Tamás Janáky

Human tear fluid contains numerous compounds, which are present in highly variable amounts owing to the dynamic and multipurpose functions of tears. A better understanding of the level and sources of variance is essential for determining the functions of the different tear components and the limitations of tear samples as a potential biomarker source. In this study, a quantitative proteomic method was used to analyze variations in the tear protein profiles of healthy volunteers. High day-to-day and inter-eye personal variances were observed in the tear volumes, protein content, and composition of the tear samples. Several normalization and outlier exclusion approaches were evaluated to decrease variances. Despite the intrapersonal variances, statistically significant differences and cluster analysis revealed that proteome profile and immunoglobulin composition of tear fluid present personal characteristics. Using correlation analysis, we could identify several correlating protein clusters, mainly related to the source of the proteins. Our study is the first attempt to achieve more insight into the biochemical background of human tears by statistical evaluation of the experimentally observed dynamic behavior of the tear proteome. As a pilot study for determination of personal protein profiles of the tear fluids of individual patients, it contributes to the application of this noninvasively collectible body fluid in personal medicine.

Background: Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptors (S1R) are expressed in platelets and endothelial cells and S1R expression is protective in diabetes. Objectives: Our aim was to examine the influence of sub-chronic, in vivo administered S1R ligands PRE-084, (S)-L1 (a new compound) and NE-100 on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. Methods: The serum level of the S1R ligands was detected by LC-MS/MS before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by RT-qPCR. The eicosanoid synthesis was examined with a radiolabelled arachidonic acid substrate and ELISA. Results: One month after the onset of STZ-induced diabetes, in vehicle-treated, diabetic rat platelet TxB₂ and aortic 6-k-PGF_1α production dropped. Sub-chronic in vivo treatment of STZ-induced diabetes in rats for one week with PRE-084 enhanced vasoconstrictor and platelet aggregator and reduced vasodilator and anti-aggregator cyclooxygenase product formation. (S)-L1 reduced the synthesis of vasodilator and anti-aggregator cyclooxygenase metabolites and promoted the recovery of physiological platelet function in diabetic rats. The S1R antagonist NE-100 produced no significant changes in platelet arachidonic acid metabolism. (S)-L1 decreased the synthesis of vasoconstrictor and platelet aggregator cyclooxygenase metabolites, whereas NE-100 increased the quantity of aortic vasodilator and anti-aggregator cyclooxygenase products and promoted the recovery of diabetic endothelial dysfunction in the aorta. The novel S1R ligand, (S)-L1 had similar effects on eicosanoid synthesis in platelets as the agonist PRE-084 and in aortas as the antagonist NE-100. Conclusions: S1R ligands regulate cellular functions and local blood circulation by influencing arachidonic acid metabolism. In diabetes mellitus, the cell-specific effects of S1R ligands have a compensatory role and aid in restoring physiological balance between the platelet and vessel.

Tear samples are considered in recent publications as easily, noninvasively collectible information sources for precision medicine. Their complex composition may aid the identification of biomarkers and the monitoring of the effectiveness of treatments for the eye and systemic diseases. Sample collection and processing are key steps in any analytical method, especially if subtle personal differences need to be detected. In this work, we evaluate the usability of a novel sample collection technique for human tear samples using phenol red threads (cotton thread treated with the pH indicator phenol red), which are efficiently used to measure tear volume in clinical diagnosis. The low invasiveness and low discomfort to the patients have already been demonstrated, but their applicability for proteomic sample collection has not yet been compared to other methods. We have shown, using various statistical approaches, the qualitative and quantitative differences in proteomic samples collected with this novel and two traditional methods using either glass capillaries or Schirmer’s paper strips. In all parameters studied, the phenol red threads proved to be equally or even more suitable than traditional methods. Based on detectability using different sampling methods, we have classified proteins in tear samples.

Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptor expressed in platelets and endothelial cells can regulate intracellular signalization. Our aim was to examine the influence of sub-chronic, in vivo administered sigma-1 receptor ligands (2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate, PRE-084; S-N-Benzyl-6,7-dimethoxy-1,2,3,4-tetrahydro-1-isoquinolineethanamine, a new compound (S)-L1; and N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)-phenyl]-ethylamine monohydrochloride, NE-100) on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. The serum level of sigma-1 receptor ligands was detected by liquid chromatography-mass spectrometry before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by reverse transcription coupled quantitative polymerase chain reaction. The eicosanoid synthesis was examined by using of radiolabeled arachidonic acid substrate and enzyme-linked immunosorbent assay. In diabetic rats, the sub-chronic, in vivo administration of the sigma-1 receptor ligands modified the transcript levels of sigma-1 receptor and cyclooxygenase-1, the concentration of cyclooxygenase in platelets and the eicosanoid synthesis in both platelets and aorta. Sigma-1 receptor ligands, by changing platelet and blood vessel eicosanoid synthesis, may play a role in modulating diabetic complications.

Biomarkers for predicting treatment response to thrombolysis in acute ischemic stroke are currently lacking. Both, animal models and clinical studies have provided evidence that the kynurenine (KYN) pathway is activated in ischemic stroke. In our pilot study, we aimed to investigate whether KYN pathway enzymes and metabolites could serve as potential biomarkers for treatment response in the hyperacute phase of ischemic stroke. We included 48 acute ischemic stroke patients who received thrombolysis. Blood samples were taken both before and 12 h after treatment. Concentrations of 11 KYN metabolites were determined using ultra-high-performance liquid chromatography-mass spectrometry. To assess the treatment response, we used early neurological improvement (ENI), calculated as the difference between the admission and discharge National Institutes of Health Stroke Scale (NIHSS) scores. We performed receiver operating characteristic (ROC) analysis for KYN pathway metabolites and enzymes that showed a correlation with ENI. In the samples taken before thrombolysis, significantly lower concentrations of kynurenic acid (KYNA) and kynurenine aminotransferase (KAT) activity were found in patients who had ENI (p = 0.01 and p = 0.002, respectively). According to the ROC analysis, the optimal cut-off value to predict ENI for KYNA was 37.80 nM (sensitivity (SN) 69.2%, specificity (SP) 68.4%) and 0.0127 for KAT activity (SN 92.3%, SP 73.7%). Our research is the first clinical pilot study to analyze changes in the KYN pathway in ischemic stroke patients who received thrombolytic treatment. Based on our results, baseline KYNA concentration and KAT activity could serve as potential biomarkers to predict early treatment response to thrombolysis.

Nasal drug delivery has become a popular research field in the last years. This is not surprising since the nose possesses unique anatomical and physical properties. Via the nasal mucosa local, systemic, and directly central nerve systemic (CNS) effect is achievable. Powders have favorable physicochemical properties over liquid formulations. Lamotrigine (LAM) is an antiepileptic agent with a relatively mild side effect spectrum, but only available in tablet form on market. Reducing the particle size to the nano range can affect the bioavailability of pharmaceutical products. The aim of this article was to continue the work started, compare the in vitro properties of a nanonized lamotrigine containing nasal powder (nanoLAMpowder) and its physical mixture (PM) that were prepared by dry milling. Moreover, to study their trans-epithelial absorption to reach the blood and target the brain by axonal transport. Due to the dry milling technique, the particle size of LAM, their surface and also their structure changed that led to higher in vitro dissolution and permeability rate. The results of the in vivo tests showed that the axonal transport of the drug was assumable by both intranasal formulations because the drug was present in the brain within a really short time, but the LAM from the nanoLAMpowder liberated even faster.