[Impact of the Reflux Origin on the Clinical Stage and Surgical Decision in Primary Varicose Veins]. / Einfluss des Refluxursprungs auf die Hämodynamik, Klinik und Therapie der primären Varikose.

Reflux and recirculation in primary varicose veins are not yet completely understood, and the contribution of perforator veins is dual.Reflux origin was assessed as junctional (JP, reflux of the greater saphenous junction or groin recurrences) with/without suspect perforator veins (SPV), or perforator phenotype (PP, reflux from SPV only or for statistical purposes from the small saphenous vein). Flow direction and intensity were recorded under Valsalva (JP) or as spontaneous/under distal compression/decompression (SPV) and weighted with one/two points as reflux/reentry, respectively, in the case of SPV. We compared the origin and extent of axial reflux and diameter/flow direction of SPV with the clinical stage by multivariate analysis.Of 107 limbs, 68 presented with JP, 49 combined with SPV, and 39 with PP. CEAP C3-C6 was associated with the presence of SPV (JP and PP) in 45/65 (11/22) limbs with primaries (recurrences) or in 3/16 (0/4), p < 0.01 (p = 0.01), without SPV. C4-C6 at first manifestation, however, was more frequent in JP and axial reflux below the knee in 14/39 limbs (p = 0.01) or above the knee in 3/11 (p = 0.12) compared with PP (5/31). SPV flow at first manifestation was reentry in the case of JP and axial reflux below the knee (estimate -1.62, p = 0.02) or above the knee (0.29, p = 0.81) compared with PP, but diameter of the most dilated perforator vein was higher in the case of JP and axial reflux above the knee (estimate 0.20, p < 0.01) or below the knee (0.04, p = 0.30) compared with PP. Predominant SPV flow was reentry/reflux during peripheral compression/decompression, respectively (p = 0.009).The data suggest that the reflux origin and extent of axial reflux are associated with diameter/flow direction of SPV and clinical stage in primary varicose veins.

Targeting Cell Senescence and Senolytics: Novel Interventions for Age-Related Endocrine Dysfunction.

Multiple changes occur in hormonal regulation with aging and across various endocrine organs. These changes are associated with multiple age-related disorders and diseases. A better understanding of responsible underling biological mechanisms could help in the management of multiple endocrine disorders over and above hormone replacement therapy (HRT). Cellular senescence is involved in multiple biological aging processes and pathologies common in elderly individuals. Cellular senescence, which occurs in many older individuals but also across the lifespan in association with tissue damage, acute and chronic diseases, certain drugs, and genetic syndromes, may contribute to such endocrine disorders as osteoporosis, metabolic syndrome, and type II diabetes mellitus (T2DM). Drugs that selectively induce senescent cell removal, "senolytics", and drugs that attenuate the tissue-destructive secretory state of certain senescent cells, "senomorphics", appear to delay the onset or alleviate multiple diseases, including but not limited to endocrine disorders such as diabetes, complications of obesity, age-related osteoporosis, and cancers as well as atherosclerosis, chronic kidney disease, neurodegenerative disorders, and many others. Over thirty clinical trials of senolytic and senomorphic agents have already been completed, are underway, or are planned for a variety of indications. Targeting senescent cells is a novel strategy that is distinct from conventional therapies such as HRT, and thus might address unmet medical needs and can potentially amplify effects of established endocrine drug regimens, perhaps allowing for dose decreases and reducing side effects.

Prevalence of tetrahydrobiopterine (BH4)-responsive alleles among Austrian patients with PAH deficiency: comprehensive results from molecular analysis in 147 patients.

Phenylketonuria (PKU, MIM 261600) is an autosomal recessive disorder caused by mutations of the phenylalanine hydroxylase gene (PAH, GenBank U49897.1, RefSeq NM_000277). To date more than 560 variants of the PAH gene have been identified. In Europe there is regional distribution of specific mutations. Due to recent progress in chaperone therapy, the prevalence of BH4-responsive alleles gained therapeutic importance. Here we report the mutational spectrum of PAH deficiency in 147 unrelated Austrian families. Overall mutation detection rate was 98.6 %. There was a total of 62 disease-causing mutations, including five novel mutations IVS4 + 6T>A, p.H290Y, IVS8-2A>G, p.A322V and p.I421S. The five most prevalent mutations found in patients were p.R408W, IVS12 + 1G>A, p.R261Q, p.R158Q and IVS2 + 5G>C. Neonatal phenylalanine levels before treatment were available in 114/147 patients. Prediction of BH4-responsiveness in patients with full genotypes was exclusively made according to published data. Among the 133 patients needing dietary treatment, 28.4 % are expected to be BH4 "non-responsive", 4.5 % are highly likely BH4-responsive, 35.8 % are probably BH4-responsive while no interpretation was possible for 31.3 %. The mutation data reflect the population history of Austria and provide information on the likely proportion of Austrian PKU patients that may benefit from BH4-therapy.

Senescent Cells: A Therapeutic Target in Cardiovascular Diseases.

Senescent cell accumulation has been observed in age-associated diseases including cardiovascular diseases. Senescent cells lack proliferative capacity and secrete senescence-associated secretory phenotype (SASP) factors that may cause or worsen many cardiovascular diseases. Therapies targeting senescent cells, especially senolytic drugs that selectively induce senescent cell removal, have been shown to delay, prevent, alleviate, or treat multiple age-associated diseases in preclinical models. Some senolytic clinical trials have already been completed or are underway for a number of diseases and geriatric syndromes. Understanding how cellular senescence affects the various cell types in the cardiovascular system, such as endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells, progenitor cells, and cardiomyocytes, is important to facilitate translation of senotherapeutics into clinical interventions. This review highlights: (1) the characteristics of senescent cells and their involvement in cardiovascular diseases, focusing on the aforementioned cardiovascular cell types, (2) evidence about senolytic drugs and other senotherapeutics, and (3) the future path and clinical potential of senotherapeutics for cardiovascular diseases.

Developing advanced models of biological membranes with hydrogenous and deuterated natural glycerophospholipid mixtures.

Cellular membranes are complex systems that consist of hundreds of different lipid species. Their investigation often relies on simple bilayer models including few synthetic lipid species. Glycerophospholipids (GPLs) extracted from cells are a valuable resource to produce advanced models of biological membranes. Here, we present the optimisation of a method previously reported by our team for the extraction and purification of various GPL mixtures from Pichia pastoris. The implementation of an additional purification step by High Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) enabled for a better separation of the GPL mixtures from the neutral lipid fraction that includes sterols, and also allowed for the GPLs to be purified according to their different polar headgroups. Pure GPL mixtures at significantly high yields were produced through this approach. For this study, we utilised phoshatidylcholine (PC), phosphatidylserine (PS) and phosphatidylglycerol (PG) mixtures. These exhibit a single composition of the polar head, i.e., PC, PS or PG, but contain several molecular species consisting of acyl chains of varying length and unsaturation, which were determined by Gas Chromatography (GC). The lipid mixtures were produced both in their hydrogenous (H) and deuterated (D) versions and were used to form lipid bilayers both on solid substrates and as vesicles in solution. The supported lipid bilayers were characterised by quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR), whereas the vesicles by small angle X-ray (SAXS) and neutron scattering (SANS). Our results show that despite differences in the acyl chain composition, the hydrogenous and deuterated extracts produced bilayers with very comparable structures, which makes them valuable to design experiments involving selective deuteration with techniques such as NMR, neutron scattering or infrared spectroscopy.

Effects of an exercise program in children with cystic fibrosis: are there differences between females and males?

OBJECTIVE: To investigate the adaptive responses of an in-patient exercise program in children with cystic fibrosis (CF) and evaluate the effects of sex. STUDY DESIGN: In total, 158 female and 186 male subjects with CF (age, 12 to 43 years) were studied during a 6-week rehabilitation course. A maximal incremental cycling test was used to determine exercise capacity and responses after 6 weeks of exercise training. Measures included lung function, peak oxygen uptake, peak workload, and peak heart rate. RESULTS: Lung function values were lower in males (P < .05). Females had a lower aerobic capacity (P < .05) at the beginning and at the end of the exercise training program. Similar training effects (P > .05) were seen between sexes in peak oxygen uptake (mL/min, mL/kg/min) and peak heart rate (beats/min) but not in peak workload (Watts, W/kg). CONCLUSIONS: The exercise program improved the fitness level similarly in females and males with CF. Basic physiological sex differences were still seen at the beginning and end of the training, despite the better lung function in females. Moreover, the finding suggested that fitness level and not lung function determined the response to training in CF, with those who were less fit at baseline having the largest response to training.

GM1 gangliosidosis and Morquio B disease: expression analysis of missense mutations affecting the catalytic site of acid beta-galactosidase.

Alterations in GLB1, the gene coding for acid beta-D-galactosidase (beta-Gal), can result in GM1 gangliosidosis (GM1), a neurodegenerative disorder, or in Morquio B disease (MBD), a phenotype with dysostosis multiplex and normal central nervous system (CNS) function. While most MBD patients carry a common allele, c.817TG>CT (p.W273L), only few of the >100 mutations known in GM1 can be related to a certain phenotype. In 25 multiethnic patients with GM1 or MBD, 11 missense mutations were found as well as one novel insertion and a transversion causing aberrant gene products. Except c.602G>A (p.R201H) and two novel alleles, c.592G>T (p.D198Y) and c.1189C>G (p.P397A), all mutants resulted in significantly reduced beta-Gal activities (<10% of normal) upon expression in COS-1 cells. Although c.997T>C (p.Y333H) expressed 3% of normal activity, the mutant protein was localized in the lysosomal-endosomal compartment. A homozygous case presented with late infantile GM1, while a heterozygous, juvenile case carried p.Y333H together with p.R201H. This allele, recently found in homozygous MBD, gives rise to rough endoplasmic reticulum (RER)-located beta-Gal precursors. Thus, unlike classical MBD, the phenotype of heterozygotes carrying p.R201H may rather be determined by poorly active, properly transported products of the counter allele than by the mislocalized p.R201H precursors.

Surfactant proteins SP-B and SP-C and their precursors in bronchoalveolar lavages from children with acute and chronic inflammatory airway disease.

BACKGROUND: The surfactant proteins B (SP-B) and C (SP-C) are important for the stability and function of the alveolar surfactant film. Their involvement and down-regulation in inflammatory processes has recently been proposed, but their level during neutrophilic human airway diseases are not yet known. METHODS: We used 1D-electrophoresis and Western blotting to determine the concentrations and molecular forms of SP-B and SP-C in bronchoalveolar lavage (BAL) fluid of children with different inflammatory airway diseases. 21 children with cystic fibrosis, 15 with chronic bronchitis and 14 with pneumonia were included and compared to 14 healthy control children. RESULTS: SP-B was detected in BAL of all 64 patients, whereas SP-C was found in BAL of all but 3 children; those three BAL fluids had more than 80% neutrophils, and in two patients, who were re-lavaged later, SP-C was then present and the neutrophil count was lower. SP-B was mainly present as a dimer, SP-C as a monomer. For both qualitative and quantitative measures of SP-C and SP-B, no significant differences were observed between the four evaluated patient groups. CONCLUSION: Concentration or molecular form of SP-B and SP-C is not altered in BAL of children with different acute and chronic inflammatory lung diseases. We conclude that there is no down-regulation of SP-B and SP-C at the protein level in inflammatory processes of neutrophilic airway disease.

Biochemical and molecular characterization of 18 patients with pyridoxine-dependent epilepsy and mutations of the antiquitin (ALDH7A1) gene.

Patients with pyridoxine dependent epilepsy (PDE) present with early-onset seizures resistant to common anticonvulsants. According to the benefit of pyridoxine (vitamin B(6)) and recurrence of seizures on pyridoxine withdrawal, patients so far have been classified as having definite, probable, or possible PDE. Recently, PDE has been shown to be caused by a defect of alpha-amino adipic semialdehyde (AASA) dehydrogenase (antiquitin) in the cerebral lysine degradation pathway. The accumulating compound piperideine-6-carboxylic acid (P6C) was shown to inactivate pyridoxalphosphate (PLP) by a Knoevenagel condensation. Pipecolic acid (PA) and AASA are markedly elevated in urine, plasma, and cerebrospinal fluid (CSF) and thus can be used as biomarkers of the disease. We have investigated 18 patients with neonatal seizure onset, who have been classified as having definite (11), probable (four), or possible (three) PDE. All patients had elevated PA and AASA in plasma (and urine) while on treatment with individual dosages of pyridoxine. Within this cohort, molecular analysis identified 10 novel mutations (six missense mutations, one nonsense mutation, two splice site mutations) within highly conserved regions of the antiquitin gene. Seven mutations were located in exonic sequences and two in introns 7 and 17. Furthermore, a novel deletion of exon 7 was identified. Two of the 36 alleles investigated require further investigation. A known mutation (p.Glu399Gln) was found with marked prevalence, accounting for 12 out of 36 alleles (33%) within our cohort. Pyridoxine withdrawal is no longer needed to establish the diagnosis of "definite" PDE. Administration of pyridoxine in PDE may not only correct secondary PLP deficiency, but may also lead to a reduction of AASA (and P6C) as presumably toxic compounds.

Oxidative changes of bronchoalveolar proteins in cystic fibrosis.

Chronic bacterial infection and severe, polymorphonuclear neutrophil-dominated endobronchial inflammation are characteristic hallmarks of cystic fibrosis (CF) lung disease. The free radicals generated can be deleterious for structure and function of many proteins. The goal of this study was to investigate the degree of oxidation of pulmonary epithelial lining fluid proteins. BAL fluid (BALF) from 55 children with CF and from 11 patients in a control group were investigated by dot-blot assay for content and by two-dimensional electrophoresis and Western blotting for the pattern of distribution of oxidized proteins. The highest level of oxidative stress, as assessed by the level of protein carbonyls, was found in patients with FEV1 < 80% of predicted or with highly elevated neutrophil counts. Compared to control subjects without lung disease, CF patients with normal lung function and CF patients with a normal neutrophil count in their BALF had significantly higher protein carbonyl levels. The extent of protein oxidation was directly related to the neutrophil granulocyte count and inversely to lung function. Our data support the hypothesis that oxidative damage of pulmonary proteins during chronic and excessive neutrophilic endobronchial inflammation may contribute to the decline of lung function in CF patients.

Sequential analysis of surfactant, lung function and inflammation in cystic fibrosis patients.

BACKGROUND: In a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time. METHODS: As part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function (median FEV1 94% of predicted) at three times over a three year period. RESULTS: There was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV1, MEF(75/25%VC), and MEF(25%VC). The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period. CONCLUSION: Our findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease.

Intragenic deletions of ALDH7A1 in pyridoxine-dependent epilepsy caused by Alu-Alu recombination.

OBJECTIVE: To investigate the role of intragenic deletions of ALDH7A1 in patients with clinical and biochemical evidence of pyridoxine-dependent epilepsy but only a single identifiable mutation in ALDH7A1. METHODS: We designed a custom oligonucleotide array with high-density probe coverage across the ALDH7A1 gene. We performed array comparative genomic hybridization in 6 patients with clinical and biochemical evidence of pyridoxine-dependent epilepsy but only a single detectable mutation in ALDH7A1 by sequence analysis. RESULTS: We found partial deletions of ALDH7A1 in 5 of 6 patients. Breakpoint analysis reveals that the deletions are likely a result of Alu-Alu recombination in all cases. The density of Alu elements within introns of ALDH7A1 suggests susceptibility to recurrent rearrangement. CONCLUSION: Patients with clinical pyridoxine-dependent epilepsy and a single identifiable mutation in ALDH7A1 warrant further investigation for copy number changes involving the ALHD7A1 gene.

Morphology and Progression in Primary Varicose Vein Disorder Due to 677C>T and 1298A>C Variants of MTHFR.

BACKGROUND: Clinical assessment and prognostic stratification of primary varicose veins have remained controversial and the molecular pathogenesis is unknown. Previous data have suggested a contribution of the MTHFR (methylenetetrahydrofolate reductase) polymorphism c.677C>T. METHODS: We collected blood and vein specimens from 159 consecutive patients undergoing varicose vein surgery, or autologous vein reconstruction for arterial occlusive disease as controls. We compared the frequencies of c.677C>T and another polymorphism of MTHFR, c.1298A>C, with morphology and types of complicated disease. Morphology was recorded as a trunk or perforator type and peripheral congestive complication was defined as chronic venous insufficiency (CEAP C3-6) associated with edema and skin manifestations. FINDINGS: Multivariate analysis of genotypes for c.677C>T and c.1298A>C indicated that c.677C>T was associated significantly with the trunk phenotype (43/53 patients, 81%, p < 0.01), while c.1298A>C was associated significantly with the perforator phenotype (18/24 patients, 75%, p < 0.01) of primary varicose veins. Accordingly, when both c.677C>T and c.1298A>C displayed a heterozygous genotype, the patients were more likely to present with both phenotypes. Additionally, c.1298A>C was found to be strongly linked to the congestive complication (34/51 patients, 67%, p < 0.01). INTERPRETATION: Both polymorphisms of MTHFR may be involved in the morphological specification of primary varicose veins and contribute to the development of complicated disease. FUNDING: None.

Beta-defensins and LL-37 in bronchoalveolar lavage fluid of patients with cystic fibrosis.

BACKGROUND: The antimicrobial peptides human beta-defensin 1 and 2 (hBD-1 and 2) and the cathelicidin LL-37/hCAP-18 are key factors in innate immune responses of the respiratory tract. The aim of this study was to determine the concentrations of these peptides in airway surface fluid of CF patients with mild lung disease. METHODS: We measured the concentrations of hBD-1, hBD-2, and LL-37 in bronchoalveolar lavage fluid of 20 patients (5-34 years) participating in the prospective BEAT-study (bronchoalveolar lavage for the evaluation of anti-inflammatory treatment) using an immuno-dot blot-assay. RESULTS: All three peptides could be detected in lavage fluid of the study population. Increased levels of inflammatory markers in bronchoalveolar lavage fluid were associated with elevated concentrations of LL-37/hCAP-18 (total cell count, P = 0.006; relative neutrophil count, P = 0.002). Deterioration of lung function, measured by MEF25 (maximal flow rate at 25% of residual forced vital capacity), correlated with decreased hBD-2 (P = 0.026), but increased LL-37/hCAP-18 concentrations (P = 0.016). CONCLUSIONS: The data suggest that concentrations of antimicrobial peptides are correlated with severity of CF lung disease: Levels of LL-37/hCAP-18 are associated with bronchial inflammation and, therefore disease severity, whereas decreased levels of beta-defensins in advanced lung disease likely contribute to a secondary defect of the local host defense.

The 12-min walk test as an assessment criterion for lung transplantation in subjects with cystic fibrosis.

Timing for the evaluation and listing of patients with cystic fibrosis who are candidates for lung transplantation is still uncertain. Our study goal was to determine the value of the 12-min walk test as a simple and easy-to-use adjunctive assessment tool for pre-transplant evaluation. A total of 34 cystic fibrosis patients (17 male, mean age 22 years) with end-stage lung disease were evaluated in this retrospective analysis. The 12-min walk test was carried out according to an established protocol. Before walking, body plethysmography was performed and a capillary sample was taken for blood gas analysis. Walking distance and SaO(2) via continuous pulse oximetry were recorded online. There was a strong correlation between survival time and walking distance (r=0.7, P=0.003). No other single parameter, such as FEV(1), SaO(2,) pCO(2) or BMI, showed a statistically significant correlation with survival time. Subjects who walked < or =700 m had a lower cumulative survival (P=0.02). There was a statistically significant positive correlation between walking distance and SaO(2) at rest and after 12 min of walking (r=0.5, P=0.001; and r=0.5, P=0.04, respectively). There was no correlation between walking distance and pCO(2) at rest, BMI, FEV(1), or degree of change in SaO(2) during the walk test. This study demonstrates that in CF patients with end-stage lung disease, walking distance during a 12-min walk test is more informative with respect to survival than single parameters such as SaO(2,) pCO(2), FEV(1) or BMI.

Pyridoxine responsiveness in novel mutations of the PNPO gene.

OBJECTIVE: To determine whether patients with pyridoxine-responsive seizures but normal biomarkers for antiquitin deficiency and normal sequencing of the ALDH7A1 gene may have PNPO mutations. METHODS: We sequenced the PNPO gene in 31 patients who fulfilled the above-mentioned criteria. RESULTS: We were able to identify 11 patients carrying 3 novel mutations of the PNPO gene. In 6 families, a homozygous missense mutation p.Arg225His in exon 7 was identified, while 1 family was compound heterozygous for a novel missense mutation p.Arg141Cys in exon 5 and a deletion c.279_290del in exon 3. Pathogenicity of the respective mutations was proven by absence in 100 control alleles and expression studies in CHO-K1 cell lines. The response to pyridoxine was prompt in 4, delayed in 2, on EEG only in 2, and initially absent in another 2 patients. Two unrelated patients homozygous for the p.Arg225His mutation experienced status epilepticus when switched to pyridoxal 5'-phosphate (PLP). CONCLUSIONS: This study challenges the paradigm of exclusive PLP responsiveness in patients with pyridoxal 5'-phosphate oxidase deficiency and underlines the importance of consecutive testing of pyridoxine and PLP in neonates with antiepileptic drug-resistant seizures. Patients with pyridoxine response but normal biomarkers for antiquitin deficiency should undergo PNPO mutation analysis.

Surfactant protein a in cystic fibrosis: supratrimeric structure and pulmonary outcome.

BACKGROUND: The state of oligomerization of surfactant associated protein-A (SP-A) monomers differs between individuals. This likely affects SP-A's functional properties and could thereby influence clinical status in patients with lung diseases. In this study we focus on SP-A structure in cystic fibrosis (CF) compared to both healthy subjects and disease controls. METHODS: SP-A composition and function were assessed in both bronchoalveolar lavage (BAL) fluid and serum of 46 CF patients with mild disease, 25 patients with chronic bronchitis and 22 healthy subjects by gel chromatography and a functional agglutination assay. Relation of SP-A agglutination ability to disease severity of the subjects was explored. RESULTS: SP-A was present in seven major oligomeric forms with the majority of SP-A being structurally organized as complex oligomeric forms. More complex oligomeric forms were associated with better SP-A function with regard to its agglutination ability. These forms were more frequently observed in BAL than in serum, but there were no differences between disease groups. In CF patients, more complex forms of SP-A were associated with better lung function. CONCLUSIONS: Organizational structure of SP-A affects its functional activity and is linked to disease severity in CF.

Motor performance is better than normal in preschool children with cystic fibrosis.

The aim of the present study was to assess the motor performance in preschool children with a reliable and valid test battery developed to identify motor dysfunction and normal motor development in children aged from 4 to 6 years. Several aspects of motor performance were examined in 29 preschool children with cystic fibrosis (CF) age range 4-6 years (mean 5.2 +/- 0.8 years), FEV(1) 97.2 +/- 15.3pred and compared to with 22 healthy children of the same age 5.5 +/- 0.8 years. All children performed the "Motoriktest fuer 4-6jaehrige Kinder" (MOT) assessing seven different aspects of motor performance. Compared to healthy children, test score "Motor Quotient" (MQ) as the mean of all test items was significantly higher (P < 0.05) in children with CF (108.1 +/- 16 vs. 93.5 +/- 17.9). In both groups, the MQ can be classified as normal. Children with CF scored higher in MOT subtests "Agility and Coordination" (P < 0.05) and "Balance" (P < 0.01) than healthy children but not in the other subtests. We speculate that chest physiotherapy in preschool children with CF may have an effect on motor performance in general and in some aspects of motor performance.