Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling.

The interaction of the human FcγRIIA with immune complexes (ICs) promotes neutrophil activation and thus must be tightly controlled to avoid damage to healthy tissue. Here, we demonstrate that a fungal-derived soluble β-1,3/1,6-glucan binds to the glycosphingolipid long-chain lactosylceramide (LacCer) to reduce FcγRIIA-mediated recruitment to immobilized ICs under flow, a process requiring high-affinity FcγRIIA-immunoglobulin G (IgG) interactions. The inhibition requires Lyn phosphorylation of SHP-1 phosphatase and the FcγRIIA immunotyrosine-activating motif.

A randomized, controlled trial evaluating the efficacy and safety of BTH1677 in combination with bevacizumab, carboplatin, and paclitaxel in first-line treatment of advanced non-small cell lung cancer.

Background: BTH1677, a beta-glucan pathogen-associated molecular pattern molecule, drives an anti-cancer immune response in combination with oncology antibody therapies. This phase II study explored the efficacy, pharmacokinetics (PK), and safety of BTH1677 combined with bevacizumab/carboplatin/paclitaxel in patients with untreated advanced non-small cell lung cancer (NSCLC).

Methods: Patients were randomized to the BTH1677 arm (N = 61; intravenous [IV] BTH1677, 4 mg/kg, weekly; IV bevacizumab, 15 mg/kg, once each 3-week cycle [Q3W]; IV carboplatin, 6 mg/mL/min Calvert formula area-under-the-curve, Q3W; and IV paclitaxel, 200 mg/m, Q3W) or Control arm (N = 31; bevacizumab/carboplatin/paclitaxel as above).

A randomized, open-label, multicenter, phase II study evaluating the efficacy and safety of BTH1677 (1,3-1,6 beta glucan; Imprime PGG) in combination with cetuximab and chemotherapy in patients with advanced non-small cell lung cancer.

Introduction BTH1677, a 1,3-1,6 beta-glucan immunomodulator, stimulates a coordinated anti-cancer immune response in combination with anti-tumor antibody therapies. This phase II study explored the efficacy, pharmacokinetics (PK), and safety of BTH1677 combined with cetuximab/carboplatin/paclitaxel in untreated stage IIIB/IV non-small cell lung cancer (NSCLC) patients. Methods Patients were randomized 2:1 to the BTH1677 arm (N=60; BTH1677, 4 mg/kg, weekly; cetuximab, initial dose 400 mg/m and subsequent doses 250 mg/m, weekly; carboplatin, 6 mg/mL/min AUC (area-under-the-curve) by Calvert formula, once each 3-week cycle [Q3W]); and paclitaxel, 200 mg/m, Q3W) or Control arm (N=30; cetuximab/carboplatin/paclitaxel as above).

Imprime PGG-Mediated Anti-Cancer Immune Activation Requires Immune Complex Formation.

Imprime PGG (Imprime), an intravenously-administered, soluble β-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-β glucan antibodies (ABA).

A Phase II Efficacy and Safety, Open-Label, Multicenter Study of Imprime PGG Injection in Combination With Cetuximab in Patients With Stage IV KRAS-Mutant Colorectal Cancer.

Background: Imprime PGG (β(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose) is an innate immune cell modulator that primes neutrophils and monocytes/macrophages to exert antitumor activity against complement opsonized tumor cells. In patients with KRAS-mutant colorectal cancer (CRC), cetuximab alone is ineffective; however, it can bind to tumor cells and induce opsonization for recognition by Imprime PGG-bound innate immune cells. The primary objective of this study was to determine the antitumor activity of Imprime PGG in combination with cetuximab in patients with KRAS-mutant metastatic CRC.

Two randomized, double-blind, placebo-controlled, dose-escalation phase 1 studies evaluating BTH1677, a 1, 3-1,6 beta glucan pathogen associated molecular pattern, in healthy volunteer subjects.

Background: BTH1677 is a beta glucan pathogen associated molecular pattern (PAMP) currently being investigated as a novel cancer therapy. Here, the initial safety and pharmacokinetic (PK) results of BTH1677 in healthy subjects are reported.

Subjects And Methods: In the Phase 1a single-dosing study, subjects were randomized (3:1 per cohort) to a single intravenous (i.

Differential regulation of oxidative burst by distinct β-glucan-binding receptors and signaling pathways in human peripheral blood mononuclear cells.

β-Glucans possess broad immunomodulatory properties, including activation of innate immune functions such as oxidative burst activity. The differential roles of complement receptor type 3 (CR3) and Dectin-1, the known β-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae-derived β-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble β-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively.

Binding of Soluble Yeast β-Glucan to Human Neutrophils and Monocytes is Complement-Dependent.

The immunomodulatory properties of yeast β-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate β-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble β-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast β-glucan, this study evaluated and characterized the binding of soluble β-glucan to human neutrophils and monocytes.

SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents.

The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane.

Kinetics of cellular and cytokine responses in a chimeric mouse model for the study of staphylococcal enterotoxin B pathogenesis.

The mechanisms by which superantigens, such as staphylococcal enterotoxin B (SEB), contribute to microbial pathogenicity have been poorly defined. The study of such pathogenic processes has been hampered by the lack of an adequate animal model. We utilized a previously described murine chimeric model to determine the cytokines and cell populations that might be involved in SEB toxicity.

The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines.

beta-Glucans stimulate leukocyte anti-infective activity, enhance murine hematopoietic recovery following bone marrow injury and mobilize murine progenitor cells from bone marrow. This study evaluated the in vitro hematopoietic potential of the beta-glucan, PGG-glucan, on human bone marrow mononuclear cells (BMMC) and CD34+ BMMC compared with protein cytokines. In the presence of submaximal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 0.

Enhanced clearance of a multiple antibiotic resistant Staphylococcus aureus in rats treated with PGG-glucan is associated with increased leukocyte counts and increased neutrophil oxidative burst activity.

PGG-Glucan [Betafectin], a highly purified soluble beta-(1-6)-branched beta-(1 3)-linked glucan isolated from Saccharomyces cerevisiae, has broad in vitro and in vivo anti-infective activities unrelated to cytokine induction. Here we present in vivo results on the anti-infective activity of PGG-Glucan against a multiple antibiotic resistant Staphylococcus aureus. PGG-Glucan (0.

In vitro and in vivo hematopoietic activities of Betafectin PGG-glucan.

Betafectin PGG-glucan is a novel beta-(1,3)glucan that has broad-spectrum anti-infective activities without cytokine induction. Here we report that PGG-glucan also has both in vitro and in vivo hematopoietic activities. In vitro studies with bone marrow target cells from the C3H/HeN mouse revealed that although PGG-glucan alone had no direct effect on hematopoietic colony-forming cell (CFC) growth, when combined with granulocyte colony-stimulating factor (CSF) or granulocyte-macrophage CSF, it increased CFC numbers 1.

Effects of particulate and soluble (1-3)-beta-glucans on Ca2+ influx in NR8383 alveolar macrophages.

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al.

Activation of rat macrophages by Betafectin PGG-glucan requires cross-linking of membrane receptors distinct from complement receptor three (CR3).

PGG-glucan (Betafectin) is a soluble, highly purified yeast (1,3)-beta-glucan with broad anti-infective and immunomodulatory activities. These studies evaluated the ability of PGG-glucan to directly elicit O2- and tumor necrosis factor alpha (TNF-alpha) production by rat leukocytes in vitro. Particulate beta-glucan stimulated O2- production by the rat NR8383 alveolar macrophage cell line and resident rat peritoneal macrophages, but soluble PGG-glucan did not.

Mobilization of peripheral blood progenitor cells by Betafectin PGG-Glucan alone and in combination with granulocyte colony-stimulating factor.

Betafectin PGG-Glucan, a novel beta-(1,6) branched beta-(1,3) glucan purified from the cell walls of Saccharomyces cerevisiae, has been shown to synergize with myeloid growth factors in vitro and to enhance hematopoietic recovery in myelosuppressed mice and primates. Here we report that PGG-Glucan is also capable of mobilizing peripheral blood progenitor cells (PBPC). PGG-Glucan (0.

Sublethal gamma irradiation increases IL-1alpha, IL-6, and TNF-alpha mRNA levels in murine hematopoietic tissues.

The effects of sublethal (7.75 Gy) 60Co gamma radiation exposure on endogenous bone marrow and splenic interleukin-1alpha (IL-1alpha), IL-6, and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein levels were assayed in B6D2F1 female mice. Bone marrow and spleen were harvested from normal and irradiated mice on days 2, 4, 7, 10, and 14 postexposure, and cytokine mRNA levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and Southern blot analysis.

Serum-free culture conditions for cells capable of producing long-term survival in lethally irradiated mice.

The goal of ex vivo culture is to expand and/or differentiate cells in culture such that they retain their functional characteristics when reinfused into a patient. The studies presented here analyzed the use of culture conditions devoid of serum to expand murine hematopoietic stem cells. Bone marrow cells from male B6D2F1/J mice were cultured for up to 28 days in serum-free medium in the absence or presence of stem cell factor (SCF), GM-CSF or a combination of the two factors.

Bone marrow and splenic granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta mRNA levels in irradiated mice.

The effects of a myeloablative sublethal 775 cGy 60C gamma radiation exposure on endogenous bone marrow (BM) and splenic granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta (TGF-beta) mRNA levels were assayed in B6D2F1 female mice. BM and spleen were harvested from normal mice and irradiated mice on days 2, 4, 7, 10, and 14 after exposure. Cytokine mRNA levels were determined using reverse transcription-polymerase chain reaction.

Comparison of interleukin-1 alpha gene expression and protein levels in the murine spleen after lethal and sublethal total-body irradiation.

To understand the effects of ionizing radiation on the production of IL-1 alpha in vivo within a hematopoietic organ, we evaluated acute changes in splenic IL-1 alpha mRNA and IL-1 alpha protein after exposing B6D2F1 mice to lethal and sublethal 60Co radiation. Results suggest that in vivo, ionizing radiation induces a time- and dose-dependent accumulation of IL-1 alpha mRNA in the mouse spleen after exposure to gamma radiation. Time-dependent increases in the level of IL-1 alpha protein were also observed, although the magnitude of increased protein expression did not complement the magnitude of the accumulation of the message.

c-kit ligand gene expression in normal and sublethally irradiated mice.

The c-kit ligand (KL; Steel factor, mast cell growth factor, stem cell factor) is a hematopoietic factor that has been shown to act as a potent cofactor for hematopoietic growth and differentiation in vitro. The in vivo effects of KL, however, have been variable. To study the hematopoietic role of KL in vivo, we evaluated KL gene expression in both normal mice and mice recovering from myelosuppressive radiation exposure using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique.

Amifostine plus granulocyte colony-stimulating factor therapy enhances recovery from supralethal radiation exposures: preclinical experience in animals models.

A murine model was used to explore whether the cytoprotective agent amifostine (WR-2721) can be used to protect a critical fraction of haemopoietic stem cells against radiation, and whether granulocyte colony-stimulating factor (G-CSF) can then be used to stimulate the protected cells to proliferate and reconstitute the haematopoietic system. Groups of C3H/HeN mice treated with 200 mg/kg amifostine i.p.

Erythroid progenitor (BFU-E, CFU-E) proliferation as inferred from 5'bromodeoxyuridine labeling.

Survival patterns of burst-forming unit-erythroid (BFU-E) and cluster-forming unit-erythroid (CFU-E) were studied in B6D2F1 mice after 5'bromodeoxycytidine (BrdCyd) infusion and near-ultraviolet (near-UV) light treatment. Comparison of the kinetics of BrdCyd sensitization of these colony formers with the kinetics of granulocyte-monocyte colony-forming cells (GM-CFC) and colony-forming unit-spleen (CFU-S) demonstrated a similar, but distinguishable, pattern of labeling between BFU-E and CFU-S. BFU-E and CFU-S each contain a subpopulation of unlabeled cells that diminishes with a half-life of 35 to 40 hours.

Therapeutic efficacy of recombinant interleukin-6 (IL-6) alone and combined with recombinant human IL-3 in a nonhuman primate model of high-dose, sublethal radiation-induced marrow aplasia.

Using a nonhuman-primate model of radiation-induced bone marrow aplasia, we examined whether the single, concomitant, or sequential administration of recombinant human interleukin-3 (IL-3) and IL-6 would promote bone marrow regeneration measured by an increase in circulating platelets (PLT) and neutrophils (PMN). Rhesus monkeys were irradiated at 450 cGy and were randomly assigned to one of five treatment protocols, receiving IL-6; IL-3; combined IL-6 and IL-3; sequential IL-3 and IL-6; or human serum albumin (HSA) as a control. Cytokines or HSA were administered at total dosages of 15 micrograms/kg/day.

Granulocyte colony-stimulating factor and amifostine (Ethyol) synergize to enhance hemopoietic reconstitution and increase survival in irradiated animals.

The reported studies tested whether amifostine could be used to protect hemopoietic stem cells, which, after irradiation, could be stimulated by granulocyte colony-stimulating factor (G-CSF) to proliferate and reconstitute the hemopoietic system. Female C3H/HeN mice were administered amifostine (Ethyol, US Bioscience, Inc, West Conshohocken, PA) (200 mg/kg intraperitoneally 30 minutes before cobalt-60 irradiation and G-CSF (125 micrograms/kg/d subcutaneously from days 1 to 16 after irradiation. Saline, G-CSF, amifostine, and amifostine plus G-CSF treatments resulted in LD50/30 values of 7.