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G protein-coupled receptors (GPCRs) not only are turned on or off to control canonical G protein signaling but also may be fine-tuned to promote qualitative/biased signaling. Qualitative signaling by M3 muscarinic acetylcholine receptors (mAChRs) has been proposed, but its impact on physiologic systems remains unclear, and currently no biased M3 mAChR ligands have been described. Herein, we identify PD 102807 as a biased M3 ligand and delineate its signaling and function in human airway smooth muscle (ASM) cells. PD 102807 induced M3-mediated β-arrestin recruitment but not calcium mobilization. PD 102807 inhibited methacholine (MCh)-induced calcium mobilization in (M3-expressing) ASM cells. PD 102807 induced phosphorylation of AMP-activated protein kinase (AMPK) and the downstream effector acetyl-coenzyme A carboxylase (ACC). PD 102807- induced phosphorylated (p)-AMPK levels were greatly reduced in ASM cells with minimal M3 expression and were not inhibited by the Gq inhibitor YM-254890. Induction of p-AMPK and p-ACC was inhibited by β-arrestin 1 or GRK2/3 knockdown. Similarly, MCh induced phosphorylation of AMPK/ACC, but these effects were Gq dependent and unaffected by GRK2/3 knockdown. Consistent with the known ability of AMPK to inhibit transforming growth factor β (TGF-β)-mediated functions, PD 102807 inhibited TGF-β-induced SMAD-Luc activity, sm-α-actin expression, actin stress fiber formation, and ASM cell hypercontractility. These findings reveal that PD 102807 is a biased M3 ligand that inhibits M3-transduced Gq signaling but promotes Gq protein-independent, GRK-/arrestin-dependent, M3-mediated AMPK signaling, which in turn regulates ASM phenotype and contractile function. Consequently, biased M3 ligands hold significant promise as therapeutic agents capable of exploiting the pleiotropic nature of M3 signaling.

The treatment of aortic valve disease in children and adolescents requires an individualized approach to provide a long-term solution with optimal hemodynamic profile. The role of aortic leaflet reconstruction techniques is evolving.We retrospectively reviewed the charts of 58 patients who underwent aortic valve tricuspidalization either by an Ozaki procedure (neo-tricuspidalization) or single leaflet reconstruction between 2015 and 2019. Immediate operative results as well as hospital and short-term outpatient follow-up data were evaluated.Fifty-eight patients underwent leaflet reconstruction with 40 (69%) receiving a neo-tricuspidalization and 18 patients (31%) undergoing single leaflet reconstruction, using either a glutaraldehyde fixed autologous pericardium or tissue engineered bovine pericardium (CardioCel; Admedus, Queensland, Australia). The median age at the time of surgery was 14.8 years (interquartile range, 10.6-16.8 years). Twenty-three patients (40%) had isolated aortic regurgitation. The peak velocity across the aortic valve decreased from 3.4 ± 1.2 meters per second (m/s) preoperatively to 2.0 ± 0.4 m/s (P .001) after surgery and remained stable (2.2 ± 0.7 m/s) during a median echocardiographic follow-up of 14.1 months (7.2-20.1 months) for the whole cohort. Freedom from reoperation or moderate and greater aortic regurgitation at 1, 2, and 3 years was 94.2% ± 3.3%, 85.0% ± 5.8%, and 79.0% ± 8.0%, respectively, with no difference between the neo-tricuspidalization and single leaflet reconstruction groups (P = .635). There were 6 late reoperations (10%) of which 3 were due to endocarditis.Aortic leaflet reconstruction provides acceptable short-term hemodynamic outcomes and proves the utility of this technique as an adjunctive strategy for surgical treatment of aortic valve disease in children and young adults.

Background Missing data are common in end-of-life care studies, but there is still relatively little exploration of which is the best method to deal with them, and, in particular, if the missing at random (MAR) assumption is valid or missing not at random (MNAR) mechanisms should be assumed. In this paper we investigated this issue through a sensitivity analysis within the ACTION study, a multicenter cluster randomized controlled trial testing advance care planning in patients with advanced lung or colorectal cancer. Methods Multiple imputation procedures under MAR and MNAR assumptions were implemented. Possible violation of the MAR assumption was addressed with reference to variables measuring quality of life and symptoms. The MNAR model assumed that patients with worse health were more likely to have missing questionnaires, making a distinction between single missing items, which were assumed to satisfy the MAR assumption, and missing values due to completely missing questionnaire for which a MNAR mechanism was hypothesized. We explored the sensitivity to possible departures from MAR on gender differences between key indicators and on simple correlations. Results Up to 39% of follow-up data were missing. Results under MAR reflected that missingness was related to poorer health status. Correlations between variables, although very small, changed according to the imputation method, as well as the differences in scores by gender, indicating a certain sensitivity of the results to the violation of the MAR assumption. Conclusions The findings confirmed the importance of undertaking this kind of analysis in end-of-life care studies.

© 2017 Elsevier B.V. Atrazine (ATR) alters female reproductive functions in different animal species. Here, we analyzed whether ATR disturbs steroidogenic and ovulatory processes in hormone-stimulated human cumulus granulosa cells and mechanism of its action. Results showed that treatment of human cumulus granulosa cells with 20 μM ATR for 48 h resulted in lower FSH-stimulated estradiol and progesterone production. ATR reduced mRNA levels of aromatase (CYP19A1), steroidogenic acute regulatory protein (STAR) and luteinizing hormone/choriogonadotropin receptor (LHCGR). Addition of hCG 48 h after FSH and ATR treatment did not trigger maximal expression of the ovulatory genes amphiregulin (AREG) and epiregulin (EREG). Mechanistic experiments showed that ATR activated cPDE and decreased cAMP level. Addition of total PDE and specific PDE4 inhibitors, IBMX and rolipram, prevented ATR's action on CYP19A1 and STAR mRNA expression in FSH-stimulated human cumulus granulosa cells. This study suggests that ATR alters steroidogenesis and ovulatory process in human cumulus granulosa cells jeopardizing female reproduction.

Background In oncology, Health Care Professionals often experience conducting Advance Care Planning (ACP) conversations as difficult and are hesitant to start them. A structured approach could help to overcome this. In the ACTION trial, a Phase III multi-center cluster-randomized clinical trial in six European countries (Belgium, Denmark, Italy, the Netherlands, Slovenia, United Kingdom), patients with advanced lung or colorectal cancer are invited to have one or two structured ACP conversations with a trained facilitator. It is unclear how trained facilitators experience conducting structured ACP conversations. This study aims to understand how facilitators experience delivering the ACTION Respecting Choices (RC) ACP conversation. Methods A qualitative study involving focus groups with RC facilitators. Focus group interviews were recorded, transcribed, anonymized, translated into English, and thematically analysed, supported by NVivo 11. The international research team was involved in data analysis from initial coding and discussion towards final themes. Results Seven focus groups were conducted, involving 28 of in total 39 trained facilitators, with different professional backgrounds from all participating countries. Alongside some cultural differences, six themes were identified. These reflect that most facilitators welcomed the opportunity to participate in the ACTION trial, seeing it as a means of learning new skills in an important area. The RC script was seen as supportive to ask questions, including those perceived as difficult to ask, but was also experienced as a barrier to a spontaneous conversation. Facilitators noticed that most patients were positive about their ACTION RC ACP conversation, which had prompted them to become aware of their wishes and to share these with others. The facilitators observed that it took patients substantial effort to have these conversations. In response, facilitators took responsibility for enabling patients to experience a conversation from which they could benefit. Facilitators emphasized the need for training, support and advanced communication skills to be able to work with the script. Conclusions Facilitators experienced benefits and challenges in conducting scripted ACP conversations. They mentioned the importance of being skilled and experienced in carrying out ACP conversations in order to be able to explore the patients’ preferences while staying attuned to patients’ needs. Trial registration International Standard Randomised Controlled Trial Number registry 63110516 (ISRCTN63110516) per 10/3/2014.

Objectives This study investigates the effects of high glucose content on patients undergoing cold crystalloid versus cold blood cardioplegia in terms of early clinical results, functional myocardial recovery and ischaemia-reperfusion injury in patients undergoing repair of acyanotic cardiac lesions. Methods Patients were randomly assigned to receive either crystalloid (n = 31) or blood cardioplegia (n = 31). Early clinical results were assessed. Changes in left ventricular fractional shortening, arterial blood lactate levels, central venous saturation, cardiac Troponin I release and blood glucose concentration were measured during the first 24 h after ischaemia. Results There was no significant difference in clinical outcomes and postoperative complication rates between groups. The postoperative changes in left ventricular function, lactate levels, central venous saturation and Troponin I were not significantly different between groups. The use of crystalloid cardioplegia was associated with significant increases in serum glucose compared with blood cardioplegia. Conclusions A high glucose content blood cardioplegia does not show any advantage compared with crystalloid cardioplegia in terms of clinical outcomes, functional recovery and the degree of ischaemic injury in infants and children undergoing repair of acyanotic heart lesions. High glucose concentration of the cardioplegic solution might potentiate ischaemia-reperfusion injury and diminish the beneficial effects of blood cardioplegia.

Introduction. Diagnosis of neonatal coarctation of the aorta (CoA) still presents a challenge in routine practice because of absence of reliable morphologic and functional parameters for early detection of this congenital heart defect in newborns. Objective. The aim of this study is to identify easy obtainable two-dimensional echocardiographic parameters for detection of the CoA in newborns. Methods. Echocardiographic evaluation was performed in 30 newborns with CoA and 20 healthy neonates (control group). Measurements of the proximal transverse arch (PTA), distal transverse arch (DTA), isthmus, distance between the left common carotid artery (LCCA) at the origin of the left subclavian artery (LSA), were obtained by two-dimensional echocardiography. Aortic arch hypoplasia was defined using Mouleart, Karl and Mee criteria, and Z-value. Index 1 was calculated as a ratio of DTA and distance between origins LCCA-LSA, Index 2 was calculated as a ratio of the ascending aorta and the distance between LCCA-LSA origins, and Index 3 was calculated as a ratio of PTA and distance between LCCA-LSA origins. Results. Index 1 was significantly lower in patients with CoA in comparison with control group (0.50 vs. 1.39; p≤0.01). A cutoff point at 0.39, for Index 1, showed a sensitivity of 92% and specificity of 99% for the diagnosis of neonatal CoA, while cut off points at 0.69 and 0.44, for Index 2 and Index 3, showed the highest sensitivity and specificity for the diagnosis of CoA in newborns. Conclusion. By using these echo indexes, two-dimensional echocardiographic aortic arch measurement becomes a simple, reliable noninvasive method for the evaluation of aortic coarctation in newborns and may lead to earlier diagnosis and subsequent surgical correction.

Although recent data suggest aristolochic acid as a putative cause of Balkan endemic nephropathy (BEN), evidence also exists in favor of ochratoxin A (OTA) exposure as risk factor for the disease. The potential role of xenobiotic metabolizing enzymes, such as the glutathione transferases (GSTs), in OTA biotransformation is based on OTA glutathione adducts (OTHQ-SG and OTB-SG) in blood and urine of BEN patients. We aimed to analyze the association between common GSTA1, GSTM1, GSTT1, and GSTP1 polymorphisms and BEN susceptibility, and thereafter performed an in silico simulation of particular GST enzymes potentially involved in OTA transformations. GSTA1, GSTM1, GSTT1 and GSTP1 genotypes were determined in 207 BEN patients and 138 non-BEN healthy individuals from endemic regions by polymerase chain reaction (PCR). Molecular modeling in silico was performed for GSTA1 protein. Among the GST polymorphisms tested, only GSTA1 was significantly associated with a higher risk of BEN. Namely, carriers of the GSTA1*B gene variant, associated with lower transcriptional activation, were at a 1.6-fold higher BEN risk than those carrying the homozygous GSTA1*A/*A genotype (OR = 1.6; p = 0.037). In in silico modeling, we found four structures, two OTB-SG and two OTHQ-SG, bound in a GSTA1 monomer. We found that GSTA1 polymorphism was associated with increased risk of BEN, and suggested, according to the in silico simulation, that GSTA1-1 might be involved in catalyzing the formation of OTHQ-SG and OTB-SG conjugates.

Introduction. Total anomalous pulmonary venous connection (TAPVC) is a rare congenital heart disease and in some variants represents the only true surgical emergency in congenital heart surgery. Basic anatomical characteristic of this anomaly is an abnormal connection of pulmonary veins with systemic venous circulation. Although the results of TAPVC repair in infancy have been markedly improved in recent years, the recurrent pulmonary venous obstruction (RPVO) remains relatively frequent complication of surgical treatment. Objective. The aim of this study was a retrospective evaluation of TAPVC repair at a single institution, identifying the risk factors associated with the increased mortality and morbidity. Methods. Between January 2001 and January 2010, 43 consecutive patients underwent repair of TAPVC at the University Children’s Hospital, with median weight of 3.8 kg (1.8-13 kg). Median age at surgery varied from 5 days to 5 years. Distribution of TAPVC types was as follows: supracardiac 19 (44%), cardiac 12 (28%), infracardiac 9 (21%), and mixed 3 (7%). Eleven patients (26%) were emergencies due to obstructed drainage. Results. Early mortality was 9.30% (4/43). An average time of followup/survival for 95% interval of confidence was 101.6±6.7 months. Kaplan-Meier cumulative survival was 83.7±5.7%. Freedom from reintervention after 10 years was 87.2±0.5%. The principal reason for reintervention was RPVO. Conclusion. Preoperative obstruction is not a risk factor of early mortality and RPVO. Low body mass (below 2.5 kg) is the only identified risk factor of early mortality. Complex morphology of the confluens, particularly in a mixed type of TAPVR, is the main risk factor of RPVO development.

Introduction. Arterial switch operation (ASO) is a cardiosurgical method of choice for complete anatomical correction of transposition of great arteries. Improvement of this procedure has made considerably improved the outcome and long-term prognosis of children born with this complex congenital heart disease. Objective. The aim of this study was to estimate the success rate of ASO through retrospective analysis of mortality and late complications. Methods. This study included 57 children operated from 1st January 2005 until 31st December 2009. Parameters that could influence the outcome of surgery were investigated. The following late complications were investigated: neopulmonary artery stenosis, neoaortic stenosis and regurgitation, as well as clinical signs of heart failure. Results. Early postoperative mortality was 15.8% (9/57 patients). During follow-up (8 to 72 months, average 36.5 months) there were no lethal outcomes. On the last echocardiography examination, 73.2% patients had neoaortic regurgitation and 67.4% patients had neopulmonary regurgitation, but all of them were mild in intensity. Neopulmonary stenosis had 32.6% of patients, but only two had moderate or severe stenosis. No one had ischemic ECG changes. Three reinterventions were performed due to serious residual problems: surgical correction of neoaortic stenosis, surgical correction of neopulmonary stenosis and transcatether balloon dilatation for aortic recoarctation. At the end of the follow-up period, only one of 46 consistently followed patients had signs of heart failure which required therapy (2.2%), while the majority of patients were without any symptoms and with good effort tolerance. Conclusion. Arterial switch operation has been successfully performed at our institution, with acceptable perioperative mortality and excellent late outcome.

Purpose To get more insight into molecular mechanisms underlying oxidative stress and its role in different types of seizure, in this study, oxidative byproducts of proteins, lipids and DNA, as well as, antioxidant enzyme activities were studied in adult patients with epilepsy. Methods Study was performed in 60 patients with epilepsy and in 25 healthy controls. Plasma protein reactive carbonyl derivatives (RCD) and protein thiol groups (P-SH), byproducts of oxidative protein damage, as well as antioxidant enzyme activities, superoxide dismutase (SOD) and glutathione peroxidase (GPX) were studied spectrophotometrically. Urinary 8-epi-prostaglandin F 2α (8-epi-PGF 2α ) and 8-hydroxy-2′-deoxyguanosine (8-OHdG), representative byproducts of lipid and DNA oxidative damage, respectively, were determined by enzyme immunoassay. Results RCD levels were significantly increased ( p =0.001), while P-SH content was decreased in patients with first seizure ( p =0.052) compared to controls, independently of the seizure type. Urinary 8-epi-PGF 2α and 8-OHdG were significantly increased in patients with epilepsy ( p =0.001 and p =0.001). Rise in 8-epi-PGF 2α was more pronounced in patients with generalized tonic–clonic seizure (GTCS) compared to those with partial seizure (PS). Both SOD and GPX activity were significantly increased in epileptic patients compared to controls ( p =0.001 and p =0.001), but only SOD activity was significantly higher in patients with GTCS than in those with PS. Conclusions Data on enhanced protein, lipid and DNA oxidation, together with upregulated antioxidant enzyme activities, confirm the existence of systemic oxidative stress in patients with epilepsy. It might be speculated that post-translational modification to existing functional proteins, particularly alterations to ion channels, might be at least partially responsible for acute early changes in neuronal networks.

Exposure to potential carcinogens is an etiologic factor for renal cell carcinoma (RCC) and transitional cell carcinoma (TCC) of the urinary bladder. Cytosolic glutathione S-transferases (GSTs) are a superfamily of enzymes that protect normal cells by catalyzing conjugation reactions of electrophilic compounds, including carcinogens, to glutathione. Some GST enzymes possess antioxidant activity against hydroperoxides. The most well characterized classes have been named alpha (GSTA), mu (GSTM), pi (GSTP) and theta (GSTT); each of these classes contains several different isoenzymes. Several types of allelic variation have been identified within classes, with GSTM1-null, GSTT1-null and GSTP1-Ile105/Ile105 conferring impaired catalytic activity. The effects of GSTM1 and GSTT1 polymorphism on susceptibility to RCC depend on exposure to specific chemicals. Individuals with the GSTM1-null genotype carry a higher risk for TCC. The roles of GSTT1 polymorphism in TCC and GSTP1 polymorphisms in both cancers are still controversial. During kidney cancerization, expression of GSTA isoenzymes tends to decrease, which promotes the pro-oxidant environment necessary for RCC growth. In the malignant phenotype of TCC of the bladder, upregulation of various GST classes occurs. Upregulation of GSTT1 and GSTP1 might have important consequences for TCC growth by providing a reduced cellular environment and inhibition of apoptotic pathways.

We aimed to discern the role of glutathione (GSH) associated enzymes in maintaining high GSH levels in renal cell carcinoma (RCC) of the clear cell type and analyze RCC enzyme antioxidant capacity. Since changes in cellular redox balance in RCC might also be related to alterations of glutathione S-transferase (GST) phenotype, GST class alpha and pi expression was also explored.Human kidney specimens of tumor and distant nontumor regions were obtained from 15 patients with RCC at the time of surgery. The activities of GSH-replenishing enzymes, gamma-glutamylcysteine synthetase (gamma-GCS), gamma-glutamyl transferase (gamma-GT), and glutathione reductase (GR), as well as the activities of antioxidant enzymes glutathione peroxidase (GPX) and catalase (CAT) were determined spectrophotometrically. GST alpha and pi class expression was determined by immunoblot.In the course of renal cancerization, significant changes appear in the activities of GSH-replenishing and antioxidant enzymes. The activity of the key enzyme of GSH synthesis, gamma-GCS, is up-regulated (P0.001), while the activities of gamma-GT and GR are down-regulated in renal tumors compared to nontumor tissue (P0.001 and P0.05, respectively). Activities of GPX and CAT were also down-regulated (P0.001 and P0.05, respectively) in RCC. Changes in enzyme antioxidant capacity in RCC were associated with decreased GST class alpha (P0.001) and unchanged GST pi expression at the protein level.Changes in redox status in RCC as a consequence of decreased enzyme antioxidant capacity, together with altered GST alpha expression, may be important factors in development and tumor growth. The up-regulation of gamma-GCS and high levels of GSH in RCC may be an attempt to limit injury caused by oxidative stress.

To compare the total fluoroscopy time (FT) based on the fluoroscopy mode used-continuous vs. pulsed-in patients who underwent percutaneous nephrolithotomy (PCNL). The study cohort evaluated 111 patients who underwent PCNL by a single surgeon. Standard (continuous) fluoroscopy of 30 frames per second (fps) was used in the first 56 cases (SF group), while the next 55 consecutive cases were performed under pulsed fluoroscopy of two fps (PF group). The presence of surgeon's previous experience decreased the possible impact of the learning curve on the outcome. In both groups, using ultrasound in combination to fluoroscopy performed the renal access. The stone complexity was determined using Guy's stone score (GSS). Complications were evaluated using Clavien-Dindo classification. Median FT was significantly lower in PF group (76.8 s) compared to SF group (155.4 s) (p

Objectives Our aim was to perform a comprehensive analysis of the antioxidant capacity of transitional cell carcinoma (TCC) of urinary bladder and discern the role of enzymes associated with glutathione (GSH) in maintaining high GSH levels in these tumours. Because the redox-sensitive protein glutathione S-transferase P1 (GSTP1) might provide an important link between high antioxidant capacity and inhibition of apoptotic pathways, we also explored how the redox state in tumour cells interacts with the expression of GSTP1. Methods We examined spectrophotometrically the specific activities of GSH-replenishing enzymes involved in GSH synthesis (γ-glutamylcysteine synthetase, γ-GCS), GSH regeneration (glutathione reductase, GR), and antioxidant protection (glutathione peroxidase, GPX; superoxide dismutase, SOD) in the cytosolic fraction of tumours and the surrounding normal tissue of 30 TCC patients. GSTP1-1 expression was also analyzed. Results We found a significant increase in the activity of both GSH-replenishing and antioxidant enzymes as well as enhanced GSTP1-1 expression in tumours in comparison with adjacent normal uroepithelium. Mean γ-GCS and GR activities in tumours were about 4- and 2-fold higher, respectively, than in corresponding normal tissue. Expression of GSTP1 correlated significantly with GSH level and γ-GCS and GR activities. GPX and SOD activities in TCC were also markedly increased. Conclusions Enhanced GSH-replenishing pathways account for increased GSH levels in TCC. Upregulated GPX and SOD also contribute to high antioxidant potential in TCC. Under such conditions, expression of redox-sensitive GSTP1 protein is upregulated.

Glutathione S-transferase (GST) isoenzyme profiles of non-tumor and tumor renal tissue of patients suffering from renal cell carcinoma (RCC) of the clear cell type were determined and compared to those of normal renal tissue. GST isoenzyme(s) were first separated on the basis of their affinity to glutathione sepharose 4B affinity column. Affinity-bound GSTs were further purified by anionic and cationic chromatofocusing. The results presented in this study show that non-tumor tissue distant from renal tumors and renal tumors have lower specific GST activity and a different isoenzyme profile than normal human kidney. Purification of normal kidney GSTs by affinity chromatography revealed the presence of two GST fractions: flow-through GST without affinity for glutathione affinity resin and GST fraction tightly bound to affinity matrix. In non-tumor kidney tissue of RCC patients, substantially less flow-through GST fraction was found, whereas renal tumors did not express flow-through GST at all. Isoelectric chromatofocusing indicated smaller numbers of GST isoenzymes in non-tumor and tumor kidney regions, with anionic forms dominating. It could be speculated that decreased expression of cationic GST isoenzymes (corresponding to class alpha) in non-tumor kidney tissue of RCC patients might be responsible for differences in sensitivity to specific carcinogens. The observations that RCCs are devoid of affinity flow-through GST and have small number of isoenzymes are further proof of low-level GST expression in RCC.

The efficiency of human recombinant epoetin in alleviating anemia in hemodialyzed patients has been well documented. However, the effects of rhEPO therapy in correction of antioxidant capacity are not completely explained. In this study we examined both extracellular (plasma) and intracellular (red blood cells) antioxidant potential in hemodialyzed patients before and after three and six months of epoetin treatment by evaluating markers of oxidative stress (malondialdehyde) and antioxidant capacity (thiol groups, superoxide dismutase, and glutathione peroxidase). Six months of treatment with epoetin was followed by significant increases in thiol groups, superoxide dismutase and glutathione peroxidase activities in both plasma and red blood cells of hemodialyzed patients. Hence, during accelerated erythropoiesis, an increase in the number of young hematopoietic cells may replenish erythrocyte superoxide dismutase and glutathione peroxidase activity. However, the consequences of an imbalance between enzymatic antioxidant system (higher superoxide dismutase and lower glutathione peroxidase activity) that exists in these patients are the very high red blood cell and plasma malondialdehyde levels. These results suggest that, in spite of epoetin treatment and improvement in red blood cells and plasma antioxidant capacity, the production of reactive oxygen species overwhelms the intracellular and extracellular antioxidant capacity.

An increased degree of oxidative stress (OS) in chronic renal failure (CRF) and a possible role of free radicals in CRF have already been described. However, data on OS after renal transplantation are scarce. The aim of the present study was to estimate the degree of OS in renal transplant patients. The study included four groups: 1) 15 haemodialysis patients (HD group), 2) 11 renal transplant patients with stable function (SF group), 3) 12 renal transplant patients with chronic biopsy-proven rejection (CR group), and 4) 10 healthy controls (C group). Markers of OS (malondialdehyde and thiol group levels) and antioxidant activity (glutathione peroxidase and Cu,Zn-superoxide dismutase) were determined in plasma and in red blood cells of all examined individuals. After successful renal transplantation a significant improvement, but not normalization, of antioxidant enzyme activities accompanied by significantly reduced lipid peroxidation were found. In the CR group the degree of OS was increased, and our results suggest that OS may be a relevant pathophysiological factor for CR development.

Background The presence of glutathione transferase (GST) M1 null genotype (GSTM1-null) in end-stage renal disease (ESRD) patients is associated with lower overall survival rate in comparison to those with GSTM1-active variants. We examined association between GSTM1 and GSTT1 deletion polymorphisms as well as SNPs in GSTA1/rs3957357 and GSTP1/rs1695 genes with overall and cause-specific cardiovascular mortality in ESRD patients. Methods Total of 199 patients undergoing hemodialysis were included in the study. Median value of time elapsed from dialysis initiation until the death, or the end of follow-up was 8 ± 5 years. The effect of GSTM1, GSTT1, GSTP1 and GSTA1 gene polymorphisms on predicting overall and specific cardiovascular outcomes (myocardial infarction, MI or stroke) was analyzed using Cox regression model, and differences in survival were determined by Kaplan-Meier. Results GSTM1-null genotype in ESRD patients was found to be independent predictor of overall and cardiovascular mortality. However, after false discovery rate and Bonferroni corrections this effect was lost. The borderline effect modification by wild-type GSTA1*A/*A genotype on associations between GSTM1-null and analyzed outcomes was found only for death from stroke. Homozygous carriers of combined GSTM1*0/GSTA1*A genotype exhibited significantly shorter time to death of stroke or MI in comparison with carriers of either GSTM1-active or at least one GSTA1*B gene variant. The best survival rate regarding cardiovascular outcome was found for ESRD patients with combined GSTM1-active and mutant GSTA1*B/*B genotype. Conclusions Combined GSTM1*0/GSTA1*A genotypes might be considered as genetic markers for cardiovascular death risk in ESRD patients, which may permit targeting of preventive and early intervention.

Inflammation plays a key role in the development of benign prostatic hyperplasia. Prostaglandin E2 (PGE2) is an important inflammation factor found in enlarged prostatic tissue that can be the main cause of inflammatory pain. The aim of this study was to investigate whether epidural anesthesia can block the negative effects of prostaglandin mediators during prostate surgery.The study included 60 patients who underwent open prostatectomy. All patients were randomly allocated to one of two study groups. The first group received general anesthesia and the second group a combination of general and epidural anesthesia. Main outcome measures were plasma concentration of PGE2, adrenaline, noradrenaline, and dopamine, before induction of anesthesia and at the time of enucleation.Preoperative serum concentrations of PGE2 were high in both groups. During enucleation, serum concentrations of adrenaline, noradrenaline, and dopamine increased, followed by a rise of systolic and diastolic blood pressure in the group of patients that received only general anesthesia. Serum concentration of PGE2 was at the same level as before induction of anesthesia in both groups.Epidural anesthesia blocks transmission of painful stimulus through the spinal cord caused by prostaglandin release and prevents the rise of catecholamines and blood pressure. Open prostatectomy can become a safer procedure performed under a combination of general and epidural anesthesia. Negative intraoperative effects of inflammatory prostate mediators during other techniques for prostate surgery could also be blocked with epidural anesthesia.

The study aimed to evaluate the results following complete repair of tetralogy of Fallot (TOF) in relation to age at surgery and to assess the role of palliation in the current era.A retrospective review of 251 consecutive patients with TOF repaired between 2003 and 2011 at the Great Ormond Street Hospital was performed. Children were divided into two groups: Group A, younger than 6 months (n = 78) and B, older than 6 months (n = 173). Early clinical outcomes and reoperation/reintervention rates were studied as well as indication for a palliation.There was 1 (0.4%) early and 1 (0.4%) late death after a median follow-up time of 4.5 years. Forty-three patients (17%) underwent repair after initial palliation with inter-stage mortality of 5%. Groups A and B were similar in terms of surgical approach, postoperative complications and length of stay. Significant differences were found in terms of more frequent use of a transannular patch (P = 0.05), longer surgeries (P = 0.02) and a greater proportion of palliated patients (P = 0.002) in older patients. There was no difference in rates of reoperation/reintervention between groups and following both primary and staged repair. Palliated patients were more symptomatic (duct-dependent pulmonary blood flow; P0.01, cyanotic spells; P0.01), had more extracardiac/genetic anomalies (P0.01), coronary anomalies (P = 0.015) and significantly smaller pulmonary annulus, right pulmonary artery (RPA) and left pulmonary artery (LPA) Z-scores (P0.01 for all).Age at complete repair was not linked to early clinical outcome or reoperation/reintervention rate. Palliative procedures postponed the timing of complete repair, but did not increase the reintervention rate.

To examine the relationship between biochemical markers and morphologic sperm characteristics, including head, neck, and tail changes.The study evaluated 154 patients who went to the Andrology Laboratory of the Clinic of Urology, Clinical Center of Serbia. Patients were divided into 4 groups: normozoospermic, oligozoospermic, severe oligozoospermic, and asthenozoospermic, according to the sperm concentration and motility.The differences in creatine kinase (CK) and CK-M levels between normozoospermic and the 2 groups of oligozoospermic patients were significantly different (P.01). The CK and CK-M levels correlated negatively with sperm concentration and sperm motility, but correlated positively with the pathologic sperm form. Patients with CK values0.093 have a total number of pathologic forms higher than 0.40 (87.5% sensitivity, 77.3% specificity, the area under the curve was 0.832, P.001). Patients with CK values0.09 U/L have normal spermatogenesis and pathologic disorder of the head15%, neck12%, and tail10%.The relation between sperm morphology and biochemical markers included in the maturation process is established during the sperm genesis process. If the results of these markers are used together with the morphology of the spermatozoa in the interpretation of infertility, it would lead us to better insight of the fertility potential of the each patient.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) represents one of the most common causes of myocardial ischemia in infants and if left untreated results in a high mortality rate. When ALCAPA coexists with other congenital malformations, particularly those associated with pulmonary hypertension, the initial presentation can be quite confusing and is often misinterpreted. We report an infant with ALCAPA associated with scimitar syndrome and aortic coarctation whose clinical course illustrates the complexities and difficulties of management with a successful outcome.

Background. Increased oxidative stress is a hallmark of end-stage renal disease (ESRD). Glutathione S-transferases (GST) are involved in the detoxification of xenobiotics and protection of oxidative damage. We hypothesized that genetic polymorphism in antioxidant enzymes GSTA1, GSTM1, GSTP1 and GSTT1 is more frequent in ESRD and modulates the degree of oxidative stress in these patients. Methods. GSTA1, GSTM1, GSTP1 and GSTT1 genotypes were determined in 199 ESRD patients and 199 age- and gender-matched controls. Markers of protein and lipid oxidative damage [thiol groups, carbonyl groups, advanced oxidative protein products, nitrotyrosine, malondialdehyde (MDA) and MDA adducts], together with total oxidant status and pro-oxidant antioxidant balance were determined. Results. Individual GST polymorphisms influence vulnerability to both protein and lipid oxidation, with GSTM1-null gene variant having the most pronounced effect. Furthermore, a strong combined effect of null/low-activity GSTM1, GSM, GSTA1 and GSTP1 genotypes in terms of susceptibility towards oxidative and carbonyl stress was found in ESRD patients. When patients were stratified according to GSTM1 and GSTT1, the highest oxidant damage was noted in those with the GSTM1-null/GSTT1-null genotype. The observed effect was even stronger in patients with the third low-activity GSTP1 or GSTA1 genotype. Finally, the level of oxidative and carbonyl stress was most pronounced in the subgroup of patients with all four null or low-activity GSTM1, GSTT1, GSTP1 and GSTA1 genotypes. Conclusions. According to the GST genotype, ESRD patients may be stratified in terms of the level of oxidative and carbonyl stress that might influence cardiovascular prognosis, but could also improve efforts towards individualization of antioxidant treatment.

Glutathione S-transferases (GSTs) are a family of enzymes involved in detoxification. Genes encoding for GSTA1, GSTM1, GSTP1, and GSTT1 proteins are polymorphic, which can result in complete or partial loss of enzyme activity. Previous studies have associated polymorphisms of GSTA1, GSTM1, and GSTP1 genes with a higher risk of bladder cancer, but this is still controversial. Potential role of GSTA1 polymorphism in susceptibility to bladder cancer in Whites is lacking. We examined association between GSTA1, GSTM1, GSTP1, and GSTT1 gene variants and bladder cancer risk and evaluated whether they were modified by smoking.A hospital-based case-control study recruited 201 incidence cases and 122 age-matched controls. Deletion polymorphism of GSTM1 and GSTT1 was identified by polymerase chain reaction method. Single nucleotide polymorphism of GSTA1 and GSTP1 was identified by restriction fragment length polymorphism method. Uniconditional multivariate logistic regression was applied to model association between genetic polymorphisms and bladder cancer risk, as well as effect modification by smoking.No significant difference was observed in the distributions of GSTM1, GSTT1, GSTA1, and GSTP1 gene variants between patients and controls. None of the examined polymorphisms was significantly associated with bladder cancer risk independently. The results of gene-smoking interaction analyses indicated a significant combined effect of smoking and all common GST polymorphisms tested (P for trend = 0.001). However, the most significant effect on bladder cancer risk was observed in smokers carrying lower activity GSTA1-AB/BB and GSTM-null genotype (OR = 3.5, P0.05) compared with GSTA1-AA and GSTM1-active non-smokers. Overall, the risk observed did not significantly differ with respect to quantity of cigarettes smoked. However, heavy smokers with GSTM1-null genotype had 2 times higher risk of bladder cancer than GSTM1-null light smokers (OR = 4.8 vs. OR = 2.0) when GSTM1-active non-smokers served as reference group. Smokers carrying both GSTM1-null and GSTA1-AB + BB genotypes exhibited the highest risk of bladder cancer (OR = 2.00, P = 0.123).Null or low-activity genotypes of the GSTA1, GSTM1, GSTT1, and GSTP1 did not contribute independently towards the risk of bladder cancer in our patients. However, in association with smoking, both low activity GSTA1 and GSTM1-null genotype increase individual susceptibility to bladder cancer.

Objectives One-stage repair of transposition of great arteries (TGA) and aortic arch obstruction (AAO) is currently advocated, but carries formidable surgical challenges. This report presents our experience and re-interventions for residual lesions over the last 10 years. Methods Twenty-two patients (19.5 ± 42.4 days; range 2-206; median 10 days, 3.5 ± 0.6 kg) diagnosed with TGA (nine patients) or double outlet right ventricle (DORV) (13 patients) and AAO underwent one-stage repair. Of the nine TGA patients (two with intact ventricular septum), AAO were: two patients hypoplastic arch, one patient discrete coarctation, four patients hypoplastic arch with coarctation and two patients interrupted aortic arch. The 13 DORV patients were all of Taussig-Bing type and one showed multiple ventricular septal defects (VSDs). The degree of AAO ranged from hypoplastic arch in five patients, coarctation two patients, combined four patients and interrupted aortic arch (IAA) two patients. Arterial switch with Lecomte ± VSD repair was performed during cooling, and aortic arch repair was performed under deep hypothermic circulatory arrest (DHCA) (35 ± 14 min at 16.9 ± 0.7 °C). Our preference was to use homograft patch-plasty for arch and direct end-to-side anastomosis for coarctation repair. Aortic-cross-clamp time was 124 ± 24 min and cardiopulmonary bypass (CPB) time 215 ± 84 min. Results Early survival was 19/22 (86%) up to 30 days without mortality in the second half of our series. Three patients required extracorporeal membrane oxygenation (ECMO) support and renal support was needed in three and preferred permanent pace maker (PPM) implantation in two. Length of stay was 21.9 ± 22.1 days. There was one late death and overall survival was 18/22 (82%) for the follow-up period of 4.8 years (0.2-9.8 years). Eight patients (44%) required re-intervention for re-coarctation. Four patients required right ventricular outflow tract (RVOT)/pulmonary artery re-interventions. At follow-up, there was no requirement for aortic valve replacement, residual VSD closure and no evidence of ventricular dysfunction. Conclusions One-stage repair of TGA/DORV and AAO can be performed safely with a good survival rate. Three important lessons that we have learnt are as follows: (1) the subpulmonary VSD may have a perimembraneous component, (2) late re-coarctation is not infrequent and (3) late residual right-sided cardiac lesions remain an issue in complex TGA repair.

Objective:We report the mid-term follow-up of patients,who underwentarterialswitch operation (ASO) for transposition of the great arteries (TGA) with intact ventricular septum and left-ventricular outflow tract obstruction (LVOTO) over a 10-year period from 2000 to 2009. Methods: Thirteen TGA patients (3.9% of our ASO cohort) with intact ventricular septum and LVOTO underwent ASO. LVOTO was defined as pulmonary valve z-score �� 2.0 (n = 3) or peak LVOT gradient � 40 mmHg with (n = 7) or without (n = 3) anatomic subvalvar stenosis on echocardiography. Median age and weight were 14 days (range, 7—130 days) and 3.2 kg (range, 2.1—4.6 kg). The LVOT abnormalities included fibromuscular narrowing (n = 5) and atrioventricular valve-related findings (n = 5). LVOT clearance was achieved by resection of accessory mitral tissue (n = 2) only. Results: Follow-up was 100% complete. There were no early or late deaths. Freedom from re-operation for neo-aortic valve regurgitation and/or LVOTO was 100% at a median follow-up of 38 months (range, 6—115 months). All patients had functional status appropriate for their age. Three patients had mild aortic regurgitation. The median Doppler estimated LVOTsystolic gradient was 12 mmHg (range, 0—18 mmHg) for the entire cohort at the latest follow-up. Conclusions: Mid-term outcomes of ASO for a highly selected group of patients with pulmonary valve annulus zscore �� 2.0 �� 0.4, resectable organic LVOTO, and dynamic peak LVOT gradient � 40 mmHg remain satisfactory, with a need for long-term follow-up.

Upregulated glutathione S-transferase P1 (GSTP1) plays an important role in the resistance to apoptosis in transitional cell carcinoma (TCC) of the urinary bladder (UB) and represents a potential target for chemotherapeutic agents. Our aim was to perform a systematic investigation of a glutathione S-transferase (GST) isoenzyme profile (GSTM, GSTP1, and GSTT1) in the upper urinary tract (UUT) TCC and compare it with the GST isoenzyme pattern of the UB TCC and normal urothelium. We examined GST activity spectrophotometrically by using substrates for the overall GST activity, GSTP1, and GSTT1 in the cytosolic fraction. GSTP1 and GSTM expression was analyzed by Western blotting. The results obtained have shown that the overall GST activity was significantly higher in UUT TCC in comparison with urothelium (P0.001), which gradually increases with tumor grade (P0.05). The mean GSTP1 and GSTT1 activities in UUT TCC were 2- and 3.6-fold higher, respectively, than in the normal urothelium (P0.001), and these values did not differ significantly from activities found in the UB TCC. GSTM was expressed in 42% of the UUT TCC and 50% of the UB TCC specimens. The level of GSTM expression was slightly increased in the UUT TCC specimens in comparison with normal urothelium (P0.05). We conclude that 3 major cytosolic GST classes, GSTM, GSTP1, and GSTT1, are expressed in the UUT TCC. The isoenzyme profile of GST in the UUT TCC is similar to that observed in the UB TCC; it shows essentially the same alteration of the GST phenotype in the course of cancerization. The association of GSTT1 and GSTP1 upregulation with the malignant phenotype of the UUT TCC might result in resistances to both chemotherapy and apoptosis.

BACKGROUND: Uncertainty exists on whether there is adjuvant benefit of percutaneous transluminal angioplasty (PTA) over supervised exercise and best medical therapy in the treatment of intermittent claudication. METHODS: Patients with symptoms of stable mild to moderate intermittent claudication (MIMIC) were randomised in two multi-centre trials, for femoropopliteal and aortoiliac arterial disease, to receive either PTA or no PTA against a background of supervised exercise and best medical therapy and followed up for 24 months. Initial claudication distance (ICD) and absolute walking distance (AWD) on treadmill were compared between randomised groups adjusting for the corresponding measure at baseline. Secondary outcomes included ankle-brachial pressure index (ABPI) and quality of life. FINDINGS: A total of 93 patients were randomised into the femoropopliteal trial (48 into PTA) and 34 into the aortoiliac trial (19 to PTA). The mean (standard deviation, SD) age was 66(9) years for the femoropopliteal trial (63% male) and 63(9) for the aortoiliac trial (65% male). At 24 months, there were significant improvements in both AWD and ICD in the PTA groups for both trials. The adjusted AWD was 38% greater in the PTA group for the femoropopliteal trial (95%; CI 1-90) (p=0.04) and 78% greater in the PTA group for the aortoiliac trial (95%; CI 0-216) (p=0.05). Further benefits were demonstrated for ABPI but not for quality of life. INTERPRETATION: PTA confers adjuvant benefit over supervised exercise and best medical therapy in terms of walking distances and ABPI 24 months after PTA in patients with stable mild to moderate intermittent claudication.

Glutathione S-transferase P1 (GSTP1) provides an important link between activity of regulatory stress kinases and apoptotic pathways. It can be hypothesized that up-regulated GSTP1, in TCC, might enhance apoptosis inhibition. We aimed to establish whether relationship between GSTP1 expression and executive (pro-caspase 3, cleaved caspase 3) and regulatory (Bcl-2) apoptotic pathways in TCC exists.Samples were obtained from 84 TCC patients (41 consecutive patient with muscle noninvasive and 43 consecutive patients with muscle invasive TCC tumors), who underwent surgery at the Institute of Urology and Nephrology, Clinical Centre of Serbia, during 2006 and 2007. Expression of GSTP1, pro-caspase 3 (CPP32), and Bcl-2, as well as cleaved caspase-3 labeling index (LI) were determined by immunocytochemistry. Levels of expression were correlated with tumor stage, grade, and invasiveness.GSTP1 protein expression was demonstrated in all tumor samples examined. According to GSTP1 status, all tumors were divided into groups with low, moderate, or high GSTP1 status. Expression of CPP32 and cleaved caspase 3 was positive in 80% of TCC patients. Their levels differed significantly between groups with various GSTP1 expression (P0.05), with the lowest CPP32 expression and cleaved caspase 3 LI in tumors with high GSTP1 status. Moreover, significant negative correlation was found between GSTP1 level and cleaved caspase 3 LI (r = -0.459, P = 0.041). The positive rate of Bcl-2 protein expression was 48%. Most of the Bcl-2 positive patients exhibited at the same time high GSTP1 positivity (P = 0.078). Significant association with tumor grade and stage was found for all examined parameters except for CPP32 regarding tumor grade.Based on results obtained, we conclude that enhanced GSTP1 expression in TCC of urinary bladder is associated with altered apoptotic pathways. Molecular interplay between GSTP1 and members of apoptotic cascade might, at least partially, play a role in development of invasive characteristics of TCC.

We aimed to study the relationship between markers of oxidative lipid or protein damage and ventricular remodeling and the validity of 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) as an indicator of disease severity in patients with ischemic chronic heart failure (CHF).We enrolled four groups of 12 patients with varying CHF according to the New York Heart Association (NYHA) classification and 25 controls. Urinary 8-epi-PGF(2alpha) and plasma malondialdehyde and protein thiol (P-SH) groups were correlated with echocardiographic indices of remodeling. The reliability of isoprostanes was analyzed by a receiver operating characteristics (ROC) curve.NYHA class III and IV patients exhibited elevated 8-epi-PGF(2alpha) levels, increased malondialdehyde concentrations and decreased P-SH groups when compared to controls and NYHA I and II patients. 8-Epi-PGF(2alpha) and P-SH groups correlated significantly with indices of remodeling. The ROC curve drawn for 8-epi-PGF(2alpha) allowed us to differentiate NYHA class III and IV patients from NYHA class I and II patients with a sensitivity of 95.8% and specificity of 95.8% (cut off 0.84 ng/mg creatinine; area under curve 0.99; P0.001).Markers of oxidative damage are unlikely to play a significant role in early stages of CHF. However, they might become important in the course of CHF when their concentrations reach critical levels. Urinary 8-epi-PGF(2alpha) is a reliable indicator of symptomatic CHF.

A 2-day-old term baby was referred to our hospital for an urgent cardiology assessment because of respiratory collapse and complete ‘white-out’ of a large left lung with mediastinal shift to the right ( Panel A ). At the time of admission the infant was on high-frequency oscillatory ventilation, inhaled nitric oxide, high-dose inotropes, and Prostin infusion. Subsequent echocardiographic examination revealed pulmonary atresia with …

To address the role of serum gamma-glutamyl transferase (GGT) as a marker of metastases in patients with renal cell carcinoma.Serum alkaline phosphatase and GGT were determined in 156 patients with localized renal cell carcinoma and 60 patients with metastases as proven by echosonography, computerized tomography and bone scan. The control group consisted of 50 healthy subjects matched for sex and age. Sensitivity and specificity of both enzymes as markers of metastatic disease were compared. In metastatic patients, enzyme activities were analyzed according to the site of metastases.Both alkaline phosphatase and GGT activities were normal in majority of patients with localized renal cell carcinoma and increased in most of the patients with metastatic disease (80% and 70%, respectively). GGT did not significantly differ from alkaline phosphatase in terms of sensitivity (70% vs 80%) and specificity (89% vs 92%). Concerning the site of metastases, high frequencies of increased GGT and alkaline phosphatase were found in patients with liver-only metastases (80% and 90%, respectively). All of the patients with both liver and bone metastases exhibited increased activity of both enzymes. Despite the fact that bone cells do not express GGT, increased activity was found in patients with bone metastases-only (45%), suggesting that enzymes might be released from tumor cells.Our data provided evidence that GGT is a sensitive marker of metastatic renal cell carcinoma. However, findings of abnormal GGT activity cannot specify the site of involvement.

Despite evidence that essential hypertension (EH) is a state of increased oxidative stress, the data on oxidative protein modifications is lacking. Besides, the role of extracellular antioxidant enzymes in EH has not been systematically studied. Study was performed in 45 subjects with EH and 25 normotensive controls. Patients were divided into three groups according to the 2003 ESH/ESC guidelines (grade 1-3). Plasma protein reactive carbonyl derivatives (RCD) and SH-groups (as byproducts of oxidative protein damage) as well as antioxidant enzyme activities superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase were studied spectrophotometrically and correlated with blood pressure (BP). RCD levels were increased in EH patients compared to controls and correlated significantly with both systolic blood pressure (SBP) (r = 0.495, P

To identify kidney glutathione S-transferase (GST) isoenzyme, which does not bind to glutathione affinity column, biochemical characterization was performed by using an array of substrates and by measuring sensitivity to inhibitors. Immunological characterization was done by immunoblotting. Affinity flow-through GST exhibited activity towards 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and cumene hydroperoxide, typical class alpha substrates and high sensitivity towards hematin, an alpha class inhibitor. It cross-reacted with antibodies against alpha class GST. Affinity flow-through GST in human kidney is an alpha class member.

Parenteral iron has been recommended for the treatment of iron deficiency in the majority of maintenance hemodialyzed (HD) patients. However, iron supplementation and consequent over saturation of transferrin and high iron levels, may aggravate oxidative stress already present in these patients. This study aimed to further clarify the role of repeated intravenous iron therapy as a supplementary cause of oxidative stress in HD patients. Markers of free radical activities (carbonyl reactive derivatives, CRD, thiol groups, SH, malondialdehyde, MDA) and antioxidant enzyme activities (superoxide dismutase, SOD and glutathione peroxidase, GPX) were determined in plasma and red blood cells (RBC) of 19 hemodialysis patients given a total iron dose of 625 mg (ferrogluconat, Ferrlecit, 62.5 mg). Blood samples were taken before the first and after the last dose of iron. Twenty apparently normal subjects served as healthy controls. Before iron treatment, HD patients exhibited increased concentrations of MDA and CRD in plasma and red blood cells, accompanied with impaired antioxidant capacity. All patients responded to iron therapy with a significant increase in their serum ferritin, serum iron, hemoglobin, and red blood cells levels. However, iron treatment resulted in enhanced oxidative stress in plasma of HD patients, since significant increase in plasma MDA and CRD concentrations, together with a decrease in nonprotein SH groups levels were detected. Supplementation with iron did not significantly influence plasma SOD and GPX activities, nor did any of the red blood cell parameters tested. Our data show that, despite improvement in hematological parameters, an increase in iron stores due to supplementation could also contribute to increased free radical production in HD patients.

To perform a systematic functional investigation of different glutathione S-transferase (GST) classes, including GST class Theta (GSTT) member GSTT1-1, in transitional cell carcinoma (TCC) and the surrounding normal uroepithelium of the same individuals. Recently, it was suggested that GSTT1-1 might be an important risk modulator for TCC.Tumor samples and surrounding normal uroepithelium were obtained from 24 patients with TCC of urinary bladder. The following substrates with differential specificities were used: 1-chloro-2,4-dinitrobenzene for overall GST activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST Alpha; 1,2-dichloro-4-nitro-benzene for GST Mu; 4-vinylpyridine for GST Pi 1-1(GSTP1-1); and 1,2-epoxy-3-(p-nitrophenoxy)propane for GSTT1-1.GSTP1-1 and GSTT1-1 activities were demonstrated in all uroepithelial and TCC samples, and GST Mu activity was detectable in 11 of 24 patients. In the tumor specimens, significant upregulation of all expressed GST subtypes was observed. The mean GSTP1-1 and GSTT1-1 level in TCC was increased 2-fold and 3.6-fold, respectively, compared with the mean level in the normal uroepithelium (P0.001). Tumor GSTT1-1 activities correlated statistically significantly with the tumor stage (P0.05).In tumors and adjacent normal uroepithelium of patients with TCC, three major cytosolic GST classes, Mu, Pi, and Theta, were expressed. Although the GST isoenzyme pattern in TCC was similar to that of the corresponding normal uroepithelium, during cancer progression a clear tendency toward an increase in all the GST subtypes expressed was noted. For the first time, distinct GSTT1-1 activity levels were demonstrated in human uroepithelium, as well as its pronounced upregulation in TCC.

The deleterious effects of free radicals in acute myocardial ischaemia/reperfusion are rather well known. However, the possibility that thrombolysis positively affects the recovery of blood antioxidant capacity in the later postinfarction period, and thus contributes to the better overall outcome of these patients, has not yet been investigated. We followed the time course of erythrocyte antioxidant activity in 45 patients with first acute myocardial infarction (AMI), who were treated with streptokinase. Success of thrombolysis was evaluated by noninvasive clinical signs of reperfusion using continuous vector cardiography. The patients were divided into two groups according to successful or unsuccessful, reperfusion, The control group consisted of 24 healthy subjects. Glutathione peroxidase (GPX) and superoxide dismutase (SOD) were determined immediately after admittance to the hospital (0 hours) and after subsequent thrombolytic therapy (1.5, 6, 12, and 24 hours after initiation of infusion of streptokinase), and 2, 4, and 8 days after AMI. Patients with AMI had decreased antioxidant enzyme activity at the time of admit- tance to the hospital, showing that the oxidative/antioxidative balance is disturbed early during the ischemic phase of AMI. In AMI patients without successful reperfusion, erythrocyte antioxidant enzyme activity remains low during the postinfarction period of 7 days. It can be concluded that prolonged ischemia reduces antioxidant enzyme activity. AMI patients with successful reperfusion have a significant rise in the activity of antioxidant enzymes within the first hours after thrombolysis, followed by a decrease until the third postinfarction day. During the subsequent postinfarction period, erythrocyte antioxidant activity gradually recovered and reached control levels. These beneficial effects of reperfusion on erythrocyte antioxidant status might contribute to the better overall prognosis of these patients.

Novel glutathione S-transferase (GST) isoenzymes, which do not bind to the glutathione (GSH) affinity column, were recently identified in dog kidney and dog renal cell lines. In humans, similar affinity flow-through GST has been previously found only in the urinary bladder. To ascertain whether these affinity flow-through GST isoenzymes also exist in the human kidney, we separated GST isoenzymes from five kidney samples on the basis of their affinity to GSH affinity resin. GSTs were further purified by anion exchange chromatography and chromatofocusing and characterized with specific substrates. Our results show that the human kidney has both affinity flow–through GST isoenzymes and those which bind tightly to the GSH affinity column. Purification of affinity-bound GST resulted in a rich profile of different isoenzymes with balanced expression of both anionic and cationic forms. Affinity flow-through GST was represented by one isoenzyme (pI-7.9) in all kidney samples tested, but one kidney specimen also contained another GST isoenzyme (pI-7.0). Our results for the first time show the presence of GST isoenzymes that do not bind to GSH-affinity resin in the human kidney. Although the assessment of similarity between the human kidney and urinary bladder affinity flow-through GST requires further elucidation, it can be speculated that these particular GSTs may play an important role in providing protection against the common carcinogens.

Purpose Optimal analgesia in ambulatory urology patients still remains a challenge. The aim of this study was to examine if the pre-emptive use of intravenous tramadol can reduce pain after ureteroscopic lithotripsy in patients diagnosed with unilateral ureteral stones. Materials and Methods This prospective pilot cohort study included 74 patients diagnosed with unilateral ureteral stones who underwent ureteroscopic lithotripsy under general anesthesia in the Urology Clinic at the Clinical Center of Serbia from March to June 2012. All patients were randomly allocated to two groups: one group (38 patients) received intravenous infusion of tramadol 100 mg in 500 mL 0.9%NaCl one hour before the procedure, while the other group (36 patients) received 500 mL 0.9%NaCl at the same time. Visual analogue scale (VAS) scores were recorded once prior to surgery and two times after the surgery (1 h and 6 h, respectively). The patients were prescribed additional postoperative analgesia (diclofenac 75 mg i.m.) when required. Pre-emptive effects of tramadol were assessed measuring pain scores, VAS1 and VAS2, intraoperative fentanyl consumption, and postoperative analgesic requirement. Results The average VAS1 score in the tramadol group was significantly lower than that in the non-tramadol group. The difference in average VAS2 score values between the two groups was not statistically significant; however, there were more patients who experienced severe pain in the non-tramadol group (p

Antioxidant enzyme activities, glutathione peroxidase (GSH-Px) and Cu, Zn superoxide dismutase (SOD-1), were investigated in erythrocytes of non-dialyzed patients with varying degrees of chronic renal failure (CRF), and of patients on regular hemodialysis treatment. The results obtained have shown that GSH-Px and SOD-1 activities were significantly higher in erythrocytes from non-dialyzed CRF patients than in corresponding age-matched healthy controls. Antioxidant enzyme activities increase with the progression of chronic renal insufficiency, and the more pronounced augmentation in GSH-Px and SOD-1 activities were found in erythrocytes from the end-stage renal patients (with Ccr less than 20 ml/min). The increase of GSH-Px and SOD-1 activities seen in non-dialyzed CRF patients were abolished in patients undergoing regular hemodialysis treatment. We propose that the increased GSH-Px and SOD-1 activities could be a protective mechanism for the cells due to the hyperproduction of free radicals in chronic renal failure. The lowering of red blood cell antioxidant activity in uremic patients on chronic dialysis may contribute to the increased oxidative damage in uremia and in the development of uremic complications.

Reduced glutathione (GSH) levels were investigated in the erythrocytes and plasma of nondialyzed patients with varying degrees of renal insufficiency and also of patients on regular hemodialysis treatment. GSH levels were from 19 to 70% higher in the erythrocytes of examined patients as compared to their corresponding age-matched controls. A correlation was found between the degree of renal insufficiency and the erythrocyte GSH level. No variations in plasma GSH levels which could be related to the degree of renal deterioration were observed. A routine hemodialysis did not significantly affect erythrocyte and plasma GSH levels. No significant differences in GSH levels between anemic and nonanemic uremic patients were observed.

Adaptability to stress is a fundamental prerequisite for survival. Mitochondria are a key component of the stress response in all cells. For steroid-hormones-producing cells, including also Leydig cells of testes, the mitochondria are a key control point for the steroid biosynthesis and regulation. However, the mitochondrial biogenesis in steroidogenic cells has never been explored. Here we show that increased mitochondrial biogenesis is the adaptive response of testosterone-producing Leydig cells from stressed rats. All markers of mitochondrial biogenesis together with transcription factors and related kinases are up-regulated in Leydig cells from rats exposed to repeated psychophysical stress. This is followed with increased mitochondrial mass. The expression of PGC1, master regulator of mitochondrial biogenesis and integrator of environmental signals, is stimulated by cAMP-PRKA, cGMP, and β-adrenergic receptors. Accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. Here we propose that all events induced by acute stress, the most common stress in human society, provoke adaptive response of testosterone-producing Leydig cells and activate PGC1, a protein required to make new mitochondria but also protector against the oxidative damage. Given the importance of mitochondria for steroid hormones production and stress response, as well as the role of steroid hormones in stress response and metabolic syndrome, we anticipate our result to be a starting point for more investigations since stress is a constant factor in life and has become one of the most significant health problems in modern societies.

ObjectivesPreoperative comorbidities (PCMs) are known risk factors for Norwood stage I (NW1). We tested the hypothesis that short-term bilateral pulmonary arterial banding (bPAB) before NW1 could improve the prognosis of these high-risk patients.MethodsFrom January 2006 to October 2011, 17 high-risk patients with hypoplastic left heart syndrome (defined as having ≥4 of the following PCMs: prolonged mechanical ventilation; older age; sepsis; necrotizing enterocolitis; hepatic, renal, or heart failure; coagulopathy; pulmonary edema; high inotropic requirements; anasarca; weight

The pathogenicity of Chlamydia species is no longer questioned and most figures indicate that these organisms are the leading cause of sexually transmitted diseases. Diagnosis by direct examination of a stained slide is not acceptable due to inadequate sensitivity. The use of monoclonal antibodies conjugated to ITCF, however, is a good method to detect chlamydial particles. Cultures are made from specimens of heavily infected cells, centrifuged with cells from the McCoy cell line treated with cycloheximide. The inclusions are visualized using immunological techniques (immunofluorescence or staining with a peroxidase-antiperoxidase reaction). By using wells of microplates, this method is less expensive and can also be used for antimicrobial susceptibility testing. Serology (by MIF or ELISA) is very helpful, especially to investigate complications originating from a lower genital tract infection. Both techniques are useful in routine screening.

Background In invertebrates, the medicinal leech is considered to be an interesting and appropriate model to study neuroimmune mechanisms. Indeed, this non-vertebrate animal can restore normal function of its central nervous system (CNS) after injury. Microglia accumulation at the damage site has been shown to be required for axon sprouting and for efficient regeneration. We characterized HmC1q as a novel chemotactic factor for leech microglial cell recruitment. In mammals, a C1q-binding protein (C1qBP alias gC1qR), which interacts with the globular head of C1q, has been reported to participate in C1q-mediated chemotaxis of blood immune cells. In this study, we evaluated the chemotactic activities of a recombinant form of HmC1q and its interaction with a newly characterized leech C1qBP that acts as its potential ligand. Methods Recombinant HmC1q (rHmC1q) was produced in the yeast Pichia pastoris. Chemotaxis assays were performed to investigate rHmC1q-dependent microglia migration. The involvement of a C1qBP-related molecule in this chemotaxis mechanism was assessed by flow cytometry and with affinity purification experiments. The cellular localization of C1qBP mRNA and protein in leech was investigated using immunohistochemistry and in situ hybridization techniques. Results rHmC1q-stimulated microglia migrate in a dose-dependent manner. This rHmC1q-induced chemotaxis was reduced when cells were preincubated with either anti-HmC1q or anti-human C1qBP antibodies. A C1qBP-related molecule was characterized in leech microglia. Conclusions A previous study showed that recruitment of microglia is observed after HmC1q release at the cut end of axons. Here, we demonstrate that rHmC1q-dependent chemotaxis might be driven via a HmC1q-binding protein located on the microglial cell surface. Taken together, these results highlight the importance of the interaction between C1q and C1qBP in microglial activation leading to nerve repair in the medicinal leech.