Tumor Necrosis Factor Toxicity Research Articles

TNFR1-induced lethal inflammation is mediated by goblet and Paneth cell dysfunction.

Tumor necrosis factor (TNF) is a powerful activator of the immune system and a well-validated target for treatment of autoimmune diseases. Injection of TNF induces systemic lethal inflammation characterized by hypothermia, induction of multiple cytokines, and extensive damage to multiple organs. Previously, we reported that TNF-induced lethal inflammation is strictly TNFR1(P55)-dependent. We also uncovered a crucial role for P55 expression levels in intestinal epithelial cells (IECs), in which P55+/+ expression is sufficient to sensitize to TNF lethality in an otherwise fully protected P55+/- background. Here, we investigated the molecular mechanism that drives TNF toxicity in IECs. Unexpectedly, we found that the degree of TNF-induced enterocyte damage and apoptosis in IECs is equally strong in TNF-sensitive P55+/+ mice and TNF-resistant P55+/- mice. Our results suggest that P55+/+-induced signaling causes goblet and Paneth cell dysfunction, leading to severe epithelial barrier dysfunction. As a result, intestinal permeability and systemic bacterial spread are induced, causing lethal systemic inflammation. In conclusion, we identified P55-induced goblet and Paneth cell dysfunction as a crucial mechanism for TNF-induced systemic and lethal inflammation.

Antisense oligonucleotides against TNFR1 prevent toxicity of TNF/IFNγ treatment in mouse tumor models

Tumor necrosis factor (TNF) has remarkable antitumor effects, but its systemic therapeutic use is prevented by its lethal inflammatory effects. TNFR1 (P55) is essential for both the antitumor and toxic effects because both of them are absent in P55-deficient mice. In previous work we demonstrated that P55+/- mice are completely resistant to TNF toxicity, while the antitumor effects induced by TNF combined with interferon gamma (IFNγ) remain fully functional in these mice. Hence, a high dose of TNF/IFNγ has an excellent therapeutic potential when P55 levels are reduced, because TNF induces tumor regression without systemic toxicity. Here, we provide proof of principle for therapeutic application of this approach by using antisense oligonucleotides (ASOs). Treatment of mice with ASOs targeting P55 resulted in a strong reduction in P55 protein levels in liver, small intestine and blood mononuclear cells. This P55 downregulation was associated with significant protection of mice against acute TNF toxicity as measured by hypothermia, systemic inflammation and lethality. This treatment also protected mice against toxicity of TNF/IFNγ treatment in several cancer models: B16Bl6, Lewis lung carcinoma and a lung colony model. Our results confirm the therapeutic value of this strategy, which could lead to the development of a safer and more effective TNF/IFNγ antitumor therapy.

Induction of autophagy, a promising approach for treating liver injury

Recent investigations have demonstrated a complex interrelationship between autophagy and cell death. A common mechanism of cell death in liver injury is tumor necrosis factor (TNF) cytotoxicity. To better delineate the in vivo function of autophagy in cell death, we examined the role of autophagy in TNF-induced hepatic injury. Atg7Δhep mice with a hepatocyte-specific knockout of the autophagy gene atg7 were generated and cotreated with D-galactosamine (GalN) and lipopolysaccharide (LPS). GalN/LPS-treated Atg7Δhep mice had increased serum alanine aminotransferase levels, histological injury, numbers of TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling)-positive cells and mortality as compared with littermate controls. Loss of hepatocyte autophagy similarly sensitized to GalN/TNF liver injury. GalN/LPS injury in knockout animals did not result from altered production of TNF or other cytokines. Atg7Δhep mice had accelerated activation of the mitochondrial death pathway and caspase-3 and −7 cleavage. Increased cell death did not occur from direct mitochondrial toxicity or a lack of mitophagy, but rather from increased activation of initiator caspase-8 causing Bid cleavage. GalN blocked LPS induction of hepatic autophagy, and increased autophagy from beclin 1 overexpression prevented GalN/LPS injury. Autophagy, therefore, mediates cellular resistance to TNF toxicity in vivo by blocking activation of caspase-8 and the mitochondrial death pathway, suggesting that autophagy is a therapeutic target in TNF-dependent tissue injury.

BDNF protects human vascular endothelial cells from TNFα-induced apoptosis

Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation that directs the quality of tissue repair. In this study, we investigated the anti-apoptotic effect of BDNF against the toxicity of tumor necrosis factor α (TNFα), which is known for its pro-apoptotic action in human microvascular endothelial cells (HMVECs). We demonstrate that BDNF attenuates TNFα-increased Annexin V-positive cells, lactic dehydrogenase (LDH) release, and intercellular adhesion molecule 1 (ICAM-1) mRNA and cleaved caspase-3 expression. In addition, biochemical analyses indicate that TNFα increases phosphatase and tensin homolog (PTEN) expression; however, it decreases phosphorylated PTEN. BDNF did not affect PTEN expression, but it did increase the phosphorylation of PTEN. BDNF-induced Akt phosphorylation was inhibited by TNFα. Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay showed that the PTEN inhibitor bpV(pic) rescues HMVECs from TNFα-induced apoptosis. In conclusion, BDNF protects HMVECs from toxicity of TNFα through the regulation of the PTEN/Akt pathway.

Autophagy protects from liver injury

Autophagy is understood as a mechanism by which subcellular debris are recycled and metabolic intermediates produced for maintaining cellular homeostasis. Moreover, autophagy can be induced under stress conditions with the goal of cell survival. On the other hand, autophagy also has a role in the cell death pathways, and the existence of a complex cross-talk between autophagy and apoptosis has recently emerged.1 For instance, key proteins originally thought to be apoptosis-specific inhibitors are also able to block autophagy,2 while some proteases involved in apoptosis block autophagy by cleaving autophagy-regulating proteins.3 In still other cases, the autophagy-related (ATG) proteins can be converted into a pro-apoptotic factor4 or can directly bind FAS-associated protein with death domain to support apoptosis.5 Unfortunately, all these findings currently fail to provide a consistent interpretation for the general function of autophagy in the apoptosis pathways. Nonetheless, an understanding of the molecular mechanisms regulating the interplay between autophagy and apoptosis promises to provide new hints on how to modulate the cell death pathways under pathological conditions such as cancer, neurodegenerative, or chronic inflammatory disorders. In this issue of the Journal, Amir et al.6 report the development of a mouse line exhibiting an inducible, hepatocyte-specific lack of the key ATG gene atg7. These mice have been challenged with 𝒟-galactosamine (GalN) and lipopolysaccharide (LPS), both of which are stimuli commonly used to study tumor necrosis factor (TNF)-dependent liver injury. With the purpose of studying autophagy in TNF-induced cytotoxicity in the liver, these investigators found that autophagy protects the hepatocytes from TNF-dependent cell death. Mice with atg7-deficient hepatocytes showed a higher rate of hepatocytic apoptosis, liver injury, and mortality as compared with the wild-type mice following combined GalN and LPS treatment. Interestingly, these effects were not due to altered, LPS-induced cytokine production by macrophages in the liver, because TNF-α, IFN-γ, IL-6, and IL-1β levels were not altered as a consequence of the ATG7 deficiency. This suggests that the accelerated TNF-induced toxicity in such knockout mice resulted from an inhibition of autophagy. Furthermore, atg7−/− mice showed features of an accelerated extrinsic apoptotic pathway in liver cells, including caspase-8 activation, increased expression of truncated Bid, and cytochrome-c release, as well as caspase-3, caspase-7, and poly (ADP-ribose) polymerase cleavage. It had previously been shown that overactivation of c-Jun N-terminal kinase (JNK) can activate caspase-8, resulting in increased TNF-dependent cell death.7 Amir et al.6 observed increased phosphorylated JNK and its downstream target c-Jun in the atg7−/− mice upon GalN/LPS stimulation, indicating that autophagy protects TNF toxicity by blocking JNK activity (Figure 1). Figure 1 Autophagy overlaps with the extrinsic apoptosis pathway in liver cells. Deficient autophagy results in overactivation of JNK, which increases caspase-8 activation and allows the induction of TNF-dependent apoptosis. How autophagy regulates JNK activity ... Interestingly, this study also showed that GalN can inhibit LPS-induced autophagy by blocking the activation of adenosine monophosphate-activated protein kinase, which in turn positively regulates autophagy. Increased autophagy resulting from Beclin 1 overexpression prevented GalN/LPS-induced liver injury. This suggests that the toxicity caused by GalN is at least partially dependent on the inhibition of autophagy. Because autophagy is crucial for the survival of the GalN/LPS-sensitized hepatocytes, its inhibition leads to extra cell death and tissue injury in the knockout mice. Therefore, it seems likely that it is the autophagic suppression of apoptosis that helps the liver cells to survive by delaying the onset of death signals under toxic conditions. Hence, upregulating the autophagic pathway might be an attractive therapeutic strategy in several inflammatory liver diseases.

Inhibition of hepatocyte autophagy increases tumor necrosis factor-dependent liver injury by promoting caspase-8 activation.

Recent investigations have demonstrated a complex interrelationship between autophagy and cell death. A common mechanism of cell death in liver injury is tumor necrosis factor (TNF) cytotoxicity. To better delineate the in vivo function of autophagy in cell death, we examined the role of autophagy in TNF-induced hepatic injury. Atg7Δhep mice with a hepatocyte-specific knockout of the autophagy gene atg7 were generated and cotreated with D-galactosamine (GalN) and lipopolysaccharide (LPS). GalN/LPS-treated Atg7Δhep mice had increased serum alanine aminotransferase levels, histological injury, numbers of TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling)-positive cells and mortality as compared with littermate controls. Loss of hepatocyte autophagy similarly sensitized to GalN/TNF liver injury. GalN/LPS injury in knockout animals did not result from altered production of TNF or other cytokines. Atg7Δhep mice had accelerated activation of the mitochondrial death pathway and caspase-3 and -7 cleavage. Increased cell death did not occur from direct mitochondrial toxicity or a lack of mitophagy, but rather from increased activation of initiator caspase-8 causing Bid cleavage. GalN blocked LPS induction of hepatic autophagy, and increased autophagy from beclin 1 overexpression prevented GalN/LPS injury. Autophagy, therefore, mediates cellular resistance to TNF toxicity in vivo by blocking activation of caspase-8 and the mitochondrial death pathway, suggesting that autophagy is a therapeutic target in TNF-dependent tissue injury.

The αVβ3/αVβ5 integrin inhibitor cilengitide augments tumor response to melphalan isolated limb perfusion in a sarcoma model

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)-α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.

Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons.

BackgroundDopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinson’s disease (PD) in part because their oxidative environment in the substantia nigra (SN) may render them vulnerable to neuroinflammatory stimuli. Chronic inhibition of soluble Tumor Necrosis Factor (TNF) with dominant-negative TNF inhibitors protects DA neurons in rat models of parkinsonism, yet the molecular mechanisms and pathway(s) that mediate TNF toxicity remain(s) to be clearly identified. Here we investigated the contribution of ceramide sphingolipid signaling in TNF-dependent toxicity.ResultsCeramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation. Using lipidomics mass spectrometry we confirmed that TNF treatment not only promotes generation of ceramide, but also leads to accumulation of several atypical deoxy-sphingoid bases (DSBs). Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons.ConclusionsWe conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons. Ceramide and atypical DSBs may represent novel drug targets for development of neuroprotective strategies that can delay or attenuate the progressive loss of nigral DA neurons in patients with PD.

Astrocytes promote TNF-mediated toxicity to oligodendrocyte precursors

Neuroinflammation and increased production of tumor necrosis factor (TNF) in the CNS have been implicated in many neurological diseases including white matter disorders periventricular leukomalacia and multiple sclerosis. However, the exact role of TNF in these diseases and how it mediates oligodendrocyte injury remain unclear. Previously, we demonstrated that lipopolysaccharide (LPS) selectively kills oligodendrocyte precursors (preOLs) in a non-cell autonomous fashion through the induction of TNF in mixed glial cultures. Here, we report that activation of oligodendroglial, but not astroglial and microglial, TNFR1 is required for LPS toxicity, and that astrocytes promote TNF-mediated preOL death through a cell contact-dependent mechanism. Microglia were the sole source for TNF production in LPS-treated mixed glial cultures. Ablation of TNFR1 in mixed glia completely prevented LPS-induced death of preOLs. TNFR1-expressing preOLs were similarly susceptible to LPS treatment when seeded into wildtype and TNFR1(-/-) mixed glial cultures, demonstrating a requirement for oligodendroglial TNFR1 in the cell death. Although exogenous TNF failed to cause significant cell death in enriched preOL cultures, it became cytotoxic when preOLs were in contact with astrocytes. Collectively, our results demonstrate oligodendroglial TNFR1 in mediating inflammatory destruction of preOLs and suggest a previously unrecognized role for astrocytes in promoting TNF toxicity to preOLs.

Purification and characterization of RGD tumor-homing peptide conjugated human tumor necrosis factor α over-expressed in Escherichia coli

A number of approaches have been investigated to enhance the selective toxicity of tumor necrosis factor α (TNFα) to permit its systemic use in cancer therapy. Because vascular targeting has been proven to be a valid strategy for improving the therapeutic index of TNFα, we prepared RGD-hTNF consisting of human TNF fused with the ACDCRGDCFCG peptide, a ligand of α vβ 3 and α vβ 5 integrins. Recombinant RGD-hTNF was produced in Escherichia coli as a polyhistidine fusion protein. Between polyhistidine tag and RGD-hTNF, a tobacco etch virus (TEV) protease cleavage site (ENLYFQG) was introduced to ensure the release of intact RGD-hTNF. The purification strategy consisted of the target protein capture step by immobilized metal affinity chromatography (IMAC), TEV protease cleavage of fusion protein, the subtractive depletion of removed His-tag by IMAC and the final gel filtration step. As a result, about 18 mg of intact RGD-hTNF was obtained from 1 l of bacteria culture. The purified RGD-hTNF was characterized by SDS-PAGE, Western blot, mass spectroscopy and gel filtration. Since the RGD-hTNF molecule retained the cytotoxic activity of the TNF moiety and the integrin binding ability of the RGD moiety, the purification method provided material for assessing its anti-tumor activity in animal model.

Survival of TNF toxicity: Dependence on caspases and NO

Tumor necrosis factor (TNF) is an endogenous pro-inflammatory cytokine, implicated in pathologies such as rheumatoid arthritis and septic shock. It was originally discovered as a factor with extraordinary antitumor activity, but its shock-inducing properties still prevent its systemic use in cancer. Clinical trials revealed hypotension as the major dose-limiting factor of TNF toxicity. When administered to mice, TNF provokes a lethal shock syndrome, where cardiovascular collapse is centrally orchestrated by nitric oxide (NO). Nevertheless, NO synthase (NOS) inhibition in animal models and septic shock patients could not improve and even aggravated outcome, suggesting a bivalent role for NO. Lymphocyte and enterocyte apoptosis has been described in septic, endotoxemic, or TNF-treated animals, as well as in septic patients. In this review, we describe our recent studies on the role of NO and caspases in TNF-induced shock in mice. In summary, we have found that both NO and caspases may exert unexpected and dual functions during TNF shock. Whereas excessive NO production provokes lethal hypotension, it also has an important anti-oxidant function, protecting organs from oxidative stress and lipid peroxidation. In addition, our results also indicate that caspases may exert an important endogenous negative feedback on oxidative stress as well.

TNF-alpha induces proliferation or apoptosis in human saphenous vein smooth muscle cells depending on phenotype.

Tumor necrosis factor (TNF)-alpha is implicated in development of restenotic and atherosclerotic vascular lesions, which are pathological processes involving both proliferation and apoptosis of vascular smooth muscle cells (VSMCs). Human VSMCs were recently found to contain heterogeneous subpopulations. We therefore examined whether TNF has different effects on distinct subpopulations of VSMCs. With the use of cloning techniques, two stable subpopulations of VSMCs were isolated from human saphenous vein: spindle- and epithelioid-shaped smooth muscle cells (Sp- and Ep-SMCs, respectively). We found that TNF stimulated growth in Sp-SMCs but had a toxic effect on Ep-SMCs. TNF did not induce apoptosis in Sp-SMCs as determined by nuclear staining and cellular DNA electrophoresis. In contrast, the reduction of viability in Ep-SMCs was associated with induction of apoptosis as characterized by cellular DNA fragmentation and nuclear condensation. Higher levels of the TNF-R1 receptor subtype were detected in membrane preparations from Ep-SMCs than in membranes from Sp-SMCs. Activation of caspase-3 was also selectively induced in Ep-SMCs but not in Sp-SMCs. Cycloheximide, an inhibitor of protein synthesis, enhanced the toxicity of TNF in Ep-SMCs. This effect of cycloheximide was not seen in Sp-SMCs. The data presented here demonstrate for the first time that TNF either promotes growth or induces apoptosis in human VSMCs depending on phenotype.

Toxicity of tumor necrosis factor (TNF)-α on human spermatozoa − Possible role in endometriosis associated infertility

TNF-α is a pleiotropic cytokine with numerous biological functions that include modulation of spermatogenesis and apoptosis in addition to acting as a pro-inflammatory cytokine. Spermatozoa may be exposed to abnormal levels of TNF-α in the male reproductive tract or along the passage in the female reproductive tract in cases of endometriosis. The objective of our study was to examine the toxic effects of TNF-α on human spermatozoa and to establish the minimum concentration that can affect both the sperm function and genomic integrity.

Selective targeted delivery of TNFα to tumor blood vessels

AbstractWe sought to enhance the selective toxicity of tumor necrosis factor α (TNFα) to permit its systemic use in cancer therapy. Because ligand-targeted therapeutics have proven successful in improving the selective toxicity of drugs, we prepared a fusion protein (L19mTNFα) composed of mouse TNFα and a high-affinity antibody fragment (L19 scFv) to the extradomain B (ED-B) domain of fibronectin, a marker of angiogenesis. L19mTNFα was expressed in mammalian cells, purified, and characterized. L19mTNFα was an immunoreactive and biologically active homotrimer. Radiolabeled L19mTNFα selectively targeted tumor neovasculature in tumor-bearing mice, where it accumulated selectively and persistently (tumor-to-blood ratio of the percentage of injected dose per gram [%ID/g] of 700, 48 hours from injection). L19mTNFα showed a greater anticancer therapeutic activity than both mTNFα and TN11mTNFα, a control fusion protein in which an antibody fragment, irrelevant in the tumor model used, substituted for L19. This activity was further dramatically enhanced by its combination with melphalan or the recently reported fusion protein L19-IL2. In conclusion, L19mTNFα allows concentrating therapeutically active doses of TNFα at the tumor level, thus opening new possibilities for the systemic use of TNFα in cancer therapy. (Blood. 2003;102:4384-4392)

Tumor cells expressing membrane-bound tumor necrosis factor activate macrophages and have a compromised growth in immunosuppressed and immunodeficient mice

Tumor necrosis factor (TNF)-α producing tumors as vaccines were demonstrated to induce a therapeutic anti-tumor immune response, but their clinical use is limited by the toxicity of soluble TNF. We investigated the growth characteristics and immunomodulatory properties of HeLa cells producing an uncleavable transmembrane form of TNF (preTNF). The growth of the transformed tumors was compromised in both immunosuppressed and severe combined immunodeficient mice; no signs of TNF toxicity were detected. Macrophages co-cultured with the transformed cells showed increased phagocytosis and cytokine production, indicating that activated macrophages may be the mediators of the anti-tumor effect. preTNF producing tumor cells are promising safe anti-tumor vaccine candidates.

Protection against TNF-Induced Lethal Shock by Soluble Guanylate Cyclase Inhibition Requires Functional Inducible Nitric Oxide Synthase

Hypotension and shock observed in sepsis, SIRS, and tumor necrosis factor (TNF) or cytokine-based cancer treatment are the consequence of excessive nitric oxide (NO) production and subsequent soluble guanylate cyclase (sGC)-mediated vascular smooth muscle relaxation. We demonstrate here that, while NO synthase (NOS) inhibitors exacerbated toxicity, inhibitors of sGC activation protected against TNF-induced lethality, bradycardia, and hypotension. Importantly, sGC inhibition did not interfere with the antitumor activity of TNF. Using NOS inhibitors or iNOS-deficient animals, we furthermore observed that no protection against TNF toxicity could be obtained in the absence of NO. These data imply that iNOS- (and not eNOS-) derived NO is an endogenous protective molecule indispensable to survive a TNF challenge and exerting this beneficial effect via sGC-independent mechanisms.

Reduced Leptin Levels in Starvation Increase Susceptibility to Endotoxic Shock

Malnutrition compromises immune function, reducing resistance to infection. We examine whether the decrease in leptin induced by starvation increases susceptibility to lipopolysaccharide (LPS)- and tumor necrosis factor (TNF)-induced lethality. In mice, fasting for 48 hours enhances sensitivity to LPS. Decreasing the fasting-induced fall in leptin by leptin administration markedly reduced sensitivity to LPS. Although fasting decreases basal leptin levels, LPS treatment increased leptin to the same extent as in fed animals. Fasting increased basal serum corticosterone; leptin treatment blunted this increase. Fasting decreased the ability of LPS to increase corticosterone; leptin restored the corticosterone response to LPS. Serum glucose levels were decreased in fasted mice and LPS induced a further decrease. Leptin treatment affected neither basal glucose nor that after LPS. LPS induced a fivefold greater increase in serum TNF in fasted mice, which was blunted by leptin replacement. In contrast, LPS induced lower levels of interferon-gamma and no differences in interleukin-1beta in fasted compared to fed animals; leptin had no effect on those cytokines. Furthermore, fasting increased sensitivity to the lethal effect of TNF itself, which was also reversed by leptin treatment. Thus, leptin seems to be protective by both inhibiting TNF induction by LPS and by reducing TNF toxicity.

Expression of the 75 kDA TNF receptor and its role in contact-mediated neuronal cell death

We previously demonstrated TNF toxicity, at high TNF doses or in the presence of actinomycin D, in the N1E-115 neuronal cell line (N1Es), which expresses only the 55 kDa TNF receptor (TNFR). To determine whether presence of the 75 kDa TNFR increases N1E sensitivity to TNF toxicity, cells were transfected with a 75 kDa TNFR expression construct. However, 75 kDa TNFR protein expression was undetectable in stably transfected N1Es. Further investigation revealed endogenous membrane-associated TNF in this neuronal line. Co-transfection with β-galactosidase and the 75 kDa TNFR or empty vector (pcDNA3) indicated cell loss in the 75 kDa TNFR-transfected population relative to vector-transfected populations, while inhibition of membrane-associated TNF with a neutralizing antibody led to increased 75 kDa TNFR expression in transiently transfected N1Es. We conclude that neutralization of membrane-associated TNF inhibits its interaction with the introduced 75 kDa TNFR, increasing neuronal survival and promoting 75 kDa TNFR expression. Induced 75 kDa TNFR expression in the presence of membrane-associated TNF and the 55 kDa TNFR results in lymphocyte cell death [J.K. Lazdins, M. Grell, M.R. Walker, K. Woods-Cook, P. Scheurich, K. Pfizenmaier, Membrane tumor necrosis factor (TNF) induced cooperative signaling of the TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells, J. Exp. Med. 185 (1997) 81–90]. This report demonstrates that membrane-associated TNF and the 75 kDa TNFR similarly contribute to neuronal cell death.

Continuous intraoperative external monitoring of perfusate leak using iodine-131 human serum albumin during isolated perfusion of the liver and limbs.

Regional isolated perfusion using tumor necrosis factor (TNF) shows significant promise for treatment of cancer which is limited to limbs or organs. The high toxicity of TNF requires very sensitive real time monitoring of leakage in order to avoid serious patient complications. Human serum albumin labeled with iodine-131 is used with an externally mounted and collimated NaI(Tl) detector to track the leakage of blood from the isolated perfusion blood circuit into the general systemic vascular space. Blood activity levels measured using the monitor demonstrated a very good correlation with blood serum samples taken concurrently with external monitoring. External monitoring can reduce the risks of perfusion leakage intraoperatively with the precision necessary to safely perform isolated perfusion using TNF.

Soluble tumor necrosis factor receptors reduce bowel ischemia-induced lung permeability and neutrophil sequestration

To evaluate the possible role of tumor necrosis factor (TNF) in the development of lung injury after bowel ischemia, and the ability of TNF-soluble receptors to negate TNF toxicity, using a rat small bowel ischemia and reperfusion model. Prospective, randomized, controlled laboratory study. Research laboratory. Forty adult male Sprague-Dawley rats weighing approximately 300 g. The rats were divided equally into four groups: a) ischemia and reperfusion alone; b) those animals receiving TNF antibodies (1 mL) before reperfusion; and c) those animals receiving 200 micrograms of human recombinant TNF soluble receptors. These 30 anesthetized rats underwent 60 mins of superior mesenteric artery occlusion per 4 hrs of reperfusion. The remaining ten animals were sham operated (laparotomy), serving as controls. Lung permeability was measured using bovine serum albumin labeled with 125I, and organ injury was assessed histologically. Thirty and 60 mins after declamping and reperfusion, plasma TNF concentrations increased to 830 +/- 66 and 173 +/- 56 pg/mL, respectively, compared with 10 pg/mL before ischemia (p < .001). In sham-operated control rats, TNF concentrations did not increase from baseline concentrations. Four hours after reperfusion, sequestration of neutrophils in the pulmonary microcirculation was noted (319 +/- 60 vs. 84 +/- 13 neutrophils/10 high-power fields in sham-operated rats [p < .04]). Pulmonary microvascular leak also occurred, as measured by translocation of radiolabeled albumin into the bronchoalveolar space and expressed as the ratio of bronchoalveolar lavage to blood concentrations. This ratio was 5.3 +/- 0.8 in ischemic control animals compared with 1.1 +/- 0.3 in sham animals (p < .03). Treatment with antibodies to TNF before reperfusion attenuated the pulmonary injury (75 +/- 6 neutrophils/10 high-power fields, permeability index 1.6 +/- 0.1) less than in ischemic controls (p < .005). A similar protection was achieved with soluble TNF receptors, which prevented bowel ischemia-induced lung neutrophil sequestration (117 +/- 35 neutrophils/10 high-power fields, pulmonary vascular leak ratio of 2.3 +/- 0.1, p < .05). The results of this study show that ischemia and subsequent reperfusion of the intestine in rats produce lung injury. This injury is mediated, at least in part, by TNF. Soluble TNF receptors are an effective tool in preventing lung TNF injury after intestinal ischemia.