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Česká republika Praha
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0032 (0)16 41 44 07
Recombinant Human Tumor Necrosis Factor alpha Mutant (TNFa Mutant)
(Cat. No.: C036)
TNF is secreted by macrophages, monocytes, neutrophils, T-cells, NK-cells following their stimulation by bacterial LPS. Cells expressing CD4 secrete TNF-alpha while CD8 cells secrete little or no TNF-alpha. The synthesis of TNF-alpha is induced by many different stimuli including interferons, IL2, GM-CSF.
The clinical use of the potent anti-tumor activity of TNF-alpha has been limited by the proinflammatory side effects including fever, dose-limiting hypotension, hepatotoxicity, intravascular thrombosis, and hemorrhage. Designing clinically applicable TNF-a mutants with low systemic toxicity has been an intense pharmacological interest. Human TNF-a, which binds to the murine TNF-R55 but not to the mouse TNF-R75, exhibits retained anti-tumor activity and reduced systemic toxicity in mice compared with murine TNF-a, which binds to both murine TNF receptors. Based on these results, many TNF-a mutants that selectively bind to TNF-R55 have been designed. These mutants displayed cytotoxic activities on tumor cell lines in vitro, and exhibited lower systemic toxicity in vivo. Recombinant
Human TNF-alpha Variant/Mutant compared with the wild-type, has an amino acid sequence deletion from a.a. 1-7, and the following a.a. substitutes Arg8, Lys9, Arg10 and Phe157 which is proven to have more activity and with less inflammatory side effect in vivo. Recombinant Human TNF-alpha Mutant produced in E. coli is a single, non-glycosylated, polypeptide chain containing 151 amino acids and having a molecular mass of 16,886 Dalton.
Biological activity: The ED50 as determined by the cytolysis of murine L929 cells in the presence of Actinomycin D is less then 0.01 ng/ml, corresponding to a Specific Activity of 1.0 x 108 IU/mg.
Purity: Greater than 95% as determined by
(a) Analysis by SEC-HPLC.
(b) Analysis by reducing and non-reducing SDS-PAGE Silver Stained gel.
Amino-Acid Sequence: The sequence of the first five N-terminal amino acids was determined and was found to be Met-Arg-Lys-Arg-Lys.
Endotoxin: Less than 0.01ng/µg (0.01IEU/µg) determined by LAL test.
Formulation: Lyophilized from a 0.2 µm filtered solution in PBS.
Storage: Lyophilized samples are stable for up to twelve months from date of receipt at -20oC to -70oC.
Please avoid freeze-thaw cycles.
Reconstitution: It is recommended to reconstitute the lyophilized TNF-alpha Mutant in sterile 18MΩ-cm H2O not less than 100µg/ml, which can then be further diluted to other aqueous solutions.
1: Mol Nutr Food Res 2009 Jan;
Silibinin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells.
[Abstract] We studied the inhibitory effect of silibinin on ochratoxin A (OTA) and LPS-mediated tumor necrosis factor alpha (TNF-alpha) release and the leakage of cytotoxic markers glutamate dehydrogenase (GLDH) and lactate dehydrogenase (LDH), from isolated blood-free perfused rat livers, and from isolated pure rat Kupffer cells. In the recirculation perfusion model at the end point 90 min, 2.5 mumol/L OTA released 2600 pg/mL TNF-alpha without effects on liver vitality. LPS at 0.1 mug/mL induced 3000 pg TNF-alpha/mL with slight leakage of GLDH and LDH. Under similar experimental conditions, the addition of silibinin 10 min prior to OTA and LPS showed dose-dependent protection against OTA or LPS-induced hepatic TNF-alpha release. High-dose of silibinin (12.5 mug/mL) also
completely restored GLDH and LDH levels in the perfusate. Pretreatment of isolated Kupffer cells with 0.02, 0.1, 0.5, 2.5, and 12.5 mug silibinin/mL 30 min prior to OTA reduced OTA-induced TNF-alpha levels to 90, 70, 25, 25, and 25% at 4 h, respectively, and abrogated any TNF-alpha release at 24 h. Similarly, in the presence of silibinin LPS-induced TNF-alpha levels decreased at 4 h to 71, 57, 18, 22, and 18%, respectively. However, after 24 h of LPS exposition the protection by silibinin vanished and TNF-alpha partially recurred into the incubation medium under LPS. In summary, silibinin had hepatoprotective effects against OTA- or LPS-mediated TNF-alpha release and also reduced the cytotoxicity of both toxins. Isolated Kupffer cells were even more sensitive to the protective effect than
perfused livers and responded to very low concentrations of silibinin with a strong inhibition of toxins-mediated TNF-alpha release.
2: PLoS ONE 2009 ;Vol 4(1)
Pleural tuberculosis in patients with early HIV infection is associated with increased TNF-alpha expression and necrosis in granulomas.
[Abstract] Although granulomas may be an essential host response against persistent antigens, they are also associated with immunopathology. We investigated whether HIV co-infection affects histopathological appearance and cytokine profiles of pleural granulomas in patients with active pleural tuberculosis (TB). Granulomas were investigated in pleural biopsies from HIV positive and negative TB pleuritis patients. Granulomas were characterised as necrotic or non-necrotic, graded histologically and investigated for the mRNA expression of IL-12, IFN-gamma, TNF-alpha and IL-4 by in situ hybridisation. In all TB patients a mixed Th1/Th2 profile was noted. Necrotic granulomas were more evident in HIV positive patients with a clear association between TNF-alpha and necrosis.
This study demonstrates immune dysregulation which may include TNF-alpha-mediated immunopathology at the site of disease in HIV infected pleural TB patients.
3: Transplantation 2009
Intragraft TNF receptor signaling contributes to activation of innate and adaptive immunity in a renal allograft model.
[Abstract] BACKGROUND: Increased levels of tumor necrosis factor (TNF) are a risk factor for allograft rejection. In vitro studies have shown that binding of TNF to its receptor activates signaling cascades that induce expression of many genes involved in inflammation. The role of intragraft TNF receptor (TNFR) signaling in activation of gene expression in allografts has not been studied. METHODS: Gene expression profiling and quantitative real-time polymerase chain reaction analysis were used to investigate the role of TNFR signaling in the early intragraft activation of cellular gene expression in renal allografts at 2 days posttransplant. RESULTS: The TNFRs play a critical role in activating intragraft expression of transcription factors controlling innate and
adaptive immunity and stress responses (interferon regulatory factor [IRF]1, IRF 8, Isgf3g, and ATF3) of cytokines and receptors mediating inflammation (TNF, interleukin [IL]-6, interferon-gamma, oncostatin M receptor [OMCR], toll-like receptor [TLR]2, and IL-2Rgamma), of chemokines and adhesion molecules that recruit inflammatory cells (Cxcl9, Cxcl11, E-selectin, and intracellular adhesion molecule [ICAM]-1), of genes involved in costimulation of T cells and processing and presentation of antigens (H2-DMb, Psmb8, and CD40), and genes that mediate the response to interferons. In addition to its proinflammatory role, TNFR signaling induces expression of SOCS3, a negative regulator of IL-6 and OSMR signaling and Nfkbie, and a negative regulator of TNFR signal transduction. CONCLUSIONS: These
studies illustrate the pleiotropic effect of TNF in both activation and down-modulation of the immune response and the complex interactions between the TNFRs and other cytokine signaling pathways in the early allograft response.
4: J Nutr Sci Vitaminol (Tokyo) 2008
Effect of dietary soy protein on tumor necrosis factor productivity in macrophages from nephritic and hepatoma-bearing rats.
[Abstract] The present study investigated the effect of dietary soy protein isolate (SPI) on tumor necrosis factor-alpha (TNF) productivity in peritoneal macrophages from nephritic and hepatoma-bearing rats. Dietary SPI significantly inhibited the elevated production of TNF by lipopolysaccharide-stimulated macrophages in nephritic and hepatoma-bearing rats compared with dietary casein, while it exerted no influence on the TNF productivity in normal rats. Removal of the minor components contained in SPI by ethanol extraction could significantly or partially restore the reduced TNF production caused by SPI in nephritic and hepatoma-bearing rats, respectively. These results suggest that dietary SPI could suppress the enhanced productivity of TNF associated with the
progression of nephritis and hepatoma, and some factors existing in the ethanol extract of SPI are suggested to be involved in suppressing TNF productivity by macrophages.
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*NOTE: ALL PRODUCTS ARE FOR RESEARCH USE ONLY