Potent TNF Research Articles

Monocytes in sarcoidosis are potent tumour necrosis factor producers and predict disease outcome.

BackgroundPulmonary sarcoidosis is an inflammatory disease characterised by granuloma formation and heterogeneous clinical outcome. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine contributing to granuloma formation and high levels of TNF have been shown to associate with progressive disease. Mononuclear phagocytes (MNPs) are potent producers of TNF and highly responsive to inflammation. In sarcoidosis, alveolar macrophages have been well studied. However, MNPs also include monocytes/monocyte-derived cells and dendritic cells, which are poorly studied in sarcoidosis, despite their central role in inflammation.ObjectiveTo determine the role of pulmonary monocyte-derived cells and dendritic cells during sarcoidosis.MethodsWe performed in-depth phenotypic, functional and transcriptomic analysis of MNP subsets from blood and bronchoalveolar lavage (BAL) fluid from 108 sarcoidosis patients and 30 healthy controls. We followed the clinical development of patients and assessed how the repertoire and function of MNP subsets at diagnosis correlated with 2-year disease outcome.ResultsMonocytes/monocyte-derived cells were increased in blood and BAL of sarcoidosis patients compared to healthy controls. Interestingly, high frequencies of blood intermediate monocytes at time of diagnosis associated with chronic disease development. RNA sequencing analysis showed highly inflammatory MNPs in BAL of sarcoidosis patients. Furthermore, frequencies of BAL monocytes/monocyte-derived cells producing TNF without exogenous stimulation at time of diagnosis increased in patients that were followed longitudinally. In contrast to alveolar macrophages, the frequency of TNF-producing BAL monocytes/monocyte-derived cells at time of diagnosis was highest in sarcoidosis patients that developed progressive disease.ConclusionOur data show that pulmonary monocytes/monocyte-derived cells are highly inflammatory and can be used as a predictor of disease outcome in sarcoidosis patients.

Metabolic Profiling of Buddleia indica Leaves using LC/MS and Evidence of their Antioxidant and Hepatoprotective Activity Using Different In Vitro and In Vivo Experimental Models.

LC-ESI-MS (Liquid Chromatography coupled with Electrospray Ionization Mass Spectrometry profiling of a methanol extract from Buddleia indica (BIM) leaves revealed 12 main peaks in which verbascoside and buddlenoid B represent the major compounds. The antioxidant and hepatoprotective activities of BIM were investigated using different in vitro and in vivo experimental models. BIM exhibited substantial in vitro antioxidant properties in DPPH· and HepG2 assays. Regarding CCl4 (carbon tetrachloride) induced hepatotoxicity in a rat model, oxidative stress markers became significantly ameliorated after oral administration of BIM. Lipid peroxide levels showed a 51.85% decline relative to CCl4-treated rats. Super oxide dismutase (SOD), total antioxidant status (TAS), and catalase (CAT) revealed a marked increase by 132.48%, 187.18%, and 114.94% relative to the CCl4 group. In a tamoxifen-induced hepatotoxicity model, BIM showed a considerable alleviation in liver stress markers manifested by a 46.06% and 40% decline in ALT (Alanine Transaminase) and AST (Aspartate Transaminase) respectively. Thiobarbituric acid reactive substances (TBARS) were reduced by 28.57% and the tumor necrosis factor alpha (TNF-α) level by 50%. A virtual screening of major secondary metabolites of BIM to TNF-alpha employing the C-docker protocol showed that gmelinoside H caused the most potent TNF- α inhibition as indicated from their high fitting scores. Thus, BIM exhibited a potent hepatoprotective activity owing to its richness in antioxidant metabolites.

Erratum to: Unexpected arterial wall and cellular inflammation in patients with rheumatoid arthritis in remission using biological therapy: a cross-sectional study.

Increasing numbers of patients (up to 40 %) with rheumatoid arthritis (RA) achieve remission, yet it remains to be elucidated whether this also normalizes their cardiovascular risk. Short-term treatment with TNF inhibitors lowers arterial wall inflammation, but not to levels of healthy controls. We investigated whether RA patients in long-term remission are characterized by normalized inflammatory activity of the arterial wall and if this is dependent on type of medication used (TNF-inhibitor versus nonbiological disease-modifying antirheumatic drugs (DMARDs)). Arterial wall inflammation, bone marrow and splenic activity (index of progenitor cell activity) was assessed with 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) in RA patients in remission (disease activity score (DAS28) 6 months) and healthy controls. We performed ex vivo characterization of monocytes using flow cytometry and a transendothelial migration assay. Overall, arterial wall inflammation was comparable in RA patients (n = 23) in long-term remission and controls (n = 17). However, RA subjects using current anti-TNF therapy (n = 13, disease activity score 1.98[1.8–2.2]) have an almost 1.2-fold higher 18F-FDG uptake in the arterial wall compared to those using DMARDs (but with previous anti-TNF therapy) (n = 10, disease activity score 2.24[1.3–2.5]), which seemed to be predominantly explained by longer duration of their rheumatic disease in a multivariate linear regression analysis. This coincided with increased expression of pro-adhesive (CCR2) and migratory (CD11c, CD18) surface markers on monocytes and a concomitant increased migratory capacity. Finally, we found increased activity in bone marrow and spleen in RA patients using anti-TNF therapy compared to those with DMARDs and controls. A subset of patients with RA in clinical remission have activated monocytes and increased inflammation in the arterial wall, despite the use of potent TNF blocking therapies. In these subjects, RA disease duration was the most important contributor to the level of arterial wall inflammation. This increased inflammatory state implies higher cardiovascular risk in these patients, who thus may require more stringent CV risk management.

Unexpected arterial wall and cellular inflammation in patients with rheumatoid arthritis in remission using biological therapy: a cross-sectional study.

BackgroundIncreasing numbers of patients (up to 40 %) with rheumatoid arthritis (RA) achieve remission, yet it remains to be elucidated whether this also normalizes their cardiovascular risk. Short-term treatment with TNF inhibitors lowers arterial wall inflammation, but not to levels of healthy controls. We investigated whether RA patients in long-term remission are characterized by normalized inflammatory activity of the arterial wall and if this is dependent on type of medication used (TNF-inhibitor versus nonbiological disease-modifying antirheumatic drugs (DMARDs)).MethodsArterial wall inflammation, bone marrow and splenic activity (index of progenitor cell activity) was assessed with 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) in RA patients in remission (disease activity score (DAS28) <2.6 for >6 months) and healthy controls. We performed ex vivo characterization of monocytes using flow cytometry and a transendothelial migration assay.ResultsOverall, arterial wall inflammation was comparable in RA patients (n = 23) in long-term remission and controls (n = 17). However, RA subjects using current anti-TNF therapy (n = 13, disease activity score 1.98[1.8–2.2]) have an almost 1.2-fold higher 18F-FDG uptake in the arterial wall compared to those using DMARDs (but with previous anti-TNF therapy) (n = 10, disease activity score 2.24[1.3–2.5]), which seemed to be predominantly explained by longer duration of their rheumatic disease in a multivariate linear regression analysis. This coincided with increased expression of pro-adhesive (CCR2) and migratory (CD11c, CD18) surface markers on monocytes and a concomitant increased migratory capacity. Finally, we found increased activity in bone marrow and spleen in RA patients using anti-TNF therapy compared to those with DMARDs and controls.ConclusionsA subset of patients with RA in clinical remission have activated monocytes and increased inflammation in the arterial wall, despite the use of potent TNF blocking therapies. In these subjects, RA disease duration was the most important contributor to the level of arterial wall inflammation. This increased inflammatory state implies higher cardiovascular risk in these patients, who thus may require more stringent CV risk management.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1008-z) contains supplementary material, which is available to authorized users.

Therapeutic intradermal delivery of tumor necrosis factor-alpha antibodies using tip-loaded dissolvable microneedle arrays

Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here, we evaluated the use of tip-loaded dissolvable microneedle arrays (MNAs) for localized intradermal delivery of anti-TNF-α Ab. MNAs with obelisk shape microneedles that incorporate the antibody cargo in the needle tips were created from carboxymethylcellulose (CMC) using a micromilling/spin-casting fabrication method. We found that anti-TNF-α Ab integrated into MNAs using this room temperature fabrication process maintained conformationally dependent TNF-α binding activity. Further, these MNAs efficiently delivered anti-TNF-α antibodies to the dermis of human skin with clinically applicable release profiles. To evaluate MNA delivered anti-TNF-α Ab function, we applied anti-TNF-α Ab containing MNAs to established psoriasiform lesions on the skin of mice. MNA anti-TNF-α Ab treatment reduced key biomarkers of psoriasiform inflammation including epidermal thickness and IL-1β expression. Taken together, these results demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans, and support the development of MNA mediated antibody delivery for clinical applications. Statement of SignificanceTumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here we demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans. These results support the development of MNA mediated antibody delivery of therapeutic antibodies for clinical applications.

Rationally designed less toxic SPD-304 analogs and preliminary evaluation of their TNF inhibitory effects.

SPD-304 was discovered as a promising tumor necrosis factor alpha (TNF) antagonist that promotes dissociation of TNF trimers and therefore blocks the interaction of TNF and its receptor. However, SPD-304 contains a potentially toxic 3-alkylindole moiety, which can be bioactivated to a reactive electrophilic intermediate. A series of SPD-304 analogs was synthesized with the aim to diminish its toxicophore groups while maintaining the binding affinity for TNF. Incorporation of electron-withdrawing substituents at the indole moiety, in conjunction with elimination of the 6'-methyl group of the 4-chromone moiety, led to a significantly less toxic and equally potent TNF inhibitor.

Optimal Xenogeneic Adoptive Transfer of Human NK Cells: Fresh NK Cells and IL-15 Administration Are Superior to Frozen NK Cells and IL-2

AbstractAbstract 346Expansion of NK cells after adoptive transfer is a major determinant of their anti-tumor efficacy but the mechanisms of their expansion are not completely understood. To study in vivo NK cell expansion and the relative merits of fresh versus frozen NK cell products, we used xenotransplantation of human GMP NK cell products (provided by two PACT centers funded by the NHLBI) into immune deficient mice. NOD/IL-2Rγc/Rag (NOG) that lack T-, B-, and NK cells and that have a macrophage defect that renders recipients highly amenable to human cell engraftment, were given 250 cGy total body irradiation, and infused with fresh or frozen NK cells. Two products were tested: 1) an enriched fresh NK cell product obtained by CD3 and CD19 depletion followed by overnight activation with IL-2 or IL-15 (U. of Minnesota) or 2) ex vivo expanded NK cells using membrane bound IL-15 and 41BB-L transduced K562 cells with 10 U/ml IL-2 in G-Rex devices for 10 days (Baylor). NK cells (1–2 million NK cells for each individual experiment) were given IV and mice were treated with no cytokines, 5 or 10 mg of IL-2 (Novartis), or 5 mg IL-15 (produced by the NCI for clinical use) as 3 injections per week for 2 weeks. Peripheral blood (Table), spleen and marrow were evaluated on days 7, 14, 21 and 28 after adoptive transfer. Counts were converted to absolute cell numbers per 100 μL of blood or the number of cells recovered per spleen or after flushing of a single femur. On day 7, the number of huNK cells in blood in the absence of cytokine administration was low with both cell products and numbers continued to diminish by day 14. In marked contrast, every other day IL-2 or IL-15 significantly increased huNK cells in murine blood. On day 7 and 14, ex vivo expanded NK cell products resulted in significantly higher numbers of huNK in mice receiving 5μg IL-2 compared to mice who received fresh NK cells activated overnight with IL-2. With 5μg IL-2, huNK number decreased between day 7 and 14 irrespective of cell product. In contrast, only IL-15 lead to increased numbers of huNK cells between days 7 and 14. Previously cryopreserved NK cells showed significantly worse survival for both cell products, with a larger fold decrease seen with ex vivo expanded cells. Blood collected from cytokine treated mice at day 14 after infusion of fresh products contained huNK cells that were fully functional as assessed by potent CD107a degranulation, TNF and IFN production after exposure to K562 target cells as well as augmented IFN production induced by IL-12 and IL-18. On day 8, Ki67+ (proliferating) huNK cells were significantly higher with IL-15 compared to IL-2 (both at 5μg) in marrow for fresh (76% vs 35%, p=<0.001) and ex vivo expanded (88% vs 53%, p=0.003) NK cells. Ki67+ proliferating NK cells had decreased by day 14 under all conditions. With fresh NK cells and IL-15 on day 8, there were 1371 huNK relative counts recovered from spleen compared to 450 recovered from marrow (p=0.01). In marked contrast, ex vivo expanded NK cells (that express a lower percentage of CD62L compared to fresh NK cells) had a reverse homing pattern with 874 huNK cells in spleen versus 2146 in marrow (p=0.07). There was a significant difference between the two cells products in terms of the absolute numbers of huNK cells found in marrow at day 8 (p=0.0007). We conclude that 1) ex vivo expanded NK cells home better to BM which may be useful in treating AML localized to BM, whereas fresh NK cells may be preferable for homing to lymphoid tissue in the periphery; 2) at early time points fresh huNK cells were less abundant than expanded cells but catch up at late time points; and 2) IL-15 is more effective than IL-2 on a mcg basis. Overall, fresh and ex vivo expanded NK cells have different properties and kinetics after infusion. This xenogeneic model results in definitive readouts to optimize the best characteristics of each cell product to tailor a product to have maximal efficacy for clinical testing.Results of experiments with fresh or ex vivo expanded NK cellsExp.Activated NK sourceIn vivo cytokine# miceAvg hu NK Day 7/100 μL muBloodAvg hu NK Day 14/100 μL muBlood1Fresh NKnone611724IL2-5 ug623853Ex vivo expandednone6281IL2-5 ug67602352Fresh NKnone421216IL2-5 ug446593IL15-5 ug45401151Ex vivo expandednone4639IL2-5 ug4888181IL15-5 ug4322129303Fresh NKnone12-1614155IL2-5 ug12-16331169IL2-10 ug12-16197182IL15-5 ug12-162661177Ex vivo expandednone12-16223IL2-5 ug12-16323106IL2-10 ug12-16372673IL15-5 ug12-167351955 Disclosures:Miller:Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

Attenuation of Inflammatory Events in Human Intervertebral Disc Cells With a Tumor Necrosis Factor Antagonist

The inflammatory responses of primary human intervertebral disc (IVD) cells to tumor necrosis factor α (TNF-α) and an antagonist were evaluated in vitro. To investigate an ability for soluble TNF receptor type II (sTNFRII) to antagonize TNF-α-induced inflammatory events in primary human IVD cells in vitro. TNF-α is a known mediator of inflammation and pain associated with radiculopathy and IVD degeneration. sTNFRs and their analogues are of interest for the clinical treatment of these IVD pathologies, although information on the effects of sTNFR on human IVD cells remains unknown. IVD cells were isolated from surgical tissues procured from 15 patients and cultured with or without 1.4 nmol/L TNF-α (25 ng/mL). Treatment groups were coincubated with varying doses of sTNFRII (12.5-100 nmol/L). Nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin-6 (IL6) levels in media were quantified to characterize the inflammatory phenotype of the IVD cells. Across all patients, TNF-α induced large, statistically significant increases in NO, PGE₂, and IL6 secretion from IVD cells compared with controls (60-, 112-, and 4-fold increases, respectively; P < 0.0001). Coincubation of TNF-α with nanomolar doses of sTNFRII significantly attenuated the secretion of NO and PGE₂ in a dose-dependent manner, whereas IL6 levels were unchanged. Mean IC₅₀ values for NO and PGE₂ were found to be 35.1 and 20.5 nmol/L, respectively. Nanomolar concentrations of sTNFRII were able to significantly attenuate the effects of TNF-α on primary human IVD cells in vitro. These results suggest this sTNFR to be a potent TNF antagonist with potential to attenuate inflammation in IVD pathology.

Novel thalidomide analogues with potent NFκB and TNF expression inhibition

A series of N-phenyl thalidomide analogues have been prepared efficiently through a Buchwald–Hartwig cross coupling reaction as the key step. Several of these compounds have extraordinary TNF expression inhibition compared to both thalidomide and the current pharmaceutical agent Revlimid (Lenalidomide).

Soluble TNFR II/IgG1 Fc fusion protein treatment in the LPS-mediated septic shock of rats

Tumor necrosis factor-α (TNF-α) is thought to play a major role in systemic inflammation associated with sepsis. A potent TNF antagonist, a recombinant fusion protein that consists of the soluble TNF receptor (p75) linked to the Fc portion of human IgG1 (sTNFR II/IgG1 Fc fusion protein, sTNFR:Fc), has been shown to provide rapid and sustained improvement in local inflammation diseases by binding TNF-α and preventing its proinflammatory activities. To explore the potential therapeutic efficacy for septic shock of sTNFR:Fc, we investigate the effect of this molecule on the survival rate, blood pressure, serum TNF-α bioactivity as well as the expression of TNF-α at mRNA level in the liver in a LPS-induced rat septic shock model. Blood pressure of the rats was monitored by multi-channel creature signal analysis system. Serum TNF-α level and bioactivity was assessed using an enzyme-linked immunoassay and a L929 cytotoxicity assay, respectively. The expression of TNF-α mRNA in liver was examined by semi-quantitative RT-PCR. sTNFR:Fc administered to rats 24 h before LPS challenge ablated the rise in serum TNF-α bioactivity that occurs in response to LPS and protected against hypotension and death. These results indicate that TNF-α is a mediator of fatal septic shock, and suggest that sTNFR:Fc offer a potential therapy of systemic infection.

Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.

1-substituted 4-aryl-5-pyridinylimidazoles: a new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency.

A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.

Uromodulin (Tamm-Horsfall glycoprotein): a renal ligand for lymphokines.

The protein portion of the immunosuppressive glycoprotein uromodulin is identical to the Tamm-Horsfall urinary glycoprotein and is synthesized in the kidney. Evidence that the glycoproteins are the same is based on amino acid sequence identity, immunologic cross-reactivity, and tissue localization to the thick ascending limb of Henle's loop. Nucleic acid sequencing of clones for uromodulin isolated from a complementary DNA bank from human kidney predicts a protein 639 amino acids in length, including a 24--amino acid leader sequence and a cysteine-rich mature protein with eight potential glycosylation sites. Uromodulin and preparations of Tamm-Horsfall glycoprotein bind to recombinant murine interleukin-1 (rIL-1) and human rIL-1 alpha, rIL-1 beta, and recombinant tumor necrosis factor (rTNF). Uromodulin isolated from urine of pregnant women by lectin adherence is more immunosuppressive than material isolated by the original salt-precipitation protocol of Tamm and Horsfall. Immunohistologic studies demonstrate that rIL-1 and rTNF bind to the same area of the human kidney that binds to antiserum specific for uromodulin. Thus, uromodulin (Tamm-Horsfall glycoprotein) may function as a unique renal regulatory glycoprotein that specifically binds to and regulates the circulating activity of a number of potent cytokines, including IL-1 and TNF.