Constitutive activity of NF-kappa B in myeloid cells drives pathogenicity of monocytes and macrophages during autoimmune neuroinflammation

The NF-κB/REL-family of transcription factors plays a central role in coordinating the expression of a wide variety of genes controlling immune responses including autoimmunity of the central nervous system (CNS). The inactive form of NF-κB consists of a heterodimer which is complexed with its inhibitor, IκB. Conditional knockout-mice for IκBα in myeloid cells (lysMCreIκBαfl/fl) have been generated and are characterized by a constitutive activation of NF-κB proteins allowing the study of this transcription factor in myelin-oligodendrocyte-glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE), a well established experimental model for autoimmune demyelination of the CNS

Apoptosis control and proliferation marker in human normal and neoplastic adrenocortical tissues

We evaluated by immunohistochemistry the expression of the Bcl-2 and p53 proteins, as markers of apoptosis control, and of MIB-1, as a marker of cell proliferation, in a series of normal and neoplastic adrenocortical tissues. The specimens were 13 normal adrenals, 13 aldosterone-producing adenomas, 13 non-functioning adenomas and 16 carcinomas. Results were calculated as percentage of immunostained cells by using specific antibodies. No p53 protein was detected in any of the adrenocortical adenomas (functioning and non functioning) or normal adrenals, while p53 was overexpressed in 15 out of 16 carcinomas. In particular, 10 adrenal cancer specimens (62.5%) showed strong staining in a high percentage (range 10–50%) of the malignant cells. The percentage of Bcl-2 positive cells was higher (P<0.05 or less) in non-functioning adenomas (8.1±1.9%) and in carcinomas (14.9±5.6%) than in normals (2.9±0.9%) and aldosterone-producing adenomas (5.3±1.3%) since four specimens of the non-functioning adenomas-group (30.7%) and six of the carcinomas-group (37.5%) showed over 10% positivity (cut-off for normal values, set at 90th percentile of our controls). MIB-1 positivity was 0.50±0.36% in normals, 0.54±0.08% in non-functioning adenomas and 0.54±0.08% in aldosterone-producing adenomas. MIB-1 was expressed in all carcinomas with values (13.7±3.1%) significantly (P<0.0006) higher than in the other groups. In conclusion, the present data indicate that the apoptosis control and proliferation activity evaluated by the p53 and MIB-1 proteins are impaired in adrenal carcinomas but preserved in adenomas, independently of their functional status. Therefore, these immunohistochemical markers, overexpressed in carcinomas only, may be useful in the diagnosis of malignancy in adrenocortical tumours. Whether Bcl-2 positivity found in some carcinomas and non-functioning adenomas may constitute, in the latter, a negative prognostic marker is still unknown

Inverted Expression Profiles of Sex-Biased Genes in Response to Toxicant Perturbations and Diseases

10.1371/journal.pone.0056668PLoS ONE82

Activation of Human Monocytes by Live Borrelia burgdorferi Generates TLR2-Dependent and -Independent Responses Which Include Induction of IFN-β

It is widely believed that innate immune responses to Borrelia burgdorferi (Bb) are primarily triggered by the spirochete's outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs) elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-β and a number of interferon-stimulated genes (ISGs), which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-α, IL-6, IL-10 and IL-1β in monocytes than did lysates. Secreted IL-18, which, like IL-1β, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-β and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs

Initial Characterization of the FlgE Hook High Molecular Weight Complex of

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility

Intermediate filament cytoskeleton of the liver in health and disease

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising ~70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion

AUTOPHAGY REGULATION AFTER DIET AND PHYSICAL ACTIVITY IN NON-ALCOHOLIC FATTY LIVER DISEASE

Megan E. Rosa1, Matthew P. Harris2, David E. Lee1, Jacob L. Brown1, Kaylee E. Poole2, Andrew Seija2, Lemuel A. Brown1, Richard A. Perry Jr.1, Tyrone A. Washington1, Joshua S. Wooten2, Nicholas P. Greene1 1University of Arkansas, 2Southern Illinois University Edwardsville; e-mail: [email protected] Obesity remains a key threat to public health. Along with other detriments associated with obesity, liver disease, specifically Non-Alcoholic Fatty Liver Disease (NAFLD) has dramatically increased. NAFLD may begin with fat accumulation on the liver, but can progress to steatosis, fibrosis, and eventual cirrhosis. With no pharmacological treatment for steatosis, lifestyle interventions appear to be vital to maintaining liver health. Previous work has shown aberrant mitochondrial quality and autophagy in models of fatty liver. Exercise is known to increase basal autophagy levels in muscle as well as mitochondrial health, thus autophagy may be a key regulatory factor for treatment of obesity induced-NAFLD. PURPOSE: The purpose of the study was to examine how lifestyle modifications impact hepatic autophagy and mitochondrial content in a murine model of NAFLD. METHODS: 48 C57BL/6J mice were evenly divided into one of 4 groups: low fat diet (LFD, 10% fat, 18 wks), high fat diet (HFD, 60% fat diet, 18 wks.), diet (D, 60% fat diet for 10 wks then 10% fat diet for 8 wks) or diet and physical activity (D/PA, 60% fat diet for 10 wks, then 10% fat diet plus a running wheel for 8 wks). After interventions, animals were humanly euthanized and livers collected and snap frozen in liquid nitrogen for later analysis. Livers were homogenized and analyzed via immunoblotting for protein content of mitochondrial and autophagy related proteins. Results were analyzed via one-way analysis of variance, (ANOVA), with α set at 0.05. RESULTS: COX-IV protein content was approximately 50% less in HFD compared to LFD, and was restored to LFD levels with D/PA. PGC-1α content was 45% lower in HFD mice, D restored PGC-1α content whereas D/PA resulted in ~60% greater PGC-1α content than LFD. p62 protein content was 2.5 fold higher in HFD animals compared to LFD, D, and D/PA, with no further differences observed. BNIP3 content was 40% lower in HFD compared to LFD; D/PA had 50% more BNIP3 compared to LFD controls. CONCLUSION: Prolonged high-fat diet causes disruptions in mitochondrial content, mitophagy and macroautophagy. Combined diet and physical activity are able to restore these derangements