UCP2 up-regulation within the course of autoimmune encephalomyelitis correlates with T-lymphocyte activation.

Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disorder of the central nervous system (CNS) associated with severe neurological disability. Reactive oxygen species (ROS) and mitochondrial dysfunction play a pivotal role in the pathogenesis of this disease. Several members of the mitochondrial uncoupling protein subfamily (UCP2-UCP5) were suggested to regulate ROS by diminishing the mitochondrial membrane potential and constitute therefore a promising pharmacological target for MS. To evaluate the role of different uncoupling proteins in neuroinflammation, we have investigated their expression patterns in murine brain and spinal cord (SC) during different stages of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. At mRNA and protein levels we found that only UCP2 is up-regulated in the SC, but not in brain. The increase in UCP2 expression was antigen-independent, reached its maximum between 14 and 21days in both OVA and MOG immunized animals and correlated with an augmented number of CD3+ T-lymphocytes in SC parenchyma. The decrease in abundance of UCP4 was due to neuronal injury and was only detected in CNS of MOG-induced EAE animals. The results provide evidence that the involvement of mitochondrial UCP2 in CNS inflammation during EAE may be mainly explained by the invasion of activated T-lymphocytes. This conclusion coincides with our previous observation that UCP2 is up-regulated in activated and rapidly proliferating T-cells and participates in fast metabolic re-programming of cells during proliferation.

Telomerase- and alternative telomere lengthening-independent telomere stabilization in a metastasis-derived human non-small cell lung cancer cell line: effect of ectopic hTERT.

In the majority of human malignancies, maintenance of telomeres is achieved by reactivation of telomerase, whereas a smaller fraction uses an alternative telomere lengthening (ALT) mechanism. Here, we used 16 non-small cell lung cancer (NSCLC) cell lines to investigate telomere stabilization mechanisms and their effect on tumor aggressiveness. Three of 16 NSCLC cell lines (VL-9, SK-LU-1, and VL-7) lacked telomerase activity, correlating with significantly reduced tumorigenicity in vitro and in vivo. Of the three telomerase-negative cell lines, only SK-LU-1 displayed characteristics of an ALT mechanism (i.e., highly heterogeneous telomeres and ALT-associated promyelocytic leukemia bodies). VL-9 cells gained telomerase during in vitro propagation, indicating incomplete immortalization in vivo. In contrast, NSCLC metastasis-derived VL-7 cells remained telomerase and ALT negative up to high passage numbers and following transplantation in severe combined immunodeficient mice. Telomeres of VL-7 cells were homogeneously short, and chromosomal instability (CIN) was comparable with most telomerase-positive cell lines. This indicates the presence of an efficient telomere stabilization mechanism different from telomerase and ALT in VL-7 cells. To test the effect of ectopic telomerase reverse transcriptase (hTERT) in these unique ALT- and telomerase-negative tumor backgrounds, hTERT was transfected into VL-7 cells. The activation of telomerase led to an excessively rapid gain of telomeric sequences resulting in very long ( approximately 14 kb), uniform telomeres. Additionally, hTERT expression induced a more aggressive growth behavior in vitro and in vivo without altering the level of CIN. These data provide further evidence for a direct oncogenic activity of hTERT not based on the inhibition of CIN.

Selective inhibition of receptor activator of NF-κB ligand (RANKL) in hematopoietic cells improves outcome after experimental myocardial infarction.

The RANK (receptor activator of nuclear factor κB)/RANKL (RANK ligand)/OPG (osteoprotegerin) axis is activated after myocardial infarction (MI), but its pathophysiological role is not well understood. Here, we investigated how global and cell compartment-selective inhibition of RANKL affects cardiac function and remodeling after MI in mice. Global RANKL inhibition was achieved by treatment of human RANKL knock-in (huRANKL-KI) mice with the monoclonal antibody AMG161. huRANKL-KI mice express a chimeric RANKL protein wherein part of the RANKL molecule is humanized. AMG161 inhibits human and chimeric but not murine RANKL. To dissect the pathophysiological role of RANKL derived from hematopoietic and mesenchymal cells, we selectively exchanged the hematopoietic cell compartment by lethal irradiation and across-genotype bone marrow transplantation between wild-type and huRANKL-KI mice, exploiting the specificity of AMG161. After permanent coronary artery ligation, mice were injected with AMG161 or an isotype control antibody over 4 weeks post-MI. MI increased RANKL expression mainly in cardiomyocytes and scar-infiltrating cells 4 weeks after MI. Only inhibition of RANKL derived from hematopoietic cellular sources, but not global or mesenchymal RANKL inhibition, improved post-infarct survival and cardiac function. Mechanistically, hematopoietic RANKL inhibition reduced expression of the pro-inflammatory cytokine IL-1ß in the cardiac cellular infiltrate. In conclusion, inhibition of RANKL derived from hematopoietic cellular sources is beneficial to maintain post-ischemic cardiac function by reduction of pro-inflammatory cytokine production. KEY MESSAGES: Experimental myocardial infarction (MI) augments cardiac RANKL expression in mice. RANKL expression is increased in cardiomyocytes and scar-infiltrating cells after MI. Global or mesenchymal cell RANKL inhibition has no influence on cardiac function after MI. Inhibition of RANKL derived from hematopoietic cells improves heart function post-MI. Hematopoietic RANKL inhibition reduces pro-inflammatory cytokines in scar-infiltrating cells.

Repression of in vivo growth of Myc/Ras transformed tumor cells by Mad1.

The Myc/Max/Mad network of transcriptional regulatory proteins plays an essential role in cell proliferation, growth, apoptosis, and differentiation. Whereas Myc proteins affect cell cycle progression positively, Mad proteins are negative regulators of cell proliferation. It has been shown in several in vitro systems that Mad proteins antagonize c-Myc functions. In this report we describe the inhibition of tumor cell outgrowth in vivo by Mad1 expression. Transformed cell lines were generated by co-transfection of c-myc, c-H-ras, and a chimeric mad1ER construct into primary rat embryo cells (MRMad1ER cells). Activation of Mad1 by 4-Hydroxy-Tamoxifen (OHT) resulted in abrogation of telomerase activity, reduced cloning efficiency, and decreased proportion of cells in S phase. Injection of MRMad1ER cells into syngenic rats induced aggressively growing tumors after a short latency period. This tumor growth was inhibited by OHT-treatment of animals, with the extent of inhibition correlating with the amount of OHT injected. No effect of OHT on tumor growth was observed with similarly transformed Myc/Ras cell lines which did not express Mad1ER. These data demonstrate that Mad1 is able to suppress Myc/Ras-mediated transformation under in vivo conditions.

Inhibitor of apoptosis protein (IAP) survivin is upregulated by oncogenic c-H-Ras.

Among the inhibitors of apoptosis proteins (IAPs), survivin has attracted special attention through its involvement in human cancer. The mechanism underlying tumour-associated survivin re-expression is not known. We found a correlation between exogenous c-H-Ras oncoprotein and endogenous survivin in a series of rat cell lines, which expressed defined oncogenes. Moreover, human HaCat cells, transfected with a constitutively activated c-H-ras gene, had significantly increased survivin levels. To study the interdependence of the two proteins, we generated a rat cell line that expressed a dexamethasone-inducible c-H-ras construct. Induction of c-H-Ras expression was followed by rapid upregulation of survivin. Conversely, downregulation of the oncoprotein resulted in prompt reduction of survivin to baseline value. c-H-Ras-induced survivin was expressed constitutively and independent of cell cycle progression or proliferation. Compromising Ras-stimulated PI3-K activity and MEK1 by chemicals abolished survivin expression and was associated with apoptotic cell death. Upregulation of survivin appeared to be an important activity of c-H-Ras oncoprotein, since cotransfection of a survivin-antisense construct into c-myc/c-H-ras-transfected primary rat embryo cells resulted in profound reduction of transformed clones. It is tempted to speculate that the frequent presence of survivin in human cancer cells might be a consequence of activated Ras-signalling pathways.

Effects of feed contaminant deoxynivalenol on plasma cytokines and mRNA expression of immune genes in the intestine of broiler chickens.

An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1ß, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κß1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1ß, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1ß, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κß1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes.