Prediction of mutant activity and its application in molecular design of tumor necrosis factor-α

Two models for prediction of the activity and stability of site-directed mutagenesis on tumor necrosis factor-α are established. The models are based on straightforward structural considerations, which do not require the elaboration of sitedirected mutagenesis on the protein core and the hydrophobic surface area by analyzing the pmperties of the mutated amino acid residues. The reliabilities of the models have been tested by analyzing the mutants of tumor necrosis factor-α (TNF-α) whose two leucine residues (L29, L157) were mutated. Based on these models, a TNFα mutant with high activity was created by molecular design.     

Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells

Two tumor necrosis factor-α mutants MT1 (32Trp157Phe) and MT2 (2Lys30Ser-32Trp 157Phe) were constructed by site-directed mutagenesis. These mutants were soluble and over-expressed inE. coli. The purity of purified mutants was above 95% by serial chromatography. The results of Western blot indicated that these mutants could be cross-reactive with monoclonal antibody against native hTNF-α. Compared to parent hTNF-α, the cytotoxicity of these mutants on murine fibrosarcoma L929 cell lines reduced 4–5 orders of magnitude but was equivalent to that of native hTNF-α on human tumor cell lines. The LD50 of mutant MT1 was reduced to 0.34% of wild type and the dose of MT2 that resulted in 30% death of mice reduced to less than 1/700 that of parent hTNF-α.     

Fusion of HSA influences TNF-α neutralizing activity of shTNFRs

Soluble human tumor necrosis factor receptors (shTNFRI and shTNFRII) are antagonists of tumor necrosis factor-α (TNF-α) and are under clinical investigation as therapy for autoimmune diseases and transplant rejection. However, shTNFRI and shTNFRII are limited for clinical usage because of their short half-lives in vivo. Recombinant TNF-α receptors (infliximab and etanercept) are used in treatment of rheumatoid arthritis and Crohn’s disease but are also being tested for a number of other autoimmune diseases. Human serum albumin (HSA) has been used to construct long-acting fusion proteins. Here, we report the effect of fusion of HSA with shTNFRI and with shTNFRII on shTNFR’s neutralizing activity against TNF-α. HSA fusion proteins were separately expressed in Pichia pastoris. Purified recombinant shTNFRI-HSA, HSA-shTNFRI and HSA-shTNFRII could block the cytolytic activity of TNF-α in L929 cells, and the fusion at N-terminus of shTNFRI could result in larger degree of activity decline than that at the C-terminus. Activity of three fusion proteins was much weaker than etanercept, which demonstrated that fusion of HSA significantly influenced TNF-α neutralizing activity of shTNFRs. Compared with Fc fragment, HSA fusion technology may therefore not be an ideal strategy in development of long-acting shTNFRs protein drugs.     

Interleukin-1, tumor necrosis factor-alpha,and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration

Introduction  

Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-β1 (TGF-β1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-β1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair.     

Activation of tumor necrosis factor receptor 1 in airway smooth muscle: a potential pathway that modulates bronchial hyper-responsiveness in asthma?

The cellular and molecular mechanisms that are involved in airway hyper-responsiveness are unclear. Current studies suggest that tumor necrosis factor (TNF)-α, a cytokine that is produced in considerable quantities in asthmatic airways, may potentially be involved in the development of bronchial hyper-responsiveness by directly altering the contractile properties of the airway smooth muscle (ASM). The underlying mechanisms are not known, but growing evidence now suggests that most of the biologic effects of TNF-α on ASM are mediated by the p55 receptor or tumor necrosis factor receptor (TNFR)1. In addition, activation of TNFR1 coupled to the tumor necrosis factor receptor-associated factor (TRAF)2-nuclear factor-κB (NF-κB) pathway alters calcium homeostasis in ASM, which appears to be a new potential mechanism underlying ASM hyper-responsiveness.     

Tumor necrosis factor-α and muscle wasting: a cellular perspective

Tumor necrosis factor-α (TNF-α) is a polypeptide cytokine that has been associated with muscle wasting and weakness in inflammatory disease. Despite its potential importance in muscle pathology, the direct effects of TNF-α on skeletal muscle have remained undefined until recently. Studies of cultured muscle cells indicate that TNF-α disrupts the differentiation process and can promote catabolism in mature cells. The latter response appears to be mediated by reactive oxygen species and nuclear factor-κB which upregulate ubiquitin/proteasome activity. This commentary outlines our current understanding of TNF-α effects on skeletal muscle and the mechanism of TNF-α action.     

Zinc supplementation attenuates thioacetamide-induced liver injury and hyperglycemia in mice

Zinc supplementation has been shown to improve not only liver dysfunction but also glucose intolerance in subjects with liver cirrhosis. In this study, we investigated the effects of zinc supplementation on the changes in circulating levels of tumor necrosis factor-α and total antioxidant capacity in mice with thioacetamide-induced liver injury. The protective effect of concurrent zinc administration for thioacetamide-induced hepatotoxicity was also examined. The results showed that zinc treatment significantly attenuated thioacetamide-induced liver injury and hyperglycemia. Furthermore, thioacetamide-induced hepatotoxicity was markedly weakened by the simultaneous zinc administration. These effects might be attributed to reduced tumor necrosis factor-α production and elevated total antioxidant capacity induced by the mineral. Our data suggest that zinc supplementation might be beneficial for the subjects with a high susceptibility to liver injury.     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor α in mice

 There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. Received: 17 February 1997 / Accepted: 5 August 1997     

Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells

Background  

Activation of nuclear factor-κB (NF-κB) is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α) induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1) can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50).     

An immunohistochemical method for the detection of tumour necrosis factor alpha in cytospins of human bronchoalveolar lavage cells

Summary An immunohistochemical method for assessing the level of tumour necrosis factor-α in alveolar macrophages obtained by brochoalveolar lavage is described. Cytospins of mixed populations of lung cells were incubated first with a monoclonal antibody to CD68 and then with a specific peroxidase-labelled second antibody in a two-step reaction for the detection of the macrophage marker CD68. A second similarly based two-step reaction for the detection of tumour necrosis factor-α followed. Both reactions were visualized, on completion, using different coloured peroxidase substrates which produced a third colour in the event of dual deposition of the substrates. Dual substrate deposition was indicative of alveolar macrophages positive for tumour necrosis factor-α. This method has provided a specific and reproducible semi-quantitative test for the presence of tumour necrosis factor-α in human activated alveolar macrophages, which can be performed retrospectively on clinical material. A range of concentrations of the cytokine has been demonstrated in individual samples. This dual detection method has the potential for detection of any cell-associated protein product by minor modification of the described method.     

Influence of <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> I5007 on the intestinal and systemic immune responses of healthy and <Emphasis Type="Italic">E. coli</Emphasis> challenged piglets

The effect of feeding Lactobacillus fermentum I5007 on the immune system of weaned pigs with or without E. coli challenge was determined. Twenty-four weaned barrows (6.07 ± 0.63 kg BW) were randomly assigned to one of four treatments (N = 6) in a factorial design experiment. The first two treatments consisted of healthy piglets with half of the pigs receiving no treatment while the other half was orally administered with L. fermentum I5007 (10⁸ CFU/ml) at a daily dose of 20 ml. Pigs in the second two treatments were challenged on the first day with 20 ml of E. coli K88ac (10⁸ CFU/ml). Half of these pigs were not treated while the remaining pigs were treated with 20 ml of L. fermentum I5007 (10⁸ CFU/ml). Peripheral blood lymphocytes subsets were determined using flow cytometry. The intestinal mucosal immunity of the pigs was monitored by real time polymerase chain reaction. The cytokine content of the pig’s serum was also analyzed. Oral administration of L. fermentum I5007 increased blood CD4⁺ lymphocyte subset percentage as well as tumor necrosis factor-α and interferon-γ expression in the ileum. Pigs challenged with E. coli had elevated jejunal tumor necrosis factor-α while interferon-γ expression was increased throughout the small intestine. There was no difference in the concentration of the cytokines interleukin-2, interleukin-6, tumor necrosis factor-α and interferon-γ in the serum. CD8⁺ and CD4⁺/CD8⁺ in peripheral blood were not affected by treatment. In conclusion, L. fermentum I5007 can enhance T cell differentiation and induce ileum cytokine expression suggesting that this probiotic strain could modulate immune function in piglets.     

Suppression of lipopolysaccharide-induced microglial activation by a benzothiazole derivative

We previously reported that KHG21834, a benzothiazole derivative, attenuates the beta-amyloid (Aβ)-induced degeneration of both cortical and mesencephalic neurons in vitro. Central nervous system inflammation mediated by activated microglia is a key event in the development of neurodegenerative disease. In this study, we show that KHG21834 suppresses inflammation-mediated cytokine upregulation. Specifically, KHG21834 induces significant reductions in the lipopolysaccharide-induced activation of microglia and production of proinflammatory mediators such as tumor necrosis factor-α, interlukin-1β, nitric oxide, and inducible nitric oxide synthase. In addition, KHG21834 blocks the expression of mitogen-activated protein kinases, including ERK, p38 MAPK, JNK, and Akt. In vivo intracerebroventricular infusion of KHG21834 also leads to decreases the level of interleukin-1β and tumor necrosis factor-α in brain. These results, in combination with our previous findings on Aβ-induced degeneration, support the potential therapeutic efficacy of KHG21834 for the treatment of neurodegenerative disorders via the targeting of key glial activation pathways.     

In vitro cytokine stimulation assay for glycolipid biosurfactant from <Emphasis Type="Italic">Rhodococcus</Emphasis><Emphasis Type="Italic">ruber</Emphasis>: role of monocyte adhesion

Glycolipid biosurfactant (GLB) from Rhodococcus ruber IEGM 231 was found to stimulate tumor necrosis factor-α (TNF-α), interleukin (IL) -1β and IL-6 production when applied as an ultrasonic emulsion to the adherent human peripheral blood monocyte culture. However, a lack of cytokine-stimulating activity was registered with the GLB applied as a hydrophobic film coating in 24-well culture plates, indicating that it may have been due to its inhibitory effect on monocyte adhesion. The mode of GLB application may therefore play an important role in in vitro assay of immunostimulatory activity of this compound as well as other bacterial glycolipids. Additionally, GLB from R. ruber displayed no cytotoxicity against human lymphocytes and therefore could be proposed as a potential immunomodulating and antitumor agent.     

Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma

Mitigation of regulatory T cell-mediated immunosuppression and elicitation of immunogenic tumor cell death are crucial events for optimal anti-tumor immune activity in vivo. This study was designed to investigate the potential synergistic activity of the combined use of cyclophosphamide (CP) and doxorubicin (DR), both of which are known to resolve these two issues. BALB/c mice were inoculated subcutaneously with CT-26 carcinoma cells in the bilateral flank and treated with an intraperitoneal injection of a low dose of CP followed by an intratumoral injection of DR into one side of the tumor. We found that, in addition to a significant suppression of growth on the DR-treated side of the tumor, combination therapy suppressed the growth of DR-untreated remote tumors in both tumor-specific and T cell-dependent manners. Mitomycin C showed no such synergistic anti-tumor activity with CP treatment. Combination therapy increased the frequency of interferon (IFN)-γ-producing T lymphocytes specific to a CT-26-associated class I-binding tumor peptide in the tumor-draining lymph nodes. Real-time PCR analysis revealed that combination therapy led to an increase in IFN-γ and tumor necrosis factor-α mRNA expression; however, levels of Foxp3 and transforming growth factor-β within the remote tumor tissues were decreased. In addition, knock down of calreticulin expression in CT-26 cells using small interfering RNA attenuated anti-tumor vaccine effects induced by DR-treated CT-26 cells. These results provide an immunological rationale for the combined use of chemotherapeutic drugs, i.e., CP and DR, and further recommend their use with current cancer vaccines.     

Effects of sulfur amino acids, <Emphasis Type="SmallCaps">l</Emphasis>-methionine, <Emphasis Type="SmallCaps">l</Emphasis>-cystine and <Emphasis Type="SmallCaps">l</Emphasis>-cysteine on lipoprotein lipase and hormone-sensitive lipase in differentiated mouse 3T3-L1 adipocytes

Rats subcutaneously implanted with AH109A hepatoma cells show hyperlipidemia with high concentrations of serum triglyceride and nonesterified fatty acid, suppression of lipoprotein lipase (LPL), and elevation of hormone-sensitive lipase (HSL) activities during the growth of the hepatoma. Supplementation of the diet with sulfur amino acids such as l-methionine (Met) and l-cystine (Cys) improved hyperlipidemia by restoring LPL and HSL activities. In the present study, we have attempted to examine the effects of sulfur amino acids on the activity and mRNA level of LPL and the activity of HSL using 3T3-L1 cells, which are known to differentiate to adipocytes. The adipocytes were incubated with various concentrations of Met, Cys or l-cysteine (CysH) in the absence or presence of tumor necrosis factor-α (TNF-α). LPL activity was suppressed by TNF-α. In the absence of TNF-α, Met, Cys and CysH did not change the LPL activity. In the presence of TNF-α, Met and Cys significantly increased the LPL activity, and Met also enhanced the LPL mRNA level. HSL activity was also suppressed by TNF-α. In the absence of TNF-α, Met enhanced the HSL activity. In the presence of TNF-α, Met, Cys and CysH suppressed the HSL activity. Sulfur amino acids such as Met, Cys and CysH affected the LPL activity, mRNA level, and HSL activity in 3T3-L1 adipocytes. Some of these effects of sulfur amino acids were different between LPL and HSL, between the absence and the presence of TNF-α, and between 3T3-L1 adipocytes and the adipose tissue from rats.     

Cytokine production by a megakaryocytic cell line

Summary The regulation of megakaryopoeisis by cytokines is not yet well understood. It is possible that autocrine loops are established during megakaryocyte growth and differentiation, aiding in the maturation of these cells. The CHRF-288-11 human megakaryoblastic cell line has been examined for cytokine production in growing cells and cells stimulated to differentiate by the addition of phorbol esters. It has been demonstrated that these cells produce RNA corresponding to the interleukins IL-1α, 1β, 3, 7, 8, and 11, granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-α (INF-α), and basic fibroblast growth factor (bFGF). Additionaly, RNA corresponding to the receptors for IL-6, GM-CSF, SCF, INF-α,β, bFGF, and monocyte colony stimulating factor (M-CSF) were also expressed by the cells. The receptor for TNF-α was detected immunologically. Analysis at the protein level demonstrated that significant amounts of INF-α, TNF-α, GM-CSF, SCF, IL-1α, and a soluble form of the IL-6 receptor were produced by the cells. Addition of phorbol esters to CHRF-288-11 cells enhances their megakaryocytic phenotype; such treatment also results in increased secretion of INF-α, TNF-α, and GM-CSF. These results suggest that potential autocrine loops are established during the differentiation of CHRF-288-11 cells, which may alter the capability of the cell to differentiate. These findings are similar to those recently obtained for marrow-derived megakaryocytes (Jiang et al.) suggesting that CHRF-288-11 cells provide a useful model system for the study of cytokine release during megakaryocyte differentiation.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.     

Proinflammatory cytokines tumor necrosis factor-α and interferon-γ modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling

Background  

The tight junction is a dynamic structure that is regulated by a number of cellular signaling processes. Occludin, claudin-1, claudin-2 and claudin-3 are integral membrane proteins found in the tight junction of MDCK cells. These proteins are restricted to this region of the membrane by a complex array of intracellular proteins which are tethered to the cytoskeleton. Alteration of these tight junction protein complexes during pathological events leads to impaired epithelial barrier function that perturbs water and electrolyte homeostasis. We examined MDCK cell barrier function in response to challenge by the proinflammatory cytokines tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ).     

Locally administered lentivirus-mediated siRNA inhibits wear debris-induced inflammation in murine air pouch model

Lentivirus(LV)-mediated small interfering RNA (siRNA) targeting tumor necrosis factor-α (TNF-α) was locally administered into the air pouch of mice to inhibit inflammation induced by titanium alloy particles. The lentiviral vector expressed green fluorescent protein (GFP) as a reporter gene. Down-regulation of TNF-α in pouch area was confirmed by real-time PCR and ELISA, resulting in significantly decreased local inflammatory responses (P < 0.01). This approach was proven safe by localized GFP fluorescence and invariant TNF-α expression in peripheral blood, liver, spleen, kidney, lung and brain of mouse. In conclusion, locally administered siRNA provides an effective and safe method for inhibiting particle-induced inflammation.