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The venomous species Deinagkistrodon acutus has been used as anti-inflammatory medicine in China for a long time. It has been proven to have anti-inflammatory activity, but its specific anti-inflammatory components have not yet been fully elucidated. Tumor necrosis factor receptor-1 (TNFR1), which participates in important intracellular signaling pathways, mediates apoptosis, and functions as a regulator of inflammation, is often used as the target to develop anti-inflammatory drugs. The small peptides of snake venom have the advantages of weak immunogenicity and strong activity. To obtain the specific TNFR1 binding peptides, we constructed a T7 phage library of D. acutus venom glands, and then performed biopanning against TNFR1 on the constructed library. After biopanning three times, several sequences with potential binding capacity were obtained and one 41-amino acid peptide was selected through a series of biological analyses including sequence length, solubility, and simulated affinity, named DAvp-1. After synthesis, the binding capacity of DAvp-1 and TNFR1 was verified using surface plasmon resonance technology (SPR). Conclusively, by applying phage display technology, this work depicts the successful screening of a promising peptide DAvp-1 from D. acutus venom that binds to TNFR1. Additionally, our study emphasizes the usefulness of phage display technology for studies on screening natural product components.

A few studies have examined biomarkers in patients with myocardial infarction (MI) and peripheral artery disease (PAD), i.e. multisite artery disease (MSAD). The aim of the study was firstly, to associate biomarkers with the occurrence of PAD/MSAD and secondly, if those can, in addition to clinical characteristics, identify MI patients with MSAD.In two prospectively observational studies including unselected patients with recent MI, PAD was defined as an abnormal ankle-brachial index (ABI) score (<0.9 or >1.4). The proximity extension assay (PEA) technique was used, simultaneously analyzing 92 biomarkers with association to cardiovascular disease. Biomarkers were tested for univariate associations with PAD. Random forest was used to identify biomarkers with a higher association to PAD. The additional discriminatory accuracy of adding biomarkers to clinical characteristics was analyzed by the c-statistics. Nine biomarkers were identified as significantly associated with MSAD/PAD in the primary patient cohort, analyzed early after the MI. In the prediction analysis, six biomarkers were identified associated with PAD. Three of these; Tumor necrosis factor receptor (TNFR-1), Tumor necrosis factor receptor 2 (TNFR-2) and Growth Differentiation Factor 15 (GDF-15) improved c-statistics when added to clinical characteristics from 0.683 (95% CI 0.610-0.756) to 0.715 (95% CI 0.645-0.784) in the primary patient cohort with a similar result, 0.729 (95% CI 0.687-0.770) to 0.752 (95% CI 0.771-0.792) in the secondary patient cohort. Biomarkers associated with inflammatory pathways are associated with MSAD in MI patients. Three biomarkers of 92; TNFR-1, TNFR-2 and GDF-15, in this exploratory added information in the prediction of MSAD and emphasis the importance of further studies.

Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11-0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis., Competing Interests: The authors have declared that no competing interests exist.

Background: Enrichment strategies improve therapeutic targeting and trial efficiency, but enrichment factors for sepsis trials are lacking. We determined whether concentrations of soluble tumor necrosis factor receptor-1 (sTNFR1), interleukin-8 (IL8), and angiopoietin-2 (Ang2) could identify sepsis patients at higher mortality risk and serve as prognostic enrichment factors., Methods: In a multicenter prospective cohort study of 400 critically ill septic patients, we derived and validated thresholds for each marker and expressed prognostic enrichment using risk differences (RD) of 30-day mortality as predictive values. We then used decision curve analysis to simulate the prognostic enrichment of each marker and compare different prognostic enrichment strategies., Measurements and Main Results: An admission sTNFR1 concentration > 8861 pg/ml identified patients with increased mortality in both the derivation (RD 21.6%) and validation (RD 17.8%) populations. Among immunocompetent patients, an IL8 concentration > 94 pg/ml identified patients with increased mortality in both the derivation (RD 17.7%) and validation (RD 27.0%) populations. An Ang2 level > 9761 pg/ml identified patients at 21.3% and 12.3% increased risk of mortality in the derivation and validation populations, respectively. Using sTNFR1 or IL8 to select high-risk patients improved clinical trial power and efficiency compared to selecting patients with septic shock. Ang2 did not outperform septic shock as an enrichment factor., Conclusions: Thresholds for sTNFR1 and IL8 consistently identified sepsis patients with higher mortality risk and may have utility for prognostic enrichment in sepsis trials.

Unlabelled: Rheumatoid arthritis (RA) is a chronic inflammatory disease with autoimmune pathogenesis. It affects mainly small joints (of the hands and feet) and has many systemic manifestations. Studying biomarkers in rheumatology intensely appeared from the need to understand the mechanisms underlying some rheumatic diseases. Discovering new biomarkers with key roles in various stages of evolution, remains a subject of interest for RA. Currently, according to the EULAR 2010 criteria, the rheumatoid factor (RF) and the anti-cyclic citrullinated peptide (anti-CCP) are used for RA diagnosis. Since 2010, new biomarkers were discovered and proved useful in identifying RA in early stages. For a more rigorous management of these cases, one of the key steps in the evolution of patients with RA is to recognize and distinguish the more aggressive forms of the disease through prognostic biomarkers. "Treat to target" recommends the use of 3 composite scores to monitor the evolution of the disease: disease activity score (DAS 28), simple disease activity index (SDAI) and clinical disease activity index (CDAI), but, a new test was developed which better monitors the disease activity. The introduction of biological therapies has revolutionized the treatment of RA. Despite these advances, 20-40% of the patients are declared nonresponders to at least one of the therapies. The patient exposure to the potential side effects and high costs requires the discovery of a biomarker that could identify those who can benefit from the pretreatment of a certain therapy., Abbreviations: RA = rheumatoid arthritis, RF = rheumatoid factor, DAS 28 = disease activity score, SDAI = simple disease activity index, CDAI = clinical disease activity index, ACR = American College of Rheumatology, EULAR = European League against Rheumatism, anti-CCP = antibodies against cyclic citrullinated proteins, anti-MCV = mutated citrullinated vimentin antibodies, anti-CarP = antibodies against carbamylated proteins, MBDA = multi biomarker disease activity test, COMP = cartilage oligomeric matrix protein, ADAs = antidrug antibodies, CDA = clinical disease activity index, SDAI = simplified disease activity index, ESR = erythrocyte sedimentation rate, CRP = C reactive protein, SAA = serum amyloid A, VCAM-1 = vascular cell adhesion molecule-1, IL-6 = interleukin-6, TNF-R1 = tumor necrosis factor receptor 1, EGF = epidermal growth factor, VEGF-A = vascular endothelial growth factor A.

Background: We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae., Methods: In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis., Results: TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups., Conclusions: Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Introduction: This prospective observational study aimed to assess the relevance of serial postoperative serum TNF-α, TNFR1 and TNFR2 measurements for predicting graft function and acute rejection episodes (AR) after transplantation., Materials and Methods: We studied 50 kidney transplant recipients (31 female, 19 male; mean age: 38.36 ± 12.88). Blood samples were collected immediately before and after surgery at day 7, month 1 and month 3. Serum TNF-α, TNFR1 and TNFR2 levels were measured by ELISA using a commercial kit (Invitrogen ELISA). Serum cystatin-C levels were measured by particle-enhanced immunonephelometric method. Glomerular filtration rate (GFR) was estimated by Chronic Kidney Disease-Epidemiology (CKD-EPI) equation. Patients were assigned to their transplant outcomes in terms of acute rejection [AR(+) and AR(-)] and slow (SGF) or immediate graft function (IGF)., Results: Among 50 recipients, six had AR(+) and 44 had AR(-), depending on graft function: 17 had SGF and 33 had IGF. Serum creatinine, cystatin-C, TNF-α, TNFR1 and TNFR2 levels demonstrated consistent significantly decreases after transplantation while GFR values had consistent increases (p = 0.001). Pretransplant levels were not statistically different between AR(+) and AR(-) groups (TNF-α: 30.79 ± 5.96 vs. 27.95 ± 2.43 pg/mL, TNFR1: 55.96 ± 21.6 vs. 40.52 ± 7.41 ng/mL, TNFR2: 58.31 ± 8.06 vs. 50.9 ± 3.34 ng/mL, respectively) (p > 0.05). Serum TNF-α, TNFR1 and TNFR2 levels on day 7 and month 1 were also significantly higher in AR(+) group compared to AR(-) (p = 0.012, p = 0.049 for TNF-α, p = 0.001, p = 0.002 for TNFR1, p = 0.001, p = 0.002 for TNFR2)., Conclusions: Our preliminary findings suggest that serum TNF-α, TNFR1 and TNFR2 levels might be considered useful markers of evaluating graft function after renal transplantation.

Inflammation is associated with cancer-prone microenvironment, leading to cancer. IL-32 is expressed in chronic inflammation-linked human cancers. To investigate IL-32α in inflammation-linked colorectal carcinogenesis, we generated a strain of mice, expressing IL-32 (IL-32α-Tg). In IL-32α-Tg mice, azoxymethane (AOM)-induced colon cancer incidence was decreased, whereas expression of TNFR1 and TNFR1-mediated apoptosis was increased. Also, IL-32α increased ROS production to induce prolonged JNK activation. In colon cancer patients, IL-32α and TNFR1 were increased. These findings indicate that IL-32α suppressed colon cancer development by promoting the death signaling of TNFR1.

Objective: Cardiovascular events are the most common cause of mortality in Cushing syndrome (CS). This study aimed to evaluate the impact of novel factors on atherosclerosis in endogenous CS., Methods: A total of 22 female patients with CS and 33 normal female controls underwent evaluation of fibrinogen, high-sensitivity C-reactive protein (hsCRP), inflammatory cytokines (interleukin [IL]-6, IL-1β, soluble tumor necrosis factor receptor [sTNFR]-1, and sTNFR2), glutathione peroxidase (GPx; measure of oxidative stress), carotid intima media thickness (CIMT; a measure of atherosclerosis), and percent change in flow-mediated vasodilation (%FMV) of the brachial artery, a measure of endothelial dysfunction. Stepwise multiple linear regressions were done after adjusting for variables in models 1 through 3 to evaluate their role in predicting CIMT and %FMV. Model 1 consisted of age and body mass index (BMI). Model 2 consisted of model 1 plus blood pressure (BP), fasting blood glucose (FBG), and 2-hour postglucose blood glucose (2hPGBG). Model 3 consisted of model 2 plus triglycerides and low- and high-density lipoprotein., Results: Females with CS had significantly higher BMI, BP, FBG, 2hPGBG, total cholesterol, triglycerides, fibrinogen, IL-6, IL-1β, sTNFR1, and GPx. CIMT and %FMV were significantly higher and lower, respectively, in CS patients. Regression analyses revealed sTNFR1 to be a consistent predictor of CIMT after adjusting for model 1 (β = 0.656; P = .004), model 2 (β = 0.571; P = .047), and model 3 (β = 0.683; P =.026). GPx was a predictor of CIMT after adjusting for model 1 (β = 0.565; P = .033) and model 3 (β = 0.756; P= .038)., Conclusion: This study highlights increased CIMT and endothelial dysfunction in CS, associated with an altered inflammatory milieu. sTNFR1 and GPx may predict CIMT in females with CS.

We have previously shown that in tumor specimens from patients with diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS), the tumor necrosis factor-α (TNF-α)-positive type correlates with a poorer prognosis compared with the TNF-α-negative type. In the present study, we further evaluated 60 lymphoma tissue specimens from patients with DLBCL, NOS by immunohistochemical staining with antibodies against TNF-α receptor 1 (TNFR1) and TNF-α receptor 2 (TNFR2). Our results demonstrated that 31 cases (52%) were positive and 29 (48%) were negative for TNFR1 and that the TNFR1-positive cases were significantly correlated with a poorer overall survival (OS; P=0.0006, log rank test) than the TNFR1-negative cases. The TNFR2-positive cases tended to have a poorer OS than the TNFR2-negative cases, although the difference was not significant. TNFR1 expression in tumor cells was a significant prognostic factor for OS and was independent of the International Prognostic Index (IPI). Among 31 TNF-α-positive DLBCL, NOS cases, 27 (87%) were positive and 4 (13%) were negative for TNFR1. Both TNF-α-positive and TNFR1-positive cases were significantly correlated with a poorer OS compared with the TNF-α-positive but TNFR1-negative cases. Twenty-seven cases (45%) with the TNF-α-positive and TNFR1-positive subtype of DLBCL, NOS had a poorer prognosis for OS and progression-free survival compared with the 33 cases (55%) with the remaining subtypes, and the TNF-α-positive and TNFR1-positive subtype of DLBCL, NOS was also shown to be independent of the IPI. In addition to the IPI, the prognosis of patients can be more accurately identified by evaluating both TNF-α and TNFR1 expression.

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

Background: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)-α on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus., Methods: Cultured PDL cells were stimulated with TNF-α and analyzed with an MMP antibody array. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP-3, -10, and -13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP-3, -10, and -13 was observed after blocking the TNFR with an antagonist. Results from real-time PCR, ELISA, and western blot were analyzed by paired t test., Results: The antibody array showed that the protein most strongly upregulated by TNF-α stimulation was MMP-3, followed by MMP-13 and MMP-10. The TNF-α receptor blocker specifically inhibited expression of MMP-3 and -13. In addition, TNF-α increased levels of MMP mRNAs in MMP-3, -13, and -10 (in decreasing order). However, ELISAs showed that MMP-13 was the most upregulated protein, followed by MMP-10 and MMP-3. Western blotting indicated that TNF-α increased MMP-3 and -13 levels but had no significant effect on the level of MMP-10, and zymography showed that TNF-α increased the activities of all forms of MMP-3 and -13, but MMP-10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1., Conclusions: TNF-α (10 ng/mL) upregulates levels of MMP-3, -10, and -13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.

Background/aim: To investigate the effect of ozone and/or taurine treatment comparatively on testicular damage due to ischemia/ reperfusion (I/R) injury in an experimental torsion model in rats., Materials and Methods: Adult Wistar rats with and without torsion/detorsion were used. In order to monitor the effect of ozone and/or taurine treatment on testicular damage due to I/R injury, following histopathological investigation apoptotic indexes were scored by TUNEL method. Moreover, tumor necrosis factor receptor 1 (TNFR1), caspase 3, caspase 8, endothelial nitric oxide synthase (eNOS), tumor necrosis factor alpha (TNFα), and cytochrome C immunostainings were performed and the levels of malondialdehyde, glutathione peroxidase, superoxide dismutase, catalase, total sulfhydryl, and nitric oxide were determined in the testicular tissue., Results: Intraperitoneal ozone and/or taurine treatment prevented both histopathological damage and increase in the apoptotic index. Torsion did not exert an effect on the levels of TNFα and cytochrome C. Ozone and/or taurine treatment prevented increases in TNFR1, caspase 3, and caspase 8. The level of oxidative stress markers was unchanged. The increases in NO level and eNOS expression were prevented by ozone and/or taurine treatment in I/R groups., Conclusion: Using ozone therapy and/or taurine before reperfusion may be a solution for germ cell degeneration resulting from testicular torsion and related infertility.

Background: Effective chemotherapeutics for primary systemic tumors have limited access to brain metastases because of the blood-brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilizing the BBB at sites of cerebral metastases., Methods: BALB/c mice were injected intracardially to induce brain metastases. After metastasis induction, either tumor necrosis factor (TNF) or lymphotoxin (LT) was administered intravenously, and 2 to 24 hours later gadolinium- diethylenetriaminepentaacetic acid, horseradish peroxidase, or radiolabeled trastuzumab ((111)In-BnDTPA-Tz) was injected intravenously. BBB permeability was assessed in vivo using gadolinium-enhanced T1-weighted magnetic resonance imaging and confirmed histochemically. Brain uptake of (111)In-BnDTPA-Tz was determined using in vivo single photon emission computed tomography/computed tomography. Endothelial expression of TNF receptors was determined immunohistochemically in both mouse and human brain tissue containing metastases. Group differences were analyzed with one-way analysis of variance followed by post hoc tests, Wilcoxon signed rank test, and Kruskal-Wallis with Dunn's multiple comparison test. All statistical tests were two-sided., Results: Localized expression of TNF receptor 1 (TNFR1) was evident on the vascular endothelium associated with brain metastases. Administration of TNF or LT permeabilized the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect at 6 hours. Metastasis-specific uptake ratio of (111)In-BnDTPA-Tz was also demonstrated after systemic TNF administration vs control (0.147±0.066 vs 0.001±0.001). Human brain metastases displayed a similar TNF receptor profile compared with the mouse model, with predominantly vascular TNFR1 expression., Conclusions: These findings describe a new approach to selectively permeabilize the BBB at sites of brain metastases to aid in detection of micrometastases and facilitate tumor-specific access of chemotherapeutic agents. We hypothesize that this permeabilization works primarily though TNFR1 activation and has the potential for clinical translation.

Background and Aims: The aim of this study was to assess the influence of anti-TNF agents on the expression of apoptosis-related proteins in Crohn's disease (CD) patients., Methods: The clinical, biochemical and endoscopic activity of CD was assessed with the use of tissue sampling before the initiation of therapy and after induction doses of infliximab and adalimumab. Additionally, the immunohistochemical expression of active caspase 3, TNFR1, Fas, Bcl-2, Bax, CD4 and CD8 proteins was estimated. Patients achieving deep remission were considered as responders., Results: Of the 35 patients qualified for the study, 60% achieved deep remission. In those patients, a significant decrease in the number of CD4 and CD8 positive cells was noted. Also observed was a significant increase in the expression of active caspase 3 in lamina propria mononuclear cells, which correlated with an increase of the pro-apoptotic Bax/Bcl-2 ratio. No change in Fas and TNFR1 expression was observed in those cells. Moreover, there was a significant decrease in active caspase 3 expression in enterocytes, observed independently of the Bax/Bcl-2 ratio. This correlated with a change in TNFR1 expression. No significant changes in the expression of the investigated proteins were noted in non-responders group., Conclusions: The efficacy of anti-TNF antibodies is, at least partly, dependent on apoptosis modulation. In lamina propria mononuclear cells, the increase of apoptosis is probably the result of the induction of the intrinsic pathway mediated by Bcl-2 family proteins. In enterocytes - the decrease of apoptosis is mediated by the extrinsic pathway, probably via TNFR1., (Copyright © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Introduction: The purpose of this study was to investigate whether in vitro stimulation of pulp cells leads to increased secretion of matrix metalloproteinases (MMPs) and, if so, to identify which MMPs are affected., Methods: Cells cultured from dental pulp were stimulated with tumor necrosis factor-α (TNF-α) (10 ng/mL) for 24 hours, and lysates were analyzed with an antibody array (Bio-Rad Laboratories, Hercules, CA). The mRNA and protein levels of MMP-3, -10, and -13 were measured by real-time polymerase chain reaction (real-time PCR), enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and zymography. In addition, tumor necrosis factor receptors in the pulp cells were assayed by flow cytometry. The ELISA and real-time PCR results were analyzed by paired t tests., Results: The expression of MMP-3, -10, and -13 was up-regulated in the pulp cells after 24 hours of stimulation with TNF-α (10 ng/mL) as seen in the antibody array, real-time PCR, and ELISA results, but MMP-10 was not detected by Western blotting or casein zymography. Flow cytometry analysis showed that the majority of the pulp cells expressed tumor necrosis factor receptor 1., Conclusions: In regions of inflammation, TNF-α may initiate the degradation of dental connective tissue by activating MMP-3 and MMP-13. These proteins may play an important pathologic role in the inflammation of dental pulp., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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.

Background: The aim of this study is to evaluate the gingival crevicular fluid (GCF), saliva, and serum concentrations of tumor necrosis factor-α (TNF-α), TNF-α receptor-1 (TNF-αR1), TNF-αR2, and interleukin-6 (IL-6) in non-obese females with polycystic ovary syndrome (PCOS) and either clinically healthy periodontium or gingivitis., Methods: Thirty-one females with PCOS and healthy periodontium, 30 females with PCOS and gingivitis, and 12 systemically and periodontally healthy females were included in the study. GCF, saliva, and serum samples were collected, and clinical periodontal measurements, body mass index, and Ferriman-Gallwey score (FGS) were recorded. Sex hormones, cortisol, and insulin levels were measured. TNF-α, TNF-αR1, TNF-αR2, and IL-6 were determined by enzyme-linked immunosorbent assay. Kruskal-Wallis followed by Bonferroni-corrected post hoc Mann-Whitney U tests were used to analyze the data., Results: The PCOS + gingivitis group revealed significantly higher GCF, saliva, and serum IL-6 concentrations than the PCOS + healthy group (P <0.0001). The two PCOS groups exhibited significantly higher saliva TNF-α concentrations than the control group (P = 0.024 and P = 0.013, respectively). The FGS index was significantly higher in the PCOS + gingivitis group than the PCOS + healthy group (P = 0.030). The PCOS + gingivitis group revealed significantly higher insulin concentration than the PCOS + healthy and control groups (P = 0.014 and P <0.0001, respectively). Serum TNF-α, TNF-αRs, and serum, GCF, and salivary IL-6 levels correlated with the clinical periodontal measurements., Conclusions: PCOS and gingival inflammation appear to act synergistically on the proinflammatory cytokines IL-6 and TNF-α. Thus, PCOS may have an impact on gingival inflammation or vice versa. Additional studies are warranted to clarify the possible relationship between PCOS and periodontal disease.

Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS), it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these variants make to disease risk has raised questions regarding their medical relevance. Here we have investigated a single nucleotide polymorphism (SNP) in the TNFRSF1A gene, that encodes tumour necrosis factor receptor 1 (TNFR1), which was discovered through GWAS to be associated with multiple sclerosis (MS), but not with other autoimmune conditions such as rheumatoid arthritis, psoriasis and Crohn’s disease. By analysing MS GWAS data in conjunction with the 1000 Genomes Project data we provide genetic evidence that strongly implicates this SNP, rs1800693, as the causal variant in the TNFRSF1A region. We further substantiate this through functional studies showing that the MS risk allele directs expression of a novel, soluble form of TNFR1 that can block TNF. Importantly, TNF-blocking drugs can promote onset or exacerbation of MS, but they have proven highly efficacious in the treatment of autoimmune diseases for which there is no association with rs1800693. This indicates that the clinical experience with these drugs parallels the disease association of rs1800693, and that the MS-associated TNFR1 variant mimics the effect of TNF-blocking drugs. Hence, our study demonstrates that clinical practice can be informed by comparing GWAS across common autoimmune diseases and by investigating the functional consequences of the disease-associated genetic variation.

Background: It is hypothesised that osteoprotegerin (OPG), as an osteoclast antagonist, may offer molecular control over the process of orthodontic root resorption. Previous work investigating OPG in a rat periodontal ligament (PDL) ankylosis model found no inhibitory effect on osteoclasts and odontoclasts when given at a recommended dosage of 2.5 mg/kg. It was considered that traumatically-induced PDL inflammation produces mediators and cytokines with the ability to stimulate clast cell differentiation and counter the effects of OPG., Aims: The present study investigated the presence of Tumour Necrosis Factor Alpha (TNF-alpha) and its receptor Tumour Necrosis Factor Receptor 1 (TNFR1) in a PDL sterile inflammatory model., Methods: Dry ice was applied for 15 minutes to the upper right first molar crown of eighteen, 8-week-old, male Sprague-Dawley rats of which 9 were injected with OPG at a dose of 2.5 mg/kg of body weight at the time of freezing. After 7 days, the rats were sacrificed and each maxilla processed for immunohistochemical identification of TNF-alpha and TNFR1., Results: Results showed the presence of root resorption in varying amounts and locations in both experimental and control rats. Reparative processes appeared greater in the OPG-treated rats, often with the presence of an ankylotic union. Immunolabelling showed the presence of TNF-alpha and TNFR1 in the sterile inflammation located mainly in the interradicular PDL area. More definitive labelling appeared in OPG-treated rats., Conclusion: The results indicated that TNF-alpha, and its receptor TNFR1, by their presence, may modify OPG effectiveness by offering an alternative pathway for osteoclast formation, which counters the anti-resorptive effects of OPG.

The mechanisms of correcting immune disorders in patients with pancreatic cancer requiring major surgery procedures by introducing perioperative immune-enhancing diet (immunonutrition) are still unclear. The purpose of our study was to investigate the effect of pancreatic cancer, extensive surgery and immunonutrition versus enteral standard nutrition on the apoptotic signaling pathways. The randomized studies were performed in 72 patients before and after pancreatic cancer resection with preoperative standard (Group I) or enteral immunonutrition (Group II). The expressions of Bcl-2, Bax, caspases-3, -9, NF-κB, PARP-1/89 kDa, TNFR1/CD120a and Fas/CD95 in peripheral blood lymphocytes were assessed by western blot analysis and flow cytometry before and on day 1, 3 and 7 after surgery. In malnourished patients before and after surgery, the expression of Bcl-2, Bax, NF-κB, PARP-1 was significantly lower, whereas the expression of caspases, as well as the percentage of cells with death receptors were significantly higher when compared with the control group. There was no difference in Bcl-2, Bax and PARP-1 expression between the control group and the patients with normal nutritional status (Group III) before surgery. In comparison to the standard nutrition, the preoperative immunonutrition increased the Bcl-2 and Bax expression inconsiderably but significantly increased the percentage of CD95- and CD120a-positive lymphocytes after surgery. In malnourished patients with pancreatic cancer, the overwhelming expression of caspases and the decrease expression of anti-apoptotic proteins may lead to inappropriate lymphocyte apoptosis and higher cell depletion. The preoperative enteral immunonutrition prevents the postoperative decrease in lymphocyte subsets, but a higher level of lymphocyte susceptibility to undergo accelerated apoptosis can also be considered.

Purpose: The purpose of this study is to clarify the significance of joint effusion (JE) on T2-weighted magnetic resonance images of the temporomandibular joint (TMJ) in comparison to various soluble cytokine receptors in the synovial fluid of patients with temporomandibular disorders (TMDs)., Patients and Methods: Magnetic resonance imaging of 55 TMJs of 55 patients with TMD was performed, and synovial fluid samples were obtained on the same day. The grade of JE was evaluated on a scale from 0 to 3, with grade 0 indicating the absence of JE and grades 1 to 3 indicating the presence of JE. Correlations were measured between JE and the concentrations of soluble tumor necrosis factor receptors I and II, interleukin (IL) 6 soluble receptor, IL-1 soluble receptor type II, and IL-1 receptor antagonist and protein in the synovial fluid samples., Results: The mean concentrations of cytokine receptors in the synovial fluid were significantly higher in the 30 joints with JE than in the 25 joints without JE. There were no correlations between the JE grade and the level of any mediators., Conclusion: Increased levels of cytokine receptors are likely to influence the expression of JE and may play important roles in the pathogenesis of TMD. These results also suggest that JE may reflect synovial inflammation of the TMJ., (Copyright 2010 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is characterized by a progressive and irreversible loss of dopaminergic neurons. Inflammatory mechanisms have been implied in the pathophysiology of PD. In this study, we assessed serum levels of TNF-alpha and the soluble forms of their receptors, sTNFR1 and sTNFR2, in 46 PD patients and 23 control subjects. Patients with PD had higher levels of sTNFR1 (p=0.048). The concentration of sTNFR1 and sTNFR2 correlated with age (p=0.006 and p=0.022, respectively). Higher levels of sTNFR1 were associated with later disease onset (p=0.048). These results corroborate the role of inflammatory events in the process of neurodegeneration in PD which can be of special relevance in the sporadic form of PD with later onset.

Background and Objective: CD40 signaling induces growth inhibition in some tumor cells in vitro, but the precise molecular mechanism remains unclear. This study was to investigate the biological effects and mechanisms of CD40 stimulation on proliferation of lung cancer cell lines NCI-H460 and A549, changes in tumor necrosis factor receptors (TNFRs) and membrane tumor necrosis factor alpha (mTNF-alpha)., Methods: The expression of CD40 on the cell surface, and changes in TNFR and mTNF-alpha expression after CD40 stimulation were detected by the immunofluorescence technique and flow cytometry. Changes in protein contents of TNFR as well as mTNF-alpha expression after CD40 stimulation were measured by western blot. The cell proliferation rate was determined by MTT assay. The content of soluble TNF-alpha(sTNF-alpha) in the supernatant of lung cancer cells was measured by ELISA assay., Results: The expression rates of CD40 in NCI-H460 and A549 were (89.0 +/- 3.2)% and (62.2 +/- 4.5)%, respectively. After 48 h of CD40 stimulation, the expression rates of TNFRI in NCI-H460 and A549 became significantly higher [(36.2 +/- 4.6)% and (38.5 +/- 5.9)%] than those in the corresponding control cells [(15.2 +/- 3.1)% and (7.2 +/- 1.9)%] (p < 0.05); while the expression rates of TNFRII were significantly lower than those in the control cells [(18.0 +/- 1.6)% and (5.8 +/- 1.2)% vs. (58.1 +/- 3.6)% and (38.8 +/- 4.3)%] (p < 0.05); the expression rates of mTNF-alpha were decreased in the two cell lines [(8.7 +/- 1.1)% and (7.0 +/- 0.9)%] as compared to those in control cells [(15.0 +/- 2.1)% and (26.5 +/- 3.2)%] (p < 0.05). The level of TNFRI protein was elevated with the downregulation of TNFRII protein in NCI-H460 and A549. The level of mTNF-alpha protein remained unchanged in the two cell lines. TNF-alpha was not detectable in the supernatant of lung cancer cells. Moreover, cell proliferation of NCI-H460 and A549 were inhibited after CD40 stimulation (p < 0.05), but the inhibition effect disappeared after blocking TNFRI. Blocking of TNF-alpha inhibited cell proliferation of the two cell lines (p < 0.05), but a synergistic effect was not observed after simultaneous stimulation with CD40., Conclusion: CD40 signaling inhibits the proliferation of CD40-positive lung cancer cells through mTNF-alpha/TNFRI in vitro.

We determined the role of interleukin-1beta (IL-1beta) signaling on tumor necrosis factor alpha-induced (TNF-alpha) lung neutrophil influx as well as neutrophil chemoattractant macrophage inflammatory protein (MIP-2) and KC and soluble TNF-alpha receptor (TNFR) levels utilizing wildtype (WT), TNF receptor double knockout (TNFR1/TNFR2 KO), and IL-1beta KO mice after oropharyngeal instillation with TNF-alpha. A significant increase in neutrophil accumulation in bronchoalveolar lavage fluid (BALF) and lung interstitium was detected in the WT mice six hours after TNF-alpha exposure. This correlated with an increase in BALF MIP-2. In contrast, BALF neutrophil numbers were not increased by TNF-alpha treatment of IL-1beta KOs, correlating with a failure to induce BALF MIP-2 and a trend toward increased BALF soluble TNFR1. TNF-alpha-instillation increased lavage and serum KC and soluble TNFR2 irrespective of IL-1beta expression. These results suggest IL-1beta contributes, in part, to TNF-alpha-mediated, chemokine release, and neutrophil recruitment to the lung, potentially associated with altered soluble TNFR1 release into the BALF.

The current study examined the role of endothelin-1 (ET-1) in mediating acute lung inflammation induced by short-term cigarette smoke exposure. Hamsters received intraperitoneal injections of ET-1, followed by a 2-hour period of smoke exposure, for 3 consecutive days. The lungs were then evaluated for inflammatory changes, using the following parameters: (1) lung histopathology, (2) neutrophil content of bronchoalveolar lavage fluid (BALF), (3) percent tumor necrosis factor receptor 1 (TNFR1)-labeled BALF macrophages, and (4) alveolar septal cell apoptosis. Results indicate that ET-1 significantly amplified the effect of smoke on each of these inflammatory markers and that these responses could be blocked by pretreatment with a novel endothelin receptor A antagonist, HJP272. In particular, exogenous ET-1 induced a marked increase in BALF neutrophils, consistent with a role for this mediator as an inflammatory cell "gatekeeper."

Aim: We reported that soluble tumour necrosis factor receptor type 2 (sTNFR2)/type 1 (sTNFR1) ratios in gingival crevicular fluid (GCF) decreased as the severity of chronic periodontitis (CP) increased. This study investigated the effects of the periodontal treatment on TNF-alpha, sTNFR1 and R2 in GCF and serum of CP patients., Material and Methods: Thirty-five serum and 90 GCF samples were obtained from 35 CP patients (23 non-smokers and 12 smokers) at baseline and after treatment. The levels of TNF-alpha, sTNFR1 and R2 in serum and GCF were quantified by enzyme-linked immunosorbant assay., Results: No significant differences were found in the serum levels of TNF-alpha, sTNFR1 and R2 and the ratio of sTNFR2/R1 between baseline and after treatment. After treatment, sTNFR1 and R2 levels in GCF of non-smokers and smokers were significantly decreased compared with baseline. However, the sTNFR2/R1 ratio was significantly increased (non-smoker: 0.56+/-0.03-0.84+/-0.03, p<0.0001; smoker: 0.59+/-0.06-0.85+/-0.04, p=0.0019). There were no significant differences between non-smoking and smoking CP groups in serum and GCF., Conclusion: The ratio of sTNFR2/R1 in GCF significantly increased after treatment, and could be related to the clinical state of CP.

Phosphoramidon blocks the formation of endothelin-1 (ET-1), a proinflammatory mediator implicated in the pathogenesis of a variety of lung diseases. To determine whether phosphoramidon can ameliorate pulmonary inflammation, our laboratory undertook a series of experiments involving treatment of hamsters with either intraperitoneal (i.p.) or aerosolized phosphoramidon prior to induction of acute lung injury by intratracheal administration of lipopolysaccharide (LPS). The results indicate that phosphoramidon significantly reduces LPS-induced pulmonary inflammation as measured by lung histology, neutrophil content of bronchoalveolar lavage (BAL) fluid, percent tumor necrosis factor receptor 1 (TNFR1)-labeled BAL macrophages, and alveolar septal cell apoptosis. In additional experiments, i.p. administration of a novel endothelin A receptor anatgonist (HJP272) similarly decreased BAL neutrophils, whereas i.p. administration of either ET-1, or its precursor peptide, "big" ET-1, had the opposite effect. These findings support further evaluation of phosphoramidon and other ET-1 suppressors as potential treatments for human inflammatory lung disease.

Unlabelled: Activation of the cytokine systems may be involved in the neuropathological changes occurring in the central nervous systems of schizophrenic patients. However, associations between the levels of cytokines and the severity of symptoms have not been completely established., Objective: It was the aim of this study to evaluate serum levels of tumor necrosis factor (TNF)-alpha and their soluble receptors (sTNFR) in schizophrenic patients and healthy controls., Methods: Forty male institutionalized schizophrenic patients (mean age +/- SD, 52.3 +/- 9.9 years) and 20 asymptomatic matched controls were recruited. The severity of symptoms was assessed using the Brief Psychiatric Rating Scale, the Positive and Negative Syndrome Scale and the Abnormal Involuntary Movement Scale. Serum levels of cytokines were measured by ELISAs., Results: Serum levels of sTNFR1 and sTNFR2 were increased in schizophrenic patients when compared with controls (all p < 0.05), but there was no difference in TNF-alpha levels. There was no correlation between the length of disease/hospitalization or the severity of symptoms and the serum levels of these molecules., Conclusion: Inflammatory markers are increased in schizophrenia but they do not correlate with symptom severity., ((c) 2008 S. Karger AG, Basel.)

Because survivin is selectively expressed in and associated with an unfavorable prognosis in transitional cell carcinoma of the bladder (TCC), we treated T-24 cells with survivin siRNA. Survivin siRNA treatment caused a profound decrease of survivin protein that was associated with decreased cell growth, a specific G2/M arrest and increased cytochrome c release. Microarray analysis of apoptosis genes showed that levels of 14/114 gene products were decreased after 72 hours treatment with survivin siRNA, including survivin, three TNF receptors, Akt, c-Abl, caspases and their related genes and Bcl-2 and NF-kappaB signaling related genes. TNFR1, pro-caspase-2 and Akt protein levels were decreased after survivin siRNA treatment for 48 and 72 hours. Downregulation of survivin causes changes in mitosis and apoptosis, which may be related to changes in TNF receptors and NF-kappaB signaling.

Background: Tumor necrosis factor-alpha (TNF-alpha) up-regulation has been associated with both low and high shear-induced arterial remodeling. To address this apparent paradox and to define the biology of TNF-alpha signaling in large arteries, we tested the hypotheses that differential temporal expression of TNF-alpha drives shear-regulated arterial remodeling., Materials and Methods: Both low- and high-shear environments in the same rabbit were surgically created for common carotid arteries. Common carotid arteries (n = 60 total) were harvested after d0, d1, d3, d7, and d14 and analyses included morphology, TNF-alpha, and IL-10 mRNA quantitation. In separate experiments, animals received pegylated soluble TNF-alpha Type 1 receptor (PEG sTNF-RI) or vehicle via either short- or long-term dosing to define the effect of TNF-alpha blockade., Results: The model yielded a 14-fold shear differential (P < 0.001) with medial thickening under low shear (P = 0.025), and evidence of outward remodeling with high shear (P = 0.007). Low shear immediately up-regulated TNF-alpha expression approximately 50 fold (P < 0.001) at d1. Conversely, high shear-induced delayed and sustained TNF-alpha expression (22-fold at d7, P = 0.012; 23-fold at d14, P = 0.007). Both low and high shear gradually induced IL-10 expression (P = 0.002 and P = 0.004, respectively). Neither short-term (5-day) nor long-term (14-day) blockage of TNF-alpha signaling resulted in treatment-induced changes in the remodeling of low- or high-shear arteries., Conclusions: Shear stress differentially and temporally regulates TNF-alpha expression in remodeling large arteries. However, TNF-alpha blockage did not substantially impact the final shear-induced morphology, suggesting that large arteries can remodel in response to flow perturbations independent of TNF-alpha signaling.

Background: Chronic infection in cystic fibrosis (CF) and airway inflammation leads to progressive lung injury. Neutrophils are considered to be responsible for the onset and promotion of the inflammatory response within the CF lung. The relationship between infection and inflammation is complex but circulating inflammatory markers may not truly reflect the local inflammatory response in the lung. The aims of this study were to investigate the change of inflammatory biomarkers and cells within sputum and blood before and after intravenous antibiotics for a pulmonary exacerbation of CF., Methods: Assays included neutrophil elastase (NE) and complex, interleukin-8 (IL-8) and soluble intercellular adhesion molecule-1 (sICAM-1), fas ligand (FAS-L), and TNFr-1. Analysis of sputum cell differential and absolute cell counts and immunocytochemistry (CD11b and CD95) on sputum and isolated blood neutrophils were carried out., Results: There were no significant differences in absolute or differential sputum cell counts or sputum sol measurements following antibiotics. There was a significant increase in the percentage of blood neutrophils with minimal CD11b staining, 28 (4.1) mean percentage (SEM) versus 41 (2.9) and a decrease in the percentage showing maximal staining 30 (0.5) versus 15 (2.5). There was a significant increase in the percentage of blood neutrophils without CD95 staining, 43 (5.4) mean percentage versus 52 (5.1)., Conclusion: These data suggest a modifiable systemic response to i.v. antibiotics but a local sustained inflammatory response in the lung., (2007 Wiley-Liss, Inc.)

Introduction: The release of tumor necrosis factor (TNF-alpha) is increased in sarcoidosis patients. TNF-alpha exerts its effect by binding to specific cell surface receptors. There are only fragmentary data concerning the expression of tumor necrosis factor receptors (TNFRs) on bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) lymphocytes. The aim of the study was to evaluate TNFRI (CD120a) and TNFRII (CD120b) expression on T cells and the level of soluble TNFRs in specimens of patients with different clinical manifestation and clinical outcome of sarcoidosis., Material and Methods: We examined 49 patients with newly diagnosed pulmonary sarcoidosis. TNFRI and TNFRII density on CD4+ and CD8+ BALF and PB cells surface was estimated using monoclonal antibodies and a flow cytometry technique. The level of TNFRs in PB serum and BALF cell culture supernatant (CCS) was measured using ELISA. Immunological analyses were also performed on PB samples collected from 10 healthy volunteers., Results: The level of soluble TNFRI (sTNFRI) in PB serum was similar in sarcoidosis patients and healthy subjects, whereas the concentration of sTNFRII in serum was significantly higher in the sarcoidosis group (P<0.001). Patients without acute symptoms of sarcoidosis, patients with radiological stage II/III as well as patients with further disease progression showed a tendency to higher levels of sTNFRs in PB serum and lower levels of sTNFRs in BALF CCS compared to Löfgren syndrome and radiological stage I subjects, and patients with spontaneous resolution of sarcoidosis. More than 80% of BALF and PB lymphocytes of sarcoidosis patients expressed both CD120a and CD120b antigens. The percentage of double-positive CD4+CD120a+ and CD4+CD120b+ cells in PB was significantly higher (P<0.005) in sarcoidosis patients than in healthy subjects. The highest percentage of CD4+CD120a+ and CD4+CD120b+ lymphocytes in BALF was determined in patients with acute disease, and in PB of patients with further spontaneous improvement., Conclusion: The evaluation of sTNFRs and TNFRs expression on T-helper cells may be useful in the estimation of sarcoidosis activity.

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)alpha, although the events through which TNFalpha induces ROS generation are not well characterized. Here, we report that TNFalpha-induced ROS production was blocked by pretreatment with internalization inhibitor monodansyl cadaverine (MDC). Similarly, a transient expression of a GTP-binding and hydrolysis-defective dynamin mutant (dynamin(K44A)) that had been shown to be defective in internalization significantly attenuated the TNFalpha-induced intracellular ROS production. Importantly, the inhibition of receptor internalization suppressed TNFalpha signaling to mitogen-activated protein kinases (MAPKs) stimulation. Together, our results suggest that receptor internalization is somehow necessary for the TNFalpha-induced ROS generation and subsequent intracellular downstream signaling in non-phagocytes.

Background: Tumor necrosis factor (TNF)-alpha and its receptors play a critical role in the inflammatory cascade after hepatic ischemia/reperfusion injury. TNF-alpha converting enzyme (TACE) or disintegrin and metalloproteinase (ADAM)-17 is a metalloproteinase disintegrin that specifically cleaves precursor TNF-alpha to its mature form and is involved in the ectodomain shedding of TNF receptors. The regulation of TACE is poorly understood and its role in liver injury and/or regeneration is unknown., Methods: Male Wistar rats were subjected to 10 or 30 min of partial warm hepatic ischemia followed by 3 to 24 hr of reperfusion. Serum and/or hepatic TACE, TNF-alpha, TNF receptor 1 (TNFR1), and interleukin (IL)-6 levels were assessed by enzyme-linked immunosorbent assay, real-time reverse-transcriptase polymerase chain reaction, and/or Western blot., Results: Low levels of TACE were detected in normal liver tissue. Both 10 and 30 min warm ischemia resulted in a rise in TACE expression which peaked six hr after reperfusion. TNF-alpha, TNFR1, and IL-6 levels were up-regulated in a pattern similar to TACE messenger RNA (mRNA) levels. Moreover, selective inhibition of TACE activity by specific inhibitor tissue inhibitor of metalloproteinase (TIMP)-3 at dosages of 100 or 1000 ng/kg body weight showed significant decrease of circulating TNF-alpha and serum alanine transferase (ALT) levels and histological improvement of hepatic ischemia/reperfusion injuries., Conclusions: TACE expression and its activity, as measured by increases in TNF-alpha, TNFR1, and IL-6 levels, are increased following hepatic ischemia/reperfusion injury, implying that TACE plays an important role in hepatic ischemia/reperfusion injury. Amelioration of hepatic ischemia/reperfusion injury after inhibition of TACE activity by TIMP-3 suggests that TACE inhibition may play an important role in preventing liver ischemia/reperfusion injury warranting further experimental and clinical study.

Background & Aims: Activation of natural killer T cells with the synthetic ligand alpha-galactosylceramide (alpha-GalCer) induced hepatotoxicity through the tumor necrosis factor (TNF) and Fas-ligand-mediated pathway in aged mice. The aim of this study was to elucidate how alpha-GalCer-activated natural killer T cells function in hepatocyte proliferation and liver regeneration in partially hepatectomized (PHx) mice., Methods: Mice were injected with alpha-GalCer at 36 hours after 70% PHx. Hepatocyte mitosis was evaluated by either mitotic figures or proliferating cell nuclear antigen staining. The role of TNF and Fas-ligand in hepatocyte mitosis also was assessed., Results: In PHx mice injected with alpha-GalCer, hepatocyte mitosis was greatly enhanced at 44 hours after surgery and the increase was more obvious in aged mice than in young mice. The expression of both TNF receptor 1 and Fas-ligand in liver natural killer T cells tended to increase after alpha-GalCer injection in PHx mice. Treatment of mice with anti-NK1.1 Ab 3 days before and just after hepatectomy greatly inhibited the effect of alpha-GalCer on hepatocyte mitosis and liver regeneration. Furthermore, pretreatment of PHx mice with either anti-TNF Ab or anti-FasL Ab 1 hour before alpha-GalCer injection mostly abrogated the increase in hepatocyte proliferation. alpha-GalCer injection did not accelerate hepatocyte proliferation in Fas-mutated lpr mice after PHx. CD1d-/- mice without alpha-GalCer injection showed decreased hepatocyte mitosis after PHx., Conclusions: Activated natural killer T cells help hepatocyte proliferation and liver regeneration after PHx via the TNF and Fas/Fas-ligand-mediated pathway.

The aim of the present study was to characterize the expression pattern of tumor necrosis factor (TNF)-alpha and its receptors in breast samples (benign diseases, in situ carcinomas and infiltrating carcinomas), and to compare these results with those obtained previously for interleukin-6, p53 and p21 using the same samples in order to elucidate the effects of these cytokines on the proliferation-apoptosis equilibrium. Immunoexpression of TNF-alpha and its receptors (TNFRI and TNFRII) were studied by western blotting and immunohistochemistry. The percentage of samples positive for TNF-alpha and TNFRII was higher in in situ carcinoma than in benign breast diseases, and TNFRII was even higher in infiltrating tumors. The percentage of samples positive for TNFRI was similar in the three groups. For the three proteins and in the three patient groups, immunoreactions were observed in the peripheral cytoplasm. In the positive samples, immunostaining for TNF-alpha was more intense in infiltrating tumors than in the other two patient groups, whereas immunostaining for both receptors was higher in in situ carcinoma than in benign breast diseases, and even higher in infiltrating tumors. Comparing the TNF-alpha results with previous results for mtp53, p21 and interleukin-6, we found an association between the expression of these four proteins and increasing malignancy. TNF-alpha might be an important factor in breast cancer promotion as its proliferation and survival effects seems to be enhanced through the increased expression of TNFRII. Also, the pro-apoptotic pathway of TNFRI could be inhibited by p21 (which appeared increased in breast cancer), altering TNFRI effects in promoting the expression of several factors, such interleukin-6, which contribute to tumor promotion.

Inflammation in the central nervous system (CNS) can be studied in experimental autoimmune encephalomyelitis (EAE). The proinflammatory cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) are implicated in EAE pathogenesis. Signals through the type 1 TNF receptor (TNFR1) are required for severe EAE to develop, whereas deficiency in IFN-gamma or its receptor result in more severe EAE. We investigated IFN-gamma expression in TNFR1-deficient (TNFR1-/-) mice. We describe here that there were more IFN-gamma-secreting T cells present in the CNS of TNFR1-/- mice during EAE compared to wild-type (WT) mice, despite that clinical symptoms were mild, with delayed onset. There was greater expression of IL-12/23p40 by antigen-presenting cells in these mice, and in vitro, TNFR1-/- antigen-presenting cells induced greater secretion of IFN-gamma but not interleukin (IL)-17 when cultured with primed T cells than did WT antigen presenting cells. TNFR1-/- mice with EAE had significantly higher expression of CXCL10 mRNA (but not CCL5 mRNA) in the CNS compared to WT mice with EAE. These data demonstrate that IFN-gamma expression is enhanced in the CNS of TNFR1-/- mice with EAE and suggest that IFN-gamma levels do not necessarily correlate with EAE severity.

Recent studies have reported a significant inflammatory reaction in the brain and the systemic circulation after traumatic brain injury (TBI). Although there is growing knowledge and understanding of the mechanisms and mediators involved in the proinflammatory reaction, little is known about the anti-inflammatory mediators in the brain. As tumor necrosis factor alpha (TNF-alpha) plays a detrimental role in the initiation and promotion of the proinflammatory reactions after TBI, the endogenous scavenger system, represented by the soluble TNF receptors (sTNFRs) p55 and p75, seems to have an important anti-inflammatory capacity by binding to circulating TNF-alpha. To evaluate this potentially anti-inflammatory response to trauma, we analyzed sTNFR p55 and p75 in paired plasma/cerebrospinal fluid (CSF) samples of 29 patients who encountered TBI. Values were compared with reference values obtained from healthy volunteers (n = 91). Patients with TBI showed significantly (P < 0.001) elevated sTNFR p55 and p75 values starting from day 2 and lasting until day 10 if compared with reference values. In contrast to the early increased plasma values p55 and p75 showed slowly increasing CSF values starting on day 4 and 3, respectively. Significantly (P < 0.001) increased CSF values of p 55 were determined on days 4 to 6 and day 9. p75 showed significantly (P < 0.001) elevated values if compared with control values on days 7 and 9. The sTNFR p55 and p75 show a distinct and long-lasting elevation in plasma of patients after TBI. In contrast, CSF values display a delayed and less intense elevation of both receptors in patients with TBI. These findings are suggestive of an imbalance of the proinflammatory and anti-inflammatory reactions of the central nervous system after trauma, with an emphasis on the proinflammatory mechanisms and a slow increase of potentially anti-inflammatory mediators such as the soluble TNFRs after TBI.

Aim: To investigate the effects of experimental partial hepatectomy and normothermic ischemia-reperfusion damage on the time course of the expression of four different growth factor receptors in liver regeneration. This is relevant due to the potential therapeutic use of growth factors in stimulating liver regeneration., Methods: For partial hepatectomy (PH) 80% of the liver mass was resected in Sprague Dawley rats. Ischemia and reperfusion (I/R) were induced by occlusion of the portal vein and the hepatic artery for 15 min. The epidermal growth factor receptor, hepatic growth factor receptor, fibroblast growth factor receptor and tumour necrosis factor receptor-1 were analysed by immunohistochemistry up to 72 h after injury. Quantitative RT-PCR was performed at the time point of minimal receptor expression (24 h)., Results: In immunohistochemistry, EGFR, HGFR, FGFR and TNFR1 showed biphasic kinetics after partial hepatectomy with a peak up to 12 h, a nadir after 24 h and another weak increase up to 72 h. During liver regeneration, after ischemia and reperfusion, the receptor expression was lower; the nadir at 24 h after reperfusion was the same. To evaluate whether this nadir was caused by a lack of mRNA transcription, or due to a posttranslational regulation, RT-PCR was performed at 24 h and compared to resting liver. In every probe there was specific mRNA for the receptors. EGFR, FGFR and TNFR1 mRNA expression was equal or lower than in resting liver, HGFR expression after I/R was stronger than in the control., Conclusion: At least partially due to a post-transcriptional process, there is a nadir in the expression of the analysed receptors 24 h after liver injury. Therefore, a therapeutic use of growth factors to stimulate liver regeneration 24 h after the damage might be not successful.

The aim of this study was to clarify the mechanism of apoptosis seen in the cortex of neural cell-specific hypoxia inducible factor-1alpha (HIF-1alpha)-deficient embryos. A previous study showed that the neural cells in the cortical area of the mutant embryos underwent apoptosis coincident with vascular regression. Through histological, immunohistochemical, and electron microscopic technique, two kinds of apoptotic cells were detected in the mutant embryonal cortex. Apoptotic cells of one type were clustered in small round structures, 10-20 mum in diameter, whereas the others, present in large numbers, were distributed in a group at the cortical plate located more to the outer side than the round structures. The histochemical and electron microscopic findings indicate that the former represented the appearance of macrophages, in which cellular fragments including vascular cells underwent oxidative stress-related, TNF receptor-mediated, caspase-2-induced apoptosis, while the latter showed c-Myc-related, caspase-3-activated apoptosis of the neural cells. These results suggest that two pathways of apoptosis are induced in neuronal and vascular cells of the cortex in the neural cell-specific HIF-1alpha-deficient mouse.

In order to elucidate the biological variance between normal ovarian surface epithelial (NOSE) and epithelial ovarian cancer (EOC) cells, and to build a molecular classifier to discover new markers distinguishing these cells, we analysed gene expression patterns of 65 primary cultures of these tissues by oligonucleotide microarray. Unsupervised clustering highlights three subgroups of tumours: low malignant potential tumours, invasive solid tumours and tumour cells derived from ascites. We selected 18 genes with expression profiles that enable the distinction of NOSE from these three groups of EOC with 92% accuracy. Validation using an independent published data set derived from tissues or primary cultures confirmed a high accuracy (87-96%). The distinctive expression pattern of a subset of genes was validated by quantitative reverse transcription-PCR. An ovarian-specific tissue array representing tissues from NOSE and EOC samples of various subtypes and grades was used to further assess the protein expression patterns of two differentially expressed genes (Msln and BMP-2) by immunohistochemistry. This study highlights the relevance of using primary cultures of epithelial ovarian cells as a model system for gene profiling studies and demonstrates that the statistical analysis of gene expression profiling is a useful approach for selecting novel molecular tumour markers.

The classic stimulus for cellular cytokine production is bacterial lipopolysaccharide (endotoxin). It was therefore hypothesized that tumor necrosis factor-alpha (TNF-alpha) may be responsible for pericoronitis. TNF-alpha and its receptors were detected by immunohistochemical staining in third molar pericoronitis in ten patients and ten healthy control samples. The percentage of TNF-alpha positive cells was high in pericoronitis (p = 0.0317). TNF receptors TNF-R1 and TNF-R2 were found in macrophage- and fibroblast-like cells, vascular endothelial cells in post-capillary venules, and basal epithelial cells in pericoronitis, but were only weakly expressed in controls. Increased expression of interleukin-1beta and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-alpha ligand-receptor interaction. Explanted tissues acquired destructive potential upon TNF-alpha stimulation, whereas TNF-alpha blockers controlled it in inflamed tissues. These findings suggest that, in pericoronitis, inflammatory and resident cells produce and respond to potent pro-inflammatory cytokine TNF-alpha, with pathogenic and potential therapeutic relevance.

Pseudomonas aeruginosa is a gram-negative bacterium that is an opportunistic pathogen in patients with cystic fibrosis and in immunocompromised hosts. This bacterium produces a variety of proteolytic enzymes, including alkaline protease (AP), which has multiple biological effects. This study investigated the effects of AP on the A549 pulmonary epithelial cell line. Results demonstrate that AP inhibited tumor necrosis factor (TNF)-alpha-induced RANTES gene expression and secretion in a concentration-dependent manner. The TNF-alpha-induced RANTES gene expression and secretion was attenuated with a neutralizing monoclonal antibody directed against the TNF receptor type 1 (TNFR1). Conversely, a neutralizing monoclonal antibody directed against TNF receptor type II had no effect, suggesting that these events were regulated through the TNFR1 receptor. In addition, we observed that soluble TNF receptor type 1 (sTNFR1) levels were significantly increased in culture supernatants of AP-treated cells in a concentration-dependent manner. Finally, membrane-associated TNFR1 was decreased after AP exposures. In these studies, the enzymatically inactive form of AP had no effect on TNF-alpha-induced RANTES secretion, shedding of sTNFR1, or membrane-associated TNFR1. These results demonstrate that AP stimulates shedding of cell-surface TNFR1, resulting in an increase in sTNFR1. Consequently, these events decrease the cells' ability to stimulate RANTES gene expression and secretion through TNFR1.

Tumor necrosis factor-alpha (TNFalpha) may play a role in the development of autoimmune thyroiditis such as Hashimoto's thyroiditis. In the present study, we examined whether TNFalpha induced its own expression in FRTL-5 rat thyroid cells. Lipopolysaccharide (LPS) markedly increased TNFalpha mRNA levels in FRTL-5 cells as assessed by semiquantitative RT-PCR. In addition, LPS-stimulated cells released TNFalpha protein into the culture medium. Similarly, TNFalpha induced its own gene and protein expression in FRTL-5 cells as assessed by RT-PCR and metabolic labeling and immunoprecipitation of TNFalpha. The autoinduction of TNFalpha gene was also observed in TNFalpha-stimulated human thyroid epithelial cells. TNFalpha induction was specific to LPS and TNFalpha since interferon-alpha or amiodarone failed to increase TNFalpha mRNA levels in FRTL-5 cells. Human TNFalpha induced rat TNFalpha gene expression, indicating that type 1 TNF receptor (TNF-R) is involved in the autoinduction. TNFalpha did not increase either type 1 or type 2 TNF-R mRNA levels, suggesting that upregulation of TNF receptors is not involved in the autoinduction of TNFalpha. Although the biological significance of autoinduction of TNFalpha remains unclear, our results suggest that thyroid epithelial cells may participate in the development of autoimmune thyroiditis through production of TNFalpha. Furthermore, inhibition of TNFalpha production in the thyroid may represent a novel approach to mitigating inflammation in autoimmune thyroiditis.

Aims: To evaluate whether plasma biomarkers of inflammation and endothelial dysfunction differed in Type 1 diabetic patients as compared with those in non-diabetic subjects, and to examine the association of these biomarkers with early stages of microvascular complications., Methods: Plasma biomarkers of inflammation [fibrinogen, hs-C-reactive protein (hs-CRP)] and endothelial dysfunction [von Willebrand factor (v-WF), intercellular adhesion molecule-1, plasminogen activator inhibitor-1 (PAI-1) activity] were measured in 88 non-smoking young patients with Type 1 diabetes without clinical macrovascular disease and in 40 healthy controls., Results: Plasma levels of hs-CRP, fibrinogen, v-WF, soluble intracellular adhesion molecule-1 (sICAM-1) and PAI-1 activity were markedly higher (P < 0.01 or less) in Type 1 diabetic patients than in healthy controls; these results were essentially unchanged when healthy controls were compared with patients without complications. After stratification by microvascular complication status, plasma biomarkers of inflammation and endothelial dysfunction were significantly increased in those with more advanced disease compared with those with early complications or without complications, respectively. However, while the significant differences in these biomarkers were little affected by adjustment for sex, age, BMI and blood pressure values, they were totally abolished after additional adjustment for diabetes duration and glycaemic control., Conclusions: These results indicate that in Type 1 diabetes there is a subclinical, chronic inflammation which is, at least partly, independent of clinically manifest macro- and microvascular complications, smoking or other traditional cardiovascular risk factors; this subclinical inflammation is closely correlated to the magnitude and duration of hyperglycaemia.

Background: It is likely that the imbalance between the pro- and anti-inflammatory cytokines will determine the outcome in infants with severe respiratory failure receiving extracorporeal membrane oxygenation (ECMO)., Aims: We determined if there was an imbalance between pro- and anti-inflammatory cytokines in serial bronchoalveolar lavage (BAL) fluid obtained from survivors and non-survivors of ECMO., Methods: We therefore measured the cellular changes and the molar ratios of pro-inflammatory and anti-inflammatory cytokines in serial BAL fluid obtained from survivors and non-survivors of ECMO. Fifteen infants surviving ECMO (median age 1 day, range 1-120) and 7 who did not (28 days, range 1-402) were studied., Results: In the lungs of survivors, the increased proportion of airway neutrophils at presentation decreased with time and was matched by a parallel increase in percent alveolar macrophages as the infants' condition improved. The pro- and anti-inflammatory pulmonary cytokine ratios were static in the survivors. In the non-survivors, the ratio of tumour necrosis factor-alpha (TNF-alpha) against soluble TNF-receptor 1 (sTNF-R1) and soluble TNF receptor 2 (sTNF-R2) was increased at days 2-3 when compared to the survivors, but the molar ratio interleukin-1beta (IL-1beta)/soluble IL-1 receptor antagonist (sIL-1RA) was largely undetectable due to undetectable IL-1beta., Conclusions: These data suggest that the infants who survive ECMO resolve their pulmonary inflammation and that in non-survivors the ratio of TNF-alpha against its receptor antagonists is increased and is associated with a poor outcome. Furthermore, this group of infants were unable to produce significant concentrations of IL-1beta.

The inflammatory response induced by the conditioning regime may be related to the outcome in allogeneic stem-cell transplantation (SCT). However, previous statements concerning the prognostic significance of cytokine measurements during conditioning have not been conclusive. We investigated a broad range of cytokines in plasma samples drawn daily immediately before start of pretransplant conditioning and during the conditioning. The presented data indicate that single-day measurements of inflammatory cytokines during conditioning may lead to unreliable conclusions concerning their prognostic significance. However, serial quantitation of soluble tumour necrosis factor receptor I (sTNFRI) is more likely to reflect the degree of inflammatory activation induced by pretransplant conditioning.