In vitro modulation of epileptiform activity in the hippocampal formation of immature rodents by GABAergic antagonists, dopamine and methylxanthines

Während des frühen Lebens stellen epileptische Anfälle schwere neurologische Zustände dar, weil sie ein großer Risikofaktor für die Manifestation der Epilepsie sind und eine hohe pharmakologische Resistenz zeigen. In meiner Doktorarbeit konzentrierte ich mich auf die Frage, wie verschiedene Neurotransmitter-Systeme und klinisch verwendete Medikamente epileptiforme Entladungen im perinatalen Hippocampus beeinflussen. rnIm ersten Teil meines Projektes untersuchte ich die Wirkung von GABA-Antagonisten und Modulatoren, die zwischen phasischen und tonischen GABAergen Strömen differenzieren, auf Feldpotentialaktivität in Hippocampusschnitten. Diese Experimente zeigten, dass im unreifen Hippocampus synaptische GABAerge Aktivität benötigt wird, um die Erregbarkeit zu begrenzen, während tonische GABAerge Ströme die Erregbarkeit verstärken können. Dies könnte darauf hinweisen, dass Antiepileptika mit einer höheren Spezifität für synaptische GABAA-Rezeptoren wirksamer zur Behandlung von epileptischen Anfällen bei Neugeborenen sein können. rnUm den Einfluss von Dopamin auf die Erregbarkeit des unreifen Hippocampus herauszufinden, untersuchte ich im zweiten Teil meiner Arbeit die Wirkung von verschiedenen Dopaminkonzentrationen und spezifische Agonisten und Antagonisten der Dopamin-Rezeptor-Subtypen auf epileptiforme Entladungen. Diese Experimente zeigten, dass niedrige Dopamin Konzentrationen eine antikonvulsive Wirkung haben, welche vom D2-ähnliche-Rezeptor-Agonisten Quinpirol nachgeahmt werden kann, während höhere Dopamin-Konzentrationen eine prokonvulsive Wirkung über Aktivierung von D1-ähnlichen Rezeptoren hervorrufen. Obwohl unsere Untersuchungen eine mögliche Verwendung von D2-ähnlichen Rezeptor-Agonisten zur Kontrolle epileptischer Anfälle in Neugeborenen nahelegen, müssen mögliche negative Auswirkungen von DAergen Agonisten und Antagonisten auf die neuronale Entwicklung berücksichtigt werden.rnIm dritten Teil meiner Arbeit untersuchte ich welche Konzentrationen von Methylxanthinen epileptische Anfälle in Hippocampuspreparationen auslösen die synaptische Übertragungen verändern können. Diese Experimente zeigten, dass sowohl Theophyllin als auch Koffein in höheren Konzentrationen die basale synaptische Übertragungen in der CA1-Region des Hippocampus modifizieren und epileptiforme Entladungen provozieren. Die Auswirkungen auf die postsynaptischen Antworten und spontanen epileptiformen Entladungen durch Koffein waren weniger ausgeprägt, was darauf hindeutet, dass diese Substanz potentiell vorteilhafter für therapeutische Anwendungen bei Frühgeborenen sein kann. rnZusammenfassend bereichern die Ergebnisse meiner Studie erheblich unser Wissen über die zugrunde liegenden Mechanismen epileptiformer Aktivität im unreifen Hippocampus und den therapeutischen Einsatz von Methylxanthinen und Pharmaka, die auf das GABAerge und DArge System einwirken.rnrn

Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in-vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme.

Efficacy of taurolidine against periodontopathic species--an in vitro study

The antimicrobial effect of taurolidine was tested against periodontopathic species in comparison to chlorhexidine digluconate in the presence or absence of serum. Minimal inhibitory concentrations (MIC), microbiocidal concentrations (MBC), as well as killing were determined against 32 different microbial strains including 3 Porphyromonas gingivalis, 3 Aggregatibacter actinomycetemcomitans, and 15 potentially superinfecting species with and without 25% v/v human serum. The MIC(50) of taurolidine against the tested microbial strains was 0.025% and the MIC(90) 0.05%. The respective values for the MBCs were 0.05% and 0.1%. Addition of 25% serum (heat-inactivated) did not change the MIC and MBC values of taurolidine. In contrast, MICs and MBCs of chlorhexidine (CHX) increased by two steps after addition of serum. Taurolidine killed microorganisms in a concentration and time-dependent manner, the killing rate of 1.6% taurolidine was 99.08% ± 2.27% in mean after 2 h. Again, killing activity of taurolidine was not affected if serum was added, whereas addition of inactivated serum clearly reduced the killing rate of all selected bacterial strains by CHX. Therefore, taurolidine possesses antimicrobial properties which are not reduced in the presence of serum as a main component in gingival crevicular fluid and wound fluid. Taurolidine may have potential as an antimicrobial agent in non-surgical and surgical periodontal treatment.

Infiltration of Natural Caries Lesions in Relation to Their Activity Status and Acid Pretreatment in vitro

This study aimed at testing how active and inactive enamel caries lesions differ by their degree of resin infiltration, and whether the choice of acid pretreatment plays a crucial role. Four examiners assessed 104 human molars and premolars with noncavitated enamel lesions and classified them as 'active' or 'inactive' using the Nyvad criteria. Forty-five teeth were included in this study after independent unanimous lesion activity assessment. Lesions were cut perpendicularly into 2 halves. Each half lesion was pretreated with either 15% hydrochloric acid or 35% phosphoric acid. The lesions were infiltrated after staining with rhodamine isothiocyanate. Thin sections of 100 µm were prepared and the specimens were bleached with 30% hydrogen peroxide. The specimens were then counterstained with sodium fluorescein, subjected to confocal laser scanning microscopy and analyzed quantitatively. Outcome parameters were maximum and average infiltration depths as well as relative penetration depths and areas. In active lesions no significant difference of percentage maximum penetration depth and percentage average penetration depth between lesions pretreated with hydrochloric or phosphoric acid could be observed. In inactive lesions, however, phosphoric acid pretreatment resulted in significantly lower penetration compared to hydrochloric acid pretreatment. Surface conditioning with hydrochloric acid led to similar infiltration results in active and inactive lesions. Moreover, inactive lesions showed greater variability in all assessed infiltration parameters than did active lesions. In conclusion, caries lesion activity and acid pretreatment both influenced the infiltration. The use of phosphoric acid to increase permeability of the surface layer of active lesions should be further explored.

Equine CD4(+) CD25(high) T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro

Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4(+) Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4(+) CD25(+) T cells, mainly in the CD4(+) CD25(high) subpopulation. Proliferation of CD4(+) CD25(-) sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co-culture with CD4(+) CD25(high) sorted cells in a dose-dependent manner. The mechanism of suppression by the CD4(+) CD25(high) cell population is mediated by close contact as well as interleukin (IL)-10 and transforming growth factor-beta1 (TGF-beta1) and probably other factors. In addition, we studied the in vitro induction of CD4(+) Treg and their characteristics compared to those of freshly isolated CD4(+) Treg cells. Upon stimulation with a combination of concanavalin A, TGF-beta1 and IL-2, CD4(+) CD25(+) T cells which express FoxP3 and have suppressive capability were induced from CD4(+) CD25(-) cells. The induced CD4(+) CD25(high) express higher levels of IL-10 and TGF-beta1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4(+) CD25(high) subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4(+) CD25(+) cells in horses and offers insights into ex vivo manipulation of Treg cells.

Application of an in vitro drug screening assay based on the release of phosphoglucose isomerase to determine the structure-activity relationship of thiazolides against Echinococcus multilocularis metacestodes

OBJECTIVES: The disease alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is fatal if treatment is unsuccessful. Current treatment options are, at best, parasitostatic, and involve taking benzimidazoles (albendazole, mebendazole) for the whole of a patient's life. In conjunction with the recent development of optimized procedures for E. multilocularis metacestode cultivation, we aimed to develop a rapid and reliable drug screening test, which enables efficient screening of a large number of compounds in a relatively short time frame. METHODS: Metacestodes were treated in vitro with albendazole, the nitro-thiazole nitazoxanide and 29 nitazoxanide derivatives. The resulting leakage of phosphoglucose isomerase (PGI) activity into the medium supernatant was measured and provided an indication of compound efficacy. RESULTS: We show that upon in vitro culture of E. multilocularis metacestodes in the presence of active drugs such as albendazole, the nitro-thiazole nitazoxanide and 30 different nitazoxanide derivatives, the activity of PGI in culture supernatants increased. The increase in PGI activity correlated with the progressive degeneration and destruction of metacestode tissue in a time- and concentration-dependent manner, which allowed us to perform a structure-activity relationship analysis on the thiazolide compounds used in this study. CONCLUSIONS: The assay presented here is inexpensive, rapid, can be used in 24- and 96-well formats and will serve as an ideal tool for first-round in vitro tests on the efficacy of large numbers of antiparasitic compounds.

Transient rhythmic network activity in the somatosensory cortex evoked by distributed input in vitro

The initiation and maintenance of physiological and pathophysiological oscillatory activity depends on the synaptic interactions within neuronal networks. We studied the mechanisms underlying evoked transient network oscillation in acute slices of the adolescent rat somatosensory cortex and modeled its underpinning mechanisms. Oscillations were evoked by brief spatially distributed noisy extracellular stimulation, delivered via bipolar electrodes. Evoked transient network oscillation was detected with multi-neuron patch-clamp recordings under different pharmacological conditions. The observed oscillations are in the frequency range of 2-5 Hz and consist of 4-12 mV large, 40-150 ms wide compound synaptic events with rare overlying action potentials. This evoked transient network oscillation is only weakly expressed in the somatosensory cortex and requires increased [K+]o of 6.25 mM and decreased [Ca2+]o of 1.5 mM and [Mg2+]o of 0.5 mM. A peak in the cross-correlation among membrane potential in layers II/III, IV and V neurons reflects the underlying network-driven basis of the evoked transient network oscillation. The initiation of the evoked transient network oscillation is accompanied by an increased [K+]o and can be prevented by the K+ channel blocker quinidine. In addition, a shift of the chloride reversal potential takes place during stimulation, resulting in a depolarizing type A GABA (GABAA) receptor response. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), N-methyl-D-aspartate (NMDA), or GABA(A) receptors as well as gap junctions prevents evoked transient network oscillation while a reduction of AMPA or GABA(A) receptor desensitization increases its duration and amplitude. The apparent reversal potential of -27 mV of the evoked transient network oscillation, its pharmacological profile, as well as the modeling results suggest a mixed contribution of glutamatergic, excitatory GABAergic, and gap junctional conductances in initiation and maintenance of this oscillatory activity. With these properties, evoked transient network oscillation resembles epileptic afterdischarges more than any other form of physiological or pathophysiological neocortical oscillatory activity.

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.

In vitro effects of thiazolides on Giardia lamblia WB clone C6 cultured axenically and in coculture with Caco2 cells

The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 microM; in comparison, NTZ and tizoxanide had IC50s of 2.4 microM, and MTZ had an IC50 of 7.8 microM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

Analgesic activity of a non-peptide imidazolidinedione somatostatin agonist: in vitro and in vivo studies in rat

Several lines of evidence support an important role for somatostatin receptors (SSTRs) in pain modulation. The therapeutic use of established SSTR peptide agonists for this indication is limited by their broad range of effects, need for intrathecal delivery, and short half-life. Therefore, the goal of the present study was to investigate the analgesic effect of SCR007, a new, highly selective SSTR2 non-peptide agonist. Behavioral studies demonstrated that paw withdrawal latencies to heat were significantly increased following intraplantar SCR007. Furthermore, both intraperitoneal and intraplantar injection of SCR007 significantly reduced formalin- and capsaicin-induced flinching and lifting/licking nociceptive behaviors. Recordings from nociceptors using an in vitro glabrous skin-nerve preparation showed that SCR007 reduced heat responses in a dose-dependent fashion, bradykinin-induced excitation, heat sensitization and capsaicin-induced excitation. In both the behavioral and single fiber studies, the SCR007 effects were reversed by the SSTR antagonist cyclo-somatostatin, demonstrating receptor specificity. In the single fiber studies, the opioid antagonist naloxone did not reverse SCR007-induced anti-nociception suggesting that SCR007 did not exert its effects through activation of opioid receptors. Analysis of cAMP/protein kinase A (PKA) involvement demonstrated that SCR007 prevented forskolin- and Sp-8-Br-cAMPS (a PKA activator)-induced heat sensitization, supporting the hypothesis that SCR007-induced inhibition could involve a down-regulation of the cAMP/PKA pathway. These data provide several lines of evidence that the non-peptide imidazolidinedione SSTR2 agonist SCR007 is a promising anti-nociceptive and analgesic agent for the treatment of pain of peripheral and/or central origin.

In vitro efficacy of nitro- and bromo-thiazolyl-salicylamide compounds (thiazolides) against Besnoitia besnoiti infection in Vero cells

Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) exhibit considerable in vitro activity against Besnoitia besnoiti tachyzoites grown in Vero cells. Real-time-PCR was used to assess B. besnoiti tachyzoite adhesion, invasion, and intracellular proliferation in vitro. A number of NTZ-derivatives, including Rm4822 and Rm4803, were generated, in which the thiazole-ring-associated nitro-group was replaced by a bromo-moiety. We here show that replacement of the nitro-group on the thiazole ring with a bromo (as it occurs in Rm4822) does not impair the efficacy of the drug, but methylation of the salicylate ring at the ortho-position in a bromo-derivative (Rm4803) results in complete abrogation of the antiparasitic activity. Treatment of extracellular B. besnoiti tachyzoites with NTZ has an inhibitory effect on host cell invasion, while treatments with TIZ, Rm4822 do not. TEM demonstrates that the effects of Rm4822 treatment upon the parasites are similar to the damage induced by NTZ. This includes increased vacuolization of the parasite cytoplasm, and loss of the structural integrity of the parasitophorous vacuole and its membrane. Thus, Rm4822, due to the absence of a potentially mutagenic nitro-group, may represent an important potential addition to the anti-parasitic arsenal for food animal production, especially in cattle.

Efficacy of gatifloxacin alone and in combination with cefepime against penicillin-resistant Streptococcus pneumoniae in a rabbit meningitis model and in vitro

Gatifloxacin penetrated well into cerebrospinal fluid (CSF) (49 +/- 11%), measured by comparison of AUC(CSF)/AUC(serum), and showed good bactericidal activity (leading to a decrease of 0.75 +/- 0.17 log10 cfu/mL/h) in the treatment of experimental meningitis in rabbits caused by a penicillin-resistant pneumococcal strain (MIC 4 mg/L). It was significantly more effective than the standard regimen, ceftriaxone with vancomycin, which led to a decrease of 0.53 +/- 0.17 log10 cfu/mL/h. The addition of cefepime to gatifloxacin slightly improved the killing rates (giving a decrease of 0.84 +/- 0.14 log10 cfu/mL/h). In vitro, synergy was demonstrated between cefepime and gatifloxacin by the chequerboard method (fractional inhibitory concentration index = 0.5) and by viable counts over 8 h.

Total synthesis of hypermodified epothilone analogs with potent in vitro antitumor activity

The convergent total synthesis of hypermodified epothilone analogs 1 and 2 has been achieved with the stereoselective cyclopropanation of allylic alcohol 17 and ring-closing olefin metathesis with diene 22 as the key steps. In spite of significant structural differences between these analogs and the natural epothilone scaffold, 1 and 2 are potent inducers of tubulin polymerization and inhibit the growth of human cancer cells in vitro with sub-nM IC50 values.