Doses Of TNF Research Articles

SYST-03. A PHASE I/II STUDY TO EVALUATE SAFETY AND PRELIMINARY ACTIVITY OF THE TUMOR-TARGETING ANTIBODY-CYTOKINE FUSION PROTEIN L19TNF IN PATIENTS WITH GLIOBLASTOMA AT FIRST RELAPSE

Abstract Treatment options for recurrent glioblastoma are limited and there is a need for novel effective therapies. We previously demonstrated encouraging anti-tumor activity with the targeted delivery of tumor necrosis factor a (TNF) in preclinical orthotopic glioma models. TNF- a is a potent pro-inflammatory cytokine which may trigger strong anti-tumor immunity. However, its systemic administration at therapeutically active doses is hampered by toxic side effects. L19TNF is a fully human antibody-cytokine fusion protein, comprising TNF- a fused to the antibody L19 that binds a tumor-specific epitope of the extracellular matrix protein fibronectin. This allows a targeted delivery of therapeutically relevant doses of TNF to the tumor site upon intravenous administration while sparing healthy tissues. In this phase I/II open label, non-randomized, monocentric study, we investigated the safety and preliminary activity of L19TNF for patients with isocitrate dehydrogenase (IDH) wildtype World Health Organization (WHO) grade III / IV glioma at first relapse. Twenty patients were enrolled from 2019-2020 and treated with intravenous infusions of L19TNF. In the phase I part of the study, 6 patients were assigned to two different dose levels of L19TNF and a dose of 13 µg/kg was established as the recommended dose. In the phase II part 14 patients were treated at the recommended dose. No dose-limiting toxicities were observed and survival follow-up is ongoing. In almost all patients, we observed treatment-associated emerging tumor necrosis. For patients that had re-surgery at progression on or after treatment with L19TNF, we demonstrated increased numbers of tumor-infiltrating lymphocytes compared to the tumor tissue obtained at primary diagnosis. Translational studies to better understand the effects of L19TNF on a molecular and immunophenotypic level are ongoing. ClinicalTrials.gov Identifier: NCT 03779230

Targeted enhancement of the therapeutic window of L19-TNF by transient and selective inhibition of RIPK1-signaling cascade.

IntroductionCytokine-based products are gaining importance for cancer immunotherapy. L19-TNF is a clinical-stage antibody-cytokine fusion protein that selectively accumulates to tumors and displays potent anticancer activity in preclinical models. Here, we describe an innovative approach to transiently inhibit off-target toxicity of L19-TNF, while maintaining antitumor activity.MethodsGSK’963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice for its ability to reduce acute toxicity associated with TNF signaling. The biological effects of L19-TNF on tumor cells, lymphocytes and tumor vessels were investigated with the aim to enable the administration of TNF doses, which would otherwise be lethal.ResultsTransient inhibition of RIPK1 allowed to increase the maximal tolerated dose of L19-TNF. The protective effect of GSK’963 did not affect the selective localization of the immunocytokine to tumors as evidenced by quantitative biodistribution analysis and allowed to reach high local TNF concentrations around tumor blood vessels, causing diffused vascular shutdown and hemorrhagic necrosis within the neoplastic mass.ConclusionsThe selective inhibition of RIPK1 with small molecule inhibitors can be used as a pharmaceutical tool to transiently mask TNF activity and improve the therapeutic window of TNF-based biopharmaceuticals. Similar approaches may be applicable to other pro-inflammatory cytokines.

Intra-pancreatic tissue-derived mesenchymal stromal cells: a promising therapeutic potential with anti-inflammatory and pro-angiogenic profiles

BackgroundHuman pancreata contain many types of cells, such as endocrine islets, acinar, ductal, fat, and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions.MethodsWe investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore, whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated.ResultsIPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5% human platelet lysate (hPL). IPTD-MSCs were found to be highly pure, with > 95% positive for CD90, CD105, and CD73, and negative for CD45, CD34, CD14, and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro, underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-α, gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased, compared to control. In contrast, treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly, a combination of TNF-α and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α contained higher levels of pro-angiogenic (VEGF, IL-6, and IL-8) compared to controls, promoting angiogenesis of human endothelial cells in vitro. In contrast, levels of MCP-1, a pro-inflammatory cytokine, were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α.ConclusionsThe results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application.

When PERK inhibitors turn out to be new potent RIPK1 inhibitors: critical issues on the specificity and use of GSK2606414 and GSK2656157.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes a state of cellular stress known as ER stress. The cells respond to ER stress by activating the unfolded protein response (UPR), a signaling network emerging from the ER-anchored receptors IRE1α, PERK and ATF6. The UPR aims at restoring ER protein-folding homeostasis, but turns into a toxic signal when the stress is too severe or prolonged. Recent studies have demonstrated links between the UPR and inflammation. Consequently, small molecule inhibitors of IRE1α and PERK have become attractive tools for the potential therapeutic manipulation of the UPR in inflammatory conditions. TNF is a master pro-inflammatory cytokine that drives inflammation either directly by promoting gene activation, or indirectly by inducing RIPK1 kinase-dependent cell death, in the form of apoptosis or necroptosis. To evaluate the potential contribution of the UPR to TNF-induced cell death, we tested the effects of two commonly used PERK inhibitors, GSK2606414 and GSK2656157. Surprisingly, we observed that both compounds completely repressed TNF-mediated RIPK1 kinase-dependent death, but found that this effect was independent of PERK inactivation. Indeed, these two compounds turned out to be direct RIPK1 inhibitors, with comparable potency to the recently developed RIPK1 inhibitor GSK'963 (about 100 times more potent than NEC-1s). Importantly, these compounds completely inhibited TNF-mediated RIPK1-dependent cell death at a concentration that did not affect PERK activity in cells. In vivo, GSK2656157 administration protected mice from lethal doses of TNF independently of PERK inhibition and as efficiently as GSK'963. Together, our results not only report on new and very potent RIPK1 inhibitors but also highlight the risk of misinterpretation when using these two PERK inhibitors in the context of ER stress, cell death and inflammation.

In TNF-stimulated cells, RIPK1 promotes cell survival by stabilizing TRAF2 and cIAP1, which limits induction of non-canonical NF-κB and activation of caspase-8.

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1(-/-) cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

Study of the level of ERM proteins in pulmonary microvascular endothelial cells induced by tumor necrosis factor-α

Objective To investigate the effect of tumor necrosis factor -α (TNF -α) on the levels of ezrin -radixin -moesin (ERM) proteins and the phosphorylated ERM proteins (p -ERM) in rat pulmonary microvascular endothelial cells (PMVEC) , and to explore Rho kinase (ROCK) influencing on modulation of the ERM proteins phosphorylation. Methods Cultured rat pulmonary microvascular endothelial cells were randomly divided into dose -dependent and time -dependent groups. In dose - dependent group, cells were cultured with different doses of TNF -α (0, 0.1 , 1 , 10 μg/LTNF -α) for 60 min. In time -dependent group, cells were cultured with TNF -α (10 μg/L) for 0, 15, 30, 60, 90, 120, 180 min. In ROCK inhibitor (Y27632) intervention group, cells were cultured with TNF -α(10 μg/L) or Y27632 (30 μmol/L) +TNF -α (10 μg/L) for 60 min respectively. The levels of ERMproteins and p -ERM were determined by western blot. One way analysis of variance (ANOVA) was employed for statistical analysis by using SPSS version 16.0 software to compare values among all groups. A significant difference was presumed as aP value <0.05. Results Western blot revealed that ERM and p-ERM proteins were present in rat PMVEC. Stimulation withTNF-α gradually up -regulated the level of p-ERM proteins in a dose -dependent manner [0 μg/LTNF-α group: (0.648 ± 0.102) , 0.1 μg/LTNF-α group: (0.728 ±0.082) , 1 μg/LTNF-α group: (0.926 ±0.121) , 10 μg/LTNF-α group: (1.245 ± 0.134) , allP =0.000 ]. In time -dependent group, the level of p -ERM proteins rose at 15 min (0.777 ± 0.151) , peaked at 90 min (1.295 ± 0.176) , then decreased gradually at 120 min (0.802 ± 0.139) , but stayed higher level at 180 min (0.669 ± 0.128) than that in un -stimulated 0 min group (0.631 ±0.123 , P =0.004 , 0.000 , 0.001, 0.016 , respectively). When PMVEC pre -incubated with ROCK inhibitor and TNF -α, the level of p -ERM proteins caused a marked attenuation of TNF-α stimulation [(0.634 ±0.112) vs. (0.875 ±0.164) , P =0.002 ] , however, there are no significant differences compared to ROCK inhibitor alone group (0.661 ±0.108) and no intervention group (0.654 ± 0.125) , P =0.973 , P =0.900 , respectively). Conclusions TNF-α could induce up -regulation of the level of the phosphorylated ERM proteins in rat PMVEC, and ROCK signal molecules might involve in modulation of the ERM proteins phosphorylation. Key words: TNF-α; Pulmonary microvascular endothelial cell; Ezrin-Radixin-Moesin; Inflammation; Acute respiratory distress syndrome; Rho kinase

Identification of Two Forms of TNF Tolerance in Human Monocytes: Differential Inhibition of NF-κB/AP-1– and PP1-Associated Signaling

The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser(536), p38, and c-jun was inhibited, and basal inhibitory p65-Ser(468) phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1-dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1-associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.

P306 Tryptophan deficiency in Crohn's disease

tuberculosis infection was confirmed by positive culture to Mycobacterium tuberculosis. Results: 225 patients received TNF alpha drugs: 136 male (60.4%), 173 (76.9%) Crohn’s disease. No patient had suffered from previous lung diseases. Tuberculin cutaneous test was documented in 190 patients, and Booster re-test in 100. Latent tuberculosis was diagnosed in 20 and 1 patients whether TCT or Booster were >5mms respectvely and all of them received oral isoniacide prophylaxis (9.3%). Seven patients suffered from active tuberculosis infection (3.1%): 4 male (57.1%) mean age 49.6 years 57.1% ulcerative colitis. Four of them had negative TCT and Booster but they were on inmunosuppresant but no corticosteroids when these tests were performed, and the other 3 patients had received oral quimioprofilaxis (3 6 months) because TCT was positive. Time from last TNF dose to symptoms and from symptoms to tuberculosis diagnose were and days respectively. Active tuberculosis were generally disseminated (85.5%) and required hospitalization until diagnose and clinical stabilization. Multidrug therapy was continued for 6 8 months but one patient eventually died. Anti TNF-alpha was no restarted in these patients. Conclusions: Active tuberculosis in IBD patients on antiTNF therapy in a high prevalence area was 3.1%. The infection was generally disseminated, required hospitalizations and even caused death. TCT test and previous oral quimioprofilaxis failed to eliminate risk of active infection in these patients, so any other tests or preventive measures should be develop.

Use of tumor necrosis factor receptor 2 (TNFR2)/p75 and TNF-α as an antitumor strategy.

e22010 Background: TNF-α has two receptors, TNFR1/p55 and TNFR2/p75, with diverse signaling roles: p55 - predominantly cytotoxic and p75 - prosurvival and proangiogenic. Compared to WT and p55KO/p75KO, growth of tumors in p75 knockout (KO) mice was decreased &gt;50% in Lewis lung carcinoma (LLC) and B16 melanomas (Sasi et al., Oncogene, 2012). This was associated with increased apoptosis and decreased capillary density in tumos. We hypothesized that selective blocking of p75 receptor in tumors may sensitize them to TNF induced apoptosis. Methods: p75 receptor was knockdown (KD) in LLCs using shRNA. LLC and p75KD LLCs were implanted into WT mice flanks. On day 8 post-inoculation, mouse recombinant (mr) TNF (12.5 ng/g/injection) or saline was injected x2 daily for 6 days. Tumor volumes (tV) were measured daily and tumor weights (tW) on day 16. Tumors, tubular bones and spleens were examined by 2 pathologists blindfolded to treatments. Results: Mouse weights remianed stable throughout the study in all groups. There was no difference in tV or tW between LLC minus(-) TNF and p75KD LLC-TNF tumors on day 16 (2 d days after last injection). Compared to 3 control groups, p75KD LLC+TNF showed &gt;2-5-fold decreases in tV (p&lt;0.001, n=8) and tW (p&lt;0.0001, n=8). There was NO difference in tV or tW (p=ns) end of study vs before injections in p75KD LLC+TNF group whereas in 3 control groups tV and tW were increased 2.7-, 4.5- and 3.2-fold (p&lt;0.01, p&lt;0.0002 and p&lt;0.0001). Pathology revealed that 1/3 of tumors in p75KD LLC+TNF group were nearly 100% necrotic. Bone marrow and spleens did not show significant TNF-induced systemic toxic effect. There was an inflammatory response in the BM and spleens of mice with necrotic tumors in +TNF group, reflected by shift of myeloid/erythroid radio in the BM and moderate increases in lymphoid follicles in the spleen. Conclusions: Our results suggest that blocking p75 expression in tumor tissue combined with administration of small exogenous doses of TNF significantly inhibits aggressive LLC tumor growth and induces nearly 100% tumor necrosis in 1/3 of the experimental group in six days, with no detectible systemic toxicity. This may represent new and effective therapy against lung neoplasms and potentially solid tumors of other origin.

Dual activation of p38MAPK and SFK pathways is required to induce endothelial permeability

We previously showed that activation of endogenous Src family kinases (SFK) by expression of a dominant negative form of Csk (DN‐Csk) is not sufficient to induce changes in endothelial monolayer permeability, but allows low doses of TNF to increase endothelial permeability. This increase induced by joint SFK activation and TNF treatment is attenuated by addition of p38 inhibitors. Thus, we hypothesized that p38MAPK activation may induce an increase in permeability of ECs already primed by activation of SFKs. To test this, we constructed a lentiviral vector to express a flag tagged constitutively active form of MKK6 under the control of a tetracycline‐regulated promoter (iMKK6E). Addition of doxycyclin (dox) to infected cells induced a dose‐dependent increase in MKK6E expression resulting in the phosphorylation and nuclear translocation of p38 as well as HSP27 phosphorylation. This treatment did not induce any change in permeability. In contrast, MKK6E expression resulted in a large drop in TEER and an increase in albumin clearance in cells with SFK activation. This was accompanied by the formation of large gaps in the monolayer but no change in actin fibers. Our results support a model in which dual activation of SFKs and p38 are required for the induction of endothelial permeability. This model could explain the tight spatio‐temporal response to inflammatory mediators during an inflammatory response.

A Protein Turnover Signaling Motif Controls the Stimulus-Sensitivity of Stress Response Pathways

Stimulus-induced perturbations from the steady state are a hallmark of signal transduction. In some signaling modules, the steady state is characterized by rapid synthesis and degradation of signaling proteins. Conspicuous among these are the p53 tumor suppressor, its negative regulator Mdm2, and the negative feedback regulator of NFκB, IκBα. We investigated the physiological importance of this turnover, or flux, using a computational method that allows flux to be systematically altered independently of the steady state protein abundances. Applying our method to a prototypical signaling module, we show that flux can precisely control the dynamic response to perturbation. Next, we applied our method to experimentally validated models of p53 and NFκB signaling. We find that high p53 flux is required for oscillations in response to a saturating dose of ionizing radiation (IR). In contrast, high flux of Mdm2 is not required for oscillations but preserves p53 sensitivity to sub-saturating doses of IR. In the NFκB system, degradation of NFκB-bound IκB by the IκB kinase (IKK) is required for activation in response to TNF, while high IKK-independent degradation prevents spurious activation in response to metabolic stress or low doses of TNF. Our work identifies flux pairs with opposing functional effects as a signaling motif that controls the stimulus-sensitivity of the p53 and NFκB stress-response pathways, and may constitute a general design principle in signaling pathways.

Isolated limb perfusion with the tumor‐targeting human monoclonal antibody–cytokine fusion protein L19‐TNF plus melphalan and mild hyperthermia in patients with locally advanced extremity melanoma

L19-TNF is a tumor-targeting immunocytokine composed of the human L19 antibody binding to extra domain B (ED-B) of fibronectin of newly formed blood vessels, and of human TNF. This exploratory trial evaluates safety and clinical activity of L19-TNF plus melphalan-containing isolated limb perfusion (ILP) in extremity melanoma patients. Seven and 10 patients received 325 µg and 650 µg of L19-TNF, respectively, during the ILP. Patients were studied for safety, tolerability, and clinical activity of this experimental L19-TNF ILP procedure. Non-hematologic toxicity of L19-TNF ILP was very low, but severe myelosuppression was seen in four patients. Although L19-TNF was administered at a TNF-equivalent dose of only 3.13 and 6.25% of the approved TNF (Beromun®) dose of 4 mg, L19-TNF ILP induced objective responses in 86 and 89% of patients, respectively, including a complete response (CR) in 5/10 patients treated with L19-TNF ILP at 650 µg that was durable at 12 months in four patients. No CR was seen at 325 µg of L19-TNF. ILP with L19-TNF had a favorable safety and a promising activity profile at a dose of 650 µg of L19-TNF, supporting the exploration of higher L19-TNF doses and a Phase II trial comparing L19-TNF ILP with standard melphalan-containing ILP.

Abstract SY27-02: In vitro and in vivo regulation of regulated necrosis induced by TNF

Abstract Cells can die by multiple ways. Two prototypes of regulated cell death have been put forward viz. apoptosis and programmed necrosis. Nowadays, programmed necrosis, also called regulated necrosis or necroptosis (1), is recognized as a highly controlled form of necrotic cell death (2). Using the L929 fibrosarcoma cellular system of TNF-induced necrotic cell death, we identified crucial subcellular events during necroptosis such as mitochondrial ROS production and lysosomal membrane permeabilization (LMP) (3). The initial events that lead to TNF-induced necrosis involve the activation of the serine threonine kinases RIPK1 and RIPK3, which initiate the formation of the socalled necrosome complex due to homotypic interaction between the RHIM domains. Also other stimuli such as TLR3 and TLR4 ligands have been shown to require RIPK3 for regulated necrosis in macrophages, while in this cellular context RIPK1 fullfils a survival function (4). Although a direct link between RIPK1/RIPK3 kinase activity and the downstream subcellular events at the mitochondrial and lysosomal level remain to be established, at the moment more is known on the regulation of the formation of necrosome complex containing FADD, caspase-8, RIPK1 and RIPK3. This TNF-induced complex formation is highly regulated by ubiquitylation/deubiquitylation balances involving ubiquitylating (a.o. IAP, A20) and deubiquitylating enzymes (CYLD), kinases (a.o. RIP1, RIP3, TAK1) and caspase activating platforms (FADD, FLIPL, caspase-8) (5,6). Recently, other stimuli like etoposide and Smac mimetics where shown to induce spontaneously a cytosolic complex called complex called ripoptosome independent of endogenous TNF production and containing FADD, caspase-8, RIPK1 and RIPK3 (7,8). These regulatory mechanisms involving ubiquitylation, phosphorylation and caspase-8-mediated proteolysis desensitize/sensitize and retard/accellerate necroptosis, like brakes and gears on the necrotic program. They determine the cellulular outcome being survival, apoptosis or necroptosis, and apparently also operate in vivo (9,10,11) and are involved in hyperinflammation in gut (12,13) and skin (14). We studied whether the regulatory mechanisms of TNF-induced necroptosis identified in vitro were also applicable for in vivo. Therefore we injected high dose of TNF causing systemic inflammatory response syndrome (SIRS), resulting in septic shock. We demonstrated that deletion of apoptotic executioner or inflammatory caspases had no impact on lethal SIRS despite lowered levels of apoptosis or circulating IL-1β. In contrast, deletion of the RIPK3 gene conferred complete survival and protection against necrotic cell death, reflected by reduced levels in the serum of mitochondrial DNA, lysosomal enzymes and other markers of organ damage. Interesting, also circulating inflammatory cytokines such as IL-6 and IL-1 were dramatically lowered in RIPK3 knockout mice, demonstrating that induction of necroptosis is preceding excessive inflammatory cytokine production. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, had a similar protective effect on mortality and resulted in reduced levels of markers of organ damage and circulating inflammatory cytokines. These results demonstrate that RIPK1/RIPK3-mediated necroptosis plays an indispensable role in TNF-induced SIRS as the determinant between life and death. The crucial role of necroptosis in SIRS in infectious sepsis was further underscored by the protective effect of RIPK3 deficiency in caecal ligation puncture (CLP) model. Altogether, our findings demonstrate that regulated necrosis or necroptosis in vivo is a crucial process and that components of the necroptotic cell death pathway are potential therapeutic targets for treatment of SIRS and sepsis (15).

IL-1Ra: its role in rheumatoid arthritis

Interleukin-1 (IL-1) is one of the pivotal cytokines in initiating and driving the processes of rheumatoid arthritis (RA), and the body's natural response, IL-1 receptor antagonist (IL-1Ra), has been shown conclusively to block its effects. IL-1 mediate several clinical symptoms of the inflammatory reaction (i.e. fever, pain, sleep disturbances). IL-1 is considered a key mediator in RA joint damage because of its greater capacity (greater than TNF) of increasing matrix degradation by inducing the production of MMPs and PGE2 in synovial cells, as well by its role as mediator of bone and cartilage destruction. In addition, IL-1 decreases the repair process by suppressing matrix synthesis and shows a strong synergism with TNF in inducing many inflammatory genes at both local and systemic level. The induced endogenous production of IL-1Ra, in presence of the RA synovitis, is too low to contrast the high affinity of IL-1 for the cell receptors. Therefore, IL-1Ra presence should result in very effective prevention of IL-1 signal transduction particularly in the inflammatory site. In laboratory and animal studies inhibition of IL-1 by either antibodies to IL-1 or IL-1Ra proved beneficial to the outcome. IL-1Ra is a member of the IL-1 superfamily. The effects of different DMARDs on IL-1Ra levels in RA patients support the important role that selected anticytokine treatments might exert in the pathophysiology of the disease. However, since anti TNF-alpha therapy it is not effective in all RA patients, nor does it fully control the arthritic process in affected joints of good responders and complete TNF suppression should be avoided, the combined treatment with intermediate doses of TNF and IL-1 blockers, reaching synergistic suppression of arthritis, seems warranted in RA.

In TNF-stimulated Cells, RIPK1 Promotes Cell Survival by Stabilizing TRAF2 and cIAP1, which Limits Induction of Non-canonical NF-κB and Activation of Caspase-8

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1(-/-) cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

TNF‐alpha signaling collaborates with Src family kinases (SFK) to promote actin rearrangement and loss of barrier function in endothelial cells

TNF and SFKs have been implicated in the regulation of barrier function and tissue damage, including acute lung injury induced by LPS. We previously showed that activation of SFKs by overexpression of DN-Csk did not increase by itself transendothelial cell electric resistance (TEER). We have not observed any activation of Src following the addition of TNF. Interestingly, we found that TNF synergizes with SFK to reduce TEER. A low dose of TNF (0.05 ng/ml) did not decrease TEER in control cells, but induced a strong decrease in cells expressing DN-Csk. LacZ- and DN-Csk-expressing cells contain a cortical actin belt and very few stress fibers (SF). Low dose TNF produced thin SF but no loss in cortical actin present at the cell-cell border. However, in cells expressing DN-Csk, TNF induced short and very thick bundles of actin and promoted the shuttling of phosphorylated Src, paxillin, FAK, PYK2 and integrin beta3 to aggregates localized at the ends of the actin bundles in a configuration that resembled v-Src-overexpressing cells, suggesting and association between actin rearrangements and TEER. Blockade of these pathways may thus prove to be beneficial for the treatment of acute lung injury. Supported by grant RO1 HL77870

TNF dose reduction and shortening of duration of isolated limb perfusion for locally advanced soft tissue sarcoma of the extremities is safe and effective in terms of long‐term patient outcome

Dose reduction and shortening of duration of perfusion in isolated limb perfusion with TNF-α and Melphalan (TM-ILP) are associated with less systemic toxicity and seem to be safe and effective on short-term. However, data on long-term patient outcome are scarce. From 1991 to 2008, 102 TM-ILPs were performed in 98 patients for a locally advanced soft tissue sarcoma of the extremity. Perfusions were categorized in three groups: (A) high dose and long duration (n = 59), (B) high dose and short duration (n = 16), and (C) low dose and short duration (n = 27). Long-term local control rates and (limb)-survival were evaluated. Limb salvage rates were in group A 76.3%, B 62.5%, and C 85.2% (P = 0.2). With a median follow up of 76 (range 4-203) months, 50 patients were still alive (51%). Disease-specific 5-year survival was not different between the three groups: A 55.4%, B 52.5%, and C 57.3% (P = 0.9). There was no difference in local recurrence-free 5-year survival (adjusted P = 0.1) and distant metastases-free survival (P = 0.9). Dose reduction and shorter duration of TM-ILP seem to be safe and effective regarding long-term patient outcome, as 5-year local control rates and (limb)-survival are not compromised.

The influence of tumor necrosis factor α (TNF) on the secretory function of bovine corpus luteum: TNF and its receptors expression during the estrous cycle

Tumor necrosis factor alpha (TNF) inversely regulates the function of bovine corpus luteum (CL). Whereas the low doses of TNF induce luteolysis, the high doses prolong CL lifespan and prevent luteolysis in vivo. We suggest that the varying effects of TNF may be caused by its action exerted on CL via multiple signaling pathways involving two distinct receptors: TNFR-I (responsible for induction of the cell death) and TNFR-II (implicated in cell proliferation). In the study, we determined CL expressions of TNF, TNFR-I and TNFR-II mRNAs during the bovine estrous cycle using semi-quantitative RT-PCR. Specific transcripts for TNF, TNFR-I and TNFR-II were found in the CL with the highest (p<0.05) expression in the regressed CL. We also examined the TNF influence on the bovine CL function in vivo. On Day 15 of the estrous cycle, cows were infused (via aorta abdominalis) with saline, TNF (1 or 10 microg) or analogue of prostaglandin (PG)F(2alpha) (aPGF(2alpha) , 500 microg; a positive control). Four hours after infusions, CLs were collected by colpotomy and luteal contents of progesterone (P(4)), stable metabolites of nitric oxide (NO; nitrite/nitrate), leukotriene (LT)C(4), luteolytic PGF(2alpha),and luteotropic PGE(2) were determined. Moreover, caspase-3 activity was measured in the CLs as an indicator of apoptosis induction. The luteal content of P(4) decreased (p<0.05) after infusion of 1 microg of TNF. TNF inversely affected PGs content in CL: the low dose increased (p<0.01) the PGF(2alpha) level and the high dose increased (p<0.05) PGE(2) level. Contents of LTC(4) and nitrite/nitrate increased (p<0.01) after the low dose of TNF. Moreover, 1 microg of TNF induced apoptosis and increased (p<0.05) caspase-3 activity in the CLs collected during the late luteal phase. In conclusion, the high expressions of TNF and TNF receptors mRNAs were observed during or just after the luteolysis. A low concentration of TNF stimulated in vivo luteolytic factors such as PGF(2alpha), LTC(4) and NO as well as induced apoptosis; whereas the high concentration of TNF stimulated a survival pathway in the bovine CL increasing luteal content of P(4) and PGE(2).

Females Exhibit Relative Resistance to Depressive Effects of Tumor Necrosis Factor-α on the Myocardium

Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in myocardial dysfunction following acute injury. It is unknown, however, if a gender-specific response to TNF infusion exists in isolated rat hearts. Elucidating such mechanisms is important to understanding the myocardial gender differences during acute injury. We hypothesize that females will exhibit a relative resistance to TNF-induced myocardial dysfunction compared to males and that menstrual cycle would influence the degree of female myocardial resistance to TNF-induced myocardial functional depression. Adult male, proestrus female, and metestrus/diestrus female hearts were subjected to 60 min of TNF infusion at 10,000 pg/mL.min via Langendorff. Myocardial contractile function (left ventricular developed pressure, and the positive/negative first derivative of pressure) was continuously recorded. 10,000 pg/mL.min of TNF markedly depressed myocardial function in males compared with other doses of TNF. Myocardial function was significantly decreased in males compared to females following TNF infusion. Additionally, both the proestrus and the metestrus/diestrus females exhibited equal resistance to TNF-induced myocardial dysfunction. Our study shows that females exhibit a significantly greater degree of resistance to TNF-induced myocardial depression. Moreover, data from this study suggest that fluctuations in estrogen during the reproductive cycle may have little to no influence on TNF-induced myocardial depression.

TNF and manipulation of the tumor cell - stromal interface: "ways to make chemotherapy effectiv"

Growth of solid tumors depends largely on the development of a functional vasculature, which has been the focus in anti-tumor therapy since Folkman in 1971 proposed that prohibiting the formation of new vessels could inhibit tumor growth. The recognition of the tumor vascular bed as an important target led to the development of 3 vascular-targeted strategies. I) The anti-angiogenesis strategy that prevents the formation of new blood vessels and normalizes the remaining vessels. II) Applying vasculo-destructive agents to induce apoptosis in the endothelium of the tumor-associated vasculature that results in vascular collapse and tumor necrosis. III) Promoting further abnormalization of the already abnormal features of the tumor-associated vasculature with vaso-active agents to enhance vessel permeability. Tumor necrosis factor alpha (TNF) is a very promising vaso-active agent because of its anti-tumor effects but its severe systemic toxicity is a major drawback. Therefore a new setting, in which the optimal therapeutic benefit of TNF could be exploited, needed to be found. Through an isolated perfusion high dose of TNF can be administered in the blood circulation of the tumor-bearing extremity or organ. Alternatively, systemically low doses can be safely administered for several times. Importantly, TNF has no anti-tumor effect by itself and the combination with a conventional chemotherapeutic drug that targets the tumor cell is a prerequisite for a good tumor response. In this dual approach, TNF enhances intratumoral accumulation of the chemotherapeutic drug resulting in an impressive tumor response.