The inflammatory signalling mediator TAK1 mediates lymphocyte recruitment to lipopolysaccharide-activated murine mesenchymal stem cells through interleukin-6.

As a response to pro-inflammatory signals mesenchymal stem cells (MSCs) secrete agents and factors leading to lymphocyte recruitment, counteracting inflammation, and stimulating immunosuppression. On a molecular level, the signalling mediator TGF-ß-activated kinase 1 (TAK1) is activated by many pro-inflammatory signals, plays a critical role in inflammation and regulates innate and adaptive immune responses as well. While the role of TAK1 as a signalling factor promoting inflammation is well documented, we also considered a role for TAK1 in anti-inflammatory actions exerted by activated MSCs. We, therefore, investigated the capacity of lipopolysaccharide (LPS)-treated murine MSCs with lentivirally modulated TAK1 expression levels to recruit lymphocytes. TAK1 downregulated by lentiviral vectors expressing TAK1 shRNA in murine MSCs interfered with the capacity of murine MSCs to chemoattract lymphocytes, indeed. Analysing a pool of 84 secreted factors we found that among 26 secreted cytokines/factors TAK1 regulated expression of one cytokine in LPS-activated murine MSCs in particular: interleukin-6 (IL-6). IL-6 in LPS-treated MSCs was responsible for lymphocyte recruitment as substantiated by neutralizing antibodies. Our studies, therefore, suggest that in LPS-treated murine MSCs the inflammatory signalling mediator TAK1 may exert anti-inflammatory properties via IL-6.

De novo hem- and lymphangiogenesis by endothelial progenitor and mesenchymal stem cells in immunocompetent mice.

Cellular pro-angiogenic therapies may be applicable for the treatment of peripheral vascular diseases. Interactions between mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) may provide such a treatment option. With the exception of some studies in man, experiments have only been performed in immunodeficient mice and rats. We studied an immunocompetent syngeneic mouse model. We isolated MSCs from bone marrow and EPCs from the lung of adult C57/Bl.6 mice and co-injected them in Matrigel subcutaneously in adult C57/Bl.6 mice. We demonstrate development of both blood vessels and lymphatics. Grafted EPCs integrated into the lining of the two vessel types, whereas MSCs usually did not incorporate into the vessel wall. Injections of each separate cell type did not, or hardly, reveal de novo angiogenesis. The release of VEGF-A by MSCs has been shown before, but its inhibitors, e.g., soluble VEGF receptors, have not been studied. We performed qualitative and quantitative studies of the proteins released by EPCs, MSCs, and cocultures of the cells. Despite the secretion of VEGF inhibitors (sVEGFR-1, sVEGFR-2) by EPCs, VEGF-A was secreted by MSCs at bioavailable amounts (350 pg/ml). We confirm the secretion of PlGF, FGF-1, MCP-1, and PDGFs by EPCs/MSCs and suggest functions for VEGF-B, amphiregulin, fractalkine, CXCL10, and CXCL16 during MSC-induced hem- and lymphangiogenesis. We assume that lymphangiogenesis is induced indirectly by growth factors from immigrating leukocytes, which we found in close association with the lymphatic networks. Inflammatory responses to the cellular markers GFP and cell-tracker red (CMPTX) used for tracing of EPCs or MSCs were not observed. Our studies demonstrate the feasibility of pro-angiogenic/lymphangiogenic therapies in immunocompetent animals and indicate new MSC/EPC-derived angiogenic factors.

Therapeutic strategies for tendon healing based on novel biomaterials, factors and cells.

The repair of tendon injuries still presents a major clinical challenge to orthopedic medicine. Tendons, like some other tissues, are poorly vascularized and heal slowly. In addition, healing often leads to the formation of fibrous tissue and scar tissue which lack flexibility and biomechanical properties. So the treatment of tendon injuries is challenging. We give an overview of the structure and composition of tendons, pathological states of tendon and natural healing, as well as therapeutic options. We focus in particular on biomaterials that have been specifically developed or suggested for the successful repair of tendon injuries. In addition, we also review factor- and cell-dependent strategies to heal tendon and ligament disorders. Although brief, we hope that this review will be helpful, particularly for those readers who are new to the field of tendon tissue engineering.

In vivo RNAi-mediated silencing of TAK1 decreases inflammatory Th1 and Th17 cells through targeting of myeloid cells.

Cells from the mononuclear phagocyte system (MPS) act as systemic and local amplifiers that contribute to the progression of chronic inflammatory disorders. Transforming growth factor-ß-activated kinase 1 (TAK1) is a pivotal upstream mitogen-activated protein kinase-kinase-kinase acting as a mediator of cytokine expression. It remains critical to determine in vivo the implication of TAK1 in controlling the innate immune system. Here, we describe a vehicle tailored to selectively deliver siRNAs into MPS cells after intravenous administration, and validate in vivo the potential of the RNAi-mediated TAK1 knock down for immunomodulation. In a mouse model of immune-mediated inflammatory disorder, we show that anti-TAK1 siRNA lipoplexes efficiently alleviate inflammation, severely impair the downstream c-Jun N-terminal kinase and nuclear factor-κB signaling pathways, and decrease the expression of proinflammatory mediators. Importantly, the systemic TAK1 gene silencing decreases the frequency of Th1 and Th17 cells, both mediating autoimmunity in experimental arthritis, demonstrating the immunomodulatory potential of TAK1. Finally, in vitro inhibition of TAK1 in myeloid cells decreases interferon-γ-producing T cells, suggesting that a delivery system able to target MPS cells and to silence TAK1 impacts on pathogenic T effector cells in autoimmunity.

Evaluation of the pharmacokinetics, biotransformation and hepatic transporter effects of troglitazone in mice with humanized livers.

The pharmacokinetics, biotransformation and hepatic transporter effects of troglitazone were investigated following daily oral dosing, at 300 and 600 mg/kg, for 7 days to control (SCID) and chimeric (PXB) mice with humanized livers. Clinical chemistry revealed no consistent pattern of changes associated with troglitazone treatment in the PXB mouse. Human MRP2 but not mouse mrp2 was down-regulated following troglitazone treatment. Pharmacokinetic analysis revealed similar T(max) values for troglitazone in both mouse groups, a mono- and bi-phasic elimination phase in PXB and SCID mice, respectively, but a 3- to 5- and 2- to 5-fold higher C(max) and AUC, respectively, in PXB mice. Oxidative and conjugative metabolic pathways were identified, with the sulfate being the predominant metabolite in PXB compared to SCID mice (4- to 13-fold increase in liver and blood, respectively). The glucuronide conjugate was predominant in SCID mice. There was no evidence of glutathione conjugation. The primary oxidative pathways were mono- and di-oxidations which may also be attributed to quinone or hydroquinone derivatives. Several metabolites were observed in PXB mice only. As the troglitazone metabolic profiles in the PXB mouse were similar to reported human data the PXB mouse model can provide a useful first insight into circulating human metabolites of xenobiotics metabolized in the liver.

The role of dopamine D(3) receptors in antipsychotic activity and cognitive functions.

Dopamine D(3) receptors have a pre- and postsynaptic localization in brain stem nuclei, limbic parts of the striatum, and cortex. Their widespread influence on dopamine release, on dopaminergic function, and on several other neurotransmitters makes them attractive targets for therapeutic intervention. The signaling pathways of D(3) receptors are distinct from those of other members of the D(2)-like receptor family. There is increasing evidence that D(3) receptors can form heteromers with dopamine D(1), D(2), and probably other G-protein-coupled receptors. The functional consequences remain to be characterized in more detail but might open new interesting pharmacological insight and opportunities. In terms of behavioral function, D(3) receptors are involved in cognitive, social, and motor functions, as well as in filtering and sensitization processes. Although the role of D(3) receptor blockade for alleviating positive symptoms is still unsettled, selective D(3) receptor antagonism has therapeutic features for schizophrenia and beyond as demonstrated by several animal models: improved cognitive function, emotional processing, executive function, flexibility, and social behavior. D(3) receptor antagonism seems to contribute to atypicality of clinically used antipsychotics by reducing extrapyramidal motor symptoms; has no direct influence on prolactin release; and does not cause anhedonia, weight gain, or metabolic dysfunctions. Unfortunately, clinical data with new, selective D(3) antagonists are still incomplete; their cognitive effects have only been communicated in part. In vitro, virtually all clinically used antipsychotics are not D(2)-selective but also have affinity for D(3) receptors. The exact D(3) receptor occupancies achieved in patients, particularly in cortical areas, are largely unknown, mainly because only nonselective or agonist PET tracers are currently available. It is unlikely that a degree of D(3) receptor antagonism optimal for antipsychotic and cognitive function can be achieved with existing antipsychotics. Therefore, selective D(3) antagonism represents a promising mechanism still to be fully exploited for the treatment of schizophrenia, cognitive deficits in schizophrenia, and comorbid conditions such as substance abuse.

Development of disease-modifying treatment of schizophrenia.

Development of disease-modifying therapies requires an innovative approach to drug development where novel drugs are designed to target mechanisms of interest rather than to produce preclinical effects similar to those of currently used antipsychotics. Application of such novel strategy will undoubtedly require a very deep understanding of the disease biology that is just starting to emerge. Alternatively, one may let environmental experiences of the diseased individual guide the repair process and use drugs only to facilitate the effects of experience. Such an approach would bring together functional experience that is age-, environment- and disease-dependent with the plasticity resources that may otherwise not be available. There are currently no preclinical drug-environment interaction models that can be claimed to have significant degrees of validity. Therefore, from a drug development perspective, principles that combine acute symptomatic and disease-modifying properties are clearly preferred. The question arises then how such treatments can be differentiated from those that have only symptomatic effects (i.e., most currently used antipsychotic medications). One expectation is that the former will show superior and broader efficacy (especially with longer treatment duration). Another possibility is that disease-modifying drugs will be particularly useful at the very earliest stages of the disease. Society and medical communities may not be ready yet to initiate the treatment as early as during the prodromal phase, but the situation may change by the time the science advances enough to bring a convincing case of a drug with disease-modification potential.

Mesenchymal stem cell-dependent formation of heterotopic tendon-bone insertions (osteotendinous junctions).

Ligament-to-bone and tendon-to-bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)-signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon-inherent interference with bone formation and may thus allow the stem cell-dependent formation of tendon-bone interfaces. The tenogenic and osteo-/chondrogenic competence was mediated by the adeno- and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage-type OTJs, while, in contrast, the viral modification of primary human bone marrow-derived mesenchymal stromal/stem cells showed evidence of fibrous tendon-bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures. These findings may contribute to the establishment of stem cell-dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually.

Coating of titanium implants with copolymer supports bone regeneration: a comparative in vivo study in rabbits.

PURPOSE: In modern orthopedics aseptic loosening caused by the formation of micro-wear particles remains a problem for endoprosthetic joint replacements as revision surgery is necessary with corresponding costs and exertions by patients. This study is devoted to the question of how the osseous ingrowth of implants can be supported. It was investigated whether the developed copolymer, p-VBP-co-GMA, coated on the surface of the implants, supports bone healing. In addition, it was analyzed whether covalent linkage of Bone Morphogenetic Protein 2 (BMP-2) to the copolymer layer enhances bone formation. METHODS: Eight adult New Zealand White Rabbits were implanted with four different foils (control, copolymer, copolymer + BMP-2, control + BMP-2) each. The histomorphometric analysis of all samples was made 28 days after implantation. RESULTS: The copolymer had a positive effect on bone remodeling compared to the control group. We observed that the copolymer group had a significantly increased bone volume per tissue volume ratio and bone density to the control group. In contrast, this in-vivo study showed that the immobilization of BMP-2 onto the copolymer layer did not enhance bone healing. The bone volume per tissue volume ratio was decreased as well as the bone density compared to control + BMP-2 group. CONCLUSION: The analysis showed that the bone remodeling process in the copolymer + BMP-2-group is in an early phase comparable to the control group. These results suggest that the coating with the developed copolymer has major potential for medical use as it enhances bone mass around the implant.

Glutamatergic (N-methyl-D-aspartate receptor) hypofrontality in schizophrenia: too little juice or a miswired brain?

Dopamine D2 receptor blockade has been an obligate mechanism of action present in all medications that effectively treat positive symptoms of schizophrenia (e.g., delusions and hallucinations) and have been approved by regulatory agencies since the 1950s. Blockade of 5-hydroxytryptamine(2A) receptors plays a contributory role in the actions of the second generation of antipsychotic drugs, the so-called atypical antipsychotics. Nevertheless, substantial unmet medical needs remain for the treatment of negative symptoms and cognitive dysfunction. Recognition that dissociative anesthetics block the N-methyl-D-aspartate (NMDA) receptor channel has inspired a search for glutamatergic therapeutic mechanisms because ketamine and phencyclidine are known to induce psychotic-like symptoms in healthy volunteers and exacerbate the symptoms of patients with schizophrenia. Current pathophysiological theories of schizophrenia emphasize that hypofunction of NMDA receptors at critical sites in local circuits modulate the function of a given brain region or control projections from one region to another (e.g., hippocampal-cortical or thalamocortical projections). The demonstration that a metabotropic glutamate 2/3 (mGlu2/3) receptor agonist prodrug decreased both positive and negative symptoms of schizophrenia raised hopes that glutamatergic mechanisms may provide therapeutic advantages. In addition to discussing the activation of mGlu2 receptors with mGlu2/3 receptor agonists or mGlu2 receptor positive allosteric modulators (PAMs), we discuss other methods that may potentially modulate circuits with hypofunctional NMDA receptors such as glycine transporter inhibitors and mGlu5 receptor PAMs. The hope is that by modulating glutamatergic neurotransmission, the dysfunctional circuitry of the schizophrenic brain (both local circuits and long-loop pathways) will be improved.

Effects of a positive allosteric modulator of group II metabotropic glutamate receptors, LY487379, on cognitive flexibility and impulsive-like responding in rats.

Orthosteric group II metabotropic glutamate receptor (mGluR) agonists are regarded as novel, effective medications for all major symptom domains of schizophrenia, including cognitive disturbances. mGluR2s also can be affected in a more subtle way by positive allosteric modulators (PAMs) characterized by a unique degree of subtype selectivity and neuronal frequency-dependent activity. Because currently available treatments for schizophrenia do not improve cognitive dysfunction, the main aim of the present study was to examine the effects of a mGluR2 PAM, N-(4-(2-methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379), on rat cognitive flexibility and impulsive-like responding, assessed in an attentional set-shifting task (ASST) and a differential reinforcement of low-rate 72 s (DRL72) schedule of food reinforcement. In addition, in vivo microdialysis was used to assess the drug's impact on cortical levels of dopamine, norepinephrine, serotonin, and glutamate. Rats treated with LY487379 (30 mg/kg) required significantly fewer trials to criteria during the extradimensional shift phase of the ASST. Under a DRL72 schedule, LY487379 (30 mg/kg) decreased the response rate and increased the number of reinforcers obtained. These effects were accompanied by the shift of the frequency distribution of responses toward longer inter-response time durations. LY487379 significantly enhanced extracellular norepinephrine and serotonin levels in the medial prefrontal cortex. In summary, the present study demonstrates that a mGluR2 PAM, LY487379, promotes cognitive flexibility and facilitates behavioral inhibition. These procognitive effects may contribute to the therapeutic efficacy of agents stimulating mGluR2 in schizophrenia.

Amyloid beta oligomers (A beta(1-42) globulomer) suppress spontaneous synaptic activity by inhibition of P/Q-type calcium currents.

Abnormal accumulation of soluble oligomers of amyloid beta (Abeta) is believed to cause malfunctioning of neurons in Alzheimer's disease. It has been shown that Abeta oligomers impair synaptic plasticity, thereby altering the ability of the neuron to store information. We examined the underlying cellular mechanism of Abeta oligomer-induced synaptic modifications by using a recently described stable oligomeric Abeta preparation called "Abeta(1-42) globulomer." Synthetically prepared Abeta(1-42) globulomer has been shown to localize to neurons and impairs long-term potentiation (Barghorn et al., 2005). Here, we demonstrate that Abeta(1-42) globulomer does not affect intrinsic neuronal properties, as assessed by measuring input resistance and discharge characteristics, excluding an unspecific alteration of membrane properties. We provide evidence that Abeta(1-42) globulomer, at concentrations as low as 8 nM, specifically suppresses spontaneous synaptic activity resulting from a reduction of vesicular release at terminals of both GABAergic and glutamatergic synapses. EPSCs and IPSCs were primarily unaffected. A detailed search for the precise molecular target of Abeta(1-42) globulomer revealed a specific inhibition of presynaptic P/Q calcium currents, whereas other voltage-activated calcium currents remained unaltered. Because intact P/Q calcium currents are needed for synaptic plasticity, the disruption of such currents by Abeta(1-42) globulomer may cause deficits in cellular mechanisms of information storage in brains of Alzheimer's disease patients. The inhibitory effect of Abeta(1-42) globulomer on synaptic vesicle release could be reversed by roscovitine, a specific enhancer of P/Q currents. Selective enhancement of the P/Q calcium current may provide a promising strategy in the treatment of Alzheimer's disease.

Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells.

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

Wnt-ligand-dependent interaction of TAK1 (TGF-beta-activated kinase-1) with the receptor tyrosine kinase Ror2 modulates canonical Wnt-signalling.

Mutations in the receptor tyrosine kinase Ror2 account for Brachydactyly type B and Robinow Syndrome. We have identified two novel factors interacting with the Ror2 intracellular domain. TAK1 (TGF-beta activated kinase 1), a MAP3K, interacts with Ror2 and phosphorylates its intracellular carboxyterminal serine/thronine/proline-rich (STP) domain. This TAK1-dependent phosphorylation of Ror2 induces phosphorylation of tyrosine-residues including a MAPK-like TGY-motif. The TAK1-dependent phosphorylation is enhanced by a second cytosolic factor, PRTB, which interacts with Ror2 and with TAK1 as well. The TAK1-dependent Tyr-phosphorylation of Ror2 is not mediated by the Ror2 tyrosine kinase domain and seems predominantly triggered by cytosolic kinases. Wnt-ligand binding differentially controls the Ror2/TAK1 interaction. Wnt1-binding displaces TAK1 from Ror2 while Wnt3a and Wnt5a are unable to do so thus modifying TAK1's capacity to cause phosphorylation of Ror2. Ror2 seems to act as a Wnt co-receptor enhancing Wnt-dependent canonical pathways while Tyr- and Ser/Thr-phosphorylation of Ror2 negatively controls the efficiency of these pathways. We propose that the level of the Wnt-ligand-regulated phosphorylation by cytosolic factors determines whether Ror2 acts as a stimulator or as an inhibitor of canonical Wnt-signalling.

Amphetamine decreases behavioral inhibition by stimulation of dopamine D2, but not D3, receptors.

Behavioral disinhibition is a manifestation of impulsive behavior that is prominent in the psychopathology of various psychiatric disorders such as addiction, attention-deficit hyperactivity disorder, mania, and personality disorders. Impulsivity may be studied by measuring anticipatory responses made before the presentation of a food-predictive, brief light stimulus in a two-choice serial reaction time task. In such serial reaction time tasks, amphetamine has been shown to produce dose-dependent increases in premature responding in a manner dependent on dopamine D(2)-like receptor stimulation. So far, it is unknown whether it is the D(2) or D(3) receptor that is involved in this form of impulsivity. In this study, rats were trained in a two-choice serial reaction time task until baseline performance was stable. Next, effects of the dopamine D(2) preferring antagonist L-741,626 and selective D(3) antagonist SB-277011 were assessed alone and in the presence of amphetamine. Neither L-741,626 nor SB-277011 affected behavioral inhibition, although the latter significantly increased reaction time at 10 mg/kg. Amphetamine dose-dependently increased impulsivity. The effect of amphetamine was attenuated by L-741,626 (3 mg/kg), whereas SB-277011 (3 mg/kg) had no effect. Therefore, amphetamine-induced behavioral disinhibition depends on D(2), but not D(3), receptor stimulation.

Inhibition of Calpain Prevents N-Methyl-D-aspartate-Induced Degeneration of the Nucleus Basalis and Associated Behavioral Dysfunction.

N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is thought to underlie a variety of neurological disorders, and inhibition of either the NMDA receptor itself, or molecules of the intracellular cascade, may attenuate neurodegeneration in these diseases. Calpain, a calcium-dependent cysteine protease, has been identified as part of such an NMDA receptor-induced excitotoxic signaling pathway. The present study addressed the question of whether inhibition of calpain can prevent neuronal cell death and associated behavioral deficits in a disease-relevant animal model, which is based on excitotoxic lesions of the cholinergic nucleus basalis magnocellularis of Meynert. Excitotoxic lesions of the nucleus basalis with NMDA induced a markedly impaired performance in the novel object recognition test. Treatment with the calpain inhibitor, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl]benzamide (A-705253), dose-dependently prevented the behavioral deficit. Subsequent analysis of choline acetyltransferase in the cortical mantle of the lesioned animals revealed that application of A-705253 dose-dependently and significantly attenuated cholinergic neurodegeneration. Calpain inhibition also significantly diminished the accompanying gliosis, as determined by immunohistochemical analysis of microglia activation. Finally, inhibition of calpain by A-705253 and the peptidic calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO did not impair long-term potentiation in hippocampal slices, indicating that calpain inhibition interrupts NMDA excitotoxicity pathways without interfering with NMDA receptor-mediated signaling involved in cognition. We conclude that inhibition of calpains may represent a valuable strategy for the prevention of excitotoxicity-induced neuronal decline without interfering with the physiological neuronal functions associated with learning and memory processes. Thus, calpain inhibition may be a promising and novel approach for the treatment of various neurodegenerative disorders.

Behavioral characterization of the mGlu group II/III receptor antagonist, LY-341495, in animal models of anxiety and depression.

There is a growing body of evidence indicating that stimulation of metabotropic glutamate type II receptors (mGlu2/3) reduces anxiety in laboratory animals and humans. Surprisingly, it was reported that mGlu2/3 receptor antagonists have antidepressant- and anxiolytic-like activities in laboratory animal studies as well. The present study aimed to resolve this controversy by characterizing behavioral effects of a selective mGlu2/3 receptor antagonist, LY-341495, in a variety of animal models sensitive to clinically used anxiolytic and antidepressant agents. In agreement with previous reports, LY-341495 (0.3-3 mg/kg, i.p.) reduced immobility in the mouse forced swim test. LY-341495 was also effective in the marble burying test in mice, although similar effects were observed after administration of various drugs including methamphetamine. Further, LY-341495 had no effects in the elevated plus maze and stress-induced hyperthermia tests in mice, as well as on punished drinking (Geller-Seifter's test) and differential reinforcement of low rates of responding (DRL) in rats. It is concluded that behavioral profile of mGlu2/3 receptor antagonists as represented by LY-341495 is different from that of conventional anxiolytic and antidepressant drugs.

Fibroblast-mediated delivery of GDNF induces neuronal-like outgrowth in PC12 cells.

HYPOTHESIS: Recombinantly modified cells deliver neurotrophic factors with the capacity to induce differentiation and the outgrowth of neurites of rat pheochromocytoma cells 12 (PC12) serving as a neuronal model. BACKGROUND: The benefit of cochlea implant (CI) is depending, among other factors, on the number of surviving spiral ganglion neurons (SGN). Studies have shown that the external application of neurotrophic factors in combination with electrical stimulation increases the survival rate of SGN after ototrauma. Therefore, functionalization of electrodes with recombinantly modified cells providing neurotrophic factors to the SGN for inducing survival mechanisms may be an approach to realize drug delivery to the cochlea. METHODS: Murine NIH3T3 cells were recombinantly modified with an infectious lentiviral monocistronic and bicistronic system to synthesize glial cell line-derived neurotrophic factor and the green fluorescent protein. Free glial cell line-derived neurotrophic factor from the supernatant of the modified NIH3T3 cells was added to rat PC12, and the neuronal-like outgrowth was determined for 10 days. RESULTS: A significant neuronal-like outgrowth appeared as early as Day 3 after the application of the supernatant. CONCLUSION: The results indicate that the established in vitro model represents a powerful basic model for determining signal pathways between neuronal-like processing PC12 cells and cellular drug delivery systems.

Mass spectrometric recommendations for Quan/Qual analysis using liquid-chromatography coupled to quadrupole time-of-flight mass spectrometry.

BACKGROUND: High-throughput simultaneous quantitative and qualitative (Quan/Qual) analysis is attractive to combine targeted with non-targeted analysis, e.g. in pharmacometabolomics and drug metabolism studies. This study aimed to investigate the possibilities and limitations of high-throughput Quan/Qual analysis by ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS), to develop a widely applicable Quan/Qual UHPLC-HRMS method and to provide recommendations for Quan/Qual method development. METHODS: A widely applicable 4.25-min UHPLC method for small-molecules was used to investigate and optimize mass spectrometric parameters of a Synapt G2S for Quan/Qual analysis. The method was applied on a rat metabolomics study investigating the effect of the fasting state and administration of a dosing vehicle on the rat plasma metabolic profile. RESULTS: Highly important parameters for high-throughput Quan/Qual analysis were the scan mode and scan rate. A negative correlation was found between the amount of qualitative information that a method can provide and its quantitative performance (accuracy, precision, sensitivity, linear dynamic range). The optimal balance was obtained using the MSE scan mode with a short scan time of 30 ms. This 4.25-min Quan/Qual analysis method enabled quantification with accuracy and precision values ≤ 20% at the lowest quality control (QC) level and ≤15% at higher QC levels for 16 out of 19 tested analytes. It provided both parent m/z values and fragmentation spectra for compound identification with limited loss of chromatographic resolution and it revealed biologically relevant metabolites in its application to the metabolomics study. CONCLUSION: Quan/Qual method development requires balancing between the amount of qualitative data, the quality of the quantitative data and the analysis time. Recommendations are provided for MS resolution, scan mode, scan rate, smoothing and peak integration in Quan/Qual method development and analysis.

Stimulation of the metabotropic glutamate 2/3 receptor attenuates social novelty discrimination deficits induced by neonatal phencyclidine treatment.

RATIONALE: Glutamatergic mechanisms are implicated in psychiatric disorders such as schizophrenia. Modulation of glutamatergic neurotransmission via stimulation of the metabotropic glutamate 2/3 receptors (mGluR2/3) has been shown to reverse a number of behavioral effects of NMDA receptor antagonists thus indicating potential antipsychotic activity of mGluR2/3 agonists. OBJECTIVES: The present study aimed to evaluate the effects of LY-354740 (mGluR2/3 agonist) and LY-487379 (mGluR2 potentiator) on social novelty discrimination in male Wistar rats that were treated with PCP (10 mg/kg, s.c.) on postnatal days 7, 9, and 11. MATERIALS AND METHODS: During each test session (twice a week, postnatal days 70-100), an adult experimental rat was presented with a juvenile, untreated rat (4 weeks old) for a period of 30 min. At the end of this period, a second (novel) juvenile rat was introduced for 5 min. RESULTS: Adult rats spent more time exploring the novel than the familiar juvenile. This capacity for social novelty discrimination was impaired in rats that received neonatal PCP treatment and the impaired discrimination could be reversed by acute treatment with antipsychotic drugs such as clozapine (0.3-3 mg/kg) and the glycine transporter GlyT1 inhibitor SSR-504734 (1-10 mg/kg). Acute pretreatment with LY-354740 (1-10 mg/kg) or LY-487379 (3-30 mg/kg) facilitated social discrimination in rats with PCP administration history without having appreciable effects in controls and without affecting total time spent in social interaction. CONCLUSIONS: These results suggest that targeting glutamatergic functions may reverse long-term developmental cognitive deficits produced by PCP.