Creation of an international registry to support discovery in schwannomatosis.

Schwannomatosis is a tumor suppressor syndrome that causes multiple tumors along peripheral nerves. Formal diagnostic criteria were first published in 2005. Variability in clinical presentation and a relative lack of awareness of the syndrome have contributed to difficulty recognizing affected individuals and accurately describing the natural history of the disorder. Many critical questions such as the mutations underlying schwannomatosis, genotype-phenotype correlations, inheritance patterns, pathologic diagnosis of schwannomatosis-associated schwannomas, tumor burden in schwannomatosis, the incidence of malignancy, and the effectiveness of current, or new treatments remain unanswered. A well-curated registry of schwannomatosis patients is needed to facilitate research in field. An international consortium of clinicians and scientists across multiple disciplines with expertise in schwannomatosis was established and charged with the task of designing and populating a schwannomatosis patient registry. The International Schwannomatosis Registry (ISR) was built around key data points that allow confirmation of the diagnosis and identification of potential research subjects to advance research to further the knowledge base for schwannomatosis. A registry with 389 participants enrolled to date has been established. Twenty-three additional subjects are pending review. A formal process has been established for scientific investigators to propose research projects, identify eligible subjects, and seek collaborators from ISR sites. Research collaborations have been created using the information collected by the registry and are currently being conducted. The ISR is a platform from which multiple research endeavors can be launched, facilitating connections between affected individuals interested in participating in research and researchers actively investigating a variety of aspects of schwannomatosis. © 2016 Wiley Periodicals, Inc.

[Multimorbidity and successful aging: the population-based KORA-Age study]. / Multimorbidität und erfolgreiches Altern: Ein Blick auf die Bevölkerung im Rahmen der KORA-Age-Studie.

BACKGROUND: The objective of the KORA-Age research consortium is to assess the determinants and consequences of multimorbidity in the elderly and to look into reasons for successful aging in the general public. PATIENTS AND METHODS: In the KORA-Age cohort study 9,197 persons were included who where born in the year 1943 or before and participants of previous KORA cohort studies conducted between 1984 and 2001 (KORA: Cooperative Health Research in the Region of Augsburg). The randomized intervention study KORINNA (Coronary infarct follow-up treatment in the elderly) tested a nurse-based case management program with 338 patients with myocardial infarct and included an evaluation in health economics. RESULTS: A total of 2,734 deaths were registered, 4,565 participants submitted a postal health status questionnaire and 4,127 participants were interviewed by telephone (response 76.2% and 68.9% respectively). A gender and age-stratified random sample of the cohort consisting of 1,079 persons took part in a physical examination (response 53.8%). CONCLUSION: The KORA-Age consortium was able to collect data in a large population-based sample and is contributing to the understanding of multimorbidity and successful aging.

[Interventional bronchoscopy in lung cancer]. / Bronchoskopische Therapieverfahren beim Lungenkarzinom.

Stenosis of central airways or hemoptysis are classical indications for interventional bronchoscopy in lung cancer. In the case of endoluminal tumor growth cryo-, laser- or brachytherapy are widely used. In the case of airway stenosis due to compression by extraluminal tumor masses balloon-dilatation and/or stenting and - with delayed effect - brachytherapy are first-choice therapies. Carcinoma in situ and early stage tumors can be treated curatively with brachytherapy or photodynamic therapy. Recently introduced bronchoscopic techniques like electro-magnetic navigation may result in new curative options for peripheral lung tumors.

Introduction of sapropterin dihydrochloride as standard of care in patients with phenylketonuria.

Sapropterin dihydrochloride, a synthetic, stable form of the tetrahydrobiopterin cofactor of phenylalanine hydroxylase, has been shown to reduce plasma phenylalanine (Phe) levels in a significant portion of patients with phenylketonuria (PKU). When we undertook introducing this medication to our PKU clinic population, the challenges of recalling and reconnecting with a variably treated and variably compliant patient population became apparent. We offered a trial of sapropterin to all of our clinic patients with PKU. In order to determine responsiveness, we used a two tier dose escalation protocol. After diet records were taken, and baseline plasma Phe levels were established, a 7-day trial of sapropterin at 10mg/kg/day was started. At day 8, plasma phenylalanine levels were measured. Patients were considered to be responders if they had a 30% reduction in plasma Phe. If they did not respond, the dose of sapropterin was increased to 20 mg/kg/day, and levels were rechecked again in 8 days. Patients who were not responders at this time continued sapropterin for a total of 30 days and had Phe levels checked one last time. Patients who were responders and who were on a Phe-restricted diet underwent gradual liberalization of their diet to the maximum tolerated natural protein intake while still maintaining plasma levels in the acceptable treatment range of 120-360 micromol/L. In our population, 36/39 patients with hyperphenylalaninemia (HPA) who were offered a trial of sapropterin elected to start sapropterin. Five of 36 patients were non-adherent with diet records and/or medication doses and we were unable to determine if they were responders. We were unable to categorize 2 of 31 of the patients who completed the trial as responders due to dietary issues, though they were probably responders. Of the 29 patients who completed the sapropterin trial and we could categorize, 18/29 (62%) were determined to be responders. Patients were classified based on their off-diet diagnostic plasma phenylalanine levels as classical PKU (>1200 micromol/L) and variant PKU (>400 and <1200 micromol/L). The group with variant PKU had a 100% response rate, and patients with classical PKU had a 27% response rate. For the patients in the responder group who were on Phe-restricted diet, we were able to liberalize most diets, in two cases to unrestricted protein intake. We also had unexpected beneficial findings in our clinic experience, including positive behavioral improvements in an adult severely affected by untreated PKU. Even in patients who were not considered to be responders, the introduction of sapropterin provided a tool to reconnect with patients and re-introduce beneficial dietary measures.

Discrimination of cancerous and non-cancerous cell lines by headspace-analysis with PTR-MS.

Proton transfer reaction mass spectrometry (PTR-MS) has been used to analyze the volatile organic compounds (VOCs) emitted by in-vitro cultured human cells. For this purpose, two pairs of cancerous and non-cancerous human cell lines were selected:1. lung epithelium cells A-549 and retinal pigment epithelium cells hTERT-RPE1, cultured in different growth media; and 2. squamous lung carcinoma cells EPLC and immortalized human bronchial epithelial cells BEAS2B, cultured in identical growth medium. The VOCs in the headspace of the cell cultures were sampled: 1. online by drawing off the gas directly from the culture flask; and 2. by accumulation of the VOCs in PTFE bags connected to the flask for at least 12 h. The pure media were analyzed in the same way as the corresponding cells in order to provide a reference. Direct comparison of headspace VOCs from flasks with cells plus medium and from flasks with pure medium enabled the characterization of cell-line-specific production or consumption of VOCs. Among all identified VOCs in this respect, the most outstanding compound was m/z = 45 (acetaldehyde) revealing significant consumption by the cancerous cell lines but not by the non-cancerous cells. By applying multivariate statistical analysis using 42 selected marker VOCs, it was possible to clearly separate the cancerous and non-cancerous cell lines from each other.

Effects of cigarette smoke extract and nicotine on bronchial tone and acetylcholine-induced airway contraction in mouse lung slices.

BACKGROUND: Tobacco smoke is a key risk factor for chronic obstructive pulmonary disease, but it may also alter the pathophysiology of asthma. In the present study, we analyzed whether tobacco smoke has acute or chronic effects on bronchial tone and whether it alters bronchial reactivity in vitro. METHODS: Airways in murine lung slices were digitally recorded and the change in cross-sectional area with time was quantified. T-bet KO mice served as a model for bronchial hyperreactivity. T-bet KO mice show a shift towards type 2 helper T lymphocytes and display histological as well as functional characteristics of asthma. Cigarette smoke extract (CSE) was obtained using commercially available cigarettes (Gauloise Blondes) by drawing cigarette smoke slowly through a water pump into a tube containing 10 mL of DMEM culture medium. RESULTS: Acute exposure to CSE led to relaxation of the airway. Acute exposure to nicotine resulted in a minor relaxation of the airway in Balb/C mice and in nonsignificant relaxation of the airway in T-bet KO mice. The nicotinic acetylcholine-receptor hexamethonium partially inhibited CSE-induced airway relaxation. Airway contraction in response to acetylcholine was stronger in T-bet KO mice than in Balb/C mice. After exposure to CSE or nicotine for 48 hours, acetylcholine-induced airway contraction was no longer different between the 2 types of mice. CONCLUSIONS: Our data indicate that acute exposure to CSE leads to airway relaxation, which is partially mediated by nicotine. Chronic exposure to CSE reverses bronchial hyperreactivity in the airways of T-bet KO mice; this effect can be mimicked by chronic exposure to nicotine.

Continuous wavelet transform in the analysis of burst synchronization in a coupled laser system.

The transition to synchronization of a pair of coupled chaotic CO2 lasers is investigated numerically in a model system. This system displays episodes of bursting of different predominant frequencies. Due to the multiple time scales present in this system, we use a complex continuous wavelet transform to perform the synchronization analysis. Thus it enables us to resolve the time of occurrence as well as the frequency of an event in a given time series up to an intrinsic uncertainty. Furthermore, due to the complex nature of that wavelet transform, it yields a direct estimate of the system's phase. We show that, as the coupling strength of the laser system is increased, the mutual coherency increases differently for different frequencies. Additionally we test our method with experimental data.

How many types of cholinergic sympathetic neuron are there in the rat stellate ganglion?

Sympathetic cholinergic postganglionic neurons are present in many sympathetic ganglia. Three classes of sympathetic cholinergic neuron have been reported in mammals; sudomotor neurons, vasodilator neurons and neurons innervating the periosteum. We have examined thoracic sympathetic ganglia in rats to determine if any other classes of cholinergic neurons exist. We could identify cholinergic sudomotor neurons and neurons innervating the rib periosteum, but confirmed that cholinergic sympathetic vasodilator neurons are absent in this species. Sudomotor neurons contained vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) and always lacked calbindin. Cholinergic neurons innervating the periosteum contained VIP and sometimes calbindin, but always lacked CGRP. Cholinergic neurons innervating the periosteum were usually surrounded by terminals immunoreactive for CGRP. We conclude that if any undiscovered populations of cholinergic neurons exist in the rat thoracic sympathetic chain, then they are indistinguishable in size, neurochemistry and inputs from sudomotor or cholinergic neurons innervating the periosteum. It may be that the latter two populations account for all cholinergic neurons in the rat thoracic sympathetic chain ganglia.

Bronchial hyperreactivity is correlated with increased baseline airway tone.

Physiologically, airways are not completely relaxed but maintain a baseline airway tone (BAT). Although not fulfilling the criteria for obstructive airway disease, increased BAT may nevertheless be important because the same amount of airway narrowing can be well tolerated or can cause severe airway obstruction depending on the starting point of the narrowing. In this study, we aimed at studying if BAT is correlated with bronchial hyperreactivity (BHR). For in vitro studies, airways in murine lung slices were digitally recorded and the change in cross-sectional area with time was quantified. BAT was measured by the amount of relaxation induced by permeabilization of the cell membrane with beta-escin in zero external calcium. BHR was induced by incubation of lung slices with interleukin-13 (IL-13). T-bet knock-out mice served as an additional model for BHR. T-bet knock-out mice show a shift towards TH2-lymphocytes and display histological as well as functional characteristics of asthma. In vivo, the specific airway resistance of healthy non-smoking volunteers was assessed before and after inhalation of formoterol and bronchial challenge was performed using methacholin. In murine lung slices that had been cultivated without serum, only a minimal BAT could be observed. But, after cultivation with 10 % new born calve serum, airways showed a BAT of approximately 13 % that could be reduced by incubation with an IL-13 receptor antagonist. Atropine, isoproterenol and indomethacin failed to relax airways regardless of cultivation with serum. Incubation of lung slices without serum but with IL-13 increased BAT as well as airway responsiveness to acetylcholine and both effects were more pronounced in small compared to large airways. In lung slices from T-bet knock-out mice, airways were hyperreactive compared to airways in slices from wild type mice and BAT was found to be increased. Again, both effects were more pronounced in small compared to large airways. In human non-smokers without airway obstruction, increased BAT was correlated with bronchial hyperreactivity. We therefore conclude that although not fulfilling the criteria for obstructive airway disease, increased airway tone may yet be relevant in asthma.

The human mast cell tryptase tetramer: a fascinating riddle solved by structure.

Tryptases, the predominant proteins of human mast cells, have been implicated as pathogenetic mediators of allergic and inflammatory conditions, most notably asthma. Until recently, the fascinating properties that distinguish tryptases among the serine proteinases, particularly their activity as a heparin-stabilized tetramer, resistance to most proteinaceous inhibitors, and preference for peptidergic over macromolecular substrates presented a riddle. This review solves this riddle with the help of the crystal structure of the human beta(2)-tryptase tetramer, but also indicates controversies between the unique quaternary architecture and some experimental data.

Ta6Br(2+)12, a tool for phase determination of large biological assemblies by X-ray crystallography.

The title compound Ta6Br(2+)12 is of interest for the analysis of biological structures as a heavy-metal derivative with great potential for the structure determination of large protein systems. In macromolecular crystallography the phases of the measured structure factor amplitudes have to be determined. The most widely used method for novel structures is isomorphous replacement by introducing electron-rich compounds into the protein crystals. These compounds produce measurable changes of the diffraction intensities, which allow phase determination. We synthetized the Ta6Br(2+)12 cluster in high yields, crystallized it, and determined its crystal structure by X-ray diffraction analysis at atomic resolution. The cluster is a regular octahedron consisting of six metal atoms with 12 bridging bromine atoms along the 12 edges of the octahedron. The cluster is compact, of approximately spherical shape with about 4.3 A radius and highly symmetrical. One Ta6Br(2+)12 ion adds 856 electrons to a protein, a considerable contribution to the scattering power even of large proteins or multimeric systems. At low resolution all atoms of the cluster scatter in phase and act as a super heavy-atom, which is easy to locate in the difference Patterson map. We investigated its binding sites in the biologically significant high-resolution structures of an antibody V(L) domain, dimethyl sulfoxide reductase, GTP-cyclohydrolase I, and the proteasome. With the randomly oriented cluster, treated as a single site scatterer, phases could be used only up to 6 A resolution. In contrast, when the cluster is correctly oriented, phases calculated from its 18 atom sites can be used to high resolution. We present the atomic structure of the Ta6Br(2+)12, describe a method to determine its localization and orientation in the unit cell of protein crystals of two different proteins, and analyse its phasing power. We show that phases can be calculated to high resolution. The phase error is lower by more than 30 degrees compared to the single site approximation, using a resolution of 2.2 A. Furthermore, Ta6Br(2+)12 has two different strong anomalous scatterers tantalum and bromine to be used for phase determination.

The structure of recombinant human annexin VI in crystals and membrane-bound.

The crystal structure of calcium-free recombinant human annexin VI was solved at a resolution of 3.2 A by using the annexin I model for Patterson search and refined to an R-factor of 19.0%. The molecule consists of two similar halves closely resembling annexin I connected by an alpha-helical segment and arranged perpendicular to each other. The calcium and membrane binding sites assigned by structural homology are therefore not located in the same plane. Analysis of the membrane-bound form of annexin VI by electron microscopy shows the two halves of the molecule coplanar with the membrane, but oriented differently to the crystal structure and suggesting a flexible arrangement. Ion channel activity has been found for annexin VI and the half molecules by electrophysiological experiments.

Cell cycle effects of radiation on human bronchial epithelium and lung carcinoma cells in monolayer cultures and a three-dimensional co-culture system.

The aim of this study was to investigate whether the three-dimensional structure of the bronchial tissue and the contact of non-malignant with malignant cells influence the effectiveness of radiotherapy. Monolayer cultures of cells of the human bronchial epithelial cell line BEAS 2B, monolayer co-cultures of BEAS 2B cells and cells of the GFP-transfected lung carcinoma cell line EPLC 32M1, organ cultures of human bronchial epithelium, and organ co-cultures with EPLC 32M1 cells were irradiated with 10 Gy, and the DNA content was analyzed using flow cytometry. In non-malignant epithelial cells, BEAS 2B monolayer cultures without tumor cells were highly radiosensitive. However, contact with tumor cells in monolayer co-cultures markedly reduced radiosensitivity. Non-malignant cells in three-dimensional organ cultures and organ co-cultures with tumor cells showed moderate radiosensitivity. In EPLC 32M1 tumor cells, proliferation was increased without irradiation when the cells were in contact with epithelial cells in both organ and monolayer co-cultures. Radiosensitivity was higher in organ co-cultures than in monolayer cultures and monolayer co-cultures. These data indicate that organ co-cultures in combination with flow cytometry allow investigation of the effects of radiation in an in vivo-like environment and that both the spatial organization and the interaction of non-malignant and tumor cells are crucial for the effectiveness of radiotherapy.

Bivalent inhibition of human beta-tryptase.

BACKGROUND: Human beta-tryptase is a mast cell specific trypsin-like serine protease that is thought to play a key role in the pathogenesis of diverse allergic and inflammatory disorders like asthma and psoriasis. The recently resolved crystal structure revealed that the enzymatically active tetramer consists of four quasi-identical monomers. The spatial display of the four identical active sites represents an ideal basis for the rational design of bivalent inhibitors. RESULTS: Based on modeling experiments homobivalent inhibitors were constructed using (i) 6A,6D-dideoxy-6A,6D-diamino-beta-cyclodextrin as a rigid template to bridge the space between the two pairs of identical active sites and (ii) 3-(aminomethyl)benzene as a headgroup to occupy the arginine/lysine specific S1 subsites. A comparative analysis of the inhibitory potencies of synthetic constructs that differ in size and type of the spacer between headgroup and template revealed that the construct contained two 3-(aminomethyl)benzenesulfonyl-glycine groups linked to the 6A,6D-diamino groups of beta-cyclodextrin as an almost ideal bivalent inhibitor with a cooperativity factor of 1.9 vs. the ideal value of 2. The bivalent binding mode is supported by the inhibitor/tetramer ratio of 2:1 required for inactivation of tryptase and by X-ray analysis of the inhibitor/tryptase complex. CONCLUSION: The results obtained with the rigid cyclodextrin template underlined the importance of a minimal loss of conformational entropy in bivalent binding, but also showed the limitations imposed by such rigid core molecules in terms of optimal occupancy of binding sites and thus of enthalpic strains in bidentate binding modes. The main advantage of bivalent inhibitors is their high selectivity for the target enzyme that can be achieved utilizing the principle of multivalency.

Monitoring of expiratory flow rates and lung volumes during a high altitude expedition.

BACKGROUND: Data on lung volumes and changes in flow-volume spirometry at high altitude are few and do not provide comprehensive assessment of the occurring changes. This study characterizes alterations of the forced expiratory flow-volume curve (FEFV-curve) and lung volumes at increasing altitude. METHODS: FEFV-curve and lung volumes at increasing altitude were characterized by daily assessment of peak expiratory flow (PEF), forced expiratory volume in one second (FEV1), forced vital capacity (FVC) and maximal expiratory flow rates (MEF 25, 50, 75) at 25%, 50% and 75% of the FEFV-curve with a portable spirometer (turbinometric method) three times a day during an expedition to Mustagh Ata (7545m) in 15 healthy mountaineers. RESULTS: With increasing altitude FVC and FEV1 were reduced by up to 25% (74.8% / 74.6% of baseline) and MEF25 was reduced to 81.5% of baseline values. PEF initially increased up to 4451m and returned to baseline values above 5000m. After descent below 2000m, all values normalized within one day. There were weak negative correlations between AMSS and FEV1, FVC and PEF (r = -0.23, p<0.001). CONCLUSIONS: We found increasing pulmonary restriction at high altitude without a marked reduction of PEF. Assessment of the FEFV-curve at high altitudes with a portable spirometer is a practical method reflecting the true field situation and may provide clinically relevant information (impending pulmonary edema).

Antireflux treatment for asthma. Improvement in patients with associated gastroesophageal reflux.

Fifteen patients with both nonallergic asthma and symptomatic gastroesophageal reflux were studied before and after an eight-week period of vigorous antireflux therapy, which included ranitidine hydrochloride, 150 mg twice a day. Pulmonary and esophageal symptoms were recorded on daily diary cards. Therapy was associated with prompt amelioration of reflux symptoms and with a less dramatic and more delayed improvement in pulmonary symptoms. Objectively, esophageal erosions healed completely in eight of the ten patients who had them at the beginning of the trial, and pulmonary function measurements improved significantly. Intraesophageal infusions of physiologic saline and 0.1N hydrochloric acid in patients and healthy controls did not significantly alter pulmonary function, as measured by standard spirometry. There is a subset of patients in whom bronchoconstriction is triggered by gastroesophageal reflux. Treatment of reflux in such patients may improve their asthma.

Design of benzamidine-type inhibitors of factor Xa.

A series of derivatives of rac-benzenesulfonyl-glycyl-phenylalanine or its ethyl ester with a combination of thioamido/amidino or amidino/amidino substituents in the benzene rings was synthesized as potential inhibitors of factor Xa (fXa). Among these, the racemic 4'-amidinobenzenesulfonyl-glycyl-4-amidinophenylalanine ethyl ester was found to exhibit the highest affinity for fXa despite the unfavored location of the amidino substituent in the para position. X-ray structural analysis of the trypsin complex with this bis-benzamidine compound revealed a retro-binding mode if compared to those of similar compounds, so far analyzed in complexes with trypsin or fXa. This noncanonical binding mode as well as its slow plasma clearance rates in rats, if compared to those of other benzamidine derivatives, suggests this compound as an interesting new lead structure for the design of fXa inhibitors.

After axotomy, substance P and vasoactive intestinal peptide expression occurs in pilomotor neurons in the rat superior cervical ganglion.

Autonomic sympathetic postganglionic neurons normally express distinct combinations of neuropeptides which are often highly correlated with the projection of the neurons. When sympathetic postganglionic neurons are axotomized, they can express quite different neuropeptides, notably substance P, vasoactive intestinal peptide or galanin. In this study, we have examined rat sympathetic postganglionic neurons in the superior cervical ganglion that project to the skin, the vasculature of the skeletal muscle or to the submandibular salivary gland, and assessed whether the neuropeptides that they express after axotomy depend on which target tissue they previously innervated. In all three populations, around half of the postganglionic neurons expressed galanin after axotomy. In contrast, only skin-projecting neurons showed a significant increase in the number of neurons that expressed substance P (22%) and vasoactive intestinal peptide (17%) following axotomy. Within the skin-projecting neurons, as judged on the basis of cell body size, substance P and vasoactive intestinal peptide were expressed predominantly in pilomotor neurons, but only rarely were the two neuropeptides present in the same nerve cell body. In conclusion, we have demonstrated that three different neuropeptides, which can be induced by axotomy in postganglionic neurons, follow quite different patterns of expression when they are viewed in relation to the function of the postganglionic neurons in the superior cervical ganglion.

Control of postganglionic neurone phenotype by the rat pineal gland.

As neurones develop they are faced with choices as to which genes to express, to match their final phenotype to their role in the nervous system. A number of processes can guide these decisions. Within the autonomic and sensory nervous systems, there are a handful of examples that suggest that one mechanism that may match phenotype to function is the presence of target-derived differentiation factors. We tested whether the rat pineal gland controls the expression of a neuropeptide (neuropeptide Y) and a calcium-binding protein (calbindin) in sympathetic postganglionic neurones that innervate it. We first showed that the chemical phenotype of sympathetic neurones innervating the rat pineal includes the expression of both neuropeptide Y and the calcium-binding protein, calbindin. After transplanting the pineal gland of neonatal rats into the submandibular salivary gland of neonatal hosts, it was innervated by sympathetic axons from the surrounding salivary gland tissue, which do not normally express neuropeptide Y and calbindin. The presence of the pineal gland led to the appearance of neuropeptide Y and calbindin in many of the postganglionic neurones that innervated the graft. From these findings we suggest that, like the rodent sweat gland, the pineal gland generates a signal that can direct the neurochemical phenotype of innervating sympathetic neurones.

Pulmonary hypersensitivity associated with pancreatin powder exposure.

A 25-year-old woman with obstructive, reversible pulmonary and nasal hypersensitivity apparently induced by casual, repeated inhalation of pancreatin powder (desiccated pork pancreas) is described. The powder was being employed as a dietary supplement for the patient's son, diagnosed as having cystic fibrosis. Two challenges of the diagnosed as having cystic fibrosis. Two challenges of the patient by reproducing home use of the powder resulted in repetition of a hypersensitivity symptom complex on both occasions. Vitalometry demonstrated an immediate and late response. Avoidance of pancreatin powder exposure resulted in subsidence of symptoms. Immunologic mechanisms are suggested but not proven.