Generierung und molekulare Charakterisierung rekombinanter humaner Zytomegalieviren mit spezifischen Mutationen im Immediate Early 1-Gen

Das humane Zytomegalievirus (HCMV) gehört zur Subfamilie der β-Herpesviren und zeichnet sich unter anderem durch eine strikte Speziesspezifität und einen langsamen Replikationszyklus aus. Trotz allgemein hoher Prävalenz des Virus beschränken sich HCMV-bedingte Gesundheitsschäden hauptsächlich auf immunsupprimierte Patienten und intrauterin infizierte Feten nach Primärinfektion von Schwangeren. Die vorhandenen Virustatika können wegen ihres ungünstigen Nebenwirkungsprofils und des Auftretens resistenter Virusstämme nicht immer erfolgreich eingesetzt werden, weshalb die Entwicklung neuer Konzepte zur antiviralen Prävention und Therapie eine wichtige Aufgabe darstellt. Hierfür sind das molekularbiologische Studium des Infektionszyklus und der Einfluss kritischer viraler Komponenten von elementarer Bedeutung. Die viralen major immediate-early (MIE)-Proteine beeinflussen als Schlüsselregulatoren in der initialen Phase der Infektion in entscheidender Weise den weiteren Verlauf der HCMV-Replikation und Pathogenese. Das 72 kDa IE1-Protein (IE1) wird dabei als erstes virales Genprodukt nach Infektion synthetisiert. Es akkumuliert in großen Mengen im Kern der Wirtszelle und moduliert dort unter anderem die Chromatin-Struktur, die zelluläre und virale Genexpression sowie die Integrität subnukleärer Multiproteinkomplexe (PML [promyelocytic leukemia]-Körper). Bisher beschriebene HCMV-Mutanten deuten auf eine wichtige, aber nicht essenzielle Rolle des IE1-Proteins für die Virusvermehrung in Zellkultur hin. Um die Konsequenzen des spezifischen Funktionsverlustes der viralen IE1-Genprodukte eindeutig zu klären, wurden im Rahmen dieser Arbeit verschiedene Typen von Mutationen sowohl in einem etablierten Laborstamm (Towne, TN), als auch in einem klinischen Primärisolat (fusion inducing factor x, FIX) in das IE1-spezifische Exon 4 der HCMV MIE-Transkriptionseinheit eingeführt. Einige davon führten über eine Exon 4-Substitution zum kompletten Verlust der Expression von IE1. Darüber hinaus ist eine genaue Kenntnis regulatorischer Proteindomänen innerhalb des IE1-Proteins für die systematische Manipulation einzelner Funktionen unabdingbar. Deshalb wurden zusätzlich, durch minimal invasiven Austausch von einem bis maximal drei Nukleotiden an relevanten Stellen des IE1-spezifischen Exon 4, diverse vermutete Protein-Motive gezielt ausgeschaltet. Zu diesem Zweck entstanden im ersten Schritt Transferplasmide mit der jeweiligen Mutation im Bereich der IE1-Sequenz, die im weiteren Verlauf mit Hilfe der neuartigen BAC (bacterial artificial chromosome)-Technologie, also über homologe Rekombination mit infektiösen HCMV-BACmiden in Escherichia coli, die Generierung IE1-mutierter HCMV-Genome ermöglichten. Diese wurden anschließend zur Rekonstitution von Viruspartikeln in permissive, primäre humane Fibroblasten transfiziert. Aus der erfolgreichen Produktion BACmid-abgeleiteter Viren konnte bereits geschlossen werden, dass die punktmutierten IE1-Genprodukte für die HCMV-Vermehrung in Zellkultur nicht absolut notwendig sind. Eine Rekonstitution der vollständig IE1-defizienten Mutanten war hingegen nicht möglich, wahrscheinlich aufgrund eines IE1-unabhängigen negativen Effekts eines transdominant wirksamen Exon 2/3-kodierten Proteins oder einer inhibitorischen Wirkung der im Austausch für das Exon 4 integrierten Genkassette. Im nächsten Schritt zeigten detaillierte Replikationskinetiken in infizierten humanen Fibroblasten deutliche Unterschiede im Ausmaß der Vermehrungskapazität einzelner Virusmutanten auf. So zog die Störung des hochkonservierten zentralen Bereichs, einschließlich eines vermuteten Leucinzippermotivs, im IE1 des TN- und FIX-Stammes (Mutanten TN/FIX_L174P und TN/FIX_K192P) eine erheblich eingeschränkte Virusreplikation nach sich. Gleiches traf für C-terminal trunkierte IE1-Mutanten zu, denen charakteristische saure Domänen fehlten (Mutanten TN/FIX_A373Stopp und TN/FIX_E421Stopp). Dagegen spielte sowohl die Ausschaltung eines putativen Zinkfingermotivs (Mutanten TN/FIX_C279A) als auch der Verlust der Bindung an das SUMO (small ubiquitin like modifier)-1-Protein (Mutante TN/FIX_K450R) für eine effektive Virusvermehrung in Zellkultur keine Rolle. Für die Virusmutante G476_Stopp, bei der die am äußersten C-Terminus gelegene Chromatinbindedomäne des IE1-Proteins deletiert wurde, ergab sich für den FIX-Stamm eine Wildtyp-ähnliche, für den TN-Stamm jedoch eine leicht attenuierte Replikationskinetik, was Spekulationen über stammspezifische Unterschiede zulässt. Desweiteren konnte mit Hilfe immunfluoreszenzmikroskopischer Kolokalisationsstudien bewiesen werden, dass keine der eingeführten Punktmutationen für eine Kolokalisation zwischen IE1 und PML-Körpern erforderlich ist. Die Ausschaltung der SUMOylierung, der Chromatinbindung sowie des putativen Zinkfingermotivs zeigte außerdem keinerlei Einfluss auf die IE1-vermittelte Auflösung der PML-Körper. Im Gegensatz dazu wurde gezeigt, dass die Auflösung der PML-Körper in strikter Abhängigkeit des vermuteten Leucinzippermotivs erfolgt und auch bei Verlust der sauren Domänen oder Störung des zentralen Bereichs im viralen Protein weitgehend eingeschränkt ist. Schließlich wurde mit geeigneten Effektorplasmiden im Dual-Luciferase Reporter Assay nachgewiesen, dass die Transaktivierung verschiedener Promotoren durch IE1 deutlich abnimmt, wenn die Struktur des C-Terminus oder des vermuteten Leucinzippermotivs gestört ist. Die Transaktivatorkapazität des viralen Proteins war jedoch bei Verlust des putativen Zinkfingermotivs oder der SUMOylierung von IE1 unbeeinflusst. Auffälligerweise waren sämtliche Mutanten mit deutlichem Wachstumsnachteil gegenüber dem Wildtyp-Virus auch in ihrer Fähigkeit zur Auflösung der PML-Körper und in ihren Transaktivierungseigenschaften beeinträchtigt. Diese Beobachtung bestätigt, dass die Zerstörung der potenziell antiviralen PML-Körper eine mögliche Erklärung für den attenuierten Phänotyp IE1-mutierter Viren darstellt. Insgesamt verdeutlichen die Ergebnisse dieser Arbeit, dass dem HCMV IE1-Protein eine wichtige Funktion im produktiven viralen Infektionszyklus zukommt, obwohl es, zumindest für die Virusvermehrung in Zellkultur, nicht absolut essenziell ist. Allerdings ist davon auszugehen, dass IE1-defiziente oder -mutierte Viren unter den stringenteren Bedingungen natürlicher HCMV-Infektionen des Menschen apathogen sind. In diesem Zusammenhang bieten sich IE1 und speziell einige der hier beschriebenen Sequenzmotive im IE1-spezifischen Exon 4 als potenzielle neue Zielstrukturen für antivirale Strategien an

Development of a longitudinal integrated clerkship at an academic medical center

In 2005, medical educators at the University of California, San Francisco (UCSF), began developing the Parnassus Integrated Student Clinical Experiences (PISCES) program, a year-long longitudinal integrated clerkship at its academic medical center. The principles guiding this new clerkship were continuity with faculty preceptors, patients, and peers; a developmentally progressive curriculum with an emphasis on interdisciplinary teaching; and exposure to undiagnosed illness in acute and chronic care settings. Innovative elements included quarterly student evaluation sessions with all preceptors together, peer-to-peer evaluation, and oversight advising with an assigned faculty member. PISCES launched with eight medical students for the 2007/2008 academic year and expanded to 15 students for 2008/2009. Compared to UCSF's traditional core clerkships, evaluations from PISCES indicated significantly higher student satisfaction with faculty teaching, formal didactics, direct observation of clinical skills, and feedback. Student performance on discipline-specific examinations and United States Medical Licensing Examination step 2 CK was equivalent to and on standardized patient examinations was slightly superior to that of traditional peers. Participants' career interests ranged from primary care to surgical subspecialties. These results demonstrate that a longitudinal integrated clerkship can be implemented successfully at a tertiary care academic medical center

Individual nutrition therapy and exercise regime: A controlled trial of injured, vulnerable elderly (INTERACTIVE trial)

© 2008 Thomas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Proximal femoral fractures are amongst the most devastating consequences of osteoporosis and injurious accidental falls with 25–35% of patients dying in the first year post-fracture. Effective rehabilitation strategies are evolving however, despite established associations between nutrition, mobility, strength and strength-related functional outcomes; there has been only one small study with older adults immediately following fragility fracture where a combination of both exercise and nutrition have been provided. The aim of the INTERACTIVE trial is to establish whether a six month, individualised exercise and nutrition program commencing within fourteen days of surgery for proximal femur fracture, results in clinically and statistically significant improvements in physical function, body composition and quality of life at an acceptable level of cost and resource use and without increasing the burden of caregivers. Methods and Design This randomised controlled trial will be performed across two sites, a 500 bed acute hospital in Adelaide, South Australia and a 250 bed acute hospital in Sydney, New South Wales. Four hundred and sixty community-dwelling older adults aged > 70 will be recruited after suffering a proximal femoral fracture and followed into the community over a 12-month period. Participants allocated to the intervention group will receive a six month individualised care plan combining resistance training and nutrition therapy commencing within 14 days post-surgery. Outcomes will be assessed by an individual masked to treatment allocation at six and 12 months. To determine differences between the groups at the primary end-point (six months), ANCOVA or logistic regression will be used with models adjusted according to potential confounders. Discussion The INTERACTIVE trial is among the first to combine nutrition and exercise therapy as an early intervention to address the serious consequence of rapid deconditioning and weight loss and subsequent ability to regain pre-morbid function in older patients post proximal femoral fracture. The results of this trial will guide the development of more effective rehabilitation programs, which may ultimately lead to reduced health care costs, and improvements in mobility, independence and quality of life for proximal femoral fracture sufferers. Trial registration Australian Clinical Trials Registry: ACTRN12607000017426

Accuracy in Diagnosis of Celiac Disease Without Biopsies in Clinical Practice

L

Multi-Organ Expression Profiling Uncovers a Gene Module in Coronary Artery Disease Involving Transendothelial Migration of Leukocytes and LIM Domain Binding 2: The Stockholm Atherosclerosis Gene Expression (STAGE) Study

Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10−27and−30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2–deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research

Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1.Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1.Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product.Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability.The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.info:eu-repo/grantAgreement/EC/FP7/20187

Tolerability of inhaled N-chlorotaurine in the pig model

<p>Abstract</p> <p>Background</p> <p>N-chlorotaurine, a long-lived oxidant produced by human leukocytes, can be applied in human medicine as an endogenous antiseptic. Its antimicrobial activity can be enhanced by ammonium chloride. This study was designed to evaluate the tolerability of inhaled N-chlorotaurine (NCT) in the pig model.</p> <p>Methods</p> <p>Anesthetized pigs inhaled test solutions of 1% (55 mM) NCT (n = 7), 5% NCT (n = 6), or 1% NCT plus 1% ammonium chloride (NH₄Cl) (n = 6), and 0.9% saline solution as a control (n = 7), respectively. Applications with 5 ml each were performed hourly within four hours. Lung function, haemodynamics, and pharmacokinetics were monitored. Bronchial lavage samples for captive bubble surfactometry and lung samples for histology and electron microscopy were removed.</p> <p>Results</p> <p>Arterial pressure of oxygen (PaO₂) decreased significantly over the observation period of 4 hours in all animals. Compared to saline, 1% NCT + 1% NH₄Cl led to significantly lower PaO₂values at the endpoint after 4 hours (62 ± 9.6 mmHg vs. 76 ± 9.2 mmHg, p = 0.014) with a corresponding increase in alveolo-arterial difference of oxygen partial pressure (AaDO₂) (p = 0.004). Interestingly, AaDO₂was lowest with 1% NCT, even lower than with saline (p = 0.016). The increase of pulmonary artery pressure (PAP) over the observation period was smallest with 1% NCT without difference to controls (p = 0.91), and higher with 5% NCT (p = 0.02), and NCT + NH₄Cl (p = 0.05).</p> <p>Histological and ultrastructural investigations revealed no differences between the test and control groups. The surfactant function remained intact. There was no systemic resorption of NCT detectable, and its local inactivation took place within 30 min. The concentration of NCT tolerated by A549 lung epithelial cells <it>in vitro </it>was similar to that known from other body cells (0.25–0.5 mM).</p> <p>Conclusion</p> <p>The endogenous antiseptic NCT was well tolerated at a concentration of 1% upon inhalation in the pig model. Addition of ammonium chloride in high concentration provokes a statistically significant impact on blood oxygenation.</p

Safety of the Deferral of Coronary Revascularization on the Basis of Instantaneous Wave-Free Ratio and Fractional Flow Reserve Measurements in Stable Coronary Artery Disease and Acute Coronary Syndromes

OBJECTIVES The aim of this study was to investigate the clinical outcomes of patients deferred from coronary revascularization on the basis of instantaneous wave-free ratio (iFR) or fractional flow reserve (FFR) measurements in stable angina pectoris (SAP) and acute coronary syndromes (ACS). BACKGROUND Assessment of coronary stenosis severity with pressure guidewires is recommended to determine the need for myocardial revascularization. METHODS The safety of deferral of coronary revascularization in the pooled per-protocol population (n = 4,486) of the DEFINE-FLAIR (Functional Lesion Assessment of Intermediate Stenosis to Guide Revascularisation) and iFR-SWEDEHEART (Instantaneous Wave-Free Ratio Versus Fractional Flow Reserve in Patients With Stable Angina Pectoris or Acute Coronary Syndrome) randomized clinical trials was investigated. Patients were stratified according to revascularization decision making on the basis of iFR or FFR and to clinical presentation (SAP or ACS). The primary endpoint was major adverse cardiac events (MACE), defined as the composite of all-cause death, nonfatal myocardial infarction, or unplanned revascularization at 1 year. RESULTS Coronary revascularization was deferred in 2,130 patients. Deferral was performed in 1,117 patients (50%) in the iFR group and 1,013 patients (45%) in the FFR group (p <0.01). At 1 year, the MACE rate in the deferred population was similar between the iFR and FFR groups (4.12% vs. 4.05%; fully adjusted hazard ratio: 1.13; 95% confidence interval: 0.72 to 1.79; p = 0.60). A clinical presentation with ACS was associated with a higher MACE rate compared with SAP in deferred patients (5.91% vs. 3.64% in ACS and SAP, respectively; fully adjusted hazard ratio: 0.61 in favor of SAP; 95% confidence interval: 0.38 to 0.99; p = 0.04). CONCLUSIONS Overall, deferral of revascularization is equally safe with both iFR and FFR, with a low MACE rate of about 4%. Lesions were more frequently deferred when iFR was used to assess physiological significance. In deferred patients presenting with ACS, the event rate was significantly increased compared with SAP at 1 year. (C) 2018 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.Peer reviewe

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation