Inhibition Of Cytokine Release Research Articles

Targeting F Box Protein Fbxo3 To Control Cytokine-Driven Inflammation

Cytokine-driven inflammation underlies the pathobiology of a wide array of infectious and immune-related disorders. The TNFR-associated factor (TRAF) proteins have a vital role in innate immunity by conveying signals from cell surface receptors to elicit transcriptional activation of genes encoding proinflammatory cytokines. We discovered that a ubiquitin E3 ligase F box component, termed Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by mediating the degradation of the TRAF inhibitory protein, Fbxl2. Analysis of the Fbxo3 C-terminal structure revealed that the bacterial-like ApaG molecular signature was indispensible for mediating Fbxl2 disposal and stimulating cytokine secretion. By targeting this ApaG motif, we developed a highly unique, selective genus of small-molecule Fbxo3 inhibitors that by reducing TRAF protein levels, potently inhibited cytokine release from human blood mononuclear cells. The Fbxo3 inhibitors effectively lessened the severity of viral pneumonia, septic shock, colitis, and cytokine-driven inflammation systemically in murine models. Thus, pharmacological targeting of Fbxo3 might be a promising strategy for immune-related disorders characterized by a heightened host inflammatory response.

Role of Interleukin 1 Receptor Like 1 (ST2) in Gram-Negative and Gram-Positive Sepsis in Mice

Interleukin 1 receptor-like 1 (ST2) has been implicated as a negative regulator of Toll-like receptor signaling. We here sought to elucidate the role of ST2 in cytokine release and systemic infection caused by two common human sepsis pathogens, Streptococcus pneumoniae (gram-positive) and Klebsiella pneumoniae (gram-negative). Whole blood leukocytes and splenocytes were harvested from ST2-deficient (st2) and wild-type (WT) mice and stimulated ex vivo with S. pneumoniae or K. pneumoniae. In addition, st2 and WT mice were infected intravenously with these bacteria, and bacterial loads and cytokine levels were measured in blood, spleens and lungs at 6, 24, and 48 h thereafter. Unexpectedly, st2 blood leukocytes and splenocytes produced lower levels of cytokines and chemokines than WT cells in response to either pathogen. In contrast, the in vivo role of ST2 during sepsis caused by these bacteria was limited, although at 6 and 24 h after infection with S. pneumoniae bacterial loads were lower in spleens of st2 mice. ST2 augments rather than inhibits cytokine release by blood leukocytes and splenocytes exposed to S. pneumoniae or K. pneumoniae, but plays a limited role in host defense during sepsis caused by these pathogens.

Chronic psychological stress induces the accumulation of myeloid-derived suppressor cells in mice.

Chronic psychological stress has been shown to adversely impact immune system functions and compromise host defenses against various infections. However, the underlying mechanisms remain elusive. Recent studies have demonstrated that myeloid-derived suppressor cells (MDSCs) play an important role in regulating immunity. It is of interest to explore whether or not chronic psychological stress plays immunosuppressive functions partially by inducing MDSCs accumulation. In this work, we report that chronic psychological stress led to the accumulation of CD11b+Gr1+ cells in the bone marrow of BALB/c mice. Repeated β-agonist infusion showed no such effect. However, β-adrenergic blockade, but not glucocorticoids blockade, partially reversed the accumulation of CD11b+Gr1+ cells under the condition of chronic psychological stress, suggesting catecholamines collaborate with other factors to induce the accumulation. Further exploration indicates that cyclooxygenase 2 (COX-2)-prostaglandin E2 (PGE2) loop might act downstream to induce the accumulation. A majority of the accumulated CD11b+Gr1+ cells were Ly6G+Ly6Clow immature neutrophils, which inhibited cytokine release of macrophages as well as T cell responsiveness. Moreover, the accumulated CD11b+Gr1+ cells under the condition of chronic psychological stress expressed multiple inhibitory molecules. Taken together, our data demonstrate for the first time that chronic psychological stress induces MDSCs accumulation in mice, which can contribute to immunosuppression.

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.

Sulforaphane enhances the activity of the Nrf2–ARE pathway and attenuates inflammation in OxyHb-induced rat vascular smooth muscle cells

A growing body of evidence indicates that the nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a protective role in many physiological stress processes such as inflammatory damage, oxidative stress, and the accumulation of toxic metabolites, which are all involved in the cerebral vasospasm following subarachnoid hemorrhage (SAH). We hypothesized that the Nrf2-ARE pathway might have a protective role in cerebral vasospasm following SAH. In our study, we investigate whether the oxyhemoglobin (OxyHb) can induce the activation of the Nrf2-ARE pathway in vascular smooth muscle cells (VSMCs), and evaluate the modulatory effects of sulforaphane (SUL) on OxyHb-induced inflammation in VSMCs. As a result, both the protein level and the mRNA level of the nuclear Nrf2 were significantly increased, while the mRNA levels of two Nrf2-regulated gene products, both heme oxygenase-1 and NAD(P)H: quinone oxidoreductase-1, were also up-regulated in VSMCs induced with OxyHb. A marked increase of inflammatory cytokines such as IL-1β, IL-6 and TNF-α release was observed at 48h after cells were treated with OxyHb. SUL enhanced the activity of the Nrf2-ARE pathway and suppressed cytokine release. Our results indicate that the Nrf2-ARE pathway was activated in OxyHb-induced VSMCs. SUL suppressed cytokine release via the activation of the Nrf2-ARE pathway in OxyHb-induced VSMCs.

AB0045 The orally available immunomodulator rhudex inhibits naÏve and memory T cell activation and proliferation and the release of pro-inflammatory TH1 and TH17 cytokines in vitro

BackgroundRhuDex, under development as an orally available immunomodulating agent, inhibits the CD80-CD28 costimulatory pathway, thus preventing the interaction between T lymphocyte and APC receptors necessary for T cell activation and...

Endothelin Receptor Antagonists Attenuate the Inflammatory Response of Human Pulmonary Vascular Smooth Muscle Cells to Bacterial Endotoxin

Bacterial infections induce exacerbations in chronic lung diseases, e.g., chronic obstructive pulmonary disease (COPD), by enhancing airway inflammation. Exacerbations are frequently associated with right heart decompensation and accelerate disease progression. Endothelin receptor antagonists (ERAs) might have therapeutic potential as pulmonary vasodilators and anti-inflammatory agents, but utility in exacerbations of chronic lung diseases is unknown. We hypothesized that cytokine releases induced by lipopolysaccharide (LPS), the major bacterial trigger of inflammation, are reduced by ERAs in pulmonary vascular smooth muscle cells (PVSMCs). Ex vivo cultivated human PVSMCs were preincubated with the endothelin-A-receptor selective inhibitor ambrisentan, with the endothelin-B-receptor selective inhibitor BQ788 [sodium (2R)-2-{[(2S)-2-({[(2R,6S)-2,6-dimethyl-1-piperidinyl]carbonyl}amino)-4,4-dimethylpentanoyl][1-(methoxycarbonyl)-d-tryptophyl]amino}hexanoate], or with the dual blocker bosentan before stimulation with smooth LPS (S-LPS), rough LPS (Re-LPS), or a mixture of long and short forms (M-LPS). Expression of cytokines and LPS receptors (TLR4, CD14) were analyzed via enzyme-linked immunosorbent assay (ELISA) and/or quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All LPS forms induced interleukin (IL)-6-, IL-8-, and granulocyte macrophage-colony stimulating factor (GM-CSF) release. Bosentan and BQ788 inhibited M-LPS-induced release of all cytokines and soluble CD14 (sCD14) but not TLR4 expression. Ambrisentan blocked M-LPS-induced IL-6 release but not IL-8, GM-CSF, or LPS receptors. IL-8 release induced by S-LPS, which requires CD14 to activate TLR4, was blocked by bosentan and BQ788. IL-8 release induced by Re-LPS, which does not require CD14 to activate TLR4, was insensitive to both bosentan and BQ788. In conclusion, PVSMCs contribute to inflammation in bacteria-induced exacerbations of chronic lung diseases. Inhibition of the endothelin-B receptor suppresses cytokine release induced by long/smooth LPS attributable to sCD14 downregulation. ERAs, particularly when targeting the endothelin-B receptor, might have therapeutic utility in exacerbations of chronic lung diseases.

Linalool: a novel natural anti-inflammatory agent?: Commentary on “Anti-inflammatory effects of linalool in RAW 264.7 macrophages and lipopolysaccharide-induced lung injury model”

DOI of original article: 10.1016/j.jss.2012.1 * Corresponding author. Department of Surg Tel.: þ1 513 558 8467; fax: þ1 513 558 3474. E-mail address: prittsta@ucmail.uc.edu (T 0022-4804/$ e see front matter a 2013 Elsev http://dx.doi.org/10.1016/j.jss.2013.02.014 Proinflammatory states are widely implicated in both acute and chronic disease processes. Severe acute systemic inflammation is at least partially responsible for shock and acute lung injury, and therapeutic options to curb the proinflammatory responses are limited. Sustained release of proinflammatory mediators by macrophages and other leukocyte subtypes is the hallmark of autoimmune illnesses. In addition to nonspecific immunosuppressants, targeted therapies to reduce inflammation via inhibition of cytokine release are available for some of these illnesses, including Crohn disease and rheumatoid arthritis. However, the subsequent risk of infection with these medications is significant, which limits their clinical use. Linalool is a terpene alcohol that occurs naturally in several edible plant species. It is used in soaps, oils, and other topical products, but has not been evaluated previously as a therapeutic. Its role as an inflammatory mediator is unknown. Based on the antimicrobial and anxiolytic properties of

Peroxisome proliferator-activated receptor-γ mediates the anti-inflammatory effect of 3-hydroxy-4-pyridinecarboxylic acid derivatives: Synthesis and biological evaluation

Seven 3-hydroxy-4-pyridinecarboxylic acid derivatives (HPs), aza-analogues of salicylic acid and structurally close to other potent inflammatory pyridine compounds such as aminopyridinylmethanols and aminopyridinamines, were synthesized, and their anti-inflammatory activity was evaluated. The synthesis was performed by adopting a general procedure involving an intramolecular Diels–Alder cycloaddition of oxazoles with acrylic acid to form various substituted pyridinic acids. The newly synthesized HPs did not exhibit cytotoxic activity on human monocytes-derived macrophages at concentrations up to 102 μM. Anti-inflammatory activity of the compounds was screened in vitro by evaluating the capability to inhibit cytokines release from lipopolysaccharide (LPS) stimulated human macrophages. 3-Hydroxy-1-methyl-4-pyridinecarboxylic acid (24) was found to be the most active HP. At 10 μM concentration, HP 24 reduced LPS-induced and nuclear factor-κB activation and cyclooxygenase-2 expression, while increased intracellular reactive oxygen species generation and peroxisome proliferator-activated receptor (PPAR-γ) mRNA transcript level. Indeed, pre-treatment of LPS-exposed human macrophages with PPAR-γ specific antagonist completely prevented HP 24-induced TNF-α and IL8 down regulation, demonstrating that the PPARγ pathway is mandatory for the HP 24 anti-inflammatory effect. Finally, daily treatment with HP 24 ameliorated the outcome of DSS-induced colitis in mice, significantly reducing colonic MPO activity and IL-1β tissue levels.

The anti-inflammatory effect of the synthetic antimicrobial peptide 19-2.5 in a murine sepsis model: a prospective randomized study

IntroductionIncreasing rates of multi-resistant bacteria are a major problem in the treatment of critically ill patients. Furthermore, conventional antibiotics lead to the release of bacterial derived membrane parts initiating pro-inflammatory cascades with potential harm to the patient. Antimicrobial peptides (AMP) may kill bacteria without releasing pro-inflammatory factors. Thus, we compared three newly developed synthetic anti-lipopolysaccharide peptides (SALPs) with a broader range of efficacy to suppress cytokine release in plasma and CD14 mRNA expression in organ tissue in a murine, polymicrobial sepsis model.MethodsA randomized, experimental trial was conducted in an animal research facility. Male NMRI mice (n = 90; 8- to 12-weeks old) were randomized to the following six groups: (i) sham operation and parenteral vehicle (NaCl 0.9%) administration (sham); (ii) cecal ligation and puncture (CLP) and vehicle infusion (sepsis-control), (iii) CLP and polymyxin B infusion (polyB), or (iv to vi) CLP and infusion of three different synthetic antimicrobial peptides Peptide 19-2.5 (Pep2.5), Peptide 19-4 (Pep4) or Peptide 19-8 (Pep8). All animals underwent arterial and venous catheterization for hemodynamic monitoring 48 hours prior to CLP or sham-operation. Physical appearance and behavior (activity), plasma cytokine levels, and CD14 mRNA expression in heart, lung, liver, spleen and kidney tissue were determined 24 hours after CLP or sham operation.ResultsOnly Pep2.5 significantly enhanced the activity after CLP, whereas none of the therapeutic regimens elevated the mean arterial pressure or heart rate. The strongly elevated IL-6, IL-10 and monocyte chemoattractant protein serum levels in septic animals were significantly reduced after Pep2.5 administration (P < 0.001, P < 0.001, and P < 0.001, respectively). Similarly, Pep2.5 significantly reduced the sepsis-induced CD14 mRNA expression in heart (P = 0.003), lung (P = 0.008), and spleen tissue (P = 0.009) but not in kidney and liver.ConclusionsStructurally variable SALPs exhibit major differences in their anti-inflammatory effect in vivo. Continuous parenteral administration of Pep2.5 is able to reduce sepsis-induced cytokine release and tissue inflammation.

Inhibitory effect of opiates on LPS mediated release of TNF and IL-8

Most patients with advanced cancer experience severe pain and are often treated with opiates. Cancer patients are especially susceptible to opportunistic infections due to treatment with immunosuppressive and cytostatic drugs. Since opiates have been demonstrated to have immunomodulatory effects, it is of clinical importance to evaluate potential differences between commonly used opiates with regard to their effect on the immune system. The aim of this study was to evaluate the effect of morphine, tramadol, fentanyl and ketobemidone on the functioning of the immune system with special reference to TNF and IL-8 release. Method. U-937 cells were preincubated with different concentrations of opioids followed by stimulation with LPS 100 μg/ml for three hours. The effect of opioids on the levels of cytokine mRNA was studied using RT-PCR. Erk and Akt phosphorylation was also measured by Western blot. Results. All opioids with the exception of fentanyl were capable of inhibiting TNF release from U-937 cells. Morphine had no effect on IL-8 release but the effect of other opiates was almost the same as the effect on TNF. All opioids with the exception of fentanyl were capable of inhibiting production of mRNA for TNF and IL-8. The observed effects of opiates were not always reversible by naloxone, suggesting that the effects might be mediated by other receptors or through a non-receptor mediated direct effect. Although preliminary evidence suggests the involvement of Erk and Akt pathways, further studies are needed to unravel the intracellular pathways involved in mediating the effects of opiates. Our data suggests that the order of potency with regard to inhibition of cytokine release is as follows: tramadol > ketobemidone > morphine > fentanyl. Conclusion. Further studies are needed to understand the clinical implications of the observed immunosuppressive effects of tramadol and ketobemidone and to improve opioid treatment strategies in patients with cancer.

P096 IFN-A protects against antigen induced arthritis through induction of antigen specific cellular tolerance

Type I interferons are pleiotropic cytokines having an important regulatory role in autoimmune diseases including rheumatoid arthritis. Our earlier data show that IFN α protects against mBSA induced arthritis (Type I IFN protects against arthritis. Eur J Immunol June 2011;41(6):1687–95) . This IFN α mediated protection was manifested by inhibition of antigen specific proliferation but antigen specific total IgG levels remained largely unaffected. To further define the altered cellular response associated with IFNa-mediated protection against arthritis, the circulating cytokines as well as cytokines produced upon antigen restimulation of leukocytes ex vivo during the development of arthritis (day 14, day 21 and on the day of termination, day 28) was determined. We also assessed the effect of IFN α on antigen-specific IgG isotypes, which may differ depending on the dominant T helper cell response. Arthritis was induced by two injections (day 1 and day 7) of mBSA and adjuvant, with or without 100–5000 U of IFNa. Arthritis was triggered by intra-articular injection of mBSA on day 21 and arthritis was scored on day 28. Presence of IFNaat the time of antigen-sensitization protected mice from arthritis in a dose-dependent manner. No significant differences were found on antigen specific serum IgG1, IgG2a and IgG2b levels regardless of IFN α treatment. The circulating cytokine analysis as measured by Luminex showed that IFN- α decreases the production of cytokines IL-6 and IL-13 in the initial phase (day 14) of arthritis induction. Furthermore, this inhibitory effect of IFNawas also apparent in antigen-specific cytokine release ex-vivo at day 14. In the arthritis phase (day 28), IFN α significantly reduced the levels of circulating, pro-inflammatory cytokines (IL-1 β , IL-17, TNF- α , IFN γ , and IL-12), which were clearly up-regulated in the control group. However, antigen-specific release of cytokines (IFN γ , IL-6, IL-17 and IL-10) after re-stimulation ex-vivo at the day of termination (day 28) showed that splenocytes from IFNatreated mice responded with higher cytokine levels than controls. Conclusion: The ability of IFNato prevent arthritis development is most likely due to its effects on cellular rather than humoral immunity. Presence of IFNaat the time of antigen sensitization (day 1 and day 7) inhibits both in vivo and ex vivo release of pro-inflammatory cytokines throughout the arthritis induction. This results in clearly lower serum concentrations of pro-inflammatory cytokines after intra-articular challenge (day 28) and likely explains the protective effect of IFNa. The inhibition of cytokine release at the time of arthritis manifestation in the IFN α treated group is in line with our earlier published data of inhibited antigen specific re-call responses upon rechallange with mBSA. The increase in ex-vivo release of cytokines from splenocytes at this time point in the IFNa-treated group may indicate that the inhibitory effect on cytokine release by IFNaadministrated on day 1 and day 7 has a time limit.

Clinical efficacy of leflunomide in primary Sjögren's syndrome is associated with regulation of T-cell activity and upregulation of IL-7 receptor α expression

ObjectivesTo investigate whether the immunomodulatory capacities of leflunomide are associated with clinical efficacy in the treatment of primary Sjögren's syndrome (SS) in a phase II pilot study.MethodsPeripheral blood mononuclear cells...

Nonenzymatic glycation of high‐density lipoprotein impairs its anti‐inflammatory effects in innate immunity

In type 2 diabetes mellitus (T2DM), the abnormal protein and lipid composition of diabetic high-density lipoprotein (HDL) could impair its anti-inflammatory functions. Whether nonenzymatic glycation directly impaired the anti-inflammatory effects of HDL in innate immunity remained unclear. Human acute monocytic leukemia cell line (THP-1) cells, mouse RAW 264.7 macrophages and primary human monocytes derived macrophages were pre-incubated with native HDL, diabetic HDL isolated from T2DM patients or HDL glycated with different doses of d-glucose in vitro and then challenged with lipopolysaccharide (LPS). The release of tumor necrosis factor (TNF)-α and IL-1β was assayed by enzyme-linked immunosorbent assay (ELISA). Phosphorylation of Iκ-Bα in cytoplasm and nuclear translocation of NF-κB were detected by western blot. Glycation levels of native HDL, glycated HDL and diabetic HDL were determined using LC-MS/MS. The potency of diabetic HDL to inhibit the release of TNF-α (p < 0.05) and IL-1β (p < 0.001) was dramatically attenuated compared with that of native HDL. Similarly, glycation of HDL in vitro impaired its ability to inhibit TNF-α and IL-1β release in a glucose dose-dependent manner. Moreover, apoHDL still effectively inhibited the release of TNF-α and IL-1β induced by LPS, but glycated apoHDL partly lost such abilities. Nonenzymatic glycation levels of glycated HDL and diabetic HDL increased 28 fold (p < 0.001) and 4 fold (p < 0.001), respectively compared with that of native HDL. In this study, we observed that diabetic HDL and HDL glycated in vitro both partly lose their protective effects to inhibit cytokines release induced by LPS in macrophages, and nonenzymatic glycation of the protein components of HDL plays key roles in these impairments.

Reflex Principles of Immunological Homeostasis

The reasoning that neural reflexes maintain homeostasis in other body organs, and that the immune system is innervated, prompted a search for neural circuits that regulate innate and adaptive immunity. This elucidated the inflammatory reflex, a prototypical reflex circuit that maintains immunological homeostasis. Molecular products of infection or injury activate sensory neurons traveling to the brainstem in the vagus nerve. The arrival of these incoming signals generates action potentials that travel from the brainstem to the spleen and other organs. This culminates in T cell release of acetylcholine, which interacts with α7 nicotinic acetylcholine receptors (α7 nAChR) on immunocompetent cells to inhibit cytokine release in macrophages. Herein is reviewed the neurophysiological basis of reflexes that provide stability to the immune system, the neural- and receptor-dependent mechanisms, and the potential opportunities for developing novel therapeutic devices and drugs that target neural pathways to treat inflammatory diseases.

Effects of Pantoprazole on Systemic and Gastric Pro- and Anti-inflammatory Cytokines in Critically Ill Patients.

Stress-related mucosal damage (SRMD) is a significant cause of morbidity and mortality in critically ill patients due to the gastrointestinal blood loss. Prophylaxis of SRMD with proton pump inhibitors or histamine-2 blockers has gained widespread use in intensive care units. Both demonstrated to be effective in reducing clinically significant bleedings, while PPIs has shown to exert some anti inflammatory effects including the inhibition of producing pro-inflammatory cytokines. As cytokines have role in developing SRMD, the aim of this study was to evaluate the effect of PPIs on the inhibition of cytokine release following the critical illness. A total of 27 critically ill patients with risk factors of developing stress ulcer and intragastric pH < 3.0 enrolled to this Randomized clinical trial study. Patients were randomly assigned in three treatment groups; group one received 40 mg of intravenous pantoprazole every 12 h for 48 h (four doses), group two received 80 mg of intravenous pantoprazole every 24 h continuous infusion for 48 h and the third group received 150 mg of ranitidine intravenously as 24 h continuous infusion for 48 h. Plasma and gastric juice samples were obtained at 0th, 12th, 24th and 48th h for the measurement of EGF, IL-1β, IL-6, IL-10 and TNF-α. Pantoprazole infusion have decreased the plasma IL-1β concentrations (p = 0.041). No other significant differences in concentrations of EGF, IL-6, IL-10 and TNF-α were detected. There were reverse correlations between the intragastric pH with gastric juice IL-1β and TNF-α concentrations and a direct correlation between the intragastric pH and gastric juice EGF in pantoprazole groups. Our data suggest that pantoprazole may have some anti-inflammatory effects on patients. However, the exact impact of this effect on patients should be assessed by further studies.

Novel 1,2,5-Oxadiazole 2-Oxide Derivatives with Analgesic and Fetal Hemoglobin Induced Properties Designed As Drug Candidate to Treat Sickle Cell Disease Symptoms

Abstract 2137 Introduction.The combination of multiple activities in the same structure using molecular modification is a powerful tool to discover more effective and safe compounds to treat sickle cell disease (SCD) symptoms. The only FDA approved drug for SCD, at present, is hydroxyurea (HU). The release of NO by HU is an important mechanism of its HbF-inducing properties. Thalidomide and some analogues are known to inhibit cytokines release, presenting important analgesic activity. Recently the drug has also been reported to induce gamma-globin expression. Our rational drug design used molecular hybridization between thalidomide and NO donor subunits, represented by 1,2,5-oxadiazole 2-oxide. The aim of this study was to evaluate the effects of eight novel hybrid compounds (3a-h) on NO donor activity, analgesic activity and γ-globin gene expression. Methods. 1. Detection of nitrite. A solution of the appropriate compound (20 μL) in DMSO was added to 2 mL of a mixture of 50 mM phosphate buffer (pH 7.4) and methanol (1:1, v:v), containing 5 mM of L-cysteine. The final concentration of the compound was 10−4 M. After 1 h at 37 °C, 1 mL of the reaction mixture was treated with 250 μL of Griess reagent. After 10 min at room temperature, the absorbance was measured at 540 nm using a spectrophotometer. Standard sodium nitrite solutions (10–80 nmol/mL) were used to construct the calibration curve. The yields of nitrite are expressed as % NO2∓ (mol/mol). 2. Antinociceptive activity. Analgesic activity was determined in vivo with the acetic-acid-induced (0.6%, 0.1 mL/10 g) abdominal constriction test in mice. Swiss mice of both sexes (18–23 g) were used. The compounds were administered orally (100 μmol/kg) as a suspension in 5% arabic gum in saline (vehicle). Dypirone (100 μmol/kg) was used as the standard drug. Acetic acid solution was administered i.p. 1 h after the administration of the compounds. Ten minutes after the i.p. acetic acid injection, the number of constrictions per animal was recorded for 20 min. The control animals received an equal volume of vehicle. Antinociceptive activity was expressed as percentage inhibition of the constrictions compared with those in the vehicle-treated control group. The data were analyzed statistically with Student’s t test at a significance level of P < 0.05. 3. Gamma-globin gene expression. Human K562 cells were maintained in DMEM with 10% FBS, Pen/Strep, in humidified air (5% CO2, 37°C). Cells (1×107cells/100mL) were incubated with compounds at different concentrations (5, 30, 60, and 100μM) for 24, 48, 72 and 96h. g-Globin gene expression was analyzed by qRT-PCR and quantified using the Gnorm program. Results are expressed as arbitrary units. Results. 1. Detection of nitrite: The quantification of the nitrite produced resulted from the oxidative reaction of NO, oxygen and water. All eight compounds were capable of inducing nitrite formation at concentrations of between 9.5% and 28%. Isosorbide dinitrate (DNS), used as the control, induced 11.7% nitrite formation. 2.Antinociceptive activity: Compounds 3c and 3d were the most active antinociceptive compounds, significantly reducing the acetic-acid-induced abdominal constrictions by 43% and 38%, respectively, while dypirone used as a control inhibited constrictions by 34%. 3. Gamma-globin gene expression: Compound 3c demonstrated inverse dose-response relationships, achieving the highest levels of γ-globin induction at 5 and 30 μM. Compound 3c achieved maximal γ-globin induction as early on as 48h after treatment ([48h; 5 μM]: 1,88±0.06 AU; control 0.78±0.1 AU; P<0.05). Testing of compound 3d is currently underway. Conclusions. Results demonstrate that molecular hybridization between a thalidomide derivative and nitric oxide donors may be an important tool to treat SCD symptoms. These compounds demonstrated NO donor and analgesic activity. Specifically, the compound 3c was capable of inducing gamma-globin expression. Testing of our lead compound 3c in a preclinical mouse model of SCD will be performed in order to evaluate its efficacy in the treatment of this hemoglobinopathy. Disclosures:No relevant conflicts of interest to declare.

Selective Inhibition of Activated B-Cell Lymphoma Cells by a Novel Small Molecule Inhibitor of NF-κB

Abstract 2498Diffuse large B-cell lymphomas (DLBCL) account for approximately 40% of lymphomas in adults, with the activated B-cell lymphoma (ABC) subtype being the least curable. ABC lymphoma cells display the phenotype of activated B-cells, which is induced by constitutive activation of the transcription factor Nuclear Factor-kappa B (NF-κB). NF-κB activation in ABC lymphoma can result from several different mutations or abnormal expression of proteins upstream of NF-κB nuclear translocation. No matter the mechanism of NF-κB constitutive activation, the resulting gene expression pattern confers an anti-apoptotic and pro-survival advantage to B-cells, hence driving the oncogenesis and supporting the survival of ABC lymphomas. Therefore, inhibiting NF-κB activity is an attractive strategy for the treatment of ABC lymphomas.Using a cell line with a reporter for NF-κB transcriptional activity in a high throughput screen of 180,000 compounds we discovered 12 compounds that inhibited NF-κB activation, suggesting these compounds negatively regulate NF-κB. Since ABC lines are generally more dependent on NF-κB signalling for cell survival than are the other types of B-cell lymphoma, the hits from the primary screen were tested in a secondary screen for the ability to selectively inhibit the growth of ABC lymphoma cells. Of the 12 hits, two compounds belonging to the same quinolone chemotype were found to selectively inhibit the growth of an ABC line compared to a non-ABC line and normal human peripheral blood mononuclear cells (PBMCs). Three more structurally related quinolones were obtained to investigate a possible limited structure activity relationship for this class of molecules, designated here as Quinolone Inhibitors of NF-κB (QINs). QIN 1 was significantly more potent (Figure A and B) and selective (Figure B and C) relative to other QINs. The limited structure activity relationship suggests that two structural regions of the chemotype may be important for potency and for ABC selectivity, hence providing impetus to further investigate QINs as possible ABC lymphoma drugs.QIN1 is more potent in inhibiting the growth of ABC lines than a known inhibitor of NF-κB activity, a commercially available selective IKK inhibitor (CAS 507475-17-4). Active IKK causes degradation of IκBα, the natural inhibitor of NF-κB, hence IKK inhibitors prevent NF-κB activation by preventing degradation of IκBα. In addition to being more potent, QIN1 retains similar ABC selectivity as the IKK inhibitor (Figure C). QIN1 and the IKK inhibitor both cause apoptosis at a similar rate and both cause G1 arrest in ABC lines at equi-toxic concentrations, initially suggesting similar mechanisms of action. Subsequently, QIN1was found to inhibit NF-κB in several different assays using the IKK inhibitor as a positive control. First, QIN1 inhibited the activation of a transcription based NF-κB reporter cell line in response to LPS, an NF-κB activator. Supporting these results, QIN1 inhibited cytokine release from human PBMCs stimulated with LPS. In addition, QIN1 prevented degradation of IκBα in response to NF-κB activating stimuli, further demonstrating the ability of QIN1 to inhibit NF-κB activity. Finally, QIN1 inhibited nuclear translocation of active NF-κB, thus preventing pro-survival gene expression.The QINs studied here all contain an alpha-beta unsaturated ketone, an electrophilic chemical moiety known to interact with cysteine thiols. Compounds containing similar electrophilic groups are known to bind a cysteine thiol in IKK, preventing its activity and hence inhibiting NF-κB activation. This suggests that this electrophilic moiety in QINs maybe responsible for the NF-κB inhibition observed in these cell lines. The presence of this electrophilic group is not necessarily detrimental to normal cells as we have shown with QIN1 in vitro (Figure A-C) and in vivo (large daily doses of QIN1 cause no observable toxicities in mice). Overall, our data indicate QIN1 inhibits IKK, a kinase immediately upstream of NF-κB nuclear translocation, allowing the majority of ABC lymphomas to be treated irrespective of the upstream mechanism of pathway activation. The selectivity for inhibition of ABC lymphoma cells by QIN1 and our in vitro data showing synergy with an inhibitor of B-cell receptor signalling, which also promotes ABC survival, provides the impetus for further preclinical development of QINs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Inhibition of Autoimmune Diabetes by TLR2 Tolerance

We have reported that apoptotic β cells undergoing secondary necrosis, called "late apoptotic (LA) β cells," stimulated APCs and induced diabetogenic T cell priming through TLR2, which might be one of the initial events in autoimmune diabetes. Indeed, diabetogenic T cell priming and the development of autoimmune diabetes were significantly inhibited in TLR2-null NOD mice, suggesting the possibility that TLR2 blockade could be used to inhibit autoimmune diabetes. Because prolonged TLR stimulation can induce TLR tolerance, we investigated whether repeated TLR2 administration affects responses to LA β cells and inhibits autoimmune diabetes in NOD mice by inducing TLR2 tolerance. Treatment of primary peritoneal macrophages with a TLR2 agonist, Pam3CSK(4), suppressed cytokine release in response to LA insulinoma cells or further TLR2 stimulation. The expression of signal transducer IRAK-1 and -4 proteins was decreased by repeated TLR2 stimulation, whereas expression of IRAK-M, an inhibitory signal transducer, was enhanced. Chronic Pam3CSK(4) administration inhibited the development of diabetes in NOD mice. Diabetogenic T cell priming by dendritic cells and upregulation of costimulatory molecules on dendritic cells by in vitro stimulation were attenuated by Pam3CSK(4) administration in vivo. Pam3CSK(4) inhibited diabetes after adoptive transfer of diabetogenic T cells or recurrence of diabetes after islet transplantation by pre-existing sensitized T cells. These results showed that TLR2 tolerance can be achieved by prolonged treatment with TLR2 agonists, which could inhibit priming of naive T cells, as well as the activity of sensitized T cells. TLR2 modulation could be used as a novel therapeutic modality against autoimmune diabetes.

Managing immunity in resistant cancer patients correlates to survival: results and discussion of a pilot study.

Many cancer patients do not die due to impaired organ functions, but as a result of reduced general conditions, such as cachexia, sarcopenia, depression, infections, or stress. Reduced general health may be caused by immune modifying cytokines released from the tumor into the body. Improvement of immunity would not only reduce cancer side effects through inhibiting cytokine release from the tumor into the blood, but also, according to a new hypothesis, modify the cancer stem cells (CSC) in the tumor, which are believed to drive cancer growth and metastasis. We reported previously several investigations with a dietary fermented soy formulation (FSWW08) in cancer patients, where we saw a) strong reduction of cancer symptoms, b) broken resistance to chemotherapy, and c) a strong reduction of chemotherapy's toxic side effects, when taken in combination. This publication reports two new findings from a pilot study with postsurgical, treatment resistant patients conducted over four years. First, neither treatment resistance nor side effects were observed. Second, more patients have survived than expected. The improved health and immunity is detected together with increased CSC differentiation, suggesting lower aggressiveness, which was corroborated by increased gene expressions, particularly of steroidal hormones, MAPkinase, NF-κB, and tumor suppressor factor p53, a typical marker of "stemness" or cell differentiation. Although limited by its small, homogenous sample size, the results of this pilot study illustrate the relationship between CSCs differentiation, and the clinical symptoms of immunity, which influence survival outcomes and raise the clinical potential of measuring CSCs in ovarian, prostate, and breast cancers. The improved survival rates are also seen in larger cohort studies, which show similar gene expression profiles, which were induced by FSWW08 in the treatment resistant cancer patients in this study.