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In JoVE (1)
- A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry
Other Publications (3)
Articles by Anna Willensdorfer in JoVE
A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry
Konstantin D. Bergmeister1,3, Marion Gröger2, Martin Aman1, Anna Willensdorfer1, Krisztina Manzano-Szalai1, Stefan Salminger1, Oskar C. Aszmann1
1CD Laboratory for the Restoration of Extremity Function, Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna, 2Core Facility Imaging, Core Facilities, Medical University Vienna, 3Department of Hand, Plastic and Reconstructive Surgery, Burn Center, BG Trauma Center Ludwigshafen, Plastic and Hand Surgery, University of Heidelberg
Other articles by Anna Willensdorfer on PubMed
Adeno-associated Virus-like Particles As New Carriers for B-cell Vaccines: Testing Immunogenicity and Safety in BALB/c Mice
Viral Immunology. Nov, 2014 | Pubmed ID: 25247267
Adeno-associated viruses (AAVs) are established vectors for gene therapy of different human diseases. AAVs are assembled of 60 capsomers, which can be genetically modified, allowing high-density display of short peptide sequences at their surface. The aim of our study was to evaluate the immunogenicity and safety of an adeno-associated virus-like particle (AAVLP)-displayed B-cell peptide epitope taking ovalbumin (OVA) as a model antigen or allergen from egg, respectively. An OVA-derived B-cell epitope was expressed as fusion protein with the AAV-2 capsid protein of VP3 (AAVLP-OVA) and for control, with the nonrelated peptide TP18 (AAVLP-TP18). Cellular internalization studies revealed an impaired uptake of AAVLP-OVA by mouse BMDC, macrophages, and human HeLa cells. Nevertheless, BALB/c mice immunized subcutaneously with AAVLP-OVA formed similarly high titers of OVA-specific IgG1 compared to mice immunized with the native OVA. The extent of the immune response was independent whether aluminum hydroxide or water in oil emulsion was used as adjuvant. Furthermore, in mice immunized with native OVA, high OVA-specific IgE levels were observed, which permitted OVA-specific mast-cell degranulation in a β-hexosaminidase release assay, whereas immunizations with AAVLP-OVA rendered background IgE levels only. Accordingly, OVA-immunized mice, but not AAVLP-OVA immunized mice, displayed an anaphylactic reaction with a significant drop of body temperature upon intravenous OVA challenge. From this mouse model, we conclude that AAVLPs that display B-cell epitope peptides on their surface are suitable vaccine candidates, especially in the field of allergy.
Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice Via the Skin Independent of Its Enzymatic Activity or TLR4 Activation
The Journal of Investigative Dermatology. Feb, 2015 | Pubmed ID: 25705851
Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin.Journal of Investigative Dermatology advance online publication, 9 April 2015; doi:10.1038/jid.2015.58.
Automated Muscle Fiber Type Population Analysis with ImageJ of Whole Rat Muscles Using Rapid Myosin Heavy Chain Immunohistochemistry
Muscle & Nerve. Aug, 2016 | Pubmed ID: 26788932
Skeletal muscle consists of different fiber types which adapt to exercise, aging, disease, or trauma. Here we present a protocol for fast staining, automatic acquisition, and quantification of fiber populations with ImageJ.