Ara h 2-specific IgE epitope-like peptides inhibit the binding of IgE to Ara h 2 and suppress lgE-dependent effector cell activation.

BACKGROUND: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. METHODS: We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. RESULTS: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. CONCLUSIONS: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.

Treating Arterial Ageing in Patients with Diabetes: From Mechanisms to Effective Drugs.

Diabetes mellitus is a major healthcare problem. It is not only characterized by hyperglycemia and chronic complications, but in longer lasting diabetes and a longer living population, it is also associated with accelerated arterial ageing, which importantly contributes to cardiovascular complications. The accelerated arterial ageing in patients with diabetes should be considered separately from arterial ageing in patients without diabetes. Basic and clinical research have allowed better insight into the mechanisms of arterial ageing. In a simplified mechanistic way, it could be considered that the three tightly connected cornerstone characteristics of arterial ageing in patients with diabetes are: phenotypic presentation as endothelial dysfunction and arterial stiffness, and the underlying basic ageing-facilitating mechanism represented as the impaired expression of genetic longevity pathways. Currently, specific drugs for preventing/treating arterial ageing are not available. Therefore, we aimed to review the capacity of available drugs, particularly antidiabetic drugs, to interfere with the arterial ageing process. In the near future, these characteristics could help to guide therapy in patients with diabetes. Overall, it appears that arterial ageing could become a new target in diabetes. The expanding knowledge regarding the capability of antidiabetic drugs and other available drugs to inhibit/delay arterial aging is therefore essential.

Expression of Longevity Genes Induced by a Low-Dose Fluvastatin and Valsartan Combination with the Potential to Prevent/Treat "Aging-Related Disorders".

The incidence of aging-related disorders may be decreased through strategies influencing the expression of longevity genes. Although numerous approaches have been suggested, no effective, safe, and easily applicable approach is yet available. Efficacy of low-dose fluvastatin and valsartan, separately or in combination, on the expression of the longevity genes in middle-aged males, was assessed. Stored blood samples from 130 apparently healthy middle-aged males treated with fluvastatin (10 mg daily), valsartan (20 mg daily), fluvastatin-valsartan combination (10 and 20 mg, respectively), and placebo (control) were analyzed. They were taken before and after 30 days of treatment and, additionally, five months after treatment discontinuation. The expression of the following longevity genes was assessed: SIRT1, PRKAA, KLOTHO, NFE2L2, mTOR, and NF-κB. Treatment with fluvastatin and valsartan in combination significantly increased the expression of SIRT1 (1.8-fold; p < 0.0001), PRKAA (1.5-fold; p = 0.262) and KLOTHO (1.7-fold; p < 0.0001), but not NFE2L2, mTOR and NF-κB. Both fluvastatin and valsartan alone significantly, but to a lesser extent, increased the expression of SIRT1, and did not influence the expression of other genes. Five months after treatment discontinuation, genes expression decreased to the basal levels. In addition, analysis with previously obtained results revealed significant correlation between SIRT1 and both increased telomerase activity and improved arterial wall characteristics. We showed that low-dose fluvastatin and valsartan, separately and in combination, substantially increase expression of SIRT1, PRKAA, and KLOTHO genes, which may be attributed to their so far unreported pleiotropic beneficial effects. This approach could be used for prevention of ageing (and longevity genes)-related disorders.

Silver Fir (Abies alba) Extracts Inhibit Enzymes Involved in Blood Glucose Management and Protect against Oxidative Stress in High Glucose Environment.

The diet rich in fruits and vegetables reduces the risk of metabolic syndrome, including diabetes development by various mechanisms of action, mainly due to the presence of polyphenolic compounds. Extracts from different conifer species are known to be a rich source of various polyphenols. In the present study we elucidated the in vitro mechanism of anti-diabetic activity of silver fir (Abies alba) wood and bark extracts and compared their activity to non-coniferous sweet chestnut wood extract and standardized maritime pine bark extract. Extracts and lignans were tested for their inhibitory activity of enzymes involved in the regulation of blood glucose in vitro. The ability of extracts to protect against oxidative stress in high glucose environment was tested on mouse myoblast cell line. Silver fir wood and bark extracts were shown to be effective inhibitors of α-glucosidase, α-amylase and dipeptidyl peptidase 4, three enzymes involved in the regulation of blood glucose levels. Coniferous extracts also showed protection against oxidative stress generated in high glucose environment. Lignans, particularly pinoresinol diglucoside, isolariciresinol and secolariciresinol were shown to be important contributors of antihyperglycemic activity through inhibition of dipeptidyl peptidase 4. This corroborates previously published in vivo results on blood glucose level obtained with silver fir wood extract and supports the use of silver fir wood and bark extracts as food supplements or functional foods in borderline diabetes.

Empagliflozin on top of metformin treatment improves arterial function in patients with type 1 diabetes mellitus.

BACKGROUND: Deteriorated arterial function and high incidence of cardiovascular events characterise diabetes mellitus. Metformin and recent antidiabetic drugs, SGLT2 inhibitors, reduce cardiovascular events. We explored the possible effects of empagliflozin's effect on top of metformin treatment on endothelial function and arterial stiffness parameters in type 1 diabetes mellitus (T1DM) patients. METHODS: Forty T1DM patients were randomised into three treatment groups: (1) empagliflozin (25 mg daily), (2) metformin (2000 mg daily) and (3) empagliflozin/metformin (25 mg daily and 2000 mg daily, respectively). The fourth group received placebo. Arterial function was assessed at inclusion and after 12 weeks treatment by: endothelial function [brachial artery flow-mediated dilation (FMD), reactive hyperaemia index (RHI)], arterial stiffness [pulse wave velocity (PWV) and common carotid artery stiffness (ß-stiffness)]. For statistical analysis one-way analysis of variance with Bonferroni post-test was used. RESULTS: Empagliflozin on top of metformin treatment significantly improved endothelial function as did metformin after 12 weeks of treatment: FMD [2.6-fold (P < 0.001) vs. 1.8-fold (P < 0.05)] and RHI [1.4-fold (P < 0.01) vs. 1.3-fold (P < 0.05)]. Empagliflozin on top of metformin treatment was superior to metformin in improving arterial stiffness parameters; it significantly improved PWV and ß-stiffness compared to metformin [by 15.8% (P < 0.01) and by 36.6% (P < 0.05), respectively]. Metformin alone did not influence arterial stiffness. CONCLUSION: Empagliflozin on top of metformin treatment significantly improved arterial stiffness compared to metformin in T1DM patients. Endothelial function was similarly improved in all treatment groups. Empagliflozin seems to possess a specific capacity to decrease arterial stiffness, which could support its cardioprotective effects observed in large clinical studies. Trial registration Clinical trial registration: NCT03639545.

Preservative efficacy of selected antimicrobials of natural origin in a cosmetic emulsion.

OBJECTIVE: To investigate the efficacy of 13 antimicrobial substances (11 substances of natural origin and two conventional preservatives as controls) at two concentrations in an o/w cosmetic emulsion. METHODS: Cosmetic formulations were analysed for total aerobic bacterial count and total combined yeast count according to the European Pharmacopoeia 8.0. Challenge tests were performed according to the ISO 11930 standard. RESULTS: The total aerobic bacterial count, the total combined yeast count and the total aerobic mesophilic microorganisms were below the acceptable limit for all cosmetic formulations. Challenge test criterion A was fulfilled by all antimicrobials of natural origin, except levulinic acid in the challenge test with Candida albicans, Lactobacillus ferment in the challenge test with C. albicans and Pseudomonas aeruginosa, and grapefruit seed extract in the challenge test with C. albicans. Phenoxyethanol and the combination of methylparaben and propylparaben were inefficient at the minimum studied concentrations. CONCLUSION: The results offer important comparative data on the level of preservative efficacy within the group of antimicrobials of natural origin and in reference to some typical, widely used conventional preservatives. Further research must be encouraged regarding cosmetic over-preservation. This article is protected by copyright. All rights reserved.

The "Rise-Peak-Fall" Pattern of Time Dependency of the Cardiovascular Pleiotropic Effects of Treatment With Low-dose Atorvastatin, Losartan, and a Combination Thereof in Rats.

Treatment with low, subtherapeutic doses of statins and sartans expresses beneficial pleiotropic effects on the arterial wall. The present study explored whether these effects depend on treatment duration. Wistar rats were randomly divided into 4 groups and received low-dose atorvastatin, low-dose losartan, their combination, or saline (control) daily. After 4, 6, 8, and 10 weeks of treatment, the animals were anesthetized, blood samples taken, and hearts and thoracic aortas isolated. Thoracic aorta endothelium-dependent relaxation and parameters of the isolated heart exposed to ischemic-reperfusion injury were assessed along with blood serum parameters and vasoactive genes expression. Low-dose atorvastatin, losartan, and especially their combination showed the characteristic time dependency of all studied parameters (thoracic aorta relaxation, isolated heart parameters, C-reactive protein values, genes encoding endothelial nitric oxide synthase, and CD40). The peak in efficacy was observed after 6 weeks of treatment and subsequently steadily declined. The peak versus control values were significant for all measured parameters. Only a combination of atorvastatin and losartan increased nitric oxide and decreased asymmetric dimethylarginine. A characteristic time-dependent "rise-peak-fall" pattern of the cardiovascular pleiotropic effects of statins and sartans in subtherapeutic low doses was revealed. Evidently, resistance to the explored treatment occurs after a certain period.

Screening of Phenolic Compounds Reveals Inhibitory Activity of Nordihydroguaiaretic Acid Against Three Enzymes Involved in the Regulation of Blood Glucose Level.

In this work we have focused on the inhibition of three different enzymes with a role in postprandial glucose management: α-amylase, α-glucosidase and dipeptidyl peptidase 4. The assortment of 29 monomeric phenolic compounds was first screened at a single concentration. Next, the IC50 values of tested compounds were evaluated for compounds that considerably inhibited any of the enzymes. Nordihydroguaiaretic acid, a phenolic compound abundant in Creosote bush Larrea tridentata, possessed inhibitory activity for all tested enzymes. This in vitro mechanism of action supports traditional use of Creosote bush in diabetes treatment.

Coenzyme Q10 supplementation decreases statin-related mild-to-moderate muscle symptoms: a randomized clinical study.

BACKGROUND: Statin use is frequently associated with muscle-related symptoms. Coenzyme Q10 supplementation has yielded conflicting results in decreasing statin myopathy. Herein, we tested whether coenzyme Q10 supplementation could decrease statin-associated muscular pain in a specific group of patients with mild-to-moderate muscle symptoms. MATERIAL/METHODS: Fifty patients treated with statins and reporting muscle pain were recruited. The Q10 group (n=25) received coenzyme Q10 supplementation over a period of 30 days (50 mg twice daily), and the control group (n=25) received placebo. The Brief Pain Inventory (BPI) questionnaire was used and blood testing was performed at inclusion in the study and after 30 days of supplementation. RESULTS: The intensity of muscle pain, measured as the Pain Severity Score (PSS), in the Q10 group was reduced from 3.9±0.4 to 2.9±0.4 (P<0.001). The Pain Interference Score (PIS) after Q10 supplementation was reduced from 4.0±0.4 to 2.6±0.4 (P<0.001). In the placebo group, PSS and PIS did not change. Coenzyme Q10 supplementation decreased statin-related muscle symptoms in 75% of patients. The relative values of PSS and PIS significantly decreased (-33.1% and -40.3%, respectively) in the Q10 group compared to placebo group (both P<0.05). From baseline, no differences in liver and muscle enzymes or cholesterol values were found. CONCLUSIONS: The present results show that coenzyme Q10 supplementation (50 mg twice daily) effectively reduced statin-related mild-to-moderate muscular symptoms, causing lower interference of statin-related muscular symptoms with daily activities.

Identification of Acyl-Protein Thioesterase-1 as a Polysorbate-Degrading Host Cell Protein in a Monoclonal Antibody Formulation Using Activity-Based Protein Profiling.

Polysorbate (PS) degradation in monoclonal antibody (mAb) formulations poses a significant challenge in the biopharmaceutical industry. PS maintains protein stability during drug product's shelf life but is vulnerable to breakdown by low-abundance residual host cell proteins (HCPs), particularly hydrolytic enzymes such as lipases and esterases. In this study, we used activity-based protein profiling (ABPP) coupled with mass spectrometry to identify acyl-protein thioesterase-1 (APT-1) as a polysorbate-degrading HCP in one case of mAb formulation with stability problems. We validated the role of APT1 by matching the polysorbate degradation fingerprint in the mAb formulation with that of a recombinant APT1 protein. Furthermore, we found an agreement between APT1 levels and PS degradation rates in the mAb formulation, and we successfully halted PS degradation using APT1-specific inhibitors ML348 and ML211. APT1 was found to co-purify with a specific mAb via hitchhiking mechanism. Our work provides a streamlined approach to identifying critical HCPs in PS degradation, supporting quality-by-design principles in pharmaceutical development.

Low-dose atorvastatin, losartan, and particularly their combination, provide cardiovascular protection in isolated rat heart and aorta.

Statins and angiotensin receptor blockers at therapeutic doses have beneficial cardiovascular effects, which can be applied for cardiovascular protection. We explored whether low doses of atorvastatin, losartan, and particularly their combination, possess important pleiotropic vasodilatory effects. Wistar rats were treated daily with low-dose atorvastatin (2 mg/kg, n = 15), low-dose losartan (5 mg/kg, n = 15), their combination (n = 15), or saline (n = 15). After 4, 6, or 8 weeks the animals were anesthetized, blood samples taken, and their hearts and thoracic aortas isolated. Two kinds of experiments were performed: the measurement of coronary flow rate after ischemia/reperfusion myocardial injury and endothelium-dependent relaxation of thoracic aorta. In both models, maximal vasodilation activity was obtained in rats treated for 6 weeks. In the ischemia/reperfusion myocardial injury model, coronary flow increased (atorvastatin or losartan 1.9-fold, P < 0.01; combination 2.4-fold, P < 0.001) compared with controls. In the thoracic aorta model, endothelium-dependent relaxation significantly increased only in the combination group compared with the control group (up to 1.4-fold; P < 0.01). Simultaneously, we detected increased anti-inflammatory activity and increased nitric oxide concentration, but no changes in lipids and blood pressure. In a rat model we showed important vasodilatory activity of low-dose atorvastatin, losartan, and particularly their combination. The effects of the low-dose combination were accompanied by, and probably at least partly achieved by, anti-inflammatory and nitric oxide pathways. Overall, these results could be valuable for the development of new vascular protective strategies focusing on a low-dose regimen of statins and sartans, and particularly their combination.

Health-Related Quality of Life Assessment in Older Patients with Type 1 and Type 2 Diabetes.

Type 1 (T1D) and type 2 diabetes (T2D) are determinants of health-related outcomes including health-related quality of life (HRQOL). We aimed to determine differences in HRQOL between older adults with T1D and T2D and specific factors influencing HRQOL in this age group. This study used a cross-sectional design with 56 age- and HbA1c-matched T1D and T2D patients (aged 68.9 ± 7.8 years; 55% had T2D). We employed several validated questionnaires (Short Form-36 (SF-36) and the EuroQol-5 Dimensions/Visual Analog Scale (VAS)) to investigate the relationships between HRQOL domains and diabetes type, glycemic control, complications, and comorbidities. T1D was associated with better self-reported general health (assessed with the SF-36 general health domain (p = 0.048) and the EuroQol-5 VAS (p = 0.002), whereas no significant differences in the other SF-36 domains, self-reported diabetes distress, anxiety, or depression were found. Most HRQOL domains were not associated with HbA1c or the presence of diabetes complications. The most significant reduction in HRQOL was experienced by patients with higher BMIs, irrespective of the diabetes type. The obtained HRQOL data could be used in clinical settings for evidence-based patient education focused on specific subgroups of patients, as well as in national healthcare policies, e.g., interventions designed to alleviate obesity.

Efficacy, safety, and patient satisfaction with oral semaglutide: first single-centre clinical experience.

OBJECTIVE: To explore the effects of oral semaglutide on glycaemic parameters, body weight, and satisfaction in the first recipient patients with type 2 diabetes mellitus in Slovenia, in a real-world clinical practice setting. METHODS: The first consecutive adult patients with type 2 diabetes who were eligible for oral semaglutide treatment were included in this prospective, open-label interventional study. Patients received increasing doses of oral semaglutide and were evaluated at inclusion, at 1 month, then 3-5 months after starting treatment. Fasting blood glucose, glycosylated haemoglobin (HbA1c), body weight, patient satisfaction with oral semaglutide treatment (using the validated Treatment Satisfaction Questionnaire for Medication), and adverse effects, were analysed. Statistical analyses were performed using one-way analysis of variance, and, when significant interactions were found, Bonferroni post-hoc test. A P-value <0.05 was considered statistically significant. RESULTS: Twenty patients (11 male: 9 female; mean age, 59.9 ± 1.5 years; mean diabetes duration, 8.5 ± 1.4 years) were included. Oral semaglutide (7 and 14 mg) significantly decreased HbA1c (from 9.4 ± 0.3% to 8.2 ± 0.2% and 7.8 ± 0.2%, respectively) and fasting plasma glucose (from 11.2 ± 0.5 mmol/L to 9.2 ± 0.7 mmol/L and 8.9 ± 0.4 mmol/L, respectively). Oral semaglutide (14 mg) significantly decreased body weight (from 100.9 ± 2.7 kg to 92.7 ± 2.4 kg). Patients reported that treatment was easy to use and expressed high global satisfaction. Mild and transient, mostly gastrointestinal, adverse effects were reported in 10 patients. CONCLUSIONS: Oral semaglutide, the first oral glucagon-like peptide 1 receptor agonist, was effective and safe, and associated with high patient satisfaction, in its first recipients in Slovenia. The results are important for daily clinical practice involving patients with type 2 diabetes, however, due to the small study population, lack of placebo control, and short exposure to oral semaglutide, the effectiveness of oral semaglutide in clinical practice requires further investigation.

Combination of Experimental and Bioinformatic Approaches for Identification of Immunologically Relevant Protein-Peptide Interactions.

Protein-peptide interactions are an essential player in cellular processes and, thus, of great interest as potential therapeutic agents. However, identifying the protein's interacting surface has been shown to be a challenging task. Here, we present a methodology for protein-peptide interaction identification, implementing phage panning, next-generation sequencing and bioinformatic analysis. One of the uses of this methodology is identification of allergen epitopes, especially suitable for globular inhaled and venom allergens, where their binding capability is determined by the allergen's conformation, meaning their interaction cannot be properly studied when denatured. A Ph.D. commercial system based on the M13 phage vector was used for the panning process. Utilization of various bioinformatic tools, such as PuLSE, SAROTUP, MEME, Hammock and Pepitope, allowed us to evaluate a large amount of obtained data. Using the described methodology, we identified three peptide clusters representing potential epitopes on the major wasp venom allergen Ves v 5.

Effectiveness of a comprehensive telemedicine intervention replacing standard care in gestational diabetes: a randomized controlled trial.

AIMS: Telemedicine improves glycemic and perinatal outcomes when used as an adjunct to standard care in gestational diabetes (GDM). Little is known about its effectiveness when used instead of standard care. We aimed to compare the outcomes of telemedicine care and the standard care in women with GDM. METHODS: In a single-center, parallel, randomized controlled trial, women were randomized to: (1) a telemedicine group, sending glucose readings via an application installed on a smartphone and monthly individual video calls replacing on-site visits or (2) standard care group with routine monthly on-site visits. The primary outcome was the effectiveness of glycemic control. The secondary outcomes were gestational weight gain (GWG) and perinatal data, including birth weight, gestational age, the incidence of the offspring large for gestational age, preterm birth, preeclampsia and cesarean section. RESULTS: A total of 106 women were randomized to the telemedicine (n = 54) and the standard care group (n = 52). The telemedicine group demonstrated less postprandial measurements above the glycemic target (10.4% [3.9-17.9] vs. 14.6% [6.5-27.1]; p = 0.015), together with lower average postprandial glucose (5.6 ± 0.3 vs. 5.9 ± 0.4; p = 0.004). Percentage of cesarean section was lower in the telemedicine group (9 (17.3%) vs. 18 (35.3%); p = 0.038). CONCLUSIONS: Telemedicine offers an effective alternative to delivering care to women with GDM. Trial registration NCT05521893, ClinicalTrials.gov Identifier URL: https://www. CLINICALTRIALS: gov/ct2/show/NCT05521893?term=NCT05521893&draw=2&rank=1.

Immunochemical properties and pathological relevance of anti-ß2-glycoprotein I antibodies of different avidity.

Despite available treatment, there is still significant morbidity and mortality present among patients with the autoimmune thrombophilic condition termed 'antiphospholipid syndrome' (Espinosa, G. and Cervera, R. 2009. Morbidity and mortality in the antiphospholipid syndrome. Curr. Opin. Pulm. Med. 15:413.). High-avidity (HAv) anti-ß(2)-glycoprotein I (anti-ß(2)GPI) antibodies, shown to correlate with thrombotic events in patients, could represent the much needed improved prognostic marker. By studying their effect on crystalline annexin A5 shield on phospholipid surfaces (one of proposed pathogenic mechanisms), with the use of atomic force microscopy, the pathogenic potential of HAv anti-ß(2)GPI antibodies was confirmed. Furthermore, by using surface plasmon resonance and enzyme-linked immunosorbent assays, unique binding characteristics of HAv antibodies in comparison with low avidity antibodies were established. HAv anti-ß(2)GPI were confirmed to (i) recognize ß(2)-glycoprotein I in a solution, (ii) interact predominantly monovalently (much lower dependency on the antigen density) and (iii) form more stable complexes with the antigen. Since enzyme-linked immunosorbent assays currently used in routine diagnostics detect anti-ß(2)GPI antibodies of unknown avidity, our observations are potentially useful for the development of improved diagnostic tests capable of detecting clinically relevant antibodies.

HWGMWSY, an unanticipated polystyrene binding peptide from random phage display libraries.

Phage display is a powerful technique for the discovery of peptide ligands that bind to various targets; however, ambiguous results often appear. Peptide HWGMWSY has been isolated repeatedly in our laboratory and by other research groups dealing with different protein and nonprotein targets, which led to a hypothesis that it may be a target-unrelated peptide interacting with polystyrene plastic surfaces. We compared binding properties and amplification rate of phage clone displaying the peptide HWGMWSY, a previously confirmed plastic binding clone WHWRLPS, and a control phage clone ASVQERK. An enzyme-linked immunosorbent assay and a phage elution assay confirmed that phage clone HWGMWSY binds to polystyrene. Surface plasmon resonance measurements on the other hand excluded the possibility of binding to bovine serum albumin, a common blocking agent in phage display experiments. Amplification rates of the above-noted phage clones were not statistically different. We therefore conclude that phage clone HWGMWSY was isolated in different selection procedures as a result of its affinity to polystyrene.

Associations among different functional and structural arterial wall properties and their relations to traditional cardiovascular risk factors in healthy subjects: a cross-sectional study.

BACKGROUND: The arterial wall possesses several functional and structural properties that define arterial health. Once they become impaired, cardiovascular risk increases. We aimed to ascertain the pattern of correlations among different arterial wall properties and to explore their relations to traditional risk factors and cardiovascular risk stratification. To allow such an investigation a middle-aged healthy population was recruited. METHODS: This cross-sectional study included 100 healthy males (aged 41.9 ± 6.4 years). Pulse wave velocity (PWV), ß-stiffness and intima-media thickness (IMT) of the carotid artery, and brachial artery flow-mediated dilation (FMD) were measured by a standardized ultrasound approach. RESULTS: No correlation between FMD and IMT was found; only relatively poor correlations between PWV (or ß-stiffness) and FMD existed, as well as between PWV (or ß-stiffness) and IMT. PWV and ß-stiffness highly correlated. Unexpectedly, only weak associations between PWV, ß-stiffness, FMD, IMT and traditional risk factors were revealed. Hence, traditional risk factors (mainly age) explained only 10-50% of variability for PWV, ß-stiffness, FMD and IMT. Although the subjects had low cardiovascular risk according to their Framingham score, their arterial wall properties were already impaired, particularly FMD. CONCLUSIONS: In healthy middle-age males we found: i) absent or poor correlations among arterial stiffness, IMT and endothelial function; ii) a low impact of traditional risk factors on the studied variables, and iii) the presence of impaired arterial wall properties despite low calculated cardiovascular risk. These results provide a deepened understanding of arterial wall properties and could help to improve cardiovascular risk stratification.