Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling

TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen-associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up-regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1- or TLR6-dependent manner. There are also di- and triacylated LP, which stimulate TLR1-deficient cells and TLR6-deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)(2)C-(VPGVG)(4)VPGKG, fibroblast-stimulating LP-1, and Pam(2)C-SK(4). Dominant-negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram-positive and Gram-negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.     

Genetic correction of PSA values using sequence variants associated with PSA levels

Measuring serum levels of the prostate-specific antigen (PSA) is the most common screening method for prostate cancer. However, PSA levels are affected by a number of factors apart from neoplasia. Notably, around 40% of the variability of PSA levels in the general population is accounted for by inherited factors, suggesting that it may be possible to improve both sensitivity and specificity by adjusting test results for genetic effects. To search for sequence variants that associate with PSA levels, we performed a genome-wide association study and follow-up analysis using PSA information from 15,757 Icelandic and 454 British men not diagnosed with prostate cancer. Overall, we detected a genome-wide significant association between PSA levels and single-nucleotide polymorphisms (SNPs) at six loci: 5p15.33 (rs2736098), 10q11 (rs10993994), 10q26 (rs10788160), 12q24 (rs11067228), 17q12 (rs4430796), and 19q13.33 [rs17632542 (KLK3: I179T)], each with P(combined) <3 × 10(-10). Among 3834 men who underwent a biopsy of the prostate, the 10q26, 12q24, and 19q13.33 alleles that associate with high PSA levels are associated with higher probability of a negative biopsy (odds ratio between 1.15 and 1.27). Assessment of association between the six loci and prostate cancer risk in 5325 cases and 41,417 controls from Iceland, the Netherlands, Spain, Romania, and the United States showed that the SNPs at 10q26 and 12q24 were exclusively associated with PSA levels, whereas the other four loci also were associated with prostate cancer risk. We propose that a personalized PSA cutoff value, based on genotype, should be used when deciding to perform a prostate biopsy.     

HIV-1 drug resistance in HIV-1-infected children in the United Kingdom from 1998 to 2004

We reviewed HIV-1 genotypes from 200 of 979 (20%) HIV-infected children in the U.K. Collaborative HIV in Pediatric Study (CHIPS) cohort (343 resistance tests). Three of 44 samples had major primary resistance mutations before antiretroviral therapy. Three-class resistance was noted in 42 samples (14.1%). Our study also highlighted underutilization of testing and the need for prompt genotyping after drug discontinuation which may have lead to an underestimation of HIV-1 resistance.     

Comparison of Calculated with Measured Oxygen Consumption in Children Undergoing Cardiac Catheterization

Our objective was to compare calculated (LaFarge) with measured oxygen consumption (VO₂) using the AS/3 TM Compact Airway Module M-CAiOVX (Datex-Ohmeda, Helsinki, Finland; AS/3 TM) in children without cardiac shunts in a prospective, observational study. VO₂ was determined at the end of the routine diagnostic and/or interventional catheterization. VO₂ was calculated according to the formula of LaFarge and Miettinen for each child and compared with the measured VO₂. Data were compared using simple regression and Bland Altman analysis. Fifty-two children aged from 0.5 to 16 years (median, 6.9 years) and weighing 3.4 to 59.4 kg (median, 22.9 kg) were investigated. Calculated VO₂ values ranged from 59.0 to 230.8 ml/min, and measured VO₂ values from 62.7 to 282.2 ml/min. Comparison of calculated versus measured VO₂ values revealed a significant correlation (r = 0.90, p < 0.0001). Bias and precision were 8.9 and 48.3 ml/min, respectively (95% limits of agreement: −39.4 to 57.2 ml/min). Comparison of calculated VO₂ in children older than 3 years (n = 41), as restricted to the formula, with measured VO₂, revealed a slightly reduced correlation (r = 0.86, p < 0.0001). Bias and precision were 10.0 and 52.5 ml/min, respectively (95% limits of agreement: –42.4 to 62.5 ml/min). We conclude that calculation of VO₂ by the LaFarge formula does not provide reliable values compared to measured values. In clinical routine, measured rather than calculated VO₂ values should be used for the estimation of cardiac output and related variables.     

Investigating circadian clock gene expression in human tendon biopsies from acute exercise and immobilization studies

Purpose

The discovery of musculoskeletal tissues, including muscle, tendons, and cartilage, as peripheral circadian clocks strongly implicates their role in tissue-specific homeostasis. Age-related dampening and misalignment of the tendon circadian rhythm and its outputs may be responsible for the decline in tendon homeostasis. It is unknown which entrainment signals are responsible for the synchronization of the tendon clock to the light–dark cycle.

Methods

We sought to examine any changes in the expression levels of core clock genes (BMAL1, CLOCK, PER2, CRY1, and NR1D1) in healthy human patellar tendon biopsies obtained from three different intervention studies: increased physical activity (leg kicks for 1 h) in young, reduced activity (2 weeks immobilization of one leg) in young, and in old tendons.

Results

The expression level of clock genes in human tendon in vivo was very low and a high variation between individuals was found. We were thus unable to detect any differences in core clock gene expression neither after acute exercise nor immobilization.

Conclusions

We are unable to find evidence for an effect of exercise or immobilization on circadian clock gene expression in human tendon samples.

     

Sleep on the Night Shift: 24-Hour EEG Monitoring of Spontaneous Sleep/Wake Behavior

The present study sought to objectively describe the spontaneous sleep/wakefulness pattern of shift workers during a 24-hour period. Portable Medilog tape-recorders were used for ambulatory EEG monitoring of 25 male papermill workers (25-55 years) during days with night and afternoon work. The results showed that sleep after night work was two hours shorter than after afternoon work. The sleep reduction affected mainly Stage 2 and REM sleep while slow wave sleep was unchanged. In connection with night work 28% of the workers took a nap in the afternoon. These naps contained a large proportion of slow wave sleep and were, apparently, caused by the sleep deficit after the short main sleep period. The EEG recordings also revealed that 20% of the participants had sleep episodes during night work. These naps were as long as the afternoon naps, were experienced as "dozing offs" rather than naps, occurred at the time of the trough of the circadian wakefulness rhythm, and were concomitant with extreme subjective sleepiness and low rated work load. It was concluded that not only the sleep of shift workers was disturbed, but also the wakefulness--to the extent that sleepiness during night work sometimes reached a level where reasonable wakefulness could not be maintained. The latter observation is probably of special importance in work situations demanding a great responsibility for human lives or for great economic values.     

Synthesis of photoreactive α-4-azidobenzoyl-ω-methoxy-poly(ethylene glycol)s and their end-on photo-grafting onto polysulfone ultrafiltration membranes

Covalent end-on grafting of poly(ethylene glycol) (PEG) onto a polysulfone (PSf) surface using α-4-azidobenzoyl-ω-methoxy-PEG (ABMPEG) is described. Photoreactive ABMPEG was synthesized by reacting monomethoxy-PEG (MPEG) with 4-azidobenzoyl chloride, yielding complete substitution of the hydroxyl groups. After adsorption from aqueous solutions, ABMPEG was photo-grafted under wet conditions onto PSf ultrafiltration (UF) membranes. Contact angle (CA) measurements showed the increased hydrophilicity of modified membranes and the irreversibility of the modification. Bovine serum albumin (BSA) adsorption decreased by 70% and the permeability decay after protein adsorption became less severe for the modified membranes compared to unmodified reference membranes.     

A prospective, multicenter, open-label assessment of atomoxetine in non-North American children and adolescents with ADHD

OBJECTIVE: The aim of this study was to study treatment response to atomoxetine in a large, multicenter study of non-North American patients with ADHD. METHODS: A total of 604 children and adolescents with ADHD were enrolled in a 10-week open-label trial with atomoxetine prior to randomization to a double-blind relapse prevention phase at 33 sites in the United Kingdom, continental Europe, Israel, South Africa, and Australia. All patients had ADHD symptom severity at least 1.5 standard deviations above United States age and gender norms for their diagnostic subtype as measured by the investigator-scored ADHD Rating Scale (ADHD RS). Outcomes were assessed by analysis of change in the ADHD RS; functional and psychosocial outcomes were assessed using the Child Health Questionnaire (CHQ). RESULTS: At endpoint, ADHD RS total scores decreased by an average of 56.7%, and 69% of patients were rated as having no or minimal symptoms. Significant improvement was observed in psychosocial and functional outcomes. Discontinuations attributed to adverse events were < 4%. CONCLUSION: These open-label data, gathered in an international setting, add to our knowledge of the value of atomoxetine in treating ADHD symptoms, as well as its safety and tolerability.     

Pharmacological regulation of the insulin receptor signaling pathway mimics insulin action in cells transduced with viral vectors

Diabetes mellitus derives from either insulin deficiency (type I) or resistance (type II). Homozygous mutations in the insulin receptor (IR) gene cause the rare leprechaunism and Rabson-Mendenhall syndromes, severe forms of hyperinsulinemic insulin resistance for which no therapy is currently available. Systems have been developed that allow protein-protein interactions to be brought under the control of small-molecule dimerizer drugs. As a potential tool to rescue glucose homeostasis at will in both insulin and insulin receptor deficiencies, we developed a recombinant chimeric insulin receptor (LFv2IRE) that can be homodimerized and activated by the small-molecule dimerizer AP20187. In HepG2 cells transduced with adeno-associated viral (AAV) vectors encoding LFv2IRE, AP20187 induces LFv2IRE homodimerization and transphosphorylation minutes after drug administration, resulting in the phosphorylation of a canonical substrate of the insulin receptor tyrosine kinase, IRS-1. AP20187 activation of LFv2IRE is dependent on the dose of drug and the amount of chimeric receptor expressed in AAV-transduced cells. Finally, AP20187-dependent activation of LFv2IRE results in insulin-like effects, such as induction of glycogen synthase activity and cellular proliferation. In vivo LFv2IRE transduction of insulin target tissues followed by AP20187 dosing may represent a therapeutic strategy to be tested in animal models of insulin resistance due to insulin receptor deficiency or of type I diabetes. This system may also represent a useful tool to dissect in vivo the independent contribution of insulin target tissues to hormone action.