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Sample records for death receptor-4 expression

  1. TRAIL Death Receptor-4 Expression Positively Correlates With the Tumor Grade in Breast Cancer Patients With Invasive Ductal Carcinoma

    SciTech Connect

    Sanlioglu, Ahter D.; Korcum, Aylin F.; Pestereli, Elif; Erdogan, Gulgun; Karaveli, Seyda; Savas, Burhan; Griffith, Thomas S.; Sanlioglu, Salih V.

    2007-11-01

    Purpose: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells, and a number of clinical trials have recently been initiated to test the safety and antitumoral potential of TRAIL in cancer patients. Four different receptors have been identified to interact with TRAIL: two are death-inducing receptors (TRAIL-R1 [DR4] and TRAIL-R2 [DR5]), whereas the other two (TRAIL-R3 [DcR1] and TRAIL-R4 [DcR2]) do not induce death upon ligation and are believed to counteract TRAIL-induced cytotoxicity. Because high levels of DcR2 expression have recently been correlated with carcinogenesis in the prostate and lung, this study investigated the importance of TRAIL and TRAIL receptor expression in breast cancer patients with invasive ductal carcinoma, taking various prognostic markers into consideration. Methods and Materials: Immunohistochemical analyses were performed on 90 breast cancer patients with invasive ductal carcinoma using TRAIL and TRAIL receptor-specific antibodies. Age, menopausal status, tumor size, lymph node status, tumor grade, lymphovascular invasion, perineural invasion, extracapsular tumor extension, presence of an extensive intraductal component, multicentricity, estrogen and progesterone receptor status, and CerbB2 expression levels were analyzed with respect to TRAIL/TRAIL receptor expression patterns. Results: The highest TRAIL receptor expressed in patients with invasive ductal carcinoma was DR4. Although progesterone receptor-positive patients exhibited lower DR5 expression, CerbB2-positive tissues displayed higher levels of both DR5 and TRAIL expressions. Conclusions: DR4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.

  2. Real-time detection of cellular death receptor-4 activation by fluorescence resonance energy transfer.

    PubMed

    Dereli-Korkut, Zeynep; Gandhok, Harmeet; Zeng, Ling Ge; Waqas, Sidra; Jiang, Xuejun; Wang, Sihong

    2013-05-01

    Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL-DR4 in live cells in real-time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4-CFP and DR4-YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4-CFP and DR4-YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4-CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4-CFP/YFP reporter cells exhibited a colocalized expression of DR4-CFP and DR4-YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi-fluorescence microscopy for FRET analysis. The real-time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time-lapse fluorescence microscopy. Therefore, DR4-CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti-cancer drug screening to identify compounds that are capable of activating TRAIL pathways.

  3. Coxsackievirus B3 induces viral myocarditis by upregulating toll-like receptor 4 expression.

    PubMed

    Zhao, Zhao; Cai, Tian-Zhi; Lu, Yan; Liu, Wen-Jun; Cheng, Man-Li; Ji, Yu-Qiang

    2015-04-01

    In the present study, we investigated the potential pathogenesis of coxsackievirus B3 (CVB3)-induced viral myocarditis and the promising protective effect of silencing RNA (small interfering RNA, siRNA). One hundred and twenty mice were included in the study, and 30 mice were intraperitoneally inoculated with CVB3 to establish an acute viral myocarditis model. The survival rate was observed for the CVB3-infected mouse model (MOD), and myocardial injury was examined by HE (hematoxylin and eosin) staining assay. Real-time PCR (RT-PCR) and Western blot assay were selected to detect the toll-like receptor 4 (TLR4) expression in myocardial tissues. The TLR4 gene was silenced for the MOD mice, and the effects of this treatment were observed. The results indicate that the expression of TLR4 mRNA and the protein significantly and persistently increased during the progression of CVB3-induced myocarditis. The activities of cardiac enzymes including CK, LDH, AST, and CK-MB were also enhanced in CVB3-induced myocardial tissues. Interestingly, when the TLR4 gene was silenced, the CVB3-induced TLR4 production was significantly decreased and the severity of myocarditis was significantly lessened. In conclusion, CVB3 may induce viral myocarditis by upregulating toll-like receptor 4 expression. The viral myocarditis can be ameliorated by silencing the TLR4 gene in the CVB3 viral myocarditis model, which may be a feasible therapeutic method for treatment of viral myocarditis.

  4. Thrombomodulin promotes diabetic wound healing by regulating toll-like receptor 4 expression.

    PubMed

    Cheng, Tsung-Lin; Lai, Chao-Han; Chen, Po-Ku; Cho, Chia-Fong; Hsu, Yun-Yan; Wang, Kuan-Chieh; Lin, Wei-Ling; Chang, Bi-Ing; Liu, Shi-Kai; Wu, Yu-Ting; Hsu, Chao-Kai; Shi, Guey-Yueh; Wu, Hua-Lin

    2015-06-01

    Keratinocyte-expressed thrombomodulin (TM) and the released soluble TM (sTM) have been demonstrated to promote wound healing. However, the effects of high glucose on TM expression in keratinocytes and the role of TM in diabetic ulcer remain unclear. In this study, we demonstrated that expressions of TM and Toll-like receptor 4 (TLR4) were both downregulated in high-glucose cultured human keratinocytes and in skin keratinocytes of diabetic patients. In addition, the wound-triggered upregulation of TM and sTM production was abolished in both high-glucose cultured human keratinocytes and streptozotocin-induced diabetic mouse skin. Furthermore, supplementation of recombinant sTM could increase TLR4 expression and promote cutaneous wound healing in both high-glucose cultured human keratinocytes and diabetic mice. However, in Tlr4-deleted mice, which exhibited delayed wound healing, the therapeutic benefit of recombinant sTM was abrogated. Moreover, our results showed that tumor necrosis factor-α (TNF-α) expression in keratinocytes was dose-dependently upregulated by glucose, and TNF-α treatment downregulated the expression of TM and TLR4. Taken together, high-glucose environment reduces the expression of TM and TLR4 in keratinocytes possibly through the action of TNF-α, and recombinant sTM can increase the TLR4 expression and promote wound healing under diabetic condition.

  5. A novel splice variant of folate receptor 4 predominantly expressed in regulatory T cells

    PubMed Central

    2012-01-01

    Background Regulatory T cells (Tregs) are required for proper maintenance of immunological self-tolerance and immune homeostasis. Folate receptor 4 (FR4) is expressed at high levels in transforming growth factor-beta (TGF-β)-induced Tregs and natural Tregs. Moreover, antibody-mediated targeting of FR4 is sufficient to mediate Treg depletion. Results In this study, we describe a novel FR4 transcript variant, FR4D3, in which exon 3 is deleted. The mRNA of FR4D3 encodes a FR4 variant truncated by 189 bp. FR4D3 was found to be predominantly expressed in CD4+CD25+ Treg cells. Overexpression of FR4D3 in CD4+CD25+ Treg cells in vitro stimulated proliferation, which may modulate the ability of these cells to bind and incorporate folic acid. Conclusions Our results suggested that high levels of FR4D3 may be critical to support the substantial proliferative capacity of Treg cells. PMID:22694797

  6. (68)Ga-Pentixafor-PET/CT for Imaging of Chemokine Receptor 4 Expression in Glioblastoma.

    PubMed

    Lapa, Constantin; Lückerath, Katharina; Kleinlein, Irene; Monoranu, Camelia Maria; Linsenmann, Thomas; Kessler, Almuth F; Rudelius, Martina; Kropf, Saskia; Buck, Andreas K; Ernestus, Ralf-Ingo; Wester, Hans-Jürgen; Löhr, Mario; Herrmann, Ken

    2016-01-01

    Chemokine receptor-4 (CXCR4) has been reported to be overexpressed in glioblastoma (GBM) and to be associated with poor survival. This study investigated the feasibility of non-invasive CXCR4-directed imaging with positron emission tomography/computed tomography (PET/CT) using the radiolabelled chemokine receptor ligand (68)Ga-Pentixafor. 15 patients with clinical suspicion on primary or recurrent glioblastoma (13 primary, 2 recurrent tumors) underwent (68)Ga-Pentixafor-PET/CT for assessment of CXCR4 expression prior to surgery. O-(2-(18)F-fluoroethyl)-L-tyrosine ((18)F-FET) PET/CT images were available in 11/15 cases and were compared visually and semi-quantitatively (SUVmax, SUVmean). Tumor-to-background ratios (TBR) were calculated for both PET probes. (68)Ga-Pentixafor-PET/CT results were also compared to histological CXCR4 expression on neuronavigated surgical samples. (68)Ga-Pentixafor-PET/CT was visually positive in 13/15 cases with SUVmean and SUVmax of 3.0±1.5 and 3.9±2.0 respectively. Respective values for (18)F-FET were 4.4±2.0 (SUVmean) and 5.3±2.3 (SUVmax). TBR for SUVmean and SUVmax were higher for (68)Ga-Pentixafor than for (18)F-FET (SUVmean 154.0±90.7 vs. 4.1±1.3; SUVmax 70.3±44.0 and 3.8±1.2, p<0.01), respectively. Histological analysis confirmed CXCR4 expression in tumor areas with high (68)Ga-Pentixafor uptake; regions of the same tumor without apparent (68)Ga-Pentixafor uptake showed no or low receptor expression. In this pilot study, (68)Ga-Pentixafor retention has been observed in the vast majority of glioblastoma lesions and served as readout for non-invasive determination of CXCR4 expression. Given the paramount importance of the CXCR4/SDF-1 axis in tumor biology, (68)Ga-Pentixafor-PET/CT might prove a useful tool for sensitive, non-invasive in-vivo quantification of CXCR4 as well as selection of patients who might benefit from CXCR4-directed therapy.

  7. A Study of the Impact of Death Receptor 4 (DR4) Gene Polymorphisms in Alzheimer’s Disease

    PubMed Central

    Edgünlü, Tuba Gökdoğan; Özge, Aynur; Yalın, Osman Özgür; Kul, Seval; Erdal, Mehmet Emin

    2013-01-01

    Background: Excessive apoptosis is believed to play a role in many degenerative and non-degenerative neurological diseases including Alzheimer’s disease (AD). Much recent data suggest that apoptotic mechanisms may represent the missing link between Aβ deposition and proteolysis of tau protein. However, there is emerging evidence that apoptotic mechanisms may play a role in Alzheimer’s Disease pathogenesis in the absence of overt apoptosis. TNF-related apoptosis inducing ligand receptor 1 (Death Receptor 4, DR4) might impair the apoptotic signal transduction and lead to dysregulation of the homeostasis between cell survival and cell death. Aims: The aim of our study was to further investigate the relationship between genetic variants of DR4 and Alzheimer’s Disease. Study Design: Case control study. Methods: Sixty-eight patients with AD were included in the study. The control group comprised 72 subjects without signs of neurodegenerative diseases, as evidenced by the examination.DNA was extracted from whole blood using the salting-out procedure. Genotypes were identified by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR-RFLP) products. Results: We observed significant differences in the genotypic distribution of the rs6557634 polymorphism in AD patients compared with controls (p<0.05); our data suggest that the GA genotype in rs6557634 could be protective against AD (p<0.05). However, there were no significant differences between AD patients and control groups in terms of the DR4 rs20575 polymorphism (p>0.05) and the DR4 rs20576 polymorphism (p>0.05). According to haplotype analysis of the DR4 gene for rs6557634, rs20575 and rs20576 polymorphisms, GCA and GCC haplotypes might be a risk factor for AD. Also, we have shown that ACA, GGC and GGA haplotypes might be protective factors against AD. Conclusion: The present results indicate for the first time the possible contribution of the DR4 gene rs6557634, rs20575, rs20576

  8. Imaging of Chemokine Receptor 4 Expression in Neuroendocrine Tumors - a Triple Tracer Comparative Approach.

    PubMed

    Werner, Rudolf A; Weich, Alexander; Higuchi, Takahiro; Schmid, Jan S; Schirbel, Andreas; Lassmann, Michael; Wild, Vanessa; Rudelius, Martina; Kudlich, Theodor; Herrmann, Ken; Scheurlen, Michael; Buck, Andreas K; Kropf, Saskia; Wester, Hans-Jürgen; Lapa, Constantin

    2017-01-01

    C-X-C motif chemokine receptor 4 (CXCR4) and somatostatin receptors (SSTR) are overexpressed in gastro-entero-pancreatic neuroendocrine tumors (GEP-NET). In this study, we aimed to elucidate the feasibility of non-invasive CXCR4 positron emission tomography/computed tomography (PET/CT) imaging in GEP-NET patients using [(68)Ga]Pentixafor in comparison to (68)Ga-DOTA-D-Phe-Tyr3-octreotide ([(68)Ga]DOTATOC) and (18)F-fluorodeoxyglucose ([(18)F]FDG). Twelve patients with histologically proven GEP-NET (3xG1, 4xG2, 5xG3) underwent [(68)Ga]DOTATOC, [(18)F]FDG, and [(68)Ga]Pentixafor PET/CT for staging and planning of the therapeutic management. Scans were analyzed on a patient as well as on a lesion basis and compared to immunohistochemical staining patterns of CXCR4 and somatostatin receptors SSTR2a and SSTR5. [(68)Ga]Pentixafor visualized tumor lesions in 6/12 subjects, whereas [(18)F]FDG revealed sites of disease in 10/12 and [(68)Ga]DOTATOC in 11/12 patients, respectively. Regarding sensitivity, SSTR-directed PET was the superior imaging modality in all G1 and G2 NET. CXCR4-directed PET was negative in all G1 NET. In contrast, 50% of G2 and 80% of G3 patients exhibited [(68)Ga]Pentixafor-positive tumor lesions. Whereas CXCR4 seems to play only a limited role in detecting well-differentiated NET, increasing receptor expression could be non-invasively observed with increasing tumor grade. Thus, [(68)Ga]Pentixafor PET/CT might serve as non-invasive read-out for evaluating the possibility of CXCR4-directed endoradiotherapy in advanced dedifferentiated SSTR-negative tumors.

  9. Betaine inhibits Toll-like receptor 4 expression in rats with ethanol-induced liver injury

    PubMed Central

    Shi, Qing-Zhao; Wang, Lu-Wen; Zhang, Wei; Gong, Zuo-Jiong

    2010-01-01

    AIM: To test whether ethanol feeding could induce Toll-like receptor 4 (TLR4) responses, assess the hepatoprotective effect of betaine and its inhibitive effect on TLR4 in animal models of alcoholic liver injury. METHODS: Forty-eight female Sprague-Dawley rats were randomly divided into four groups as control, model, low and high dose betaine groups. Except control group, all rats were fed with high fat-containing diet plus ethanol and fish oil gavages for 8 wk. Betaine was administered intragastrically after exposure of ethanol for 4 wk. The changes of liver histology were examined. The expression of TLR4 mRNA and protein was detected by RT-PCR and Western blotting, respectively. The serum aminotransferase activity [alanine transarninase (ALT), aspartate aminotransferase (AST)], serum endotoxin, and liver inflammatory factors [tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-18 (IL-18)] were also assayed. RESULTS: Compared with control group, rats of model group developed marked liver injury, accompanied by an increase of ALT (159.41 ± 7.74 U/L vs 59.47 ± 2.34 U/L, P < 0.0001), AST (248.25 ± 1.40 U/L vs 116.89 ± 3.48 U/L, P < 0.0001), endotoxin (135.37 ± 30.17 ng/L vs 44.15 ± 7.54 ng/L, P < 0.0001), TNF-α (20.81 ± 8.58 pg/mL vs 9.34 ± 2.57 pg/mL, P = 0.0003), IFN-γ (30.18 ± 7.60 pg/mL vs 16.86 ± 9.49 pg/mL, P = 0.0039) and IL-18 (40.99 ± 8.25 pg/mL vs 19.73 ± 9.31 pg/mL, P = 0.0001). At the same time, the expression of TLR4 mRNA and protein was markedly induced in the liver after chronic ethanol consumption (1.45 ± 0.07 vs 0.44 ± 0.04, P < 0.0001; 1.83 ± 0.13 vs 0.56 ± 0.08, P < 0.0001). Compared with model group, betaine feeding resulted in significant decreases of ALT (64.93 ± 6.06 U/L vs 159.41 ± 7.74 U/L, P < 0.0001), AST (188.73 ± 1.11 U/L vs 248.25 ± 1.40 U/L, P < 0.0001), endotoxin (61.80 ± 12.56 ng/L vs 135.37 ± 30.17 ng/L, P < 0.0001), TNF-α (9.79 ± 1.32 pg/mL vs 20.81 ± 8.58 pg/mL, P = 0.0003), IFN-γ (18

  10. High expression of C-X-C chemokine receptor 4 and Notch1 is predictive of lymphovascular invasion and poor prognosis in lung adenocarcinoma.

    PubMed

    Cong, Zhuangzhuang; Wu, Haiwei; Guo, Zhong; Qin, Tao; Xu, Yang; Jing, Hua; Wang, Yanqing; Shen, Yi

    2017-06-01

    C-X-C chemokine receptor 4 and Notch1 have been shown to play oncogenic role individually. This study aimed to determine the combinatorial role of C-X-C chemokine receptor 4 and Notch1 in lung adenocarcinoma. Expression of C-X-C chemokine receptor 4 and Notch1 was detected in resected tumor samples from 185 patients with lung adenocarcinoma at stage I-IIIa by immunohistochemistry. Correlations of their immunoscores with clinicopathological characteristics and disease-specific survival were retrospectively investigated. A three-dimensional capillary-like sprouting model was established to assess the effects of C-X-C chemokine receptor 4 and Notch1 on angiogenesis in vitro. The results revealed that expression of C-X-C chemokine receptor 4 and Notch1 was elevated in lung adenocarcinoma tissues. The high co-expression of C-X-C chemokine receptor 4 and Notch1 was significantly correlated with tumor size, tumor status, nodal status, tumor stage, and lymphovascular invasion, as well as decreased disease-specific survival. Multivariate analysis showed that lymphovascular invasion (hazard ratio: 0.205, 95% confidence interval: 0.086-0.491, p < 0.0001) and co-expression of C-X-C chemokine receptor 4 and Notch1 (hazard ratio: 0.293, 95% confidence interval: 0.168-0.510, p < 0.0001) were independent indicators of poor prognosis in lung adenocarcinoma. Furthermore, Notch1 enhanced the effects of C-X-C chemokine receptor 4 to promote angiogenesis by regulating Flt1 and Flt4 in vitro. In conclusion, co-expression of C-X-C chemokine receptor 4 and Notch1 is associated with tumor progression and lymphovascular invasion and is an independent indicator of poor survival in lung adenocarcinoma. In lung adenocarcinoma patients with high C-X-C chemokine receptor 4 and Notch1 expression, simultaneous inhibition of both factors might be an effective treatment strategy.

  11. A tissue microarray study of toll-like receptor 4, decoy receptor 3, and external signal regulated kinase 1/2 expressions in astrocytoma.

    PubMed

    Lin, Chih-Kung; Ting, Chun-Chieh; Tsai, Wen-Chiuan; Chen, Yuan-Wu; Hueng, Dueng-Yuan

    2016-01-01

    Decoy receptor 3 (DcR3) functions as a death decoy inhibiting apoptosis mediated by the tumor necrosis factor receptor family. It is highly expressed in many tumors and its expression can be regulated by the MAPK/ERK signaling pathway and ERK is a vital member of this pathway. Toll-like receptor 4 (TLR4) is expressed on immune cells. Increased TLR4 expression has been associated with various types of cancers. The study was conducted to investigate the expression of DcR3, ERK1/2, and TLR4 in astrocytomas and evaluate if they are validating markers for discriminating glioblastoma from anaplastic astrocytoma in limited surgical specimen. Expression of DcR3, ERK1/2, and TLR4 was determined by immunohistochemical staining of tissue microarray from 48 paraffin-embedded tissues. A binary logistic regression method was used to generate functions that discriminate between anaplastic astrocytomas and glioblastomas. The expression of TLR4 and DcR3 was significantly higher in glioblastomas than in anaplastic astrocytomas. DcR3 could discriminate anaplastic astrocytomas from glioblastomas with high sensitivity (93.8%), specificity (90%), and accuracy (92.3%). Our results suggest that DcR3 may be a useful marker for discriminating anaplastic astrocytomas from glioblastomas.

  12. Bradykinin promotes Toll like receptor-4 expression in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Arreguín-Cano, Juan Antonio; Hernández-Bermúdez, Cristina

    2012-12-01

    Bacterial infections are a potent mechanism for enzymatic generation of kinins such as bradykinin (BK), a universal mediator for inducing inflammatory reaction by associating with the B2 receptor and stimulating liberation of arachidonic acid and synthesis of prostaglandin E2 (PGE2). In this study we evaluate the role of bradykinin in regulating the expression of TLR4 receptor in human gingival fibroblasts. We examine the ability of bradykinin to modulate inflammatory response of human gingival fibroblasts to Gram-negative components and evaluated the role of Toll-like receptors (TLR)-4 in the co-operation between bradykinin and bacterial pathogens. We show that treatment with bradykinin promotes TLR4 receptor expression in human gingival fibroblasts (HGF) and amplifies inflammatory responses to the bacterial components of Gram-negative bacteria. The TLR4 expression induced by bradykinin was blocked with Hoe 140, a B2R antagonist. When HGF cells were incubated with BK resulted of an increased in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 synthesis. Bradykinin and lipopolysaccharide, a specific TLR4 ligand stimulated COX-2 expression. In other series of experiments we found that ERK, phosphatidylinositol-3 kinase, protein kinase C and NFkB are involved in BK promoted-increased in TLR4 expression. The results demonstrate that bradykinin up-regulates the expression of TLR4 and promotes an additive increase in inflammatory responses to lipopolysaccharides.

  13. Lipopolysaccharide stimulation upregulated Toll-like receptor 4 expression in chicken cerebellum.

    PubMed

    Ansari, Abdur Rahman; Wen, Le; Huang, Hai-Bo; Wang, Ji-Xiang; Huang, Xi-Yao; Peng, Ke-Mei; Liu, Hua-Zhen

    2015-08-15

    Toll-like receptors (TLRs) play crucial roles in innate and adaptive immune responses to invading pathogens. TLR4 is responsible for the recognition of bacterial lipopolysaccharide (LPS) in different parts of central nervous system of many vertebrates. To better understand the functions of TLR4 in cerebellum of chicken, present study was designed to identify the cell types that express TLR4 during postnatal stages as well as the changes in its expression in response to LPS challenge. For this purpose, cerebella were collected from chicken aged 1, 14 and 40 days (n=7 in each group) to analyze TLR4 distribution pattern. The cerebella from 14 chickens injected with LPS or sterilizing saline were also collected at Day 14 (n=7 in each group) to investigate changes in TLR4 expression. This expression was analyzed by immunohistochemistry using an anti-TLR4 antibody. TLR4 was constitutively expressed in the Purkinje cell layer, pia mater, neurons in medulla and blood vessels in the cerebellum and LPS stimulation significantly up-regulated TLR4 expression on Day 14 in the chicken cerebellum. This study provides evidence that neurons in chicken cerebellum can express TLR4 in vivo and suggests that these neurons may play an important role in initiating a defense reaction via activation of TLR4. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Artesunate ameliorates severe acute pancreatitis (SAP) in rats by inhibiting expression of pro-inflammatory cytokines and Toll-like receptor 4.

    PubMed

    Cen, Yanyan; Liu, Chao; Li, Xiaoli; Yan, Zifei; Kuang, Mei; Su, Yujie; Pan, Xichun; Qin, Rongxin; Liu, Xin; Zheng, Jiang; Zhou, Hong

    2016-09-01

    Severe acute pancreatitis (SAP) is a severe clinical condition with significant morbidity and mortality. Multiple organs dysfunction (MOD) is the leading cause of SAP-related death. The over-release of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α is the underlying mechanism of MOD; however, there is no effective agent against the inflammation. Herein, artesunate (AS) was found to increase the survival of SAP rats significantly when injected with 3.5% sodium taurocholate into the biliopancreatic duct in a retrograde direction, improving their pancreatic pathology and decreasing serum amylase and pancreatic lipase activities along with substantially reduced pancreatic IL-1β and IL-6 release. In vitro, AS-pretreatment strongly inhibited IL-1β and IL-6 release and their mRNA expressions in the pancreatic acinar cells treated with lipopolysaccharide (LPS) but exerted little effect on TNF-α release. Additionally, AS reduced the mRNA expressions of Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) p65 as well as their protein expressions in the pancreatic acinar cells. In conclusion, our results demonstrated that AS could significantly protect SAP rats, and this protection was related to the reduction of digestive enzyme activities and pro-inflammatory cytokine expressions via inhibition of TLR4/NF-κB signaling pathway. Therefore, AS may be considered as a potential therapeutic agent against SAP.

  15. [Expression and significance of Toll like receptor 2 and Toll like receptor 4 in chronic rhinosinusitis].

    PubMed

    Wang, Xin; Ji, Wenjun; Xu, Yuan; Guo, Huamin; Zhao, Chunyuan

    2014-09-01

    To explore the role of the innate immune factors TLR2 and TLR4 in the pathogenesis of chronic rhinosinusitis (CRS) by detecting their expression in different clinical types of CRS and the normal control group. Immunohistochemistry was used to detect the expression of TLR2 and TLR4 respectively in 21 cases (chronic rhinosinusitis with nasal polyps, CRSwNP) group, 15 cases (chronic rhinosinusitis without nasal polyos, CRSsNP) group, 11 cases recurrent CRSwNP group and 13 cases control group. Positive cells were counted under the microscope artificially, Mann-Whitney U analysis was applied for the ranked data, and one-way anova analysis was adopted to analyze the experimental group and control group. (1) TLR2 and TLR4 expression had the same characteristics. Expression mainly concentrated in parts of the whole layer of epithelial basement membrane, cytoplasm of glandular cells, very few inflammatory cells such as monocytes and plasma cells in the cytoplasm, sometimes unknown cell nuclei positive expression. (2) The glandular cells were stained manual counting and color grading. TLR2 and TLR4 packet application Wilcoxon rank test Mann-Whitney U test analysis was not statistically significant (P > 0.05), measurement data within the group variance statistical difference between the groups (P < 0.05). The Nasal mucosa can produce the innate immune factors TLR2 and TLR4. The different expression of TLR2 and TLR4 in the various clinical types of CRS suggests that they play the certain role in the pathogenesis of CRS.

  16. Lithium Ameliorates LPS-Induced Astrocytes Activation Partly via Inhibition of Toll-Like Receptor 4 Expression.

    PubMed

    Li, Nana; Zhang, Xiang; Dong, Hongquan; Zhang, Susu; Sun, Jie; Qian, Yanning

    2016-01-01

    Astrocytes are critical for the development of postoperative cognitive dysfunction (POCD). In addition, astrocytes express toll-like receptors 4 (TLR4) and build up responses to innate immune triggers by releasing pro-inflammatory molecules. The pathogenesis of neurological disorders often involves the activation of astrocytes and associated inflammatory processes. Lithium, a primary drug for the treatment of bipolar disorder, has recently been suggested to have a role in neuroprotection during neurodegenerative diseases. In this study, we aimed to investigate whether lithium can ameliorate LPS-induced astrocytes activation via inhibition of TLR4 expression. Primary astrocytes cells were pretreated with lithium and stimulated with lipopolysaccharide (LPS). Cellular activation, cytokine production, and TLR4 expression, were assessed. Lithium significantly inhibited LPS-induced astrocytes activation and pro-inflammatory cytokine production, as well as LPS-induced TLR4 expression. Lithium can inhibit LPS-induced TLR4 expression and astrocytes activation. These results indicate that lithium plays an important role in astrocytes activation and neuroinflammation-related diseases, which may open new avenues for neuroscience and biomedical research, and also offers new insight into the treatment of POCD. © 2016 The Author(s) Published by S. Karger AG, Basel.

  17. Scutellarin Attenuates Hypertension-Induced Expression of Brain Toll-Like Receptor 4/Nuclear Factor Kappa B

    PubMed Central

    Chen, Xingyong; Shi, Xiaogeng; Zhang, Xu; Lei, Huixin; Long, Simei; Su, Huanxing; Pei, Zhong; Huang, Ruxun

    2013-01-01

    Hypertension is associated with low-grade inflammation, and Toll-like receptor 4 (TLR4) has been shown to be linked to the development and maintenance of hypertension. This study aimed to investigate the effects of scutellarin (administered by oral gavage daily for 2 weeks) on brain TLR4/nuclear factor kappa B-(NF-κB-) mediated inflammation and blood pressure in renovascular hypertensive (using the 2-kidney, 2-clip method) rats. Immunofluorescence and western immunoblot analyses revealed that hypertension contributed to the activation of TLR4 and NF-κB, accompanied by significantly enhanced expression of proinflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18). Furthermore, expression of the antiapoptotic protein, myeloid cell leukemia-1 (Mcl1), was decreased, and the pro-apoptotic proteins, Bax and cleavedcaspase-3 p17 were increased in combined cerebral cortical/striatal soluble lysates. Scutellarin significantly lowered blood pressure and attenuated the number of activated microglia and macrophages in brains of hypertensive rats. Furthermore, scutellarin significantly reduced the expression of TLR4, NF-κB p65, TNF-α, IL-1β, IL-18, Bax and cleaved-caspase-3 p17, and increased the expression of Mcl1. Overall, these results revealed that scutellarin exhibits anti-inflammatory and anti-apoptotic properties and decreases blood pressure in hypertensive rats. Therefore, scutellarin may be a potential therapeutic agent in hypertension-associated diseases. PMID:24223475

  18. A Comparative Review of Toll-Like Receptor 4 Expression and Functionality in Different Animal Species

    PubMed Central

    Vaure, Céline; Liu, Yuanqing

    2014-01-01

    Toll-like receptors (TLRs) belong to the pattern recognition receptor (PRR) family, a key component of the innate immune system. TLRs detect invading pathogens and initiate an immediate immune response to them, followed by a long-lasting adaptive immune response. Activation of TLRs leads to the synthesis of pro-inflammatory cytokines and chemokines and the expression of co-stimulatory molecules. TLR4 specifically recognizes bacterial lipopolysaccharide, along with several other components of pathogens and endogenous molecules produced during abnormal situations, such as tissue damage. Evolution across species can lead to substantial diversity in the TLR4’s affinity and specificity to its ligands, the TLR4 gene and cellular expression patterns and tissue distribution. Consequently, TLR4 functions vary across different species. In recent years, the use of synthetic TLR agonists as adjuvants has emerged as a realistic therapeutic goal, notably for the development of vaccines against poorly immunogenic targets. Given that an adjuvanted vaccine must be assessed in pre-clinical animal models before being tested in humans, the extent to which an animal model represents and predicts the human condition is of particular importance. This review focuses on the current knowledge on the critical points of divergence between human and the mammalian species commonly used in vaccine research and development (non-human primate, mouse, rat, rabbit, swine, and dog), in terms of molecular, cellular, and functional properties of TLR4. PMID:25071777

  19. Toll-like receptor 4 expression in the epithelium of inflammatory periapical lesions. An immunohistochemical study.

    PubMed

    Leonardi, R; Perrotta, R E; Loreto, C; Musumeci, G; Crimi, S; Dos Santos, J N; Rusu, M C; Bufo, P; Barbato, E; Pannone, G

    2015-10-26

    Toll-like receptors (TLR) are essential for the innate immune response against invading pathogens and have been described in immunocompetent cells of areas affected by periapical disease. Besides initiating the inflammatory response, they also directly regulate epithelial cell proliferation and survival in a variety of settings. This study evaluates the in situ expression of TLR4 in periapical granulomas (PG) and radicular cysts, focusing on the epithelial compartment. Twenty-one periapical cysts (PC) and 10 PG were analyzed; 7 dentigerous non-inflamed follicular cyst (DC) served as control. TLR4 expression was assessed by immunohistochemistry. TLR4 immunoreaction products were detected in the epithelium of all specimens, with a higher percentage of immunostained cells in PG. Although TLR4 overexpression was detected in both PG and PC, there were differences that seemed to be related to the nature of the lesion, since in PG all epithelial cells of strands, islands and trabeculae were strongly immunoreactive for TLR4, whereas in PC only some areas of the basal and suprabasal epithelial layers were immunostained. This staining pattern is consistent with the action of TLR4: in PG it could promote formation of epithelial cell rests of Malassez and in epithelial strands and islands the enhancement of cell survival, proliferation and migration, whereas in PC TLR4 could protect the lining epithelium from extensive apoptosis. These findings go some way towards answering the intriguing question of why many epithelial strands or islands in PG and the lining epithelium of apical cysts regress after non-surgical endodontic therapy, and suggest that TLR4 plays a key role in the pathobiology of the inflammatory process related to periapical disease.

  20. Histone Deacetylase 7 Promotes Toll-like Receptor 4-dependent Proinflammatory Gene Expression in Macrophages*

    PubMed Central

    Shakespear, Melanie R.; Hohenhaus, Daniel M.; Kelly, Greg M.; Kamal, Nabilah A.; Gupta, Praveer; Labzin, Larisa I.; Schroder, Kate; Garceau, Valerie; Barbero, Sheila; Iyer, Abishek; Hume, David A.; Reid, Robert C.; Irvine, Katharine M.; Fairlie, David P.; Sweet, Matthew J.

    2013-01-01

    Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target. PMID:23853092

  1. Histone deacetylase 7 promotes Toll-like receptor 4-dependent proinflammatory gene expression in macrophages.

    PubMed

    Shakespear, Melanie R; Hohenhaus, Daniel M; Kelly, Greg M; Kamal, Nabilah A; Gupta, Praveer; Labzin, Larisa I; Schroder, Kate; Garceau, Valerie; Barbero, Sheila; Iyer, Abishek; Hume, David A; Reid, Robert C; Irvine, Katharine M; Fairlie, David P; Sweet, Matthew J

    2013-08-30

    Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target.

  2. Toll-Like Receptor 4 Expression in the Epithelium of Inflammatory Periapical Lesions.

    PubMed Central

    Leonardi, R.; Perrotta, R.E.; Musumeci, G.; Crimi, S.; dos Santos, J.N.; Rusu, M.C.; Bufo, P.; Barbato, E.; Pannone, G.

    2015-01-01

    Toll-like receptors (TLR) are essential for the innate immune response against invading pathogens and have been described in immunocompetent cells of areas affected by periapical disease. Besides initiating the inflammatory response, they also directly regulate epithelial cell proliferation and survival in a variety of settings. This study evaluates the in situ expression of TLR4 in periapical granulomas (PG) and radicular cysts, focusing on the epithelial compartment. Twenty-one periapical cysts (PC) and 10 PG were analyzed; 7 dentigerous non-inflamed follicular cyst (DC) served as control. TLR4 expression was assessed by immunohistochemistry. TLR4 immunoreaction products were detected in the epithelium of all specimens, with a higher percentage of immunostained cells in PG. Although TLR4 overexpression was detected in both PG and PC, there were differences that seemed to be related to the nature of the lesion, since in PG all epithelial cells of strands, islands and trabeculae were strongly immunoreactive for TLR4, whereas in PC only some areas of the basal and suprabasal epithelial layers were immunostained. This staining pattern is consistent with the action of TLR4: in PG it could promote formation of epithelial cell rests of Malassez and in epithelial strands and islands the enhancement of cell survival, proliferation and migration, whereas in PC TLR4 could protect the lining epithelium from extensive apoptosis. These findings go some way towards answering the intriguing question of why many epithelial strands or islands in PG and the lining epithelium of apical cysts regress after non-surgical endodontic therapy, and suggest that TLR4 plays a key role in the pathobiology of the inflammatory process related to periapical disease. PMID:26708181

  3. Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

    SciTech Connect

    Sabatino, María Eugenia; Sosa, Liliana del Valle; Petiti, Juan Pablo; Mukdsi, Jorge Humberto; Mascanfroni, Iván Darío; Pellizas, Claudia Gabriela; Gutiérrez, Silvina; Torres, Alicia Inés; De Paul, Ana Lucía

    2013-11-15

    Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K

  4. Expression of Toll-like receptor 4 in lungs of immune-suppressed rat with Acinetobacter baumannii infection

    PubMed Central

    Wang, Yanmei; Zhang, Xiaohong; Feng, Xuanlin; Liu, Xiaoshu; Deng, Lei; Liang, Zong-An

    2016-01-01

    Toll-like receptor 4 (TLR4) is involved in the regulation of host responses to Acinetobacter baumannii (A. baumannii). The aim of the present study was to examine the function of TLR4 in lung inflammation in immune-suppressed rats with A. baumannii infection. A total of 72 Sprague-Dawley male rats were randomly divided into the control, A. baumannii infection and immune-suppressed infection groups. The immune-suppressed infection group was treated with 100 mg/kg hydrocortisone by subcutaneous injection every other day for 2 weeks prior to A. baumannii infection. Lung tissue was obtained on the 3rd and 7th day after tracheal inoculation with A. baumannii. The expression of TLR4 in bronchial and alveolar epithelial cells, and alveolar macrophage was examined using immunohistochemistry. The levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid were detected using ELISA. The results showed that in the control group, the expression of TLR4 was upregulated in the bronchial and alveolar epithelial, and alveolar macrophages, and the levels of IL-6 and TNF-α were increased in the early phase of A. baumannii infection. On the 7th day, no significant difference in the levels of IL-6 and TNF-α was observed between the A. baumannii infection and control groups. Conversely, the expression of TLR4 was downregulated in the immune-suppressed group, and the levels of IL-6 and TNF-α were reduced on the 3rd day after infection. In the subsequent observation period, the expression of TLR4 was upregulated and the levels of IL-6 and TNF-α were increased. In conclusion, the results show a critical role of TLR4 in mediating effective immune response in the lung of rat with A. baumannii infection. PMID:27703512

  5. Toll-like receptor 4 gene polymorphism downregulates gene expression and involves in susceptibility to bladder cancer.

    PubMed

    Shen, Yizhen; Bu, Meimei; Zhang, Aimin; Liu, Yi; Fu, Baochen

    2015-04-01

    Bladder cancer is the ninth most frequent malignancy in China. Toll-like receptor 4 (TLR4) is expressed on various cells and greatly involves in immune responses. Genetic polymorphism may affect the pathogenesis of diseases through various pathways. In the current study, we evaluated the association between genetic polymorphisms in TLR4 and risk of bladder cancer. We also examined the effect of the polymorphisms on gene expression. The TLR4 -729G/C and -260G/C polymorphisms were genotyped in 282 bladder cancer patients and 298 healthy controls in the Chinese population. Results showed that subjects with -729GC genotype are at significantly higher risk of bladder cancer than those with GG genotype [odds ratio (OR) = 2.50, 95% confidence interval (CI) = 1.39-4.48, P = 0.002]. Similarly, TLR4 -729C allele revealed a positive association with the disease (OR = 2.39, P = 0.002). The other polymorphism, TLR4 -260G/C, did not present clear correlations with bladder cancer. To understand the function of the polymorphisms, we evaluated TLR4 messenger RNA (mRNA) and protein levels in CD4+ T cells, CD8+ T cells, and monocytes from subjects carrying different TLR4 genotypes. Results revealed that subjects carrying -729GC genotype had significantly downregulated mRNA and protein levels of TLR4 in CD4+ T cells, CD8+ T cells, and monocytes compared to those carrying GG genotype. However, subjects with -260G/C polymorphism did not show any differences in gene expression from immune cells These data suggest that TLR4 polymorphism is associated with increased susceptibility to bladder cancer possibly by downregulating gene expression in various immune cells.

  6. Hypoxia preconditioning increases survival and decreases expression of Toll-like receptor 4 in pulmonary artery endothelial cells exposed to lipopolysaccharide.

    PubMed

    Ali, Irshad; Nanchal, Rahul; Husnain, Fouad; Audi, Said; Konduri, G Ganesh; Densmore, John C; Medhora, Meetha; Jacobs, Elizabeth R

    2013-09-01

    Abstract Pulmonary or systemic infections and hypoxemic respiratory failure are among the leading causes of admission to intensive care units, and these conditions frequently exist in sequence or in tandem. Inflammatory responses to infections are reproduced by lipopolysaccharide (LPS) engaging Toll-like receptor 4 (TLR4). Apoptosis is a hallmark of lung injury in sepsis. This study was conducted to determine whether preexposure to LPS or hypoxia modulated the survival of pulmonary artery endothelial cells (PAECs). We also investigated the role TLR4 receptor expression plays in apoptosis due to these conditions. Bovine PAECs were cultured in hypoxic or normoxic environments and treated with LPS. TLR4 antagonist TAK-242 was used to probe the role played by TLR4 receptors in cell survival. Cell apoptosis and survival were measured by caspase 3 activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) incorporation. TLR4 expression and tumor necrosis factor α (TNF-α) production were also determined. LPS increased caspase 3 activity in a TAK-242-sensitive manner and decreased MTT incorporation. Apoptosis was decreased in PAECs preconditioned with hypoxia prior to LPS exposure. LPS increased TNF-α production, and hypoxic preconditioning blunted it. Hypoxic preconditioning reduced LPS-induced TLR4 messenger RNA and TLR4 protein. TAK-242 decreased to baseline the LPS-stimulated expression of TLR4 messenger RNA regardless of environmental conditions. In contrast, LPS followed by hypoxia substantially increased apoptosis and cell death. In conclusion, protection from LPS-stimulated PAEC apoptosis by hypoxic preconditioning is attributable in part to reduction in TLR4 expression. If these signaling pathways apply to septic patients, they may account for differing sensitivities of individuals to acute lung injury depending on oxygen tensions in PAECs in vivo.

  7. Hypoxia preconditioning increases survival and decreases expression of Toll-like receptor 4 in pulmonary artery endothelial cells exposed to lipopolysaccharide

    PubMed Central

    Nanchal, Rahul; Audi, Said; Konduri, G. Ganesh; Medhora, Meetha

    2013-01-01

    Abstract Pulmonary or systemic infections and hypoxemic respiratory failure are among the leading causes of admission to intensive care units, and these conditions frequently exist in sequence or in tandem. Inflammatory responses to infections are reproduced by lipopolysaccharide (LPS) engaging Toll-like receptor 4 (TLR4). Apoptosis is a hallmark of lung injury in sepsis. This study was conducted to determine whether preexposure to LPS or hypoxia modulated the survival of pulmonary artery endothelial cells (PAECs). We also investigated the role TLR4 receptor expression plays in apoptosis due to these conditions. Bovine PAECs were cultured in hypoxic or normoxic environments and treated with LPS. TLR4 antagonist TAK-242 was used to probe the role played by TLR4 receptors in cell survival. Cell apoptosis and survival were measured by caspase 3 activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) incorporation. TLR4 expression and tumor necrosis factor α (TNF-α) production were also determined. LPS increased caspase 3 activity in a TAK-242-sensitive manner and decreased MTT incorporation. Apoptosis was decreased in PAECs preconditioned with hypoxia prior to LPS exposure. LPS increased TNF-α production, and hypoxic preconditioning blunted it. Hypoxic preconditioning reduced LPS-induced TLR4 messenger RNA and TLR4 protein. TAK-242 decreased to baseline the LPS-stimulated expression of TLR4 messenger RNA regardless of environmental conditions. In contrast, LPS followed by hypoxia substantially increased apoptosis and cell death. In conclusion, protection from LPS-stimulated PAEC apoptosis by hypoxic preconditioning is attributable in part to reduction in TLR4 expression. If these signaling pathways apply to septic patients, they may account for differing sensitivities of individuals to acute lung injury depending on oxygen tensions in PAECs in vivo. PMID:24618542

  8. Both intrinsic and extrinsic apoptotic pathways are involved in Toll-like receptor 4 (TLR4)-induced cell death in monocytic THP-1 cells.

    PubMed

    Liu, Bei; Sun, Ruili; Luo, Hongbo; Liu, Xueting; Jiang, Manli; Yuan, Chuang; Yang, Li; Hu, Jinyue

    2017-02-01

    Our previous study showed that TLR3 induces apoptosis via both death receptors and mitochondial in human endothelial cells. We report here that the activation of TLR4 induced dose- and time-dependent cell death in moncytic THP-1 cells. LPS treatment of THP-1 cells induced the activation of both caspase 8 and 9, suggesting the involvement of intrinsic and extrinsic apoptosis pathways. TNFα was induced by TLR4 activation at both mRNA and protein levels, but its neutralization did not down-regulated TLR4-induced cell death. TLR4 activation also induced the up-regulation of TRAIL and its receptors DR4 and DR5, and the neutralization of TRAIL ameliorated TLR4 induced apoptosis, suggesting the involvement of TRAIL and its receptors DR4 and DR5 in LPS-induced cell death. Meanwhile, LPS treatment down-regulated the expression of FLICE inhibitory protein (FLIP), a suppressor of death receptor-induced cell death. In addition, TLR4 activation down-regulated the anti-apoptotic protein bcl-2, and up-regulated the pro-apoptotic proteins Noxa and Puma, suggesting that mitochondrial apoptotic pathway was also involved in LPS-induced cell death. Furthermore, we found that TAP63α might confer to the activation of intrinsic and extrinsic apoptotic pathways. The treatment of THP-1 cells with LPS induced the translocation of TAP63α from cytoplasm to nucleus. Taken together, our study suggested that both death receptors and mitochondial were involved in TLR4-induced cell death, and TAP63α may be a target for the prevention of LPS-induced cell death.

  9. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment.

    PubMed

    Okito, Asuka; Nakahama, Ken-Ichi; Akiyama, Masako; Ono, Takashi; Morita, Ikuo

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment.

  10. Retinoic Acid Facilitates Toll-Like Receptor 4 Expression to Improve Intestinal Barrier Function through Retinoic Acid Receptor Beta.

    PubMed

    Li, Yingying; Gao, Yuan; Cui, Ting; Yang, Ting; Liu, Lan; Li, Tingyu; Chen, Jie

    2017-07-17

    Vitamin A (VA) protects the intestinal epithelial barrier by improving cell migration and proliferation. Our previous studies demonstrated that VA deficiency (VAD) during pregnancy suppresses the systemic and mucosal immune responses in the intestines of offspring and that VA supplementation (VAS) during early life can increase immune cell counts. However, little is known about the mechanisms by which VA regulates intestinal epithelial barrier function. Caco-2 cells were treated with all-trans retinoic acid (ATRA) for 24 hours to determine the optimum ATRA concentration to which the cells in question respond. Caco-2 cells were infected with recombinant adenoviruses carrying retinoic acid receptor beta (Ad-RARβ) and small interfering RARβ(siRARβ) to assess the effects of RARβ signalling on the expression of specific proteins. A siTLR4 lentivirus was used to knockdown Toll-like receptor 4 (TLR4) in Caco-2 cells to determine its role in the protective effects of VA on the intestinal epithelial barrier, and experiments involving TLR4-knock-out mice were performed to verify the effect of TLR4. VA normal (VAN), VAD and VAS rat models were established to confirm that changes in RARβ, TLR4 and ZO-2 expression levels that occurred following decreases or increases in retinol concentrations in vivo, and the permeability of the Caco-2 cell monolayer, as well as that of the epithelial barrier of the rat intestine was detected by measuring transepithelial resistance (TER) or performing enzyme-linked immunosorbent assay (ELISA). Retinoic acid receptor (RAR), toll like receptor (TLR) and tight junction (TJ) mRNA and protein expression levels in Caco-2 cells and the colon monolayers in the rat and mouse models were measured by PCR and western blotting, respectively. Co-immunoprecipitation (co-IP) and immunofluorescence staining were performed to assess the interactions among RARβ, TLR4 and zonula occluden-2 (ZO-2) in Caco-2 cells, and chromatin immunoprecipitation (Ch

  11. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    SciTech Connect

    Okito, Asuka; Nakahama, Ken-ichi; Akiyama, Masako; Ono, Takashi; Morita, Ikuo

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

  12. Akt-phosphorylated mitogen-activated kinase-activating death domain protein (MADD) inhibits TRAIL-induced apoptosis by blocking Fas-associated death domain (FADD) association with death receptor 4.

    PubMed

    Li, Peifeng; Jayarama, Shankar; Ganesh, Lakshmy; Mordi, David; Carr, Ryan; Kanteti, Prasad; Hay, Nissim; Prabhakar, Bellur S

    2010-07-16

    MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies.

  13. Impaired Cd14 and Cd36 expression, bacterial clearance, and Toll-like receptor 4-Myd88 signaling in caveolin-1-deleted macrophages and mice.

    PubMed

    Tsai, Tsung-Huang; Chen, Shu-Fen; Huang, Tai-Yu; Tzeng, Chun-Fu; Chiang, Ann-Shyn; Kou, Yu Ru; Lee, Tzong-Shyuan; Shyue, Song-Kun

    2011-01-01

    An overwhelming immune response, particularly from macrophages, with gram-negative bacteria-induced sepsis plays a critical role in survival of and organ damage in infected patients. Caveolin-1 (Cav-1), a major structure protein of caveolae, regulates many cellular functions. We examined the vital role of Cav-1 in the response of macrophages and mice to bacteria or LPS exposure. Deletion of Cav-1 decreased the expression of CD14 and CD36 during macrophage differentiation and suppressed their phagocytotic ability. As well, the ability to kill bacteria was inhibited in Cav-1 macrophages and mice peritoneal cavity, tissue, and plasma, which was partly attributed to hindered expression of iNOS induced by bacteria or LPS. Furthermore, deletion of Cav-1 attenuated the expression of Toll-like receptor 4 and myeloid differentiation factor 88 and the activation of nuclear factor κB, all of which impeded the production of inflammatory cytokines in response to bacterial exposure in Cav-1 macrophages and mice. Thus, Cav-1 participates in the regulation of CD14, CD36, Toll-like receptor 4 and myeloid differentiation factor 88 protein expression and is crucial for the immune response of macrophages to bacterial infection. Cav-1 may be a therapeutic target in the treatment of sepsis.

  14. Surface expression of toll-like receptor 4 on THP-1 cells is modulated by Bu-Zhong-Yi-Qi-Tang and Shi-Quan-Da-Bu-Tang.

    PubMed

    Mita, Y; Dobashi, K; Shimizu, Y; Nakazawa, T; Mori, M

    2002-03-01

    Human Toll-like receptor 4 (TLR4) has recently been identified and has been shown to be the main protein involved in recognizing Gram-negative bacteria. We examined the regulation of TLR4 surface expression in a human monocytic cell line (THP-1 cells) by two traditional Chinese herbal medicines. Bu-Zhong-Yi-Qi-Tang (TJ-41) and Shi-Quan-Da-Bu-Tang (TJ-48). TJ-41 and TJ-48 upregulated TLR4 surface expression in THP-1 cells, as well as enhanced TLR4 surface expression in these cells both dose- and time-dependently. These findings suggest that TJ-41 and TJ-48 increase the receptor involved in the response to Gram-negative bacteria and may enhance defenses against these pathogens.

  15. The association of down-regulated toll-like receptor 4 expression with airflow limitation and emphysema in smokers

    PubMed Central

    2012-01-01

    Background An association between innate immunity including Toll-like receptors (TLRs) and COPD is reported recently; TLR4 deficiency in lung can cause emphysema in animals, which is not evident in humans. We analyzed the association of TLR4 expression, airflow limitation and emphysema in smokers. Methods We enrolled patients of ≥40years old with smoking histories of ≥10 pack-years and who had undergone lung resection. We measured TLR4 expression in lung lysates. The severity of emphysema was evaluated on computed tomography. TLR4 expression was also evaluated immunohistochemically. Results In total, 53 patients were enrolled. Forced expiratory volume in one second per forced vital capacity (FEV1/FVC) increased (P=0.03) and emphysema score decreased (P=0.01) as TLR4 expression increased. These were still significant, in multiple regression analysis including sex, age, tuberculosis history, smoking history and inhaled corticosteroid (ICS) usage. We also classified patients as high, intermediate, and low expressers according to TLR4 expression. Although no differences in age, gender, tuberculosis, or smoking history were observed among the groups, emphysema severity increased significantly (P = 0.02) and FEV1/FVC decreased significantly (P = 0.006) in TLR4 low expresser. The difference in TLR4 expression based on immunohistochemistry was most prominent in bronchial and alveolar epithelial cells. Conclusion Down-regulated TLR4 expression in lung was associated with emphysema and airflow limitation in smokers. PMID:23170858

  16. INVOLVEMENT OF TOLL-LIKE RECEPTOR 4 AND MAPK PATHWAYS IN LPS-INDUCED CD40 EXPRESSION IN MONOCYTIC CELLS

    EPA Science Inventory

    CD40 is a co-stimulatory surface molecule actively expressed on mature dendritic cells (DC). Recent studies suggest that endotoxin (LPS) inhalation induces DC maturation in the airways of healthy volunteers. To characterize the effect of LPS on CD40 expression and underlying mech...

  17. INVOLVEMENT OF TOLL-LIKE RECEPTOR 4 AND MAPK PATHWAYS IN LPS-INDUCED CD40 EXPRESSION IN MONOCYTIC CELLS

    EPA Science Inventory

    CD40 is a co-stimulatory surface molecule actively expressed on mature dendritic cells (DC). Recent studies suggest that endotoxin (LPS) inhalation induces DC maturation in the airways of healthy volunteers. To characterize the effect of LPS on CD40 expression and underlying mech...

  18. Saturated fatty acids up-regulate COX-2 expression in prostate epithelial cells via toll-like receptor 4/NF-κB signaling.

    PubMed

    Liu, Jie; Hu, Shuai; Cui, Yun; Sun, Meng-Kui; Xie, Feng; Zhang, Qian; Jin, Jie

    2014-04-01

    Cyclooxygenase-2 (COX-2) has been implicated in prostate carcinogenesis, and recently it has been confirmed to be a molecular target of saturated fatty acids (SFAs). In the present study, we investigated the effect of stearic acid (SA) and palmitic acid (PA), two of the most abundant SFAs contained in dietary fat, on COX-2 expression in prostate epithelial cells and the signaling transduction pathway involved. First, we demonstrated that both SA and PA increased the mRNA and protein expression of COX-2, and consistently induced the activation of NF-κB in RWPE-1, BPH-1 and PC-3 prostate epithelial cell lines. The effect of SA and PA on COX-2 over-expression and NF-κB activation was in a dose-dependent manner, and PA was more potent than SA at the same concentration. Then, we demonstrated inhibition of NF-κB using its specific inhibitor strikingly attenuated PA-induced COX-2 expression. Toll-like receptor 4 (TLR4) was revealed to be expressed on RWPE-1, BPH-1 and PC-3 cell lines by PCR and immunofluorescence staining, and blocking its signaling significantly inhibited PA induced COX-2 over-expression and NF-κB activation. Taken together, we demonstrated that SFAs can up-regulate COX-2 expression in prostate epithelial cells, and this effect was mediated mainly through the TLR4/NF-κB signaling pathway.

  19. Expression of Toll-like receptor 4 in the prostate gland and its association with the severity of prostate cancer.

    PubMed

    Gatti, Gerardo; Quintar, Amado A; Andreani, Virginia; Nicola, Juan P; Maldonado, Cristina A; Masini-Repiso, Ana Maria; Rivero, Virginia E; Maccioni, Mariana

    2009-09-15

    Chronic inflammation has been postulated to be an important driving force to prostate carcinoma. Toll-like receptors (TLRs) compose a family of receptors mainly expressed on immune cells. Recently, functional TLRs have been shown to be also expressed in numerous cancer cells, but their significance has only recently begun to be explored. The purpose of this study was to investigate the putative role of TLR4 expression in prostate carcinoma. To determine if there is an association between TLR4 expression and the malignancy of the tumor, 35 prostate carcinoma samples showing different Gleason grades were analyzed by immunohistochemistry. Also, to explore the functionality of the receptors expressed on the epithelium, we analyzed the type of cytokine response elicited and the signaling pathways involved after TLR4 triggering in the human prostate adenocarcinoma cell line, DU-145. TLR4 is expressed in the normal prostate gland in both stroma and epithelium. TLR4 expression significantly drops to negative values as the Gleason grade augments in both, stroma and epithelium. Moreover, DU-145 cells also exhibit TLR4 expression and respond to TLR4 agonists, activating the transcription factor NF-kappaB and increasing the expression of pro-inflammatory mediators. Inhibition of the molecular adaptors MyD88 and MAL by overexpression of dominant-negative mutants diminished LPS-induced activation of NF-kappaB, showing that DU-145 cells activate the NF-kappaB through MyD88-dependent signaling pathways. We hypothesize that TLR4 in prostate cells could synergize with innate immune cells contributing to an eventual inflammatory process, which in genetically prone individuals could promote carcinogenesis. Prostate 69: 1387-1397, 2009. (c) 2009 Wiley-Liss, Inc.

  20. High expression of Toll-like receptor 4/myeloid differentiation factor 88 signals correlates with poor prognosis in colorectal cancer

    PubMed Central

    Wang, E L; Qian, Z R; Nakasono, M; Tanahashi, T; Yoshimoto, K; Bando, Y; Kudo, E; Shimada, M; Sano, T

    2010-01-01

    Background: The Toll-like receptor (TLR) 4 signalling pathway has been shown to have oncogenic effects in vitro and in vivo. To demonstrate the role of TLR4 signalling in colon tumourigenesis, we examined the expression of TLR4 and myeloid differentiation factor 88 (MyD88) in colorectal cancer (CRC). Methods: The expression of TLR4 and MyD88 in 108 CRC samples, 15 adenomas, and 15 normal mucosae was evaluated by immunohistochemistry, and the correlations between their immunoscores and clinicopathological variables, including disease-free survival (DFS) and overall survival (OS), were analysed. Results: Compared with normal mucosae and adenomas, 20% cancers displayed high expression of TLR4, and 23% cancers showed high expression of MyD88. The high expression of TLR4 and MyD88 was significantly correlated with liver metastasis (P=0.0001, P=0.0054). In univariate analysis, the high expression of TLR4 was significantly associated with shorter OS (hazard ratio (HR): 2.17; 95% confidence interval (95% CI): 1.15–4.07; P=0.015). The high expression of MyD88 expression was significantly associated with poor DFS and OS (HR: 2.33; 95% CI: 1.31–4.13; P=0.0038 and HR: 3.03; 95% CI: 1.67–5.48; P=0.0002). The high combined expression of TLR4 and MyD88 was also significantly associated with poor DFS and OS (HR: 2.25; 95% CI: 1.27–3.99; P=0.0053 and HR: 2.97; 95% CI: 1.64–5.38; P=0.0003). Multivariate analysis showed that high expressions of TLR4 (OS: adjusted HR: 1.88; 95% CI: 0.99–3.55; P=0.0298) and MyD88 (DFS: adjusted HR: 1.93; 95% CI: 1.01–3.67; P=0.0441; OS: adjusted HR: 2.25; 95% CI: 1.17–4.33; P=0.0112) were independent prognostic factors of OS. Furthermore, high co-expression of TLR4/MyD88 was strongly associated with both poor DFS and OS. Conclusion: Our findings suggest that high expression of TLR4 and MyD88 is associated with liver metastasis and is an independent predictor of poor prognosis in patients with CRC. PMID:20145615

  1. Association of Toll-Like Receptor 4 Gene Polymorphism and Expression with Urinary Tract Infection Types in Adults

    PubMed Central

    Yin, Xiaolin; Hou, Tianwen; Liu, Ying; Chen, Jing; Yao, Zhiyan; Ma, Cuiqing; Yang, Lijuan; Wei, Lin

    2010-01-01

    Background Innate immunity of which Toll-like receptor (TLR) 4 and CXCR1 are key elements plays a central role in the development of urinary tract infection (UTI). Although the relation between the genetics of TLR4 and CXCR1 and UTI is investigated partly, the polymorphisms and expression of TLR4 and CXCR1 in different types of UTI in adults are not extremely clear. Methodology/Principal Findings This study investigates the presence of TLR4 A (896) G and CXCR1 G (2608) C polymorphisms in 129 UTI patients using RFLP-PCR. Gene and allelic prevalence were compared with 248 healthy controls. Flow cytometry was used to detect TLR4 and CXCR1 expression in the monocytes of UTI patients and healthy controls. TLR4 (896) AG genotype and TLR4 (896) G allele had higher prevalence in UTI (especially in acute cystitis and urethritis) patients, whereas CXCR1 (2608) GC genotype and CXCR1 (2608) C allele had lower prevalence in UTI patients than controls. TLR4 expression was significantly lower in chronic UTI patients than in acute pyelonephritis or healthy controls. CXCR1 expression was similar in both controls and patients. TLR4 expression in chronic UTI patients after astragalus treatment was higher than pre-treatment. Conclusions The results indicate the relationship between the carrier status of TLR4 (896) G alleles and the development of UTI, especially acute cystitis and urethritis, in adults. TLR4 expression levels are correlated with chronic UTI. PMID:21151974

  2. Toll-like receptor 4 expression is increased in circulating mononuclear cells of patients with immunoglobulin A nephropathy

    PubMed Central

    Coppo, R; Camilla, R; Amore, A; Peruzzi, L; Daprà, V; Loiacono, E; Vatrano, S; Rollino, C; Sepe, V; Rampino, T; Dal Canton, A

    2010-01-01

    We investigated Toll-like receptors (TLR-3, -4 and -7) expression in circulating mononuclear cells of patients with immunoglobulin A nephropathy (IgAN), a disease with debated relationships with mucosal immunity. TLR-4 expression (detected by fluorescence activated cell sorter) and mRNA transcriptional levels (Taqman) were significantly higher in patients with IgAN than in healthy controls (P = 0·00200 and P = 0·0200). TLR-3 and TLR-7 were not modified significantly. In IgAN patients proteinuria was correlated significantly with TLR-4 expression (P = 0·0312). In a group of nephrotic syndromes, TLR-3, -4 and -7 expression was similar to healthy controls. A significant difference in TLR-4 expression and mRNA levels was found between very active IgAN patients (proteinuria > 1 g/1·73 m2/day in association with severe microscopic haematuria) and inactive patients (proteinuria < 0·5 g/1·73 m2/day, with absent or minimal haematuria). No correlation with levels of aberrantly glycosylated IgA1, age, renal biopsy features or therapy was found. This study shows for the first time an up-regulation of TLR-4 in circulating mononuclear cells of patients with IgAN, particularly in association with proteinuria and heavy microscopic haematuria. PMID:19891659

  3. Gene expression of somatostatin receptor 4 predicts clinical outcome of patients with metastatic neuroendocrine tumors treated with somatostatin analogs.

    PubMed

    Slaby, Ondrej; Sachlova, Milana; Bednarikova, Marketa; Fabian, Pavel; Svoboda, Marek; Vytopilova, Simona; Valik, Dalibor; Vyzula, Rostislav

    2010-04-01

    Somatostatin analogs (SSA) are the standard diagnostic and treatment tools in the clinical management of patients with neuroendocrine tumors (NETs) expressing somatostatin receptors (SSTRs). Although symptomatic and biochemical control is obtained with SSA in the majority of functional NETs, antineoplastic effects of SSA are partial and of limited duration. The aim of this study was to quantify expression levels of five SSTR subtypes (SSTR1-SSTR5) and correlate them with the clinical outcomes of patients with NETs who underwent SSA therapy. The expression levels were analyzed using real-time polymerase chain reaction in a series of 22 metastatic NETs with a median time of 10 months on the SSA therapy (range 2-82 months). The median duration of disease stabilization in patients who developed progression (n = 14) was 9 months (range 3-92 months). The median survival period for all patients was 44 months (range 3-175 months). According to RECIST criteria, one (5%) partial objective tumor response was obtained, disease stabilization was achieved in 10 (45%) patients, and progressive disease was observed in 11 (50%). Analysis of mRNA expression of the SSTR subtypes showed that SSTR2 and SSTR5 were expressed in all of the studied NETs; SSTR1 and SSTR4 in all but 3 tumors (86%); and SSTR3 in only 10 NETs (49%). Interestingly, our preliminary data suggest that only the levels of SSTR4, though it has the lowest affinity for SSA of all SSTR subtypes, were significantly associated with the stabilization of disease during SSA therapy (p = 0.0357). These levels correlated with time to progression (p = 0.0015) and overall survival (p = 0.0017) in NET patients.

  4. Influence of the fibroblast growth factor receptor 4 expression and the G388R functional polymorphism on Cushing's disease outcome.

    PubMed

    Brito, Luciana Pinto; Lerário, Antonio Marcondes; Bronstein, Marcello Delano; Soares, Iberê Cauduro; Mendonca, Berenice Bilharinho; Fragoso, Maria Candida Barisson Villares

    2010-10-01

    Abnormal FGFR4 expression has been detected in pituitary tumors, especially in larger and invasive adenomas. In addition, the FGFR4 functional polymorphism G388R has been associated with poor outcome in several human malignancies. Then, we hypothesized that FGFR4 expression and genotype could be markers of adverse outcome of Cushing's disease after transsphenoidal surgery. The objective was to investigate whether there is an association between the postoperative outcome of Cushing's disease (remission/recurrence) and the FGFR4 G388R genotype or the FGFR4 expression in corticotrophinomas. Clinical, hormonal, and pathological data of 76 patients who underwent the first transsphenoidal surgery were retrospectively reviewed. All patients were genotyped for G388R polymorphism. FGFR4 expression was assessed by real-time PCR in 18 corticotrophinomas. The outcome measures included the FGFR4 G388R genotype and FGFR4 expression in postoperative remission and recurrence of Cushing's disease. Homozygosis for FGFR4 glycine (Gly(388)) allele was associated with reduced disease-free survival, in the univariate analysis (hazard ratio of 6.91; 95% confidence interval of 1.14-11.26; P = 0.028). Male gender (P = 0.036), lack of pathology confirmation (P = 0.009), and cortisol levels more than 2 μg/dl in the early postoperative period (P < 0.001) were also significant predictors of Cushing's disease recurrence in the univariate analysis. FGFR4 overexpression was found in 44% of the corticotrophinomas, and it was associated with lower postoperative remission rate (P = 0.009). Our data suggest that homozygosis for FGFR4 Gly(388) allele and FGFR4 overexpression are associated with higher frequency of postoperative recurrence and persistence of Cushing's disease, respectively.

  5. Impact of probiotics on toll-like receptor 4 expression in an experimental model of ulcerative colitis.

    PubMed

    Yang, Xia; Fu, Yu; Liu, Jun; Ren, Hong-yu

    2013-10-01

    Toll-like receptors (TLRs) are key components of the innate immune system which trigger antimicrobial host defense responses. This study aimed to investigate the impact of probiotics (Lactobacillus, Bifidobacterium) on the expression of TLR4 and tumor necrosis factor-alpha (TNF-α) in the colon mucosa of rat experimental ulcerative colitis model induced by trinitrobenzene sulfonic acid (TNBS)/ethanol and immune complexes. The gross and histological changes of the colonic mucosa were observed and assessed by the means-standard deviation and independent samples t-test. The protein expression levels of TLR4 and TNF-α were detected by using immunohistochemistry and Western blotting, respectively. It was revealed that there was visible infiltration of inflammatory cells, formation of crypt abscess, and the reduction of goblet cells in the colon tissue of experimental models. As compared with the control group, the levels of TLR4 and TNF-α protein were significantly increased in the model group (P<0.01 for both). No significant difference was found in the expression of TLR4 and TNF-α between the two-week probiotics treatment group and the model group (P>0.05), whereas significant reductions were shown in rats which were treated with probiotics for four weeks as compared with the model group (P<0.01). There was no significant difference between two probiotics-treated groups. Our results implied that probiotics were likely to play a key role in protecting ulcerative colitis by reducing the inflammatory factor TNF-α expression through inhibiting the TLR4 expression in the colon tissue of experimental models.

  6. Involvement of L-type Ca²⁺ channel and toll-like receptor-4 in nickel-induced interleukin-8 gene expression.

    PubMed

    Lin, Chia-Hsien; Chung, Chih-Ang; Wong, Jhen-Hong; Chen, Ben-Kuen; Chiu, Siou-Jin; Klahan, Sukhontip; Lee, Yi-Chao; Chang, Wei-Chiao

    2016-01-01

    The metal nickel (Ni(2+)) is found everywhere in our daily lives, including coins, costume jewelry, and even nuts and chocolates. Nickel poisoning can cause inflammatory reactions, respiratory diseases, and allergic contact dermatitis. To clarify the mechanism by which nickel induces mediators of inflammation, we used the human acute monocytic leukemia THP-1 cell line as a model. Interleukin (IL)-8 promoter activity as well as gene expression were tested by luciferase assay and real-time polymerase chain reaction. The underlying mechanisms of nickel-induced IL-8 were investigated. We found that nickel induced IL-8 gene expression via the L-type Ca(2+) channel, Toll-like receptor-4 (TRL-4) and nuclear factor NF-κB signal transduction pathways. Nickel activated NF-κB expression through extracellular signal-regulated kinase 1/2 phosphorylation and then increased IL-8 expression. Thus, the L-type Ca(2+) channel and TRL-4 play important roles in nickel-induced inflammatory gene expressions.

  7. Toll‐like receptor 4 induced FcγR expression potentiates early onset of joint inflammation and cartilage destruction during immune complex arthritis: Toll‐like receptor 4 largely regulates FcγR expression by interleukin 10

    PubMed Central

    van Lent, P L E M; Blom, A B; Grevers, L; Sloetjes, A; van den Berg, W B

    2007-01-01

    Objective To study the role of Toll‐like receptor (TLR)2 and 4 in the onset of joint inflammation and cartilage destruction during immune complex‐mediated arthritis (ICA), and its relationship with FcγR expression. Materials and methods ICA was induced in knee joints of TLR2−/− and TLR4−/− mice and their wild‐type controls. Joint inflammation and cartilage destruction were measured in the knee joint using histology. mRNA levels were determined in synovial specimens and macrophages using quantitative polymerase chain reaction and cytokine protein levels in synovial washouts using Bioplex. Results Joint inflammation and cartilage destruction were not different in arthritic TLR2−/− and wild‐type mice. By contrast, at day 1 after ICA induction, joint swelling and proteoglycan depletion in knee joints of TLR4−/− mice were considerably lower (inflammation 68–79% and proteoglycan depletion 27–76%) when compared with wild‐type controls. Cytokine production at this time point was markedly reduced in TLR4−/− mice (interleukin (IL)1, IL6, macrophage inflammatory chemokine (MIP)‐1α and keratinocyte‐derived chemokine 49%, 72%, 68% and 84%, respectively). In arthritic synovia of TLR4−/− mice, and also after injection of the antigen poly‐l‐lysine (PLL) lysozyme alone, mRNA levels of FcγR, and the FcγR regulating cytokine IL10 were considerably lower. Stimulation of peritoneal macrophages with PLL lysozyme up regulated mRNA levels of FcγR and IL10, whereas neutralisation by anti‐IL10 antibodies largely blocked FcγR up regulation. At day 4, joint inflammation and cartilage destruction were comparable in TLR4−/− mice and wild‐type controls. Conclusion TLR4 regulates early onset of joint inflammation and cartilage destruction during ICA arthritis by up regulation of FcγR expression and enhanced cytokine production. TLR4‐mediated up regulation of FcγR is largely mediated by IL10. PMID:17068066

  8. Curcumin inhibiting Th17 cell differentiation by regulating the metabotropic glutamate receptor-4 expression on dendritic cells.

    PubMed

    Zhao, Guangming; Liu, Ying; Yi, Xiaying; Wang, Yupeng; Qiao, Song; Li, Zhen; Ni, Jing; Song, Zhiqi

    2017-05-01

    Th17 cells have been categorized as a new lineage of CD4+ T cells, and played a crucial role in the pathogenesis of numerous autoimmune disorders. Type 4 metabotropic glutamate receptor (mGluR4), a member of group III mGluRs, recently has been found to be expressed in many types of immune cells and mediate adaptive immunity. Curcumin has been shown to exhibit potent anti-inflammatory, antimutagenic and anticarcinogenic properties. For the past few years, it has gradually been regarded as an pluripotent immunomodulatory agent that can regulate the activation of immune cells. In the present study, we investigated the efficacy and mechanism of curcumin on Th17 cells. Treatment with curcumin significantly reduced IL-6 and IL-23 production by dendritic cells (DC). Additionally, it had a dramatic reduction in the proliferation of CD4+ T cells co-cultured with DC. Furthermore, expression of the Th17 cells related cytokine profiles (IL-17A and RORγt) was dramatically decreased in curcumin-treated groups. These findings indicated that curcumin inhibited the differentiation and development of Th17 cells. Besides, we found that mGluR4 was constitutively expressed in mouse bone marrow derived DC (BMDC) for the first time. In addition, mGluR4 siRNA-transfected BMDC tipped the balance of T cell differentiation in favor of the Th17 phenotype. We first reported that curcumin increased the mGluR4 expression in mouse BMDC activated with LPS, which likely contributed to the mechanism of inhibiting the Th17 cell differentiation. Our findings suggest that curcumin might be a potential candidate for Th17 related autoimmune disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Activity-regulated Somatostatin Expression Reduces Dendritic Spine Density and Lowers Excitatory Synaptic Transmission via Postsynaptic Somatostatin Receptor 4*

    PubMed Central

    Hou, Zai-Hua; Yu, Xiang

    2013-01-01

    Neuronal activity regulates multiple aspects of the morphological and functional development of neural circuits. One mechanism by which it achieves this is through regulation of gene expression. In a screen for activity-induced genes, we identified somatostatin (SST), a neuropeptide secreted by the SST subtype of interneurons. Using real time quantitative PCR and ELISA, we showed that persistent elevation of neuronal activity increased both the gene expression and protein secretion of SST over a relatively prolonged time course of 48 h. Using primary hippocampal neuronal cultures, we found that SST treatment for 1 day significantly reduced the density of dendritic spines, the morphological bases of excitatory synapses. Furthermore, the density of pre- and postsynaptic markers of excitatory synapses was significantly lowered following SST treatment, whereas that of inhibitory synapses was not affected. Consistently, SST treatment reduced the frequency of miniature excitatory postsynaptic currents, without affecting inhibition. Finally, lowering the endogenous level of SST receptor subtype 4 in individual hippocampal pyramidal neurons significantly blocked the effect of SST in reducing spine density and excitatory synaptic transmission in a cell autonomous fashion, suggesting that the effect of SST in regulating excitatory synaptic transmission is mainly mediated by SST receptor subtype 4. Together, our results demonstrated that activity-dependent release of SST reduced the density of dendritic spines and the number of excitatory synapses through postsynaptic activation of SST receptor subtype 4 in pyramidal neurons. To our knowledge, this is the first demonstration of the long term effect of SST on neuronal morphology. PMID:23233668

  10. Pyrrolidine Dithiocarbamate Inhibits Nuclear Factor κB and Toll-Like Receptor 4 Expression in Rats with Acute Necrotizing Pancreatitis

    PubMed Central

    Xu, Min; Wang, Kun-Ning; Wu, Kai; Wang, Xing-Peng

    2015-01-01

    Background/Aims To investigate the expression of Toll-like receptor 4 (TLR4) in the pancreases of rats with acute necrotizing pancreatitis (ANP) and any changes upon treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor κB (NF-κB), as well as to determine the relationship between TLR4 and NF-κB in ANP pathogenesis. Methods A total of 72 SD rats were randomly divided into three groups, namely, the control (sham-operation), ANP, and ANP with PDTC pretreatment groups. The PDTC-pretreated group was intraperitoneally injected with PDTC at a dose of 100 mg/kg 1 hour before the induction of ANP. The expressions of TLR4 and NF-κB in pancreatic tissue were evaluated by immunohistochemistry and Western blot analysis. The mRNA levels of cytokines tumor necrosis factor α, interleukin (IL)-1β, and IL-6 were measured by reverse transcription polymerase chain reaction. Results The expressions of TLR4, NF-κB, and cytokine (NF-κB target) genes in the pancreatic tissue increased more significantly in the ANP groups than in the sham-operation group at 3, 6, and 12 hours. Pretreatment with PDTC alleviated the inflammatory activation in the pancreas with ANP, causing a significant decrease in the expressions of TLR4, NF-κB, and cytokine genes in the pancreatic tissue. Conclusions The expressions of TLR4 and NF-κB were increased in the pancreases of rats with ANP. PDTC not only inhibits NF-κB but also suppresses the expression of TLR4 and downregulates the expression of the related cytokine genes. PMID:25287011

  11. Toll-like receptor 4 and CD14 mRNA expression are lower in resistive exercise-trained elderly women.

    PubMed

    Flynn, Michael G; McFarlin, Brian K; Phillips, Melody D; Stewart, Laura K; Timmerman, Kyle L

    2003-11-01

    The purpose of this study was to examine the influence of resistive exercise training and hormone status on mRNA expression of toll-like receptor 4 (TLR4), CD14, IL-1beta, IL-6, and TNF-alpha. Resistive exercise-trained women on "traditional" hormone replacements [hormone replacement therapy (HRT), n = 9], not taking hormones (NHR, n = 6), or taking medications known to influence bone (MIB, n = 7) were compared with untrained subjects not taking supplemental hormones (Con, n = 6). Blood was taken from trained subjects before, immediately after, and 2 h after resistive exercise (same time points for resting Con). TLR4 mRNA expression (RT-PCR) was not different among groups or across time but was significantly (P = 0.044) lower (1.9-fold) when trained groups were collapsed and compared with Con. There was also a significant group effect (P < 0.0001) for TLR4 mRNA when expressed per monocyte. CD14 expression was significantly (P = 0.006) lower (2.3-fold) for training groups collapsed and compared with Con. CD14 mRNA, expressed per monocyte, was significantly lower immediately after resistive exercise for NHR, HRT, and MIB compared with Con. There were few significant effects detected for IL-6, IL-1beta, and TNF-alpha mRNA, but there was a significant group effect (P < 0.0001) for TNF-alpha mRNA expressed per monocyte (Con > HRT, NHR, MIB). These findings suggest that there may be a resistive exercise training-induced reduction in TLR4/CD14 expression in older women. Further research is needed to determine whether lower TLR4/CD14 could explain the lower LPS-stimulated inflammatory cytokines observed in these women.

  12. Expressing death risk as condensed life experience and death intensity.

    PubMed

    Ioannidis, John P A

    2013-08-01

    Some risk exposures, including many medical and surgical procedures, typically carry hazards of death that are difficult to convey and appreciate in absolute terms. I propose presenting the death risk as a condensed life experience (i.e., the equivalent amount of life T that would carry the same cumulative mortality hazard for a person of the same age and sex based on life tables). For example, if the risk of death during an elective 1-hour procedure is 0.01%, and same-age and same-sex people have a 0.01% death risk over 1 month, one can inform the patient that "this procedure carries the same death risk as living 1 month of normal life." Comparative standards from other risky activities or from a person with the same disease at the same stage and same predictive profile could also be used. A complementary metric that may be useful to consider is the death intensity. The death intensity λ is the hazard function that shows the fold-risk estimate of dying compared with the reference person. The death intensity can vary substantially for different phases of the event, operation, or procedure (e.g., intraoperative, early postoperative, late postoperative), and this variability may also be useful to convey. T will vary depending on the time window for which it is computed. I present examples for calculating T and λ using literature data on accidents, ascent to Mount Everest, and medical and surgical procedures.

  13. Pu-erh Tea Reduces Nitric Oxide Levels in Rats by Inhibiting Inducible Nitric Oxide Synthase Expression through Toll-Like Receptor 4

    PubMed Central

    Xu, Yang; Wang, Guan; Li, Chunjie; Zhang, Min; Zhao, Hang; Sheng, Jun; Shi, Wei

    2012-01-01

    Pu-erh tea undergoes a unique fermentation process and contains theabrownins, polysaccharides and caffeine; although it is unclear about which component is associated with the down regulation of nitric oxide levels or how this process is mediated. To address this question we examined the effects of pu-erh tea on nitric oxide synthase (NOS) genes. Cohorts of rats were separately given four-week treatments of water as control, pu-erh tea, or the tea components: theabrownins, caffeine or polysaccharides. Five experimental groups were injected with lipopolysaccharides (LPS) to induce nitric oxide (NO) production, while the corresponding five control groups were injected with saline as a negative control. The serum and liver NO concentrations were examined and the NOS expression of both mRNA and protein was measured in liver. The results showed that the rats which were fed pu-erh tea or polysaccharides had lower levels of NO which corresponded with the down-regulation of inducible nitric oxide synthase (iNOS) expression. We further demonstrate that this effect is mediated through reduction of Toll-like receptor 4 (TLR4) signaling. Thus we find that the polysaccharide components in pu-erh tea reduce NO levels in an animal model by inhibiting the iNOS expression via signaling through TLR4. PMID:22837686

  14. Association between Toll-Like Receptor 4 Expression and Neural Stem Cell Proliferation in the Hippocampus Following Traumatic Brain Injury in Mice

    PubMed Central

    Ye, Yuqin; Xu, Hongyu; Zhang, Xin; Li, Ze; Jia, Yanfeng; He, Xiaosheng; Huang, Jason H.

    2014-01-01

    Whether or how neural stem cells (NSCs) respond to toll-like receptor 4 (TLR4) in an inflammatory environment caused by traumatic brain injury (TBI) has not been understood. In the present study, association between TLR4 expression and NSCs proliferation in the hippocampus was investigated in a mouse model of TBI using controlled cortical impact (CCI). Hippocampal proliferating cells were labeled with the thymidine analog 5-bromo-2-deoxyuridine (BrdU). In order to identify NSCs, the proliferating cells were further co-labeled with BrdU/sex determination region of Y chromosome related high mobility group box gene 2 (SOX2). Morphological observation on the expression of BrdU, SOX2, and TLR4 in the hippocampus was performed by inmmunofluorescence (IF). Relative quantification of TLR4 expression at the protein and mRNA level was performed using Western blotting and real-time polymerase chain reaction (PCR). It was observed that BrdU+/SOX2+ cells accounted for 95.80% ± 7.91% among BrdU+ cells; several BrdU+ cells and SOX2+ cells in the hippocampus were also TLR4-positive post injury, and that BrdU+ cell numbers, together with TLR4 expression at either protein or mRNA level, increased significantly in TBI mice over 1, 3, 7, 14, and 21 days survivals and changed in a similar temporal pattern with a peak at 3 day post-injury. These results indicate that hippocampal proliferating cells (suggestive of NSCs) expressed TLR4, and that there was a potential association between increased expression of TLR4 and the proliferation of NSCs post TBI. It is concluded that hippocampal TLR4 may play a potential role in endogenous neurogenesis after TBI. PMID:25036030

  15. Lipopolysaccharides impair insulin gene expression in isolated islets of Langerhans via Toll-Like Receptor-4 and NF-κB signalling.

    PubMed

    Amyot, Julie; Semache, Meriem; Ferdaoussi, Mourad; Fontés, Ghislaine; Poitout, Vincent

    2012-01-01

    Type 2 diabetes is characterized by pancreatic β-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that the signalling cascade activated by lipopolysaccharides (LPS) binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. In this study, we tested the hypothesis that LPS alters insulin gene expression via TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in islets. A 24-h exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of pancreas-duodenum homebox-1 (PDX-1) and mammalian homologue of avian MafA/l-Maf (MafA). Accordingly, LPS exposure also decreased glucose-induced insulin secretion. LPS repression of insulin, PDX-1 and MafA expression, as well as its inhibition of insulin secretion, were not observed in islets from TLR4-deficient mice. LPS inhibition of β-cell gene expression in rat islets was prevented by inhibition of the NF-κB pathway, but not the p38 mitogen-activated protein kinase (p38 MAPK) pathway. Our findings demonstrate that LPS inhibit β-cell gene expression in a TLR4-dependent manner and via NF-κB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function.

  16. Expression of Toll-Like Receptor 4 and Downstream Effectors in Selected Cecal Cell Subpopulations of Chicks Resistant or Susceptible to Salmonella Carrier State▿

    PubMed Central

    Chaussé, Anne-Marie; Grépinet, Olivier; Bottreau, Elisabeth; Le Vern, Yves; Menanteau, Pierrette; Trotereau, Jérome; Robert, Vincent; Wu, Zhiguang; Kerboeuf, Dominique; Beaumont, Catherine; Velge, Philippe

    2011-01-01

    Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1β (IL-1β), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1β, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ. PMID:21628520

  17. Differential Effects of Cream, Glucose, and Orange Juice on Inflammation, Endotoxin, and the Expression of Toll-Like Receptor-4 and Suppressor of Cytokine Signaling-3

    PubMed Central

    Deopurkar, Rupali; Ghanim, Husam; Friedman, Jay; Abuaysheh, Sanaa; Sia, Chang Ling; Mohanty, Priya; Viswanathan, Prabhakar; Chaudhuri, Ajay; Dandona, Paresh

    2010-01-01

    OBJECTIVE We have recently shown that a high-fat high-carbohydrate (HFHC) meal induces an increase in plasma concentrations of endotoxin (lipopolysaccharide [LPS]) and the expression of Toll-like receptor-4 (TLR-4) and suppresser of cytokine signaling-3 (SOCS3) in mononuclear cells (MNCs) in addition to oxidative stress and cellular inflammation. Saturated fat and carbohydrates, components of the HFHC meal, known to induce oxidative stress and inflammation, also induce an increase in LPS, TLR-4, and SOCS3. RESEARCH DESIGN AND METHODS Fasting normal subjects were given 300-calorie drinks of either glucose, saturated fat as cream, orange juice, or only water to ingest. Blood samples were obtained at 0, 1, 3, and 5 h for analysis. RESULTS Indexes of inflammation including nuclear factor-κB (NF-κB) binding, and the expression of SOCS3, tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β in MNCs, increased significantly after glucose and cream intake, but TLR-4 expression and plasma LPS concentrations increased only after cream intake. The intake of orange juice or water did not induce any change in any of the indexes measured. CONCLUSIONS Although both glucose and cream induce NF-κB binding and an increase in the expression of SOCS3, TNF-α, and IL-1β in MNCs, only cream caused an increase in LPS concentration and TLR-4 expression. Equicaloric amounts of orange juice or water did not induce a change in any of these indexes. These changes are relevant to the pathogenesis of atherosclerosis and insulin resistance. PMID:20067961

  18. Whole body hyperthermia treatment increases interleukin 10 and toll-like receptor 4 expression in patients with ankylosing spondylitis: a pilot study.

    PubMed

    Zauner, Dorothea; Quehenberger, Franz; Hermann, Josef; Dejaco, Christian; Stradner, Martin H; Stojakovic, Tatjana; Angerer, Hannes; Rinner, Beate; Graninger, Winfried B

    2014-09-01

    Exposure to increased environmental temperatures is commonly used as a non-pharmacological treatment modality in ankylosing spondylitis (AS). We aimed to investigate systemic immunological effects of moderate whole body hyperthermia in patients with AS compared to healthy control subjects. Ten healthy control subjects and six AS patients underwent whole body hyperthermia treatment with 38.7-39 °C body core temperature over 60 min. Numbers of polymorphonuclear leucocytes and lymphocyte subsets, plasma concentrations of several acute phase reactants and cytokines, and gene expression levels of toll-like receptor 4 (TLR-4), interleukin 10 (IL-10) and heat shock protein beta 1 (HSPB1) were determined during and up to 24 h after treatment. TLR-4, IL-10 and HSPB1 gene expression increased significantly up to 3 h post treatment, with an earlier, higher and more pronounced increase of IL-10 in patients with AS. An increase of natural killer cells and CD8+ T lymphocytes was noted during active heating, with a subsequent decrease up to 2 h after treatment. CD4+ T lymphocytes showed a short increase during active treatment in AS patients, while decreasing immediately after start of treatment in control subjects. Neutrophil granulocytes increased significantly up to 3 h after treatment, monocytes and B lymphocytes remained unchanged. Likewise, no significant changes were found concerning systemic cytokine concentrations and acute phase reactants. Our data support the concept of systemic immunological effects of moderate whole body hyperthermia in patients with AS.

  19. Binge alcohol drinking is associated with GABAA α2-regulated Toll-like receptor 4 (TLR4) expression in the central amygdala

    PubMed Central

    Liu, Juan; Yang, Andrew R.; Kelly, Timothy; Puche, Adam; Esoga, Chioma; June, Harry L.; Elnabawi, Ahmed; Merchenthaler, Istvan; Sieghart, Werner; June, Harry L.; Aurelian, Laure

    2011-01-01

    Binge drinking (blood-alcohol levels ≥ 0.08 g% in a 2-h period), is a significant public health burden in need of improved treatment. Gene therapy may offer beneficial alternatives to current psychosocial and pharmacotherapeutic interventions, but identification of the target genes is a clinical challenge. We report that a GABAA α2 siRNA vector (pHSVsiLA2) infused into the central nucleus of the amygdala (CeA) of alcohol-preferring (P) rats caused profound and selective reduction of binge drinking associated with inhibition of α2 expression, decreased GABAA receptor density, and inhibition of Toll-like receptor 4 (TLR4). CeA infusion of a TLR4 siRNA vector (pHSVsiLTLR4a) also inhibited binge drinking, but neither vector functioned when infused into the ventral pallidum. Binge drinking was inhibited by a GABAA α1 siRNA vector (pHSVsiLA1) infused into the ventral pallidum, unrelated to TLR4. The vectors did not alter sucrose intake and a scrambled siRNA vector was negative. The data indicate that GABAA α2-regulated TLR4 expression in the CeA contributes to binge drinking and may be a key early neuroadaptation in excessive drinking. PMID:21368176

  20. Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216.

    PubMed

    Kumazoe, Motofumi; Nakamura, Yuki; Yamashita, Mai; Suzuki, Takashi; Takamatsu, Kanako; Huang, Yuhui; Bae, Jaehoon; Yamashita, Shuya; Murata, Motoki; Yamada, Shuhei; Shinoda, Yuki; Yamaguchi, Wataru; Toyoda, Yui; Tachibana, Hirofumi

    2017-03-10

    Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3″Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Lithium ameliorates lipopolysaccharide-induced microglial activation via inhibition of toll-like receptor 4 expression by activating the PI3K/Akt/FoxO1 pathway.

    PubMed

    Dong, Hongquan; Zhang, Xiang; Dai, Xiaonan; Lu, Shunmei; Gui, Bo; Jin, Wenjie; Zhang, Susu; Zhang, Shu; Qian, Yanning

    2014-08-14

    Lithium, an effective mood stabilizer for the treatment of bipolar disorders, has been recently suggested to have a role in neuroprotection during neurodegenerative diseases. The pathogenesis of neurological disorders often involves the activation of microglia and associated inflammatory processes. Thus, in this study, we aimed to understand the role of lithium in microglial activation and to elucidate the underlying mechanism(s). Primary microglial cells were pretreated with lithium and stimulated with lipopolysaccharide (LPS). The cells were assessed regarding the responses of pro-inflammatory cytokines, and the associated signaling pathways were evaluated. Lithium significantly inhibited LPS-induced microglial activation and pro-inflammatory cytokine production. Further analysis showed that lithium could activate PI3K/Akt signaling. Analyses of the associated signaling pathways demonstrated that the lithium pretreatment led to the suppression of LPS-induced toll-like receptor 4 (TLR4) expressions via the PI3K/Akt/FoxO1 pathway. This study demonstrates that lithium can inhibit LPS-induced TLR4 expression and microglial activation through the PI3K/Akt/FoxO1 signaling pathway. These results suggest that lithium plays an important role in microglial activation and neuroinflammation-related diseases, which may lead to a new therapeutic strategy for the treatment of neuroinflammation-related disorders.

  2. Toll-Like Receptor 4 Promotes NO Synthesis by Upregulating GCHI Expression under Oxidative Stress Conditions in Sheep Monocytes/Macrophages.

    PubMed

    Deng, Shoulong; Yu, Kun; Zhang, Baolu; Yao, Yuchang; Wang, Zhixian; Zhang, Jinlong; Zhang, Xiaosheng; Liu, Guoshi; Li, Ning; Liu, Yixun; Lian, Zhengxing

    2015-01-01

    Many groups of Gram-negative bacteria cause diseases that are harmful to sheep. Toll-like receptor 4 (TLR4), which is critical for detecting Gram-negative bacteria by the innate immune system, is activated by lipopolysaccharide (LPS) to initiate inflammatory responses and oxidative stress. Oxidation intermediates are essential activators of oxidative stress, as low levels of free radicals form a stressful oxidative environment that can clear invading pathogens. NO is an oxidation intermediate and its generation is regulated by nitric oxide synthase (iNOS). Guanosine triphosphate cyclohydrolase (GCHI) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, which is essential for the production of inducible iNOS. Previously, we made vectors to overexpress the sheep TLR4 gene. Herein, first generation (G1) of transgenic sheep was stimulated with LPS in vivo and in vitro, and oxidative stress and GCHI expression were investigated. Oxidative injury caused by TLR4 overexpression was tightly regulated in tissues. However, the transgenic (Tg) group still secreted nitric oxide (NO) when an iNOS inhibitor was added. Furthermore, GCHI expression remained upregulated in both serum and monocytes/macrophages. Thus, overexpression of TLR4 in transgenic sheep might accelerate the clearance of invading microbes through NO generation following LPS stimulation. Additionally, TLR4 overexpression also enhances GCHI activation.

  3. Mutation at the folate receptor 4 locus modulates gene expression profiles in the mouse uterus in response to preconceptual folate supplementation

    PubMed Central

    Salbaum, J. michael; Kruger, Claudia; Kappen, Claudia

    2013-01-01

    Periconceptional supplementation of folic acid to the diet of women is considered a great success for a public health intervention. Higher folate status, either by supplementation, or via the mandatory fortification of grain products in the United States, has lead to significant reduction in the incidence of neural tube defects. Besides birth defects, folate deficiency has been linked to a variety of morbidities, most notably to increased risk for cancer. However, recent evidence suggests that excess folate may be detrimental - for birth defect incidence or in the progression of cancer. How folate mediates beneficial or detrimental effects is not well understood. It is also unknown what molecular responses are elicited in women taking folate supplements, and thus experience a bolus of folate on top of the status achieved by fortification. To characterize the response to a preconceptional regimen of supplementation with folinic acid, we performed gene expression profiling experiments on uterus tissue of pregnant mice with either wildtype alleles or targeted disruption at the folate receptor 4 locus. We observed that, depending on the genetic background, folinic acid supplementation affects expression of genes that contribute to lipid metabolism, protein synthesis, mitochondrial function, cell cycle, and cell activation. The extent of the response is strongly modulated by the genetic background. Finally, we provide evidence that folinic acid supplementation in the mutant paradigm affects histone methylation status, a potential mechanisms of gene regulation in this model. PMID:23651732

  4. Eotaxin-2 increased toll-like receptor 4 expression in endothelial cells in vitro and exacerbates high-cholesterol diet-induced atherogenesis in vivo

    PubMed Central

    Tsai, Chien-Sung; Huang, Chun-Yao; Chen, Chien-Ho; Lin, Yi-Wen; Shih, Chun-Ming; Tsao, Nai-Wen; Chiang, Kuang-Hsing; Lee, Chi-Yuan; Jeng, Hellen; Lin, Feng-Yen

    2016-01-01

    Eotaxin-2 is a potent chemoattractant. High concentration of eotaxin-2 triggers the inflammation and tumor metastasis. Inhibition of eotaxin-2 may protect experimental atherogenesis although the mechanism is still unclear. Toll-like receptor 4 (TLR4) plays a major role mediating vascular inflammation, which is related to atherogenesis. In the results, we demonstrated that eotaxin-2 potentially impairs the tube formation capacity of human coronary artery endothelial cells (HCAECs). Eotaxin-2 augments the monocytic adhesion in lipopolysaccharides (LPS)-induced HCAECs, and which were reversed by TLR4 siRNA. Thus this study was conducted to investigate whether eotaxin-2 increases TLR4 expression, and then enhances the sensitivity of cells to antigen stimulation in HCAECs, which mediates the increasing of the development of serious atherosclerosis. In fact, we showed that JNK/SAPK, p38 MAPK, and ERK1/2 activation contribute to the transcriptional signaling pathway, JNK/SAPK and p38 MAPK regulate post-transcriptional modification, as well as protein-trafficking pathway in eotaxin-2-treated HCAECs TLR4 expression. RNA binding proteins, such as human antigen R (HuR) and tristetraprolin (TTP) mediate stability of TLR4 mRNA and chaperone, such as PRAT4A (a protein associated with TLR4) regulate trafficking of TLR4 protein might confer eotaxin-2 responsiveness. Eotaxin-2 administration led to a significant elevation of high cholesterol diet-induced atherosclerosis, and of TLR4 expression in B6.129S7-Ldlrtm1Her/J but not Ldlr-/--/-/ Tlr4-/- mice. Our results revealed that eotaxin-2 induced overexpression TLR4 via mitogen-activated protein kinases (MAPK) signaling pathways, RNA binding proteins-mediated mRNA stabilization, and PRAT4A-regulated trafficking in HCAECs. These effects may lead to amplification of inflammatory responses contribute to the pathogenesis of cardiovascular disorders. PMID:28078007

  5. Coenzyme Q10 supplementation downregulates the increase of monocytes expressing toll-like receptor 4 in response to 6-day intensive training in kendo athletes.

    PubMed

    Shimizu, Kazuhiro; Kon, Michihiro; Tanimura, Yuko; Hanaoka, Yukichi; Kimura, Fuminori; Akama, Takao; Kono, Ichiro

    2015-06-01

    This study examined changes in toll-like receptor 4 (TLR-4)-expressing monocytes and lymphocyte subpopulations in response to continuous intensive exercise training in athletes, as well as the effect of coenzyme Q10 (CoQ10) supplementation on these changes. Eighteen male elite kendo athletes in Japan were randomly assigned to a CoQ10-supplementation group (n = 9) or a placebo-supplementation group (n = 9) using a double-blind method. Subjects in the CoQ10 group took 300 mg CoQ10 per day for 20 days. Subjects in the placebo group took the same dosage of placebo. All subjects practiced kendo 5.5 h per day for 6 consecutive days during the study period. Blood samples were collected 2 weeks before training, on the first day (day 1), third day (day 3), and fifth day of training (day 5), and 1 week after the training period (post-training) to ascertain TLR-4(+)/CD14(+) monocyte and lymphocyte subpopulations (CD3(+), CD4(+), CD8(+), CD28(+)/CD4(+), CD28(+)/CD8(+), and CD56(+)/CD3(-) cells) using flow cytometry analysis. The group × time interaction for TLR-4(+)/CD14(+) cells did not reach significance (p = 0.08). Within the CoQ10 group, the absolute number of TLR-4(+)/CD14(+) cells was significantly higher only at day 5. The placebo group showed a significant increase in the absolute number of TLR-4(+)/CD14(+) cells at day 3, day 5, and post-training (p < 0.05). There was no significant group × time interaction for any lymphocyte subpopulation. CD3(+), CD8(+), and CD56(+)/CD3(-) cells were significantly reduced at day 3 in both groups (p < 0.05). In conclusion, CoQ10 supplementation might downregulate the increase of TLR-4-expressing monocytes in response to continuous strenuous exercise training in kendo athletes.

  6. The GroEL protein of Porphyromonas gingivalis regulates atherogenic phenomena in endothelial cells mediated by upregulating toll-like receptor 4 expression

    PubMed Central

    Huang, Chun-Yao; Shih, Chun-Ming; Tsao, Nai-Wen; Lin, Yi-Wen; Shih, Chun-Che; Chiang, Kuang-Hsing; Shyue, Song-Kun; Chang, Yu-Jia; Hsieh, Chi-Kun; Lin, Feng-Yen

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is a bacterial species that causes periodontitis. GroEL from P. gingivalis may possess biological activity and may be involved in the destruction of periodontal tissues. However, it is unclear whether P. gingivalis GroEL enhances the appearance of atherogenic phenomena in endothelial cells and vessels. Here, we constructed recombinant GroEL from P. gingivalis to investigate its effects in human coronary artery endothelial cells (HCAECs) in vitro and on aortas of high-cholesterol (HC)-fed B57BL/6 and B57BL/6-Tlr4lps-del mice in vivo. The results showed that GroEL impaired tube-formation capacity under non-cytotoxic conditions in HCAECs. GroEL increased THP-1 cell/HCAEC adhesion by increasing the expression of intracellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 in endothelial cells. Additionally, GroEL increased DiI-oxidized low density lipoprotein (oxLDL) uptake, which may be mediated by elevated lectin-like oxLDL receptor (LOX)-1 but not scavenger receptor expressed by endothelial cells (SREC) and scavenger receptor class B1 (SR-B1) expression. Furthermore, GroEL interacts with toll-like receptor 4 (TLR4) and plays a causal role in atherogenesis in HCAECs. Human antigen R (HuR), an RNA-binding protein with a high affinity for the 3’ untranslated region (3’UTR) of TLR4 mRNA, contributes to the up-regulation of TLR4 induced by GroEL in HCAECs. In a GroEL animal administration study, GroEL elevated ICAM-1, VCAM-1, LOX-1 and TLR4 expression in the aortas of HC diet-fed wild C57BL/6 but not C57BL/6-Tlr4lps-del mice. Taken together, our findings suggest that P. gingivalis GroEL may contribute to cardiovascular disorders by affecting TLR4 expression. PMID:27158334

  7. EPA and DHA exposure alters the inflammatory response but not the surface expression of toll-like receptor 4 in macrophages

    PubMed Central

    Honda, Kaori L.; Lamon-Fava, Stefania; Matthan, Nirupa R.; Wu, Dayong; Lichtenstein, Alice H.

    2014-01-01

    Dietary intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and their respective enrichment in cell membranes have been negatively associated with atherosclerotic lesion development. This effect may be mediated, in part, by dampened inflammatory response of macrophages triggered by toll-like receptor 4 (TLR4) activation. This study investigated the influence of membrane fatty acid profile on TLR4-mediated inflammation in RAW 264.7 macrophages. Cells pretreated with myristic acid (MA), EPA, DHA or vehicle control for 24 h were stimulated with ultra-pure LPS, a specific TLR4 agonist, for 6 h or 24 h, corresponding to early and late stages of TNFα and IL-6 protein induction. Treatment significantly increased cell membrane MA, EPA, and DHA by 4.5-, 20.6-, and 8.9-fold, respectively. MA significantly increased IL-6 secretion 6 h post-exposure to the fatty acid, but did not change TNFα secretion in response to any other treatment condition. EPA and DHA significantly reduced TNFα secretion by 36% and 41%, respectively, in cells stimulated for 24 h but not 6 h. In contrast, EPA and DHA significantly reduced IL-6 secretion at both 6 h (67% and 72%, respectively) and 24 h (69% and 72%, respectively). MA or DHA treatment had no significant effect compared to vehicle on factors influencing cellular LPS recognition, including LPS-cell association, and cell surface expression of TLR4, TLR4-MD2 complex, and CD14. These data suggest that membrane fatty acid profiles influence the TLR4-mediated inflammatory response in macrophages, via mechanisms that occur downstream of TLR4 receptor activation. PMID:25408476

  8. [Effect of high volume hemofiltration on expression of Toll-like receptor 4 in myocardium in endotoxin induced shock in dogs].

    PubMed

    Zeng, Zhen-guo; Zhang, Jian-guo; Jiang, Rong; Xia, Liang; Qian, Ke-jian; Wang, Chun-ting

    2010-05-01

    To investigate the effects of high volume hemofiltration (HVHF) on the expression of Toll-like receptor 4 (TLR4) mRNA in myocardium in endotoxin (lipopolysaccharide, LPS) induced shock in dogs. Sixteen healthy male dogs were injected with LPS 650 microg/kg via central vein to reproduce the model of endotoxin shock. All dogs were divided randomly into two groups: control group and therapy group, with 8 dogs in each group. Contents of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10 in circulation were measured by radioimmunological method. The expression levels of TLR4 mRNA in all group were measured by reverse transcription-polymerase chain reaction (RT-PCR). Change in myocardial histopathology was observed and analyzed with the aid of electron microscope. The contents of TNF-alpha (microg/L: 0.59+/-0.15, 0.51+/-0.12, 0.41+/-0.10), IL-6 (ng/L: 11.08+/-2.83, 9.82+/-2.58, 8.25+/-2.05), IL-10 (microg/L: 57.28+/-5.93, 53.81+/-5.83, 50.67+/-6.33) in therapy group were found to have decreased significantly at 1, 2, and 4 hours after HVHF compared with those when the model was completed [(0.84+/-0.16) microg/L, (16.97+/-2.50) ng/L, (70.86+/-5.43) microg/L], showing a continuous trend of lowering (all P<0.01). The contents of TNF-alpha, IL-6, IL-10 in therapy group were lower than those in control group significantly at any time point [TNF-alpha (microg/L): 0.75+/-0.14, 0.74+/-0.11, 0.72+/-0.11, IL-6 (ng/L): 15.33+/-3.20, 14.66+/-3.24, 14.20+/-3.33, IL-10 (microg/L): 71.54+/-4.73, 70.71+/-4.34, 69.35+/-4.60, all P<0.01]. Compared with control group, HVHF treatment group could down-regulate mRNA expression of TLR4 in myocardium (t=3.58, P<0.01). Correlation analysis revealed significant positive-correlation between tissue TLR4 mRNA expression and contents of TNF-alpha, IL-6, IL-10 in circulation (r(1)=0.785, r(2)=0.569, r(3)=0.653, all P<0.05). Injury to the myocardium was significantly ameliorated in therapy group compared with control group as shown by

  9. Campylobacter jejuni Increases Flagellar Expression and Adhesion of Noninvasive Escherichia coli: Effects on Enterocytic Toll-Like Receptor 4 and CXCL-8 Expression

    PubMed Central

    Reti, Kristen L.; Tymensen, Lisa D.; Davis, Shevaun P.; Amrein, Matthias W.

    2015-01-01

    Campylobacter jejuni is the most common cause of bacterium-induced gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with postinfectious intestinal disorders, including flares in patients with inflammatory bowel disease and postinfectious irritable bowel syndrome (PI-IBS), via mechanisms that remain obscure. Based on the hypothesis that acute campylobacteriosis may cause pathogenic microbiota dysbiosis, we investigated whether C. jejuni may activate dormant virulence genes in noninvasive Escherichia coli and examined the epithelial pathophysiological consequences of these alterations. Microarray and quantitative real-time PCR analyses revealed that E. coli adhesin, flagellum, and hemolysin gene expression were increased when E. coli was exposed to C. jejuni-conditioned medium. Increased development of bacterial flagella upon exposure to live C. jejuni or C. jejuni-conditioned medium was observed under transmission electron microscopy. Atomic force microscopy demonstrated that the forces of bacterial adhesion to colonic T84 enterocytes, and the work required to rupture this adhesion, were significantly increased in E. coli exposed to C. jejuni-conditioned media. Finally, C. jejuni-modified E. coli disrupted TLR4 gene expression and induced proinflammatory CXCL-8 gene expression in colonic enterocytes. Together, these data suggest that exposure to live C. jejuni, and/or to its secretory-excretory products, may activate latent virulence genes in noninvasive E. coli and that these alterations may directly trigger proinflammatory signaling in intestinal epithelia. These observations shed new light on mechanisms that may contribute, at least in part, to postcampylobacteriosis inflammatory disorders. PMID:26371123

  10. Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

    SciTech Connect

    Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

    2008-04-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha} and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms

  11. Ketamine inhibits tumor necrosis factor-alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation.

    PubMed

    Wu, Gone-Jhe; Chen, Ta-Liang; Ueng, Yune-Fang; Chen, Ruei-Ming

    2008-04-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through

  12. Cutting Edge: Roles of Toll-Like Receptor 4 and IL-23 in IL-17 Expression in Response to Klebsiella pneumoniae Infection1

    PubMed Central

    Happel, Kyle I.; Zheng, Mingquan; Young, Erana; Quinton, Lee J.; Lockhart, Euan; Ramsay, Alistair J.; Shellito, Judd E.; Schurr, Jill R.; Bagby, Gregory J.; Nelson, Steve; Kolls, Jay K.

    2010-01-01

    Local production of IL-17 is a significant factor in effective host defense against Gram-negative bacteria. However, the proximal events mediating IL-17 elaboration by T cells remain unclear. In this study, we show in vivo that intact Toll-like receptor 4 signaling in the lung is required for induction of both the p19 transcript of IL-23 and IL-17 protein elaboration in response to Klebsiella pneumoniae. Although IL-17 is widely considered a CD4+ T cell product, we also demonstrate significant in vitro IL-17 production by CD8+ T cells after culture in medium from dendritic cells exposed to these bacteria. The dominant portion of this IL-17-inducing activity for both CD4+ and CD8+ T cells is IL-23. These data demonstrate the critical signaling pathway for IL-17 induction in the host response to Gram-negative pulmonary infection and suggest a direct role for IL-23 in CD8+ T cell IL-17 production. PMID:12707317

  13. Resistin-induced stromal cell-derived factor-1 expression through Toll-like receptor 4 and activation of p38 MAPK/ NFκB signaling pathway in gastric cancer cells.

    PubMed

    Hsieh, Yung-Yu; Shen, Chien-Heng; Huang, Wen-Shih; Chin, Chih-Chien; Kuo, Yi-Hung; Hsieh, Meng Chiao; Yu, Hong-Ren; Chang, Te-Sheng; Lin, Tseng-Hsi; Chiu, Yung-Wei; Chen, Cheng-Nan; Kuo, Hsing-Chun; Tung, Shui-Yi

    2014-06-14

    Stromal cell-derived factor-1 (SDF-1) (CXC chemokine ligand-12)/CXC chemokine receptor 4 (CXCR4) is involved in the carcinogenesis of human gastric cancer, where it stimulates angiogenesis and favors metastasis of tumor cells to distant organs. In addition, resistin is suggested to be an important link between obesity and the development of gastric cancer. Resistin has identified as an important player in inflammatory responses, and emerged as a mediator in inflammation-associated cancer. A limited number of studies have investigated the association of resistin and SDF-1 with gastric cancer. Herein, we investigated the molecular mechanisms by which resistin influences the expression of SDF-1 in gastric carcinoma cells. Human gastric cancer cell lines were exposed to doses of resistin; SDF-1 expression and secretion levels were then determined. Real-time polymerase chain reaction and western blotting analyses were performed to clarify molecular changes. Inhibition of Toll-like receptor 4 (TLR4) by a competitive antagonist inhibited resistin-induced SDF-1 expression. Pharmacological inhibitors and small interfering RNA (siRNA) demonstrated that activation of the p38 mitogen-activated protein kinase (MAPK) pathway is critical for resistin-induced SDF-1 expression mediated by TLR4. The promoter activity and transcription factor enzyme-linked immunosorbent assay revealed that resistin induced expression of SDF-1 mediated by NF-κB in gastric cancer cells. Inhibition of p38 MARK activation blocked the SDF-1-induced expression and the SDF-1 promoter activity in the cancer gastric cells. Chromatin immunoprecipitation assay revealed that inhibition of p38 MARK activation also blocked the resistin-increased NF-κB-DNA-binding activity. Resistin-induced SDF-1 upregulation by activation of TLR4, p38 MARK and NF-κB may explain a new role of resistin in the link of obesity and gastric cancer.

  14. Resistin-induced stromal cell-derived factor-1 expression through Toll-like receptor 4 and activation of p38 MAPK/ NFκB signaling pathway in gastric cancer cells

    PubMed Central

    2014-01-01

    Background Stromal cell-derived factor-1 (SDF-1) (CXC chemokine ligand-12)/CXC chemokine receptor 4 (CXCR4) is involved in the carcinogenesis of human gastric cancer, where it stimulates angiogenesis and favors metastasis of tumor cells to distant organs. In addition, resistin is suggested to be an important link between obesity and the development of gastric cancer. Resistin has identified as an important player in inflammatory responses, and emerged as a mediator in inflammation-associated cancer. A limited number of studies have investigated the association of resistin and SDF-1 with gastric cancer. Herein, we investigated the molecular mechanisms by which resistin influences the expression of SDF-1 in gastric carcinoma cells. Results Human gastric cancer cell lines were exposed to doses of resistin; SDF-1 expression and secretion levels were then determined. Real-time polymerase chain reaction and western blotting analyses were performed to clarify molecular changes. Inhibition of Toll-like receptor 4 (TLR4) by a competitive antagonist inhibited resistin-induced SDF-1 expression. Pharmacological inhibitors and small interfering RNA (siRNA) demonstrated that activation of the p38 mitogen-activated protein kinase (MAPK) pathway is critical for resistin-induced SDF-1 expression mediated by TLR4. The promoter activity and transcription factor enzyme-linked immunosorbent assay revealed that resistin induced expression of SDF-1 mediated by NF-κB in gastric cancer cells. Inhibition of p38 MARK activation blocked the SDF-1-induced expression and the SDF-1 promoter activity in the cancer gastric cells. Chromatin immunoprecipitation assay revealed that inhibition of p38 MARK activation also blocked the resistin-increased NF-κB-DNA-binding activity. Conclusions Resistin-induced SDF-1 upregulation by activation of TLR4, p38 MARK and NF-κB may explain a new role of resistin in the link of obesity and gastric cancer. PMID:24929539

  15. The revival of death: expression, expertise and governmentality.

    PubMed

    Arnason, Arnar; Hafsteinsson, Sigurjón Baldur

    2003-03-01

    This paper discusses Walter's (1994) assertion that death in the West has recently undergone a revival. In particular it focuses on his claim that this revival is composed of two different strands: a late modern strand and a postmodern strand. The former, according to Walter, is driven by experts who seek to control death, the latter by ordinary people who seek to express their emotions freely. Describing the history and work of Cruse Bereavement Care, the largest bereavement counselling organization in the UK, we question Walter's distinction. We then problematize Walter's suggestion that the revival of death is caused by general social transformations. In contrast we evoke Rose's (1996) work on 'subjectification' and seek to link recent changes in the management of death and grief to permutations in governmental rationality.

  16. Mincle suppresses Toll-like receptor 4 activation.

    PubMed

    Greco, Stephanie H; Mahmood, Syed Kashif; Vahle, Anne-Kristin; Ochi, Atsuo; Batel, Jennifer; Deutsch, Michael; Barilla, Rocky; Seifert, Lena; Pachter, H Leon; Daley, Donnele; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu R; Miller, George

    2016-07-01

    Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle(-/-) leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation.

  17. PI3K/Akt contributes to increased expression of Toll-like receptor 4 in macrophages exposed to hypoxic stress

    SciTech Connect

    Kim, So Young; Jeong, Eunshil; Joung, Sun Myung; Lee, Joo Young

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer Hypoxic stress-induced TLR4 expression is mediated by PI3K/Akt in macrophages. Black-Right-Pointing-Pointer PI3K/Akt regulated HIF-1 activation leading to TLR4 expression. Black-Right-Pointing-Pointer p38 mitogen-activated protein kinase was not involved in TLR4 expression by hypoxic stress. Black-Right-Pointing-Pointer Sulforaphane suppressed hypoxia-mediated TLR4 expression by inhibiting PI3K/Akt. -- Abstract: Toll-like receptors (TLRs) play critical roles in triggering immune and inflammatory responses by detecting invading microbial pathogens and endogenous danger signals. Increased expression of TLR4 is implicated in aggravated inflammatory symptoms in ischemic tissue injury and chronic diseases. Results from our previous study showed that TLR4 expression was upregulated by hypoxic stress mediated by hypoxia-inducible factor-1 (HIF-1) at a transcriptional level in macrophages. In this study, we further investigated the upstream signaling pathway that contributed to the increase of TLR4 expression by hypoxic stress. Either treatment with pharmacological inhibitors of PI3K and Akt or knockdown of Akt expression by siRNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl{sub 2}. Phosphorylation of Akt by hypoxic stress preceded nuclear accumulation of HIF-1{alpha}. A PI3K inhibitor (LY294002) attenuated CoCl{sub 2}-induced nuclear accumulation and transcriptional activation of HIF-1{alpha}. In addition, HIF-1{alpha}-mediated upregulation of TLR4 expression was blocked by LY294002. Furthermore, sulforaphane suppressed hypoxia- and CoCl{sub 2}-induced upregulation of TLR4 mRNA and protein by inhibiting PI3K/Akt activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1{alpha}. However, p38 was not involved in HIF-1{alpha} activation and TLR4 expression induced by hypoxic stress in macrophages. Collectively, our results demonstrate that PI3K

  18. Decreased expression of Toll-like receptor 4 and 5 during progression of prostate transformation in transgenic adenocarcinoma of mouse prostate mice.

    PubMed

    Han, Ju-Hee; Park, Jong-Hwan; Kim, Bo-Yeon; Chang, Seo-Na; Kim, Tae-Hyoun; Park, Jae-Hak; Kim, Dong-Jae

    2015-01-01

    Chronic inflammation has been considered an important risk factor for development of prostate cancer. Toll-like receptors (TLRs) recognize microbial moieties or endogenous molecules and play an important role in the triggering and promotion of inflammation. In this study, we examined whether expression of TLR4 and TLR5 was associated with progression of prostate transformation in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The expression of TLR4 and TLR5 was evaluated by immunohistochemisty in formalin-fixed paraffin-embedded prostate tissue from wild-type (WT) and TRAMP mice. Normal prostate tissue from WT mice showed strong expression of TLR4 and TLR5. However, TLR4 expression in the prostate tissue from TRAMP mice gradually decreased as pathologic grade became more aggressive. TLR5 expression in the prostate tissue from TRAMP mice also decreased in low-grade prostate intraepithelial neoplasia (PIN), high-grade PIN and poorly differentiated adenocarcinoma. Overall, our results suggest that decreased expression of TLR4 and TLR5 may contribute to prostate tumorigenesis.

  19. Correlated gene expression encoding serotonin (5-HT) receptor 4 and 5-HT transporter in proximal colonic segments of mice across different colonization states and sexes.

    PubMed

    Reigstad, C S; Linden, D R; Szurszewski, J H; Sonnenburg, J L; Farrugia, G; Kashyap, P C

    2016-09-01

    The production and handling of serotonin (5-HT) is an important determinant of colonic motility and has been reported to be altered in gastrointestinal (GI) disorders such as irritable bowel syndrome (IBS). Recent studies suggest that the intestinal microbiota and sex of the host can influence expression of genes involved in 5-HT biosynthesis and signaling. While expression of genes in serotonergic pathways has been shown to be variable, it remains unclear whether genes within this pathway are coregulated. As a first step in that direction, we investigated potential correlations in relative mRNA expression of serotonergic genes, in the proximal colon isolated from male and female mice in different states of microbial association: germ-free (GF), humanized (ex-germ-free colonized with human gut microbiota, HM), and conventionally raised (CR) mice. Among the 10 pairwise comparisons conducted between five serotonergic transcripts, Tph1, Chga, Maoa, Slc6a4, and Htr4, we found a strong, positive correlation between colonic expression of Slc6a4 and Htr4 across different colonization states and sexes. We also identified a positive correlation between the expression of Tph1 and Chga; however, there were no correlations observed between any other tested pair of 5-HT-related transcripts. These data suggest that correlated expression of Slc6a4 and Htr4 likely involves coregulation of genes located on different chromosomes which modulate serotonergic activity in the gut. Further work will need to be done to understand the pathways and cell types responsible for this correlated expression, given the important role of 5-HT in gastrointestinal physiology.

  20. Medium dose ultraviolet A1 phototherapy and mRNA expression of interleukin 8, interferon γ, and chemokine receptor 4 in acute skin lesions in atopic dermatitis

    PubMed Central

    Malinowska, Karolina; Sysa-Jedrzejowska, Anna; Wozniacka, Anna

    2016-01-01

    Introduction Mechanisms responsible for UVA1 efficacy in atopic dermatitis (AD) are not fully elucidated. Aim To investigate IL-8, CCR-4, and IFN-γ mRNA expression in AD before and after UVA1, to identify correlations among them, and to determine whether and to what degree mRNA expression is influenced by UVA1. Material and methods Twenty-five patients with AD underwent medium dose UVA1-phototherapy at daily dosages of 10, 20, 30, 45, and then continuing 45 J/cm2 up to 20 days, from Monday to Friday for 4 weeks. Before and after UVA1, biopsies from acute skin lesions were studied using reverse-transcription and RT-PCR. Results The levels of CCR-4 mRNA correlated with those of IFN-γ, both before and after UVA1 phototherapy (p < 0.05). A significant correlation was found after UVA1 between mRNA levels of IL-8 and IFN-γ (p < 0.05). After UVA1 an increase in IL-8 mRNA expression in comparison to the baseline assessment (p = 0.02) was found, while no significant difference was revealed in the expression of CCR-4 and IFN-γ mRNA. UVA1 improved both SCORAD and severity of AD (p < 0.001). SCORAD and the severity of AD did not correlate with the degree of expression of measured cytokine mRNA, neither before nor after UVA1. Conclusions CCR-4 is expressed in parallel with IFN-γ in acute skin lesions of patients with AD both before and after UVA1 phototherapy. UVA1 significantly improves SCORAD index, lessens the severity of AD and increases the expression of IL-8, with no direct effects on other studied molecules. PMID:27512350

  1. Toll-Like Receptor 4 (TLR4) and Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) Activation Balance Astrocyte Polarization into a Proinflammatory Phenotype.

    PubMed

    Rosciszewski, Gerardo; Cadena, Vanesa; Murta, Veronica; Lukin, Jeronimo; Villarreal, Alejandro; Roger, Thierry; Ramos, Alberto Javier

    2017-05-25

    Astrocytes react to brain injury with a generic response known as reactive gliosis, which involves activation of multiple intracellular pathways including several that may be beneficial for neuronal survival. However, by unknown mechanisms, reactive astrocytes can polarize into a proinflammatory phenotype that induces neurodegeneration. In order to study reactive gliosis and astroglial polarization into a proinflammatory phenotype, we used cortical devascularization-induced brain ischemia in Wistar rats and primary astroglial cell cultures exposed to oxygen-glucose deprivation (OGD). We analyzed the profile of TLR4 expression and the consequences of its activation by gain- and loss-of-function studies, and the effects produced by the activation of triggering receptor expressed on myeloid cells-2 (TREM-2), a negative regulator of TLR4 signaling. Both OGD exposure on primary astroglial cell cultures and cortical devascularization brain ischemia in rats induced TLR4 expression in astrocytes. In vivo, astroglial TLR4 expression was specifically observed in the ischemic penumbra surrounding necrotic core. Functional studies showed that OGD increased the astroglial response to the TLR4 agonist lipopolysaccharide (LPS), and conversely, TLR4 knockout primary astrocytes had impaired nuclear factor kappa-B (NF-κB) activation when exposed to LPS. In gain-of-function studies, plasmid-mediated TLR4 over-expression exacerbated astroglial response to LPS as shown by sustained NF-κB activation and increased expression of proinflammatory cytokines IL-1β and TNFα. TREM-2 expression, although present in naïve primary astrocytes, was induced by OGD, LPS, or high-mobility group box 1 protein (HMGB-1) exposure. TREM-2 activation by antibody cross-linking or the overexpression of TREM-2 intracellular adaptor, DAP12, partially suppressed LPS-induced NF-κB activation in purified astrocytic cultures. In vivo, TREM-2 expression was observed in macrophages and astrocytes located in the

  2. The expression of functional Toll-like receptor 4 is associated with proliferation and maintenance of stem cell phenotype in endothelial progenitor cells (EPCs).

    PubMed

    He, Jin; Xiao, Zhilin; Chen, Xiaobin; Chen, Meifang; Fang, Li; Yang, Mei; Lv, Qingshan; Li, Yuehui; Li, Guancheng; Hu, Jinyue; Xie, Xiumei

    2010-09-01

    Endothelial dysfunction is involved in various cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs) contribute to re-endothelialization and neo-vascularization, and the increase of EPCs in peripherial circulation benefits the prognosis of cardiovascular disease. However, little is known about the biological stimuli that initiate the proliferation and the maintenance of stem cell phenotype of EPCs. Here we reported that human umbilical vein blood derived EPCs expressed gene transcripts coding for Toll-like receptor (TLR) 1-6, TLR8-10, TLR4 co-receptor CD14, and myeloid differentiation factor 88 (MyD88), a TLR adaptor molecule. Protein expression of TLR2, 4, CD14, and MyD88 was also detected by FACS or Western blot. The activation of TLR4 by LPS modulated the expression of TLRs, induced the phosphorylation of NF-kappaB, P38, and ERK42/44, and up-regulated the gene expression of cytokines IL-8, IFN-alpha, IFN-beta, and TNF-alpha, suggesting EPCs expressed functional TLR4. Unexpectedly LPS treatment failed to induce apoptosis in EPCs, but instead promoted cell proliferation of EPCs. Furthermore, the treatment of EPCs with LPS up-regulated stem cell markers AC133 and CD34 in both mRNA and protein levels, and down-regulated the protein expression of differential marker eNOS. These results suggested that TLR4 functions to maintain the stem cell phenotype of EPCs and enlarge its population, which reveals a novel aspect of the multiple-faced TLR biology, and may open new prospects for using TLR4 agonists to promote the production of EPCs for clinical use.

  3. Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts

    PubMed Central

    2014-01-01

    Background Toll-like receptors (TLRs) are recognized as important contributors to the initiation and modulation of the inflammatory response in the eye. This study investigated the precise expression patterns and functionality of TLRs in human corneal epithelial cells (HCE) and in conjunctival fibroblasts (HCF). Methods The cell surface expression of TLRs 2-4, TLR7 and TLR9 in HCE and HCF was examined by flow cytometry with or without stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C). The mRNA expression of the TLRs was determined by real-time PCR. The protein content levels of interleukin (IL)-6, IL-8, IL-1β and tumor necrosis factor-α (TNF-α) were measured in HCE and HCF using multiplex fluorescent bead immunoassay (FBI). Results The surface expression of TLR3 and TLR4 was detected on both HCE and HCF. Following incubation with LPS, the percentage of HCE cells staining for TLR4 decreased from 10.18% to 0.62% (P < 0.001). Incubation with poly I:C lowered the percentage of HCE cells positive for TLR3 from 10.44% to 2.84% (P < 0.001). The mRNA expression of TLRs2, 4, 7 and 9 was detected in HCE only. Activation of HCE with LPS complex elicited protein secretion up to 4.51 ± 0.85-fold higher levels of IL-6 (P < 0.05), 2.5 ± 0.36-fold IL-8 (P > 0.05), 4.35 ± 1.12-fold IL-1β (P > 0.05) and 29.35 ± 2.3-fold TNFα (P < 0.05) compared to cells incubated in medium. Conclusions HCF and HCE both express TLRs that respond to specific ligands by increasing cytokine expression. Following activation, the surface expression of TLR3 and TLR4 on HCE is decreased, thus creating a negative feedback loop, mitigating the effect of TLR activation. PMID:24491080

  4. Specific vaginal lactobacilli suppress the inflammation induced by lipopolysaccharide stimulation through downregulation of toll-like receptor 4 expression in human embryonic intestinal epithelial cells

    PubMed Central

    TOBITA, Keisuke; WATANABE, Itsuki; SAITO, Masanori

    2016-01-01

    Vaginal lactobacilli (VLB) spread from the mother to the infant during vaginal delivery. However, the effects of VLB on infant intestinal function remain unclear. We investigated the probiotic function and immune effects of VLB on the human embryonic intestinal epithelial cell line INT-407. VLB survived artificial gastric juice and adhered to INT-407 cells. Exposure of INT-407 cells to VLB attenuated both the lipopolysaccharide (LPS)-induced stimulation of interleukin-8 and tumor necrosis factor alpha production and the LPS-stimulated upregulation of TLR4 expression. These results suggest that specific VLB suppresses the inflammation induced by LPS stimulation through downregulation of TLR4 expression in human embryonic intestinal epithelial cells. PMID:28243550

  5. Apigenin suppresses migration and invasion of transformed cells through down-regulation of C-X-C chemokine receptor 4 expression

    SciTech Connect

    Wang, Lei; Kuang, Lisha; Hitron, John Andrew; Son, Young-Ok; Wang, Xin; Budhraja, Amit; Lee, Jeong-Chae; Pratheeshkumar, Poyil; Chen, Gang; Zhang, Zhuo; Luo, Jia; Shi, Xianglin

    2013-10-01

    Environmental exposure to arsenic is known to cause various cancers. There are some potential relationships between cell malignant transformation and C-X-C chemokine receptor type 4 (CXCR4) expressions. Metastasis, one of the major characteristics of malignantly transformed cells, contributes to the high mortality of cells. CXCR4 and its natural chemokine ligand C-X-C motif ligand 12 (CXCL12) play a critical role in metastasis. Therefore, identification of nutritional factors which are able to inhibit CXCR4 is important for protection from environmental arsenic-induced carcinogenesis and for abolishing metastasis of malignantly transformed cells. The present study demonstrates that apigenin (4′,5,7-trihydroxyflavone), a natural dietary flavonoid, suppressed CXCR4 expression in arsenic-transformed Beas-2B cells (B-AsT) and several other types of transformed/cancer cells in a dose- and time-dependent manner. Neither proteasome nor lysosome inhibitor had any effect in reducing the apigenin-induced down-regulation of CXCR4, indicating that apigenin-induced down-regulation of CXCR4 is not due to proteolytic degradation. The down-regulation of CXCR4 is mainly due to the inhibition of nuclear factor κB (NF-κB) transcriptional activity. Apigenin also abolished migration and invasion of transformed cells induced by CXCL12. In a xenograft mouse model, apigenin down-regulated CXCR4 expression and suppressed tumor growth. Taken together, our results show that apigenin is a novel inhibitor of CXCR4 expression. This dietary flavonoid has the potential to suppress migration and invasion of transformed cells and prevent environmental arsenic-induced carcinogenesis. - Highlights: • Apigenin has a potential in preventing environmental arsenic induced carcinogenesis. • Apigenin suppresses CXCR4 in malignant transformed cells in vitro and in vivo. • The down-regulation of CXCR4 is mainly due to inhibition of NF-κB activity.

  6. Apigenin suppresses migration and invasion of transformed cells through down-regulation of C-X-C chemokine receptor 4 expression

    PubMed Central

    Wang, Lei; Kuang, Lisha; Hitron, John Andrew; Son, Young-Ok; Wang, Xin; Budhraja, Amit; Lee, Jeong-Chae; Poyil, Pratheeshkumar; Chen, Gang; Zhang, Zhuo; Luo, Jia; Shi, Xianglin

    2013-01-01

    Environmental exposure to arsenic is known to cause various cancers. There are some potential relationships between cell malignant transformation and C-X-C chemokine receptor type 4 (CXCR4) expressions. Metastasis, one of the major characteristics of malignantly transformed cells, contributes to the high mortality of cells. CXCR4 and its natural chemokine ligand C-X-C motif ligand 12 (CXCL12) play a critical role in metastasis. Therefore, identification of nutritional factors which are able to inhibit CXCR4 is important for protection from environmental arsenic-induced carcinogenesis and for abolishing metastasis of malignantly transformed cells. The present study demonstrates that apigenin (4′, 5, 7-trihydroxyflavone), a natural dietary flavonoid, suppressed CXCR4 expression in arsenic-transformed Beas-2B cells (B-AsT) and several other type of transformed/cancer cells in a dose- and time-dependent manner. Neither proteasome nor lysosome inhibitor had any effect in reducing the apigenin-induced down-regulation of CXCR4, indicating that apigenin-induced down-regulation of CXCR4 is not due to proteolytic degradation. The down-regulation of CXCR4 is mainly due to the inhibition of nuclear factor κB (NF-κB) transcriptional activity. Apigenin also abolished migration and invasion of transformed cells induced by CXCL12. In a xenograft mouse model, apigenin down-regulated CXCR4 expression and suppressed tumor growth. Taken together, our results show that apigenin is a novel inhibitor of CXCR4 expression. This dietary flavonoid has the potential to suppress migration and invasion of transformed cells and prevent environmental arsenic-induced carcinogenesis. PMID:23743303

  7. Expression and Polymorphism of Toll-Like Receptor 4 and Effect on NF-κB Mediated Inflammation in Colon Cancer Patients.

    PubMed

    Semlali, Abdelhabib; Reddy Parine, Narasimha; Arafah, Maha; Mansour, Lamjed; Azzi, Arezki; Al Shahrani, Omair; Al Amri, Abdullah; Shaik, Jilani P; Aljebreen, Abdulrahman M; Alharbi, Othman; Almadi, Majid A; Azzam, Nahla Ali; Kohailan, Muhammad; Rouabhia, Mahmoud; Alanazi, Mohammad Saud

    2016-01-01

    Our aim was to evaluate the association between the expression and the polymorphism of TLR4/NF-κB pathways and colon cancer. TLR4 (rs4986790, rs10759932, rs10759931 and rs2770150) were genotyped in blood samples from Colorectal patients and healthy controls. TLR4 and cytokines inflammatory expression were evaluated by real time PCR on 40 matching normal and colon tissues and the protein level by Immunohistochemistry. The high level of TLR4 expression in colon cancer tissues is mainly due to infections by bacteria in the human colon and leads to induction of an acute secretion of inflammatory cytokines mediated by NF-κB. Also, we report here a clear evidence for an association between TLR4 rs10759931 polymorphism (OR = 0.086, CI: 0.04-0.18, P = <0.00001). This polymorphism affects the entire population without being specific to either gender or to any age group. In contrast, the rs2770150 is associated with colon cancer in women aged over 50 years and is closely linked with the decreased levels of female sex hormones during the post-menopausal period (OR = 0.188, CI: 0.074-0.48, P = <0.00084). rs10759932 and rs4986790 appear to have any association with colon cancer. Our data suggest that TLR4 SNPs could possibly serve as biomarkers for decision making in colon cancer treatment.

  8. Expression and Polymorphism of Toll-Like Receptor 4 and Effect on NF-κB Mediated Inflammation in Colon Cancer Patients

    PubMed Central

    Semlali, Abdelhabib; Reddy Parine, Narasimha; Arafah, Maha; Mansour, Lamjed; Azzi, Arezki; Al Shahrani, Omair; Al Amri, Abdullah; Shaik, Jilani P.; Aljebreen, Abdulrahman M.; Alharbi, Othman; Almadi, Majid A.; Azzam, Nahla Ali; Kohailan, Muhammad; Rouabhia, Mahmoud; Alanazi, Mohammad Saud

    2016-01-01

    Our aim was to evaluate the association between the expression and the polymorphism of TLR4/NF-κB pathways and colon cancer. TLR4 (rs4986790, rs10759932, rs10759931 and rs2770150) were genotyped in blood samples from Colorectal patients and healthy controls. TLR4 and cytokines inflammatory expression were evaluated by real time PCR on 40 matching normal and colon tissues and the protein level by Immunohistochemistry. The high level of TLR4 expression in colon cancer tissues is mainly due to infections by bacteria in the human colon and leads to induction of an acute secretion of inflammatory cytokines mediated by NF-κB. Also, we report here a clear evidence for an association between TLR4 rs10759931 polymorphism (OR = 0.086, CI: 0.04–0.18, P = <0.00001). This polymorphism affects the entire population without being specific to either gender or to any age group. In contrast, the rs2770150 is associated with colon cancer in women aged over 50 years and is closely linked with the decreased levels of female sex hormones during the post-menopausal period (OR = 0.188, CI: 0.074–0.48, P = <0.00084). rs10759932 and rs4986790 appear to have any association with colon cancer. Our data suggest that TLR4 SNPs could possibly serve as biomarkers for decision making in colon cancer treatment. PMID:26771524

  9. PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2.

    PubMed

    Darehgazani, Reyhaneh; Peymani, Maryam; Hashemi, Motahare-Sadat; Omrani, Mir Davood; Movafagh, Abolfazl; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

    2016-08-01

    TLR4 is transmembrane pattern-recognition receptor that initiates signals in response to diverse pathogen-associated molecular patterns especially LPS. Recently, there have been an increasing number of studies about the role of TLRs in the pathogenesis of several disorders as well as the therapeutic potential of TLR intervention in such diseases. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a ligand-activated transcription factor with numerous biological effects. PPARγ has been shown to exert a potential anti-inflammatory effect through suppression of TLR4-mediated inflammation. Therefore, PPARγ agonists may have a potential to combat inflammatory conditions in pathologic states. The current study aims to show the decrease of inflammation by overexpression of PPARγ in a cell reporter model. To reach this goal, recombinant pBudCE4.1 (+) containing encoding sequences of human TLR4 and MD2 was constructed and used to transfect HEK cells. Subsequently, inflammation was induced by LPS treatment as control group. In the treatment group, overexpression of PPARγ prior to inflammation was performed and the expression of inflammatory markers was assessed in this condition. The expression of inflammatory markers (TNFα and iNOS) was defined by quantitative real time PCR and the amount of phosphorylated NF-κB was measured by western blot. Data indicated expression of TNFα and iNOS increased in LPS induced inflammation of stably transformed HEK cells with MD2 and TLR4. In this cell reporter model overexpression of PPARγ dramatically prevented LPS-induced inflammation through the blocking of TLR4/NF-κB signaling. PPARγ was shown to negatively regulate TLR4 activity and therefore exerts its anti-inflammatory action against LPS induced inflammation.

  10. Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination.

    PubMed

    Polycarpou, Anastasia; Holland, Martin J; Karageorgiou, Ioannis; Eddaoudi, Ayad; Walker, Stephen L; Willcocks, Sam; Lockwood, Diana N J

    2016-01-01

    Toll-like receptor (TLR)-1 and TLR2 have been shown to be receptors for Mycobacterium leprae (M. leprae), yet it is unclear whether M. leprae can signal through alternative TLRs. Other mycobacterial species possess ligands for TLR4 and genetic association studies in human populations suggest that people with TLR4 polymorphisms may be protected against leprosy. Using human embryonic kidney (HEK)-293 cells co-transfected with TLR4, we demonstrate that M. leprae activates TLR4. We used human macrophages to show that M. leprae stimulation of cytokine production is diminished if pre-treated with TLR4 neutralizing antibody. TLR4 protein expression was up-regulated on macrophages derived from non-bacillus Calmette-Guerin (BCG) vaccinated healthy volunteers after incubation with M. leprae, whereas it was down-regulated in macrophages derived from BCG-vaccinated donors. Finally, pre-treatment of macrophages derived from BCG-naive donors with BCG reversed the effect of M. leprae on TLR4 expression. This may be a newly described phenomenon by which BCG vaccination stimulates "non-specific" protection to the human immune system.

  11. Increased Expression of Toll-Like Receptors 4, 5, and 9 in Small Bowel Mucosa from Patients with Irritable Bowel Syndrome

    PubMed Central

    Zakikhany, Katherina; Acevedo, Nathalie; D'Amato, Mauro; Lindberg, Greger

    2017-01-01

    The aim of our study was to compare patients with irritable bowel syndrome (IBS) and healthy controls regarding the expression of toll-like receptors 2, 4, 5, and 9 (TLR2, TLR4, TLR5, and TLR9), the primary mucosal receptors of bacterial components, in small and large bowel mucosa. Methods. We analysed biopsies from jejunum and sigmoid colon of 22 patients (17 females) with IBS aged 18–66 (median: 39) years and 14 healthy volunteers (12 females) aged 22–61 (median: 42) years. Eight patients had constipation-predominant IBS (C-IBS), 7 had diarrhoea-predominant IBS (D-IBS), and 7 had IBS without predominance of constipation or diarrhoea. We analysed mRNA levels for TLRs using quantitative PCR and distribution of TLRs in mucosa using immunohistochemistry. Results. We found increased mRNA expression of TLR4 (mean fold change 1.85 ± 0.31 versus 1.0 ± 0.20; p < 0.05), TLR5 (1.96 ± 0.36 versus 1.0 ± 0.20; p < 0.05) and TLR9 (2.00 ± 0.24 versus 1.0 ± 0.25; p < 0.01) but not of TLR2 in the small bowel mucosa from patients with IBS compared to the controls. There was no significant difference in mRNA levels for TLRs in colon mucosa between patients and controls. Conclusion. Upregulation of TLR4, TLR5, and TLR9 suggests the involvement of bacteria or dysregulation of the immune response to commensal flora in small bowel mucosa in IBS patients. PMID:28246611

  12. Betaine protects against high-fat-diet-induced liver injury by inhibition of high-mobility group box 1 and Toll-like receptor 4 expression in rats.

    PubMed

    Zhang, Wei; Wang, Lu-Wen; Wang, Li-Kun; Li, Xun; Zhang, Hong; Luo, Li-Ping; Song, Jin-Chun; Gong, Zuo-Jiong

    2013-11-01

    Previous studies have shown that betaine prevents alcohol-induced liver injury and improves liver function. The purpose of this study was to investigate the hepatoprotective effects of betaine on nonalcoholic fatty liver disease (NAFLD) and to observe changes of HMGB1/TLR4 signaling. Thirty rats were randomly divided into control, model, and betaine groups. The rats in the model and betaine groups were fed a high-fat diet for 12 weeks to induce an animal model of NAFLD. The rats in the betaine group were then intragastrically administered betaine solution at a dose of 400 mg/kg per day for four weeks. Liver histology was examined. Serum levels of ALT, AST, TC, TG, HDL-C, LDL-C, FFA, HMGB1, NF-κB, TLR4, and tHcy were determined and intrahepatic TC, TG, and Hcy levels were assayed. mRNA expression and protein levels of HMGB1, NF-κB, and TLR4 in liver tissue were also determined. Compared with the control group, rats in the model group developed severe liver injury, accompanied by significant increases in serum levels of ALT, AST, TC, TG, LDL-C, FFA, HMGB1, NF-κB, and TLR4, intrahepatic TC, TG, and Hcy content, histological scores for steatosis, inflammation, and necrosis, and mRNA expression and protein levels of HMGB1, NF-κB, and TLR4, and a significant decrease in serum HDL-C (P < 0.05). Compared with the model group, all these indicators were significantly improved by administration of betaine (P < 0.05). Betaine effectively protects against high-fat-diet-induced NAFLD and improves liver function; the mechanism is probably related to inhibition of HMGB1/TLR4 signaling pathways.

  13. Lipopolysaccharide decreases single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) expression by suppressing specificity protein 1 (Sp1) via the Toll-like receptor 4 (TLR4)-p38 pathway in monocytes and neutrophils.

    PubMed

    Ueno-Shuto, Keiko; Kato, Kosuke; Tasaki, Yukihiro; Sato, Miki; Sato, Keizo; Uchida, Yuji; Sakai, Hiromichi; Ono, Tomomi; Suico, Mary Ann; Mitsutake, Kazunori; Tokutomi, Naofumi; Kai, Hirofumi; Shuto, Tsuyoshi

    2014-06-27

    Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Lipopolysaccharide Decreases Single Immunoglobulin Interleukin-1 Receptor-related Molecule (SIGIRR) Expression by Suppressing Specificity Protein 1 (Sp1) via the Toll-like Receptor 4 (TLR4)-p38 Pathway in Monocytes and Neutrophils*

    PubMed Central

    Ueno-Shuto, Keiko; Kato, Kosuke; Tasaki, Yukihiro; Sato, Miki; Sato, Keizo; Uchida, Yuji; Sakai, Hiromichi; Ono, Tomomi; Suico, Mary Ann; Mitsutake, Kazunori; Tokutomi, Naofumi; Kai, Hirofumi; Shuto, Tsuyoshi

    2014-01-01

    Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells. PMID:24821721

  15. Programmed cell death ligand 1 expression in osteosarcoma.

    PubMed

    Shen, Jacson K; Cote, Gregory M; Choy, Edwin; Yang, Pei; Harmon, David; Schwab, Joseph; Nielsen, G Petur; Chebib, Ivan; Ferrone, Soldano; Wang, Xinhui; Wang, Yangyang; Mankin, Henry; Hornicek, Francis J; Duan, Zhenfeng

    2014-07-01

    Programmed cell death ligand 1 (PDL1, also known as B7H1) is a cell-surface protein that suppresses the cytotoxic CD8(+) T-cell-mediated immune response. PDL1 expression and its clinical relevance in sarcomas are not well understood. Therefore, we sought to measure RNA expression levels for PDL1 in 38 clinically annotated osteosarcoma tumor samples and aimed to determine if PDL1 expression correlates with clinical features and tumor-infiltrating lymphocytes (TIL). Quantitative real-time RT-PCR for PDL1 was optimized in 18 cell lines, of which 5 were osteosarcoma derived. qRT-PCR results were validated via flow cytometry and immunohistochemistry (IHC) in select cell lines. Total RNA was isolated from 38 human osteosarcoma samples for qRT-PCR analysis. Clinical data were sorted, and significance was determined by the Student t test. TILs were examined in patient samples by tissue microarray hematoxylin-eosin staining. We confirmed the constitutive PDL1 mRNA expression in cell lines by qRT-PCR, flow cytometry, and IHC. Across human osteosarcoma samples, PDL1 mRNA gene expression ranged over 4 log (>5,000-fold difference). Relative expression levels were evaluated against clinical factors such as age/gender, metastasis, recurrence, chemotherapy, percentage of necrosis, and survival; no significant associations were identified. The presence of TILs was associated with high PDL1 expression (R(2) = 0.37; P = 0.01). In summary, we developed an RNA-based assay to determine PDL1 expression levels, and we show, for the first time, that high levels of PDL1 are expressed in a subset of osteosarcoma, and PDL1 expression is positively correlated with TILs. Multiple agents targeting PD1/PDL1 are in clinical development, and this may be a novel immunotherapeutic strategy for osteosarcoma clinical trials.

  16. Increased Ubqln2 expression causes neuron death in transgenic rats.

    PubMed

    Huang, Bo; Wu, Qinxue; Zhou, Hongxia; Huang, Cao; Xia, Xu-Gang

    2016-10-01

    Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While over-expression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats over-expressing wild-type human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats over-expressing rat's own Ubqln2. When over-expressed in rats, both human and rat wild-type Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Over-expressed wild-type UBQLN2 formed protein inclusions attracting the autophagy substrate sequestosome-1 and the proteasome component 26S proteasome regulatory subunit 7. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis. Pathogenic mutation in ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wild-type UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that over-expression of human or rat wild-type Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis

  17. Oral administration of synthetic porcine beta-defensin-2 improves growth performance and cecal microbial flora and down-regulates the expression of intestinal toll-like receptor-4 and inflammatory cytokines in weaned piglets challenged with enterotoxigenic Escherichia coli.

    PubMed

    Tang, Zhiru; Xu, Ling; Shi, Baoshi; Deng, Huang; Lai, Xin; Liu, Jingyan; Sun, Zhihong

    2016-10-01

    Synthetic porcine beta-defensin-2 (pBD-2) was tested as an alternative to antimicrobial growth-promoters in pig production. Thirty 21-day weaned piglets were challenged with enterotoxigenic Escherichia coli, and orally dosed with either sterile water (CON), pBD-2 (BD) or neomycin sulphate (NS) twice daily for 21 days. pBD-2 and NS led to higher growth performance, jejunum villus height and increased expression of insulin-like growth factor-I compared with the CON group (P < 0.05). Hemolytic E. coli scores from rectal swabs, and copy numbers of E. coli, Bacteroides fragilis and Streptococcus in the cecal digesta of the BD- or NS-treated piglets were lower than those in the CON group (P < 0.05). Messenger RNA levels of toll-like receptor 4, tumor necrosis factor-α, interleukin (IL)-1β, and IL-8 in the jejunum mucosa of the BD and NS groups were lower than those in the CON group (P < 0.05). Copy numbers of Lactobacilli and Bifidobacteria in the cecal digesta of the BD group were higher than those of the CON and NS groups (P < 0.05). Therefore, pBD-2 has antimicrobial activity in piglets, and it can improve growth performance, reduce inflammatory cytokine expression and affect intestinal morphological indices in the same way as probiotics. © 2015 Japanese Society of Animal Science.

  18. CXC chemokine receptor 4 expression and stromal cell-derived factor-1α-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10

    PubMed Central

    Jinquan, T; Quan, S; Jacobi, H H; Madsen, H O; Glue, C; Skov, P S; Malling, H-J; Poulsen, L K

    2000-01-01

    We report that interleukin (IL)-4 and IL-10 can significantly up- or down-regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell-derived factor-1α (SDF-1α)-induced CD4+ T-lymphocyte chemotaxis was also correspondingly regulated by IL-4 and IL-10. IL-4 and IL-10 up- or down-regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd ≈ 6·3 nm), and ≈ 70 000 SDF-1α-binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1 ≈ 4·4 nm and Kd2 ≈ 14·6 nm), and a total of ≈ 130 000 SDF-1α-binding sites per cell, among IL-4-stimulated CD4+ T lymphocytes. The regulation of CXCR4 expression in CD4+ T lymphocytes by IL-4 and IL-10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP- and cGMP-dependent protein kinase (H-8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both cAMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL-4 and IL-10 on CXCR4 expression implied that the capacity of IL-4 and IL-10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium-mobilization stimulation. These results indicate that the effects of IL-4 and IL-10 on the CXCR4–SDF-1 receptor–ligand pair may be of particular importance in the cytokine/chemokine environment concerning the inflammatory processes and in the progression of human immunodeficiency virus (HIV) infection. PMID:10712670

  19. MD-2 is involved in the stimulation of matrix metalloproteinase-1 expression by interferon-γ and high glucose in mononuclear cells - a potential role of MD-2 in Toll-like receptor 4-independent signalling.

    PubMed

    Lu, Zhongyang; Li, Yanchun; Samuvel, Devadoss J; Jin, Junfei; Zhang, Xiaoming; Lopes-Virella, Maria F; Huang, Yan

    2013-11-01

    We reported recently that treatment of diabetic apolipoprotein E-deficient mice with the Toll-like receptor 4 (TLR4) antagonist Rs-LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs-LPS antagonizes TLR4 by targeting TLR4 co-receptor MD-2, this finding indicates that MD-2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD-2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon-γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD-2 is involved in IFN-γ and high-glucose-regulated gene expression in mononuclear cells. Results showed that IFN-γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP-1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs-LPS inhibited the MMP-1 stimulation. To provide more evidence for a role of MD-2 in IFN-γ-stimulated MMP-1, studies using antibodies and small interfering RNA demonstrated that MD-2 blockade or knockdown attenuated the effect of IFN-γ on MMP-1. Furthermore, studies using PCR arrays showed that MD-2 blockade had a similar effect as IFN-γ receptor blockade on the inhibition of IFN-γ-stimulated pro-inflammatory molecules. Although these findings indicate the involvement of MD-2 in IFN-γ signalling, we also observed that MD-2 was up-regulated by IFN-γ and high glucose. We found that MD-2 up-regulation by IFN-γ played an essential role in the synergistic effect of IFN-γ and LPS on MMP-1 expression. Taken together, these findings indicate that MD-2 is involved in IFN-γ signalling and IFN-γ-augmented MMP-1 up-regulation by LPS. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  20. Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels.

    PubMed

    Nimmanapalli, R; Perkins, C L; Orlando, M; O'Bryan, E; Nguyen, D; Bhalla, K N

    2001-01-15

    We have demonstrated that Apo-2 ligand (Apo-2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of human prostate cancer PC-3, DU145, and LNCaP cells in a dose-dependent manner, with PC-3 cells displaying the greatest sensitivity to Apo-2L/TRAIL. Susceptibility of the prostate cancer cell types to Apo-2L/TRAIL-induced apoptosis did not appear to correlate with the levels of the Apo-2L/TRAIL receptors death receptor (DR) 4 (TRAIL receptor 1) or DR5 (TRAIL receptor 2), decoy receptor (DcR) 1 and DcR2, Flame-1, or the inhibitors of apoptosis proteins family of proteins. Apo-2L/TRAIL-induced apoptosis of PC-3 cells was associated with the processing of caspase-8, caspase-10, and the proapoptotic Bid protein, resulting in the cytosolic accumulation of cytochrome c as well as the processing of procaspase-9 and procaspase-3. Cotreatment with the caspase-8 inhibitor z-IETD-fmk or DR4:Fc significantly inhibited Apo-2L/TRAIL-induced apoptosis. Treatment with paclitaxel or taxotere increased DR4 and/or DR5 protein levels (up to 8-fold) without affecting the protein levels of DcR1 and DcR2, Apo-2L/TRAIL, Fas, or Fas ligand. Up-regulation of DR4 and DR5 was not preceded by the induction of their mRNA levels but was inhibited by cotreatment with cycloheximide. Importantly, sequential treatment of PC-3, DU145, and LNCaP cells with paclitaxel followed by Apo-2L/TRAIL induced significantly more apoptosis than Apo-2L/TRAIL treatment alone (P < 0.01). This was also associated with greater processing of procaspase-8 and Bid, as well as greater cytosolic accumulation of cytochrome c and the processing of caspase-3. These findings indicate that up-regulation of DR4 and DR5 protein levels by treatment with paclitaxel enhances subsequent Apo-2L/TRAIL-induced apoptosis of human prostate cancer cells.

  1. Higher expression of galectin-3 and galectin-9 in periapical granulomas than in radicular cysts and an increased toll-like receptor-2 and toll-like receptor-4 expression are associated with reactivation of periapical inflammation.

    PubMed

    de Oliveira, Rita de Cássia Medeiros; Beghini, Marcela; Borges, Cláudia Renata Bibiano; Alves, Polyanna Miranda; de Araújo, Marcelo Sivieri; Pereira, Sanívia Aparecida de Lima; Rodrigues, Virmondes; Rodrigues, Denise Bertulucci Rocha

    2014-02-01

    Cysts and periapical granulomas are inflammatory reactions that develop in response to periapical infection by microbial species in dental root canal. It is known that toll-like receptors (TLRs) are pathogen recognition molecules and that galectins are lectins that can be associated with the inflammatory process, stimulating or inhibiting the immune system. The objective of this study was to evaluate the in situ expression of TLRs and galectins in radicular cysts and periapical granulomas. We analyzed 62 cases (30 radicular cysts, 27 periapical granulomas, and 5 control cases). Indirect immunohistochemistry was used to evaluate the expression of TLRs (TRL-2 and TLR-4) and galectins (Gal-3 and Gal-9). The expression of Gal-3 and Gal-9 was significantly higher in periapical granulomas and radicular cysts than in the control group. Similarly, both Gal-3 and Gal-9 were expressed significantly more in periapical granulomas than in radicular cysts. The expression of TLR-2 was significantly higher in periapical granulomas and radicular cysts than in the control group, and it was also significantly higher in radicular cysts with sinus tract than in the cases without sinus tract. Furthermore, the expression of TLR-4 was significantly higher in the cases of periapical granulomas with sinus tract than in the cases without sinus tract. Gal-3/Gal-9 and TLR-2/TLR-4 expression in the periapical granulomas and radicular cysts is associated with reactive periapical inflammation. Pathobiology of periapical disease is a very complex interplay of many bioactive molecules involved in immunoinflammatory responses. Up-regulation of these bioactive molecules might be an important modulator of inflammatory periapical lesions. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  2. Elevated Expression of Programmed Death-1 and Programmed Death Ligand-1 Negatively Regulates Immune Response against Cervical Cancer Cells

    PubMed Central

    Chen, Zhifang; Pang, Nannan; Du, Rong; Zhu, Yuejie; Fan, Lingling; Cai, Donghui

    2016-01-01

    The present study is to measure the expression of programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), as well as its clinical significance in cervical cancer patients. Our results showed that different T cell subsets in patients with cervical cancer had high expression of PD-1, and DCs had high expression of PD-L1. High expression of PD-1 on Treg cells in cervical cancer patients facilitated the production of TGF-β and IL-10 but inhibited the production of IFN-γ. Cervical cancer elevated the expression of PD-1 and PD-L1 in mRNA level. PD-1 expression in peripheral blood of cervical cancer patients was related with tumor differentiation, lymph node metastasis, and invasiveness. PD-1/PD-L1 pathway inhibited lymphocyte proliferation but enhanced the secretion of IL-10 and TGF-β in vitro. In summary, our findings demonstrate that elevated levels of PD-1/PD-L1, TGF-β, and IL-10 in peripheral blood of cervical cancer patients may negatively regulate immune response against cervical cancer cells and contribute to the progression of cervical cancer. Therefore, PD-1/PD-L1 pathway may become an immunotherapy target in the future. PMID:27721577

  3. Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

    PubMed

    Shang, Hung-Sheng; Liu, Jia-You; Lu, Hsu-Feng; Chiang, Han-Sun; Lin, Chia-Hain; Chen, Ann; Lin, Yuh-Feng; Chung, Jing-Gung

    2016-11-14

    Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca(2+) productions, level of mitochondria membrane potential (ΔΨm ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨm , and Ca(2+) , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells.

  4. Should a Toll-like receptor 4 (TLR-4) agonist or antagonist be designed to treat cancer? TLR-4: its expression and effects in the ten most common cancers

    PubMed Central

    Mai, Chun Wai; Kang, Yew Beng; Pichika, Mallikarjuna Rao

    2013-01-01

    Toll-like receptor 4 (TLR-4) is well known for its host innate immunity. Despite the fact that TLR-4 activation confers antitumor responses; emerging evidence suggests that TLR-4 is associated with tumor development and progression. It is now clear that overactivation of TLR-4, through various immune mediators, may cause immune response dysfunction, resulting in tumorigenesis. Different cancers could have different extents of TLR-4 involvement during tumorigenesis or tumor progression. In this review, we focus on infection- and inflammation-related TLR-4 activation in noncancer and cancer cells, as well as on the current evidence about the role of TLR-4 in ten of the most common cancers, viz, head and neck cancer, lung cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, skin cancer, breast cancer, ovarian cancer, cervical cancer, and prostate cancer. PMID:24235843

  5. Novel Toll-like receptor-4 antagonist (+)-naloxone protects mice from inflammation-induced preterm birth.

    PubMed

    Chin, Peck Yin; Dorian, Camilla L; Hutchinson, Mark R; Olson, David M; Rice, Kenner C; Moldenhauer, Lachlan M; Robertson, Sarah A

    2016-11-07

    Toll-like receptor 4 (TLR4) activation by bacterial infection, or by sterile inflammatory insult is a primary trigger of spontaneous preterm birth. Here we utilize mouse models to investigate the efficacy of a novel small molecule TLR4 antagonist, (+)-naloxone, the non-opioid isomer of the opioid receptor antagonist (-)-naloxone, in infection-associated preterm birth. Treatment with (+)-naloxone prevented preterm delivery and alleviated fetal demise in utero elicited by i.p. LPS administration in late gestation. A similar effect with protection from preterm birth and perinatal death, and partial correction of reduced birth weight and postnatal mortality, was conferred by (+)-naloxone administration after intrauterine administration of heat-killed E. coli. Local induction by E. coli of inflammatory cytokine genes Il1b, Il6, Tnf and Il10 in fetal membranes was suppressed by (+)-naloxone, and cytokine expression in the placenta, and uterine myometrium and decidua, was also attenuated. These data demonstrate that inhibition of TLR4 signaling with the novel TLR4 antagonist (+)-naloxone can suppress the inflammatory cascade of preterm parturition, to prevent preterm birth and perinatal death. Further studies are warranted to investigate the utility of small molecule inhibition of TLR-driven inflammation as a component of strategies for fetal protection and delaying preterm birth in the clinical setting.

  6. Novel Toll-like receptor-4 antagonist (+)-naloxone protects mice from inflammation-induced preterm birth

    PubMed Central

    Chin, Peck Yin; Dorian, Camilla L.; Hutchinson, Mark R.; Olson, David M.; Rice, Kenner C.; Moldenhauer, Lachlan M.; Robertson, Sarah A.

    2016-01-01

    Toll-like receptor 4 (TLR4) activation by bacterial infection, or by sterile inflammatory insult is a primary trigger of spontaneous preterm birth. Here we utilize mouse models to investigate the efficacy of a novel small molecule TLR4 antagonist, (+)-naloxone, the non-opioid isomer of the opioid receptor antagonist (−)-naloxone, in infection-associated preterm birth. Treatment with (+)-naloxone prevented preterm delivery and alleviated fetal demise in utero elicited by i.p. LPS administration in late gestation. A similar effect with protection from preterm birth and perinatal death, and partial correction of reduced birth weight and postnatal mortality, was conferred by (+)-naloxone administration after intrauterine administration of heat-killed E. coli. Local induction by E. coli of inflammatory cytokine genes Il1b, Il6, Tnf and Il10 in fetal membranes was suppressed by (+)-naloxone, and cytokine expression in the placenta, and uterine myometrium and decidua, was also attenuated. These data demonstrate that inhibition of TLR4 signaling with the novel TLR4 antagonist (+)-naloxone can suppress the inflammatory cascade of preterm parturition, to prevent preterm birth and perinatal death. Further studies are warranted to investigate the utility of small molecule inhibition of TLR-driven inflammation as a component of strategies for fetal protection and delaying preterm birth in the clinical setting. PMID:27819333

  7. Gene expression reprogramming protects macrophage from septic-induced cell death.

    PubMed

    Melo, Edielle Sant'Anna; Barbeiro, Denise F; Gorjão, Renata; Rios, Ester Correia Sarmento; Vasconcelos, Dewton; Velasco, Irineu T; Szabo, Csaba; Curi, Rui; de Lima-Salgado, Thais Martins; Soriano, Francisco Garcia

    2010-10-01

    Sepsis induces a systemic inflammatory response leading to tissue damage and cell death. LPS tolerance affects inflammatory response. To comprehend potential new mechanisms of immune regulation in endotoxemia, we examined macrophage mRNA expression by macroarray affected by LPS tolerance. LPS tolerance was induced with subcutaneous administration of 1 mg/kg/day of LPS over 5 days. Macrophages were isolated from the spleen and the expression of 1200 genes was quantitatively analyzed by the macroarray technique. The tolerant group displayed relevant changes in the expression of 84 mRNA when compared to naïve mice. A functional group of genes related to cell death regulation was identified. PARP-1, caspase 3, FASL and TRAIL genes were confirmed by RT-PCR to present lower expression in tolerant mice. In addition, reduced expression of the pro-inflammatory genes TNF-α and IFN-γ in the tolerant group was demonstrated. Following this, animals were challenged with polymicrobial sepsis. Flow cytometry analysis showed reduced necrosis and apoptosis in macrophages from the tolerant group compared to the naïve group. Finally, a survival study showed a significant reduction in mortality in the tolerant group. Thus, in the current study we provide evidence for the selective reprogramming of the gene expression of cell death pathways during LPS tolerance and link these changes to protection from cell death and enhanced survival rates. Copyright © 2010 Elsevier Ltd. All rights reserved.

  8. Brain death induces the alteration of liver protein expression profiles in rabbits.

    PubMed

    Du, Bing; Li, Ling; Zhong, Zhibiao; Fan, Xiaoli; Qiao, Bingbing; He, Chongxiang; Fu, Zhen; Wang, Yanfeng; Ye, Qifa

    2014-08-01

    At present, there is no accurate method for evaluating the quality of liver transplant from a brain-dead donor. Proteomics are used to investigate the mechanisms involved in brain death‑induced liver injury and to identify sensitive biomarkers. In the present study, age‑ and gender‑matched rabbits were randomly divided into the brain death and sham groups. The sham served as the control. A brain‑death model was established using an intracranial progressive pressurized method. The differentially expressed proteins extracted from the liver tissues of rabbits that were brain‑dead for 6 h in the two groups were determined by two‑dimensional gel electrophoresis and matrix‑assisted laser desorption ionization time of flight mass spectrometry. Although there was no obvious functional and morphological difference in 2, 4 and 6 h after brain death, results of the proteomics analysis revealed 973±34 and 987±38 protein spots in the control and brain death groups, respectively. Ten proteins exhibited a ≥2‑fold alteration. The downregulated proteins were: aldehyde dehydrogenase, runt‑related transcription factor 1 (RUNX1), inorganic pyrophosphatase, glutamate‑cysteine ligase regulatory subunit and microsomal cytochrome B5. By contrast, the expression of dihydropyrimidinase-related protein 4, peroxiredoxin‑6, 3‑phosphoinositide‑dependent protein kinase‑1, 3-mercaptopyruvate and alcohol dehydrogenase were clearly upregulated. Immunohistochemistry and western blot analysis results revealed that the expression of RUNX1 was gradually increased in a time‑dependent manner in 2, 4, and 6 h after brain death. In conclusion, alteration of the liver protein expression profile induced by brain death indicated the occurrence of complex pathological changes even if no functional or morphological difference was identified. Thus, RUNX1 may be a sensitive predict factor for evaluating the quality of brain death donated liver.

  9. Programmed Death-Ligand 1 Expression and Response to the Anti–Programmed Death 1 Antibody Pembrolizumab in Melanoma

    PubMed Central

    Wolchok, Jedd D.; Robert, Caroline; Hwu, Wen-Jen; Weber, Jeffrey S.; Ribas, Antoni; Hodi, F. Stephen; Joshua, Anthony M.; Kefford, Richard; Hersey, Peter; Joseph, Richard; Gangadhar, Tara C.; Dronca, Roxana; Patnaik, Amita; Zarour, Hassane; Roach, Charlotte; Toland, Grant; Lunceford, Jared K.; Li, Xiaoyun Nicole; Emancipator, Kenneth; Dolled-Filhart, Marisa; Kang, S. Peter; Ebbinghaus, Scot; Hamid, Omid

    2016-01-01

    Purpose Expression of programmed death-ligand 1 (PD-L1) is a potential predictive marker for response and outcome after treatment with anti–programmed death 1 (PD-1). This study explored the relationship between anti–PD-1 activity and PD-L1 expression in patients with advanced melanoma who were treated with pembrolizumab in the phase Ib KEYNOTE-001 study (clinical trial information: NCT01295827). Patients and Methods Six hundred fifty-five patients received pembrolizumab10 mg/kg once every 2 weeks or once every 3 weeks, or 2 mg/kg once every 3 weeks. Tumor response was assessed every 12 weeks per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 by independent central review. Primary outcome was objective response rate. Secondary outcomes included progression-free survival (PFS) and overall survival (OS). Membranous PD-L1 expression in tumor and tumor-associated immune cells was assessed by a clinical trial immunohistochemistry assay (22C3 antibody) and scored on a unique melanoma (MEL) scale of 0 to 5 by one of three pathologists who were blinded to clinical outcome; a score ≥ 2 (membranous staining in ≥ 1% of cells) was considered positive. Results Of 451 patients with evaluable PD-L1 expression, 344 (76%) had PD-L1–positive tumors. Demographic and staging variables were equally distributed among PD-L1–positive and –negative patients. An association between higher MEL score and higher response rate and longer PFS (hazard ratio, 0.76; 95% CI, 0.71 to 0.82) and OS (hazard ratio, 0.76; 95% CI, 0.69 to 0.83) was observed (P < .001 for each). Objective response rate was 8%, 12%, 22%, 43%, 57%, and 53% for MEL 0, 1, 2, 3, 4, and 5, respectively. Conclusion PD-L1 expression in pretreatment tumor biopsy samples was correlated with response rate, PFS, and OS; however, patients with PD-L1–negative tumors may also achieve durable responses. PMID:27863197

  10. Programmed Death-Ligand 1 Expression and Response to the Anti-Programmed Death 1 Antibody Pembrolizumab in Melanoma.

    PubMed

    Daud, Adil I; Wolchok, Jedd D; Robert, Caroline; Hwu, Wen-Jen; Weber, Jeffrey S; Ribas, Antoni; Hodi, F Stephen; Joshua, Anthony M; Kefford, Richard; Hersey, Peter; Joseph, Richard; Gangadhar, Tara C; Dronca, Roxana; Patnaik, Amita; Zarour, Hassane; Roach, Charlotte; Toland, Grant; Lunceford, Jared K; Li, Xiaoyun Nicole; Emancipator, Kenneth; Dolled-Filhart, Marisa; Kang, S Peter; Ebbinghaus, Scot; Hamid, Omid

    2016-12-01

    Purpose Expression of programmed death-ligand 1 (PD-L1) is a potential predictive marker for response and outcome after treatment with anti-programmed death 1 (PD-1). This study explored the relationship between anti-PD-1 activity and PD-L1 expression in patients with advanced melanoma who were treated with pembrolizumab in the phase Ib KEYNOTE-001 study (clinical trial information: NCT01295827). Patients and Methods Six hundred fifty-five patients received pembrolizumab10 mg/kg once every 2 weeks or once every 3 weeks, or 2 mg/kg once every 3 weeks. Tumor response was assessed every 12 weeks per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 by independent central review. Primary outcome was objective response rate. Secondary outcomes included progression-free survival (PFS) and overall survival (OS). Membranous PD-L1 expression in tumor and tumor-associated immune cells was assessed by a clinical trial immunohistochemistry assay (22C3 antibody) and scored on a unique melanoma (MEL) scale of 0 to 5 by one of three pathologists who were blinded to clinical outcome; a score ≥ 2 (membranous staining in ≥ 1% of cells) was considered positive. Results Of 451 patients with evaluable PD-L1 expression, 344 (76%) had PD-L1-positive tumors. Demographic and staging variables were equally distributed among PD-L1-positive and -negative patients. An association between higher MEL score and higher response rate and longer PFS (hazard ratio, 0.76; 95% CI, 0.71 to 0.82) and OS (hazard ratio, 0.76; 95% CI, 0.69 to 0.83) was observed ( P < .001 for each). Objective response rate was 8%, 12%, 22%, 43%, 57%, and 53% for MEL 0, 1, 2, 3, 4, and 5, respectively. Conclusion PD-L1 expression in pretreatment tumor biopsy samples was correlated with response rate, PFS, and OS; however, patients with PD-L1-negative tumors may also achieve durable responses.

  11. Jasmonates induce nonapoptotic death in high-resistance mutant p53-expressing B-lymphoma cells

    PubMed Central

    Fingrut, Orit; Reischer, Dorit; Rotem, Ronit; Goldin, Natalia; Altboum, Irit; Zan-Bar, Israel; Flescher, Eliezer

    2005-01-01

    Mutations in p53, a tumor suppressor gene, occur in more than half of human cancers. Therefore, we tested the hypothesis that jasmonates (novel anticancer agents) can induce death in mutated p53-expressing cells. Two clones of B-lymphoma cells were studied, one expressing wild-type (wt) p53 and the other expressing mutated p53. Jasmonic acid and methyl jasmonate (0.25–3 mM) were each equally cytotoxic to both clones, whereas mutant p53-expressing cells were resistant to treatment with the radiomimetic agent neocarzinostatin and the chemotherapeutic agent bleomycin. Neocarzinostatin and bleomycin induced an elevation in the p53 levels in wt p53-expressing cells, whereas methyl jasmonate did not. Methyl jasmonate induced mostly apoptotic death in the wt p53-expressing cells, while no signs of early apoptosis were detected in mutant p53-expressing cells. In contrast, neocarzinostatin and bleomycin induced death only in wt p53-expressing cells, in an apoptotic mode. Methyl jasmonate induced a rapid depletion of ATP in both clones. In both clones, oligomycin (a mitochondrial ATP synthase inhibitor) did not increase ATP depletion induced by methyl jasmonate, whereas inhibition of glycolysis with 2-deoxyglucose did. High glucose levels protected both clones from methyl jasmonate-induced ATP depletion (and reduced methyl jasmonate-induced cytotoxicity), whereas high levels of pyruvate did not. These results suggest that methyl jasmonate induces ATP depletion mostly by compromising oxidative phosphorylation in the mitochondria. In conclusion, jasmonates can circumvent the resistance of mutant p53-expressing cells towards chemotherapy by inducing a nonapoptotic cell death. PMID:16170329

  12. Can dead bacterial cells be defined and are genes expressed after cell death?

    PubMed

    Trevors, J T

    2012-07-01

    There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Taip2 is a novel cell death-related gene expressed in the brain during development

    SciTech Connect

    Yamada, Kazumi; Akiyama, Nobutake; Yamada, Shuichi; Tanaka, Hiromitsu; Saito, Saburo; Hiraoka, Masahiro; Kizaka-Kondoh, Shinae

    2008-05-02

    TAIP2 was isolated as one of the homologous genes of TAIP3 (TGF-{beta}-up-regulated apoptosis-inducing-protein chromosome 3). The transcript of the mouse counterpart of TAIP2, designated mTaip2, was detected in several tissue specimens from embryos to adults, while mTaip2 was dominantly expressed in the embryonic brain. The overexpression of the full-length mTaip2 induced cell death in various cell lines. An analysis of mTaip2 deletion mutants revealed that the N-terminal half of mTaip2, but not the C-terminal half, had nuclear localization and cell death-inducing activities. The results indicate that mTaip2 is a novel cell death-related gene dominantly expressed in the embryonic brain, thus suggesting that mTaip2 may play a role in development of the brain.

  14. Myocardial Cx43 expression in the cases of sudden death due to dilated cardiomyopathy.

    PubMed

    Chen, Xinshan; Zhang, Yigu

    2006-10-16

    Probing into myocardial connexin (Cx) 43 expression in the cases of sudden death due to dilated cardiomyopathy (DCM) and relationship between Cx43 expression and sudden death. Myocardial Cx43 was detected with immunohistochemical staining in the cases of 11 sudden death caused by DCM and 14 cases of control group who died of violent reasons and other diseases, which were autopsied in our department from 1997 to 2003. Of 11 cases of DCM, there were 10 men and 1 woman with ranging in age from 7 to 49 years old (x (37.8) years old for 9 adult cases). Of 14 cases in the control group, there were 10 men and 4 women with ranging in age from 11 to 53 years old (x (29.9) years old for 11 adult cases). Myocardial Cx43 expression was obviously decreased in DCM group. Positive dyeing spots were different in size, distribution, color and disparity, some of them were distributed in the form of particle. Obvious change had not been observed in the cases of control group or with only slight changes in coloring degree and expressive area. The quantitative data showed that there was significant difference between two groups (p=0.0075) about Cx43 expressive area, but there was no difference between the left and right ventricles (p>0.05) in each group itself. And there was not difference between the two groups about average optical density of expression. Myocardial Cx43 expression is obviously reduced in the patients with DCM who die suddenly. The alteration of quantity and distribution of myocardial Cx43 expression is probably related to sudden death of the patients with DCM.

  15. bcl-2 transgene expression can protect neurons against developmental and induced cell death.

    PubMed Central

    Farlie, P G; Dringen, R; Rees, S M; Kannourakis, G; Bernard, O

    1995-01-01

    The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life. Images Fig. 1 Fig. 2 Fig. 4 PMID:7753817

  16. The role of calpain-myosin 9-Rab7b pathway in mediating the expression of Toll-like receptor 4 in platelets: a novel mechanism involved in α-granules trafficking.

    PubMed

    Tsai, Jui-Chi; Lin, Yi-Wen; Huang, Chun-Yao; Lin, Chih-Yuan; Tsai, Yi-Ting; Shih, Chun-Min; Lee, Chung-Yi; Chen, Yung-Hsiang; Li, Chi-Yuan; Chang, Nen-Chung; Lin, Feng-Yen; Tsai, Chien-Sung

    2014-01-01

    Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.

  17. The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

    PubMed Central

    Tsai, Jui-Chi; Lin, Yi-Wen; Huang, Chun-Yao; Lin, Chih-Yuan; Tsai, Yi-Ting; Shih, Chun-Min; Lee, Chung-Yi; Chen, Yung-Hsiang; Li, Chi-Yuan; Chang, Nen-Chung; Lin, Feng-Yen; Tsai, Chien-Sung

    2014-01-01

    Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation. PMID:24489676

  18. Expression of the human tumor suppressor p53 induces cell death in Pichia pastoris.

    PubMed

    Abdelmoula-Souissi, Salma; Mabrouk, Imed; Gargouri, Ali; Mokdad-Gargouri, Raja

    2012-02-01

    The human tumor suppressor p53 is known as guardian of genome because of its involvement in many signals related to cell life or death. In this work, we report that human p53 induces cell death in the yeast Pichia pastoris. We showed a growth inhibition effect, which increased with the p53 protein expression level in recombinant Mut(s) (methanol utilization slow) strain of Pichia. However, no effect of p53 was observed in recombinant strain of Mut(+) (methanol utilization plus) phenotype. Interestingly, human p53 induces cell death in recombinant strains Mut(s) with characteristic markers of apoptosis such as DNA fragmentation, exposure of phosphatidylserine, and reactive oxygen species generation. Taken together, our results strongly suggest that human p53 is biologically active in this heterologous context. Thus, we propose that P. pastoris could be a useful tool to better understand the biological function of human p53.

  19. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy.

    PubMed

    Apetoh, Lionel; Ghiringhelli, François; Tesniere, Antoine; Obeid, Michel; Ortiz, Carla; Criollo, Alfredo; Mignot, Grégoire; Maiuri, M Chiara; Ullrich, Evelyn; Saulnier, Patrick; Yang, Huan; Amigorena, Sebastian; Ryffel, Bernard; Barrat, Franck J; Saftig, Paul; Levi, Francis; Lidereau, Rosette; Nogues, Catherine; Mira, Jean-Paul; Chompret, Agnès; Joulin, Virginie; Clavel-Chapelon, Françoise; Bourhis, Jean; André, Fabrice; Delaloge, Suzette; Tursz, Thomas; Kroemer, Guido; Zitvogel, Laurence

    2007-09-01

    Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.

  20. Expression of heat shock proteins (hsp) 27 and 70 in various organ systems in cases of death due to fire.

    PubMed

    Doberentz, E; Genneper, L; Böker, D; Lignitz, E; Madea, B

    2014-11-01

    The expression of heat shock proteins (hsp) increases in case of variable types of endogenous and exogenous cellular stress, as for example thermal stress. Immunohistochemical staining with hsp antibodies can visualize these stress proteins. Fifty-three cases of death due to heat and a control group of 100 deaths without any antemortem thermic stress were examined regarding hsp27 and hsp70 expression in myocardial, pulmonary, and renal tissues. The results revealed a correlation between hsp expression, survival time, and cause of death. In cases of death due to fire, the expression of hsp is more extensive than in the control group, especially in pulmonary and renal tissues. The immunohistochemical investigation of an hsp expression can support the proof of vitality in cases of death related to fire.

  1. Altered gene expression and possible immunodeficiency in cases of sudden infant death syndrome.

    PubMed

    Ferrante, Linda; Rognum, Torleiv O; Vege, Åshild; Nygård, Ståle; Opdal, Siri H

    2016-07-01

    A large number of studies have tried to uncover a genetic predisposition for sudden infant death syndrome (SIDS), but there is still uncertainty concerning the pathogenesis of these deaths. The purpose of this study was to investigate mRNA gene expression in SIDS cases and controls, in order to uncover genes that are differentially expressed in the two groups. Tissue from brain, heart, and liver from 15 SIDS cases and 15 controls were included in the study, and mRNA expression was determined using the Illumina whole genome gene expression DASL HT assay. Seventeen genes showed significantly altered expression compared to controls, after correction for multiple testing. Three genes involved in the immune system were of particular interest, including the downregulation of MyD88 in tissue from SIDS brains, as well as the downregulation of the genes encoding CCL3 and UNC13 in the liver. These findings indicate that there is an altered expression of genes involved in the inflammatory process in a proportion of SIDS cases, which further strengthen the hypothesis that impaired immune response play a role in this syndrome.

  2. Glutamate mediates the function of melanocortin receptor 4 on sim1 neurons in body weight regulation

    USDA-ARS?s Scientific Manuscript database

    The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing ...

  3. Hydrogen Peroxide Activates Cell Death and Defense Gene Expression in Birch1

    PubMed Central

    Pellinen, Riikka I.; Korhonen, Minna-Sisko; Tauriainen, Airi A.; Palva, E. Tapio; Kangasjärvi, Jaakko

    2002-01-01

    The function of hydrogen peroxide (H2O2) as a signal molecule regulating gene expression and cell death induced by external stresses was studied in birch (Betula pendula). Ozone (O3), Pseudomonas syringae pv syringae (Pss), and wounding all induced cell death of various extents in birch leaves. This was temporally preceded and closely accompanied by H2O2 accumulation at, and especially surrounding, the lesion sites. O3 and Pss, along with an artificial H2O2 producing system glucose (Glc)/Glc oxidase, elicited elevated mRNA levels corresponding to genes encoding reactive oxygen species detoxifying enzymes, Pal, Ypr10, and mitochondrial phosphate translocator 1. In addition to the regulation of gene expression, Glc/Glc oxidase also induced endogenous H2O2 production in birch leaves, accompanied by cell death that resembled O3 and Pss damage. Wound-induced gene expression differed from that induced by O3 and Pss. Thus, it appears that at least two separate defense pathways can be activated in birch leaves by stress factors, even though the early H2O2 accumulation response is common among them all. PMID:12376624

  4. Expression analysis of KDEL-CysEPs programmed cell death markers during reproduction in Arabidopsis.

    PubMed

    Zhou, Liang-Zi; Höwing, Timo; Müller, Benedikt; Hammes, Ulrich Z; Gietl, Christine; Dresselhaus, Thomas

    2016-09-01

    CEP cell death markers. Programmed cell death (PCD) is essential for proper plant growth and development. Plant-specific papain-type KDEL-tailed cysteine endopeptidases (KDEL-CysEPs or CEPs) have been shown to be involved in PCD during vegetative development as executors for the last step in the process. The Arabidopsis genome encodes three KDEL-CysEPs: AtCEP1, AtCEP2 and AtCEP3. With the help of fluorescent fusion reporter lines, we report here a detailed expression analysis of KDEL-CysEP (pro)proteins during reproductive processes, including flower organ and germline development, fertilization and seed development. AtCEP1 is highly expressed in different reproductive tissues including nucellus cells of mature ovule and the connecting edge of anther and filament. After fertilization, AtCEP1 marks integument cell layers of the seeds coat as well as suspensor and columella cells of the developing embryo. Promoter activity of AtCEP2 is detected in the style of immature and mature pistils, in other floral organs including anther, sepal and petal. AtCEP2 mainly localizes to parenchyma cells next to xylem vessels. Although there is no experimental evidence to demonstrate that KDEL-CysEPs are involved in PCD during fertilization, the expression pattern of AtCEPs, which were previously shown to represent cell death markers during vegetative development, opens up new avenues to investigate PCD in plant reproduction.

  5. Hydrogen peroxide activates cell death and defense gene expression in birch.

    PubMed

    Pellinen, Riikka I; Korhonen, Minna-Sisko; Tauriainen, Airi A; Palva, E Tapio; Kangasjärvi, Jaakko

    2002-10-01

    The function of hydrogen peroxide (H(2)O(2)) as a signal molecule regulating gene expression and cell death induced by external stresses was studied in birch (Betula pendula). Ozone (O(3)), Pseudomonas syringae pv syringae (Pss), and wounding all induced cell death of various extents in birch leaves. This was temporally preceded and closely accompanied by H(2)O(2) accumulation at, and especially surrounding, the lesion sites. O(3) and Pss, along with an artificial H(2)O(2) producing system glucose (Glc)/Glc oxidase, elicited elevated mRNA levels corresponding to genes encoding reactive oxygen species detoxifying enzymes, Pal, Ypr10, and mitochondrial phosphate translocator 1. In addition to the regulation of gene expression, Glc/Glc oxidase also induced endogenous H(2)O(2) production in birch leaves, accompanied by cell death that resembled O(3) and Pss damage. Wound-induced gene expression differed from that induced by O(3) and Pss. Thus, it appears that at least two separate defense pathways can be activated in birch leaves by stress factors, even though the early H(2)O(2) accumulation response is common among them all.

  6. The Effects of Antidepressants “Fluoxetine and Imipramine” on Vascular Abnormalities and Toll Like Receptor-4 Expression in Diabetic and Non-Diabetic Rats Exposed to Chronic Stress

    PubMed Central

    Habib, Mohamed; Shaker, Safaa; El-Gayar, Nesreen; Aboul-Fotouh, Sawsan

    2015-01-01

    Several studies reveal that diabetes doubles the odds of comorbid depression with evidence of a pro-inflammatory state underlying its vascular complications. Indeed, little information is available about vascular effects of antidepressant drugs in diabetes. Method: We investigated the effect of chronic administration of fluoxetine “FLU” and imipramine “IMIP” on behavioral, metabolic and vascular abnormalities in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Results: Both diabetes and CRS induced depressive-like behavior which was more prominent in diabetic/depressed rats; this was reversed by chronic treatment with FLU and IMIP in a comparable manner. Diabetic and non-diabetic rats exposed to CRS exhibited abnormalities in glucose homeostasis, lipid profile and vascular function, manifested by decreased endothelium-dependent relaxation, increased systolic blood pressure and histopathological atherosclerotic changes. Vascular and metabolic dysfunctions were associated with significant increase in aortic expression of TLR-4, and pro-inflammatory cytokines (TNF-α and IL-1ß). FLU ameliorated these metabolic, vascular and inflammatory abnormalities, while IMIP induced either no change or even worsening of some parameters. Conclusion: FLU has favorable effect over IMIP on metabolic, vascular and inflammatory aberrations associated with DM and CRS in Wistar rats, clarifying the preference of FLU over IMIP in management of comorbid depression in diabetic subjects. PMID:25826421

  7. Programmed death-ligand 1, 2 expressions are decreased in the psoriatic epidermis.

    PubMed

    Kim, Dae Suk; Je, Jung Hwan; Kim, Sung Hee; Shin, Dongyun; Kim, Tae-Gyun; Kim, Do Young; Kim, Soo Min; Lee, Min-Geol

    2015-08-01

    Psoriatic keratinocytes are one of the key components that amplify and maintain chronic inflammation. We hypothesized that lack of proper regulatory functions of keratinocytes can be responsible for chronic inflammation in psoriasis. Programmed death-ligands (PD-L) 1, 2 are expressed on keratinocytes, and expressions by nonlymphoid cells are important for mediating peripheral T cell tolerance. In our study, we investigated whether PD-L1, 2 expressions are altered in keratinocytes of psoriatic epidermis compared to normal epidermis. Epidermis was separated and analyzed for PD-L1, 2 expressions in mRNA and protein levels. Immunohistochemical stainings were done in skin biopsy samples from psoriasis, normal skin, allergic contact dermatitis (ACD), pityriasis rosea (PR) and lichen planus (LP). Expressions of PD-L1, 2 mRNA levels were significantly decreased in psoriatic epidermis compared to normal epidermis. In protein levels, PD-L1 expression was significantly decreased in psoriatic epidermis. However, PD-L2 expression was not detected in both normal and psoriatic epidermis. Immunohistochemical stainings revealed significantly less PD-L1 expression in psoriatic epidermis compared to normal epidermis. Even compared to other cutaneous inflammatory diseases, psoriatic epidermis showed less expression than ACD, PR and LP. PD-L2 expression was minimally detected in normal epidermis and not in psoriatic epidermis, but its expression was increased in ACD, PR and LP. In conclusion, we demonstrated that PD-L1, 2 are decreased in psoriatic epidermis in mRNA and protein levels. In addition, we showed that their expression was significantly lower than other inflammatory skin diseases. We suggest that decreased expression of PD-L1, 2 on psoriatic epidermis can contribute to its chronic unregulated inflammatory characteristics.

  8. The significance of programmed cell death ligand 1 expression in resected lung adenocarcinoma.

    PubMed

    Wu, Shafei; Shi, Xiaohua; Sun, Jian; Liu, Yuanyuan; Luo, Yufeng; Liang, Zhiyong; Wang, Jinghui; Zeng, Xuan

    2017-03-07

    Lung adenocarcinoma (AD) is a common variant of non-small cell lung cancer (NSCLC). Programmed cell death protein 1/programmed cell death ligand 1 (PD1/PD-L1) are promising immunotherapy targets and its expression may be an important biomarker of predicting clinical response. In this study, we evaluated PD-L1 expression in conjunction with clinicopathological characteristics and outcomes in resected lung adenocarcinoma. This study included 133 cases of lung adenocarcinoma. PD-L1 expression rate in lung adenocarcinoma was 16.5% at the mRNA level and 13.5% at the protein level, and the kappa coefficient of the two examination methods was 0.824 (P = 0.219, highly correlated). PD-L1 was highly expressed in male patients and smokers with lung adenocarcinoma (P = 0.019 and 0.002, respectively), while no associations were identified between PD-L1 expression and age, tumor size, clinical stage, positive pleural invasion, lymph node metastasis, or therapy methods. Overexpression of PD-L1 was a significant indicator of shorter recurrence free survival time and overall survival (P = 0.000 and 0.000, respectively). Multivariate analysis revealed that PD-L1 expression was an independent risk factor for poor recurrence free survival and overall survival (P = 0.009 and 0.016, respectively). Expression of PD-L1 was examined with immunohistochemistry, using the VENTANA PD-L1 (SP263) rabbit monoclonal antibody. mRNA levels of PD-L1 were evaluated using in situ hybridization. PD-L1 overexpression is more frequently observed in male patients and smokers in lung adenocarcinoma. PD-L1 expression is an indicator of worse prognosis in surgically resected lung adenocarcinoma patients.

  9. Reduced Frequency of a CD14+ CD16+ Monocyte Subset with High Toll-Like Receptor 4 Expression in Cord Blood Compared to Adult Blood Contributes to Lipopolysaccharide Hyporesponsiveness in Newborns

    PubMed Central

    Pedraza-Sánchez, Sigifredo; Hise, Amy G.; Ramachandra, Lakshmi; Arechavaleta-Velasco, Fabian

    2013-01-01

    The human innate immune response to pathogens is not fully effective and mature until well into childhood, as exemplified by various responses to Toll-like receptor (TLR) agonists in newborns compared to adults. To better understand the mechanistic basis for this age-related difference in innate immunity, we compared tumor necrosis factor alpha (TNF-α) production by monocytes from cord blood (CB) and adult blood (AB) in response to LAM (lipoarabinomannan from Mycobacterium tuberculosis, a TLR2 ligand) and LPS (lipopolysaccharide from Escherichia coli, a TLR4 ligand). LPS or LAM-induced TNF-α production was 5 to 18 times higher in AB than in CB monocytes, whereas interleukin-1α (IL-1α) stimulated similar levels of TNF-α in both groups, suggesting that decreased responses to LPS or LAM in CB are unlikely to be due to differences in the MyD88-dependent signaling pathway. This impaired signaling was attributable, in part, to lower functional TLR4 expression, especially on CD14+ CD16+ monocytes, which are the primary cell subset for LPS-induced TNF-α production. Importantly, the frequency of CD14+ CD16+ monocytes in CB was 2.5-fold lower than in AB (P < 0.01). CB from Kenyan newborns sensitized to parasite antigens in utero had more CD14+ CD16+ monocytes (P = 0.02) and produced higher levels of TNF-α in response to LPS (P = 0.004) than CB from unsensitized Kenyan or North American newborns. Thus, a reduced CD14+ CD16+ activated/differentiated monocyte subset and a correspondingly lower level of functional TLR4 on monocytes contributes to the relatively low TNF-α response to LPS observed in immunologically naive newborns compared to the response in adults. PMID:23595503

  10. Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

    SciTech Connect

    Shin, Jung Ar; Chung, Jin Sil; Cho, Sang-Ho; Kim, Hyung Jung; Yoo, Young Do

    2013-09-20

    Highlights: •Romo1 mediates oxidative stress-induced mitochondrial ROS production. •Romo1 induction by oxidative stress plays an important role in oxidative stress-induced apoptosis. •Romo1 overexpression correlates with epithelial cell death in patients with IPF. -- Abstract: Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H{sub 2}O{sub 2}) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H{sub 2}O{sub 2} treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.

  11. Programmed death ligand 1 expression in hepatocellular carcinoma: Relationship With clinical and pathological features.

    PubMed

    Calderaro, Julien; Rousseau, Benoît; Amaddeo, Giuliana; Mercey, Marion; Charpy, Cécile; Costentin, Charlotte; Luciani, Alain; Zafrani, Elie-Serge; Laurent, Alexis; Azoulay, Daniel; Lafdil, Fouad; Pawlotsky, Jean-Michel

    2016-12-01

    The prognosis of hepatocellular carcinoma (HCC) remains poor, with only one third of patients eligible for curative treatments and very limited survival benefits with the use of sorafenib, the current standard of care for advanced disease. Recently, agents targeting the programmed death ligand 1 (PD-L1)/programmed death receptor 1 (PD-1) immune checkpoint were shown to display impressive antitumor activity in various solid or hematological malignancies, including HCC. PD-L1 immunohistochemical expression is thought to represent a biomarker predictive of drug sensitivity. Here, we investigated PD-L1 expression in a series of 217 HCCs and correlated our results with clinical and histological features and immunohistochemical markers (PD-1, cytokeratin 19, glutamine synthetase, and β-catenin expression). PD-L1 expression by neoplastic cells was significantly associated with common markers of tumor aggressiveness (high serum alpha-fetoprotein levels, P = 0.038; satellite nodules, P < 0.001; macrovascular invasion, P < 0.001; microvascular invasion, P < 0.001; poor differentiation, P < 0.001) and with the progenitor subtype of HCC (cytokeratin 19 expression, P = 0.031). High PD-L1 expression by inflammatory cells from the tumor microenvironment also correlated with high serum alpha-fetoprotein levels (P < 0.001), macrovascular invasion (P = 0.001), poor differentiation (P = 0.001), high PD-1 expression (P < 0.001), and the so-called lymphoepithelioma-like histological subtype of HCC (P = 0.003).

  12. Mutant SOD1-expressing astrocytes release toxic factors that trigger motoneuron death by inducing hyperexcitability

    PubMed Central

    Fritz, Elsa; Izaurieta, Pamela; Weiss, Alexandra; Mir, Franco R.; Rojas, Patricio; Gonzalez, David; Rojas, Fabiola; Brown, Robert H.; Madrid, Rodolfo

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by dysfunction and degeneration of motoneurons starting in adulthood. Recent studies using cell or animal models document that astrocytes expressing disease-causing mutations of human superoxide dismutase 1 (hSOD1) contribute to the pathogenesis of ALS by releasing a neurotoxic factor(s). Neither the mechanism by which this neurotoxic factor induces motoneuron death nor its cellular site of action has been elucidated. Here we show that acute exposure of primary wild-type spinal cord cultures to conditioned medium derived from astrocytes expressing mutant SOD1 (ACM-hSOD1G93A) increases persistent sodium inward currents (PCNa), repetitive firing, and intracellular calcium transients, leading to specific motoneuron death days later. In contrast to TTX, which paradoxically increased twofold the amplitude of calcium transients and killed motoneurons, reduction of hyperexcitability by other specific (mexiletine) and nonspecific (spermidine and riluzole) blockers of voltage-sensitive sodium (Nav) channels restored basal calcium transients and prevented motoneuron death induced by ACM-hSOD1G93A. These findings suggest that riluzole, the only FDA-approved drug with known benefits for ALS patients, acts by inhibiting hyperexcitability. Together, our data document that a critical element mediating the non-cell-autonomous toxicity of ACM-hSOD1G93A on motoneurons is increased excitability, an observation with direct implications for therapy of ALS. PMID:23486205

  13. p53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death

    PubMed Central

    Feng, Xi; Liu, Xing; Zhang, Wei; Xiao, Wuhan

    2011-01-01

    Hypoxia stabilizes the tumour suppressor p53, allowing it to function primarily as a transrepressor; however, the function of p53 during hypoxia remains unclear. In this study, we showed that p53 suppressed BNIP3 expression by directly binding to the p53-response element motif and recruiting corepressor mSin3a to the BNIP3 promoter. The DNA-binding site of p53 must remain intact for the protein to suppress the BNIP3 promoter. In addition, taking advantage of zebrafish as an in vivo model, we confirmed that zebrafish nip3a, a homologous gene of mammalian BNIP3, was indeed induced by hypoxia and p53 mutation/knockdown enhanced nip3a expression under hypoxia resulted in cell death enhancement in p53 mutant embryos. Furthermore, p53 protected against hypoxia-induced cell death mediated by p53 suppression of BNIP3 as illustrated by p53 knockdown/loss assays in both human cell lines and zebrafish model, which is in contrast to the traditional pro-apoptotic role of p53. Our results suggest a novel function of p53 in hypoxia-induced cell death, leading to the development of new treatments for ischaemic heart disease and cerebral stoke. PMID:21792176

  14. Effect of Heat Shock Protein 72 Expression on Etoposide-induced Cell Death of Rat Retinal Ganglion Cells

    PubMed Central

    Sohn, Seongsoo; Im, Ji-Eun; Kim, Tae Eun

    2013-01-01

    Purpose To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. Methods Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. Results Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. Conclusions Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease. PMID:23372380

  15. Therapeutic implications of continuing bonds expressions following the death of a pet.

    PubMed

    Packman, Wendy; Carmack, Betty J; Ronen, Rama

    Through the exploration of 12 continuing bonds expressions (CBE), this current study investigated the grief reaction and continuing impact of the death of a pet. Thirty-three individuals were interviewed to determine the degree of connection maintained with the deceased pet and how that affects their coping. Findings emphasize that the majority of respondents frequently maintain ongoing meaningful ties with their deceased pet through the use of CBE such as fond memories, rituals, dreams. The findings suggest that it is not the number of CBE but the degree of adaptability that is significant. The importance of recognizing the unique, total experience of those grieving the death of a pet is addressed. Implications for those working with and supporting those in grief are included. Future directions for research are described.

  16. Decrease in doublecortin expression without neuronal cell death in rat retrosplenial cortex after stress exposure.

    PubMed

    Kutsuna, Nobuo; Suma, Takeshi; Takada, Yoshiyuki; Yamashita, Akiko; Oshima, Hideki; Sakatani, Kaoru; Yamamoto, Takamitsu; Katayama, Yoichi

    2012-03-07

    Exposure to acute stress by forced swim impairs spatial learning and memory in rats. The retrosplenial cortex plays an important role in spatial learning and memory. A cell population that expresses immature neuronal markers, including doublecortin (DCX), plays a key role in plasticity of the adult brain through formation of new neurons. Here, we aimed to determine whether rats exposed to acute stress showed changes in DCX expression in retrosplenial cortex cells. Twelve male Sprague-Dawley rats were used. Six were subjected to acute stress by forced swim (group S), and the remaining six served as controls (group C). Immunohistochemical staining was performed for DCX, neuron-specific nuclear protein, parvalbumin, calbindin, calretinin, and somatostatin. Newly generated cells were immunohistochemically detected by daily administration of 5-bromo-2'-deoxyuridine for 1 week. Fluoro-Jade B staining was performed to detect cell death. Group S showed lower number of DCX-expressing cells than group C (P<0.001). The proportion of DCX-expressing cells showing neuron-specific nuclear protein co-localization (24% in group S; 27% in group C) or parvalbumin co-localization (65% in group S; 61% in group C) remained unchanged after acute stress exposure. Neither 5-bromo-2'-deoxyuridine-positive nor Fluoro-Jade B-positive cells were found in the retrosplenial cortex of groups S and C. DCX-expressing cells in the retrosplenial cortex decreases markedly without cell death after acute stress exposure. Neuronal differentiation of these cells toward gamma aminobutyric acidergic interneurons appears to be unaltered. The decrease in DCX expression may reduce plasticity potential within the retrosplenial cortex and attenuate spatial learning and memory function.

  17. Increased expression of programmed death ligand 1 (PD-L1) in human pituitary tumors

    PubMed Central

    Greenwald, Noah F.; Du, Ziming; Agar, Nathalie Y. R.; Kaiser, Ursula B.; Woodmansee, Whitney W.; Reardon, David A.; Freeman, Gordon J.; Fecci, Peter E.; Laws, Edward R.; Santagata, Sandro; Dunn, Gavin P.; Dunn, Ian F.

    2016-01-01

    Purpose Subsets of pituitary tumors exhibit an aggressive clinical courses and recur despite surgery, radiation, and chemotherapy. Because modulation of the immune response through inhibition of T-cell checkpoints has led to durable clinical responses in multiple malignancies, we explored whether pituitary adenomas express immune-related biomarkers that could suggest suitability for immunotherapy. Specifically, programmed death ligand 1 (PD-L1) has emerged as a potential biomarker whose expression may portend more favorable responses to immune checkpoint blockade therapies. We thus investigated the expression of PD-L1 in pituitary adenomas. Methods PD-L1 RNA and protein expression were evaluated in 48 pituitary tumors, including functioning and non-functioning adenomas as well as atypical and recurrent tumors. Tumor infiltrating lymphocyte populations were also assessed by immunohistochemistry. Results Pituitary tumors express variable levels of PD-L1 transcript and protein. PD-L1 RNA and protein expression were significantly increased in functioning (growth hormone and prolactin-expressing) pituitary adenomas compared to non-functioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary adenomas harbored higher levels of PD-L1 mRNA compared to recurrent tumors. Tumor infiltrating lymphocytes were observed in all pituitary tumors and were positively correlated with increased PD-L1 expression, particularly in the functional subtypes. Conclusions Human pituitary adenomas harbor PD-L1 across subtypes, with significantly higher expression in functioning adenomas compared to non-functioning adenomas. This expression is accompanied by the presence of tumor infiltrating lymphocytes. These findings suggest the existence of an immune response to pituitary tumors and raise the possibility of considering checkpoint blockade immunotherapy in cases refractory to conventional management. PMID:27655724

  18. Gene Expression Analysis Implicates a Death Receptor Pathway in Schizophrenia Pathology

    PubMed Central

    Catts, Vibeke Sørensen; Shannon Weickert, Cynthia

    2012-01-01

    An increase in apoptotic events may underlie neuropathology in schizophrenia. By data-mining approaches, we identified significant expression changes in death receptor signaling pathways in the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia, particularly implicating the Tumor Necrosis Factor Superfamily member 6 (FAS) receptor and the Tumor Necrosis Factor [ligand] Superfamily member 13 (TNFSF13) in schizophrenia. We sought to confirm and replicate in an independent tissue collection the noted mRNA changes with quantitative real-time RT-PCR. To test for regional and diagnostic specificity, tissue from orbital frontal cortex (OFC) was examined and a bipolar disorder group included. In schizophrenia, we confirmed and replicated significantly increased expression of TNFSF13 mRNA in the DLPFC. Also, a significantly larger proportion of subjects in the schizophrenia group had elevated FAS receptor expression in the DLPFC relative to unaffected controls. These changes were not observed in the bipolar disorder group. In the OFC, there were no significant differences in TNFSF13 or FAS receptor mRNA expression. Decreases in BH3 interacting domain death agonist (BID) mRNA transcript levels were found in the schizophrenia and bipolar disorder groups affecting both the DLPFC and the OFC. We tested if TNFSF13 mRNA expression correlated with neuronal mRNAs in the DLPFC, and found significant negative correlations with interneuron markers, parvalbumin and somatostatin, and a positive correlation with PPP1R9B (spinophilin), but not DLG4 (PSD-95). The expression of TNFSF13 mRNA in DLPFC correlated negatively with tissue pH, but decreasing pH in cultured cells did not cause increased TNFSF13 mRNA nor did exogenous TNFSF13 decrease pH. We concluded that increased TNFSF13 expression may be one of several cell-death cytokine abnormalities that contribute to the observed brain pathology in schizophrenia, and while increased TNFSF13 may be associated with lower

  19. Transgenic cloned sheep overexpressing ovine toll-like receptor 4.

    PubMed

    Deng, Shoulong; Li, Guiguan; Zhang, Jinlong; Zhang, Xiaosheng; Cui, Maosheng; Guo, Yong; Liu, Guoshi; Li, Guangpeng; Feng, Jianzhong; Lian, Zhengxing

    2013-07-01

    An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance.

  20. Sudden infant death syndrome: no significant expression of heat-shock proteins (HSP27, HSP70).

    PubMed

    Doberentz, Elke; Führing, Sarah; Madea, Burkhard

    2016-03-01

    In industrialized countries, sudden infant death is the most common cause of death in young children. Although prone sleeping position is a well-known risk factor, hyperthermia might also be important. Pathognomonic findings of premortem hyperthermia do not exist. During stress, including thermal effects, heat-shock protein (HSP) expression increases. This study investigated hyperthermia as a contributing or pathogenic factor for sudden infant death syndrome (SIDS). Immunohistochemical staining for HSP27 and HSP70 in the kidney, heart, and lung from 120 SIDS cases was examined. HSP70 immunostaining was negative in kidney, heart, and lung tissues in all cases and in tissues from the control group. HSP27 staining was positive in the kidney from one case, and was positive in the lungs (respiratory epithelia in 27% of cases; vascular endothelia in 19% of cases) and was negative in the heart. In the control group HSP27 was positive in 8% of renal tubular tissues and in 29% of renal vascular endothelia. Staining for HSP27 in lung tissues was positive in respiratory epithelia in 8% of cases and for vascular endothelia in 29%, whereas tissues from the heart were positive in only 4%. The hypothesis of hyperthermia being a pathogenic factor for SIDS was not supported by immunohistochemical visualization of HSP70 or HSP27.

  1. Programmed death ligand-1 expression and its prognostic role in esophageal squamous cell carcinoma

    PubMed Central

    Kim, Ryul; Keam, Bhumsuk; Kwon, Dohee; Ock, Chan-Young; Kim, Miso; Kim, Tae Min; Kim, Hak Jae; Jeon, Yoon Kyung; Park, In Kyu; Kang, Chang Hyun; Kim, Dong-Wan; Kim, Young Tae; Heo, Dae Seog

    2016-01-01

    AIM To investigate the expression and prognostic role of programmed death ligand-1 (PD-L1) in locally advanced esophageal squamous cell carcinoma (ESCC). METHODS A total of 200 patients with ESCC who underwent radical esophagectomy with standard lymphadenectomy as the initial definitive treatment in Seoul National University Hospital from December 2000 to April 2013 were eligible for this analysis. Tissue microarrays were constructed by collecting tissue cores from surgical specimens, and immunostained with antibodies directed against PD-L1, p16, and c-Met. Medical records were reviewed retrospectively to assess clinical outcomes. Patients were divided into two groups by PD-L1 status, and significant differences in clinicopathologic characteristics between the two groups were assessed. RESULTS Tumor tissues from 67 ESCC patients (33.5%) were PD-L1-positive. Positive p16 expression was observed in 21 specimens (10.5%). The H-score for c-Met expression was ≥ 50 in 42 specimens (21.0%). Although PD-L1-positivity was not significantly correlated with any clinical characteristics including age, sex, smoking/alcoholic history, stage, or differentiation, H-scores for c-Met expression were significantly associated with PD-L1-positivity (OR = 2.34, 95%CI: 1.16-4.72, P = 0.017). PD-L1 expression was not significantly associated with a change in overall survival (P = 0.656). In contrast, the locoregional relapse rate tended to increase (P = 0.134), and the distant metastasis rate was significantly increased (HR = 1.72, 95%CI: 1.01-2.79, P = 0.028) in patients with PD-L1-positive ESCC compared to those with PD-L1-negative ESCC. CONCLUSION PD-L1 expression is positively correlated with c-Met expression in ESCC. PD-L1 may play a critical role in distant failure and progression of ESCC. PMID:27729745

  2. High programmed death 1 expression on T cells in aplastic anemia.

    PubMed

    Zhao, Wanhong; Zhang, Yilin; Zhang, Pengyu; Yang, Juan; Zhang, Longjin; He, Aili; Zhang, Wanggang; Hideto, Tamura

    2017-03-01

    Programmed death 1 (PD-1) has been reported to be associated with aberrant regulation of T cells activation in aplastic anemia (AA). However, the connection between PD-1 expression status and AA needs to be further explored. The aim of this study is to investigate PD-1 expression status on T cells in AA patients and to explore the effect of PD-1 on apoptosis of T cells and BMHSCs. The concentration of platelet, lymphocyte and hemoglobin in peripheral blood of AA patients and healthy volunteers was detected by automatic blood-counter system. Mononuclear cells (MNCs) of peripheral blood and marrow were isolated from AA patients and healthy volunteers. PD-1 expression level on CD3+, CD4+, CD8+, CD4+CD25+FOXP3+ T cells and bone marrow hematopoietic stem cells (BMHSCs) and B7-H1 expression level on peripheral blood mononuclear cells (PBMCs) and BMHSCs were detected by flow cytometry (FCM). Cell apoptosis on T cells and BMHSCs was also detected by FCM. PD-1, B7-H1, Bax and Bcl-2 mRNA expression levels on T cells of peripheral blood were detected by real-time PCR. MNCs from healthy volunteers were treated with ammonium pyrrolidine dithiocarbamate (PDTC) to block the nuclear factor (NF)-кB pathway. Our results showed that in AA patients, T cells had high levels of PD-1 expression and apoptosis rate. In BMHSCs, apoptosis rate was also higher than healthy volunteers. The expression of PD-1 on T cells was correlated with lymphocyte count and hemoglobin level. PD-1 was highly expressed on CD4+CD25+FOXP3+ T cells of AA patients and PD-1 expression was negatively correlated with the percentage of CD4+CD25+FOXP3+ T cells in AA patients. B7-H1, the ligand of PD-1, was also highly expressed on T cells, B cells, PBMCs and BMHSCs. When the NF-κB pathway was blocked by PDTC, the apoptosis of T cells and BMHSCs was reduced in a dose-dependent manner and PD-1 expression was also decreased in a dose-dependent manner. In conclusion, PD-1 was up-regulated in T cells in AA patients

  3. Expression level of P2X7 receptor is a determinant of ATP-induced death of mouse cultured neurons.

    PubMed

    Ohishi, A; Keno, Y; Marumiya, A; Sudo, Y; Uda, Y; Matsuda, K; Morita, Y; Furuta, T; Nishida, K; Nagasawa, K

    2016-04-05

    Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7R-expressing HEK293T cell systems. Treatment of pure neuron cultures with 5mM ATP for 2h, followed by 3-h incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7R-specific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death.

  4. Expression of programmed death 1 is correlated with progression of osteosarcoma.

    PubMed

    Zheng, Wenjie; Xiao, Hong; Liu, Huan; Zhou, Yue

    2015-02-01

    Accumulating bodies of evidence indicate that immune dysregulation plays a key role in the development of osteosarcoma (OS). Programmed death 1 (PD-1) is a surface receptor expressed on activated and exhausted T cells, which mediate T-cell inhibition upon binding with its ligand. Researches on PD-1 and OS remain extremely limited. Here, we investigated whether PD-1 could be involved in the development of OS. Expression of PD-1 was measured by flow cytometry on peripheral CD4+ and CD8+ T cells from 56 OS cases and 42 healthy controls. Data revealed that percentages of PD-1 were significantly upregulated on both peripheral CD4+ and CD8+ T cells from OS patients (p < 0.001 and p < 0.001, respectively). Patients with different tumor locations did not present obvious variations in PD-1 level. However, patients with metastasis showed significantly higher level of PD-1 on CD4+ T cells than those without metastasis (p < 0.001). Furthermore, PD-1 expression on CD4+ T cells started to increase in stage III, whereas PD-1 expression on CD8+ T cells started to increase in stage II. In addition, patients with pathological fracture were observed to have elevated PD-1 on both CD4+ and CD8+ T cells. These data suggest that PD-1 is involved in the pathogenesis of OS, especially in the progression of disease.

  5. Influence of high-frequency electromagnetic fields on different modes of cell death and gene expression.

    PubMed

    Port, M; Abend, M; Römer, B; Van Beuningen, D

    2003-09-01

    International thresholds for exposure to non-ionizing radiation leading to non-thermal effects were conservatively set by the International Commission on Non-Ionizing Radiation Protection (ICNIRP). The aim of this study was to examine whether biological effects such as different modes of cell death and gene expression modifications related to tumorgenesis are detectable above the threshold defined. Human leukaemia cells (HL-60) grown in vitro were exposed to electromagnetic fields (EMF; t 1/2(r) about 1 ns; field strength about 25 times higher than the ICNIRP reference levels for occupational exposure) leading to non-thermal effects using a high-voltage-improved GTEM cell 5302 (EMCO) connected to a pulse generator NP20 (C = 1 nF, U(Load) = 20kV). HL-60 cells were harvested at 0, 24, 48 and 72 h after radiation exposure. Micronuclei, apoptosis and abnormal cells (e.g. necrosis) were determined using morphological criteria. In parallel, the expression of 1176 genes was measured using Atlas Human 1.2. Array. Based on high data reproducibility calculated from two independent experiments (> 99%), array analysis was performed. No significant change in apoptosis, micronucleation, abnormal cells and differential gene expression was found. Exposure of HL-60 cells to EMFs 25 times higher than the ICNIRP reference levels for occupational exposure failed to induce any changes in apoptosis, micronucleation, abnormal morphologies and gene expression. Further experiments using EMFs above the conservatively defined reference level set by the ICNIRP may be desirable.

  6. Molecular cloning, expression and characterization of programmed cell death 10 from sheep (Ovis aries).

    PubMed

    Yang, Yong-Jie; Liu, Zeng-Shan; Lu, Shi-Ying; Li, Chuang; Hu, Pan; Li, Yan-Song; Liu, Nan-Nan; Tang, Feng; Xu, Yun-Ming; Zhang, Jun-Hui; Li, Zhao-Hui; Feng, Xiao-Li; Zhou, Yu; Ren, Hong-Lin

    2015-03-01

    Programmed cell death 10 (PDCD10) is a highly conserved adaptor protein. Its mutations result in cerebral cavernous malformations (CCMs). In this study, PDCD10 cDNA from the buffy coat of Small Tail Han sheep (Ovis aries) was cloned from a suppression subtractive hybridization cDNA library, named OaPDCD10. The full-length cDNA of OaPDCD10 was 1343bp with a 639bp open reading frame (ORF) encoding 212 amino acid residues. Tissue distribution of OaPDCD10 mRNA determined that it was ubiquitously expressed in all tested tissue samples, and the highest expression was observed in the heart. The differential expression of OaPDCD10 between infected sheep (challenged with Brucella melitensis) and vaccinated sheep (vaccinated with Brucella suis S2) was also investigated. The results revealed that, compared to the control group, the expression of OaPDCD10 from infected and vaccinated sheep was both significantly up-regulated (p<0.05). Moreover, the expression levels of OaPDCD10 from the vaccinated sheep were significantly higher than the infected sheep (p<0.05) after 30days post-inoculation. The recombinant OaPDCD10 (rOaPDCD10) protein was expressed in Escherichia coli BL21 (DE3), and then purified by affinity chromatography. The rOaPDCD10 protein was demonstrated to induce apoptosis and promote cell proliferation. Our studies are intended to discover potential diagnostic biomarkers of brucellosis to discern infected sheep from vaccinated sheep, and OaPDCD10 could be considered as a potential diagnostic biomarker of brucellosis.

  7. Toll-like receptor 4 promotes proliferation and apoptosis resistance in human papillomavirus-related cervical cancer cells through the Toll-like receptor 4/nuclear factor-κB pathway.

    PubMed

    Jiang, Ninghong; Xie, Feng; Guo, Qisang; Li, Ming-Qing; Xiao, Jingjing; Sui, Long

    2017-06-01

    Toll-like receptor 4 is overexpressed in various tumors, including cervical carcinoma. However, the role of Toll-like receptor 4 in cervical cancer remains controversial, and the underlying mechanisms are largely elusive. Therefore, Toll-like receptor 4 in cervical cancer and related mechanisms were investigated in this study. Quantitative reverse transcription polymerase chain reaction and western blot analyses were used to detect messenger RNA and protein levels in HeLa, Caski, and C33A cells with different treatments. Proliferation was quantified using Cell Counting Kit-8. Cell cycle distribution and apoptosis were assessed by flow cytometry. Higher levels of Toll-like receptor 4 expression were found in human papillomavirus-positive cells compared to human papillomavirus-negative cells. Proliferation of HeLa and Caski cells was promoted in lipopolysaccharide-stimulated groups but suppressed in short hairpin RNA-transfected groups. Apoptosis rates were lower in lipopolysaccharide-stimulated groups relative to short hairpin RNA-transfected groups. In addition, G2-phase distribution was enhanced when Toll-like receptor 4 was downregulated. Moreover, the pNF-κBp65 level was positively correlated with the Toll-like receptor 4 level in HeLa and Caski cells, though when an nuclear factor-κB inhibitor was applied to lipopolysaccharide-stimulated groups, the patterns of proliferation and apoptosis were opposite to those of the lipopolysaccharide-stimulated groups without inhibitor treatment. In conclusion, these data suggest that Toll-like receptor 4 promotes proliferation and apoptosis resistance in human papillomavirus-related cervical cancer cells at least in part through the Toll-like receptor 4/nuclear factor-κB pathway, which may be correlated with the occurrence and development of cervical carcinoma.

  8. Enhanced expression of Programmed cell death 1 (PD-1) protein in benign vascular anomalies.

    PubMed

    Amaya, Clarissa N; Wians, Frank H; Bryan, Brad A; Torabi, Alireza

    2017-04-01

    Programmed cell death 1 (PD-1) and its ligands have been shown to play a significant role in evasion of malignant tumour cells from the immune system. Last year, the United States Food and Drug Administration (FDA) approved anti-PD-1 inhibitors for treatment of non-small cell lung carcinoma and recently has approved anti-PD-L1 blocker for treatment of metastatic urothelial cell carcinoma. However, the role that the immune system might have on benign tumours including vascular anomalies has received less attention. In this study, we evaluated PD-1 and PD-L1 expression on two benign vascular anomalies: infantile haemangiomas and venous malformations. Tissue microarrays (TMAs) from these two entities were stained for PD-1 and PD-L1 antibodies. Blood vessels from normal tissue were used as control. The endothelial cells in both infantile haemangioma and venous malformation showed high expression of PD-1 but were negative for PD-L1. Endothelial cells within the blood vessels in normal tissues were negative for both PD-1 and PD-L1. Our results showed over-expression of PD-1 in subsets of vascular anomalies, while PD-L1 was negative. This would raise the possibility of immunotherapy in benign vascular tumour when other options are exhausted.

  9. Expression and biological function of programmed death ligands in human placenta mesenchymal stem cells.

    PubMed

    Wang, Guoyan; Zhang, Siying; Wang, Feifei; Li, Guangyun; Zhang, Lixia; Luan, Xiying

    2013-02-01

    Mesenchymal stem cells (MSCs) play important roles in tissue regeneration due to their self-renewal, multilineage differentiation and immunosuppression abilities. MSCs can be isolated from various kinds of tissue, such as umbilical cord, cord blood and placenta. Human placenta mesenchymal stem cells (hPMSCs) possess stronger immunosuppressive properties, such as the ability to inhibit T-cell activation and proliferation, than human bone marrow MSCs. We have investigated that the roles of the programmed death ligands 1 and 2 (PDL1 and PDL2) in hPMSC adhesion, migration and immunosuppression were investigated. PDL1 and PDL2 were highly expressed by hPMSCs. Knockdown of PDL1 and/or PDL2 by siRNA increased hPMSC adhesion, but greatly decreasing migration. PDL1 and PDL2 expressed on hPMSCs inhibited T-cell proliferation by arresting the cell cycle. Knockdown of PDL1 and/or PDL2 in hPMSCs, however, had no effect on the expression of CD69, a T-cell early activation marker found on both CD4(+) and CD8(+) T-cell subsets. In summary, the roles of the negative co-stimulators PDL1 and PDL2 is on the adhesion, migration and immunosuppression of hPMSCs. These findings may be useful regarding the potential use of hPMSCs in clinical cell.

  10. Premature death and neurologic abnormalities in transgenic mice expressing a mutant huntingtin exon-2 fragment

    PubMed Central

    Tebbenkamp, Andrew T.N.; Swing, Debbie; Tessarollo, Lino; Borchelt, David R.

    2011-01-01

    Huntington's disease (HD) is a fatal neurodegenerative disease characterized pathologically by aggregates composed of N-terminal fragments of the mutant form of the protein huntingtin (htt). The role of these N-terminal fragments in disease pathogenesis has been questioned based in part on studies in transgenic mice. In one important example, mice that express an N-terminal fragment of mutant htt terminating at the C-terminus of exon 2 (termed the Shortstop mouse) were reported to develop robust inclusion pathology without developing phenotypic abnormalities seen in the R6/2 or N171-82Q models of HD, which are also based on expression of mutant N-terminal htt fragments. To further explore the capacity of mutant exon-2 htt fragments to produce neurologic abnormalities (N-terminal 118 amino acids; N118), we generated transgenic mice expressing cDNA that encodes htt N118-82Q with the mouse prion promoter vector. In mice generated in this manner, we demonstrate robust inclusion pathology accompanied by early death and failure to gain weight. These phenotypes are the most robust abnormalities identified in the R6/2 and N171-82Q models. We conclude that the lack of an overt phenotype in the initial Shortstop mice cannot be completely explained by the properties of mutant htt N118 fragments. PMID:21307087

  11. Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice.

    PubMed

    Qi, Yaocheng; Wang, Hongjuan; Zou, Yu; Liu, Cheng; Liu, Yanqi; Wang, Ying; Zhang, Wei

    2011-01-03

    In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H(2)O(2)-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ(m)) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ(m) relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ(m) and inhibiting the amplification of ROS. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. High mobility group box 1/toll-like receptor 4/myeloid differentiation factor 88 signaling promotes progression of gastric cancer.

    PubMed

    Yue, Yanqiu; Zhou, Tao; Gao, Yanjing; Zhang, Zongli; Li, Li; Liu, Lin; Shi, Wenna; Su, Lihui; Cheng, Baoquan

    2017-03-01

    High mobility group box 1 and toll-like receptor 4/myeloid differentiation factor 88 signaling pathway have been indicated to have oncogenic effects in many cancers. However, the role of high mobility group box 1/toll-like receptor 4/myeloid differentiation factor 88 signaling pathway in the development of gastric cancer remains unclear. In this study, we demonstrated that high mobility group box 1, toll-like receptor 4, and myeloid differentiation factor 88 were overexpressed in gastric cancer tumors compared with the adjacent non-tumor tissues. The overexpression of high mobility group box 1, toll-like receptor 4, and myeloid differentiation factor 88 were correlated with tumor-node-metastasis stage (p = 0.0068, p = 0.0063, p = 0.0173) and lymph node metastasis (p = 0.0272, p = 0.0382, and p = 0.0495). Furthermore, we observed that knockdown of high mobility group box 1 by high mobility group box 1-small interfering RNA suppressed the expression of toll-like receptor 4 and myeloid differentiation factor 88. Blockage of high mobility group box 1/toll-like receptor 4/myeloid differentiation factor 88 signaling by high mobility group box 1-small interfering RNA resulted in elevation of apoptotic ratio and inhibition of cell growth, migration, and invasion by upregulating Bax expression and downregulating Bcl-2, matrix metalloproteinase-2, nuclear factor kappa B/p65 expression, and the nuclear translocation of nuclear factor kappa B/p65 in gastric cancer cells. Our findings suggest that high mobility group box 1/toll-like receptor 4/myeloid differentiation factor 88 signaling pathway may contribute to the development and progression of gastric cancer via the nuclear factor kappa B pathway and it also represents a novel potential therapeutic target for gastric cancer.

  13. Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice.

    PubMed

    Chen, Songbiao; Songkumarn, Pattavipha; Venu, R C; Gowda, Malali; Bellizzi, Maria; Hu, Jinnan; Liu, Wende; Ebbole, Daniel; Meyers, Blake; Mitchell, Thomas; Wang, Guo-Liang

    2013-02-01

    Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta-expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta-expressed cell death-inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.

  14. Microglia modulate brainstem serotonergic expression following neonatal sustained hypoxia exposure: implications for sudden infant death syndrome.

    PubMed

    MacFarlane, P M; Mayer, C A; Litvin, D G

    2016-06-01

    Neonatal sustained hypoxia exposure modifies brainstem microglia and serotonin expression. The altered brainstem neurochemistry is associated with impaired ventilatory responses to acute hypoxia and mortality. The deleterious effects of sustained hypoxia exposure can be prevented by an inhibitor of activated microglia. These observations demonstrate a potential cause of the brainstem serotonin abnormalities thought to be involved in sudden infant death syndrome. We showed previously that the end of the second postnatal week (days P11-15) represents a period of development during which the respiratory neural control system exhibits a heightened vulnerability to sustained hypoxia (SH, 11% O2 , 5 days) exposure. In the current study, we investigated whether the vulnerability to SH during the same developmental time period is associated with changes in brainstem serotonin (5-HT) expression and whether it can be prevented by the microglia inhibitor minocycline. Using whole-body plethysmography, SH attenuated the acute (5 min) hypoxic ventilatory response (HVR) and caused a high incidence of mortality compared to normoxia rats. SH also increased microglia cell numbers and decreased 5-HT immunoreactivity in the nucleus of the solitary tract (nTS) and dorsal motor nucleus of the vagus (DMNV). The attenuated HVR, mortality, and changes in nTS and DMNV immunoreactivity was prevented by minocycline (25 mg kg(-1) /2 days during SH). These data demonstrate that the 5-HT abnormalities in distinct respiratory neural control regions can be initiated by prolonged hypoxia exposure and may be modulated by microglia activity. These observations share several commonalities with the risk factors thought to underlie the aetiology of sudden infant death syndrome, including: (1) a vulnerable neonate; (2) a critical period of development; (3) evidence of hypoxia; (4) brainstem gliosis (particularly the nTS and DMNV); and (5) 5-HT abnormalities. © 2015 The Authors. The Journal of

  15. Expression and regulation of antioxidant enzymes in the developing limb support a function of ROS in interdigital cell death.

    PubMed

    Schnabel, Denhí; Salas-Vidal, Enrique; Narváez, Verónica; Sánchez-Carbente, María del Rayo; Hernández-García, David; Cuervo, Rodrigo; Covarrubias, Luis

    2006-03-15

    Vertebrate limb development is a well-studied model of apoptosis; however, little is known about the intracellular molecules involved in activating the cell death machinery. We have shown that high levels of reactive oxygen species (ROS) are present in the interdigital 'necrotic' tissue of mouse autopod, and that antioxidants can reduce cell death. Here, we determined the expression pattern of several antioxidant enzymes in order to establish their role in defining the areas with high ROS levels. We found that the genes encoding the superoxide dismutases and catalase are expressed in autopod, but they are downregulated in the interdigital regions at the time ROS levels increased and cell death was first detected. The possible role of superoxide and/or peroxide in activating cell death is supported by the protective effect of a superoxide dismutase/catalase mimetic. Interestingly, we found that peroxidase activity and glutathione peroxidase-4 gene (Gpx4) expression were restricted to the non-apoptotic tissue (e.g., digits) of the developing autopod. Induction of cell death with retinoic acid caused an increase in ROS and decrease in peroxidase activity. Even more inhibition of glutathione peroxidase activity leads to cell death in the digits, suggesting that a decrease in antioxidant activity, likely due to Gpx4, caused an increase in ROS levels, thus triggering apoptosis.

  16. Toll-Like Receptor 4 Wild Type Homozygozity of Polymorphisms +896 and +1196 Is Associated with High Gastrin Serum Levels and Peptic Ulcer Risk.

    PubMed

    Pohjanen, Vesa-Matti; Koivurova, Olli-Pekka; Huhta, Heikki; Helminen, Olli; Mäkinen, Johanna M; Karhukorpi, Jari M; Joensuu, Tapio; Koistinen, Pentti O; Valtonen, Jarno M; Niemelä, Seppo E; Karttunen, Riitta A; Karttunen, Tuomo J

    2015-01-01

    Toll-like receptor 4 is a part of the innate immune system and recognizes Helicobacter pylori lipopolysaccharide. The goal of this study was to analyze the role of Toll-like receptor 4 polymorphisms +896 (rs4986790) and +1196 (rs4986791) in the pathogenesis of Helicobacter pylori related gastroduodenal diseases in relation to gastric secretion and inflammation. Toll-like receptor 4 polymorphisms, serum gastrin-17 and pepsinogen I and II concentrations were determined, and gastroscopies with histopathological analyses were performed to 216 dyspeptic patients. As genotype controls, 179 controls and 61 gastric cancer patients were studied. In our study, the Toll-like receptor 4 +896 and +1196 polymorphisms were in total linkage disequilibrium. The homozygous wild types displayed higher gastrin-17 serum concentrations than the mutants (p = 0.001) and this effect was independent of Helicobacter pylori. The homozygous wild types also displayed an increased risk for peptic ulcers (OR: 4.390). Toll-like receptor 4 genotypes did not show any association with Helicobacter pylori positivity or the features of gastric inflammation. Toll-like receptor 4 expression was seen in gastrin and somatostatin expressing cells of antral mucosa by immunohistochemistry. Our results suggest a role for Toll-like receptor 4 in gastric acid regulation and that the Toll-like receptor 4 +896 and +1196 wild type homozygozity increases peptic ulcer risk via gastrin secretion.

  17. Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

    PubMed Central

    O’Connor, Roddy S.; Thangavelu, Govindarajan; Lovitch, Scott B.; Dandamudi, Durga Bhavani; Vincent, Benjamin G.; Tkachev, Victor; Pawlicki, Jan M.; Furlan, Scott N.; Kean, Leslie S.; Aoyama, Kazutoshi; Taylor, Patricia A.; Panoskaltsis-Mortari, Angela; Foncea, Rocio; Ranganathan, Parvathi; Devine, Steven M.; Burrill, Joel S.; Guo, Lili; Sacristan, Catarina; Snyder, Nathaniel W.; Blair, Ian A.; Milone, Michael C.; Dustin, Michael L.; Riley, James L.; Bernlohr, David A.; Murphy, William J.; Fife, Brian T.; Munn, David H.; Miller, Jeffrey S.; Serody, Jonathan S.; Freeman, Gordon J.; Sharpe, Arlene H.; Turka, Laurence A.

    2016-01-01

    Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1–/– donors. PD-L1–deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1–/– donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell–mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD. PMID:27294527

  18. Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality.

    PubMed

    Saha, Asim; O'Connor, Roddy S; Thangavelu, Govindarajan; Lovitch, Scott B; Dandamudi, Durga Bhavani; Wilson, Caleph B; Vincent, Benjamin G; Tkachev, Victor; Pawlicki, Jan M; Furlan, Scott N; Kean, Leslie S; Aoyama, Kazutoshi; Taylor, Patricia A; Panoskaltsis-Mortari, Angela; Foncea, Rocio; Ranganathan, Parvathi; Devine, Steven M; Burrill, Joel S; Guo, Lili; Sacristan, Catarina; Snyder, Nathaniel W; Blair, Ian A; Milone, Michael C; Dustin, Michael L; Riley, James L; Bernlohr, David A; Murphy, William J; Fife, Brian T; Munn, David H; Miller, Jeffrey S; Serody, Jonathan S; Freeman, Gordon J; Sharpe, Arlene H; Turka, Laurence A; Blazar, Bruce R

    2016-07-01

    Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

  19. Time course of programmed cell death, which included autophagic features, in hybrid tobacco cells expressing hybrid lethality.

    PubMed

    Ueno, Naoya; Nihei, Saori; Miyakawa, Naoto; Hirasawa, Tadashi; Kanekatsu, Motoki; Marubashi, Wataru; van Doorn, Wouter G; Yamada, Tetsuya

    2016-12-01

    PCD with features of vacuolar cell death including autophagy-related features were detected in hybrid tobacco cells, and detailed time course of features of vacuolar cell death were established. A type of interspecific Nicotiana hybrid, Nicotiana suaveolens × N. tabacum exhibits temperature-sensitive lethality. This lethality results from programmed cell death (PCD) in hybrid seedlings, but this PCD occurs only in seedlings and suspension-cultured cells grown at 28 °C, not those grown at 36 °C. Plant PCD can be classified as vacuolar cell death or necrotic cell death. Induction of autophagy, vacuolar membrane collapse and actin disorganization are each known features of vacuolar cell death, but observed cases of PCD showing all these features simultaneously are rare. In this study, these features of vacuolar cell death were evident in hybrid tobacco cells expressing hybrid lethality. Ion leakage, plasma membrane disruption, increased activity of vacuolar processing enzyme, vacuolar membrane collapse, and formation of punctate F-actin foci were each evident in these cells. Transmission electron microscopy revealed that macroautophagic structures formed and tonoplasts ruptured in these cells. The number of cells that contained monodansylcadaverine (MDC)-stained structures and the abundance of nine autophagy-related gene transcripts increased just before cell death at 28 °C; these features were not evident at 36 °C. We assessed whether an autophagic inhibitor, wortmannin (WM), influenced lethality in hybrid cells. After the hybrid cell began to die, WM suppressed increases in ion leakage and cell deaths, and it decreased the number of cells containing MDC-stained structures. These results showed that several features indicative of autophagy and vacuolar cell death were evident in the hybrid tobacco cells subject to lethality. In addition, we documented a detailed time course of these vacuolar cell death features.

  20. Expression of FADD and cFLIPL balances mitochondrial integrity and redox signaling to substantiate apoptotic cell death.

    PubMed

    Ranjan, Kishu; Pathak, Chandramani

    2016-11-01

    FADD and cFLIP both are pivotal components of death receptor signaling. The cellular signaling of apoptosis accomplished with death receptors and mitochondria follows independent pathways for cell death. FADD and cFLIP both have an important role in the regulation of apoptotic and non-apoptotic functions. Dysregulated expression of FADD and cFLIP is associated with resistance to apoptosis in cancer cells. Mitochondria are known to play critical role in maintaining cellular respiration and homeostasis in the cells as well as transduces various signals to determine the fate of cell death. However, involvement of FADD and cFLIP in regulation of mitochondrial integrity and programmed cell death signaling to define the fate of cells remains elusive. In the present study, we explored that, induced expression of FADD challenges the mitochondrial integrity and pulverizes the membrane potential by altering the expression of Bcl-2 and cytochrome c. In contrast, mutant of FADD was unable to affect the mitochondrial integrity. Interestingly, expression of FADD and cFLIP helps to balance redox potential by regulating the anti-oxidant levels. Further, we noticed that, knockdown of cFLIPL and induced expression of FADD rapidly accumulate intracellular ROS accompanied by JNK1 activation to substantiate apoptosis. Notably, the ectopic expression of cFLIPL resists the sensitivity of cancer cells against apoptosis inducers Etoposide and HA14-1. Altogether, our findings suggest that FADD and cFLIPL are important modulators of mitochondrial-associated apoptosis apart from the death receptor signaling.

  1. The expression of Death Inducer-Obliterator (DIDO) variants in Myeloproliferative Neoplasms.

    PubMed

    Berzoti-Coelho, Maria Gabriela; Ferreira, Aline Fernanda; de Souza Nunes, Natalia; Pinto, Mariana Tomazini; Júnior, Maurício Cristiano Rocha; Simões, Belinda Pinto; Martínez-A, Carlos; Souto, Elizabeth Xisto; Panepucci, Rodrigo Alexandre; Covas, Dimas Tadeu; Kashima, Simone; Castro, Fabíola Attié

    2016-07-01

    Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation without cell maturation impairment. CML pathogenesis is associated with the Ph chromosome leading to BCR-ABL tyrosine-kinase constitutive expression. The Ph negative MPN (PV, ET and PMF) are characterized by the mutation JAK2(V617F) of the JAK2 protein in the auto-inhibitory JH2 domain, which is found in most PV patients and in approximately half of ET and PMF patients. Considerable effort is being made to understand the role of JAK2(V617F) at the MPN initiation and to clarify the pathogenesis and apoptosis resistance in CML, PV, ET and PMF patients. In the present investigation, we evaluated the Death Inducer-Obliterator (DIDO) (variants DIDO 1, 2 and 3) levels in CML, PV, ET and PMF patients. Our data reported the DIDO 1, 2 and 3 differential expressions in Myeloproliferative Neoplasms. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana

    PubMed Central

    Pitino, Marco; Armstrong, Cheryl M.; Cano, Liliana M.; Duan, Yongping

    2016-01-01

    Candidatus Liberibacter asiaticus “Las” is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta. PMID:27458468

  3. Expression of programmed cell death 1 and programmed cell death ligand 1 in extranodal NK/T-cell lymphoma, nasal type.

    PubMed

    Jo, Jae-Cheol; Kim, Misung; Choi, Yunsuk; Kim, Hyun-Jung; Kim, Ji Eun; Chae, Seoung Wan; Kim, Hawk; Cha, Hee Jeong

    2017-01-01

    Programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) are new therapeutic targets in cancer immunotherapy. The aim of this study was to investigate the clinicopathological characteristics of PD-1 and PD-L1 expression in extranodal natural killer/T‑cell lymphoma, nasal type (ENKTL). We performed PD-1 and PD-L1 immunostaining in 79 ENKTL biopsy samples and retrospectively analyzed medical records of all 79 patients from four tertiary referral hospitals. The expression of PD-1 and PD-L1 by tumor cells and/or infiltrating immune cells was evaluated. The expression rates of PD-L1 in tumor cells and infiltrating immune cells were 79.7 and 78.5 %, respectively, whereas PD-1 in tumor cells and infiltrating immune cells were 1.3 and 11.4 %. The PD-L1 positivity in tumor cells and infiltrating immune cells was significantly associated with low international prognostic index (IPI) (P = 0.044 and 0.037, respectively). Patients with normal range of serum lactate dehydrogenase demonstrated a significantly higher PD-L1 positivity in tumor cells (P = 0.020). PD-L1-positive patients had a trend toward better overall survival compared with that in patients with PD-L1-negative in tumor cells and infiltrating immune cells (P = 0.498 and 0.435, respectively). The expression rate of PD-L1 was up to 79.7 % in ENKTL, while PD-1 expression rate was very low. This is the first report describing the clinicopathological features and survival outcome according to expression of PD-1 and PD-L1 in ENKTL.

  4. Galectin-3 expression in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia, and its inhibition by hypothermia.

    PubMed

    Satoh, Kunio; Niwa, Masayuki; Goda, Wael; Binh, Nguyen Huy; Nakashima, Masaya; Takamatsu, Manabu; Hara, Akira

    2011-03-25

    The ischemic damage in the hippocampal CA1 sector following transient ischemia, delayed neuronal death, is a typical apoptosis, but the mechanism underlying the delayed neuronal death is still far from fully understood. Galectin-3 is a β-galactosidase-binding lectin which is important in cell proliferation and apoptotic regulation. Galectin-3 is expressed by microglial cells in experimental models of adult stroke. It has been reported that activated microglial cells are widely observed in the brain, including in the hippocampal CA1 region after transient ischemic insult. In the present study, time course expression of galectin-3 following transient forebrain ischemia in gerbils was examined by immunohistochemistry, combined with Iba-1 immunostaining (a specific microglial cell marker), hematoxylin and eosin staining (for morphological observation), and in situ terminal dUTP-biotin nick end labeling of DNA fragments method (for determination of cell death). Following transient ischemia, we observed a transient increase of galectin-3 expression in CA1 region, which was maximal 96h after reperfusion. Galectin-3 expression was predominately localized within CA1 region and observed only in cells which expressed Iba-1. The galectin-3-positive microglial cells emerge after the onset of neuronal cell damage. Expressions of galectin-3 and Iba-1 were strongly reduced by hypothermia during ischemic insult. Prevention of galectin-3 and Iba-1 expression in microglia by hypothermia has led us to propose that hypothermia either inhibits microglial activation or prevents delayed neuronal death itself. Our results indicate that galectin-3 might exert its effect by modulating the neuronal damage in delayed neuronal death.

  5. Dinitro-o-cresol induces apoptosis-like cell death but not alternative oxidase expression in soybean cells.

    PubMed

    Aranha, Márcia M; Matos, Ana R; Teresa Mendes, Ana; Vaz Pinto, Vera; Rodrigues, Cecília M P; Arrabaça, João D

    2007-06-01

    In plants, programmed cell death is thought to be activated during differentiation and in response to biotic and abiotic stresses. Although its mechanisms are far less clear, several morphological and biochemical features have been described in different experimental systems, including DNA laddering and cytosolic protease activation. Moreover, plant mitochondria have an alternative terminal oxidase (AOX), which is thought to be involved in protection against increased reactive oxygen species production, perhaps representing a mechanism to prevent programmed cell death. In this study, we analysed cell death induced by the herbicide dinitro-o-cresol (DNOC) in soybean (Glycine max) suspension cell cultures and evaluated biochemical and molecular events associated with programmed cell death. AOX capacity and expression were also determined. DNOC-treated cells showed fragmented nuclear DNA as assessed by an in situ assay that detects 3'-OH ends. In addition, specific colorimetric assays and immunoblot analysis revealed activation of caspase-3-like proteins and release of cytochrome c from mitochondria, respectively, confirming the apoptotic-like phenotype. Surprisingly, AOX capacity and protein levels decreased in DNOC-treated cells, suggesting no association between cell death and AOX under these experimental conditions. In conclusion, the results show that DNOC induces programmed cell death in soybean cells, suggesting that plants and animals might share similar pathways. Further, the role of AOX in cell death has not been confirmed, and may depend on the nature and intensity of stress conditions.

  6. Roles of Defense Hormones in the Regulation of Ozone-Induced Changes in Gene Expression and Cell Death.

    PubMed

    Xu, Enjun; Vaahtera, Lauri; Brosché, Mikael

    2015-12-07

    Apoplast, the diffusional space between plant cell plasma membranes, is an important medium for signaling within and between the cells. Apoplastic reactive oxygen species (ROS) are crucial signaling molecules in various biological processes. ROS signaling is interconnected with the response to several hormones, including jasmonic acid (JA), salicylic acid (SA) and ethylene. Using ozone (O3) to activate apoplastic ROS signaling, we performed global and targeted analysis of transcriptional changes and cell death assays to dissect the contribution of hormone signaling and various transcription factors (TFs) in the regulation of gene expression and cell death. The contributions of SA, JA, and ethylene were assessed through analysis of single, double, and triple mutants deficient in biosynthesis or signaling for all three hormones. Even in the triple mutant, the global gene expression responses to O3 were mostly similar to the wild-type. Cell death in the JA receptor mutant coi1-16 was suppressed by impairment of the NADPH oxidase RBOHF, suggesting a role for a ROS signal in limiting the spread of cell death. In response to apoplastic ROS, there is not a single signaling pathway that regulates gene expression or cell death. Instead, several pathways regulate the apoplastic ROS response via combinatorial or overlapping mechanisms. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  7. Effect of medium/ω-6 long chain triglyceride-based emulsion on leucocyte death and inflammatory gene expression

    PubMed Central

    Cury-Boaventura, M F; Gorjão, R; Martins de Lima, T; Fiamoncini, J; Godoy, A B P; Deschamphs, F C; Soriano, F G; Curi, R

    2011-01-01

    Lipid emulsion (LE) containing medium/ω-6 long chain triglyceride-based emulsion (MCT/ω-6 LCT LE) has been recommended in the place of ω-6 LCT-based emulsion to prevent impairment of immune function. The impact of MCT/ω-6 LCT LE on lymphocyte and neutrophil death and expression of genes related to inflammation was investigated. Seven volunteers were recruited and infusion of MCT/ω-6 LCT LE was performed for 6 h. Four volunteers received saline and no change was found. Blood samples were collected before, immediately afterwards and 18 h after LE infusion. Lymphocytes and neutrophils were studied immediately after isolation and after 24 and 48 h in culture. The following determinations were carried out: plasma-free fatty acids, triacylglycerol and cholesterol concentrations, plasma fatty acid composition, neutral lipid accumulation in lymphocytes and neutrophils, signs of lymphocyte and neutrophil death and lymphocyte expression of genes related to inflammation. MCT/ω-6 LCT LE induced lymphocyte and neutrophil death. The mechanism for MCT/ω-6 LCT LE-dependent induction of leucocyte death may involve changes in neutral lipid content and modulation of expression of genes related to cell death, proteolysis, cell signalling, inflammatory response, oxidative stress and transcription. PMID:21682721

  8. Host programmed death ligand 1 is dominant over programmed death ligand 2 expression in regulating graft-versus-host disease lethality

    PubMed Central

    Saha, Asim; Aoyama, Kazutoshi; Taylor, Patricia A.; Koehn, Brent H.; Veenstra, Rachelle G.; Panoskaltsis-Mortari, Angela; Munn, David H.; Murphy, William J.; Azuma, Miyuki; Yagita, Hideo; Fife, Brian T.; Sayegh, Mohammed H.; Najafian, Nader; Socie, Gerard; Ahmed, Rafi; Freeman, Gordon J.; Sharpe, Arlene H.

    2013-01-01

    Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, play an important role in the maintenance of peripheral tolerance. We explored the role of PD-1 ligands in regulating graft-versus-host disease (GVHD). Both PD-L1 and PD-L2 expression were upregulated in the spleen, liver, colon, and ileum of GVHD mice. Whereas PD-L2 expression was limited to hematopoietic cells, hematopoietic and endothelial cells expressed PD-L1. PD-1/PD-L1, but not PD-1/PD-L2, blockade markedly accelerated GVHD-induced lethality. Chimera studies suggest that PD-L1 expression on host parenchymal cells is more critical than hematopoietic cells in regulating acute GVHD. Rapid mortality onset in PD-L1-deficient hosts was associated with increased gut T-cell homing and loss of intestinal epithelial integrity, along with increased donor T-cell proliferation, activation, Th1 cytokine production, and reduced apoptosis. Bioenergetics profile analysis of proliferating alloreactive donor T-cells demonstrated increased aerobic glycolysis and oxidative phosphorylation in PD-L1-deficient hosts. Donor T-cells exhibited a hyperpolarized mitochondrial membrane potential, increased superoxide production, and increased expression of a glucose transporter in PD-L1-deficient hosts. Taken together, these data provide new insight into the differential roles of host PD-L1 and PD-L2 and their associated cellular and metabolic mechanisms controlling acute GVHD. PMID:24030385

  9. Catching up with solid tumor oncology: what is the evidence for a prognostic role of programmed cell death-ligand 1/programmed cell death-1 expression in B-cell lymphomas?

    PubMed Central

    McClanahan, Fabienne; Sharp, Thomas G.; Gribben, John G.

    2016-01-01

    Therapeutic strategies targeting the programmed cell death-ligand 1/programmed cell death-1 pathway have shown significant responses and good tolerability in solid malignancies. Although preclinical studies suggest that inhibiting programmed cell death-ligand 1/programmed cell death-1 interactions might also be highly effective in hematological malignancies, remarkably few clinical trials have been published. Determining patients who will benefit most from programmed cell death-ligand 1/programmed cell death-1-directed immunotherapy and whether programmed cell death-ligand 1/programmed cell death-1 are adequate prognostic markers becomes an increasingly important clinical question, especially as aberrant programmed cell death-ligand 1/programmed cell death-1 expression are key mediators of impaired anti-tumor immune responses in a range of B-cell lymphomas. Herein, we systematically review the published literature on the expression and prognostic value of programmed cell death-ligand 1/programmed cell death-1 in these patients and identify considerable differences in expression patterns, distribution and numbers of programmed cell death-ligand 1+/programmed cell death-1+cells, both between and within lymphoma subtypes, which is reflected in conflicting findings regarding the prognostic value of programmed cell death-ligand 1+/programmed cell death-1+ cells. This can be partly explained by differences in methodologies (techniques, protocols, cutoff values) and definitions of positivity. Moreover, lymphomagenesis, disease progression, and prognosis appear to be determined not only by the presence, numbers and distribution of specific subtypes of T cells, but also by other cells and additional immune checkpoints. Collectively, our findings indicate that programmed cell death-ligand 1/programmed cell death-1 interactions play an essential role in B-cell lymphoma biology and are of clinical importance, but that the overall outcome is determined by additional components

  10. Disseminated cysticercosis: clinical spectrum, Toll-like receptor-4 gene polymorphisms and role of albendazole

    PubMed Central

    Qavi, Abdul; Garg, Ravindra Kumar; Malhotra, Hardeep Singh; Jain, Amita; Kumar, Neeraj; Malhotra, Kiran Preet; Srivastava, Pradeep Kumar; Verma, Rajesh; Sharma, Praveen Kumar

    2016-01-01

    Abstract In this study, we describe clinical and imaging spectrum, and the natural course of patients with disseminated cysticercosis. How albendazole affects the course of disease has also been evaluated. We assessed the Toll-like receptor-4 gene polymorphisms, to know the reason for the apparently higher prevalence of disseminated cysticercosis in India. Sixty consecutive patients with disseminated cysticercosis were enrolled. Sixty age-and-sex-matched healthy controls were also enrolled for the purpose of genetic study. Twenty patients, who gave consent, were treated with albendazole along with corticosteroids. Forty patients did not give consent for antiparasitic therapy. Assessment for Toll-like receptor-4 gene polymorphisms (Asp299Gly and Thr399Ile genes) was done. Patients were followed for 6 months. We also performed a literature search of cases published in English language using PubMed electronic database and analyzed 56 cases thus available. There was an increased risk (6.63 fold and 4.61 fold) of disseminated cysticercosis in the presence of Asp299Gly and Thr399Ile polymorphisms in Toll-like receptor-4, respectively. The allelic frequency of Gly (11% vs. 3%, P = 0.024, odds ratio [OR] = 3.52) and Ile alleles (11% vs. 2%, P = 0.009, OR = 4.738) in disseminated cysticercosis was high. Albendazole resulted in complete disappearance of all cerebral lesions in 35% (7/20) patients and reduction in lesion load in remaining 65% (13/20) patients. No significant change in number of cysticercal lesion was noted in patients who did not receive albendazole. No major adverse reaction following antiparasitic treatment was noted. Three deaths were recorded in patients who did not receive antiparasitic treatment. Of the 56 cases reported in PubMed, 33 patients received antiparasitic treatment with follow-up data available for 31 patients. Most (24) of these patients received albendazole. A significant clinical and/or imaging improvements, on follow up, were observed in

  11. Astrocytes expressing mutant SOD1 and TDP43 trigger motoneuron death that is mediated via sodium channels and nitroxidative stress

    PubMed Central

    Rojas, Fabiola; Cortes, Nicole; Abarzua, Sebastian; Dyrda, Agnieszka; van Zundert, Brigitte

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by dysfunction and degeneration of motor neurons. Multiple disease-causing mutations, including in the genes for SOD1 and TDP-43, have been identified in ALS. Astrocytes expressing mutant SOD1 are strongly implicated in the pathogenesis of ALS: we have shown that media conditioned by astrocytes carrying mutant SOD1G93A contains toxic factor(s) that kill motoneurons by activating voltage-sensitive sodium (Nav) channels. In contrast, a recent study suggests that astrocytes expressing mutated TDP43 contribute to ALS pathology, but do so via cell-autonomous processes and lack non-cell-autonomous toxicity. Here we investigate whether astrocytes that express diverse ALS-causing mutations release toxic factor(s) that induce motoneuron death, and if so, whether they do so via a common pathogenic pathway. We exposed primary cultures of wild-type spinal cord cells to conditioned medium derived from astrocytes (ACM) that express SOD1 (ACM-SOD1G93A and ACM-SOD1G86R) or TDP43 (ACM-TDP43A315T) mutants; we show that such exposure rapidly (within 30–60 min) increases dichlorofluorescein (DCF) fluorescence (indicative of nitroxidative stress) and leads to extensive motoneuron-specific death within a few days. Co-application of the diverse ACMs with anti-oxidants Trolox or esculetin (but not with resveratrol) strongly improves motoneuron survival. We also find that co-incubation of the cultures in the ACMs with Nav channel blockers (including mexiletine, spermidine, or riluzole) prevents both intracellular nitroxidative stress and motoneuron death. Together, our data document that two completely unrelated ALS models lead to the death of motoneuron via non-cell-autonomous processes, and show that astrocytes expressing mutations in SOD1 and TDP43 trigger such cell death through a common pathogenic pathway that involves nitroxidative stress, induced at least in part by Nav channel activity. PMID:24570655

  12. Melatonin enhances arsenic trioxide-induced cell death via sustained upregulation of Redd1 expression in breast cancer cells.

    PubMed

    Yun, Sun-Mi; Woo, Sang Hyeok; Oh, Sang Taek; Hong, Sung-Eun; Choe, Tae-Boo; Ye, Sang-Kyu; Kim, Eun-Kyu; Seong, Min Ki; Kim, Hyun-A; Noh, Woo Chul; Lee, Jin Kyung; Jin, Hyeon-Ok; Lee, Yun-Han; Park, In-Chul

    2016-02-15

    Melatonin is implicated in various physiological functions, including anticancer activity. However, the mechanism(s) of its anticancer activity is not well understood. In the present study, we investigated the combined effects of melatonin and arsenic trioxide (ATO) on cell death in human breast cancer cells. Melatonin enhanced the ATO-induced apoptotic cell death via changes in the protein levels of Survivin, Bcl-2, and Bax, thus affecting cytochrome c release from the mitochondria to the cytosol. Interestingly, we found that the cell death induced by co-treatment with melatonin and ATO was mediated by sustained upregulation of Redd1, which was associated with increased production of reactive oxygen species (ROS). Combined treatment with melatonin and ATO induced the phosphorylation of JNK and p38 MAP kinase downstream from Redd1 expression. Rapamycin and S6K1 siRNA enhanced, while activation of mTORC1 by transfection with TSC2 siRNA suppressed the cell death induced by melatonin and ATO treatment. Taken together, our findings suggest that melatonin enhances ATO-induced apoptotic cell death via sustained upregulation of Redd1 expression and inhibition of mTORC1 upstream of the activation of the p38/JNK pathways in human breast cancer cells.

  13. Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death

    PubMed Central

    1996-01-01

    Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point. PMID:8642331

  14. Protective Effect of Ginsenosides Rg1 and Re on Lipopolysaccharide-Induced Sepsis by Competitive Binding to Toll-Like Receptor 4

    PubMed Central

    Su, Fei; Xue, Yin; Wang, Yuemin; Zhang, Lili; Chen, Wangxue

    2015-01-01

    We previously demonstrated that ginsenosides Rg1 and Re enhanced the immune response in C3H/HeB mice but not in C3H/HeJ mice carrying a mutation in the Tlr4 gene. The results of the present study showed that both Rg1 and Re inhibited mRNA expression and production of proinflammatory mediators that included tumor necrosis factor α, interleukin-1β, interleukin-6, cyclooxygenase-2, and inducible nitric oxide synthase from lipopolysaccharide (LPS)-stimulated macrophages. Rg1 was found to be distributed both extracellularly and intracellularly but Re was located only extracellularly to compete with LPS for binding to Toll-like receptor 4. Preinjection of Rg1 and Re into rats suppressed LPS-induced increases in body temperature, white blood cell counts, and levels of serum proinflammatory mediators. Preinjection of Rg1 and Re into mice prevented the LPS-induced decreases in total white blood cell counts and neutrophil counts, inhibited excessive expression of multiple proinflammatory mediators, and successfully rescued 100% of the mice from sepsis-associated death. More significantly, when administered after lethal LPS inoculation, Rg1, but not Re, still showed a potent antisepsis effect and protected 90% of the mice from death. The better protection efficacy of Rg1 could result from its intracellular distribution, suggesting that Rg1 may be an ideal antisepsis agent. PMID:26149990

  15. Protective effect of ginsenosides Rg1 and Re on lipopolysaccharide-induced sepsis by competitive binding to Toll-like receptor 4.

    PubMed

    Su, Fei; Xue, Yin; Wang, Yuemin; Zhang, Lili; Chen, Wangxue; Hu, Songhua

    2015-09-01

    We previously demonstrated that ginsenosides Rg1 and Re enhanced the immune response in C3H/HeB mice but not in C3H/HeJ mice carrying a mutation in the Tlr4 gene. The results of the present study showed that both Rg1 and Re inhibited mRNA expression and production of proinflammatory mediators that included tumor necrosis factor α, interleukin-1β, interleukin-6, cyclooxygenase-2, and inducible nitric oxide synthase from lipopolysaccharide (LPS)-stimulated macrophages. Rg1 was found to be distributed both extracellularly and intracellularly but Re was located only extracellularly to compete with LPS for binding to Toll-like receptor 4. Preinjection of Rg1 and Re into rats suppressed LPS-induced increases in body temperature, white blood cell counts, and levels of serum proinflammatory mediators. Preinjection of Rg1 and Re into mice prevented the LPS-induced decreases in total white blood cell counts and neutrophil counts, inhibited excessive expression of multiple proinflammatory mediators, and successfully rescued 100% of the mice from sepsis-associated death. More significantly, when administered after lethal LPS inoculation, Rg1, but not Re, still showed a potent antisepsis effect and protected 90% of the mice from death. The better protection efficacy of Rg1 could result from its intracellular distribution, suggesting that Rg1 may be an ideal antisepsis agent. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Expression of TRAIL and death receptor DR4 in Palmer type 2 TFCC lesions.

    PubMed

    Unglaub, Frank; Thomas, Susanne B; Kroeber, Markus W; Dragu, Adrian; Fellenberg, Jörg; Mittlmeier, Thomas; Wolf, Maya B; Horch, Raymund E

    2010-10-01

    Degenerative articular disc perforations of the triangular fibrocartilage (TFC) of the wrist are characterized by fibrocartilage cell loss and are often associated with ulna-plus situations. Apoptosis has been found to play a crucial role in fibrocartilage cell loss, however, the molecular mechanism and mediators are still poorly understood. The purpose of this study was to identify receptors to apoptosis in degenerative disc lesions. Included in the study were 17 patients with degenerative articular disc tears of the TFC (Palmer type 2C). Following arthroscopic debridement of the TFC, histological sections were examined to assess the presence of apoptosis. Apoptosis was determined using TRAIL and death receptor DR4 agonists for immunohistochemical analyses. The number of cells positive for apoptosis was then correlated with ulna length. Cells positive for TRAIL and DR4 were found in all specimens. The number of cells positive for TRAIL was significantly increased in specimens of patients with an ulna positive variance (P = 0.040). However, DR4 was not significantly increased in ulna plus (P > 0.05). Both, TRAIL and DR4 positive cells were found to be evenly distributed throughout each specimen. There was no accumulation of any type of cells in any particular zone of the biopsies. This is the first study that shows that TFCC cells express TRAIL and DR4, which suggests that apoptosis, as well as, mechanical trauma are involved in the development of disc perforation. The TRAIL/DR4 receptor system is a molecular mediator of apoptosis induction in TFC cells and therefore plays a role in cell loss in degenerative disc lesions.

  17. Targeting breast to brain metastatic tumours with death receptor ligand expressing therapeutic stem cells.

    PubMed

    Bagci-Onder, Tugba; Du, Wanlu; Figueiredo, Jose-Luiz; Martinez-Quintanilla, Jordi; Shah, Khalid

    2015-06-01

    Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study, we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging, we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next, we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain, we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective, potent and secretable variant of a TRAIL, S-TRAIL, and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore, the incorporation of pro-drug converting enzyme, herpes simplex virus thymidine kinase, into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications. © The Author (2015). Published by Oxford University Press on

  18. Osteopontin Expression in the Brain Triggers Localized Inflammation and Cell Death When Immune Cells Are Activated by Pertussis Toxin

    PubMed Central

    Marcondes, Maria Cecilia Garibaldi; Ojakian, Ryan; Bortell, Nikki; Flynn, Claudia; Conti, Bruno; Fox, Howard S.

    2014-01-01

    Upregulation of osteopontin (OPN) is a characteristic of central nervous system pathologies. However, the role of OPN in inflammation is still controversial, since it can both prevent cell death and induce the migration of potentially damaging inflammatory cells. To understand the role of OPN in inflammation and cell survival, we expressed OPN, utilizing an adenoviral vector, in the caudoputamen of mice deficient in OPN, using beta-galactosidase- (β-gal-) expressing vector as control. The tissue pathology and the expression of proinflammatory genes were compared in both treatments. Interestingly, inflammatory infiltrate was only found when the OPN-vector was combined with a peripheral treatment with pertussis toxin (Ptx), which activated peripheral cells to express the OPN receptor CD44v6. Relative to β-gal, OPN increased the levels of inflammatory markers, including IL13Rα1, CXCR3, and CD40L. In Ptx-treated OPN KOs, apoptotic TUNEL+ cells surrounding the OPN expression site increased, compared to β-gal. Together, these results show that local OPN expression combined with a peripheral inflammatory stimulus, such as Ptx, may be implicated in the development of brain inflammation and induction of cell death, by driving a molecular pattern characteristic of cytotoxicity. These are characteristics of inflammatory pathologies of the CNS in which OPN upregulation is a hallmark. PMID:25525298

  19. T-cell infiltration and clonality correlate with programmed cell death protein 1 and programmed death-ligand 1 expression in patients with soft tissue sarcomas.

    PubMed

    Pollack, Seth M; He, Qianchuan; Yearley, Jennifer H; Emerson, Ryan; Vignali, Marissa; Zhang, Yuzheng; Redman, Mary W; Baker, Kelsey K; Cooper, Sara; Donahue, Bailey; Loggers, Elizabeth T; Cranmer, Lee D; Spraker, Matthew B; Seo, Y David; Pillarisetty, Venu G; Ricciotti, Robert W; Hoch, Benjamin L; McClanahan, Terrill K; Murphy, Erin; Blumenschein, Wendy M; Townson, Steven M; Benzeno, Sharon; Riddell, Stanley R; Jones, Robin L

    2017-09-01

    Patients with metastatic sarcomas have poor outcomes and although the disease may be amenable to immunotherapies, information regarding the immunologic profiles of soft tissue sarcoma (STS) subtypes is limited. The authors identified patients with the common STS subtypes: leiomyosarcoma, undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS), well-differentiated/dedifferentiated liposarcoma, and myxoid/round cell liposarcoma. Gene expression, immunohistochemistry for programmed cell death protein (PD-1) and programmed death-ligand 1 (PD-L1), and T-cell receptor Vβ gene sequencing were performed on formalin-fixed, paraffin-embedded tumors from 81 patients. Differences in liposarcoma subsets also were evaluated. UPS and leiomyosarcoma had high expression levels of genes related to antigen presentation and T-cell infiltration. UPS were found to have higher levels of PD-L1 (P≤.001) and PD-1 (P≤.05) on immunohistochemistry and had the highest T-cell infiltration based on T-cell receptor sequencing, significantly more than SS, which had the lowest (P≤.05). T-cell infiltrates in UPS also were more oligoclonal compared with SS and liposarcoma (P≤.05). A model adjusted for STS histologic subtype found that for all sarcomas, T-cell infiltration and clonality were highly correlated with PD-1 and PD-L1 expression levels (P≤.01). In the current study, the authors provide the most detailed overview of the immune microenvironment in sarcoma subtypes to date. UPS, which is a more highly mutated STS subtype, provokes a substantial immune response, suggesting that it may be well suited to treatment with immune checkpoint inhibitors. The SS and liposarcoma subsets are less mutated but do express immunogenic self-antigens, and therefore strategies to improve antigen presentation and T-cell infiltration may allow for successful immunotherapy in patients with these diagnoses. Cancer 2017;123:3291-304. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc

  20. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death.

    PubMed

    Guida, Natascia; Laudati, Giusy; Anzilotti, Serenella; Secondo, Agnese; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2015-11-01

    Resveratrol (3,5,4'-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Neurotropin suppresses inflammatory cytokine expression and cell death through suppression of NF-κB and JNK in hepatocytes.

    PubMed

    Zhang, Bi; Roh, Yoon Seok; Liang, Shuang; Liu, Cheng; Naiki, Mitsuru; Masuda, Koichi; Seki, Ekihiro

    2014-01-01

    Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.

  2. Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia

    PubMed Central

    Liu, Hui; Li, Lu; Meng, Haitao; Qian, Qijun

    2013-01-01

    An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis. PMID:23765161

  3. Cell death processes during expression of hybrid lethality in interspecific F1 hybrid between Nicotiana gossei Domin and Nicotiana tabacum.

    PubMed

    Mino, Masanobu; Maekawa, Kenji; Ogawa, Ken'ichi; Yamagishi, Hiroshi; Inoue, Masayoshi

    2002-12-01

    Hybrid lethality, a type of reproductive isolation, is a genetically controlled event appearing at the seedling stage in interspecific hybrids. We characterized the lethality of F(1) hybrid seedlings from Nicotiana gossei Domin and Nicotiana tabacum cv Bright-Yellow 4 using a number of traits including growth rate, microscopic features of tissues and cells, ion leakage, DNA degradation, reactive oxygen intermediates including superoxide radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and expression of stress response marker genes. Lethal symptoms appeared at 4 d after germination in the basal hypocotyl and extended toward both the hypocotyl and root of the plants grown at 26 degrees C. Microscopic analysis revealed a prompt lysis of cell components during cell death. Membrane disruption and DNA degradation were found in the advanced stage of the lethality. The death of mesophyll cells in the cotyledon was initiated by the vascular bundle, suggesting that a putative factor inducing cell death diffused into surrounding cells from the vascular tissue. In contrast, these symptoms were not observed in the plants grown at 37 degrees C. Seedlings grown at 26 degrees C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37 degrees C. A number of stress response marker genes were expressed at 26 degrees C but not at 37 degrees C. We proposed that a putative death factor moving systemically through the vascular system induced a prompt and successive lysis of the cytoplasm of cells and that massive cell death eventually led to the loss of the hybrid plant.

  4. Cell Death Processes during Expression of Hybrid Lethality in Interspecific F1 Hybrid between Nicotiana gossei Domin and Nicotiana tabacum

    PubMed Central

    Mino, Masanobu; Maekawa, Kenji; Ogawa, Ken'ichi; Yamagishi, Hiroshi; Inoue, Masayoshi

    2002-01-01

    Hybrid lethality, a type of reproductive isolation, is a genetically controlled event appearing at the seedling stage in interspecific hybrids. We characterized the lethality of F1 hybrid seedlings from Nicotiana gossei Domin and Nicotiana tabacum cv Bright-Yellow 4 using a number of traits including growth rate, microscopic features of tissues and cells, ion leakage, DNA degradation, reactive oxygen intermediates including superoxide radical (O2−) and hydrogen peroxide (H2O2), and expression of stress response marker genes. Lethal symptoms appeared at 4 d after germination in the basal hypocotyl and extended toward both the hypocotyl and root of the plants grown at 26°C. Microscopic analysis revealed a prompt lysis of cell components during cell death. Membrane disruption and DNA degradation were found in the advanced stage of the lethality. The death of mesophyll cells in the cotyledon was initiated by the vascular bundle, suggesting that a putative factor inducing cell death diffused into surrounding cells from the vascular tissue. In contrast, these symptoms were not observed in the plants grown at 37°C. Seedlings grown at 26°C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37°C. A number of stress response marker genes were expressed at 26°C but not at 37°C. We proposed that a putative death factor moving systemically through the vascular system induced a prompt and successive lysis of the cytoplasm of cells and that massive cell death eventually led to the loss of the hybrid plant. PMID:12481061

  5. Assessment of programmed death-ligand 1 expression and tumor-associated immune cells in pediatric cancer tissues.

    PubMed

    Majzner, Robbie G; Simon, Jason S; Grosso, Joseph F; Martinez, Daniel; Pawel, Bruce R; Santi, Mariarita; Merchant, Melinda S; Geoerger, Birgit; Hezam, Imene; Marty, Virginie; Vielh, Phillippe; Daugaard, Mads; Sorensen, Poul H; Mackall, Crystal L; Maris, John M

    2017-10-01

    Programmed death 1 (PD-1) signaling in the tumor microenvironment dampens immune responses to cancer, and blocking this axis induces antitumor effects in several malignancies. Clinical studies of PD-1 blockade are only now being initiated in pediatric patients, and little is known regarding programmed death-ligand 1 (PD-L1) expression in common childhood cancers. The authors characterized PD-L1 expression and tumor-associated immune cells (TAICs) (lymphocytes and macrophages) in common pediatric cancers. Whole slide sections and tissue microarrays were evaluated by immunohistochemistry for PD-L1 expression and for the presence of TAICs. TAICs were also screened for PD-L1 expression. Thirty-nine of 451 evaluable tumors (9%) expressed PD-L1 in at least 1% of tumor cells. The highest frequency histotypes comprised Burkitt lymphoma (80%; 8 of 10 tumors), glioblastoma multiforme (36%; 5 of 14 tumors), and neuroblastoma (14%; 17 of 118 tumors). PD-L1 staining was associated with inferior survival among patients with neuroblastoma (P = .004). Seventy-four percent of tumors contained lymphocytes and/or macrophages. Macrophages were significantly more likely to be identified in PD-L1-positive versus PD-L1-negative tumors (P < .001). A subset of diagnostic pediatric cancers exhibit PD-L1 expression, whereas a much larger fraction demonstrates infiltration with tumor-associated lymphocytes. PD-L1 expression may be a biomarker for poor outcome in neuroblastoma. Further preclinical and clinical investigation will define the predictive nature of PD-L1 expression in childhood cancers both at diagnosis and after exposure to chemoradiotherapy. Cancer 2017;123:3807-3815. © 2017 American Cancer Society. © 2017 American Cancer Society.

  6. Attitudes of Children towards Aging, the Elderly, and Death & Dying as Expressed through the Arts.

    ERIC Educational Resources Information Center

    Zaki, Gamal; Zaki, Sylvia

    The purpose of this study was to explore the conceptions, feelings and attitudes of elementary and junior high school students toward the topics of aging, the elderly, death, and dying. To gather data, an announcement was made to all schools within the state that the Rhode Island Gerontology Center would sponsor a contest for all school children…

  7. Programmed cell death ligand 1 (PD-L1) expression on gastric cancer and its relationship with clinicopathologic factors.

    PubMed

    Zhang, Lin; Qiu, Miaozhen; Jin, Ying; Ji, Jiao; Li, Baoxia; Wang, Xueping; Yan, Shumei; Xu, Ruihua; Yang, Dajun

    2015-01-01

    Targeting the immune checkpoints in solid tumors becomes hot recently. Programmed cell death ligand 1 (PD-L1) is ligand for programmed death 1 (PD-1), which is known to negatively regulate T-cell activation. In the present study, we investigated the expression of PD-L1 in tumor specimens of gastric cancer and its relationships with clinicopathological variables and survival. The expression of PD-L1 in 132 surgically resected specimens of stage II and III gastric cancer was evaluated by immunohistochemistry in microarray tissue. Expression of PD-L1 was observed in 50.8% (67/132) of gastric cancer tumor specimens. Patients whose tumor size over 5cm had a higher positive rate of PD-L1 expression. There was no relationship between the expression of PD-L1 and other clinicopathological variables including age, gender, clinical stage, location as well as histological differentiation. PD-L1 positive patients had significantly poorer survival than negative patients. The 5-year survival rates was 83.1% in those with PD-L1 negative patients and 50.7% for PD-L1 positive patients (P<0.001). The multivariate analysis indicated that both PD-L1 positive and Tumor-node-metastasis stage were independent prognostic factors in gastric cancer patients (P=0.001 and 0.025, respectively). The expression of PD-L1 was found in half of stages II and III gastric cancer patients. Positive of PD-L1 expression indicated poor survival in Chinese stages II and III gastric adenocarcinoma patients. These results may provide the clue for immunotherapy in the adjuvant treatment setting of gastric cancer patients.

  8. Influence of antiretroviral therapy on programmed death-1 (CD279) expression on T cells in lymph nodes of human immunodeficiency virus-infected individuals.

    PubMed

    Ehrhard, Simone; Wernli, Marion; Dürmüller, Ursula; Battegay, Manuel; Gudat, Fred; Erb, Peter

    2009-10-01

    Human immunodeficiency virus infection leads to T-cell exhaustion and involution of lymphoid tissue. Recently, the programmed death-1 pathway was found to be crucial for virus-specific T-cell exhaustion during human immunodeficiency virus infection. Programmed death-1 expression was elevated on human immunodeficiency virus-specific peripheral blood CD8+ and CD4+ T cells and correlated with disease severity. During human immunodeficiency infection, lymphoid tissue acts as a major viral reservoir and is an important site for viral replication, but it is also essential for regulatory processes important for immune recovery. We compared programmed death-1 expression in 2 consecutive inguinal lymph nodes of 14 patients, excised before antiretroviral therapy (antiretroviral therapy as of 1997-1999) and 16 to 20 months under antiretroviral therapy. In analogy to lymph nodes of human immunodeficiency virus-negative individuals, in all treated patients, the germinal center area decreased, whereas the number of germinal centers did not significantly change. Programmed death-1 expression was mostly found in germinal centers. The absolute extent of programmed death 1 expression per section was not significantly altered after antiretroviral therapy resulting in a significant-relative increase of programmed death 1 per shrunken germinal center. In colocalization studies, CD45R0+ cells that include helper/inducer T cells strongly expressed programmed death-1 before and during therapy, whereas CD8+ T cells, fewer in numbers, showed a weak expression for programmed death-1. Thus, although antiretroviral therapy seems to reduce the number of programmed death-1-positive CD8+ T lymphocytes within germinal centers, it does not down-regulate programmed death-1 expression on the helper/inducer T-cell subset that may remain exhausted and therefore unable to trigger immune recovery.

  9. Declines in Drp1 and parkin expression underlie DNA damage-induced changes in mitochondrial length and neuronal death.

    PubMed

    Wang, David B; Garden, Gwenn A; Kinoshita, Chizuru; Wyles, Cody; Babazadeh, Nasim; Sopher, Bryce; Kinoshita, Yoshito; Morrison, Richard S

    2013-01-23

    Maintaining proper mitochondrial length is essential for normal mitochondrial function in neurons. Mitochondrial fragmentation has been associated with neuronal cell death caused by a variety of experimental toxic stressors. Despite the fact that oxidative stress is a hallmark of neurodegenerative conditions and aging and the resulting activation of p53 is believed to contribute to the neuropathology, little is still known regarding changes in mitochondrial morphology in p53-dependent neuronal death. Therefore, we specifically addressed the relationship between genotoxic stress, p53 activation, and the regulation of mitochondrial morphology in neurons. In cultured postnatal mouse cortical neurons, treatment with the DNA-damaging agent camptothecin (CPT) resulted in elongated mitochondria, in contrast to fragmented mitochondria observed upon staurosporine and glutamate treatment. In fibroblasts, however, CPT resulted in fragmented mitochondria. CPT treatment in neurons suppressed expression of the mitochondrial fission protein Drp1 and the E3 ubiquitin ligase parkin. The presence of elongated mitochondria and the declines in Drp1 and parkin expression occurred before the commitment point for apoptosis. The CPT-induced changes in Drp1 and parkin were not observed in p53-deficient neurons, while p53 overexpression alone was sufficient to reduce the expression of the two proteins. Elevating Drp1 or parkin expression before CPT treatment enhanced neuronal viability and restored a normal pattern of mitochondrial morphology. The present findings demonstrate that genotoxic stress in neurons results in elongated mitochondria in contrast to fission induced by other forms of stress, and p53-dependent declines in Drp1 and parkin levels contribute to altered mitochondrial morphology and cell death.

  10. Expression of apoptosis and proliferating cell nuclear antigen (PCNA) in the cardiac conduction system of crib death (SIDS).

    PubMed

    Matturri, L; Ottaviani, G; Lavezzi, A M; Turconi, P; Cazzullo, A; Rossi, L

    2001-07-01

    Aim of this study is to determine the expression of apoptosis and Proliferating Cell Nuclear Antigen (PCNA) in the cardiac conduction system in crib death and explained death (ED) cases. Postnatal morphogenesis of the conducting tissue is an important part of its normal development. In the atrio-ventricular node (AVN) and His bundle (HB) it consists of degeneration, cell death and replacing in an orderly programmed way. However, its nature and its relation to crib death is not yet fully explained. Apoptosis and PCNA were investigated in 8 heart conduction systems of infants dying of crib death and in 3 conduction systems of infants dying of ED as controls. The cardiac conduction system was removed in two blocks: the first included the sino-atrial node (SAN) and the crista terminalis, the second contained the atrio-ventricular node (AVN), His bundle (HB), bifurcation, and bundle branches. In the conduction systems as well as in the common myocardium the PCNA Labeling Index (PCNA-LI) was found to be negative in all cases. The apoptotic indices (AI) in SIDS and in ED were found to have no statistically significant differences (p>0.05). The SAN, in both groups, showed an AI similar to the one detected in common myocardium. In almost all cases, TUNEL labeling was detected in peripheral region of the AVN, close to the atrial myocardium. The AI was higher in the AVN, HB and the initial tract of bundle branches than in the common myocardium (p<0.05; Student's t test).

  11. Clinical Use of Programmed Cell Death-1 and Its Ligand Expression as Discriminatory and Predictive Markers in Ovarian Cancer.

    PubMed

    Chatterjee, Jayanta; Dai, Wei; Aziz, Nor Haslinda Abd; Teo, Pei Yun; Wahba, John; Phelps, David L; Maine, Christian J; Whilding, Lynsey M; Dina, Roberto; Trevisan, Giorgia; Flower, Kirsty J; George, Andrew J T; Ghaem-Maghami, Sadaf

    2017-07-01

    Purpose: We aimed to establish whether programmed cell death-1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, in ovarian cancer tumor tissue and blood, could be used as biomarkers for discrimination of tumor histology and prognosis of ovarian cancer.Experimental Design: Immune cells were separated from blood, ascites, and tumor tissue obtained from women with suspected ovarian cancer and studied for the differential expression of possible immune biomarkers using flow cytometry. PD-L1 expression on tumor-associated inflammatory cells was assessed by immunohistochemistry and tissue microarray. Plasma soluble PD-L1 was measured using sandwich ELISA. The relationships among immune markers were explored using hierarchical cluster analyses.Results: Biomarkers from the discovery cohort that associated with PD-L1(+) cells were found. PD-L1(+) CD14(+) cells and PD-L1(+) CD11c(+) cells in the monocyte gate showed a distinct expression pattern when comparing benign tumors and epithelial ovarian cancers (EOCs)-confirmed in the validation cohort. Receiver operating characteristic curves showed PD-L1(+) and PD-L1(+) CD14(+) cells in the monocyte gate performed better than the well-established tumor marker CA-125 alone. Plasma soluble PD-L1 was elevated in patients with EOC compared with healthy women and patients with benign ovarian tumors. Low total PD-1(+) expression on lymphocytes was associated with improved survival.Conclusions: Differential expression of immunological markers relating to the PD-1/PD-L1 pathway in blood can be used as potential diagnostic and prognostic markers in EOC. These data have implications for the development and trial of anti-PD-1/PD-L1 therapy in ovarian cancer. Clin Cancer Res; 23(13); 3453-60. ©2016 AACR. ©2016 American Association for Cancer Research.

  12. TRPV channel expression in human skin and possible role in thermally induced cell death.

    PubMed

    Radtke, Christine; Sinis, Nektarios; Sauter, Michael; Jahn, Sabrina; Kraushaar, Udo; Guenther, Elke; Rodemann, H Peter; Rennekampff, Hans-Oliver

    2011-01-01

    Cell death via necrosis and apoptosis is a hallmark of deep dermal to full-thickness cutaneous burn injuries. Keratinocytes might act as thermosensory cells that transmit information regarding ambient temperature via heat-gated transient receptor potential vanilloid (TRPV) ion channels. The aim of this study was to investigate the distribution of TRPV1, 2, 3, and 4 in uninjured and thermally burned skin. The authors investigated warmth-evoked currents in keratinocytes and cell kinetics of thermally injured keratinocytes in culture with agonists and antagonists of TRPV channels. Specimens of uninjured normal skin and discarded tissue of thermally injured skin were stained for TRPV1, 2, 3, and 4. Cultured primary human keratinocytes were heated for 5 minutes at the following temperatures: 37°C (control), 42°C, and 60°C and thereafter cultured for 24 or 48 hours at 37°C. Thermally stressed cells were treated with TRPV antagonists capsazepine or ruthenium red, and cell viability capacity was determined. TRPV1, TRPV2, TRPV3, and TRPV4 immunoreactivity was differentially identified on basal and suprabasal keratinocytes of healthy human skin. Patch clamp analysis showed a functional response of human keratinocytes at temperatures >40°C. Cell death of keratinocytes after heating at 42°C was reduced by 15 and 5% with ruthenium red and by 20 and 30% by capsazepine at 24 and 48 hours, respectively. Cell death after treatment at 60°C was significantly reduced at 24 hours with capsazepine (22%) or ruthenium red (18%) but only minimally affected after 48 hours postinjury. Interaction with TRPV channels on keratinocytes may offer a new strategy to counteract cell death after thermal injury.

  13. A Greek perspective on concepts of death and expression of grief, with implications for practice.

    PubMed

    Mystakidou, Kyriaki; Tsilika, Eleni; Parpa, Efi; Katsouda, Emmanuela; Vlahos, Lambros

    2003-12-01

    Death has been conceptualised in different ways by different cultures and civilizations. It is increasingly entering into the public consciousness and society is now more ready to discuss and lessen the fear of dying and grief than it has been in the past few decades. In Greece, by Classical times there was an increase in burial rituals and commemorative practices compared to earlier periods. When Christianity was introduced into Greece it attempted to change the way the dead were mourned, preaching immortality of the soul and resurrection of the dead. Nevertheless, the way people grieve and bury their dead in Greece has not changed greatly since before the introduction of Christianity, except for the difficulty experienced in witnessing burial procedures observed in the large cities. Burial and bereavement traditions were introduced to help Greeks cope with death and bereavement. In Greece today beliefs about grief and death are based both on the ancient and the Christian Orthodox traditions. Healthcare professionals need to develop cultural competence to improve nursing and future health care. If care is culturally informed and tailored its quality is improved.

  14. Cytokines, Chaperones and Neuroinflammatory Responses in Heroin-Related Death: What Can We Learn from Different Patterns of Cellular Expression?

    PubMed Central

    Neri, Margherita; Panata, Laura; Bacci, Mauro; Fiore, Carmela; Riezzo, Irene; Turillazzi, Emanuela; Fineschi, Vittorio

    2013-01-01

    Heroin (3,6-diacetylmorphine) has various effects on the central nervous system with several neuropathological alterations including hypoxic-ischemic brain damage from respiratory depressing effects and neuroinflammatory response. Both of these mechanisms induce the release of cytokines, chemokines and other inflammatory mediators by the activation of many cell types such as leucocytes and endothelial and glial cells, especially microglia, the predominant immunocompetent cell type within the central nervous system. The aim of this study is to clarify the correlation between intravenous heroin administration in heroin related death and the neuroinflammatory response. We selected 45 cases among autopsies executed for heroin-related death (358 total cases); immunohistochemical studies and Western blotting analyses were used to investigate the expression of brain markers such as tumor necrosis factor-α, oxygen-regulated protein 150, (interleukins) IL-1β, IL-6, IL-8, IL-10, IL-15, cyclooxygenase-2, heat shock protein 70, and CD68 (MAC387). Findings demonstrated that morphine induces inflammatory response and cytokine release. In particular, oxygen-regulated protein 150, cyclooxygenase-2, heat shock protein 70, IL-6 and IL-15 cytokines were over-expressed with different patterns of cellular expression. PMID:24084728

  15. Lipophilic but not hydrophilic statins selectively induce cell death in gynaecological cancers expressing high levels of HMGCoA reductase.

    PubMed

    Kato, S; Smalley, S; Sadarangani, A; Chen-Lin, K; Oliva, B; Brañes, J; Carvajal, J; Gejman, R; Owen, G I; Cuello, M

    2010-05-01

    Recent reports have suggested that statins induce cell death in certain epithelial cancers and that patients taking statins to reduce cholesterol levels possess lower cancer incidence. However, little is known about the mechanisms of action of different statins or the effects of these statins in gynaecological malignancies. The apoptotic potential of two lipophilic statins (lovastatin and simvastatin) and one hydrophilic statin (pravastatin) was assessed in cancer cell lines (ovarian, endometrial and cervical) and primary cultured cancerous and normal tissues. Cell viability was studied by MTS assays and apoptosis was confirmed by Western blotting of PARP and flow cytometry. The expressions of key apoptotic cascade proteins were analysed. Our results demonstrate that both lovastatin and simvastatin, but not pravastatin, selectively induced cell death in dose- and time-dependent manner in ovarian, endometrial and cervical cancers. Little or no toxicity was observed with any statin on normal cells. Lipophilic statins induced activation of caspase-8 and -9; BID cleavage, cytochrome C release and PARP cleavage. Statin-sensitive cancers expressed high levels of HMG-CoA reductase compared with resistant cultures. The effect of lipophilic statins was dependent on inhibition of enzymatic activity of HMG-CoA reductase since mevalonate pre-incubation almost completely abrogated the apoptotic effect. Moreover, the apoptotic effect involved the inhibition of synthesis of geranylgeranyl pyrophosphate rather than farnesyl pyrophosphate. In conclusion, lipophilic but not hydrophilic statins induce cell death through activation of extrinsic and intrinsic apoptotic cascades in cancerous cells from the human female genital tract, which express high levels of HMG-CoA reductase. These results promote further investigation in the use of lipophilic statins as anticancer agents in gynaecological malignancies.

  16. Lipophilic but not hydrophilic statins selectively induce cell death in gynaecological cancers expressing high levels of HMGCoA reductase

    PubMed Central

    Kato, S; Smalley, S; Sadarangani, A; Chen-Lin, K; Oliva, B; Brañes, J; Carvajal, J; Gejman, R; Owen, G I; Cuello, M

    2010-01-01

    Abstract Recent reports have suggested that statins induce cell death in certain epithelial cancers and that patients taking statins to reduce cholesterol levels possess lower cancer incidence. However, little is known about the mechanisms of action of different statins or the effects of these statins in gynaecological malignancies. The apoptotic potential of two lipophilic statins (lovastatin and simvastatin) and one hydrophilic statin (pravastatin) was assessed in cancer cell lines (ovarian, endometrial and cervical) and primary cultured cancerous and normal tissues. Cell viability was studied by MTS assays and apoptosis was confirmed by Western blotting of PARP and flow cytometry. The expressions of key apoptotic cascade proteins were analysed. Our results demonstrate that both lovastatin and simvastatin, but not pravastatin, selectively induced cell death in dose- and time-dependent manner in ovarian, endometrial and cervical cancers. Little or no toxicity was observed with any statin on normal cells. Lipophilic statins induced activation of caspase-8 and -9; BID cleavage, cytochrome C release and PARP cleavage. Statin-sensitive cancers expressed high levels of HMG-CoA reductase compared with resistant cultures. The effect of lipophilic statins was dependent on inhibition of enzymatic activity of HMG-CoA reductase since mevalonate pre-incubation almost completely abrogated the apoptotic effect. Moreover, the apoptotic effect involved the inhibition of synthesis of geranylgeranyl pyrophosphate rather than farnesyl pyrophosphate. In conclusion, lipophilic but not hydrophilic statins induce cell death through activation of extrinsic and intrinsic apoptotic cascades in cancerous cells from the human female genital tract, which express high levels of HMG-CoA reductase. These results promote further investigation in the use of lipophilic statins as anticancer agents in gynaecological malignancies. PMID:19432822

  17. Expression of the Streptococcus pneumoniae yoeB chromosomal toxin gene causes cell death in the model plant Arabidopsis thaliana.

    PubMed

    Bakar, Fauziah Abu; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

    2015-04-12

    Bacterial toxin-antitoxin systems usually comprise of a pair of genes encoding a stable toxin and its cognate labile antitoxin and are located in the chromosome or in plasmids of several bacterial species. Chromosomally-encoded toxin-antitoxin systems are involved in bacterial stress responses and activation of the toxins usually leads to cell death or dormancy. Overexpression of the chromosomally-encoded YoeB toxin from the yefM-yoeB toxin-antitoxin locus of the Gram-positive bacterium Streptococcus pneumoniae has been shown to cause cell death in S. pneumoniae as well as E. coli. Induction of a YoeB-GFP fusion protein using a 17-β-estradiol-inducible plant expression system in Arabidopsis thaliana Col 0, was lethal in all T2 progeny. Examination of plants by fluorescent confocal microscopy showed GFP fluorescence in all parts of the leaves at 24 hours after 17-β-estradiol induction, continuing up to plant death. Quantitative RT-PCR analysis revealed that the expression of the yoeB toxin gene peaked at 3 days after induction with 17-β-estradiol, coinciding with the onset of visible effects on the plants. Moreover, we detected DNA laddering in the transgenic plants at 24 hours after yoeB induction, indicative of apoptosis. Expression of the YoeB toxin from Streptococcus pneumoniae is lethal in Arabidopsis. We believe this is the first report of a toxin from a bacterial toxin-antitoxin system functioning in plants. The results presented here mark an important milestone towards the development of a cell ablation based bio-containment strategy, which may be useful for functional studies and for the control of spread of transgenic plants.

  18. Fucoidan induces Toll-like receptor 4-regulated reactive oxygen species and promotes endoplasmic reticulum stress-mediated apoptosis in lung cancer.

    PubMed

    Hsu, Hsien-Yeh; Lin, Tung-Yi; Lu, Mei-Kuang; Leng, Pei-Ju; Tsao, Shu-Ming; Wu, Yu-Chung

    2017-03-23

    Fucoidan, a sulfated polysaccharide extracted from brown algae, exhibits anti-cancer activity. However, the effects and mechanism of fucoidan-induced apoptosis via endoplasmic reticulum (ER) stress is unclear. In this study, we demonstrated that fucoidan prevents tumorigenesis and reduces tumor size in LLC1-xenograft male C57BL/6 mice. Fucoidan induces an ER stress response by activating the PERK-ATF4-CHOP pathway, resulting in apoptotic cell death in vitro and in vivo. Furthermore, ATF4 knockdown abolishes fucoidan-induced CHOP expression and rescues cell viability. Specifically, fucoidan increases intracellular reactive oxygen species (ROS), which increase ATF4 and CHOP in lung cancer cells. Using the ROS scavenger N-acetyl-l-cysteine (NAC), we found that ROS generation is involved in fucoidan-induced ER stress-mediated apoptosis. Moreover, via Toll-like receptor 4 (TLR4) knockdown, we demonstrated that fucoidan-induced ROS and CHOP expression were attenuated. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan. We showed that fucoidan inhibits tumor viability by activating the TLR4/ROS/ER stress axis and the downstream PERK-ATF4-CHOP pathway, leading to apoptosis and suppression of lung cancer cell progression. Together, these results indicate that fucoidan is a potential preventive and therapeutic agent for lung cancer that acts via activation of ROS-dependent ER stress pathways.

  19. Fucoidan induces Toll-like receptor 4-regulated reactive oxygen species and promotes endoplasmic reticulum stress-mediated apoptosis in lung cancer

    PubMed Central

    Hsu, Hsien-Yeh; Lin, Tung-Yi; Lu, Mei-Kuang; Leng, Pei-Ju; Tsao, Shu-Ming; Wu, Yu-Chung

    2017-01-01

    Fucoidan, a sulfated polysaccharide extracted from brown algae, exhibits anti-cancer activity. However, the effects and mechanism of fucoidan-induced apoptosis via endoplasmic reticulum (ER) stress is unclear. In this study, we demonstrated that fucoidan prevents tumorigenesis and reduces tumor size in LLC1-xenograft male C57BL/6 mice. Fucoidan induces an ER stress response by activating the PERK-ATF4-CHOP pathway, resulting in apoptotic cell death in vitro and in vivo. Furthermore, ATF4 knockdown abolishes fucoidan-induced CHOP expression and rescues cell viability. Specifically, fucoidan increases intracellular reactive oxygen species (ROS), which increase ATF4 and CHOP in lung cancer cells. Using the ROS scavenger N-acetyl-l-cysteine (NAC), we found that ROS generation is involved in fucoidan-induced ER stress-mediated apoptosis. Moreover, via Toll-like receptor 4 (TLR4) knockdown, we demonstrated that fucoidan-induced ROS and CHOP expression were attenuated. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan. We showed that fucoidan inhibits tumor viability by activating the TLR4/ROS/ER stress axis and the downstream PERK-ATF4-CHOP pathway, leading to apoptosis and suppression of lung cancer cell progression. Together, these results indicate that fucoidan is a potential preventive and therapeutic agent for lung cancer that acts via activation of ROS-dependent ER stress pathways. PMID:28332554

  20. Toll Like Receptor 4 Affects the Cerebral Biochemical Changes Induced by MPTP Treatment.

    PubMed

    Conte, Carmela; Roscini, Luca; Sardella, Roccaldo; Mariucci, Giuseppina; Scorzoni, Stefania; Beccari, Tommaso; Corte, Laura

    2017-02-01

    The etiology and pathogenesis of Parkinson's disease (PD) are still unclear. However, multiple lines of evidence suggest a critical role of the toll like receptor 4 (TLR4) in inflammatory response and neuronal death. Neuroinflammation may be associated with the misfolding and aggregation of proteins accompanied by a change in their secondary structure. Recent findings also suggest that biochemical perturbations in cerebral lipid content could contribute to the pathogenesis of central nervous system (CNS) disorders, including PD. Thus, it is of great importance to determine the biochemical changes that occur in PD. In this respect, Fourier Transform Infrared (FTIR) spectroscopy represents a useful tool to detect molecular alterations in biological systems in response to stress stimuli. By relying upon FTIR approach, this study was designed to elucidate the potential role of TLR4 in biochemical changes induced by methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin in a mouse model of PD. The analysis of the FTIR spectra was performed in different brain regions of both wild type (WT) and toll like receptor 4-deficient (TLR4(-/-)) mice. It revealed that each brain region exhibited a characteristic molecular fingerprint at baseline, with no significant differences between genotypes. Conversely, WT and TLR4(-/-) mice showed differential biochemical response to MPTP toxicity, principally related to lipid and protein composition. These differences appeared to be characteristic for each brain area. Furthermore, the present study showed that WT mice resulted more vulnerable than TLR4(-/-) animals to striatal dopamine (DA) depletion following MPTP treatment. These results support the hypothesis of a possible involvement of TLR4 in biochemical changes occurring in neurodegeneration.

  1. Surgery increases cell death and induces changes in gene expression compared with anesthesia alone in the developing piglet brain.

    PubMed

    Broad, Kevin D; Kawano, Go; Fierens, Igor; Rocha-Ferreira, Eridan; Hristova, Mariya; Ezzati, Mojgan; Rostami, Jamshid; Alonso-Alconada, Daniel; Chaban, Badr; Hassell, Jane; Fleiss, Bobbi; Gressens, Pierre; Sanders, Robert D; Robertson, Nicola J

    2017-01-01

    In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia.

  2. Surgery increases cell death and induces changes in gene expression compared with anesthesia alone in the developing piglet brain

    PubMed Central

    Fierens, Igor; Rocha-Ferreira, Eridan; Hristova, Mariya; Ezzati, Mojgan; Rostami, Jamshid; Alonso-Alconada, Daniel; Chaban, Badr; Hassell, Jane; Fleiss, Bobbi; Gressens, Pierre; Sanders, Robert D.; Robertson, Nicola J.

    2017-01-01

    In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia. PMID:28355229

  3. O6.09PROSTAGLANDIN E RECEPTOR-4 ACTIVATION REGULATES TRYPTOPHAN METABOLISM IN HUMAN MALIGNANT GLIOMAS

    PubMed Central

    Ochs, K.; Ott, M.; Rauschenbach, K.J.; Sahm, F.; Opitz, C.A.; von Deimling, A.; Wick, W.; Platten, M.

    2014-01-01

    Malignant gliomas generate a local immunosuppressive microenvironment as well as systemic immunosuppression. Tryptophan-2,3-dioxygenase (TDO)-mediated tryptophan metabolism and the production of immunosuppressive prostaglandins relevantly contribute to this inhibition of anti-glioma immune responses. We now connect these two critical immunosuppressive pathways by demonstrating that prostaglandins enhance TDO expression and enzymatic activity in malignant gliomas via activation of prostaglandin E receptor-4 (EP4). Stimulation with prostaglandin E2 (PGE2) concentration-dependently upregulates TDO-mediated kynurenine release in human glioma cell lines, while knockdown of the PGE2 receptor EP4 inhibits TDO expression and activity. In tissue of human malignant gliomas expression of the PGE2-producing enzyme cyclooxygenase-2 (COX-2) and its receptor EP4 are associated with TDO expression both on transcript and protein level. Of clinical relevance, high expression of EP4 correlates with poor survival in patients with gliomas of the WHO grades III and IV. Importantly, treatment of glioma cells with an EP4 inhibitor decreased TDO expression and activity. In summary targeting EP4 may inhibit both immunosuppressive COX-2 signaling as well as tryptophan degradation and thus could provide a novel immunotherapeutic avenue for the treatment of malignant gliomas.

  4. Heat stress and sudden infant death syndrome--stress gene expression after exposure to moderate heat stress.

    PubMed

    Rohde, Marianne Cathrine; Corydon, Thomas Juhl; Hansen, Jakob; Pedersen, Christina Bak; Schmidt, Stinne P; Gregersen, Niels; Banner, Jytte

    2013-10-10

    The aim of the present study was to investigate stress gene expression in cultured primary fibroblasts established from Achilles tendons collected during autopsies from sudden infant death syndrome (SIDS) cases, and age-matched controls (infants dying in a traumatic event). Expression of 4 stress responsive genes, HSPA1B, HSPD1, HMOX1, and SOD2, was studied by quantitative reverse transcriptase PCR analysis of RNA purified from cells cultured under standard or various thermal stress conditions. The expression of all 4 genes was highly influenced by thermal stress in both SIDS and control cells. High interpersonal variance found in the SIDS group indicated that they represented a more heterogeneous group than controls. The SIDS group responded to thermal stress with a higher expression of the HSPA1B and HSPD1 genes compared to the control group, whereas no significant difference was observed in the expression of SOD2 and HMOX1 between the two groups. The differences were related to the heat shock treatment as none of the genes were expressed significantly different in SIDS at base levels at 37 °C. SOD2 and HMOX1 were up regulated in both groups, for SOD2 though the expression was lower in SIDS at all time points measured, and may be less related to heat stress. Being found dead in the prone position (a known risk factor for SIDS) was related to a lower HSPA1B up-regulation in SIDS compared to SIDS found on their side or back. The study demonstrates the potential usefulness of gene expression studies using cultured fibroblasts established from deceased individuals as a tool for molecular and pathological investigations in forensic and biomedical sciences. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Abnormal serotonin receptor expression in DBA/2 mice associated with susceptibility to sudden death due to respiratory arrest.

    PubMed

    Uteshev, Victor V; Tupal, Srinivasan; Mhaskar, Yashanad; Faingold, Carl L

    2010-02-01

    Previous studies indicate that DBA/2 mice may be a useful model of human sudden unexpected death in epilepsy (SUDEP), since these mice exhibit generalized convulsive seizures followed by respiratory arrest (RA). Respiratory deficits, following generalized convulsive seizures, are observed prior to SUDEP in patients. RA that occurs in DBA/2 mice following sound-induced seizures can be prevented by treatments that activate serotonin (5-HT) receptors. 5-HT receptor subtypes in brainstem respiratory centers are important in regulating normal respiration. The present study compared the expression of 5-HT subtype receptor proteins in excised brainstem tissue, containing the rostral ventral medulla respiratory region in DBA/2 mice vs. seizure-resistant C57BL/6J mice, using Western blot analysis. The results indicate that expression of specific 5-HT(2C), 5-HT(3), and 5-HT(4) receptor proteins in the brainstem tissue of DBA/2 mice is significantly diminished, while expression of the 5-HT(2B) receptors is significantly enhanced as compared to C57BL/6J mice. No difference in expression of 5-HT transporter protein is seen. These findings suggest that the DBA/2 mice are susceptible to RA, in part, because of the altered expression of 5-HT receptors. Preliminary studies indicate that 5-HT(2C) receptors may be particularly important, since a 5-HT(2C) agonist is very effective in blocking RA in DBA/2 mice.

  6. The Gfi-1 protooncoprotein represses Bax expression and inhibits T-cell death

    PubMed Central

    Grimes, H. Leighton; Gilks, C. Blake; Chan, Tung O.; Porter, Susan; Tsichlis, Philip N.

    1996-01-01

    The Gfi-1 protooncogene encodes a nuclear zinc-finger protein that carries a novel repressor domain, SNAG, and functions as a position- and orientation-independent active transcriptional repressor. The Gfi-1 repressor allows interleukin 2 (IL-2)-dependent T cells to escape G1 arrest induced by IL-2 withdrawal in culture and collaborates with c-myc and pim-1 for the induction of retrovirus-induced lymphomas in animals. Here we show that overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL-2-deficient media. Similarly, induction of Gfi-1 in primary thymocytes from mice carrying a metal-inducible Gfi-1 transgene inhibits cell death induced by cultivation in vitro. The protein and mRNA levels of the proapoptotic regulator Bax are down-regulated by Gfi-1 in both immortalized T-cell lines and primary transgenic thymocytes. The repression is direct and depends on several Gfi-1-binding sites in the p53-inducible Bax promoter. In addition to Bax, Gfi-1 also represses Bak, another apoptosis-promoting member of the Bcl-2 gene family. Therefore, Gfi-1 may inhibit apoptosis by means of its repression of multiple proapoptotic regulators. The antiapoptotic properties of Gfi-1 provide a potential explanation for its strong collaboration with c-myc during oncogenesis. PMID:8962093

  7. On the reception of the concept of the death drive in Germany: expressing and resisting an 'evil principle'?

    PubMed

    Frank, Claudia

    2015-04-01

    With Beyond the Pleasure Principle, Freud attempted 'to describe and to account for the facts of daily observation in our field of study' (1920, p. 7), in particular concerning destructive clinical phenomena that confront us in the analytic situation: traumatic neuroses, melancholic states, negative-therapeutic reactions, masochism, repetition compulsion and so on. The author demonstrates in the first section how Freud's own resistance - later self-diagnosed - to recognizing these unwelcome facts was expressed in the terminological and conceptual ambiguities of the death drive hypothesis then introduced, ambiguities that to some extent continue to impede the reception of its clinical usefulness to this day. As soon as Freud had demonstrated the connection with clinical practice more directly in The Ego and the Id (1923), some contemporaries adopted it as a helpful clinical concept, while others believed that they could (and must) refute it. The second part outlines its reception in the 1920s and 1930s, which was part of an international discussion that was, of course, initially conducted mainly in German. The beginnings of an important further development of the death drive hypothesis are described in a separate section because it originated from Melanie Klein's earliest experiences in analysing children in Berlin in the early to mid-1920s. She referred at that time to an 'evil principle', and in 1932 published her view of the death drive hypothesis, which was further developed in subsequent decades by her and her followers in London. In this period, conditions changed dramatically: in Germany Freud's books (among others) were burnt, crimes against humanity were instigated and psychoanalysis ceased to exist in this country. Almost all the analysts who published on the death drive had to emigrate. From then on, entirely different discourses took place in the various regions. In Germany, the death drive hypothesis was (largely) disregarded or rejected for decades

  8. A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

    PubMed Central

    Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

    2013-01-01

    Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

  9. Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema.

    PubMed

    Kasahara, Y; Tuder, R M; Cool, C D; Lynch, D A; Flores, S C; Voelkel, N F

    2001-03-01

    Emphysema due to cigarette smoking is characterized by a loss of alveolar structures. We hypothesize that the disappearance of alveoli involves apoptosis of septal endothelial cells and a decreased expression of lung vascular endothelial growth factor (VEGF) and its receptor 2 (VEGF R2). By terminal transferase dUTP nick end labeling (TUNEL) in combination with immunohistochemistry, we found that the number of TUNEL+ septal epithelial and endothelial cells/lung tissue nucleic acid (microg) was increased in the alveolar septa of emphysema lungs (14.2 +/- 2.0/microg, n = 6) when compared with normal lungs (6.8 +/- 1.3/microg, n = 7) (p < 0.01) and with primary pulmonary hypertensive lungs (2.3 +/- 0.8/microg, n = 5) (p < 0.001). The cell death events were not significantly different between healthy nonsmoker (7.4 +/- 1.9/microg) and smoker (5.7 +/- 0.7/microg) control subjects. The TUNEL results were confirmed by single-stranded DNA and active caspase-3 immunohistochemistry, and by DNA ligation assay. Emphysema lungs (n = 12) had increased levels of oligonucleosomal-length DNA fragmentation when compared with normal lungs (n = 11). VEGF, VEGF R2 protein, and mRNA expression were significantly reduced in emphysema. We propose that epithelial and endothelial alveolar septal death due to a decrease of endothelial cell maintenance factors may be part of the pathogenesis of emphysema.

  10. Enhanced expression of proapoptotic and autophagic proteins involved in the cell death of glioblastoma induced by synthetic glycans.

    PubMed

    Faried, Ahmad; Arifin, Muhammad Zafrullah; Ishiuchi, Shogo; Kuwano, Hiroyuki; Yazawa, Shin

    2014-06-01

    Glioblastoma is the most aggressive malignant brain tumor, and overall patient survival has not been prolonged even by conventional therapies. Previously, the authors found that chemically synthesized glycans could be anticancer agents against growth of a series of cancer cells. In this study, the authors examined the effects of glycans on the growth of glioblastoma cells both in vitro and in vivo. The authors investigated not only the occurrence of changes in the cell signaling molecules and expression levels of various proteins related to cell death, but also a mouse model involving the injection of glioblastoma cells following the administration of synthetic glycans. Synthetic glycans inhibited the growth of glioblastoma cells, induced the apoptosis of the cells with cleaved poly (adenosine diphosphate-ribose) polymerase (PARP) expression and DNA fragmentation, and also caused autophagy, as shown by the detection of autophagosome proteins and monodansylcadaverine staining. Furthermore, tumor growth in the in vivo mouse model was significantly inhibited. A dramatic induction of programmed cell death was found in glioblastoma cells after treatment with synthetic glycans. These results suggest that synthetic glycans could be a promising novel anticancer agent for performing chemotherapy against glioblastoma.

  11. Toll-Like Receptor 4 Activation in Cancer Progression and Therapy

    PubMed Central

    Oblak, Alja; Jerala, Roman

    2011-01-01

    Cancer immunotherapy has been the focus of intense research since the late 19th century when Coley observed that bacterial components can contribute to cancer regression by eliciting an antitumor immune response. Successful activation and maturation of tumor-specific immune cells is now known to be mediated by bacterial endotoxin, which activates Toll-like receptor 4 (TLR4). TLR4 is expressed on a variety of immune as well as tumor cells, but its activation can have opposing effects. While TLR4 activation can promote antitumor immunity, it can also result in increased tumor growth and immunosuppression. Nevertheless, TLR4 engagement by endotoxin as well as by endogenous ligands represents notable contribution to the outcome of different cancer treatments, such as radiation or chemotherapy. Further research of the role and mechanisms of TLR4 activation in cancer may provide novel antitumor vaccine adjuvants as well as TLR4 inhibitors that could prevent inflammation-induced carcinogenesis. PMID:22110526

  12. Computational Approaches to Toll-Like Receptor 4 Modulation.

    PubMed

    Billod, Jean-Marc; Lacetera, Alessandra; Guzmán-Caldentey, Joan; Martín-Santamaría, Sonsoles

    2016-07-30

    Toll-like receptor 4 (TLR4), along with its accessory protein myeloid differentiation factor 2 (MD-2), builds a heterodimeric complex that specifically recognizes lipopolysaccharides (LPS), which are present on the cell wall of Gram-negative bacteria, activating the innate immune response. Some TLR4 modulators are undergoing preclinical and clinical evaluation for the treatment of sepsis, inflammatory diseases, cancer and rheumatoid arthritis. Since the relatively recent elucidation of the X-ray crystallographic structure of the extracellular domain of TLR4, research around this fascinating receptor has risen to a new level, and thus, new perspectives have been opened. In particular, diverse computational techniques have been applied to decipher some of the basis at the atomic level regarding the mechanism of functioning and the ligand recognition processes involving the TLR4/MD-2 system at the atomic level. This review summarizes the reported molecular modeling and computational studies that have recently provided insights into the mechanism regulating the activation/inactivation of the TLR4/MD-2 system receptor and the key interactions modulating the molecular recognition process by agonist and antagonist ligands. These studies have contributed to the design and the discovery of novel small molecules with promising activity as TLR4 modulators.

  13. Hoxb8 regulates expression of microRNAs to control cell death and differentiation.

    PubMed

    Salmanidis, M; Brumatti, G; Narayan, N; Green, B D; van den Bergen, J A; Sandow, J J; Bert, A G; Silke, N; Sladic, R; Puthalakath, H; Rohrbeck, L; Okamoto, T; Bouillet, P; Herold, M J; Goodall, G J; Jabbour, A M; Ekert, P G

    2013-10-01

    Hoxb8 overexpression immortalises haematopoietic progenitor cells in a growth-factor-dependant manner and co-operates with interleukin-3 (IL-3) to cause acute myeloid leukaemia. To further understand how Hoxb8 contributes to myeloid cell immortalisation, we generated IL-3-dependant myeloid cells expressing Hoxb8 under the control of an inducible promoter. Downregulation of Hoxb8, in the presence of IL-3, caused cell-cycle arrest and apoptosis in the majority of cells. Apoptosis was dependant on Bax and Bak and, in part, on Bim, which was repressed by Hoxb8. Deletion of the miR-17∼92 seed sequences in the Bim 3'UTR abolished Hoxb8-dependant regulation of Bim reporter constructs. Expression of all six miRNAs from this cluster were elevated when Hoxb8 was overexpressed. The miR-17∼92 cluster was required for repression of Bim in Hoxb8-immortalised cells and deletion of the miR-17∼92 cluster substantially inhibited Hoxb8, but not Hoxa9, mediated survival and proliferation. Hoxb8 appears to promote miR-17∼92 expression through c-Myc, a known transcriptional regulator of the miR-17∼92 cluster. We have uncovered a previously unrecognised link between Hoxb8 expression and microRNAs that provides a new insight into the oncogenic functions of Hoxb8.

  14. Chronic Social Stress and Ethanol Increase Expression of KLF11, a Cell Death Mediator, in Rat Brain.

    PubMed

    Duncan, Jeremy; Wang, Niping; Zhang, Xiao; Johnson, Shakevia; Harris, Sharonda; Zheng, Baoying; Zhang, Qinli; Rajkowska, Grazyna; Miguel-Hidalgo, Jose Javier; Sittman, Donald; Ou, Xiao-Ming; Stockmeier, Craig A; Wang, Jun Ming

    2015-07-01

    Major depressive disorder and alcoholism are significant health burdens that can affect executive functioning, cognitive ability, job responsibilities, and personal relationships. Studies in animal models related to depression or alcoholism reveal that the expression of Krüppel-like factor 11 (KLF11, also called TIEG2) is elevated in frontal cortex, which suggests that KLF11 may play a role in stress- or ethanol-induced psychiatric conditions. KLF11 is a transcriptional activator of monoamine oxidase A and B, but also serves other functions in cell cycle regulation and apoptotic cell death. In the present study, immunohistochemistry was used to quantify intensity of nuclear KLF11, combined with an unbiased stereological approach to assess nuclei in fronto-limbic, limbic, and other brain regions of rats exposed chronically to social defeat or ethanol. KLF11 immunoreactivity was increased significantly in the medial prefrontal cortex, frontal cortex, and hippocampus of both stressed rats and rats fed ethanol. However, expression of KLF11 protein was not significantly affected in the thalamus, hypothalamus, or amygdala in either treatment group compared to respective control rats. Triple-label immunofluorescence revealed that KLF11 protein was localized in nuclei of neurons and astrocytes. KLF11 was also co-localized with the immunoreactivity of cleaved caspase-3. In addition, Western blot analysis revealed a significant reduction in anti-apoptotic protein, Bcl-xL, but an increase of caspase-3 expression in the frontal cortex of ethanol-treated rats compared to ethanol-preferring controls. Thus, KLF11 protein is up-regulated following chronic exposure to stress or ethanol in a region-specific manner and may contribute to pro-apoptotic signaling in ethanol-treated rats. Further investigation into the KLF11 signaling cascade as a mechanism for neurotoxicity and cell death in depression and alcoholism may provide novel pharmacological targets to lessen brain damage and

  15. The correlation between programmed death-ligand 1 expression and driver gene mutations in NSCLC.

    PubMed

    Yang, Haitao; Chen, Huijuan; Luo, Shuimei; Li, Lina; Zhou, Sijing; Shen, Ruifen; Lin, Heng; Xie, Xianhe

    2017-04-04

    This study aimed to evaluate the correlation between positive PD-L1 expression and driver gene mutations in NSCLC and to seek preliminary evidence in favor of the strategy of PD-L1 inhibitors plus targeted agents. The overall analyses revealed that positive PD-L1 expression had a significant relationship with KRAS status (RR = 1.26; 95% CI, 1.06-1.50, P = 0.010), but no correlation with clinical characteristics (gender, smoking status, histological types), driver gene status (EGFR, ALK) and overall survival (OS): male versus female (RR = 1.16; 95% CI, 0.95-1.42; P = 0.15), never smoking versus former/current smoking (RR = 0.79; 95% CI, 0.56-1.11; P = 0.17), adenocarcinoma versus non-adenocarcinoma (RR = 0.94; 95% CI, 0.63-1.41; P = 0.77), EGFR mutation versus EGFR wild type (RR = 0.74; 95% CI, 0.52-1.06; P = 0.10), ALK positive versus ALK negative (RR = 1.02; 95% CI, 0.75-1.38; P = 0.91), OS of positive PD-L1 expression versus that of negative PD-L1 expression (HR = 1.31, 95% CI, 0.90-1.90; P = 0.15), respectively. Noteworthily, subgroup analyses exhibited that in Chinese cohort studies, positive PD-L1 expression was significantly correlated with OS (HR = 1.75, 95% CI, 1.36-2.24, P < 0.0001); and in the studies using PD-L1 monoclonal antibodies (McAbs), positive PD-L1 expression was significantly correlated with KRAS mutation (RR = 1.32, 95% CI, 1.06-1.65, P = 0.01) and EGFR mutation (RR = 0.51, 95% CI, 0.28-0.93, P = 0.03). After thoroughly searching PubMed, EMBASE and Cochrane Library databases, 11 relevant studies incorporating 3128 cases were identified. The pooled data were analyzed via Review manager 5.3 software. PD-L1 inhibitors probably was a potential promising option to manage advanced NSCLC harboring KRAS mutation.

  16. Programmed death ligand 1 expression in triple-negative breast cancer is associated with tumour-infiltrating lymphocytes and improved outcome.

    PubMed

    Beckers, Rhiannon K; Selinger, Christina I; Vilain, Ricardo; Madore, Jason; Wilmott, James S; Harvey, Kate; Holliday, Anne; Cooper, Caroline L; Robbins, Elizabeth; Gillett, David; Kennedy, Catherine W; Gluch, Laurence; Carmalt, Hugh; Mak, Cindy; Warrier, Sanjay; Gee, Harriet E; Chan, Charles; McLean, Anna; Walker, Emily; McNeil, Catriona M; Beith, Jane M; Swarbrick, Alexander; Scolyer, Richard A; O'Toole, Sandra A

    2016-07-01

    Triple-negative breast cancer (TNBC) patients generally have a poor outcome; there is a pressing need to identify more effective therapeutic strategies. Clinical trials targeting programmed death 1/programmed death ligand 1 (PD1/PDL1) in melanoma and non-small-cell lung cancer have reported high response rates, and tumoral PDL1 expression has been suggested as a potential biomarker to enrich for patient response to these treatments. There are only very limited data to date reporting the expression of PDL1 in TNBC. PDL1 immunohistochemistry was performed on 161 primary TNBCs and assessed in the tumour as well as immune cells in the stromal compartment. PDL1 expression was very common in TNBC, expressed in the tumour cell membrane (64%), cytoplasm (80%) and stromal (93%) cellular compartments. Cytoplasmic tumoral expression of PDL1 was associated with a lower risk of breast cancer-specific death [hazard ratio (HR) 0.45, P = 0.035] while stromal PDL1 expression was associated with a lower rate of deaths from all causes (HR 0.305, P = 0.0042). Membranous expression of PDL1 was not associated with outcome. While both PDL1 expression and tumour-infiltrating lymphocytes were associated with a better outcome, only lymphovascular invasion and high tumour-infiltrating lymphocytes were independently prognostic for breast cancer-specific death. While PDL1 expression is frequent in TNBC, it was not independently prognostic. There were differences in outcome depending on the cellular compartment of PDL1 expression. These data provide further impetus for investigating the utility of immune checkpoint therapies in TNBC, given the clinical significance of tumour-infiltrating lymphocytes (TILs) and PDL1 expression in this cohort. © 2015 John Wiley & Sons Ltd.

  17. Lactobacillus zeae Protects Caenorhabditis elegans from Enterotoxigenic Escherichia coli-Caused Death by Inhibiting Enterotoxin Gene Expression of the Pathogen

    PubMed Central

    Zhou, Mengzhou; Yu, Hai; Yin, Xianhua; Sabour, Parviz M.; Chen, Wei; Gong, Joshua

    2014-01-01

    Background The nematode Caenorhabditis elegans has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a C. elegans life-span assay to preselect probiotic bacteria for controlling K88+ enterotoxigenic Escherichia coli (ETEC), a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by Lactobacillus. Methodology/Principal Findings Life-span of C. elegans was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB). Among 13 LAB isolates that varied in their ability to protect C. elegans from death induced by ETEC strain JG280, Lactobacillus zeae LB1 offered the highest level of protection (86%). The treatment with Lactobacillus did not reduce ETEC JG280 colonization in the nematode intestine. Feeding E. coli strain JFF4 (K88+ but lacking enterotoxin genes of estA, estB, and elt) did not cause death of worms. There was a significant increase in gene expression of estA, estB, and elt during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode. However, the elt clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and E. coli DH5α in-vitro. Conclusions/Significance The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies). Heat-stable enterotoxins appeared to be the main factors responsible for the death of C. elegans. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the mechanism of protection

  18. Fibroblast growth factor receptor 4 induced resistance to radiation therapy in colorectal cancer

    PubMed Central

    Ahmed, Mohamed A.; Selzer, Edgar; Dörr, Wolfgang; Jomrich, Gerd; Harpain, Felix; Silberhumer, Gerd R.; Müllauer, Leonhard; Holzmann, Klaus; Grasl-Kraupp, Bettina; Grusch, Michael; Berger, Walter; Marian, Brigitte

    2016-01-01

    In colorectal cancer (CRC), fibroblast growth factor receptor 4 (FGFR4) is upregulated and acts as an oncogene. This study investigated the impact of this receptor on the response to neoadjuvant radiotherapy by analyzing its levels in rectal tumors of patients with different responses to the therapy. Cellular mechanisms of FGFR4-induced radioresistance were analyzed by silencing or over-expressing FGFR4 in CRC cell line models. Our findings showed that the FGFR4 staining score was significantly higher in pre-treatment biopsies of non-responsive than responsive patients. Similarly, high expression of FGFR4 inhibited radiation response in cell line models. Silencing or inhibition of FGFR4 resulted in a reduction of RAD51 levels and decreased survival in radioresistant HT29 cells. Increased RAD51 expression rescued cells in the siFGFR4-group. In radiosensitive SW480 and DLD1 cells, enforced expression of FGFR4 stabilized RAD51 protein levels resulting in enhanced clearance of γ-H2AX foci and increased cell survival in the mismatch repair (MMR)-proficient SW480 cells. MMR-deficient DLD1 cells are defective in homologous recombination repair and no FGFR4-induced radioresistance was observed. Based on our results, FGFR4 may serve as a predictive marker to select CRC patients with MMR-proficient tumors who may benefit from pre-operative radiotherapy. PMID:27650548

  19. Toll-like receptor 4 signalling attenuates experimental allergic conjunctivitis.

    PubMed

    Chung, S-H; Choi, S H; Cho, K J; Joo, C-K

    2011-05-01

    Allergic conjunctivitis from an allergen-driven T helper type 2 (Th2) response is characterized by conjunctival eosinophilic infiltration. Association between signalling through Toll-like receptor 4 (TLR-4) and adaptive immune responses has been observed in allergic airway disease. We examined whether administration of bacterial lipopolysaccharide (LPS), a prototypic bacterial product that activates immune cells via TLR-4, could affect the development of allergic conjunctivitis and modify the immune response to ovalbumin (OVA) allergen in an experimental allergic conjunctivitis (EAC) model. Mice were challenged with two doses of OVA via conjunctival sac after systemic challenge with OVA in alum. Several indicators for allergy were evaluated in wild-type and TLR-4(-/-) mice with or without adding of different doses of LPS into OVA in alum. Mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. Of interest, LPS administration markedly suppressed immunoglobulin (Ig)E-mediated and eosinophil-dependent conjunctival inflammation. In addition, mice sensitized with OVA plus LPS had less interleukin (IL)-4, IL-5 and eotaxin secretion than mice sensitized with OVA only. The suppression of allergic response by LPS administration was due to Th1 shift. In contrast, the presence of LPS during sensitization with OVA had no effect on severity of allergic conjunctivitis and Th2 responses in TLR4-4(-/-) mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses via the TLR-4-dependent pathway in the EAC model. © 2011 The Authors; Clinical and Experimental Immunology © 2011 British Society for Immunology.

  20. Toll-like receptor 4 signalling attenuates experimental allergic conjunctivitis

    PubMed Central

    Chung, S-H; Choi, S H; Cho, K J; Joo, C-K

    2011-01-01

    Allergic conjunctivitis from an allergen-driven T helper type 2 (Th2) response is characterized by conjunctival eosinophilic infiltration. Association between signalling through Toll-like receptor 4 (TLR-4) and adaptive immune responses has been observed in allergic airway disease. We examined whether administration of bacterial lipopolysaccharide (LPS), a prototypic bacterial product that activates immune cells via TLR-4, could affect the development of allergic conjunctivitis and modify the immune response to ovalbumin (OVA) allergen in an experimental allergic conjunctivitis (EAC) model. Mice were challenged with two doses of OVA via conjunctival sac after systemic challenge with OVA in alum. Several indicators for allergy were evaluated in wild-type and TLR-4−/− mice with or without adding of different doses of LPS into OVA in alum. Mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. Of interest, LPS administration markedly suppressed immunoglobulin (Ig)E-mediated and eosinophil-dependent conjunctival inflammation. In addition, mice sensitized with OVA plus LPS had less interleukin (IL)-4, IL-5 and eotaxin secretion than mice sensitized with OVA only. The suppression of allergic response by LPS administration was due to Th1 shift. In contrast, the presence of LPS during sensitization with OVA had no effect on severity of allergic conjunctivitis and Th2 responses in TLR4-4−/− mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses via the TLR-4-dependent pathway in the EAC model. PMID:21391988

  1. The MOC31PE immunotoxin reduces cell migration and induces gene expression and cell death in ovarian cancer cells

    PubMed Central

    2014-01-01

    Background The standard treatment of ovarian cancer with chemotherapy often leads to drug resistance and relapse of the disease, and the need for development of novel therapy alternatives is obvious. The MOC31PE immunotoxin binds to the cell surface antigen EpCAM, which is expressed by the majority of epithelial cancers including ovarian carcinomas, and we studied the cytotoxic effects of MOC31PE in ovarian cancer cells. Methods Investigation of the effects of MOC31PE treatment on protein synthesis, cell viability, proliferation and gene expression of the ovarian cancer cell lines B76 and HOC7. Results MOC31PE treatment for 24 h caused a dose-dependent reduction of protein synthesis with ID50 values of less than 10 ng/ml, followed by reduced cell viability. In a gene expression array monitoring the expression of 84 key genes in cancer pathways, 13 of the genes were differentially expressed by MOC31PE treatment in comparison to untreated cells. By combining MOC31PE and the immune suppressor cyclosporin A (CsA) the MOC31PE effect on protein synthesis inhibition and cell viability increased tenfold. Cell migration was also reduced, both in the individual MOC31PE and CsA treatment, but even more when combining MOC31PE and CsA. In tumor metastasis PCR arrays, 23 of 84 genes were differentially expressed comparing CsA versus MOC31PE + CsA treatment. Increased expression of the tumor suppressor KISS1 and the nuclear receptor NR4A3 was observed, and the differential candidate gene expression was confirmed in complementary qPCR analyses. For NR4A3 this was not accompanied by increased protein expression. However, a subcellular fractionation assay revealed increased mitochondrial NR4A3 in MOC31PE treated cells, suggesting a role for this protein in MOC31PE-induced apoptotic cell death. Conclusion The present study demonstrates that MOC31PE may become a new targeted therapy for ovarian cancer and that the MOC31PE anti-cancer effect is potentiated by CsA. PMID:24528603

  2. Rosiglitazone negatively regulates c-Jun N-terminal kinase and toll-like receptor 4 proinflammatory signalling during initiation of experimental aortic aneurysms.

    PubMed

    Pirianov, Grisha; Torsney, Evelyn; Howe, Franklyn; Cockerill, Gillian W

    2012-11-01

    Development and rupture of aortic aneurysms (AA) is a complex process involving inflammation, cell death, tissue and matrix remodelling. The thiazolidinediones (TZDs) including Rosiglitazone (RGZ) are a family of drugs which act as agonists of the nuclear peroxisome proliferator-activated receptors and have a broad spectrum of effects on a number of biological processes in the cardiovascular system. In our previous study we have demonstrated that RGZ has a marked effect on both aneurysm rupture and development, however, the precise mechanism of this is unknown. In the present study, we examined possible targets of RGZ action in the early stages of Angiotensin II-induced AA in apolipoprotein E-deficient mice. For this purpose we employed immunoblotting, ELISA and antibody array approaches. We found that RGZ significantly inhibited c-Jun N-terminal kinase (JNK) phosphorylation and down-regulated toll-like receptor 4 (TLR4) expression at the site of lesion formation in response to Angiotensin II infusion in the initiation stage (6-72 h) of experimental AA development. Importantly, this effect was also associated with a decrease of CD4 antigen and reduction in production of TLR4/JNK-dependant proinflammatory chemokines MCP-1 and MIP-1α. These data suggest that RGZ can modulate inflammatory processes by blocking TLR4/JNK signalling in initiation stages of AA development. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Expression of programmed cell death protein 4 (PDCD4) and miR-21 in urothelial carcinoma

    SciTech Connect

    Fischer, Nicolas; Goeke, Friederike; Splittstoesser, Vera; Lankat-Buttgereit, Brigitte; Mueller, Stefan C.; Ellinger, Joerg

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer The tumor suppressor gene PDCD4 is down-regulated in many tumorous entities. Black-Right-Pointing-Pointer We investigate the impact of PDCD4 and its regulating factor miR-21 in urothelial carcinoma. Black-Right-Pointing-Pointer We confirm PDCD4 as a tumor suppressor gene and it could be a diagnostic marker for this tumor. -- Abstract: Background: We investigated the role of the programmed cell death 4 (PDCD4) tumor suppressor gene in specimens of transitional cell carcinoma and of healthy individuals. Methods: PDCD4 immunohistochemical expression was investigated in 294 cases in histologically proven transitional cell carcinoma in different tumorous stages (28 controls, 122 non-muscle invasive urothelial carcinoma, stages Tis-T1, 119 invasive transitional cell carcinoma stages T2-T4 and 25 metastases). MiR-21 expression, an important PDCD4 regulator, was assessed with real-time PCR analysis and showed inverse correlation to tissue PDCD4 expression. Results: Nuclear and cytoplasmatic PDCD4 immunostaining decreased significantly with histopathological progression of the tumor (p < 0001). Controls showed strong nuclear and cytoplasmatic immunohistochemical staining. MiR-21 up regulation in tissue corresponded to PDCD4 suppression. Conclusions: These data support a decisive role for PDCD4 down regulation in transitional cell carcinoma and confirm miR-21 as a negative regulator for PDCD4. Additionally, PDCD4 immunohistochemical staining turns out to be a possible diagnostic marker for transitional cell carcinoma.

  4. Axin expression reduces staurosporine-induced mitochondria-mediated cell death in HeLa cells.

    PubMed

    Shin, Jee-Hye; Kim, Hyun-wook; Rhyu, Im Joo; Song, Ki-Joon; Kee, Sun-Ho

    2012-10-01

    Cytoplasmic axin expression frequently produces punctuate structures in cells, but the nature of axin puncta has not been fully elucidated. In an effort to analyze cytoplasmic axin puncta, we established HeLa cells expressing axin in a doxycycline-inducible manner (HeLa-Axin). We observed that axin accumulated in an aggregate-like pattern in perinuclear areas and appeared to be associated with mitochondria, Golgi apparatus, and endoplasmic reticulum (ER), but not lysosomes. Further biochemical analysis suggested that some part of the cytoplasmic axin pool was associated with mitochondria. In addition, mitochondrial proteins [i.e., cytochrome oxidase IV (CoxIV) and cytochrome c] were slightly higher in HeLa-Axin cells than in HeLa-EV cells, suggesting altered mitochondrial degradation. HeLa-Axin cells were then treated with staurosporine (STS) to determine if the mitochondria-induced apoptosis pathway was altered. Compared to STS-treated control cells (HeLa-EV), HeLa-Axin cells had less STS-induced cytotoxicity and reduced caspase-3 activation and PARP cleavage. Given that mitochondria outer membrane potential was unchanged, HeLa-Axin cells might be relatively resistant to STS-mediated mitochondrial damage. Mitochondria associated with axin aggregates were resistant to detergent-mediated permeabilization. These results suggest that axin forms aggregate-like structures in association with mitochondria, which render mitochondria resistant to STS-induced membrane damage and cytotoxicity.

  5. DNA Repair Biomarker Profiling of Head & Neck Cancer: Ku80 Expression Predicts Locoregional Failure and Death Following Radiotherapy

    PubMed Central

    Moeller, Benjamin J.; Yordy, John S.; Williams, Michelle D.; Giri, Uma; Raju, Uma; Molkentine, David P.; Byers, Lauren A.; Heymach, John V.; Story, Michael D.; Lee, J. Jack; Sturgis, Erich M.; Weber, Randal S.; Garden, Adam S.; Ang, K. Kian; Schwartz, David L.

    2011-01-01

    Purpose Radiotherapy plays an integral role in the treatment of head and neck squamous cell carcinoma (HNSCC). Although proteins involved in DNA repair may predict HNSCC response to radiotherapy, none has been validated in this context. We examined whether differential expression of double-strand DNA break (DSB) repair proteins in HNSCC, the chief mediators of DNA repair following irradiation, predict for treatment outcomes. Experimental Design Archival HNSCC tumor specimens (n = 89) were assembled onto a tissue microarray and stained with antibodies raised against 38 biomarkers. The biomarker set was enriched for proteins involved in DSB repair, in addition to established mechanistic markers of radioresistance. Staining was correlated with treatment response and survival alongside established clinical and pathologic covariates. Results were validated in an independent intramural cohort (n = 34). Results Ku80, a key mediator of DSB repair, correlated most closely with clinical outcomes. Ku80 was overexpressed in half of all tumors, and its expression was independent of all other covariates examined. Ku80 overexpression was an independent predictor for both locoregional failure and mortality following radiotherapy (P < 0.01). The predictive power of Ku80 overexpression was confined largely to HPV-negative HNSCC, where it conferred a 9-fold greater risk of death at 2 years. Conclusions Ku80 overexpression is a common feature of HNSCC, and is a candidate DNA repair-related biomarker for radiation treatment failure and death, particularly in patients with high-risk HPV-negative disease. It is a promising, mechanistically rational biomarker to select individual HPV-negative HNSCC patients for strategies to intensify treatment. PMID:21349997

  6. BURN-INDUCED ACUTE LUNG INJURY REQUIRES A FUNCTIONAL TOLL-LIKE RECEPTOR 4

    PubMed Central

    Krzyzaniak, Michael; Cheadle, Gerald; Peterson, Carrie; Loomis, William; Putnam, James; Wolf, Paul; Baird, Andrew; Eliceiri, Brian; Bansal, Vishal; Coimbra, Raul

    2014-01-01

    The role of the Toll-like receptor 4 (TLR4), a component of the innate immune system, in the development of burn-induced acute lung injury (ALI) has not been completely defined. Recent data suggested that an intact TLR4 plays a major role in the development of organ injury in sterile inflammation. We hypothesized that burn-induced ALI is a TLR4-dependent process. Male C57BL/6J (TLR4 wild-type [WT]) and C57BL/10ScN (TLR4 knockout [KO]) mice were subjected to a 30% total body surface area steam burn. Animals were killed at 6 and 24 h after the insult. Lung specimens were harvested for histological examination after hematoxylin-eosin staining. In addition, lung myeloperoxidase (MPO) and intercellular adhesion molecule 1 immunostaining was performed. Lung MPO was measured by an enzymatic assay. Total lung keratinocyte-derived chemoattractant (IL-8) content was measured by enzyme-linked immunosorbent assay. Western blot was performed to quantify phosphorylated IκBα, phosphorylated nuclear factor κB p65 (NF-κBp65), and high mobility group box 1 expression. Acute lung injury, characterized by thickening of the alveolar-capillary membrane, hyaline membrane formation, intraalveolar hemorrhage, and neutrophil infiltration, was seen in WT but not KO animals at 24 h. Myeloperoxidase and intercellular adhesion molecule 1 immunostaining of KO animals was also similar to sham but elevated in WT animals. In addition, a reduction in MPO enzymatic activity was observed in KO mice as well as a reduction in IL-8 levels compared with their WT counterparts. Burn-induced ALI develops within 24 h after the initial thermal insult in our model. Toll-like receptor 4 KO animals were clearly protected and had a much less severe lung injury. Our data suggest that burn-induced ALI is a TLR4-dependent process. PMID:21330948

  7. Burn-induced acute lung injury requires a functional Toll-like receptor 4.

    PubMed

    Krzyzaniak, Michael; Cheadle, Gerald; Peterson, Carrie; Loomis, William; Putnam, James; Wolf, Paul; Baird, Andrew; Eliceiri, Brian; Bansal, Vishal; Coimbra, Raul

    2011-07-01

    The role of the Toll-like receptor 4 (TLR4), a component of the innate immune system, in the development of burn-induced acute lung injury (ALI) has not been completely defined. Recent data suggested that an intact TLR4 plays a major role in the development of organ injury in sterile inflammation. We hypothesized that burn-induced ALI is a TLR4-dependent process. Male C57BL/6J (TLR4 wild-type [WT]) and C57BL/10ScN (TLR4 knockout [KO]) mice were subjected to a 30% total body surface area steam burn. Animals were killed at 6 and 24 h after the insult. Lung specimens were harvested for histological examination after hematoxylin-eosin staining. In addition, lung myeloperoxidase (MPO) and intercellular adhesion molecule 1 immunostaining was performed. Lung MPO was measured by an enzymatic assay. Total lung keratinocyte-derived chemoattractant (IL-8) content was measured by enzyme-linked immunosorbent assay. Western blot was performed to quantify phosphorylated IκBα, phosphorylated nuclear factor κB p65 (NF-κBp65), and high mobility group box 1 expression. Acute lung injury, characterized by thickening of the alveolar-capillary membrane, hyaline membrane formation, intraalveolar hemorrhage, and neutrophil infiltration, was seen in WT but not KO animals at 24 h. Myeloperoxidase and intercellular adhesion molecule 1 immunostaining of KO animals was also similar to sham but elevated in WT animals. In addition, a reduction in MPO enzymatic activity was observed in KO mice as well as a reduction in IL-8 levels compared with their WT counterparts. Burn-induced ALI develops within 24 h after the initial thermal insult in our model. Toll-like receptor 4 KO animals were clearly protected and had a much less severe lung injury. Our data suggest that burn-induced ALI is a TLR4-dependent process.

  8. Toll-like receptor 4 dependent responses to lung injury in a murine model of pulmonary contusion

    PubMed Central

    Hoth, J. Jason; Wells, Jonathan D.; Brownlee, Noel A.; Hiltbold, Elizabeth M.; Meredith, J. Wayne; McCall, Charles E.; Yoza, Barbara K.

    2010-01-01

    Blunt chest trauma resulting in pulmonary contusion with an accompanying acute inflammatory response is a common but poorly understood injury. We previously demonstrated that toll-like receptor 2 participates in the inflammatory response to lung injury. We hypothesized that the toll-like receptor 4, in a MyD88-dependent manner, may also participate in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated post injury lung function, pulmonary neutrophil recruitment and the systemic innate immune response. Comparisons were made between wild type mice and mice deficient in toll like receptor 4 or MyD88. We found toll-like receptor 4 dependent responses to pulmonary contusion that include hypoxemia, edema, and neutrophil infiltration. Increased expression of interleukin 6 and chemokine (C-X-C motif) ligand-1 in the bronchoalveolar lavage and serum was also dependent on TLR4 activation. We further demonstrated that these responses to pulmonary contusion were dependent on MyD88, an adapter protein in the signal transduction pathway mediated by toll-like receptors. These results show that toll-like receptors have a primary role in the response to acute lung injury. Lung inflammation and systemic innate immune responses are dependent on toll-like receptor activation by pulmonary contusion. PMID:18665044

  9. Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

    PubMed Central

    Bowman, James; Rodgers, Mary A.; Shi, Mude; Amatya, Rina; Hostager, Bruce; Iwai, Kazuhiro; Gao, Shou-Jiang

    2015-01-01

    ABSTRACT Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. PMID:26578682

  10. Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response.

    PubMed

    Paramo, Teresa; Tomasio, Susana M; Irvine, Kate L; Bryant, Clare E; Bond, Peter J

    2015-12-09

    Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the "membrane-like" nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics.

  11. Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response

    PubMed Central

    Paramo, Teresa; Tomasio, Susana M.; Irvine, Kate L.; Bryant, Clare E.; Bond, Peter J.

    2015-01-01

    Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the “membrane-like” nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics. PMID:26647780

  12. Fibroblast Growth Factor Receptor-4 and Prostate Cancer Progression

    DTIC Science & Technology

    2006-10-01

    variant arising from a cryptic promoter (ptd- FGFR-4) is expressed in pituitary adenomas and that expression of this variant resulted in increased inva...siveness of pituitary cells. Expression of the ptd-FGFR- 4 variant decreased adhesion to collagen IV in pituitary cells and NIH3T3 fibroblasts...formation of NCAM and N- cadherin complexes in pituitary cells which could affect adhesion to collagen IV. On the other hand, Coppolino and Dedhar [37

  13. Fibroblast Growth Factor Receptor-4 and Prostate Cancer Progression

    DTIC Science & Technology

    2007-10-01

    al. [24] have reported that a cytoplasmic FGFR-4 variant arising from a cryptic promoter (ptd- FGFR-4) is expressed in pituitary adenomas and that...expression of this variant resulted in increased inva- siveness of pituitary cells. Expression of the ptd-FGFR- 4 variant decreased adhesion to collagen...IV in pituitary cells and NIH3T3 fibroblasts compared to full length FGFR-4 in a ligand independentmanner. These authors provide evidence that

  14. CXC motif chemokine receptor 4 gene polymorphism and cancer risk

    PubMed Central

    Wu, Yang; Zhang, Chun; Xu, Weizhang; Zhang, Jianzhong; Zheng, Yuxiao; Lu, Zipeng; Liu, Dongfang; Jiang, Kuirong

    2016-01-01

    Abstract Background: Previous epidemiological studies have reported the relationship between CXC motif chemokine receptor 4 (CXCR4) synonymous polymorphism (rs2228014), and risk of cancer, but the results remained conflicting and controversial. Therefore, this study was devised to evaluate the genetic effects of the rs2228014 polymorphism on cancer risk in a large meta-analysis. Methods: The computer-based databases (EMBASE, Web of Science, and PubMed) were searched for all relevant studies evaluating rs2228014 and susceptibility to cancer. In the analysis, pooled odds ratios (ORs) with its corresponding 95% confidence intervals (CIs) were calculated in 5 genetic models to assess the genetic risk. Egger regression and Begg funnel plots test were conducted to appraise the publication bias. Results: Data on rs2228014 polymorphism and overall cancer risk were available for 3684 cancer patients and 5114 healthy controls participating in 11 studies. Overall, a significantly increased risk of cancer was associated with rs2228014 polymorphism in homozygote model (OR = 2.01, 95% CI: 1.22–3.33) and in recessive model (OR = 1.97, 95% CI: 1.23–3.16). When stratified by ethnicity, the results were positive only in Asian populations (heterozygote model: OR = 1.36, 95% CI: 1.13–1.65; homozygote model: OR = 2.43, 95% CI: 1.21–4.91; dominant model: OR = 1.47, 95% CI: 1.13–1.90; recessive model: OR = 2.25, 95% CI: 1.13–4.48; and allele model: OR = 1.48, 95% CI: 1.10–1.99). Besides, in the subgroup analysis by source of control, the result was significant only in population-based control (homozygote model: OR = 2.39, 95% CI: 1.06–5.40; recessive model: pooled OR = 2.24, 95% CI: 1.02–4.96). Conclusion: In general, our results first indicated that the rs2228014 polymorphism in CXCR4 gene is correlated with an increased risk of cancer, especially among Asian ethnicity. Large, well-designed epidemiological studies are required to verify the current findings. PMID

  15. Janus kinase 3 is expressed in erythrocytes, phosphorylated upon energy depletion and involved in the regulation of suicidal erythrocyte death.

    PubMed

    Bhavsar, Shefalee K; Gu, Shuchen; Bobbala, Diwakar; Lang, Florian

    2011-01-01

    Janus kinase 3, a tyrosine kinase expressed in haematopoetic tissues, plays a decisive role in T-lymphocyte survival. JAK3 deficiency leads to (Severe) Combined Immunodeficiency (SCID) resulting from enhanced lymphocyte apoptosis. JAK3 is activated by phosphorylation. Nothing is known about expression of JAK3 in erythrocytes, which may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure and cell shrinkage. Triggers of eryptosis include energy depletion. The present study utilized immunohistochemistry and confocal microscopy to test for JAK3 expression and phosphorylation, and FACS analysis to determine phosphatidylserine exposure (annexin binding) and cell volume (forward scatter). As a result, JAK3 was expressed in erythrocytes and phosphorylated following 24h and 48h glucose depletion. Forward scatter was slightly but significantly smaller in erythrocytes from JAK3-deficient mice (jak3(-/-)) than in erythrocytes from wild type mice (jak3(+/+)). Annexin V binding was similarly low in both genotypes. The JAK3 inhibitors WHI-P131/JANEX-1 (4-(4'-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 156μM) and WHI-P154 (4-[(3'-Bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 11.2μM) did not significantly modify annexin V binding or forward scatter. Glucose depletion increased annexin V binding, an effect significantly blunted in jak3(-/-) erythrocytes and in the presence of the JAK3 inhibitors. The observations disclose a completely novel role of Janus kinase 3, i.e. the triggering of cell membrane scrambling in energy depleted erythrocytes.

  16. High immune activation and abnormal expression of cytokines contribute to death of SHIV89.6-infected Chinese rhesus macaques.

    PubMed

    Tian, Ren-Rong; Zhang, Ming-Xu; Zhang, Lin-Tao; Zhang, Xiao-Liang; Zheng, Hong-Yi; Zhu, Lin; Pang, Wei; Zhang, Gao-Hong; Zheng, Yong-Tang

    2015-08-01

    Chinese rhesus macaques (CRMs) are ideal experimental animals for studying the pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) and for vaccine research. SHIV89.6 has been reported to be an attenuated virus because, in most cases, SHIV89.6 infection only causes limited alteration of immune cells and tissues, and it has been used commonly for vaccine research. After two serial passages in vivo, SHIV (SHIV-89.6P) induces CD4 lymphopenia and an AIDS-like disease with wasting and opportunistic infections. However, the pathogenic ability of SHIV89.6 is not well understood. In this study, we found that 6 of 14 SHIV89.6-infected CRMs died within 127 weeks after infection. We found especially high immune activation, low IFN-α expression, and distinctive cytokine expression profiles in the infected and dead (ID) group of monkeys, while there was only few change in the CD4(+) T counts and distribution of T cell subsets in the ID group monkeys. Also, there was a similar dynamic of viral load between infected and surviving (IS) and ID group monkeys. Furthermore, we found various correlations among immune activation, IFN-α expression, and frequencies of cytokine-secreting cells. These results suggest that SHIV89.6 infections have pathogenic potential in CRMs and that high immune activation and abnormal expression of cytokines contribute to death of SHIV89.6-infected CRMs. This also implies that high immune activation may be relevant to dysfunction of immune cells. It is proposed that high immune activation and dysfunction of immune cells may be good predictors for disease progression and markers for therapy.

  17. Diet-Induced Obesity Enhances Progression of Hepatocellular Carcinoma through Tenascin-C/Toll-Like Receptor 4 Signaling.

    PubMed

    Benbow, Jennifer H; Thompson, Kyle J; Cope, Heidi L; Brandon-Warner, Elizabeth; Culberson, Catherine R; Bossi, Krista L; Li, Ting; Russo, Mark W; Gersin, Keith S; McKillop, Iain H; deLemos, Andrew S; Schrum, Laura W

    2016-01-01

    Obesity is an independent risk factor for the development of liver fibrosis/cirrhosis and hepatocellular carcinoma (HCC). Tenascin-C (TnC), an extracellular matrix protein, is transiently expressed during tissue injury and plays a role in fibrogenesis and tumorigenesis. However, the mechanistic role of TnC signaling in the development of HCC remains unknown. We developed a diet-induced obesity HCC mouse model and examined TnC expression and liver injury. To determine the cellular mechanism of TnC signaling in promoting inflammation and hepatocyte epithelial-mesenchymal transition and migration, we used primary hepatocytes and hepatoma and macrophage cell lines. Further, to determine whether elevated TnC expression correlated with obesity-associated HCC, we measured plasma TnC in obese patients with various levels of liver injury. Increased tissue inflammation accompanied with elevated hepatic stellate cell-derived TnC and Toll-like receptor 4 expression was observed in the diet-induced obesity HCC animal model. In vitro studies found enhanced Toll-like receptor 4 signaling activated by TnC, promoting an increased inflammatory response, hepatocyte transformation, and migration. Further, obese patients with cirrhosis alone and in combination with HCC showed significant increases in plasma TnC compared with healthy volunteers and patients with less severe liver injury. Overall, these studies suggest TnC/Toll-like receptor 4 signaling as an important regulator in HCC; inhibiting this signaling axis may be a viable therapeutic target for impeding HCC. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  18. Time course analysis of apoptotic cell death during expression of hybrid lethality in hybrid tobacco cells (Nicotiana suaveolens x N. tabacum).

    PubMed

    Masuda, Yu; Yamada, Tetsuya; Marubashi, Wataru

    2003-04-01

    Hybrid cells from the cross Nicotiana suaveolens x N. tabacum expressed hybrid lethality at 28 degrees C in a thin layer cell culture system. Features characteristic of apoptosis, such as DNA fragmentation, chromatin condensation and nuclear fragmentation, were detected during expression of hybrid lethality. Actinomycin D (ActD) or cycloheximide (CHX) added to the medium suppressed apoptotic cell death during hybrid lethality. This indicates that hybrid lethality requires de novo transcription and translation, and is thus under genetic control. To estimate the time course of apoptotic cell death during the expression of hybrid lethality, we determined when factors controlling hybrid lethality were expressed by observing the point of no return. When cells were exposed to 28 degrees C for 2 h or less in inhibitor-free medium before addition of ActD or CHX, the percentage of dead cells did not increase. However, when cells were exposed to 28 degrees C for 4 h before the addition of inhibitor, the percentage of dead cells increased. When cells were exposed to 28 degrees C for 3 h before the addition of inhibitor, the percentage of dead cells varied from experiment to experiment. These data indicate that the factors controlling hybrid lethality are expressed 3 h after induction of hybrid lethality. In addition, we found a time difference between the expression of cell death and nuclear fragmentation. This suggests that the factor controlling cell death is different from the one controlling nuclear fragmentation.

  19. [Regulatory effects of glutamine on Toll-like receptor 4 in neonatal rats with necrotizing enterocolitis].

    PubMed

    Li, Wei; Zheng, Xiao-Hui; Zhou, Wei; Rong, Xiao; Huang, Long-Guang

    2011-05-01

    To study the expression of Toll-like receptor 4 (TLR-4) and caspase-3 in the intestine of neonatal rats with necrotizing enterocolitis (NEC), and explore the protective effects and possible regulatory mechanisms of glutamine (Gln) in NEC. Sixty premature rats were randomly divided into three groups (n=20 each): control, NEC model and Gln intervention group. NEC model was prepared by formula feeding, hypoxia and cold stress. The Gln intervention group was also subjected to hypoxia and cold stress but was fed with formula containing Gln (0.3 g/kg). Two days later, the rats were sacrificed and the intestine tissues were obtained. The histological changes of ileal tissues were observed by hemetoxylin and eosin staining. The expression of caspase-3 and TLR-4 protein in the jejunum, ileum and colon were detected by inmunohistochemistry. The expression of TLR-4 mRNA in the jejunum, ileum and colon were detected by RT-PCR. Compared with the control group, the histological score of ileal tissues, and the expression of caspase-3, TLR-4 protein and TLR-4 mRNA in the NEC model group increased significantly (P<0.01). Gln intervention decreased significantly the histological score of ileal tissues, and the expression of caspase-3, TLR-4 protein and TLR-4 mRNA compared with the NEC model group (P<0.05). TLR-4 might be involved in the pathogenesis of NEC. Gln may provide protective effects on intestine possibly through reducing the TLR-4 expression and then decreasing the apoptosis of intestinal epithelial cells.

  20. Expression of the fetal Alz-50 clone 1 protein induces apoptotic cell death

    SciTech Connect

    Strachan, Gordon D.; Ostrow, Liya Avshalumov; Jordan-Sciutto, Kelly L. . E-mail: Jordan@path.dental.upenn.edu

    2005-10-21

    The fetal Alz-50 clone 1 (FAC1) protein exhibits altered expression patterns in neurodegenerative disease. Though it has been shown to bind DNA in a site-specific, phosphorylation-dependent manner, its cellular function remains unknown. Here, we demonstrate that overexpression of FAC1 in PT67 fibroblasts induces nuclear condensation and cleavage of caspase 3 to its active form indicating induction of apoptosis. The amino-terminal domain of FAC1 is necessary and sufficient to induce both nuclear condensation and activation of caspase 3. Disruption of FAC1 interaction with a known binding partner, kelch-like ECH-associated protein 1 (Keap1), enhances activation of caspase 3. Keap1 is known to block activation of the antioxidant response gene products by direct interaction with the transcriptional activator, Nrf2. Disruption of the Keap1:Nrf2 interaction enhances FAC1 induction of apoptosis. These findings suggest a role for FAC1 in apoptosis following release of Nrf2 from Keap1 in response to oxidative stress.

  1. Actinomycin D Down-regulates SOX2 Expression and Induces Death in Breast Cancer Stem Cells.

    PubMed

    Das, Tuhin; Nair, Rajesh R; Green, Ryan; Padhee, Shruti; Howell, Mark; Banerjee, Jit; Mohapatra, Shyam S; Mohapatra, Subhra

    2017-04-01

    One of the major hurdles in the treatment of breast cancers is the inability of anti-cancer drugs to eliminate the breast cancer stem cells (BCSCs) population, which leads to disease relapse. The dearth in anti-cancer drugs that target BCSCs can be attributed to the absence of in vitro screening models that can not only recapitulate the tumor microenvironment consisting of BCSCs but also preserve the 3-dimensional (3D) architecture of in vivo tumors. In our present study, we have developed a 3D cell culture system that shows: (i) enrichment of BCSCs, (ii) increased drug resistance, and (iii) generation of hypoxic conditions similar to tumors. Using this model, we were able to screen a FDA-approved diversity set and identify as well as validate actinomycin D as a potential anti-breast cancer agent. Interestingly, we show that actinomycin D specifically targets and down-regulates the expression of the stem cell transcription factor, Sox-2. Additionally, down-regulation of Sox-2 leads to depletion of the stem-cell population resulting in the inability of breast cancer cells to initiate tumor progression. This study demonstrates the utility of an in vivo-like 3D cell culture system for the identification and validation of anti-cancer agents that will have a better probability of success in the clinic. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  2. Expression of brain-derived neurotrophic factor and TrkB receptor in the sudden infant death syndrome brainstem.

    PubMed

    Tang, Samantha; Machaalani, Rita; Waters, Karen A

    2012-01-15

    This study compared the expression of BDNF (proBDNF and rhBDNF forms) and its receptor TrkB, in the medulla of sudden infant death syndrome (SIDS) infants and infants who died from known causes (non-SIDS). This study also evaluated these markers in association with SIDS clinical risk factors including, sleep position, cigarette smoke exposure and gender. Brainstem tissue was immunohistochemically stained and quantitative analyses were made for eight nuclei of the caudal and rostral medulla. Compared to non-SIDS, SIDS infants had lower rhBDNF in the caudal nucleus of the solitary tract and higher TrkB in the caudal dorsal motor nucleus of the vagus. Within the SIDS cohort, prone sleep position was associated with lower rhBDNF in the caudal arcuate nucleus, and cigarette smoke exposure was associated with lower rhBDNF and TrkB in the inferior olivary nucleus. Abnormal expression of BDNF and TrkB suggests that neuroprotective functions of the BDNF/TrkB system may be reduced in respiratory-related nuclei of SIDS infants.

  3. IGF-1 protects against Aβ25-35-induced neuronal cell death via inhibition of PUMA expression and Bax activation.

    PubMed

    Hou, Xunyao; Jin, Yan; Chen, Jian; Hong, Yan; Luo, Dingzhen; Yin, Qingqing; Liu, Xueping

    2017-01-10

    Amyloid-β-peptide (Aβ) is considered to be the toxic species in AD and causes cell death in the affected areas of patient's brain. Insulin-like growth factor 1 (IGF-1) has been reported to attenuate Aβ toxicity in neuronal cells. However, the molecular mechanisms involved in the neuroprotective function of IGF-1 remain largely unknown. In the present study, we for the first time demonstrated that IGF-1 protects against Aβ-induced neurotoxicity via inhibition of PUMA expression and Bax activation. We found that IGF-1 could activate Akt, which in turn inhibited Aβ-induced FOXO3a nuclear translocation and thus decreased the binding ability of FOXO3a to PUMA promoter, leading to decreased PUMA expression. In addition, IGF-1 inhibited the translocation of Bax to the mitochondria induced by Aβ. Notably, addition of wortmannin, a specific inhibitor of PI3K, significantly abolished the neuroprotective effect of IGF-1, suggesting that IGF-1 exerts its anti-apoptotic effect depend on PI3K activity. Our findings may provide new insights into molecular mechanisms mediated by IGF-1 in cell survival against Aβ-induced apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Homologs of genes associated with programmed cell death in animal cells are differentially expressed during senescence of Ipomoea nil petals.

    PubMed

    Yamada, Tetsuya; Ichimura, Kazuo; Kanekatsu, Motoki; van Doorn, Wouter G

    2009-03-01

    In senescent petals of Ipomoea nil, we investigated the expression of genes showing homology to genes involved in animal programmed cell death (PCD). Three encoded proteins were homologous to apoptotic proteins in animals: Bax inhibitor-1 (BI-1), a vacuolar processing enzyme (VPE; homologous to caspases) and a monodehydroascorbate reductase [MDAR; homologous to apoptosis-inducing factor (AIF)]. AIFs harbor an oxidoreductase domain and an apoptotic domain. MDARs exhibit homology to the AIF oxidoreductase domain, not to the apoptotic domain. The three other genes studied relate to autophagy. They encode homologs to vacuolar protein sorting 34 (VPS34) and to the Arabidopsis autophagy-related proteins 4b and 8a (ATG4b and ATG8a). The transcript abundance of MDAR decreased continuously, whereas that of the other genes studies exhibited a transient increase, except ATG4b whose abundance stayed high after an increase. Treatment with ethylene advanced the time to visible petal senescence, and hastened the changes in expression of each of the genes studied. In order to assess the role of VPS34 in petal senescence, we studied the effect of its inhibitor 3-methyladenine (3-MA). 3-MA reduced the time to visible petal senescence, and also accelerated the time to DNA degradation. Remarkably, 3-MA increased the time to nuclear fragmentation, indicating that the time to visible petal senescence was independent of nuclear fragmentation. The data on 3-MA might suggest the idea that autophagy is not a cause of PCD, but part of the remobilization process.

  5. Endogenous DUX4 expression in FSHD myotubes is sufficient to cause cell death and disrupts RNA splicing and cell migration pathways

    PubMed Central

    Rickard, Amanda M.; Petek, Lisa M.; Miller, Daniel G.

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation that results in aberrant expression of the transcription factor Double Homeobox 4 (DUX4). DUX4 protein is present in a small subset of FSHD muscle cells, making its detection and analysis of its effects historically difficult. Using a DUX4-activated reporter, we demonstrate the burst expression pattern of endogenous DUX4, its method of signal amplification in the unique shared cytoplasm of the myotube, and FSHD cell death that depends on its activation. Transcriptome analysis of DUX4-expressing cells revealed that DUX4 activation disrupts RNA metabolism including RNA splicing, surveillance and transport pathways. Cell signaling, polarity and migration pathways were also disrupted. Thus, DUX4 expression is sufficient for myocyte death, and these findings suggest mechanistic links between DUX4 expression and cell migration, supporting recent descriptions of phenotypic similarities between FSHD and an FSHD-like condition caused by FAT1 mutations. PMID:26246499

  6. Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

    PubMed

    Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

    2016-04-01

    Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are

  7. Expression of Inflammatory and Cell Death Program Genes and Comet DNA Damage Assay Induced by Escherichia coli in Layer Hens

    PubMed Central

    Mehaisen, Gamal M. K.; Eshak, Mariam G.; El Sabry, M. I.; Abass, Ahmed O.

    2016-01-01

    Modern methods of industrial poultry and egg production systems involve stressful practices that stimulate Escherichia coli (E. coli) activity causing endotoxic shock. This investigation was conducted to evaluate the expression of pro-inflammatory cytokines and cell death program genes and DNA damage induced by E. coli in the brain and liver tissues of laying hens. A total of two hundred and ten H&N brown layer hens with 20 week age, were used in this research. First, preliminary experiments were designed (60 hens in total) to establish the optimal exposure dose of E. coli and to determine the nearest time of notable response to be used in the remainder studies of this research. At 35-wk of age, 150 hens were randomly assigned into 2 groups with 3 replicates of 25 birds each; the first group was injected in the brachial wing vein with 107 E. coli colony/hen, while the second group was injected with saline and served as a control. The body temperature and plasma corticosterone concentration were measured 3 hr after injection. Specimens of liver and brain were obtained from each group and the gene expression of p38 mitogen-activated protein kinase, interlukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), Bax, and caspase-3 genes were measured by quantitative real-time PCR. DNA damage in the brain and liver tissues were also measured by comet assay. Hens treated with E. coli showed significant (P<0.05) increase of body temperature and plasma corticosterone (42.6°C and 14.5 ng/ml, respectively) compared to the control group (41.1°C and 5.5 ng/ml, respectively). Additional remarkable over-inflammation gene expression of p38, IL-1β and TNF-α.genes were also detected in the brain (2.2-fold, 2.0-fold and 3.3-fold, respectively) and the liver (2.1-fold, 1.9-fold and 3.0-fold, respectively) tissues of the infected chickens. It is also important to note that hens injected with E. coli showed an increase in DNA damage in the brain and liver cells (P<0.05). These

  8. Liver and kidney damage induced by 4-aminopyridine in a repeated dose (28 days) oral toxicity study in rats: gene expression profile of hybrid cell death.

    PubMed

    Frejo, María Teresa; Del Pino, Javier; Lobo, Margarita; García, Jimena; Capo, Miguel Andrés; Díaz, María Jesús

    2014-03-03

    4-Aminopyridine (4-AP) is an orphan drug indicated for the treatment of neuromuscular disorders. There is a great controversy around the use of this drug because of its narrow safety index and because a large number of adverse effects have been reported. Moreover, it was shown to induce cell death in different cell lines, being reported mainly apoptosis and necrosis as the principal pathways of cell death mediated by blockage of K channels or the Na, K-ATPase, but until now it was not described in vivo cell death induced by 4-aminipyridine. To provide new subchronic toxicity data and specifically, evaluate if 4-AP is able to induce in vivo cell death process and the main pathways related to it, a repeated dose (28 days) oral toxicity study, at therapeutic range of doses, was conducted in rats. The anatomical pathology, the biochemical and hematological parameters were analyzed and a real-time PCR array analysis was developed with an Ingenuity Pathway Analysis (IPA). The leucocytes number, the lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) enzymatic activity were increased at all dose but the erythrocytes number, the hemoglobin concentration, the alkaline phosphatase (FAL) and alanine aminotransferase (ALT) enzymatic activity were increased only at highest dose studied. However, glucose levels decreased at all doses. The biochemical results are indicative of hepatic damage. The anatomy pathology studies showed cell death only on liver and kidney, and the real-time PCR array on liver tissue expressed a gene expression profile of necrotic and apoptotic induced cell death. The present work shows for the first time in vivo cell death on liver and kidney with features of apoptosis and necrosis induced by 4-AP and the gene expression profile shows that the cell death is mediated by necrotic and apoptotic pathways that support this finding.

  9. Aspirin Use and Colorectal Cancer Survival According to Tumor CD274 (Programmed Cell Death 1 Ligand 1) Expression Status.

    PubMed

    Hamada, Tsuyoshi; Cao, Yin; Qian, Zhi Rong; Masugi, Yohei; Nowak, Jonathan A; Yang, Juhong; Song, Mingyang; Mima, Kosuke; Kosumi, Keisuke; Liu, Li; Shi, Yan; da Silva, Annacarolina; Gu, Mancang; Li, Wanwan; Keum, NaNa; Zhang, Xuehong; Wu, Kana; Meyerhardt, Jeffrey A; Giovannucci, Edward L; Giannakis, Marios; Rodig, Scott J; Freeman, Gordon J; Nevo, Daniel; Wang, Molin; Chan, Andrew T; Fuchs, Charles S; Nishihara, Reiko; Ogino, Shuji

    2017-06-01

    Purpose Blockade of the programmed cell death 1 (PDCD1, PD-1) immune checkpoint pathway can improve clinical outcomes in various malignancies. Evidence suggests that aspirin (a widely used nonsteroidal anti-inflammatory drug) not only prolongs colorectal cancer survival, but can also activate T cell-mediated antitumor immunity and synergize with immunotherapy through inhibition of prostaglandin E2 production. We hypothesized that the survival benefit associated with aspirin might be stronger in colorectal carcinoma with a lower CD274 (PDCD1 ligand 1, PD-L1) expression level that resulted in lower signaling of the immune checkpoint pathway. Patients and Methods Using data from 617 patients with rectal and colon cancer in the Nurses' Health Study and the Health Professionals Follow-Up Study, we examined the association of postdiagnosis aspirin use with patient survival in strata of tumor CD274 expression status measured by immunohistochemistry. We used multivariable Cox proportional hazards regression models to control for potential confounders, including disease stage, microsatellite instability status, CpG island methylator phenotype, long interspersed nucleotide element-1 methylation, cyclooxygenase-2 (PTGS2), and CDX2 expression, and KRAS, BRAF, and PIK3CA mutations. Results The association of postdiagnosis aspirin use with colorectal cancer-specific survival differed by CD274 expression status ( Pinteraction < .001); compared with aspirin nonusers; multivariable-adjusted hazard ratios for regular aspirin users were 0.16 (95% CI, 0.06 to 0.41) in patients with low CD274 and 1.01 (95% CI, 0.61 to 1.67) in patients with high CD274. This differential association seemed consistent in patients with microsatellite-stable or PIK3CA wild-type disease and in strata of PTGS2 expression, CDX2 expression, tumor-infiltrating lymphocytes, or prediagnosis aspirin use status. Conclusion The association of aspirin use with colorectal cancer survival is stronger in patients with

  10. A Matrix Metalloproteinase Gene Is Expressed at the Boundary of Senescence and Programmed Cell Death in Cucumber1

    PubMed Central

    Delorme, Valérie G.R.; McCabe, Paul F.; Kim, Dae-Jae; Leaver, Christopher J.

    2000-01-01

    Cell-cell and extracellular cell matrix (ECM) interactions provide cells with information essential for controlling morphogenesis, cell-fate specification, and cell death. In animals, one of the major groups of enzymes that degrade the ECM is the matrix metalloproteinases (MMPs). Here, we report the characterization of the cucumber (Cucumis sativus L. cv Marketmore) Cs1-MMP gene encoding such an enzyme likely to play a role in plant ECM degradation. Cs1-MMP has all the hallmark motif characteristics of animal MMPs and is a pre-pro-enzyme having a signal peptide, propeptide, and zinc-binding catalytic domains. Cs1-MMP also displays functional similarities with animal MMPs. For example, it has a collagenase-like activity that can cleave synthetic peptides and type-I collagen, a major component of animal ECM. Cs1-MMP activity is completely inhibited by a hydroxamate-based inhibitor that binds at the active site of MMPs in a stereospecific manner. The Cs1-MMP gene is expressed de novo at the end stage of developmental senescence, prior to the appearance of DNA laddering in cucumber cotyledons leaf discs and male flowers. As the steady-state level of Cs1-MMP mRNA peaks late in senescence and the pro-enzyme must undergo maturation and activation, the protease is probably not involved in nutrient remobilization during senescence but may have another function. The physiological substrates for Cs1-MMP remain to be determined, but the enzyme represents a good candidate for plant ECM degradation and may be involved in programmed cell death (PCD). Our results suggest that PCD occurs only at the culmination of the senescence program or that the processes are distinct with PCD being triggered at the end of senescence. PMID:10889240

  11. Multidestructive pathways triggered in photoreceptor cell death of the rd mouse as determined through gene expression profiling.

    PubMed

    Rohrer, Baerbel; Pinto, Francisco R; Hulse, Kathryn E; Lohr, Heather R; Zhang, Li; Almeida, Jonas S

    2004-10-01

    In the rd/rd mouse, photoreceptor degeneration is due to a mutation of the rod-specific enzyme cGMP phosphodiesterase, resulting in permanently opened cGMP-gated cation channels in the rod outer segment membrane that allow Na(+) and Ca(2+) ions to enter the cell, resulting in possibly toxic levels of Ca(2+). To identify pathways involved in cell death of the rd/rd rods, we evaluated gene expression in the rd/rd and wild type (wt) mouse retina (U74A oligonucleotide arrays (Affymetrix)) over the known time course of photoreceptor degeneration. 181 genes passed the selection criteria (low standard deviation and high correlation between replicates), falling into six clusters. For any given pair of genes, an expression profile correlation distance and a semantic distance (one for each class of gene ontology terms) were established using newly designed software. Gene expression in rd/rd started to deviate from wt by postnatal day 10. The reduction in photoreceptor-specific genes followed the known time course of photoreceptor degeneration. Likewise the increase in transcription factors and apoptosis- and neuroinflammation-specific genes followed the kinetics of the rise in intracellular cGMP in the rod photoreceptors. In addition, genes coding for calcium-binding proteins and those implicated in tissue and vessel remodeling were increased. These results suggest that photoreceptor degeneration in the rd/rd mouse is a process starting with Ca(2+) toxicity followed by secondary insults involving multidestructive pathways such as apoptosis and neuroinflammation, presumably boosting morphological changes. All of these components need to be addressed if rods are to be successfully protected.

  12. Characterization of Cyclin E Expression in Multiple Myeloma and Its Functional Role in Seliciclib-Induced Apoptotic Cell Death

    PubMed Central

    Shimoni, Avichai; Ostrovsky, Olga; Samookh, Michal; Peled, Amnon; Nagler, Arnon

    2012-01-01

    Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator – cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator - cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion–mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy. PMID:22558078

  13. Fibroblast growth factor receptor 4 promotes progression and correlates to poor prognosis in cholangiocarcinoma

    SciTech Connect

    Xu, Yun-Fei; Yang, Xiao-Qing; Lu, Xiao-Fei; Guo, Sen; Liu, Yi; Iqbal, Mohammad; Ning, Shang-Lei; Yang, Hui; Suo, Ning; Chen, Yu-Xin

    2014-03-28

    Highlights: • FGFR4 is significantly related with N stage in IHCC, with T stage and TNM stage in PHCC. • FGFR4 is an independent prognostic factor in IHCC and PHCC. • FGFR4 promotes proliferation, invasion and EMT in cholangiocarinoma cell lines. • Inhibitor AP24354 can decrease proliferation, invasion and induce apoptosis of CCA. - Abstract: Fibroblast growth factor receptor 4 (FGFR4) is related to poor prognosis of several cancers, but the correlation between FGFR4 expression and cholangiocarcinoma (CCA) has not been well elucidated. We investigated the expression of FGFR4 in 83 intrahepatic cholangiocarcinomas (IHCCs), 75 perihilar cholangiocarcinomas (PHCCs) and 41 distal cholangiocarcinomas (DCCs) by immunohistochemistry (IHC), and subsequently evaluated association of FGFR4 with clinicopathologic parameters and survival rate. The rate of FGFR4 higher expression was 61.4% (51/83) in IHCCs, 53.3% (40/75) in PHCCs and 56.1% (23/41) in DCCs. FGFR4 expression was significantly related to poor prognosis of IHCC (P = 0.002) and PHCC (P = 0.019) with univariate analysis, and also identified as an independent prognostic factor in IHCC (P = 0.045) and PHCC (P = 0.049) with multivariate analysis. Additionally, with functional assays in vitro, we found FGFR4 can induce proliferation, invasion and epithelial–mesenchymal transition (EMT) of CCA cell lines with FGF19 stimulation. Moreover, FGFR4 inhibitor AP24354 can suppress proliferation, invasion and induce apoptosis of CCA cells. In conclusion, FGFR4 expression can be identified as a significant independent prognostic biomarker of IHCC and PHCC. FGFR4 played a pivotal role in proliferation, invasion and EMT of CCA. FGFR4 inhibitor can suppress proliferation, invasion and induce apoptosis of CCA, indicating that FGFR4 may act as a potential therapeutic target.

  14. Ubenimex inhibits cell proliferation, migration and invasion by inhibiting the expression of APN and inducing autophagic cell death in prostate cancer cells.

    PubMed

    Wang, Xiaoqing; Niu, Zhihong; Jia, Yang; Cui, Meng; Han, Liping; Zhang, Yongfei; Liu, Zheng; Bi, Dongbin; Liu, Shuai

    2016-04-01

    Prostate cancer is the second most frequently diagnosed cancer in males worldwide and is commonly associated with metastasis. Moreover, in prostate cancer, aminopeptidase N (APN) expression is closely correlated with metastasis. Ubenimex, an APN inhibitor, is widely used as an adjunct therapy for cancer, enhancing the function of immunocompetent cells and conferring antitumor effects. However, due to the low expression of APN, it is rarely used to treat prostate cancer. Recently, the induction of autophagy as a molecular mechanism has been strongly connected with tumor cell death. Thus, we investigated whether ubenimex could inhibit cell proliferation, migration and invasion by downregulating APN expression to induce autophagic cell death in prostate cancer cells. The LNCaP and PC-3 cell lines were treated with different doses of ubenimex. Cell viability was measured using growth curve analysis and WST-8 proliferation assay. Autophagic cell death was assessed using fluorescence microscopy and acridine orange/ethidium bromide (AO/EB) staining. Protein expression was assessed by immunofluorescence and western blot analyses. Autophagosomes were evaluated using transmission electron microscopy. Wound-healing migration assays were performed to determine the migratory ability of the PC-3 cells. In addition, nude mice were used in the present study to examine PC-3 cell proliferation in vivo. The results revealed that APN expression differed between the metastatic and non-metastatic prostate cancer cells. In addition, ubenimex inhibited APN expression in the prostate cancer cells. Ubenimex increased prostate cancer cell death, as determined using the lactate dehydrogenase (LDH) cytotoxicity assay. This effect was accompanied by increased levels of LC3B. Furthermore, ubenimex inhibited PC-3 cell proliferation in vivo and in vitro. Ubenimex inhibited the cell migration and invasion in prostate cancer cells by downregulating APN expression. Finally, ubenimex induced

  15. Inhibitory effect of CXC chemokine receptor 4 antagonist AMD3100 on bleomycin induced murine pulmonary fibrosis.

    PubMed

    Song, Jeong Sup; Kang, Chun Mi; Kang, Hyeon Hui; Yoon, Hyung Kyu; Kim, Young Kyoon; Kim, Kwan Hyung; Moon, Hwa Sik; Park, Sung Hak

    2010-06-30

    CXC chemokine receptor 4 (CXCR4), which binds the stromal cell-derived factor-1 (SDF-1), has been shown to play a critical role in mobilizing the bone marrow (BM)-derived stem cells and inflammatory cells. We studied the effects of AMD3100, CXCR4 antagonist, on a murine bleomycin-induced pulmonary fibrosis model. Treatment of mice with AMD3100 in bleomycin-treated mice resulted in the decrease of SDF-1 in bronchoalveolar lavage (BAL) fluids at an early stage and was followed by the decrease of fibrocytes in the lung. AMD3100 treatment decreased the SDF-1 mRNA expression, fibrocyte numbers in the lung at an early stage (day 3) and CXCR4 expression at the later stage (day 7 and 21) after bleomycin injury. The collagen content and pulmonary fibrosis were significantly attenuated by AMD3100 treatment in later stage of bleomycin injury. AMD3100 treatment also decreased the murine mesenchymal and hematopoietic stem cell chemotaxis when either in the stimulation with bleomycin treated lung lysates or SDF-1 in vitro. In BM stem cell experiments, the phosphorylation of p38 MAPK which was induced by SDF-1 was significantly blocked by addition of AMD3100. Our data suggest that AMD3100 might be effective in preventing the pulmonary fibrosis by inhibiting the fibrocyte mobilization to the injured lung via blocking the SDF-1/CXCR4 axis.

  16. Upregulation of epidermal growth factor receptor 4 in oral leukoplakia

    PubMed Central

    Kobayashi, Hiroshi; Kumagai, Kenichi; Gotoh, Akito; Eguchi, Takanori; Yamada, Hiroyuki; Hamada, Yoshiki; Suzuki, Satsuki; Suzuki, Ryuji

    2013-01-01

    In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP). The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP. PMID:23492901

  17. Reduction of trophic support enhances apoptosis in PC12 cells expressing Alzheimer's APP mutation and sensitizes cells to staurosporine-induced cell death.

    PubMed

    Leutz, Steffen; Steiner, Barbara; Marques, Celio A; Haass, Christian; Müller, Walter E; Eckert, Anne

    2002-06-01

    Mutations in the amyloid precursor protein (APP) gene are known as causative factors in the pathogenesis of early-onset familial Alzheimer's disease (FAD). In this study, the influence of the Swedish double-mutation form of APP (APPsw; KM670/671NL) on apoptosis regulation in PC12 cells was investigated. APPsw-transfected PC12 cells were compared with wild-type APP (APPwt)-expressing and vector-transfected PC12 cells with regard to their susceptibility to cell death induced by the reduction of trophic support or by additional treatment with staurosporine. Expression of APPsw markedly enhanced the level of apoptotic PC12 cells induced by serum reduction. A similar hypersensitivity of APPsw-expressing PC12 cells could be detected after differentiation with nerve growth factor under serum-reduced conditions. Likewise, the expression of APPsw rendered PC12 cells more vulnerable to staurosporine but only under serum-reduced conditions. This APPsw-effect disappeared in high serum-containing medium. Thus, expression of APPsw seems to enhance cellular sensitivity not in general but after the reduction of trophic factors probably by causing oxidative stress. This, in turn, may sensitize cells to secondary apoptotic stimuli. Moreover, the mutation-specific increase in vulnerability to cell death was only seen at the stage of apoptotic nuclei, but not using methods measuring cell death by determining metabolic activity or membrane integrity. Therefore, the expression of APPsw seems to affect specifically apoptotic cell death rather than overall cell death in vitro. Our study further emphasizes the pathogenic role of mutant APP and may provide new insights in the mechanisms underlying the massive neurodegeneration in brain from patients bearing the APPsw mutation.

  18. Inhibition of never in mitosis A (NIMA)-related kinase-4 reduces survivin expression and sensitizes cancer cells to TRAIL-induced cell death.

    PubMed

    Park, So Jung; Jo, Doo Sin; Jo, Se-Young; Shin, Dong Woon; Shim, Sangmi; Jo, Yoon Kyung; Shin, Ji Hyun; Ha, Ye Jin; Jeong, Seong-Yun; Hwang, Jung Jin; Kim, Young Sam; Suh, Young-Ah; Chang, Jong Wook; Kim, Jin Cheon; Cho, Dong-Hyung

    2016-10-04

    The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells. However, many tumors are resistant to TRAIL-induced apoptosis, and resistance mechanisms are not fully understood. To identify novel regulatory molecules of TRAIL resistance, we screened a siRNA library targeting the human kinome, and NEK4 (NIMA-related kinase-4) was identified. Knockdown of NEK4 sensitized TRAIL-resistant cancer cells and in vivo xenografts to cell death. In contrast, over expression of NEK4 suppressed TRAIL-induced cell death in TRAIL-sensitive cancer cells. In addition, loss of NEK4 resulted in decrease of the anti-apoptotic protein survivin, but an increase in apoptotic cell death. Interestingly, NEK4 was highly upregulated in tumor tissues derived from patients with lung cancer and colon cancer. These results suggest that inhibition of NEK4 sensitizes cancer cells to TRAIL-induced apoptosis by regulation of survivin expression.

  19. Inhibition of never in mitosis A (NIMA)-related kinase-4 reduces survivin expression and sensitizes cancer cells to TRAIL-induced cell death

    PubMed Central

    Park, So Jung; Jo, Doo Sin; Jo, Se-Young; Shin, Dong Woon; Shim, Sangmi; Jo, Yoon Kyung; Shin, Ji Hyun; Ha, Ye Jin; Jeong, Seong-Yun; Hwang, Jung Jin; Kim, Young Sam; Suh, Young-Ah; Chang, Jong Wook; Kim, Jin Cheon; Cho, Dong-Hyung

    2016-01-01

    The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells. However, many tumors are resistant to TRAIL-induced apoptosis, and resistance mechanisms are not fully understood. To identify novel regulatory molecules of TRAIL resistance, we screened a siRNA library targeting the human kinome, and NEK4 (NIMA-related kinase-4) was identified. Knockdown of NEK4 sensitized TRAIL-resistant cancer cells and in vivo xenografts to cell death. In contrast, over expression of NEK4 suppressed TRAIL-induced cell death in TRAIL-sensitive cancer cells. In addition, loss of NEK4 resulted in decrease of the anti-apoptotic protein survivin, but an increase in apoptotic cell death. Interestingly, NEK4 was highly upregulated in tumor tissues derived from patients with lung cancer and colon cancer. These results suggest that inhibition of NEK4 sensitizes cancer cells to TRAIL-induced apoptosis by regulation of survivin expression. PMID:27602754

  20. Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer.

    PubMed

    Turkington, R C; Longley, D B; Allen, W L; Stevenson, L; McLaughlin, K; Dunne, P D; Blayney, J K; Salto-Tellez, M; Van Schaeybroeck, S; Johnston, P G

    2014-02-06

    The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

  1. CXC Chemokine Receptor 4 (CXCR4) Antagonist, a Novel Pathway to Prevent Chronic Allograft Nephropathy.

    PubMed

    Xu, Yue; Zhang, Qiang; Xue, Wenrui; Zeng, Song; Zhang, Zijian; Zhang, Xiaodong; Hu, Xiaopeng

    2016-11-25

    BACKGROUND Chronic allograft nephropathy (CAN) remains a major problem for long-term graft survival and different pathways participate in its development. CXC chemokine receptor 4 (CXCR4) is significantly upregulated following renal injury and fibrotic response. We investigated the effect of AMD3100, a CXCR4 antagonist, on the development of CAN in rat models. MATERIAL AND METHODS CAN rat models (n=20) were established using male Fisher 344 to Lewis rats. Rats in the experimental group (n=10) were treated with AMD3100 (1 mg/kg/day subcutaneously, 0-12 weeks), rats in the control group (n=10) were treated with saline. The serum creatinine levels were monitored every week. Kidney grafts were harvested 12 weeks after modeling for histological analysis. We used chronic allograft damage index (CADI) scores to evaluate each group. Q-PCR and Western blotting were used to measure CXCR4, TGF-β1/Smad3 signaling pathway and α-smooth muscle actin (α-SMA) expression in renal allograft tissue. RESULTS CXCR4 expression was increased significantly in the control group which developed intense chronic changes after 12 weeks. Histological changes of CAN in the experimental group were ameliorated by AMD3100 which also had better graft function compare to the control group. AMD3100 significantly blunted the increase in the mRNA expression level of CXCR4, TGF-β1/Smad3, and α-SMA. A significant reduction in TGF-β1 and α-SMA protein content was observed only in the experimental group as shown in a representative Western blot. CONCLUSIONS Based on these findings, CXCR4 expression may mediate in part the development of CAN. AMD3100 may ameliorate histological changes of CAN and maintain better allograft function. It blunts downstream effects of TGF-β1 signaling and fibroblast activation. Therefore, antagonism of CXCR4 may provide a novel way to prevent the development of CAN.

  2. Immunostimulatory bioactivity of algal polysaccharides from Chlorella pyrenoidosa activates macrophages via Toll-like receptor 4.

    PubMed

    Hsu, Hsien-Yeh; Jeyashoke, Narumon; Yeh, Chin-Hsi; Song, Yuan-Jaw; Hua, Kuo-Feng; Chao, Louis Kuoping

    2010-01-27

    Much research suggests that a dietary supplement of Chlorella pyrenoidosa may be helpful to human health, but the molecular mechanism involved remains unclear. The aim of this research was to investigate the effects of certain hot-water-soluble polysaccharides from Chlorella pyrenoidosa (CWSP) on cytokine production, human leukocyte antigen (HLA) expression, and costimulatory molecule expression in macrophages. We demonstrated that CWSP induced IL-1beta secretion in macrophages via Toll-like receptor 4 (TLR4) mediated protein kinase signaling pathways. In addition, CWSP also stimulated the cell surface expression of HLA-DA, -DB, and -DC, and HLA-DR, -DP, and -DQ as well as the expression of costimulatory family molecules such as CD80 and CD86 in macrophages. Furthermore, we demonstrated that preinjection of C57BL/6J mice with CWSP increased lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha and IL-1beta secretion into serum in vivo. This outcome was consistent with the corresponding outcome for cells treated with CWSP in vitro. Our current results provide support for the possible use of CWSP as a modulation agent of immune responses in humans and certain animal species. Finally, in using GC-MS to analyze the polysaccharides, we found that the major monosaccharides of CWSP were rhamnose (31.8%), glucose (20.42%), galactose (10.28%), mannose (5.23%), and xylose (1.27%). This study is the first to report the molecular mechanism of immune-modulated signal transduction in vitro from the polysaccharides of Chlorella pyrenoidosa.

  3. Fibroblast Growth Factor Receptor-4 and Prostate Cancer Progression

    DTIC Science & Technology

    2005-10-01

    promoter. Note log scale. FGFR4 Arg388 transgenics FGFR4 Gfy388 transgenics 4 After establishment of transgenic lines, we sacrificed mice at 2 month...8 PNT1a- FGFR4 PNT1a- FGFR4 Gly/Gly Arg/Arg To analyze expression of Ehm2 at the protein level in vivo we performed immunohistochemical analysis of Ehm2

  4. The expression of '150-kDa oxygen regulated protein (ORP-150)' in human brain and its relationship with duration time until death.

    PubMed

    Ikematsu, Kazuya; Tsuda, Ryouichi; Kondo, Toshikazu; Kondo, Hisayoshi; Ozawa, Kentaro; Ogawa, Satoshi; Nakasono, Ichiro

    2004-04-01

    The expression of oxygen regulated protein 150-kDa (ORP-150) was strongly induced in human brain under the hypoxic conditions. We examined the expression of ORP-150 in the brain samples, and discussed its significance in forensic practice. The cerebral cortexes of 31 cases (asphyxia: 9 cases, hypothermia: 4, exsanguinations: 5, CO intoxication (CO): 6, sudden cardiac death (SCD): 7) were used for this study. Each tissue section was incubated with anti-ORP-150 polyclonal antibody and the number of ORP-150 positive cells were counted. In the multiple linear regression method, the estimated regression coefficient of ORP-150 on age was significant (P=0.039) thus, we could find that the ORP-150 expression level depended on age. Using analysis of covariance, we compared the means of ORP-150, LSMEAN, which means hypothetic average value excluding influence of age, for each cause of death. The LSMEAN+/-SE was 84.74+/-9.03 in hypothermia, 57.52+/-6.34 in asphyxia, 46.68+/-6.70 in CO, 24.84+/-8.05 in exsanguinations, and 16.24+/-7.35 in SCD. As a result of the analysis, the LSMEAN of the ORP-150 expression level was related to the cause of death. There might be differences in the duration of brain ischemia before death. For example, SCD is presumed to be instant death, while brain ischemia continues for several minutes in asphyxia, CO and exsanguinations, and for several hours in hypothermia cases. Therefore, the immunohistochemical and morphometrical analysis of ORP-150 in the brain may be very useful to determine the duration of brain ischemia before death in forensic autopsy cases.

  5. Faces in the face of death: effects of exposure to life-threatening events and mortality salience on facial expression recognition in combat and noncombat military veterans.

    PubMed

    Anaki, David; Brezniak, Tamar; Shalom, Liron

    2012-08-01

    Soldiers in war zones often experience life-threatening events that put their lives at stake. The present study examined how these exposures shape soldiers' social behavior, manifested by recognition of facial expressions. In addition, we investigated how explicit awareness of one's eventual death affects sensitivity to facial expressions. Veterans of elite military combat units were exposed to conditions of mortality or pain salience and later requested to label the emotions depicted in threatening and nonthreatening faces. Combat veterans were more accurate than noncombat veterans in identifying threatening expressions, both in mortality or pain salience induction (experiment 1) or under no induction at all (experiment 2). In addition, noncombat veterans primed with mortality salience identified fear expressions more accurately than those primed with pain salience. Finally, mortality salience improved accuracy for nonthreatening expressions for all veterans. The present results demonstrate that fear of death, resulting from exposure to concrete life-endangering perils or from thoughts on human's inevitable death, influences perception of facial expressions, which is critical for successful interpersonal communication.

  6. [Relation between expression of cerebral beta-APP in the chronic alcoholism rats and death caused by TSAH].

    PubMed

    Wei, Lai; Lei, Huai-Cheng; Yu, Xiao-Jun; Lai, Xiao-Ping; Qian, Hong; Xu, Xiao-Hu; Zhu, Fang-Cheng

    2013-04-01

    By observing the cerebral beta-amyloid precursor protein (beta-APP) expression in the chronic alcoholism rats with slight cerebral injury, to discuss the correlation of chronic alcoholism and death caused by traumatic subarachnoid haemorrhage (TSAH). Sixty male SD rats were randomly divided into watering group, watering group with strike, alcoholism group and alcoholism group with strike. Among them, the alcohol was used for continuous 4 weeks in alcoholism groups and the concussion was made in groups with strike. In each group, HE staining and immunohistochemical staining of the cerebral tissues were done and the results were analyzed by the histopathologic image system. In watering group, there was no abnormal. In watering group with strike, mild neuronic congestion was found. In alcoholism group, vascular texture on cerebral surface was found. And the neurons arranged in disorder with dilated intercellular space. In alcoholism group with strike, diffuse congestion on cerebral surface was found. And there was TSAH with thick-layer patches around brainstem following irregular axonotmesis. The quantity of beta-APP IOD in alcoholism group was significantly higher in the frontal lobe, hippocampus, cerebellum, brainstem than those in watering group with strike and alcoholism group with strike. The cerebral tissues with chronic alcoholism, due to the decreasing tolerance, could cause fatal TSAH and pathological changes in cerebral tissues of rats under slight cerebral injury.

  7. Early embryonic death-associated changes in genome-wide gene expression profiles in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo.

    PubMed

    Oishi, Masahito; Gohma, Hiroshi; Hashizume, Kazuyoshi; Taniguchi, Yukio; Yasue, Hiroshi; Takahashi, Seiya; Yamada, Takahisa; Sasaki, Yoshiyuki

    2006-04-01

    Successful somatic nuclear transfer-derived cloning has been reported in cattle; however, the cloned embryo is highly susceptible to death around day 60 of gestation leading to early embryonic loss. The early embryonic death is postulated to possibly arise in part from an atypical placentation. We have performed cDNA macroarray analysis using 3,353 of the previously cataloged 4,165 genes, in order to characterize the early embryonic death-associated changes in genome-wide gene expression profiles in the fetal placenta of the cow carrying somatic nuclear transfer-derived cloned embryo. A more marked difference in the expression profiles was observed between the fetal placentas of the cows with the cloned immotile embryo (CD) and with the cloned motile embryo (CL) or artificial insemination-derived motile embryo (AI), as compared to between the CL and AI placentas, suggesting an aberration of the expression profile in the CD placenta among the three placentas. Further, 291 and 77 genes showed more than twofold elevation and less than 50% reduction, respectively, in either or both of two CD (CD1 and CD2) placentas in comparison with the CL placenta, but no differential expression between the CL and AI placentas. The expression patterns of six genes in the AI, CL, and CD placentas were confirmed in an experiment with an additional sample for each of the three placentas. Among the placental genes showing the early embryonic death-associated changes of expression in the cow with the cloned embryo, IGF2 (elevated gene), and HBA1, HBA2, SPTB, and SPTBN1 genes (reduced gene) are intriguing in that the changes of expression in these genes were observed in an additional sample of CD placenta as well as the CD1 and CD2 placentas, and in that overexpression (for IGF2) and dysfunction or deficiency (for HBA1, HBA2, SPTB, and SPTBN1) result in embryonic lethality. Copyright 2006 Wiley-Liss, Inc.

  8. Targeting N-cadherin through fibroblast growth factor receptor-4: distinct pathogenetic and therapeutic implications.

    PubMed

    Ezzat, Shereen; Zheng, Lei; Winer, Daniel; Asa, Sylvia L

    2006-11-01

    Several molecular aberrations have been implicated in the pathogenesis of pituitary tumors, but few have proven thus far to be of therapeutic value. Pituitary tumor-derived fibroblast growth factor receptor-4 (ptd-FGFR4) is an alternatively transcribed cytoplasmic isoform lacking most of the extracellular domain. This oncogene recapitulates the morphological features of human pituitary tumors in transgenic mice. To investigate the therapeutic potential of targeting ptd-FGFR4, we examined the impact of FGFR4 tyrosine kinase inhibition in xenografted mice. GH4 pituitary cells expressing ptd-FGFR4 develop into invasive tumors. Systemic treatment of mice bearing ptd-FGFR4 tumors with the FGFR-selective inhibitor PD173074 resulted in recovery of membranous N-cadherin staining and a significant reduction in tumor volume with less invasive growth behavior. Mutation of tyrosine Y754F in ptd-FGFR4 abrogated the effect of PD173074-mediated inhibition. The pivotal role of N-cadherin as a mediator of this pituitary cell growth was demonstrated by small interfering RNA mediated down-regulation, which promoted invasive growth in xenografted mice. To validate this model in primary human pituitary tumors, we examined the expression of ptd-FGFR4, N-cadherin, and clinical behavior. Loss of membranous N-cadherin correlated with cytoplasmic FGFR4 expression and with tumor invasiveness in surgically resected human pituitary tumors. Primary human pituitary tumor cells treated with PD173074 showed restoration of N-cadherin to the membrane with dephosphorylation of retinoblastoma protein. These data highlight the pathogenetic significance of N-cadherin misexpression and emphasize the importance of FGFR partnership in mediating its functions.

  9. Endothelial cell Toll-like receptor 4 regulates fibrosis associated angiogenesis in liver

    PubMed Central

    Jagavelu, K; Routray, C; Shergill, U; O’Hara, SP; Faubion, W; Shah, VH

    2010-01-01

    Angiogenesis defines the growth of new blood vessels from pre-existing vascular endothelial networks and corresponds with the wound healing process that is typified by the process of liver fibrosis. Liver fibrosis is also associated with increased endotoxin within the gut lumen and its associated portal circulation. However, the interrelationship of gut endotoxin and its receptor, Toll-like receptor 4 (TLR4), with liver fibrosis and associated angiogenesis remains incompletely defined. RESULT Here we provide evidence, using complementary genetic, molecular, and pharmacologic approaches that the pattern recognition receptor that recognizes endotoxin, TLR4, expressed on liver endothelial cells (LEC), regulates angiogenic responses both in vitro and in vivo. Mechanistic studies reveal a key role for a cognate TLR4 effector protein, MyD88 in this process which culminates in extracellular protease production that regulates LEC invasive capacity, a key step in angiogenesis. Furthermore TLR4 dependent angiogenesis in vivo corresponds with fibrosis in complementary liver models of fibrosis. CONCLUSION These studies provide evidence that the TLR4 pathway in LEC regulates angiogenesis through its MyD88 effector protein by regulating extracellular protease production and that this process is linked to the development of liver fibrosis. PMID:20564354

  10. Death Imagery and Death Anxiety.

    ERIC Educational Resources Information Center

    McDonald, Rita T.; Hilgendorf, William A.

    1986-01-01

    Investigated the relationship between death imagery and death anxiety among 179 undergraduate students. Results reveal subjects with low death anxiety scores had more positive death images. Subjects who imagined death to be young had a more positive image of death. Death was seen as male by majority of respondents. (Author/BL)

  11. Cancer Cell Growth Inhibitory Effect of Bee Venom via Increase of Death Receptor 3 Expression and Inactivation of NF-kappa B in NSCLC Cells

    PubMed Central

    Choi, Kyung Eun; Hwang, Chul Ju; Gu, Sun Mi; Park, Mi Hee; Kim, Joo Hwan; Park, Joo Ho; Ahn, Young Jin; Kim, Ji Young; Song, Min Jong; Song, Ho Sueb; Han, Sang-Bae; Hong, Jin Tae

    2014-01-01

    Our previous findings have demonstrated that bee venom (BV) has anti-cancer activity in several cancer cells. However, the effects of BV on lung cancer cell growth have not been reported. Cell viability was determined with trypan blue uptake, soft agar formation as well as DAPI and TUNEL assay. Cell death related protein expression was determined with Western blotting. An EMSA was used for nuclear factor kappaB (NF-κB) activity assay. BV (1–5 μg/mL) inhibited growth of lung cancer cells by induction of apoptosis in a dose dependent manner in lung cancer cell lines A549 and NCI-H460. Consistent with apoptotic cell death, expression of DR3 and DR6 was significantly increased. However, deletion of DRs by small interfering RNA significantly reversed BV induced cell growth inhibitory effects. Expression of pro-apoptotic proteins (caspase-3 and Bax) was concomitantly increased, but the NF-κB activity and expression of Bcl-2 were inhibited. A combination treatment of tumor necrosis factor (TNF)-like weak inducer of apoptosis, TNF-related apoptosis-inducing ligand, docetaxel and cisplatin, with BV synergistically inhibited both A549 and NCI-H460 lung cancer cell growth with further down regulation of NF-κB activity. These results show that BV induces apoptotic cell death in lung cancer cells through the enhancement of DR3 expression and inhibition of NF-κB pathway. PMID:25068924

  12. A major suppressor of cell death, slm1, modifies the expression of the maize (Zea mays L.) lesion mimic mutation les23.

    PubMed

    Penning, Bryan W; Johal, Gurmukh S; McMullen, Michael D

    2004-10-01

    Disease lesion mimics provide an excellent biological system to study the genetic basis of cell death in plants. Many lesion mimics show variation in phenotype expression in different genetic backgrounds. Our goal was to identify quantitative trait loci (QTL) modifying lesion mimic expression thereby identifying genetic modifiers of cell death. A recessive lesion mimic, les23, in a severe-expressing line was crossed to the maize inbred line Mo20W, a lesion-suppressing line, and an F(2) population was developed for QTL analysis. In addition to locating les23 to the short arm of chromosome 2, this analysis detected significant loci for modification of lesion expression. One highly significant locus was found on the long arm of chromosome 2. The Mo20W allele at this QTL significantly delayed initiation of the lesion phenotype and decreased the final lesion severity. Other QTL with lesser effect affected severity of lesion expression without affecting lesion initiation date. Our results demonstrate that dramatic change in lesion phenotype can be controlled by a single major QTL. The presumed function of this QTL in normal plants is to regulate some aspect of the cell death pathway underlying the les23 phenotype.

  13. In vivo expression of p53 and Bcl-2 and their role in programmed cell death in premalignant and malignant lung lesions.

    PubMed

    Koty, Patrick P; Zhang, Haifan; Franklin, Wilbur A; Yousem, Samuel A; Landreneau, Rodney; Levitt, Mark L

    2002-02-01

    Forty-four specimens of non-malignant and malignant human lung tissue, taken from patients with non-small cell lung cancer (NSCLC), were examined for the expression of wild-type p53, mutant p53, and bcl-2 and the occurrence of programmed cell death (apoptosis). Wild-type p53 expression peaked in peritumoral and metaplastic samples, whereas mutant p53, bcl-2 and apoptosis were first detected in metaplasia and increased with progression to carcinoma. Bcl-2 positive samples had lower levels of apoptosis than bcl-2 negative samples and was independent of wild-type or mutant p53 expression. These results suggest that the over-expression of wild-type p53 may be an early cellular response to an alteration in normal cellular homeostasis. The ensuing increase in apoptosis appears to be relatively independent of mutant or wild-type p53 expression, but does not occur in cells expressing bcl-2.

  14. Whither brain death?

    PubMed

    Bernat, James L

    2014-01-01

    The publicity surrounding the recent McMath and Muñoz cases has rekindled public interest in brain death: the familiar term for human death determination by showing the irreversible cessation of clinical brain functions. The concept of brain death was developed decades ago to permit withdrawal of therapy in hopeless cases and to permit organ donation. It has become widely established medical practice, and laws permit it in all U.S. jurisdictions. Brain death has a biophilosophical justification as a standard for determining human death but remains poorly understood by the public and by health professionals. The current controversies over brain death are largely restricted to the academy, but some practitioners express ambivalence over whether brain death is equivalent to human death. Brain death remains an accepted and sound concept, but more work is necessary to establish its biophilosophical justification and to educate health professionals and the public.

  15. ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

    PubMed

    Miyamae, Yuka; Mochizuki, Satsuki; Shimoda, Masayuki; Ohara, Kentaro; Abe, Hitoshi; Yamashita, Shuji; Kazuno, Saiko; Ohtsuka, Takashi; Ochiai, Hiroki; Kitagawa, Yuko; Okada, Yasunori

    2016-05-01

    ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells.

  16. Asparagine reduces the mRNA expression of muscle atrophy markers via regulating protein kinase B (Akt), AMP-activated protein kinase α, toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling in weaning piglets after lipopolysaccharide challenge.

    PubMed

    Wang, Xiuying; Liu, Yulan; Wang, Shuhui; Pi, Dingan; Leng, Weibo; Zhu, Huiling; Zhang, Jing; Shi, Haifeng; Li, Shuang; Lin, Xi; Odle, Jack

    2016-10-01

    Pro-inflammatory cytokines are critical in mechanisms of muscle atrophy. In addition, asparagine (Asn) is necessary for protein synthesis in mammalian cells. We hypothesised that Asn could attenuate lipopolysaccharide (LPS)-induced muscle atrophy in a piglet model. Piglets were allotted to four treatments (non-challenged control, LPS-challenged control, LPS+0·5 % Asn and LPS+1·0 % Asn). On day 21, the piglets were injected with LPS or saline. At 4 h post injection, piglet blood and muscle samples were collected. Asn increased protein and RNA content in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). However, Asn had no effect on the protein abundance of MAFbx and MuRF1. In addition, Asn decreased muscle AMP-activated protein kinase (AMPK) α phosphorylation, but increased muscle protein kinase B (Akt) and Forkhead Box O (FOXO) 1 phosphorylation. Moreover, Asn decreased the concentrations of TNF-α, cortisol and glucagon in plasma, and TNF-α mRNA expression in muscles. Finally, Asn decreased mRNA abundance of muscle toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) signalling-related genes, and regulated their negative regulators. The beneficial effects of Asn on muscle atrophy may be associated with the following: (1) inhibiting muscle protein degradation via activating Akt and inactivating AMPKα and FOXO1; and (2) decreasing the expression of muscle pro-inflammatory cytokines via inhibiting TLR4 and NOD signalling pathways by modulation of their negative regulators.

  17. Sialomucin expression is associated with erbB-2 oncoprotein overexpression, early recurrence, and cancer death in non-small-cell lung cancer.

    PubMed

    Yu, C J; Shun, C T; Yang, P C; Lee, Y C; Shew, J Y; Kuo, S H; Luh, K T

    1997-04-01

    Mucin production, when heavily sialylated, can promote cancer cell invasion and metastasis, and modulate the immune recognition system of the host. To explore the prognostic implication of sialomucin expression in lung cancer, we studied 116 patients with non-small-cell lung cancer (NSCLC). Tumor specimens were stained immunohistochemically with monoclonal antibodies (mAbs) against mucin glycoprotein (17Q2, HMFG2, SM3), and histochemically with periodic acid-Schiff/alcian blue to differentiate neutral mucin from acid mucin, and with high-iron diamine/alcian blue to differentiate sialomucin from sulfomucin. The expression status of two established molecular prognostic factors, the p53 and erbB-2 oncoproteins, were evaluated immunohistochemically. The staining was performed on two separately archived, paraffin-embedded tumor blocks for each patient, with normal lung as a control. Correlations were subsequently made among stains and various clinicopathologic factors. All analyses were blinded, and included Kaplan-Meier survival estimates with Cox proportional hazards regression modeling. Associations were established among adenocarcinoma histotype and erbB-2 overexpression, sialomucin expression, and 17Q2 and HMFG2 immunohistochemical positivity (p < 0.05). Sialomucin expression was closely linked to erbB-2 overexpression (p = 0.01). Significant univariate predictors (p < 0.05) of recurrence and cancer death were surgical stage, p53 expression, erbB-2 overexpression, and sialomucin expression. These four factors remained as independent predictors of early recurrence (p < 0.05) after multivariate analysis. For cancer death prediction, p53 and sialomucin expression had a marginal effect. We concluded that sialomucin expression is also a poor indicator of prognosis, which is associated with erbB-2 oncoprotein overexpression, early postoperative recurrence, and cancer death in NSCLC.

  18. Toll-like receptor 4 deficiency causes pulmonary emphysema.

    PubMed

    Zhang, Xuchen; Shan, Peiying; Jiang, Ge; Cohn, Lauren; Lee, Patty J

    2006-11-01

    TLRs have been studied extensively in the context of pathogen challenges, yet their role in the unchallenged lung is unknown. Given their direct interface with the external environment, TLRs in the lungs are prime candidates to respond to air constituents, namely particulates and oxygen. The mechanism whereby the lung maintains structural integrity in the face of constant ambient exposures is essential to our understanding of lung disease. Emphysema is characterized by gradual loss of lung elasticity and irreversible airspace enlargement, usually in the later decades of life and after years of insult, most commonly cigarette smoke. Here we show Tlr4(-/-) mice exhibited emphysema as they aged. Adoptive transfer experiments revealed that TLR4 expression in lung structural cells was required for maintaining normal lung architecture. TLR4 deficiency led to the upregulation of what we believe to be a novel NADPH oxidase (Nox), Nox3, in lungs and endothelial cells, resulting in increased oxidant generation and elastolytic activity. Treatment of Tlr4(-/- )mice or endothelial cells with chemical NADPH inhibitors or Nox3 siRNA reversed the observed phenotype. Our data identify a role for TLR4 in maintaining constitutive lung integrity by modulating oxidant generation and provide insights into the development of emphysema.

  19. Luminal Cathepsin G and Protease-Activated Receptor 4

    PubMed Central

    Dabek, Marta; Ferrier, Laurent; Roka, Richard; Gecse, Krisztina; Annahazi, Anita; Moreau, Jacques; Escourrou, Jean; Cartier, Christel; Chaumaz, Gilles; Leveque, Mathilde; Ait-Belgnaoui, Afifa; Wittmann, Tibor; Theodorou, Vassilia; Bueno, Lionel

    2009-01-01

    Impairment of the colonic epithelial barrier and neutrophil infiltration are common features of inflammatory bowel disease. Luminal proteases affect colonic permeability through protease-activated receptors (PARs). We evaluated: (i) whether fecal supernatants from patients with ulcerative colitis (UC) trigger alterations of colonic paracellular permeability and inflammation, and (ii) the roles of cathepsin G (Cat-G), a neutrophil serine protease, and its selective receptor, PAR4, in these processes. Expression levels of both PAR4 and Cat-G were determined in colonic biopsies from UC and healthy subjects. The effects of UC fecal supernatants on colonic paracellular permeability were measured in murine colonic strips. Involvement of Cat-G and PAR4 was evaluated using pepducin P4pal-10 and specific Cat-G inhibitor (SCGI), respectively. In addition, the effect of PAR4-activating peptide was assessed. UC fecal supernatants, either untreated or pretreated with SCGI, were infused into mice, and myeloperoxidase activity was determined. PAR4 was found to be overexpressed in UC colonic biopsies. Increased colonic paracellular permeability that was triggered by UC fecal supernatants was blocked by both SCGI (77%) and P4pal-10 (85%). Intracolonic infusion of UC fecal supernatants into mice increased myeloperoxidase activity. This effect was abolished by SCGI. These observations support that both Cat-G and PAR4 play key roles in generating and/or amplifying relapses in UC and provide a rationale for the development of new therapeutic agents in the treatment of this disease. PMID:19528350

  20. Toll-like receptor 4 promotes fibrosis in bleomycin-induced lung injury in mice.

    PubMed

    Li, X X; Jiang, D Y; Huang, X X; Guo, S L; Yuan, W; Dai, H P

    2015-12-21

    The specific role of Toll-like receptor 4 (TLR4) in bleomycin-induced lung fibrosis of mice, a model of human idiopathic pulmonary fibrosis, has not been characterized. We injected bleomycin intratracheally into TLR4 knockout (TLR4(-/-)) and wild-type (WT) mice. Twenty-one days after injection, mice were sacrificed and their lungs were harvested for pathological, hydroxyproline, mRNA expression, and collagen I analyses. Body weight changes and mortality were observed. Light microscopy showed that lung fibrosis was minimal in TLR4(-/-) compared to that in WT mice on day 21 after bleomycin instillation. The Ashcroft score was significantly lower in TLR4(-/-) than in WT mice (3.667 ± 0.730 vs 4.945 ± 0.880, P < 0.05). Hydroxyproline content was significantly lower in TLR4(-/-) than in WT mice on day 21 after bleomycin injection (0.281 ± 0.022 vs 0.371 ± 0.047, P < 0.05). Compared to WT mice, bleomycin-treated TLR4(-/-) mice expressed significantly lower type I collagen mRNA levels (mesenchymal marker; 11.069 ± 2.627 vs 4.589 ± 1.440, P < 0.05). Collagen I was significantly lower in TLR4(-/-) than in WT mice (0.838 ± 0.352 vs 2.427 ± 0.551, P < 0.05). Bleomycin-treated TLR4(-/-) mice had a significantly lower mortality rate on day 21 than WT mice (33 vs 75%, P < 0.05). Body weight reduction was lower in TLR4(-/-) mice than in WT mice; this difference was not statistically significant (-3.735 ± 5.276 vs -6.698 ± 3.218, P > 0.05). Thus, bleomycin-induced pulmonary fibrosis is TLR4-dependent and TLR4 promoted fibrosis in bleomycin-challenged mice.

  1. Toll-like receptor 4 regulates chronic stress-induced visceral pain in mice.

    PubMed

    Tramullas, Monica; Finger, Beate C; Moloney, Rachel D; Golubeva, Anna V; Moloney, Gerard; Dinan, Timothy G; Cryan, John F

    2014-08-15

    Functional gastrointestinal disorders, which have visceral hypersensitivity as a core symptom, are frequently comorbid with stress-related psychiatric disorders. Increasing evidence points to a key role for toll-like receptor 4 (TLR4) in chronic pain states of somatic origin. However, the central contribution of TLR4 in visceral pain sensation remains elusive. With pharmacological and genetic approaches, we investigated the involvement of TLR4 in the modulation of visceral pain. The TLR4-deficient and wild-type mice were exposed to chronic stress. Visceral pain was evaluated with colorectal distension. Protein expression levels for TLR4, Cd11b, and glial fibrillary acidic protein (glial cells markers) were quantified in the lumbar region of the spinal cord, prefrontal cortex (PFC), and hippocampus. To evaluate the effect of blocking TLR4 on visceral nociception, TAK-242, a selective TLR4 antagonist, was administered peripherally (intravenous) and centrally (intracerebroventricular and intra-PFC) (n = 10-12/experimental group). The TLR4 deficiency reduced visceral pain and prevented the development of chronic psychosocial stress-induced visceral hypersensitivity. Increased expression of TLR4 coupled with enhanced glia activation in the PFC and increased levels of proinflammatory cytokines were observed after chronic stress in wild-type mice. Administration of a TLR4 specific antagonist, TAK-242, attenuated visceral pain sensation in animals with functional TLR4 when administrated centrally and peripherally. Moreover, intra-PFC TAK-242 administration also counteracted chronic stress-induced visceral hypersensitivity. Our results reveal a novel role for TLR4 within the PFC in the modulation of visceral nociception and point to TLR4 as a potential therapeutic target for the development of drugs to treat visceral hypersensitivity. © 2013 Society of Biological Psychiatry Published by Society of Biological Psychiatry All rights reserved.

  2. Pharmacological characterization of LPS and opioid interactions at the toll-like receptor 4.

    PubMed

    Stevens, C W; Aravind, S; Das, S; Davis, R L

    2013-03-01

    Previous work in our laboratory showed opioid agents inhibit cytokine expression in astrocytes. Recently, Watkins and colleagues hypothesized that opioid agonists activate toll-like receptor 4 (TLR4) signalling, which leads to neuroinflammation. To test this hypothesis, we characterized LPS and opioid effects on TLR4 signalling in reporter cells. NF-κB reporter cells expressing high levels of TLR4 were used to compare LPS and opioid effects on NF-κB activation, a pathway activated by TLR4 stimulation. LPS increased TLR4 signalling in a concentration-dependent manner and was antagonized by LPS antagonist (LPS-RS, from Rhodobacter sphaeroides). A concentration ratio analysis showed that LPS-RS was a competitive antagonist. The opioid agonists, morphine and fentanyl, produced minor activation of TLR4 signalling when given alone. When tested following LPS stimulation, opioid agonists inhibited NF-κB activation but this inhibition was not blocked by the general opioid antagonist, naloxone, nor by the selective μ opioid receptor antagonist, β-FNA. Indeed, both naloxone and β-FNA also inhibited NF-κB activation in reporter cells. Further examination of fentanyl and β-FNA effects revealed that both opioid agents inhibited LPS signalling in a non-competitive fashion. These results show that LPS-RS is a competitive antagonist at the TLR4 complex, and that both opioid agonists and antagonists inhibit LPS signalling in a non-competitive fashion through a non-GPCR, opioid site(s) in the TLR4 signalling pathway. If confirmed, existing opioid agents or other drug molecules more selective at this novel site may provide a new therapeutic approach to the treatment of neuroinflammation. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  3. Targeting Toll-like receptor 4 prevents cobalt-mediated inflammation.

    PubMed

    Lawrence, Helen; Mawdesley, Amy Elizabeth; Holland, James Patrick; Kirby, John Andrew; Deehan, David John; Tyson-Capper, Alison Jane

    2016-02-16

    Cobalt-chrome alloy is a widely used biomaterial in joint replacements, dental implants and spinal rods. Although it is an effective and biocompatible material, adverse reactions to metal debris (ARMD) have arisen in a minority of patients, particularly in those with metal-on-metal bearing hip replacements. There is currently no treatment for ARMD and once progressive, early revision surgery of the implant is necessary. Therapeutic agents to prevent, halt or reverse ARMD would therefore be advantageous. Cobalt ions activate Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to bacterial lipopolysaccharide (LPS) resulting in the production of pro-inflammatory cytokines and chemokines. We hypothesised that anti-TLR4 neutralising antibodies, reported to inhibit TLR4-mediated inflammation, could prevent the inflammatory response to cobalt ions in an in vitro macrophage cell culture model. This study shows that a monoclonal anti-TLR4 antibody inhibited cobalt-mediated increases in pro-inflammatory IL8, CCL20 and IL1A expression, as well as IL-8 secretion. In contrast, a polyclonal antibody did not prevent the effect of cobalt ions on either IL-8 or IL1A expression, although it did have a small effect on the CCL20 response. Interestingly, both antibodies inhibited cobalt-mediated neutrophil migration although the greater effect was observed with the monoclonal antibody. In summary our data shows that a monoclonal anti-TLR4 antibody can inhibit cobalt-mediated inflammatory responses while a polyclonal antibody only inhibits the effect of specific cytokines. Anti-TLR4 antibodies have therapeutic potential in ARMD although careful antibody design is required to ensure that the LPS response is preserved.

  4. Targeting Toll-like receptor 4 prevents cobalt-mediated inflammation

    PubMed Central

    Lawrence, Helen; Mawdesley, Amy Elizabeth; Holland, James Patrick; Kirby, John Andrew; Deehan, David John; Tyson-Capper, Alison Jane

    2016-01-01

    Cobalt-chrome alloy is a widely used biomaterial in joint replacements, dental implants and spinal rods. Although it is an effective and biocompatible material, adverse reactions to metal debris (ARMD) have arisen in a minority of patients, particularly in those with metal-on-metal bearing hip replacements. There is currently no treatment for ARMD and once progressive, early revision surgery of the implant is necessary. Therapeutic agents to prevent, halt or reverse ARMD would therefore be advantageous. Cobalt ions activate Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to bacterial lipopolysaccharide (LPS) resulting in the production of pro-inflammatory cytokines and chemokines. We hypothesised that anti-TLR4 neutralising antibodies, reported to inhibit TLR4-mediated inflammation, could prevent the inflammatory response to cobalt ions in an in vitro macrophagecell culture model. This study shows that a monoclonal anti-TLR4 antibody inhibited cobalt-mediated increases in pro-inflammatory IL8, CCL20 and IL1A expression, as well as IL-8 secretion. In contrast, a polyclonal antibody did not prevent the effect of cobalt ions on either IL-8 or IL1A expression, although it did have a small effect on the CCL20 response. Interestingly, both antibodies inhibited cobalt-mediated neutrophil migration although the greater effect was observed with the monoclonal antibody. In summary our data shows that a monoclonal anti-TLR4 antibody can inhibit cobalt-mediated inflammatory responses while a polyclonal antibody only inhibits the effect of specific cytokines. Anti-TLR4 antibodies have therapeutic potential in ARMD although careful antibody design is required to ensure that the LPS response is preserved. PMID:26840091

  5. A toll-like receptor 4 (TLR4) variant is associated with asthma severity

    PubMed Central

    Zhang, Long; Xu, Ai-Guo; Zhao, Wei; Xu, Qin-Fu; Zhao, Yu-Miao; Li, Dan-Dan; Shi, Xiao-Ya; Zhao, Jun-Jie

    2015-01-01

    Asthma is a complex airways disease resulting from the input of both biological and environmental factors. Previous studies of single-nucleotide polymorphisms in toll-like receptor 4 (TLR4), which produces a protein involved in regulating T cell populations, have presented conflicting results regarding its role in asthma severity. In the current study, individuals with asthma were genotyped for variants of TLR4, and the genotypes were compared with asthma severity and T cell subpopulations. TLR4 rs11536879 (A>G) and rs1927907 (G>A) genotypes were determined in 350 asthma patients using TaqMan. Asthma severity was graded by clinical symptoms, and blood markers and lung function measures were also collected. T cell subpopulations were identified from peripheral blood by flow cytometry. No significant correlations were observed between genotypes at TLR4 rs11536879 or rs1927907 and eosinophil counts, total serum IgE, serum hypersensitive C-reactive protein, forced expiratory volume in 1 second (FEV1%), or FEV1/forced vital capacity (FVC) in asthma patients (P > 0.05). However, the GG genotype of rs1927907 was correlated with higher asthma severity (P < 0.05). No associations were detected between genotypes at rs11536879 or rs1927907 and CD4+CD25high regulatory T cell counts in peripheral blood from asthmatic patients (P > 0.05), but the rs1927907 genotype was associated with TLR4 expression on the surface of CD4+CD25high regulatory T cells (P < 0.05). Therefore, the TLR4 variant rs1927907 appears to be related to asthma severity and TLR4 expression on the surface of CD4+CD25high regulatory T cells, suggesting the potential influence of TLR4 on T cell population balances. PMID:26221339

  6. Regulation of Fasting Fuel Metabolism by Toll-Like Receptor 4

    PubMed Central

    Pang, Shanshan; Tang, Haiqing; Zhuo, Shu; Zang, Ying Qin; Le, Yingying

    2010-01-01

    OBJECTIVE Toll-like receptor 4 (TLR4) has been reported to induce insulin resistance through inflammation in high-fat–fed mice. However, the physiological role of TLR4 in metabolism is unknown. Here, we investigated the involvement of TLR4 in fasting metabolism. RESEARCH DESIGN AND METHODS Wild-type and TLR4 deficient (TLR4−/−) mice were either fed or fasted for 24 h. Glucose and lipid levels in circulation and tissues were measured. Glucose and lipid metabolism in tissues, as well as the expression of related enzymes, was examined. RESULTS Mice lacking TLR4 displayed aggravated fasting hypoglycemia, along with normal hepatic gluconeogenesis, but reversed activity of pyruvate dehydrogenase complex (PDC) in skeletal muscle, which might account for the fasting hypoglycemia. TLR4−/− mice also exhibited higher lipid levels in circulation and skeletal muscle after fasting and reversed expression of lipogenic enzymes in skeletal muscle but not liver and adipose tissue. Adipose tissue lipolysis is normal and muscle fatty acid oxidation is increased in TLR4−/− mice after fasting. Inhibition of fatty acid synthesis in TLR4−/− mice abolished hyperlipidemia, hypoglycemia, and PDC activity increase, suggesting that TLR4-dependent inhibition of muscle lipogenesis may contribute to glucose and lipid homeostasis during fasting. Further studies showed that TLR4 deficiency had no effect on insulin signaling and muscle proinflammatory cytokine production in response to fasting. CONCLUSIONS These data suggest that TLR4 plays a critical role in glucose and lipid metabolism independent of insulin during fasting and identify a novel physiological role for TLR4 in fuel homeostasis. PMID:20855545

  7. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos

    USGS Publications Warehouse

    Toomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.E.; Di Giulio, R.T.

    2001-01-01

    Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC50 and LD50 of TCDD for Fundulus embryos were ???19.7??9.5 pg TCDD/??l (water bath) and 0.25??0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction. Copyright ?? 2001 Elsevier Science B.V.

  8. Extracellular nucleotide inhibits cell proliferation and negatively regulates Toll-like receptor 4 signalling in human progenitor endothelial cells.

    PubMed

    Xiao, Zhilin; Yang, Mei; Fang, Li; Lv, Qingshan; He, Qing; Deng, Minjie; Liu, Xueting; Chen, Xiaobin; Chen, Meifang; Xie, Xiumei; Hu, Jinyue

    2012-07-01

    Extracellular nucleotides mediate a wide range of physiological effects by interacting with plasma membrane P2 purinergic receptors. P2 receptors are expressed in certain kinds of stem cells, and function to induce cytokine expression and to modulate cell proliferation. We have analysed the expression and the function of P2 receptors in human umbilical cord blood-derived EPCs (endothelial progenitor cells). EPCs expressed P2X4,6,7 and P2Y2,4,11,13,14 receptors and extracellular ATP inhibited EPCs proliferation. As in a previous study, EPCs expressed functional TLR4 (Toll-like receptor 4) and activation of TLR4 by LPS (lipopolysaccharide) evoked a pro-inflammatory immune response. When human EPCs were stimulated with LPS and nucleotides, ATP or UTP inhibited the expression of pro-inflammatory cytokines including MCP-1 (monocyte chemoattractant protein-1), IFNα (interferon α), TNFα (tumour necrosis factor α) and adhesion molecule VCAM-1 (vascular cell adhesion molecule 1) induced by LPS. ATP and UTP also down-regulated the gene expression of TLR4, CD14 and MyD88 (myeloid differentiation factor 88), a TLR adaptor molecule, and protein expression of CD14 and MyD88. Moreover, the phosphorylation of NF-κB (nuclear factor κB) p65 induced by TLR4 activation was inhibited partly by ATP or UTP at concentrations of 1-5 μM. These results suggest that extracellular nucleotides negatively regulate EPCs proliferation and TLR4 signalling.

  9. Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxiainducible factor-1alpha expression via Poly(ADP-ribose) polymerase -1

    PubMed Central

    Pan, Rong; Chen, Chen; Liu, Wenlan; Liu, Ke Jian

    2013-01-01

    Aim Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study testes the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Methods Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-hour hypoxic treatment. Results Although 3-hr hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc concentration dependent manner. Exposure of astrocytes to hypoxia for 3-hr remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Conclusions Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. PMID:23582235

  10. Induction of antigen-positive cell death by the expression of perforin, but not DTa, from a DNA vaccine enhances the immune response.

    PubMed

    Gargett, Tessa; Grubor-Bauk, Branka; Garrod, Tamsin J; Yu, Wenbo; Miller, Darren; Major, Lee; Wesselingh, Steve; Suhrbier, Andreas; Gowans, Eric J

    2014-04-01

    The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response.

  11. Programmed death 1 expression in variant immunoarchitectural patterns of nodular lymphocyte predominant Hodgkin lymphoma: comparison with CD57 and lymphomas in the differential diagnosis.

    PubMed

    Churchill, Hywyn R O; Roncador, Giovanna; Warnke, Roger A; Natkunam, Yasodha

    2010-12-01

    Recent studies have exploited an antibody directed against programmed death 1 expressed by follicular helper T-cells in the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma. We had previously described clinically relevant, variant immunoarchitectural patterns of nodular lymphocyte predominant Hodgkin lymphoma and, in this study, sought to address the diagnostic utility of programmed death 1 in comparison with CD57 in variant nodular lymphocyte predominant Hodgkin lymphoma. Immunohistologic staining for programmed death 1 was carried out on biopsies of 67 patients with variant nodular lymphocyte predominant Hodgkin lymphoma. Thirty-four additional cases of nodular lymphocyte predominant Hodgkin lymphoma with associated diffuse areas, de novo T-cell and histiocyte-rich large B-cell lymphoma, and lymphocyte-rich classic Hodgkin lymphoma were also studied. Our results show that programmed death 1 positivity was found in the majority of nodular lymphocyte predominant Hodgkin lymphoma cases with a classic nodular architecture (87%) as compared with 50% for CD57 and was particularly helpful in identifying extranodular large atypical cells. Nodular lymphocyte predominant Hodgkin lymphoma with diffuse areas showed a gradual decrease in programmed death 1 reactivity from nodular to diffuse areas, although a significant proportion (40%-50%) of cases retained programmed death 1 positivity also in diffuse areas. In addition, T-cell and histiocyte-rich large B-cell lymphoma and lymphocyte-rich classic Hodgkin lymphoma displayed programmed death 1 positivity in a significant subset of cases (33%-40%). In conclusion, our study supports the utility of programmed death 1 in the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma and shows greater sensitivity of staining of programmed death 1 as compared with CD57 across all variants of nodular lymphocyte predominant Hodgkin lymphoma. Loss of programmed death 1 reactivity did not correlate with diffuse areas

  12. Proapoptotic BH3-Only Bcl-2 Family Member Bik/Blk/Nbk Is Expressed in Hemopoietic and Endothelial Cells but Is Redundant for Their Programmed Death

    PubMed Central

    Coultas, Leigh; Bouillet, Philippe; Stanley, Edouard G.; Brodnicki, Thomas C.; Adams, Jerry M.; Strasser, Andreas

    2004-01-01

    The BH3-only members of the Bcl-2 protein family are essential for initiation of programmed cell death and stress-induced apoptosis. We have determined the expression pattern in mice of the BH3-only protein Bik, also called Blk or Nbk, and examined its physiological function by gene targeting. We found that Bik is expressed widely in the hematopoietic compartment and in endothelial cells of the venous but not arterial lineages. Nevertheless, its loss did not increase the numbers of such cells in mice or protect hematopoietic cells in vitro from apoptosis induced by cytokine withdrawal or diverse other cytotoxic stimuli. Moreover, whereas loss of the BH3-only protein Bim rescued mice lacking the prosurvival protein Bcl-2 from fatal polycystic kidney disease and lymphopenia, loss of Bik did not. These results indicate that any function of Bik in programmed cell death and stress-induced apoptosis must overlap that of other BH3-only proteins. PMID:14749373

  13. Testicular orphan nuclear receptor 4 is associated with the radio-sensitivity of prostate cancer.

    PubMed

    Yu, Shicheng; Wang, Mingchao; Ding, Xianfan; Xia, Liqun; Chen, Bide; Chen, Yicheng; Zhang, Zhigen; Niu, Yuanjie; Li, Gonghui; Chang, Chawnshang

    2015-10-01

    It is well known that a significant number of prostate cancers (PCa) showed different extents of radio-resistance and the tumor may recur after treatment. Recent studies demonstrated that Testicular orphan nuclear receptor 4 (TR4) could play a critical role in anti-oxidative stress responses and might modulate the DNA damage repair. The objective of this study is to investigate the role of TR4 in the radiotherapy for PCa. The TR4 expression in tissue samples from PCa patients treated with brachytherapy was measured by immunohistochemistry (IHC). Cell survival test and colony formation assay were applied to test the radio-sensitivity of PCa cells with modulated TR4 gene expression upon irradiation. PCa patients with biochemical recurrence (BCR) after brachytherapy tend to have higher TR4 expression (80%, n = 30) as compared to those without BCR (36.67%, n = 30). Survival analysis demonstrated a significant higher BCR occurrence in patients with high level of TR4 expression (HR = 3.474, 95%CI 1.678-7.192, P = 0.0008). Multivariate analysis showed that the TR4 staining score on IHC was the only significant variable for predicting the PCa patients' clinical outcomes after radiotherapy (OR = 9.919, 95% CI 2.516-39.101, P = 0.001). Using cell survival test and colony forming assay, we found that the addition of functional TR4 in PC3 cells lead to elevated radio-resistance. In contrast, knocking-down TR4 in LNCaP cells resulted in increased radio-sensitivity. The γH2AX foci kinetic analysis suggested that knocking down TR4 might delay the PCa cell's DNA damage repair which would enhance the radio-sensitivity. TR4 could mediate the PCa cells' radio-sensitivity and might become a prognostic indicator for PCa patients received radiotherapy. This study provides a novel approach to manipulate radio-sensitivity of PCa cells, and may bring a promoted therapeutic outcome of radiotherapy to battle PCa in future. © 2015 Wiley Periodicals, Inc.

  14. Dimorphic ovary differentiation in honeybee (Apis mellifera) larvae involves caste-specific expression of homologs of ark and buffy cell death genes.

    PubMed

    Dallacqua, Rodrigo Pires; Bitondi, Márcia Maria Gentile

    2014-01-01

    The establishment of the number of repeated structural units, the ovarioles, in the ovaries is one of the critical events that shape caste polyphenism in social insects. In early postembryonic development, honeybee (Apis mellifera) larvae have a pair of ovaries, each one consisting of almost two hundred ovariole primordia. While practically all these ovarioles continue developing in queen-destined larvae, they undergo massive programmed cell death (PCD) in worker-destined larvae. So as to gain insight into the molecular basis of this fundamental process in caste differentiation we used quantitative PCR (qPCR) and fluorescent in situ hybridization (FISH) to investigate the expression of the Amark and Ambuffy genes in the ovaries of the two honeybee castes throughout the fifth larval instar. These are the homologs of ark and buffy Drosophila melanogaster genes, respectively, involved in activating and inhibiting PCD. Caste-specific expression patterns were found during this time-window defining ovariole number. Amark transcript levels were increased when ovariole resorption was intensified in workers, but remained at low levels in queen ovaries. The transcripts were mainly localized at the apical end of all the worker ovarioles, but appeared in only a few queen ovarioles, thus strongly suggesting a function in mediating massive ovariolar cell death in worker larvae. Ambuffy was mainly expressed in the peritoneal sheath cells covering each ovariole. The levels of Ambuffy transcripts increased earlier in the developing ovaries of queens than in workers. Consistent with a protective role against cell death, Ambuffy transcripts were localized in practically all queen ovarioles, but only in few worker ovarioles. The results are indicative of a functional relationship between the expression of evolutionary conserved cell death genes and the morphological events leading to caste-specific ovary differentiation in a social insect.

  15. Increase of galectin-3 expression in microglia by hyperthermia in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia.

    PubMed

    Satoh, Kunio; Niwa, Masayuki; Binh, Nguyen Huy; Nakashima, Masaya; Kobayashi, Kazuhiro; Takamatsu, Manabu; Hara, Akira

    2011-10-31

    The ischemic damage in the hippocampal CA1 region following transient forebrain ischemia, delayed neuronal death, is a typical apoptotic response, but the underlying mechanisms are not fully understood. We have reported that mild hyperthermia (38 °C) accelerates DNA fragmentation of the gerbil CA1 pyramidal neurons following transient forebrain ischemia. Recently, we reported that galectin-3, a β-galactosidase-binding lectin, is spatio-temporally expressed only by activated microglial cells located within CA1 region following transient forebrain ischemia in gerbils. Furthermore, expression of galectin-3 and Iba-1 (a specific microglial cell marker) are strongly reduced by hypothermia during ischemic insult. To further elucidate the effect of hyperthermia on the expression of galectin-3 by micloglia in delayed neuronal death, we examined immunohistochemical expression of galectin-3 and Iba-1, in situ terminal dUTP-biotin nick end labeling of DNA fragmentation (for determination of cell death) and hematoxylin and eosin staining (for morphological observation). We observed that between 37 °C and 39 °C, there was a temperature-dependent enhancement of galectin-3 expression in microglial cells in the CA1 region following transient ischemia. Apoptotic DNA fragmentation, detected by TUNEL staining, was observed in CA1 region in normothermia. This TUNEL staining was enhanced by hyperthermia at 37.5 °C and 38 °C, but not at 39 °C. Ischemia-induced neuronal degeneration in CA1 region in gerbil hippocampus subjected to hyperthermia (37.5 °C, 38 °C and 39 °C) observed by HE staining is similar to that in normothermic gerbils. These findings imply that galectin-3 expression in microglia may influence the survival of CA1 pyramidal neurons in cases such as hyperthermia-related neuronal injury.

  16. Inhibition of methionine adenosyltransferase II induces FasL expression, Fas-DISC formation and caspase-8-dependent apoptotic death in T leukemic cells.

    PubMed

    Jani, Tanvi S; Gobejishvili, Leila; Hote, Prachi T; Barve, Aditya S; Joshi-Barve, Swati; Kharebava, Giorgi; Suttles, Jill; Chen, Theresa; McClain, Craig J; Barve, Shirish

    2009-03-01

    Methionine adenosyltransferase II (MAT II) is a key enzyme in cellular metabolism and catalyzes the formation of S-adenosylmethionine (SAMe) from L-methionine and ATP. Normal resting T lymphocytes have minimal MAT II activity, whereas activated proliferating T lymphocytes and transformed T leukemic cells show significantly enhanced MAT II activity. This work was carried out to examine the role of MAT II activity and SAMe biosynthesis in the survival of leukemic T cells. Inhibition of MAT II and the resultant decrease in SAMe levels enhanced expression of FasL mRNA and protein, and induced DISC (Death Inducing Signaling Complex) formation with FADD (Fas-associated Death Domain) and procaspase-8 recruitment, as well as concomitant increase in caspase-8 activation and decrease in c-FLIP(s) levels. Fas-initiated signaling induced by MAT II inhibition was observed to link to the mitochondrial pathway via Bid cleavage and to ultimately lead to increased caspase-3 activation and DNA fragmentation in these cells. Furthermore, blocking MAT 2A mRNA expression, which encodes the catalytic subunits of MAT II, using a small-interfering RNA approach enhanced FasL expression and cell death, validating the essential nature of MAT II activity in the survival of T leukemic cells.

  17. The histone deacetylase inhibitor LBH589 inhibits expression of mitotic genes causing G2/M arrest and cell death in head and neck squamous cell carcinoma cell lines.

    PubMed

    Prystowsky, Michael B; Adomako, Alfred; Smith, Richard V; Kawachi, Nicole; McKimpson, Wendy; Atadja, Peter; Chen, Quan; Schlecht, Nicolas F; Parish, Joanna L; Childs, Geoffrey; Belbin, Thomas J

    2009-08-01

    Head and neck squamous cell carcinoma represents a complex set of neoplasms arising in diverse anatomical locations. The site and stage of the cancer determine whether patients will be treated with single or multi-modality therapy. The HDAC inhibitor LBH589 is effective in treating some haematological neoplasms and shows promise for certain epithelial neoplasms. As with other human cancer cell lines, LBH589 causes up-regulation of p21, G2/M cell cycle arrest, and cell death of human HNSCC cell lines, as measured using flow cytometry and cDNA microarrays. Global RNA expression studies following treatment of the HNSCC cell line FaDu with LBH589 reveal down-regulation of genes required for chromosome congression and segregation (SMC2L1), sister chromatid cohesion (DDX11), and kinetochore structure (CENP-A, CENP-F, and CENP-M); these LBH589-induced changes in gene expression coupled with the down-regulation of MYC and BIRC5 (survivin) provide a plausible explanation for the early mitotic arrest and cell death observed. When LBH589-induced changes in gene expression were compared with gene expression profiles of 41 primary HNSCC samples, many of the genes that were down-regulated by LBH589 showed increased expression in primary HNSCC, suggesting that some patients with HNSCC may respond to treatment with LBH589. (c) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  18. Conditional Expression of E2A-HLF Induces B-Cell Precursor Death and Myeloproliferative-Like Disease in Knock-In Mice

    PubMed Central

    Duque-Afonso, Jesús; Smith, Kevin S.; Cleary, Michael L.

    2015-01-01

    Chromosomal translocations are driver mutations of human cancers, particularly leukemias. They define disease subtypes and are used as prognostic markers, for minimal residual disease monitoring and therapeutic targets. Due to their low incidence, several translocations and their biological consequences remain poorly characterized. To address this, we engineered mouse strains that conditionally express E2A-HLF, a fusion oncogene from the translocation t(17;19) associated with 1% of pediatric B-cell precursor ALL. Conditional oncogene activation and expression were directed to the B-cell compartment by the Cre driver promoters CD19 or Mb1 (Igα, CD79a), or to the hematopoietic stem cell compartment by the Mx1 promoter. E2A-HLF expression in B-cell progenitors induced hyposplenia and lymphopenia, whereas expression in hematopoietic stem/progenitor cells was embryonic lethal. Increased cell death was detected in E2A-HLF expressing cells, suggesting the need for cooperating genetic events that suppress cell death for B-cell oncogenic transformation. E2A-HLF/Mb1.Cre aged mice developed a fatal myeloproliferative-like disorder with low frequency characterized by leukocytosis, anemia, hepatosplenomegaly and organ-infiltration by mature myelocytes. In conclusion, we have developed conditional E2A-HLF knock-in mice, which provide an experimental platform to study cooperating genetic events and further elucidate translational biology in cross-species comparative studies. PMID:26588248

  19. Proline and glycinebetaine induce antioxidant defense gene expression and suppress cell death in cultured tobacco cells under salt stress.

    PubMed

    Banu, Nasrin Akhter; Hoque, Anamul; Watanabe-Sugimoto, Megumi; Matsuoka, Ken; Nakamura, Yoshimasa; Shimoishi, Yasuaki; Murata, Yoshiyuki

    2009-01-30

    Salt stress causes oxidative damage and cell death in plants. Plants accumulate proline and glycinebetaine (betaine) to mitigate detrimental effects of salt stress. The aim of this study was to investigate the protective effects of proline and betaine on cell death in NaCl-unadapted tobacco (Nicotiana tabacum) Bright Yellow-2 suspension-cultured cells subjected to salt stress. Salt stress increased reactive oxygen species (ROS) accumulation, lipid peroxidation, nuclear deformation and degradation, chromatin condensation, apoptosis-like cell death and ATP contents. Neither proline nor betaine affected apoptosis-like cell death and G(1) phase population, and increased ATP contents in the 200mM NaCl-stressed cells. However, both of them effectively decreased ROS accumulation and lipid peroxidation, and suppressed nuclear deformation and chromatin condensation induced by severe salt stress. Evans Blue staining experiment showed that both proline and betaine significantly suppressed increment of membrane permeability induced by 200mM NaCl. Furthermore, among the ROS scavenging antioxidant defense genes studied here, mRNA levels of salicylic acid-binding (SAbind) catalase (CAT) and lignin-forming peroxidase (POX) were found to be increased by proline and betaine under salt stress. It is concluded that both proline and betaine provide a protection against NaCl-induced cell death via decreasing level of ROS accumulation and lipid peroxidation as well as improvement of membrane integrity.

  20. Activation of Toll-like Receptor 4 (TLR4) Attenuates Adaptive Thermogenesis via Endoplasmic Reticulum Stress*

    PubMed Central

    Okla, Meshail; Wang, Wei; Kang, Inhae; Pashaj, Anjeza; Carr, Timothy; Chung, Soonkyu

    2015-01-01

    Adaptive thermogenesis is the cellular process transforming chemical energy into heat in response to cold. A decrease in adaptive thermogenesis is a contributing factor to obesity. However, the molecular mechanisms responsible for the compromised adaptive thermogenesis in obese subjects have not yet been elucidated. In this study we hypothesized that Toll-like receptor 4 (TLR4) activation and subsequent inflammatory responses are key regulators to suppress adaptive thermogenesis. To test this hypothesis, C57BL/6 mice were either fed a palmitate-enriched high fat diet or administered with chronic low-dose LPS before cold acclimation. TLR4 stimulation by a high fat diet or LPS were both associated with reduced core body temperature and heat release. Impairment of thermogenic activation was correlated with diminished expression of brown-specific markers and mitochondrial dysfunction in subcutaneous white adipose tissue (sWAT). Defective sWAT browning was concomitant with elevated levels of endoplasmic reticulum (ER) stress and autophagy. Consistently, TLR4 activation by LPS abolished cAMP-induced up-regulation of uncoupling protein 1 (UCP1) in primary human adipocytes, which was reversed by silencing of C/EBP homologous protein (CHOP). Moreover, the inactivation of ER stress by genetic deletion of CHOP or chemical chaperone conferred a resistance to the LPS-induced suppression of adaptive thermogenesis. Collectively, our data indicate the existence of a novel signaling network that links TLR4 activation, ER stress, and mitochondrial dysfunction, thereby antagonizing thermogenic activation of sWAT. Our results also suggest that TLR4/ER stress axis activation may be a responsible mechanism for obesity-mediated defective brown adipose tissue activation. PMID:26370079

  1. Toll-like receptor 4 signalling pathway activation in a rat model of Acanthamoeba Keratitis.

    PubMed

    Ren, M Y; Wu, X Y

    2011-01-01

    The pathogenesis of Acanthamoeba keratitis (AK) is complicated. In our previous studies, TLR4 was found involved in the process of infection by Acanthamoeba in human corneal cells. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signalling pathway in Wistar rats challenged with Acanthamoeba. The rat model of AK was established. Corneas were collected and analysed by real-time PCR to assess the mRNA levels of TLR 2, 4, myeloid differentiation protein (MyD)88, nuclear factor (NF)-κB, extracellular signal-regulated kinase (ERK), interleukin (IL)-8, tumour necrosis factor (TNF)-α and interferon (IFN) -β. Immunocytochemistry and Western blot were conducted to examine the proteins of TLR2, TLR4, p-Erk1/2 and p-IκB. Specific inhibitors PDTC and U0126 were used to pretreat the animals to determine the exact receptor and signalling pathway involved in pathogenesis. Expressions of TLR4, MyD88, all three cytokines, NF-κB, p-IκB and p-Erk1/2 were increased in Acanthamoeba-treated rat corneas. PDTC inhibited the production of IL-8 and TNF-α, while U0126 inhibited the synthesis of IFN-β. TLR4 was involved in sensing the challenge of Acanthamoeba and inducing production of cytokines through TLR4-NF-κB and TLR4-Erk1/2 pathways in corneas of Wistar rats. © 2010 Blackwell Publishing Ltd.

  2. Acute toll-like receptor 4 activation impairs rat renal microvascular autoregulatory behaviour.

    PubMed

    Van Beusecum, J P; Zhang, S; Cook, A K; Inscho, E W

    2017-05-25

    Little is known about how toll-like receptor 4 (TLR4) influences the renal microvasculature. We hypothesized that acute TLR4 stimulation with lipopolysaccharide (LPS) impairs afferent arteriole autoregulatory behaviour, partially through reactive oxygen species (ROS). We assessed afferent arteriole autoregulatory behaviour after LPS treatment (1 mg kg(-1) ; i.p.) using the in vitro blood-perfused juxtamedullary nephron preparation. Autoregulatory behaviour was assessed by measuring diameter responses to stepwise changes in renal perfusion pressure. TLR4 expression was assessed by immunofluorescence, immunohistochemistry and Western blot analysis in the renal cortex and vasculature. Baseline arteriole diameter at 100 mmHg averaged 15.2 ± 1.2 μm and 12.2 ± 1.0 μm for control and LPS groups (P < 0.05) respectively. When perfusion pressure was increased in 15 mmHg increments from 65 to 170 mmHg, arteriole diameter in control kidneys decreased significantly to 69 ± 6% of baseline diameter. In the LPS-treated group, arteriole diameter remained essentially unchanged (103 ± 9% of baseline), indicating impaired autoregulatory behaviour. Pre-treatment with anti-TLR4 antibody or the TLR4 antagonist, LPS-RS, preserved autoregulatory behaviour during LPS treatment. P2 receptor reactivity was normal in control and LPS-treated rats. Pre-treatment with Losartan (angiotensin type 1 receptor blocker; (AT1 ) 2 mg kg(-1) ; i.p.) increased baseline afferent arteriole diameter but did not preserve autoregulatory behaviour in LPS-treated rats. Acute exposure to Tempol (10(-3) mol L(-1) ), a superoxide dismutase mimetic, restored pressure-mediated vasoconstriction in kidneys from LPS-treated rats. These data demonstrate that TLR4 activation impairs afferent arteriole autoregulatory behaviour, partially through ROS, but independently of P2 and AT1 receptor activation. © 2017 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  3. Novel CC chemokine receptor 4 antagonist RS-1154 inhibits ovalbumin-induced ear swelling in mice.

    PubMed

    Nakagami, Yasuhiro; Kawashima, Kayo; Yonekubo, Kazuki; Etori, Maki; Jojima, Takaaki; Miyazaki, Shojiro; Sawamura, Ryoko; Hirahara, Kazuki; Nara, Futoshi; Yamashita, Makoto

    2009-12-10

    CC chemokine ligand 17 (CCL17/thymus and activation-regulated chemokine: TARC) and CCL22 (macrophage-derived chemokine: MDC) selectively bind to CC chemokine receptor 4 (CCR4). The CCR4 system is considered to be responsible for the pathology of allergic diseases such as atopic dermatitis. To find and develop potential medicines against allergic diseases, we screened an in-house library to search for compounds having a profile as a CCR4 antagonist. From among the screening hits, we focused on 3-{2-[(2R)-2-phenyl-4-(4-pyridin-4-ylbenzyl)morpholin-2-yl]ethyl}quinazoline-2,4(1H,3H)-dione (named RS-1154), which had been newly synthesized in our laboratory. This compound inhibited the binding of [(125)I]CCL17 to human CCR4-expressing CHO cells with an IC(50) value of 27.7 nM and moreover inhibited CCL17-induced migration of DO11.10 mice-derived T helper 2 cells with an IC(50) value of 1.5 nM in vitro. We then examined the effect of RS-1154 in an ovalbumin-induced ear swelling assay. The ear thickness was decreased by intravenous administration of anti-CCL17 or anti-CCL22 antibodies, suggesting that the CCR4 system is involved in the ear swelling. Though partially, the oral administration of RS-1154 also significantly ameliorated the ear swelling at the doses of 30 and 100 mg/kg. Furthermore, the serum level of interleukin-4 decreased after the administration of RS-1154. In this study, we succeeded in obtaining a newly-synthesized compound, RS-1154, which has a potential to inhibit the chemotaxis of T helper 2 cells in vitro and to ameliorate ovalbumin-induced ear swelling in vivo. These results raise the possibility that RS-1154 or one of derivatives might become a therapeutic agent for atopic dermatitis patients.

  4. Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

    SciTech Connect

    Zhang, X.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

    2009-07-01

    Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidative stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.

  5. Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression.

    PubMed

    Zhang, X; Li, L; Zhang, L; Borowitz, J L; Isom, G E

    2009-07-01

    Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidative stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H2O2 generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H2O2 generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H2O2 accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.

  6. Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain

    PubMed Central

    Fleiss, Bobbi; Kawano, Go; Ezzati, Mojgan; Rocha-Ferreira, Eridan; Hristova, Mariya; Bennett, Kate; Fierens, Igor; Burnett, Ryan; Chaban, Badr; Alonso-Alconada, Daniel; Oliver-Taylor, Aaron; Tachsidis, Ilias; Rostami, Jamshid; Gressens, Pierre; Sanders, Robert D.

    2016-01-01

    Exposure of the brain to general anesthesia during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex, incompletely understood and may be sexually dimorphic, but include developmentally inappropriate apoptosis, inflammation and a disruption to cognitively salient gene expression. We investigated the effects of a 6h isoflurane exposure on cell death, microglial activation and gene expression in the male neonatal piglet brain. Piglets (n = 6) were randomised to: (i) naive controls or (ii) 6h isoflurane. Cell death (TUNEL and caspase-3) and microglial activation were recorded in 7 brain regions. Changes in gene expression (microarray and qPCR) were assessed in the cingulate cortex. Electroencephalography (EEG) was recorded throughout. Isoflurane anesthesia induced significant increases in cell death in the cingulate and insular cortices, caudate nucleus, thalamus, putamen, internal capsule, periventricular white matter and hippocampus. Dying cells included both neurons and oligodendrocytes. Significantly, microglial activation was observed in the insula, pyriform, hippocampus, internal capsule, caudate and thalamus. Isoflurane induced significant disruption to the expression of 79 gene transcripts, of these 26 are important for the control of transcription and 23 are important for the mediation of neural plasticity, memory formation and recall. Our observations confirm that isoflurane increases apoptosis and inflammatory responses in the neonatal piglet brain but also suggests novel additional mechanisms by which isoflurane may induce adverse neural and cognitive development by disrupting the expression of genes mediating activity dependent development of neural circuits, the predictive adaptive responses of the brain, memory formation and recall. PMID:27898690

  7. Death Cafe.

    PubMed

    Miles, Lizzy; Corr, Charles A

    2017-06-01

    This article explains the meaning of the phrase Death Cafe and describes what typically occurs at a Death Cafe gathering. The article traces the history of the Death Cafe movement, explores some reasons why people take part in a Death Cafe gathering, and gives examples of what individuals think they might derive from their participation. In addition, this article notes similarities between the Death Cafe movement and three other developments in the field of death, dying, and bereavement. Finally, this article identifies two provisional lessons that can be drawn from Death Cafe gatherings and the Death Cafe movement itself.

  8. Gene expression in the twilight of death: The increase of thousands of transcripts has implications to transplantation, cancer, and forensic research.

    PubMed

    Pozhitkov, Alexander E; Noble, Peter A

    2017-09-01

    After a vertebrate dies, many of its organ systems, tissues, and cells remain functional while its body no longer works as a whole. We define this state as the "twilight of death" - the transition from a living body to a decomposed corpse. We claim that the study of the twilight of death is important to ethical, legal and medical science. We examined gene expression at the twilight of death in the zebrafish and mouse reaching the conclusion that apparently thousands of transcripts significantly increase in abundance from life to several hours/days postmortem relative to live controls. Transcript dynamics of different genes provided "proof-of-principle" that models accurately predict an individual's elapsed-time-of-death (i.e. postmortem interval). While many transcripts were associated with survival and stress compensation, others were associated with epigenetic factors, developmental control, and cancer. Future studies are needed to determine whether the high incidence of cancer in transplant recipients is due to the postmortem processes in donor organs. © 2017 WILEY Periodicals, Inc.

  9. Status epilepticus decreases glutamate receptor 2 mRNA and protein expression in hippocampal pyramidal cells before neuronal death

    PubMed Central

    Grooms, Sonja Y.; Opitz, Thoralf; Bennett, Michael V. L.; Zukin, R. Suzanne

    2000-01-01

    Kainic acid (KA)-induced status epilepticus in adult rats leads to delayed, selective death of pyramidal neurons in the hippocampal CA1 and CA3. Death is preceded by down-regulation of glutamate receptor 2 (GluR2) mRNA and protein [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors] in CA1 and CA3, as indicated by in situ hybridization, immunolabeling, and quantitative Western blotting. GluR1 mRNA and protein are unchanged or slightly increased before cell death. These changes could lead to formation of GluR2-lacking, Ca2+-permeable AMPA receptors and increased toxicity of endogenous glutamate. GluR2 immunolabeling is unchanged in granule cells of the dentate gyrus, which are resistant to seizure-induced death. Thus, formation of Ca2+-permeable AMPA receptors may be a critical mediator of delayed neurodegeneration after status epilepticus. PMID:10725374

  10. FTY720 increases CD74 expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death

    PubMed Central

    Alinari, Lapo; Mahoney, Emilia; Patton, John; Zhang, Xiaoli; Huynh, Lenguyen; Earl, Christian T.; Mani, Rajeswaran; Mao, Yicheng; Yu, Bo; Quinion, Carl; Towns, William H.; Chen, Ching-Shih; Goldenberg, David M.; Blum, Kristie A.; Byrd, John C.; Muthusamy, Natarajan

    2011-01-01

    Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phospho-Akt and cyclin D1 and subsequent cell-cycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti-CD74 mAb. Treatment of MCL cell lines and primary tumor cells with FTY720 and milatuzumab resulted in statistically significant enhanced cell death, which was synergistic in blastic variant MCL cell lines. Significant in vivo therapeutic activity of combination treatment was also demonstrated in a preclinical, in vivo model of MCL. These findings support clinical evaluation of this combination in patients with MCL. PMID:22042694

  11. The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy

    PubMed Central

    Lohberger, Birgit; Steinecker-Frohnwieser, Bibiane; Stuendl, Nicole; Kaltenegger, Heike; Leithner, Andreas; Rinner, Beate

    2016-01-01

    High grade chondrosarcoma is characterized by its lack of response to conventional cytotoxic chemotherapy, the tendency to develop lung metastases, and low survival rates. Research within the field prioritizes the development and expansion of new treatment options for dealing with unresectable or metastatic diseases. Numerous clinical trials using the proteasome inhibitor bortezomib have shown specific efficacy as an active antitumor agent for treating a variety of solid tumors. However, as of yet the effect of bortezomib on chondrosarcoma has not been investigated. In our study, bortezomib decreased cell viability and proliferation in two different chondrosarcoma cell lines in a time- and dose dependent manner. FACS analysis, mRNA- and protein expression studies illustrated that induction of apoptosis developed through the intrinsic mitochondria-caspase dependent pathway. Furthermore, bortezomib treatment significantly increased expression of the death receptors TRAILR-1 and TRAILR-2 in chondrosarcoma cells. An increased expression of the autophagy markers Atg5/12, Beclin, and LC3BI-II supports the interpretation that bortezomib functions as a trigger for autophagy. Our results demonstrated for the first time that bortezomib reduced viability and proliferation of chondrosarcoma cells, induced apoptosis via the mitochondria-caspase dependent pathway and enhanced death receptor expression and autophagy. PMID:27978543

  12. Tumor programmed cell death ligand 1 expression correlates with nodal metastasis in patients with cutaneous squamous cell carcinoma of the head and neck.

    PubMed

    García-Pedrero, Juana María; Martínez-Camblor, Pablo; Diaz-Coto, Susana; Munguia-Calzada, Pablo; Vallina-Alvarez, Aitana; Vazquez-Lopez, Francisco; Rodrigo, Juan Pablo; Santos-Juanes, Jorge

    2017-09-01

    Binding of tumor-expressed programmed cell death ligand 1 (PD-L1) to the programmed cell death 1 (PD-1) surface receptor blocks T-cell activation thereby leading to immune evasion. Tumor PD-L1 expression has been associated with poor outcome in a wide variety of cancers; however, data in cutaneous squamous cell carcinoma (cSCC) are scarce and conflicting. To investigate the relationship of tumor PD-L1 expression with the clinicopathologic features and prognosis of cSCC. PD-L1 expression was analyzed by immunohistochemistry on paraffin-embedded tissue samples from 100 patients with cSCC. Cumulative/dynamic receiver operating characteristic curve was used to determine the optimal PD-L1 threshold. Kaplan-Meier estimators and Cox proportional hazards regression models were also used. On the basis of cumulative/dynamic receiver operating characteristic curves, we defined the cut-off score for PD-L1 expression as ≥25% of tumor cells positively stained. PD-L1 expression was a significant risk factor for nodal metastasis with crude and adjusted hazard ratios of 3.39 (1.71-6.65) and 6.54 (2.28-18.78), respectively. This is a retrospective study limited to cSCC of the head and neck. These findings indicate that tumor PD-L1 expression predicts increased risk for nodal metastasis in patients with cSCC. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  13. A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4.

    PubMed

    Sangawa, Takeshi; Nogi, Terukazu; Takagi, Junichi

    2008-10-01

    Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.

  14. RS-1748, a novel CC chemokine receptor 4 antagonist, inhibits ovalbumin-induced airway inflammation in guinea pigs.

    PubMed

    Nakagami, Yasuhiro; Kawase, Yumi; Yonekubo, Kazuki; Nosaka, Emi; Etori, Maki; Takahashi, Sakiko; Takagi, Nana; Fukuda, Takeshi; Kuribayashi, Takeshi; Nara, Futoshi; Yamashita, Makoto

    2010-01-01

    CC chemokine receptor 4 (CCR4) is generally recognized as a preferential marker for T helper 2 cells, and we have previously reported morpholine-derivative CCR4 antagonists, RS-1154 and RS-1269. Here, we investigate the pharmacological profiles of a novel pyrimidine-derivative CCR4 antagonist, 2-{4-[2-(diethylamino)ethoxy]phenyl}-N-(2,4-difluorobenzyl)-5-fluoropyrimidin-4-amine (RS-1748), which showed potency to inhibit the bindings of [(125)I]CCL17 and [(35)S]GTPgammaS to human CCR4-expressing Chinese hamster ovary (CHO) cells with IC(50) values of 59.9 nM and 18.4 nM, respectively. Furthermore, RS-1748 inhibited ovalbumin-induced airway inflammation in guinea pigs at a dose of 10 mg/kg. These results indicate that RS-1748 would be a promising lead compound for developing a therapeutic agent against asthma.

  15. Aging and amyloid β oligomers enhance TLR4 expression, LPS-induced Ca(2+) responses, and neuron cell death in cultured rat hippocampal neurons.

    PubMed

    Calvo-Rodríguez, María; de la Fuente, Carmen; García-Durillo, Mónica; García-Rodríguez, Carmen; Villalobos, Carlos; Núñez, Lucía

    2017-01-31

    Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer's disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca(2+) dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence. Ca(2+) imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca(2+)] and on apoptosis as well as on expression of TLR4. LPS increases cytosolic [Ca(2+)] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca(2+) responses and neuron cell death. Aging and amyloid β oligomers, the neurotoxin involved in Alzheimer's disease, enhance TLR4 expression as well as LPS-induced Ca(2+) responses and neuron cell death in rat hippocampal neurons aged in vitro.

  16. Preparation of cell-lines for conditional knockdown of gene expression and measurement of the knockdown effects on E4orf4-induced cell death.

    PubMed

    Brestovitsky, Anna; Sharf, Rakefet; Kleinberger, Tamar

    2012-10-21

    Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways(1,2). However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time(3-5). Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein(6). Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured(7-9). The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

  17. Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

    PubMed Central

    Brestovitsky, Anna; Sharf, Rakefet; Kleinberger, Tamar

    2012-01-01

    Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9. The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal. PMID:23117279

  18. TRPV4 regulates insulin mRNA expression and INS-1E cell death via ERK1/2 and NO-dependent mechanisms.

    PubMed

    Billert, M; Skrzypski, M; Sassek, M; Szczepankiewicz, D; Wojciechowicz, T; Mergler, S; Strowski, M Z; Nowak, K W

    2017-07-01

    TRPV4 is a Ca(2+)-permeable, nonselective cation channel. Recently, TRPV4 was implicated in controlling peripheral insulin sensitivity, insulin secretion and apoptosis of pancreatic beta cells. Here, we characterize the role and potential mechanisms of TRPV4 in regulating insulin mRNA expression and cell death in insulin producing INS-1E cells and rat pancreatic islets. TRPV4 protein production was downregulated by siRNA. Intracellular calcium level was measured using Fluo-3 AM. Gene expression was studied by real-time PCR. Phosphorylation of extracellular signal-regulated kinase (ERK1 and ERK2) was detected by Western blot. Nitric oxide (NO) production was assessed by chemiluminescent reaction. Reactive oxygen species (ROS) level was analysed using a fluorogenic dye (DCFDA). Cell death was evaluated by determination of cytoplasmic histone-associated DNA fragments. Downregulation of TRPV4 neither affected insulin mRNA expression nor INS-1E cell growth. By contrast, pharmacological TRPV4 activation by 100nmol/l GSK1016790A increased Ca(2+) levels in INS-1E cells and enhanced insulin mRNA expression after 1 and 3h, whereas a suppression of insulin mRNA expression was detected after 24h incubation. GSK1016790A increased ERK1/2 phosphorylation and NO production but not ROS production. Pharmacological blockade of ERK1/2 attenuated GSK1016790A-induced insulin mRNA expression. Inhibition of NO synthesis by l-NAME failed to affect insulin mRNA expression in GSK1016790A treated INS-1E cells. Furthermore, inhibition of NO production attenuated GSK1016790A-induced INS-1E cell death. In pancreatic islets, 100nmol/l GSK1016790A increased insulin mRNA levels after 3h without inducing cytotoxicity after 24h. In conclusion, TRPV4 differently regulates insulin mRNA expression in INS-1E cells via ERK1/2 and NO-dependent mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. MyD88-dependent Toll-like receptor 4 signal pathway in intervertebral disc degeneration

    PubMed Central

    Qin, Chuqiang; Zhang, Bo; Zhang, Liang; Zhang, Zhi; Wang, Le; Tang, Long; Li, Shuangqing; Yang, Yixi; Yang, Fuguo; Zhang, Ping; Yang, Bo

    2016-01-01

    Lower back pain (LBP) is a common and remitting problem. One of the primary causes of LBP is thought to be degeneration of the intervertebral disc (IVD). The aim of the present study was to investigate the role of the myeloid differentiation primary-response protein 88 (MyD88)-dependent Toll-like receptor 4 (TLR4) signal pathway in the mechanism of IVD degeneration. IVD nucleus pulposus cells isolated and cultured from the lumbar vertebrae of Wistar rats were stimulated by various doses of lipopolysaccharide (LPS; 0.1, 1, 10 and 100 µg/ml) to simulate IVD degeneration. Cells were rinsed and cultured in serum-free Dulbecco's modified Eagle's medium/F12. Reverse transcription-quantitative polymerase chain reaction was used to determine the levels of TLR4, MyD88, tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β) mRNA expression after 1, 3, 6, 9 and 12 h of incubation. Additionally, western blot and enzyme-linked immunosorbent assay analyses were used to determine the levels of TLR4, MyD88, TNFα, and IL-1β protein expression after 24, 48 and 72 h of incubation. The levels of TLR4, MyD88, TNFα and IL-1β mRNA all increased in the cells stimulated by 10 µg/ml LPS at 3, 6 and 9 h (all P<0.001). Furthermore, the levels of TLR4, MyD88, TNFα and IL-1β protein all increased at 24, 48 and 72 h (all P<0.001). Additionally, the mRNA and protein levels of TLR4, MyD88, TNFα and IL-1β increased significantly in the cells stimulated by 1, 10 and 100 µg/ml LPS compared with the control group, and reached a peak in the 10 µg/ml LPS group (all P<0.001). These results suggest that the MyD88-dependent TLR4 signal pathway is a target pathway in IVD degeneration. This pathway is time phase- and dose-dependent, and when activated can lead to the release of inflammatory factors that participate in IVD degeneration. PMID:27446251

  20. Nuclear receptor 4A1 as a drug target for breast cancer chemotherapy.

    PubMed

    Hedrick, Erik; Lee, Syng-Ook; Doddapaneni, Ravi; Singh, Mandip; Safe, Stephen

    2015-10-01

    The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in mammary tumors and breast cancer cell lines. The functional activity of this receptor was investigated by RNA interference with oligonucleotides targeted to NR4A1 (siNR4A1) and by treatment with NR4A1 antagonists. Breast cancer cells were treated with NR4A1 antagonists or transfected with siNR4A. Effects on cell proliferation and apoptosis as well as specific genes associated with these responses were investigated in MCF-7, SKBR3, and MDA-MB-231 cells, and in athymic nude mice bearing MDA-MB-231 cells as xenografts. Transfection of MCF-7, MDA-MB-231, and SKBR3 breast cancer cells with siNR4A1 decreased cell proliferation and induced apoptosis in these cell lines. Transfection of breast cancer cells with siNR4A1 also decreased expression of Sp-regulated genes including survivin, bcl-2, and epidermal growth factor receptor, inhibited mTOR signaling in MCF-7 cells that express WT p53, and activated oxidative and endoplasmic reticulum stress through downregulation of thioredoxin domain-containing 5 and isocitrate dehydrogenase 1. 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) are NR4A1 ligands that act as NR4A1 antagonists. Treatment with selected analogs also inhibited breast cancer cell and tumor growth and induced apoptosis. The effects of C-DIM/NR4A1 antagonists were comparable to those observed after NR4A1 knockdown. Results with siNR4A1 or C-DIMs/NR4A1 antagonists in breast cancer cells and tumors were similar to those previously reported in pancreatic, lung, and colon cancer cells. They demonstrate the potential clinical applications of NR4A1 antagonists in patients with tumors that overexpress this receptor. © 2015 Society for Endocrinology.

  1. Hepatocyte Toll-like receptor 4 regulates obesity-induced inflammation and insulin resistance

    USDA-ARS?s Scientific Manuscript database

    Chronic low-grade inflammation is a hallmark of obesity and thought to contribute to the development of obesity-related insulin resistance. Toll-like receptor 4 (Tlr4) is a key mediator of pro-inflammatory responses. Mice lacking Tlr4s are protected from diet-induced insulin resistance and inflammat...

  2. Cyanide-induced Death of Dopaminergic Cells is Mediated by Uncoupling Protein-2 Up-regulation and Reduced Bcl-2 Expression

    PubMed Central

    Zhang, X.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

    2009-01-01

    Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidative stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H2O2 generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H2O2 generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H2O2 accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that overexpression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death. PMID:19361538

  3. Changes in the expression level of MAPK pathway components induced by monosodium glutamate-administration produce neuronal death in the hippocampus from neonatal rats.

    PubMed

    Cervantes, Martha Catalina Rivera; Basulto, José Jaime Jarero; Velasco, Alfredo Ignacio Feria; Zárate, Carlos Beas; Meza, Mónica Navarro; López, Mariana Berenice González; Cabrera, Graciela Gudiño; Rodríguez, Julio Cesar García

    2017-09-24

    Excessive Glutamate (Glu) release may trigger excitotoxic cellular death by the activation of intracellular signaling pathways that transduce extracellular signals to the cell nucleus, which determines the onset of a death program. One such signaling pathway is the mitogen-activated protein kinases (MAPK), which is involved in both survival and cell death. Experimental evidences from the use of specific inhibitors supports the participation of some MAPK pathway components in the excitotoxicity mechanism, but the complete process of this activation, which terminates in cell damage and death, is not clearly understood. In the present work, we investigated the changes in the expression level of some MAPK-pathway components in hippocampal excitotoxic cell death in the neonatal rats using an experimental model of subcutaneous monosodium glutamate (MSG) administration on postnatal days (PD) 1, 3, 5 and 7. Data were collected at different ages through PD 14. Cell viability was evaluated using fluorescein diacetate mixed with propidium iodide (FDA-PI), and the Nissl-staining technique was used to evaluate histological damage. Transcriptional changes were also investigated in 98 components of the MAPK pathway that are associated with cell damage. These results are an evidence of that repetitive use of MSG, in neonatal rats, induces cell damage-associated transcriptional changes of MAPK components, that might reflect a differential stage of both biochemical and molecular brain maturation. This work also suggests that some of the proteins evaluated such as phosphorylated retinoblastoma (pRb) protein, which was up-regulated, could regulate the response to excitotoxic through modulation of the process of re-entry into the cell cycle in the hippocampus of rats treated with MSG. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Induction of cell death by stimulation of protein kinase C in human epithelial cells expressing a mutant ras oncogene: a potential therapeutic target.

    PubMed Central

    Hall-Jackson, C. A.; Jones, T.; Eccles, N. G.; Dawson, T. P.; Bond, J. A.; Gescher, A.; Wynford-Thomas, D.

    1998-01-01

    Ras oncogene activation is a key genetic event in several types of human cancer, making its signal pathways an ideal target for novel therapies. We previously showed that expression of mutant ras sensitizes human thyroid epithelial cells to induction of cell death by treatment with phorbol 12-myristate 13-acetate (PMA) and other phorbol esters. We have now investigated further the nature and mechanism of this cell death using both primary and cell line models. The cytotoxic effect of PMA could be blocked by bisindolylmaleimide (GF 109203X), a well-characterized inhibitor of c and n protein kinase C (PKC) isoforms, and by prior down-regulation of PKC, indicating that it is mediated by acute stimulation, rather than down-regulation. Western analysis identified two candidate isoforms--alpha and epsilon--both of which showed PMA-induced subcellular translocation, either or both of which may be necessary for PMA-induced cell death. Immunofluorescence showed that PMA induced a rapid nuclear translocation of p42 MAP kinase of similar magnitude in the presence or absence of mutant ras expression. Cell death exhibited the microscopic features (chromatin condensation, TdT labelling) and DNA fragmentation typical of apoptosis but after a surprising lag (4 days). Taken together with recent models of ras-modulated apoptosis, our data suggest that activation of the MAPK pathway by PMA tips the balance of pro- and anti-apoptotic signals generated by ras in favour of apoptosis. The high frequency of ras mutations in some cancers, such as cancer of the pancreas, which are refractory to conventional chemotherapy, together with the potential for stimulating PKC by cell-permeant pharmacological agents, makes this an attractive therapeutic approach. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 7 Figure 6 Figure 8 PMID:9744505

  5. A prognostic model of recurrence and death in stage I non-small cell lung cancer utilizing presentation, histopathology, and oncoprotein expression.

    PubMed

    Harpole, D H; Herndon, J E; Wolfe, W G; Iglehart, J D; Marks, J R

    1995-01-01

    In order to construct a multivariate model for predicting early recurrence and cancer death for patients with stage I non-small cell lung cancer, 271 consecutive patients (mean age, 63 +/- 8 years) who were diagnosed, treated, and followed at one institution were studied. All patients were clinical stage I with head and chest/abdominal computed tomograms and radionuclide bone scans without evidence of metastatic disease. Pathological material after resection was reviewed to verify histological staging. Follow-up documented the time and location of any recurrence, was a median 56 months in duration, and was complete in all cases. Data recorded included age, sex, smoking history, presenting symptoms, pathological description, and oncoprotein staining for erbB-2 (HER-2/neu), p53, and KI-67 proliferation protein. Immunohistochemistry of oncogene expression was performed on two separate archived paraffin tumor blocks for each patient, with normal lung as control. All analyses were blinded and included Kaplan-Meier survival estimates with Cox proportional hazards regression modeling. Data, including immunohistochemistry, were complete for all 271 patients. Actual 5-year survival was 63% and actuarial 10-year survival was 58%. Significant univariate predictors (P < 0.05) of early recurrence and cancer-death were: male sex; the presence of symptoms; chest pain; type of cough; hemoptysis; tumor size > 3 cm diameter (T2); poor differentiation; vascular invasion; erbB-2 expression; p53 expression; and a higher KI-67 proliferation index (> 5%). An additive oncogene expression curve demonstrated a 5-year survival of 72% for 136 patients without p53 or erbB-2, 58% for 108 patients who expressed either oncogene, and 38% for 27 who expressed both (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization.

    PubMed

    Pesce, Silvia; Greppi, Marco; Tabellini, Giovanna; Rampinelli, Fabio; Parolini, Silvia; Olive, Daniel; Moretta, Lorenzo; Moretta, Alessandro; Marcenaro, Emanuela

    2017-01-01

    Programmed death 1 (PD-1) is an immunologic checkpoint that limits immune responses by delivering potent inhibitory signals to T cells on interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms. Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known about the expression/function of PD-1 on human natural killer (NK) cells. We sought to clarify whether human NK cells can express PD-1 and analyze their phenotypic/functional features. We performed multiparametric cytofluorimetric analysis of PD-1(+) NK cells and their functional characterization using degranulation, cytokine production, and proliferation assays. We provide unequivocal evidence that PD-1 is highly expressed (PD-1(bright)) on an NK cell subset detectable in the peripheral blood of approximately one fourth of healthy subjects. These donors are always serologically positive for human cytomegalovirus. PD-1 is expressed by CD56(dim) but not CD56(bright) NK cells and is confined to fully mature NK cells characterized by the NKG2A(-)KIR(+)CD57(+) phenotype. Proportions of PD-1(bright) NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production on interaction with tumor targets. We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD-1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma. They display low proliferative responses and impaired antitumor activity that can be partially restored by antibody-mediated disruption of PD-1/programmed death ligand interaction. Copyright © 2016 American Academy of Allergy, Asthma

  7. Oncogenic Ras and B-Raf proteins positively regulate death receptor 5 expression through co-activation of ERK and JNK signaling.

    PubMed

    Oh, You-Take; Yue, Ping; Zhou, Wei; Balko, Justin M; Black, Esther P; Owonikoko, Taofeek K; Khuri, Fadlo R; Sun, Shi-Yong

    2012-01-02

    Oncogenic mutations of ras and B-raf frequently occur in many cancer types and are critical for cell transformation and tumorigenesis. Death receptor 5 (DR5) is a cell surface pro-apoptotic death receptor for tumor necrosis factor-related apoptosis-inducing ligand and has been targeted in cancer therapy. The current study has demonstrated induction of DR5 expression by the oncogenic proteins Ras and B-Raf and revealed the underlying mechanisms. We demonstrated that both Ras and B-Raf induce DR5 expression by enforced expression of oncogenic Ras (e.g. H-Ras12V or K-Ras12V) or B-Raf (i.e. V600E) in cells and by analyzing gene expression array data generated from cancer cell lines and from human cancer tissues. This finding is further supported by our results that knockdown of endogenous K-Ras or B-Raf (V600E) reduced the expression of DR5. Importantly, we have elucidated that Ras induces DR5 expression through co-activation of ERK/RSK and JNK signaling pathways and subsequent cooperative effects among the transcriptional factors CHOP, Elk1, and c-Jun to enhance DR5 gene transcription. Moreover, we found that the majority of cancer cell lines highly sensitive to the DR5 agonistic antibody AMG655 have either Ras or B-Raf mutations. Our findings warrant further study on the biology of DR5 regulation by Ras and B-Raf, which may provide new insight into the biology of Ras and B-Raf, and on the potential impact of Ras or B-Raf mutations on the outcome of DR5-targeted cancer therapy.

  8. Expression of Ik6 and Ik8 Isoforms and Their Association with Relapse and Death in Mexican Children with Acute Lymphoblastic Leukemia.

    PubMed

    Reyes-León, Adriana; Juárez-Velázquez, Rocío; Medrano-Hernández, Alma; Cuenca-Roldán, Teresa; Salas-Labadía, Consuelo; Del Pilar Navarrete-Meneses, María; Rivera-Luna, Roberto; López-Hernández, Gerardo; Paredes-Aguilera, Rogelio; Pérez-Vera, Patricia

    2015-01-01

    Expression of the 6 and 8 dominant-negative Ikaros isoforms in pediatric patients with acute lymphoblastic leukemia has been associated with a high risk of relapse and death; due to these isoforms disrupting the differentiation and proliferation of lymphoid cells. The aim of this study was to know the frequency of Ik6 and Ik8 in 113 Mexican ALL-children treated within the National Popular Medical Insurance Program to determine whether there was an association with relapse-free survival, event-free survival and overall survival, and to assess its usefulness in the initial stratification of patients. The expression of these isoforms was analyzed using specific primer sets and nested RT-PCR. The detected transcripts were classified according to the isoforms's sizes reported. A non-expected band of 300 bp from one patient was analyzed by sequencing. Twenty-six patients expressed Ik6 and/or Ik8 and one of them expressed a variant of Ik8 denominated Ik8-deleted. Although the presence of them was not statistically associated with lower relapse free survival (p = 0.432), event free survival (p = 0.667) or overall survival (p = 0.531), inferior overall survival was observed in patients that expressed these isoforms and showed high or standard risk by age and white blood-cell count at diagnosis. Of the 26 patients Ik6+ and/or Ik8+, 14 did not present adverse events; from them 6 were exclusively Ik6+ and/or Ik8+, and 8 were positive for the other Ikaros isoforms (Ik1, Ik2, Ik5, Ik3A, Ik4, Ik4A, Ik7). In the patients studied, the expression of Ik6 and Ik8 did not constitute an independent prognostic factor for relapse or death related to disease; therefore, they could not be used in the initial risk stratification.

  9. Expression of Ik6 and Ik8 Isoforms and Their Association with Relapse and Death in Mexican Children with Acute Lymphoblastic Leukemia

    PubMed Central

    Reyes-León, Adriana; Juárez-Velázquez, Rocío; Medrano-Hernández, Alma; Cuenca-Roldán, Teresa; Salas-Labadía, Consuelo; del Pilar Navarrete-Meneses, María; Rivera-Luna, Roberto; López-Hernández, Gerardo; Paredes-Aguilera, Rogelio; Pérez-Vera, Patricia

    2015-01-01

    Expression of the 6 and 8 dominant-negative Ikaros isoforms in pediatric patients with acute lymphoblastic leukemia has been associated with a high risk of relapse and death; due to these isoforms disrupting the differentiation and proliferation of lymphoid cells. The aim of this study was to know the frequency of Ik6 and Ik8 in 113 Mexican ALL-children treated within the National Popular Medical Insurance Program to determine whether there was an association with relapse-free survival, event-free survival and overall survival, and to assess its usefulness in the initial stratification of patients. The expression of these isoforms was analyzed using specific primer sets and nested RT-PCR. The detected transcripts were classified according to the isoforms’s sizes reported. A non-expected band of 300 bp from one patient was analyzed by sequencing. Twenty-six patients expressed Ik6 and/or Ik8 and one of them expressed a variant of Ik8 denominated Ik8-deleted. Although the presence of them was not statistically associated with lower relapse free survival (p = 0.432), event free survival (p = 0.667) or overall survival (p = 0.531), inferior overall survival was observed in patients that expressed these isoforms and showed high or standard risk by age and white blood-cell count at diagnosis. Of the 26 patients Ik6+ and/or Ik8+, 14 did not present adverse events; from them 6 were exclusively Ik6+ and/or Ik8+, and 8 were positive for the other Ikaros isoforms (Ik1, Ik2, Ik5, Ik3A, Ik4, Ik4A, Ik7). In the patients studied, the expression of Ik6 and Ik8 did not constitute an independent prognostic factor for relapse or death related to disease; therefore, they could not be used in the initial risk stratification. PMID:26131904

  10. NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains.

    PubMed

    Lucena, Pedro I; Faget, Douglas V; Pachulec, Emilia; Robaina, Marcela C; Klumb, Claudete E; Robbs, Bruno K; Viola, João P B

    2016-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4(+) T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions.

  11. NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains

    PubMed Central

    Lucena, Pedro I.; Faget, Douglas V.; Pachulec, Emilia; Robaina, Marcela C.; Klumb, Claudete E.

    2015-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4+ T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions. PMID:26483414

  12. Voodoo death.

    PubMed

    Lester, David

    2009-01-01

    Scholarly writing on voodoo death is reviewed. Criticisms that voodoo deaths in indigenous societies have never been well documented are refuted with cases medically documented in developed nations. The work of Cannon and Richter on sudden death in animals is reviewed and dismissed as irrelevant for understanding voodoo death. The role of starvation and dehydration is discussed, and it is suggested that the given-up/giving-up hypothesis best fits the phenomenon of voodoo death. Hypotheses for future research are suggested.

  13. Nuclear Respiratory Factor-1 (NRF-1) Regulates Transcription of the CXC Receptor 4 (CXCR4) in the Rat Retina.

    PubMed

    Chen, Pei; Cai, Xiaoxiao; Yang, Ying; Chen, Zhao; Qiu, Jin; Yu, Na; Tang, Mingjun; Wang, Qiyun; Ge, Jian; Yu, Keming; Zhuang, Jing

    2017-09-01

    The CXC receptor 4 (CXCR4) is required for various physiologic and pathologic processes in the eye, including stem cell trafficking, neuronal development, immune responses, and ocular neovascularization. Here, we used the rat retina models to determine the mechanisms driving CXCR4 transcription. The expression pattern of CXCR4 and nuclear respiratory factor-1 (NRF-1) were profiled in the rat retina during the course of development. Chromatin immunoprecipitation (CHiP) assay determined the transcriptional mechanism of CXCR4 in rat retina. A rat model of oxygen-induced retinopathy (OIR) that mimics retinal ischemia-reperfusion injury was established. Under either normoxic or hypoxic conditions, CXCR4 and NRF-1 expression in rat retinas was tracked by RT-PCR and Western analysis. Immunofluorescence staining localized CXCR4 and NRF-1. Both CXCR4 and NRF-1 were highly expressed in the neonatal rat retina, down-regulated in parallel, and silenced in fully developed retinas (1 month of age). ChIP assays revealed that NRF-1 was required for CXCR4 promoter activity in rat retinas. In the OIR rat model, retinal hypoxia induced up-regulation of CXCR4 and NRF-1 concurrently. Our findings suggest that NRF-1 regulates the expression of CXCR4 in normal retinal development and in pathologic processes of retinal hypoxia and neovascularization.

  14. Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

    PubMed Central

    Cao, Li Juan; Liu, Xin He; Liu, Zhu Hui; Wang, Xiao Qun; Chen, Qiu Jin; Lu, Lin; Shen, Wei Feng; Liu, Yan

    2014-01-01

    Oxidized low-density lipoprotein (oxLDL)-regulated secretion of inflammatory cytokines in smooth muscle cells (SMCs) is regarded as an important step in the progression of atherosclerosis; however, its underlying mechanism remains unclear. This study investigated the role of toll-like receptor 4 (TLR4) in oxLDL-induced expression of inflammatory cytokines in SMCs both in vivo and in vitro. We found that the levels of TLR4, interleukin 1-β (IL1-β), tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase-2 (MMP-2) expression were increased in the SMCs of atherosclerotic plaques in patients with femoral artery stenosis. In cultured primary arterial SMCs from wild type mice, oxLDL caused dose- and time-dependent increase in the expression levels of TLR4 and cytokines. These effects were significantly weakened in arterial SMCs derived from TLR4 knockout mice (TLR4−/−). Moreover, the secretion of inflammatory cytokines was blocked by TLR4-specific antibodies in primary SMCs. Ox-LDL induced activation of p38 and NFκB was also inhibited in TLR4−/− primary SMCs or when treated with TLR4-specific antibodies. These results demonstrated that TLR4 is a crucial mediator in oxLDL-induced inflammatory cytokine expression and secretion, and p38 and NFκB activation. PMID:24755612

  15. MiR-7a is an important mediator in Fas-associated protein with death domain (FADD)-regulated expression of focal adhesion kinase (FAK)

    PubMed Central

    Liu, Yingting; Cui, Hongen; Huang, Xianjie; Zhu, Bo; Guan, Shengwen; Cheng, Wei; Lai, Yueyang; Zhang, Xiaoxin; Hua, Zi-Chun

    2016-01-01

    Fas-associated protein with death domain (FADD), a classical adaptor protein mediating apoptotic stimuli-induced cell death, has been reported to engage in several non-apoptotic processes such as T cell and cardiac development and tumorigenesis. Recently, there are several reports about the FADD's involvement in cell migration, however the underlying mechanism remains elusive. Here, we present a new finding that FADD could regulate the expression of FAK, a non-receptor protein tyrosine kinase overexpressed in many cancers, and played an important role in cell migration in murine MEF and melanoma cells with different metastatic potential, B16F10 and B16F1. Moreover, miR-7a, a tumor suppressor which prohibits cell migration and invasion, was up-regulated in FADD-deficient cells. And FAK was verified to be the direct target gene of miR-7a in B16F10 cells. Furthermore, we demonstrate that miR-7a was a necessary mediator in FADD-regulated FAK expression. In contrast to its classical apoptotic role, FADD interference could reduce the rate of cell migration, which could be rescued by inhibiting miR-7a expression. Taken together, our data provide a novel explanation regarding how FADD regulates cell migration in murine melanoma cells. PMID:27286445

  16. c-CBL E3 Ubiquitin Ligase is Over-Expressed in Cutaneous T-Cell Lymphoma: Its Inhibition Promotes Activation Induced Cell Death

    PubMed Central

    Wu, Jianqiang; Salva, Katrin A.; Wood, Gary S.

    2014-01-01

    Mycosis fungoides (MF) and Sezary syndrome (SS) are two major forms of cutaneous T-cell lymphoma (CTCL) characterized by resistance to apoptosis. A central pathway for T-cell apoptosis is activation-induced cell death (AICD) which is triggered through the T-cell receptor (TCR). This results in upregulation of FAS-ligand (FASL) and subsequent apoptosis through the FAS death receptor pathway. It has been known for more than a decade that TCR signaling is defective in CTCL; however, the underlying mechanism has not been apparent. In this report, we show that the E3 ubiquitin ligase, c-CBL, is over-expressed in CTCL and that its knockdown overcomes defective TCR signaling resulting in phosphorylation of PLCg1, calcium influx, ROS generation, up-regulation of FASL and extrinsic pathway apoptosis in CTCL cells expressing adequate FAS. In CTCL cells with suboptimal FAS expression, FAS can be upregulated epigenetically by derepression of the FAS promoter using methotrexate (MTX) which we showed previously has activity as a DNA methylation inhibitor. Using these combined strategies, FAS-low as well as FAS-high CTCL cells can be killed effectively. PMID:25140833

  17. A bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-Fas antibody injection

    PubMed Central

    1996-01-01

    Stimulation of the Fas (APO-1, CD95) receptor, which is present on a variety of cells, usually triggers a process of programmed cell death. Systemic injection of anti-Fas antibody into mice leads to fulminant liver destruction resulting from massive hepatocyte apoptosis, and to rapid death. Hepatocytes bear Fas but do not express Bcl-2, a protein that plays, in a number of conditions, a protective role against apoptosis. We have generated mice whose liver expresses Bcl-2 as the result of bcl-2 transgene placed under the control of the hepatocyte- specific alpha1-anti-trypsin gene promoter, but is otherwise not distinguishable from that of normal mice. These mice display a marked to almost total resistance to liver damage induced by anti-Fas antibody injection. This protective effect of Bcl-2 occurs in the absence of significant variations, in the stimulated livers, in the level of expression of other proteins also involved in resistance or sensitivity to apoptosis, namely Bcl-x, Bax, Bad, Bak, and p53. Mice with protected livers, however, die almost as rapidly as normal mice, which indicates that acute lethality results from stimulation of Fas receptors present on other target organs or cells. PMID:8642244

  18. Spermatozoal cell death-inducing DNA fragmentation factor-α-like effector A (CIDEA) gene expression and DNA fragmentation in infertile men with metabolic syndrome and normal seminogram.

    PubMed

    Elsamanoudy, Ayman Z; Abdalla, Hussein Abdelaziz; Hassanien, Mohammed; Gaballah, Mohammad A

    2016-01-01

    This is the first study to investigate spermatozoal cell death-inducing DNA fragmentation factor-α-like effector A (CIDEA) gene expression and DNA fragmentations in the spermatozoa of men diagnosed with metabolic syndrome (MS) who have normal seminograms with unexplained infertility, and to correlate these parameters with seminal glucose concentration. This study included 120 participants: 75 male subjects with MS (38 fertile and 37 infertile), and a control group of 45 fertile males without MS. HOMA-IR, semen analysis, and biochemical measurement of seminal plasma insulin and glucose levels were carried out. Spermatozoal insulin gene and CIDEA gene expressions were performed by the RT-PCR method. The percentage of spermatozoal DNA fragmentation was also estimated. The spermatozoal insulin and CIDEA gene expression, as well as the DNA fragmentation, were significantly higher in the infertile MS group than in the fertile MS group, and significantly higher in both the MS groups than in the control group. Seminal glucose concentration showed significant positive correlations with seminal insulin level, spermatozoa insulin, CIDEA gene expression, and DNA fragmentation. Moreover, there was a positive correlation between spermatozoa CIDEA gene expression and DNA fragmentation. It can be concluded that MS may affect male fertility at the molecular level, through its possible inducing effect of spermatozoa CIDEA and insulin gene expression, DNA fragmentation, and increased seminal glucose.

  19. Plant programmed cell death caused by an autoactive form of Prf is suppressed by co-expression of the Prf LRR domain.

    PubMed

    Du, Xinran; Miao, Min; Ma, Xinrong; Liu, Yongsheng; Kuhl, Joseph C; Martin, Gregory B; Xiao, Fangming

    2012-09-01

    In tomato, the NBARC-LRR resistance (R) protein Prf acts in concert with the Pto or Fen kinase to determine immunity against Pseudomonas syringae pv. tomato (Pst). Prf-mediated defense signaling is initiated by the recognition of two sequence-unrelated Pst-secreted effector proteins, AvrPto and AvrPtoB, by tomato Pto or Fen. Prf detects these interactions and activates signaling leading to host defense responses including localized programmed cell death (PCD) that is associated with the arrest of Pst growth. We found that Prf variants with single amino acid substitutions at D1416 in the IHD motif (isoleucine-histidine-aspartic acid) in the NBARC domain cause effector-independent PCD when transiently expressed in leaves of Nicotiana benthamiana, suggesting D1416 plays an important role in activation of Prf. The N-terminal region of Prf (NPrf) and the LRR domain are required for this autoactive Prf cell death signaling but dispensable for accumulation of the Prf(D1416V) protein. Significantly, co-expression of the Prf LRR but not NPrf, with Prf(D1416V), AvrPto/Pto, AvrPtoB/Pto, an autoactive form of Pto (Pto(Y207D)), or Fen completely suppresses PCD. However, the Prf LRR does not interfere with PCD caused by Rpi-blb1(D475V), a distinct R protein-mediated PCD signaling event, or that caused by overexpression of MAPKKKα, a protein acting downstream of Prf. Furthermore, we found the Prf(D1416V) protein is unable to accumulate in plant cells when co-expressed with the Prf LRR domain, likely explaining the cell death suppression. The mechanism for the LRR-induced degradation of Prf(D1416V) is unknown but may involve interference in the intramolecular interactions of Prf or to binding of the unattached LRR to other host proteins that are needed for Prf stability.

  20. Silymarin modulates doxorubicin-induced oxidative stress, Bcl-xL and p53 expression while preventing apoptotic and necrotic cell death in the liver

    SciTech Connect

    Patel, Nirav; Joseph, Cecil; Corcoran, George B.; Ray, Sidhartha D.

    2010-06-01

    The emergence of silymarin (SMN) as a natural remedy for liver diseases, coupled with its entry into NIH clinical trial, signifies its hepatoprotective potential. SMN is noted for its ability to interfere with apoptotic signaling while acting as an antioxidant. This in vivo study was designed to explore the hepatotoxic potential of Doxorubicin (Dox), the well-known cardiotoxin, and in particular whether pre-exposures to SMN can prevent hepatotoxicity by reducing Dox-induced free radical mediated oxidative stress, by modulating expression of apoptotic signaling proteins like Bcl-xL, and by minimizing liver cell death occurring by apoptosis or necrosis. Groups of male ICR mice included Control, Dox alone, SMN alone, and Dox with SMN pre/co-treatment. Control and Dox groups received saline i.p. for 14 days. SMN was administered p.o. for 14 days at 16 mg/kg/day. An approximate LD{sub 50} dose of Dox, 60 mg/kg, was administered i.p. on day 12 to animals receiving saline or SMN. Animals were euthanized 48 h later. Dox alone induced frank liver injury (> 50-fold increase in serum ALT) and oxidative stress (> 20-fold increase in malondialdehyde [MDA]), as well as direct damage to DNA (> 15-fold increase in DNA fragmentation). Coincident genomic damage and oxidative stress influenced genomic stability, reflected in increased PARP activity and p53 expression. Decreases in Bcl-xL protein coupled with enhanced accumulation of cytochrome c in the cytosol accompanied elevated indexes of apoptotic and necrotic cell death. Significantly, SMN exposure reduced Dox hepatotoxicity and associated apoptotic and necrotic cell death. The effects of SMN on Dox were broad, including the ability to modulate changes in both Bcl-xL and p53 expression. In animals treated with SMN, tissue Bcl-xL expression exceeded control values after Dox treatment. Taken together, these results demonstrated that SMN (i) reduced, delayed onset, or prevented toxic effects of Dox which are typically associated

  1. Adenovirus expressing dual c-Met-specific shRNA exhibits potent antitumor effect through autophagic cell death accompanied by senescence-like phenotypes in glioblastoma cells

    PubMed Central

    Yoo, Ji Young; Choi, Kyeong Sook; Yoon, Mi Jin; Yun, Chae-Ok

    2015-01-01

    c-Met, a cognate receptor tyrosine kinase of hepatocyte growth factor, is overexpressed and/or mutated in number of tumors. Therefore, abrogation of c-Met signaling may serve as potential therapeutic targets. In this study, we generated Ads expressing single shRNA specific to c-Met (shMet) (dl/shMet4 and dl/shMet5) or dual shRNAs specific to c-Met (dl/shMet4+5); and examined the therapeutic potential of these newly engineered Ads in targeting c-Met, and delineated their mechanism of action in vitro and in vivo. Ads expressing shMet induced knock-down in c-Met, and phenotypically resulted in autophagy-like features including appearance of membranousvacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein1 light chain 3 to autophagosomes. Ads expressing shMet also suppressed Akt phosphorylation and increased number of senescence-related gene products including SM22, TGase II, and PAI-1. These changes resulted in inhibition of cell proliferation and G2/M arrest of U343 cells. In vivo, intratumoral injection with dl/shMet4+5 resulted in a significant reduction of tumor growth with corresponding increasing overall survival. Histopathological analysis of these treated tumors revealed that Atg5 was highly up-regulated, indicating the therapeutic induction of autophagy. In sum, these results reveal that autophagic cell death induced by shMet-expressing Ads provide a novel strategy for targeting c-Met-expressing tumors through non-apoptotic mechanism of cell death. PMID:25726528

  2. Acute ethanol administration inhibits Toll-like receptor 4 signaling pathway in rat intestinal epithelia.

    PubMed

    Zhou, Chao; Zhao, Ji; Li, Jing; Wang, Haiying; Tang, Chengwei

    2013-05-01

    Excess alcohol intake, as in binge drinking, increases susceptibility to microbial pathogens. Alcohol impairs macrophage function by suppression of the Toll-like receptor 4 (TLR4) pathway. This study investigated the effects of acute ethanol intake on the TLR4 pathway in rat intestinal epithelia, which usually encounters luminal antigens at first and participates in the development of intestinal immunity. Twenty Wistar rats were randomly assigned to an ethanol group given ethanol as a 25% (v/v) solution in water at 7.5 g/kg, or a control group given saline, by oral gavage daily for 3 days. The epithelial histology and ultrastructure, the intestinal microflora, peripheral and portal venous plasma lipopolysaccharide (LPS) levels, and somatostatin (SST) levels in the peripheral plasma and small intestine were evaluated. Somatostatin receptor 2 (SSTR2), TLR4, TANK binding kinase-1 (TBK1), activated nuclear factor-κB (NF-κB), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the intestinal mucosa were assayed. LPS responsiveness with or without SST pretreatment was assayed in vitro by quantification of TLR4, TBK1, activated NF-κB, IFN-γ and TNF-α in isolated intestinal epithelia. Mucosal damage was observed in the ethanol group by light and electron microscopy. Escherichia coli cultures were unchanged in rat intestine of the ethanol group compared with controls, but lactobacilli cultures were reduced (p < 0.05). LPS levels increased in peripheral and portal venous plasma (p < 0.05), but mucosal TLR4, TBK1, nuclear NF-κB, IFN-γ and TNF-α were unchanged in the ethanol group. LPS treatment in vitro up-regulated the level of TLR4, TBK1 and nuclear NF-κB as well as the production of IFN-γ and TNF-α in isolated intestinal epithelia in the control (p < 0.05), but not the ethanol group. The stimulatory effects of LPS on intestinal epithelia isolated from the control group were significantly inhibited by SST pretreatment (p < 0.05). The

  3. Hydrogen sulfide donor NaHS induces death of alveolar epithelial L2 cells that is associated with cellular shrinkage, transgelin expression and myosin phosphorylation.

    PubMed

    Fujii, Yusuke; Funakoshi, Takeshi; Unuma, Kana; Noritake, Kanako; Aki, Toshihiko; Uemura, Koichi

    2016-01-01

    Hydrogen sulfide (H2S) is a highly toxic gaseous molecule that causes death to humans exposed to high concentrations. H2S is absorbed into the body through the alveolar epithelium and other tissues. The aim of this study is to evaluate the molecular mechanism underling acute lung injury caused by the inhalation of high concentrations of H2S. Rat lung epithelium-derived L2 cells were exposed to a H2S donor, NaHS, at concentrations of 2-4 mM for 1-6 hr. NaHS caused shrinkage and death of the cells without caspase activation. An actin-binding protein, transgelin, was identified as one of the NaHS-inducible proteins in the cells. NaHS increased myosin light chain (MLC) phosphorylation, indicating that actomyosin-mediated cellular contractility and/or motility could be increased after NaHS exposure. The administration of ML-7, a myosin light chain kinase (MLCK) inhibitor, accelerated cell death after NaHS exposure. Based on these data, we conclude that the increase in MLC phosphorylation in response to NaHS exposure is a cellular protective reaction against NaHS toxicity. Enhancements in smooth muscle cell properties such as transgelin expression and actomyosin-mediated contractility/motility might be involved in cell survival after NaHS exposure.

  4. Cutting edge: the "death" adaptor CRADD/RAIDD targets BCL10 and suppresses agonist-induced cytokine expression in T lymphocytes.

    PubMed

    Lin, Qing; Liu, Yan; Moore, Daniel J; Elizer, Sydney K; Veach, Ruth A; Hawiger, Jacek; Ruley, H Earl

    2012-03-15

    The expression of proinflammatory cytokines and chemokines in response to TCR agonists is regulated by the caspase-recruitment domain membrane-associated guanylate kinase 1 (CARMA1) signalosome through the coordinated assembly of complexes containing the BCL10 adaptor protein. We describe a novel mechanism to negatively regulate the CARMA1 signalosome by the "death" adaptor protein caspase and receptor interacting protein adaptor with death domain (CRADD)/receptor interacting protein-associated ICH-1/CED-3 homologous protein with a death domain. We show that CRADD interacts with BCL10 through its caspase recruitment domain and suppresses interactions between BCL10 and CARMA1. TCR agonist-induced interaction between CRADD and BCL10 coincides with reduction of its complex formation with CARMA1 in wild-type, as compared with Cradd-deficient, primary cells. Finally, Cradd-deficient spleen cells, CD4(+) T cells, and mice respond to T cell agonists with strikingly higher production of proinflammatory mediators, including IFN-γ, IL-2, TNF-α, and IL-17. These results define a novel role for CRADD as a negative regulator of the CARMA1 signalosome and suppressor of Th1- and Th17-mediated inflammatory responses.

  5. High expression of IMPACT protein promotes resistance to indoleamine 2,3-dioxygenase-induced cell death.

    PubMed

    Habibi, Darya; Jalili, Reza B; Forouzandeh, Farshad; Ong, Christopher J; Ghahary, Aziz

    2010-10-01

    Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO-induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non-derepressible-2(GCN2) kinase stress-responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO-induced low tryptophan environment. IDO-expressing human fibroblasts were co-cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO-expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO-induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co-cultured with IDO-expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT-expressing vector, was rescued significantly in tryptophan-deficient but not IDO-expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO-mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO-generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non-immune cells such as skin cells acts as a protective mechanism against IDO-induced GCN2 activation, therefore, makes them resistant to the amino acid-deprived environment caused by IDO.

  6. The Expression and Prognostic Impact of CD95 Death Receptor and CD20, CD34 and CD44 Differentiation Markers in Pediatric Acute Lymphoblastic Leukemia

    PubMed Central

    M. Kamazani, Fatemeh; Bahoush-Mehdiabadi, Gholamreza; Aghaeipour, Mahnaz; Vaeli, Shahram; Amirghofran, Zahra

    2014-01-01

    Objective: This study investigated the expression and prognostic significance of the CD95 death receptor and CD20, a B cell-lineage associated marker, along with CD34 and CD44 non-lineage associated molecules in Iranian children with acute lymphoblastic leukemia (ALL). Methods: We performed immunophenotyping for expressions of the molecules in blood samples from children diagnosed with ALL by using a panel of monoclonal antibodies for flow cytometry analysis. The expression of markers was evaluated in relation to clinical and paraclinical features as well as response to treatment in the patients. Findings : CD95 showed a higher expression in T-ALL compared to B-ALL (P<0.001). Analysis of the clinical and laboratory findings at diagnosis in the group of B-ALL patients revealed an association between CD95 expression with lower white blood cell (WBC) numbers and bone marrow blasts (P<0.05). We detected a positive correlation between the expressions of CD95 and CD44 (r=0.445, P<0.01) in B-ALL patients. There was an association between CD20 expression and several poor prognostic factors that included increased extramedullary involvement (EMI) and decreased platelet numbers (P<0.008). The mean expression of CD34 in B-ALL was higher than T-ALL (P=0.004). At follow-up, complete remission duration (CRD) and survival duration did not significantly differ between patients who were positive or negative for each marker. Conclusion: Association of the studied molecules with several prognostic factors implies the significance of CD95 molecule as favorable and CD20 as unfavorable prognostic markers for childhood ALL. PMID:25755857

  7. Expression of programmed death 1 ligand 1 on periodontal tissue cells as a possible protective feedback mechanism against periodontal tissue destruction.

    PubMed

    Zhang, Jiehua; Wang, Chieh-Mei; Zhang, Ping; Wang, Xiaoqian; Chen, Jiao; Yang, Jun; Lu, Wanlu; Zhou, Wenjie; Yuan, Wenwen; Feng, Yun

    2016-03-01

    Programmed death 1 ligand 1 (PD‑L1) is a negative co‑stimulatory molecule in immune responses. Previous reports have indicated that inflammatory cytokines can upregulate the expression of PD‑L1 in tumor cells, which in turn suppresses host immune responses. Periodontitis is characterized by persistent inflammation of the periodontium, which is initiated by infection with oral bacteria and results in damage to cells and the matrices of the periodontal connective tissues. In the present study, the expression and function of PD‑L1 in periodontal tissue destruction were examined. Periodontal ligament cells (PDLCs) were stimulated by inflammatory cytokines and periodontal pathogens. The expression and function of PD‑L1 on the surface of PDLCs was investigated using flow cytometry in vitro. Periodontal disease was induced by the injection of Porphyromonas gingivalis in mouse models. The expression levels of PD‑L1 in the periodontal tissues of the mice were analyzed using flow cytometry and immunohistochemistry. PD‑L1 was inducibly expressed on the PDLCs by the inflammatory cytokines and periodontal pathogens. The inflammation‑induced expression of PD‑L1 was shown to cause the apoptosis of activated T lymphocytes and improve the survival of PDLCs. Furthermore, in the mouse model of experimental periodontitis, the expression of PD‑L1 in severe cases of periodontitis was significantly lower, compared with that in mild cases. By contrast, no significant differences were observed between the healthy control and severe periodontitis groups. The results of the present study showed that the expression of PD‑L1 may inhibit the destruction of periodontal tissues, indicating the involvement of a possible protective feedback mechanism against periodontal infection.

  8. Oocyte-secreted growth differentiation factor 9 inhibits BCL-2-interacting mediator of cell death-extra long expression in porcine cumulus cell.

    PubMed

    Wang, Xian-Long; Wang, Kun; Zhao, Shuan; Wu, Yi; Gao, Hui; Zeng, Shen-Ming

    2013-09-01

    Oocyte-secreted factors (OSFs) maintain the low incidence of cumulus cell apoptosis. In this report, we described that the presence of oocytes suppressed the expression of proapoptotic protein BCL-2-interacting mediator of cell death-extra long (BIMEL) in porcine cumulus cells. Atretic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cumulus cells strongly expressed BIMEL protein. The healthy cumulus- oocyte complex exhibited a low BIMEL expression in cumulus cell while the removal of oocyte led to an about 2.5-fold (P < 0.5) increased expression in oocytectomized complex (OOX). Coculturing OOXs with denuded oocytes decreased BIMEL expression to the normal level. The similar expression pattern could also be achieved in OOXs treated with exogenous recombinant mouse growth differentiation factor 9 (GDF9), a well-characterized OSF. This inhibitory action of GDF9 was prevented by the addition of a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Luciferase assay further demonstrated that BIM gene expression was forkhead box O3a (FOXO3a)-dependent because mutation of FOXO3a-binding site on the BIM promoter inhibited luciferase activities. Moreover, the activity of BIM promoter encompassing the FOXO3a-binding site could be regulated by GDF9. Additionally, we found that GDF9 elevated the levels of phosphorylated AKT and FOXO3a, and this process was independent of the SMAD signal pathway. Taken together, we concluded that OSFs, particularly GDF9, maintained the low level of BIMEL expression in cumulus cell through activation of the PI3K/FOXO3a pathway.

  9. Promotion of the Unfolding Protein Response in Orexin/Dynorphin Neurons in Sudden Infant Death Syndrome (SIDS): Elevated pPERK and ATF4 Expression.

    PubMed

    Hunt, Nicholas J; Waters, Karen A; Machaalani, Rita

    2016-10-29

    We previously demonstrated that sudden infant death syndrome (SIDS) infants have decreased orexin immunoreactivity within the hypothalamus and pons compared to non-SIDS infants. In this study, we examined multiple mechanisms that may promote loss of orexin expression including programmed cell death, impaired maturation/structural stability, neuroinflammation and impaired unfolding protein response (UPR). Immunofluorescent and immunohistochemical staining for a number of markers was performed in the tuberal hypothalamus and pons of infants (1-10 months) who died from SIDS (n = 27) compared to age- and sex-matched non-SIDS infants (n = 19). The markers included orexin A (OxA), dynorphin (Dyn), cleaved caspase 3 (CC3), cleaved caspase 9 (CC9), glial fibrillary acid protein (GFAP), tubulin beta chain 3 (TUBB3), myelin basic protein (MBP), interleukin 1β (IL-1β), terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL), c-fos and the UPR activation markers: phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (pPERK), and activating transcription factor 4 (ATF4). It was hypothesised that pPERK and ATF4 would be upregulated in Ox neurons in SIDS compared to non-SIDS. Within the hypothalamus, OxA and Dyn co-localised with a 20 % decrease in expression in SIDS infants (P = 0.001). pPERK and ATF4 expression in OxA neurons were increased by 35 % (P = 0.001) and 15 % (P = 0.001) respectively, with linear relationships between the decreased OxA/Dyn expression and the percentages of co-localised pPERK/OxA and ATF4/OxA evident (P = 0.01, P = 0.01). No differences in co-localisation with CC9, CC3, TUNEL or c-fos, nor expression of MBP, TUBB3, IL-1β and GFAP, were observed in the hypothalamus. In the pons, there were 40 % and 20 % increases in pPERK expression in the locus coeruleus (P = 0.001) and dorsal raphe (P = 0.022) respectively; ATF4 expression was not changed. The findings that decreased orexin levels in SIDS infants

  10. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    PubMed Central

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

  11. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells.

    PubMed

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-03-01

    Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer.

  12. Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork

    PubMed Central

    Hernandez, Humberto; Medina-Ortiz, Wanda E.; Luan, Tomi; Clark, Abbot F.; McDowell, Colleen M.

    2017-01-01

    Purpose The trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. Regulation of the extracellular matrix (ECM) by TGFβ2 signaling pathways in the TM has been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in liver, kidney, lung, and skin. Here, we investigated the role of TGFβ2–TLR4 signaling crosstalk in the regulation of the ECM in the TM and ocular hypertension. Methods Cross sections of human donor eyes, primary human TM cells in culture, and dissected mouse TM rings were used to determine Tlr4 expression in the TM. Trabecular meshwork cells in culture were treated with TGFβ2 (5 ng/mL), TLR4 inhibitor (TAK-242, 15 μM), and a TLR4 ligand (cellular fibronectin isoform [cFN]-EDA). A/J (n = 13), AKR/J (n = 7), BALBc/J (n = 8), C3H/HeJ (n = 20), and C3H/HeOuJ (n = 10) mice were injected intravitreally with adenovirus 5 (Ad5).hTGFβ2c226s/c228s in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Results Toll-like receptor 4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFβ2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFβ2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFβ2c226s/c228s induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Conclusions These studies identify TGFβ2–TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data further explain the complex mechanisms involved in the development of glaucomatous TM damage. PMID:28346614

  13. Yokukansan, a Kampo Medicine, Protects PC12 Cells from Glutamate-Induced Death by Augmenting Gene Expression of Cystine/Glutamate Antiporter System Xc−

    PubMed Central

    Kanno, Hitomi; Kawakami, Zenji; Mizoguchi, Kazushige; Ikarashi, Yasushi; Kase, Yoshio

    2014-01-01

    Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc−, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc− subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook. PMID:25551766

  14. New transgenic ALS/FTD models on the rat-walk: An Editorial Highlight for 'Increased Ubqln2 expression causes neuron death in transgenic rats'.

    PubMed

    Stegmüller, Judith; Synofzik, Matthis

    2016-10-01

    This Editorial highlights a study by Huang and colleagues in the current issue of Journal of Neurochemistry. The authors introduce a novel ALS-FTD (amyotrophic lateral sclerosis-frontotemporal dementia) rat model to explore the role of the UBLQN2 gene that has previously been associated with familial ALS-FTD. Over-expression of ubiquilin 2 in the cortex (CTX) and hippocampus of the rat results in ubiquilin 2 aggregates and neurodegeneration together with cognitive deficits. The new rat model not only gives insight into potential molecular underpinnings of ALS-FTD, but also represents an important new tool for future research and therapeutic approaches. Read the highlighted article 'Increased Ubqln2 expression causes neuron death in transgenic rats' on page 285. © 2016 International Society for Neurochemistry.

  15. Zingerone ameliorates lipopolysaccharide-induced acute kidney injury by inhibiting Toll-like receptor 4 signaling pathway.

    PubMed

    Song, Jie; Fan, Hao-jun; Li, Hui; Ding, Hui; Lv, Qi; Hou, Shi-ke

    2016-02-05

    Acute kidney injury (AKI) is a serious complication of sepsis. Zingerone, a phenolic alkanone isolated from ginger, has been reported to have anti-inflammatory effect. The aim of this study was to investigate the therapeutic effects of zingerone on lipopolysaccharide (LPS)-induced AKI in mice. Zingerone was administrated 1h after LPS challenge. The production of blood urea nitrogen (BUN) and creatinine were measured in this study. The expressions of inflammatory cytokines in serum and kidney tissues were detected by ELISA. The expressions of Toll-like receptor 4 (TLR4), MyD88, TRIF, Nuclear factor Kappa B (NF-κB) and IκB were measured by Western blotting. The results showed that zingerone suppressed LPS-induced BUN, creatinine, and inflammatory cytokines TNF-α, IL-6 and IL-1β levels in a dose-dependent manner. Zingerone also attenuated LPS-induced kidney histopathologic changes. Furthermore, zingerone was found to inhibit LPS-induced TLR4, MyD88, TRIF expression and NF-κB activation. In conclusion, the current study demonstrated that zingerone inhibited LPS-induced AKI by suppressing TLR4/NF-κB signaling pathway.

  16. Disabled-2 is a negative immune regulator of lipopolysaccharide-stimulated Toll-like receptor 4 internalization and signaling

    PubMed Central

    Hung, Wei-Shan; Ling, Pin; Cheng, Ju-Chien; Chang, Shy-Shin; Tseng, Ching-Ping

    2016-01-01

    Toll-like receptor 4 (TLR4) plays a pivotal role in the host response to lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Here, we elucidated whether the endocytic adaptor protein Disabled-2 (Dab2), which is abundantly expressed in macrophages, plays a role in LPS-stimulated TLR4 signaling and trafficking. Molecular analysis and transcriptome profiling of RAW264.7 macrophage-like cells expressing short-hairpin RNA of Dab2 revealed that Dab2 regulated the TLR4/TRIF pathway upon LPS stimulation. Knockdown of Dab2 augmented TRIF-dependent interferon regulatory factor 3 activation and the expression of subsets of inflammatory cytokines and interferon-inducible genes. Dab2 acted as a clathrin sponge and sequestered clathrin from TLR4 in the resting stage of macrophages. Upon LPS stimulation, clathrin was released from Dab2 to facilitate endocytosis of TLR4 for triggering the TRIF-mediated pathway. Dab2 functions as a negative immune regulator of TLR4 endocytosis and signaling, supporting a novel role for a Dab2-associated regulatory circuit in controlling the inflammatory response of macrophages to endotoxin. PMID:27748405

  17. Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4.

    PubMed

    Lager, Susanne; Gaccioli, Francesca; Ramirez, Vanessa I; Jones, Helen N; Jansson, Thomas; Powell, Theresa L

    2013-03-01

    Obese women have an increased risk to deliver large babies. However, the mechanisms underlying fetal overgrowth in these pregnancies are not well understood. Obese pregnant women typically have elevated circulating lipid levels. We tested the hypothesis that fatty acids stimulate placental amino acid transport, mediated via toll-like receptor 4 (TLR4) and mammalian target of rapamycin (mTOR) signaling pathways. Circulating NEFA levels and placental TLR4 expression were assessed in women with varying prepregnancy body mass index (BMI). The effects of oleic acid on system A and system L amino acid transport, and on the activation of the mTOR (4EBP1, S6K1, rpS6), TLR4 (IĸB, JNK, p38 MAPK), and STAT3 signaling pathways were determined in cultured primary human trophoblast cells. Maternal circulating NEFAs (n = 33), but not placental TLR4 mRNA expression (n = 16), correlated positively with BMI (P < 0.05). Oleic acid increased trophoblast JNK and STAT3 phosphorylation (P < 0.05), whereas mTOR activity was unaffected. Furthermore, oleic acid doubled trophoblast system A activity (P < 0.05), without affecting system L activity. siRNA-mediated silencing of TLR4 expression prevented the stimulatory effect of oleic acid on system A activity. Our data suggest that maternal fatty acids can increase placental nutrient transport via TLR4, thereby potentially affecting fetal growth.

  18. Improved Survival and Neurological Outcomes after Cardiopulmonary Resuscitation in Toll-like Receptor 4-mutant Mice

    PubMed Central

    Xu, Li; Zhang, Qing; Zhang, Qing-Song; Li, Qian; Han, Ji-Yuan; Sun, Peng

    2015-01-01

    Background: Toll-like receptor 4 (TLR4) is a crucial receptor in the innate immune system and noninfectious immune responses. It has been reported that TLR4 participates in the pathological course of ischemia/reperfusion (I/R) injury. However, the role of TLR4 in the process of I/R injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) is still unknown. In this study, we investigated the effects of TLR4 mutation on survival and neurological outcome in a mouse model of CA/CPR. Methods: A model of potassium-induced CA was performed on TLR4-mutant mice (C3H/HeJ) and wild-type mice (C3H/HeN). After 3 min of untreated CA, resuscitation was attempted with chest compression, ventilation, and intravenous epinephrine. Behavioral tests were performed on mice on day 3 after CPR. The morphological changes in hippocampal neurons were assessed by light and electron microscopy. Expressions of TLR4 and intercellular adhesion molecule-1 (ICAM-1) were detected by Western blot. Levels of tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) were measured with enzyme-linked immunosorbent assay (ELISA). Results: On day 3 after resuscitation the overall mortality was 33.33% in C3H/HeJ group compared with 53.33% in C3H/HeN group (P < 0.05). And there was much higher central tendency in C3H/HeJ group than C3H/HeN group during open field test (P < 0.05). Meanwhile, the percentage of nonviable neurons was 21.16% in C3H/HeJ group compared with 53.11% in C3H/HeN group (P < 0.05). And there were significantly lower levels of hippocampal TNF-α and MPO in C3H/HeJ mice (TNF-α: 6.85±1.19 ng/mL, MPO: 0.33±0.11 U/g) than C3H/HeN mice (TNF-α: 11.36±2.12 ng/mL, MPO: 0.54±0.17 U/g) (all P < 0.01). CPR also significantly increased the expressions of TLR4 and ICAM-1 in C3H/HeN group. However, the expression of ICAM-1 was much lower in C3H/HeJ group than in C3H/HeN group after CPR (P < 0.01). Conclusion: TLR4 signaling is involved in brain damage and in inflammation

  19. A novel quinone-based derivative (DTNQ-Pro) induces apoptotic death via modulation of heat shock protein expression in Caco-2 cells

    PubMed Central

    Gomez-Monterrey, Isabel; Campiglia, Pietro; Bertamino, Alessia; Aquino, Claudio; Sala, Marina; Grieco, Paolo; Dicitore, Alessandra; Vanacore, Daniela; Porta, Amalia; Maresca, Bruno; Novellino, Ettore; Stiuso, Paola

    2010-01-01

    Background and purpose: The resistance of human colon adenocarcinoma cells to antineoplastic agents may be related to the high endogenous expression of stress proteins, including the family of heat shock proteins (HSPs). Recently, a quinone-based pentacyclic derivative, DTNQ-Pro, showed high cytotoxic activity in human colon carcinoma cell lines. The aim of the present study was to determine the precise cellular mechanisms of this cytotoxic action of DTNQ-Pro. Experimental approach: Using human colorectal carcinoma-derived Caco-2 cells as a model, we studied the effects of DTNQ-Pro on cellular viability and oxidative stress; HSP70 and HSP27 accumulation; and cell cycle, differentiation and apoptosis. Key results: Incubation of Caco-2 cells with DTNQ-Pro reduced cell growth and increased the levels of reactive oxygen species in mitochondria. After 48 h of treatment, cells surviving showed an increased expression of Mn-superoxide dismutase (SOD), nitric oxide production and membrane lipid peroxidation. Treatment with DTNQ-Pro decreased HSP70 expression, and redistributed HSP27 and vimentin within the cell. DTNQ-Pro down-regulated the expression of A and B cyclins with arrest of the cell cycle in S phase and increased cellular differentiation. A second treatment of Caco-2 cells with DTNQ-Pro induced cellular death by activation of the apoptotic pathway. Conclusions and implications: DTNQ-Pro causes Caco-2 cell death by induction of apoptosis via inhibition of HSP70 accumulation and the intracellular redistribution of HSP27. These findings suggest the potential use of DTNQ-Pro in combination chemotherapy for colon cancer. PMID:20590589

  20. Cot Deaths.

    ERIC Educational Resources Information Center

    Tyrrell, Shelagh

    1985-01-01

    Addresses the tragedy of crib deaths, giving particular attention to causes, prevention, and medical research on Sudden Infant Death Syndrome (SIDS). Gives anecdotal accounts of coping strategies used by parents and families of SIDS infants. (DT)

  1. Cot Deaths.

    ERIC Educational Resources Information Center

    Tyrrell, Shelagh

    1985-01-01

    Addresses the tragedy of crib deaths, giving particular attention to causes, prevention, and medical research on Sudden Infant Death Syndrome (SIDS). Gives anecdotal accounts of coping strategies used by parents and families of SIDS infants. (DT)

  2. Pesticide exposure during pregnancy, like nicotine, affects the brainstem α7 nicotinic acetylcholine receptor expression, increasing the risk of sudden unexplained perinatal death.

    PubMed

    Lavezzi, Anna Maria; Cappiello, Achille; Pusiol, Teresa; Corna, Melissa Felicita; Termopoli, Veronica; Matturri, Luigi

    2015-01-15

    This study indicates the impact of nicotine and pesticides (organochlorine and organophosphate insecticides used in agriculture) on neuronal α7-nicotinic acetylcholine receptor expression in brainstem regions receiving cholinergic projections in human perinatal life. An in-depth anatomopathological examination of the autonomic nervous system and immunohistochemistry to analyze the α7-nicotinic acetylcholine receptor expression in the brainstem from 44 fetuses and newborns were performed. In addition, the presence of selected agricultural pesticides in cerebral cortex samples of the victims was determined by specific analytical procedures. Hypodevelopment of brainstem structures checking the vital functions, frequently associated with α7-nicotinic acetylcholine receptor immunopositivity and smoke absorption in pregnancy, was observed in high percentages of victims of sudden unexpected perinatal death. In nearly 30% of cases however the mothers never smoked, but lived in rural areas. The search for pesticides highlighted in many of these cases traces of both organochlorine and organophosphate pesticides. We detain that exposition to pesticides in pregnancy produces homologous actions to those of nicotine on neuronal α7-nicotinic acetylcholine receptor, allowing to developmental alterations of brainstem vital centers in victims of sudden unexplained death. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Possible involvement of auxin-induced ethylene in an apoptotic cell death during temperature-sensitive lethality expressed by hybrid between Nicotiana glutinosa and N. repanda.

    PubMed

    Yamada, T; Marubashi, W; Nakamura, T; Niwa, M

    2001-09-01

    Interspecific hybrids of Nicotiana glutinosa L. x N. repanda Willd. express temperature-sensitive lethality induced by apoptotic cell death. Hybrid seedlings cultured at 28 degrees C began to exhibit lethal symptoms during early growth stages, and then they showed a high level of endogenous auxin compared with those of parental seedlings. Meanwhile, the level of auxin in hybrid seedlings cultured at 32 degrees C, which is a condition suppressing the lethality of this cross combination, was equal to or lower than those of parental seedlings. Administration of 2,3,5-triiodobenzoic acid (TIBA) as an auxin transport inhibitor into the hybrid seedlings suppressed lethal symptoms and had a life-extending effect. Additionally, TIBA has an effect to suppress DNA fragmentation, which is one of characteristics of apoptosis and has been detected in the hybrid seedlings expressing the lethality. Administration of aminooxyacetic acid (AOA) as an ethylene synthesis inhibitor, which could inhibit ethylene production, also showed the same effects as TIBA for the lethality. From these results, we suggested that auxin and ethylene were involved in an apoptotic cell death during the lethality, and the abnormal increase of endogenous auxin may lead to the ethylene production in hybrid seedlings during early growth stages.

  4. Up-Regulation of Programmed Death-1 Expression on Beryllium-Specific CD4+ T Cells in Chronic Beryllium Disease1

    PubMed Central

    Palmer, Brent E.; Mack, Douglas G.; Martin, Allison K.; Gillespie, May; Mroz, Margaret M.; Maier, Lisa A.; Fontenot, Andrew P.

    2015-01-01

    Chronic beryllium disease (CBD) is caused by workplace exposure to beryllium and is characterized by the accumulation of memory CD4+ T cells in the lung. These cells respond vigorously to beryllium salts in culture by producing proinflammatory Th1-type cytokines. The presence of these inflammatory cytokines leads to the recruitment of alveolar macrophages, alveolitis, and subsequent granuloma development. It has been shown that chronic exposure to conventional Ags leads to up-regulation in the expression of negative regulators of T cells such as programmed death-1 (PD-1). Due to the persistence of beryllium in the lung after the cessation of exposure, aberrant regulation of the PD-1 pathway may play an important role in CBD development. In the present study, PD-1 expression was measured on blood and bronchoalveolar lavage (BAL) CD4+ T cells from beryllium-sensitized and CBD subjects. PD-1 expression was significantly higher on BAL CD4+ T cells compared with those cells in blood, with the highest expression on the beryllium-specific T cell subset. In addition, the expression of PD-1 on BAL CD4+ T cells directly correlated with the severity of the T cell alveolitis. Increased expression of the PD-1 ligands, PD-L1 and PD-L2, on BAL CD14+ cells compared with blood was also seen. The addition of anti-PD-1 ligand mAbs augmented beryllium-induced CD4+ T cell proliferation, and an inverse correlation was seen between PD-1 expression on beryllium-specific CD4+ T cells and beryllium-induced proliferation. Thus, the PD-1 pathway is active in beryllium-induced disease and plays a key role in controlling beryllium-induced T cell proliferation. PMID:18250483

  5. Programmed death-1 ligand 1 and 2 are highly expressed in pleomorphic carcinomas of the lung: Comparison of sarcomatous and carcinomatous areas.

    PubMed

    Kim, Sehui; Kim, Moon-Young; Koh, Jaemoon; Go, Heounjeong; Lee, Dong Soo; Jeon, Yoon Kyung; Chung, Doo Hyun

    2015-11-01

    Pleomorphic carcinoma (PC) of the lung is a rare type of poorly differentiated non-small cell lung carcinoma (NSCLC) that belongs to sarcomatoid carcinoma (SC). It exhibits aggressive behaviour and resistance to chemotherapy and radiotherapy. Recently, immunotherapy targeting the programmed death-1 (PD-1)/PD ligand 1 (PD-L1) pathway has demonstrated favourable clinical outcomes in NSCLC. However, the expression patterns of PD-1-related molecules in pulmonary PC remain elusive. PD-L1 and PD-L2 expression was estimated in 41 cases of PC using immunohistochemistry. CD8(+) and PD-1(+) tumour-infiltrating lymphocytes (TILs) were also evaluated. PD-L1 and PD-L2 were highly expressed in pulmonary PCs (90.2% [37/41)]; 87.8% [36/41]). The amount of CD8(+) or PD-1(+) TILs and the ratio of PD-1(+)/CD8(+) TILs in PC were higher in males, smokers and older patients. PD-L1-positive PCs were infiltrated by higher numbers of CD8(+) TILs compared to PD-L1-negative cases (P=0.006). Of note, PD-L1 expression in pulmonary PCs was significantly higher in sarcomatous areas than in the carcinomatous portion (P=0.006). PC patients with a high ratio of PD-1(+)/CD8(+) TILs showed a shorter progression-free survival (P=0.036), whereas PD-L1 and PD-L2 expression had no prognostic implications. Our study demonstrates that pulmonary PCs very frequently express PD-L1 and PD-L2. Moreover, their expression is higher in sarcomatous cells than in carcinomatous areas. Thus, targeting the PD-1/PD-L1 pathway may represent a potential therapeutic candidate for this aggressive tumour.

  6. Decreased GABABR expression and increased neuronal cell death in developing rat brain after PTZ-induced seizure.

    PubMed

    Naseer, Muhammad Imran; Ullah, Ikram; Al-Qahtani, Mohammed H; Karim, Sajjad; Ullah, Najeeb; Ansari, Shakeel Ahmed; Kim, Myeong Ok; Bibi, Fehmida

    2013-04-01

    The objective of this study was to evaluate the PTZ-induced seizures effects on GABAB receptor (R) expression and to observe its neurodegenerative effect in hippocampal part of developing rat brain. In the present study, high dose of pentylenetetrazol (PTZ 40 mg/kg) was injected in developing rats of age 5 weeks having average weight of 60-65 g for 4 days. Further, baclofen (B 3 mg/kg i.p) agonist and phaclofen (P 30 μg/rat) antagonist of GABABR were injected along with PTZ. Western blot analysis was used to elucidate expression of GABABR protein upon PTZ, baclofen and phaclofen exposure in the developing rat brain. Furthermore, PTZ-induced apoptotic neurodegeneration was also observed through the release of caspase-3 antibody and propidium iodide (PI) staining using confocal microscopy. Seizure was confirmed using electroencephalography (EEG) data obtained from the Laxtha EEG-monitoring device in the EEG recording room and EEG was monitored 5-15 min after PTZ injection. The results of the present study showed that PTZ-induced seizure significantly decreased GABABR expression and induced neuronal apoptosis in cortical and hippocampal part of brain. While, baclofen reverse the effect of PTZ by increasing the expression of GABABR as compared to the PTZ- , PTZ plus B- and PTZ plus P-treated groups. Our findings indicated that PTZ-induced seizure showed not only decrease in GABABR expression but also cause neuronal apoptosis in the developing rat brain.

  7. Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death

    SciTech Connect

    Porter, Holly A.; Carey, Gregory B.; Keegan, Achsah D.

    2012-08-15

    The adapters IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determine the role of IRS1 and IRS2 in the sensitivity of cells to chemotherapy, we treated 32D cells lacking or expressing IRS proteins with various concentrations of chemotherapeutic agents. We found that expression of IRS1, in contrast to IRS2, enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. When IRS2 was expressed with IRS1, the cells no longer showed enhanced sensitivity. Expression of IRS1 did not alter the expression of pro- and anti-apoptotic proteins; however, 32D-IRS1 cells expressed higher levels of Annexin A2. In 32D-IRS1 cells, IRS1 and Annexin A2 were both located in cytoplasmic and membrane fractions. We also found that IRS1 coprecipitated with Annexin A2, while IRS2 did not. Decreasing Annexin A2 levels reduced 32D-IRS1 cell sensitivity to chemotherapy. These results suggest IRS1 enhances sensitivity to chemotherapy in part through Annexin A2. -- Highlights: Black-Right-Pointing-Pointer IRS1 enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. Black-Right-Pointing-Pointer This sensitivity is abrogated by the expression of IRS2. Black-Right-Pointing-Pointer Expressing IRS1 in 32D cells increased levels of Annexin A2. Black-Right-Pointing-Pointer Both IRS1 and Annexin A2 were located in cytoplasmic and membrane fractions. Black-Right-Pointing-Pointer Decreasing Annexin A2 in 32D-IRS1 cells abated their sensitivity to chemotherapy.

  8. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

    PubMed

    Liu, Youhong; Chen, Lin; Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M; Sun, Lunquan; Li, Xiong

    2015-02-20

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future.

  9. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells

    PubMed Central

    Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M.; Sun, Lunquan; Li, Xiong

    2015-01-01

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future. PMID:25605010

  10. Somatically expressed germ-granule components, PGL-1 and PGL-3, repress programmed cell death in C. elegans

    PubMed Central

    Al-Amin, Mohammad; Min, Hyemin; Shim, Yhong-Hee; Kawasaki, Ichiro

    2016-01-01

    We previously reported that germline apoptosis in C. elegans increased by loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, from germ cells in adult hermaphrodite gonads. In this study, we found that somatic apoptosis was reduced in synthetic multivulva class B (synMuv B) mutants due to ectopic expression of PGL-1 and PGL-3 in the soma. In synMuv B-mutant somatic cells, CED-4 expression level was reduced due to ectopic expression of PGL-1. Furthermore, in contrast to wild type, somatic apoptosis in synMuv B mutants increased following DNA damage in a SIR-2.1-dependent manner. Intriguingly, somatic apoptosis was repressed not only in synMuv B mutants but also by ectopically expressing pgl-1 and/or pgl-3 transgenes in wild-type somatic cells. Our study demonstrates that germ-granule components, PGL-1 and PGL-3, can serve as negative regulators of apoptosis not only in the germline but also in the soma in C. elegans. PMID:27650246

  11. PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca(2+)-permeable AMPA receptors.

    PubMed

    Gerace, E; Masi, A; Resta, F; Felici, R; Landucci, E; Mello, T; Pellegrini-Giampietro, D E; Mannaioni, G; Moroni, F

    2014-10-01

    An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG, 100μM for 5min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD(+) and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca(2+)-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca(2+) permeability of AMPA channels, thus affecting

  12. Differential expression of TRPM2 and TRPV4 channels and their potential role in oxidative stress-induced cell death in organotypic hippocampal culture.

    PubMed

    Bai, Ji-Zhong; Lipski, Janusz

    2010-03-01

    TRPM2 and TPPV4 channels, two members of TRP channel family, are known to be widely expressed in the brain but their exact expression pattern and function are not well understood. Due to their high Ca(2+) permeability and gating by reactive oxygen species (TRPM2), or cell swelling, low pH and high temperature (TRPV4), they are likely to be involved in cell damage associated with various brain pathologies. The aim of this study was to investigate the expression of these channels and their potential role in oxidative stress-induced cell damage in organotypic hippocampal slice cultures, a model that retains the complex interaction between neurons and astrocytes. Channel expression was confirmed with RT-PCR and western blotting, while immunocytochemistry demonstrated TRPM2 in CA1-CA3 pyramidal neurons and TRPV4 in astrocytes. Oxidative stress induced by exogenous application of H(2)O(2) (600 microM) caused preferential damage of pyramidal neurons, while oxidative stress evoked with mercaptosuccinate (MCS; 400 microM) or buthionine sulfoximine (BSO; 4 microM) mainly damaged astrocytes, as identified by propidium iodide fluorescence. Antioxidants (Trolox 500 microM; MitoE 2 microM) reduced both neuronal and astrocytic cell death. Blockers of TRPV4 channels (Gd(3+) 500 microM; Ruthenium red 1 microM) increased the viability of astrocytes following MCS or BSO treatments, consistent with the expression pattern of these channels. Blockers of TRPM2 channels clotrimazole (20 microM), N-(p-amylcinnomoyl)anthranilic acid (ACA, 25 microM) or flufenamic acid (FFA, 200 microM) failed to protect pyramidal neurons from damage caused by exogenous H(2)O(2), and increased damage of these neurons caused by MCS and BSO. The differential expression of stress-sensitive TRPM2 and TRPV4 channels in hippocampal neurons and astrocytes that show distinct differences in vulnerability to different forms of oxidative stress suggests the specific involvement of these channels in oxidative stress

  13. Effects of the knockdown of death-associated protein 3 expression on cell adhesion, growth and migration in breast cancer cells.

    PubMed

    Wazir, Umar; Sanders, Andrew J; Wazir, Ahmad M A; Ye, Lin; Jiang, Wen G; Ster, Irina C; Sharma, Anup K; Mokbel, Kefah

    2015-05-01

    The death-associated protein 3 (DAP3) is a highly conserved phosphoprotein involved in the regulation of autophagy. A previous clinical study by our group suggested an association between low DAP3 expression and clinicopathological parameters of human breast cancer. In the present study, we intended to determine the role of DAP3 in cancer cell behaviour in the context of human breast cancer. We developed knockdown sub-lines of MCF7 and MDA-MB-231, and performed growth, adhesion, invasion assays and electric cell-substrate impedance sensing (ECIS) studies of post-wound migration of the cells. In addition, we studied the mRNA expression of caspase 8 and 9, death ligand signal enhancer (DELE), IFN-β promoter stimulator 1 (IPS1), cyclin D1 and p21 in the control and knockdown sub-lines. The knockdown sub-lines of MCF7 and MDA-MB-231 had significantly increased adhesion and decreased growth when compared to the controls. Furthermore, invasion and migration were significantly increased in the MDA-MB-231DAP3kd cells vs. the controls. The expression of caspase 9 and IPS1, known components of the apoptosis pathway, were significantly reduced in the MCF7DAP3kd cells (p=0.05 and p=0.003, respectively). We conclude that DAP3 silencing contributes to breast carcinogenesis by increasing cell adhesion, migration and invasion. It is possible that this may be due to the activity of focal adhesion kinase further downstream of the anoikis pathway. Further research in this direction would be beneficial in increasing our understanding of the mechanisms underlying human breast cancer.

  14. Effect of cobalt-mediated Toll-like receptor 4 activation on inflammatory responses in endothelial cells

    PubMed Central

    Holland, James P.; Kirby, John A.; Deehan, David J.; Tyson, Alison J.

    2016-01-01

    Cobalt-containing metal-on-metal hip replacements are associated with adverse reactions to metal debris (ARMD), including inflammatory pseudotumours, osteolysis, and aseptic implant loosening. The exact cellular and molecular mechanisms leading to these responses are unknown. Cobaltions (Co2+) activate human Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to Gram negative bacterial lipopolysaccharide (LPS). We investigated the effect of Co2+-mediated TLR4 activation on human microvascular endothelial cells (HMEC-1), focusing on the secretion of key inflammatory cytokines and expression of adhesion molecules. We also studied the role of TLR4 in Co2+-mediated adhesion molecule expression in MonoMac 6 macrophages. We show that Co2+ increases secretion of inflammatory cytokines, including IL-6 and IL-8, in HMEC-1. The effects are TLR4-dependent as they can be prevented with a small molecule TLR4 antagonist. Increased TLR4-dependent expression of intercellular adhesion molecule 1 (ICAM1) was also observed in endothelial cells and macrophages. Furthermore, we demonstrate for the first time that Co2+ activation of TLR4 upregulates secretion of a soluble adhesion molecule, sICAM-1, in both endothelial cells and macrophages. Although sICAM-1 can be generated through activity of matrix metalloproteinase-9 (MMP-9), we did not find any changes in MMP9 expression following Co2+ stimulation. In summary we show that Co2+ can induce endothelial inflammation via activation of TLR4. We also identify a role for TLR4 in Co2+-mediated changes in adhesion molecule expression. Finally, sICAM-1 is a novel target for further investigation in ARMD studies. PMID:27835611

  15. Toll like receptor 4 facilitates invasion and migration as a cancer stem cell marker in hepatocellular carcinoma.

    PubMed

    Liu, Wen-Ting; Jing, Ying-Ying; Yu, Guo-feng; Han, Zhi-peng; Yu, Dan-dan; Fan, Qing-Min; Ye, Fei; Li, Rong; Gao, Lu; Zhao, Qiu-Dong; Wu, Meng-Chao; Wei, Li-Xin

    2015-03-28

    Cancer stem cells (CSCs) or tumor-initiating cells (TICs), a small subset of tumor cells, are involved in tumor initiation, progression, recurrence and metastasis. In human hepatocellular carcinoma (HCC), TICs are enriched with cell surface markers and play a key role in chemotherapy resistance, tumor invasion and migration. Toll like receptor 4 (TLR4), acting as a receptor for lipopolysaccharide (LPS), has been reported to be responsible for carcinogenesis, invasion, metastasis and cancer progression. In our study, two HCC cell lines and a splenic vein metastasis of the nude mouse model were used to study the invasive ability of TLR4 positive HCC cells in vitro and in vivo. Stem-like features were also detected in TLR4 positive HCC cells. A total of 88 clinical samples from HCC patients were used to evaluate the association of TLR4 and stem-cell marker expression, and the relationship between TLR4 expression and clinicopathological characteristics was analyzed. The in vitro and in vivo experiments indicated that TLR4 positive HCC cells displayed significantly enhanced invasion and migration, and stem-like properties were also detected in TLR4 positive HCC cells. Clinically, TLR4 expression levels were found to be significantly higher in HCC tissues with microvascular invasion. Additionally, high expression of TLR4 in HCC tissues was strongly associated with both early recurrence and poor survivals in patients. Our results indicated that there was a relationship between TLR4 expression and CSC's features, TLR4 may act as a CSC marker, prompting tumor invasion and migration, which contributes to the poor prognosis of HCC.

  16. Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure.

    PubMed

    Ben Ari, Ziv; Avlas, Orna; Pappo, Orit; Zilbermints, Veacheslav; Cheporko, Yelena; Bachmetov, Larissa; Zemel, Romy; Shainberg, Asher; Sharon, Eran; Grief, Franklin; Hochhauser, Edith

    2012-01-01

    Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-κ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.

  17. Albumin induces neuroprotection against ischemic stroke by altering Toll-like receptor 4 and regulatory T cells in mice.

    PubMed

    Wang, Min; Wang, Yongming; He, Jing; Wei, Siyu; Zhang, Na; Liu, Fengyong; Liu, Xin; Kang, Yi; Yao, Xiaomei

    2013-03-01

    The objective of this study was to characterize the effect of albumin therapy on the expression of Toll-like receptor 4 (TLR 4), anti-inflammation cytokines and CD4(+)CD25(+)forkhead box P3 (Foxp3)(+) regulatory T lymphocytes (Treg cells) in the ischemic brain and peripheral immune system after Middle Cerebral Artery Occlusion (MCAO). Adult male Kunming mice were subjected to MCAO, the suture was withdrawn 2 h later followed by an intravenous administration of 25% albumin (1.25 g/kg) or saline (5 ml/kg) through caudal vein. We demonstrated that albumin administration elevated the serum albumin level supranormally at 6 h and 24 h after MCAO in mice. In addition, we showed that both in the ischemic brain and in the spleen, albumin administration significantly depressed the increase of TLR 4 mRNA expression by quantitative real-time polymerase chain reaction (QRT-PCR), and significantly increase the anti-inflammatory related interleukin-10 (IL-10) and transforming growth factor beta1 (TGF-β1) mRNA expression by transcription-polymerase chain reaction (RT-PCR) after MCAO. In the spleen, compared to sham group, strong TLR 4 immunoreactivity was noted in the saline group; while compared to saline group, albumin administration markedly reduced the immunoreactivity of TLR 4 after MCAO by immunohistochemistry. Moreover, albumin administration significantly increased the percentage of Treg in spleen CD4(+) cells by flow cytometry. In conclusion, the decrease of TLR 4 expression and the increase of Treg cell, IL-10, and TGF-β1 expression may partly contribute to the neuroprotective effect of albumin therapy on MCAO induced immune inflammatory responses.

  18. Characterization and functional analysis of toll-like receptor 4 in Chinese soft-shelled turtle Pelodiscus sinensis.

    PubMed

    Zhou, Yingshan; Liang, Quan; Li, Weifen; Gu, Yuanxing; Liao, Xun; Fang, Weihuan; Li, Xiaoliang

    2016-10-01

    Mammalian Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) in initiating the innate immune responses. Early studies indicate that turtles are more resistant to LPS challenge than mammals. It remains unknown if turtles express TLR4 and why they are more resistant to LPS. In this study, TLR4 gene from Chinese soft-shelled turtle, Pelodiscus sinensis, was cloned and characterized. The full length cDNA of turtle TLR4 (tTLR4) consists of 3396 base pairs with an 2499-bp open reading frame, encoding 833 amino acids. Phylogenetic and syntenic analyses suggest that tTLR4 is to be orthologous to human TLR4. Its mRNA expression was up-regulated in spleen and blood of turtles upon Aeromonas hydrophila infection. Stimulation of turtle peripheral blood monocytes with LPS significantly upregulated tTLR4 mRNA and inflammation-related gene expression, such as Interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2). In tTLR4-expressing HEK293 cells, higher concentration of LPS exposure could enhance the activity of the NF-κB promoter, but not the INF-β promoter. Such activity required co-expression of turtle myeloid differentiation factor 2 (tMD2) and cluster of differentiation 14 (tCD14). These results provide evidence for a functional TLR4 in reptiles and, together with the syntenic analysis, support the idea that the TLR4 receptor for LPS recognition may have arisen after reptiles. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Pressure overload induces IL-18 and IL-18R expression, but markedly suppresses IL-18BP expression in a rabbit model. IL-18 potentiates TNF-α-induced cardiomyocyte death.

    PubMed

    Yoshida, Tadashi; Friehs, Ingeborg; Mummidi, Srinivas; del Nido, Pedro J; Addulnour-Nakhoul, Solange; Delafontaine, Patrice; Valente, Anthony J; Chandrasekar, Bysani

    2014-10-01

    Recurrent or sustained inflammation plays a causal role in the development and progression of left ventricular hypertrophy (LVH) and its transition to failure. Interleukin (IL)-18 is a potent pro-hypertrophic inflammatory cytokine. We report that induction of pressure overload in the rabbit, by constriction of the descending thoracic aorta induces compensatory hypertrophy at 4weeks (mass/volume ratio: 1.7±0.11) and ventricular dilatation indicative of heart failure at 6weeks (mass/volume ratio: 0.7±0.04). In concordance with this, fractional shortening was preserved at 4weeks, but markedly attenuated at 6weeks. We cloned rabbit IL-18, IL-18Rα, IL-18Rβ, and IL-18 binding protein (IL-18BP) cDNA, and show that pressure overload, while enhancing IL-18 and IL-18R expression in hypertrophied and failing hearts, markedly attenuated the level of expression of the endogenous IL-18 antagonist IL-18BP. Cyclical mechanical stretch (10% cyclic equibiaxial stretch, 1Hz) induced hypertrophy of primary rabbit cardiomyocytes in vitro and enhanced ANP, IL-18, and IL-18Rα expression. Further, treatment with rhIL-18 induced its own expression and that of IL-18Rα via AP-1 activation, and induced cardiomyocyte hypertrophy in part via PI3K/Akt/GATA4 signaling. In contrast, IL-18 potentiated TNF-α-induced cardiomyocyte death, and by itself induced cardiac endothelial cell death. These results demonstrate that pressure overload is associated with enhanced IL-18 and its receptor expression in hypertrophied and failingrabbit hearts. Since IL-18BP expression is markedly inhibited, our results indicate a positive amplification in IL-18 proinflammatory signaling during pressure overload, and suggest IL-18 as a potential therapeutic target in pathological hypertrophy and cardiac failure.

  20. Roles of Toll-Like Receptor 4, IκB Kinase, and the Proteasome in the Intestinal Alterations Caused by Sepsis.

    PubMed

    Gonzalo, Sergio; Valero, Marta Sofía; Martínez de Salinas, Fernando; Vergara, Claudia; Arruebo, María Pilar; Plaza, Miguel Ángel; Murillo, María Divina; Grasa, Laura

    2015-05-01

    Lipopolysaccharide decreases intestinal contractility and induces the production of cytokines, which play an important role in the pathogenesis of sepsis. The objective of the present study was to examine the role of Toll-like receptor 4, IκB kinase, and the proteasome in the intestinal alterations induced by lipopolysaccharide. Sepsis was induced in rabbits by intravenous injection of lipopolysaccharide. Contractility studies of rabbit duodenum were performed in an organ bath. Expressions of interleukin-1β, interleukin-6, interleukin-8, interleukin-10, IκB kinase-α, IκB kinase-β, IκB kinase-γ, and the proteasome mRNA were determined by RT-PCR on rabbit duodenum. Neomycin and polymyxin B (Toll-like receptor 4 inhibitors), IKK NBD peptide (IκB kinase complex inhibitor), and MG-132 (proteasome inhibitor) blocked partially the effects of lipopolysaccharide on the acetylcholine-, prostaglandin E2-, substance P-, and KCl-induced contractions in the longitudinal and circular smooth muscle of rabbit duodenum. Lipopolysaccharide increased the mRNA expression of interleukin-6 and interleukin-8 in duodenal tissue, and this effect was partly reversed by neomycin, polymyxin B, IKK NBD peptide, and MG-132. IκB kinase-α, IκB kinase-β, IκB kinase-γ, and the proteasome mRNA expressions was not affected by lipopolysaccharide treatment. Toll-like receptor 4, the IκB kinase complex, and the proteasome could be therapeutic targets in the treatment of sepsis symptoms in the intestine.

  1. Expression of TRAIL and the death receptors DR4 and DR5 correlates with progression of degeneration in human intervertebral disks.

    PubMed

    Bertram, Helge; Nerlich, Andreas; Omlor, Georg; Geiger, Florian; Zimmermann, Gerald; Fellenberg, Joerg

    2009-07-01

    Intervertebral disks degenerate far earlier than other musculoskeletal tissues and apoptosis has been suggested to have a vital function in promoting the degeneration process that is strongly associated with back pain. However, the molecular mediators of apoptosis in the intervertebral disk are poorly understood. Fas/FasL, TRAIL/DR4, TRAIL/DR5 and TNF-alpha/TNFR1 are ligand/receptor pairs of the tumor necrosis factor/nerve growth factor family, which are able to induce apoptosis by trimerization of the receptor by its corresponding ligand. We investigated which of these molecules are expressed in intervertebral disks and whether their expression correlates to disk degeneration. Intervertebral disks from 28 donors (age 12-70 years) suffering from scoliosis, vertebrae fracture or disk degeneration were scored histologically for degeneration and analyzed for gene expression of FasL/Fas, TRAIL/DR4, TNF-alpha/TNFR1 and caspase 8. Protein expression of FasL and TRAIL was assessed by immunohistology and apoptotic cell death was quantified by poly(ADP-ribose) polymerase (PARP) p85 staining. Isolated disk cells were analyzed by flow cytometry for Fas, FasL, TRAIL, DR4 and DR5 expression. Gene expression of TRAIL (P=0.002) and caspase 8 (P=0.027) significantly correlated with degeneration. TRAIL expression further correlated with cellularity (P=0.04), muccoid matrix changes (P=0.009) and tears and cleft formation (P=0.019). FasL and TRAIL expression was confirmed by immunohistology and PARP cleavage was significantly associated with degeneration (P=0.027). Flow cytometry on isolated disk cells revealed correlations between DR4 and degeneration (P=0.014), DR4/DR5 double-positive cells and degeneration (P=0.019), as well as DR5 and changes in tissue granularity (P=0.03). This is the first study that shows that intervertebral disk cells express TRAIL, DR4 and DR5, which correlate to the degenerative state of the disk. Therefore, disk cells inherit the molecular machinery to

  2. Curcumin Induces Downregulation of E2F4 Expression and Apoptotic Cell Death in HCT116 Human Colon Cancer Cells; Involvement of Reactive Oxygen Species.

    PubMed

    Kim, Kyung-Chan; Lee, Chuhee

    2010-12-01

    E2F transcription factors and their target genes have been known to play an important role in cell growth control. We found that curcumin, a polyphenolic phytochemical isolated from the plant Curcuma longa, markedly suppressed E2F4 expression in HCT116 colon cancer cells. Hydrogen peroxide was also found to decrease E2F4 protein level, indicating the involvement of reactive oxygen species (ROS) in curucmin-induced downregulation of E2F4 expression. Involvement of ROS in E2F4 downregulation in response to curcumin was confirmed by the result that pretreatment of cells with N-acetylcystein (NAC) before exposure of curcumin almost completely blocked the reduction of E2F4 expression at the protein as well as mRNA level. Anti-proliferative effect of curcumin was also suppressed by NAC which is consistent to previous reports showing curcumin-superoxide production and induction of poly (ADP-ribose) polymerase (PARP) cleavage as well as apoptosis. Expression of several genes, cyclin A, p21, and p27, which has been shown to be regulated in E2F4-dependent manner and involved in the cell cycle progression was also affected by curcumin. Moreover, decreased (cyclin A) and increased (p21 and p27) expression of these E2F4 downstream genes by curcumin was restored by pretreatment of cells with NAC and E2F4 overexpression which is induced by doxycycline. In addition, E2F4 overexpression was observed to partially ameliorate curcumin-induced growth inhibition by cell viability assay. Taken together, we found curcumin-induced ROS down-regulation of E2F4 expression and modulation of E2F4 target genes which finally lead to the apoptotic cell death in HCT116 colon cancer cells, suggesting that E2F4 appears to be a novel determinant of curcumin-induced cytotoxicity.

  3. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    PubMed Central

    Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

    2014-01-01

    Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

  4. The prevalence and clinicopathological features of programmed death-ligand 1 (PD-L1) expression: a pooled analysis of literatures.

    PubMed

    Lin, Ziying; Xu, Yutong; Zhang, Yaxiong; He, Qihua; Zhang, Jianrong; He, Jianxing; Liang, Wenhua

    2016-03-22

    Programmed death-ligand 1 (PD-L1) has been recognized as a critical and promising target in therapies that direct immune escape of cancers. However, its association with aggressive clinicopathological features in solid tumors remains unclear. We investigated this question by synthesizing published articles. Electronic databases were searched for relevant studies. Outcomes of interest included age, gender, tumor size, tumor size, lymph node metastasis and tumor cell differentiation. A total of 61 studies involving 17 types of malignancies were included. The overall expression rate of PD-L1 was 44.5% (95% CI, 37.5% to 51.6 %). Patients with regional lymph node metastases (OR 1.38; P < 0.01), large size tumor (OR 1.89; P < 0.01) or poor differentiated tumors (OR 1.71; P < 0.01) were associated with higher PD-L1 expression rate. However, no significant association was observed between young and elder patients (OR 1.04; P = 0.58), or male and female patients (OR 1.13; P = 0.06). A numerically higher PD-L1 expression rate was detected in polyclonal antibodies (57.2%) than monoclonal antibodies (39.6%). In addition, the PD-L1 expression rate reported by studies from Asian areas (52.3%) was numerically higher than those from non-Asian areas, namely Caucasians (32.7%). This meta-analysis indicated that patients with larger tumors, regional lymph node metastases, or poor-differentiated tumors were associated with a higher PD-L1 expression rate; in addition the expression rate of PD-L1 in Asians might be higher than that of Caucasians. This information might be useful in screening candidates for relevant tests and treatments.

  5. The prevalence and clinicopathological features of programmed death-ligand 1 (PD-L1) expression: a pooled analysis of literatures

    PubMed Central

    He, Qihua; Zhang, Jianrong; He, Jianxing; Liang, Wenhua

    2016-01-01

    Background & Aims Programmed death-ligand 1 (PD-L1) has been recognized as a critical and promising target in therapies that direct immune escape of cancers. However, its association with aggressive clinicopathological features in solid tumors remains unclear. We investigated this question by synthesizing published articles. Methods Electronic databases were searched for relevant studies. Outcomes of interest included age, gender, tumor size, tumor size, lymph node metastasis and tumor cell differentiation. Results A total of 61 studies involving 17 types of malignancies were included. The overall expression rate of PD-L1 was 44.5% (95% CI, 37.5% to 51.6 %). Patients with regional lymph node metastases (OR 1.38; P < 0.01), large size tumor (OR 1.89; P < 0.01) or poor differentiated tumors (OR 1.71; P < 0.01) were associated with higher PD-L1 expression rate. However, no significant association was observed between young and elder patients (OR 1.04; P = 0.58), or male and female patients (OR 1.13; P = 0.06). A numerically higher PD-L1 expression rate was detected in polyclonal antibodies (57.2%) than monoclonal antibodies (39.6%). In addition, the PD-L1 expression rate reported by studies from Asian areas (52.3%) was numerically higher than those from non-Asian areas, namely Caucasians (32.7%). Conclusions This meta-analysis indicated that patients with larger tumors, regional lymph node metastases, or poor-differentiated tumors were associated with a higher PD-L1 expression rate; in addition the expression rate of PD-L1 in Asians might be higher than that of Caucasians. This information might be useful in screening candidates for relevant tests and treatments. PMID:26930715

  6. Smad8 is expressed in the anterior necrotic zone: evidence for a role of bone morphogenetic proteins/SMAD signaling in the activation of a molecular cascade that culminates in cell death.

    PubMed

    Abarca-Buis, René F; Bustamante, Marcia; Cuervo, Rodrigo; Aguilar-Fernández-de-Lara, Dante; Chimal-Monroy, Jesús

    2011-08-01

    Bone morphogenetic proteins (BMPs) play a crucial role in programmed cell death (PCD), a biological process required for the sculpturing of the embryonic limbs. However, it is unknown if BMP signaling directly promotes cell death, or if it induces a molecular cascade that culminates in cell death. Given that Smad8, which encodes one component of BMP signaling, is expressed during the regression of interdigital tissue and responds to BMPs, we presumed that it may be expressed in other cell death areas during chick limb development such as the anterior and posterior necrotic zones (ANZ and PNZ). The present study found that the Smad8 expression pattern in the anterior mesoderm of the hindlimb is very similar to that observed in limbs stained to detect cell death. Also, BMPs and retinoic acid, which act as apoptosis-promoting factors, induced expression of Smad8 before the onset of cell death, while sonic hedgehog protein, acting as a survival factor, inhibited Smad8 expression in the ANZ. However, although there was correlation between Smad8 expression patterns and PCD in the ANZ, phosphorylated forms of SMAD1/5/8 and TUNEL staining did not co-localize in dying cells. Interestingly, a short pulse of BMP was sufficient to trigger cell death. On the other hand, most dying cells were located in the avascular region, while many cells expressing Smad8 were located in the vascular region of the ANZ. These results suggest that BMPs mediated by SMAD signaling activate a molecular cascade that culminates in PCD.

  7. Effects of lentiviral short hairpin RNA silencing of Toll-like receptor 4 on the lens epithelial cell line HLEC.

    PubMed

    Yu, H T; Lu, P R

    2016-06-03

    The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.

  8. CCL-34, a synthetic toll-like receptor 4 activator, modulates differentiation and maturation of myeloid dendritic cells.

    PubMed

    Fu, Shu-Ling; Lin, Chun-Cheng; Hsu, Ming-Ling; Liu, Sheng-Hung; Huang, Yu-Chuen; Chen, Yu-Jen

    2016-03-08

    CCL-34, a synthetic α-galactosylceramide analog, has been reported as an activator of toll-like receptor 4 (TLR4) in macrophages. TLR4 is highly expressed in dendritic cell (DC) and several TLR4 agonists are known to trigger DC maturation. We herein evaluated the effect of CCL-34 on DC maturation. Human CD14+ monocyte-derived immature DC were treated with CCL-34, its inactive structural analog CCL-44, or LPS to assess the DC maturation. CCL-34 induced DC maturation according to their characteristically dendrite-forming morphology, CD83 expression and IL-12p70 production. The allostimulatory activity of DC on proliferation of naive CD4+CD45+RA+ T cells and their secretion of interferon-γ was increased by CCL-34. Phagocytosis, an important function of immature DC, was reduced after CCL-34 treatment. All these effects related to DC maturation were evidently induced by positive control LPS but not by CCL-44 treatment. TLR4 neutralization impaired human DC maturation triggered by CCL-34. The induction of IL-12, a hallmark of DC maturation, by CCL-34 and LPS was only evident in TLR4-competent C3H/HeN, but not in TLR4-defective C3H/HeJ mice. CCL-34 could further elicit the antigen presentation capability in mice inoculated with doxorubicin-treated colorectal cancer cells. In summary, CCL-34 triggers DC maturation via a TLR4-dependent manner, which supports its potential application as an immunostimulator.

  9. Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

    PubMed

    Tiong, Kai Hung; Tan, Boon Shing; Choo, Heng Lungh; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Tan, Si Hoey; Khor, Nelson Tze Woei; Wong, Shew Fung; See, Sze-Jia; Tan, Yuen-Fen; Rosli, Rozita; Cheong, Soon-Keng; Leong, Chee-Onn

    2016-09-06

    Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

  10. Fibrinogen, an endogenous ligand of Toll-like receptor 4, activates monocytes in pre-eclamptic patients.

    PubMed

    Al-ofi, Ebtisam; Coffelt, Seth B; Anumba, Dilly O

    2014-06-01

    Pre-eclampsia (PE) remains the leading cause of pregnancy-associated mortality and morbidity, urging the need for a better understanding of its aetiology and pathophysiological progression. A key characteristic of PE is a systemic, exaggerated, inflammatory condition involving abnormal cytokine levels in serum, altered immune cell phenotype and Th1/Th2-type immunological imbalance. However, it is unknown how this heightened inflammatory condition manifests. We previously reported increased expression of the lipopolysaccharide receptor, Toll-like receptor 4 (TLR4), on monocytes from PE patients compared with normotensive, pregnant patients (NP). This upregulation of TLR4 on PE monocytes was accompanied by a hyper-responsiveness to bacterial TLR4 ligands. To determine whether non-microbial, endogenous TLR4 ligands also activate monocytes from PE patients, we investigated the expression of host-derived TLR4 ligands and the response of monocytes to these endogenous ligands. Plasma levels of fibrinogen - but not fibronectin or heparan sulphate - were higher in PE patients than in NP. Exposure to fibrinogen was associated with significantly increased production of inflammatory cytokines by monocytes from PE patients. Interestingly, this effect was not observed with NP monocytes. Our findings suggest that the fibrinogen-TLR4 axis might play an important role in the atypical activation of monocytes observed in PE patients that may contribute to the exaggerated inflammatory condition.

  11. Human platelet interaction with E. coli O111 promotes tissue-factor-dependent procoagulant activity, involving Toll like receptor 4.

    PubMed

    Matus, Valeria; Valenzuela, J Guillermo; Hidalgo, Patricia; Pozo, L María; Panes, Olga; Wozniak, Aniela; Mezzano, Diego; Pereira, Jaime; Sáez, Claudia G

    2017-01-01

    Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

  12. Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells

    PubMed Central

    Arbon, Kate S.; Christensen, Cody M.; Harvey, Wendy A.; Heggland, Sara J.

    2012-01-01

    Recent reports of cadmium in electronic waste and jewelry have increased public awareness regarding this toxic metal. Human exposure to cadmium is associated with the development of osteoporosis. We previously reported cadmium induces apoptosis in human tumor-derived Saos-2 osteoblasts. In this study, we examine the extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC) pathways in cadmium-induced apoptosis and altered osteoblast gene expression. Saos-2 osteoblasts were cultured in the presence or absence of 10 μM CdCl2 for 2–72 hours. We detected significant ERK activation in response to CdCl2 and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKCα activation was not observed after exposure to CdCl2 and pretreatment with the PKC inhibitor, Calphostin C, was unable to rescue cells from cadmium-induced apoptosis. Gene expression studies were conducted using qPCR. Cells exposed to CdCl2 exhibited a significant decrease in the bone-forming genes osteopontin (OPN) and alkaline phosphatase (ALP) mRNA. In contrast, SOST, whose protein product inhibits bone formation, significantly increased in response to CdCl2. Pretreatment with PD98059 had a recovery effect on cadmium-induced changes in gene expression. This research demonstrates cadmium can directly inhibit osteoblasts via ERK signaling pathway and identifies SOST as a target for cadmium-induced osteotoxicity. PMID:22019892

  13. Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells.

    PubMed

    Arbon, Kate S; Christensen, Cody M; Harvey, Wendy A; Heggland, Sara J

    2012-02-01

    Recent reports of cadmium in electronic waste and jewelry have increased public awareness regarding this toxic metal. Human exposure to cadmium is associated with the development of osteoporosis. We previously reported cadmium induces apoptosis in human tumor-derived Saos-2 osteoblasts. In this study, we examine the extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC) pathways in cadmium-induced apoptosis and altered osteoblast gene expression. Saos-2 osteoblasts were cultured in the presence or absence of 10μM CdCl(2) for 2-72h. We detected significant ERK activation in response to CdCl(2) and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKCα activation was not observed after exposure to CdCl(2) and pretreatment with the PKC inhibitor, Calphostin C, was unable to rescue cells from cadmium-induced apoptosis. Gene expression studies were conducted using qPCR. Cells exposed to CdCl(2) exhibited a significant decrease in the bone-forming genes osteopontin (OPN) and alkaline phosphatase (ALP) mRNA. In contrast, SOST, whose protein product inhibits bone formation, significantly increased in response to CdCl(2). Pretreatment with PD98059 had a recovery effect on cadmium-induced changes in gene expression. This research demonstrates cadmium can directly inhibit osteoblasts via ERK signaling pathway and identifies SOST as a target for cadmium-induced osteotoxicity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. N-Acetylcysteine Attenuates Hexavalent Chromium-Induced Hypersensitivity through Inhibition of Cell Death, ROS-Related Signaling and Cytokine Expression

    PubMed Central

    Huang, Chien-Cheng; Sheu, Hamm-Ming; Tsai, Jui-Chen; Lin, Chia-Ho; Wang, Ying-Jan; Wang, Bour-Jr

    2014-01-01

    Chromium hypersensitivity (chromium-induced allergic contact dermatitis) is an important issue in occupational skin disease. Hexavalent chromium (Cr (VI)) can activate the Akt, Nuclear factor κB (NF-κB), and Mitogen-activated protein kinase (MAPK) pathways and induce cell death, via the effects of reactive oxygen species (ROS). Recently, cell death stimuli have been proposed to regulate the release of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). However, the exact effects of ROS on the signaling molecules and cytotoxicity involved in Cr(VI)-induced hypersensitivity have not yet been fully demonstrated. N-acetylcysteine (NAC) could increase glutathione levels in the skin and act as an antioxidant. In this study, we investigated the effects of NAC on attenuating the Cr(VI)-triggered ROS signaling in both normal keratinocyte cells (HaCaT cells) and a guinea pig (GP) model. The results showed the induction of apoptosis, autophagy and ROS were observed after different concentrations of Cr(VI) treatment. HaCaT cells pretreated with NAC exhibited a decrease in apoptosis and autophagy, which could affect cell viability. In addition, Cr (VI) activated the Akt, NF-κB and MAPK pathways thereby increasing IL-1α and TNF-α production. However, all of these stimulation phenomena could be inhibited by NAC in both of in vitro and in vivo studies. These novel findings indicate that NAC may prevent the development of chromium hypersensitivity by inhibiting of ROS-induced cell death and cytokine expression. PMID:25248126

  15. N-acetylcysteine attenuates hexavalent chromium-induced hypersensitivity through inhibition of cell death, ROS-related signaling and cytokine expression.

    PubMed

    Lee, Yu-Hsuan; Su, Shih-Bin; Huang, Chien-Cheng; Sheu, Hamm-Ming; Tsai, Jui-Chen; Lin, Chia-Ho; Wang, Ying-Jan; Wang, Bour-Jr

    2014-01-01

    Chromium hypersensitivity (chromium-induced allergic contact dermatitis) is an important issue in occupational skin disease. Hexavalent chromium (Cr (VI)) can activate the Akt, Nuclear factor κB (NF-κB), and Mitogen-activated protein kinase (MAPK) pathways and induce cell death, via the effects of reactive oxygen species (ROS). Recently, cell death stimuli have been proposed to regulate the release of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). However, the exact effects of ROS on the signaling molecules and cytotoxicity involved in Cr(VI)-induced hypersensitivity have not yet been fully demonstrated. N-acetylcysteine (NAC) could increase glutathione levels in the skin and act as an antioxidant. In this study, we investigated the effects of NAC on attenuating the Cr(VI)-triggered ROS signaling in both normal keratinocyte cells (HaCaT cells) and a guinea pig (GP) model. The results showed the induction of apoptosis, autophagy and ROS were observed after different concentrations of Cr(VI) treatment. HaCaT cells pretreated with NAC exhibited a decrease in apoptosis and autophagy, which could affect cell viability. In addition, Cr (VI) activated the Akt, NF-κB and MAPK pathways thereby increasing IL-1α and TNF-α production. However, all of these stimulation phenomena could be inhibited by NAC in both of in vitro and in vivo studies. These novel findings indicate that NAC may prevent the development of chromium hypersensitivity by inhibiting of ROS-induced cell death and cytokine expression.

  16. 1B/(−)IRE DMT1 Expression during Brain Ischemia Contributes to Cell Death Mediated by NF-κB/RelA Acetylation at Lys310

    PubMed Central

    Ingrassia, Rosaria; Lanzillotta, Annamaria; Sarnico, Ilenia; Benarese, Marina; Blasi, Francesco; Borgese, Laura; Bilo, Fabjola; Depero, Laura; Chiarugi, Alberto; Spano, Pier Franco; Pizzi, Marina

    2012-01-01

    The molecular mechanisms responsible for increasing iron and neurodegeneration in brain ischemia are an interesting area of research which could open new therapeutic approaches. Previous evidence has shown that activation of nuclear factor kappa B (NF-κB) through RelA acetylation on Lys310 is the prerequisite for p50/RelA-mediated apoptosis in cellular and animal models of brain ischemia. We hypothesized that the increase of iron through a NF-κB-regulated 1B isoform of the divalent metal transporter-1 (1B/DMT1) might contribute to post-ischemic neuronal damage. Both in mice subjected to transient middle cerebral artery occlusion (MCAO) and in neuronally differentiated SK-N-SH cells exposed to oxygen-glucose-deprivation (OGD), 1A/DMT1 was only barely expressed while the 1B/DMT1 without iron-response-element (−IRE) protein and mRNA were early up-regulated. Either OGD or over-expression of 1B/(−)IRE DMT1 isoform significantly increased iron uptake, as detected by total reflection X-ray fluorescence, and iron-dependent cell death. Iron chelation by deferoxamine treatment or (−)IRE DMT1 RNA silencing displayed significant neuroprotection against OGD which concomitantly decreased intracellular iron levels. We found evidence that 1B/(−)IRE DMT1 was a target gene for RelA activation and acetylation on Lys310 residue during ischemia. Chromatin immunoprecipitation analysis of the 1B/DMT1 promoter showed there was increased interaction with RelA and acetylation of H3 histone during OGD exposure of cortical neurons. Over-expression of wild-type RelA increased 1B/DMT1 promoter-luciferase activity, the (−)IRE DMT1 protein, as well as neuronal death. Expression of the acetylation-resistant RelA-K310R construct, which carried a mutation from lysine 310 to arginine, but not the acetyl-mimic mutant RelA-K310Q, down-regulated the 1B/DMT1 promoter, consequently offering neuroprotection. Our data showed that 1B/(−)IRE DMT1 expression and intracellular iron influx are

  17. Increased Amyloid Precursor Protein and Tau Expression Manifests as Key Secondary Cell Death in Chronic Traumatic Brain Injury.

    PubMed

    Acosta, Sandra A; Tajiri, Naoki; Sanberg, Paul R; Kaneko, Yuji; Borlongan, Cesar V

    2017-03-01

    In testing the hypothesis of Alzheimer's disease (AD)-like pathology in late stage traumatic brain injury (TBI), we evaluated AD pathological markers in late stage TBI model. Sprague-Dawley male rats were subjected to moderate controlled cortical impact (CCI) injury, and 6 months later euthanized and brain tissues harvested. Results from H&E staining revealed significant 33% and 10% reduction in the ipsilateral and contralateral hippocampal CA3 interneurons, increased MHCII-activated inflammatory cells in many gray matter (8-20-fold increase) and white matter (6-30-fold increased) regions of both the ipsilateral and contralateral hemispheres, decreased cell cycle regulating protein marker by 1.6- and 1-fold in the SVZ and a 2.3- and 1.5-fold reductions in the ipsilateral and contralateral dentate gyrus, diminution of immature neuronal marker by two- and onefold in both the ipsilateral and contralateral SVZ and dentate gyrus, and amplified amyloid precursor protein (APP) distribution volumes in white matter including corpus callosum, fornix, and internal capsule (4-38-fold increase), as well as in the cortical gray matter, such as the striatum hilus, SVZ, and dentate gyrus (6-40-fold increase) in TBI animals compared to controls (P's < 0.001). Surrogate AD-like phenotypic markers revealed a significant accumulation of phosphorylated tau (AT8) and oligomeric tau (T22) within the neuronal cell bodies in ipsilateral and contralateral cortex, and dentate gyrus relative to sham control, further supporting the rampant neurodegenerative pathology in TBI secondary cell death. These findings indicate that AD-like pathological features may prove to be valuable markers and therapeutic targets for late stage TBI. J. Cell. Physiol. 232: 665-677, 2017. © 2016 Wiley Periodicals, Inc.

  18. Mode of cell death induced by the HSP90 inhibitor 17-AAG (tanespimycin) is dependent on the expression of pro-apoptotic BAX

    PubMed Central

    Powers, Marissa V; Valenti, Melanie; Miranda, Susana; Maloney, Alison; Eccles, Suzanne A.; Thomas, George; Clarke, Paul A; Workman, Paul

    2013-01-01

    Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) are of considerable current interest as targeted cancer therapeutic agents because of the ability to destabilize multiple oncogenic client proteins. Despite their resulting pleiotropic effects on multiple oncogenic pathways and hallmark traits of cancer, resistance to HSP90 inhibitors is possible and their ability to induce apoptosis is less than might be expected. Using an isogenic model for BAX knockout in HCT116 human colon carcinoma cells, we demonstrate the induction of BAX-dependent apoptosis at pharmacologically relevant concentrations of the HSP90 inhibitor 17-AAG both in vitro and in tumor xenografts in vivo. Removal of BAX expression by homologous recombination reduces apoptosis in vitro and in vivo but allows a lower level of cell death via a predominantly necrotic mechanism. Despite reducing apoptosis, the loss of BAX does not alter the overall sensitivity to 17-AAG in vitro or in vivo. The results indicate that 17-AAG acts predominantly to cause a cytostatic antiproliferative effect rather than cell death and further suggest that BAX status may not alter the overall clinical response to HSP90 inhibitors. Other agents may be required in combination to enhance tumor-selective killing by these promising drugs. In addition, there are implications for the use of apoptotic endpoints in the assessment of the activity of molecularly targeted agents. PMID:24185264

  19. Tomato ribonuclease LX with the functional endoplasmic reticulum retention motif HDEF is expressed during programmed cell death processes, including xylem differentiation, germination, and senescence.

    PubMed

    Lehmann, K; Hause, B; Altmann, D; Köck, M

    2001-10-01

    We have studied the subcellular localization of the acid S-like ribonuclease (RNase) LX in tomato (Lycopersicon esculentum Mill.) cells using a combination of biochemical and immunological methods. It was found that the enzyme, unexpectedly excluded from highly purified vacuoles, accumulates in the endoplasmic reticulum. The evidence that RNase LX is a resident of the endoplasmic reticulum (ER) is supported by an independent approach showing that the C-terminal peptide HDEF of RNase LX acts as an alternative ER retention signal in plants. For functional testing, the cellular distribution of chimeric protein constructs based on a marker protein, Brazil nut (Bertholletia excelsa) 2S albumin, was analyzed immunochemically in transgenic tobacco (Nicotiana tabacum) plants. Here, we report that the peptide motif is necessary and sufficient to accumulate 2S albumin constructs of both vacuolar and extracellular final destinations in the ER. We have shown immunochemically that RNase LX is specifically expressed during endosperm mobilization and leaf and flower senescence. Using immunofluorescence, RNase LX protein was detected in immature tracheary elements, suggesting a function in xylem differentiation. These results support a physiological function of RNase LX in selective cell death processes that are also thought to involve programmed cell death. It is assumed that RNase LX accumulates in an ER-derived compartment and is released by membrane disruption into the cytoplasma of those cells that are intended to undergo autolysis. These processes are accompanied by degradation of cellular components supporting a metabolic recycling function of the intracellular RNase LX.

  20. Tomato Ribonuclease LX with the Functional Endoplasmic Reticulum Retention Motif HDEF Is Expressed during Programmed Cell Death Processes, Including Xylem Differentiation, Germination, and Senescence1

    PubMed Central

    Lehmann, Karin; Hause, Bettina; Altmann, Dorit; Köck, Margret

    2001-01-01

    We have studied the subcellular localization of the acid S-like ribonuclease (RNase) LX in tomato (Lycopersicon esculentum Mill.) cells using a combination of biochemical and immunological methods. It was found that the enzyme, unexpectedly excluded from highly purified vacuoles, accumulates in the endoplasmic reticulum. The evidence that RNase LX is a resident of the endoplasmic reticulum (ER) is supported by an independent approach showing that the C-terminal peptide HDEF of RNase LX acts as an alternative ER retention signal in plants. For functional testing, the cellular distribution of chimeric protein constructs based on a marker protein, Brazil nut (Bertholletia excelsa) 2S albumin, was analyzed immunochemically in transgenic tobacco (Nicotiana tabacum) plants. Here, we report that the peptide motif is necessary and sufficient to accumulate 2S albumin constructs of both vacuolar and extracellular final destinations in the ER. We have shown immunochemically that RNase LX is specifically expressed during endosperm mobilization and leaf and flower senescence. Using immunofluorescence, RNase LX protein was detected in immature tracheary elements, suggesting a function in xylem differentiation. These results support a physiological function of RNase LX in selective cell death processes that are also thought to involve programmed cell death. It is assumed that RNase LX accumulates in an ER-derived compartment and is released by membrane disruption into the cytoplasma of those cells that are intended to undergo autolysis. These processes are accompanied by degradation of cellular components supporting a metabolic recycling function of the intracellular RNase LX. PMID:11598219

  1. Expression of the cellular FLICE-inhibitory protein (c-FLIP) protects Hodgkin's lymphoma cells from autonomous Fas-mediated death.

    PubMed

    Dutton, A; O'Neil, J D; Milner, A E; Reynolds, G M; Starczynski, J; Crocker, J; Young, L S; Murray, P G

    2004-04-27

    Hodgkin's lymphoma (HL) is characterized by the presence of malignant so-called Hodgkin's/Reed-Sternberg (HRS) cells, which display resistance to certain apoptotic stimuli, including a lack of sensitivity to Fas-mediated cell death. However, the mechanisms responsible for their resistance to apoptosis inducers have not been elucidated. Here we confirm that both HL-derived cell lines and the HRS cells of primary HL tissues express Fas ligand (FasL) along with the inhibitory c-FLIP protein. Down-regulation of cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) through the use of specific small inhibitory RNAs (siRNAs) leads to reduced viability of the L428 and L591 HL-derived cell lines. To determine whether endogenous FasL was responsible for the reduction in cell viability observed after down-regulation of c-FLIP, L428 and L591 cells were treated with c-FLIP-specific siRNAs with and without siRNAs directed to FasL. Treatment of these cells with both c-FLIP- and FasL-specific siRNAs in combination restored cell viability to near control levels. Our results provide a mechanism whereby HRS cells are protected from autonomous FasL-mediated cell death while preserving their ability to evade immunosurveillance. Targeting c-FLIP could provide a novel approach to the treatment of HL.

  2. Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells

    PubMed Central

    Reinecke, Fimmie; Levanets, Oksana; Olivier, Yolanda; Louw, Roan; Semete, Boitumelo; Grobler, Anne; Hidalgo, Juan; Smeitink, Jan; Olckers, Antonel; Van Der Westhuizen, Francois H.

    2006-01-01

    The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells. PMID:16402917

  3. Programmed Death Ligand 1 (PD-L1) Tumor Expression Is Associated with a Better Prognosis and Diabetic Disease in Triple Negative Breast Cancer Patients

    PubMed Central

    Botti, Gerardo; Collina, Francesca; Scognamiglio, Giosuè; Rao, Federica; Peluso, Valentina; De Cecio, Rossella; Piezzo, Michela; Landi, Gabriella; De Laurentiis, Michelino; Cantile, Monica; Di Bonito, Maurizio

    2017-01-01

    Triple Negative Breast Cancers (TNBC) subtype is an aggressive disease with poor clinical outcome. The only treatment available is surgery followed by chemotherapy or radiotherapy. Programmed death-ligand 1 (PD-L1) is a trans-membrane protein expressed on a wide variety of cells including immune cells, epithelial and vascular endothelial cells. Recently, PD-1/PD-L1 pathway signaling was described as an adaptive immune resistance mechanism enacted by the tumor cells to evade the immune response. Its presence on tumor cell membranes, acquired for this reason, through time, is an important prognostic value. However, data available in the literature about PD-L1 immunohistochemical expression in breast cancer are often discordant and not uniform, probably for the use of different antibodies clones and the high molecular heterogeneity of the different tumor types. The absence of target therapies, in particular for TNBC, has shifted the clinical attention mainly on the role of PD-L1 in this subtype of breast cancer. In this study, we evaluated tumor and TIL (tumor infiltrating lymphocytes) PDL-1 expression in a series of TNBC, included in Tissue Micro Arrays (TMAs), to define its real prognostic value, optimizing immunohistochemistry method with an “approved for diagnostic assay” antibody. PD-L1 expression directly correlated with proliferation index (Ki-67), glycemia, the presence of diabetes and indirectly with menopausal status, presence of lymph node metastasis and relapse. The analysis of Kaplan–Meier showed that an increased PD-L1 expression was strongly associated with better disease-free survival (DFS) but not correlated with overall survival (OS). Our data confirmed that PD-L1 could be an important marker for prognostic stratification and for planning immune checkpoint inhibitors therapies in patients with TNBC. PMID:28230773

  4. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    PubMed

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the f