Endre Sulyok

Functional analyses indicate a pathogenic role of factor H autoantibodies in atypical haemolytic uraemic syndrome

BACKGROUNDnAtypical haemolytic uraemic syndrome (aHUS) is associated with defective complement regulation. Recently, an autoimmune aHUS form has been described that is associated with complement factor H (CFH) autoantibodies. The aim of this study was to address the pathologic relevance of CFH autoantibodies in aHUS.nnnMETHODSnCFH autoantibodies were identified and antibody levels were analysed in three aHUS patients during the disease course by the ELISA method. Epitope mapping was performed using recombinant factor H fragments and domain-mapped monoclonal antibodies. The effect of the antibodies on cell-protective activity of CFH was measured by haemolytic assays. CFH:autoantibody complexes were analysed by ELISA.nnnRESULTSnAll three autoantibodies bound to the C-terminal domain of CFH, which is essential for CFH binding to cell surfaces. In patient 1, plasma exchanges and immune adsorption temporarily reduced the autoantibody titre and led to temporary clinical improvement. In patient 2, plasma exchanges and long-term immunosuppression strongly reduced the CFH autoantibody level, and induced a stable remission of aHUS. Patient 3 had lower autoantibody levels that decreased during the follow-up and is in good clinical condition. The patients plasma samples caused enhanced lysis of sheep erythrocytes, and the degree of lysis correlated with the CFH autoantibody titre and the amount of CFH:autoantibody complexes. An addition of purified CFH to aHUS plasma or removal of IgG inhibited the haemolytic activity.nnnCONCLUSIONnThese results support a direct role of the autoantibodies in aHUS pathology by inhibiting the regulatory function of CFH at cell surfaces and suggest that reduction of the autoantibody titre is beneficial for the patients.

Fetal development of membrane water channel proteins aquaporin-1 and aquaporin-4 in the human brain

Aquaporin‐1 and aquaporin‐4, water channel membrane proteins reported in both experimental animals and in adult humans, have been detected in different, non‐overlapping areas of the central nervous system. This immunohistochemical study describes the developmental expression pattern of the water channel membrane proteins, aquaporin‐1 and aquaporin‐4, in various structures of human fetal brain over the gestational period of 14–40 weeks.

Determination of cerebrospinal fluid concentrations of arginine and dimethylarginines in patients with subarachnoid haemorrhage

Elevated cerebrospinal fluid (CSF) concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), are assumed to be related to delayed vasospasm after subarachnoid haemorrhage (SAH). However, data on CSF concentrations of L-arginine, ADMA and its structural isomer symmetric dimethylarginine (SDMA) are very sparse in humans. We here present a new hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS-MS) method for the precise determination of these substances in CSF. The method requires only minimal sample preparation and features isotope labeled internal standards. First data of patients with SAH showed that on the day of admission CSF concentration values of L-arginine and ADMA were not significantly different from controls, but increased markedly during the course of the hospital stay. The decrease of the L-arginine to ADMA ratio points to a progressive impairment of the NO production rate in the brain after SAH which is confirmed by a simultaneous decrease in nitrate and nitrite concentrations in CSF.

Plasma levels of asymmetric dimethylarginine in premature neonates: its possible involvement in developmental programming of chronic diseases

Aim: The endothel dysfunction in early life may play a role in developmental programming of cardiovascular morbidity. The changes of dimethylarginines plasma levels during the first month among preterm infants and their determinants had been investigated in our study.

Characterization of ouabain-like immunoreactivity in human urine

Objective To characterize ouabain-like immunoreactivity in human urine Methods Sensitive radioimmunoassay for ouabain characterized by high- performance liquid chromatography Results Serial dilution of urinary immunoreactive ouabain paralleled the standard curve, but not so plasma immunoreactive ouabain. Intravenous administration of 86nmol (62.5 μg) ouabain caused a rapid rise in ouabain immunoreactivity in plasma of healthy volunteers with a maximum of 1.7nmol/l 8min after injection and returned to basal levels after 6 h. Ouabain immunoreactivity rose to 36 nmol/l in urine, suggesting that exogenously administered ouabain can be measured reliably in plasma and urine. Analytical reversed-phase high-performance liquid chromatography (isopropanol-propanol biphasic gradient; linear acetonitrile gradient) of sample extracts before assay demonstrated measurable amounts of ouabain-related material only in native urine, but not in plasma. When plasma and urine were spiked with ouabain standard or normal volunteers were injected with ouabain, the assay reliably measured ouabain Conclusion: A substance closely related to ouabain can be detected in urine, but circulates, if at all, in small amounts in human plasma

Aquaporin-4 distribution in control and stressed astrocytes in culture and in the cerebrospinal fluid of patients with traumatic brain injuries.

Distribution of aquaporin-4 (AQP4) was studied by western analysis and immunofluorescence in rat astrocytes exposed to either hypothermic (30xa0°C) or hyperosmolar (0.45xa0M sucrose) stress, and in the cerebrospinal fluid (CSF) of patients who suffered traumatic brain injury (TBI). CSF was obtained from 5 healthy subjects and from 20 patients suffering from severe TBI. CSF samples were taken at admission and on days 3 and 5–7. Here we report that, in response to both hypothermia and hyperosmolar stress, AQP4 was markedly reduced in cultured astrocytes. We also found that AQP4 significantly increased in patients with severe brain injury in respect to healthy subjects (Pxa0<xa00.002). AQP4 in CSF remained unchanged in patients with elevated intracranial pressure (ICP), whereas there was a clear tendency to further increase in those patients whose ICP could be controlled within the normal range. We conclude that AQP4 levels in CSF are elevated after TBI and it might serve as a useful biochemical marker to assess brain water metabolism in clinical settings.

Acylcarnitine esters profiling of serum and follicular fluid in patients undergoing in vitro fertilization

BackgroundL-carnitine-mediated beta-oxidation of fatty acids has a well established role in energy supply of oocytes and embryos. Disturbed carnitine metabolism may impair the reproductive potential in IVF and can serve as a biomarker of pregnancy outcome.MethodsOur study was performed between March 24, 2011 and May 9, 2011. We performed 44 unselected IVF cycles, (aged 23–40 years (mean: 32.3+/−5.1 years) and had BMI of 17.3-34.7 (mean: 23.80+/−4.9). Samples were also obtained from 18 healthy women of similar age admitted for minor elective surgery to serve as control for plasma carnitine profile. Serum and follicular fluid (FF) free carnitine (FC) and 20 major acylcarnitines (ACs) were measured by ESI/MS/MS method.ResultsSerum FC and AC levels in IVF patients were comparable to those in healthy control women. In FF FC and short-chain AC concentrations were similar to those in maternal serum, however, the levels of medium-chain, and long-chain AC esters were markedly reduced (p<0.05). The serum to FF ratio of individual carnitine compounds increased progressively with increasing carbon chain length of AC esters (p<0.05). There was a marked reduction in total carnitine, FC and AC levels of serum and FF in patients with oocyte number of >9 and/or with embryo number of >6 as compared to the respective values of <9 and/or <6 (p<0.05).ConclusionsIn IVF patients with better reproductive potential the carnitine/AC pathway appears to be upregulated that may result in excess carintine consumption and relative depletion of carnitine pool. Consequently, IVF patients may benefit from carnitine supplementation.

Birth by cesarean section is associated with elevated neonatal plasma levels of dimethylarginines

Background:u2002 This study was undertaken to compare the effects of vaginal delivery and cesarean section on the l‐arginine‐nitric oxide system by measuring levels of l‐arginine, an endogenous nitric oxide synthase antagonist asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) in the cord blood and postnatally.

Effect of hemodialysis session on the dynamics of carnitine ester profile changes in l-carnitine pretreated end-stage renal disease patients

PurposeCarnitine deficiency is common in end-stage renal disease (ESRD) patients receiving hemodialysis (HD) treatment. We investigated the effects of l-carnitine supplementation on acyl carnitine (AC) profile and the changes of distinct ACs during a single HD session in long-term l-carnitine pretreated ESRD patients.MethodsTwenty non-diabetic adult patients and 37 healthy controls were studied. Blood samples were drawn before and after 12xa0weeks of carnitine supplementation, then hourly during an HD session, as well as 30 min after the end of the session. Free and individual AC plasma levels were determined by using ESI MS/MS technique.ResultsHD patients showed lower free- and total carnitine levels and elevated ACs and acyl/free carnitine ratio before carnitine supplementation. The l-carnitine supplementation resulted in dramatic elevation of all carnitine esters. The HD session induced a progressive decline in free, short-chain, and dicarboxylic ACs (~80xa0% of pre-HD amount was washed out); the decrease of medium-chain ACs proved to be more moderate (~60xa0% washed out), whereas the long-chain ACs remained unaffected. Already 30xa0min after HD, a substantial increase was seen in free carnitine concentration (reaching 26xa0% of predialysis level) and the ACs also started to replenish (to 21–52xa0% of predialysis levels), without further exogenous carnitine load.ConclusionsThe washout induced by HD session results in variable depletion of short-, medium-, and long-chain carnitine esters in carnitine-pretreated patients; the recovery of the circulating carnitine esters from the body stores occurs within 30xa0min after the cessation of the HD procedure.

Dynamic adaptive changes of the serum carnitine esters during and after L-carnitine supplementation in patients with maintenance haemodialysis

Abstract Background. Here we report the serum carnitine ester profile during and after 1g iv/day L-carnitine supplementation in haemodialysis patients. Materials and methods. Seven patients were studied over 29 weeks. After a control day, 12 weeks of replacement therapy was introduced followed by 17 weeks of washout period. The serum acylcarnitine concentrations were determined by isotope dilution ESI MS/MS technique. Results. At baseline significantly decreased free carnitine (48%, p < 0.01) and a 1.5–16-fold elevation of 16 out of 27 acylcarnitines were detected in HD patients compared with the controls. On the last day of L-carnitine supplementation a 1.6–4.8-fold increase was observed in the acylcarnitine levels compared with day 0; the increase-profile was achieved in four different patterns. The increase rate was rapid and early saturable for C5, C5OH, C6DC, C8:1, C10DC and C18:2 esters, slower for C2, C4, C6, C18 and C18:1 esters, it was slowest and reached a late plateau for C3, C8DC, C14:2, C16 and C16:1, and finally almost gradual increase was seen for 11 acylcarnitines. Three months after the cessation of carnitine treatment marked concentration drops were found for almost all acylcarnitines (by 11–74 % of week 12, p < 0.05); the values further decreased over the five remaining weeks of the observation period. Conclusion. Carnitine administration affected the levels of circulating esters in different dynamics and kinetics suggesting a regulated, non-random adaptive reallocation of nutrients. A considerable washout was achieved 3 months after discontinuation of the supplementation; however, the profile still was suggestive for presence of rest of accumulated supplement.