the long-standing use of intra-articular corticosteroids, there is an ongoing debate about their benefits and safety. This is an update of a Cochrane review first published in 2005. OBJECTIVES To determine the benefits and harms of intra-articular corticosteroids compared with sham or no intervention in people with knee osteoarthritis in terms of pain, physical function, quality of life, and safety. SEARCH METHODS We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, and EMBASE (from inception to 3 February 2015), checked trial registers, conference proceedings, reference lists, and contacted authors. SELECTION CRITERIA We included randomised or quasi-randomised controlled trials that compared intra-articular corticosteroids with sham injection or no treatment in people with knee osteoarthritis. We applied no language restrictions. DATA COLLECTION AND ANALYSIS We calculated standardised mean differences (SMDs) and 95% confidence intervals (CI) for pain, function, quality of life, joint space narrowing, and risk ratios (RRs) for safety outcomes. We combined trials using an inverse-variance random-effects meta-analysis. MAIN RESULTS We identified 27 trials (13 new studies) with 1767 participants in this update. We graded the quality of the evidence as 'low' for all outcomes because treatment effect estimates were inconsistent with great variation across trials, pooled estimates were imprecise and did not rule out relevant or irrelevant clinical effects, and because most trials had a high or unclear risk of bias. Intra-articular corticosteroids appeared to be more beneficial in pain reduction than control interventions (SMD -0.40, 95% CI -0.58 to -0.22), which corresponds to a difference in pain scores of 1.0 cm on a 10-cm visual analogue scale between corticosteroids and sham injection and translates into a number needed to treat for an additional beneficial outcome (NNTB) of 8 (95% CI 6 to 13). An I(2) statistic of 68% indicated considerable between-trial heterogeneity. A visual inspection of the funnel plot suggested some asymmetry (asymmetry coefficient -1.21, 95%CI -3.58 to 1.17). When stratifying results according to length of follow-up, benefits were moderate at 1 to 2 weeks after end of treatment (SMD -0.48, 95% CI -0.70 to -0.27), small to moderate at 4 to 6 weeks (SMD -0.41, 95% CI -0.61 to -0.21), small at 13 weeks (SMD -0.22, 95% CI -0.44 to 0.00), and no evidence of an effect at 26 weeks (SMD -0.07, 95% CI -0.25 to 0.11). An I(2) statistic of ≥ 63% indicated a moderate to large degree of between-trial heterogeneity up to 13 weeks after end of treatment (P for heterogeneity≤0.001), and an I(2) of 0% indicated low heterogeneity at 26 weeks (P=0.43). There was evidence of lower treatment effects in trials that randomised on average at least 50 participants per group (P=0.05) or at least 100 participants per group (P=0.013), in trials that used concomittant viscosupplementation (P=0.08), and in trials that used concomitant joint lavage (P≤0.001).Corticosteroids appeared to be more effective in function improvement than control interventions (SMD -0.33, 95% CI -0.56 to -0.09), which corresponds to a difference in functions scores of -0.7 units on standardised Western Ontario and McMaster Universities Arthritis Index (WOMAC) disability scale ranging from 0 to 10 and translates into a NNTB of 10 (95% CI 7 to 33). An I(2) statistic of 69% indicated a moderate to large degree of between-trial heterogeneity. A visual inspection of the funnel plot suggested asymmetry (asymmetry coefficient -4.07, 95% CI -8.08 to -0.05). When stratifying results according to length of follow-up, benefits were small to moderate at 1 to 2 weeks after end of treatment (SMD -0.43, 95% CI -0.72 to -0.14), small to moderate at 4 to 6 weeks (SMD -0.36, 95% CI -0.63 to -0.09), and no evidence of an effect at 13 weeks (SMD -0.13, 95% CI -0.37 to 0.10) or at 26 weeks (SMD 0.06, 95% CI -0.16 to 0.28). An I(2) statistic of ≥ 62% indicated a moderate to large degree of between-trial heterogeneity up to 13 weeks after end of treatment (P for heterogeneity≤0.004), and an I(2) of 0% indicated low heterogeneity at 26 weeks (P=0.52). We found evidence of lower treatment effects in trials that randomised on average at least 50 participants per group (P=0.023), in unpublished trials (P=0.023), in trials that used non-intervention controls (P=0.031), and in trials that used concomitant viscosupplementation (P=0.06).Participants on corticosteroids were 11% less likely to experience adverse events, but confidence intervals included the null effect (RR 0.89, 95% CI 0.64 to 1.23, I(2)=0%). Participants on corticosteroids were 67% less likely to withdraw because of adverse events, but confidence intervals were wide and included the null effect (RR 0.33, 95% CI 0.05 to 2.07, I(2)=0%). Participants on corticosteroids were 27% less likely to experience any serious adverse event, but confidence intervals were wide and included the null effect (RR 0.63, 95% CI 0.15 to 2.67, I(2)=0%).We found no evidence of an effect of corticosteroids on quality of life compared to control (SMD -0.01, 95% CI -0.30 to 0.28, I(2)=0%). There was also no evidence of an effect of corticosteroids on joint space narrowing compared to control interventions (SMD -0.02, 95% CI -0.49 to 0.46). AUTHORS' CONCLUSIONS Whether there are clinically important benefits of intra-articular corticosteroids after one to six weeks remains unclear in view of the overall quality of the evidence, considerable heterogeneity between trials, and evidence of small-study effects. A single trial included in this review described adequate measures to minimise biases and did not find any benefit of intra-articular corticosteroids.In this update of the systematic review and meta-analysis, we found most of the identified trials that compared intra-articular corticosteroids with sham or non-intervention control small and hampered by low methodological quality. An analysis of multiple time points suggested that effects decrease over time, and our analysis provided no evidence that an effect remains six months after a corticosteroid injection.