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Exosomes are nanometer-sized vesicles secreted by a diverse range of live cells that probably have physiological roles in modulating cellular immunity. The extracellular factors that regulate the quantity and phenotype of exosomes produced are poorly understood, and the properties of exosomes that dictate their immune functions are not yet clear. We investigated the effect of cellular stress on the exosomes produced by B-lymphoblastoid cell lines. Under steady-state conditions, the exosomes were positive for hsp27, hsc70, hsp70 and hsp90, and other recognised exosome markers such as MHC class I, CD81, and LAMP-2. Exposing cells to heat stress (42°C for up to 3 hours), resulted in a marked increase in these heat shock proteins (hsps), while the expression of other stress proteins such as hsp60 and gp96 remained negative, and other exosome markers remained unchanged. Stress also triggered a small increase in the quantity of exosomes produced [with a ratio of 1.245±0.07 to 1 (mean±s.e.m., n=20) of 3-hour-stress-exosomes to control-exosomes]. Flow-cytometric analysis of exosome-coated beads and immuno-precipitation of intact exosomes demonstrated that hsps were located within the exosome lumen, and not present at the exosome-surface, suggesting that such exosomes may not interact with target cells through cell-surface hsp-receptors. Functional studies further supported this finding, in that exosomes from control or heat-stressed B cells did not trigger dendritic cell maturation, assessed by analysis of dendritic-cell-surface phenotype, and cytokine secretion profile. Our findings demonstrate that specific alterations in exosome phenotype are a hitherto unknown component of the cellular response to environmental stress and their extracellular function does not involve the direct activation of dendritic cells.

Interest has recently been shown in adapting the microwave oven heating technique for antigen retrieval to routine diagnostic immunocytochemical practice. Although it has proved effective as a specialist method for individual antigen localization in many laboratories, it has certain drawbacks which have hampered its wider routine application. These include the need to monitor the sections during the microwave treatment to prevent damage or drying, the limited number of sections that can be accommodated in the microwave oven, and the inevitable alteration in nuclear morphology induced by the microwaves. In order to obviate these difficulties, we have modified the wet autoclave method of Shin et al. (Lab Invest 1991; 64: 693-702) as a routine technique for retrieval of a variety of cell surface, cytoplasmic, and nuclear antigens in formalin-fixed and paraffin-embedded tissue. The technique produces even enhancement of several refractory antigens in anatomically different sites and has the potential to handle reliably up to 200 sections at a time without significant damage to the section or to nuclear morphology.

High-temperature preheating of sections in the presence of a salt (e.g., citrate) or a protein denaturant (e.g., urea) solution has been shown recently to provide a reliable alternative to tissue proteolysis for antigen retrieval from formaldehyde-fixed, paraffin-embedded tissues. However, the underlying mechanism of action of this form of pretreatment remains highly speculative. In this study, we show that calcium chelating agents such EDTA and EGTA are more effective than citrate in the retrieval of a citrate-sensitive nuclear antigen, Ki-67. Also, sodium carbonate and another calcium precipitating agent are both able to effect antigen retrieval at high temperatures. The overall data therefore suggest that either the chelation or the precipitation of tissue-bound calcium ions, and perhaps also other divalent metal cations, is a critical step in salt-mediated antigen retrieval. As a corollary, it is suggested that tight complexing of calcium ions or other divalent metal cations with proteins during formaldehyde tissue fixation is responsible for the masking of certain antigens.

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To ensure the accuracy and reproducibility of immunohistochemical assays for determining HER-2/neu status of patients with breast cancer, a reliable standard for monitoring assay sensitivity is necessary. We optimally fixed and paraffin processed human ovarian and breast carcinoma cell lines SKOV-3, MDA-MB-453, BT-20, and MCF-7 in quantities sufficient to meet the needs of a laboratory for the foreseeable future. The material was tested, alongside HercepTest kit cell lines (DAKO, Carpinteria, CA), by 7 breast cancer centers in the United Kingdom and France with different immunohistochemical assays and markers. The cell lines also were analyzed by fluorescence in situ hybridization (FISH) by 2 centers using HER-2/neu kits. FISH produced 100% agreement between the 2 centers: SKOV-3 and MDA-MB-453 showed HER-2/neu amplification and BT-20 and MCF-7 did not. Immunohistochemical analysis and a common evaluation method produced 100% agreement that SKOV-3 and MCF-7 showed 3+ and zero HER-2/neu overexpression, respectively. For MDA-MB-453, there was 71% (5/7) concordance of 2+ immunohistochemical staining and 86% (6/7) concordance of zero or 1 + staining for BT-20. The cell lines provide a valuable standard for gauging HER-2/neu assay sensitivity irrespective of the antibody, antigen retrieval system, detection system, or method of evaluation used.