Multiple Agonists Research Articles

Glaucocalyxin A alleviates LPS-mediated septic shock and inflammation via inhibiting NLRP3 inflammasome activation

Glaucocalyxin A (GLA) is a bioactive ent-kauranoid diterpenoid derived from the herbal medicine, Rabdosia japonica var. glaucocalyx, and it has been reported to possess marked anti-inflammatory properties. However, the underlying mechanisms are not fully understood. Here, we reported that GLA dramatically inhibited canonical and non-canonical NLRP3 inflammasome activation induced by multiple agonists. In addition, GLA also blocked NLRC4 inflammasome activation but had no effect on AIM2 inflammasome. Furthermore, we found that GLA inhibited NLRP3 or NLRC4 agonists-induced ASC oligomerization, which is an upstream event of the inflammasomes assembly. Most importantly, administration of GLA significantly reduced lipopolysaccharide (LPS)-induced mortality in septic-shock mouse model. Additionally, GLA dose-dependently inhibited the production of interleukin (IL)-1β, but had no effect on NLRP3-independent TNF-α production induced by LPS in vivo. In conclusion, our study suggests that GLA alleviates LPS-induced septic shock and inflammation via inhibiting NLRP3 inflammasome activation and provides a promising candidate drug for the treatment of NLRP3-driven diseases.

Airway profile of bioactive lipids predicts early progression of lung disease in cystic fibrosis

BackgroundPreviously, we showed that abnormal levels of bioactive lipids in bronchoalveolar lavage fluid (BALF) from infants with cystic fibrosis (CF) correlated with early structural lung damage. MethodTo extend these studies, BALF bioactive lipid measurement by mass spectrometry and chest computed tomography (CT, combined with the sensitive PRAGMA-CF scoring method) were performed longitudinally at 2-year intervals in a new cohort of CF children (n = 21, aged 1–5 yrs). ResultsPRAGMA-CF, neutrophil elastase activity, and myeloperoxidase correlated with BALF lysolipids and isoprostanes, markers of oxidative stress, as well as prostaglandin E2 and combined ceramide precursors (Spearman's Rho > 0.5; P < 0.01 for all). Multiple protein agonists of inflammation and tissue remodeling, measured by Olink protein array, correlated positively (r = 0.44–0.79, p < 0.05) with PRAGMA-CF scores and bioactive lipid levels. Notably, levels of lysolipids, prostaglandin E2 and isoprostanes at first BALF predicted the evolution of PRAGMA-CF scores 2 years later. In wild-type differentiated primary bronchial epithelial cells, and in CFTR-inducible iCFBE cells, treatment with a lysolipid receptor agonist (VPC3114) enhanced shedding of pro-inflammatory and pro-fibrotic proteins. ConclusionsTogether, our findings suggest that bioactive lipids in BALF correlate with and possibly predict structural lung disease in CF children, which supports their use as biomarkers of disease progression and treatment efficacy. Furthermore, our data suggest a causative role of airway lysolipids and oxidative stress in the progression of early CF lung disease, unveiling potential therapeutic targets.

Triggering of Toll-like Receptors in Old Individuals. Relevance for Vaccination.

Aging is characterized by a general decline in a range of physiological functions, with a consequent increase in the risk of developing a variety of chronic diseases and geriatric syndromes. Additionally, increasing age is accompanied by a progressive decline in both innate and acquired immune system, referred to as immunosenescence. This impaired ability to mount an efficient immune response after exposure to microorganisms or vaccines represents a major challenge in acquiring protection against pathogens in aging. Therefore, there is still a great need for vaccines that are tailored to optimally stimulate the aged immune system, thus promoting more successful aging. Various strategies can be used to improve vaccine efficacy in old people. Despite this, metaanalyses have clearly shown that the magnitude of protection obtained remains lower in older adults. Recent studies show that stimulation of Toll-like receptors, using stimulatory ligands, can enhance vaccine efficacy by a number of mechanisms, including the activation of innate immune cells and the consequent production of inflammatory cytokines. Therefore, a possible strategy for more effective vaccination in the older population is the triggering of multiple TLRs, using a combined adjuvant for the synergistic activation of cellular immunity. Preliminary in vitro data suggest that in humans the presence of multiple TLR agonists can result in the greater stimulation of antigen-specific immune responses in immune cells both in the young healthy and in the immune senescent older donors. These data suggest that appropriately selected combinations of TLR agonists could enhance the efficacy of vaccination mediated immunity in older people.

Transient Receptor Potential Cation Channels in Cancer Therapy.

In mammals, the transient receptor potential (TRP) channels family consists of six different families, namely TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPA (ankyrin), that are strictly connected with cancer cell proliferation, differentiation, cell death, angiogenesis, migration, and invasion. Changes in TRP channels’ expression and function have been found to regulate cell proliferation and resistance or sensitivity of cancer cells to apoptotic-induced cell death, resulting in cancer-promoting effects or resistance to chemotherapy treatments. This review summarizes the data reported so far on the effect of targeting TRP channels in different types of cancer by using multiple TRP-specific agonists, antagonists alone, or in combination with classic chemotherapeutic agents, microRNA specifically targeting the TRP channels, and so forth, and the in vitro and in vivo feasibility evaluated in experimental models and in cancer patients. Considerable efforts have been made to fight cancer cells, and therapies targeting TRP channels seem to be the most promising strategy. However, more in-depth investigations are required to completely understand the role of TRP channels in cancer in order to design new, more specific, and valuable pharmacological tools.

Detection and Analysis of Gene Mutations in Patients with Glanzmann's Thrombasthenia

Background: Glanzmann thrombasthenia (GT) is a rare inherited disorder of bleeding, and it is characterized by the impaired or absent platelet aggregation to multiple physiologic agonists such as collagen, adenosine diphosphate (ADP), arachidonic acid(AA), but normal reaction to ristocetin. There is qualitative or quantitative defect in platelet integrin αIIbβ3(GPIIb/IIIa). Pathogenic variants of either αIIb or β3 unit could cause GT. The database of gene mutations is continuously updated on the Internet (http://www.hgmd.org); it totally lists 236 variants of ITGA2B gene and 170 variants of ITGB3 gene. Aim: To characterize the clinical manifestation and molecular basis of GT patients in China, and update the pathogenic variants database. Method: Clinical features are evaluated in 104 patients with GT. New generation sequencing was performed with a custom designed panel for the bleeding and platelet disease involving 76 genes, while ITGA2B and ITGB3 were enrolled. Result: The initial bleeding occurred before 1 age in most patients. Incidence of consanguinity is 12.5%. Symptoms lessened with age in about 30% patients. Female patients suffered more severe bleeding than male patients. Fifty different mutations were detected, among which 15 were novel. Most patients were compound heterozygotes and most mutations detected were missense mutations. Among 15 novel mutations, there were 7 missense mutations, 2 nonsense mutations, 2 splicing mutations, 4 frameshift mutations. Pathogenicity of all novel mutations were evaluated according to the standards and guidelines of ACMG. All variants detected were pathogenic or likely pathogenic. Furthermore, c.1750C&gt;T [p.R584X] and c.2333A&gt;C [p.Q778P] in ITGA2B were detected in 10 and 16 unrelated families, strongly suggesting a founder effect. Conclusion: Our study reports the largest cohort of GT in China, describing the clinical, laboratory and genetic characteristics of 104 patients. We found 15 novel pathogenic mutations in ITGA2B and ITGB3 causing GT. Theses novel findings expand the GT mutation spectrum. Disclosures No relevant conflicts of interest to declare.

Evaluation of Operational Models of Agonism and Allosterism at Receptors with Multiple Orthosteric Binding Sites

Current operational models of agonism and allosterism quantify ligand actions at receptors where agonist concentration-response relationships are nonhyperbolic by introduction of a transducer slope that relates receptor occupancy to response. However, for some receptors nonhyperbolic concentration-response relationships arise from multiple endogenous agonist molecules binding to a receptor in a cooperative manner. Thus, we developed operational models of agonism in systems with cooperative agonist binding and evaluated the models by simulating data describing agonist effects. The models were validated by analyzing experimental data demonstrating the effects of agonists and allosteric modulators at receptors where agonist binding follows hyperbolic (M4 muscarinic acetylcholine receptors) or nonhyperbolic relationships (metabotropic glutamate receptor 5 and calcium-sensing receptor). For hyperbolic agonist concentration-response relationships, no differences in estimates of ligand affinity, efficacy, or cooperativity were observed when the slope was assigned to either a transducer slope or agonist binding slope. In contrast, for receptors with nonhyperbolic agonist concentration-response relationships, estimates of ligand affinity, efficacy, or cooperativity varied depending on the assignment of the slope. The extent of this variation depended on the magnitude of the slope value and agonist efficacy, and for allosteric modulators on the magnitude of cooperativity. The modified operational models described herein are well suited to analyzing agonist and modulator interactions at receptors that bind multiple orthosteric agonists in a cooperative manner. Accounting for cooperative agonist binding is essential to accurately quantify agonist and drug actions. SIGNIFICANCE STATEMENT: Some orthosteric agonists bind to multiple sites on a receptor, but current analytical methods to characterize such interactions are limited. Herein, we develop and validate operational models of agonism and allosterism for receptors with multiple orthosteric binding sites, and demonstrate that such models are essential to accurately quantify agonist and drug actions. These findings have important implications for the discovery and development of drugs targeting receptors such as the calcium-sensing receptor, which binds at least five calcium ions.

Simultaneous Detection of Multiple β-Adrenergic Agonists with 2-Directional Lateral Flow Strip Platform.

Clenbuterol (CL), salbutamol (SAL) and ractopamine (RAC) are the three common β-adrenergic agonists, which are the main hazards in food safety and affect human health through the food chain. A convenient and efficient method is urgently required to perform on-site detection of multiple β-adrenergic agonists to avoid frequent poisoning incidents. In this paper, a 2-directional lateral flow strip technique (2-directional LFS) is developed for rapid and simultaneous detection of CL, SAL and RAC with single sampling. Compared to the conventional lateral flow strip, this 2-directional LFS technique can realize simultaneous detection of three or more target analytes without any change of intrinsic simplicity of LFS. Furthermore, this 2-directional LFS can effectively avoid the potential intrinsic cross-reactivity among the reagents to analogues. Under the optimized conditions, CL, SAL and RAC were all successfully determined with satisfactory results in both buffer and urine samples with the detection limit as low as 0.5 ng/mL. This 2-directional LFS technique can revolutionize the commercial single-analyte LFS products and can effectively widen the applications of the classic LFS in various fields.

Activation of AMPK promotes thyroid cancer cell migration through its interaction with PKM2 and β-catenin

AMP-activated protein kinase (AMPK) is induced by the exhaustion of cellular energy and activates adaptive alterations in cellular metabolism, which is the basis for cell survival during different environmental stresses. We aimed to investigate the biological functions of AMPK and its molecular mechanism in regulating thyroid cancer (TC) progression. In current study, we found that activation of AMPK by multiple agonists suppresses TC cell proliferation. However, AMPK activation also led to TC cell migration at the same time. Depletion of AMPK abolished the effect of its agonist on cell multiplication and migration. Mechanistic investigations revealed that the impact of AMPK in terms of cell migration is dependent on its nuclear translocation, since site mutation of AMPK in its nuclear translocation domain (K244A) abolished TC cell migration but did not affect the inhibition of cell proliferation by AMPK agonist. Moreover, the nuclear AMPK recruits PKM2 and β-catenin by their interaction, which promotes the transcription of cell migration related genes, including MMP7 and c-Myc. Furthermore, depletion of PKM2/β-catenin abolished the migration effect of AMPK agonists, but did not affect their effects on suppression of cell proliferation. Our results provided a novel function of AMPK in cancer migration, and suggested that a combination of AMPK activation and PKM2 depletion or inhibition can be a new strategy to achieve better therapeutic effects for TC patients.

Co-encapsulation of synthetic lipidated TLR4 and TLR7/8 agonists in the liposomal bilayer results in a rapid, synergistic enhancement of vaccine-mediated humoral immunity

To increase vaccine immunogenicity, modern vaccines incorporate adjuvants, which serve to enhance immune cross-protection, improve humoral and cell-mediated immunity, and promote antigen dose sparing. Pattern recognition receptors (PRRs), including the Toll-like receptor (TLR) family are promising targets for development of agonist formulations for use as vaccine adjuvants. Combinations of co-delivered TLR4 and TLR7/8 ligands have been demonstrated to have synergistic effects on innate and adaptive immune response. Here, we create liposomes that stably co-encapsulate CRX-601, a synthetic TLR4 agonist, and UM-3004, a lipidated TLR7/8 agonist, within the liposomal bilayer in order to achieve co-delivery, allow tunable physical properties, and induce in vitro and in vivo immune synergy. Co-encapsulation demonstrates a synergistic increase in IL-12p70 cytokine output in vitro from treated human peripheral blood mononuclear cells (hPBMCs). Further, co-encapsulated formulations give significant improvement of early IgG2a antibody titers in BALB/c mice following primary vaccination when compared to single agonist or dual agonists delivered in separate liposomes. This work demonstrates that co-encapsulation of TLR4 and lipidated TLR7/8 agonists within the liposomal bilayer leads to innate and adaptive immune synergy which biases a Th1 immune response. Thus, liposomal co-encapsulation may be a useful and flexible tool for vaccine adjuvant formulation containing multiple TLR agonists.

A novel missense variant in the RASGRP2 gene in patients with moderate to severe bleeding disorder

Inherited platelet function disorder-18 (IPD-18) is a relatively new non-syndromic autosomal recessive bleeding disorder. It is characterized by deficient or dysfunctional CalDAG-GEFI protein. The distinctive feature of the disease is impaired platelet aggregation in response to multiple physiologic agonists. We here report a family with a platelet-type bleeding disorder and a novel mutation in the RASGRP2 gene. The overall bleeding score for the affected individuals was 15 and 12. Based on the initial diagnosis of Glanzmann thrombasthenia, targeted sequencing of integrin subunit alpha 2b and integrin subunit beta 3 encoding genes ITGA2B and ITGB3 were carried out in both affected members of a family. Sequence alignment failed to identify the disease-causing variant(s) in both genes. Therefore, whole exome sequencing in one affected individual was performed. Data analysis detected a novel homozygous missense variant (c.956C>T; p.Pro319Leu) in the exon 9 of the RASGRP2 gene. Five additional individuals of a family including both parents, an affected individual and two asymptomatic individuals were Sanger sequenced for the variant (c.956C>T). The variant segregates in the family in an autosomal recessive manner. Several in silico tools predicted the variant as pathogenic. Protein modeling studies suggest that the mutation (p.Pro319Leu) cause a conformational change in the loop structure of the RasGEF domain of the CalDAG-GEFI protein. Reported variants in the RasGEF domain impair expression and/or nucleotide exchange activity of CalDAG-GEFI protein and thus inhibit the activation of Rap1 protein required for platelet adhesion and hemostatic plug formation.

Abstract P2010: Silencing Epidermal Growth Factor Receptor Reduces P44/42 Mitogen-Activated Protein Kinase Signaling in the Hypothalamic Paraventricular Nucleus and Ameliorates Sympathetic Excitation in Rats With Heart Failure After Myocardial Infarction

The p44/42 mitogen-activated protein kinase (MAPK) signaling pathway is activated in the brain in myocardial infarction (MI)-induced heart failure (HF). Activation of brain p44/42 MAPK signaling can induce endoplasmic reticulum (ER) stress, increase proinflammatory cytokine expression and upregulate renin-angiotensin system (RAS) activity in the brain, contributing to sympathetic excitation. Multiple excitatory agonists (e.g., angiotensin II, aldosterone and pro-inflammatory cytokines) activate brain MAPK signaling in HF, but the mechanisms are poorly understood. We tested the involvement of the epidermal growth factor receptor (EGFR), which has recently been shown to mediate MAPK signaling in peripheral cardiovascular tissues. Male rats received bilateral microinjections of a lentiviral vector encoding a small interfering RNA (siRNA) for EGFR, or a scrambled siRNA, into the hypothalamic paraventricular nucleus (PVN), a critical cardiovascular and autonomic center. One week later, some rats were euthanized to confirm the effectiveness of EGFR knockdown in PVN. mRNA for EGFR was significantly (*P&lt;0.05) lower in rats that received the EGFR siRNA (0.54±0.17* vs 1.10±0.20, fold change). Other rats underwent coronary artery ligation (CL) to induce HF. Four weeks later, HF rats treated with siRNA for EGFR, compared with HF rats treated with scrambled siRNA, had lower protein levels of phosphorylated p44/42 MAPK (0.20±0.08* vs 0.34±0.06), lower mRNA levels of the ER stress markers BIP (0.77±0.10* vs 1.00±0.12) and ATF4 (0.71±0.14* vs 1.03±0.25), the inflammatory mediators TNF-α (0.73±0.11* vs 1.01±0.15), IL-1β (0.65±0.14* vs 1.02±0.21) and COX-2 (0.71±0.16* vs 1.00±0.14), and the RAS components ACE (0.68±0.26* vs 1.06±0.37) and AT 1a R (0.70±0.11* vs 1.03±0.28) in the PVN, lower urinary norepinephrine levels (28±9* vs 39±7 ng/ml), and improved peripheral manifestations of HF as indicated by reduced right ventricular/body weight and lung/body weight and left ventricular end-diastolic pressure. These results suggest that EGFR in the PVN activates p44/42 MAPK, as well as RAS, neuroinflammation and ER stress, in MI-induced HF. Brain EGFR may be a novel target for therapeutic intervention in the MI-induced HF syndrome.

Cargo-less nanoparticles program innate immune cell responses to toll-like receptor activation

Developing biomaterials to control the responsiveness of innate immune cells represents a clinically relevant approach to treat diseases with an underlying inflammatory basis, such as sepsis. Sepsis can involve activation of Toll-like receptor (TLR) signaling, which activates numerous inflammatory pathways. The breadth of this inflammation has limited the efficacy of pharmacological interventions that target a single molecular pathway. Here, we developed cargo-less particles as a single-agent, multi-target platform to elicit broad anti-inflammatory action against innate immune cells challenged by multiple TLR agonists. The particles, prepared from poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA), displayed potent molecular weight-, polymer composition-, and charge-dependent immunomodulatory properties, including downregulation of TLR-induced costimulatory molecule expression and cytokine secretion. Particles prepared using the anionic surfactant poly(ethylene-alt-maleic acid) (PEMA) significantly blunted the responses of antigen presenting cells to TLR4 (lipopolysaccharide) and TLR9 (CpG-ODN) agonists, demonstrating broad inhibitory activity to both extracellular and intracellular TLR ligands. Interestingly, particles prepared using poly(vinyl alcohol) (PVA), a neutrally-charged surfactant, only marginally inhibited inflammatory cytokine secretions. The biochemical pathways modulated by particles were investigated using TRanscriptional Activity CEll aRrays (TRACER), which implicated IRF1, STAT1, and AP-1 in the mechanism of action for PLA-PEMA particles. Using an LPS-induced endotoxemia mouse model, administration of PLA-PEMA particles prior to or following a lethal challenge resulted in significantly improved mean survival. Cargo-less particles affect multiple biological pathways involved in the development of inflammatory responses by innate immune cells and represent a potentially promising therapeutic strategy to treat severe inflammation.

G protein–coupled receptors activate p38 MAPK via a non-canonical TAB1–TAB2– and TAB1–TAB3–dependent pathway in endothelial cells

Endothelial dysfunction is induced by inflammatory mediators including multiple G protein-coupled receptor (GPCR) agonists. However, the GPCR signaling pathways that promote endothelial dysfunction are incompletely understood. We previously showed that thrombin promotes endothelial barrier disruption through autophosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via a non-canonical transforming growth factor-β-activated protein kinase-1-binding protein-1 (TAB1) and TAB2-dependent pathway rather than the canonical three-tiered kinase cascade. Here, we sought to determine whether other GPCR agonists stimulate p38 MAPK activation via this non-canonical pathway in human endothelial cells derived from different vascular beds. Using primary human umbilical vein endothelial cells (HUVECs), HUVEC-derived EA.hy926 cells, and human dermal microvascular endothelial cells (HDMECs), we found that both non-canonical and canonical p38 activation pathways components are expressed in these various endothelial cell types, including TAB3, a structurally-related TAB2 homolog. Moreover, multiple GPCRs agonists, including thrombin, histamine, prostaglandin E2, and ADP, stimulated robust p38 autophosphorylation, whereas phosphorylation of the upstream MAPKs MAP kinase kinase 3 (MKK3) and MKK6, was virtually undetectable, indicating that non-canonical p38 activation may exist for other GPCRs. Indeed, in EA.hy926 cells, thrombin- and histamine-stimulated p38 activation depended on TAB1-TAB2, whereas in primary HUVECs, both TAB1-TAB2 and TAB1-TAB3 were required for p38 activation. In HDMECs, thrombin-induced p38 activation depended on TAB1-TAB3, but histamine-induced p38 activation required TAB1-TAB2. Moreover, thrombin- and histamine-stimulated interleukin-6 production required both TAB1-TAB2 and TAB1-TAB3 in HUVEC. We conclude that multiple GPCR agonists utilize non-canonical TAB1-TAB2 and TAB1-TAB3-dependent p38 activation to promote endothelial inflammatory responses.

Identification of a novel scaffold for a small molecule GPR139 receptor agonist

GPR139 is an orphan G protein-coupled receptor (GPCR) that is primarily expressed in the brain in regions known to regulate motor control and metabolism. Here, we screened a diverse 4,000 compound library in order to identify GPR139 agonists. We identified 11 initial hits in a calcium mobilization screen, including one compound, AC4, which contains a different chemical scaffold to what has previously been described for GPR139 agonists. Our mutagenesis data shows that AC4 interacts with the same hotspots in the binding site of GPR139 as those reported to interact with the reference agonists 1a and 7c. We additionally tested and validated 160 analogs in a calcium mobilization assay and found 5 compounds with improved potency compared to AC4. In total, we identified 36 GPR139 agonists with potencies in the nanomolar range (90–990 nM). The most potent compounds were confirmed as GPR139 agonists using an orthogonal ERK phosphorylation assay where they displayed a similar rank order of potency. Accordingly, we herein introduce multiple novel GPR139 agonists, including one with a novel chemical scaffold, which can be used as tools for future pharmacological and medicinal chemistry exploration of GPR139.

Abstract B178: Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy

Abstract Background: The first cancer immunotherapy was developed in the 1890s by Dr. William Coley, who observed tumor regressions after injecting cancer patients with a heat-killed mixture of Gram-positive and negative pathogenic bacteria. Dr. Coley determined that the Gram-negative bacteria were the principal contributor to antitumor activity and that the product would likely be most effective when administered intravenously (i.v.), but was too toxic in this setting, limiting use to intramuscular and intratumoral administration. Coley’s toxins, as it was known, was credited with curing hundreds of late-stage cancer patients over 70 years. However, lack of knowledge regarding mechanism of action made it difficult to optimize or standardize, producing high variability. The use of multiple, local administration routes also likely contributed to variability in response, leading the FDA to refuse to grandfather in the product in 1963. We now know the mechanism of action of Coley’s toxins and the source of the i.v. toxicity. Gram-negative bacteria contain immune system danger signals, including multiple TLR agonists (activating TLRs 2, 4, 5 and 9), which directly and indirectly, via induction of cytokine and chemokine secretion, participate in the activation of most of the cellular mediators of innate and adaptive immune responses. Lipopolysaccharide (LPS), which activates TLR4, has been identified as a major contributor to both the antitumor activity and i.v. toxicity of Gram-negative bacteria. Decoy’s hypothesis is that significant reduction without complete elimination of LPS activity, in conjunction with killing and stabilization of nonpathogenic, Gram-negative bacteria, may produce a multiple TLR product that can safely and effectively induce antitumor immune responses via i.v. administration. Methods and Results: Nonpathogenic, Gram-negative E. coli were treated with polymyxin B and glutaraldehyde under conditions to kill and stabilize the cells, producing &amp;gt;90% reduction in LPS endotoxin activity and pyrogenicity. Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate (LAL) and in vivo rabbit assays. Bacterial integrity was assessed by electron and light microscopy. Antitumor activity was determined using standard syngeneic and xenograft tumor models. Decoy-treated bacteria exhibited a 3-fold reduction in acute in vivo toxicity relative to untreated bacteria. Surprisingly, induction of antitumor cytokine secretion by murine and human peripheral blood mononuclear cells (PBMCs) was not compromised, relative to untreated bacteria. Treatment with Decoy bacteria (i.v.) produced significant single-agent antitumor activity against orthotopic murine colorectal carcinoma and metastatic murine pancreatic carcinoma. Synergistic combination activity, including eradication of established tumors, with a therapeutic index of up to 10-fold, was observed in combination with IL-2 or low-dose cyclophosphamide (LDC) in murine colorectal carcinoma models, with LDC in a subcutaneous (s.c.), murine non-Hodgkin’s lymphoma (NHL) model and with LDC plus rituximab in a s.c., human NHL model. Synergistic antitumor activity was also observed in combination with a low-dose, nonsteroidal anti-inflammatory drug (NSAID) in a metastatic, murine pancreatic carcinoma model. In addition, tumor eradications were observed in combination with NSAID and were enhanced by addition of anti-PD-1 therapy in a s.c., murine hepatocellular carcinoma model. Optimal (80-100%) tumor eradication was shown to be mediated by natural killer (NK), CD4+ and CD8+ T-cells. Immunologic memory (80-100% and partial), determined by rejection of subsequent tumor challenge, was demonstrated in both immune competent and innate only settings, respectively. Conclusion: We have developed an i.v. administered multiple TLR agonist, based on Coley’s toxins, that can be combined with a wide variety of existing agents to induce curative, antitumor immune responses. Citation Format: Michael J. Newman. Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B178.

Platelet dysfunction during trauma involves diverse signaling pathways and an inhibitory activity in patient-derived plasma.

Trauma-induced coagulopathy occurs in about 25% of injured patients and accounts for about 10% of deaths worldwide. Upon injury, hemostatic function may decline due to vascular dysfunction, clotting factor deficiencies, hyperfibrinolysis, and/or platelet dysfunction. We investigated agonist-induced calcium signaling in platelets obtained over time from trauma patients. Platelets from trauma patients and healthy donors were monitored via intracellular calcium mobilization and flow cytometry markers (α2bβ3 activation, P-selectin display, and phosphatidylserine exposure) following stimulation with a panel of agonists (adenosine 5'-diphosphate sodium salt, U46619, convulxin, PAR-1/4 activating peptides, iloprost) used in isolation or in pairwise tests. Furthermore, healthy donor platelets were tested in heterologous plasma isolated from healthy subjects and trauma patients. When exposed to agonists over the first 24 hours postinjury, trauma patient platelets mobilized less calcium in comparison to healthy platelets. Partial recovery of platelet activity was observed in about a third of patients after 120 hours, although not fully obtaining healthy baseline function. Flow cytometry markers of trauma platelets were similar to healthy platelets prior to stimulation, but were depressed in trauma platelets stimulated with adenosine 5'-diphosphate sodium salt or convulxin. Also, washed healthy platelets showed a significant reduction in calcium mobilization when reconstituted in plasma from trauma patients, relative to healthy plasma, at all plasma doses tested. Platelet dysfunction in trauma patients included poor response to multiple agonists relevant to hemostatic function. Furthermore, the inhibitor effect of patient plasma on healthy platelets suggests that soluble plasma species may downregulate endogenous or transfused platelets during trauma.

Prostaglandins and Bone.

Prostaglandins (PGs) are highly bioactive fatty acids. PGs, especially prostaglandin E2 (PGE2), are abundantly produced by cells of both the bone-forming (osteoblast) lineage and the bone-resorbing (osteoclast) lineage. The inducible cyclooxygenase, COX-2, is largely responsible for most PGE2 production in bone, and once released, PGE2 is rapidly degraded in vivo. COX-2 is induced by multiple agonists - hormones, growth factors, and proinflammatory factors - and the resulting PGE2 may mediate, amplify, or, as we have recently shown for parathyroid hormone (PTH), inhibit responses to these agonists. In vitro, PGE2 can directly stimulate osteoblast differentiation and, indirectly via stimulation of RANKL in osteoblastic cells, stimulate the differentiation of osteoclasts. The net balance of these two effects of PGE2 in vivo on bone formation and bone resorption has been hard to predict and, as expected for such a widespread local factor, hard to study. Some of the complexity of PGE2 actions on bone can be explained by the fact that there are four receptors for PGE2 (EP1-4). Some of the major actions of PGE2 in vitro occur via EP2 and EP4, both of which can stimulate cAMP signaling, but there are other distinct signaling pathways, important in other tissues, which have not yet been fully elucidated in bone cells. Giving PGE2 or agonists of EP2 and EP4 to accelerate bone repair has been examined with positive results. Further studies to clarify the pathways of PGE2 action in bone may allow us to identify new and more effective ways to deliver the therapeutic benefits of PGE2 in skeletal disorders.

Bioactive Lipids in Cancer, Inflammation and Related Diseases : Acute and Chronic Mild Traumatic Brain Injury Differentially Changes Levels of Bioactive Lipids in the CNS Associated with Headache.

Headache is a common complaint after mild traumatic brain injury (mTBI). Changes in the CNS lipidome were previously associated with acrolein-induced headache in rodents. mTBI caused similar headache-like symptoms in rats; therefore, we tested the hypothesis that mTBI might likewise alter the lipidome. Using a stereotaxic impactor, rats were given either a single mTBI or a series of 4 mTBIs 48 h apart. 72 h later for single mTBI and 7 days later for repeated mTBI, the trigeminal ganglia (TG), trigeminal nucleus (TNC), and cerebellum (CER) were isolated. Using HPLC/MS/MS, ~80 lipids were measured in each tissue and compared to sham controls. mTBI drove widespread alterations in lipid levels. Single mTBI increased arachidonic acid and repeated mTBI increased prostaglandins in all 3 tissue types. mTBI affected multiple TRPV agonists, including N-arachidonoyl ethanolamine (AEA), which increased in the TNC and CER after single mTBI. After repeated mTBI, AEA increased in the TG, but decreased in the TNC. Common to all tissue types in single and repeated mTBI was an increase the AEA metabolite, N-arachidonoyl glycine, a potent activator of microglial migration. Changes in the CNS lipidome associated with mTBI likely play a role in headache and in long-term neurodegenerative effects of repeated mTBI.

Comparing a New Bovine Source Heparin to the Clinically Used Porcine Heparin for Platelet Function Effects and Heparin-Induced Thrombocytopenia Potential

BackgroundHeparin is a sulfated polysaccharide obtained from intestinal mucosa with anticoagulant properties that is widely used as a standard clinical therapeutic agent to treat and prevent thrombosis. Heparin is known to affect platelet function, and among its side effects is heparin-induced thrombocytopenia (HIT) that can occur in about 1% of patients exposed to heparin. Presently, only porcine source heparin is approved for use in the United States. The aims of this study were to determine if platelet activation by physiological agonists and platelet aggregation induced by HIT antibodies would be equivalent in the presence of bovine source heparin and porcine source heparin.Materials and MethodsSeven lots of bovine heparin from Eurofarma and 3 lots of commercial clinical grade porcine heparin (Pfizer/Hospira) were evaluated. The USP Reference Standard for porcine heparin was used to determine anti-Xa and anti-IIa potencies of the bovine heparins. For each study, blood was collected from healthy volunteers (n=5 per test group), anticoagulated with sodium citrate, and centrifuged to obtain platelet rich plasma (PRP). Platelet aggregation responses were assessed using the BioData PAP-8 platelet aggregometer. For the first aim to evaluate platelet function, PRP was combined with heparin at final concentrations of 10.0, 1.0, and 0.1 µg/mL, covering both therapeutic and prophylactic ranges. Platelet agonists included adenosine diphosphate (ADP), collagen, epinephrine, arachidonic acid, and thrombin receptor agonist peptide (TRAP). The aggregation response was quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). For the second aim to evaluate the HIT potential, antibodies to the complex of heparin-platelet factor 4 (H-PF4) from banked HIT patient apheresis fluid were combined with donor PRP and heparin. Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. PS and FA results were recorded. For all data, comparisons were analyzed with 2-Way ANOVA using SigmaPlot software.ResultsIn the presence of either bovine (BMH) or porcine heparin (PMH), the normal platelet aggregation response of all donors was not altered from that obtained with saline (see representative aggregation tracing in the image below). All heparin concentrations produced the same response. There were no significant differences between the bovine and porcine heparins for each of the 4 platelet aggregation parameters for ADP, arachidonic acid, collagen, epinephrine, and TRAP. Variation in the PS for arachidonic acid and collagen need to be assessed in a larger pool of donors to assure the lack of significant difference. Platelet activation to H-PF4 antibodies was strong at 0.1 to 1 U/mL concentrations with the expected inhibition observed when using 100 U/mL heparin. The HIT potential between bovine heparin and porcine heparin demonstrated no significant difference between the heparins (see MA responses in the image below). There were no lot to lot differences for the bovine heparins or the porcine heparins in either the platelet aggregation studies or the assessment for HIT.ConclusionIn these studies of platelet function, the bovine and porcine source heparins were comparable with regards to their effects on platelet aggregation induced by multiple different agonists and their HIT potential. [Display omitted] DisclosuresWalenga:Eurofarma: Research Funding.

Thrombin Generation Impairments in Platelet Function Disorders

Many platelet function disorders (PFD) present as uncharacterized disorders that impair aggregation responses to multiple agonists and/or cause non-syndromic dense granule deficiency (DGD). We postulated that some of these commonly encountered PFD might also impair the ability of platelets to support thrombin generation, given that an important subgroup are caused by mutations in transcription factors, such as RUNX1, that impair multiple aspects of platelet function. Accordingly, we initiated a study of thrombin generation (TG) in a prospective cohort of individuals presenting with an uncharacterized PFD. In addition, we reassessed how Quebec platelet disorder (QPD, previously called Factor V Quebec) affects coagulation as this disorder (which is caused by a duplication mutation of PLAU) triggers intraplatelet (but not systemic) plasmin generation and proteolysis of platelet but not plasma factor V (FV), and the normal plasma FV might potentially compensate for the loss of platelet FV in QPD.The study was conducted with written informed consent of participants and with the approval of the Hamilton Integrated Research Ethics Board and the Research Ethics Board of Centre Hospitalier Universitaire Sainte Justine. Participants included: 1) five individuals with QPD; 2) eighteen individuals presenting with an uncharacterized PFD and confirmed reduced maximal aggregation responses to ≥2 agonists and/or confirmed DGD, including five that had a pathogenic RUNX1 mutation; and 3) eighteen similarly-aged general population controls. TG was assessed by the calibrated automated thrombogram (CAT) procedure on a Fluoroskan (Thermo Fisher Scientific AG, Reinach, Switzerland) using Thrombinoscope software (Synapse BV, Maastricht, The Netherlands), manufacturer- and ISTH-recommended protocols and reagents for testing platelet poor plasma (PPP) and platelet rich plasma (PRP). Endpoints included: endogenous thrombin potential (ETP, nM·min), peak thrombin concentration (nM), time-to-peak (min) and lag time (min). Platelet lysate and plasma FV concentrations were determined by enzyme-linked immunoassay. Data were analyzed using Mann-Whitney tests with Bonferroni correction for multiple comparisons.All TGA endpoints for PPP were comparable for controls and participants with PFD, including QPD (p values ≥0.10). In TGA with PRP, most PFD participants had findings similar to controls, however, the subgroups with pathogenic RUNX1 mutations or QPD had significantly reduced ETP and peak thrombin concentration compared to controls (data as median [range]: ETP: controls: 1860 [1530-2630]; RUNX1 mutation subgroup: 1520 [805-1640], p=0.004; QPD: 1370 [981-2010], p=0.01; peak thrombin concentration: controls: 111 [65-152]; RUNX1 mutations: 66 [32-93], p=0.006; QPD: 59 [41-91], p=0.005). Plasma FV levels were similar in all subjects (µg/ml PPP, median [range]: controls: 7.7 [6.3-10.7]; QPD: 8.4 [7.0-10.2], p=0.33; other PFD: 7.5 [5.4-12], p=0.74) and showed no significant association to TG endpoints for PPP or PRP (p values ≥0.14). Only QPD subjects had platelet FV deficiency (µg FV/mg platelet protein, median [range]: controls: 0.89 [0.63-1.54]; QPD: 0.35 [0.18-0.46], p<0.001; other PFD: 0.83 [0.47-1.75], p=0.48; RUNX1 mutation subgroup: 0.73 [0.50-1.53], p=0.41). In QPD, but not other participants, platelet FV showed a significant association to ETP (R2=0.81, p=0.04) and peak TG endpoints (R2=0.88, p=0.01).Our study illustrates that platelet-dependent thrombin generation, evaluated by CAT, is abnormal in QPD but normal in many uncharacterized PFD with defective aggregation and/or DGD except for the important subgroup with pathogenic RUNX1 mutations, which impair TG but do not reduce platelet FV (unlike QPD). In QPD, the platelet-dependent TG defect shows a unique relationship to the platelet FV deficiency, which suggests that the normal levels of plasma FV are insufficient to compensate for the platelet procoagulant abnormalities in this PFD. DisclosuresNo relevant conflicts of interest to declare.