RETROVIRUS-MEDIATED TNF-α GENE TRANSFER INTO TCA8113 CELLS

Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) can be used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cells in vitro with retroviral vector carring genes for both TNF-α and Neo^R. After that, presence and expression of exogenous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate of the cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique, ELISA technique, FACS technique, ^60 Co irradiation inactivation test, cryopreservation test, and Ames test, respectively. Results The presence of both TNF-α and Neo^R gene and expression of TNF-α gene were demonstrated in transgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parental cells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, all the cells died by d14 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h was no different from that cryopreserved for I week after irradiation, the level of TNF-α secreted by the cryopreserved cells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cells have no mutagenic activity. Conclusion TNF-α gene can be transduced into Tco8113 cells with retroviral vector, and the cells can express TNF-α. Expression of HLA Ⅰ,Ⅱ antigens on Tco8113 cells can be increased by TNF-Ⅱ gene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservation method for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.     

TNF-α and IL-8 of the Patients with Allergic Asthma

The levels of serum TNF-α and IL-8 in the patients with allergic asthma during acute attack period and remission period, and the effects of glucocorticoid (GC) on them were investigated. By using ELISA, the levels of TNF-α and IL-8 were detected in the healthy volunteers (group C, n=40), the patients with allergic asthma (n=40) during acute attack period (group A) and remission period (group B) and those taking GC for a week (n=28). The results were compared among them. It was found that the levels of TNF-α and IL-8 in group A were higher than in group B and group C. In the patients subject to GC therapy, the levels of TNF-α and IL-8 were decreased as compared with those in group A. In group B, the level of TNF-α was higher than in group C, but there was no significant difference in the level of IL-8 between group B and group C. It was concluded that the inflammatory cytokines, TNF-α and 11.-8, played important roles in the bronchus allergic inflammation. GC could reduce the levels of serum TNF-α and IL-8 to exert the anti-inflammatory effects.     

Expression of IL-βα and TNF-α in MCMV Myocarditis and Its Role

In order to study the expression of IL-β and TNF-α in the myocardium of MCMV myocarditis and their role in the myocardial damages, 60 BALB/C mice of 4 weeks were randomly divided into two groups: 36 were injected intraperitoneally with MCMV and 24 served as control group. Immunohistochemistry was used to detect IL-β and TNF-α expression in the myocardium, and myocardial lesions were observed histopathologically. Histopathological study on the myocardium from infected mice revealed focal or diffuse lesions characterized by inflammatory cells and degeneration or necrosis of myocytes. The myocardial lesion score showed the degree of inflammatory cell infiltration was slight in MCMV myocarditis. The positive staining signals for IL-β and TNF-α proteins which mainly located in the infiltrating inflammatory cells and degenerative or necrotic myocytes were markedly detectable whereas there were no positive findings in the myocardium of control mice. IL-β and TNF-α was expressed in the myocardium of viral myocarditis murine model induced by MCMV. IL-β and TNF-α may play an important role in the pathogenesis of viral myocarditis.     

Antitumor Effects of the Fibroblasts Transfected TNF-α Gene and its Mutants

Summary: To compare the anti-tumor effects of transmembrane TNF-α (TM-TNF) and secreted TNF-α (S-TNF) in vivo, mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-α (Wt-TNF), TM-TNF mutant (TM-TNFm), S-TNF mu tant (S-TNFm). Southern blot, RT-PCR, FACS and bioassay were used to investigate TNF-α gene integration, expression and its biological activity. It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF, the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-α or its mutants and effectively kill H22 in vitro. The trans fected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation ac cording to a ratio of 5: 1 or 1: 1 (effector/target cells, E/T) after the third day of H22 challenge,respectively. At the E/T= 5 : 1, the NIH3T3/TM-TNFm induced the highest tumor regression,while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T= 1 .: 1 in vivo. No obvious side effects were noted throughout the course of treatment. The results suggest that both TM-TNF and S-TNF could cause tumor regression. The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio.     

Influence of Radix Isatidis on the Endotoxin-induced Release of TNF-α and IL-8 from HL-60 Cells

Radix Isatidisis one of the most commonlyused antipyretic and detoxicant agents in tradition-al Chinese medicine.Its original source was identi-fied to be the dried roots ofIsatidis indigoticaFort.(Cruciferae).The antipyretic and detoxicantagents in traditional Chinese medicine are a type ofsynthetic therapeutic medicine.It can comprehen-sively activate the ability of anti-infection and re-pair of the human body.There are many pharma-cological effects such as antibacteria,antivirus,antipyre…     

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 TNF-α and curcumin on the expression of VEGF in Raji and U937 cells and on angiogenesis in ECV304 cells

Background To better understand the possibilities of antiangiogenic tumor therapy and to assess possible side effects, we investigated the effect of tumour necrosis factor （TNF）-α and curcumin on the expression of vascular endothelial growth factor （VEGF） in U937 and Raji cell lines and their effect on angiogenesis in a human umbilical vein endothelial cell （HUVECs）-derived cell line （ECV304）, and also the relationship between Notchl and VEGF. The aim of this study was to elucidate potential mechanisms controlling tumor neovascularization. Methods VEGF secreted by U937 and Raji cell lines was determined by ELISA. Angiogenesis was tested by network formation of endothelial cells on Matrigel. Levels of VEGF mRNA in U937 and Raji cells and Notchl mRNA levels in EV304 cells were determined by RT-PCR. Results Secretion of VEGF by U937 and Raji cells was increased by TNF-α treatment and suppressed by curcumin （P 〈 0. 01 ）. The mRNA expression of VEGF165 and VEGF121 （containing 165 and 121 amino acid residues, respectively） were detected in any fractions. TNF-α augmented the expression of VEGF165 and VEGF121 mRNA and curcumin reduced the expression （P 〈0. 01 ）. No networks or cords formed in control and curcumin groups. There was tube formation on matrigel in the supernatants of the Raji culture group and the supernatants groups treated by VEGF group and TNF-α in Raji cell. Notch1 mRNA was detected but there was no significant change in the VEGF group compared with control （P 〉 0. 05）. Conclusions Expressions of VEGF mRNA in U937 and Raji cells were increased by TNF-α and suppressed by curcumin. VEGF and TNF-α can induce angiogenesis, and curcumin can inhibit angiogenesis in ECV304 cells.     

A Simple Method of Detecting Chlamydia Trachomatis Using Enzymatically Amplified DNA and Immobilized Probes on Microtiter Plate

We have developed a simple and economical method for Chlamydia trachomatis detecting, called microtiter plate hybridization (PCR-MPtt) , which may replace standard PCR. This method is similar to that of an ELISA. Briefly, the PCR products labeled at the 5‘ termini with biotin were hybridized with probes immobilized on a microtiter well     

The Expression of TNF-α and ICAM-1 in Lesions of Lichen Planus and Its Implication

In order to investigate the role of TNF-α and ICAM-1 in the pathogenesis of lichen planus, immunohistochemistry was used to detect the expression of TNF-α and ICAM-1 in skin le- sions of the patients with lichen planus and skin tissues of normal subjects. The results showed that positive rates of TNF-α and ICAM-1 expressions in lichen planus were significantly higher than those in normal skins (both P<0.05). Meanwhile, there was a obvious correlation between the in- crease of TNF-α and that of ICAM-1 in lichen planus. The expression of TNF-α and ICAM-1 might play an important role in the development of lichen planus.     

Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha -induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65,IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB.TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.     

Assay of sIL-2R and TNF-α in experimental allergic encephalomyelitis

目的 :研究 s IL- 2 R和 TNF- α在实验性变态反应性脑脊髓炎 (EAE)免疫发病机制中的变化与作用。方法 :应用豚鼠诱导 EAE动物模型。在 MBP+CFA免疫豚鼠的第 8、15、2 2天处死动物 ,取脾细胞 ,加入 Con A诱生培养 ,收集上清液 ,采用 ELISA方法测定 s IL- 2 R水平 ,采用生物活性测定法检测TNF- α水平。结果 :EAE组的 s IL- 2 R与 TNF- α水平明显高于正常对照组。结论 :s IL- 2 R及 TNF- α在EAE免疫发病机制中具有重要作用     

Immunohistochemical Analysis of Growth Factor Expression and Localization in Gastric Coronary Vein of Cirrhotic Patients

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Experimental study of changes of TNF-α in rabbits with steroid-induced avascular necrosis of femoral head

INTRODUCTION Avascularnecrosisoffemoralhead(ANFH)isawell recognizedconditionofunknownaetiology wherebythecirculationofthebloodtofemoralheadis impaired.Eventuallytheinvolvedareaofbonediesand necrosisdevelops.Itmaybeidiopathicorassociated withsomediseases,suchastrauma,useofsteroids.ANFHinducedbysteroidisgettingmoreandmorewith theunreliableapplicationofsteroidinclinic.However,theexactmechanismofitsoccurrence,sofar,is uncertain.Theexperimenttriestoexploretheeffectof tumornecrosisfactorα(…     

Alteration of NF-kB and TNF-α mRNA and protein in hippocampus in the chronic constrictive injury model of rats

Objective To investigate the alteration of nuclear factor kappa B( NF-κB) and tumor necrosis factor-a (TNF-α) mRNA and protein in hippocampus in chronic constrictive injury (CCI) model of rats. Methods Seventy-six male Wistar rats were randomly divided into 2 groups ( n = 38): the CCI group which received the chronic constriction injury and the sham group which received the sham operation as control. The mechanical and thermal nociceptive thresholds were assessed with paw withdrawal latency (PWL) to von Frey filaments and radiant heat at 1d before and ld,4d,7d,14d and 28d after CCI operation. Five animals were sacrificed at each time point for real-time polymerase chain reaction (real-time PCR) and another three animals sacrificed at 7d postoperation for immunofluorescence histochemical staining. Results The thresholds to mechanical and thermal stimuli decreased obviously after operation in CCI group. The expressions of TNF-α and NF-κB mRNA began to increase at ld( (2.079 ±0. 104)times and 4d( ( 1.640 ± 0.064) times) after operation and reached the peak at 7d ((2.748 ±0.147)times, (2.010 ±0.096)times) ,then the expressions of TNF-a mRNA began to decrease,while the expressions of NF-kB mRNA maintained at a high level throughout the experiment. The result of immunofluorescence histochemical staining revealed that NF-kB and TNF-α protein expressions at 7 day increased significantly on the hippocampus,which was consisted with NF-κB and TNF-a mRNA levels. Conclusion The activation NF-κB and TNF-α in hippocampus may be involved in the procession of neuropathic pain.     

Expression of IL-1β and TNF-α in MCMV Myocarditis and Its Role

Autoi mmune myocarditis in Coxsackievirus B3(CB3)-infected mice is associated with infiltrationof the heart by inflammatory cells that secreteTNF-αand IL-1[1].In this study we examine theexpression of cytokines , TNF-αand IL-1βpro-duced locallyinthe heart inresponse to MCMVin-fectionin order to determine their potential role inthe development of acute myocarditis .1MATERIALS AND METHODS1 .1Reagents and AntibodiesMonoclonal goat anti-mouse IL-1β, TNF-αanti-body and SABC …