NF-B Activation Research Articles

The Suppressive Effect of NF-kB Activation After Eicosapentaenoic Acid Intake Before Surgery.

To ascertain whether preoperative neo-adjuvant nutritional therapy (NANT) using eicosapentaenoic acid (EPA) supplementation can provoke a rise in blood levels of EPA capable of restricting NF-B nuclear translocation in resected specimens. Patients were allocated to two groups depending on individual preference: Patients in the treatment group received 2 g of EPA daily for two weeks prior to surgery (NANT group, n=18). Patients in the control group had a normal diet (CONT group, n=26). NF-B translocation rate, in specimens collected, was investigated by histopathology. Five hundred malignant cells were counted, and tissues with 10% or higher NF-B nuclear translocation were determined to be positive. The EPA blood concentration rose significantly in the NANT group (p<0.01). The positive rate of NF-B nuclear translocation in cancer cells was 11.1% in the NANT group compared with 50% in the CONT group. This difference was statistically significant (p<0.01). Increased blood concentrations of EPA after preoperative supplementation was associated with suppression of NF-B nuclear translocation in malignant cells. These results suggest that intake of EPA-containing supplements before surgery can control NF-B activation and by extension, cancer aggressiveness.

Protective Effects and Potential Mechanisms of Dietary Resveratrol Supplementation on the Spleen of Broilers Under Heat Stress.

Resveratrol (RSV) is a natural polyphenolic compound with potent antioxidant and anti-inflammatory properties. This study aimed to investigate the protective effects of RSV supplementation on the inflammatory responses of broilers during heat stress. A total of 432 28-d-old white-feathered broilers (817 crossbred chicken) with an average weight of 549 ± 4 g were randomly allotted to 4 equal groups (Half male and half female, 6 replicates/group, 18 chickens/replicate), including normal temperature (NT) group (24 ± 2°C for 24 h/d, basal diet), NT+RSV group (24 ± 2°C for 24 h/d, basal diet + RSV), heat stress (HT) group (37 ± 2°C for 8 h/d, basal diet), and HT+RSV group (37 ± 2°C for 8 h/d, basal diet + RSV). Serum samples were collected on d 7 and 14 of heat stress, and thymus, spleen, jejunum, and bursa of Fabricius samples were collected and analyzed on d14. RSV treatment decreased the feed conversion ratio, partially reversed the negative alternations in body weight, average daily gain, and average daily feed intake caused by heat stress. RSV treatment also decreased the elevated levels of corticosterone on d 14, adrenocorticotropic hormone, and triiodothyronine in serum on d 7 caused by heat stress, and significantly increased the villus height to crypt depth ratio in the jejunum on d 14. Dietary RSV also reduced heat stress-induced splenic pro-inflammatory cytokine concentrations. TUNEL assay showed that RSV significantly reduced heat stress-induced the number of apoptotic cells. Remarkably, RSV down-regulated some splenic related genes for apoptosis genes, including BCL-2, Apaf-1, and MDM2 mRNA levels induced by heat stress. According to GO and KEGG enrichment analyses, the differential genes between HT and HT + RSV groups were mainly associated with immune system process, hematopoietic or lymphoid organ development, and toll-like receptor signaling pathway. The relative mRNA expression of NF-κB, heat shock protein 70 (HSP70), and p38 MAPK were markedly decreased by the combination of RSV and heat stress. These findings showed that RSV might reduce the splenic inflammatory response in heat-stressed white-feather broilers by inhibiting heat stress-induced activation of NF-B, MAPK, and HSP70, as well as inhibiting the activation of mitochondrial apoptotic pathways.

Sars-COV-2 infection leads to neurodegenerative or neuropsychiatric diseases

The current COVID-19 epidemic caused by the new SARS-CoV-2 has severely harmed global healthcare (severe acute respiratory syndrome coronavirus). COVID-19's pulmonary and cardiovascular effects have been known from its inception, but its causes, mechanisms, and neuropath logical consequences remain unknown. Our research focused on neurological problems in COVID-19 patients, as well as probable SARS-CoV-2 infection routes like hematogenous, direct/neuronal, lymphatic tissue, cerebrospinal fluid, or infiltration by infected immune cells. Late December 2019 in Wuhan, China, a mysterious viral pneumonia struck. The disease was caused by a new corona virus. Corona virus infection spread rapidly from person to person in 2019. The WHO has called it a global public health emergency (WHO). Activation of NF-B in SARS-CoV-2 infection may be linked to immune cell pathogenicity, cytokine storms, and multi-organ failure. COVID-19's inhibition of the NF-B signaling pathway shows promising therapeutically. Inhibiting IKK phosphorylation, a critical downstream consequence of the NF-B signaling cascade, reduces COVID-19 levels. All three disorders have been linked to COVID-19 gene mutations. This study provides a biological basis for future research on COVID-19-related neurological disease. W

Andrographis Paniculata in the Treatment of Upper Respiratory Tract Infections

Andrographis paniculata is a species of Andrographis paniculata (Burm. F) The herbaceous plant Nees, sometimes known as "king of bitters," is a member of the Acanthaceae family. It's commonly used to treat sore throats, flu, and upper respiratory tract infections in China, India, Thailand, and Malaysia. In numerous studies, andrographolide, a significant bioactive chemical ingredient of the plant, has shown anticancer potentialIn experimental models of asthma, stroke, and arthritis, andrographolide and its derivatives show anti-inflammatory properties. Pharmaceutical chemists have recently synthesized a number of andrographolide derivatives with important pharmacological properties such as anti-inflammatory, antibacterial, anticancer, antidiabetic, anti-HIV, antifeedant, and antiviral properties. The summary of the effects of andrographolide on cardiovascular illness, platelet activation, infertility, and NF-B activation in this research is remarkable. In traditional Indian and Chinese medicine, a variety of plant items were frequently recommended as medications. We investigate the potency of four selected phytochemicals from the A. paniculata plant, namely andrographolide (AGP1), 14-deoxy 11,12-didehydro andrographolide (AGP2), neoandrographolide (AGP3), and 14-deoxy andrographolide (AGP4), against four key targets, including three non-structural protein (3 L main protease (3CLpro), Papain like proteinase (PLpro) and RNA-directed RNA polymerase (RdRp)) and a structural protein (spike protein (S)) of the virus which are responsible for replication, transcription and host cell recognition. This paper intended to know the mechanism of A.Panniculata bioactive compounds and its effect to relief the upper respiratory tract infection symptomps.

Antcamphin M Inhibits TLR4-Mediated Inflammatory Responses by Upregulating the Nrf2/HO-1 Pathway and Suppressing the NLRP3 Inflammasome Pathway in Macrophages.

The medicinal mushroom Antrodia cinnamomea has been demonstrated to have anti-inflammatory properties. However, the bioactive compounds in A. cinnamomea need further investigation. The present study aimed to understand the mechanism of action of antcamphin M, an ergostanoid isolated from A. cinnamomea mycelium and to clarify its underlying mechanisms of action. RAW264.7 cells were pretreated with the indicated concentrations of antcamphin M, prior to stimulation with lipopolysaccharide (LPS). Cell viability, production of nitric oxide (NO), prostaglandin E2 (PGE, cytokines, and chemokines, as well as the inflammation-related signaling pathways were investigated. The study revealed that antcamphin M significantly decreased the LPS-induced production of NO, PGE2, pro-inflammatory cytokines, and keratinocyte chemoattractant CXCL1 (KC), along with the levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins without significant cytotoxicity, indicating it had a better anti-inflammatory activity than that of gisenoside Rb1 and Rg1. Additionally, antcamphin M significantly inhibited the activation of MAPKs (p38, ERK, and JNK), NFB, and components of the NLRP3 inflammasome (NLRP3, ASC, and caspase-1) signaling pathways and also increased the levels of nuclear factor erythroid-2-related factor (Nrf2) and heme oxygenase-1 (HO-1). These findings suggest that antcamphin M possesses potent anti-inflammatory activities and could be a potential candidate for the development of anti-inflammatory drugs.

Abstract 570: Cystathionine γ-Lyase Modulates Flow-dependent Vascular Remodeling

Disturbed flow causes endothelial dysfunction and development of atherosclerotic lesions. The gaseous signaling molecule H 2 S and cystathionine γ-lyase (CSE), its major enzymatic source in the vasculature, protect against cardiovascular diseases including atherosclerosis, peripheral artery disease, and cardiac ischemia in a nitric oxide (NO) dependent manner. Therefore, we sought to investigate the role of CSE in the endothelial response to disturbed flow. Wild-type C57Bl/6 (WT) and CSE knockout (CSE-/-) mice underwent partial carotid ligation to induce disturbed flow in the left carotid with the right carotid serving as an internal control. Additionally, endothelial cells isolated from WT and CSE-/- mice were exposed to oscillatory flow, a model of disturbed flow, in vitro. While disturbed flow decreased endothelial CSE mRNA expression, CSE protein expression showed strong induction under disturbed flow conditions both in vitro and in vivo. This induction correlated with enhanced free sulfide and sulfane sulfur production in WT but not in CSE-/- mice. Intimal mRNA isolated 2 days post-ligation showed increased VCAM-1 and ICAM-1 expression in WT mice which was prevented in CSE-/- mice. Similarly, endothelial cells isolated from CSE-/- mice show reduced NF-B activation and proinflammatory gene expression in response to oscillatory flow in vitro. Morphometric analysis of carotid arteries collected 7 days post-ligation revealed reduced macrophage infiltration and medial thickening in the ligated carotid of CSE-/- mice. Interestingly, ligation increased the carotid nitrite level in WT mice but not in CSE-/- mice. However, nitrite level of the non-ligated carotid was significantly higher in the CSE-/- mice compared to WT mice. Shear induced phosphorylation of eNOS Ser1179 in vitro was not different between WT and CSE knockout endothelial cells, suggesting alternative regulatory mechanisms. Collectively, CSE in mouse carotid arteries plays a critical role in flow dependent vascular remodeling, which may be mediated by CSE derived free sulfide and sulfane sulfur. CSE deficiency completely inhibits disturbed flow-induced NF-κB activation and macrophage recruitment, consistent with the role of inflammation in vascular remodeling.

Telomerase extratelomeric effects in hepatocellular carcinoma: Molecular targeting to impair tumoral networks in HCC

Background: The molecular mechanisms involved in HCC tumorigenesis are still poorly understood. Telomerase activation is one of the earliest events in HCC tumoral transformation. Telomerase is responsible for telomeres elongation leading to indefinite cell proliferation. Recently telomerase has been linked to several mechanisms characteristics of tumoral cells. It has been hypothesized a crosstalk between Telomerase and NFB and WNTcatenin pathways not yet experimentally demonstrated in HCC. Aim: To elucidate the molecular interactions between the Telomerase, NFB and WNTcatenin pathways. Methods: Telomerase catalytic subunit (hTERT) was silenced with 25nM of siRNA for 72h. To assess NFB activation, 200ng of total DNA consisting of IL-8 promoter−1498/ +44 hIL-8/Luc and pRL-CMV (ratio 49:1) were transfected into Huh7 and JHH6. Mutant −162/ +44 hIL-8 NFB/Luc was used as negative control. 200ng of M51 Super 8XTOP-FLASH and pRL-CMV (ratio 49:1) were transfected to evaluate the WNTcatenin pathway activation. FOP-FLASH was used as negative control. Cell proliferation, cell viability, cell cycle arrest and apoptosis were also evaluated after hTERT silencing. Results: hTERT silencing resulted in a decreased mRNA expression (p<0.001) and enzymatic activity (p<0.001). TOPFLASH/FOPFLASH reporter activity was inhibited after hTERT silencing (p<0.01) even in presence of 10mM liCl. Luciferase reporter assays showed a reduced activation of the NFB pathway (p<0.01) corresponding to the decreased hTERT expression and activity. A significant inhibition of LPS (5 g/mL) induced signal was also observed in silenced cells (p<0.001). The reduced NFB and catenin activity impaired the downstream pathway gene activation of cycD1 (p<0.05), birc5 (p<0.05), cmyc (p<0.01) resulting in a cell cycle arrest (p<0.001 for Huh7; p<0.01 for JhH6), and an increased apoptosis (p<0.01 for Huh7; p<0.001 for JHH6). Conclusions: This study demonstrates the important coactivatory role of Telomerase in NFB and WNTcatenin pathways inHCC. hTERT inhibitionaffects not only telomeresmaintenance but also cancer cell pathways. SupportedbyPAR-FSC 2007-13decree n◦3984/LAVFOR.ISTR/2014.

참당귀 추출분말이 in vitro and in vivo model에서 MMPs 조절 기전

골관절염에 대한 참당귀 추출분말 (AGE)의 치료효과를 검토하고자 토끼연골세포와 흰쥐의 monosodium iodoacetate (MIA)로 유발된 골관절염 부위에서 시료를 채취하여 MMPs의 발현에 대한 AGE의 억제 효능을 검토하였다. 고 농도의 AGE (50 &#x3BC;g/mL) 투여에서 세포독성은 관찰되지 않았으며 면역 및 염증반응과 관련된 여러 인자의 전사인자인 NF-&#x03BA;B 활성화를 효과적으로 억제시켰다. 토끼연골 세포에서 MMP-2와 MMP-9의 활성을 확인해본 결과 AGE는 MMP-9의 활성을 효과적으로 억제시키는 것을 확인하였다. AGE를 토끼연골세포에 처리하여 분석한 결과, 주요성분인 decursin과 decursinol angelate가 3.62&#xB1;0.47 &#x3BC;g/mg protein와 2.14&#xB1;0.36 &#x3BC;g/mg protein으로 검출되었다. 동물실험을 위하여, 골관절염은 MIA를 흰쥐 무릎관절에 처리하여 동물모델을 만들었으며, 매일 25, 50와 100 mg/kg의 AGE를 3주 동안 먹인 결과 흰쥐의 연골 조직에서 MMPs가 억제되는 것을 확인하였다. 연골조직으로부터 RT-PCR을 통해 collagen Type I, collagen Type II, aggrecan 및 MMPs (MMP-3, MMP-9, MMP-13)의 mRNA를 확인해본 결과 AGE는 collagen Type I, collagen Type II, aggrecan은 증가시키며 MMPs는 감소시키는 효과를 얻었다. 결론적으로 AGE는 MMPs 억제를 통하여 골관절염의 발생을 억제한다. The precise mechanism underlying the therapeutic efficacy of an extraction powder of Angelica gigas (AGE) for the treatment of degenerative osteoarthritis was investigated in primary cultured rabbit chondrocytes and in a monosodium-iodoacetate (MIA)-induced osteoarthritis rat model. The treatment with AGE (50 &#x3BC;g/mL) effectively inhibited NF-B activation. The anti-inflammatory mechanism was clarified by gelatin zymography and western blotting measurements of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities. The AGE (50 &#x3BC;g/mL) treatment significantly reduced MMP-9 activity. The constituents of AGE&#x2014; decursinol, decursin, and decursinol angelate&#x2014;were determined by LC-MS/MS after a 24 hr treatment of rabbit chondrocytes. The contents of the major products, decursin and decursinol angelate, were 3.62&#xB1;0.47 and 2.14 &#xB1;0.36 &#x3BC;g/mg protein, respectively in AGE-treated (50 &#x3BC;g/mL) rabbit chondrocytes. An in vivo animal study on rats fed a diet containing 25, 50, and 100 mg/kg AGE for 3 weeks revealed a significant inhibition of the MMPs in the MIA-induced rat articular cartilage. The genetic expression of arthritic factors in the articular cartilage was examined by RT-PCR of collagen Type I, collagen Type II, aggrecan, and MMP (MMP3, MMP-9, MMP13). Specifically, AGE up-regulated the expression of collagen Type I, collagen Type II, and aggrecan and inhibited MMP levels at all tested concentrations. Collectively, AGE showed a strong specific site of action on MMP regulation and protected against the degeneration of articular cartilage via cellular regulation of MMP expression both in vitro and in vivo.

Immunomodulation of Inflammatory Markers in Activated Macrophages by Leaf Extracts of Gingko Biloba

Much recent attention has been given to traditional medicines and natural products with potential and promising anti-inflammatory properties. Leaf extract of Ginkgo biloba GbE-761, possesses several clinical beneficial effects and is now used in a broad spectrum of pharmacological actions. However, mechanisms associated with its anti-inflammatory effect are not very much clear. Previously we established that GbE-761 exerts a neuroprotective effect against ischemic brain injury through an anti-apoptotic mechanism. In this study we investigated the anti-inflammatory effect of GbE-761 in LPS-activated murine macrophage. Our results demonstrate that GbE-761 potently inhibited LPS-induced NO and PGE2 production and these observations were consistent with the inhibition in the protein and mRNA expression levels of inducible iNOS and COX-2 enzymes in a dose dependent manner. Not only this, GbE-761 attenuated the production of pro-inflammatory cytokines like IL-1 , IL-6 and TNFin LPS-activated murine macrophages in concentration dependent manner. In addition we reported inhibition in the protein and mRNA expression levels of IL-1 , IL-6 and TNF. Furthermore, GbE-761 inhibited the NFB activation induced by LPS. Together these results suggest that anti-inflammatory properties of GbE-761 extract might be the results of inhibition of iNOS and COX-2 by upregulating anti-oxidative enzymes and inhibition of pro-inflammatory cytokines (IL-1 , IL-6 and TNF) expression through the down-regulation of NF-kB activation in murine macrophages.

Demographics and disparities in sub-sternal goiters in the united states: a distinct entity

INTRODUCTION: Over 50% of papillary thyroid cancers carry the BRAFV600E mutation, which triggers constitutive activation of the MAPK pathway. Activation of NF-B is also associated with thyroid cancer tumorigenesis. We aimed to examine the effectiveness of the dual targeting of MAPK and NF-B signaling pathways using clinically safe selective inhibitors, the MEK inhibitor AZD-6244 and the NF-B-targeting proteasome inhibitor Bortezomib, for aggressive thyroid cancers.

Poxvirus Protein MC132 from Molluscum Contagiosum Virus Inhibits NF-B Activation by Targeting p65 for Degradation.

Molluscum contagiosum virus (MCV) is unique in being the only known extant, human-adapted poxvirus, yet to date, it is very poorly characterized in terms of host-pathogen interactions. MCV causes persistent skin lesions filled with live virus, but these are generally immunologically silent, suggesting the presence of potent inhibitors of human antiviral immunity and inflammation. Fewer than five MCV immunomodulatory genes have been characterized in detail, but it is likely that many more remain to be discovered given the density of such sequences in all well-characterized poxviruses. Following virus infection, NF-B activation occurs in response to both pattern recognition receptor (PRR) signaling and cellular activation by virus-elicited proinflammatory cytokines, such as tumor necrosis factor (TNF). As such, NF-B activation is required for virus detection, antiviral signaling, inflammation, and clearance of viral infection. Hence, we screened a library of MCV genes for effects on TNF-stimulated NF-B activation. This revealed MC132, a unique protein with no orthologs in other poxviral genomes, as a novel inhibitor of NF-B. Interestingly, MC132 also inhibited PRR- and virus-activated NF-B, since MC132 interacted with the NF-B subunit p65 and caused p65 degradation. Unbiased affinity purification to identify host targets of MC132 revealed that MC132 acted by targeting NF-B p65 for ubiquitin-dependent proteasomal degradation by recruiting p65 to a host Cullin-5/Elongin B/Elongin C complex. These data reveal a novel mechanism for poxviral inhibition of human innate immunity and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity to persist in skin lesions.

The Emotional Pictures Application to Subthreshold Depression by Evaluating REP Changes

Purpose: Eupatorium fortunei has long been used to treat nausea and poor appetite, and has been prescribed as a diuretic and detoxifying drug in Chinese medicine. Recent studies have demonstrated that E. fortunei possesses antibacterial, anti-oxidant, and anti-diabetic activities, as well as cytotoxicity to human leukemia cells. However, at non-toxic concentrations, the effects of an aqueous extract of E. fortunei (WEF) on themetastatic and angiogenic potential ofmalignant tumor cells have not been reported. Methods: The inhibitory effect of WEF on the metastatic and angiogenic properties of malignant tumor cells in vitro and in vivo was examined. Furthermore, detailed underlying mechanism of the anti-metastatic and angiogenic activity of WEF was elucidated. Results: We found that WEF suppressed the metastatic properties, including anchorage-independent colony formation, migration, and invasion, by downregulating the proteolytic activity of MMP-9. NFB activation and the phosphorylation of p38 and JNKwere reduced significantly byWEF. Additionally, WEF inhibited tumor-induced angiogenesis markedly, affecting HUVEC migration, tube formation by HUVECs, and microvessel sprouting from rat aortic rings via a reduction in VEGF in tumors. In a pulmonary metastasis model, daily administration of WEF at 50mg/kg markedly decreased metastatic colonies of intravenously injected B16F10 cells on the lung surface in C57BL/6J mice. Further, none of the WEFadministered mice exhibited systemic toxicity. Conclusion: Taken together, our results indicate that WEF is a potential therapeutic herbal product that may be useful for controlling malignant metastatic cancer. Contact: Aeyung Kim, aykim71@kiom.re.kr

Abstract 1727: Development of novel TAK1 inhibitors for pancreatic cancer

Abstract Transforming growth factor- (TGF-) activation kinase (TAK1, MAP3K7) is a member of MAPKKK family [1]. It was well established that TAK1 plays a key role in both TNFα and IL-1 pathway. Knock down of TAK1 impaired the activation of IKK and JNK by TNFα and IL-1 [2, 3]. NF-B activation induced by TNFα and IL-1 are severely impaired in TAK1 knock out MEF cells [4]. TAK1 acts as the kinase of IKK and therefore regulates NF-κB activation. Our previous work has demonstrated that TAK1 is upregulated and plays an important role in chemotherapeutic drug resistance in pancreatic cancer cells. Block TAK1 activity can reverse pancreatic cancer cell resistance to chemotherapeutic drug and reduce tumor size in Orthotopic Xenograft models [5]. These findings underscore the significance of TAK1 in mediating drug resistance. Strategies to target TAK1 may play a critical role in the treatment of this deadly malignancy. Utilizing computational-based structural modeling and a recently published crystal structure of TAK1, compounds were designed to inhibit the ATP-binding pocket of the TAK1. Synthetic templates were proposed for building a urea based library of potential inhibitors. A multiple stage docking involving both pre- and post-filtering and progressively more stringent docking criteria led to approximately 1,000 structures. The resulting scores from two docking programs were fed into workflow software that combined the results and calculated ADME properties. To provide better consistency in scoring and less bias in setting the filtering cutoffs, we also included docking decoys and utilized a more statistically relevant Z-score. After application of a final binding mode filter, there were 22 structures that were selected and ranked. A seven-step synthesis was developed and a focused library of compounds was synthesized from those top-ranked compounds. Note: This abstract was not presented at the meeting. Citation Format: Paul J. Chiao, Zhuonan Zhuang, Qianghua Xia, Paul T. Schuber, Duoli Sun, Zhenghong Peng, David S. Maxwell, William G. Bornmann. Development of novel TAK1 inhibitors for pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1727. doi:10.1158/1538-7445.AM2014-1727

Tenofovir induced renal damage is associated with activation of NF-κB inflammatory signaling pathway and PARP overactivation

Background Tenofovir is recommended as a first-line therapy in HIV treatment. However, its long term use is associated with proximal tubular injury and renal dysfunction. Tenofovir has been shown to target the proximal tubular mitochondria, resulting in severe mitochondrial injury and overproduction of ROS and RNS. ROS are potent stimuli for the activation of NFB, a key transcription factor, which is known to mediate inflammation.The NFB response proinflammatory genes include iNOS, COX, TNFa, and others such as PARP-1. In the present study, we investigated whether NFB inflammatory signaling pathway plays a role in tenofovir nephrotoxicity.

Abstract 256: Activation of Cell Surface GRP78 by Anti-GRP78 Autoantibodies Accelerates Lesion Development by Promoting Endothelial Cell Activation

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is a molecular chaperone localized to the endoplasmic reticulum (ER) in healthy cells that functions to assist in the correct folding of newly synthesized proteins and to prevent aggregation of folding intermediates. In addition, GRP78 is also present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a membrane signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies isolated from the serum of cancer patients. We have demonstrated previously that high levels of anti-GRP78 autoantibodies accelerate lesion development in apoE -/- mice. However, the role of anti-GRP78 autoantibody activation of cell surface GRP78 on endothelial cells and how this contributes to atherogenesis is unknown. The objective of this study is to identify factors that mediate surface GRP78 expression on endothelial cells and to determine the mechanism by which surface GRP78 activation on endothelial cells contributes to atherogenesis. Here we demonstrate that induction of ER stress by tunicamycin increased surface GRP78 expression in cultured human aortic endothelial cells. We also show that activation of surface GRP78 on endothelial cells by anti-GRP78 autoantibodies significantly increases gene expression of the adhesion molecules ICAM-1 and VCAM-1. Pretreatment of these cells with a calcium chelator or an inhibitor of NF-B activation attenuated the anti-GRP78 autoantibody-induced promotion of ICAM-1 and VCAM-1. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation, an important initiating event in atherogenesis. These findings further our understanding of the role of anti-GRP78 autoantibodies and the activation of surface GRP78 in endothelial cell function and lesion development. Furthermore, inhibiting the interaction of anti-GRP78 autoantibodies with surface GRP78 could present a novel therapeutic strategy to modulate lesion growth and thereby reduce the risk for atherosclerosis and cardiovascular disease.

Pathobiology of Paget's Disease of Bone.

Paget's disease of bone is characterized by highly localized areas of increased bone resorption accompanied by exuberant, but aberrant new bone formation with the primary cellular abnormality in osteoclasts. Paget's disease provides an important paradigm for understanding the molecular mechanisms regulating both osteoclast formation and osteoclast-induced osteoblast activity. Both genetic and environmental etiologies have been implicated in Paget's disease, but their relative contributions are just beginning to be defined. To date, the only gene with mutations in the coding region linked to Paget's disease is sequestosome-1 (SQSTM1), which encodes the p62 protein, and these mutations lead to elevated cytokine activation of NF-B in osteoclasts but do not induce a "pagetic osteoclast" phenotype. Further, genetic mutations linked to Paget's appear insufficient to cause Paget's disease and additional susceptibility loci or environmental factors may be required. Among the environmental factors suggested to induce Paget's disease, chronic measles (MV) infection has been the most studied. Expression of the measles virus nucleocapsid gene (MVNP) in osteoclasts induces pagetic-like osteoclasts and bone lesions in mice. Further, mice expressing both MVNP in osteoclasts and germline mutant p62 develop dramatic pagetic bone lesions that were strikingly similar to those seen in patients with Paget's disease. Thus, interactions between environmental and genetic factors appear important to the development of Paget's disease. In this article we review the mechanisms responsible for the effects of mutant p62 gene expression and MVNP on osteoclast and osteoblast activity, and how they may contribute to the development of Paget's disease of bone.

HTLV-1 Tax disrupts the host epigenome by interacting with a Polycomb group protein EZH2

ATL is a highly aggressive T-cell neoplasm caused by HTLV-1. We have recently demonstrated that EZH2, a catalytic enzyme of histone H3K27 methylation, is overexpressed in ATL cells, which contributes to persistent NF-B activation by repressing a tumor-suppressive miRNA, miR-31. This suggests that epigenetic abnormalities are closely associated with the molecular hallmarks of leukemic cells. However, the mechanisms by which epigenetic disruption is caused and sustained in HTLV-1 infected cells remain to be clarified. In the present study, we first found that Tax directly interacts with EZH2 in HTLV-1 infected cells. Given that epigenetic state is directly affected by cell lineage-dependent transcription factors and genetic background, we assessed the functional interconnection between HTLV-1 Tax and the histone methyltransferases in an appropriate model. Using a lentivirus vector, we introduced tax gene into peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells from healthy donors and established Tax-transduced T-cells. In this model, we observed overexpression of EZH2, aberrant accumulation of H3K27 trimethylation, and epigenetic silencing of p21 cip1/waf1 ,a ll of which have been observed in primary ATL cells. In addition, EZH2 inhibition reduced the viability of Tax-transduced T-cells. Our results strongly suggest that Tax epigenetically affects gene expression through its interaction with EZH2, and that Tax-dependent epigenetic abnormalities may be involved in determining the molecular characteristics of HTLV-1-infected cells as well as ATL cells. Since epigenetic marks are potentially reversible, development of genuine epigenetic-targeted drugs will hold great promise in treatment of HTLV-1-related diseases.

NF-&amp;kappa;B as a Target for Modulating Inflammatory Responses

The inflammatory response is of major importance in host defence, but is involved in all acute and chronic diseases. Multiple inflammatory cells and molecules are involved. Among the latter, the Nuclear Factor -κB (NF-κB) has been found to be most important and present in all cell types. NF-B regulates the expression of a large number of genes involved in inflammation. NF-κB plays a key role in the orchestration of the multifaceted inflammatory response, not only in the first pro-inflammatory phase, but also later in the regulation of the resolution of inflammation, when anti-inflammatory genes are expressed and apoptosis is induced. The review describes NF-κB and its two pathways: the canonical, mediated by the p65 and p50 subunits, and the non-canonical, mediated by the subunits RelB, p52 and p50. The relevance of the kinases and interactions leading to NF-κB activation is considered in different primary cells (i.e. macrophages, dendritic cells, fibroblasts, cells from inflammatory tissues), together with the response induced and the ligand involved. Then we overview the different steps to NF-B activation that can be targeted (IKKs, IκBα or NF-κB subunits themselves) with various technologies available i.e. small molecules peptides or nucleic acids. A rationale is provided for possible targets to consider, in the light that NF-κB signaling pathways regulates both pro-inflammatory and anti-inflammatory responses. The possibility of using NF-κB targeted dendritic cells in immunotherapy is considered.

2.47 p53 Homolog p63 Promotes Apoptosis and Chemosensitivity in Chronic Lymphocytic Leukemia B-Cells

in cell culture medium were measured by enzyme-linked immunosorbent assay (ELISA) and activation of STAT3 and NFB was indicated by the ratio of p-STAT3/STAT3 and p-NFB/NFB. Patients with high levels of p-STAT3/STAT3 and/or p-NFB/ NFB had higher levels of IL-6 production. The level of IL-6 production significantly correlated with STAT3 activation (R 0.856, p 0.001) and NFB activation (R 0.815, p 0.001). Inhibition of constitutive STAT3 and NFB activation by the STAT3 phosphorylation inhibitor, diphenylporphyrin, and the NFB inhibitor, caffeic acid phenethyl ester (CAPE), blocked IL-6 production by 40% on average (p 0.004, p 0.003), active STAT3 or NFB significant increase IL-6 secretion. Analyzing the level of autocrine IL-6 production with in vitro spontaneous apoptosis in CLL cells from 38 patients, we found that patients with higher levels of IL-6 production (more than median) had significantly less apoptosis compared with those below the median (23% vs. 42.2% in average, p 0.0001). Correlation between the levels of autocrine IL-6 production and cell viability in 38 patients (CLL cells were incubated for 48 hours in vitro) was significant (R 0.72, p 0.0001). High IL-6 production was also associated with a shorter average time to first treatment (0.5 vs. 3 years, p 0.0023). This study demonstrates that autocrine IL-6 production correlates with STAT3 and NFB activation and apoptosis resistance in CLL. Furthermore, higher IL-6 production is associated with biological markers of poor prognosis and earlier treatment.

2.14 The Kinetics of Phosphorylation of the CD5 Regulatory Domain in Chronic Lymphocytic Leukemia B Cells Reflects Ongoing Activation of the B-Cell Receptor

gene transcription. Similarly, the ataxia telangiectasia mutated kinase (ATM), which senses double strand breaks, is required for DNA damage–induced activation of NFB in breast cancer cells. Furthermore, other reports showed that the DNA damage–signalling enzyme, DNA-dependent protein kinase (DNA-PK) activates NFB by regulating MEK/ERK signalling, the IKK complex, and the p50 subunit. We previously demonstrated that inhibition of DNA-PK chemosensitises CLL cells and that DNA-PK is highly expressed in CLL cells from patients with a poor prognosis. These data collectively led us to hypothesise that, like ATM, part of the DNA-PK–mediated DNA damage response involves regulation of NFB activity. First, we quantified p50, p65, p52, c-rel, and Rel-B subunits in 68 CLL samples (standardised enzyme-linked immunosorbent assay) to determine their role in disease progression and correlation with prognostic markers and resistance. High levels of p50 and p65 subunits correlated with shorter overall survival (hazard ratio 5.8 and 5.7, respectively) and identified patients harbouring del(17p) or del(11q). High levels of p52 and c-Rel had no significant effect on outcome, but in 17 cases of known ATM mutation status, Rel-B levels were significantly higher (p 0.005) in patients with ATM mutations. Increased NFB activity correlated with ex vivo chemoresistance (XTT assay) to fludarabine (p 0.001), chlorambucil (p 0.0001), and mitoxantrone (p 0.03). Of note, in 4 cases, drug-induced activity of the p50 and p65 NFB subunits in fresh CLL cells was abrogated by at least 50% by DNA-PK inhibition (using 1 M of NU7441), indicating that catalytic activity of DNAPKcs is required for drug-induced NFB signalling. In support of this concept, human glioblastoma cells deficient in DNA-PKcs (M059J) were 3-fold more sensitive to the NFB antagonist, BAY 11-7082, than the DNA-PKcs–proficient counterpart (M059J Fus1). These data indicate that in addition to its role in double strand break repair, DNA-PKcs can mediate chemosensitisation by decreasing NFB activity. Pilot data using chromatin immunoprecipitation indicate that NFB subunits are capable of binding to the promoter region of DNA-PKcs, and future studies using knockdown of DNA-PKcs will elucidate the role of DNA-PK on NFB– dependent gene transcription. Collectively, these data suggest that targeting DNA damage activated NFB activity represents a novel approach to overcoming chemoresistance in patients with CLL who have increased levels of the p65 and p50 NFB subunits.