23 research outputs found
Analyse der Funktion der NS-Proteine von klinischen HRSV-Isolaten
HRSV ist eine hĂ€ufige und weltweit verbreitete Ursache von Infektionen des Respirationstraktes. Es fĂŒhrt zu einer entzĂŒndlichen Erkrankung der respiratorischen SchleimhĂ€ute mit Mukosaödem, Hypersekretion und Bronchospasmus. Die Ăbertragung des viralen Erregers erfolgt durch Tröpfcheninfektion oder Kontakt mit kontaminierten GegenstĂ€nden.
HRSV-Infektionen zeigen die höchste Inzidenz bei SÀuglingen, vor allem in den ersten zwei bis sechs Lebensmonaten. Bei 25% bis 40% dieser SÀuglinge nimmt die Erkrankung einen schweren Verlauf mit Befall des unteren Respirationstraktes in Form einer HRSV-Bronchiolitis oder -Pneumonie. Bei 0,5% bis 2,0% ist eine stationÀre Behandlung im Krankenhaus erforderlich. Die Inzidenz nimmt wegen des zunehmend effektiveren Immunsystems mit dem Alter ab. Erwachsene und Àltere Kinder zeigen meist keine Symptome bzw. Symptome einer leichten ErkÀltung. Reinfektionen im Laufe des Lebens sind hÀufig.
Eine effektive kausale Therapie bei HRSV-Infektionen steht derzeit nicht zur VerfĂŒgung. Bei Patienten mit leichtem Krankheitsverlauf ist keine spezielle Behandlung erforderlich, therapiert wird symptomatisch. Aktuell ist keine spezifische PrĂ€vention in Form einer aktiven Impfung oder als effektive antivirale Therapie etabliert. Angesichts der hohen Inzidenz von HRSV-Infektionen und -Reinfektionen sowie der enormen gesundheitlichen und wirtschaftlichen Auswirkungen ist ein effektiver Impfstoff gegen HRSV als Forschungsziel vorrangig.
Das Genom von HRSV, das zur Ordnung der Mononegavirales gehört, besteht aus einem negativ-orientierten RNA-Einzelstrang mit einer LĂ€nge von 15 222 Nukleotiden (beim A2-Stamm) und kodiert fĂŒr zehn subgenomische mRNAs in der Reihenfolge 3â-leader, NS1, NS2, N, P, M, SH, G, F, M2(1+2), L, trailer-5â, die zur Expression von elf viralen Proteinen fĂŒhren: fĂŒnf RNP-assoziierte Proteine, das sind das Nukleoprotein N, das Phosphoprotein P, die groĂe katalytische Untereinheit L der RNA-Polymerase und der Transkriptionselongationsfaktor M2-1 sowie das nicht essentielle M2-2-Protein; vier HĂŒllproteine, dazu zĂ€hlen das nicht-glykosylierte Matrixprotein M und drei OberflĂ€chenproteine, im Einzelnen das Fusionsprotein F, das Anheftungsprotein G und das kleine hydrophobe Protein SH; zwei Nicht-Strukturproteine NS1 und NS2.
NS1 und NS2 zeichnen die Pneumoviren vor allen anderen Viren der Ordnung der Mononegavirales aus. Beide NS-Proteine sind im Virion nur in Spuren nachweisbar, wĂ€hrend sie in infizierten Zellen akkumulieren. Die beiden fĂŒr die Proteine NS1 und NS2 kodierenden, nichtĂŒberlappenden Gene liegen am 3â-Ende des Genoms direkt im Anschluss an die leader-Region. NS1 und NS2 stimmen in den vier carboxyterminalen AminosĂ€uren ĂŒberein, ansonsten weisen sie keine SequenzĂ€hnlichkeiten auf. Das NS1-Gen hat eine LĂ€nge von 552 nt und kodiert fĂŒr ein leicht saures Protein von 139 AS und 15,7 kD. Das NS2-Gen ist 503 nt lang und kodiert fĂŒr ein basisches Protein von 124 AS und 14,7 kD.
Die fĂŒr die Ordnung der Mononegavirales charakteristische progressive Attenuation der Transkription sowie die Genlokalisation von NS1 und NS2 am 3â-Ende lassen auf die höchste Transkriptionsrate fĂŒr NS1- und NS2-mRNA unter den zehn HSRV-mRNA schlieĂen, was auf eine bedeutende Rolle der NS1- und NS2-Proteine in infizierten Zellen hindeutet. NS1 und NS2 antagonisieren im Zusammenwirken die durch alpha-IFN und beta-IFN induzierte antivirale Antwort des Wirtsorganismus. HierfĂŒr ist eine Koexpression beider NS-Proteine unbedingt erforderlich, ein NS-Protein allein zeigt keine derartige AktivitĂ€t. Der Mechanismus, mit dem HRSV die IFN-Antwort des Wirtsorganismus umgeht, ist unklar.
In dieser Arbeit wurde die Funktion der NS-Proteine von klinischen HRSV-Isolaten aus fĂŒnf bis fĂŒnfzehn Monate alten Kindern untersucht. Durch die Anzucht der klinischen HRSV-Isolate in HEp-2-Zellkultur unter identischen Bedingungen wurden zunĂ€chst patientenabhĂ€ngige Faktoren ausgeschaltet und damit die Grundlage fĂŒr die Vergleichbarkeit der Wachstumseigenschaften der Isolate geschaffen. In den daraufhin erstellten Wachstumskurven konnten deutlich voneinander abweichende Wachstumverhalten der Isolate aufgezeigt werden. Der Befund, dass 3/4 der Bronchiolitis hervorrufenden HRS-Viren hohe infektiöse Titer (>106 infektiöse Viruspartikel/ ml an Tag 3) erreichten, wĂ€hrend dies nur bei 1/3 der Bronchitis verursachenden Viren zu beobachten war, könnte auf eine Korrelation zwischen Wachstum in vitro und PathogenitĂ€t in vivo hindeuten. Um dies zu belegen, mĂŒsste eine gröĂere Zahl von klinischen Isolaten analysiert werden.
Die beiden Nicht-Strukturproteine versetzen HRSV in die Lage, die antivirale IFN-Antwort der Wirtszelle zu umgehen. Durch Behandlung von Virus-infizierten Zellkulturen mit IFN lieĂ sich nachweisen, dass alle klinischen HRSV-Isolate die Eigenschaft der IFN-Resistenz gleichermaĂen besitzen und erst durch unphysiologisch hohe IFN Dosen eine wesentliche Inhibierung der Virusreplikation erreicht werden kann. Die in gleicher Weise ausgeprĂ€gte α-IFN-Resistenz bei den in Virulenz und Wachstumsgeschwindigkeit unterschiedlichen Viren deutete bereits darauf hin, dass diese Resistenz essentiell fĂŒr alle klinischen RSV-Isolate ist, und dass zusĂ€tzliche Faktoren fĂŒr das MaĂ der AggressivitĂ€t der Erreger verantwortlich sind.
Mittels Nukleotid- und AminosĂ€uresequenzanalysen von NS1 und NS2 konnte dies weitgehend bestĂ€tigt werden. Anhand von RNA aus den HRSV-Isolaten wurde mit Hilfe des Enzyms Reverse Transkriptase cDNA von NS1 und NS2 synthetisiert, die nach dem Prinzip der PCR in vitro amplifiziert wurde. In anschlieĂenden Klonierungsarbeiten wurden aus dem Vektor pBluescript II SK (â) und NS1-DNA bzw. NS1+NS2-DNA als Insert Plasmide konstruiert, in denen die Gensequenzen von NS1 und NS2 ermittelt und rechnergestĂŒtzt in die entsprechenden AminosĂ€uresequenzen translatiert wurden.
Die Analyse der NS-Sequenzen zeigte eine ĂŒberraschend hohe Konservierung. Die Isolate waren einschlieĂlich des Long-Stamms diesbezĂŒglich untereinander sehr Ă€hnlich. Diese Beobachtung stimmt mit der IFN-Resistenz ĂŒberein und zeigt die Bedeutung der NS-Proteine. Die Ergebnisse dieser Arbeit deuten darauf hin, dass Abweichungen in den Sequenzen der ĂŒbrigen Gene sowie patientenbezogene Faktoren wie Abwehrlage und anatomische Beschaffenheit des Respirationstraktes als Grund fĂŒr die Unterschiede im Schweregrad der HRSV-Infektion eine Rolle spielen. Angesichts der stabilen Koexpression beider Nicht-Strukturproteine und des dadurch bedingten effektiven IFN-Escape sichern die Gene NS1 und NS2 die ĂberlebensfĂ€higkeit von HRSV in vivo und stellen ebenso geeignete wie interessante Angriffspunkte in der Entwicklung eines attenuierten Lebendimpfstoffs dar
Prostaglandin E Positively Modulates Endothelial Progenitor Cell Homeostasis: An Advanced Treatment Modality for Autologous Cell Therapy
Aims: The mobilization of endothelial progenitor cells (EPC) and their functioning in postnatal neovascularization are tightly regulated. To identify new modulators of EPC homeostasis, we screened biologically active prostaglandin E compounds for their effects on EPC production, trafficking and function. Methods and Results: We found that EPC are a rich source for prostaglandin E 2 (PGE 2), stimulating their number and function in an auto- and paracrine manner. In vivo blockade of PGE 2 production by selective cyclooxygenase-2 inhibition virtually abrogated ischemia-induced EPC mobilization demonstrating its crucial role in EPC homeostasis following tissue ischemia. Conversely, ex vivo treatment of isolated EPC with the clinically approved PGE 1 analogue alprostadil enhanced EPC number and function. These effects were mediated by increased expression of the chemokine receptor CXCR4 and were dependent on nitric oxide synthase activity. Most importantly, ex vivo PGE 1 pretreatment of isolated EPC significantly enhanced their neovascularization capacity in a murine model of hind limb ischemia as assessed by laser Doppler analysis, exercise stress test and immunohistochemistry. Conclusions: The conserved role for PGE in the regulation of EPC homeostasis suggests that ex vivo modulation of the prostaglandin pathway in isolated progenitor cells may represent a novel and safe strategy to facilitate cell-based therapies. Copyright (C) 2009 S. Karger AG, Base
Preoperative Imaging for Thoracic Branch of Supraclavicular Artery Flap A Comparative Study of Contrast-Enhanced Ultrasound With Three-Dimensional Reconstruction and Color Duplex Ultrasound
Objective: The thoracic branch of supraclavicular artery (TBSA) flap has been widely used to reconstruct face and neck defects. However, the branches of the supraclavicular artery (SCA) exhibit considerable anatomical variations. The aim of this study was to evaluate and compare the role of contrast-enhanced ultrasound (CEUS) with 3-dimensional (3D) reconstruction and regular color duplex ultrasonography (CDUS) in the preoperative assessment of TBSA flap. Methods: From May 2009 to October 2013, 20 patients (involving 26 flaps) receiving anterior chest flaps for lower face and neck reconstruction underwent both CDUS and CEUS with 3D reconstruction preoperatively for detecting the TBSAs. The number of TBSAs, their caliber, peak systolic velocity (PSV), and course were recorded. In case of an absent TBSA, the second and third perforators of the internal mammary artery were detected. The preoperative imaging data were compared with the intraoperative findings to evaluate the value of CDUS and CEUS with 3D reconstruction for planning and performing the TBSA flaps. All patients were followed up for more than 1 year. Results: A total of 37 TBSAs in 16 flaps were found by CDUS with a mean caliber of 0.6 +/- 0.1 mm and a mean PSV of 13.1 +/- 1.6 cm/s, whereas 48 TBSAs in 20 flaps were found by CEUS with a mean caliber of 0.8 +/- 0.2 mm and a mean PSVof 12.5 +/- 2.1 cm/sec. In 18 flaps with TBSA PSV above 10 cm/s, pedicled TBSA flaps were performed, whereas pedicled or free internal mammary artery flaps were chosen as alternative for the remaining 8 flaps. All 48 TBSAs were found intraoperatively and their origin from the SCA confirmed, indicating specificity and positive predictive value of both CDUS and CEUS were 100% in localizing TBSA preoperatively, whereas sensitivity and negative predictive value of CEUS were higher than using CDUS. Conclusions: The branches of SCA have marked anatomical variations. CEUS with 3D reconstruction has advantages over CDUS for the preoperative assessment of the donor-site vascular supply of TBSA flaps
Autologous temporomandibular joint reconstruction independent of exogenous additives: a proof-of-concept study for guided self-generation
Joint defects are complex and difficult to reconstruct. By exploiting the body's own regenerative capacity, we aimed to individually generate anatomically precise neo-tissue constructs for autologous joint reconstruction without using any exogenous additives. In a goat model, CT scans of the mandibular condyle including articular surface and a large portion of the ascending ramus were processed using computer-aided design and manufacturing. A corresponding hydroxylapatite negative mold was printed in 3D and temporarily embedded into the transition zone of costal periosteum and perichondrium. A demineralized bone matrix scaffold implanted on the contralateral side served as control. Neo-tissue constructs obtained by guided self-generation exhibited accurate configuration, robust vascularization, biomechanical stability, and function. After autologous replacement surgery, the constructs showed stable results with similar anatomical, histological, and functional findings compared to native controls. Further studies are required to assess long-term outcome and possible extensions to other further applications. The absence of exogenous cells, growth factors, and scaffolds may facilitate clinical translation of this approach
99mTc-MAG3 scintigraphy for the longitudinal follow-up of kidney function in a mouse model of renal ischemia-reperfusion injury
Nephroprotective effects of enalapril after [177Lu]-DOTATATE therapy using serial renal scintigraphies in a murine model of radiation-induced nephropathy
Background: Radiation-induced nephropathy is still dose limiting in radionuclide therapy of neuroendocrine tumors. We investigated the nephroprotective potential of the angiotensine converting enzyme inhibiting drug enalpril after [177Lu]-DOTATATE therapy in a murine model of radiation-induced nephropathy by renal scintigraphy. At first, the appropriate therapy activity to induce nephropathy was identified. Baseline scintigraphy (n = 12) entailed 12-min dynamic acquisitions after injection of 25 MBq [99mTc]-MAG3, which was followed by radionuclide therapy at four escalating activities of [177Lu]-DOTATATE: group (Gp) 1: 10 MBq;Gp 2: 20 MBq;Gp 3: 40 MBq;Gp 4: 65 MBq. Follow-up [99mTc]-MAG3 scintigraphy was carried out at days 9, 23, 44, and 65. The treatment activity for the intervention arm was selected on the basis of histological examination and declining renal function. In the second part, daily administration by gavage of 10 mg/kg/d enalapril or water (control group) was initiated on the day of radionuclide therapy. Follow-up scintigraphy was carried out at days 9, 23, 44, 65, and 86. We also created a non-therapy control group to detect therapy-independent changes of renal function over time. For all scintigraphies, mean renogram curves were analyzed and the "fractional uptake rate" (FUR;%I.D./min +/- SEM) of the tracer by the kidneys was calculated as an index of renal clearance. Results: At day 65 of follow-up, no significant change in the FUR relative to baseline (11.0 +/- 0.3) was evident in radionuclide therapy groups 1 (11.2 +/- 0.5) and 2 (10.1 +/- 0.6), but FUR was significantly reduced in groups 3 (8.93 +/- 0.6, p < 0.05) and 4 (6.0 +/- 0.8, p < 0.01);we chose 40 MBq [177Lu]-DOTATATE (Gp 3) for the intervention study. Here, at the last day of follow-up (day 86), FUR was unaltered in enalapril-treated mice (11.8 +/- 0.5) relative to the baseline group (12.4 +/- 0.3) and non-therapy group (11.9 +/- 0.8), whereas FUR in the control group had undergone a significant decline (9.3 +/- 0.5;p < 0.01). Histological examination revealed prevention of kidney damage by enalapril treatment. Conclusions: Treatment with enalapril is effective for nephroprotection during radionuclide therapy with [177Lu]-DOTATATE in mice. Although these results are only limitedly transferable to human studies, enalapril might serve as a promising drug in the mitigation of nephropathy following treatment with [177Lu]-DOTATATE
Cadmium Telluride Quantum Dots as a Fluorescence Marker for Adipose Tissue Grafts
Plastic and reconstructive surgeons increasingly apply adipose tissue grafting in a clinical setting, although the anticipation of graft survival is insecure. There are only few tools for tracking transplanted fat grafts in vivo. Murine adipose tissue clusters were incubated with negatively charged, mercaptoproprionic acid-coated cadmium telluride quantumdots (QDs) emitting in the dark red or near infrared. The intracellular localization of QDs was studied by confocal laser scanning microscopy. As a result, the adipose tissue clusters showed a proportional increase in fluorescence with increasing concentrations (1, 10, 16, 30, 50 nM) of cadmium telluride QDs. Laser scanning microscopy demonstrated a membrane bound localization of QDs. Vacuoles and cell nuclei of adipocytes were spared by QDs. We conclude that QDs were for the first time proven intracellular in adult adipocytes and demonstrate a strong fluorescence signal. Therefore, they may play an essential role for in vivo tracking of fat grafts
Bioengineered human tissue regeneration and repair using endogenous stem cells
We describe a general approach to produce bone and cartilaginous structures utilizing the self-regenerative capacity of the intercostal rib space to treat a deformed metacarpophalangeal joint and microtia. Anatomically precise 3D molds were positioned on the perichondro-periosteal or perichondral flap of the intercostal rib without any other exogenous elements. We find anatomically precise metacarpal head and auricle constructs within the implanted molds after 6 months. The regenerated metacarpal head was used successfully to surgically repair the deformed metacarpophalangeal joint. Auricle reconstructive surgery in five unilateral microtia patients yielded good aesthetic and functional results. Long-term follow-up revealed the auricle constructs were safe and stable. Single-cell RNA sequencing analysis reveal early infiltration of a cell population consistent with mesenchymal stem cells, followed by IL-8-stimulated differentiation into chondrocytes. Our results demonstrate the repair and regeneration of tissues using only endogenous factors and a viable treatment strategy for bone and tissue structural defects.</p
X-Ray Phase-Contrast Tomography of Renal Ischemia-Reperfusion Damage
Purpose: The aim of the study was to investigate microstructural changes occurring in unilateral renal ischemia-reperfusion injury in a murine animal model using synchrotron radiation. Material and Methods: The effects of renal ischemia-reperfusion were investigated in a murine animal model of unilateral ischemia. Kidney samples were harvested on day 18. Grating-Based Phase-Contrast Imaging (GB-PCI) of the paraffin-embedded kidney samples was performed at a Synchrotron Radiation Facility (beam energy of 19 keV). To obtain phase information, a two-grating Talbot interferometer was used applying the phase stepping technique. The imaging system provided an effective pixel size of 7.5 mu m. The resulting attenuation and differential phase projections were tomographically reconstructed using filtered back-projection. Semi-automated segmentation and volumetry and correlation to histopathology were performed. Results: GB-PCI provided good discrimination of the cortex, outer and inner medulla in non-ischemic control kidneys. Post-ischemic kidneys showed a reduced compartmental differentiation, particularly of the outer stripe of the outer medulla, which could not be differentiated from the inner stripe. Compared to the contralateral kidney, after ischemia a volume loss was detected, while the inner medulla mainly retained its volume (ratio 0.94). Post-ischemic kidneys exhibited severe tissue damage as evidenced by tubular atrophy and dilatation, moderate inflammatory infiltration, loss of brush borders and tubular protein cylinders. Conclusion: In conclusion GB-PCI with synchrotron radiation allows for non-destructive microstructural assessment of parenchymal kidney disease and vessel architecture. If translation to lab-based approaches generates sufficient density resolution, and with a time-optimized image analysis protocol, GB-PCI may ultimately serve as a non-invasive, non-enhanced alternative for imaging of pathological changes of the kidney
In Vivo Monitoring of Parathyroid Hormone Treatment after Myocardial Infarction in Mice with [68Ga]Annexin A5 and [18F]Fluorodeoxyglucose Positron Emission Tomography
[68Ga]Annexin A5 positron emission tomography (PET) reveals the externalization of phosphatidylserine as a surrogate marker for apoptosis. We tested this technique for therapy monitoring in a murine model of myocardial infarction (MI) including parathyroid hormone (PTH) treatment. MI was induced in mice, and they were assigned to the saline or the PTH group. On day 2, they received [68Ga]annexin A5 PET or histofluorescence TUNEL staining. Mice had 2-deoxy-2-[18F]fluoro-D-glucose (FDG)-PET examinations on days 6 and 30 for calculation of the left ventricular ejection fraction and infarct area. [68Ga]Annexin A5 uptake was 7.4 ± 1.3 %ID/g within the infarction for the controls and 4.5 ± 1.9 %ID/g for the PTH group (p = .013). TUNEL staining revealed significantly more apoptotic cells in the infarct area on day 2 in the controls (64 ± 9%) compared to the treatment group (52 ± 4%; p = .045). FDG-PET revealed a significant decrease in infarct size in the treatment group and an increase in the controls. Examinations of left ventricular ejection fraction on days 6 and 30 did not reveal treatment effects. [68Ga]Annexin A5 PET can detect the effects of PTH treatment as a marker of apoptosis 2 days after MI; ex vivo examination confirmed significant rescue of myocardiocytes. FDG-PET showed a small but significant reduction in infarct size but no functional improvement