V.E. Varga

Reference genes for quantitative real time PCR in UVB irradiated keratinocytes.

Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a sensitive and highly reproducible method often used for determining mRNA levels. To enable proper comparison of gene expression genes expressed at stabile levels within the cells in the studied experimental system need to be identified and used as reference. Ultraviolet B (UVB) radiation is an exogenous carcinogenic stimulus in keratinocytes, and UVB elicited changes have extensively been studied by qRT-PCR, yet a comparison of commonly used reference genes in UVB treatment is lacking. To find the best genes for compensating slight inter-sample variations in keratinocytes in UVB experiments and to understand the potential effects of improper reference gene (RG) selection we have analyzed the mRNA expression of 10 housekeeping genes in neonatal human epidermal keratinocytes (NHEK) after UVB treatment. The biological effect of the used UVB light source was validated by trypane blue exclusion, MTT and comet assays. 20-40mJ/cm(2) dose was chosen for the experiments. The stability of the 10 RGs was assessed by the GeNorm and Normfinder software tools. Regardless of their slightly different algorithm the programs found succinate dehydrogenase complex subunit A (SDHA) to be the best individual RG and SDHA and phosphoglycerate kinase-1 (PGK1) as the most suitable combination. Analysis of the expression of tumor necrosis factor alpha (TNFalpha) and vascular endothelial growth factor (VEGF) found that while the perception of changes in TNF-alpha, a gene undergoing marked upregulation after UVB irradiation is independent of the used RG, changes seen in the more modestly upregulated VEGF are greatly effected by reference gene selection. These findings highlight the importance of reference gene selection in UVB irradiation experiments, and provide evidence that using SDHA or the combination of SDHA and PGK1 as standards could be a reliable method for normalizing qRT-PCR results in keratinocytes after UVB treatment.

Photosensitivity skin disorders in childhood

Photosensitivity in childhood is caused by a diverse group of diseases. It usually indicates idiopathic photodermatoses, first of all polymorphic light eruption. It may be an early symptom of genetic disorders such as porphyria or very rare genophotodermatoses. Photosensitivity secondary to topical or systemic external agents as well as photoexacerbated dermatoses is not so frequent in childhood. Here we present our experience with childhood photosensitivity skin diseases collected over a 40‐year period.

Hemochromatosis (HFE) gene mutations and hepatitis C virus infection as risk factors for porphyria cutanea tarda in Hungarian patients

Aim: It is not clear whether the mutations in hemochromatosis (HFE) gene and hepatitis C virus (HCV) infection act independently in the pathogenesis of porphyria cutanea tarda (PCT). The prevalence of both risk factors varies greatly in different parts of the world. PCT patients from Hungary were evaluated to assess both factors.

Different phenotypes in identical twins with cerebrotendinous xanthomatosis: case series

Cerebrotendinous xanthomatosis (CTX) is a rare, genetically determined error of metabolism. The characteristic clinical symptoms are diarrhea, juvenile cataracts, tendon xanthomas and neuropsychiatric alterations. The aim of this study is to present a pair of identical adult twins with considerable differences in the severity of phenotype. With regards to neuropsychiatric symptoms, the predominant features were severe Parkinsonism and moderate cognitive dysfunctions in the more-affected individual, whereas these alterations in the less-affected patient were only very mild and mild, respectively. The characteristic increase in the concentrations of serum cholestanol and the lesion volumes in dentate nuclei in the brain assessed with magnetic resonance imaging were quite similar in both cases. The lifestyle conditions, including eating habits of the twin pair, were quite similar as well; therefore, currently unknown genetic modifiers or certain epigenetic factors may be responsible for the differences in severity of phenotype. This case series serves as the first description of an identical twin pair with CTX presenting heterogeneous clinical features.

The alteration of Irisin-Brain-derived neurotrophic factor axis parallels severity of distress disorder in bronchial asthma patients

Distress disorder (a collective term for generalized anxiety disorder and major depressive disorder) is a well-known co-morbidity of bronchial asthma. The irisin—brain-derived neurotrophic factor (BDNF) axis is a pathway that influences several neurobehavioral mechanisms involved in the pathogenesis of distress disorder. Thus, the aim of the present study was to quantify the serum irisin and BDNF concentrations in order to investigate the possible link between the irisin/BDNF axis and distress disorder in an asthma patient cohort. Data of 167 therapy-controlled asthma patients were analyzed. Demographic, anthropometric, and anamnestic data were collected, routine laboratory parameters supplemented with serum irisin and BDNF levels were determined, pulmonary function test was performed using whole-body plethysmography, and quality of life was quantified by means of the St. Georges Respiratory Questionnaire (SGRQ). Correlation analysis as well as simple and multiple linear regression were used to assess the relationship between the irisin level and the Impacts score of SGRQ, which latter is indicative of the presence and severity of distress disorder. We have found a significant, positive linear relationship between the Impacts score and the reciprocal of irisin level. This association was stronger in patients whose BDNF level was higher, and it was weaker (and statistically non-significant) in patients whose BDNF level was lower. Our results indicate that higher serum irisin level together with higher serum BDNF level are associated with milder (or no) distress disorder. This finding suggests that alteration of the irisin/BDNF axis influences the presence and severity of distress disorder in asthma patients.

Paraoxonase-1 arylesterase activity is an independent predictor of myeloperoxidase levels in overweight patients with or without cardiovascular complications.

OBJECTIVES Myeloperoxidase (MPO), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were shown to contribute to atherogenesis, while human paraoxonase-1 (PON1) protects against oxidative stress. Although several studies investigated these biomarkers, their associations have not been completely clarified yet. We aimed to investigate these parameters in overweight hyperlipidemic, lipid-lowering therapy-naive patients (n=167) with and without vascular complications. DESIGN AND METHODS MPO, MMP-9 and TIMP-1 levels were measured by ELISA. PON1 activities were detected spectrophotometrically. PON1 phenotype was calculated by using a dual substrate method. RESULTS Patients with vascular complications (VC) had significantly higher MPO and TIMP-1 levels compared to those without (patients with no vascular complications; NVC) (728 (367.25-1177.90) mg/ml vs. 315.9 (176.05-687.40) mg/ml; p<0.001; and 172.7 (157.7-197.7) ng/ml vs. 152.6 (129.3-172.3) ng/ml; p<0.0001; respectively). MPO levels showed a significant negative correlation with PON1 arylesterase activity (whole patient group (W): r=0.42, p<0.0001; VC: r=0.44, p=0.01; NVC: r=0.39, p<0.0001) and positive correlations with MMP-9 (W: r=0.37, p<0.0001; VC: r=0.29, p=0.07; NVC: r=0.42, p<0.0001) and TIMP-1 (W: r=0.42, p<0.0001; VC: r=0.33, p<0.05; NVC: r=0.41, p<0.0001), respectively. PON1 arylesterase activity was found to be an independent predictor of MPO levels in the whole patient group (β=-0.350, p<0.0001) or when studied separately in the subgroups with or without cardiovascular complications (VC: β=-0.57, p<0.05; NVC: β=-0.33, p<0.0001). CONCLUSIONS Our results suggest that parallel investigation of MPO, MMP-9 and TIMP-1 levels and PON1 arylesterase activity may be a more accurate indicator of atherosclerosis, which may allow earlier treatment and therefore, improvement of treatment efficacy.

Changes in serum afamin and vitamin E levels after selective LDL apheresis: VARGA et al.

Afamin is a plasma vitamin E‐binding glycoprotein partially associated with ApoA1‐containing high‐density lipoprotein (HDL) subfractions. In a previous study, the serum vitamin E decreased after low‐density lipoprotein (LDL) apheresis, while vitamin E/cholesterol ratio increased. We aimed to study the effect of LDL apheresis on serum afamin level.

Paraoxonase-1 and myeloperoxidase correlate with vascular biomarkers in overweight patients with newly diagnosed untreated hyperlipidaemia

BACKGROUND In hyperlipidaemic state, increased levels of myeloperoxidase (MPO) and decreased paraoxonase-1 (PON1) activity have been reported; however, their relationships with other atherosclerotic biomarkers have not been completely clarified. PATIENTS AND METHODS Serum concentrations of lipid and inflammatory parameters, MPO levels, and PON1 activities were investigated in 167 untreated hyperlipidaemic patients with and without vascular complications and in 32 healthy controls. Additionally, levels of CD40 ligand (sCD40L) and asymmetric dimethyl arginine (ADMA), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1, and oxidized LDL were determined. RESULTS We found elevated C-reactive protein (CRP), ADMA, sCD40L, sICAM-1 concentrations, and higher MPO levels in patients with vascular complications compared to those without. The PON1 arylesterase activity correlated negatively with sCD40L, ADMA, and sICAM-1 levels, respectively. In contrast, MPO concentrations showed positive correlations with sCD40L, ADMA, and sICAM-1 levels, respectively. CONCLUSIONS It can therefore be stated that PON1 activity and MPO level correlate strongly with the vascular biomarkers, highlighting the importance of the HDL-associated pro- and antioxidant enzymes in the development of endothelial dysfunction and atherogenesis.

Egy ritka, veleszületett neurodegeneratív betegség: a cerebrotendinosus xanthomatosis laboratóriumi diagnosztikája@@@Laboratory diagnosis of a rare congenital neurodegenerative disease: cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis is a rare neurodegenerative disease characterized by the accumulation of cholesterol and cholestanol in the brain and the tendons caused by mutations of the gene encoding sterol 27-hydroxylase (CYP27A1), which is involved in bile acid synthesis. The diagnosis is often missed and delayed because of the variable clinical presentation of the disease. Blood testing for cerebrotendinous xanthomatosis is routinely performed using gas chromatography-mass spectrometry measurement of elevated cholestanol level, and the diagnosis is confirmed by molecular genetic analysis. Early recognition and initiation of chenodeoxycholic acid therapy with hydoxymethyl‑glutaryl‑Coenzyme-A reductase inhibitors is critical to prevent irreversible neurological damage and permanent disability. The authors summarize the current knowledge about the pathomechanism, laboratory diagnosis and therapeutic options of cerebrotendinous xanthomatosis.

[Laboratory diagnosis of a rare congenital neurodegenerative disease: cerebrotendinous xanthomatosis].

Cerebrotendinous xanthomatosis is a rare neurodegenerative disease characterized by the accumulation of cholesterol and cholestanol in the brain and the tendons caused by mutations of the gene encoding sterol 27-hydroxylase (CYP27A1), which is involved in bile acid synthesis. The diagnosis is often missed and delayed because of the variable clinical presentation of the disease. Blood testing for cerebrotendinous xanthomatosis is routinely performed using gas chromatography-mass spectrometry measurement of elevated cholestanol level, and the diagnosis is confirmed by molecular genetic analysis. Early recognition and initiation of chenodeoxycholic acid therapy with hydoxymethyl‑glutaryl‑Coenzyme-A reductase inhibitors is critical to prevent irreversible neurological damage and permanent disability. The authors summarize the current knowledge about the pathomechanism, laboratory diagnosis and therapeutic options of cerebrotendinous xanthomatosis.