Medical School ResearchTonya Smikal2020-10-20T09:07:28-05:00
Medical School Research
Research Index – MG Beta Glucan exclusive to NSC IMMUNITION Products
Research Index Titles with Brief Summaries
Cellular Immunology (2015) 104-114 PMID 26549577; Full Text; K Berner, S.A. duPre, D. Redelman, K.W. Hunter: “Microparticulate B-glucan vaccine conjugates phagocytized by dendritic cells activate both naive CD4 and CD8 T cells in vitro”
Quote:“Microparticulate Beta Glucan [MG} joined with pathogenic antigens ingested by dendritic immune cells activate both helper T-Cells and Cytotoxic Killer T-cells in vitro.”
Research in a peer reviewed article in Cellular Immunology reports MG Beta Glucan has been studied extensively for its immunostimulatory properties with those properties providing an interaction between the MG Beta Glucan and dendritic cell receptors for beta glucan that serve as an activating signal promoting anti-fungal immunity. The report from the U. of Nevada School of Medicine, Dept of Microbiology and Immunology under the direction of Kenneth W. Hunter ScD further reports MG Beta Glucan, “…also has a long history of use as an adjuvant to promote immune responses to tumors and other microorganisms.”
The results of the study show Microparticulate glucan (MG) acts as an adjuvant, or an immunological agent that enhances the effects of other agents present, to conjugated [joined] vaccine antigens with the result to enhance antigen presentation by dendritic immune cells to CD4 (Helper) and CD8 (Cytotoxic killer) T-cells. Nutritional Scientific Corporation supported the research in part by a grant, but was not involved in the design or interpretation of the experiments.
“Oral MG Crosses the Small Intestinal Epithelial Layer and is Phagocytized by Mucosal Dendritic Cells”, MG Glucan Research Findings – U. of Nevada School of Medicine, Reno, 2013, Kenneth W. Hunter Jr, ScD, Research Director, Dept of Microbiology and Immunology:
Quote: ‘For oral MG (microparticulate glucan) to cause changes in the immune system, it must be absorbed across the epithelium of the small intestine. There are those who say that particulate b-glucan cannot be absorbed. Giving a dose equivalent to the MG Beta 1,3/1,6 glucan, 10 mg capsule, it was found that fluorescently labeled MG Microparticulate Glucan crosses the small intestinal epithelium and is significantly phagocytized by mucosal dendritic cells(DC). In the mucosal associated immune system (MALT), the mucosal dendritic cell is the gatekeeper for almost all immune responses. …100% of viable macrophages had phagocytized MG by 4 h[ours]. Note: the particulate MG Beta 1,3/1,6 glucan utilized was provided by Nutritional Scientific Corporation (NSC).
Applied Microbiology and Biotechnology (2008) 1053-1061 PMID 18677470; Berner VK, Sura ME, Hunter KW Jr.: “Conjugation of protein antigen to microparticulate B-glucan [MG] from Saccharomyces cerevisiae : a new adjuvant for intradermal and oral immunizations.” Dept of Microbiology and Immunology, U. Of Nevada School of Medicine, Reno, NV USA.
Quote: “Our laboratory has prepared and characterized a novel microparticulate beta-glucan (MG)…we hypothesized that MG could serve as a vaccine adjuvant to enhance specific immune responses. …When used to immunize mice by the intradermal route, these conjugates enhanced the primary IgG antibody response to BSA in a manner comparable to the prototypic complete Freund’s adjuvant....These results suggest that protein antigens can be conjugated to MG via a carabondiimide linkage and that these conjugates provide an adjuvant effect for stimulating the antibody response to the protein antigens.”
Quote: “In the present study, we have tested a new microparticulate form of (1-3)-d-glucan (MG) from Saccharomyces cerevisiae for its ability to induce proinflammatory cytokine secretion in mouse peritoneal macrophages in vitro, and we have examined the effect of IFN-. MG was rapidly phagocytized by peritoneal macrophages and these MG-treated macrophages upregulated TNF- IL-6,and IL-1 mRNAs and secreted these proinflammatory cytokines. Note: The orange color in macrophage cells shown on the cover of a study presented to the Nevada State Legislature [shown above] is ingested and phagocytized MG Beta Glucan from NSC used in this study.
Immunology Letters 93 (2004) 71-78: Microparticulate B-glucan upregulates the expression of B7.1, B7.2, B7-H1, but not B7-DC on cultured murine peritoneal macrophages
U.S. Patent 6,474,003 entitled, “Improved Method for Preparing Small Particle Size Glucan in a Dry Material” with F.M. Jordan, K.W. Hunter and R.A. Gault
U.S. Patent Application Serial No. 09/707,436 entitled, “Improved Method for Preparing Small Particle Glucan” with F.M. Jordan, K.W. Hunter and R.A. Gault
U.S. Patent Application Serial No. 09/707,437 entitled, “Improved Method for Preparing Small Particle Glucan in a Finely Dispersed Powder” with F.M. Jordan, K.W. Hunter and R.A. Gault
U.S. Patent Application Serial No. 09/707,582 entitled, “The Use of Beta 1,3 -Glucan-Containing Compositions to Potentiate Immune Responses by Upregulating the Expression of Costimulatory Molecules” with F.M. Jordan, K.W. Hunter and R.A. Gault
U.S. Patent Application Serial No. 60/400,377 N-Acetylglucosamine Containing Microparticulate Beta-Glucan for use as a Vaccine Adjuvant & Method of Manufacture & Use, Conjugates of the Adjuvant & Vaccine, & Pharmaceutical Formulation of Such Conjugates” with F.M. Jordan, K.W. Hunter and R.A. Gault
Medical School Research – Full Studies
Immunology Letters 98 (2005) 115–122
IFN-y primes macrophages for enhanced TNF-a expression in response to stimulatory and non-stimulatory amounts of microparticulate B-glucan
Mathew D. Berner, Michael E. Sura, Bryce N. Alves, Kenneth W. Hunter Jr.
Department of Microbiology and Immunology, University of Nevada School of Medicine, Applied Research Facility,
MS-199, Reno, NV 89557, USA
Beta-(1→3)-d-Glucan is an integral cell wall component of a variety of fungi, plants, and bacteria. Like the prototypic inflammatory mediator lipopolysaccharide (LPS), some Beta-(1→3)-d-glucan-containing preparations have been shown to induce the production of proinflammatory cytokines by macrophages. In the present study, we have tested a new microparticulate form of -(1→3)-d-glucan (MG) from Saccharomyces cerevisiae for its ability to induce proinflammatory cytokine secretion in mouse peritoneal macrophages in vitro, and we have examined the effect of IFN-..MG was rapidly phagocytized by peritoneal macrophages, and these MG-treated macrophages upregulated TNF-, IL-6, and IL-1 mRNAs and secreted these proinflammatory cytokines.
IFN-. treatment alone did not induce unstimulated macrophages to produce TNF-. However, a 4 h IFN-. pretreatment augmented TNF- secretion by peritoneal macrophages subsequently treated with an optimally stimulatory dose of MG. IFN-. pretreatment for 2 h followed by thorough washing and a further 2 h incubation without IFN-. still resulted in enhanced TNF- production in response to MG, suggesting that IFN-. can prime macrophages for a subsequent proinflammatory response. Most interestingly, we found that IFN-. pretreatment of peritoneal macrophages enhanced the TNF- response to amounts of MG that were poorly stimulatory or non-stimulatory in the absence of IFN-. priming. These data suggest that a synergy between IFN-. and -glucan may have evolved to lower the threshold of sensitivity of the innate immune response to fungal pathogens.
Beta-(1→3)-d-Glucan ( -glucan) is an integral cell wall constituent found in a variety of plants, bacteria, and fungi [1–3]. Macrophages and several other cell-types of the innate immune system have evolved pattern recognition receptors that bind to this conserved structure on pathogenic fungi [4–7]. Interaction of B-glucan with its receptor on macrophages causes these cells to produce proinflammatory mediators [8–10], and to upregulate the expression of costimulatory molecules . We have recently prepared MG, a microparticulate form of B-glucan from the yeast Saccharomyces cerevisiae for use as a vaccine adjuvant . We ∗ Corresponding author. Tel.: +1 7753275255; fax: +1 7757841142.
Researchers were interested in studying its effect on the proinflammatory cytokine response of murine peritoneal macrophages in vitro. Moreover, because it has been shown that the proinflammatory response of alveolar macrophages to soluble B-glucan is markedly enhanced by IFN-y [13,14], we wanted to see if the MG-mediated proinflammatory cytokine response of peritoneal macrophages would be enhanced by IFN-y. We demonstrate that MG induces proinflammatory gene expression and protein secretion in murine peritoneal macrophages in a dose-dependent manner, and that IFN-y can prime these cells for an enhanced response. Moreover, IFN-y pretreated macrophages demonstrated a striking enhancement of TNF- secretion to poorly or non-stimulatory amounts of MG, suggesting that the response threshold of macrophages to B-glucan can be lowered by IFN-y priming.
2. Materials and methods
Female BALB/c mice aged 6–8 weeks and weighing 18–24 g were obtained from Charles River Laboratories (Wilmington, MA). All animals were housed in microisolator cages in a temperature-controlled facility kept on a 12 h photoperiod. Food and water were provided ad libitum. All research described in this paper was done under a protocol approved by the University of Nevada, Reno Institutional Animal Care and Use Committee.
Sterile filtered RPMI 1640 (with l-glutamine and 25mM HEPES) was obtained from Biowhittaker (Walkersville, MD). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT), and was also sterile filtered. Lipopolysaccharide (LPS) from Escherichia coli 011:B4 was obtained from Sigma Chemical Company (St. Louis, MO). Starting material for the preparation of MG was a B-glucan-rich extract from S. cerevisiae obtained from Nutritional Supply Corporation (Carson City, NV).
2.3. Microparticulate B-glucan
MG was prepared as previously described
. Briefly, S. cerevisiae yeast cells were subjected to a series of chemical extractions to remove most protein, lipid, nucleic acid, and non-glucan carbohydrate, resulting in a particulate material with >93% B-(1→3)-d-glucan. However, examination of this dried yeast extract in aqueous buffer revealed aggregates of small particles ranging from 5 to 50 um. A sonication/spray drying procedure was used to make a microparticulate preparation consisting essentially of individual particles with an average diameter of 1.7±0.3 um that upon rehydration remained in the microparticulate form. Concentrations of MG used in this study are based on dry weight.
2.4. Preparation of fluorophore-labeled MG Fluorescein isothiocyanate (FITC) was conjugated to bovine serum albumin (BSA) in sodium carbonate buffer, pH 9.0 for 8 h at 4 ◦C in the dark. After extensive dialysis in PBS to remove any unbound FITC molecules, an analysis of the FITC–BSA conjugate revealed a fluorescein to protein (F/P) ratio of 0.71. The conjugation of FITC–BSA to MG involved the use of water-soluble carbodiimide [N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride]. Carbodiimide first binds to the primary NH2 functionality in partially deacetylated chitin on the outside of MG. Following incubation, any unbound carbodiimide was removed by centrifugal washes (350×g). With the addition of FITC–BSA to the reaction mixture, the carbodiimide binds a free carboxyl group from BSA, thereby resulting in the MG:FITC–BSA conjugate. The MG:FITC–BSA was washed extensively by centrifugal washes to remove any unbound FITC–BSA.
2.5. Endotoxin testing
All reagents contacting live cells were tested for endotoxin levels prior to use. To avoid cross-reaction in the standard limulus amoebocyte lysate (LAL) test, the endospecy® endotoxin-specific test (Seikagaku America Inc. via Associates of Cape Cod Inc.) was used to evaluate all B-glucan-containing preparations. All reagents used during cell culture, including the MG, were determined to be below 0.15 IU/mL.
2.6. Macrophage isolation and cell culture
Mice were euthanized by halothane inhalation, then 7–10 ml ice-cold RPMI 1640 supplemented with 10% heat-inactivated FBS was injected into their peritoneal cavities. Lavage fluids were pooled from several mice and large, macrophage-like cells were counted on a hemacytometer. The cells were then placed in 6- or 24-well tissue culture plates (Costar), or 8-well chambered slides (Nunc) at 3.0×105/ml. Plates were then placed in a 37 ◦C incubator supplemented with 5% CO2. Macrophages were allowed to adhere for 4 h, at which point the wells were washed three times with endotoxin-free, phosphate buffered saline (PBS) to rinse away most non-adherent cells.
Flow cytometric analysis of these cell preparations routinely revealed 85% F4/80 positive macrophages, 15% CD19- positive B lymphocytes, and less than 1% other cells . Since dendritic cells can also be F4/80 positive, we have stained the adherent cells with antibodies to CD11c and find less than 0.5% positive cells, indicating that the F4/80 positive cells are macrophages. One milliliter fresh RPMI 1640 supplemented with 10% FBS was then added to each well and incubated for 6–12 h prior to stimulation.
2.7. Enzyme linked immunosorbent assay (ELISA)
All cytokines were measured using ELISA kits (Pharmingen, San Diego, CA) as per the manufacturers instructions. Briefly, 96-well microtiter plates (Nunc) were coated with purified monoclonal capture antibodies for TNF-, IL-6, and IL-1 . The plates were then incubated overnight at 4 ◦C. Recombinant standards or samples were added to the wells for 2 h. The wells were then incubated with detection antibodies conjugated to horseradish peroxidase and allowed to bind for 1 h at RT. The wells were then incubated with TMB substrate solution (Sigma Chemical Co., St. Louis, MO) for 30 min, and the reaction stopped with 2N H2SO4.
Absorbance was read at 450 nm on a thermomax microplate reader and analyzed on Softmax Pro® software (Molecular
All materials used in the mRNA extraction were purchased in an endotoxin-free/RNase-free state. Macrophages were detached from wells using cell lifters (Costar) and cytoplasmic extract was obtained using an RNeasyTM kit (Qiagen). Briefly, cells were resuspended in 175 ml of plasma membrane lysis buffer (50mM Tris–HCl pH 8.0, 140mM NaCl, 1.5mMMgCl2, 0.5% Nonidet P-40, 1.0mMDTT, and 1000 U/ml RNase inhibitor) in a 1.5 ml centrifuge tube, and allowed to lyse for 5 min on ice. The lysate was then spun down at 300×g for 2 min to pellet the nuclei. The supernate, which contains cytoplasmic residue including mRNA, was decanted into a new 1.5 ml centrifuge tube, at which point
600 l of a proprietary buffer from the RNeasy TM kit containing 1.0% (v/v) -mercaptoethanolwas added. Isolation of mRNA was achieved using an ExpressDirect TM mRNA capture system (Pierce, Rockford, IL) as per the manufacturers instructions. Briefly, 50 l of lysate was incubated in a 0.2 ml PCR tube in which oligo(dT) had been covalently bonded to the inside.
The mRNA was then allowed to bind for 15 min at room temperature. Contaminating DNA as well as other cytoplasmic residue was eliminated by washing twice using a high salt buffer and then a low salt buffer provided in the kit. Reverse transcription was then performed at 37 ◦C for 60 min in a thermocycler (Perkin-Elmer) using the reagents provided in the kit: M-MuLV reverse transcriptase, buffer, dNTPs, and RNase inhibitor. The use of extra oligo(dT)’s was not necessary as the covalently bonded oligo(dT) functioned as the primer. PCR was then performed by first removing the residual RT reagents. The new cDNA was hybridized to them RNA which was attached to the covalently-linked oligo (dT) and therefore remained in the tube. A 45 l aliquot of Platinum Taq SuperMix TM (GibcoBRL) and 1.0 M of each primer, forward and reverse, were then placed into the tube. Primers used in these experiments and amplimer sizes were as follows:
TNF- forward 5-CCAGACCCTCACACTCAGAT-3 and reverse 5-AACACCCATTCCCTTCACAG-3 (498 bp),IL-6 forward 5-TGGGACTGATGCTGGTGAC-3 and reverse 5-TCTGCAAGTGCATCATCGTT-3 (211 bp), IL-1 forward 5-GAGCC-TGTGTTTCCTCCTTG-3 and reverse 5-CAAGTGCAAGGCTATGACCA-3 (172 bp).
PCR reactions were run in a GeneAmpTM PCR System 2400 (Perkin-Elmer) with a 3 min, 94 ◦C pre-heat, followed by 94 ◦C for 30 s, 55 ◦C for 1 min, and 71 ◦C for 1 min, with a 7 min final extension at 71 ◦C. The appropriate number of cycles was determined for each individual primer set in order to determine an appropriate endpoint within the exponential phase of amplification. Ten microlitre of PCR product was run on a 2% agarose gel and stained with ethidium bromide. Bands were visualized using a Gel Doc® system and analyzed using Quantity One® software (Bio-Rad Laboratories, Hercules, CA).
3.1. MG is rapidly phagocytized by mouse peritoneal macrophages in vitro We first verified that MG is phagocytized by non-activated peritoneal macrophages. Our initial observations with unlabeled MG suggested that these particles were avidly phagocytized by non-activated peritoneal macrophages. However, to eliminate the possibility that the particles were only adherent to the surface of the cells, MG was conjugated with FITC–BSA as described, and then fluorescent particles were incubated for various time periods with cultured macrophages at a final concentration of 100 mg/mL/106 cells. The number of MG phagocytized per macrophage was then determined by fluorescence microscopy. Under the conditions described, 100% of viable macrophages had phagocytized MG by 4 h, with an average of 14.4±8.7 particles/macrophage (Fig. 1). Quenching the fluorescence of non-internalized particles by addition of 0.1M trypan blue dye (pH 4.4) revealed that most particles were internalized, and therefore counts made under brightfield microscopy accurately reflected phagocytosis.
3.2. MG induces proinflammatory cytokine mRNA and protein secretion by mouse peritoneal macrophages To verify that our new MG preparation upregulates the genes and enhances the secretion of proinflammatory cytokines by mouse peritoneal macrophages, cultures with 106 cells were incubated for 4 h with 100 mg/mLMG or medium, then the cells were lysed and RNA harvested for RT-PCR analysis of cytokine mRNAs. At 4 h, when the macrophages had ingested on average more than a dozen MG, mRNAs for TNF-, IL-6, and to a lesser extent, IL-1 were upregulated.
(Fig. 2). Supernatants harvested from cultures treated with 100 g/mL MG for 12 h were tested by ELISA and found to have significant levels of TNF- and IL-6 proteins. A lower level of IL-1 protein was seen, but this was significantly higher than the level found in unstimulated cultures (Fig. 2).
Note that 100mg/mL of a similar particulate B-(1→3)-d glucan-containing preparation from S. cerevisiae was shown by others to induce maximal levels of proinflammatory cytokines by mouse alveolar macrophages . Further studies reported in this paper concentrate on the induction of TNF.
3.3. Effect of IFN- on TNF-secretion by mouse peritoneal macrophages treated in vitro with different concentrations of MG or LPS It has been reported that IFN-y treatment of macrophages enhances their proinflammatory cytokine response to – 118 M.D. Berner et al. / Immunology Letters 98 (2005) 115–122
Fig. 1. MG is phagocytized by macrophages in a time-dependent manner. Macrophages were incubated for 4 h with FITC–BSA coated MG (100 mg/mL) then examined by (A) brightfield or (B) epifluorescence microscopy. Ingestion was established by quenching all non-internalized particles with trypan blue (pH 4.4). (C) At 30 min intervals 50 macrophages were examined by fluorescence microscopy and the mean number of ingested MG particles± S.D. was calculated. (D) The percent of macrophages with ≥1 particle, and ≥5 particles was also determined. glucan [13,14], and we wanted to know if IFN-y would also enhance the proinflammatory response to MG. Cultures of mouse peritoneal macrophages (2.5×105/well) were left untreated or treated for 4 h with 2 ng/mL of IFN-y. At this time, various doses of MG (0.01–1000 g/mL) or LPS
(0.01–1000 ng/mL) were added and the macrophages incubated for an additional 4 h. Supernatants were then harvested and analyzed by ELISA for TNF- (Fig. 3, black bars). The optimal stimulatory doses of LPS and MG for TNF- production were 1.0 ng/mL and 100 g/mL, respectively. However, at optimal concentrations LPS induced nearly 10-fold more TNF- than did MG (3291 pg/mL versus 340 pg/mL). It thus appears that while our new MG preparation upregulates the expression of TNF- it is less potent than the prototypic inflammatory mediator LPS. [3rd Party Note: LPS is toxic to humans] IFN-y pretreatment alone did not cause the peritoneal macrophages to secrete TNF- (Fig. 3, gray bars).
However,the optimally stimulatory doses of LPS and MG were enhanced 5- and 3.5-fold, respectively, by IFN-y pretreatment. Interestingly, doses of both MG and LPS that were unstimulatory or poorly stimulatory caused the secretion of TNF- if the macrophages had been pretreated with IFN-y. For example, 0.1 g/mL dose of MG did not induce TNF-secretion above control levels, but IFN-y pretreatment resulted in an 8.6-fold increase. The highest dose of MG tested (1000mg/mL) showed a poor stimulation of TNF- secretion, and many macrophages were dead or had released from the substrate, suggesting toxicity. However, IFN-y pretreatment caused robust secretion of TNF- presumably by the residual live macrophages. Finally, we asked whether the priming effect of IFN-y required that the cytokine be present in the culture milieu during stimulation with MG and LPS. As shown in Fig. 4, removal of IFN-y after 2 h of pretreatment did not significantly reduce macrophage priming for subsequent MG or LPS-induced TNF- production. These data suggest that the biochemical changes induced by IFN-y persisted for at least 2 h after removal of the cytokine.
In the present study we show that MG, a new microparticulate form of B-glucan derived from the yeast S. cerevisiae , is rapidly phagocytized by mouse peritoneal macrophages. MG treatment caused peritoneal macrophages to upregulate the genes for TNF-, IL-6, and IL-1 , and to secrete these proinflammatory cytokines. The dose–response and kinetics of in vitro cytokine production were similar to those described by Hoffman et al.
 for rat alveolar
Fig. 2. MG activates macrophages to secrete inflammatory cytokines. (A) Macrophages (3.0×105/mL) were exposed to MG for 4 h after which the lysate was assayed by RT-PCR for TNF-, IL-6, IL-1 , and -actin. (B) Supernatant was collected after 12 h and assayed by ELISA for TNF-, IL-6, and IL-1 . RT-PCR results are from one of three independent experiments with similar results. ELISA results are the mean±S.D. of duplicate samples from one of three representative experiments.
Fig. 3. IFN-y enhances the macrophage TNF- response to both MG and LPS. Peritoneal macrophages were incubated for 4 h with various doses of (A) MG or (B) LPS with (gray bars) or without (black bars) a 4 h preincubation with IFN-. (2.0 ng/mL). The data represent the mean±S.D. of culture supernatants run in triplicate, and are representative of two independent experiments in (A) and three in (B). macrophages and by Abel and Czop  for human monocytes treated with particulate B-glucans. Interpretation of published B-glucan studies requires attention to the source and the physical/chemical form used (i.e., soluble or particulate, linear or branched chain), as significant functional differences have been reported [16–19]. As an oversimplification, most particulate and some soluble B-glucan-containing preparations induce proinflammatory mediators.
While it is tempting to Fig. 4. IFN-y primes the macrophage TNF- response to both MG and LPS. To determine whether the enhanced TNF- response of macrophages to MG and LPS was dependent upon the continued presence of IFN-y in the culture milieu, the cultures were treated with 2 ng/mL IFN-y for 2 h then washed three times with pyrogen-free PBS. The wells were refilled with fresh media without IFN-y and allowed to incubate for 2 h. The cells were then treated with media, MG (100 g/mL), or LPS (100 ng/mL) for 4 h. The supernatants were then analyzed in triplicate by ELISA for the presence of TNF- protein. Shown are representative results (mean±S.D.) from one of two independent experiments.
120 M.D. Berner et al. / Immunology Letters 98 (2005) 115–122 describe structure-function relationships, some apparent differences may be caused by LPS contamination; surprisingly few B-glucan studies have adequately dealt with this problem. Because B-glucan triggers the standard Limulus assay, we analyzed our MG preparations with a LPS-specific Endospecy® assay and specifically excluded any preparation with more than 1.5 IU/mL. In our study, we compared the effects of MG with LPS and found that the optimal dose of LPS was 1000-fold lower than MG, yet LPS still induced nearly 10-fold more secreted TNF- protein. Perhaps this explains why septic shock syndrome with high serum TNF- levels and significant morbidity and mortality is not usually described for fungal infections even though large amounts of B-glucan can be demonstrated in the serum [20,21]. Indeed, it has been suggested that B-glucan released from fungal pathogens down regulates the production of TNF- in response to LPS .
It has been reported that grifolan, a soluble -glucan preparation, stimulated more TNF- than did LPS , however this unusual result may have been due to the use of a less potent LPS preparation. IFN-y is a potent macrophage activator, and this cytokine synergizes with LPS to enhance proinflammatory mediator production by macrophages [23–27], and it has been suggested that IFN-y is a principal mediator of the toxic effects of LPS in vivo [28–32]. The combined effect of IFN-y and LPS is probably more physiologically relevant than LPS alone, as IFN-. is secreted by NK cells and T cells in response to LPS . Sakurai et al.  demonstrated that a soluble B-glucan preparation enhanced proinflammatory cytokine production in murine alveolar macrophages in vitro.
When these alveolar macrophages were treated with IFN-y either before or at the same time as B-glucan, they had a markedly augmented proinflammatory cytokine response and also showed enhanced production of nitric oxide. In a later in vivo study they suggested that IFN-. produced by activated T lymphocytes may contribute to the deleterious inflammatory response to fungal pathogens in the lung . Ohno et al.  found that various soluble and particulate B-glucan preparations administered to mice induced nitric oxide production by macrophages ex vivo. However, in vitro cultures of peritoneal macrophages and the macrophage-like cell line RAW 264.7 failed to produce nitric oxide unless they were treated with IFN-. and B-glucan simultaneously, suggesting that the B-glucan-induced production of this proinflammatory mediator was IFN-. dependent.
We performed experiments to determine whether IFN-y would have a synergistic effect with our new MG preparation and found that TNF- protein secretion by peritoneal macrophages treated with a optimally stimulatory dose of MG was increased 3.5-fold by pretreatment with IFN-y. IFN-y alone did not result in secretion of any measurable amount of TNF- protein, a finding consistent with a previous report suggesting that IFN-y does not induce proinflammatory cytokines in the absence of another proinflammatory signal . We also discovered that the priming effect of IFN-y was still present if the IFN-y was washed away 2 h before MG or LPS stimulation, suggesting that IFN-.-induced biochemical changes in the macrophages are stable for at least 2 h after exposure. This is consistent with the ability of IFN-y to prime macrophages for enhanced nitric oxide production in response to LPS . It is certainly possible that IFN-y remains attached to the IFN-y receptors after the bulk is washed away.
The possibility also exists that the macrophage, once exposed to IFN-., begins to produce just enough of its own IFN-y to remain primed. This idea is supported by studies showing that macrophages secrete low levels of IFN-y constitutively  and more in response to LPS . This priming effect was not limited to TNF- as IFN-y also enhanced IL-6 secretion by MG- and LPS-treated macrophages (data not shown). Gifford and Lohmann-Matthes  reported that IFN-y treated mouse and human macrophages showed enhanced TNF- responses to doses of LPS that were non-stimulatory without IFN-y priming. In the present study, we verified these previous findings for LPS and found that IFN-y priming significantly augmented TNF- production in peritoneal macrophages treated with poorly stimulatory and even nonstimulatory doses of MG. We also found that high doses of MG were toxic to macrophages and caused a significant inhibition of TNF- secretion. This was consistent with results from Olson et al.  who reported that high doses of a particulate B-glucan derived from Pneumocystis carinii blocked proinflammatory cytokine production in alveolar macrophages.
However, we found that when macrophages were pretreated with IFN-., a significant amount of TNF- was secreted in response to these high doses of MG, presumably by enhancing cytokine secretion by a minority population of live cells in the cultures. The mechanism by which IFN-. primes macrophages for enhanced proinflammatory mediator production in response to second signals remains unknown, though it has been reported that IFN-. upregulates the LPS receptor molecule TLR-4  and increases the number of B-glucan binding sites [13,14]. In their study of IFN-y priming of TNF-a secretion by LPS, Held et al.  demonstrated that IFN-y caused the degradation of IB chain, thus allowing NF-B to more quickly translocate to the nucleus and bind DNA. It has been demonstrated that although B-glucan binds to different receptors than LPS [4–7], it apparently signals through the NF-B pathway [39–41].
Therefore, the mechanism whereby IFN-. primes macrophages for enhanced TNF-a production in response to B-glucan may be similar to that of LPS. It is interesting to note that Sherwood et al.  showed that B-glucan potentiates IFN-y expression in response to LPS, possibly demonstrating a reciprocal communication between the IFN-y signaling pathway  and the NF-B pathway.
In summary, we have demonstrated that a new microparticulate form of B-glucan induces a dose-dependent production of proinflammatory cytokines in mouse peritoneal macrophages in vitro, and that IFN-y primes these cells for enhanced cytokine production in response to both stimulatory and non-stimulatory amounts of MG. The synergy between IFN-y and B-glucan may have evolved to lower the sensitivity threshold of the innate immune response to fungal pathogens.
This work was supported in part by grants from the Henry Rushing Fund, and by funds from Nutritional Supply Corporation (Carson City, NV). Brandon Carter provided excellent technical support.
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Immunology Letters 93 (2004) 71-78
Microparticulate B-glucan upregulates the expression of B7.1, B7.2, B7-H1, but not B7-DC on cultured murine peritoneal macrophages
Kenneth W. Hunter, Jr.a, Sally duPre’a and Doug Redelmanb
Immunology Letters 93 (2004) 71-78 [Published May 2004]
a Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, NV 89557, USA; b Department of Biology, University of Nevada, Reno, NV 89557, USA
Beta-1,3-()-glucan from a variety of biological sources has been shown to enhance both humoral and cellular immune responses to a variety of antigens, infectious agents, and tumors. Nevertheless, its mode of action has not been fully defined. We sought to determine whether a 1–2 micron diameter microparticulate form of beta-glucan (MG) from the yeast Saccharomyces cerevisiae could regulate expression of B7 family glycoproteins on resident peritoneal macrophages from BALB/c mice. We discovered that MG [microparticulate Beta -1,3-(D)-glucan] upregulated B7.2 mRNA expression and enhanced the surface membrane expression of B7.2 glycoprotein.
Although B7.1 mRNA was not upregulated above constitutive levels, MG [microparticulate Beta -1,3-(D)-glucan] treatment enhanced B7.1 glycoprotein expression on the macrophages, albeit to a lesser extent than B7.2. At the same time, the gene and cell surface expression of B7-H1, a putative negative regulator of T cell activity, was also upregulated by MG. The expression of B7-DC, another B7 family molecule with negative regulatory activity, was not affected by incubation with MG.
This study has demonstrated that a microparticulate form of <beta-glucan can enhance B7 co-stimulatory molecule expression on macrophages, thereby enabling these antigen-presenting cells to deliver the second signal to T-lymphocytes that express CD28. In addition, because MG [microparticulate Beta -1,3-(D)-glucan] also induces the expression of B7-H1, it may enable macrophages to provide a concomitant downregulatory signal to T-lymphocytes expressing PD-1 or related receptors.
Excerpt from page 77, Immunology Letters 93 (2004) 71-78
The upregulation of B7.1 and B7.2 co-stimulatory molecule expression demonstrated in this study suggests that MG [microparticulate Beta -1,3-(D)-glucan] can generate the second signal needed for T cell activation. Interestingly, MG [microparticulate Beta -1,3-(D)-glucan] also upregulates the expression of B7-H1 surface molecules that can interact with PD-1 and perhaps unknown receptors on activated T cells predominantly to send downregulatory signals.
The simultaneous presence of T cell upregulatory (B7.1/B7.2) and downregulatory (B7-H1) molecules on APCs would seem to be a conundrum. However, it is possible that activation or suppression is determined not by the differential expression of B7 family molecules on APCs, but rather by the differential expression of their respective ligands, CD28 and PD-1, on T cells.
MG [microparticulate Beta -1,3-(D)-glucan] may therefore serve as an adjuvant, upregulating critical co-stimulatory responses, while at the same time upregulating cell surface molecules on macrophages that can downregulate T cell responses, or perhaps channel them in different directions.
Funds from Nutritional Supply Corporation, Carson City, NV, The Henry Rushing Fund and a matching grant from the State of Nevada Applied Research Initiative supported this work.
Note: The MG Beta Glucan referenced in the study is produced by a U.S. Patent exclusive to NSC Immunition Products.
NIH Grant Application – Abstract Excerpt – January 2003
Microparticulate Glucan (MG) as a Vaccine Adjuvant
Kenneth W. Hunter Jr.
Department of Microbiology and Immunology, University of Nevada School of Medicine, Applied Research Facility, MS-199, Reno, NV 89557, USA
As part of an Abstract related to Microparticulate Glucan as a Vaccine Adjuvant (Jan 2003), Kenneth W. Hunter, Jr. ScD from the University of Reno School of Medicine, Dept of Microbiology reported, “Nutritional Supply Corporation has recently developed a novel microparticulate form of beta-1,3-(D)-glucan (MG) from Saccharomyces cerevisiae…The uniform 1-2 micron diameter MG is rapidly enocytosed by APC’s (macrophages and dendritic cells), and most importantly, upregulates the expression of B7 family co-stimulatory molecules in the APC’s. Without co-stimulation, APC’s not only fail to activate T lymphocytes, but they may actually induce an unresponsive state called tolerance.”
APC’s are antigen presenting cells which are essential to a proper immune response wherein T Cells are properly activated. “Enocytosis” is the process of cellular ingestion in which the plasma membrane folds inward to bring substances into the cell.
Research Presentation – Stanford University Western Conference on Immunology – 2002
MG Beta Glucan Research Presented at Stanford University at Western Conference on Immunology
David Berner from the University of Nevada School of Medicine, Department of Microbiology, presented research results at Stanford University at the prestigious Western Conference in Immunology. The research report findings were based on nonaggregated, microparticulate glucan’s (MG glucan) superior potentiation of murine macrophages.
The presentation highlighted non-proprietary parts of the extensive four year research project utilizing MG Glucan provided by Nutritional Scientific Corporation. Various research aspects include dosage studies, mode of potentiation and results of potentiation comparing glucans that are microparticulate and nonaggregated to those globular in size which do reaggregate when exposed to hydration in the digestive process.
According to participants in the Conference, the research presentation by David Berner, under the direction of Kenneth Hunter, ScD, was well received and the nutritional potentiation capabilities of MG glucan involving the immune response confirmed by the scientific research performed by David Berner and others at the University of Nevada School of Medicine, Department of Microbiology.
The medical school research has demonstrated microparticulate Beta 1/3,1/6 Glucan is twice as potent in creating the “nitric oxide burst” in the macrophage cells as all other globular or reaggregated glucans. The nitric oxide burst measurement is a definitive marker for the immune response potentiation by any given glucan.
Letters in Applied Microbiology
Volume 35 Issue 4 Page 267 – October 2002
Preparation of microparticulate B-glucan from Saccharomyces cerevisiae for use in immune potentiation
K.W. Hunter Jr, R.A. Gault and M.D. Berner
Aims: To develop a method for the preparation of an immunologically active, homogeneous, nonaggregated, microparticulate -glucan-containing material from the budding yeast Saccharomyces cerevisiae. Methods and Results: Using a combination of sonication and spray-drying, a homogeneous preparation of 1-2-µ diameter -glucan-containing particles was made from alkali- and acid-insoluble yeast cell wall material. This microparticulate -glucan remained in suspension longer and, following oral administration at 0·1 mg kg 1 for 14 d, enhanced phagocytosis of mouse peritoneal macrophages significantly better than did aggregated -glucan particles. Conclusions: A new sonication and spray-drying method can be employed to overcome the problem of aggregation of -glucan microparticles in aqueous media.
Significance and Impact of the Study: A process to address aggregation of microparticulate form of -glucan that remains in suspension longer for pharmaceutical applications and has superior immune potentiation characteristics has been developed.
β-glucans are polymers of -(1,3)-D-glucose [with or without -(1,6)-D-glucose side chains] found in the cell walls of many bacteria, plants and yeasts. There is an extensive literature describing the immunomodulating effects of both water soluble and insoluble -glucans, with macrophages as the principal target cells (Reynolds et al. 1980; DiLuzio 1983; Gallin1992; Cleary et al. 1999). While various soluble and particulate -glucans have been used in pharmaceutical applications (Williams et al. 1992; Chihara 1992; Babineau et al. 1994) particulate -glucan preparations derived from the yeast Saccharomyces cerevisiae are widely used as over-the-counter nutritional supplements. Examination of several commercially available products consistently revealed a predominant ‘globular’ morphology consisting of aggregated -glucan particles ranging in size from 5 to 100-µ diameter, with some unaggregated single particles in the 1-2-µ range.
While globular -glucan preparations have immune potentiating activity, it was thought that a homogeneous preparation of smaller particles would be more efficient at activating macrophages, as well as more suitable for incorporation into pharmaceutical and cosmetic formulations. As 1-2-µ diameter particles are optimally phagocytized by macrophages (Tabata and Ikada 1988), our goal was to increase the number of microparticles in this size range in the -glucan preparations.
However, even after extensive grinding and sieving of dried -glucan extracted from yeast cell walls, it was discovered that the -glucan particles formed aggregates when suspended in aqueous media. Therefore, we devised a sonication and spray drying method that yielded a consistent 1-2-µ diameter particle that remained dispersed upon hydration. Although both aggregated and microparticulate glucans enhanced peritoneal macrophage activation when administered orally to mice, the microparticulate glucan was significantly better than the aggregated form.
Materials and Methods
Processing of yeast glucan
The starting S. cerevisiae -glucan material was obtained from Nutritional Supply Corporation (Carson City, NV, USA). This material was processed from common baker’s yeast using the following procedure. Active dry yeast was added to 0·1 mol l 1 of NaOH and stirred for 30 min at 60 °C. The material was then heated to 115 °C at 8·5 psi for 45 min and then allowed to settle for 72 h. The sediment was resuspended and washed in distilled H2O by centrifugation (350 g for 20 min). The alkali insoluble solids were combined with 0·1 mol l 1 acetic acid and heated to 85 °C for 1 h, then allowed to settle at 38 °C.
The acid insoluble solids were drawn off and centrifuged as above. The compacted solid material was mixed with 3% H2O2 and refrigerated for 3 h with periodic mixing. The material was then centrifuged and the pellet washed twice with distilled H2O followed by two washes in 100% acetone. The harvested solid material was dispersed on drying trays and dried under vacuum at 38 °C for 2 h in the presence of Ca2SO4, and then further dried overnight under vacuum at room temperature. This procedure yielded a white powder with less than 5% protein, lipid and nucleic acid. Carbohydrate analysis revealed 85% hexoses (using the anthrone method) with 4·5% chitin (measured as N-acetylglucosamine).
Preparation of microparticulate -glucan
Examination of the -glucan-containing powder dispersed in saline revealed aggregates ranging from 5 to 100 µ in diameter (20 µ on average). To make a uniform 1-2-µ diameter particulate preparation, the aggregated -glucan material was first hydrated in distilled H2O overnight at 4 °C. A 1·5% suspension of the hydrated material was subjected to sonic energy via a 19-mm probe utilizing a 300-V/T Sonic Dismembrator (BioLogics, Gainesville, VA, USA). Using an ultrasonic output frequency of 20 kilohertz per s at 192 watts, the glucan suspensions were sonicated on ice for 12 min (12 48-s cycles of sonication with a 12-s pause between cycles). The sonicated -glucan suspension was spray-dried using a Buchi 190 Mini-Spray Dryer (Buchi, Germany) with an inlet air temperature of 110-170 °C, an outlet air temperature of 90-120 °C and an atomizer pressure of 30-100 psi. Using flow cytometric analysis with an EPICS XL-MCL Flow Cytometer (Coulter Electronics, Hialeah, FL, USA), 1 mg of sonicated glucan consisted of 1·81 1011 microparticles.
Morphology and sedimentation of the -glucan preparations
-glucan preparations were suspended in normal saline and viewed with a Nikon Eclipse E400 microscope under bright-field illumination. Photomicrographs were taken with a Kodak DC digital camera. Dried -glucan samples were placed on an s.e.m. specimen holder and sputter-coated with gold to an approximately 200-Å thickness. Prepared samples were viewed on a JEOL TSM T300 Scanning Electron Microscope (s.e.m.).
Suspensions of aggregated -glucan and microparticulate -glucan were prepared in distilled H2O (1·5% w/v). Each suspension was vortexed for 10 s and allowed to settle in one gravity in a 15-ml test tube for 0, 2, 5, 10, 20, 30 or 60 min. Photographs were taken of the sedimentation using a Kodak digital camera.
Results and Discussion
-glucan-containing material resulting from the chemical extraction process detailed in the Methods section was examined by light microscopy after hydration in distilled H2O or saline. This material was determined to be a heterogeneous mixture of individual microparticles (1-2 µ in diameter) and glucan particle aggregates ranging from 5 to 100-µ diameters (Fig. 1a). As there was evidence that macrophages, key target cells for the immunopharmacological activity of -glucans, preferentially ingest particles in the 1-2-µ diameter size range (Tabata and Ikada 1988), we wanted to develop a method for making microparticulate glucan. However, initial attempts to disrupt the aggregates by vigorous vortexing, heating (100 °C), or treatment with strong acid (2 N HCl) or strong base (2 N NaOH) failed (data not shown). Because ultrasonic energy has been used to prepare microparticles in other systems (Hata et al. 2000), we investigated sonication as a method of disrupting the glucan aggregates.
Although disaggregation was accomplished by sonication using the optimized conditions outlined in the Methods section (Fig. 1b), when the sonicated material was air-dried (either directly or after addition of various organic solvents such as acetone) the resulting dry material had the consistency of cardboard. This material could be ground into a fine powder with a mortar and pestle, but upon hydration in distilled H2O or saline it demonstrated significant aggregation (Fig. 1c). To overcome this re-aggregation problem, we employed a spray-drying technique. The fine powder resulting from this spray-drying process when hydrated in distilled H2O or saline resulted in a homogeneous suspension of 1-2-µ diameter particles with very few small aggregates (Fig. 1d).
Interestingly, the addition of an excipient like maltodextrin did not significantly improve the process. A similar ultrasonic approach was used by Levis and Deasy (2001) to achieve particle size reduction of microcrystalline cellulose. These authors discovered that re-aggregation in aqueous media was substantially reduced by spray-drying, with or without the addition of a surfactant. Just how sonication and spray-drying alters the chemical or physical attributes of particles to mitigate against re-aggregation remains to be determined.
The aggregated -glucan and the microparticles obtained following the sonication and spray-drying procedure were gold shadowed and examined with a s.e.m. . Figure 1(e) shows the morphology of a typical aggregate with a diameter of approximately 35 µ. Note that this aggregate appears to be composed of subunits in the 1-2-µ diameter size range (). Sonication and spray drying results in separate and discrete microparticles in the 1-2-µ diameter size range (Fig. 1f). This analysis indicates that the aggregated -glucan is composed of discrete subcomponents that can be disrupted into 1-2-µ diameter microparticles by a combination of sonication and spray-drying. The chemical composition (-glucan and chitin) and size of the -glucan microparticles suggest that they may be yeast bud scars (Bacon et al. 1969; Manners et al. 1973). We are presently investigating this notion.
To demonstrate that the microparticulate -glucan preparation had a lower sedimentation rate, we performed the experiment shown in Fig. 2. As can be seen from this figure, after 1 h of sedimentation at 1 g the microparticulate -glucan demonstrated very little settling, where as the aggregated -glucan preparation had nearly sedimented fully. Indeed, some settling of the aggregated -glucan was observed at even the earliest time point. Because the microparticulate -glucan remains in aqueous suspension longer, it can be more easily formulated into gels and creams for dermatological applications.
-glucans bind to glucan receptors on phagocytic cells (Goldman 1988; Czop and Kay 1991; Brown and Gordon 2001) and cause these cells to become ‘activated’ (DiLuzio 1983). Earlier studies by Suzuki et al. (1990) in mice showed that oral administration of a -1,3-glucan derived from the fungus Sclerotinia sclerotiorum enhanced the phagocytic activity of peritoneal macrophages. Therefore, we compared the ability of orally administered microparticulate and aggregated -glucan preparations given at 0·1 mg kg 1 daily for 14 d to enhance peritoneal macrophage phagocytosis. Note that this dosage is equivalent to a 10-mg capsule of -glucan given orally to a 75-kg human. As shown in Table 1, cultured peritoneal macrophages taken from mice treated with either microparticulate -glucan or aggregated -glucan increased the percentage of peritoneal macrophages ingesting bioparticles over the vehicle control (P< 0·05).
In addition, the microparticulate -glucan was more stimulatory than the aggregated -glucan (P=0·06). Also, the number of bioparticles ingested/cell was increased over controls by both aggregated -glucan and microparticulate -glucan (P< 0·05), and macrophages from microparticulate -glucan-treated mice ingested more bioparticles/cells than macrophages from mice treated with aggregated -glucan (P=0·06). These data imply that both microparticulate and aggregated -glucan can survive transit through the gastrointestinal tract in forms capable of being absorbed and ultimately of interacting with -glucan receptors on the surfaces of resident peritoneal macrophages. It appears that the same dose of microparticulate -glucan is better able to enhance macrophage phagocytosis than aggregated -glucan.
In conclusion, we have developed a new method for preparing homogeneous, nonaggregated, 1-2-µ diameter -glucan-containing particles from yeast cell walls. Compared with the aggregated form of -glucan, the -glucan microparticles remain in suspension longer for pharmaceutical applications and are more effective at enhancing phagocytosis by peritoneal macrophages following oral administration.
Research Summary Release – Mode of Action of B-Glucan Immunopotentiators – January 2001
Activation of Immune Defense Against Infectious Disease
Hunter K, Gault R, Jordan F
Department of Microbiology, University of Nevada School of Medicine
“MG Beta Glucan has been shown to enhance the envelopment and digestion (phagocytosis) of pathogenic microorganisms that cause infectious disease…The Beta-1,3/1,6 glucans additionally enhance the ability of macrophages, one of the most important cells in the immune system, to kill tumor cells. Laboratory studies have revealed the new MG Glucan is significantly effective at activating macrophages, and via the macrophages, the entire immune cascade including T-Cells and B-Cells.”
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