Birch Pollen Induces Toll-Like Receptor 4-Dependent Dendritic Cell Activation Favoring T Cell Responses.

Seasonal exposure to birch pollen (BP) is a major cause of pollinosis. The specific role of Toll-like receptor 4 (TLR4) in BP-induced allergic inflammation and the identification of key factors in birch pollen extracts (BPE) initiating this process remain to be explored. This study aimed to examine (i) the importance of TLR4 for dendritic cell (DC) activation by BPE, (ii) the extent of the contribution of BPE-derived lipopolysaccharide (LPS) and other potential TLR4 adjuvant(s) in BPE, and (iii) the relevance of the TLR4-dependent activation of BPE-stimulated DCs in the initiation of an adaptive immune response. In vitro, activation of murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs by BPE or the equivalent LPS (nLPS) was analyzed by flow cytometry. Polymyxin B (PMB), a TLR4 antagonist and TLR4-deficient BMDCs were used to investigate the TLR4 signaling in DC activation. The immunostimulatory activity of BPE was compared to protein-/lipid-depleted BPE-fractions. In co-cultures of BPE-pulsed BMDCs and Bet v 1-specific hybridoma T cells, the influence of the TLR4-dependent DC activation on T cell activation was analyzed. In vivo immunization of IL-4 reporter mice was conducted to study BPE-induced Th2 polarization upon PMB pre-treatment. Murine and human DC activation induced by either BPE or nLPS was inhibited by the TLR4 antagonist or by PMB, and abrogated in TLR4-deficient BMDCs compared to wild-type BMDCs. The lipid-free but not the protein-free fraction showed a reduced capacity to activate the TLR4 signaling and murine DCs. In human DCs, nLPS only partially reproduced the BPE-induced activation intensity. BPE-primed BMDCs efficiently stimulated T cell activation, which was repressed by the TLR4 antagonist or PMB, and the addition of nLPS to Bet v 1 did not reproduce the effect of BPE. In vivo, immunization with BPE induced a significant Th2 polarization, whereas administration of BPE pre-incubated with PMB showed a decreased tendency. These findings suggest that TLR4 is a major pathway by which BPE triggers DC activation that is involved in the initiation of adaptive immune responses. Further characterization of these BP-derived TLR4 adjuvants could provide new candidates for therapeutic strategies targeting specific mechanisms in BP-induced allergic inflammation.

Changes in Posttraumatic Brain Edema in Craniectomy-Selective Brain Hypothermia Model Are Associated With Modulation of Aquaporin-4 Level.

Both hypothermia and decompressive craniectomy have been considered as a treatment for traumatic brain injury. In previous experiments we established a murine model of decompressive craniectomy and we presented attenuated edema formation due to focal brain cooling. Since edema development is regulated via function of water channel proteins, our hypothesis was that the effects of decompressive craniectomy and of hypothermia are associated with a change in aquaporin-4 (AQP4) concentration. Male CD-1 mice were assigned into following groups (n = 5): sham, decompressive craniectomy, trauma, trauma followed by decompressive craniectomy and trauma + decompressive craniectomy followed by focal hypothermia. After 24 h, magnetic resonance imaging with volumetric evaluation of edema and contusion were performed, followed by ELISA analysis of AQP4 concentration in brain homogenates. Additional histopathological analysis of AQP4 immunoreactivity has been performed at more remote time point of 28d. Correlation analysis revealed a relationship between AQP4 level and both volume of edema (r 2 = 0.45, p < 0.01, **) and contusion (r 2 = 0.41, p < 0.01, **) 24 h after injury. Aggregated analysis of AQP4 level (mean ± SEM) presented increased AQP4 concentration in animals subjected to trauma and decompressive craniectomy (52.1 ± 5.2 pg/mL, p = 0.01; *), but not to trauma, decompressive craniectomy and hypothermia (45.3 ± 3.6 pg/mL, p > 0.05; ns) as compared with animals subjected to decompressive craniectomy only (32.8 ± 2.4 pg/mL). However, semiquantitative histopathological analysis at remote time point revealed no significant difference in AQP4 immunoreactivity across the experimental groups. This suggests that AQP4 is involved in early stages of brain edema formation after surgical decompression. The protective effect of selective brain cooling may be related to change in AQP4 response after decompressive craniectomy. The therapeutic potential of this interaction should be further explored.

Anti-inflammatory Effects of Fungal Metabolites in Mouse Intestine as Revealed by In vitro Models.

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-1ß, 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-1ß (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.

Impact of protein kinase CK2 inhibitors on proliferation and differentiation of neural stem cells.

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.

Isolation of high-purity myenteric plexus from adult human and mouse gastrointestinal tract.

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.

Vascular and neural stem cells in the gut: do they need each other?

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.

Maintenance of the enteric stem cell niche by bacterial lipopolysaccharides? Evidence and perspectives.

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.

The mesenterially perfused rat small intestine: A versatile approach for pharmacological testings.

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.

Do different assays for human acylated ghrelin concentrations provide comparable results?

BACKGROUND: 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. METHODS: 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. RESULTS: 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, p<0.01). Bland-Altman plotting revealed complementary aspects concerning the agreement of the two tested methods at low and high concentrations. CONCLUSIONS: 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.

Granulocyte-colony stimulating factor: a new player for the enteric nervous system.

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.

Changes of the enteric nervous system in amyloid-ß protein precursor transgenic mice correlate with disease progression.

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-ß protein precursor (AßPP) 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 AßPP 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-ß was located in myenteric neurons. Myenteric networks showed a reduction of the neuronal density in AßPP compared to wild-type mice, which was functionally relevant as revealed by motility analysis. The ENS undergoes significant changes during the early onset of AßPP 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.

Developmental changes of the protein repertoire in the rat auditory brainstem: a comparative proteomics approach in the superior olivary complex and the inferior colliculus with DIGE and iTRAQ.

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.

The microenvironment in the Hirschsprung's disease gut supports myenteric plexus growth.

INTRODUCTION: 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. METHODS: 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-ß-1 (TGFß-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. RESULTS AND CONCLUSIONS: 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.

Smooth muscle proteins from Hirschsprung's disease facilitates stem cell differentiation.

BACKGROUND AND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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 milk contains relevant neurotrophic factors and cytokines for enteric nervous system development.

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.

Hyperphosphorylation of autoantigenic targets of paraproteins is due to inactivation of PP2A.

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.

Simultaneous tuberculous meningoencephalitis in two siblings.

UNLABELLED: 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/microl and lymphocytic predominance, hyponatremia, and possible hydrocephalus. This notion is especially true for children originating from high-endemicity 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.

Acquired intestinal aganglionosis after a lytic infection with varicella-zoster virus.

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.

Expression of intermediate filament proteins and neuronal markers in the human fetal gut.

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.