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

Das Odeo Cornaro in Padua

Die Entstehungsgeschichte des Odeo Cornaro in Padua steht in engem Zusammenhang mit der Person Alvise Cornaros und dessen Interessensfeld, welches mit dem humanistischen Bildungsmodus des 16. Jahrhunderts gleichzusetzen ist. Durch sein Engagement entsteht eine Institution, welche zu einem vielfältigen Spielraum für Theater, Musik, Literatur und bildender Kunst wird. Der künstlerischen Ausstattung liegen mehrere Variablen zugrunde, die in Padua erstmalig in Erscheinung treten. So findet in den Landschaftsfresken eine facettenreiche Synthese statt, welche das kunsthistorische Panorama auf innovative Weise aufzeigt und auf die kulturelle Bedeutsamkeit des Odeo hinweist. Sie sind Werk des Malers Lambert Sustris und entstehen in dessen früher Karrierephase um 1540. Dies ist das Ergebnis einer vergleichenden Untersuchung, welche die Zuschreibung unter Berücksichtigung der künstlerischen Anleihen stabilisiert

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

Cadherin 2-Related Arrhythmogenic Cardiomyopathy Prevalence and Clinical Features

Background:Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by fibrofatty replacement of the right and left ventricle, often causing ventricular dysfunction and life-threatening arrhythmias. Variants in desmosomal genes account for up to 60% of cases. Our objective was to establish the prevalence and clinical features of ACM stemming from pathogenic variants in the nondesmosomal cadherin 2 (CDH2), a novel genetic substrate of ACM.Methods:A cohort of 500 unrelated patients with a definite diagnosis of ACM and no disease-causing variants in the main ACM genes was assembled. Genetic screening of CDH2 was performed through next-generation or Sanger sequencing. Whenever possible, cascade screening was initiated in the families of CDH2-positive probands, and clinical evaluation was performed.Results:Genetic screening of CDH2 led to the identification of 7 rare variants: 5, identified in 6 probands, were classified as pathogenic or likely pathogenic. The previously established p.D407N pathogenic variant was detected in 2 additional probands. Probands and family members with pathogenic/likely pathogenic variants in CDH2 were clinically evaluated, and along with previously published cases, altogether contributed to the identification of gene-specific features (13 cases from this cohort and 11 previously published, for a total of 9 probands and 15 family members). Ventricular arrhythmic events occurred in most CDH2-positive subjects (20/24, 83%), while the occurrence of heart failure was rare (2/24, 8.3%). Among probands, sustained ventricular tachycardia and sudden cardiac death occurred in 5/9 (56%).Conclusions:In this worldwide cohort of previously genotype-negative ACM patients, the prevalence of probands with CDH2 pathogenic/likely pathogenic variants was 1.2% (6/500). Our data show that this cohort of CDH2-ACM patients has a high incidence of ventricular arrhythmias, while evolution toward heart failure is rare.</p

Accuracy in Diagnosis of Celiac Disease Without Biopsies in Clinical Practice

L

Tolerability of inhaled N-chlorotaurine in an acute pig streptococcal lower airway inflammation model

<p>Abstract</p> <p>Background</p> <p>Inhalation of N-chlorotaurine (NCT), an endogenous new broad spectrum non-antibiotic anti-infective, has been shown to be very well tolerated in the pig model recently. In the present study, inhaled NCT was tested for tolerability and efficacy in the infected bronchopulmonary system using the same model.</p> <p>Methods</p> <p>Anesthetized pigs were inoculated with 20 ml of a solution containing approximately 10⁸CFU/ml <it>Streptococcus pyogenes </it>strain d68 via a duodenal tube placed through the tracheal tube down to the carina. Two hours later, 5 ml of 1% NCT aqueous solution (test group, n = 15) or 5 ml of 0.9% NaCl (control group, n = 16) was inhaled via the tracheal tube connected to a nebulizer. Inhalation was repeated every hour, four times in total. Lung function and haemodynamics were monitored. Bronchoalveolar lavage samples were removed for determination of colony forming units (CFU), and lung samples for histology.</p> <p>Results</p> <p>Arterial pressure of oxygen (PaO₂) decreased rapidly after instillation of the bacteria in all animals and showed only a slight further decrease at the end of the experiment without a difference between both groups. Pulmonary artery pressure increased to a peak 1-1.5 h after application of the bacteria, decreased in the following hour and remained constant during treatment, again similarly in both groups. Histology demonstrated granulocytic infiltration in the central parts of the lung, while this was absent in the periphery. Expression of TNF-alpha, IL-8, and haemoxygenase-1 in lung biopsies was similar in both groups. CFU counts in bronchoalveolar lavage came to 170 (10; 1388) CFU/ml (median and 25 and 75 percentiles) for the NCT treated pigs, and to 250 (10; 5.5 × 10⁵) CFU/ml for NaCl treated pigs (p = 0.4159).</p> <p>Conclusions</p> <p>Inhaled NCT at a concentration of 1% proved to be very well tolerated also in the infected bronchopulmonary system. This study confirms the tolerability in this delicate body region, which has been proven in healthy pigs previously. Regarding efficacy, no conclusions can be drawn, mainly because of the limited test period of the model.</p

Commissioning Plan of the IFMIF-DONES Accelerator

IFMIF-DONES (International Fusion Materials Irradiation Facility- DEMO-Oriented Neutron Early Source) - a powerful neutron irradiation facility for studies and certification of materials to be used in fusion reactors - is planned as part of the European roadmap to fusion electricity. Its main goal will be to characterize and to qualify materials under irradiation in a neutron field similar to the one faced in a fusion reactor. The intense neutron source is produced by impinging deuterons, from high-power linear deuteron accelerator, on a liquid lithium curtain. The facility has accomplished the preliminary design phase and is currently in its detailed design phase. At the present stage, it is important to have a clear understanding of how the commissioning of the facility will be performed, especially the commissioning of a 5 MW CW deuteron beam, together with the lithium curtain and the beam optimization for the neutron irradiation. In this contribution, the present plans for the hardware and beam commissioning of the accelerator will be given, focusing on the most critical aspects of the tiered approach and on the integration of the procedure with the lithium and tests systems

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