Tumor necrosis factor-alpha(TNF-α) in spotted halibut Verasper variegatus at the embryonic and metamorphic stages

As an aquatic fish,the spotted halibut Verasper variegatus is highly susceptible to bacterial and virus infections.Tumor necrosis factor-alpha(TNF-α)as a cytokine could control the inflammatory responses.The functions of TNF-αin many species have been widely studied,particularly in mammals.However,little is known about the TNF-αfunctions in V.variegatus.We first cloned and sequenced the TNF-αgene in V.variegatus(VvTNF-α).The two conserved cysteine residues,transmembrane sequence,Thr-Leu motif,and TNF family signature,as well as the TA-rich motifs of its proteins related to inflammatory responses had high similarity to those of the other teleost and mammalian TNF-α.The phylogenetic analysis showed that VvTNF-αwas consistent with TNF-αgenes of other vertebrates.The VvTNF-αtranscripts were extensively distributed in the peripheral blood leukocytes(PBLs),spleen,and gill,indicating that the VvTNF-αhad a role in immune function.Furthermore,treatment with pathogen-associated molecular patterns(PAMPs)could induce a rapid and significant increase of VvTNF-αin the PBLs,which reveals that VvTNF-αdoes participate in the host immune responses against bacterial and viral pathogens.We found that VvTNF-αhad an interesting expression pattern during metamorphosis,showing that the flatfish TNF-αmay have some novel functions during specific developmental stages.In addition,the 3 D structure prediction of VvTNF-αprovided an indication of how it is likely to interact with other proteins.Therefore,VvTNF-αhas multiple functions,and provides valuable information to explore novel functions of TNF-α.

Increased expression of tumor necrosis factor-a is associated with advanced colorectal cancer stages

AIM:To detect the expression of tumor necrosis factor-a(TNF-a)in colorectal cancer(CRC)cells among Saudi patients,and correlate its expression with clinical stages of cancer.METHODS:Archival tissue specimens were collected from 30 patients with CRC who had undergone surgical intervention at King Khalid University Hospital.Patient demographic information,including age and gender,tumor sites,and histological type of CRC,was recorded.To measure TNF-a m RNA expression in CRC,total RNA was extracted from tumor formalin-fixed,paraffinembedded,and adjacent normal tissues.Reverse transcription and reverse transcription polymerase chain reaction were performed.Colorectal tissue microarrays were constructed to investigate the protein expression of TNF-a by immunohistochemistry.RESULTS:The relative expression of TNF-a m RNA in colorectal cancer was significantly higher than that seen in adjacent normal colorectal tissue.High TNF-a gene expression was associated with StageⅢandⅣneoplasms when compared with earlier tumor stages(P=0.004).Eighty-three percent of patients(25/30)showed strong TNF-a positive staining,while only 10%(n=3/30)of patients showed weak staining,and 7%(n=2/30)were negative.We showed the presence of elevated TNF-a gene expression in cancer cells,which strongly correlated with advanced stages of tumor.CONCLUSION:High levels of TNF-a expression could be an independent diagnostic indicator of colorectal cancer,and targeting TNF-a might be a promising prognostic tool by assessment of the clinical stages of CRC.

Effects of erythropoietin on the expression of tumor necrosis factor-alpha and Bax after facial nerve axotomy in rats

This study sought to evaluate the effect of high-dose erythropoietin （EPO; 5 000 IU/kg） on the expression of tumor necrosis factor-alpha （TNF-α） and Bax in the facial nucleus after facial nerve transection in rats. A total of 42 Wistar rats of both genders were used in this study, and 40 rats were randomly divided into 2 groups： EPO group and model group. The EPO group was treated with EPO once a day for 5 days at a dose of 5 000 IU/kg body weight. The model group was treated with saline of the same amount. At day 3 after EPO （or saline） treatment, the right facial nerves of the 40 rats were transected at the level of the stylomastoid foramen, with the left sides untreated. The remaining 2 rats that did not undergo axotomy served as the control group. The surviving motor neurons in operated rats were counted in coronal paraffin sections of the facial nucleus. The expression of TNF-a and Bax in the facial nucleus was detected by immunohistochemical staining at days 3, 7, 14, 21, and 28 after axotomy. At days 14, 21, and 28 after facial nerve axotomy, a significantly greater proportion of facial motor neurons survived in the EPO group than in the model group. After axotomy, the expression of TNF-a and Bax increased in motor neurons in both the EPO and the model groups. TNF-o expression reached its peak level at day 14 after axotomy, while Bax expression reached its peak level at day 21. TNF-α expression was much lower in the EPO group than in the model group at all time points. No significant difference in Bax expression was found between the EPO and the model groups. These results indicate that high-dose EPO treatment attenuates the increase in TNF-α expression in the facial nucleus and reduces the loss of motor neurons after facial nerve transection in rats. However, high-dose EPO treatment has little effect on Bax expression.

Up-regulation of tumor necrosis factor-a pathway survival genes and of the receptor TNFR2 in gastric cancer

BACKGROUND Gastric carcinogenesis can be induced by chronic inflammation triggered by Helicobacter pylori(H. pylori) infection. Tumor necrosis factor(TNF)-α and its receptors(TNFR1 and TNFR2) regulate important cellular processes, such as apoptosis and cell survival, and the disruption of which can lead to cancer. This signaling pathway is also modulated by microRNAs(miRNAs), altering gene expression.AIM To evaluate the mRNA and miRNAs expression involved in the TNF-α signaling pathway in gastric cancer(GC) tissues and its relationship.METHODS Quantitative polymerase chain reaction(qPCR) by TaqMan? assay was used to quantify the RNA transcript levels of TNF-α signaling pathway(TNF, TNFR1,TNFR2, TRADD, TRAF2, CFLIP, NFKB1, NFKB2, CASP8, CASP3) and miRNAs that targets genes from this pathway(miR-19 a, miR-34 a, miR-103 a, miR-130 a,miR-181 c) in 30 GC fresh tissue samples. Molecular diagnosis of H. pylori was performed by nested PCR for gene HSP60. A miRNA:mRNA interaction network was construct using Cytoscape v3.1.1 from the in silico analysis performed using public databases.RESULTS Up-regulation of cellular survival genes as TNF, TNFR2, TRADD, TRAF2, CFLIP,and NFKB2, besides CASP8 and miR-34 a was observed in GC tissues, whereas mediators of apoptosis such as TNFR1 and CASP3 were down-regulated. When the samples were stratified by histological type, the expression of miR-103 a and miR-130 a was significantly increased in the diffuse-type of GC compared to the intestinal-type. However, no influence of H. pylori infection was observed on the expression levels of mRNA and miRNAs analyzed. Moreover, the miRNA:mRNA interaction network showed several interrelations between the miRNAs and their target genes, highlighting miR-19 a and miR-103 a, which has as predicted or validated target a large number of genes in the TNF-α pathway,including TNF, TNFR1, TNFR2, CFLIP, TRADD, CASP3 and CASP8.CONCLUSION Our findings show that cell survival genes mediated by TNF/TNFR2 binding is up-regulated in GC favoring its pro-tumoral effect, while pro-apoptotic genes as CASP3 and TNFR1 are down-regulated, indicating disbalance between apoptosis and cell proliferation processes in this neoplasm. This process can also be influenced by an intricate regulatory network of miRNA:mRNA.

Anti Cervix Cancer Activity of Co-immobilized Tumor Necrosis Factor-α and Interferon-γ

Tumor necrosis factor α （TNF-α） and interferon-γ （IFN-γ） are cytokines with strong antitumor activities. They were reacted with a photoactive arylazide-4-azidobenzoic acid, resulting in photoactive TNF-α and IFN-γ. The infrared （IR） spectra of these products showed the characteristic absorption of an azido group at 2127 cm^-1. By photo-immobilization, this modified TNF-α and IFN-γ were immobilized on polystyrene membranes for cell culture to prepare biomaterials. The micro-morphology of photoactive cytokines was observed with a scanning electron microscope （SEM）. The inhibitory effect on growth of Hela cells and inducing apoptosis activity of these two cytokines were analyzed by growth curve, transmission electron microscope （TEM） and fluorescence active cell sorter （FACS）. The results showed that co-immobilization of IFN-γ and TNF-α had significant inhibitory effect on growth of Hela cells, inhibitory rate up to 82%, and IFN-γ had obviously synergistic action.

Secoemestrin C Ameliorates Psoriasis-like Skin Inflammation in Mice by Suppressing the TNF-α/NF-κB Signaling Pathway

Objective Secoemestrin C(SC),an epitetrathiodioxopiperazine isolated from Aspergillus nidulans,has been previously reported to have immunomodulatory and hepatoprotective effects against acute autoimmune hepatitis.However,the effect of SC on regulating the inflammation and its underlying mechanisms in the pathogenesis of psoriasis remain unclear.This study aimed to evaluate the effects of SC on inflammatory dermatosis both in vitro and in vivo.Methods In vitro,HaCaT cells were induced with tumor necrosis factor-alpha(TNF-α,10 ng/mL)to establish an inflammatory injury model,and the expression of nuclear transcription factor-κB(NF-κB)pathway components was measured using qRT-PCR and Western blotting.An in vivo mouse model of imiquimod(IMQ)-induced psoriasis-like skin inflammation was used to evaluate the effectiveness of SC in alleviating psoriasis.Results SC significantly blocked the activation of NF-κB signaling in TNF-α-stimulated HaCaT cells.In addition,systemic and local administration of SC improved psoriatic dermatitis in the IMQ-induced mouse model.SC reduced skin scale and significantly inhibited the secretion of inflammatory factors in skin lesions.Conclusion The protective effect of SC against psoriatic-associated inflammation reveals its potential therapeutic value for treating psoriasis.

Roles of tumor necrosis factor alpha on sperm acrosin activity and acrosome reaction

Aim: To study the roles of tumor necrosis factor alpha (TNF-α) on the sperm acrosin activity and acrosome reaction. Methods: The sperm acrosin activity was tested by the method of BAEE/ ADH Unity and the acrosome reaction by the Triple-stain technique. Results: TNF-α decreased the sperm acrosin activity and acrosome reaction (P<0.01; P<0.01, respectively); it also inhibited the Ca2+-ATPase activity and SOD activity in sperm (P< 0.05; P<0.001, respectively), but increased the NOS activity and the amount of NO in sperm (P<0.001; P<0.001, respectively). While it had a less significant effect on the activity of Na+-K+-ATPase (P>0.05). Conclusion: TNF-α inhibits the sperm acrosin activity and acrosome reaction.

Relationship of Tumor Necrosis Factor-α and Nitrogen Oxide with Treatment of Frequent Relapse Nephrotic Syndrome by Shenkangling(肾康灵)Granule in Children

Objective: To observe the relationship of tumor necrosis factor-o (TNF-a) and nitrogen oxide (NO) with the treatment of frequent relapse nephrotic syndrome (FRNS) and to explore the patho-genesis of FRNS and the therapeutic mechanism of Shenkangling (肾康灵,SKL) Granule in children. Methods: Sixty children suffering from FRNS were randomly divided into the treated group and control group, 30 in each, and the other 30 healthy children were taken as healthy group. The patients were treated with prednisone for a long-term course, and those with no effect or partial effect shown were treated with additional Tripterygium or Cytoxan in the control group, while in the treated group patients were treated with prednisone and additional SKL. The two groups were compared as to their changes of TNF-a, NO before and after treatment, and the relapses after treatment. Results: The levels of TNF-a and NO in the sick children before treatment were markedly higher than those after treatment and normal group (P< 0. 01). The positive correlation between TNF-o of FRNS cases and relapse risk displayed more significance than that between the relapse of FRNS and NO. The difference between treated group and control group was significant (P<0. 01). Conclusion: TNF-a can be regarded as the monitoring parameter of the active phase in FRNS, and the higher the level, the more possible the relapse would occur. SKL could markedly reduce the relapse rate of FRNS in children.

THE CHANGE OF SERUM TUMOR NECROSIS FACTOR-α LEVEL AND ITS CLINICAL SIGNIFICANCE DURING THE TREATMENT OF CHRONIC HEPATITIS B WITH LAMIVUDINE

Objective To evaluate the effect of lamivudine on immunity of chronic hepatitis B by observing the sequential changes of serum TNF-α and HBV-DNA level. Methods 31 CHB patients with elevated serum ALT/AST level and HBVDNA level higher than 106 copies/mL were treated with lamivudine (100mg/day) for one year. The sequential serum samples, which were taken at the 0, 3 rd, 6 th, 12 th month after initiation of therapy, were used to detect serum level of TNF-α and quantity of HBV-DNA respectively. Results ① The serum TNF-α levels were higher than normal value before treatment in all patients; ② At In the 3 rd month of treatment, The the serum HBV-DNA level began to decline and became negative in the 54.9% of all patients. At the end of treatment, HBV-DNA was negative in 48.4% of all patients; ③ The decrease of TNF-α level was later than HBVDNA level drop. TNF-α level began to decline after 6 months treatment. At the end of treatment, TNF-α level was lower than that at in 6 th month, TNF-α level returned to normal in the 38.7% of all patients; ④ The TNF-α level decreased significantly after 6 months treatment in the patients with ALT>80IU/L at the beginning of treatment. But in the patients with ALT≤80IU/L, the TNF-α level decreased just after 12 months treatment; ⑤ TNF-α level fell obviously and early in patients whose HBVDNA became negative at in the 3 rd month. Conclusion Lamivudine can suppress the replication of HBVDNA quickly, and decrease TNF-α level in the serum TNF-α level. It suggests that lamivudine can modulate immune response directly or indirectly. The changes of serum TNF-α level may be used to evaluate the clinical efficacy of lamivudine.

Promising Effects of Zerumbone on the Regulation of Tumor-promoting Cytokines Induced by TNF-α-activated Fibroblasts

Inflammation plays an important role in the development of several cancers.Inflammatory cytokines,including tumor necrosis factor-α(TNF-α),are associated with the induction of inflammation.Chronic inflammation contributes to the progression of cancer through several mechanisms,including increased cytokine production and activation of transcription factors,such as nuclear factor-κB(NF-κB).Zerumbone(ZER),a component of subtropical ginger(Zingiber zerumbet Smith),seems to have anti-inflammatory,anti-cancer,and antioxidant activities.In this study,we aimed to explore the protective function and mechanisms of ZER against TNF-α-induced cancer-promoting cytokines.We found that the viability of stimulated human fibroblast cell lines was reduced after treatment with ZER(IC50=18µmol/L),compared to un-stimulated fibroblasts(IC50=40µmol/L).Besides,ZER inhibited mRNA expression and protein secretion of transforming growth factor-β(TGF-β),interleukin-33(IL-33),monocyte chemoattractant protein-1(MCP-1),and stromal cell-derived factor 1(SDF-1),which were produced by TNF-α-induced fibroblasts,as measured by quantitative real time-PCR(qRT-PCR)and ELISA assays.The mRNA expression levels of TGF-β,IL-33,SDF-1,and MCP-1 showed 8,5,2.5,and 4-fold reductions,respectively.Moreover,secretion of TGF-β,IL-33,SDF-1,and MCP-1 was reduced to 3.65±0.34 ng/mL,6.3±0.26,1703.6±295.2,and 5.02±0.18 pg/mL,respectively,compared to the untreated group.In addition,the conditioned media(CM)of TNF-α-stimulated fibroblasts increased the NF-κB expression in colorectal cancer cell lines(HCT-116 and Sw48),while in the vicinity of ZER,the expression of NF-κB was reversed.Considering the significant effects of ZER,this component can be used as an appropriate alternative herbal treatment for cancer-related chronic inflammation.

酸脂清胶囊对尿酸性肾病大鼠肾功能及TNF-α的影响

目的:观察酸脂清胶囊对尿酸性肾病大鼠肾功能及TNF-α的影响,探讨其作用的机理。方法:将6周龄SD雄性大鼠分为5组,分别为正常组、模型组、生理盐水组、酸脂清治疗组、别嘌醇治疗组。后4组用腺嘌呤、乙胺丁醇造模,成功后分别给予生理盐水、酸脂清、别嘌醇灌胃给药,3周后检测各组大鼠血中尿素氮(BUN)、肌酐(Cr)、尿酸(UA),同时用酶联反应法(Elisa法)测量各组肾脏组织中肿瘤坏死因子α(TNF-α)的含量。结果:1)造模2周后,与正常组比较,模型组大鼠血中BUN、Cr、UA均明显升高(P<0.05),证实造模成功。2)治疗3周后,与生理盐水组相比,酸脂清治疗组、别嘌醇治疗组大鼠血中BUN、UA均明显下降(P<0.05),酸脂清治疗组和别嘌醇治疗组Cr均有下降,但只有酸脂清治疗组有统计学意义(P<0.05)。3)酸脂清治疗组和别嘌醇治疗组血中BUN、Cr、UA相比,差异无统计学意义(P>0.05)。4)Elisa法测定各组TNF-α的浓度,与正常组相比,生理盐水组TNF-α明显升高(P<0.05);与生理盐水组相比,酸脂清治疗组、别嘌醇治疗组TNF-α含量明显减少(P<0.05);5)肾组织病理切片显示:与模型组相比,酸脂清治疗组、别嘌醇治疗组尿酸盐结晶沉积、炎性细胞浸润均明显减少,肾小管扩张改善。结论:酸脂清胶囊能够降低尿酸性肾病大鼠血中BUN、Cr、UA的含量,减少尿酸盐结晶在肾小管中沉积及炎性细胞浸润,降低TNF-α在肾组织中的含量,从而达到治疗的作用。

Expression and distribution of TNF-α and PGE_2 of periodontal tissues in rat periodontitis model

Objective:To simulate the expression of TNF-α and PGE_2 of periodontal tissues in rat periodontitis model.Methods:40 Wistar rats were randomly divided into the periodontitis group and the control group(n=20).After the successful establishment of periodontitis rat model,raising for six weeks before the animals were sacrificed.The periodontal tissues were obtained and made into slices.Observed the histopathological changes of the periodontal tissues and measured TNF-α,PGE_2 levels change by immunohistochemistry.Western blot analysis and EI.ISA.Results:TNF-α,PGE_2 expression of the periodontitis group was significantly higher than that in the control group,the difference was significant(P<0.0S).Conclusions:The TNF-α.PGE_2expression of the rat periodontal tissue in the periodontitis group was significantly higher than the control group.

Atorvastatin Attenuates TNF-alpha Production via Heme Oxygenase-1 Pathway in LPS-stimulated RAW264.7 Macrophages

Objective To assess the effect of atorvastatin on lipopolysaccharide （LPS）-induced TNF-a production in RAW264.7 macrophages. Methods RAW264.7 macrophages were treated in different LPS concentrations or at different time points with or without atorvastatin. TNF-a level in supernatant was measured. Expressions of TNF-a mRNA and protein and heme oxygenase-1 （HO-1） were detected by ELISA, PCR, and Western blot, respectively. HO activity was assayed. Results LPS significantly increased the TNF-a expression and secretion in a dose- and time-dependent manner. The HO-1 activity and HO-1 expression level were significantly higher after atorvastatin treatment than before atorvastatin treatment and attenuated by SB203580 and PD98059 but not by SP600125, suggesting that the ERK and p38 mitogen-activated protein kinase （MAPK） pathways participate in regulating the above-mentioned effects of atorvastatin. Moreover, the HO-1 activity suppressed by SnPP or the HO-1 expression inhibited by siRNA significantly attenuated the effect of atorvastatin on TNF-c~ expression and production in LPS-stimulated macrophages. Conclusion Atorvastatin can attenuate LPS-induced TNF-e expression and production by activating HO-1 via the ERK and p38 MAPK pathways, suggesting that atorvastatin can be used in treatment of inflammatory diseases such as sepsis, especially in those with atherosclerotic diseases.

TNF-α gene polymorphisms: association with age-related macular degeneration in Russian population

AIM: To study polymorphisms in promotor regions of tumor necrosis factor(TNF)-α TNF-863 A/C(rs1800630), TNF-308 A/G(rs1800629), and TNF-238 A/G(rs361525) in patients with age-related macular degeneration(AMD) and associations of complex TNF-α genotypes with AMD. METHODS: One hundred and two patients(82 women, 20 men; mean age 64.2±1.2 y) with AMD and 100 healthy age-and sex-matched controls(82 women, 18 men; 60±1.4 y) were included in the study. All subjects were Caucasian, all subjects and their parents were inhabitants of Russia. Genomic DNA was obtained from EDTA-preserved blood using the standard phenol-chloroform method. Polymorphisms were detected by polymerase chain reaction followed by the restriction fragment length polymorphism method. The following TNF-α genotypes were studied: TNF-α-238 AA, GA, GG, TNF-α-308 AA, GA, GG, TNF-α-863 AA, CA, CC. RESULTS: Differences in TNF-α-863 and TNF-α-238 genotypes frequencies in patients with AMD and healthy controls were not found. The distribution of TNF-α-308 AA and TNF-α-308 GA genotypes was significantly different between the studied group and the controls [odds ratios(OR) =0.22, P=0.0287 and OR=2.91, P=0.0063, respectively]. TNF-863 CC/TNF-308 GA and TNF-308 GA/TNF-238 GG genotypes were associated with the increased risk of AMD(OR=2.48, P=0.0332 and OR=2.51, P=0.0187, respectively). Five genotypes combinations appeared to be protective. CONCLUSION: In the present study, single nucleotide polymorphisms and complex polymorphisms of one of the key inflammatory cytokines TNF-α, and a number of significant associations of these polymorphisms with AMD in Russian population have been shown. Complexanalysis of genotypes could be important in AMD risk factors detection and studying pathogenesis.

Utility of serum TNF-a,infliximab trough level,and antibody titers in inflammatory bowel disease

AIM:To assess tumor necrosis factor-a(TNF-a),infliximab(IFX)concentrations,and antibodies against IFX molecules in patients with inflammatory bowel disease(IBD)who develop loss of response,side effects,or allergic reaction during anti TNF-a therapy.METHODS:Blood samples of 36 patients with response loss,side effects,or hypersensitivity to IFX therapy(Group?Ⅰ)and 31 patients in complete clinical remission(GroupⅡ)selected as a control group were collected to measure trough serum TNF-a level,IFX,and anti-IFX antibody(ATI)concentration.We examined the correlation between loss of response,the development of side effects or hypersensitivity,and serum TNF-a,IFX trough levels,and ATI concentrations.RESULTS:The serum TNF-a level was shown to be correlated with the presence of ATI;ATI positivity was significantly correlated with low trough levels of IFX.ATIs were detected in 25%of IBD patients with loss of response,side effects,or hypersensitivity,however no association was revealed between these patients and antibody positivity or lower serum IFX levels.Previous use of IFX correlated with the development of ATI,although concomitant immunosuppression did not have any impact on them.CONCLUSION:On the basis of the present study,we suggest that the simultaneous measurement of serum TNF-a level,serum anti TNF-a concentration,and antibodies against anti TNF-a may further help to optimize the therapy in critical situations.

Inhibitory Effect of Oxymatrine on Quartz-induced Secretion of TNF-α by the Pulmonary Alveolar Macrophages in the Fibroblast Proliferation

To study the inhibitory effect of oxymatrine （OM） on quartz-induced secretion of TNF-α in the fibroblast proliferation, a given amount of quartz powder and OM of different concentrations were put into the media of pure culture containing macrophages. After 24 h of the culture, the TNF-α in the media was measured by double-antibody sandwich ELISA. The TNF-α （10 ng/mL） and OM of different concentrations were added into the media containing the fibroblasts of the 4th generations from neonate rats. The y values of cAMP and cGMP in fibroblasts were determined by the radioimmunoassay and the concentrations of cAMP and cGMP were calculated according to standard curve. The intracellular Ca^2＋ was determined by flow cytometry and cell proliferation was detected by MTT. Our results showed that at the concentrations between 200 μg/mL-1600 μg/mL, OM inhibited the secretion of TNF-α by alveolar macrophages （AM） in a dose-dependent manner. Especially, there were significant differences, to various degrees, in the inhibitory effect of OM between the concentration range of 800 μg/mL-1600 μg/mL and the concentration of 10 ng/mL TNF-α. When compared with 10 ng/mL TNF-α, OM of different concentrations could dose-independently increased the level of intracellular cAMP and decreased the level of cGMP, thereby raising the ratio of cAMP/cGMP and lowering the concentrations of intracellular Ca^2＋. Moreover, OM of 800 μg/mL had the strongest inhibitory effect on cell proliferation and at this concentration, the cAMP/cGMP was highest and Ca^2＋ was at the lowest level. We are led to conclude that OM can antagonize the damaging effect of quartz on the membrane of AM and the effect of TNF-α promoting the proliferation of fibroblasts. It achieves its inhibitory effect on the promoting effect of TNF-α on fibroblast proliferation by elevating the cAMP level and decreasing the release of Ca^2＋.

Study of renaturation condition of anti-TNF-α recombinant antibodies

Objective: To study the rules in the renaturation of recombinant antibody. Methods: Anti-TNF-a single domain (Sd) and single chain fragment variety(SCFv) antibodies were separated and purified under four conditions, including cosolvent redoxing, surface active solvent inducting, denaturant solvent inducting and the antigen of rhu TNFL-a inducting. Results: The dissolubility of renaturation products were between 6% - 11 %. Conclusion: These several conditions were good enough to the 2 antibody proteins and the best in them is the combination of the antigen inducting with affinity chromatography.

TIME-AND DOSE-DEPENDENT UP-REGULATION OF TNF-α mRNA AFTER IRRADIATION OF HUMAN NSCLC CELL LINES IN VITRO

Objective： Even though radiotherapy plays a major role in the local treatment of non-small cell lung cancer （NSCLC）, little is known about the molecular effects of irradiation in this tumor. In the present study, we examined two NSCLC cell lines for their endogenous production of TNF-α after irradiation. To investigate the radiation-induced TNF-α production in NSCLC cell lines. Methods： Two human NSCLC cell lines （A549： squamous; NCI-H596： adenosquamous） were investigated for their TNF-α mRNA （real-time RT-PCR） after exposure to different irradiation doses （2, 5, 10, 20, 30, 40 Gy） and time intervals （1, 3, 6, 12, 24, 48 or 72 h）. The TNF-α mRNA expression was quantified by real-time RT-PCR. The clonogenic survival was evaluated after irradiation with 2, 4, 6 and 8 Gy. Results： Non-irradiated NSCLC cells exhibited no or very low TNF-α expression. For the NCI-H596 cell line, TNF-α expression was significantly elevated 1～12 h （maximum 6h： 568fold increase relative to unirradiated cells） in a time-dependent manner. The radiation-induced increase could be observed after irradiation with 2 Gy reaching maximal at 40 Gy, with 83 times higher than normal controls. The clonogenic survival of these cell lines was nearly identical. Conclusion： NCI-H596 cells produce significant quantities of TNF-α following irradiation in a time- and dose-dependent manner. The pro-inflammatory cytokine TNF-α is a key mediator for the pathogenesis of radiation pneumonitis. Radiation-induced endogenous TNF-α expression in NSCLC cells may affect the normal lung adjacent to the tumor and may be associated with an adverse clinical outcome of the patient.

The effect of spinal cord injury on the expression of TGF-β and TNF-α in rat articular cartilage

Objective： To observe the expression of TGF-β and TNF-α in the spinal cord injured rat model and discuss the significance of the articular cartilage metabolism. Methods： 36 SD female rats were randomly divided into 2 groups： Rats models of spinal cord injury were implemented by Allen method. T10 laminectomy was performed in the control group. Both groups of rats were killed respectively in 1w, 3w and 6w. Hematoxylin-eosin stain was given to each slice in the model group and control group. Immunohistochemical stain was applied by using ABC method in the expression of TGF-β and TNF-α. Those expressed level were performed in image analysis and statistics process. Results： TGF-β and TNF-α were mainly distributed on the surface layer of the articular cartilage, with a weak expression in control group. The expression of TNF-α in the model group was more significant than that in the control group in the lw, and still remained an evident difference with that in control group until the 6w（P 〈 0.05）. TGF-β expression of the model group had no remarkable difference with the control group in the lw （P 〉 0.05） and prominently became stronger at 6w（P 〈 0.05）. Conclusion： The expression of TNF-α occurred early in the development of spinal cord injury, and the expression of TGF-β became stronger with the revival of spinal neural function. Both expressions were strengthened in articular cartilage in the 3rd week.

Effects of morphine and fentanyl on tumor necrosis factor-α and interleukin-6 concentrations in human whole blood in vitro

Cytokines are essential for hematopoiesis and immune responses, and play a key role in the defense against infections. Lipopolysaccharide ( LPS) is a potent inducer of the agents involved in the pathogenesis of inflammation responses. Tumor necrosis factor-α ( TNF-α ) and interleukin-6 (IL-6) are two important proinflammatory