ABSTRACT
Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases.
Subject(s)
Extracellular Matrix/metabolism , Granzymes/metabolism , Skin/metabolism , Skin/radiation effects , Ultraviolet Rays , Animals , Cell Count , Collagen/metabolism , Decorin/metabolism , Dermis/pathology , Dermis/radiation effects , Dose-Response Relationship, Radiation , Female , Fibroblasts/enzymology , Fibroblasts/radiation effects , Fibronectins/metabolism , Granzymes/deficiency , Mast Cells/enzymology , Mast Cells/radiation effects , Matrix Metalloproteinase 1/metabolism , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Protein Transport/radiation effects , Proteolysis/radiation effects , Skin Aging/radiation effectsABSTRACT
In the field of molecular analysis of cancer, there exists a need for a clinical device that can automate protocols for immunohistochemical and in situ hybridization diagnostic staining on tissue microarrays. The tissue microarray antibody spotter (TMAS) has been developed to provide fundamental improvements over current histological staining techniques by enabling precision application of reagents to individual biopsies within a tissue microarray. This allows for multiplexed reactions on a single slide and promises to significantly reduce costs associated with immunohistochemistry and in situ hybridization based assays. Additionally, because TMAS allows for testing of different biomarkers on each element of a tissue array, a complete cancer profile can be obtained from a single TMA slide. Ultimately this may lead to cost-effective, faster and more accurate diagnosis of the patient.
Subject(s)
Biopsy , Immunohistochemistry/instrumentation , Neoplasms/diagnosis , Tissue Array Analysis/instrumentation , Humans , Nanotechnology , Neoplasms/pathology , Staining and Labeling/instrumentationABSTRACT
No routine test exists to determine the quality of blood platelet transfusions although every year millions of patients require platelet transfusions to survive cancer chemotherapy, surgery or trauma. A new, portable dynamic light scattering instrument is described that is suitable for the measurement of turbid solutions of large particles under temperature-controlled conditions. The challenges of small sample size, short light path through the sample and accurate temperature control have been solved with a specially designed temperature-controlled sample holder for small diameter, disposable capillaries. Efficient heating and cooling is achieved with Peltier elements in direct contact with the sample capillary. Focusing optical fibres are used for light delivery and collection of scattered light. The practical use of this new technique was shown by the reproducible measurement of latex microspheres and the temperature-induced morphological changes of human blood platelets. The measured parameters for platelet transfusions are platelet size, number of platelet-derived microparticles and the response of platelets to temperature changes. This three-dimensional analysis provides a high degree of confidence for the determination of platelet quality. The experimental data are compared to a matrix and facilitate automated, unbiased quality testing.
