ovalbumin and Ascites

Epithelial ovarian cancer (EOC) is typically diagnosed at advanced stages, and is associated with a high relapse rate. Patients in remission are ideal candidates for immunotherapy aimed at cure or prolonging disease-free periods. However, immunosuppressive pathways in the tumor microenvironment are obstacles to durable anti-tumor immunity. In a metastatic syngeneic mouse model of EOC, immunosuppressive macrophages and myeloid-derived suppressor cells (MDSCs) accumulate in the local tumor environment. In addition, resident peritoneal macrophages from non-tumor-bearing mice were highly immunosuppressive, abrogating stimulated T cell proliferation in a cell contact-dependent manner. Immunization with microparticles containing TLR9 and NOD-2 ligands (MIS416) significantly prolonged survival in tumor-bearing mice. The strategy of MIS416 immunization followed by anti-CD11b administration further delayed tumor progression, thereby establishing the proof of principle that myeloid depletion can enhance vaccine efficacy. In patients with advanced EOC, ascites analysis showed substantial heterogeneity in the relative proportions of myeloid subsets and their immunosuppressive properties. Together, these findings point to immunosuppressive myeloid cells in the EOC microenvironment as targets to enhance vaccination. Further studies of myeloid cell accumulation and functional phenotypes in the EOC microenvironment may identify patients who are likely to benefit from vaccination combined with approaches that deplete tumor-associated myeloid cells.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Ascites; Cancer Vaccines; Carcinoma, Ovarian Epithelial; CD11b Antigen; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Disease Progression; Female; Humans; Ligands; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Macrophages; Mice, Inbred C57BL; Mice, Transgenic; Myeloid Cells; Neoplasms, Glandular and Epithelial; Nod2 Signaling Adaptor Protein; Ovalbumin; Ovarian Neoplasms; T-Lymphocytes; Time Factors; Toll-Like Receptor 9; Tumor Escape; Tumor Microenvironment; Vaccination

A large quantity of polyclonal anti-ovalbumin antibodies was obtained from mice by a simple modification of the method described by Kurpisz et al. (1988). In addition, the cells from ascitic fluid were used to produce monoclonal antibodies. Egg ovalbumin hyperimmunized BALB/c mice were injected successively with pristane, antigen and a non-antibody secreting myeloma cell line: the production of ascitic fluid containing antiovalbumin antibody activity was observed after 10-25 days. Cells from ascitic fluid were harvested, washed and fused together with polyethylene glycol to produce monoclonal antibodies. Two fusions were performed and a large number of monoclonal anti-ovalbumin antibodies was obtained. This method is simple, reproducible, allows many fusions to be obtained from one mouse, and allows the use of ascitic B cells rather than the more, frequently used splenic B cells.

Topics: Animals; Antibodies, Monoclonal; Antibody Formation; Ascites; Immunization; Mice; Mice, Inbred BALB C; Ovalbumin

Avidin conjugated to the fluorescent dyes rhodamine or fluorescein binds to mast cell granules in rodent and human skin. Sequential staining of tissue mast cells first with conjugated avidin, and then with a metachromatic stain demonstrated that both techniques identify the same mast cell granules. Specificity for mast cells was confirmed by the absence of avidin-positive cells in the skin of mast cell-deficient (W/Wv) mice. Binding of conjugated avidin to mast cells was inhibited by pretreating tissue specimens with unconjugated avidin but not by pretreating conjugated avidin with biotin, indicating that avidin does not bind to biotin or a biotin-like molecule. Within murine dermis, unique patterns of mast cell distributions were observed, with a prominent perivascular localization in ear skin, and a complete absence of mast cells underlying the scales in tail skin. In tissue sections of guinea pig skin undergoing basophil hypersensitivity reactions and in murine and human skin specimens infiltrated with eosinophils, conjugated avidin selectively stained only dermal mast cells, demonstrating specificity for mast cells in sites of inflammation. Conjugated avidin also readily stained rat peritoneal mast cells, demonstrating its utility for identifying extracutaneous mast cells. Unlike the metachromatic stains, avidin binding to mast cells in tissues is not limited by methods of fixation or special embedding and cutting procedures. Thus, mast cell identification with conjugated avidin is a reliable, specific, and simple method with important clinical and investigative applications.

Topics: Animals; Ascites; Avidin; Fluorescent Dyes; Guinea Pigs; Humans; Mast Cells; Mice; Mice, Inbred BALB C; Ovalbumin; Rats; Rats, Inbred Strains; Skin; Species Specificity

Topics: Animals; Antigens; Ascites; Ascitic Fluid; Benzoates; Cell Count; Culture Techniques; Glycogen; Guinea Pigs; Hypersensitivity, Delayed; Immunization, Passive; Lymph Nodes; Lymphocytes; Macrophages; Ovalbumin; Skin Tests; Tuberculin Test