Tumor Necrosis Factor and Regulatory T Cells

CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg) cells play major roles in the maintenance of immune homeostasis. In this review, we comprehensively describe the relationship between tumor necrosis factor (TNF) and Treg cells, focusing on the effects of TNF on Treg cells and on TNF-producing Treg cells. Contradictory results have been reported for the effect of TNF on the suppressive activity of Treg cells. In patients with rheumatoid arthritis, TNF has been shown to reduce the suppressive activity of Treg cells. Meanwhile, however, TNF has also been reported to maintain the suppressive activity of Treg cells via a TNFR2-mediated mechanism. In addition, Treg cells have been found to acquire the ability to produce TNF under inflammatory conditions, such as acute viral hepatitis. These TNF-producing Treg cells exhibit T helper 17-like features and hold significance in various human diseases.

TNF-α/TNFR1 Signaling is Required for the Full Expression of Acute and Chronic Itch in Mice via Peripheral and Central Mechanisms / 神经科学通报·英文版

Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha (TNF-α) and its receptors TNF receptor subtype-1 (TNFR1) and TNFR2 in acute and chronic itch in mice. Compared to wild-type (WT) mice, TNFR1-knockout (TNFR1-KO) and TNFR1/R2 double-KO (DKO), but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine (CQ). Application of the TNF-synthesis inhibitor thalidomide and the TNF-α antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-α was not sufficient to evoke scratching, but potentiated itch induced by compound 48/80, but not CQ. In addition, compound 48/80 induced TNF-α mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia (DRG) and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etanercept and in TNFR1/R2 DKO mice. Dry skin induced TNF-α expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-α/TNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be beneficial for chronic itch treatment.

Sputum Inflammatory Mediators Are Increased in Aspergillus fumigatus Culture-Positive Asthmatics

Aspergillus fumigatus sensitization and culture in asthma are associated with disease severity and lung function impairment, but their relationship with airway inflammation is poorly understood. We investigated the profile of 24 sputum inflammatory mediators in A. fumigatus culture-positive or-negative moderate-to-severe asthmatics. Fifty-two subjects were recruited from a single center. A. fumigatus was cultured from 19 asthmatics. Asthma control, symptom score, lung function, and sputum cell count were not significantly different between the asthmatics with and without a positive A. fumigatus culture. All of the sputum mediators were numerically increased in subjects with a positive versus negative sputum A. fumigatus culture. Sputum TNF-R2 was significantly elevated (P=0.03) and the mediator that best distinguished A. fumigatus culture-positive from culture-negative subjects (receiver-operator characteristic area under the curve 0.66 [95% CI: 0.51 to 0.82, P=0.045]). A. fumigates-positive culture in moderate-to-severe asthma is associated with increased inflammatory sputum mediators.

Oxidative stress and skeletal muscle dysfunction are present in healthy smokers

Chronic exposure to cigarette smoke seems to be related to an increase of pro-inflammatory cytokines, oxidative stress and changes in muscular and physical performances of healthy smokers. However, these parameters have not yet been evaluated simultaneously in previous studies. The participants of this study were healthy males divided into two groups: smokers (n=20) and non-smokers (n=20). Inflammation was evaluated by measuring plasma levels of the cytokines IL-10, IL-6 e TNF-α, and of the soluble receptors sTNFR1 and sTNFR2. Oxidative stress was evaluated by determination of thiobarbituric acid reactive substances (TBARS) plasma levels, total antioxidant capacity of plasma and erythrocytes activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase. Muscular performance was evaluated by measuring the peak torque of knee flexors and extensors, and by determining the total work of the knee extensors. Physical performance was assessed by measuring the peak oxygen uptake (VO2 peak), the maximum heart rate (HRmax) and the walking distance in the shuttle walking test. Smokers showed an increase in the levels of the sTNFR1 and TBARS and a decrease in the total antioxidant capacity of plasma, in the catalase activity and in the total work (P<0.05). IL-6, IL-10, sTNFR2, SOD, peak torque, VO2 peak, HRmax and walking distance were similar between groups. Smokers presented increased oxidative stress and skeletal muscle dysfunction, demonstrating that the changes in molecular and muscular parameters occur simultaneously in healthy smokers.

Association of TNFRSF1B +676 gene polymorphism with the risk of rheumatoid arthritis in Han Chinese population in Hunan / 中南大学学报(医学版)

OBJECTIVE@#To study the association of TNFRSF1B +676 gene (rs1061622) polymorphism with the risk of rheumatoid arthritis (RA ) in Han Chinese population of Hunan.@*METHODS@#A total of 112 patients with RA from Han Chinese population in Hunan were recruited, along with 129 healthy controls. TNFRSF1B +676 (rs1061622) gene polymorphisms were examined by PCR-RFLP. Serum levels of soluble TNFR II were analyzed by ELISA.@*RESULTS@#RA patients displayed a similar TNFRSF1B +676 genotype to controls (GG/TG/TT: 5/62/45 vs 9/56/64, P=0.167), but signifi cant diff erence was found between female RA patients and female controls (GG/TG/TT: 3/49/24 vs 8/28/48, P0.05). RA patients showed a signifi cantly higher level of serum soluble tumor necrosis factor receptor II (sTNFR II) than controls [(7.83±2.61) ng/mL vs (4.32±1.67) ng/mL, P0.05). No association was found between TNFRSF1B+676 gene polymorphism and RA clinical characteristics.@*CONCLUSION@#In Han Chinese population of Hunan province, TNFRSF1B+676 gene polymorphisms are not associated with the genetic risk of RA .

Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.

Avaliacao comparativa entre os metodos objetivo e subjetivo em laminas coradas pela imuno-histoquimica / Comparative assessment between objective and subjective methods in slides stained by immunohistochemistry

Métodos objetivos de avaliação são frequentemente cobrados em estudos científicos. Exames histológicos com coloração imuno-histoquímica podem ser avaliados por meio de fotometria. OBJETIVO: Comparar este método objetivo com a avaliação subjetiva realizada por três observadores independentes, utilizando lâminas de colesteatoma adquirido da orelha média. MÉTODO: Foram selecionadas um total de 54 imagens de colesteatomas imuno-histoquimicamente coradas pelos anticorpos anti-TNF-R2 (32 lâminas) e anti-TGF-α; (22 lâminas). O anticorpo secundário utilizado nos dois grupos foi o Max Polimer Detection System (Kit Novo Link, Novocastra®, UK). As amostras foram processadas por um scanner digital de lâminas (modelo ScanScope - Aperio). As áreas selecionadas foram submetidas à análise por fotometria. RESULTADOS: A avaliação objetiva por fotometria foi comparada com a avaliação subjetiva por três observadores e submetidas à análise estatística. A análise estatística revelou reprodutibilidade moderada (K valores entre 0,41 e 0,60) para os dois grupos. CONCLUSÃO: O presente estudo demonstrou que as características irregulares das lâminas de colesteatoma da orelha média coradas pela imuno-histoquímica impossibilita a sua adequada avaliação objetiva, enquanto a avaliação subjetiva por observadores experientes se mostrou mais confiável. .

Objective methods of assessment are often required in scientific studies. Histological tests with immunohistochemical staining can be assessed by photometry. OBJECTIVE: To compare this objective method with the subjective evaluation performed by three independent examiners, using slides of acquired middle ear cholesteatomas. METHOD: We selected a total of 54 cholesteatoma images, immunohistochemically stained by anti-TNF-R2 (32 slides) and anti-TGF-α, (22 slides). The secondary antibody used in the two groups was the Max Polymer Detection System (Novo Link Kit, Novocastra®, UK). The samples were processed by a digital slide scanner (ScanScope - Aperio). The selected sites were analyzed by photometry. RESULTS: The objective assessment by photometry was compared with the subjective evaluation by three examiners and subjected to statistical analysis. The Statistical analysis revealed moderate reproducibility (K values between 0.41 and 0.60) for both groups. CONCLUSION: Our study showed that the irregular characteristics of middle ear cholesteatoma slides stained by immunohistochemistry prevents its proper objective evaluation, while the subjective assessment by experienced examiners was more reliable. .

Serum levels of soluble TNF-a receptors but not BDNF are associated with apathy symptoms in mild Alzheimer's disease and amnestic mild cognitive impairment / Níveis Séricos de receptores Solúveis Do TNF-a mas não de BDNF estão associados a sintomas de apatia na doença de Alzheimer leve e no comprometimento cognitivo leve amnéstico

Apathy is intimately associated with dementia. Unfortunately, its pathophysiology remains poorly understood. The motivational impairment that characterizes this disorder might share the same inflammatory mechanisms, as suggested by the sickness behavior theory. OBJECTIVE: The primary aim of this study was to investigate the association between apathy symptoms and serum levels of tumor necrosis factor alpha (TNF-a) and its soluble receptors. Brain-derived neurotrophic factor (BDNF) levels were also analyzed since these have been associated with depression, a condition which shares abulic features with apathy. METHODS: The sample consisted of 27 subjects with mild Alzheimer's disease or amnestic mild cognitive impairment, who were submitted to specific apathy evaluation using the Apathy Scale (AS) and provided blood samples for biomarker analysis. Participants were categorized into two groups according to median AS scores (17 points). RESULTS: Subjects with higher apathy symptoms (n=13) displayed higher levels of TNF-a soluble receptors (type 1: p=0.03; type 2: p=0.04). No other difference was found between groups. CONCLUSION: These findings point to the involvement of inflammatory mediators in the genesis of apathy symptoms, as suggested by the sickness behavior theory.

Apatia está intimamente associada à demência. Lamentavelmente, sua fisiopatologia ainda é pouco compreendida. O comprometimento motivacional que caracteriza este transtorno poderia compartilhar mecanismos inflamatórios como sugere a teoria do comportamento associado à doença. OBJETIVO: O principal objetivo deste estudo foi investigar a associação entre apatia e os níveis séricos do fator de necrose tumoral alfa (TNF-a) e de seus receptores solúveis. Os níveis de fator neurotrófico derivado do cérebro também foram analisados já que estes foram associados à depressão, que compartilha aspectos abúlicos com a apatia. MÉTODOS: A amostra consistiu de 27 indivíduos com doença de Alzheimer leve ou com comprometimento cognitivo leve amnéstico, que foram submetidos à avaliação de apatia pela Escala de Apatia (EA), e proveram amostra de sangue para análise de biomarcadores. De acordo com a mediana de escores na EA (17 pontos), a amostra foi divida em dois grupos. RESULTADOS: O grupo com mais sintomas de apatia apresentou maiores níveis séricos de receptores solúveis de TNF-a (tipo 1: p=0,03 ; tipo 2: p=0,04). Nenhuma outra diferença foi encontrada entre os grupos.CONCLUSÃO: Estes achados sugerem o envolvimento de mediadores inflamatórios na gênese de sintomas de apatia, assim como sugere a teoria do comportamento associado à doença.

Comparison of two types of cell cultures for preparation of sTNFRII-gAD fusion protein / 生物工程学报

In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.

Predictive Value of Conjointly Examined IL-1ra, TNF-R I, TNF-R II, and RANTES in Patients with Primary Glomerulonephritis

Interleukin-1 receptor antagonist (IL-1ra), tumor necrosis factor soluble receptors (sTNF-R) type I and II, and regulated upon activation, normal T-cell expressed and secreted (RANTES) play an important role in the modulation of primary glomerulonephritis (GN) course. The aim of the study was to assess whether pre-treatment measurements of IL-1ra, sTNF-R, and RANTES assessed conjointly may be useful as predicting factors in patients with GN. In 84 patients (45 males and 39 female) serum concentration (pg/mL) and urinary excretion (pg/mgCr) of cytokines were measured. After 12 months of therapy with steroids and cyclophosphamide the patients were divided into two subgroups: Responders (R) and Non-Responders (NR) according to the treatment results. The urinary IL-1ra, TNF-RI and RII were significantly higher in R than NR (1,732 vs 646 with P < 0.001, 13.1 vs 6.3 with P = 0.005, and 33.6 vs 14.4 with P = 0.012). The urinary RANTES excretion was increased in NR (79.6 vs 28.5; P < 0.001). The multivariable analysis showed that if conjointly assessed, only urinary IL-1ra, TNF-R I and R II, RANTES with 85% probability pointed the feature remission (R). In conclusion, the urinary excretion of IL-1ra, TNF-R I and R II, and RANTES examined conjointly are effective in predicting favorable response to immunosuppressive treatment in patients with GN.

Increased serum levels of soluble tumor necrosis factor receptor-2 (sTNFR2) in patients with active toxoplasmic retinochoroiditis

This study aimed to investigate the serum levels of the cytokine TNF-α and its soluble receptors (sTNFR1 and sTNFR2) in patients with toxoplasmosis retinochoroidits (TR) and controls. 37 patients with TR and 30 subjects with positive serology for toxoplasmosis but without history and signs of uveitis were included in this study. Serum concentrations of TNF-α, sTNFR1, and sTNFR2 were determined by ELISA. Serum concentrations of TNF-α and sTNFR1 were similar in controls (mean ± SD median values; 56.57 ± 141.96 and 504.37 ± 163.87, respectively) and TR patients (mean ± SD values, 121.62 ± 217.56 and 511.15 ± 189.30, respectively). Serum concentrations of sTNFR2 were higher in the uveitis group when compared to the control group (respectively, mean ± SD values, 1734.84 ± 379.32 and 1442.75 ± 309.47; p=0.002). There was no association between the serum levels of the molecules and the time of first symptoms, severity of vitreous haze, size or localization of active lesions, levels of visual acuity, and presence of vasculitis. These results suggest that TR is associated with changes in the circulating levels of inflammatory biomarkers, but they are not correlated with local/ocular signs.

TNF-R2 expression in acquired middle ear cholesteatoma / Expressão do TNF-R2 no colesteatoma adquirido da orelha média

Acquired middle ear cholesteatoma is a disease which promotes bone erosion resulting in potentially serious complications. The tumor necrosis factor alpha (TNF-α) is present in cholesteatoma and it is related to bone erosion, as shown by different authors. To understand the aggressiveness characteristics of cholesteatoma is necessary, however, to better address the presence and distribution of their receptors. OBJECTIVE: To evaluate the expression of type 2 TNF-α receptor (TNF-R2) in fragments of cholesteatoma and correlate it to the degree of inflammation present. MATERIAL AND METHODS: observational cross-sectional study, which analyzed 33 fragments of cholesteatomas through histological analysis and immunohistochemistry (using as primary antibody to TNF-R2 LabVision® brand). The evaluation was performed by means of a qualitative and semi-quantitative agreement with the observed intensity. For statistical analysis we used the Fisher exact test and Spearman´s correlation coefficient (considered statistically significant when p < 0. 05). RESULTS: The expression of TNF-R2 was present in all fragments, however a statistical analysis showed no correlation or association between inflammation and the expression of TNF-R2. CONCLUSIONS: TNF-R2 is present in cholesteatoma of the middle ear, however, its expression is not directly related to the degree of inflammation observed in patients with this disease.

O colesteatoma adquirido da orelha média promove erosão óssea, ocasionando complicações potencialmente graves. O fator de necrose tumoral alfa (TNF-α) está presente no colesteatoma adquirido da orelha média e relaciona-se com a erosão óssea, como demonstraram diferentes autores. Para que se compreenda as características de agressividade do colesteatoma, é necessário que se estude a presença e a distribuição seus receptores. OBJETIVO: Avaliar a expressão do receptor tipo dois do TNF-α (TNF-R2) em fragmentos de colesteatoma e relacioná-lo com o grau de inflamação. MATERIAL E MÉTODOS: Estudo observacional do tipo transversal. Foram analisados 33 fragmentos de colesteatomas, submetidos à análise histológica e imunoistoquímica (utilizando o TNF-R2 da marca Labvision®). A avaliação foi realizada de forma qualitativa e semiquantitativa, de acordo com a intensidade observada. Para a análise estatística, foram utilizados o teste exato de Fischer e o coeficiente de correlação de Spearman (estatisticamente significativo quando p < 0,05). RESULTADOS: A expressão do TNF-R2 estava presente em todos os fragmentos, entretanto a estatística não evidenciou correlação, nem associação entre o processo inflamatório e a expressão do TNF-R2. CONCLUSÕES: O TNF-R2 está presente no colesteatoma adquirido da orelha média. Entretanto, a sua expressão não está relacionada ao grau de inflamação.

Moxibustion treatment restoring the intestinal epithelium barrier in rats with Crohn's disease by down-regulating tumor necrosis factor alpha, tumor necrosis factor receptor 1, and tumor necrosis factor receptor 2 / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate whether moxibustion regulates tumor necrosis factor alpha (TNF-α), tumor necrosis factor receptor 1 (TNFR1), and TNFR2 in the intestinal mucosa and to explore whether moxibustion could be used by means of this mechanism, to repair the intestinal epithelium barrier disruption in Crohn's disease (CD).</p><p><b>METHODS</b>The CD rat models were established by trinitrobenzene sulfonic acid (TNBs), randomly divided into a model control (MC) group, an herb-partition moxibustion (HPM) group, a mild-warm moxibustion (MWM) group, and a salicylazosulfapyridine (SASP) group, and all were compared with a normal control (NC) group. The HPM and MWM groups were treated by moxibustion at Tianshu (ST25) and Qihai (RN6) for 14 days, and the SASP group obtained the SASP solution orally for the same period of time. The intestinal epithelium morphology and TNF-α, TNFR1, and TNFR2 contents were observed by the transmission electron microscopy and enzyme linked immunosorbent assay.</p><p><b>RESULTS</b>The severity of morphological changes in CD intestinal epithelium was obviously improved, and the levels of TNF-α, TNFR1, and TNFR2 in the intestinal mucosa all significantly decreased in the HPM and MWM groups. However, there were no significant differences between the HPM and MWM groups.</p><p><b>CONCLUSION</b>The moxibustion therapies (HPM and MWM) could reduce intestinal inflammation and restore intestinal epithelium barrier disruption in CD, which might be due to down-regulating TNF-α, TNFR1, and TNFR2 in intestinal mucosa and improving intestinal epithelium morphology.</p>

Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system / 生物工程学报

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

Role of Clusterin and Tumor Necrosis Factor Receptors on the Apoptosis of Prostate Cancer Cells / 대한남성과학회지

PURPOSE: In prostate cancer, the anti-apoptotic mechanism of sulfated glycoprotein-2 (clusterin) against tumor necrosis factor-alpha (TNF-alpha) receptors and the action of type 2 TNF-alpha receptor (TNFR2) were investigated. MATERIALS AND METHODS: TNF-alpha, agonistic-TNF type 1 receptor (TNFR1) antibody, agonistic-TNF-R2 antibody and their combination were treated in PC3 cell line with or without anti-clusterin. Cytotoxicity was assessed by trypan blue dye exclusion assay. By using flowcytometric analysis, the exact amount of apoptosis and their changes were assessed. RESULTS: Apoptosis was significantly increased in both agonistic-TNFR1 antibody and TNF-alpha treated cases after blocking the activity of clusterin. The more the anti-clusterin antibody added, the more the apoptosis occurred. The increase of total apoptosis was greater in TNF-alpha treated cells than in agonistic-TNFR1 antibody treated ones. However, there was no increase of apoptosis in agonistic-TNFR2 antibody and TNF-alpha with agonistic-TNFR2 antibody treated cases, respectively. CONCLUSIONS: Clusterin prevents TNF-alpha induced apoptosis by affecting TNFR1. The difference in degree of apoptosis between agonistic-TNFR1 antibody treated cells and TNF-alpha treated ones suggests the possibility of the action of TNFR2. It may be associated with affinity of TNF-alpha to the tumor cell surface.

Effect of intra-articular ozone injection on serum and synovial TNF-α, TNFR I, and TNFR II contents in rats with rheumatoid arthritis / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effects of intra-articular ozone injection at different concentrations on the contents of tumor necrosis factor-α (TNF-α), TNF receptor I (TNFR I), and TNFR II in the serum and synovium of rats with rheumatoid arthritis (RA) and explore the therapeutic mechanism of ozone in RA treatment.</p><p><b>METHODS</b>Forty-eight Wistar rats were randomized into 8 groups, including 5 ozone groups receiving intra-articular injection of 10, 20, 30, 40 or 50 µg/ml ozone, a blank control group, an oxygen group and a RA model group. All the rats, except for those in the blank control group, were subjected to hypodermic injection of bovine collagen II and complete Freunds adjuvant to induce RA. Ozone treatment was administered once weekly for 3 weeks starting at 21 days after the modeling. The swelling and thickness of the hind paws were observed, and the serum and synovial contents of TNF-α, TNFR I, and TNFR II were detected.</p><p><b>RESULTS</b>At the end of treatment, the paw thickness was reduced significantly in rats with 40 µg/ml ozone injection compared with that in the model RA group (P<0.01). The serum contents of TNF-α, TNFR I and TNFR II showed no significant difference between the RA model group, oxygen group and the ozone groups, but their synovial contents showed significant reductions in rats with 40 and 50 µg/ml ozone injection (P<0.01); the synovial TNFR I was significantly higher in 40 µg/ml ozone group than in the model group (P<0.05).</p><p><b>CONCLUSION</b>Intra-articular injection of 40 µg/ml ozone can attenuate synovitis in rats with RA, the mechanism of which may involve the inhibition of TNF-α and TNFR II activity and enhancement of TNFR I activity in the synovium.</p>

Eukaryotic expression and bioactivity determination of the fusion protein sTNFRII-gAD consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin / 生物工程学报

In order to get soluble TNF receptor (sTNFR) II with good neutralizing activity against TNFalpha, we constructed the fusion gene sTNFRII-gAD, which encoded human sTNFR II and the globular domain of adiponectin (gAD), and then expressed it in mammalian cells and analyzed its anti-TNFalpha activity. First, sTNFRII cDNA was obtained by RT-PCR from the total RNA of human peripheral blood lymphocytes, and fused in frame with gAD gene. Then, the fusion gene sTNFRII-gAD was cloned into the expression vector pAAV2neo to result in the plasmid pAAV2neo-sTNFRII-gAD. By immunofluorescent staining with monoclonal antibody either against TNFRII or against adiponectin, we demonstrated that the pAAV2neo-s7NFRII-gAD-transiently-transfected BHK-21S cells were positive. To obtain G418-resistant BHK-21S/pAAV2neo-sTNFRII-gAD cells, we cultured the transfected BHK-21S cells above in 10% FBS containing DMEM media with 800 microg/mL G418 for 15 days, and changed the serum-containing culture media to a serum-free chemically defined media so as to change the cells culturing style from adhesion to suspension. 24 hours later, we harvested the supernatant of the culture for sTNFRII-gAD fusion protein characterization and anti-TNFalpha activity analysis. With monoclonal antibody either against TNFRII or against adiponectin, the Western blotting analysis showed that the sTNFRII-gAD fusion protein was expressed and existed as monomer, trimer and multimer forms in the supernatant. The bioactivity assay demonstrated that the sTNFRII-gAD fusion protein had the ability to neutralize TNFalpha so as to inhibit the cytotoxicity of TNFalpha on L929 cells. Put together, this study has laid the groundwork for large-scale preparation of sTNFRII-gAD fusion protein.

Soluble inflammatory markers as predictors of hepatocellular damage and therapeutic response in chronic hepatitis C

Hepatitis C is an important burden worldwide being an important cause of cirrhosis and liver cancer in different parts of the world. Host immune response, especially T helper type 1 (Th1) cell-mediated, seems to play an important role in disease progression but is also crucial for viral elimination following specific therapy. Immune activation can be evaluated using peripheral levels of different cytokines, such as different chemokines (e.g. CCL5, CXCL10) and tumor necrosis factor alpha (TNF-á), and their soluble receptors (e.g. soluble TNF-á receptors 1 (sTNF-R1) and 2 (sTNF-R2). This review article focuses on the potential use of peripheral inflammatory markers as predictors of liver histological changes and therapeutic response among patients with chronic hepatitis C.

Serum levels of TNF-alpha, TNF-alpha RI, TNF-alpha RII and IL-12 in treated Rheumatoid Arthritis patients

Rheumatoid arthritis [RA] is a chronic multisystem autoimmune disease common in all races and ethnics. Cytokines and cytokines receptors play an important role in RA pathogenesis and clinical presentation. To investigate the serum levels of TNF-alpha, TNF-alpha RI, TNF-alpha RII and IL-12 in RA patients and healthy control group. In this study 43 patients fulfilling the revised criteria of American College of Rheumatology [ACR] for RA and 13 healthy cases as a control group were selected for TNF-alpha, TNF-alpha RI, TNF-alpha RII and IL-12 serum level analysis. The patients' age was 42.2 +/- 22 and the age of healthy group was 40.1 +/- 19.2 years [p=0.1]. The patients had an active disease with at least six swollen and ten tender joints. Minimum ESR was 28 mm at first hours of the morning. Early morning stiffness in patients lasted longer than 45 minutes. Our study showed that IL-12 serum level of the patients [91.69 +/- 43.07 [rho]g/ml] and control [61.79 +/- 40.08 [rho]g/ml] group was significantly different [p<0.001]. The serum level of TNF-alpha RI was 2.36 +/- 0.77 ng/ml in the patient and 1.73 +/- 0.37 ng/ml in the control group [p<0.01]. TNF-alpha RII serum concentration in patients was 8.89 +/- 2.3 ng/ml, while that of control group was 7.06 +/- 1.30 ng/ml [p=0.03]. The serum level of TNF-alpha in patients was 32.90 +/- 19.27 [rho]g/ml and that of the control group was 24.27 +/- 8.28 [rho]g/ml [p=0.08] with no significant difference between the two. It is concluded that IL-12, TNF-alpha RI and TNF-alpha RII serum concentrations are more important and better predictive factors than TNF-alpha in RA course and in the active forms of the disease

Induction of apoptosis by tumor necrosis factor receptors 2 transgene in human laryngeal squamous cacinoma in nude mice animal model / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the effect of tumor necrosis factor receptors II (TNFR II) in vivo transgene with topical injection of TNF alpha in inducing apoptosis and cell killing of laryngeal squamous carcinoma in nude mice animal model.@*METHOD@#Laryngeal carcinoma implantation animal model was established on nude mice. In vivo gene transfection of TNFR II was carried out using liposome as a carrier. TNF alpha was topically injected into tumor. Goss measurement of tumor, flow cytometry, immunohistochemistry, tunel and transmission electron microscopy were conducted to observe the expression of TNFR II protein and the apoptosis of tumor cells, and the effects of tumor killing and growth inhibition was objectively evaluated.@*RESULT@#Nude mice models bearing laryngeal carcinoma was established in 94.5% animals. After in vivo gene transfection, the expression of TNFR II protein reach the highest level at 48 hours, and remain in a substantially high level within 72 hours. Immunohistochemistry showed the expression of TNFR II is mainly on the cell membrane of the transfected tumor cells. Topical injection of 2000 U TNF alpha was most efficient in inducing tumor cell apoptosis, cell inhibition and cell killing. The tumor volume, weight, and tumor/body ratio in TNFR II transfected group were (1161.333 +/- 166.555) mm3, (1.100 +/- 0.832) g and 0.044 +/- 0.332, respectively, with a corresponding high level of tumor cell apoptosis rate (38.226 +/- 13.671) %, all of which were significantly higher than that in non-transfected group. Tunel and ultrastructural observations demonstrated apoptosis-related changes in the transfected tumor cells.@*CONCLUSION@#Up-regulation of TNFR II expression by in vivo gene transfection on tumor cells can remarkably enhance the tumor cell killing effect of topical injection of TNF-alpha. In vivo transgene of TNFR II in combination with topical injection of TNF alpha may become a effective gene therapy method in treating laryngeal cancer.