Multiple sclerosis in pediatric patients in Slovenia.

AIM: The purpose of this study is to review the Slovenian experience with the diagnostics, treatment and outcome in pediatric multiple sclerosis (MS) patients. METHODS: Children and adolescent diagnosed with MS and followed by Department of Child, Adolescent and Developmental Neurology, University Childrens' Hospital Ljubljana, between 1 January 2000 and 31 December 2012 were included. Data from patients' documentation were analyzed retrospectively to record demographic data, clinical presentation, paraclinical findings, disability progression, relapse rate and treatment strategies. RESULTS: The study includes 38 patients up to 18 years with MS diagnosis, with female: male ratio 2.8:1 and the incidence of 0.81 per 100.000 children of 0-18 years. The mean age at the time of diagnosis was 15 years 4 months. Most frequent presenting symptoms were sensory, motor, brain-stem, visual and ataxia and 65% of patients had a relapse in the first year. The value of paraclinical findings was asessed. 74% of patients with definite MS and 36% of those with clinically isolated syndrome received disease modifyng therapy and 68% of them was not affected at the follow-up. INTERPRETATION: The characteristics of pediatric MS patients in Slovenia disclose higher annual relapse rates than in adults but also favorable impact of disease modifying treatment on a clinical course. Our data suggest a good treatment tolerance but also the influence of the formulation on a decision to start or switch the treatment.

Dynamics of Rear Stagnant Cap formation at the surface of spherical bubbles rising in surfactant solutions at large Reynolds numbers under conditions of small Marangoni number and slow sorption kinetics.

On the surface of bubbles rising in a surfactant solution the adsorption process proceeds and leads to the formation of a so called Rear Stagnant Cap (RSC). The larger this RSC is the stronger is the retardation of the rising velocity. The theory of a steady RSC and steady retarded rising velocity, which sets in after a transient stage, has been generally accepted. However, a non-steady process of bubble rising starting from the initial zero velocity represents an important portion of the trajectory of rising, characterized by a local velocity profile (LVP). As there is no theory of RSC growth for large Reynolds numbers Re ¼ 1 so far, the interpretation of LVPs measured in this regime was impossible. It turned out, that an analytical theory for a quasi-steady growth of RSC is possible for small Marangoni numbers Ma « 1, i.e. when the RSC is almost completely compressed, which means a uniform surface concentration Γ(θ)=Γ(∞) within the RSC. Hence, the RSC angle ψ(t) is obtained as a function of the adsorption isotherm parameters and time t. From the steady velocity v(st)(ψ), the dependence of non-steady velocity on time is obtained by employing v(st)[ψ(t)] via a quasi-steady approximation. The measurement of LVP creates a promising new opportunity for investigation of the RSC dynamics and adsorption kinetics. While adsorption and desorption happen at the same localization in the classical methods, in rising bubble experiments desorption occurs mainly within RSC while adsorption on the mobile part of the bubble surface. The desorption flux from RSC is proportional to αΓ(∞), while it is usually αΓ. The adsorption flux at the mobile surface above RSC can be assumed proportional to ßC0, while it is usually ßC0(1-Γ/Γ(∞)). These simplifications may become favorable in investigations of the adsorption kinetics for larger molecules, in particular for globular proteins, which essentially stay at an interface once adsorbed.

Electrochemotherapy of mouse sarcoma tumors using electric pulse trains with repetition frequencies of 1 Hz and 5 kHz.

Electrochemotherapy is an efficient local treatment of tumors that combines administration of a chemotherapeutic drug with the subsequent application of electric pulses to the tumor. Although no difference in clinical response of the treated tumors to the electrochemotherapy when using 1 Hz or 5 kHz repetition frequency was observed, it is mandatory to be aware of possible differences in the effectiveness of electrochemotherapy when using suboptimal doses of the drugs. Therefore, this study compares the antitumor effectiveness of electrochemotherapy using electric pulse trains with repetition frequencies of 1 Hz and 5 kHz at suboptimal drug doses of bleomycin or cisplatin. Electrochemotherapy of fibrosarcoma SA-1 subcutaneous tumors transplanted in A/J mice resulted in good antitumor effectiveness, but antitumor effectiveness was significantly better at 1 Hz repetition frequency than at 5 kHz. The platinum content was higher in tumors treated with a 1 Hz repetition frequency. The application of electric pulses to the tumors at a 5 kHz repetition frequency induced an immediate reduction in tumor perfusion, comparable to the reduction at 1 Hz but with faster reperfusion. The greater effectiveness of electrochemotherapy using electric pulse trains of 1 Hz compared to 5 kHz is due to the greater electroporative effect and longer time in which electroporated tumors are exposed to the two chemotherapeutic drugs. These differences are observed at suboptimal drug doses, whereas at optimal drug doses of bleomycin or cisplatin the antitumor effectiveness is the same, as demonstrated in clinical trials.

Kinetic and pharmacological properties of [(3)H]-histamine transport into cultured type 1 astrocytes from neonatal rats.

OBJECTIVE AND DESIGN: Astrocytes actively participate in the inactivation of neurotransmitters. In this work we elucidated the contribution of astrocytes in clearance of histamine, a process which has not yet been fully clarified. METHODS: The characteristics of [(3)H]-histamine uptake were determined in cultured neonatal rat type 1 astrocytes and histamine-N-methyl-transferase expression was determined using RT-PCR. RESULTS: These cells transport [(3)H]-histamine in a time- and concentration-dependent manner. The histamine clearance by astrocytes was described by a mathematical model including two processes: electrodiffusion and active transport. A further analysis of kinetic parameters of a carrier-operated transport revealed a single transport system with Michaelis constant (K(m)) of 3.5 +/- 0.8 microM and a maximal uptake rate (V(max)) of 7.9 +/- 0.3 pmol/mg protein/min. From drugs tested amitriptyline, desipramine, mepyramine and cimetidine significantly decreased [(3)H]-histamine uptake. Taken-up histamine could be metabolically degraded in cultured astrocytes, since they express mRNA for enzyme histamine-N-methyltransferase. CONCLUSIONS: Astrocytes participate in the clearance of extracellular histamine by electrodiffusion and active transport by a yet not identified carrier. Taken up histamine can be converted to tele-methylhistamine within astrocytes thus indicating the involvement of astrocytes not only in clearance but also in the inactivation of histamine.

Influence of surface active substances on bubble motion and collision with various interfaces.

Bubble motion as a function of distance from a point of its detachment and phenomena occurring during the bubble approach and collision with liquid/gas and liquid/solid interfaces in pure water and solutions of various surface active substances are described and discussed. It is showed that presence of surface active substance has a profound influence on values of the terminal velocity and profiles of the local velocity. At low solutions concentrations there are three distinct stages in the bubble motion: (i) a rapid acceleration, (ii) a maximum velocity value followed by its monotonic decrease, and (iii) attainment of the terminal velocity, while at high concentrations (and in pure water) there are only stages (i) and (iii). It is showed that the bubble terminal velocity decreases rapidly at low surfactant concentration, but there can be found some characteristic concentrations (adsorption coverage's) above which the velocity almost stopped to decrease. Immobilization of the bubble surface resulting from adsorption of the surface active substances (surface tension gradients inducement) causes over twofold lowering of the bubble velocity. Presence of the maximum on the local velocity profiles is an indication that a stationary non-uniform distribution of adsorption coverage (needed for immobilization the bubble interface) was not established there. When the rising bubble arrives at liquid/gas interface or liquid/solid interface there can be formed either foam or wetting film or three-phase contact (TPC). It is showed that prior to the foam and/or wetting film formation the bubble colliding with the interfaces can bounce backward and simultaneously its shape pulsates rapidly with a frequency over 1000 Hz. It is rather unexpected that even in the case of the free surface the bubble's shape and consequently its surface area can vary so rapidly. It shows straightforward that on such a rapidly distorted interface the adsorption coverage can be very different from that at equilibrium. This fact should be taken into account more appropriately in the discussion of the mechanism of formation and stabilization of various dispersed systems (e.g. foams, emulsions). Bubble collision with solids and formation of the three-phase contact is a necessary condition for flotation separation. It is rather common understanding that immediate attachment should occur in the case of hydrophobic surface, while there should be no attachment in the case of the hydrophilic ones. It is reported that even in the case of such hydrophobic solid surface as Teflon, the bubble attachment did not need to occur at first collision and in distilled water the bubble can bounce a few times without attachment. Presence of frother facilitates the bubble attachment to hydrophobic solid surface. Time scale of the TPC formation is very short, of an order of single ms. It was observed that presence of a micro-bubble at the solid surface facilitated drastically an attachment of the colliding bubble. Roughness of Teflon surface increases probability of the bubble attachment-most probably-as a result of higher probability of micro- and/or nano-bubbles presence at the solid surface.

Activation of histamine H1-receptor enhances neurotrophic factor secretion from cultured astrocytes.

OBJECTIVE: Histamine stimulates nerve growth factor (NGF) secretion from cultured astrocytes. Histamine H(1)-receptor antagonists completely block its effect. In the present study, we determined the involvement of histamine-receptor subtypes in this process. MATERIALS AND METHODS: Radioligand-binding assay was used to establish the presence of histamine H(1)- and H(2)-receptors on new-born rat cortical astrocytes in primary culture. Histamine H(1)-, H(2)- and H(3)/H(4)-receptor ligands, and highly selective protein kinase C (PKC) inhibitor were used to influence NGF secretion from cultured astrocytes. NGF, released into the culture medium, was measured by NGF-ELISA. RESULTS: Histamine H(1)-receptor agonists (histamine, selected histaprodifens) increased the secretion of NGF from cultured astrocytes in a concentration-dependent manner. H(1)-receptor antagonists/inverse agonists (mepyramine, triprolidine) and PKC inhibitor completely blocked the effect of histamine. Histamine H(2)- and H(3)-receptor agonists did not enhance NGF secretion significantly. In addition, H(2)- and H(3)/H(4)-receptor antagonists did not diminish histamine-stimulated NGF release. CONCLUSIONS: Our results indicate that histamine H(1)-receptor and PKC are involved in the signal transduction pathway, responsible for histamine-stimulated NGF secretion from cultured astrocytes.

Regulatory role of extracellular Na+ and Ca2+ ions in nerve growth factor induced histamine secretion from rat mast cells.

OBJECTIVE AND DESIGN: This study was aimed to investigate effects of extracellular Na+ and Ca2+ ions on nerve growth factor (NGF) induced histamine release from mast cells. MATERIAL: Isolated peritoneal mast cells were obtained from male Wistar rats. METHODS: Cells were suspended in solution with different concentration of Na+ and Ca2+ ions and stimulated with NGF. Histamine release was assayed spectrofluorometrically. RESULTS: NGF (0.001-1 microg/ml) dose-dependently releases histamine from mast cell at physiological extracellular Na+ (134 mM) and Ca2+ (1 mM) conditions. Lowering extracellular Ca2+ ions to 0.1 mM reduced histamine response to nearly basal level. However, the removal of extracellular Na+ ions significantly enhanced the secretion provoked by NGF (0.6 microg/ml) in low Ca2+ medium. Amiloride, an inhibitor of Na+/Ca2+ and Na/H+ exchangers inhibited the potentiating effect of sodium free conditions. CONCLUSIONS: Our results suggest that the activity of Na+/Ca2+ and/or Na+/H+ exchange mechanisms could be of particular importance in the secretory process of mast cells induced by NGF.

Interleukin-1 beta, but not IL-1 alpha, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor.

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1 beta (IL-1 beta). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1 alpha and IL-1 beta) and the antagonist (IL-1ra)-members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [(125)I] IL-1 beta to cultured astrocytes was saturable and of high affinity. Mean values for the K(D) and B(max) were calculated to be 60.7+/-7.4 pM and 2.5+/-0.1 fmol mg(-1) protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1 alpha (K(i) of 341.1 pM) and IL-1 beta (K(i) 59.9 pM), as well as the antagonist IL-1ra (K(i) 257.6 pM), displaced specific [(125)I] IL-1 beta binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [(125)I] IL-1 beta binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1 alpha and IL-1 beta revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1 beta (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1 alpha by itself had no stimulatory action on NGF release. The simultaneous application of IL-1 alpha and IL-1 beta elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1 beta. We concluded that IL-1 beta-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1 alpha and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.

Serotonin regulation of nerve growth factor synthesis in neonatal and adult astrocytes: comparison to the beta-adrenergic agonist isoproterenol.

Although serotonin regulates synthesis of the neurotrophic factor S-100 beta by astrocytes, its ability to affect nerve growth factor (NGF) synthesis has never been examined. We report here that there is a correlation between the effect of serotonin on cyclic adenosine monophosphate (cAMP) content and on NGF content in neonatal astrocytes but not in adult astrocytes. In neonatal striatal astrocytes, serotonin increases both cAMP and NGF, whereas, in neonatal cerebellar astrocytes, serotonin decreases both. The increase in neonatal cortical astrocyte cAMP appeared to be too small (45%) to increase NGF significantly. The beta-adrenergic agonist isoproterenol increased cAMP and NGF in both cortical and striatal astrocytes derived from neonatal rats. In contrast, there was a dissociation between cAMP changes and NGF changes in astrocytes derived from adult rats. Both serotonin and isoproterenol increased cAMP in adult cortical astrocytes, without any effect on NGF content. However, adult striatal astrocytes responded to serotonin with an elevation of both cAMP and NGF, whereas isoproterenol could only enhance cAMP, without affecting NGF. Thus, in neonatal astrocytes, a change of sufficient magnitude in cAMP was correlated with a comparable change in NGF, in response to activation of either serotonergic or beta-adrenergic receptors; in cerebellar astrocytes, the decrease in cAMP was accompanied by a decrease in NGF. In contrast, adult astrocytes were not responsive: Although cAMP changes were large, NGF synthesis was increased only in striatal astrocytes and only in response to serotonin. J. Neurosci. Res. 64:261-267, 2001. Published 2001 Wiley-Liss, Inc.

Prognostic value of motor evoked potentials elicited by multipulse magnetic stimulation in a surgically induced transitory lesion of the supplementary motor area: a case report.

Surgery involving the supplementary motor area (SMA) places the patient at risk of transient motor deficit. To predict outcome in patients with early postoperative hypokinesis would be relevant to both the patient and the surgical team. A 15 year old girl with a large left thalamic tumour removed through a left transcallosal approach is described. Despite intraoperatively preserved muscle motor evoked potentials (mMEPs) from all limbs, elicited by multipulse electrical stimulation, she awoke with a right hemiplegia and mutism. On the first postoperative day, neurophysiological evaluation using a multipulse magnetic stimulation technique, with a train of four magnetic stimuli, confirmed the presence of mMEPs from the hemiplegic right limbs. Slight spontaneous motor activity of the right limbs and initial speech were seen later on the same day with dramatic improvement over subsequent days. It is concluded that multiple rather than single magnetic stimulation techniques may be needed to elicit mMEPs for an early postoperative differential diagnosis of SMA damage versus injury to the primary motor cortex or the corticospinal tract.

Cytokine-regulated secretion of nerve growth factor from cultured rat neonatal astrocytes.

Interactions involved in the regulation of nerve growth factor (NGF) release by inflammatory cytokines: interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) were examined in rat neonatal cortical astrocytes in primary culture. Exposure of cultured astrocytes to IL-1beta, IL-6, and TGF-beta1 resulted in the stimulation of NGF secretion. Treatment of cells for 24 h with IL-1beta (10 U/ml), IL-6 (5 ng/ml) and TGF-beta1 (5 ng/ml) caused 3-, 1.8-, and 2.8-fold increase in NGF secretion as compared to the control cells. In contrast, TNF-alpha (30 ng/ml) by itself had no stimulatory action on NGF release whereas co-stimulation of astrocytes with IL-1beta and TNF-alpha showed a synergistic interaction. Co-treatment of astrocytes with IL-1beta and TGF-beta1 increased NGF secretion in an additive way, whereas simultaneous application of IL-1beta and IL-6 resulted in the inhibitory effect on NGF secretion. Our results suggest that interactions between cytokines used cause the stimulation of NGF secretion through different regulatory mechanisms.

The effects of histamine and interleukin-6 on NGF release from cortical astrocytes in primary culture.

The influence of neurotransmitter histamine and cytokine interleukin-6 (IL-6) on nerve growth factor (NGF) release was studied in rat neonatal cortical astrocytes in primary culture. Exposure of astrocytes to either histamine or IL-6 resulted in the stimulation of NGF release. Maximal stimulation of NGF release was obtained using histamine at concentration 100 nM after 24 h of treatment (2.3 fold increase over the basal secretion from the control cells). IL-6 (30 ng/ml) induced NGF secretion was 1.66 fold over the basal level. Time course of NGF release, after histamine or IL-6 treatment, showed elevation of NGF level in the culture medium after 8 h or 24 h, respectively. IL-6 antibody effectively blocked the IL-6 stimulatory effect on NGF release, but did not influence NGF release, evoked by histamine. IL-6 antibody alone did not show any influence on NGF release. Our results suggest that IL-6 and histamine stimulate release of NGF by two different and independent molecular pathways.