Ultrasound measurement of joint cartilage thickness in large and small joints in healthy children: a clinical pilot study assessing observer variability

<p>Abstract</p> <p>Background</p> <p>Loss of joint cartilage is a feature of destructive disease in JIA. The cartilage of most joints can be visualized with ultrasonography (US). Our present study focuses on discriminant validity of US in children. We studied reproducibility between and within a skilled and a non-skilled investigator of US assessment of cartilage thickness in small and large joints in healthy children.</p> <p>Methods and results</p> <p>In 11 healthy children (5 girls/6 boys), aged 9.6 years (9.3–10 years), 110 joints were examined. Cartilage thickness of the right and left hip, knee, ankle, 2^ndmetacarpophalangeal (MCP), and 2^ndproximal interphalangeal (PIP) joint independently. The joints were examined twice, two days apart by a skilled and a non-skilled investigator. Mean cartilage thickness in the five joints was: hip 2.59 ± 0.41, knee 3.67 ± 0.64, ankle 1.08 ± 0.31, MCP 1.52 ± 0.27 and PIP 0.73 ± 0.15 mm. We found the same mean differences in CTh of 0.6 mm in the inter-observer part with regard of the PIP joint. Within investigators (intra-observer), the smallest mean difference of CTh was found in the MCP joint with -0.004 (skilled) and 0.013 mm (non-skilled).</p> <p>Conclusion</p> <p>We found the level of agreement between observers within a 95% Confidence Interval in assessment of cartilage thickness in hip-, knee-, ankle-, MCP-, and PIP joints in healthy children. Observer variability seems not to relate to joint size but to the positioning of the joints and the transducer. These factors seem to be of major importance for reproducible US measurements. The smallest difference in measurement of cartilage thickness <it>between observers </it>was found in the PIP joint, and <it>within observers </it>in the MCP joint and it seems that using EULAR standard US guidelines is feasible for a pediatric setting. The use of US in children is promising. Studies on larger groups of children are needed to confirm the validation and variability of US in children as well as determining the smallest detectable difference of US measures.</p

Hedgehog pathway mutations drive oncogenic transformation in high-risk T-cell acute lymphoblastic leukemia.

The role of Hedgehog signaling in normal and malignant T-cell development is controversial. Recently, Hedgehog pathway mutations have been described in T-ALL, but whether mutational activation of Hedgehog signaling drives T-cell transformation is unknown, hindering the rationale for therapeutic intervention. Here, we show that Hedgehog pathway mutations predict chemotherapy resistance in human T-ALL, and drive oncogenic transformation in a zebrafish model of the disease. We found Hedgehog pathway mutations in 16% of 109 childhood T-ALL cases, most commonly affecting its negative regulator PTCH1. Hedgehog mutations were associated with resistance to induction chemotherapy (P = 0.009). Transduction of wild-type PTCH1 into PTCH1-mutant T-ALL cells induced apoptosis (P = 0.005), a phenotype that was reversed by downstream Hedgehog pathway activation (P = 0.007). Transduction of most mutant PTCH1, SUFU, and GLI alleles into mammalian cells induced aberrant regulation of Hedgehog signaling, indicating that these mutations are pathogenic. Using a CRISPR/Cas9 system for lineage-restricted gene disruption in transgenic zebrafish, we found that ptch1 mutations accelerated the onset of notch1-induced T-ALL (P = 0.0001), and pharmacologic Hedgehog pathway inhibition had therapeutic activity. Thus, Hedgehog-activating mutations are driver oncogenic alterations in high-risk T-ALL, providing a molecular rationale for targeted therapy in this disease

Improving prescribing of antihypertensive and cholesterol-lowering drugs: a method for identifying and addressing barriers to change

BACKGROUND: We describe a simple approach we used to identify barriers and tailor an intervention to improve pharmacological management of hypertension and hypercholesterolaemia. We also report the results of a post hoc exercise and survey we carried out to evaluate our approach for identifying barriers and tailoring interventions. METHODS: We used structured reflection, searched for other relevant trials, surveyed general practitioners and talked with physicians during pilot testing of the intervention. The post hoc exercise was carried out as focus groups of international researchers in the field of quality improvement in health care. The post hoc survey was done by telephone interviews with physicians allocated to the experimental group of a randomised trial of our multifaceted intervention. RESULTS: A wide range of barriers was identified and several interventions were suggested through structured reflection. The survey led to some adjustments. Studying other trials and pilot testing did not lead to changes in the design of the intervention. Neither the post hoc focus groups nor the post hoc survey revealed important barriers or interventions that we had not considered or included in our tailored intervention. CONCLUSIONS: A simple approach to identifying barriers to change appears to have been adequate and efficient. However, we do not know for certain what we would have gained by using more comprehensive methods and we do not know whether the resulting intervention would have been more effective if we had used other methods. The effectiveness of our multifaceted intervention is under evaluation in a randomised controlled trial

Inter -and intraobserver variation of ultrasonographic cartilage thickness assessments in small and large joints in healthy children

<p>Abstract</p> <p>Background</p> <p>There is an increasing interest among pediatric rheumatologist for using ultrasonography (US) in the daily clinical examination of children with juvenile idiopathic arthritis (JIA). Loss of joint cartilage may be an early feature of destructive disease in JIA. However, US still needs validation before it can be used as a diagnostic bedside tool in a pediatric setting. This study aims to assess the inter- and intraobserver reliability of US measurements of cartilage thickness in the joints of healthy children.</p> <p>Methods</p> <p>740 joints of 74 healthy Caucasian children (27 girls/47 boys), aged 11.3 (7.11 – 16) years were examined with bilateral US in 5 preselected joints to assess the interobserver variability. In 17 of these children (6 girls/11 boys), aged 10.1(7.11–11.1) years, 170 joints was examined in an intraobserver sub study, with a 2 week interval between the first and second examination.</p> <p>Results</p> <p>In this study we found a good inter- and intraobserver agreement expressed as a coefficient of variation (CV) less than 10% in the knee (CV = 9.5%_{interobserver}and 5.9%_{intraobservserI}, 9.3%_{intraobserverII}respectively for the two intraobserver measurements) and fairly good for the MCP joints (CV = 11.9%_{interobserver}, 12.9%_{intraobserverI}and 11.9%_{intraobsevrerII}). In the ankle and PIP joints the inter- and intraobserver agreement was within an acceptable limit (CV<20%) but not for the wrist joint (CV>26%). We found no difference in cartilage thickness between the left and right extremity in the investigated joints.</p> <p>Conclusion</p> <p>We found a good inter -and intraobserver agreement when measuring cartilage thickness with US. The inter- and intraobserver variation seemed not to be related to joint size. These findings suggest that positioning of the joint and the transducer is of major importance for reproducible US measurements. We found no difference in joint cartilage thickness between the left and right extremity in any of the examined joint of the healthy children. This is an important finding giving the opportunity of using the non-affected extremity as a reference when assessing articular joint cartilage damage in JIA.</p

Role of mitochondrial raft-like microdomains in the regulation of cell apoptosis

Lipid rafts are envisaged as lateral assemblies of specific lipids and proteins that dissociate and associate rapidly and form functional clusters in cell membranes. These structural platforms are not confined to the plasma membrane; indeed lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named raft-like microdomains. In addition, some components of raft-like microdomains are present within ER-mitochondria associated membranes. This review is focused on the role of mitochondrial raft-like microdomains in the regulation of cell apoptosis, since these microdomains may represent preferential sites where key reactions take place, regulating mitochondria hyperpolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These structural platforms appear to modulate cytoplasmic pathways switching cell fate towards cell survival or death. Main insights on this issue derive from some pathological conditions in which alterations of microdomains structure or function can lead to severe alterations of cell activity and life span. In the light of the role played by raft-like microdomains to integrate apoptotic signals and in regulating mitochondrial dynamics, it is conceivable that these membrane structures may play a role in the mitochondrial alterations observed in some of the most common human neurodegenerative diseases, such as Amyotrophic lateral sclerosis, Huntington's chorea and prion-related diseases. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents in these pathologies

Inhibition of Iron Uptake Is Responsible for Differential Sensitivity to V-ATPase Inhibitors in Several Cancer Cell Lines

Many cell lines derived from tumors as well as transformed cell lines are far more sensitive to V-ATPase inhibitors than normal counterparts. The molecular mechanisms underlying these differences in sensitivity are not known. Using global gene expression data, we show that the most sensitive responses to HeLa cells to low doses of V-ATPase inhibitors involve genes responsive to decreasing intracellular iron or decreasing cholesterol and that sensitivity to iron uptake is an important determinant of V-ATPase sensitivity in several cancer cell lines. One of the most sensitive cell lines, melanoma derived SK-Mel-5, over-expresses the iron efflux transporter ferroportin and has decreased expression of proteins involved in iron uptake, suggesting that it actively suppresses cytoplasmic iron. SK-Mel-5 cells have increased production of reactive oxygen species and may be seeking to limit additional production of ROS by iron

Role of lipid apheresis in changing times

During the last decades, LDL-apheresis was established as an extracorporeal treatment option for patients with severe heterozygous or homozygous familial hypercholesterolemia (FH) that is resistant to conventional treatment strategies such as diet, drugs, and changes in lifestyle. Nearly half a century ago, the first LDL-apheresis treatment was performed by plasma exchange in a child with homozygous FH

Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue

Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity