Dectin-1 multimerization and signaling depends on fungal ß-glucan structure and exposure.

Dectin-1A is a C-type lectin innate immunoreceptor that recognizes ß-(1,3;1,6)-glucan, a structural component of Candida species cell walls. ß-Glucans can adopt solution structures ranging from random coil to insoluble fiber due to tertiary (helical) and quaternary structure. Fungal ß-glucans of medium and high molecular weight are highly structured, but low molecular weight glucan is much less structured. Despite similar affinity for Dectin-1, the ability of glucans to induce Dectin-1A-mediated signaling correlates with degree of structure. Glucan denaturation experiments showed that glucan structure determines agonistic potential, but not receptor binding affinity. We explored the impact of glucan structure on molecular aggregation of Dectin-1A. Stimulation with glucan signaling decreased Dectin-1A diffusion coefficient. Fluorescence measurements provided direct evidence of ligation-induced Dectin-1A aggregation, which positively correlated with increasing glucan structure content. In contrast, Dectin-1A is predominantly in a low aggregation state in resting cells. Molecular aggregates formed during interaction with highly structured, agonistic glucans did not exceed relatively small (<15 nm) clusters of a few engaged receptors. Finally, we observed increased molecular aggregation of Dectin-1A at fungal particle contact sites in a manner that positively correlated with the degree of exposed glucan on the particle surface. These results indicate that Dectin-1A senses the solution conformation of ß-glucans through their varying ability to drive receptor dimer/oligomer formation and activation of membrane proximal signaling events.

Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects ß-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate ß-glucan polymers, Dectin-1 signalling is only activated by particulate ß-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.

Modification of the degree of branching of a beta-(1,3)-glucan affects aggregation behavior and activity in an oxidative burst assay.

Scleroglucan is a ß-(1,3)-glucan which is highly branched at the 6-position with a single glucose residue. Acid hydrolysis of a high molecular weight scleroglucan gave a medium molecular weight, freely soluble material. Linkage analysis by the partially methylated alditol acetate method showed that the solubilized material had 30% branching. When the material was subjected to partial Smith degradations, the percent branching was reduced accordingly to 12% or 17%. After the percent branching was reduced, the average molecular weight of the samples increased considerably, indicating the assembly of higher ordered aggregate structures. An aggregate number distribution analysis was applied to confirm the higher aggregated structures. These aggregated structures gave the material significantly enhanced activity in an in vitro oxidative burst assay compared to the highly branched material.

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

ß-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 ß-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae-derived ß-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble ß-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively. Oxidative burst as measured by the formation of superoxide (SO) was detected in PBMC in response to WGP and ISG. SO induction with WGP was concluded to be Dectin-1-mediated and required Src family kinases, phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, the SO induction generated by ISG was CR3-mediated and required focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The study results support the hypothesis that human PBMC, specifically monocytes, utilize distinct receptors and overlapping, but distinct, signaling pathways for the oxidative burst in response to challenge by different physical forms of ß-glucan.

Imprime PGG Enhances Anti-Tumor Effects of Tumor-Targeting, Anti-Angiogenic, and Immune Checkpoint Inhibitor Antibodies.

Imprime PGG (Imprime) is in late-stage clinical development as a combinatorial agent with several therapeutic modalities. Here we present pre-clinical mechanistic data supportive of Imprime, a soluble yeast ß-1,3/1,6-glucan pathogen-associated molecular pattern able to prime innate immune cells in a Dectin-1dependent manner. In tumor-free mice, Imprime evoked broad innate immune responses (type I interferon signature, mobilization of myeloid cells, dendritic cell and monocyte/macrophage expression of co-stimulatory ligands like CD86, and activation of natural killer cells). Imprime-mediated activation of myeloid cells also resulted in functional priming of antigen-specific CD8 T cell response. In tumor-bearing mice, Imprime monotherapy further resulted in activation of systemic and tumor infiltrating macrophages and enhanced cytotoxic CD8 T cell trafficking. Imprime enhanced the anti-tumor activity of several combinatorial agents in mouse cancer models; anti-tyrosinase-related protein 1 antibody in B16F10 melanoma experimental lung metastasis model, anti-vascular endothelial growth factor receptor 2 antibody in H1299 and H441 lung cancer, and anti-programmed cell death protein 1 antibody in MC38 colon cancer models. Mechanistically, combining Imprime with these combinatorial therapeutic agents elicited enhanced innate immune activation, supporting immunological synergy. Finally, Imprime treatment induced similar in vitro phenotypic and functional activation of human innate immune cells. Collectively, these data demonstrate Imprime's potential to orchestrate a broad, yet coordinated, anti-cancer immune response and complement existing cancer immunotherapies.

Total synthesis of (-)-callipeltoside A.

A total synthesis of (-)-callipeltoside A (1) has been achieved. The core macrocycle was made via a dual macrolactonization/pyran hemiketal formation reaction, developed to circumvent issues related to the reversible nature of acylketene formation from ß-keto lactone substrates. Initial approaches to the core of the natural product that revolved around ring-closing metathesis (RCM) and relay ring-closing metathesis (RRCM) reactions are also described.

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

Imprime PGG (Imprime), an intravenously-administered, soluble ß-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-ß glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy.

Whole glucan particles as a vaccine against systemic coccidioidomycosis.

We reported previously that yeast-derived whole glucan particles (WGPs), with or without conjugation to BSA, used as a vaccine protected against systemic aspergillosis in mice. Here, we examined their utility as a potential vaccine against coccidioidomycosis. WGPs were prepared from Saccharomyces cerevisiae; conjugation with BSA (WGP-BSA) was done using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-mediated conjugation. Heat-killed S. cerevisiae (HKY) was used as a positive-control vaccine. CD-1 mice were vaccinated with WGPs or WGP-BSA, HKY or PBS once weekly, beginning 21 days prior to infection. Mice were infected intravenously with arthroconidia of Coccidioides posadasii. In the low-mortality study, 50 % of PBS-treated controls died. Only WGP-BSA at 0.6 mg per dose induced significant protection compared with PBS treatment. All surviving mice were infected in all three organs examined. Those given WGP-BSA at 0.6 mg per dose had fewer c.f.u. in liver and lungs (P = 0.04), and those given WGPs at 6 mg per dose had fewer in lungs (P < 0.02), compared with PBS. In the high-mortality study, 90 % of PBS mice died. Vaccination with HKY, and WGPs or WGP-BSA at 6 or 12 mg per dose significantly prolonged survival (P ≤ 0.05). No surviving mice were free of infection. HKY and WGP-BSA at 12 mg per dose reduced c.f.u. in the liver and lungs (P < 0.05) and WGP-BSA at 6 mg per dose reduced c.f.u. in the lungs (P < 0.05); unconjugated WGPs did not reduce infection. WGPs or WGP-BSA acted as a vaccine that protected against mortality caused by coccidioidomycosis. Thus, WGP protection against coccidioidomycosis and aspergillosis provides the basis for development of a pan-fungal vaccine.

Whole glucan particles as a vaccine against murine aspergillosis.

Vaccination with heat-killed Saccharomyces cerevisiae (HKY) protects against experimental infection by pathogenic fungi of five genera. Here we tested whether purified Saccharomyces cell wall ß-glucan could induce protection against systemic aspergillosis. CD-1 mice were given three weekly vaccine doses subcutaneously prior to intravenous infection with Aspergillus fumigatus. Mice received PBS, 2.5 mg HKY, whole glucan particles (WGP), WGP conjugated to BSA (0.06 to 12 mg per dose), a soluble medium molecular mass (MMW) ß-glucan alone or MMW-BSA (≤24 mg per dose). Survival and c.f.u. were determined, and cytokine induction and anti-ß-glucan antibodies were assessed in vaccinated mice. Neither soluble MMW glucan, nor MMW-BSA was effective. HKY protected in two studies (survival and c.f.u. were reduced in brain and kidney organs, P<0.004). Six or 12 mg WGP or WGP-BSA prolonged survival (P≤0.004) and reduced c.f.u. in each organ (P≤0.015) in both experiments; 0.6 mg WGP or WGP-BSA prolonged survival (P≤0.015) and reduced c.f.u. (P≤0.015) in one experiment. Cytokine profiles in serum and bronchoalveolar lavage from uninfected vaccinated mice showed an innate and adaptive immune profile (i.e. upregulation of colony stimulating factors, interferons, TNF-α, chemokines such as MCP-1, MIP-1α, RANTES and KC, and Th17-activating cytokines such as IL-6, IL-1ß, IL-17). No anti-ß-glucan antibodies were in the sera, suggesting an adaptive T cell-mediated, not a B cell-mediated, protective response. Vaccination with WGP or WGP-BSA proved protective against systemic aspergillosis, equivalent to that of HKY, supporting the potential of particulate ß-glucans, alone or conjugated, as vaccines against aspergillosis.

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

The immunomodulatory properties of yeast ß-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 ß-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble ß-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast ß-glucan, this study evaluated and characterized the binding of soluble ß-glucan to human neutrophils and monocytes. The results demonstrated that soluble ß-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble ß-glucan in these cells. Binding of soluble ß-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble ß-glucan was demonstrated by detection of iC3b, the complement opsonin on ß-glucan-bound cells, as well as by the direct binding of iC3b to ß-glucan in the absence of cells. Binding of ß-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding.

Enzymatic method to measure ß-1,3-ß-1,6-glucan content in extracts and formulated products (GEM assay).

An enzymatic method to measure ß-glucan content (GEM assay) is applicable in a variety of matrices. The method is composed of swelling the sample with KOH and initial digestion with a lyticase, which is followed by treatment with a mixture of exo-1,3-ß-d-glucanase and ß-glucosidase that converts the ß-glucan to glucose. The glucose generated by the enzymatic hydrolysis is measured by another enzymatic method. The method is shown to be accurate and precise. The method is selective and applicable to both highly branched and unbranched ß-1,3-glucans.

Deprotonation and regioselective addition of 2H-pyrazolo[3,4-c]quinolines to electrophiles.

The deprotonation and regioselective reaction of 2H-pyrazolo[3,4-c]quinolines with a variety of electrophiles is described. Electrophiles include benzaldehyde, DMF, carbon dioxide, and iodine. This method provides a direct route to a class of pharmacologically interesting compounds.