Protein Profiling of the secretome of Fc sigma RI Activated RBL-2H3.1 cells

Secretory proteins of IgE receptor activated mast cells and basophils play a pivotal role in the generation of immediate and long term immune responses in allergy and type I hypersensitivity. The present study aims to generate a 2-D map and profile of proteins secreted from a high secretory variant of the rat basophilic leukemia cell line, RBL-2H3.1, which in view of the difficulty associated with gaining adequate numbers of pure primary mast cell and basophiles, represents an accepted model system for the study and standardization of the methodology to characterize the secretome of these cell types. A 2-D map of secretory proteins was generated by 2-D PAGE and a shotgun mass spectrometric approach carried out for protein identification. Study resulted into identification of 299 proteins released from resting and IgE receptor activated RBL-2H3.1 cells after 90 s, 30 min and 3 h antigen challenge. Further sequence analysis identified 53% of total proteins as secretory proteins which could be attributed to classical and non-classical secretory pathways. Additionally, functional classification of classic secretory proteins verified the presence of proteins belonged to cytokines, receptors, membrane proteins, lysosomal proteins and proteins associated with specific sub-cellular localizations such as endoplasmic reticulum, mitochondria, nucleus, cytoplasm and ribosome. According to this data the presence of some secretory proteins such as cytokines [e.g. MCP-2, PF-4, CSF-1 and TGF-beta1] are all subject to Ag challenge which may point to their importance toward pathogenesis in allergic diseases. In view of both a beneficial and adverse role of mast cell mediators in health and disease, an identification of temporal changes in the secretory pattern may form the basis for future tailor made intervention strategies that may enable us to harvest the therapeutic potential inherent in mast cell exocytosis while inhibiting/attenuating negative outcomes

Angiogenesis following three-dimensional culture of isolated human endometrial stromal cells

Endometriosis is the presence of endometrial tissue outside of the uterine cavity and is the most common gynecologic disorder in women of reproductive age. We have preliminary evidence that in the presence of a 3-dimensional [3-D] fibrin matrix, human endometrial glands, stroma, and neovascularization can develop in vitro, mimicking the earliest stages of endometriosis. The aim of the present study was to determine if angiogenesis can be developed in a 3-D culture of human stromal cells in vitro. This was an in vitro study of human endometrial biopsies in 3-D culture of fibrin matrix and conducted at a university affiliated infertility center. Biopsies were taken from ten normal ovulating women undergoing infertility treatment. The samples obtained from fundus of the uterine cavity were minced, stromal cells isolated and placed in a 3-D fibrin matrix culture system. Degree of proliferation of stromal cells, invasion of the fibrin matrix, gland formation, vessel sprouting and immunohistochemical characterization of cellular components were recorded. Three-dimensional culture of human stromal cells formed sheets of cells in the fibrin matrix. By 3-4 weeks, endothelial cell branching was observed and rudimentary capillary-like structures formed and endothelial cells confirmed by CD31 immunostaining. These data show that stromal cells from endometrial explants can proliferate and invade a fibrin matrix in vitro generating new vessels. This procedure represents a controlled, quantifiable model for the study of angiogenesis during the menstrual cycle, and in conditions such as endometriosis and cancer