Neurotrophins are expressed in giant cell arteritis lesions and may contribute to vascular remodeling

Introduction

Giant cell arteritis (GCA) is characterized by intimal hyperplasia leading to ischaemic manifestations that involve large vessels. Neurotrophins (NTs) and their receptors (NTRs) are protein factors for growth, differentiation and survival of neurons. They are also involved in the migration of vascular smooth muscle cells (VSMCs). Our aim was to investigate whether NTs and NTRs are involved in vascular remodelling of GCA.

Methods

We included consecutive patients who underwent a temporal artery biopsy for suspected GCA. We developed an enzymatic digestion method to obtain VSMCs from smooth muscle cells in GCA patients and controls. Neurotrophin protein and gene expression and functional assays were studied from these VSMCs. Neurotrophin expression was also analysed by immunohistochemistry in GCA patients and controls.

Results

Whereas temporal arteries of both GCA patients (n = 22) and controls (n = 21) expressed nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB) and sortilin, immunostaining was more intense in GCA patients, especially in the media and intima, while neurotrophin-3 (NT-3) and P75 receptor (P75^NTR) were only detected in TA from GCA patients. Expression of TrkB, a BDNF receptor, was higher in GCA patients with ischaemic complications. Serum NGF was significantly higher in GCA patients (n = 28) vs. controls (n = 48), whereas no significant difference was found for BDNF and NT-3. NGF and BDNF enhanced GCA-derived temporal artery VSMC proliferation and BDNF facilitated migration of temporal artery VSMCs in patients with GCA compared to controls.

Conclusions

Our results suggest that NTs and NTRs are involved in vascular remodelling of GCA. In GCA-derived temporal artery VSMC, NGF promoted proliferation and BDNF enhanced migration by binding to TrkB and p75^NTR receptors. Further experiments are needed on a larger number of VSMC samples to confirm these results.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0487-z) contains supplementary material, which is available to authorized users.     

Association of HLA-DRB1 amino acid residues with giant cell arteritis: genetic association study,meta-analysis and geo-epidemiological investigation

IntroductionGiant cell arteritis (GCA) is an autoimmune disease commonest in Northern Europe and Scandinavia. Previous studies report various associations with HLA-DRB1*04 and HLA-DRB1*01; HLA-DRB1 alleles show a gradient in population prevalence within Europe. Our aims were (1) to determine which amino acid residues within HLA-DRB1 best explained HLA-DRB1 allele susceptibility and protective effects in GCA, seen in UK data combined in meta-analysis with previously published data, and (2) to determine whether the incidence of GCA in different countries is associated with the population prevalence of the HLA-DRB1 alleles that we identified in our meta-analysis.MethodsGCA patients from the UK GCA Consortium were genotyped by using single-strand oligonucleotide polymerization, allele-specific polymerase chain reaction, and direct sequencing. Meta-analysis was used to compare and combine our results with published data, and public databases were used to identify amino acid residues that may explain observed susceptibility/protective effects. Finally, we determined the relationship of HLA-DRB1*04 population carrier frequency and latitude to GCA incidence reported in different countries.ResultsIn our UK data (225 cases and 1378 controls), HLA-DRB1*04 carriage was associated with GCA susceptibility (odds ratio (OR) = 2.69, P = 1.5×10⁻¹¹), but HLA-DRB1*01 was protective (adjusted OR = 0.55, P = 0.0046). In meta-analysis combined with 14 published studies (an additional 691 cases and 4038 controls), protective effects were seen from HLA-DR2, which comprises HLA-DRB1*15 and HLA-DRB1*16 (OR = 0.65, P = 8.2×10⁻⁶) and possibly from HLA-DRB1*01 (OR = 0.73, P = 0.037). GCA incidence (n = 17 countries) was associated with population HLA-DRB1*04 allele frequency (P = 0.008; adjusted R² = 0.51 on univariable analysis, adjusted R² = 0.62 after also including latitude); latitude also made an independent contribution.ConclusionsWe confirm that HLA-DRB1*04 is a GCA susceptibility allele. The susceptibility data are best explained by amino acid risk residues V, H, and H at positions 11, 13, and 33, contrary to previous suggestions of amino acids in the second hypervariable region. Worldwide, GCA incidence was independently associated both with population frequency of HLA-DRB1*04 and with latitude itself. We conclude that variation in population HLA-DRB1*04 frequency may partly explain variations in GCA incidence and that HLA-DRB1*04 may warrant investigation as a potential prognostic or predictive biomarker.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0692-4) contains supplementary material, which is available to authorized users.     

Comparison of Macular Integrity Assessment (MAIA ?), MP-3, and the Humphrey Field Analyzer in the Evaluation of the Relationship between the Structure and Function of the Macula

Purpose

This study was conducted in order to compare relationships between the macular visual field (VF) mean sensitivity measured by MAIA^TM (Macular Integrity Assessment), MP-3, or Humphry field analyzer (HFA) and the ganglion cell and inner plexiform layer (GCA) thicknesses.

Methods

This cross-sectional study examined 73 glaucoma patients and 19 normal subjects. All subjects underwent measurements for GCA thickness by Cirrus HD-OCT and static threshold perimetry using MAIA^TM, MP-3, or HFA. VF and OCT in the retinal view were used to examine both the global relationship between the VF sensitivity and GCA thickness, and the superior hemiretina and inferior hemiretina. The relationship between the GCA thickness and macular sensitivity was examined by Spearman correlation analysis.

Results

For each instrument, statistically significant macular VF sensitivity (dB) and GCA thickness relationships were observed using the decibel scale (R = 0.547–0.687, all P < 0.001). The highest correlation for the global (R = 0.682) and the superior hemiretina (R = 0.594) GCA thickness-VF mean sensitivity was observed by the HFA. The highest correlation for the inferior hemiretina (R = 0.687) GCA thickness-VF mean sensitivity was observed by the MP-3. Among the three VF measurement instruments, however, no significant differences were found for the structure-function relationships.

Conclusions

All three VF measurement instruments found similar structure-function relationships in the central VF.     

Current halo structures in high-current plasma experiments: θ-pinch

Experimental data elucidating mechanisms for halo formation in θ-pinch discharges are presented and discussed. The experiments were performed with different gases (H₂, D₂, He, and Ar) in a theta-pinch device with a porcelain vacuum chamber and an excitation coil 15 cm in diameter and 30 cm in length. The stored energy, the current in the excitation coil, and the current half-period were W = 10 kJ, I = 400 kA, and T/2 = 14 μs, respectively. It is found that the plasma rings (halos) surrounding the pinch core arise as a result of coaxial pinch stratification due to both the excitation of closed currents (inductons) inside the pinch and the radial convergence of the plasma current sheaths produced after the explosion of T-layers formed near the wall in the initial stage of the discharge. It is concluded that halo structures observed in pinches, tokamaks, and other high-current devices used in controlled fusion research have the same nature.     

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Abstract

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H₂O₂. After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H₂O₂ concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.     

Assessment of lexical semantic judgment abilities in alcohol-dependent subjects: An fMRI study

Neuropsychological studies have shown that alcohol dependence is associated with neurocognitive deficits in tasks requiring memory, perceptual motor skills, abstraction and problem solving, whereas language skills are relatively spared in alcoholics despite structural abnormalities in the language-related brain regions. To investigate the preserved mechanisms of language processing in alcohol-dependents, functional brain imaging was undertaken in healthy controls (n=18) and alcohol-dependents (n=16) while completing a lexical semantic judgment task in a 3 T MR scanner. Behavioural data indicated that alcohol-dependents took more time than controls for performing the task but there was no significant difference in their response accuracy. fMRI data analysis revealed that while performing the task, the alcoholics showed enhanced activations in left supramarginal gyrus, precuneus bilaterally, left angular gyrus, and left middle temporal gyrus as compared to control subjects. The extensive activations observed in alcoholics as compared to controls suggest that alcoholics recruit additional brain areas to meet the behavioural demands for equivalent task performance. The results are consistent with previous fMRI studies suggesting compensatory mechanisms for the execution of task for showing an equivalent performance or decreased neural efficiency of relevant brain networks. However, on direct comparison of the two groups, the results did not survive correction for multiple comparisons; therefore, the present findings need further exploration.     

Allopregnanolone levels are reduced in temporal cortex in patients with Alzheimer's disease compared to cognitively intact control subjects

The neurosteroid allopregnanolone has pronounced neuroprotective actions, increases myelination, and enhances neurogenesis. Evidence suggests that allopregnanolone dysregulation may play a role in the pathophysiology of Alzheimer's disease (AD) and other neurodegenerative disorders. Our prior data demonstrate that allopregnanolone is reduced in prefrontal cortex in male patients with AD compared to male cognitively intact control subjects, and inversely correlated with neuropathological disease stage (Braak and Braak). We therefore determined if allopregnanolone levels are also reduced in AD patients compared to control subjects in temporal cortex, utilizing a larger set of samples from both male and female patients. In addition, we investigated if neurosteroids are altered in subjects who are APOE4 allele carriers. Allopregnanolone, dehydroepiandrosterone (DHEA), and pregnenolone levels were determined in temporal cortex postmortem samples by gas chromatography/mass spectrometry, preceded by high performance liquid chromatography (40 subjects with AD/41 cognitively intact control subjects). Allopregnanolone levels are reduced in temporal cortex in patients with AD (median 2.68 ng/g, n = 40) compared to control subjects (median 5.64 ng/g, n = 41), Mann–Whitney p = 0.0002, and inversely correlated with Braak and Braak neuropathological disease stage (Spearman r = − 0.38, p = 0.0004). DHEA and pregnenolone are increased in patients with AD compared to control subjects. Patients carrying an APOE4 allele demonstrate reduced allopregnanolone levels in temporal cortex (Mann–Whitney p = 0.04). In summary, our findings indicate that neurosteroids are altered in temporal cortex in patients with AD and related to neuropathological disease stage. In addition, the APOE4 allele is associated with reduced allopregnanolone levels. Neurosteroids may be relevant to the neurobiology and therapeutics of AD.     

Venous Thromboembolism and Cerebrovascular Events in Patients with Giant Cell Arteritis: A Population-Based Retrospective Cohort Study

Objective

To investigate the incidence of venous thromboembolism (VTE) and cerebrovascular events in a community-based incidence cohort of patients with giant cell arteritis (GCA) compared to the general population.

Methods

A population-based inception cohort of patients with incident GCA between January 1, 1950 and December 31, 2009 in Olmsted County, Minnesota and a cohort of non-GCA subjects from the same population were assembled and followed until December 31, 2013. Confirmed VTE and cerebrovascular events were identified through direct medical record review.

Results

The study population included 244 patients with GCA with a mean ± SD age at diagnosis of 76.2 ± 8.2 years (79% women) and an average length of follow-up of 10.2 ± 6.8 years. Compared to non-GCA subjects of similar age and sex, patients diagnosed with GCA had a higher incidence (%) of amaurosis fugax (cumulative incidence ± SE: 2.1 ± 0.9 versus 0, respectively; p = 0.014) but similar rates of stroke, transient ischemic attack (TIA), and VTE. Among patients with GCA, neither baseline characteristics nor laboratory parameters at diagnosis reliably predicted risk of VTE or cerebrovascular events.

Conclusion

In this population-based study, the incidence of VTE, stroke and TIA was similar in patients with GCA compared to non-GCA subjects.     

A Large-Scale Genetic Analysis Reveals a Strong Contribution of the HLA Class II Region to Giant Cell Arteritis Susceptibility

We conducted a large-scale genetic analysis on giant cell arteritis (GCA), a polygenic immune-mediated vasculitis. A case-control cohort, comprising 1,651 case subjects with GCA and 15,306 unrelated control subjects from six different countries of European ancestry, was genotyped by the Immunochip array. We also imputed HLA data with a previously validated imputation method to perform a more comprehensive analysis of this genomic region. The strongest association signals were observed in the HLA region, with rs477515 representing the highest peak (p = 4.05 × 10⁻⁴⁰, OR = 1.73). A multivariate model including class II amino acids of HLA-DRβ1 and HLA-DQα1 and one class I amino acid of HLA-B explained most of the HLA association with GCA, consistent with previously reported associations of classical HLA alleles like HLA-DRB1^∗04. An omnibus test on polymorphic amino acid positions highlighted DRβ1 13 (p = 4.08 × 10⁻⁴³) and HLA-DQα1 47 (p = 4.02 × 10⁻⁴⁶), 56, and 76 (both p = 1.84 × 10⁻⁴⁵) as relevant positions for disease susceptibility. Outside the HLA region, the most significant loci included PTPN22 (rs2476601, p = 1.73 × 10⁻⁶, OR = 1.38), LRRC32 (rs10160518, p = 4.39 × 10⁻⁶, OR = 1.20), and REL (rs115674477, p = 1.10 × 10⁻⁵, OR = 1.63). Our study provides evidence of a strong contribution of HLA class I and II molecules to susceptibility to GCA. In the non-HLA region, we confirmed a key role for the functional PTPN22 rs2476601 variant and proposed other putative risk loci for GCA involved in Th1, Th17, and Treg cell function.     

Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test

The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm.     

Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15'-monooxygenase 1 single nucleotide polymorphisms

The mechanisms as well the genetics underlying the bioavailability and metabolism of carotenoids in humans remain unclear. To begin to address these questions, we used cluster analysis to examine individual temporal responses of plasma carotenoids from a controlled-diet study of subjects who consumed carotenoid-rich beverages. Treatments, given daily for 3 weeks, were watermelon juice at two levels (20-mg lycopene, 2.5-mg β-carotene, n=23 and 40-mg lycopene, 5-mg β-carotene, n=12) and tomato juice (18-mg lycopene, 0.6-mg β-carotene, n=10). Cluster analysis revealed distinct groups of subjects differing in the temporal response of plasma carotenoids and provided the basis for classifying subjects as strong responders or weak responders for β-carotene, lycopene, phytoene and phytofluene. Individuals who were strong or weak responders for one carotenoid were not necessarily strong or weak responders for another carotenoid. Furthermore, individual responsiveness was associated with genetic variants of the carotenoid metabolizing enzyme β-carotene 15,15'-monooxygenase 1. These results support the concept that individuals absorb or metabolize carotenoids differently across time and suggest that bioavailability of carotenoids may involve specific genetic variants of β-carotene 15,15'-monooxygenase 1.     

Current Efavirenz (EFV) or Ritonavir-Boosted Lopinavir (LPV/r) Use Correlates with Elevate Markers of Atherosclerosis in HIV-Infected Subjects in Addis Ababa,Ethiopia

Background

HIV patients on antiretroviral therapy have shown elevated incidence of dyslipidemia, lipodystrophy, and cardiovascular disease (CVD). Most studies, however, focus on cohorts from developed countries, with less data available for these co-morbidities in Ethiopia and sub-Saharan Africa.

Methods

Adult HIV-negative (n = 36), treatment naïve (n = 51), efavirenz (EFV)-treated (n = 91), nevirapine (NVP)-treated (n = 95), or ritonavir-boosted lopinavir (LPV/r)-treated (n=44) subjects were recruited from Black Lion Hospital in Addis Ababa, Ethiopia. Aortic pressure, augmentation pressure, and pulse wave velocity (PWV) were measured via applanation tonometry and carotid intima-media thickness (cIMT) and carotid arterial stiffness, and brachial artery flow-mediated dilation (FMD) were measured via non-invasive ultrasound. Body mass index, waist-to-hip circumference ratio (WHR), skinfold thickness, and self-reported fat redistribution were used to quantify lipodystrophy. CD4+ cell count, plasma HIV RNA levels, fasting glucose, total-, HDL-, and LDL-cholesterol, triglycerides, hsCRP, sVCAM-1, sICAM-1, leptin and complete blood count were measured.

Results

PWV and normalized cIMT were elevate and FMD impaired in EFV- and LPV/r-treated subjects compared to NVP-treated subjects; normalized cIMT was also elevated and FMD impaired in the EFV- and LPV/r-treated subjects compared to treatment-naïve subjects. cIMT was not statistically different across groups. Treated subjects exhibited elevated markers of dyslipidemia, inflammation, and lipodystrophy. PWV was associated with age, current EFV and LPV/r used, heart rate, blood pressure, triglycerides, LDL, and hsCRP, FMD with age, HIV duration, WHR, and glucose, and cIMT with age, current EFV use, skinfold thickness, and blood pressure.

Conclusions

Current EFV- or LPV/r-treatment, but not NVP-treatment, correlated with elevated markers of atherosclerosis, which may involve mechanisms distinct from traditional risk factors.     

Nucleoid halo expansion indirectly measures DNA damage in single cells

A simple test has been developed that measures how much DNA damage has occurred in a single mammalian cell. The procedure is based on the microscopic examination of "halos" of nucleoids that adhere to coverslips. Nucleoids are produced by flowing salt solutions containing detergents over the attached cells. The nucleoid halos are thought to be a tangle of loops of free DNA that emanate from the remnants of the nucleus. When visualized by staining with ethidium bromide the nucleoid halos first expand, and then contract as the concentration of ethidium increases. Exposure of nucleoids to very low levels of DNA chain-breaking treatments results in the incremental expansion of the halos to a maximum of 15 microns or more. Our assay is based upon quantitating the degree of halo expansion. If intact cells are exposed to DNA-damaging treatments, then allowed increasing periods of post-treatment growth before forming nucleoids, the DNA repair processes result first in expanded and then in contracted halos. By admixing a supercoiled plasma DNA of known length (38 kb) to nucleoids with contracted halos, the fractional halo expansion and the fraction of surviving plasmid supercoils can be measured from the same solution. Use of photodynamic DNA damage showed that the halo expansion was 11.6 times more sensitive than plasmid relaxation. Use of gamma-irradiation showed that the halo expansion was 3.6 times more sensitive than plasmid relaxation. The latter value demonstrates that one break per 137,000 bp results in the expansion of the halos to 63% of their maximal value. We estimate that this method will detect about 5000 breaks per nucleus containing 5 x 10(9) bp.     

The Value of MRI in the Diagnosis of Primary Biliary Cirrhosis and Assessment of Liver Fibrosis

Objectives

To evaluate MRI findings in patients with primary biliary cirrhosis (PBC) and to determine the value of MRI in the diagnosis of PBC and assessment of liver fibrosis.

Materials and Methods

This study reviewed the prevalence of MRI abnormalities seen in 45 PBC patients in the past four years, including 33 patients who underwent liver biopsy. Correlation between the MRI findings and the pathological stage was determined.

Results

There were 33 patients who underwent liver biopsy. Twenty-five patients (75.8%) had non-homogeneous changes in the liver signal intensity, 25 (75.8%) had a periportal halo sign, and 29 (87.9%) had lymphadenopathy. The short axis of the enlarged lymph nodes was a mean of 1.2±0.3 cm. A strong positive correlation was observed between histological stage and the inhomogeneity of liver signal intensity (P<0.001). There were significant differences among the four histological stages based on the periportal halo sign (P=0.034), and the grading of the periportal halo sign was found to be significantly correlated with the histological stage (P<0.001). Grading of the periportal halo sign was significantly different at stage II versus III, and stage III versus IV; no significant difference was found between stages I and II. There were also no significant differences among the four histological states in the occurrence and size of enlarged lymph nodes (P=0.674 and P=0.394).

Conclusion

MRI is valuable in the diagnosis of PBC, and the periportal halo sign and liver signal intensity help to evaluate the degree of liver fibrosis.     

Brain Function Alterations in Patients with Diabetic Nephropathy Complicated by Retinopathy Under Resting State Conditions Assessed by Voxel-Mirrored Homotopic Connectivity

Objective: The voxel-mirrored homologous connection (VHMC) technique was applied to detect resting brain function alterations in patients with diabetic nephropathy and retinopathy (DNR), and their relationships with clinical manifestations in the kidneys and eyes are discussed.Methods: Twenty-two patients with DNR and 22 healthy controls (HCs) similarly matched in age, sex, and educational background were recruited. Resting-state functional magnetic resonance imaging scans were performed for all subjects. Retinal fundus photography and renal biopsy were employed to observe the clinical features of the kidney and retina. Pearson correlation analysis was used to analyze the relationship between clinical manifestations and experimental results.Results: Compared with the HCs, patients with DNR showed decreased mean VMHC values in the bilateral middle temporal gyrus, bilateral middle occipital gyrus (BMOG), and bilateral medial frontal gyrus. The receiver operating characteristic curve analysis of each brain region confirmed that the accuracy of the area under the curve was excellent. The results showed that the average VHMC value of BMOG signals was positively correlated with the urinary protein to creatinine ratio in female subjects (r = 0.626; P<.05). Nonetheless, no such correlation was noted among the male subjects.Conclusion: There were significant changes in brain function in DNR patients compared to the control group. Changes in the central nervous system in patients with DNR were mainly due to the dual negative effects of kidney function and diabetes mellitus.Abbreviations: ACR = albumin/creatinine ratio; BMFG = bilateral medial frontal gyrus; BMOG = bilateral middle occipital gyrus; BMTG = bilateral middle temporal gyrus; DN = diabetic nephropathy; DNR = diabetic nephropathy complicated by retinopathy; DR = diabetic retinopathy; fMRI = functional magnetic resonance imaging; HC = healthy control; MRI = magnetic resonance imaging; PCR = protein to creatinine ratio; ROC = receiver operating characteristic; VHMC = voxel-mirrored homologous connection     

Vascular Dysfunction in Obstructive Sleep Apnea and Type 2 Diabetes Mellitus

Despite the high prevalence of obstructive sleep apnea (OSA) in type 2 diabetes mellitus (DM), the attributable vascular risk from each condition is unknown. We hypothesize that OSA may have a similar effect on vascular function as type 2 diabetes does. Healthy normal‐weight subjects, healthy obese subjects, subjects with type 2 diabetes, and obese subjects with OSA were enrolled. Vascular function was assessed with brachial artery ultrasound for flow‐mediated dilatation (FMD) and in skin microcirculation by laser Doppler flowmetry. One hundred fifty‐three subjects were studied: healthy normal‐weight controls (NCs) (n = 14), healthy obese controls (OCs) (n = 33), subjects with DM (n = 68), and obese subjects with OSA (n = 38). The DM group did not undergo sleep study and thus may have had subclinical OSA. The OSA and type 2 diabetes groups had impaired FMD as compared to both the normal‐weight and OC groups (5.8 ± 3.8%, 5.4 ± 1.6% vs. 9.1 ± 2.5%, 8.3 ± 5.1%, respectively, P < 0.001, post hoc Fischer test). When referenced to the NC group, a multiple linear regression model adjusting for covariates found that baseline brachial artery diameter (β = ?3.75, P < 0.001), OSA (β = ?2.45, P = 0.02) and type 2 diabetes status (β = ?2.31, P = 0.02), negatively predicted % FMD. OSA status did not seem to affect nitroglycerin‐induced vasodilation (endothelium‐independent) of the brachial artery or vascular function in the skin microcirculation. OSA impairs endothelial function in the brachial artery to a similar degree as type 2 diabetes does. OSA, however, does not appear to affect brachial endothelium‐independent vasodilation or skin microcirculatory function. Treatment of OSA in patients with concomitant type 2 diabetes, therefore, may be a potential therapeutic option to improve macro‐, but not microvascular outcomes.     

High mobility group box 1 levels in large vessel vasculitis are not associated with disease activity but are influenced by age and statins

IntroductionTakayasu arteritis (TA) and giant cell arteritis (GCA) are large vessel vasculitides (LVV) that usually present as granulomatous inflammation in arterial walls. High mobility group box 1 (HMGB1) is a nuclear protein that acts as an alarmin when released by dying or activated cells. This study aims to evaluate whether serum HMGB1 can be used as a biomarker in LVV.MethodsTwenty-nine consecutive TA patients with 29 healthy controls (HC) were evaluated in a cross-sectional study. Eighteen consecutive GCA patients with 16 HC were evaluated at the onset of disease and some of them during follow-up. Serum HMGB1 levels were measured by enzyme-linked immunosorbent assay.ResultsIn GCA patients at disease onset mean serum HMGB1 levels did not differ from HC (5.74 ± 4.19 ng/ml vs. 4.17 ± 3.14 ng/ml; p = 0.230). No differences in HMGB1 levels were found between GCA patients with and without polymyalgia rheumatica (p = 0.167), ischemic manifestations (p = 0.873), systemic manifestations (p = 0.474) or relapsing disease (p = 0.608). During follow-up, no significant fluctuations on serum HMGB1 levels were observed from baseline to 3 months (n = 13) (p = 0.075), 12 months (n = 6) (p = 0.093) and at the first relapse (n = 4) (p = 0.202). Serum HMGB1 levels did not differ between TA patients and HC [1.19 (0.45–2.10) ng/ml vs. 1.46 (0.89–3.34) ng/ml; p = 0.181] and no difference was found between TA patients with active disease and in remission [1.31 (0.63–2.16) ng/ml vs. 0.75 (0.39–2.05) ng/ml; p = 0.281]. HMGB1 levels were significantly lower in 16 TA patients on statins compared with 13 patients without statins [0.59 (0.29–1.46) ng/ml vs. 1.93 (0.88–3.34) ng/ml; p = 0.019]. Age was independently associated with higher HMGB1 levels regardless of LVV or control status.ConclusionsPatients with TA and GCA present similar serum HMGB1 levels compared with HC. Serum HMGB1 is not useful to discriminate between active disease and remission. In TA, use of statins was associated with lower HMGB1 levels. HMGB1 is not a biomarker for LVV.     

Effect of postural change on common femoral artery volume flow,measured by duplex ultrasound,in normal subjects and patients with peripheral vascular disease

Regulatory peripheral vasoconstriction occurs in response to adoption of the erect posture. Mildly ischaemic limbs are thought to exhibit near normal responses, but patients with rest pain show increases in blood flow on limb dependency. Previous methods of limb blood flow quantification (xenon clearance and venous occlusion plethysmography) have inherent difficulties when applied in these situations. We studied orthostatic responses in 12 normal subjects (aged 22–74 years (median 52)) and 16 patients (aged 21–83 (median 48)) with mild and severe peripheral vascular disease, using a duplex flowmeter system. In the normal subjects changes in the 60s mean, common femoral artery volume flow values were as follows (mlmin⁻¹ (1 SD)): 77 (83), − 78 (116) and − 190 (136), for elevation, dependency, and standing respectively. For claudicants (n = 7) the values were 18 (37), − 112 (123) and − 216 (103) respectively. In rest pain patients (n = 9) the responses were reversed, being − 252 (124), 131 (89) and 184 (85) respectively. Significant differences were apparent between elevation, dependency and standing flows in each of the three groups (P < 0.0001). Normal subject and claudicant responses on elevation and dependency differed significantly (P < 0.0001 and 0.03 respectively). On standing, the responses were similarly significantly different. The rest pain group displayed characteristically different responses compared with both normal subjects and claudicants for each postural change (P < 0.0001 in all cases). Duplex ultrasound volume flowmetry is non-invasive and offers an excellent method of quantifying physiological changes.     

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.