Cytokine Production In Whole Blood Research Articles

LPS-induced whole-blood cytokine production and depressive symptoms in dementia spousal caregivers: The moderating effect of childhood trauma

Dementia spousal caregivers are at risk for adverse mental and physical health outcomes. Caregiver burden, anticipatory grief, and proinflammatory cytokine production may contribute to depressive symptoms among caregivers. People who report childhood trauma are more likely to have exaggerated stress responses that may also contribute to depressive symptoms in adulthood. This study aimed to test whether the relationship between whole-blood cytokine production and depressive symptoms is strongest in caregivers who report high levels of childhood trauma. MethodsA sample of 103 dementia spousal caregivers provided self-report data on demographics, health information, caregiver burden, anticipatory grief, and depressive symptoms. We also determined lipopolysaccharide-induced whole-blood cytokine production as the primary measure of immune cell reactivity. We measured interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) and converted z-scores of each cytokine into a composite panel. We regressed depressive symptoms on proinflammatory cytokine production, caregiver burden, and anticipatory grief, adjusting for demographic and health-related covariates. ResultsWhole-blood cytokine production and childhood trauma were associated with depressive symptoms. Childhood trauma moderated the relationship between whole-blood cytokine production and depressive symptoms. Whole-blood cytokine production was only associated with depressive symptoms at mean and high levels of childhood trauma, but not at low levels of childhood trauma. The main effects of burden and anticipatory grief on depressive symptoms were strongest for caregivers reporting high levels of childhood trauma. DiscussionChildhood trauma has lasting impacts on psychosocial experiences later in life and has effects that may confer susceptibility to inflammation-related depression. Our findings contribute to ongoing efforts to identify risk factors for adverse mental health in dementia spousal caregivers.

Effect of a novel anti-PD-1-proIL-2 bifunctional fusion protein on potent anti-tumor activity via PD-1 checkpoint inhibition and conditional IL-2R agonism.

e14697 Background: HD IL-2 was approved by FDA for metastatic melanoma (mM) and renal cell carcinoma (mRCC) but the treatment is associated with severe toxicities. PD-1 blockade has improved the overall survival, but only in 20-30% of cancer patients and many of them experience frequent relapses. Interestingly, HD IL-2 therapy displayed durable anti-tumor activity in mM and mRCC patients who progressed on anti-PD-1. Furthermore, combination therapy of IL-2 and pembrolizumab in mRCC has shown durable 70% response rate, while the ORR of monotherapy is 20% and 33% for IL-2 and pembrolizumab, respectively. Thus, combining IL-2 receptor activation and PD-1 blockade may represent a promising strategy in the post PD-1 era to overcome the resistance and improve the clinical efficacy. PTX-912 is a novel bifunctional PD-1-proIL-2v fusion protein designed to simultaneously harness PD-1 blockade and PD-1-cis directed IL-2R agonism conditionally in TME without the peripheral toxicities associated with IL-2. Methods: The biological functions were demonstrated in human PBMC assays. Anti-tumor efficacy and biomarker studies were evaluated in mouse / human PBMC xenograft models. Tox studies were performed in NHP. Results: PTX-912 shows dose-dependent inhibition of PD-1 pathway measured by GM-CSF and IFNgamma production in an MLR assay. Protease activated PTX-912, but not the intact drug, stimulates the phosphorylation of STAT5 of NK and T cells, the proliferation of CD8 T cells as well as the cytokine production in human whole blood. The potency of pSTAT5 by protease activated product is enhanced by > 10 folds on PD-1+ T cells than T cells without PD-1. When tested in mouse tumor models resistant to PD-1 blockade, PTX-912 demonstrated high potency of tumor inhibition at as low as 0.1 mg/kg dose. In contrast, non-targeted proIL-2v at molar equivalent or higher dose showed minimal antitumor effect in either monotherapy or combination with anti-PD-1. Analysis of PTX-912 treated mice showed increased tumor infiltrating CD4+ and CD8+ T cells, including PD-1+Tcf-1+ and PD-1+ IL-18Ra+TIM3- CD8+ T cells, referred to “stem-like” progenitor effector cells and “better” effector cells, respectively. Activation of the IL-2v in PTX-912 was observed in mouse tumor tissues but not in the plasma. In monkey GLP-compliant 4-week repeat-dose toxicity study, PTX-912 demonstrated excellent safety profiles without overt irAEs that are characteristic of IL-2. Conclusions: Our preclinical data demonstrated that PTX-912 effectively stimulated the expansion and reinvigoration of antigen specific T cells in the tumor tissue resulting in strong anti-tumor efficacy while it did not cause significant peripheral toxicity. Phase 1 clinical trial of PD-1-proIL-2v is planned to start in early 2024 to evaluate the safety, pharmacokinetics and anti-tumor activity in patients with advanced solid tumors.

Abstract 2473: A novel anti-PD-1 and proIL-2 bifunctional fusion protein delivers potent anti-tumor activity via PD-1 checkpoint inhibition and tumor selective IL-2R agonism preferentially on antigen specific T cells

Abstract High-dose IL-2 (HD IL-2) induces complete and durable responses in RCC and melanoma but the treatment is associated with severe toxicities. PD-(L)1 blockade represents a recent breakthrough in immunotherapy with solid tumors, but only 20-30% of patients can benefit from the therapy and most responders eventually develop resistance. In more recent clinical studies with small number of subjects, HD IL-2 has shown promising responses in patients who progressed on anti-PD-1 therapy and combination therapy of HD-IL-2 and PD-1 blockade has resulted in high rate of objective response. In an attempt to maximize the clinical benefit of these immunotherapies to a broader patient population with deeper response and in the meantime to minimize the severe toxicity of HD IL-2, we have generated a novel bifunctional PD-1-proIL-2v fusion protein harnessing the mechanisms of PD-1 blockade and PD-1-cis directed tumor conditional IL-2R activation. PD-1-proIL-2v shows dose dependent inhibition of PD-1 immune checkpoint pathway measured by GM-CSF and IFNγ production in an MLR assay. Protease activated PD-1-proIL-2v, but not the intact drug, stimulates the phosphorylation of STAT5 of NK and T cells, the proliferation of CD8 T cells as well as the cytokine production in human whole blood. The potency of pSTAT5 by protease activated product is enhanced by 10-20 folds on PD-1+ T cells than T cells without PD-1. When tested in mouse tumor models resistant to PD-1 blockade, PD1-proIL-2v demonstrated high potency of tumor inhibition at as low as 0.1 mg/kg dose. In contrast, non-targeted proIL-2v at molar equivalent or higher dose showed minimal antitumor effect in either monotherapy or combination with anti-PD-1. Analysis of PD-1-proIL-2v treated mice showed dramatically increased tumor infiltrating CD4+ and CD8+ T cells, including PD-1+Tcf-1+ and PD-1+ IL-18Ra+TIM3− CD8+ T cells, referred to “stem-like” progenitor effector cells and “fresh” effector cells, respectively. Activation of the IL-2 procytokine in PD-1-proIL-2v was observed in mouse tumor tissues but not in the plasma. In monkey GLP-compliant 4-week repeat-dose toxicity study, PD-1-proIL-2v demonstrated excellent safety profiles without overt irAEs that are characteristic of IL-2. In conclusion, our preclinical data demonstrated that PD-1-proIL-2v effectively stimulated the expansion and reinvigoration of antigen specific T cells in the tumor tissue resulting in strong anti-tumor efficacy while it did not cause significant peripheral toxicity. Phase 1 clinical trial of PD-1-proIL-2v is planned to start in early 2024 to evaluate the safety, pharmacokinetics and anti-tumor activity in advanced solid tumors. Citation Format: Zijuan Li, Feifei Zhang, Ze Zhang, Harry Zhou. A novel anti-PD-1 and proIL-2 bifunctional fusion protein delivers potent anti-tumor activity via PD-1 checkpoint inhibition and tumor selective IL-2R agonism preferentially on antigen specific T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2473.

The role of cortisol in immunosuppression in subarachnoid haemorrhage

BackgroundWe sought to determine the extent to which cortisol suppressed innate and T cell-mediated cytokine production and whether it could be involved in reducing peripheral cytokine production following subarachnoid haemorrhage (SAH).MethodsWhole blood from healthy controls, patients with SAH and healthy volunteers was stimulated with lipopolysaccharide (LPS), to stimulate innate immunity, or phytohaemagglutinin (PHA), to stimulate T cell-mediated immunity. Varying concentrations of cortisol were included, with or without the cortisol antagonist RU486. Concentration of interleukin-6 (IL-6), IL-1β and tumour necrosis factor-alpha) TNFα were determined as a measure of innate immunity. IL-6, IL-17 (interferon gamma) IFNƔ and IL-17 were determined as an indicator of T cell-mediated immunity.ResultsSuppression of innate responses to LPS was apparent in whole blood from SAH patients, relative to healthy controls, and TNFα production was inversely correlated with plasma cortisol concentration. Cytokine production in whole blood from healthy volunteers was inhibited by cortisol concentrations from 0.33 µM, or 1 µM and above, and these responses were effectively reversed by the cortisol antagonist RU-486. In SAH patients, RU-486 reversed suppression of innate TNF-α and IL-6 responses, but not IL-1ß or T cell-mediated responses.ConclusionThese data suggest that cortisol may play a role in reducing innate, but not T cell-mediated immune responses in patients with injuries such as SAH and that cortisol antagonists could be effective in boosting early innate responses.

Effects of Caffeine and 5-Caffeoylquinic Acid on Blood Cell In Vitro Cytokine Production in Response to Lipopolysaccharide Stimulation

Previous in vitro studies the have suggested anti-inflammatory properties of caffeine. Coffee is also rich in phenolic acids with several potential health benefits (chlorogenic acids), mainly 5-caffeoylquinic acid (5-CQA). The aim of the present study was to determine the effect of caffeine and 5-CQA, using concentrations comparable to typical human consumption, on LPS-induced cytokine production in whole blood in vitro cultures. Whole blood samples (n = 10) were stimulated for 24 h with LPS in the presence of caffeine or 5-CQA. The concentrations of various cytokines, malondialdehyde (MDA) and adenosine-3′,5′-cyclic monophosphate (cAMP) were determined in the culture media. Significant effects of caffeine were found only for the stimulated production of TNF-α (p = 0.014) and cAMP (p = 0.001). However, only the highest caffeine concentration (50 μM) induced lower TNF-α (p = 0.016) and higher cAMP (p = 0.006) levels in comparison to the control. The 5-CQA only influenced the stimulated production of IL-6 (p = 0.002), with lower values for the highest 5-CQA concentrations (2 μM: p = 0.012; 20 μM: p < 0.001). Neither caffeine (p = 0.227) nor 5-CQA (p = 0.259) influenced MDA concentrations. In conclusion, both caffeine and 5-CQA exerted limited in vitro anti-inflammatory effects. For caffeine, these effects were attained with high physiological concentrations. However, for 5-CQA, supraphysiological concentrations were needed.

Effect of the p38 MAPK inhibitor doramapimod on the systemic inflammatory response to intravenous lipopolysaccharide in horses.

BackgroundDoramapimod, a p38 MAPK inhibitor, is a potent anti‐inflammatory drug that decreases inflammatory cytokine production in equine whole blood in vitro. It may have benefits for treating systemic inflammation in horses.ObjectiveTo determine whether doramapimod is well tolerated when administered IV to horses, and whether it has anti‐inflammatory effects in horses in a low‐dose endotoxemia model.AnimalsSix Standardbred horses.MethodsTolerability study, followed by a blinded, randomized, placebo‐controlled cross‐over study. Horses were given doramapimod, and clinical and clinicopathological variables were monitored for 24 hours. Horses then were treated with doramapimod or placebo, followed by a low dose infusion of lipopolysaccharide (LPS). Clinical variables (heart rate, rectal temperature, noninvasive blood pressure), leukocyte count and tumor necrosis factor alpha (TNF‐α) and interleukin‐1 beta (IL‐1β) concentrations were measured at multiple time points until 6 hours post‐LPS infusion.ResultsNo adverse effects or clinicopathological changes were seen in the safety study. When treated with doramapimod as compared to placebo, horses had significantly lower heart rates (P = .03), rectal temperatures (P = .03), and cytokine concentrations (P = .03 for TNF‐α and IL‐1β), and a significantly higher white blood cell count (P = .03) after LPS infusion.Conclusions and Clinical ImportanceDoramapimod has clinically relevant anti‐inflammatory effects in horses, likely mediated by a decrease in leukocyte activation and decrease in the release of pro‐inflammatory cytokines. To evaluate its potential as a novel treatment for systemic inflammatory response syndrome in horses, clinical trials will be necessary to determine its efficacy in naturally occurring disease.

OP0046 TARGETING IRAK1 AND 4 SIGNALING WITH R835, A NOVEL ORAL SMALL MOLECULE INHIBITOR: A POTENTIAL NEW TREATMENT FOR SYSTEMIC LUPUS ERYTHEMATOSUS

Background:Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by loss of immunological tolerance, hyperactivation of immune cells, proinflammatory cytokine production and, ultimately, end organ damage due to immune complex deposition. Toll-like receptors (TLRs), which are essential to the innate immune response to microbes and other danger signals, play a key role in the pathogenesis of SLE by recognition of self-molecules (1). Interleukin receptor associated kinases (IRAK)1 and 4 are responsible for initiating MyD88-dependent signaling from most TLRs and Interleukin-1 Receptors (IL-1R) and represent attractive targets for the therapeutic treatment of SLE (2). We have identified a potent and selective IRAK1/4 inhibitor, R835, that substantially suppressed the elevation of LPS (TLR4 agonist)-induced serum cytokines in healthy human volunteers in a recently completed phase 1 study.Objectives:The aim of our study was to investigate the effect of IRAK1/4 selective inhibition as potential therapeutic approach for SLE. We evaluated the effect of our clinical candidate R835 on TLR7 signaling and in a mouse model of lupus-like disease.Methods:Human primary dendritic cells and whole blood were stimulated with gardiquimod (TLR7 agonist) to evaluate the effect of R835 on Interferon-alpha (IFN-α) production. R835 was further evaluated for its efficacy on survival and disease progression in lupus-prone NZB/W F1 mice with early or active signs of disease.Results:R835 inhibited TLR7-induced cytokine production in human dendritic cells and whole blood. Given orally to mice, R835 dose-dependently decreased serum IFN-α in response to administration of a TLR7 agonist. Furthermore, treatment of NZB/W F1 lupus-prone mice with R835 reversed the progression of lupus-like disease and the establishment of a pro-inflammatory environment, as demonstrated by decreased levels of proteinuria, blood urea nitrogen and autoantibodies, and reversal of renal pathology.Conclusion:To our knowledge, R835 is the first dual IRAK1/4 inhibitor to enter clinical development and provides an attractive approach to treat a range of autoimmune and rheumatic diseases, including lupus.

Natural cannabinoids suppress the cytokine storm in sepsis-like in vitro model

Natural cannabinoids may have beneficial effects on various tissues and functions including a positive influence on the immune system and the inflammatory process. The purpose of this study was to investigate the effects of natural cannabinoids on the production of pro-inflammatory cytokines by lipopolysaccharide (LPS)-stimulated whole human blood cells. Levels of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were measured before and after exposure of LPS-stimulated whole blood to different concentrations of Cannabidiol (CBD) or a combination of CBD and Tetrahydrocannabinol (THC) extract. LPS stimulated the production of the pro-inflammatory cytokines. Exposure to both CBD and CBD/THC extracts significantly suppressed cytokine production in a dose-dependent manner. Exposure to cannabinoid concentrations of 50 μg/mlor 100 μg/mlresulted in a near-complete inhibition of cytokine production. This study demonstrates that natural cannabinoids significantly suppress pro-inflammatory cytokine production in LPS-stimulated whole blood in a dose-dependent manner. The use of human whole blood, rather than isolated specific cells or tissues, may closely mimic an in vivo sepsis environment. These findings highlight the role that natural cannabinoids may play in suppressing inflammation and call for additional studies of their use as possible novel therapeutic agents for acute and chronic inflammation.

Inflammation and coagulation crosstalk induced by BJcuL, a galactose-binding lectin isolated from Bothrops jararacussu snake venom

Inflammation and coagulopathies are important systemic events following snakebite. Snake venom galactoside-binding lectins (SVgalLs) are known modulators of the immune response with no direct effect on hemostasis. Considering the crosstalk between inflammation and coagulation, the present study investigated how BJcuL, a proinflammatory SVgalL isolated from Bothrops jararacussu venom, mediated the inflammation-induced procoagulant activity. We examined the proinflammatory cytokine production and procoagulant tissue factor (TF) activity in human whole blood and monocyte-rich cell suspension (MR-PBMC) treated with BJcuL. This lectin increased production of the cytokines TNF-α, IL-1β and IL-6, upregulated TF expression on the cell surface, and induced procoagulant activity. The proinflammatory behavior was mediated by the direct interaction between the lectin and toll-like receptor 4, via binding to β-galactoside-containing glycoconjugates on the cell surface, and activation of NFκ-B signaling. Interestingly, the BJcuL-induced inflammation was directly associated with the procoagulant activity of MR-PBMC cells. In whole blood culture, the lectin exhibited similar behavior, i.e. it induced cytokine production and MR-PBMC TF-mediated procoagulant activity. Therefore, the present study is the first report on the inflammation-induced procoagulant activity of SVgalLs, and it indicates that BJcuL is an important factor associated with coagulopathy in patients with snake envenomation.

Antibacterial action of synthetic antilipopolysaccharide peptides (SALP) involves neutralization of both membrane-bound and free toxins.

Increasing failure of conventional antibiotics to combat bacterial infections requires the urgent development of new antibacterial drugs; a promising class of new drugs based on antimicrobial peptides. Here, we studied the molecular interaction of polycationic synthetic antilipopolysaccharide peptides (SALPs) with various gram-negative and gram-positive bacteria, including resistant strains. The analysis of antimicrobial activity by conventional techniques and atomic force microscopy showed a strict dependence on amino acid (aa) sequences, with the type of amino acid, its position within the primary structure, and the sequence length being critical parameters. By monitoring lipopolysaccharide (LPS)- or bacteria-induced cytokine production in human mononuclear cells and whole blood, we found a direct link between the binding of the lead compound Pep19-2.5 to Salmonella enterica and the anti-inflammatory activity of the peptide. Thermodynamic analysis of Pep19-2.5 binding to the bacterial cell envelope showed an exothermic reaction with saturation characteristics, whereas small-angle X-ray scattering data indicated a direct attachment of Pep19-2.5 to the bacterial cell envelope. This binding preferentially takes place to the LPS outer monolayer, as evidenced by the change in the LPS acyl chain and phosphate vibrational bands seen by Fourier-transform infrared spectroscopy. We report here that the anti-inflammatory activity of Pep19-2.5 is not only connected with neutralization of cell-free bacterial toxins but also with a direct binding of the peptide to the outer leaflet of the bacterial outer membrane.

Clinical relevance of single nucleotide polymorphisms in the CXCL1 and CXCL12 genes in patients with major trauma

Genetic backgrounds have been recognized as significant determinants of susceptibility to sepsis. CXC chemokines play a significant role in innate immunity against infectious diseases. Genetic polymorphisms of CXC chemokine genes have been widely studied in inflammatory and infectious diseases but not in sepsis. Thus, we aimed to investigate the clinical relevance of CXC chemokine gene polymorphisms and susceptibility to sepsis in a traumatically injured population. Thirteen tag single nucleotide polymorphisms were selected from CXC chemokine genes using a multimarker tagging algorithm in the Tagger software. Three independent cohorts of injured patients (n = 1700) were prospectively recruited. Selected single nucleotide polymorphisms were genotyped using an improved multiplex ligation detection reaction method. Cytokine production in lipopolysaccharide-stimulated whole blood was measured using an enzyme-linked immunosorbent assay. Among the 13 tag single nucleotide polymorphisms, four single nucleotide polymorphisms (rs1429638, rs266087, rs2297630, and rs2839693) were significantly associated with the susceptibility to sepsis, and three (rs3117604, rs1429638, and rs4074) were significantly associated with an increased multiple organ dysfunction score in the derivation cohort. However, only the clinical relevance of rs1429638 and rs266087 was confirmed in the validation cohorts. In addition, rs2297630 was significantly associated with interleukin 6 production. The rs1429638 polymorphism in the CXCL1 gene and the rs2297630 polymorphism in the CXCL12 gene were associated with altered susceptibility to sepsis and might be used as important genetic markers to assess the risks of sepsis in trauma patients. Prognostic and epidemiologic study, level II.

Effect of immunosuppressive drugs on cytokine production in canine whole blood stimulated with lipopolysaccharide or a combination of ionomycin and phorbol 12-myristate 13-acetate.

A pharmacodynamic assay has been previously developed to monitor ciclosporin treatment in dogs by assessing inhibition of cytokine transcription after whole blood stimulation with 12‐myristate 13‐1 acetate and ionomycin (PMA/I). In this study, whole blood stimulation with either PMA/I or lipopolysaccharide (LPS) was used to assess the effect of multiple drugs (azathioprine, ciclosporin, mycophenolate, leflunomide and prednisone) after a 7‐day treatment course on production of cytokines measured with a multiplex assay in healthy dogs (n = 4 for each treatment). Interleukin‐10 (IL‐10), interferon gamma (IFN γ) and tumour necrosis factor alpha (TNF α) were significantly activated by PMA/I stimulation and IL‐6, IL‐10 and TNF α by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL‐10, IFN γ and TNF α production was significantly reduced after stimulation with PMA/I compared to pre‐treatment. After prednisone treatment, TNF α production was significantly reduced after stimulation with PMA/I or LPS compared to pre‐treatment. No significant change was observed after treatment with azathioprine, leflunomide or mycophenolate. This methodology may be useful to monitor dogs not only treated with ciclosporin, but also with prednisone or a combination of both. Further studies are needed to assess the use of this assay in a clinical setting.

Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel.

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC50 = 10 nM) with reduced hERG activity (14 μM).

Abstract LB-291: EOS100850, an insurmountable and non-brain penetrant A2A receptor antagonist, inhibits adenosine-mediated T cell suppression, demonstrates anti-tumor activity and exhibits best-in class characteristics

Abstract High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor (A2AR), suppresses the Th1 cytokine production of T cells and monocytes and cytolytic activity of T and NK cells. We demonstrated that A2AR antagonists initially designed for Parkinson's disease but repurposed for immuno-oncology dramatically lost potency in a high adenosine environment. We therefore developed EOS100850, a novel, non-brain penetrant and highly selective inhibitor of A2AR with sub-nanomolar Ki. Using experimental conditions that mimic tumor environment, we have shown that EOS100850 potently inhibited A2AR signalling in human T lymphocytes independently of adenosine concentrations, and rescued cytokine production, even in the presence of high concentrations of A2AR agonists. iTeos A2AR antagonist potently rescued Th1 cytokine production in human whole blood treated by A2AR agonists, and increased CD8+ T cell cytotoxicity in a co-culture assay of effector CD8+ T cells and target cancer cells. An in vivo pharmacodynamic assay based on phosphorylation of CREB (pCREB) in mouse peripheral CD8+ and CD4+ T was developed and validated as a readout for A2AR activation. EOS100850, 30 minutes after oral gavage at doses ranging from 0,03 to 1mg/kg demonstrated 80 to 100% inhibition of pCREB induced by the ex-vivo addition of A2AR agonist. Remarkably, 12 hours after gavage at 1 and 3mg/kg, when the EOS100850 antagonist was no longer detectable in the plasma, more than 50% of inhibition of pCREB was still observed. These results demonstrate that EOS100850 has a PD activity that extends well beyond its PK based on a long residence time. iTeos's A2AR antagonist, uniquely designed to address the challenge of counteracting elevated adenosine concentrations in tumors, was tested for the first time in a mouse A20 lymphoma model. iTeos's A2AR antagonist in combination with anti-PD-L1 demonstrated significant tumor growth suppression (p=0.0008) compared with anti-PD-L1 alone, with a 6 fold decrease in tumor volume compared to anti-PD-L1 alone (median tumor volume =105±79 mm3 vs 696±427mm3 on day 23, respectively). EOS100850 represents a novel, potent, insurmountable and best-in-class A2AR blocker, specifically optimized for immuno-oncology indications, that deserves studies in Human. Citation Format: Erica Houthuys, Reece Marillier, Théo Deregnaucourt, margreet Brouwer, Paola Basilico, Romain Pirson, Joao Marchante, Shruthi Prasad, Annelise Hermant, Florence Nyawouame, Julie Preillon, Kim Frederix, Anne Catherine Michaux, Florence Lambolez, Jakub Swiercz, Gregory Driessens, Noemie Wald, Chiara Martinoli, Veronique Bodo, Michel Detheux, Xavier Leroy, Stefano Crosignani. EOS100850, an insurmountable and non-brain penetrant A2A receptor antagonist, inhibits adenosine-mediated T cell suppression, demonstrates anti-tumor activity and exhibits best-in class characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-291.

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of "intrinsic" patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native "intrinsic pathogenic" disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Anti-human CD14 monoclonal antibody improves survival following sepsis induced by endotoxin, but not following polymicrobial infection

Cluster of differentiation 14 (CD14), a pattern recognition receptor expressed on myeloid cells and a critical component of the innate immune system, mediates local and systemic host responses to gram-negative bacterial products, including lipopolysaccharide (LPS). Therefore, CD14 is an attractive target for development of sepsis therapies, and several monoclonal anti-CD14 antibodies have been reported. In this study, we prepared an anti-human CD14 monoclonal antibody, F1024-1-3, which suppressed LPS-induced upregulation of pro-inflammatory cytokines and an adhesion molecule in human peripheral mononuclear cells and human vascular endothelial cells. Half-maximal inhibitory concentrations in these assays ranged from 0.1 to 1μg/ml. In rabbits, intravenous administration (3mg/kg) as well as in vitro exposure of F1024-1-3 suppressed LPS-induced cytokine production in whole blood. In endotoxemia models generated by three sequential injections of LPS, intravenous administration of F1024-1-3 at 0.3–3mg/kg sharply reduced pro-inflammatory responses in a dose-dependent manner and moderately attenuated pro-coagulant responses; at 1mg/kg, the protein protected rabbits from lethality even when administered 2h after the initial LPS injection. However, F1024-1-3 (10mg/kg) given 2h post-surgery did not prevent death of rabbits in a cecal ligation and puncture model. Thus, suppression of CD14-mediated activation of leukocytes and endothelial cells alone may not be clinically efficacious for the treatment of severe sepsis and septic shock.

Inflammation-Induced Changes in Circulating T-Cell Subsets and Cytokine Production During Human Endotoxemia.

Observational clinical studies suggest the initial phase of sepsis may involve impaired cellular immunity. In the present study, we investigated temporal changes in T-cell subsets and T-cell cytokine production during human endotoxemia. Endotoxin (Escherichia coli lipopolysaccharide 4 ng/kg) was administered intravenously in 15 healthy volunteers. Peripheral blood and bronchoalveolar lavage fluid (BALF) were collected at baseline and after 2, 4, 6, 8, and 24 hours for flow cytometry. CD4+CD25+CD127lowFoxp3+ regulatory T cells (Tregs), CD4+CD161+ cells, and activated Human leukocyte antigen, HLA-DR+CD38+ T cells were determined. Ex vivo whole-blood cytokine production and Toll-like receptor (TLR)-4 expression on Tregs were measured. Absolute number of CD3+CD4+ (P = .026), CD3+CD8+ (P = .046), Tregs (P = .023), and CD4+CD161+ cells (P = .042) decreased after endotoxin administration. The frequency of anti-inflammatory Tregs increased (P = .033), whereas the frequency of proinflammatory CD4+CD161+ cells decreased (P = .034). Endotoxemia was associated with impaired whole-blood production of tumor necrosis factor-α, interleukin-10, IL-6, IL-17, IL-2, and interferon-γ in response to phytohaemagglutinin but did not affect TLR4 expression on Tregs. No changes in the absolute count or frequency of BALF T cells were observed. Systemic inflammation is associated with lymphopenia, a relative increase in the frequency of anti-inflammatory Tregs, and a functional impairment of T-cell cytokine production.

Molecular Design of Squalene/Squalane Countertypes via the Controlled Oligomerization of Isoprene and Evaluation of Vaccine Adjuvant Applications.

The potential to replace shark-derived squalene in vaccine adjuvant applications with synthetic squalene/poly(isoprene) oligomers, synthesized by the controlled oligomerization of isoprene is demonstrated. Following on from our previous work regarding the synthesis of poly(isoprene) oligomers, we demonstrate the ability to tune the molecular weight of the synthetic poly(isoprene) material beyond that of natural squalene, while retaining a final backbone structure that contained a minimum of 75% of 1,4 addition product and an acceptable polydispersity. The synthesis was successfully scaled from the 2 g to the 40 g scale both in the bulk (i.e., solvent free) and with the aid of additional solvent by utilizing catalytic chain transfer polymerization (CCTP) as the control method, such that the target molecular weight, acceptable dispersity levels, and the desired level of 1,4 addition in the backbone structure and an acceptable yield (∼60%) are achieved. Moreover, the stability and in vitro bioactivity of nanoemulsion adjuvant formulations manufactured with the synthetic poly(isoprene) material are evaluated in comparison to emulsions made with shark-derived squalene. Emulsions containing the synthetic poly(isoprene) achieved smaller particle size and equivalent or enhanced bioactivity (stimulation of cytokine production in human whole blood) compared to corresponding shark squalene emulsions. However, as opposed to the shark squalene-based emulsions, the poly(isoprene) emulsions demonstrated reduced long-term size stability and induced hemolysis at high concentrations. Finally, we demonstrate that the synthetic oligomeric poly(isoprene) material could successfully be hydrogenated such that >95% of the double bonds were successfully removed to give a representative poly(isoprene)-derived squalane mimic.

Anti-endotoxic and antibacterial effects of a dermal substitute coated with host defense peptides

Biomaterials used during surgery and wound treatment are of increasing importance in modern medical care. In the present study we set out to evaluate the addition of thrombin-derived host defense peptides to human acellular dermis (hAD, i.e. epiflex®). Antimicrobial activity of the functionalized hAD was demonstrated using radial diffusion and viable count assays against Gram-negative Escherichia coli, Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. Electron microscopy analyses showed that peptide-mediated bacterial killing led to reduced hAD degradation. Furthermore, peptide-functionalized hAD displayed endotoxin-binding activity in vitro, as evidenced by inhibition of NF-κB activation in human monocytic cells (THP-1 cells) and a reduction of pro-inflammatory cytokine production in whole blood in response to lipopolysaccharide stimulation. The dermal substitute retained its anti-endotoxic activity after washing, compatible with results showing that the hAD bound a significant amount of peptide. Furthermore, bacteria-induced contact activation was inhibited by peptide addition to the hAD. E. coli infected hAD, alone, or after treatment with the antiseptic substance polyhexamethylenebiguanide (PHMB), yielded NF-κB activation in THP-1 cells. The activation was abrogated by peptide addition. Thus, thrombin-derived HDPs should be of interest in the further development of new biomaterials with combined antimicrobial and anti-endotoxic functions for use in surgery and wound treatment.