ABSTRACT
OBJECTIVE: Physiological mechanical loading reduces inflammatory signalling in numerous cell types including articular chondrocytes however the mechanism responsible remains unclear. This study investigates the role of chondrocyte primary cilia and associated intraflagellar transport (IFT) in the mechanical regulation of interleukin-1ß (IL-1ß) signalling. DESIGN: Isolated chondrocytes and cartilage explants were subjected to cyclic mechanical loading in the presence and absence of the cytokine IL-1ß. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were used to monitor IL-1ß signalling whilst Sulphated glycosaminoglycan (sGAG) release provided measurement of cartilage degradation. Measurements were made of HDAC6 activity and tubulin polymerisation and acetylation. Effects on primary cilia were monitored by confocal and super resolution microscopy. Involvement of IFT was analysed using ORPK cells with hypomorphic mutation of IFT88. RESULTS: Mechanical loading suppressed NO and PGE2 release and prevented cartilage degradation. Loading activated HDAC6 and disrupted tubulin acetylation and cilia elongation induced by IL-1ß. HDAC6 inhibition with tubacin blocked the anti-inflammatory effects of loading and restored tubulin acetylation and cilia elongation. Hypomorphic mutation of IFT88 reduced IL-1ß signalling and abolished the anti-inflammatory effects of loading indicating the mechanism is IFT-dependent. Loading reduced the pool of non-polymerised tubulin which was replicated by taxol which also mimicked the anti-inflammatory effects of mechanical loading and prevented cilia elongation. CONCLUSIONS: This study reveals that mechanical loading suppresses inflammatory signalling, partially dependent on IFT, by activation of HDAC6 and post transcriptional modulation of tubulin.
Subject(s)
Chondrocytes/metabolism , Histone Deacetylase 6/metabolism , Interleukin-1beta/metabolism , Stress, Mechanical , Tubulin/metabolism , Animals , Biomarkers/metabolism , Blotting, Western , Cartilage, Articular/metabolism , Cattle , Cells, Cultured , Cilia/metabolism , Dinoprostone/metabolism , Humans , Microscopy, Confocal , Nitric Oxide/metabolism , Sensitivity and Specificity , Signal TransductionABSTRACT
BACKGROUND: National Confidential Enquiry into Patient Outcome and Death (NCEPOD) 'Measuring the Units' (June 2013) identified significant organisational and attitudinal deficits in hospital care of patients with alcohol-related liver disease (ARLD), care being recognised as good in less than 50% of patients. METHOD: We surveyed over 700 consultants and trainees in acute medical and intensive therapy specialties to examine their perceptions of the NCEPOD findings. RESULTS: A total of 178 responded. In keeping with the NCEPOD findings, their perception was of lack of 24-hour access to specialty advice for patients with liver disease and inequity of access to high-dependency units. Their explanations include lack of resources, therapeutic nihilism and prejudicial judgements that would not be made of other patient groups. CONCLUSION: There is an urgent need for robust mechanisms to ensure equity of access to specialist liver advice and intensive therapy unit resources, and to counter negative and prejudicial attitudes to these patients.
Subject(s)
Attitude of Health Personnel , Health Services Accessibility , Liver Diseases, Alcoholic/therapy , Physicians/psychology , Critical Care , England , Gastroenterology , Health Resources , Humans , Internship and Residency , Medical Futility , Prejudice , Referral and Consultation , Surveys and QuestionnairesABSTRACT
PURPOSE: Primary ciliary dyskinesia (PCD) is characterised by repeated upper and lower respiratory tract infections, neutrophilic airway inflammation and obstructive airway disease. Different ultrastructural ciliary defects may affect lung function decline to different degrees. Lung clearance index (LCI) is a marker of ventilation inhomogeneity that is raised in some but not all patients with PCD. We hypothesised that PCD patients with microtubular defects would have worse (higher) LCI than other PCD patients. METHODS: Spirometry and LCI were measured in 69 stable patients with PCD. Age at testing, age at diagnosis, ethnicity, ciliary ultrastructure, genetic screening result and any growth of Pseudomonas aeruginosa was recorded. RESULTS: Lung clearance index was more abnormal in PCD patients with microtubular defects (median 10.24) than those with dynein arm defects (median 8.3, p = 0.004) or normal ultrastructure (median 7.63, p = 0.0004). Age is correlated with LCI, with older patients having worse LCI values (p = 0.03, r = 0.3). CONCLUSION: This study shows that cilia microtubular defects are associated with worse LCI in PCD than dynein arm defects or normal ultrastructure. The patient's age at testing is also associated with a higher LCI. Patients at greater risk of obstructive lung disease should be considered for more aggressive management. Differences between patient groups may potentially open avenues for novel treatments.
Subject(s)
Cilia/ultrastructure , Ciliary Motility Disorders/complications , Lung Diseases/etiology , Lung/physiopathology , Lung/ultrastructure , Microtubules/ultrastructure , Mucociliary Clearance , Adolescent , Adult , Age Factors , Child , Child, Preschool , Ciliary Motility Disorders/genetics , Ciliary Motility Disorders/pathology , Ciliary Motility Disorders/physiopathology , Female , Forced Expiratory Volume , Humans , Infant , Infant, Newborn , Lung Diseases/pathology , Lung Diseases/physiopathology , Male , Maximal Midexpiratory Flow Rate , Microscopy, Electron, Transmission , Risk Factors , Spirometry , Young AdultABSTRACT
Tissue engineering-based therapies targeting cartilage diseases, such as osteoarthritis, require in vitro expansion of articular chondrocytes. A major obstacle for these therapies is the dedifferentiation and loss of phenotype accompanying chondrocyte expansion. Recent studies suggest that manipulation of hedgehog signalling may be used to promote chondrocyte re-differentiation. Hedgehog signalling requires the primary cilium, a microtubule-based signalling compartment, the integrity of which is linked to the cytoskeleton. We tested the hypothesis that alterations in cilia expression occurred as consequence of chondrocyte dedifferentiation and influenced hedgehog responsiveness. In vitro chondrocyte expansion to passage 5 (P5) was associated with increased actin stress fibre formation, dedifferentiation and progressive loss of primary cilia, compared to primary (P0) cells. P5 chondrocytes exhibited ~50 % fewer cilia with a reduced mean length. Cilia loss was associated with disruption of ligand-induced hedgehog signalling, such that P5 chondrocytes did not significantly regulate the expression of hedgehog target genes (GLI1 and PTCH1). This phenomenon could be recapitulated by applying 24 h cyclic tensile strain, which reduced cilia prevalence and length in P0 cells. LiCl treatment rescued cilia loss in P5 cells, partially restoring hedgehog signalling, so that GLI1 expression was significantly increased by Indian hedgehog. This study demonstrated that monolayer expansion disrupted primary cilia structure and hedgehog signalling associated with chondrocyte dedifferentiation. This excluded the possibility to use hedgehog ligands to stimulate re-differentiation without first restoring cilia expression. Furthermore, primary cilia loss during chondrocyte expansion would likely impact other cilia pathways important for cartilage health and tissue engineering, including transforming growth factor (TGF), Wnt and mechanosignalling.
Subject(s)
Chondrocytes/cytology , Cilia/metabolism , Hedgehog Proteins/metabolism , Signal Transduction , Actins/metabolism , Animals , Cartilage, Articular/cytology , Cattle , Cell Dedifferentiation/drug effects , Cell Proliferation/drug effects , Chondrocytes/drug effects , Chondrocytes/metabolism , Ligands , Lithium Chloride/pharmacology , Phenotype , Polymerization , Signal Transduction/drug effects , Weight-BearingABSTRACT
PURPOSE: To compare three separate assessment stations used for selection to Core Medical Training (CMT) and to determine the effect of reducing the number from three to two. METHODS: Quantitative analysis of candidates' assessment station scores, financial analysis of costs of the selection process and quantitative and qualitative surveys of candidates and assessors. RESULTS: The assessment stations used for selection to CMT were reliable and valid for assessing suitability for employment as a CMT trainee. There was no significant difference in candidate ranking if only two assessment stations were used rather than three, i.e. there was no change in the likelihood of receiving a job offer. All of the assessment stations were perceived to have face validity by candidates and assessors. The efficiency of the selection process could be improved without loss of quality if two stations were used rather than three. CONCLUSIONS: Using two assessment stations rather than three would appear to improve the efficiency and maintain the quality of the CMT selection process while reducing costs.
Subject(s)
Education, Medical, Graduate , Educational Measurement/standards , School Admission Criteria , Costs and Cost Analysis , Educational Measurement/economics , Employment , Humans , Quality Control , Reproducibility of ResultsABSTRACT
... The limbs on the right side are stronger. [The] cause may be ... [that] ... motion, and abilities of moving, are somewhat holpen from the liver, which lieth on the right side. (Sir Francis Bacon, Sylva sylvarum (1627).)Fifty per cent of people with primary ciliary dyskinesia (PCD) (also known as immotile cilia syndrome or Siewert-Kartagener syndrome) have situs inversus, which is thought to result from absent nodal ciliary rotation and failure of normal symmetry breaking. In a study of 88 people with PCD, only 15.2% of 46 individuals with situs inversus, and 14.3% of 42 individuals with situs solitus, were left handed. Because cerebral lateralization is therefore still present, the nodal cilia cannot be the primary mechanism responsible for symmetry breaking in the vertebrate body. Intriguingly, one behavioural lateralization, wearing a wrist-watch on the right wrist, did correlate with situs inversus.
Subject(s)
Functional Laterality/physiology , Kartagener Syndrome/complications , Models, Biological , Situs Inversus/etiology , Humans , Logistic Models , Surveys and QuestionnairesABSTRACT
Primary ciliary dyskinesia (PCD) is a phenotypically and genetically heterogeneous condition in which three genetic mutations have already been identified. The primary defect is in the ultrastructure or function of cilia, highly complex organelles that are structurally related to the flagella of sperm and protozoa. The clinical features of PCD include recurrent sinopulmonary infections, subfertility and laterality defects; the latter due to ciliary dysfunction at the embryological node. Completion of the human genome sequence has accelerated the identification and characterisation of disease genes, and the current molecular strategy in PCD includes candidate gene analysis, positional cloning, model organism analysis and proteomic analysis. The identification of these genes will provide new insights into the molecular mechanisms involved in the assembly and function of cilia and the pathway that determines left-right axis in man. This may also allow the development of new methods for diagnosis, prevention and treatment of PCD.
Subject(s)
Genetic Predisposition to Disease , Kartagener Syndrome/genetics , Mutation , Animals , Child, Preschool , Cilia/physiology , Ciliary Motility Disorders/diagnosis , Ciliary Motility Disorders/genetics , Female , Genome, Human , Humans , Infant , Infant, Newborn , Kartagener Syndrome/diagnosis , Male , Molecular Biology , Phenotype , Rodentia , Sensitivity and SpecificitySubject(s)
Chromosomes, Human, Pair 15/genetics , Chromosomes, Human, Pair 16/genetics , Ciliary Motility Disorders/genetics , Genetic Heterogeneity , Chromosome Mapping , Ciliary Motility Disorders/epidemiology , Consanguinity , Female , Geography , Humans , Israel/epidemiology , Israel/ethnology , Lod Score , Male , Netherlands/ethnology , Norway/ethnology , Pedigree , Software , United Kingdom/ethnologyABSTRACT
BACKGROUND: We have previously reported, in an uncontrolled trial, an improvement in fatigue scores in patients with primary biliary cirrhosis given oral antioxidant supplementation. We now present data from a controlled trial. PATIENTS AND METHODS: Sixty-one patients with primary biliary cirrhosis-associated fatigue were randomized into a double-blind, placebo-controlled, cross-over trial. Participants received 12 weeks each of placebo and antioxidant supplementation (vitamins A, C and E, selenium, methionine and ubiquinone) in random order, separated by a 4-week washout period. The primary trial outcome (fatigue) was assessed using the Fisk scale. Other symptoms of primary biliary cirrhosis were measured using Likert and visual analogue scales. RESULTS: Forty-four patients completed both arms of the trial. No significant changes in fatigue were recorded in the active phase of treatment (median improvement in Fisk score, 1; P = 0.61). Small improvements in Fisk scores were recorded during placebo therapy (median improvement, 4; P = 0.03). Neither medication was associated with improvement in any other symptoms related to primary biliary cirrhosis. Adverse effects were more common during active therapy and were mild and self-limiting. One patient died from unrelated causes during active treatment. CONCLUSIONS: Although oral antioxidant supplementation appears to be safe, we could not find any evidence for a beneficial effect on fatigue or other liver-related symptoms.
Subject(s)
Antioxidants/administration & dosage , Dietary Supplements , Fatigue/prevention & control , Liver Cirrhosis, Biliary/complications , Administration, Oral , Ascorbic Acid/administration & dosage , Cross-Over Studies , Double-Blind Method , Fatigue/etiology , Humans , Methionine/administration & dosage , Selenium/administration & dosage , Ubiquinone/administration & dosage , Vitamin A/administration & dosage , Vitamin E/administration & dosageABSTRACT
In the past decade there have been significant advances in our understanding of the molecular genetic basis of the neuronal ceroid lipofuscinoses, a clinically and genetically heterogeneous group of childhood neurodegenerative storage disorders. Recent research progress is reviewed here, to summarize new disease gene identification, diagnostics, treatment, protein functional studies and investigations into the underlying molecular pathogenesis of these devastating disorders.
Subject(s)
Membrane Glycoproteins , Molecular Chaperones , Neuronal Ceroid-Lipofuscinoses/genetics , Animals , Child , Chromosome Mapping , Genotype , Humans , Infant , Membrane Proteins/genetics , Models, Genetic , Neuronal Ceroid-Lipofuscinoses/diagnosis , Neuronal Ceroid-Lipofuscinoses/therapy , Phenotype , Phosphoprotein Phosphatases/genetics , Proteins/genetics , Thiolester HydrolasesABSTRACT
Batten disease, the juvenile-onset form of neuronal ceroid lipofuscinosis (NCL), is a progressive neurodegenerative disorder of childhood with an age of onset of 5-10 years of age. JNCL is caused by mutations in the CLN3 gene which encodes a membrane protein of unknown function. Magnetic resonance imaging of the brain of juvenile NCL patients has revealed changes in signal intensity and tissue atrophy, predominantly in the cortex and cerebellum. A mouse model for Batten disease was created by targeted disruption of the murine Cln3 gene in order to further understanding of the pathophysiology of Batten disease and to evaluate potential therapeutic approaches. Several features of the disease are displayed by Cln3 mice including accumulation of characteristic storage material in neurons. The aim of this work was to investigate neurodegeneration in the Cln3 mouse model using high resolution magnetic resonance imaging to measure signal intensity ratios in selected regions of interest. Global changes were observed in the brains of 12-month-old mutant mice that mirror those seen in juvenile NCL patients. There is a decrease in signal intensity ratio in grey matter regions including cortex, hippocampus and cerebellum, tissues where neuronal storage accumulation and cell loss have been seen in the mouse model. The alterations seen in Cln3 mutant mice support the validity of further imaging studies and suggest that this method will have application in assessment of therapeutic approaches in the study of mutant mouse models of NCL including the Cln3 mouse.
Subject(s)
Brain/pathology , Magnetic Resonance Imaging , Membrane Glycoproteins , Molecular Chaperones , Neuronal Ceroid-Lipofuscinoses/pathology , Proteins/genetics , Animals , Atrophy , Disease Models, Animal , Male , Mice , Mice, Mutant Strains , Neuronal Ceroid-Lipofuscinoses/geneticsABSTRACT
The transcription factor FOXJ1 (alias HFH-4 or FKHL13) of the winged-helix/forkhead family is expressed in cells with cilia or flagella, and seems to be involved in the regulation of axonemal structural proteins. The knockout mouse Foxj1(-/-) shows abnormalities of organ situs, consistent with random determination of left-right asymmetry, and a complete absence of cilia. The human FOXJ1 gene which maps to chromosome 17q, is thus an excellent candidate gene for Kartagener Syndrome (KS), a subphenotype of Primary Ciliary Dyskinesia (PCD), characterized by bronchiectasis, chronic sinusitis and situs inversus. We have collected samples from 61 PCD families, in 31 of which there are at least two affected individuals. Two families with complete aciliogenesis, and six families, in which the affected members have microsatellite alleles concordant for a locus on distal chromosome 17q, were screened for mutations in the two exons and intron-exon junctions of the FOXJ1 gene. No sequence abnormalities were observed in the DNAs of the affected individuals of the selected families. These results demonstrate that the FOXJ1 gene is not responsible for the PCD/KS phenotype in the families examined.
Subject(s)
Ciliary Motility Disorders/genetics , DNA-Binding Proteins , Mutation/genetics , Trans-Activators/genetics , Alleles , Amino Acid Sequence , Base Sequence , DNA Mutational Analysis , Databases as Topic , Exons/genetics , Forkhead Transcription Factors , Genotype , Humans , Introns/genetics , Kartagener Syndrome/genetics , Microsatellite Repeats/genetics , Molecular Sequence Data , Phenotype , Polymorphism, Genetic/geneticsSubject(s)
Alcoholism/economics , Cost of Illness , National Health Programs , Adult , Alcoholism/complications , Alcoholism/therapy , Female , Humans , Male , Medical Audit , Patient Admission , United KingdomABSTRACT
Primary ciliary dyskinesia is an autosomal recessive condition characterised by chronic sinusitis, bronchiectasis, and subfertility. Situs inversus occurs in 50% of cases (Kartagener syndrome). It has an estimated incidence of 1 in 20 000 live births. The clinical phenotype is caused by defective ciliary function associated with a range of ultrastructural abnormalities including absent dynein arms, absent radial spokes, and disturbed ciliary orientation. The molecular genetic basis is unknown. A genome scan was performed in five Arabic families. Using GENEHUNTER, a maximal multipoint lod score (HLOD) of 4.4 was obtained on chromosome 19q13.3-qter at alpha (proportion of linked families) = 0.7. A 15 cM critical region is defined by recombinations at D19S572 and D19S218. These data provide significant evidence for a PCD locus on chromosome 19q and confirm locus heterogeneity.
Subject(s)
Chromosomes, Human, Pair 19 , Ciliary Motility Disorders/genetics , Adult , Chromosome Mapping , Ciliary Body/ultrastructure , Ciliary Motility Disorders/physiopathology , Female , Humans , Male , Microsatellite Repeats , Pedigree , Sinusitis/etiology , Situs Inversus/etiologyABSTRACT
Deficiency in palmitoyl protein thioesterase (PPT) results in the rapid death of neocortical neurons in human. Very little is known about the developmental and cell-specific expression of this lysosomal enzyme. Here we show that PPT is expressed as a major 2.65 kb and a minor 1.85 kb transcript in the mouse brain. Transcript levels gradually increase between postnatal days 10 and 30. In situ hybridization analysis revealed that PPT transcripts are found widely but not homogeneously in the brain. The most intense signal was detected in the cerebral cortex (layers II, IV-V), hippocampal CA1-CA3 pyramidal cells, dentate gyrus granule cells and the hypothalamus. Immunostaining of PPT was localized in the cell soma, axons and dendrites, especially in the pyramidal and granular cells of the hippocampus, correlating well, both spatially and temporally, with the immunoreactivity of a presynaptic vesicle membrane protein, synaptophysin. In whole embryos, at embryonic day 8, the PPT mRNA expression was most apparent throughout the neuroepithelium, and from day 9 onwards it was seen in all tissues. The expression pattern of PPT suggests its general significance for the brain cells and reflects the response to maturation and growth of the neural networks. Strong PPT immunoreactivity in the axons and dentrites would imply that PPT may not be exclusively a lysosomal enzyme. A notable correlation with synaptophysin would suggest that PPT may have a role in the function of the synaptic machinery.
Subject(s)
Aging/metabolism , Animals, Newborn/metabolism , Brain/embryology , Brain/metabolism , Fetus/metabolism , Thiolester Hydrolases/metabolism , Animals , Animals, Newborn/growth & development , Embryonic and Fetal Development , Fetus/physiology , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred Strains , RNA, Messenger/metabolism , Reference Values , Thiolester Hydrolases/genetics , Tissue DistributionABSTRACT
Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease.
Subject(s)
Hippocampus/pathology , Membrane Glycoproteins , Molecular Chaperones , Neuronal Ceroid-Lipofuscinoses/pathology , Neurons/pathology , Proteins/genetics , Animals , Disease Models, Animal , Female , Genotype , Hippocampus/metabolism , Hippocampus/ultrastructure , Humans , Hypertrophy , Interneurons/pathology , Kidney/metabolism , Kidney/pathology , Male , Mice , Mice, Knockout , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/physiopathology , Neurons/metabolism , Neurons/ultrastructure , Proteins/physiology , Restriction Mapping , Reverse Transcriptase Polymerase Chain Reaction , Tripeptidyl-Peptidase 1ABSTRACT
The neuronal ceroid lipofuscinoses (NCLs), also referred to as Batten disease, are a group of neurodegenerative disorders characterised by the accumulation of an autofluorescent lipopigment in many cell types. Different NCL types are distinguished according to age of onset, clinical phenotype, ultrastructural characterisation of the storage material, and chromosomal location of the disease gene. At least eight genes underlie the NCLs, of which four have been isolated and mutations characterised: CLN1, CLN2, CLN3, CLN5. Two of these genes encode lysosomal enzymes, and two encode transmembrane proteins, at least one of which is likely to be in the lysosomal membrane. The basic defect in the NCLs appears to be associated with lysosomal function.
Subject(s)
Membrane Glycoproteins , Membrane Proteins/genetics , Molecular Chaperones , Neuronal Ceroid-Lipofuscinoses/enzymology , Peptide Hydrolases/genetics , Proteins/genetics , Thiolester Hydrolases/genetics , Aminopeptidases , DNA Mutational Analysis , Databases, Factual , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases , Endopeptidases , Humans , Hydrogen-Ion Concentration , Intracellular Membranes/metabolism , Lipofuscin/metabolism , Lysosomal Membrane Proteins , Lysosomes/enzymology , Lysosomes/genetics , Lysosomes/metabolism , Mutation , Neuronal Ceroid-Lipofuscinoses/genetics , Peptide Hydrolases/metabolism , Polymorphism, Genetic , Serine Proteases , Thiolester Hydrolases/metabolism , Tripeptidyl-Peptidase 1ABSTRACT
Two distinct clinical subtypes of neuronal ceroid lipofuscinosis caused by mutations in the PPT gene, INCL and vJNCL/GROD, occur at a high frequency in the central region of Scotland. In this paper we summarize the clinical details and the molecular basis underlying the disease in the Scottish patients. Comparison of the combination of mutations in the different clinical types reveals a clear genotype-phenotype correlation.