Proliferative kidney disease in rainbow trout: time- and temperature-related renal pathology and parasite distribution

Proliferative kidney disease is a parasitic infection of salmonid fishes caused by Tetracapsuloides bryosalmonae. The main target organ of the parasite in the fish is the kidney. To investigate the influence of water temperature on the disease in fish, rainbow trout Oncorhynchus mykiss infected with T bryosalmonae were kept at 12 degrees C and 18 degrees C. The number of parasites, the type and degree of lesions in the kidney and the mortality rate was evaluated from infection until full development of disease. While mortality stayed low at 12 degrees C, it reached 77% at 18 degrees C. At 12 degrees C, pathological lesions were dominated by a multifocal proliferative and granulomatous interstitial nephritis. This was accompanied by low numbers of T. bryosalmonae, mainly located in the interstitial lesions. With progression of the disease, small numbers of parasites appeared in the excretory tubuli, and parasite DNA was detected in the urine. Parasite degeneration in the interstitium was observed at late stages of the disease. At 18 degrees C, pathological lesions in kidneys were more severe and more widely distributed, and accompanied by significantly higher parasite numbers. Distribution of parasites in the renal compartments, onset of parasite degeneration and time course of appearance of parasite DNA in urine were not clearly different from the 12 degrees C group. These findings indicate that higher mortality at 18 degrees C compared to 12 degrees C is associated with an enhanced severity of renal pathology and increased parasite numbers.

Time courses and quantitative analysis of atrial fibrillation episode number and duration after circular plus linear left atrial lesions: trigger elimination or substrate modification: early or delayed cure?

OBJECTIVES We sought to analyze the time course of atrial fibrillation (AF) episodes before and after circular plus linear left atrial ablation and the percentage of patients with complete freedom from AF after ablation by using serial seven-day electrocardiograms (ECGs). BACKGROUND The curative treatment of AF targets the pathophysiological corner stones of AF (i.e., the initiating triggers and/or the perpetuation of AF). The pathophysiological complexity of both may not result in an "all-or-nothing" response but may modify number and duration of AF episodes. METHODS In patients with highly symptomatic AF, circular plus linear ablation lesions were placed around the left and right pulmonary veins, between the two circles, and from the left circle to the mitral annulus using the electroanatomic mapping system. Repetitive continuous 7-day ECGs administered before and after catheter ablation were used for rhythm follow-up. RESULTS In 100 patients with paroxysmal (n = 80) and persistent (n = 20) AF, relative duration of time spent in AF significantly decreased over time (35 +/- 37% before ablation, 26 +/- 41% directly after ablation, and 10 +/- 22% after 12 months). Freedom from AF stepwise increased in patients with paroxysmal AF and after 12 months measured at 88% or 74% depending on whether 24-h ECG or 7-day ECG was used. Complete pulmonary vein isolation was demonstrated in <20% of the circular lesions. CONCLUSIONS The results obtained in patients with AF treated with circular plus linear left atrial lesions strongly indicate that substrate modification is the main underlying pathophysiologic mechanism and that it results in a delayed cure instead of an immediate cure.

Studying changes in life circumstances and personality: It's about time

Most theories of personality development posit that changes in life circumstances (e.g. due to major life events) can lead to changes in personality, but few studies have examined the exact time course of these changes. In this article, we argue that time needs to be considered explicitly in theories and empirical studies on personality development. We discuss six notions on the role of time in personality development. First, people can differ before the event. Second, change can be non-linear and discontinuous. Third, change can be reversible. Fourth, change can occur before the event. Fifth, control groups are needed to disentangle age-related and event-related changes. Sixth, we need to move beyond examining single major life events and study the effects of non-normative events, non-events, multiple events, and minor events on personality. We conclude by summarizing the methodological and theoretical implications of these notions.

Transcriptional profiles of cytokine/chemokine factors of immune cell-homing to the parasitic lesions: a comprehensive one-year course study in the liver of E. multilocularis-infected mice.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

Phenotypes of childhood asthma: are they real?

It has been suggested that there are several distinct phenotypes of childhood asthma or childhood wheezing. Here, we review the research relating to these phenotypes, with a focus on the methods used to define and validate them. Childhood wheezing disorders manifest themselves in a range of observable (phenotypic) features such as lung function, bronchial responsiveness, atopy and a highly variable time course (prognosis). The underlying causes are not sufficiently understood to define disease entities based on aetiology. Nevertheless, there is a need for a classification that would (i) facilitate research into aetiology and pathophysiology, (ii) allow targeted treatment and preventive measures and (iii) improve the prediction of long-term outcome. Classical attempts to define phenotypes have been one-dimensional, relying on few or single features such as triggers (exclusive viral wheeze vs. multiple trigger wheeze) or time course (early transient wheeze, persistent and late onset wheeze). These definitions are simple but essentially subjective. Recently, a multi-dimensional approach has been adopted. This approach is based on a wide range of features and relies on multivariate methods such as cluster or latent class analysis. Phenotypes identified in this manner are more complex but arguably more objective. Although phenotypes have an undisputed standing in current research on childhood asthma and wheezing, there is confusion about the meaning of the term 'phenotype' causing much circular debate. If phenotypes are meant to represent 'real' underlying disease entities rather than superficial features, there is a need for validation and harmonization of definitions. The multi-dimensional approach allows validation by replication across different populations and may contribute to a more reliable classification of childhood wheezing disorders and to improved precision of research relying on phenotype recognition, particularly in genetics. Ultimately, the underlying pathophysiology and aetiology will need to be understood to properly characterize the diseases causing recurrent wheeze in children.

Impact of differential P450c17 phosphorylation by cAMP stimulation and by starvation conditions on enzyme activities and androgen production in NCI-H295R cells

CYP17A1 plays a pivotal role in the biosynthesis of androgens in the adrenals and the gonads. Although this enzyme catalyzes two different reactions on one single active site, its specific activities are regulated independently. Although the 17alpha-hydroxylase activity is rather constant and regulated by gene expression, the 17,20-lyase activity varies significantly with the amount of cofactors or by protein phosphorylation. cAMP increases CYP17A1 expression, P450c17 phosphorylation, and androgen production. However, the exact mechanism(s) and the specific regulators of CYP17A1 remain unknown. Therefore, we studied the regulation of adrenal androgen biosynthesis in human adrenal H295R cells focusing on CYP17A1. We analyzed androgen production and P450c17 activities in H295R cells grown under normal and serum-free conditions and/or after stimulation with 8-bromoadenosine-cAMP. H295R cells grown in starvation medium produced more androgens and had decreased HSD3B2 expression and activity but increased P450c17-17,20-lyase activity and serine phosphorylation. Although starvation increased serine phosphorylation of P450c17 specifically, cAMP stimulation enhanced threonine phosphorylation exclusively. Time-course experiments revealed that a short cAMP stimulation augmented threonine phosphorylation of P450c17 but did not increase 17,20-lyase activity. By contrast, long cAMP stimulation increased androgen production through increased P450c17 activities by enhancing CYP17A1 gene expression. We conclude that serum withdrawal shifts steroidogenesis of H295R cells towards androgen production, providing a suitable model for detailed studies of androgen regulation. In addition, our study shows that starvation and cAMP stimulation regulate P450c17 phosphorylation differentially and that an increase in P450c17 phosphorylation does not necessarily lead to enhanced enzyme activity and androgen production.

In vivo assessment of HCN channel current (I(h)) in human motor axons

The "Trond" protocol of nerve excitability tests has been used widely to assess axonal function in peripheral nerve. In this study, the routine Trond protocol was expanded to refine assessment of cAMP-dependent, hyperpolarization-activated current (I(h)) activity. I(h) activity is generated by hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels in response to hyperpolarization. It limits activity-dependent hyperpolarization, contributes to neuronal automaticity, and is implicated in chronic pain states. Published data regarding I(h) activity in motor nerve are scant. We used additional strong, prolonged hyperpolarizing conditioning stimuli in the threshold electrotonus component of the Trond protocol to demonstrate the time-course of activation of I(h) in motor axons. Fifteen healthy volunteers were tested on four occasions during 1 week. I(h) action was revealed in the threshold electrotonus by the limiting and often reversal, after about 100 ms, of the threshold increase caused by strong hyperpolarizing currents. Statistical analysis by repeated-measures analysis of variance enabled confidence limits to be established for variation between subjects and within subjects. The results demonstrate that, of all the excitability parameters, those dependent on I(h) were the most characteristic of an individual, because variance between subjects was more than four times the variance within subjects. This study demonstrates a reliable method for in vivo assessment of I(h,) and also serves to document the normal variability in nerve excitability properties within subjects.

Effect of two beta-alanine dosing protocols on muscle carnosine synthesis and washout

Carnosine (β-alanyl-L-histidine) is found in high concentrations in skeletal muscle and chronic β-alanine (BA) supplementation can increase carnosine content. This placebo-controlled, double-blind study compared two different 8-week BA dosing regimens on the time course of muscle carnosine loading and 8-week washout, leading to a BA dose-response study with serial muscle carnosine assessments throughout. Thirty-one young males were randomized into three BA dosing groups: (1) high-low: 3.2 g BA/day for 4 weeks, followed by 1.6 g BA/day for 4 weeks; (2) low-low: 1.6 g BA/day for 8 weeks; and (3) placebo. Muscle carnosine in tibialis-anterior (TA) and gastrocnemius (GA) muscles was measured by 1H-MRS at weeks 0, 2, 4, 8, 12 and 16. Flushing symptoms and blood clinical chemistry were trivial in all three groups and there were no muscle carnosine changes in the placebo group. During the first 4 weeks, the increase for high-low (TA 2.04 mmol/kgww, GA 1.75 mmol/kgww) was ~twofold greater than low-low (TA 1.12 mmol/kgww, GA 0.80 mmol/kgww). 1.6 g BA/day significantly increased muscle carnosine within 2 weeks and induced continual rises in already augmented muscle carnosine stores (week 4-8, high-low regime). The dose-response showed a carnosine increase of 2.01 mmol/kgww per 100 g of consumed BA, which was only dependent upon the total accumulated BA consumed (within a daily intake range of 1.6-3.2 g BA/day). Washout rates were gradual (0.18 mmol/kgww and 0.43 mmol/kgww/week; ~2%/week). In summary, the absolute increase in muscle carnosine is only dependent upon the total BA consumed and is not dependent upon baseline muscle carnosine, the muscle type, or the daily amount of supplemented BA.

Phosphorylation of sst2 receptors in neuroendocrine tumors after octreotide treatment of patients

Somatostatin analogues, which are used to treat neuroendocrine tumors, target the high levels of somatostatin receptor subtype 2 (SSTR1; alias sst2) expressed in these cancers. However, some tumors are resistant to somatostatin analogues, and it is unknown whether the defect lies in sst2 activation or downstream signaling events. Because sst2 phosphorylation occurs rapidly after receptor activation, we examined whether sst2 is phosphorylated in neuroendocrine tumors. The sst2 receptor phosphorylation was evaluated by IHC and Western blot analysis with the new Ra-1124 antibody specific for the sst2 receptor phosphorylated at Ser341/343 in receptor-positive neuroendocrine tumors obtained from 10 octreotide-treated and 7 octreotide-naïve patients. The specificity, time course, and subcellular localization of sst2 receptor phosphorylation were examined in human embryo kinase-sst2 cell cultures by immunofluorescence and confocal microscopy. All seven octreotide-naïve tumors displayed exclusively nonphosphorylated cell surface sst2 expression. In contrast, 9 of the 10 octreotide-treated tumors contained phosphorylated sst2 that was predominantly internalized. Western blot analysis confirmed the IHC data. Octreotide treatment of human embryo kinase-sst2 cells in culture demonstrated that phosphorylated sst2 was localized at the plasma membrane after 10 seconds of stimulation and was subsequently internalized into endocytic vesicles. These data show, for the first time to our knowledge, that phosphorylated sst2 is present in most gastrointestinal neuroendocrine tumors from patients treated with octreotide but that a striking variability exists in the subcellular distribution of phosphorylated receptors among such tumors.

Radial extracorporeal shock wave therapy (rESWT) induces new bone formation in vivo: results of an animal study in rabbits

The aim of this study was to investigate if radial extracorporeal shock wave therapy (rESWT) induces new bone formation and to study the time course of ESWT-induced osteogenesis. A total of 4000 impulses of radial shock waves (0.16 mJ/mm²) were applied to one hind leg of 13 New Zealand white rabbits with the contralateral side used for control. Treatment was repeated after 7 days. Fluorochrome sequence labeling of new bone formation was performed by subcutaneous injection of tetracycline, calcein green, alizarin red and calcein blue. Animals were sacrificed 2 weeks (n = 4), 4 weeks (n = 4) and 6 weeks (n = 5) after the first rESWT and bone sections were analyzed by fluorescence microscopy. Deposits of fluorochromes were classified and analyzed for significance with the Fisher exact test. rESWT significantly increased new bone formation at all time points over the 6-week study period. Intensity of ossification reached a peak after 4 weeks and declined at the end of the study. New bone formation was significantly higher and persisted longer at the ventral cortex, which was located in the direction to the shock wave device, compared with the dorsal cortex, emphasizing the dose-dependent process of ESWT-induced osteogenesis. No traumata, such as hemorrhage, periosteal detachment or microfractures, were observed by histologic and radiologic assessment. This is the first study demonstrating low-energy radial shock waves to induce new bone formation in vivo. Based on our results, repetition of ESWT in 6-week intervals can be recommended. Application to bone regions at increased fracture risk (e.g., in osteoporosis) are possible clinical indications.

Analgesic and anti-inflammatory actions of robenacoxib in acute joint inflammation in dog

The objectives of this study were to establish dose-response and blood concentration-response relationships for robenacoxib, a novel nonsteroidal anti-inflammatory drug with selectivity for inhibition of the cyclooxygenase (COX)-2 isoenzyme, in a canine model of synovitis. Acute synovitis of the stifle joint was induced by intra-articular injection of sodium urate crystals. Robenacoxib (0.25, 0.5, 1.0, 2.0 and 4.0 mg/kg), placebo and meloxicam (0.2 mg/kg) were administered subcutaneously (s.c.) 3 h after the urate crystals. Pharmacodynamic endpoints included data from forceplate analyses, clinical orthopaedic examinations and time course of inhibition of COX-1 and COX-2 in ex vivo whole blood assays. Blood was collected for pharmacokinetics. Robenacoxib produced dose-related improvement in weight-bearing, pain and swelling as assessed objectively by forceplate analysis (estimated ED(50) was 1.23 mg/kg for z peak force) and subjectively by clinical orthopaedic assessments. The analgesic and anti-inflammatory effects of robenacoxib were significantly superior to placebo (0.25-4 mg/kg robenacoxib) and were non-inferior to meloxicam (0.5-4 mg/kg robenacoxib). All dosages of robenacoxib produced significant dose-related inhibition of COX-2 (estimated ED(50) was 0.52 mg/kg) but no inhibition of COX-1. At a dosage of 1-2 mg/kg administered s.c., robenacoxib should be at least as effective as 0.2 mg/kg of meloxicam in suppressing acute joint pain and inflammation in dogs.

Evolution of gene expression changes in newborn rats after mechanical ventilation with reversible intubation

Mechanical ventilation (MV) is life-saving but potentially harmful for lungs of premature infants. So far, animal models dealt with the acute impact of MV on immature lungs, but less with its delayed effects. We used a newborn rodent model including non-surgical and therefore reversible intubation with moderate ventilation and hypothesized that there might be distinct gene expression patterns after a ventilation-free recovery period compared to acute effects directly after MV. Newborn rat pups were subjected to 8 hr of MV with 60% oxygen (O(2)), 24 hr after injection of lipopolysaccharide (LPS), intended to create a low inflammatory background as often recognized in preterm infants. Animals were separated in controls (CTRL), LPS injection (LPS), or full intervention with LPS and MV with 60% O(2) (LPS + MV + O(2)). Lungs were recovered either directly following (T:0 hr) or 48 hr after MV (T:48 hr). Histologically, signs of ventilator-induced lung injury (VILI) were observed in LPS + MV + O(2) lungs at T:0 hr, while changes appeared similar to those known from patients with chronic lung disease (CLD) with fewer albeit larger gas exchange units, at T:48 hr. At T:0 hr, LPS + MV + O(2) increased gene expression of pro-inflammatory MIP-2. In parallel anti-inflammatory IL-1Ra gene expression was increased in LPS and LPS + MV + O(2) groups. At T:48 hr, pro- and anti-inflammatory genes had returned to their basal expression. MMP-2 gene expression was decreased in LPS and LPS + MV + O(2) groups at T:0 hr, but no longer at T:48 hr. MMP-9 gene expression levels were unchanged directly after MV. However, at T:48 hr, gene and protein expression increased in LPS + MV + O(2) group. In conclusion, this study demonstrates the feasibility of delayed outcome measurements after a ventilation-free period in newborn rats and may help to further understand the time-course of molecular changes following MV. The differences obtained from the two time points could be interpreted as an initial transitory increase of inflammation and a delayed impact of the intervention on structure-related genes.

Expression of antagonists of WNT and BMP signaling after non-rigid fixation of osteotomies

Delayed fracture healing and non-unions represent rare but severe complications in orthopedic surgery. Further knowledge on the mechanisms of the bone repair process and of the development of a pseudoarthrosis is essential to predict and prevent impaired healing of fractures. The present study aimed at elucidating differences in gene expression during the repair of rigidly and non-rigidly fixed osteotomies. For this purpose, the MouseFix™ and the FlexiPlate™ systems (AO Development Institute, Davos, CH), allowing the creation of well defined osteotomies in mouse femora, were employed. A time course following the healing process of the osteotomy was performed and bones and periimplant tissues were analyzed by high-resolution X-ray, MicroCT and by histology. For the assessment of gene expression, Low Density Arrays (LDA) were done. In animals with rigid fixation, X-ray and MicroCT revealed healing of the osteotomy within 3 weeks. Using the FlexiPlate™ system, the osteotomy was still visible by X-ray after 3 weeks and a stabilizing cartilaginous callus was formed. After 4.5 weeks, the callus was remodeled and the osteotomy was, on a histological level, healed. Gene expression studies revealed levels of transcripts encoding proteins associated with inflammatory processes not to be altered in tissues from bones with rigid and non-rigid fixation, respectively. Levels of transcripts encoding proteins of the extracellular matrix and essential for bone cell functions were not increased in the rigidly fixed group when compared to controls without osteotomy. In the FlexiPlate™ group, levels of transcripts encoding the same set of genes were significantly increased 3 weeks after surgery. Expression of transcripts encoding BMPs and BMP antagonists was increased after 3 weeks in repair tissues from bones fixed with FlexiPlate™, as were inhibitors of the WNT signaling pathways. Little changes only were detected in transcript levels of tissues from rigidly fixed bones. The data of the present study suggest that rigid fixation enables accelerated healing of an experimental osteotomy as compared to non-rigid fixation. The changes in the healing process after non-rigid fixation are accompanied by an increase in the levels of transcripts encoding inhibitors of osteogenic pathways and, probably as a consequence, by temporal changes in bone matrix synthesis.

Uncovering the dynamics of cardiac systems using stochastic pacing and frequency domain analyses

Alternans of cardiac action potential duration (APD) is a well-known arrhythmogenic mechanism which results from dynamical instabilities. The propensity to alternans is classically investigated by examining APD restitution and by deriving APD restitution slopes as predictive markers. However, experiments have shown that such markers are not always accurate for the prediction of alternans. Using a mathematical ventricular cell model known to exhibit unstable dynamics of both membrane potential and Ca2+ cycling, we demonstrate that an accurate marker can be obtained by pacing at cycle lengths (CLs) varying randomly around a basic CL (BCL) and by evaluating the transfer function between the time series of CLs and APDs using an autoregressive-moving-average (ARMA) model. The first pole of this transfer function corresponds to the eigenvalue (λalt) of the dominant eigenmode of the cardiac system, which predicts that alternans occurs when λalt≤−1. For different BCLs, control values of λalt were obtained using eigenmode analysis and compared to the first pole of the transfer function estimated using ARMA model fitting in simulations of random pacing protocols. In all versions of the cell model, this pole provided an accurate estimation of λalt. Furthermore, during slow ramp decreases of BCL or simulated drug application, this approach predicted the onset of alternans by extrapolating the time course of the estimated λalt. In conclusion, stochastic pacing and ARMA model identification represents a novel approach to predict alternans without making any assumptions about its ionic mechanisms. It should therefore be applicable experimentally for any type of myocardial cell.

Genome-wide prediction of childhood asthma and related phenotypes in a longitudinal birth cohort

Childhood wheezing and asthma vary greatly in clinical presentation and time course. The extent to which phenotypic variation reflects heterogeneity in disease pathways is unclear.