Evaluation of the novel folate receptor ligand [18F]fluoro-PEG-folate for macrophage targeting in a rat model of arthritis

Introduction

Detection of (subclinical) synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging (sub)clinical arthritis was demonstrated by targeting the translocator protein in activated macrophages using (R)-[¹¹C]PK11195 and positron emission tomography (PET). Images, however, also showed significant peri-articular background activity. The folate receptor (FR)-β is a potential alternative target for imaging activated macrophages. Therefore, the PET tracer [¹⁸F]fluoro-PEG-folate was synthesized and evaluated in both in vitro and ex vivo studies using a methylated BSA induced arthritis model.

Methods

[¹⁸F]fluoro-PEG-folate was synthesized in a two-step procedure. Relative binding affinities of non-radioactive fluoro-PEG-folate, folic acid and naturally circulating 5-methyltetrahydrofolate (5-Me-THF) to FR were determined using KB cells with high expression of FR. Both in vivo [¹⁸F]fluoro-PEG-folate PET and ex vivo tissue distribution studies were performed in arthritic and normal rats and results were compared with those of the established macrophage tracer (R)-[¹¹C]PK11195.

Results

[¹⁸F]fluoro-PEG-folate was synthesized with a purity >97%, a yield of 300 to 1,700 MBq and a specific activity between 40 and 70 GBq/µmol. Relative in vitro binding affinity for FR of F-PEG-folate was 1.8-fold lower than that of folic acid, but 3-fold higher than that of 5-Me-THF. In the rat model, [¹⁸F]fluoro-PEG-folate uptake in arthritic knees was increased compared with both contralateral knees and knees of normal rats. Uptake in arthritic knees could be blocked by an excess of glucosamine-folate, consistent with [¹⁸F]fluoro-PEG-folate being specifically bound to FR. Arthritic knee-to-bone and arthritic knee-to-blood ratios of [¹⁸F]fluoro-PEG-folate were increased compared with those of (R)-[¹¹C]PK11195. Reduction of 5-Me-THF levels in rat plasma to those mimicking human levels increased absolute [¹⁸F]fluoro-PEG-folate uptake in arthritic joints, but without improving target-to-background ratios.

Conclusions

The novel PET tracer [¹⁸F]fluoro-PEG-folate, designed to target FR on activated macrophages provided improved contrast in a rat model of arthritis compared with the accepted macrophage tracer (R)-[¹¹C]PK11195. These results warrant further exploration of [¹⁸F]fluoro-PEG-folate as a putative PET tracer for imaging (sub)clinical arthritis in RA patients.     

[18F]DPA-714: Direct Comparison with [11C]PK11195 in a Model of Cerebral Ischemia in Rats

Purpose

Neuroinflammation is involved in several brain disorders and can be monitored through expression of the translocator protein 18 kDa (TSPO) on activated microglia. In recent years, several new PET radioligands for TSPO have been evaluated in disease models. [¹⁸F]DPA-714 is a TSPO radiotracer with great promise; however results vary between different experimental models of neuroinflammation. To further examine the potential of [¹⁸F]DPA-714, it was compared directly to [¹¹C]PK11195 in experimental cerebral ischaemia in rats.

Methods

Under anaesthesia, the middle cerebral artery of adult rats was occluded for 60 min using the filament model. Rats were allowed recovery for 5 to 7 days before one hour dynamic PET scans with [¹¹C]PK11195 and/or [¹⁸F]DPA-714 under anaesthesia.

Results

Uptake of [¹¹C]PK11195 vs [¹⁸F]DPA-714 in the ischemic lesion was similar (core/contralateral ratio: 2.84±0.67 vs 2.28±0.34 respectively), but severity of the brain ischemia and hence ligand uptake in the lesion appeared to vary greatly between animals scanned with [¹¹C]PK11195 or with [¹⁸F]DPA-714. To solve this issue of inter-individual variability, we performed a direct comparison of [¹¹C]PK11195 and [¹⁸F]DPA-714 by scanning the same animals sequentially with both tracers within 24 h. In this direct comparison, the core/contralateral ratio (3.35±1.21 vs 4.66±2.50 for [¹¹C]PK11195 vs [¹⁸F]DPA-714 respectively) showed a significantly better signal-to-noise ratio (1.6 (1.3–1.9, 95%CI) fold by linear regression) for [¹⁸F]DPA-714.

Conclusions

In a clinically relevant model of neuroinflammation, uptake for both radiotracers appeared to be similar at first, but a high variability was observed in our model. Therefore, to truly compare tracers in such models, we performed scans with both tracers in the same animals. By doing so, our result demonstrated that [¹⁸F]DPA-714 displayed a higher signal-to-noise ratio than [¹¹C]PK11195. Our results suggest that, with the longer half-life of [¹⁸F] which facilitates distribution of the tracer across PET centre, [¹⁸F]DPA-714 is a good alternative for TSPO imaging.     

MARCH2: Comparative Assessment of Therapeutic Effects of Acarbose and Metformin in Newly Diagnosed Type 2 Diabetes Patients

Background

The data of MARCH (Metformin and AcaRbose in Chinese as the initial Hypoglycaemic treatment) trial demonstrated that acarbose and metformin have similar efficacy as initial therapy for hemoglobin A1c (HbA1c) reduction in Chinese patients with newly diagnosed type 2 diabetes. We investigated whether the therapeutic efficacy was diversified under different body mass index (BMI) status.

Methods

All 784 subjects were divided into normal-weight group (BMI<24 kg/m²), overweight group (BMI 24–28 kg/m²) and obese group (BMI≥28 kg/m²). Patients were assigned to 48 weeks of therapy with acarbose or metformin, respectively. The clinical trial registry number was ChiCTR-TRC-08000231.

Results

The reduction of HbA1c levels and the proportion of patients with HbA1c of 6.5% or less were similar in the three groups after acarbose and metformin treatment. In overweight group, fasting blood glucose (FBG) after metformin treatment showed greater decline compared to acarbose group at 48 weeks [−1.73 (−1.99 to −1.46) vs. −1.37 (−1.61 to −1.12), P<0.05), however the decrease of 2 h post-challenge blood glucose (PBG) after acarbose treatment at 48 weeks was bigger compared to metformin group [−3.34 (−3.83 to−2.84) vs. −2.35 (−2.85 to −1.85), P<0.01 ]. Both acarbose and metformin treatment resulted in a significant decrease in waist circumference, hip circumference, weight and BMI in the three groups (all P<0.05).

Conclusion

Acarbose and metformin decreased HbA1c levels similarly regardless of BMI status of Chinese type 2 diabetic patients. Acarbose and metformin resulted in a significant and modest improvement of anthropometric parametres in different BMI status. Thus, acarbose treatment may contribute a similar effect on plasma glucose control compared to metformin, even in obesity patients.

Trial Registration

ChiCTR.org ChiCTR-TRC-08000231     

Deficiency of C5L2 Increases Macrophage Infiltration and Alters Adipose Tissue Function in Mice

Background

Obesity is considered as a systemic chronic low grade inflammation characterized by increased serum pro-inflammatory proteins and accumulation of macrophages within white adipose tissue (WAT) of obese patients. C5L2, a 7-transmembrane receptor, serves a dual function, binding the lipogenic hormone acylation stimulating protein (ASP), and C5a, involved in innate immunity.

Aim

We evaluated the impact of C5L2 on macrophage infiltration in WAT of wildtype (Ctl) and C5L2 knock-out (C5L2^−/−) mice over 6, 12 and 24 weeks on a chow diet and moderate diet-induced obesity (DIO) conditions.

Results

In Ctl mice, WAT C5L2 and C5a receptor mRNA increased (up to 10-fold) both over time and with DIO. By contrast, in C5L2^−/−, there was no change in C5aR in WAT. C5L2^−/− mice displayed higher macrophage content in WAT, varying by time, fat depot and diet, associated with altered systemic and WAT cytokine patterns compared to Ctl mice. However, in all cases, the M1 (pro-) vs M2 (anti-inflammatory) macrophage proportion was unchanged but C5L2^−/− adipose tissue secretome appeared to be more chemoattractant. Moreover, C5L2^−/− mice have increased food intake, increased WAT, and altered WAT lipid gene expression, which is reflected systemically. Furthermore, C5L2^−/− mice have altered glucose/insulin metabolism, adiponectin and insulin signalling gene expression in WAT, which could contribute to development of insulin resistance.

Conclusion

Disruption of C5L2 increases macrophage presence in WAT, contributing to obesity-associated pathologies, and further supports a dual role of complement in WAT. Understanding this effect of the complement system pathway could contribute to targeting treatment of obesity and its comorbidities.     

Common Variants of FTO Are Associated with Childhood Obesity in a Cross-Sectional Study of 3,126 Urban Indian Children

Background

FTO variants are robustly associated with obesity and related traits in many population and shown to have variable impact during life course. Although studies have shown association of FTO variants with adiposity in adult Indian, its association in Indian children is yet to be confirmed.

Methods

Here we examined association of FTO variants (rs9939609 and rs8050136) with obesity and related anthropometric and biochemical traits in 3,126 Indian children (aged 11–17 years) including 2,230 normal-weight and 896 over-weight/obese children. We also compared effects observed in the present study with that observed in previous studies on South Asian adults and children of other ethnic groups.

Results

The variant rs9939609 showed significant association with risk of obesity [OR = 1.21, P = 2.5×10⁻³] and its measures BMI, weight, waist circumference and hip circumference [β range = 0.11 to 0.14 Z-score units; P range = 1.3×10⁻⁴ to 1.6×10⁻⁷] in children. The observed effect sizes in Indian children were similar to those reported for European children. Variant rs9939609 explained 0.88% of BMI variance in Indian children. The effect sizes of rs9939609 on BMI and WC were ∼2 fold higher in children than adults. Interestingly rs9939609 was also associated with serum levels of thyroid stimulating hormone (TSH) [β = 0.10 Z-score, P = 5.8×10⁻³]. The other variant rs8050136 was in strong linkage disequilibrium with rs9939609 (r² = 0.97) and provided similar association results.

Conclusion

The study provides first report of association of FTO variants with obesity and related anthropometric traits in Indian children with higher impact in children compared to adults. We also demonstrated association of FTO variant with serum levels of TSH, indicating putative influence of FTO in hypothalamic-pituitary-thyroid axis.     

In Vivo Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists [11C]A-582941 and [11C]A-844606 in Mice and Conscious Monkeys

Background

The α7 nicotinic acetylcholine receptors (nAChRs) play an important role in the pathophysiology of neuropsychiatric diseases such as schizophrenia and Alzheimer''s disease. The goal of this study was to evaluate the two carbon-11-labeled α7 nAChR agonists [¹¹C]A-582941 and [¹¹C]A-844606 for their potential as novel positron emission tomography (PET) tracers.

Methodology/Principal Findings

The two tracers were synthesized by methylation of the corresponding desmethyl precursors using [¹¹C]methyl triflate. Effects of receptor blockade in mice were determined by coinjection of either tracer along with a carrier or an excess amount of a selective α7 nAChR agonist (SSR180711). Metabolic stability was investigated using radio-HPLC. Dynamic PET scans were performed in conscious monkeys with/without SSR180711-treatment. [¹¹C]A-582941 and [¹¹C]A-844606 showed high uptake in the mouse brain. Most radioactive compounds in the brain were detected as an unchanged form. However, regional selectivity and selective receptor blockade were not clearly observed for either compound in the mouse brain. On the other hand, the total distribution volume of [¹¹C]A-582941 and [¹¹C]A-844606 was high in the hippocampus and thalamus but low in the cerebellum in the conscious monkey brain, and reduced by pretreatment with SSR180711.

Conclusions/Significance

A nonhuman primate study suggests that [¹¹C]A-582941 and [¹¹C]A-844606 would be potential PET ligands for imaging α7 nAChRs in the human brain.     

Altered Plasma Apolipoprotein Modifications in Patients with Pancreatic Cancer: Protein Characterization and Multi-Institutional Validation

Background

Among the more common human malignancies, invasive ductal carcinoma of the pancreas has the worst prognosis. The poor outcome seems to be attributable to difficulty in early detection.

Methods

We compared the plasma protein profiles of 112 pancreatic cancer patients with those of 103 sex- and age-matched healthy controls (Cohort 1) using a newly developed matrix-assisted laser desorption/ionization (oMALDI) QqTOF (quadrupole time-of-flight) mass spectrometry (MS) system.

Results

We found that hemi-truncated apolipoprotein AII dimer (ApoAII-2; 17252 m/z), unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z), and their summed value were significantly decreased in the pancreatic cancer patients [P = 1.36×10⁻²¹, P = 4.35×10⁻¹⁴, and P = 1.83×10⁻²⁴ (Mann-Whitney U-test); area-under-curve values of 0.877, 0.798, and 0.903, respectively]. The significance was further validated in a total of 1099 plasma/serum samples, consisting of 2 retrospective cohorts [Cohort 2 (n = 103) and Cohort 3 (n = 163)] and a prospective cohort [Cohort 4 (n = 833)] collected from 8 medical institutions in Japan and Germany.

Conclusions

We have constructed a robust quantitative MS profiling system and used it to validate alterations of modified apolipoproteins in multiple cohorts of patients with pancreatic cancer.     

Conventional Hemodynamic Resuscitation May Fail to Optimize Tissue Perfusion: An Observational Study on the Effects of Dobutamine,Enoximone, and Norepinephrine in Patients with Acute Myocardial Infarction Complicated by Cardiogenic Shock

Aim

To investigate the effects of inotropic agents on parameters of tissue perfusion in patients with cardiogenic shock.

Methods and Results

Thirty patients with cardiogenic shock were included. Patients received dobutamine, enoximone, or norepinephrine. We performed hemodynamic measurements at baseline and after titration of the inotropic agent until cardiac index (CI) ≥2.5 L.min⁻¹.m⁻² or mixed-venous oxygen saturation (SvO₂) ≥70% (dobutamine or enoximone), and mean arterial pressure (MAP) ≥70 mmHg (norepinephrine). As parameters of tissue perfusion, we measured central-peripheral temperature gradient (delta-T) and sublingual perfused capillary density (PCD). All patients reached predefined therapeutic targets. The inotropes did not significantly change delta-T. Dobutamine did not change PCD. Enoximone increased PCD (9.1 [8.9–10.2] vs. 11.4 [8.4–13.9] mm.mm⁻²; p<0.05), and norepinephrine tended to decrease PCD (9.8 [8.5–11.9] vs. 8.8 [8.2–9.6] mm.mm⁻², p = 0.08). Fifteen patients (50%) died within 30 days after admission. Patients who had low final PCD (≤10.3 mm.mm⁻²; 64%) were more likely to die than patients who had preserved PCD (>10.3 mm.mm⁻²; mortality 72% vs. 17%, p = 0.003).

Conclusion

This study demonstrates the effects of commonly used inotropic agents on parameters of tissue perfusion in patients with cardiogenic shock. Despite hemodynamic optimization, tissue perfusion was not sufficiently restored in most patients. In these patients, mortality was high. Interventions directed at improving microcirculation may eventually help bridging the gap between improved hemodynamics and dismal patient outcome in cardiogenic shock.     

Combining [(11)C]-AnxA5 PET Imaging with Serum Biomarkers for Improved Detection in Live Mice of Modest Cell Death in Human Solid Tumor Xenografts

Background

In vivo imaging using Annexin A5-based radioligands is a powerful technique for visualizing massive cell death, but has been less successful in monitoring the modest cell death typically seen in solid tumors after chemotherapy. Here we combined dynamic positron emission tomography (PET) imaging using Annexin A5 with a serum-based apoptosis marker, for improved sensitivity and specificity in assessment of chemotherapy-induced cell death in a solid tumor model.

Methodology/Principal Findings

Modest cell death was induced by doxorubicin in a mouse xenograft model with human FaDu head and neck cancer cells. PET imaging was based on ¹¹C-labeled Sel-tagged Annexin A5 ([¹¹C]-AnxA5-ST) and a size-matched control. 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]-FDG) was utilized as a tracer of tissue metabolism. Serum biomarkers for cell death were ccK18 and K18 (M30 Apoptosense® and M65). Apoptosis in tissue sections was verified ex vivo for validation. Both PET imaging using [¹¹C]-AnxA5-ST and serum ccK18/K18 levels revealed treatment-induced cell death, with ccK18 displaying the highest detection sensitivity. [¹⁸F]-FDG uptake was not affected by this treatment in this tumor model. [¹¹C]-AnxA5-ST gave robust imaging readouts at one hour and its short half-life made it possible to perform paired scans in the same animal in one imaging session.

Conclusions/Significance

The combined use of dynamic PET with [¹¹C]-AnxA5-ST, showing specific increases in tumor binding potential upon therapy, with ccK18/K18 serum measurements, as highly sensitive markers for cell death, enabled effective assessment of modest therapy-induced cell death in this mouse xenograft model of solid human tumors.     

Association of Cytokines in Individuals Sensitive and Insensitive to Dust Mites in a Brazilian Population

Introduction

Allergic reaction to dust mites is a relatively common condition among children, triggering cutaneous and respiratory responses that have a great impact on the health of this population. Anaphylactic hypersensitivity is characterized by an exacerbated response involving the production of regulatory cytokines responsible for stimulating the production of IgE antibodies.

Objective

To investigate an association of variants in cytokine genes (IL1A ⁻⁸⁸⁹, IL1B ^{−511, +3962}, IL1R ¹⁹⁷⁰, IL1RA ¹¹¹⁰⁰, IL4RA ⁺¹⁹⁰², IL12 ⁻¹¹⁸⁸, IFNG ⁺⁸⁷⁴, TGFB1 ^{codon 10, codon 25}, TNFA ^{−308, −238}, IL2 ^{−330, +166}, IL4 ^{−1098, −590, −33}, IL6 ^{−174, nt565}, and IL10 ^{−1082, −819, −592}) between patients sensitive to dust mites and a control group.

Methods

A total of 254 patients were grouped as atopic and non-atopic according to sensitivity as evaluated by the Prick Test and to cytokine genotyping by the polymerase chain reaction-sequence specific primers (PCR-SSP) method using the Cytokine Genotyping Kit.

Results

A comparison between individuals allergic to Dermatophagoides farinae, Dermatophagoides pteronyssinus, and Blomia tropicalis and a non-atopic control group showed significant differences between allele and genotype frequencies in the regulatory regions of cytokine genes, with important evidence for IL4 ⁻⁵⁹⁰ in T/C (10.2% vs. 43.1%, odd ratio [OR] = 0.15, p = 5.2 10⁻⁸, pc = 0.0000011, and 95% confidence interval [95%CI] = 0.07–0.32) and T/T genotypes (42.9% vs. 13.8%, OR = 4.69, p = 2.5 10⁻⁶, pc = 0.000055, and 95%CI = 2.42–9.09). Other associations were observed in the pro-inflammatory cytokines IL1A ⁻⁸⁸⁹ (T/T, C, and T) and IL2 ⁻³³⁰ (G/T and T/T) and the anti-inflammatory cytokines IL4RA ⁺¹⁹⁰² (A and G), IL4 ⁻⁵⁹⁰ (T/C, T/T, C, and T), and IL10 ⁻⁵⁹² (A/A, C/A, A, and C).

Conclusion

Our results suggest a possible association between single nucleotide polymorphisms (SNPs) in cytokine genes and hypersensitivity to dust mites.     

The Effect of Neoadjuvant Chemoradiotherapy on Whole-Body Physical Fitness and Skeletal Muscle Mitochondrial Oxidative Phosphorylation In Vivo in Locally Advanced Rectal Cancer Patients – An Observational Pilot Study

Background

In the United Kingdom, patients with locally advanced rectal cancer routinely receive neoadjuvant chemoradiotherapy. However, the effects of this on physical fitness are unclear. This pilot study is aimed to investigate the effect of neoadjuvant chemoradiotherapy on objectively measured in vivo muscle mitochondrial function and whole-body physical fitness.

Methods

We prospectively studied 12 patients with rectal cancer who completed standardized neoadjuvant chemoradiotherapy, recruited from a large tertiary cancer centre, between October 2012 and July 2013. All patients underwent a cardiopulmonary exercise test and a phosphorus magnetic resonance spectroscopy quadriceps muscle exercise-recovery study before and after neoadjuvant chemoradiotherapy. Data were analysed and reported blind to patient identity and clinical course. Primary variables of interest were the two physical fitness measures; oxygen uptake at estimated anaerobic threshold and oxygen uptake at Peak exercise (ml.kg⁻¹.min⁻¹), and the post-exercise phosphocreatine recovery rate constant (min⁻¹), a measure of muscle mitochondrial capacity in vivo.

Results

Median age was 67 years (IQR 64–75). Differences (95%CI) in all three primary variables were significantly negative post-NACRT: Oxygen uptake at estimated anaerobic threshold −2.4 ml.kg⁻¹.min⁻¹ (−3.8, −0.9), p = 0.004; Oxygen uptake at Peak −4.0 ml.kg⁻¹.min⁻¹ (−6.8, −1.1), p = 0.011; and post-exercise phosphocreatine recovery rate constant −0.34 min⁻¹ (−0.51, −0.17), p<0.001.

Conclusion

The significant decrease in both whole-body physical fitness and in vivo muscle mitochondrial function raises the possibility that muscle mitochondrial mechanisms, no doubt multifactorial, may be important in deterioration of physical fitness following neoadjuvant chemoradiotherapy. This may have implications for targeted interventions to improve physical fitness pre-surgery.

Trial Registration

Clinicaltrials.gov registration {"type":"clinical-trial","attrs":{"text":"NCT01859442","term_id":"NCT01859442"}}NCT01859442     

Clinical Profile of PiB-Positive Corticobasal Syndrome

Background

Corticobasal syndrome (CBS) is a multifaceted neurodegenerative disorder characterized by a combination of motor and cognitive deficits. Several different pathological entities, including Alzheimer’s pathology, have been described in association with CBS. The present study aimed to establish clinical, neuropsychological, and neuroimaging features that could be useful in the distinction of CBS due to AD pathology from other CBS cases in life based on [¹¹C] Pittsburgh Compound B positron emission tomography (PiB-PET) status.

Methods

Patients with CBS were prospectively recruited from a specialized cognitive disorders clinic. All patients underwent detailed clinical and neuropsychological assessment, with structural imaging using voxel-based analysis of magnetic resonance imaging. Alzheimer’s pathology was detected using PiB-PET imaging, and PiB-positive and PiB-negative groups were compared.

Results

Fourteen CBS patients meeting defined criteria were included (7 male, 7 female; mean age 66.1+/−6.9 years; median symptom duration was 35.5+/−22.6 months) and compared to 20 matched control subjects. Of the 14 patients, 4 were PiB-positive and 10 PiB-negative. There were no significant differences between PiB-positive and PiB-negative CBS patients in age, gender, education, symptom duration, or motor features. PiB-positive patients had greater visuospatial deficits, a higher rate of sentence repetition impairment, and more functional decline. Voxel-based morphometry analyses demonstrated extensive peri-insular and post-central atrophy in both groups, but PiB-positive patients had atrophy that extended to include the posterior part of the left superior temporal gyrus.

Conclusions

Visuospatial function, aspects of language, and the pattern of cerebral atrophy may be useful in distinguishing patients with CBS due to underlying AD pathology.     

Osteoprotegerin Inhibits Aortic Valve Calcification and Preserves Valve Function in Hypercholesterolemic Mice

Background

There are no rigorously confirmed effective medical therapies for calcific aortic stenosis. Hypercholesterolemic Ldlr ^−/− Apob ^100/100 mice develop calcific aortic stenosis and valvular cardiomyopathy in old age. Osteoprotegerin (OPG) modulates calcification in bone and blood vessels, but its effect on valve calcification and valve function is not known.

Objectives

To determine the impact of pharmacologic treatment with OPG upon aortic valve calcification and valve function in aortic stenosis-prone hypercholesterolemic Ldlr ^−/− Apob ^100/100 mice.

Methods

Young Ldlr ^−/− Apob ^100/100 mice (age 2 months) were fed a Western diet and received exogenous OPG or vehicle (N = 12 each) 3 times per week, until age 8 months. After echocardiographic evaluation of valve function, the aortic valve was evaluated histologically. Older Ldlr ^−/− Apob ^100/100 mice were fed a Western diet beginning at age 2 months. OPG or vehicle (N = 12 each) was administered from 6 to 12 months of age, followed by echocardiographic evaluation of valve function, followed by histologic evaluation.

Results

In Young Ldlr ^−/− Apob ^100/100 mice, OPG significantly attenuated osteogenic transformation in the aortic valve, but did not affect lipid accumulation. In Older Ldlr ^−/− Apob ^100/100 mice, OPG attenuated accumulation of the osteoblast-specific matrix protein osteocalcin by ∼80%, and attenuated aortic valve calcification by ∼ 70%. OPG also attenuated impairment of aortic valve function.

Conclusions

OPG attenuates pro-calcific processes in the aortic valve, and protects against impairment of aortic valve function in hypercholesterolemic aortic stenosis-prone Ldlr ^−/− Apob ^100/100 mice.     

Genetic Variants at 20p11 Confer Risk to Androgenetic Alopecia in the Chinese Han Population

Background

Androgenetic alopecia (AGA) is a well-characterized type of progressive hair loss commonly seen in men, with different prevalences in different ethnic populations. It is generally considered to be a polygenic heritable trait. Several susceptibility genes/loci, such as AR/EDA2R, HDAC9 and 20p11, have been identified as being involved in its development in European populations. In this study, we aim to validate whether these loci are also associated with AGA in the Chinese Han population.

Methods

We genotyped 16 previously reported single nucleotide polymorphisms (SNPs) with 445 AGA cases and 546 healthy controls using the Sequenom iPlex platform. The trend test was used to evaluate the association between these loci and AGA in the Chinese Han population. Conservatively accounting for multiple testing by the Bonferroni correction, the threshold for statistical significance was P ≤3.13×10⁻³.

Results

We identified that 5 SNPs at 20p11 were significantly associated with AGA in the Chinese Han population (1.84×10⁻¹¹≤P≤2.10×10⁻⁶).

Conclusions

This study validated, for the first time, that 20p11 also confers risk for AGA in the Chinese Han population and implicated the potential common genetic factors for AGA shared by both Chinese and European populations.     

Development of TaqMan-Based Quantitative PCR for Sensitive and Selective Detection of Toxigenic Clostridium difficile in Human Stools

Background

Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.

Methods

TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).

Results

The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA⁺TcdB⁺, TcdA⁻TcdB⁺, and TcdA⁻TcdB⁻ types), TcdB-producing strains (TcdA⁺TcdB⁺ and TcdA⁻TcdB⁺ types), and TcdA-producing strains (TcdA⁺TcdB⁺ type), respectively, with a lower detection limit of 10³ cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA⁺TcdB⁺ strain in six specimens and TcdA⁻TcdB⁻ strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 10^4.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.

Conclusion

Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.     

A Meta-Analysis of Core Stability Exercise versus General Exercise for Chronic Low Back Pain

Objective

To review the effects of core stability exercise or general exercise for patients with chronic low back pain (LBP).

Summary of Background Data

Exercise therapy appears to be effective at decreasing pain and improving function for patients with chronic LBP in practice guidelines. Core stability exercise is becoming increasingly popular for LBP. However, it is currently unknown whether core stability exercise produces more beneficial effects than general exercise in patients with chronic LBP.

Methods

Published articles from 1970 to October 2011 were identified using electronic searches. For this meta-analysis, two reviewers independently selected relevant randomized controlled trials (RCTs) investigating core stability exercise versus general exercise for the treatment of patients with chronic LBP. Data were extracted independently by the same two individuals who selected the studies.

Results

From the 28 potentially relevant trials, a total of 5 trials involving 414 participants were included in the current analysis. The pooling revealed that core stability exercise was better than general exercise for reducing pain [mean difference (−1.29); 95% confidence interval (−2.47, −0.11); P = 0.003] and disability [mean difference (−7.14); 95% confidence interval (−11.64, −2.65); P = 0.002] at the time of the short-term follow-up. However, no significant differences were observed between core stability exercise and general exercise in reducing pain at 6 months [mean difference (−0.50); 95% confidence interval (−1.36, 0.36); P = 0.26] and 12 months [mean difference (−0.32); 95% confidence interval (−0.87, 0.23); P = 0.25].

Conclusions

Compared to general exercise, core stability exercise is more effective in decreasing pain and may improve physical function in patients with chronic LBP in the short term. However, no significant long-term differences in pain severity were observed between patients who engaged in core stability exercise versus those who engaged in general exercise.

Systematic Review Registration

http://www.crd.york.ac.uk/PROSPERO PROSPERO registration number: CRD42011001717.     

In Vivo Detection of Activated Platelets Allows Characterizing Rupture of Atherosclerotic Plaques with Molecular Magnetic Resonance Imaging in Mice

Background

Early and non-invasive detection of platelets on micro atherothrombosis provides a means to identify unstable plaque and thereby allowing prophylactic treatment towards prevention of stroke or myocardial infarction. Molecular magnetic resonance imaging (mMRI) of activated platelets as early markers of plaque rupture using targeted contrast agents is a promising strategy. In this study, we aim to specifically image activated platelets in murine atherothrombosis by in vivo mMRI, using a dedicated animal model of plaque rupture.

Methods

An antibody targeting ligand-induced binding sites (LIBS) on the glycoprotein IIb/IIIa-receptor of activated platelets was conjugated to microparticles of iron oxide (MPIO) to form the LIBS-MPIO contrast agent causing a signal-extinction in T2*-weighted MRI. ApoE^−/− mice (60 weeks-old) were fed a high fat diet for 5 weeks. Using a small needle, the surface of their carotid plaques was scratched under blood flow to induce atherothrombosis. In vivo 9.4 Tesla MRI was performed before and repetitively after intravenous injection of either LIBS-MPIO versus non-targeted-MPIO.

Results

LIBS-MPIO injected animals showed a significant signal extinction (p<0.05) in MRI, corresponding to the site of plaque rupture and atherothrombosis in histology. The signal attenuation was effective for atherothrombosis occupying ≥2% of the vascular lumen. Histology further confirmed significant binding of LIBS-MPIO compared to control-MPIO on the thrombus developing on the surface of ruptured plaques (p<0.01).

Conclusion

in vivo mMRI detected activated platelets on mechanically ruptured atherosclerotic plaques in ApoE^−/− mice with a high sensititvity. This imaging technology represents a unique opportunity for noninvasive detection of atherothrombosis and the identification of unstable atherosclerotic plaques with the ultimate promise to prevent strokes and myocardial infarctions.     

The Risk of Familial Mediterranean Fever in MEFV Heterozygotes: A Statistical Approach

Background

Familial Mediterranean fever (FMF) is an autosomal recessive autoinflammatory disorder due to MEFV mutations and one of the most frequent Mediterranean genetic diseases. The observation of many heterozygous patients in whom a second mutated allele was excluded led to the proposal that heterozygosity could be causal. However, heterozygosity might be coincidental in many patients due to the very high rate of mutations in Mediterranean populations.

Objective

To better delineate the pathogenicity of heterozygosity in order to improve genetic counselling and disease management.

Methods

Complementary statistical approaches were used: estimation of FMF prevalence at population levels, genotype comparison in siblings from 63 familial forms, and genotype study in 557 patients from four Mediterranean populations.

Results

At the population level, we did not observe any contribution of heterozygosity to disease prevalence. In affected siblings of patients carrying two MEFV mutations, 92% carry two mutated alleles, whereas 4% are heterozygous with typical FMF diagnosis. We demonstrated statistically that patients are more likely to be heterozygous than healthy individuals, as shown by the higher ratio heterozygous carriers/non carriers in patients (p<10⁻⁷–p<0.003). The risk for heterozygotes to develop FMF was estimated between 2.1×10⁻³ and 5.8×10⁻³ and the relative risk, as compared to non carriers, between 6.3 and 8.1.

Conclusions

This is the first statistical demonstration that heterozygosity is not responsible for classical Mendelian FMF per se, but constitutes a susceptibility factor for clinically-similar multifactorial forms of the disease. We also provide a first estimate of the risk for heterozygotes to develop FMF.     

The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

Background

The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh^−/− mice to oxidative stress.

Methodology/Principal Findings

The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS). The Mutyh^−/− phenotpe was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh^−/− mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh^−/− mice. Lymphoid hyperplasia and a significant reduction in Foxp3⁺ regulatory T cells were observed only in Mutyh^−/− mice.

Conclusions

The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.     

Haplotypes in IL-8 Gene Are Associated to Age-Related Macular Degeneration: A Case-Control Study

Background

Age-related macular degeneration (AMD) is the main cause of blindness in the developed world. The etiology of AMD is multifactorial due to the interaction between genetic and environmental factors. IL-8 has a role in inflammation and angiogenesis; we report the genetic characterization of IL-8 allele architecture and evaluate the role of SNPs or haplotypes in the susceptibility to wet AMD, case-control study.

Methods

Case-control study including 721 AMD patients and 660 controls becoming from Italian population. Genotyping was carried out by Real Time-PCR. Differences in the frequencies were estimated by the chi-square test. Direct sequencing was carried out by capillary electrophoresis trough ABI3130xl.

Results

rs2227306 showed a p–value of 4.15*10⁻⁵ and an Odds Ratio (OR) for T allele of 1.39 [1.19–1.62]. After these positive results, we sequenced the entire IL-8 regulatory and coding regions of 60 patients and 30 controls stratified for their genotype at rs2227306. We defined two different haplotypes involving rs4073 (A/T), rs2227306 (C/T), rs2227346 (C/T) and rs1126647 (A/T): A-T-T-T (p-value: 2.08*10⁻⁹; OR: 1.68 [1.43–1.97]) and T-C-C-A (p-value: 7.07*10⁻¹¹; OR: 0.60 [0.51–0.70]). To further investigate a potential functional role of associated haplotypes, we performed an expression study on RNA extracted from whole blood of 75 donors to verify a possible direct correlation between haplotype and gene expression, failing to reveal significant differences.

Conclusions

These results suggest a possible secondary role of IL-8 gene in the development of the disease. This paper outlines the importance of association between inflammation and AMD. Moreover IL-8 is a new susceptibility genomic biomarker of AMD.