Browsing by Subject "Tumor necrosis factor"

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    Examination of epithelial tissue cytokine response to natural peste des petits ruminants virus (PPRV) infection in sheep and goats by immunohistochemistry
    (F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2012) Tarik Atmaca, Hasan; Kul, Oguz
    In this study, we aimed to evaluate expression of IL-4, IL-10, TNF-α, IFN-γ and iNOS in lingual, buccal mucosa and lung epithelial tissue using immunoperoxidase technique and to compare with the tissues of control animals. The tissues used in the study were collected from 17 PPRV-affected and 5 healthy sheep and goats. In PPRV positive animals, the lungs, lingual and buccal mucosa had significantly higher iNOS, IFN-γ and TNF-α expressions compared to control group animals. There was no significant difference between PPRV positive and control groups for IL-4 and IL-10 expressions of epithelial tissues. In conclusion, the epithelial tissues infected by PPRV showed significant iNOS, IFN-γ and TNF-α expressions and they might play an important role in the initiation and regulation of cytokine response, as they take place in the first host barrier to be in contact with PPRV. It is suggested that the more epithelial damage produced by PPRV the more cytokine response may result in the infected epithelial cells. The first demonstration of iNOS expression and epithelial cytokine response to PPRV in natural cases is important because it may contribute to an early initiation of systemic immunity against PPRV infection, in addition to direct elimination of the virus during the initial epithelial phase of the infection
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    Post-transcriptional regulation of cytokine genes in fish: A role for conserved AU-rich elements located in the 3′-untranslated region of their mRNAs
    (Elsevier, 2006-04-03) Cayuela Fuentes, Maria Luisa; Secombes, Chris. J.; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
    The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.
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    Signaling pathways mediated by tumor necrosis factor a
    (Murcia : F. Hernández, 2000) Leong, K.G.; Karsan, A.
    Tumor necrosis factor a (TNFa) has been shown to trigger many signaling pathways. Following oligomerization by TNFa, the receptors TNF-RI and TNF-RI1 associate with adapter molecules via specific protein-protein interactions. The subsequent recruitment of downstream molecules to the receptor complex enables propagation of the TNFa signal. l k o cellular responses to TNFa have been well documented, the induction of cell death and the activation of gene transcription for cell survival. TNFa-induced apoptosis involves the activation of caspase cascades, which culminate in the cleavage of specific cellular substrates to effect cell death. TNFa has also been implicated in various caspase-independent cell death processes. Two transcription factors activated by TNFa are nuclear factor KB (NFKB) and activating protein 1 (AP-1). Pathways that promote the activation of these transcription factors involve signaling molecules such as kinases, phospholipases, and sphingomyelinases. In addition to increased survival (anti-apoptotic) gene expression, NFKB and AP-1 also induce the expression of genes involved in inflammation, cell growth, and signal regulation. The past decade has witnessed the identification of numerous signaling intermediates implicated in TNFa cellular responses. This article reviews the molecular mechanisms of TNFa signal transduction. In particular, pathways involved in cell death and transcription factor activation are discussed.
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    Turbot TNFα gene: molecular characterization and biological activity of the recombinant protein
    (Elsevier, 2006-04-17) Ordas, M. C.; Costa, Maria del Mar; Lopez-Castejón, Gloria; Meseguer Peñalver, J.; Figueras, Antonio; Novoa, Beatriz; Mulero Méndez, Victoriano Francisco; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
    The tumor necrosis factor (TNF) superfamily is composed by several proteins with similar structure and functions. One of the main representatives of this family is TNF-alpha (TNFα), a proinflammatory cytokine which is produced by different immune cells and presents a wide variety of activities. Using the RACE technique, we have cloned and sequenced the turbot TNF cDNA. The analysis of its sequence showed several conserved motifs characteristic of members of the TNFα family. A phylogenetic tree constructed with different TNFs of fish and mammals grouped our sequence within the fish TNFα cluster. Therefore, the turbot TNF here studied was identified as TNFα. The complete TNFα gene was obtained by gene walking, and, similarly to the other known fish TNFα genes, presented three introns and four exons. A PCR was designed to study the turbot TNFα expression in vivo using as stimulus the bacteria Vibrio pelagius strain Hq222 and virus VHSV. The expression of the cytokine happened early after injection, and it was dependent on the pathogen injected and organ analyzed. Virus induced a higher TNFα expression, but this response was shorter in time than that induced by bacteria. In addition, TNFα expression was in general higher in kidney than in liver, as expected since the former is the haematopoietic organ of fish. The turbot recombinant TNFα (rTNFα) was obtained by IPTG induction of bacteria transformed with the pET15b-TNFα construct, and it was purified in native conditions. The recombinant protein was approximately 20 kDa in size, and its biological activity was assessed in vitro. No effect of the rTNFα neither alone nor in combination with LPS was observed on the chemiluminescence activity of turbot macrophages at any time tested. However, NO production was enhanced by the recombinant protein alone or with LPS 72 h after the addition of the treatments. Finally, turbot rTNFα was able to recruit and activate inflammatory cells when injected in gilthead seabream, although to a lesser extent than gilthead seabream rTNFα.
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    Voluntary oral feeding of rats not requiring a very high fat diet is a clinically relevant animal model of non-alcoholic fatty liver disease (NAFLD)
    (Murcia : F. Hernández, 2009) Tipoe, G.L.; Ho, C.T.; Liong, Emily C.; Leung, T.M.; Lau, T.Y.H.; Fung, M.L.; Nanji, A.A.
    Animal models used to study the pathogenesis of non-alcoholic fatty liver disease (NAFLD) are, in general, either genetically altered, or fed with a diet that is extremely high in fat or carbohydrates. Recent findings support the role of oxidative stress, lipid peroxidation and inflammation as probable causative factors. We hypothesize that not only the amount of dietary fat, but the quality of fat is also important in inducing NAFLD. Based on previous observations that female rats fed a diet comprising unsaturated fatty acids are susceptible to liver injury, we proposed that female rats fed with a diet containing fish oil and dextrose would develop pathological and biochemical features of NAFLD. We fed a highly unsaturated fat diet (30% fish oil) to female SpragueDawley rats (180-200g), consumed ad libitum for 8 weeks (NAFLD; n=6-8 ). Control animals (CF; n=6-8) were fed with an isocaloric regular rat chow. At killing, blood and liver samples were collected for serum alanine aminotransferase (ALT), histology and molecular analysis. Each histological sample was evaluated for fatty liver (graded from 0 to 4+ according to the amount of fatty change), necrosis (number of necrotic foci (no./mm2 ) and inflammation (cells per mm2 ). The amount of collagen formation was estimated based on the amount of Sirius Red staining. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out for tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), adiponectin, glutathione peroxidase (GPx), superoxide dismutase (Cu/Zn SOD) and catalase (CAT). Western Blot analysis was done for cyclooxygenases-2 (COX-2), inducible nitric oxide synthase (iNOS) and nitrotyrosine. Electrophoretic mobility shift assay was performed for nuclear factor-kappa B (NF-κB) activity. NAFLD rats had a significantly higher serum ALT level, amount of collagen formation, fatty liver, necrosis and inflammation when compared with the chow-fed control rats. mRNA and protein levels of NF-κB regulated genes, which included TNF-alpha, COX-2 and iNOS were also significantly (p<0.01; p<0.01; p<0.05 respectively) upregulated in the NAFLD group when compared with the chow-fed control rats. mRNA levels of antioxidants CAT and GPX were reduced by 35% and 50% respectively in the NAFLD group. However, Cu/Zn SOD mRNA was similar in both groups. The mRNA level of adiponectin was also reduced in NAFLD group. NF-κB activity was markedly increased in the NAFLD rats (p<0.01). The level of oxidative stress, represented by the formation of nitrotyrosine, was significantly elevated in the NAFLD rats (p<0.01). We conclude that NAFLD rats demonstrated several features of NAFLD, which included fatty liver, inflammation, necrosis, increased oxidative stress, an imbalance between pro and antioxidant enzymes mRNAs, reduced adiponectin levels and upregulation of pro-inflammatory mediators. We propose that female rats fed with a diet containing highly unsaturated fatty acids are an extremely useful model for the study of NAFLD.