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Aims/hypothesis: The proportion of children with type 1 diabetes (T1D) who have experience with low-carbohydrate diet (LCD) is unknown. Our goal was to map the frequency of LCD among children with T1D and to describe their clinical and laboratory data., Methods: Caregivers of 1040 children with T1D from three centers were addressed with a structured questionnaire regarding the children's carbohydrate intake and experience with LCD (daily energy intake from carbohydrates below 26% of age-recommended values). The subjects currently on LCD were compared to a group of non-LCD respondents matched to age, T1D duration, sex, type and center of treatment., Results: A total of 624/1040 (60%) of the subjects completed the survey. A total of 242/624 (39%) subjects reported experience with voluntary carbohydrate restriction with 36/624 (5.8%) subjects currently following the LCD. The LCD group had similar HbA1c (45 vs. 49.5, p = 0.11), lower average glycemia (7.0 vs. 7.9, p = 0.02), higher time in range (74 vs. 67%, p = 0.02), lower time in hyperglycemia >10 mmol/L (17 vs. 20%, p = 0.04), tendency to more time in hypoglycemia <3.9 mmol/L(8 vs. 5%, p = 0.05) and lower systolic blood pressure percentile (43 vs. 74, p = 0.03). The groups did not differ in their lipid profile nor in current body height, weight or BMI. The LCD was mostly initiated by the parents or the subjects themselves and only 39% of the families consulted their decision with the diabetologist., Conclusions/interpretation: Low carbohydrate diet is not scarce in children with T1D and is associated with modestly better disease control. At the same time, caution should be applied as it showed a tendency toward more frequent hypoglycemia.

Background: Imaging methods bring new possibilities for describing the brain plasticity processes that underly the improvement of clinical function after physiotherapy in people with multiple sclerosis (pwMS). Although these processes have been described mainly in connection with task-oriented physiotherapy and aerobic training, they have not been properly verified in neuroproprioceptive "facilitation, inhibition" (facilitation) approaches., Aim: The study determined whether facilitation physiotherapy could enhance brain plasticity, compared two facilitation methods and looked for any relation to clinical improvement in pwMS., Design: The study was designed as parallel group randomized comparison of two kinds of physiotherapeutic interventions referred to healthy controls., Setting: Thirty-eight outpatients were involved in the study., Population: The study had 80 participants (38 pwMS and 42 healthy controls)., Methods: PwMS were divided into two groups and underwent a two-month physiotherapy program: Vojta reflex locomotion (VRL) or Motor program activating therapy (MPAT), (1 hour, twice a week). Functional magnetic resonance imaging (fMRI) and clinical examination was performed before and after therapy. Healthy controls underwent one fMRI examination., Results: Physiotherapy in pwMS leads to extension of brain activity in specific brain areas (cerebellum, supplementary motor areas and premotor areas) in connection with the improvement of the clinical status of individual patients after therapy (P=0.05). Greater changes (P=0.001) were registered after MPAT than after VRL. The extension of activation was a shift to the examined activation of healthy controls, whose activation was higher in the cerebellum and secondary visual area (P=0.01)., Conclusions: Neuroproprioceptive "facilitation, inhibition" physiotherapy may enhance brain activity and could involve processes connected with the processing of motion activation., Clinical Rehabilitation Impact: The study showed that facilitation approach can modulate brain activity. This could be useful for developing of effective physiotherapeutic treatment in MS.

Background : Only few studies have monitored the potential of physical activity training and physical therapy to modulate the reaction of the endocrine system. In this study, the effect of neuroproprioceptive facilitation and inhibition physical therapy on clinical outcomes and neuroactive steroids production in people with multiple sclerosis was evaluated. Moreover, we were interested in the factors that influence the treatment effect., Methods: In total, 44 patients with multiple sclerosis were randomly divided into two groups. Each group underwent a different kind of two months ambulatory therapy (Motor program activating therapy and Vojta's reflex locomotion). During the following two months, participants were asked to continue the autotherapy. Primary (serum level of cortisol, cortisone, 7α-OH-DHEA, 7β-OH-DHEA, 7-oxo-DHEA, DHEA) and secondary (balance, cognition and patient-reported outcomes) outcomes were examined three times (pre, post, and washout assessments)., Results: In both groups, there is a decreasing trend of 7-oxo-DHEA concentration in post-assessment and 7β-OH-DHEA in washout versus pre-assessment. A higher impact on neuroactive steroids is visible after Vojta's reflex locomotion. As for clinical outcomes, the Paced Auditory Serial Addition Test and Multiple Sclerosis Impact Scale significantly improved between post-assessment and washout assessment. The improvement was similar for both treatments., Conclusions: Neuroproprioceptive facilitation and inhibition improved the clinical outcomes and led to non-significant changes in neuroactive steroids. Trial registration (NCT04379193).

Urate transporters, which are located in the kidneys, significantly affect the level of uric acid in the body. We looked at genetic variants of genes encoding the major reabsorption proteins GLUT9 ( SLC2A9 ) and URAT1 ( SLC22A12 ) and their association with hyperuricemia and gout. In a cohort of 250 individuals with primary hyperuricemia and gout, we used direct sequencing to examine the SLC22A12 and SLC2A9 genes. Identified variants were evaluated in relation to clinical data, biochemical parameters, metabolic syndrome criteria, and our previous analysis of the major secretory urate transporter ABCG2. We detected seven nonsynonymous variants of SLC2A9 . There were no nonsynonymous variants of SLC22A12 . Eleven variants of SLC2A9 and two variants of SLC22A12 were significantly more common in our cohort than in the European population ( p = 0), while variants p.V282I and c.1002+78A>G had a low frequency in our cohort ( p = 0). Since the association between variants and the level of uric acid was not demonstrated, the influence of variants on the development of hyperuricemia and gout should be evaluated with caution. However, consistent with the findings of other studies, our data suggest that p.V282I and c.1002+78A>G ( SLC2A9 ) reduce the risk of gout, while p.N82N ( SLC22A12 ) increases the risk.

Background: Functional electric stimulation (FES) is recommended for foot drop in multiple sclerosis, although little is known about its therapeutic effect., Aim: The aim of this study is to evaluate a therapeutic effect immediately and two months after program termination (persistent and delayed effect) of a new approach using FES in combination with correcting the patients' postural system. More specifically, we evaluate the effects of this approach on the patients' clinical functions and compared it with individual physiotherapy., Design: Parallel randomized blind trial., Setting: Two-month-long treatments, functional electric stimulation in posturally corrected position (group 1) and neuroproprioceptive facilitation and inhibition physiotherapy called motor program activating therapy (group 2)., Population: Forty-four subjects with multiple sclerosis., Methods: Primary outcomes: gait (the 2-Minute Walk Test; Timed 25-Foot Walk test; Multiple Sclerosis Walking Scale-12) and balance (by e.g. Berg Balance Scale [BBS], the Activities-Specific Balance Confidence Scale [ABC], Timed Up-and-Go Test [TUG])., Secondary Outcomes: mobility, cognition, fatigue and subjects' perceptions (e.g. Multiple Sclerosis Impact Scale [MSIS], Euroqol-5 dimensions-5 levels [EQ-5D-5L])., Results: Group 1 showed immediate therapeutic effect in BBS (P=0.008), ABC (P=0.04) and EQ-5D-5L (self-care, P=0.019, mobility P=0.005). The improvement in EQ-5D-5L persisted and in TUG-cognitive we documented a delayed effect (P=0.005). Group 2 showed an immediate improvement in BBS (P=0.025), MSIS (P=0.043) and several aspects of daily life (the effect on health today was significantly higher than in group 1, significant difference between groups P=0.038)., Conclusions: FES in the posturally corrected position has an immediate therapeutic effect on balance and patients' perceptions comparable to motor program activating therapy, and higher persistent and even delayed therapeutic effect., Clinical Rehabilitation Impact: The study results point to the importance of correcting the patients' posture when applying FES, the possibility to treat foot drop by individual physiotherapy and the activation of the patients' auto reparative processes.

Aims: The development of the immune phenotype in patients with type 1 diabetes (T1D) during the first year following disease onset remains poorly described, and studies analysing the longitudinal development of a complex set of immunological and metabolic parameters are missing. Thus, we aim to provide such complex view in a cohort of 38 children with new onset T1D who were prospectively followed for 1 year., Methods: All subjects were tested for a set of immunological parameters (complete blood count; serum immunoglobulins; and T, B and dendritic cells), HbA1c and daily insulin dose at baseline and at 6 and 12 months after T1D diagnosis. A mixed meal tolerance test was administered to each of the subjects 12 months after diagnosis, and the C-peptide area under the curve (AUC) was noted and was then tested for association with all immunological parameters., Results: A gradual decrease in leukocytes (adjusted p = 0.0012) was reflected in a significant decrease in neutrophils (adjusted p = 0.0061) over the post-onset period, whereas Tregs (adjusted p = 0.0205) and originally low pDCs (adjusted p < 0.0001) increased. The expression of the receptor for BAFF (BAFFR) on B lymphocytes (adjusted p = 0.0127) markedly increased after onset. No immunological parameters were associated with C-peptide AUC; however, we observed a linear increase in C-peptide AUC with the age of the patients (p < 0.0001)., Conclusions: Our study documents substantial changes in the innate and adaptive immune system over the first year after disease diagnosis but shows no association between immunological parameters and residual beta-cell activity. The age of patients remains the best predictor of C-peptide AUC, whereas the role of the immune system remains unresolved.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F 420 H 2 -dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F 420 H 2 -dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.

Objectives: We analyzed primary school performance of girls with Turner syndrome (TS) in two distinct countries to ascertain if the cognitive phenotype of TS causes selective learning difficulties., Methods: The cohort comprised of 44 Czech and 50 Egyptian girls with TS who attended public schools. School reports from grades 1 to 9 were obtained retrospectively from Czech participants with TS. Only recent school reports were obtained from Egyptian participants. Two controls per participant were requested - biological sisters and/or female classmates. The results were converted into a 5-point scale (1-excellent; 5-unsatisfactory)., Results: Analysis of longitudinal Czech data displayed a strong time component in both subjects and controls. Showing better points in lower grades with its gradual worsening as the education complexity increased. In contrast, there was a strong statistically significant difference between groups in Mathematics (p=0.0041, p=0.0205 after Bonferroni correction) and this difference increased over time. The points for Mathematics did not differ in grades 1+2 (0.05 difference in mean grade between the two groups), however, they differed by 0.28 in grades 6+7 and by 0.32 in grades 8+9. While slightly different in character (cross-sectional vs. longitudinal), the Egyptian cohort data confirmed our findings, showing no difference in general school performance but having similar trends in Mathematics (grades 1+2: 0.11, grades 6+7: 0.54, grades 8+9: 0.68; p=0.0058, p=0.029 after Bonferroni correction)., Conclusion: Excluding results in Mathematics, which showed pronounced worsening in relation to age in comparison with unaffected controls, girls with TS performed similarly to their controls., (Copyright© of YS Medical Media ltd.)

Background: Maternal characteristics may be associated with human milk macronutrients but no definite conclusions have been made to date., Aim: This study aimed to determine the relationship of maternal-associated factors on the content of macronutrients in human milk for the first six weeks after preterm delivery., Study Design: Prospective observational cohort study., Subjects: Milk samples were collected from mothers after premature birth between 24 + 0-35 + 6 weeks., Outcome Measures: Macronutrients and energy content were analyzed by mid-infrared transmission spectroscopy. Demographic and anthropometric data from mothers were systematically recorded., Results: A total 1.558 human milk samples from 192 mothers were analyzed. Colostrum: higher protein (p = 0.001) and lower carbohydrate content (p = 0.003) were present in primiparous compared to multiparous milk. Vaginal birth was associated with increased carbohydrate content (p = 0.021). Fat and energy content in colostrum was not related to any maternal characteristics. Mature human milk: similarly to colostrum, higher protein content (p = 0.001) and lower carbohydrates content (p = 0.022) were observed in primiparous compared to multiparous milk. The mode of delivery was found to be another factor possibly influencing protein and carbohydrate levels (p = 0.036, p = 0.003, respectively). Pre-pregnancy obesity was associated with increased fat (p = 0.030) and energy content (p = 0.020) in human milk. On the contrary, smoking had a negative relationship to fat and energy content (p = 0.026, p = 0.007, respectively)., Conclusion: Human milk macronutrient concentration after preterm delivery is associated with pre-pregnancy obesity, parity, mode of delivery and smoking. The impact of maternal factors on human milk composition should be taken into account in a strategy of feeding in premature infants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In patients with gout, the serum uric acid (SUA) is usually lower during acute gouty attacks than during intercritical periods. It has been suggested that systemic inflammatory response can cause this phenomenon. The objective is to determine whether therapy with TNF inhibitors (TNFis) affects SUA levels in patients with systemic autoimmune rheumatic diseases (SARDs) and whether SUA changes correlate with pro-inflammatory cytokines or with the oxidative stress marker allantoin. In this study, SUA, CRP, creatinine, MCP-1, IFN-α2, IFN-γ, Il-1β, IL-6, IL-8, IL-10, IL-12, IL-17a, IL-18, IL-23, IL-33, TNF-α, and allantoin levels were measured prior to and after 3 months of TNFis treatment in patients with SARDs. The values obtained in the biochemical assays were then tested for associations with the patients' demographic and disease-related data. A total of 128 patients (rheumatoid arthritis, n = 44; ankylosing spondylitis, n = 45; psoriatic arthritis, n = 23; and adults with juvenile idiopathic arthritis, n = 16) participated in this study. Among the entire patient population, SUA levels significantly increased 3 months after starting treatment with TNFis (279.5 [84.0] vs. 299.0 [102.0] μmol/l, p < 0.0001), while the levels of CRP, IL-6, IL-8, and MCP-1 significantly decreased. Male sex was the most powerful baseline predictor of ΔSUA in univariate and multivariate models. None of the measured laboratory-based parameters had statistically significant effects on the magnitude of ΔSUA. 3 months of anti-TNF therapy increased the levels of SUA in patients with SARDs, but neither the measured pro-inflammatory cytokines nor the oxidation to allantoin appeared responsible for this effect.

Background Females with Turner syndrome (TS) are prone to develop autoimmune diseases (AIDs). The X chromosome contains several immune-related genes. Growth hormone (GH) and estrogens modulate the immune system. We aimed to clarify whether the loss of a specific X chromosome gene locus and the administration of GH and estradiol facilitate the development of AIDs in TS females. Methods Retrospective data on clinical course, AIDs, karyotype and treatment were analyzed from a cohort of 286 Czech females with TS (current age 2.8-43.3 years; median age 18.7 years). The karyotypes were sorted using two different classification systems: a mosaicism-focused and an isochromosome (isoXq)-focused approach. Karyotype subgroups with a significantly higher prevalence of AIDs were further evaluated. Data of common therapies were correlated with the prevalence of AIDs. Results The most frequent AIDs were autoimmune thyroid disease (AITD; 37.4%; n = 107) and celiac disease (CD; 8.7%; n = 25). All karyotype subgroups were prone to develop AIDs. Females with an isolated Xp deletion had a significantly higher prevalence of AITD and CD compared to all other individuals with TS (AITD: 66.0% vs. 31.5%, p < 0.0001; CD: 17.4% vs. 7.2%; p = 0.04, respectively). We observed no link between the mean age at initiation as well as the duration of GH and/or estrogen administration and the occurrence of AIDs. Conclusions Isolated Xp deletion contributes to the development of AIDs in TS patients. The haploinsufficiency of genes located in Xpter-p11.2 may explain this observation. Common therapies used in TS do not modify the risk of AIDs.

Background: ABCG2 is a high-capacity urate transporter that plays a crucial role in renal urate overload and extra-renal urate underexcretion. Previous studies have suggested an association between hyperuricemia and gout susceptibility relative to dysfunctional ABCG2 variants, with rs2231142 (Q141K) being the most common. In this study, we analyzed the ABCG2 gene in a hyperuricemia and gout cohort focusing on patients with pediatric-onset, i.e., before 18 years of age., Method: The cohort was recruited from the Czech Republic (n = 234) and consisted of 58 primary hyperuricemia and 176 gout patients, with a focus on pediatric-onset patients (n = 31, 17 hyperuricemia/14 gouts); 115 normouricemic controls were used for comparison. We amplified, sequenced, and analyzed 15 ABCG2 exons. The chi-square goodness-of-fit test was used to compare minor allele frequencies (MAF), and the log-rank test was used to compare empirical distribution functions., Results: In the pediatric-onset cohort, two common (p.V12M, p.Q141K) and three very rare (p.K360del, p.T421A, p.T434M) allelic ABCG2 variants were detected. The MAF of p.Q141K was 38.7% compared to adult-onset MAF 21.2% (OR = 2.4, P = 0.005), to the normouricemic controls cohort MAF 8.5% (OR = 6.8, P <  0.0001), and to the European population MAF 9.4% (OR = 5.7, P <  0.0001). The MAF was greatly elevated not only among pediatric-onset gout patients (42.9%) but also among patients with hyperuricemia (35.3%). Most (74%) of the pediatric-onset patients had affected family members (61% were first-degree relatives)., Conclusion: Our results show that genetic factors affecting ABCG2 function should be routinely considered in a hyperuricemia/gout diagnosis, especially in pediatric-onset patients. Genotyping of ABCG2 is essential for risk estimation of gout/hyperuricemia in patients with very early-onset and/or a family history.

Natural pyrrolobenzodiazepines (PBDs) form a large and structurally diverse group of antitumour microbial metabolites produced through complex pathways, which are encoded within biosynthetic gene clusters. We sequenced the gene cluster of limazepines and proposed their biosynthetic pathway based on comparison with five available gene clusters for the biosynthesis of other PBDs. Furthermore, we tested two recombinant proteins from limazepine biosynthesis, Lim5 and Lim6, with the expected substrates in vitro. The reactions monitored by LC-MS revealed that limazepine biosynthesis involves a new way of 3-hydroxyanthranilic acid formation, which we refer to as the chorismate/DHHA pathway and which represents an alternative to the kynurenine pathway employed for the formation of the same precursor in the biosynthesis of other PBDs. The chorismate/DHHA pathway is presumably also involved in the biosynthesis of PBD tilivalline, several natural products unrelated to PBDs, and its part is shared also with phenazine biosynthesis. The similarities between limazepine and phenazine biosynthesis indicate tight evolutionary links between these groups of compounds.

Objectives: Common dysfunctional variants of ATP binding cassette subfamily G member 2 (Junior blood group) (ABCG2), a high-capacity urate transporter gene, that result in decreased urate excretion are major causes of hyperuricemia and gout. In the present study, our objective was to determine the frequency and effect on gout of common and rare non-synonymous and other functional allelic variants in the ABCG2 gene., Methods: The main cohort recruited from the Czech Republic consisted of 145 gout patients; 115 normouricaemic controls were used for comparison. We amplified, directly sequenced and analysed 15 ABCG2 exons. The associations between genetic variants and clinical phenotype were analysed using the t-test, Fisher's exact test and a logistic and linear regression approach. Data from a New Zealand Polynesian sample set and the UK Biobank were included for the p.V12M analysis., Results: In the ABCG2 gene, 18 intronic (one dysfunctional splicing) and 11 exonic variants were detected: 9 were non-synonymous (2 common, 7 rare including 1 novel), namely p.V12M, p.Q141K, p.R147W, p.T153M, p.F373C, p.T434M, p.S476P, p.D620N and p.K360del. The p.Q141K (rs2231142) variant had a significantly higher minor allele frequency (0.23) in the gout patients compared with the European-origin population (0.09) and was significantly more common among gout patients than among normouricaemic controls (odds ratio = 3.26, P < 0.0001). Patients with non-synonymous allelic variants had an earlier onset of gout (42 vs 48 years, P = 0.0143) and a greater likelihood of a familial history of gout (41% vs 27%, odds ratio = 1.96, P = 0.053). In a meta-analysis p.V12M exerted a protective effect from gout (P < 0.0001)., Conclusion: Genetic variants of ABCG2, common and rare, increased the risk of gout. Non-synonymous allelic variants of ABCG2 had a significant effect on earlier onset of gout and the presence of a familial gout history. ABCG2 should thus be considered a common and significant risk factor for gout., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Background: Understanding the organisational set-up of physiotherapy services across different countries is increasingly important as clinicians around the world use evidence to improve their practice. This also has to be taken into consideration when multi-centre international clinical trials are conducted. This survey aimed to systematically describe organisational aspects of physiotherapy services for people with multiple sclerosis (MS) across Europe., Methods: Representatives from 72 rehabilitation facilities within 23 European countries completed an online web-based questionnaire survey between 2013 and 2014. Countries were categorised according to four European regions (defined by United Nations Statistics). Similarities and differences between regions were examined., Results: Most participating centres specialized in rehabilitation (82 %) and neurology (60 %), with only 38 % specialising in MS. Of these, the Western based Specialist MS centres were predominately based on outpatient services (median MS inpatient ratio 0.14), whilst the Eastern based European services were mostly inpatient in nature (median MS inpatient ratio 0.5). In almost all participating countries, medical doctors - specialists in neurology (60 %) and in rehabilitation (64 %) - were responsible for referral to/prescription of physiotherapy. The most frequent reason for referral to/prescription of physiotherapy was the worsening of symptoms (78 % of centres). Physiotherapists were the most common members of the rehabilitation team; comprising 49 % of the team in Eastern countries compared to approximately 30 % in the rest of Europe. Teamwork was commonly adopted; 86 % of centres based in Western countries utilised the interdisciplinary model, whilst the multidisciplinary model was utilised in Eastern based countries (p = 0.046)., Conclusion: This survey is the first to provide data about organisational aspects of physiotherapy for people with MS across Europe. Overall, care in key organisational aspects of service provision is broadly similar across regions, although some variations, for example the models of teamwork utilised, are apparent. Organisational framework specifics should be considered anytime a multi-centre study is conducted and results from such studies are applied.

The paper presented the possibility of recognition by adult the comfort and discomfort state of 3 and 6 months old infant's on the base of their vocalizations. The acoustic features of the vocalizations that are important for the recognition of the infant state of the characteristics of voice was described. It is shown that discomfort vocalizations differ from comfort ones on the basis of the average and maximum values of pitch, pitch values in the central and final part of the vocalization. A mathematical model is proposed and described a classification function signal of discomfort and comfort. Was found that the vocalizations of infants attributable adults with a probability of 0.75 and above the categories of comfort and discomfort with high reliability are recognized by the mathematical model based on a classification function.

Objective: Using European descent Czech populations, we performed a study of SLC2A9 and SLC22A12 genes previously identified as being associated with serum uric acid concentrations and gout. This is the first study of the impact of non-synonymous allelic variants on the function of GLUT9 except for patients suffering from renal hypouricemia type 2., Methods: The cohort consisted of 250 individuals (150 controls, 54 nonspecific hyperuricemics and 46 primary gout and/or hyperuricemia subjects). We analyzed 13 exons of SLC2A9 (GLUT9 variant 1 and GLUT9 variant 2) and 10 exons of SLC22A12 by PCR amplification and sequenced directly. Allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. The functional studies were analyzed using the non-parametric Wilcoxon and Kruskall-Wallis tests; the association study used the Fisher exact test and linear regression approach., Results: We identified a total of 52 sequence variants (12 unpublished). Eight non-synonymous allelic variants were found only in SLC2A9: rs6820230, rs2276961, rs144196049, rs112404957, rs73225891, rs16890979, rs3733591 and rs2280205. None of these variants showed any significant difference in the expression of GLUT9 and in urate transport. In the association study, eight variants showed a possible association with hyperuricemia. However, seven of these were in introns and the one exon located variant, rs7932775, did not show a statistically significant association with serum uric acid concentration., Conclusion: Our results did not confirm any effect of SLC22A12 and SLC2A9 variants on serum uric acid concentration. Our complex approach using association analysis together with functional and immunohistochemical characterization of non-synonymous allelic variants did not show any influence on expression, subcellular localization and urate uptake of GLUT9.

Regulatory T cells have been well described and the factors regulating their development and function have been identified. Recently, a growing body of evidence has documented the existence of interleukin-10 (IL-10) -producing B cells, which are called regulatory B10 cells. These cells attenuate autoimmune, inflammatory and transplantation reactions, and the main mechanism of their inhibitory action is the production of IL-10. We show that the production of IL-10 by lipopolysaccharide-stimulated B cells is significantly enhanced by IL-12 and interferon-γ and negatively regulated by IL-21 and transforming growth factor-β. In addition, exogenous IL-10 also inhibits B-cell proliferation and the expression of the IL-10 gene in lipopolysaccharide-stimulated B cells. The negative autoregulation of IL-10 production is supported by the observation that the inclusion of anti-IL-10 receptor monoclonal antibody enhances IL-10 production and the proliferation of activated B cells. The effects of cytokines on IL-10 production by B10 cells did not correlate with their effects on B-cell proliferation or on IL-10 production by T cells or macrophages. The cytokine-induced changes in IL-10 production occurred on the level of IL-10 gene expression, as confirmed by increased or decreased IL-10 mRNA expression in the presence of a particular cytokine. The regulatory cytokines modulate the number of IL-10-producing cells rather than augmenting or decreasing the secretion of IL-10 on a single-cell level. Altogether these data show that the production of IL-10 by B cells is under the strict regulatory control of cytokines and that individual cytokines differentially regulate the development and activity of regulatory T cells and IL-10-producing regulatory B cells., (© 2013 John Wiley & Sons Ltd.)

The MAPK/ERK/p38 are signal transduction pathways that couple intracellular responses to the external stimuli. Contrary to ERK protein which is part of the survival route, presence of p38 could have an impact on cell injury. Tolerance induced by ischemic preconditioning (IPC) is a phenomenon of tissue adaptation, which results in increased tolerance to lethal ischemia-reperfusion injury (IRI). Paper describes changes in MAPK protein pathways after brain IPC. Ischemia was induced by 4-vessels occlusion and rats were preconditioned by sub-lethal ischemia. Western blot and immunohistochemistry identified ERK/p38 proteins in injured areas. The highest level of the pERK was detected at 24 h in IPC groups. A contrary pattern of MAPK/p38 activation was observed in this group, where the lowest level of p38 was displayed at 24 h after ischemia. This suggests that the MAPK signal transduction might have a potential role in tissues response subjected to IRI and in the phenomenon of tolerance.

Introduction and Hypothesis: Polypropylene meshes are frequently used in abdominal and vaginal reconstructive surgery. Recently, several authors have claimed that mesh-associated complications may be linked to mesh shrinkage. We have performed a prospective study with postoperative follow-up by ultrasound examination at two time points after Prolift anterior implantation to assess changes in the ultrasound appearance of mesh implants over time., Methods: We assessed 36 patients who had undergone mesh implantation with Prolift anterior mesh for the correction of symptomatic anterior vaginal wall prolapse. During the surgery, we measured the actual midline length of the mesh (initial length). On the fourth postoperative day, we performed a vaginal ultrasound examination (US) to measure mesh length in the midsagittal plane. A second US was performed 3-5 months after surgery to repeat this measurement., Results: There was a significant difference in mesh length determined before and 4 days after surgery (90.3 vs. 57.1 mm, P = <0.0001) indicating intraoperative folding. On comparing early and late postoperative ultrasound measurements, there was a reduction in length from 57.1 to 48.3 mm (P < 0.0001), indicating possible shrinkage or retraction., Conclusions: Intraoperative folding seems to be responsible for a large part of the difference between preoperative (in vitro) and postoperative (US) measurements of mesh dimensions, suggesting that surgical techniques may require adjustment.

The study determines whether hyperhomocysteinemia (risk factor of brain ischemia) alone or in combination with ischemic preconditioning (IPC) affects the ischemia-induced changes in gene expression of secretory pathways Ca(2+)-ATPase (SPCA1). Hyperhomocysteinemia was induced by subcutaneous administration of homocysteine (Hcy; 0.45 µmol/g body weight) twice a day at 8 h intervals for 14 days. Rats were preconditioned by 5 min ischemia and 2 days later, 15 min of global forebrain ischemia was induced by four vessel occlusion. We observed that hyperhomocysteinemia significantly decreased the level of SPCA1 mRNA in the cortex. Pre-ischemic challenge was noticeable in both brain areas. In the cortex, pre-ischemia in Hcy group led to the abrupt stimulation of the mRNA expression by 249% within the Hcy ischemic group and by 321% in the Hcy control. Values further exceeded those observed in the naive control. In the hippocampus, the differences between naive and Hcy groups were not observed. IPC initiated elevation of mRNA expression to 159% (p < 0.05) of control with Hcy and to 131% (p < 0.01) of ischemia with Hcy, respectively. Documented response of SPCA gene to IPC in hyperhomocysteinemic group might suggest a correlation of SPCA expression consistent with the role of cross-talks between intracellular Ca(2+) stores including secretory pathways in the tolerance phenomenon.

Objective: To evaluate and quantify early and late changes in mesh length after anterior vaginal repair with implants with ultrasound., Type of Study: Prospective interventional study., Methods: Ultrasound assessment of 35 patient randomized in two groups both with Gynemesh insertion. In one group with Gynemesh Prolift anterior kit and second group with individualized size of the Gynemesh. Ultrasound measurement of the mesh in sagittal plane was performed postoperative day 4 and after 3-4 month. Measured values were put in proportions, together with the original size of the mesh. Results are expressed as a percentage of shortening., Results: In comparison of measurements from the late ultrasound scans with original size the shortening of the Prolift was 45% vs. 25% in Mesh group. When we compared the late and early ultrasound scans, there was no difference in the shrinking of the mesh in both group 16% vs 20%., Conclusion: Insufficient spreading and anchoring of the anterior Prolift has a major impact on the final length of the mesh. The tissue reaction expressed as shrinking of the mesh is only 16-20%.

Objective: We aimed to assess the occurrence of stress urinary incontinence (SUI) and urgency (U) before and after an operation to treat anterior compartment defect, and to ascertain whether there is a correlation between the position or mobility of the urethro-vesical junction (UVJ) and the lowest point of bladder base (N) and SUI and U before and after surgical treatment of the defect, using various procedures., Design: Prospective, randomized study., Settings: Department of Gynecology and Obstetrics, First Medical Faculty, Charles University and General Teaching Hospital, Prague., Materials and Methods: 87 women were enrolled who had proven symptomatic Pelvic Organ Prolapse POP > or = II (Pelvic Organ Prolapse Quantitative--POP-Q)--specifically anterior compartment defect cystocele; they were then randomized into three group according to the type of surgical procedure planned: the BM group, treated with the traditional Barnett-Macků technique of anterior vaginal plastic surgery (BM; n=18); the Gynemesh group, treated with anterior plastic surgery with free insertion of individualized mesh (Mesh; n=33); and the Prolift group, treated with an original kit with pre-set standard-size mesh which is anchored to the lower arm of pubis (Prolift; n=36). SUI tests were carried out for the women before the operation and 3-4 months afterwards, using International Consultation on Incontinence Questionnaire - Short form (ICIQ-UI SF) and objective assessment by cough-test, while we also took into account the urge symptom. Before and after the operation patients were also examined by 4D imaging (GE Voluson 730 Expert), with emphasis on the position of UVJ and N point at rest and at maximum Valsalva. Data were processed and analysed in open computer environment, R language, version 2.9.1., Results: The different groups of patients did not show statistically significant differences in demographic data. The results also show that there is no statistical difference between individual operation groups regarding occurrence of SUI: objectively this was established for 33% of patients, and according to ICIQ for 79%. Among women where SUI was not objectively proven, 74% felt SUI, while among women with objectively proved SUI, only one did not feel the urine leakage. This means that SUI is much more often subjectively felt than objectively proven. After the operation objective improvement of SUI occurred for 6% (5) patients, while it worsened for 16% (12) patients. The differences are not statistically significant. 78% (65 patients; n=83) felt incontinent before the operation compared with 66% (54 patients; n=82) after the operation, according to the ICIQ questionnaire. 18% (14) patients showed improvement and 5% (4) deterioration. In subjective assessment of the symptoms, improvement of SUI is more often recorded than worsening, to a statistically significant degree. 34% (30) patients in total suffered from urge before the operation (for three of them urge incontinence, for the others just urgency) and 8% (7) patients after the operation (of which one suffered from urge incontinence and 6 just urgency). This means that improvement occurred for 32% (25) and deterioration for just 3% (2) if patients. We have not ascertained any correlation between UVJ mobility, N point and urinary incontinence before and after the operation., Conclusions: The results of our study imply that the presence of SUI and U before an operation to treat anterior compartment defect is one of the main symptoms accompanying prolapse. While the operation may solve the SUI problem, it very often does not, as it deals mainly with eliminating the prolapse, or para-vaginal effect. We also failed to establish any correlation between mobility of the UVJ or N point and occurrence of SUI before and after the operation. We may, however, state that elevation of the N point--bladder base due to the operation results in mitigating U. Therefore, for reconstructive surgeries that do not treat SUI it is necessary that the operation is followed by a tape procedure in the second stage, ideally after the first operation has healed, i.e., 3 months at minimum.

Introduction and Hypothesis: The hypotheses of this study were that the TVT-SECUR procedure restricts urethral mobility, which leads to a greater likelihood of curative effect, and that the restriction might change over time., Methods: Analyses of the position of the urethra and the tape of 85 patients who underwent the TVT-S procedure were performed using perineal ultrasonography. The efficacy of the TVT-S procedure was evaluated by cough test and by the questionnaire ICIQ-UI SF., Results: Objectively, 53/85 (62%) of patients had a negative cough test, and in 32/85 (38%) of patients leakage of urine persisted. The TVT-S procedure restricts urethral mobility, and a higher degree of obstruction is associated with a higher likelihood of cure. The restriction weakens within the first 3 months after surgery., Conclusions: The objective cure rate of TVT-S procedure is low irrespective of placement technique. This may be due to insufficient restriction of urethral mobility.

Objective: The aim of this study is to present our first experience with a novel modification of the tension-free sling idea and to evaluate the safety and efficacy of this new procedure for the treatment of stress urinary incontinence in women., Study Design: Eighty-five women with previously untreated stress urinary incontinence were recruited to participate in a clinical study. The efficacy of this surgical procedure was evaluated perioperatively and 3 months (+/-1 week) after operation--objectively by cough test and subjectively by the questionnaires Pelvic Organ Prolapse/Urinary Incontinence Sexual Function Questionnaire and the International Consultation on Incontinence Questionnaire-Short Form. All data were processed and statistical analyses performed in statistical environment R, version 2.5.1., Results: From our results we conclude that there were no perioperative complications, objectively 62% of these patients were completely dry and 25% of patients improved. We observed a higher proportion of vaginal wall erosion (7/85) and urgency de novo (5/85) in the learning period group with respect to the routine period group., Conclusions: Our first experience with the tension-free vaginal tape secur system procedure is that it has a low percentage of perioperative complications. The learning curve has to be taken in account with reference to postoperative complications.

Objective: The objective of this study is to evaluate the efficacy of the procedure of shortening the tape in the treatment of persistent stress urinary incontinence when the tension-free vaginal tape (TVT) SECUR procedure proves unsuccessful., Design: Retrospective study., Settings: Department of Gynecology and Obstetrics, First Faculty of Medicine, Charles University and General Teaching Hospital, Prague., Materials and Methods: Eight patients with persistent stress urinary incontinence (SUI) after the TVT-S procedure were included in our study. In order to resolve their condition, the tape was shortened. We used the overlap and the cut-off technique. Pre- and postoperative urodynamic and ultrasound (US) examinations were carried out, and the efficacy of this procedure was evaluated by cough test, pad-weighting test (PWT) and by the questionnaires Pelvic Organ Prolapse/Urinary Incontinence Sexual Function Questionnaire (PISQ12) and the International Consultation on Incontinence Questionnaire--Short form (ICIQ-UI SF). All data were processed and statistical analyses performed in statistical environment R, version 2.5.1., Results: In six patients the cough test and PWT were negative after reoperation, indicating no stress urinary incontinence, while in two patients mild leakage of urine persisted. The reoperation significantly increased the value of Maximum Urethral Closure Pressure (MUCP) while coughing. From the US examination we can conclude that the tape is tightened closer to the urethra and pubic bone., Conclusions: We describe a method--shortening the tape--for the treatment of persistent stress urinary incontinence when the TVT-S procedure fails. Based on our results we have established that this procedure is simple and as effective as inserting a new tape, but cheaper.

Objective: The aims of this study were to compare ultrasonographic findings of the urethra and the tape position and mobility following the tension-free vaginal tape secure system (TVT-S) procedure and to correlate these data with clinical signs of cure and failure of this procedure in the treatment of stress urinary incontinence (SUI) in women. Another aim of our study was to determine indications for particular position of the tape (the "hammock" or "U" position) based on ultrasonographic findings of the urethra and the tape position following TVT-S., Design: Prospective, randomized study., Settings: Department of Gynecology and Obstetrics, First faculty of Medicine, Charles University and General Teaching Hospital, Prague., Materials and Methods: Analyses of the position of the urethra and the tape of 85 patients were performed using perineal ultrasonography. The efficacy of the TVT-S procedure was evaluated by cough test and by the International Consultation on Incontinence Questionnaire - Short Form (ICIQ-UI SF)., Results: Objective assessment by cough test showed that 53 (62%) of our patients were completely dry, and in 32 (38%) patients leakage of urine persisted after the operation. From the US examination and results we can conclude that 3 months after the operation the mobility of the urethra and bladder neck are restricted. A correlation between cure effect and the restriction of the movement of the urethra was found between the position at the middle of the urethra before and after the operation. Subjective assessment of the cure effect of this operation by the ICIQ-UI SF questionnaires (n=81) showed that 39 (48%) of our patients were completely dry and 24 (30%) of patients improved, which means that in 42 (52%) patients leakage of urine persists after the operation. There are differences in the restriction of urethral mobility between the cured patients and those where leakage persisted. The middle of the urethra and UVJ are more restricted in movement in cured patients. We found no statistically significant differences between the US measurements of the position of the urethra of patients with the tape in the "hammock" and the "U" position. The only statistically significant difference found is a slightly higher tension of the "U" tape three months after the operation during the Valsalva maneuver; the median width of the tape in the "hammock" position is 10.1 mm, while in the "U" position it is 8.1 (Wilcoxon test p = 0.0056). There were six patients diagnosed with urgency de novo, without urge incontinence in this series. In seven cases we found vaginal erosion., Conclusions: From the US examination and results we can conclude that after the TVT-S operation, mobility of the urethra and bladder neck is restricted. After comparing ultrasonographic findings of the urethra and the tape position following TVT- Secure we can determine that there is no certain indication for placement of the tape in the "hammock" or the "U" position. The cure effect for different positions of the tape is similar. In some respects the slightly higher tension of the tape in the "U" position suggests that this tape position may be preferred in patients with intrinsic sphincter defect (ISD) of the urethra. A comparison of the cure effect of the TVT-S procedure with other tape procedures suggests a lower cure effect of this operation.

4-hydroxynonenal (HNE) is a highly toxic product of lipid peroxidation (LPO). Its role in the inhibition of cytochrome c oxidase activity and oxidative modifications of mitochondrial lipids and proteins were investigated. The exposure of mitochondria isolated from rat heart to HNE resulted in a time- and concentration-dependent inhibition of cytochrome c oxidase activity with an IC50 value of 8.3 +/- 1.0 microM. Immunoprecipitation-Western blot analysis showed the formation of HNE adducts with cytochrome c oxidase subunit I. The loss of cytochrome c oxidase activity was also accompanied by reduced thiol group content and increased HNE-lysine fluorescence. Furthermore, there was a marked increase in conjugated diene formation indicating LPO induction by HNE. Fluorescence measurements revealed the formation of bityrosines and increased surface hydrophobicity of HNE-treated mitochondrial membranes. Superoxide dismutase + catalase and the HO* radical scavenger mannitol partially prevented inhibition of cytochrome c oxidase activity and formation of bityrosines. These findings suggest that HNE induces formation of reactive oxygen species and its damaging effect on mitochondria involves both formation of HNE-protein adducts and oxidation of membrane lipids and proteins by free radicals.

We have identified 21 different alpha-galactosidase A gene (GLA) mutations in 22 unrelated Czech and Slovak families with Fabry disease. Eleven of these mutations were novel (point mutations D93N, A135V, D155H, G171R, Q280K, G360S, Q330X, splicing errors c.194ins14, c.801ins36 and deletions c.674&#95;732del59, g.3405&#95;6021del2617). Genotyping of family members for family-specific mutations revealed 55 heterozygotes that manifested clinical symptoms of different severity. To examine the contribution of X-inactivation skewing to disease manifestation in Fabry heterozygotes, we have adopted the Mainz severity scoring scheme and compared the score values with the X-inactivation status in 39 carriers in an age-dependent manner. The age-score trendline of Fabry females who had a predominantly inactivated X-chromosome bearing a wild-type GLA allele (10 of 38 females) was markedly steeper than in the rest of the cohort. One female carrier with an inactivated mutated allele had a low score value when compared to the other heterozygotes of the same age. These data suggest that X-inactivation is indeed a major factor determining the severity of clinical involvement in Fabry heterozygotes. There was a statistically significant difference between the severity score values of heterozygotes with random and non-random X-chromosome inactivation at the 5% level of significance. Further studies will show if the degree of the wildtype allele inactivation will be useful as a predictive marker of severity of phenotype in Fabry heterozygotes. Although the correlation between X-inactivation skewing and presentation of the disease in Fabry heterozygotes has previously been suggested in the literature, this report is among the first attempts to examine this relationship systematically.