Djordje Miljković

Novel platinum(IV) complexes induce rapid tumor cell death in vitro

The anticancer activity of platinum complexes has been known since the discovery of classical Pt(II)‐based drug cisplatin. However, Pt(IV) complexes have greater inertness than corresponding Pt(II) complexes, thus allowing the oral administration and reducing the toxicity associated with platinum‐based chemotherapy. Here, we describe the in vitro antitumor activity of some novel Pt(IV)‐based agents against mouse fibrosarcoma L929 cells and human astrocytoma U251 cells. The cytotoxicity of 2 Pt(IV) complexes with bidentate ethylenediamine‐N,N′‐di‐3‐propanoato esters was found to be markedly higher than that of their Pt(II) counterparts and comparable to the antitumor action of cisplatin. In contrast to cisplatin, which caused oxidative stress‐independent apoptotic cell death of tumor cells, these Pt(IV) complexes induced oxygen radical‐mediated tumor cell necrosis. Importantly, the cytotoxic action of novel Pt(IV) complexes was markedly more rapid than that of cisplatin, indicating their potential usefulness in anticancer therapy.

Uric acid levels in sera from patients with multiple sclerosis

Abstract The levels of uric acid (UA), a natural peroxynitrite scavenger, were measured in sera from 240 patients with multiple sclerosis (MS) and 104 sex- and age-matched control patients with other neurological diseases (OND). The mean serum UA concentration was lower in the MS than in the OND group, but the difference did not reach the level of statistical significance (P=0.068). However, the mean serum UA level from patients with active MS (202.6+67.1 μmol/l) was significantly lower than that in inactive MS patients (226.5+78.6 μmol/l; P=0.046) and OND controls (P=0.007). We found a significant inverse correlation of serum UA concentration with female gender (P=0.0001), disease activity (P=0.012) and duration (P=0.017), and a trend towards an inverse correlation with disability as assessed by EDSS score, which did not reach statistical significance (P=0.067). Finally, multivariate linear regression analyses showed that UA concentration was independently correlated with gender (P=0.0001), disease activity (P=0.014) and duration of the disease (P=0.043) in MS patients. These findings suggest that serum UA might serve as a possible marker of disease activity in MS. They also provide support to the potential beneficial therapeutic effect of radical-scavenging substances in MS.

Interleukin-17 stimulates inducible nitric oxide synthase activation in rodent astrocytes

The effect of interleukin-17 (IL-17) on production of nitric oxide (NO) in rodent astrocytes was investigated. While IL-17 by itself did not induce NO production, it caused a dose-dependent enhancement of IFN-gamma-triggered NO synthesis in both mouse and rat primary astrocytes. In contrast, IL-17 was unable to stimulate NO synthesis in either murine or rat macrophages. IFN-gamma-triggered expression of mRNA for iNOS, but not for its transcription factor interferon regulatory factor-1 (IRF-1), was markedly elevated in IL-17-treated astrocytes. The induction of iNOS mRNA by IL-17 in IFN-gamma-pretreated astrocytes was abolished by antagonists of nuclear factor-kappaB (NF-kappaB) activation--a proteasome inhibitor MG132 and an antioxidant agent PDTC, as well as with specific p38 MAP kinase inhibitor SB203580. While IL-17 stimulated both IL-1beta and IL-6 production in astrocytes, only IL-1 was partly responsible for IL-17-induced NO release. Finally, IL-17 synergized with exogenous IL-1beta and TNF-alpha for astrocyte NO production. Having in mind a well-known neurotoxic action of NO, these results suggest a possible role for IL-17 in the inflammatory diseases of the CNS.

Astrocyte-induced regulatory T cells mitigate CNS autoimmunity.

Although astrocytes presumably participate in maintaining the immune privilege of the central nervous system (CNS), the mechanisms behind their immunoregulatory properties are still largely undefined. In this study, we describe the development of regulatory T cells upon contact with astrocytes. Rat T cells pre‐incubated with astrocytes completely lost the ability to proliferate in response to mitogenic stimuli. The cells were blocked in G0/G1 phase of the cell cycle, expressed less IL‐2R, and produced significantly lower amounts of interferon‐γ (IFN‐γ), but not interleukin‐2 (IL‐2), IL‐10, or tumor necrosis factor (TNF). These anergic cells completely prevented mitogen‐induced growth of normal T lymphocytes, as well as CNS antigen‐driven proliferation of autoreactive T cells. The suppressive activity resided in both CD4+ and CD8+ T‐cell compartments. Heat‐sensitive soluble T‐cell factors, not including transforming growth factor‐β (TGF‐β) or IL‐10, were solely responsible for the observed suppression, as well as for the transfer of suppressive activity to normal T cells. The administration of astrocyte‐induced regulatory T cells markedly alleviated CNS inflammation and clinical symptoms of CNS autoimmunity in rats with experimental allergic encephalomyelitis. Finally, the cells with suppressive properties were readily generated from human lymphocytes after contact with astrocytes. Taken together, these data indicate that astrocyte‐induced regulatory T cells might represent an important mechanism for self‐limitation of excessive inflammation in the brain.

Macrophage migration inhibitory factor (MIF) is necessary for progression of autoimmune diabetes mellitus

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine of the innate immune system that plays a major role in the induction of immunoinflammatory responses. To examine the role of endogenous MIF in the pathogenesis of type 1 diabetes (TID) we evaluated the effects of administration of neutralizing anti‐MIF antibodies to NOD mice with accelerated forms of diabetes induced by injection of cyclophosphamide or by transfer of diabetogenic spleen cells. Both accelerated forms of diabetes were markedly reduced by anti‐MIF antibody. Furthermore, MIF‐deficient (MIF−/−) mice were less susceptible to the induction of immunoinflammatory diabetes, insulitis and apoptosis within the endocrine pancreas by multiple low doses of streptozotocin (MLD‐STZ) than genetically matched wild type (WT) mice. MIF deficiency resulted in lower proliferation and lymphocyte adhesion, as well as reduced production from the spleens and peritoneal cells of a variety of inflammatory mediators typically associated with development of the disease including IL‐12, IL‐23, TNF‐α, and IL‐1β. Furthermore, MIF deletion affected the production of IL‐18, TNF‐α, IL‐1β, and iNOS in the islets of Langerhans. These data, along with the higher expression of IL‐4 and TGF‐β observed in the periphery and in the pancreas of MLD‐STZ‐challenged MIF−/− mice as compared to WT controls suggest that MIF deficiency has induced an immune deviation towards protective type 2/3 response. These results suggest that MIF participates in T1D by controlling the functional activity of monocytes/macrophages and T cells and modulating their secretory capacity of pro‐ and anti‐inflammatory molecules. J. Cell. Physiol. 215: 665–675, 2008.

Anticancer properties of the novel nitric oxide-donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid-nitric oxide in vitro and in vivo.

Preclinical studies have shown that nitric oxide (NO)–donating nonsteroidal anti-inflammatory drugs possess anticancer activities. Here, we report in vitro and in vivo studies showing the antitumor effect of the NO-donating isoxazole derivative (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid (GIT-27NO). GIT-27NO, but not the NO-deprived parental compound VGX-1027, significantly affected viability of both rodent (L929, B16, and C6) and human (U251, BT20, HeLa, and LS174) tumor cell lines. GIT-27NO triggered either apoptotic cell death (e.g., L929 cells) or autophagic cell death (C6 and B16 cells). Moreover, GIT-27NO hampered the viability of cisplatin-resistant B16 cells. NO scavenger hemoglobin completely prevented GIT-27NO-induced death, indicating that NO release mediated the tumoricidal effect of the compound. Increase in intracellular NO upon on the treatment was associated with intensified production of reactive oxygen species, whereas their neutralization by antioxidant N-acetylcysteine resulted in partial recovery of cell viability. The antitumor activity of the drug was mediated by the selective activation of mitogen-activated protein kinases in a cell-specific manner and was neutralized by their specific inhibitors. In vivo treatment with GIT-27NO significantly reduced the B16 melanoma growth in syngeneic C57BL/6 mice. The therapeutic effect occurred at dose (0.5 mg/mouse) up to 160 times lower than those needed to induce acute lethality (80 mg/mouse). In addition, a dose of GIT-27NO five times higher than that found effective in the melanoma model was well tolerated by the mice when administered for 4 consecutive weeks. These data warrant additional studies to evaluate the possible translation of these findings to the clinical setting. [Mol Cancer Ther 2008;7(3):510–20]

Macrophage migration inhibitory factor stimulates interleukin-17 expression and production in lymph node cells

Interleukin (IL)‐17 is a pro‐inflammatory cytokine produced by recently described T helper type 17 (Th17) cells, which have critical role in immunity to extracellular bacteria and the pathogenesis of several autoimmune disorders. IL‐6 and transforming growth factor (TGF)‐β are crucial for the generation of Th17 cells in mice, while the production of IL‐17 is supported by various cytokines, including IL‐23, IL‐1β, IL‐21, IL‐15 and tumour necrosis factor (TNF)‐α. In this study, the influence of a multifunctional cytokine, macrophage migration inhibitory factor (MIF), on IL‐17 production in mice was investigated. Treatment of lymph node cells (LNCs) with recombinant MIF up‐regulated mitogen‐stimulated IL‐17 expression and secretion. Additionally, LNCs from MIF knockout mice (mif−/−) had severely impaired production of IL‐17, as well as of IL‐1β, IL‐6, IL‐23 and TGF‐β. When stimulated with recombinant IL‐1β, IL‐23 or TNF‐α, mitogen‐triggered mif−/− LNCs were fully able to achieve the IL‐17 production seen in wild‐type (WT) LNCs, while the addition of IL‐6 and TGF‐β had no effect. Finally, after injection of mice with complete Freund’s adjuvant, secretion of IL‐17 as well as the number of IL‐17‐positive cells was significantly lower in the draining lymph nodes of mif−/− mice in comparison with WT mice. The effect of MIF on IL‐17 production was dependent on p38, extracellular signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK) and Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/STAT3), and not on nuclear factor (NF)‐κB and nuclear factor of activated T cells (NFAT) signalling. Bearing in mind the contribution of MIF and IL‐17 to the pathology of inflammatory and autoimmune disorders, from the results presented here it seems plausible that targeting MIF biological activity could be a valid therapeutic approach for the treatment of such diseases.

Astrocytes in the tempest of multiple sclerosis

Astrocytes are the most abundant cell population within the CNS of mammals. Their glial role is perfectly performed in the healthy CNS as they support functions of neurons. The omnipresence of astrocytes throughout the white and grey matter and their intimate relation with blood vessels of the CNS, as well as numerous immunity‐related actions that these cells are capable of, imply that astrocytes should have a prominent role in neuroinflammatory disorders, such as multiple sclerosis (MS). The role of astrocytes in MS is rather ambiguous, as they have the capacity to both stimulate and restrain neuroinflammation and tissue destruction. In this paper we present some of the proved and the proposed functions of astrocytes in neuroinflammation and discuss the effect of MS therapeutics on astrocytes.

Strain difference in susceptibility to experimental autoimmune encephalomyelitis between Albino Oxford and Dark Agouti rats correlates with disparity in production of IL-17, but not nitric oxide

Albino Oxford (AO) rats, unlike Dark Agouti (DA) rats are resistant to the induction of experimental autoimmune encephalomyelitis (EAE). The reason for the resistance could be some restraining mechanism preventing auto‐aggressive cell activation at the level of draining lymph nodes (DLN) during the induction phase of the disease. Such a mechanism could be anti‐proliferative action of nitric oxide (NO), which has already been shown of importance for the resistance of several rat strains to the induction of the disease. Importantly, number of AO DLN cells (DLNC) is markedly lower and with lower proliferative response to myelin basic protein (MBP) ex vivo in comparison to DA DLNC in the inductive phase of EAE, thus implying that in AO rats DLNC do not proliferate as extensively as in DA rats. We show that AO rats do not produce larger quantities of NO than DA rats after immunization. Further, DLNC of immunized AO rats have significantly lower mRNA expression and synthesis of interferon (IFN)‐γ and interleukin (IL)‐17 compared to DLNC of DA rats. Collectively, these results suggest that there is a substantial difference between EAE‐resistant AO rats and EAE‐prone DA rats in the initiation of autoimmune response. This difference seems to be independent of anti‐proliferative actions of NO, but correlates with impaired IL‐17 production in AO rats.

Multiple Sclerosis: Molecular Mechanisms and Therapeutic Opportunities

The pathophysiology of multiple sclerosis (MS) involves several components: redox, inflammatory/autoimmune, vascular, and neurodegenerative. All of them are supported by the intertwined lines of evidence, and none of them should be written off. However, the exact mechanisms of MS initiation, its development, and progression are still elusive, despite the impressive pace by which the data on MS are accumulating. In this review, we will try to integrate the current facts and concepts, focusing on the role of redox changes and various reactive species in MS. Knowing the schedule of initial changes in pathogenic factors and the key turning points, as well as understanding the redox processes involved in MS pathogenesis is the way to enable MS prevention, early treatment, and the development of therapies that target specific pathophysiological components of the heterogeneous mechanisms of MS, which could alleviate the symptoms and hopefully stop MS. Pertinent to this, we will outline (i) redox processes involved in MS initiation; (ii) the role of reactive species in inflammation; (iii) prooxidative changes responsible for neurodegeneration; and (iv) the potential of antioxidative therapy.