Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.

Reticulocyte counting using flow cytometry and a membrane-permeable fluorescent dye, thiazole orange, was evaluated as an alternative to the conventional manual method. The flow cytometric method was more precise (mean c.v. of 4.3%) than the imprecise manual technique (mean c.v. of 22.4%). Linearity was highly acceptable (r = 0.99) over the reticulocyte count range of 1.8-30.1%. Flow cytometry allows processing of large numbers of samples with reduction in technologist time, and the improved precision and tenfold increase in the number of cells counted should considerably improve the clinical utility of the reticulocyte count.

The changes in plasma fibronectin and IgG, and monocyte- and lymphocyte-associated fibronectin were studied in patients undergoing elective cardiopulmonary bypass surgery. A significant fall (P = less than 0.0005) in plasma fibronectin occurred during bypass, resulting largely from hemodilution as assessed by IgG concentrations, but also related to consumption of fibronectin. Plasma levels were still reduced 48 hours following the operative procedure, despite variable amounts of blood components infused in the immediate post-bypass period. Monocyte-associated fibronectin increased significantly (P = less than 0.05) during bypass, and lymphocyte-associated fibronectin levels decreased. Our studies confirm a reduction in circulating fibronectin in cardiac surgery, with accompanying fall in lymphocyte-associated levels presumed to reflect nonspecific adsorption. In contrast, the increased binding to monocytes may be an important functional aspect requiring further investigation, together with assessment of monocyte-macrophage function, before empiric use of cryoprecipitate therapy in these patients is recommended.