Modified human beta 2-microglobulin (desLys(58)) displays decreased affinity for the heavy chain of MHC class I and induces nitric oxide production and apoptosis.

Beta2-microglobulin (beta2m) is the light chain of major histocompatibility complex class I (MHC-I) molecules, and is a prerequisite for the binding of peptides to the heavy chain and their presentation to CD8+ T cells. beta2m can be modified in vivo and in vitro by proteolytic cleavage by complement C1 and subsequent carboxypeptidase B-like activity--processes that lead to the generation of desLys(58) beta2m (dbeta2m). This work aims to study the effect of dbeta2m on peptide binding to MHC-I, the influence of dbeta2m on the binding of beta2m to the MHC-I heavy chain and the biological activity of dbeta2m. Both beta2m and dbeta2m are able to support the generation of MHC-I/peptide complexes at 18 degrees C, but complexes formed in the presence of dbeta2m destabilize at 37 degrees C. Moreover, a 250 times higher concentration of dbeta2m than of beta2m is needed to displace MHC-I associated beta2m from the cell surface. In addition, only beta2m is able to restore MHC-I/peptide complex formation on acid-treated cells whereas dbeta2m appears to bind preferentially to denatured MHC-I heavy chains. In cell cultures, exogenously added dbeta2m, but not beta2m, induces apoptotic cell death in monocytic leukaemic cell lines but spares other kinds of leukaemic cells. Additionally, the presence of dbeta2m, and to a lesser extent beta2m, enhances IFN-gamma-induced NO production by monocytic leukaemic cells. In conclusion, these data show that dbeta2m is not able to support the formation of a stable tri-molecular MHC-I complex at physiological temperature and that dbeta2m exerts other biological functions compared to beta2m when bound to cells.

Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis.

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G(0)/G(1) phase without inducing significant cell death. In confluent C6 cultures, on the other hand, metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP-activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251, while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin.

Iron down-regulates macrophage anti-tumour activity by blocking nitric oxide production.

Although the inhibitory effect of iron on macrophage production of tumoricidal free radical nitric oxide (NO) has been reported, its possible influence on macrophage anti-tumour activity has not been established. In the present study, FeSO4 markedly reduced IFN-gamma + LPS-induced NO synthesis in mouse and rat macrophages. The effect of iron coincided with the loss of macrophage cytotoxic activity against NO-sensitive C6 rat astrocytoma and L929 mouse fibrosarcoma cell lines, as measured by MTT assay for cellular respiration and the crystal violet test for cell viability. Tumour cell survival did not improve further in the presence of FeSO4 if macrophage NO release and cytotoxicity were already blocked by aminoguanidine. In accordance with the results obtained with exogenous iron, cell membrane permeable iron chelator o-phenanthroline enhanced both macrophage NO release and anti-tumour activity. Iron also down-regulated NO production and increased the viability of L929 fibrosarcoma cells stimulated with IFN-gamma + LPS in the absence of macrophages. However, neither NO release nor cell viability was affected by iron addition to cultures of the C6 astrocytoma cell line. Iron was unable to prevent L929 and C6 cell death induced by the NO releasing chemicals SNP and SIN-1, indicating that iron-mediated inhibition of NO synthesis, rather than interference with its cytotoxic action, was responsible for the protection of tumour cells. Collectively, these results indicate that iron might protect tumour cells by reducing both macrophage and tumour cell-derived NO release.

Induction of experimental autoimmune encephalomyelitis in Dark Agouti rats without adjuvant.

Experimental autoimmune encephalomyelitis (EAE) is a well-recognized model for multiple sclerosis (MS) in humans. However, adjuvants used with encephalitogens to induce EAE produce non-specific effects interfering with the mechanisms involved in the autoimmune response to the central nervous system (CNS) tissue. It is therefore important to establish a more suitable model of EAE for analysis of autoimmune phenomena resembling those operative in MS. Here we report that EAE can be induced regularly in Dark Agouti (DA) strain of rats with spinal cord tissue without any adjuvant, as judged by both clinical and histological parameters. The incidence and severity of EAE depended on the origin of the encephalitogen, the rat versus guinea pig spinal cord homogenate being more efficient. Furthermore, EAE could be reinduced in animals which had recovered from disease that had been induced actively with encephalitogen alone, suggesting the role of adjuvant-generated non-specific mechanisms in resistance to reinduction of EAE. Thus, EAE induced in DA rats with encephalitogen alone provides a reproducible model for defining pathogenically relevant events in CNS autoimmunity devoid of the potentially misleading effects of adjuvants.

Aloe-emodin prevents cytokine-induced tumor cell death: the inhibition of auto-toxic nitric oxide release as a potential mechanism.

Aloe-emodin (AE) is a plant-derived hydroxyanthraquinone with potential anticancer activity. We investigated the ability of AE to modulate survival of mouse L929 fibrosarcoma and rat C6 astrocytoma cells through interference with the activation of inducible nitric oxide (NO) synthase (NOS) and subsequent production of tumoricidal free radical NO. Somewhat surprisingly, AE in a dose-dependent manner rescued interferon-gamma + interleukin-1-stimulated L929 cells from NO-dependent killing by reducing their autotoxic NO release. The observed protective effect was less pronounced in C6 cells, due to their higher sensitivity to a direct toxic action of the drug. AE-mediated inhibition of tumor cell NO release coincided with a reduction in cytokine-induced accumulation of transcription and translation products of genes encoding inducible NOS and its transcription factor IRF-1, while activation of NF-kappaB remained unaltered. These data indicate that the influence of AE on tumor growth might be more complex that previously recognized, the net effect being determined by the balance between the two opposing actions of the drug: its capacity to directly kill tumor cells, but also to protect them from NO-mediated toxicity.

Taxol activates inducible nitric oxide synthase in rat astrocytes: the role of MAP kinases and NF-kappaB.

Taxol is a microtubule-stabilizing agent that has recently been shown effective in the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. As astrocytes could modulate central nervous system (CNS) autoimmunity through inducible nitric oxide synthase (iNOS)-mediated production of immunoregulatory free radical nitric oxide (NO), we investigated the effect of taxol on NO synthesis in rat astrocytes. Taxol, either alone or in combination with interferon-gamma, induced NO generation in primary astrocytes and astrocytoma C6 cells in a dose- and time-dependent manner. Accordingly, the drug markedly up-regulated the expression of both iNOS mRNA and protein in astrocytes. The observed effect of taxol was mediated through induction of iNOS transcription factors NF-kappaB and IRF-1, and required the activation of p38 MAP kinase and JNK. Finally, NO release by taxol-stimulated astrocytes was blocked with the microtubule-depolymerizing agent colchicine, suggesting the involvement of a microtubule-stabilizing activity of taxol in the observed effect.