Impact of corset bracing on 3D spine kinematics during ADL in children with Spondylolysis.

Spondylolysis is a stress fracture of the vertebral pars interarticularis that frequently affects adolescents involved in sports. Conservative bracing methods may assist the clinician in treating spondylolysis, though there is a need to further validate these techniques. The goal of this study was to evaluate differences in the 3D movements of the thoracic and lumbar spine before and after bracing. Five patients (mean age 14.4 ± 1.3 years) with spondylogenic back pain were evaluated for kinematic measurements using a Vicon motion capture system. Patients performed activities both with and without a lumbar corset brace including walking, kneeling, standing from a chair, standing from the floor, ascending and descending stairs, and lifting. Patients were evaluated for differences in thoracic and lumbar range of motion (ROM) in the braced and unbraced condition. While wearing the brace, patients demonstrated reduced extension ROM of the thoracic spine while walking (mean reduction = 0.4°), ascending stairs (3.0°), descending stairs (2.1°), lifting (14.8°), standing from a chair (4.1°), standing from the floor (16.7°), and kneeling (8.4°). Patients also exhibited reduced extension ROM of the total lumbar spine while ascending stairs (mean reduction = 1.8°), lifting (12.7°), standing from a chair (9.5°), standing from the floor (11.8°), and kneeling (4.7°). These results provide evidence that bracing reduces stress on the pars interarticularis and relieves symptoms in the athlete with spondylogenic back pain, thereby facilitating a return to sports.

Variability in hepatic expression of organic anion transporter 7/SLC22A9, a novel pravastatin uptake transporter: impact of genetic and regulatory factors.

Human organic anion transporter 7 (OAT7, SLC22A9) is a hepatic transport protein poorly characterized so far. We therefore sought to identify novel OAT7 substrates and factors contributing to variable hepatic OAT7 expression. Using OAT7-expressing cells, pravastatin was identified as a substrate. Hepatic SLC22A9/OAT7 mRNA and protein expression varied 28-fold and 15-fold, respectively, in 126 Caucasian liver samples. Twenty-four variants in SLC22A9 were genotyped, including three rare missense variants (rs377211288, rs61742518, rs146027075), which occurred only heterozygously. No variant significantly affected hepatic SLC22A9/OAT7 expression. The three missense variants, however, showed functional consequences when expressed in vitro. Hepatic nuclear factor 4-alpha (HNF4α) emerged as a major transcriptional regulator of SLC22A9 by a series of in silico and in vitro analyses. In conclusion, pravastatin is the first identified OAT7 drug substrate. Substantial inter-individual variability in hepatic OAT7 expression, majorly driven by HNF4α, may contribute to pravastatin drug disposition and might affect response.The Pharmacogenomics Journal advance online publication, 4 August 2015; doi:10.1038/tpj.2015.55.

Cell-mediated reduction of human ß-defensin 1: a major role for mucosal thioredoxin.

Human ß-defensin 1 (hBD-1) is an antimicrobial peptide expressed by epithelia and hematopoietic cells. We demonstrated recently that hBD-1 shows activity against enteric commensals and Candida species only after its disulfide bonds have been reduced by thioredoxin (TRX) or a reducing environment. Here we show that besides TRX, glutaredoxin (GRX) is also able to reduce hBD-1, although with far less efficacy. Moreover, living intestinal and lymphoid cells can effectively catalyze reduction of extracellular hBD-1. By chemical inhibition of the TRX system or specific knockdown of TRX, we demonstrate that cell-mediated reduction is largely dependent on TRX. Quantitative PCR in intestinal tissues of healthy controls and inflammatory bowel disease patients revealed altered expression of some, although not all, redox enzymes, especially in ulcerative colitis. Reduced hBD-1 and TRX localize to extracellular colonic mucus, suggesting that secreted or membrane-bound TRX converts hBD-1 to a potent antimicrobial peptide in vivo.

What does the nature of the MECP2 mutation tell us about parental origin and recurrence risk in Rett syndrome?

The MECP2 mutations occurring in the severe neurological disorder Rett syndrome are predominantly de novo, with rare familial cases. The aims of this study were to provide a precise estimate of the parental origin of MECP2 mutations using a large Chinese sample and to assess whether parental origin varied by mutation type. The parental origin was paternal in 84/88 [95.5%, (95% confidence interval 88.77-98.75)] of sporadic Chinese cases. However, in a pooled sample including data from the literature the spectrum of mutations occurring on maternally and paternally derived chromosomes differed significantly. The excess we found of 'single base pair gains or losses' on maternally derived MECP2 gene alleles suggests that this mutational category is associated with an elevated risk of gonadal mosaicism, which has implications for genetic counseling.

Spatio-temporal expression analysis of the calcium-binding protein calumenin in the rodent brain.

Calumenin is a Ca(2+)-binding protein that belongs to the CREC superfamily. It contains six EF-hand domains that exhibit a low affinity for Ca(2+) as well as an endoplasmic reticulum retention signal. Calumenin exhibits a broad and relatively high expression in various brain regions during development as demonstrated by in situ hybridization. Signal intensity of calumenin is highest during the early development and then declines over time to reach a relatively low expression in adult animals. Immunohistochemistry indicates that at the P0 stage, calumenin expression is most abundant in migrating neurons in the zones around the lateral ventricle. In the brain of adult animals, it is expressed in various glial and neuronal cell types, including immature neurons in subgranular zone of hippocampal dentate gyrus. At the subcellular level, calumenin is identified in punctuate and diffuse distribution mostly in somatic regions where it co-localizes with endoplasmic reticulum (ER) and partially Golgi apparatus. Upon subcellular fractionation, calumenin is enriched in fractions containing membranes and is only weakly present in soluble fractions. This study points to a possible important role of calumenin in migration and differentiation of neurons, and/or in Ca(2+) signaling between glial cells and neurons.

A pitfall of glomerular sieving: profibrotic and matrix proteins derive from the Bowman's capsule and not the glomerular tuft in rats with renovascular hypertension.

BACKGROUND: The glomeruli in the non-clipped kidney of rats with 2-kidney, 1-clip hypertension are a classical model for studying the mechanisms of glomerular injury. METHODS: In the present study, we compared the glomerular expression of PAI-1 and collagen I alpha1 mRNA from glomeruli isolated by the classic technique of sieving with the recently developed technique of tissue laser microdissection. For quantification of mRNA from both methods, real-time PCR was used. RESULTS: Real-time PCR revealed a 9.0 +/- 1.3- and a 7.1 +/- 0.2-fold induction of PAI-1 and collagen I alpha 1, respectively, in the glomeruli from hypertensive rats isolated by sieving. However, in situ hybridization and microdissection revealed that expression of both mRNAs was mainly from the Bowman's capsule and not from the glomerular tuft (10.7 +/- 1.3- and 7.2 +/- 0.6-fold higher induction in whole glomeruli compared with tuft alone). CONCLUSION: This emphasizes that studies focusing on processes in the mesangium, endothelial cells or podocytes should not rely on glomeruli obtained by sieving. Rather, a technique like the laser microdissection or in situ hybridization should be applied which allows the clear separation of different glomerular and periglomerular compartments.

Antihypertensive therapy upregulates renin and (pro)renin receptor in the clipped kidney of Goldblatt hypertensive rats.

Recently, a (pro)renin receptor has been identified which mediates profibrotic effects independent of angiotensin II. Because antihypertensive therapy induces renal injury in the clipped kidney of two kidney-1-clip hypertensive rats, we examined the regulation of renin and the (pro)renin receptor in this model. Hypertensive Goldblatt rats were treated with increasing doses of the vasopeptidase inhibitor AVE 7688 after which the plasma renin and prorenin as well as the renal renin and (pro)renin receptor expression were measured. The vasopeptidase inhibitor dose-dependently lowered blood pressure, which was associated with a massive increase in plasma prorenin and renin as well as increased renal renin expression. The (pro)renin receptor was upregulated in the clipped kidney of the Goldblatt rat indicating a parallel upregulation of renin and its receptor in vivo. Immunohistochemistry showed a redistribution of renin upstream from the glomerulus in preglomerular vessels and renin staining in tubular cells. Expression of the (pro)renin receptor was increased in the vessels and tubules. This upregulation was associated with thickening of renin-positive vessels and tubulointerstitial damage. We propose that renin and the (pro)renin receptor may play a profibrotic role in the clipped kidney of Goldblatt rats treated for hypertension.

[Back pain and acne conglobata: SAPHO syndrome]. / Junge Frau mit Rückenschmerzen und Akne: SAPHO-Syndrom (Synovitis, Akne, Pustulose, Hyperostose und Osteitis).

We report on a young woman suffering from SAPHO syndrome with back pain and arthritis of the sternoclavicular joints. This inflammatory disorder of the osteoarticular system (synovitis, osteitis, and hyperostosis) is associated with severe acne or palmoplantar pustulosis. The patient was treated with pamidronate, NSAID and physiotherapy which improved the musculoskeletal symptoms completely. The acne was treated with isotretinoin.

Antihypertensive therapy induces compartment-specific chemokine expression and a Th1 immune response in the clipped kidney of Goldblatt hypertensive rats.

The present study examined the pathogenesis of interstitial inflammation and fibrosis in antihypertensively treated rats with two-kidney, one-clip hypertension. Hypertensive rats were randomized into four groups: no treatment and moderate, intermediate, and intensified lowering of blood pressure with increasing doses of a vasopeptidase inhibitor for 6 wk. The vasopeptidase inhibitor dose dependently lowered blood pressure. The tubulointerstitial damage was accompanied by a diffuse infiltration of mononuclear cells and circumscript mononuclear inflammatory cell cluster formation consisting mainly of T cells and to a lesser degree of macrophages and B cells. Real-time PCR analyses showed a dose-dependent induction of MCP-1 and the Th1-type chemokines IP10 and Mig as well as their receptor CXCR3 and the Th1 cytokine IFN-gamma. In situ hybridization and laser microdissection revealed a strong expression of these Th1-associated transcripts in the clusters and, in the case of MCP-1, also diffusely in the interstitium. The inflammation was accompanied by the appearance of myofibroblasts and synthesis of the fibrogenic factor plasminogen activator inhibitor-1 as well as the collagenase matrix metalloproteinase-2, leading to collagen I upregulation and interstitial scarring. No inflammation or fibrosis was found in normotensive rats treated with the vasopeptidase inhibitor. The renal injury in the clipped kidney is accompanied by compartment-specific chemokine expression and cell cluster formation of Th1 specificity associated with upregulation of fibrogenic proteins and matrix metalloproteinases. These findings suggest that the Th1 chemokines IP10 and Mig as well as their receptor CXCR3 are potential targets for therapeutic interventions in ischemic nephropathy.

Start-up delays of infusion syringe pumps.

BACKGROUND: We performed a bench experiment to investigate the extent of start-up delays in fluid delivery for four different syringe pumps after initially placing the infusion syringe in the syringe pump. METHODS: Pump performance was determined at an infusion rate of 1 ml.h-1 with and without a fluid bolus delivered by the infusion pump prior to connecting the infusion line to the simulated patient. RESULTS: The time (mean +/- SD) from starting the pump up to first fluid delivery (t1) differed considerably between pumps (from 6.75 +/- 4.4 to 57.2 +/- 28.6 min) as did the time to steady state fluid delivery (t2) (from 19.6 +/- 9.3 to 76.3 +/- 29.0 min). Applying an initial bolus of 2 ml before connecting the line to the simulated patient practically eliminated the delay in fluid delivery (t1 ranging from 0.3 +/- 0.1 to 1.1 +/- 0.8 min). This manoeuvre also reduced the time to steady flow delivery (t2 from 6.0 +/- 3.1 to 11.1 +/- 4.3 min, P<0.001) and minimized the differences between syringe pumps. CONCLUSIONS: Syringe pump design affects start-up delay times because of free play of the syringe. These delays can be eliminated by a start-up bolus of 2 ml prior to connecting the infusion line to the patient.

Clonidine decreases propofol requirements during anaesthesia: effect on bispectral index.

Assessment of the effect of clonidine on depth of anaesthesia is difficult because clonidine combines analgesic, sedative and direct haemodynamic effects. We thus evaluated the influence of clonidine on the bispectral index (BIS) and its potential dose-sparing effect on propofol. After induction of anaesthesia with target-controlled infusion of propofol and obtaining an unchanged bispectral index (pre-BIS), clonidine 4 microg kg(-1) or placebo was administered randomly to 50 patients in a double-blind manner. Subsequently, if there was a decrease in BIS we reduced the target concentration of propofol until pre-BIS was reached. The pre-BIS was maintained and a remifentanil infusion was added during surgery. The courses of the BIS, heart rate and blood pressure were recorded and the total amounts of intra-operative propofol and remifentanil were determined. Assessment of implicit memory during anaesthesia was performed with an auditory implicit memory test consisting of item sequences. Administration of clonidine resulted in a decrease in the BIS from 45 (SD 4) to 40 (6) (P<0.001), which allowed a reduction of propofol target concentration from 3.3 (0.6) to 2.7 (0.7) microg ml(-1) (P<0.001) and measured propofol concentration from 2.9 (0.6) to 2.5 (0.7) kg ml(-1) (P=0.009) in order to maintain the pre-BIS value. During subsequent surgery, propofol requirements were reduced by 20% (P=0.002) in the clonidine group and a similar amount of remifentanil was used in each group. The increase in anaesthetic depth given by clonidine can therefore be measured with bispectral EEG analysis and allows reduction of the propofol dose to achieve a specific depth of anaesthesia.

Corticosteroids impair intestinal epithelial wound repair mechanisms in vitro.

BACKGROUND: Despite enormous progress in the medical treatment of inflammatory bowel diseases (IBD), corticosteroids still represent the most effective drugs in the management of acute IBD. Unfortunately, surgical intervention under concomitant therapy with corticosteroids is often complicated by impaired intestinal wound healing. Our aim was to assess the effects of the corticosteroids prednisolone and budesonide on different aspects of intestinal epithelial wound healing in vitro to identify potential causes for impaired intestinal wound healing under corticosteroid therapy. METHODS: The effects of both corticosteroids on intestinal epithelial cell function were studied in non-transformed small intestinal epithelial intestinal epithelial cell line IEC-6 cells and human colon cancer-derived HT-29 cells. Effects on epithelial migration were assessed using an in vitro wounding model. Effects on epithelial cell proliferation were assessed using colorimetric 3(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assays. Transforming Growth Factor beta (TGFbeta) mRNA and protein expression were determined by semi-quantitative RT-PCR and ELISA. RESULTS: Prednisolone and budesonide caused a significant dose-dependent inhibition of intestinal epithelial cell migration and proliferation in IEC-6 and HT-29 cells. Both corticosteroids induced apoptosis of intestinal epithelial cells in a dose-dependent fashion. Neither corticosteroid modulated the expression of TGFbeta mRNA and the synthesis of TGFbeta peptide. However, both corticosteroids stimulated the bioactivation of latent TGFbeta peptide. CONCLUSIONS: Prednisolone and budesonide inhibit intestinal epithelial cell restitution and proliferation in vitro. Both processes play a key role in the rapid resealing of the mucosal barrier following intestinal injury. Thus, impaired intestinal epithelial wound healing under corticosteroid therapy in vivo may be caused by inhibition of intestinal epithelial cell restitution and proliferation.

Evaluation of the FASTSTART mode for reducing start-up delay in syringe pump infusion systems.

OBJECTIVE: The aim of the study was to evaluate the IVAC P7000 FASTSTART mode with regard to start-up performance in a 50-ml infusion syringe at a flow rate of 1 ml.h-1. METHODS: The time from depression of the start button to first fluid flow (T1) and to establishment of a pre-set flow rate (T2) were gravimetrically recorded with and without FASTSTART and with and without priming of the infusion system with a 1-ml fluid bolus prior to connection of the infusion line to the patient. RESULTS: FASTSTART significantly reduced start-up times in the unprimed syringe pump infusion system from (mean [SD]) 9.4 (6.0) to 2.5 (3.5) min for T1 and from 21.8 (9.8) to 9.4 (6.2) min for T2 (all p < 0.001). The greatest improvement in shortening of T1 and T2 was obtained when the system was primed prior to starting (p < 0.0001). After priming the infusion system, FASTSTART shortened T2 by some 50% from 1.4 (1.4) to 0.7 (0.6) min. CONCLUSION: Our data indicate that the FASTSTART procedure is effective and that substantial improvements can be obtained by priming the system prior to starting.

A constitutively open potassium channel formed by KCNQ1 and KCNE3.

Mutations in all four known KCNQ potassium channel alpha-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the beta-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current I(Ks) that is affected in some forms of cardiac arrhythmia. Here we show that the novel beta-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.

Organization and expression of bovine TSPY.

We have isolated genomic sequences as well as transcripts from the bovine homolog of the human testis-specific protein, Y-encoded, TSPY which-in both species-is located on the Y Chromosome (Chr), organized as a gene family with a variable number of members, and expressed exclusively in the testis. 1266 bp of bovine TSPY specific sequence have been isolated from a testis cDNA library, by RT-PCR analyses and by Rapid Amplification of cDNA Ends (RACE). A bovine TSPY gene 4 is organized in seven exons, and transcripts are polyadenylated at various 3' ends. Consensus polyadenylation signals AAUUAAA are missing. Microheterogeneous sequence variation is found between TSPY family members. In addition, homologies to other Y-located repeated sequence families, BRY, have been discovered; these sequences are presumably derived from ancient members of the TSPY cluster, now forming a separate, probably nonfunctional subfamily. Bovine TSPY is subject to differential splicing. In the adult, it is expressed in early germ-cell stages, and expression could also be detected in fetal testis. Comparison with the human homolog shows the highest degree of similarity in the coding regions of exons 2, 3, and 4, which are also precisely conserved regarding their length.

Dendritic localization of rat vasopressin mRNA: ultrastructural analysis and mapping of targeting elements.

Transcripts encoding the vasopressin precursor are located in axons and dendrites of rat hypothalamic magnocellular neurons. While the axonal vasopressin mRNA has been extensively characterized both at the biochemical and morphological level, little is known about those transcripts residing in dendrites of magnocellular neurons. As revealed by in situ hybridization at the electron microscopic level, the mRNA is located in proximal and distal dendritic segments and is exclusively confined to regions containing rough endoplasmic reticulum. These results suggest that dendrites of hypothalamic neurons may be capable of local precursor synthesis independent of that occurring in the cell somata. A heterologous system has been employed to define cis-acting elements within the vasopressin mRNA which may be involved in dendritic compartmentalization. Expression vector constructs consisting of the cytomegalovirus promoter coupled to the rat vasopressin cDNA have been injected into the cell nuclei of cultured neurons derived from embryonic rat superior cervical ganglia. Vector-encoded vasopressin transcripts were also sorted to dendrites of these neurons indicating that the molecular determinants of dendritic mRNA transport are not cell specific. Mapping of the targeting elements revealed two segments within the vasopressin mRNA that are able to confer dendritic compartmentalization to alpha-tubulin mRNA which is normally confined to the cell body.

Sequence analysis of a cDNA encoding an isotocin precursor and localization of the corresponding mRNA in the brain of the cartilaginous fish Torpedo marmorata.

The nucleotide sequence of a cDNA encoding an isotocin hormone precursor has been elucidated by analyzing a lambda ZAPII library constructed using poly(A)+ RNA from the brain of the cartilaginous fish Torpedo marmorata. The sequence predicts a precursor of 126 amino acid residues that consists of a signal peptide, the isotocin moiety, and a neurophysin carrier protein. In contrast to other known fish isotocin precursor sequences, the Torpedo neurophysin moiety is not extended at its carboxy-terminus by a copeptin-like sequence. The T. marmorata isotocin precursor exhibits highest amino acid sequence identity (61%) to the toad mesotocin precursor. As demonstrated by in situ hybridization, the isotocin mRNA is present in neurons of the preoptic area of the Torpedo brain.

Distribution of somatostatin receptor subtype 1 mRNA in the developing cerebral hemispheres of the rat.

Somatostatin (SST) is one of the major peptide transmitters in the mammalian central nervous system and also seems to exert specific functions during brain development. In contrast to ligand binding experiments, by which two pharmacologically different binding sites were characterized, molecular cloning techniques have led to the identification of at least five different receptor subtypes (SSTR1-5), which according to RNA blot analyses seem to be differentially distributed and regulated in the developing brain. In order to provide more precise data on the distribution of SSTR1 during ontogenesis, we have performed an in situ hybridization analysis, using a 35S-labelled RNA probe, in the developing rat cortex between embryonic day (E)12 and adulthood. Within the cortical plate, expression of SSTR1 gene was first detected in parallel with the establishment of the deep laminae V/VI at E16, thereby following the characteristic morphogenetic gradients of cortical plate construction. Thus, with the subsequent addition of cells along the radial dimension, e.g. the deposition of the supragranular neurons beyond E18, the hybridization signal spreads as an uniform homogenous band through the entire cortical plate, whereby silver grains reach their peak density around birth. Similar developmental gradients were observed along the lateromedial and frontooccipital dimension, whereby SSTR1 transcripts were detected near the frontal pole and the lateral cortical areas roughly 2 days before they appeared in the occipital and medial cortical anlage, respectively. From the initially homogenous distribution, two distinct SSTR1 mRNA-positive bands coextensive with laminae V/VI and II/III, respectively, and sparing lamina IV evolved during the first postnatal week, the grain density of which decreased during further postnatal development. Within the hippocampal formation, SSTR1 transcripts were initially observed at E18 in the subicular complex, and after birth also extending into the neighboring CA1 region. During the 1st and 2nd postnatal week, silver grains were observed over the pyramidal cell layer of CA2 and CA3 and as a faint supragranular band in the dentate gyrus. Similar to the isocortex, grain density decreased thereafter. Hypothetically, the pronounced temporospatial regulation of SSTR1 gene expression during brain development can be correlated with (1) the establishment and eventual reduction of transient cortical SSTergic neuron populations described for late pregnancy and early postnatal development and (2) a receptor subtype exchange during maturation as evidenced by the late (from postnatal day 7 onward) appearance of e.g. SSTR3.

Expression patterns of rat somatostatin receptor genes in pre- and postnatal brain and pituitary.

The relative abundances of mRNAs encoding four different somatostatin receptors were examined using PCR techniques during postnatal development of the rat brain and hypophysis. In most tissues, somatostatin receptor 1 and 4 mRNAs are more abundant than those encoding somatostatin receptor 2 and 3. Transcript levels of somatostatin receptor subtype 4 are relatively high in the cortex, hippocampus, and striatum, those of subtype 1 in the cortex and brainstem, and those of subtype 3 in the cerebellum. In situ hybridization revealed the presence of significant amounts of somatostatin receptor 1 mRNA, as early as prenatal day 14, in the trigeminal ganglion and in the neuroepithelial layers surrounding the lateral, third, and fourth ventricles. In the developing cortex a morphological change in the sites of somatostatin receptor 1 gene expression occurs; mRNA is present superficially in the cortex at prenatal stages, appears in all layers shortly after birth, and in adult rats is restricted to the deep cortical layers. In the cerebellum, somatostatin receptor 1 mRNA levels are highest around birth, declining thereafter. In contrast, cerebellar somatostatin receptor 3 transcripts are absent at birth, become detectable around postnatal day 7, and reach a maximal level during maturation.