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Nigerooligosaccharides (NOS), a mixture of nigerose and nigerosylmaltooligosaccharides, consists of immunopotentiating oligosaccharides found in foodstuffs. We have previously reported that activation of peripheral blood mononuclear cells (PBMC) in response to concanavalin A (Con A) or a streptococcal preparation of OK-432 is augmented in healthy young adults and elderly subjects after the intake of NOS-supplemented syrup. A reappraisal of the data suggests that NOS augments proliferation but partly suppresses activation-induced apoptosis of PBMC in response to these mitogens. To confirm this hypothesis, PBMC from healthy male subjects were stimulated with Con A or OK-432 in the presence of nigerose at the concentrations at which it was detected in the blood of subjects who had ingested NOS-supplemented syrup. Cellular activation, specifically metabolic demand, viability and proliferation, was assessed from glucose consumption, by WST-1 colorimetry and by 5-bromo-2'-deoxy-uridine incorporation assay, respectively. The Con A-induced activation of PBMC in each measurement was significantly augmented by nigerose. OK-432-induced decreases in the viability of PBMC were significantly inhibited by nigerose. Stimulation of PBMC with Con A or OK-432 induced apoptosis, but nigerose suppressed such activation-induced cell death. These results indicated that nigerose activated PBMC in vitro in a manner similar to the process observed in vivo, providing further evidence for the effectiveness of consumption of NOS-supplemented syrup.

Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1(-/-) mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1(-/-) thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1(-/-) thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1(+/-) thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1(-/-) thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones.

In CD4+ UE160 cells with inducible expression of gp160, mechanisms of apoptosis induced by human immunodeficiency virus (HIV) Env protein were analyzed. Induction of gp160 caused intracellular calcium increase followed by the release of cytochrome c from mitochondria, which was inhibited by calcineurin inhibitors. Association of BAD with Bcl-xL was observed, and a portion of BAD was dephosphorylated after induction of gp160. These data suggested that calcineurin plays a role in the HIV Env-induced apoptosis in a mitochondrion-dependent way.

Apaf1 is a critical molecule in the mitochondria-dependent apoptotic pathway. Here we show that Apaf1-deficient embryonic fibroblasts died at a later phase of apoptotic induction, although these cells were resistant to various apoptotic stimulants at an early phase. Neither caspase 3 activation nor nuclear condensation was observed during this cell death of Apaf1-deficient cells. Electron microscopic examination revealed that death in response to apoptotic stimulation resembled necrosis in that nuclei were round and swollen with low electron density. Necrosis-like cell death was also observed in wild-type cells treated with z-VAD-fmk. Mitochondria were not only morphologically abnormal but functionally affected, since mitochondrial transmembrane potential (ΔΨm) was lost even in cells with intact plasma membrane integrity. These mitochondrial alterations were also observed in the wild-type cells dying of apoptosis. Combined, these data suggest that cells without caspase activation, such as Apafl-deficient cells or cells treated with caspase inhibitors, die of necrosis-like cell death with mitochondrial damage in response to “apoptotic stimulation.” [ABSTRACT FROM AUTHOR]