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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.

The high morbidity and mortality of tuberculous meningoencephalitis (TBM) warrants an early diagnosis and treatment. BCG vaccine has been proven to reduce the incidence of disseminated disease in children. We report on two siblings (2-year-old boy and 4-year-old girl) with simultaneous TBM, whose parents originated from Kosovo, Albania, but presently reside in Germany. Early diagnosis of TBM was delayed, and at first the misdiagnosis of viral meningoencephalitis was made. Antituberculosis treatment was not initiated despite profound hyponatremia, hydrocephalus, and signs of inflammatory cerebral disease. After establishing the diagnosis of TBM, the boy died from antituberculosis, drug-induced hepatic failure; the sister survived with severe neurological deficits. Contact tracing revealed that TB had been transmitted by a household contact person with proven pulmonary TB who had refused antituberculosis treatment. A thorough contact investigation including tuberculin skin testing to identify children at risk for TB in the vicinity of this patient was not carried out. These case reports demonstrate an unusual simultaneous occurrence of TBM in a brother and sister. It draws attention to the importance of TBM as a differential diagnosis in children with suspected viral meningoencephalitis. CONCLUSIONS: To prevent severe neurological sequelae, early antituberculosis therapy should be considered in infants and children with a clinical impression of meningitis in the context of cerebrospinal fluid white blood cell count of less than 500 cells/μl and lymphocytic predominance, hyponatremia, and possible hydrocephalus. This notion is especially true for children originating from highendemicity countries for TB. A rigid implementation of antituberculosis treatment of infected individuals and contact tracing is mandatory in order to prevent dissemination of TB in the community. The use of BCG vaccine should be considered in children at high risk for TB infection because of its potential to reduce disseminated TB.

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.

Motility disorders of the human intestine are so variable that they cannot be diagnosed by just one technique. Their aetiology is obviously so varied that they have to be approached with a broad range of technical methods. These reach from the simple haematoxylin-stained section to the isolation of stem or precursor cells. In this study, various methods to investigate the enteric nervous system and its surrounding tissue are demonstrated. While sections from paraffin-embedded material or cryostat sections provide only a two-dimensional perspective of the ENS, the whole-mount method yields three-dimensional perspectives of large areas of the gut wall. The three-dimensional impression can even be enhanced by electron microscopy of the isolated ENS. Dynamical aspects of ENS development can be tackled by in vitro studies. The myenteric plexus can be isolated and cultivated under the influence of the microenvironment (protein extracts). Although the postnatal myenteric plexus is not fully developed, the choice of embryological neuronal cells seems to be more effective for certain approaches. They can be isolated from the embryonic mouse gut and cultivated under the influence of various factors. This method seems to us a valuable tool for the investigation of the aetiology of motility disorders, although only a "complete" approach which considers all available methods will yield at the end a clear understanding which might lead to new therapeutical concepts.

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.

Enteric neurons and blood vessels form intricate networks throughout the gastrointestinal tract. To support the hypothesis of a possible interaction of both networks, we investigated whether primary mesenteric vascular cells (MVCs) and enteric nervous system (ENS)-derived cells (ENSc) depend on each other using two- and three-dimensional in vitro assays. In a confrontation assay, both cell types migrated in a target-oriented manner towards each other. The migration of MVCs was significantly increased when cultured in ENSc-conditioned medium. Co-cultures of ENSc with MVCs resulted in an improved ENSc proliferation and differentiation. Moreover, we analysed the formation of the vascular and nervous system in developing mice guts. It was found that the patterning of newly formed microvessels and neural stem cells, as confirmed by nestin and SOX2 stainings, is highly correlated in all parts of the developing gut. In particular in the distal colon, nestin/SOX2-positive cells were found in the tissues adjacent to the capillaries and in the capillaries themselves. Finally, in order to provide evidences for a mutual interaction between endothelial and neural cells, the vascular patterns of a RET((-/-)) knockout mouse model as well as human Hirschsprung's cases were analysed. In the distal colon of postnatal RET((-/-)) knockout mice, the vascular and neural networks were similarly disrupted. In aganglionic zones of Hirschsprung's patients, the microvascular density was significantly increased compared with the ganglionic zone within the submucosa. Taken together, these findings indicate a strong interaction between the enteric nervous and vascular system.

Different assays have been used in investigations on human ghrelin blood concentrations. The range of human ghrelin blood concentrations varies markedly between different studies. The variance of reported ghrelin concentrations might be due to patient specific factors, differences in sample processing, different analytical methods and different manufacturers of the assays. It is unknown how well ghrelin concentrations measured by different analytical methods are comparable and few data exist on the validity (for external consistency) of ghrelin assays.We analyzed 256 human plasma samples for acylated ghrelin concentrations with a commercially available enzyme-linked immunoassay (ELISA) and a multiplex analysis kit using Luminex(®) technology.Both methods yielded ghrelin concentration within the same range. Concentrations measured by ELISA were systematically higher (median 1.4-fold). The measured concentrations of both methods correlated well as shown by a high Pearson's correlation coefficient (0.753, p0.01). Bland-Altman plotting revealed complementary aspects concerning the agreement of the two tested methods at low and high concentrations.We conclude that the two investigated techniques yield results with an acceptable agreement. The agreement of both measurements indicates a good external consistency and reliability of both analytical methods. In the absence of a gold standard for ghrelin measurement, our data are a cross-validation for both methods.

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.

Pharmaceutical compounds enter the body via several major natural gateways; i.e. the lung, the skin and the gastrointestinal tract. Drug application during surgical operations can lead to severe impairment of gastrointestinal motility, which can contribute to a paralytic ileus. Here we investigated an ex vivo perfused small intestine model that allows us to ascertain the influence of pharmaceuticals upon the gut. Corresponding segments from the proximal jejunum of adult rats were used. Their mesenteric arteries and veins were cannulated and the jejunal segment excised. The individual segments were placed in a custom designed perfusion chamber and perfusion performed through the intestinal lumen as well as the mesenteric superior artery. Three test drugs, which are commonly used in anesthesiology; i.e. pentobarbital, propofol and ketamine were administered via the blood vessels. Their effects upon gastrointestinal motility patterns were evaluated by optical measurements. Longitudinal and pendular movements were distinguishable and separately analyzed. Pharmacological effects of the individual substances could be investigated. Propofol (50-200 μg/ml) was found to decrease intestinal motility, especially longitudinal movements in a dose dependent manner. Pentobarbital decreased intestinal motility only at high concentrations, above 2.5 mg/ml. A dose of 2.5 mg/ml lead to an increase in longitudinal- and pendular movements in comparison to control, while ketamine (2.5-10 mg/ml) did not alter intestinal motility at all. Histological examination of the perfused segments revealed only minor changes in tissue morphology after perfusion. The perfusion approach shown here allows for the identification of compounds which interfere with gut motility in a highly sophisticated way. It is suitable for characterization of drug and dose specific changes in motility patterns and can be used in drug development and preclinical studies.

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.

Background and Purpose In this report, we present the first case of an immunologically impaired child surviving a lytic varicella-zoster virus infection affecting the enteric nervous system. In histological findings, myenteric and submucous enteric ganglia were nearly completely absent owing to virus infection. Methods A 3-year-old girl with acute lymphoblastic leukemia and generalized varicella-zoster infection developed an ileus. She underwent multiple laparotomies in which histological sections of the entire small intestine could be obtained. Results The histological evaluation of these samples showed a generalized aganglionosis with inflammatory residuals. A more detailed immunohistochemical analysis using neuronal (PGP, enolase), glial (S100), and lymphocytic (LCA) antibodies demonstrated a nearly complete neuronal loss. Conclusion To our knowledge, this is the first case of a secondary intestinal aganglionosis after varicella-zoster virus infection.

The enteric nervous system (ENS) controls and modulates gut motility and responds to food intake and to internal and external stimuli such as toxins or inflammation. Its plasticity is maintained throughout life by neural progenitor cells within the enteric stem cell niche. Granulocyte-colony stimulating factor (G-CSF) is known to act not only on cells of the immune system but also on neurons and neural progenitors in the central nervous system (CNS). Here, we demonstrate, for the first time, that G-CSF receptor is present on enteric neurons and progenitors and that G-CSF plays a role in the expansion and differentiation of enteric neural progenitor cells. Cultured mouse ENS-neurospheres show increased expansion with increased G-CSF concentrations, in contrast to CNS-derived spheres. In cultures from differentiated ENS- and CNS-neurospheres, neurite outgrowth density is enhanced depending on the amount of G-CSF in the culture. G-CSF might be an important factor in the regeneration and differentiation of the ENS and might be a useful tool for the investigation and treatment of ENS disorders.

In Alzheimer's disease (AD), fatal neuronal cell loss occurs long before relevant evidence can lead to a reliable diagnosis. If characteristic pathological alterations take place in the enteric nervous system (ENS), it could be one of the most promising targets for an early diagnosis, using submucosal biopsies from the gut. We therefore investigated time- and spatial-dependent changes in an amyloid-beta protein precursor (A beta PP) overexpressing transgenic mouse model to examine early changes within the ENS. Wholemount preparations and paraffin sections were analyzed for the expression of neuronal, glial, and innate immunity markers. Isolated myenteric networks were screened for differences in overall protein expression, and a motility analysis delivered functional data. The level of A beta PP in the gut was significantly higher in the AD mouse model than in wild-type mice and also higher in the gut than in the brain at all ages investigated. The transcriptional level of Nestin, GFAP, and TLR4 increased with age with a peak at 3 months. At the protein level, human amyloid-beta was located in myenteric neurons. Myenteric networks showed a reduction of the neuronal density in A beta PP compared to wild-type mice, which was functionally relevant as revealed by motility analysis. The ENS undergoes significant changes during the early onset of A beta PP expression in AD mouse models that appear before those seen in the brain as demonstrated in this study. Thus, there is a chance of determining similar alterations in the human gut of AD patients, which could be used to develop early diagnostic approaches.

Protein profiles of developing neural circuits undergo manifold changes. The aim of this proteomic analysis was to quantify postnatal changes in two auditory brainstem areas in a comparative approach. Protein samples from the inferior colliculus (IC) and the superior olivary complex (SOC) were obtained from neonatal (P4) and young adult (P60) rats. The cytosolic fractions of both areas were examined by 2-D DIGE, and the plasma membrane-enriched fraction of the IC was analyzed via iTRAQ. iTRAQ showed a regulation in 34% of the quantified proteins. DIGE revealed 12% regulated spots in both the SOC and IC and, thus, numeric congruency. Although regulation in KEGG pathways displayed a similar pattern in both areas, only 13 of 71 regulated DIGE proteins were regulated in common, implying major area-specific differences. 89% of regulated glycolysis/gluconeogenesis and citrate cycle proteins were up-regulated in the SOC or IC, suggesting a higher energy demand in adulthood. Seventeen cytoskeleton proteins were regulated, consistent with complex morphological reorganization between P4 and P60. Fourteen were uniquely regulated in the SOC, providing further evidence for area-specific differences. Altogether, we provide the first elaborate catalog of proteins involved in auditory brainstem development, several of them possibly of particular developmental relevance.

Ultrasound is widely used in clinical practice, mostly in diagnostic studies, but increasingly in therapeutic applications as well. This may be the case in acceleration of wound healing or treatment of cancer. Still, little is known about the direct effect of frequency or energy density of the ultrasound upon the cells themselves. We therefore investigated the impact of three different protocols using high, medium and low energy densities at three different frequencies on normal endothelial and epithelial as well as carcinoma cell lines (neuroblastoma and adenocarcinoma cell lines). Proliferation of endothelial and epithelial cell lines was significantly increased depending on the frequency and energy density applied. No influence on actin cytoskeleton formation was seen in these cells after treatment, while a significant decrease in the density of microvilli and the length of filopodia in the epithelial cell line could be noted. The proliferation rate of the carcinoma cell lines was reduced and cells destroyed. Apoptosis was induced in the adenocarcinoma cells after ultrasound exposure. Additionally, the expression of neurofilament was increased in neuroblastoma cells as evidence of beginning differentiation. So, different settings of frequency and energy density in an ultrasonic treatment protocol lead to different impacts on proliferation, morphology and differentiation and might be used to stimulate or inhibit the growth of individual cell types.

The transplantation of neural crest derived stem cells (NCSC) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. Influences of HSCR-smooth-muscle-protein extracts upon isolated myenteric plexus cells, dissociated dorsal root ganglia and NCSC were studied in vitro to investigate the quality of this microenvironment effects. Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) and transforming-growth-factor-β-1 (TGFβ-1) concentration were measured in the homogenates from the individual segment using ELISA. Myenteric plexus and dissociated dorsal root ganglia (DRG) cultures, as well as NCSCs were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival, and branching was evaluated. The amount of the factors varied considerably between the individual segments and also from patient to patient. Four major expression patterns could be detected. While all extracts tested lead to a significant increase in neurite outgrowth compared to the control, extracts from proximal segments tended to have more prominent effects. In one experiment, extracts from all individual segments of a single patient were tested. Neurite outgrowth, neuronal survival, and branching pattern varied from segment to segment, but all HSCR-muscle-protein extracts increased neuronal survival and network formation. Smooth muscle protein from aganglionic bowel supports the survival and outgrowth of myenteric neurons and NCSCs and is so an appropriate target for neural stem cell treatment.

The transplantation of neural crest derived stem cells (NCSC’s) is a potent alternative for the treatment of Hirschsprung’s disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. To investigate the quality of this microenvironment, effects of HSCR-smooth-muscle-protein extracts upon NCSC’s were studied in vitro. Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) concentration was measured in the homogenates from the individual segment using ELISA. NCSC’s were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival and branching was evaluated. The amount of the factors varied considerably between the proximal and distal segments, and also from patient to patient. While extracts from proximal segments tended to have more prominent effects, all HSCR-muscle-protein extracts increased neuronal survival and network formation. Muscle protein from aganglionic bowel supports the survival and outgrowth of NCSC’s and is so an appropriate target for neural stem cell treatment.

Breast-feeding plays an important role for the development of the newborn. Non-breast fed premature born infants show a significantly higher risk of developing diseases like infantile diarrhoea and necrotizing enterocolitis. In this study, the content of neurotrophic factors and cytokines, which might influence the postnatal development of the enteric nervous system (ENS), was determined in human breast milk. Glial cell-line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) as well as a panel of cytokines were analyzed using single factor or multiplex ELISA. In order to link their presence in milk with possible effects on the development of the ENS, rat myenteric neurons were cultured in protein extracts from breast milk. Neurite outgrowth, neuron survival and nestin expression in glial cells were measured. Growth factors and cytokines were found in all breast milk samples at varying concentrations. It could be demonstrated that protein extracts of breast milk increased the amount of surviving enteric neurones as well as neurite outgrowth. Additionally it was shown, that the number of nestin and S100-expressing glial cells increased significantly after incubating in breast milk protein extracts. The data suggest that milk-born proteins support the development of the enteric nervous system.

of the disease. There is an unmet need for diagnostic biomarkers for FTD. The objective of this hypothesis generating study was to discover new candidate protein markers for early, differential diagnosis of FTD that may improve the performance of current diagnostic assays. Methods: Surface Enhanced Laser Desorption/Ionization (SELDI) TOF-MS and three different chromatographic surfaces were used to differentially profile proteins and peptides in CSF samples from 20 patients with FTD (age range 43-76 yrs) and 34 healthy controls (age range 42-86 yrs). Diagnosis was made using DSM-IV and ICD-10 criteria. Results: A total of 33 candidate biomarkers separating FTD from healthy aging were found with ROC values above 0.7 and p values below 0.01. Among them were fragments of the neurosecretory protein VGF as well as the neuroendochrine peptide Chromogranin B and a member of the complement system C3a. Conclusions: This novel panel of biomarkers could potentially be used to improve early diagnosis of FTD as well as to provide complementary information to help decision making in the development of disease modifying compounds.

Background Pancreatic neuropathy in chronic pancreatitis (CP) and pancreatic cancer (PCa) is characterized by pancreatic neuropathy, i.e. increased neural density and hypertrophy, which are associated with neuropathic pain. To better understand the mechanism of these neuropathic alterations, we aimed at achieving an in-vitro simulation of the intrapancreatic neuroplasticity. Methods Dissociated myenteric plexus (MP) and dorsal root ganglia (DRG) neurons of newborn rats were treated with normal human pancreas (NP), CP or PCa tissue extracts. Furthermore, MP and DRG neurons were cultured in supernatants from different pancreatic cancer cell lines (PCC) and human pancreatic stellate cells (hPSC) obtained from either CP or PCa tissues. For analysis, the neurite density, outgrowth, neuronal branching capacity and perikaryonal size were quantified. Key Results Myenteric plexus and DRG neurons grown in CP and PCa tissue extracts built denser networks than in NP extracts. Both neuronal types showed a strong neurite outgrowth, more complex branching pattern and a somatic hypertrophy in CP and PCa extracts. Pancreatic cancer cell supernatants induced a prominent neurite outgrowth, increased neurite density and perikaryonal hypertrophy in MP and DRG neurons. Supernatants of CP-derived hPSC strongly stimulated neurite outgrowth. Glial density in MP cultures was strikingly increased by PCa tissue extracts. Conclusions & Inferences Intrapancreatic microenvironment in CP and PCa induces neuroplastic alterations under in-vitro conditions, leading to increased neural density and hypertrophy. Thus, due to its neurotrophic attributes, the intrapancreatic microenviroment in CP and PCa seems to be a key player in the generation of pancreatic neuropathy and neuroplasticity.

The enteric nervous system with its intricate network of neurons and glia shows a high plasticity, which not only changes during pre- and postnatal development, but also with disease or changing dietary habits. FGF as a potent neurotrophic factor in the central nervous system might also play a specific role for the ENS development, FGF-2 knockout and corresponding wild-type mice were histologically and functionally analyzed. FGF-2 knockout mice are viable and thrive normally and do apparently not display any obvious neurological deficit. Morphological differences were studied on whole mount preparations of muscle and submucous layer using either cuprolinic blue or immunohistochemical stainings for the neuronal marker PGP 9.5. Ussing-chamber and isometric muscle contraction experiments were performed on isolated gut wall, respectively muscle preparations. Intravital microscopy with GFP-transfected E. coli bacteria was used to investigate influences upon bacterial translocation. In additional experiments the protein pattern of the isolated myenteric plexus of knockout and wild-type mice were compared using 2D-DIGE technology. The morphometric analysis of the myenteric plexus revealed significant differences between FGF-2 knockout and wild-type animals, resulting in larger neurons in the knock out animals, embedded in less densely packed enteric ganglia. While muscle contractility appeared not to be affected, there was a significant difference in bacterial translocation as well as differences in basal chloride secretion to be seen. The observed morphological differences were reflected in the varying protein patterns, which were revealed by 2D-DIGE. A large number of differentially expressed proteins were found in both colonic and duodenal samples. FGF obviously influences the development of well established gastrointestinal functions by various means, thus leading to minor but significant deficiencies. Whether the revealed deficits in the mucous barrier are indebted to the morphological alterations in the ENS cannot yet be proved, but is very likely.

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.

Autoantibodies in patients with paraneoplastic neurological syndromes (PNS) have been reported to be predominantly IgG1 and IgG3 isotypes. However, no data are available about the IgG subclass distribution of the total serum IgG in these patients. Therefore, we investigated the IgG subclass distribution (given as percentage of total IgG) in 15 anti-Hu positive PNS patients, 15 patients with small cell lung cancer (SCLC) without PNS and 23 healthy controls using a commercial enzyme-linked immunosorbant assay test. Although IgG1 (and to a lower extent IgG3) are the predominant subclasses of the anti-Hu antibodies, PNS and SCLC showed a significant decrease in IgG1 and a concomitant increase in IgG2 compared with healthy controls (P < 0.05, respectively). In contrast, only SCLC patients, but not PNS patients, had higher IgG3 and IgG4 values compared with controls (P < 0.05, respectively). There was no correlation between IgG subclass levels and the titre or the predominant isotype of the antineuronal antibodies. PNS patients with autonomic disturbances had lower IgG4 levels than PNS patients without autonomic disturbances (P < 0.05). Our study demonstrates a disturbance in the IgG subclass distribution in PNS patients which is partly different from SCLC patients. The isotype regulation of the anti-Hu antibody seems to be independent from this phenomenon.

This study introduces a new experimental setup for particle detection within the nasal airways and describes intranasal deposition of particles at various regions of the nasal cavity and the nasopharynx. During respiration of an aerosol of starch particles the nondeposited particles in the air were detected in 11 volunteers by a transnasally placed suction probe at numerous sites of the nasal cavity and nasopharynx. Another, identical suction probe measured the initial number of inhaled particles at the nostril. The two suction probes were connected to two identical laser particle counters and allowed calculation of particle deposition. Particles 1-3 microm in size were deposited to about 60% within the entire nasal cavity, whereas most of the particles 4-30 microm in size were deposited within the entire nasal cavity. Between 80% and 90% of the particles retained in the nasal cavity were deposited at the anterior nasal segment. Studies on deposition of various drugs within the nasal cavity using this experimental set-up are conceivable.

The treatment of paraneoplastic neurological syndromes (e.g., tumor therapy, immunosuppressive therapy, plasmapheresis) rarely leads to an improvement in the neurological symptoms. We treated four patients suffering from paraneoplastic neurological syndromes with intravenous immunoglobulins. All four had high titers of antineuronal antibodies in serum and CSF. Two of the patients, one suffering from paraneoplastic cerebellar degeneration and the other from paraneoplastic brain stem encephalitis and polyneuropathy, received intravenous immunoglobulin treatment within 3 weeks of the onset of neurological symptoms. Both patients showed clinical improvement within 2 weeks after the initiation of therapy. They also showed a decline in the intrathecal antibody synthesis of the antineuronal antibody. Two other patients, who had suffered from paraneoplastic neuropathy for 3 and 6 months showed no improvement with the intravenous immunoglobulin therapy. In these cases there was no effect on intrathecal antibody synthesis. When started early, intravenous immunoglobulins may be of therapeutical value in treating paraneoplastic neurological syndromes. Specific intrathecal antibody synthesis may be a better measure of clinical course that autoantibody serum titers.

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.