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This article provides a brief overview of electrochemical potential-biological activity relationships of natural and synthetic cyclic sulfur-containing molecules against Steinernema feltiae, Botrytis cinerea, and Neuro 2a cell line (from murine neuroblastoma). This article finds natural cyclic sulfur-containing molecules and their synthetic analogues were more reducing than glutathione (GSH) and therefore apparently did not react with GSH. The nematicidal assay indicated that cyclic disulfide compound of 1 (3-vinyl-4H-1,2-dithiin, 1,2-VDT) was more active against Steinernema feltiae with the LD50 value 151.93 ± 1.3 μM, while dithiole thione group compounds showed moderate activity against this nematode. The article also finds compound 7 (3H-1,2-dithiole-3-thione or dithiolethione, DT) has a strong activity against all different strains of Botrytis cinerea in the range concentration of 0.1–0.5 mM. This article also finds that compounds 3 (1,2-dithiane, 1,2-DT), 4 (1,5-dithiacyclooctane, 1,5-DTCO), and 7 (3H-1,2-dithiole-3-thione or dithiolethione, DT) possess some moderate activity on Neuro 2a cell lines. Antinematode, antifungal, and anticancer activity of cyclic sulfur-containing molecules indicated that they could be promising candidates for “green pesticides” or phytoprotectans and for cancer prevention.

Therapeutic ultrasound is a widely used application in clinics. The response to ultrasound is tissue specific and the parameters of ultrasonic treatments are decisive. Here, the responses of primary enteric (ENS) and central nervous system (CNS) neural stem cells were investigated to different frequency – energy density – combination treatments between 510 kHz–3 W s/cm2 and 4.36 MHz–25 W s/cm2. Responses to ultrasonic treatments were both frequency and energy density dependent. Ultrasound enhanced the expansion and showed enlarged neurospheres for both ENS and CNS neural stem cell cultures. Differentiation was not impaired in regard to neuronal and glial cell number as well as neurite and glial fiber outgrowth. Ultrasound is a promising tool to expand neural stem cells in vitro neither influencing neurogenesis nor gliogenesis by applying specific frequency – energy density – combinations. Keywords: Enteric nervous system, Ultrasound, Neural stem cells

Inflammatory bowel diseases (IBD), which include Crohn's disease and ulcerative colitis, are chronic inflammatory disorders that can affect the whole gastrointestinal tract or the colonic mucosal layer. Current therapies aiming to suppress the exaggerated immune response in IBD largely rely on compounds with non-satisfying effects or side-effects. Therefore, new therapeutical options are needed. In the present study, we investigated the anti-inflammatory effects of the fungal metabolites, galiellalactone, and dehydrocurvularin in both an in vitro intestinal inflammation model, as well as in isolated myenteric plexus and enterocyte cells. Administration of a pro-inflammatory cytokine mix through the mesenteric artery of intestinal segments caused an up-regulation of inflammatory marker genes. Treatment of the murine intestinal segments with galiellalactone or dehydrocurvularin by application through the mesenteric artery significantly prevented the expression of pro-inflammatory marker genes on the mRNA and the protein level. Comparable to the results in the perfused intestine model, treatment of primary enteric nervous system (ENS) cells from the murine intestine with the fungal compounds reduced expression of cytokines such as IL-6, TNF-α, IL-1β, and inflammatory enzymes such as COX-2 and iNOS on mRNA and protein levels. Similar anti-inflammatory effects of the fungal metabolites were observed in the human colorectal adenocarcinoma cell line DLD-1 after stimulation with IFN-γ (10 ng/ml), TNF-α (10 ng/ml), and IL-1β (5 ng/ml). Our results show that the mesenterially perfused intestine model provides a reliable tool for the screening of new therapeutics with limited amounts of test compounds. Furthermore, we could characterize the anti-inflammatory effects of two novel active compounds, galiellalactone, and dehydrocurvularin which are interesting candidates for studies with chronic animal models of IBD.

Paratarg-7, a frequent autoantigenic target, and all other autoantigenic targets of human paraproteins molecularly defined to date are hyperphosphorylated in the respective patients compared with healthy controls, suggesting that hyperphosphorylation of autoantigenic paraprotein targets is a general mechanism underlying the pathogenesis of these paraproteins. We now show that hyperphosphorylation of paratarg-7 occurs because of an additional phosphorylation of Ser17, which is located within the paraprotein-binding epitope. Coimmunoprecipitation identified phosphokinase C ζ (PKCζ) as the kinase responsible for the phosphorylation of most, and phosphatase 2A (PP2A) as the phosphatase responsible for the dephosphorylation of all hyperphosphorylated autoantigenic targets of paraproteins. Single-nucleotide polymorphisms (SNPs) or mutations of PKCζ and PP2A were excluded. However, PP2A was inactivated by phosphorylation of its catalytic subunit at Y307. Stimulation of T cells from healthy carriers of wild-type paratarg-7 induced a partial and transient hyperphosphorylation between days 4 and 18, which was maintained by incubation with inhibitors of PP2A, again indicating that an inactivation of PP2A is responsible for the hyperphosphorylation of autoantigenic paraprotein targets. We conclude that the genetic defect underlying the dominantly inherited hyperphosphorylation of autoantigenic paraprotein targets is not in the PP2A itself, but in genes or proteins controlling PP2A activity by phosphorylation of its catalytic subunit.

Objective: Despite the frequently reported assumption that VEGFR and Aquaporin4 (AQP4) expression seem to correlate with peritumoral brain edema formation in meningiomas, the molecular pathogenesis of edema formation is still elusive. Therefore, the authors analyzed meningioma edema formation in correlation to typical chromosomal deletions and the expression of VEGFR and Aquaporin4. Methods: Tissue specimens of 69 meningiomas were obtained from surgery between 2008 and 2010. Locus specific FISH analysis detecting the chromosomes 1, 9, 14, 18 and 22 were performed. Moreover, immunoblotting was performed to detect the expression of VEGFR1 and VEGFR2. Aquaporin4 expression was investigated by ELISA. The results were correlated with the peritumoral brain edema in the preoperative MRI. Statistical analysis including Pearson correlation and multivariate statistical analysis was performed. Results: In contrast to previous reports, a significant correlation to the brain edema in meningiomas could be identified only for VEGFR2 ( p

The enteric nervous system (ENS) orchestrates a broad range of important gastrointestinal functions such as intestinal motility and gastric secretion. The ENS can be affected by environmental factors, diet and disease. Changes due to these alterations are often hard to evaluate in detail when whole gut samples are used. Analyses based on pure ENS tissue can more effectively reflect the ongoing changes during pathological processes. Here, we present an optimized approach for the isolation of pure myenteric plexus (MP) from adult mouse and human. To do so, muscle tissue was individually digested with a purified collagenase. After incubation and a gentle mechanical disruption step, MP networks could be collected with anatomical integrity. These tissues could be stored and used either for immediate genomic, proteomic or in vitro approaches and enteric neurospheres could be generated and differentiated. In a pilot experiment, the influence of bacterial lipopolysaccharide on human MP was analyzed using 2-dimensional gel electrophoresis. The method also allows investigation of factors that are secreted by myenteric tissue in vitro. The isolation of pure MP in large amounts allows new analytical approaches that can provide a new perspective in evaluating changes of the ENS in experimental models, human disease and aging.

Background and aims: Achalasia is a disease of unknown aetiology. An immune mechanism has been suggested on the basis of previous morphological observations. The objective of this study was to test whether the serum of achalasia patients could reproduce the phenotype and functional changes that occur with disease progression in an ex vivo human model.Methods: Specimens of normal human fundus were maintained in culture in the presence of serum from patients with achalasia, gastro-oesophageal reflux disease (GORD), or healthy subjects (controls). Immunohistochemical detection of choline acetyltransferase (ChAT), neurone specific enolase (NSE), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), and substance P was carried out in whole mounts of gastric fundus myenteric plexus. In addition, the effects of achalasia serum on electrical field stimulation (EFS) induced contractions were measured in circular muscle preparations.Results: Serum from achalasia patients did not affect the number of myenteric neurones. Tissues incubated with serum from achalasia patients showed a decrease in the proportion of NOS (-26% of NSE positive neurones; p = 0.016) and VIP (-54%; p = 0.09) neurones, and a concomitant increase in ChAT neurones (+ 16%; p Conclusions: Serum from achalasia patients can induce phenotypic and functional changes which reproduce the characteristics of the disease. Further identification of putative seric factors and mechanisms involved could lead to the development of novel diagnostic and/or therapeutic strategies in achalasia.

Background Protein kinases play central roles in cell and tissue development. Protein kinase CK2, an ubiquitously expressed serine/threonine kinase has severe impacts on embryo- and spermatogenesis. Since its role in neurogenesis has so far only been investigated in very few studies, we analysed the role of CK2 in neural stem cells by using two specific inhibitors. Methods Neural stem cells were isolated from the subventricular zone of neonatal mice, using a neurosphere approach. Proliferation of the neurospheres, as well as their differentiation was investigated with and without inhibition of CK2. Changes in proliferation were assessed by counting the number and measuring the diameter of the neurospheres. Furthermore, the absolute cell numbers within the neurospheres were estimated. Differentiation was induced by retinoic acid in single cells after dissociation of the neurospheres. CK2 was inhibited at consecutive time points after induction of the differentiation process. Results CK2 inhibition reduced the amount and size of proliferating neurospheres dose dependently. Adding the CK2 inhibitor CX–4945 at the start of differentiation we observed a dose-dependent effect of CX-4945 on cell viability and glia cell differentiation. Adding quinalizarin, a second CK2 inhibitor, at the start of differentiation led to an elevated level of apoptosis, which was accompanied by a reduced neural differentiation. Adding the CK2 inhibitors at 72 h after the start of differentiation had no effect on stem cell differentiation. Conclusion: Inhibition of CK2 influences early gliogenesis in a time point and concentration dependent manner. General significance The use of a CK2 inhibitor significantly affects the neural stem cell niche.

The enteric nervous system (ENS) has to respond to continuously changing microenvironmental challenges within the gut and is therefore dependent on a neural stem cell niche to keep the ENS functional throughout life. In this study, we hypothesize that this stem cell niche is also affected during inflammation and therefore investigated lipopolysaccharides (LPS) effects on enteric neural stem/progenitor cells (NSPCs). NSPCs were derived from the ENS and cultured under the influence of different LPS concentrations. LPS effects upon proliferation and differentiation of enteric NSPC cultures were assessed using immunochemistry, flow cytometry, western blot, Multiplex ELISA and real-time PCR. LPS enhances the proliferation of enteric NSPCs in a dose-dependent manner. It delays and modifies the differentiation of these cells. The expression of the LPS receptor toll-like receptor 4 on NSPCs could be demonstrated. Moreover, LPS induces the secretion of several cytokines. Flow cytometry data gives evidence for individual subgroups within the NSPC population. ENS-derived NSPCs respond to LPS in maintaining at least partially their stem cell character. In the case of inflammatory disease or trauma where the liberation and exposure to LPS will be increased, the expansion of NSPCs could be a first step towards regeneration of the ENS. The reduced and altered differentiation, as well as the induction of cytokine signalling, demonstrates that the stem cell niche may take part in the LPS-transmitted inflammatory processes in a direct and defined way.

Background . Autoantibodies against nervous system structures have been proven to be a prognostic factor in small cell lung cancer. However, little is known about humoral autoimmunity in non-small cell lung cancer (NSCLC) and its prognostic significance. Methods . We examined antineural antibodies (AnAb) and antinuclear antibodies (ANA) in the sera of 61 patients with NSCLC (histologically: 29 adenocarcinoma, 32 squamous cell carcinoma). Twenty-one patients had stage I NSCLC, 11 stage II, and 29 patients stage III. Autoantibody detection was done by immunofluorescence test; Western blotting was used as a confirmation test. Results . Of the NSCLC patients, 27.8% were antineural antibody positive, and 32.7% had ANA. No differences were found between the histological groups. AnAb-positive patients showed a better survival in all patients ( p = 0.005). There was also a higher survival of ANA-positive patients, but this was only significant in stage III ( p = 0.0025). Cox regression analysis showed that antineural and antinuclear antibodies are a stage-independent prognostic factor in NSCLC. Conclusions . Antineural and antinuclear autoantibodies are a stage-independent prognostic factor in patients with NSCLC and may represent an effective immune response to the tumor.

Paraneoplastic neurological syndromes (PNS) are often associated with antineuronal autoantibodies and many of them could be identified in the recent years. However, there are still new antineuronal binding patterns with yet unidentified autoantigens. We here describe a new autoantibody associated with paraneoplastic sensorimotor and autonomic neuropathy in a patient with small cell lung cancer. In indirect immunofluorescence test, the patients serum colocalised with the synaptic protein synaptophysin in the cerebellum and myenteric plexus of the gut. Immunoblotting showed a 38 kDa reactivity, which is also the molecular weight of synaptophysin. Therefore a Western Blot with recombinant synaptophysin has been used and revealed reactivity of the serum against synaptophysin. In patients with non-paraneoplastic neuropathies or healthy controls, anti-synaptophysin autoantibodies were not detectable. In 20 SCLC patients without neurological syndromes, one patient had low-titer anti-synaptophysin autoantibodies. Interestingly, the PNS patient´s serum and IgG fraction showed marked cytotoxicity to primary cultured myenteric plexus neurons.We conclude that synaptophysin is an autoantigen in paraneoplastic neurological syndromes.

The human enteric nervous system (ENS) derives from migrating neural crest cells (NCC) and is structured into different plexuses embedded in the gastrointestinal tract wall. During development of the NCC, a rearrangement of various cytoskeletal intermediate filaments such as nestin, peripherin, or alpha-internexin takes place. Although all are related to developing neurons, nestin is also used to identify neural stem cells. Until now, information about the prenatal development of the human ENS has been very restricted, especially concerning potential stem cells. In this study the expression of nestin, peripherin, and alpha-internexin, but also of neuronal markers such as protein gene product (PGP) 9.5 and tyrosine hydroxylase, were investigated in human fetal and postnatal gut. The tissue samples were rapidly removed and subsequently processed for immunohistochemistry or immunoblotting. Nestin could be detected in all samples investigated with the exception of the 9th and the 12th week of gestation (WOG). Although the neuronal marker PGP9.5 was coexpressed with nestin at the 14th WOG, this could no longer be observed at later time points. Alpha-internexin and peripherin expression also did not appear before the 14th WOG, where they were coexpressed with PGP9.5. This study reveals that the intermediate filament markers investigated are not suitable to detect early neural crest stem cells.

Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins might play a role in the pathogenesis of these neoplasms. By screening a commercially available recombinant human cDNA expression macroarray we identified paratarg-7 as the antigenic target of the paraprotein from 14% (35/252) of all patients analyzed (Preuss et al. Int J Cancer 2009). The high frequency of hyperphosphorylated paratarg-7 as the antigenic target of paraproteins from MM/MGUS patients suggests that the modified protein is involved in the development of sporadic and familial MGUS/MM by acting as a chronic antigenic stimulus for the respective B-cell clones. With a relative risk of 7.9, the carrier state of hyperphosphorylated paratarg-7 (pP-7) is the strongest risk factor for the development of MGUS/MM. Moreover, the analysis of consanguineous relatives of MM/MGUS patients with an anti-paratarg-7 specific paraprotein showed that pP7 is inherited in a dominant fashion (Grass et al., Lancet Oncology, 2009). Here we present data showing that the hyperphosphorylation of paratarg-7 is due to an additional phosphorylation of this protein at the identical phosphorylation site when compared to normal paratarg-7. We identified protein kinase C zeta as responsible for this hyperphosphorylation which specifically occurs on Ser17 which is part of the 15 amino acid region identified as the paraprotein-binding epitope in all paratarg-7-immunopositive patients. In additional experiments no DNA modification of PKCzeta was detected. As shown by Western blot experiments and quantitative PCR no overexpression or copy number variation of PKCzeta was shown, suggesting that other not yet identified enzyme(s) must be involved in the process of paratarg-7 hyperphosphorylation or the maintenance of the hyperphosphorylated state. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4772.