92 research outputs found
Proteomic and Postproteomic Characterization of Keratan Sulfate-Glycanated Isoforms of Thyroglobulin and Transferrin Uniquely Elaborated by Papillary Thyroid Carcinomas.
No full textPrevious studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients
Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine.
No full textRecombinant poxviruses encoding tumor-associated antigens (TAA) are attractive as candidate cancer vaccines. Their effectiveness, however, will depend upon expression of the TAA in appropriate antigen-presenting cells. We have used a murine model in which the TAA is beta-galactosidase (beta-gal) and a panel of recombinant vaccinia viruses (rVV) in which beta-gal was expressed under early or late promoters at levels that varied over 500-fold during productive infections in tissue culture cells. Remarkably, only those rVV employing early promoters were capable of prolonging the survival of mice bearing established tumors expressing the model TAA. Late promoters were ineffective regardless of their determined promoter strength. The best results were obtained when beta-gal was regulated by a strong early promoter coupled to a strong late promoter. When a variety of cell types were infected with the panel of viruses in vitro, dendritic cells were found to express beta-gal only under the control of the early promoters even though late promoters were intrinsically more active in other cell types. Furthermore, in a functional assay, dendritic cells infected in vitro with rVV encoding beta-gal regulated by an early promoter activated beta-gal-specific cytotoxic T lymphocytes, whereas similar rVV with a late promoter-regulated gene did not. These data indicate that promoter strength per se is not the most critical quality of a recombinant poxvirus-based tumor vaccine and that the use of promoters capable of driving the production of TAA in "professional" antigen presenting cells may be crucial
Functionally fused antibodies--a novel adjuvant fusion system.
No full textInternational audienceAntibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radioactivity- or effector-domain delivery. There is now a growing interest in using anti-idiotypic antibodies or other antigen mimics to induce potent immune responses against antigen structures in question. We have earlier reported on the functional rescue of antibodies that are active when fused to the phage, but inactive as soluble protein [Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F. and Kristensen, P. (2002) Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem. Biophys. Res. Commun. 298, 566-73.]. The rescue was accomplished by maintaining the fusion between the antibody fragment and portions of the filamentous bacteriophage coat protein 3, as present in the original antibody-displaying phage. In the present study, we have applied this system in an attempt to improve immunogenicity of anti-idiotypic antibodies isolated by phage display. Here we demonstrate that by preserving linkage between phage antibody and the N-terminal domain of phage coat protein 3, we induce multimerization of the antibody fragments, and improve their immunogenicity. This immunization approach allows induction of anti-idiotypic antibodies in mice, and facilitates the use of antibodies that are non-functional as non-fused soluble protein
Vakzine gegen konformationsabhaengige Antigene sowie gegen Antigene, die keine oder nicht ausschliesslich Proteine oder Peptide sind
No full textA phase I study of PankoMab-GEX, a humanised glyco-optimised monoclonal antibody to a novel tumour-specific MUC1 glycopeptide epitope in patients with advanced carcinomas
No full textAbstractBackgroundA phase I open-label dose-escalation study was conducted to define the safety, tolerability, and pharmacokinetics (PK) of PankoMab-GEX, a glyco-optimised humanised IgG1, with high affinity to a novel tumour-specific glycopeptide epitope of MUC1 (TA-MUC1) with excellent preclinical anti-tumour activity.Patients and methodsSeventy-four patients with advanced TA-MUC1-positive carcinomas received PankoMab-GEX intravenously every 3 (Q3W), 2 (Q2W), or 1 (QW) week in doses of 1–2200 mg in a three-plus-three dose-escalation design until disease progression (NCT01222624).ResultsNo maximum tolerated dose was reached. Adverse events were mainly mild-to-moderate infusion-related reactions (IRRs) by the first infusion in 45% of patients. Only one dose-limiting toxicity, a grade III IRR, was observed. PankoMab-GEX exhibited linear PK over all doses. Mean terminal half-life was 189 ± 66 h (Q3W), without dose dependency. A target trough level ≥50 μg/mL was reached after one infusion with doses ≥1700 mg Q3W in 80% of patients. Clinical benefit in 60 evaluable patients included one complete response in a patient with ovarian cancer treated 483 d and confirmed disease stabilisation in 19 patients lasting a median (range) of 23 (10–109) weeks. All but two of the patients with clinical benefit had received a compounded total dose ≥700 mg over a 3-week period, including 8 of 12 (67%) patients with ovarian cancer.ConclusionPankoMab-GEX is safe, well tolerated, and showed promising anti-tumour activity in advanced disease. A phase IIb study is ongoing evaluating the efficacy of PankoMab-GEX as a maintenance therapy in advanced ovarian cancer
What makes MUC1 a tumor antigen?
No full textThe epithelial mucin 1 (MUC1) is an accepted serum tumor marker and cellular tumor antigen. We discuss recent views on the difference(s) between normal and tumor MUC1, and its implication for the development of cancer vaccines and antibody therapies, with special emphasis on the role of glycosylation
Intermodal urban mobility: users, uses, and use cases
No full textCities are growing nowadays and so is their citizens’ demand for mobility. On a global scale, motorized individual traffic is hardly capable of meeting this need due to its ownership costs and due to the lack of an accordingly large infrastructure. Besides, motorized individual traffic is responsible for the majority of today's traffic burdens, such as air pollution, traffic jams, noise, and accidents. It is thereby assumed that only a combination of soft transport modes and public and private modes of motorized transport with different capacities, time schedules, and operation times can achieve what is called “sustainable cities”. Besides the need for coordinated transport services, their cooperation must be assured to obtain seamless interchanges and consequently undisturbed, fast, and reliable travelling. Such a use of different transport modes within a single journey is called “intermodality” and is a work topic fostered in a national, European, and world-wide context. This report shows the initial results of the project “UrMo” (“Urban Mobility”) that is being performed at three institutes of the German Aerospace Center (DLR). The report gives an introduction to the topic by evaluating how socio-demographics and space structure influence intermodal behavior. In addition to this, the subsequent project steps are outlined
Supplementary Materials: Laminin β4 is a constituent of the cutaneous basement membrane zone and additional autoantigen of anti-p200 pemphigoid_JAAD
No full textSupplemental information for the manuscript published in the Journal of the American Academy of Dermatology (JAAD): "Laminin β4 is a constituent of the cutaneous basement membrane zone and additional autoantigen of anti-p200 pemphigoid"THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV
Isolierung und Charakterisierung equiner mesenchymaler Stammzellen für einen möglichen Einsatz im Tissue Engineering
No full textOsteoarthritiden und Sehnenverletzungen sind häufige Gründe für eine erhöhte Morbidität und verminderte Leistungsfähigkeit bei Pferden oder Kleintieren.Aufgrund des langen Zeitraums, die das Gewebe für die Eigenregeneration benötigt, ist eine Auswahl an Therapiemöglichkeiten mit niedrigerer Regenerationszeit und somit mit schnellerer Belastbarkeit der Strukturen unabdingbar.
Zu den mittlerweile gebräuchlichen Therapieansätzen, die sich von der konservativen Therapie bis hin zu Ersatzmaterialien belaufen, wäre es eine interessante und vorteilhafte Alternative, körpereigene Zellen aufzuarbeiten und diese in Form eines Transplantates in den Defekt einzubringen. Zu den möglichen körpereigenen Zellen gehören mesenchymale Stammzellen (MSC) aus dem Knochenmark.
Das Ziel dieser Studie war, die aus dem Pferd gewonnenen und isolierten equinen Mesenchymalen Stammzellen (eMSC) näher zu charakterisieren. Um die Potenz der MSC´s für einen therapeutischen Einsatz zu untersuchen, wurden morphologische Untersuchungen mit diesen isolierten Stammzellen ex vivo durchgeführt und anschließend beurteilt.Nach Trennung der Knochenmarkflüssigkeit in ihre Bestandteile, Zellen, Blut und Restbestandteile, konnten die Zellen in einer Zellkultur aufgereinigt und expandiert werden. Im Zuge der Expansion wurden verschiedene Seren und Medien, zu Anreicherung, im Hinblick auf Proliferation getestet. Zur Beurteilung des Proliferationsverhaltens wurde ein Colony Forming Unit Assay (CFU-Assay) durchgeführt. Anhand diesem sollte gezeigt werden, wie sich das Koloniewachstum in Abhängigkeit der Zellzahl entwickelt.
Um das Proliferationspotential, sowohl im Moment der Zellteilung, als auch in einem Zeitraum von 24 Stunden darzustellen, wurden die Zellen mit Ki 67 zum Zeitpunkt der Fixierung und mit BrDU über 24 Stunden konfrontiert.Nach erfolgreicher Isolation und Expansion, wurden die Zellen in flüssigem Stickstoff kryokonserviert. Diesen Vorgang überstanden die Zellen ohne Alterationen aufzuweisen. Selbst nach einer Kryokonservierung von 1,5 Jahren stellten sich die Zellen unverändert dar.
Zur Überprüfung der Vitalität der Zellen wurden die eMSC bei unterschiedlichen Bedingungen, zum einen auf Eis und zum anderen bei 4° C, gelagert. Bei diesem Versuchsaufbau sollte geklärt werden, ob es eine Möglichkeit ist, die Zellen gekühlt zur Tierarztpraxis/-klinik zu senden. Dabei stellte sich heraus, daß eine Lagerung der Zellen unter beiden Bedingungen für zwei Tage mit nur geringem Zellverlust möglich ist.
Da bekannt ist, daß im Knochenmark eine niedrige Sauerstoffspannung herrscht, wurden die Kulturbedingungen dahingehend variiert, daß der Sauerstoffgehalt von 21% auf 3% in einer Hypoxiekammer herabgesetzt wurde. Die dadurch gesteigerte Proliferationsfähigkeit der eMSC konnte sowohl mit Ki 67 als auch mit BrDU visualisiert werden.
Auf den Stammzellmarker THY 1 / CD 90, der in anderen morpholgischen Studien routinemäßig eingesetzt wird, reagierten die Zellen positiv. Ein weiterer Stammzellmarker, Oct 4, konnte mittels RT-PCR nachgewiesen werden.Im Rahmen einer immunhistochemischen Charakterisierung konnte der Nachweis erbracht werden, daß die Zellen in ihrer Extrazellularmatrix (EZM) Fibronektin, Perlecan, Aggrecan und Kollagen IV exprimieren. Es konnte weiterhin gezeigt werden, daß die Interaktion mit der Extrazellularmatrix über das transmembranäre Protein ß1 Integrin erfolgt.Weitere Untersuchungen hinsichtlich der Pluripotenz der eMSC zeigten die Differenzierbarkeit der Zellen in die verschiedenen Richtungen wie Fett, Knorpel und Knochen, sowohl vor wie nach der Kryokonservierung.
Untersuchungen zum Migrationsverhalten zeigten, daß eine deutliche Zellmotilität vorhanden war. Dies wurde mit Hilfe einer Phalloidin-Färbung visualisiert. Die Migration der Zellen konnte durch Zugabe von Interleukin 6 positiv beeinflußt werden.
Die Transduktion der Zellen, mit einem adenoviralen Vektor, mit der Sequenz für das grün fluoreszierende Protein (GFP), zur Markierung der Zellen, konnte ebenfalls erfolgreich durchgeführt werden. Diese Markierung soll bei einer späteren Detektion der Zellen im Zielgewebe helfen.
Im Hinblick auf einen therapeutischen Einsatz im tissue engineering ist die Kultivierung auf verschiedenen Trägermaterialien untersucht worden. Auf allen getesteten Trägermaterialien (Kollagen-Gel, Microcarrier und Fibrinkügelchen) war eine Kultivierung für mindestens drei Wochen möglich, ohne daß die Vitalität der Zelle abnimmt. Desweiteren wurden die Zellen in einer Rotationskultur auf Microcarriern kultiviert, einer Versuchsanordnung, die die Schwerelosigkeit imitiert und eine gleichmäßige Versorgung der Zellen garantiert.
Der therapeutischer Einsatz von entnommenen, isolierten und expandierten Zellen wurde bereits erfolgreich durchgeführt. Mit Hilfe der Zellinjektion konnte ultrasonographisch eine verbesserte Durchbauung eines Sehnendefektes nachgewiesen werden. Die Pferde, bei denen eine solche Zellinjektion stattgefunden hatte, zeigten eine deutliche Besserung, bis hin zum Verschwinden, der klinischen Symptome. Der klinische Einsatz von mit Zellen beschickten Trägermaterialen soll noch in weiterführenden Studien getestet werden.Osteoarthritis and tendon injuries are often the reason for a high morbidity rate and a reduced ability in horses and small animals.Due to the long regeneration phase that is required by the tissue for self-renewal, a range of critical options with a shorter regeneration phase enabling a swifter recovery is indispensable.
In addition to conventional therapy practice ranging from the more classical methods as well as the use of substitute materials, an interesting alternative would be to process autologous cells with the aim to transplant them into the injured tissues. For this purpose mesenchymal stem cells (MSC) isolated from the bone marrow stroma would be an autologous cell source.The purpose of this paper is to further characterize mesenchymal stem cells (eMSC) from horses.
In order to examine the potency of MSCs for therapeutic purposes, morphologic examinations with isolated stem cells ex vivo were carried out and evaluated.
The cells were isolated from the bone marrow fluid with the aim of cultivation and expansion in the cell culture laboratory.During the expansion period different serumbatches and methods for accumulation were tested with regards to their impact on proliferation.
To evaluate the characteristics of proliferation a colony forming unit assay (CFU assay) was performed.To show the characteristics of proliferation in both, the moment of cell division and the period of 24 hours, proliferation marker Ki 67 was applied at the time of fixation as was Bromodeoxyuridine (BrDU) over the period of 24 hours.
After a successful isolation und expansion, the cells were cryoconserved in liquid nitrogen. The cells survived the treatment without alteration. Even after cryoconservation for up to 1.5 years, the cells remained unaltered.To examine the vitality of the cells the eMSC were stored under different conditions both, on ice and at 4° C. This experimental setup was to approve the possibility to transport refrigerated cells to the veterinarians practice / clinic . Both methods approved that storage for two days is possible with only a limited loss of cells.
Since the oxygen content in the bone marrow is lower than in the sorrounding tissue, the cell culture conditions were adjusted by reducing the oxygen concentration accordingly from 21% to only 3%. The effect was achieved by reducing the oxygen content in a hypoxy chamber.
The visualization of the proliferation potential was achieved using the BrDU assay or immunocytochemically with the Ki 67 proliferation marker.The eMSCs were immunopositive for the stem cell marker CD 90 furthermore the stem cell marker Oct-4 could be detected using RT-PCR.Stem cell characteristics were investigated immunocytochemically by detecting the expression of Fibronectin, Perlecan, Aggrecan and Collagen IV as a part of its extracellular matrix (ECM).Further on, it could be shown that the interaction of cells with its ECM is regulated via the transmembrane protein ß1-Integrin. Test results about the pluripotency of cells showed the differentiation between fat, cartilage and bone was successful before and even after cyroconservation.
Studies on the migratory behavior proved that cells can be stimulated to perform a cell motility. The visualization of the migration was achieved using the phalloidin staining. Furthermore, the migration of the cells was positively influenced by the addition of Interleukin 6.
The transduction of the cells with an adenoviral vector, with the GFP sequence to mark the cells, was also carried out successfully. The transduction was carried out in order to support the future detection of cells in the tissue of the recipient.
Cultivation on different carrier materials was intended with regard to a therapeutic application in tissue engineering. Cultivation of cells on all carrier materials tested (collagen gel, collagen micro carriers and the fibrin sealant Tissucol) was possible and the viability of cells could be demonstrated for up to 3 weeks. Furthermore, the cells were cultivated in a rotary cell culture system on microcarriers, a test arrangement which imitates microgravity and ensures an optimal supply of trophic substances.The therapeutic application of extracted, isolated and expandated cells had been carried out successfully.The application of this treatment made a recovery of the tendon damage possible and outlined ultrasonographically.The tests on horses that had received an injection of cells over nine month observation period showed a definite improvement and in some cases even a disappearance of the clinical symptoms.The clinical application of carrier materials has yet to be tested
A novel series of anti-human glycophorin A (CD235a) antibodies defining five extra- and intracellular epitopes
No full textGlycophorin A (GPA, CD235a) is a major membrane glycoprotein and marker of cells of the erythroid lineage. It is also the target of Plasmodium falciparum and of influenza virus. We describe a novel series of 10 antibodies towards GPA, recognizing four extra- and intracellular peptide epitopes of this molecule (defined by epitope mapping) and one mixed peptide/carbohydrate epitope. All antibodies bind better to the desialylated than to the fully sialylated molecule, including those specific for the intracellular epitope. For some of the antibodies (representing all five epitopes) functional binding constants were determined by Surface Plasmon Resonance. The new panel complements the already known anti-glycophorin antibodies and offers several potential applications for, e.g., differential diagnosis of erythroleukemias, lineage analyses of erythroid cells, isolation of senescent erythrocytes, or a highly sensitive neuraminidase assay
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