S100B Protein but Not 3-Nitrotyrosine Positively Correlates with Plasma Ammonia in Patients with Inherited Hyperammonemias: A New Promising Diagnostic Tool?

Individuals with inherited hyperammonemias often present developmental and intellectual deficiencies which are likely to be exaggerated by hyperammonemia episodes in long-term outcomes. In order to find a new, systemic marker common to the course of congenital hyperammonemias, we decided to measure the plasma level of S100 calcium-binding protein B (S100B), which is associated with cerebral impairment. Further, we analyzed three mechanistically diverged but linked with oxidative-nitrosative stress biochemical parameters: 3-nitrotyrosine (3-NT), a measure of plasma proteins' nitration; advanced oxidation protein products (AOPP), a measure of protein oxidation; and glutathione peroxidase (GPx) activity, a measure of anti-oxidative enzymatic capacity. The plasma biomarkers listed above were determined for the first time in congenital hyperammonemia. Also, the level of pro- and anti-inflammatory mediators (i.e., IL-12, IL-6, IL-8, TNF-α, IL-1ß, and IL-10) and chemokines (IP-10, MCP-1, MIG, and RANTES) were quantified. S100B was positively correlated with plasma ammonia level, while noticeable levels of circulating 3-NT in some of the patients' plasma did not correlate with ammonia concentration. Overall, the linear correlation between ammonia and S100B but not standard oxidative stress-related markers offers a unique perspective for the future identification and monitoring of neurological deficits risk-linked with hyperammonemia episodes in patients with inherited hyperammonemias. The S100B measure may support the development of therapeutic targets and clinical monitoring in these disorders.

Attenuation of initial pilocarpine-induced electrographic seizures by methionine sulfoximine pretreatment tightly correlates with the reduction of extracellular taurine in the hippocampus.

OBJECTIVE: Initiation and development of early seizures by chemical stimuli is associated with brain cell swelling resulting in edema of seizure-vulnerable brain regions. We previously reported that pretreatment with a nonconvulsive dose of glutamine (Gln) synthetase inhibitor methionine sulfoximine (MSO) mitigates the intensity of initial pilocarpine (Pilo)-induced seizures in juvenile rats. We hypothesized that MSO exerts its protective effect by preventing the seizure-initiating and seizure-propagating increase of cell volume. Taurine (Tau) is an osmosensitive amino acid, whose release reflects increased cell volume. Therefore, we tested whether the poststimulus rise of amplitude of Pilo-induced electrographic seizures and their attenuation by MSO are correlated with the release of Tau from seizure-affected hippocampus. METHODS: Lithium-pretreated animals were administered MSO (75 mg/kg ip) 2.5 h before the induction of convulsions by Pilo (40 mg/kg ip). Electroencephalographic (EEG) power was analyzed during 60 min post-Pilo, at 5-min intervals. Extracellular accumulation of Tau (eTau) served as a marker of cell swelling. eTau, extracellular Gln (eGln), and extracellular glutamate (eGlu) were assayed in the microdialysates of the ventral hippocampal CA1 region collected at 15-min intervals during the whole 3.5-h observation period. RESULTS: The first EEG signal became apparent at ~10 min post-Pilo. The EEG amplitude across most frequency bands peaked at ~40 min post-Pilo, and showed strong (r ~ .72-.96) temporal correlation with eTau, but no correlation with eGln or eGlu. MSO pretreatment delayed the first EEG signal in Pilo-treated rats by ~10 min, and depressed the EEG amplitude across most frequency bands, to values that remained strongly correlated with eTau (r > .92) and moderately correlated (r ~ -.59) with eGln, but not with eGlu. SIGNIFICANCE: Strong correlation between attenuation of Pilo-induced seizures and Tau release indicates that the beneficial effect of MSO is due to the prevention of cell volume increase concurrent with the onset of seizures.

Inhibition of Glutamate Release, but Not of Glutamine Recycling to Glutamate, Is Involved in Delaying the Onset of Initial Lithium-Pilocarpine-Induced Seizures in Young Rats by a Non-Convulsive MSO Dose.

Initial seizures observed in young rats during the 60 min after administration of pilocarpine (Pilo) were delayed and attenuated by pretreatment with a non-convulsive dose of methionine sulfoximine (MSO). We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was administered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-13C]acetate and [U-13C]glucose were i.p.-injected either together with Pilo (short period) or 15 min before sacrifice (long period). Their conversion to Glu and Gln in the hippocampus and entorhinal cortex was followed using [13C] gas chromatography-mass spectrometry. Release of in vitro loaded Glu surrogate, [3H]d-Asp from ex vivo brain slices was monitored in continuously collected superfusates. [3H]d-Asp uptake was tested in freshly isolated brain slices. At no time point nor brain region did MSO modify incorporation of [13C] to Glu or Gln in Pilo-treated rats. MSO pretreatment decreased by ~37% high potassium-induced [3H]d-Asp release, but did not affect [3H]d-Asp uptake. The results indicate that MSO at a non-convulsive dose delays the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.

Decreased Expression and Uncoupling of Endothelial Nitric Oxide Synthase in the Cerebral Cortex of Rats with Thioacetamide-Induced Acute Liver Failure.

Acute liver failure (ALF) is associated with deregulated nitric oxide (NO) signaling in the brain, which is one of the key molecular abnormalities leading to the neuropsychiatric disorder called hepatic encephalopathy (HE). This study focuses on the effect of ALF on the relatively unexplored endothelial NOS isoform (eNOS). The cerebral prefrontal cortices of rats with thioacetamide (TAA)-induced ALF showed decreased eNOS expression, which resulted in an overall reduction of NOS activity. ALF also decreased the content of the NOS cofactor, tetrahydro-L-biopterin (BH4), and evoked eNOS uncoupling (reduction of the eNOS dimer/monomer ratio). The addition of the NO precursor L-arginine in the absence of BH4 potentiated ROS accumulation, whereas nonspecific NOS inhibitor L-NAME or EDTA attenuated ROS increase. The ALF-induced decrease of eNOS content and its uncoupling concurred with, and was likely causally related to, both increased brain content of reactive oxidative species (ROS) and decreased cerebral cortical blood flow (CBF) in the same model.

Pretreatment with a glutamine synthetase inhibitor MSO delays the onset of initial seizures induced by pilocarpine in juvenile rats.

The contribution of glutamatergic transmission to generation of initial convulsive seizures (CS) is debated. We tested whether pretreatment with a glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), affects the onset and progression of initial CS by cholinergic stimulus in juvenile rats. Male rats (24 days old, Sprague Dawley) sequentially received i.p. injections of lithium-carbonate, MSO, methyl-scopolamine, and pilocarpine (Pilo). Pilo was given 150 min after MSO. Animals were continuously monitored using the Racine scale, EEG/EMG and intrahippocampal glutamate (Glu) biosensors. GS activity as measured in hippocampal homogenates, was not altered by MSO at 150 min, showed initial, varied inhibition at 165 (15 min post-Pilo), and dropped down to 11% of control at 60 min post-Pilo, whereas GS protein expression remained unaltered throughout. Pilo did neither modulate the effect of MSO on GS activity nor affect GS activity itself, at any time point. MSO reduced from 32% to 4% the number of animals showing CS during the first 12 min post-Pilo, delayed by ~6 min the appearance of electrographic seizures, and tended to decrease EMG power during ~15 min post-Pilo. The results indicate that MSO impairs an aspect of glutamatergic transmission involved in the transition from the first cholinergic stimulus to the onset of seizures. A continuous rise of extracellular Glu lasting 60 min was insignificantly affected by MSO, leaving the nature of the Glu pool(s) involved in altered glutamatergic transmission undefined.

Local Treatment of Burns with Cell-Based Therapies Tested in Clinical Studies.

Effective wound management is an important determinant of the survival and prognosis of patients with severe burns. Thus, novel techniques for timely and full closure of full-thickness burn wounds are urgently needed. The purpose of this review is to present the current state of knowledge on the local treatment of burn wounds (distinguishing radiation injury from other types of burns) with the application of cellular therapies conducted in clinical studies. PubMed search engine and ClinicalTrials.gov were used to analyze the available data. The analysis covered 49 articles, assessing the use of keratinocytes (30), keratinocytes and fibroblasts (6), fibroblasts (2), bone marrow-derived cells (8), and adipose tissue cells (3). Studies on the cell-based products that are commercially available (Epicel®, Keraheal™, ReCell®, JACE, Biobrane®) were also included, with the majority of reports found on autologous and allogeneic keratinocytes. Promising data demonstrate the effectiveness of various cell-based therapies; however, there are still scientific and technical issues that need to be solved before cell therapies become standard of care. Further evidence is required to demonstrate the clinical efficacy and safety of cell-based therapies in burns. In particular, comparative studies with long-term follow-up are critical.

The Status of Bile Acids and Farnesoid X Receptor in Brain and Liver of Rats with Thioacetamide-Induced Acute Liver Failure.

Acute liver failure (ALF) leads to neurological symptoms defined as hepatic encephalopathy (HE). Although accumulation of ammonia and neuroinflammation are generally accepted as main contributors to HE pathomechanism, a buildup of bile acids (BA) in the blood is a frequent component of liver injury in HE patients. Recent studies have identified the nuclear farnesoid X receptor (FXR) acting via small heterodimer partner (SHP) as a mediator of BA-induced effects in the brain of ALF animals. The present study investigated the status of the BA-FXR axis in the brain and the liver, including selective changes in pertinent genes in thioacetamide (TAA)-induced ALF in Sprague-Dawley rats. FXR was found in rat neurons, confirming earlier reports for mouse and human brain. BA accumulated in blood but not in the brain tissue. Expression of mRNAs coding for Fxr and Shp was reduced in the hippocampus and of Fxr mRNA also in the cerebellum. Changes in Fxr mRNA levels were not followed by changes in FXR protein. The results leave open the possibility that mobilization of the BA-FXR axis in the brain may not be necessarily pathognomonic to HE but may depend upon ALF-related confounding factors.

S-Adenosylmethionine Deficiency and Brain Accumulation of S-Adenosylhomocysteine in Thioacetamide-Induced Acute Liver Failure.

BACKGROUND: Acute liver failure (ALF) impairs cerebral function and induces hepatic encephalopathy (HE) due to the accumulation of neurotoxic and neuroactive substances in the brain. Cerebral oxidative stress (OS), under control of the glutathione-based defense system, contributes to the HE pathogenesis. Glutathione synthesis is regulated by cysteine synthesized from homocysteine via the transsulfuration pathway present in the brain. The transsulfuration-transmethylation interdependence is controlled by a methyl group donor, S-adenosylmethionine (AdoMet) conversion to S-adenosylhomocysteine (AdoHcy), whose removal by subsequent hydrolysis to homocysteine counteract AdoHcy accumulation-induced OS and excitotoxicity. METHODS: Rats received three consecutive intraperitoneal injections of thioacetamide (TAA) at 24 h intervals. We measured AdoMet and AdoHcy concentrations by HPLC-FD, glutathione (GSH/GSSG) ratio (Quantification kit). RESULTS: AdoMet/AdoHcy ratio was reduced in the brain but not in the liver. The total glutathione level and GSH/GSSG ratio, decreased in TAA rats, were restored by AdoMet treatment. CONCLUSION: Data indicate that disturbance of redox homeostasis caused by AdoHcy in the TAA rat brain may represent a deleterious mechanism of brain damage in HE. The correction of the GSH/GSSG ratio following AdoMet administration indicates its therapeutic value in maintaining cellular redox potential in the cerebral cortex of ALF rats.