GLUCAN A Bibliographic Collection

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GGGLLLUUUCCCAAANNN IIImmmmmmuuunnnooossstttiiimmmuuulllaaannnttt AAAccctttiiivvviiitttyyy

Glucans are natural polysaccharides compounded by glucopyranosic units and can be

found in grain, seaweed, cell wall of bacteria and cell wall of fungi.

Glucans derived from various types of grain, are essential components of human diet but

it was determined that purified Beta-Glucans, specifically Beta 1-3 Glucan, solubilized

from yeast, are strong stimulants of the immune system1.

They have the capacity to activate macrophages, neutrophils, and other cells that

possess specific receptors for these compounds. The activation of these cells by glucan

stimulates the body’s non-specific immune response.

Other polysaccharides, like as mannans and Alfa 1-4 or Beta 1-4 polymers of glucose

don’t possess this activity. Glucan strengthens the body’s defence mechanism under

various conditions. It was demonstrated that the reason for this strengthening is due to

the increased rate of phagocytosis through glucan-activated macrophages2.

1 Kokashis, P.L., Williams, D.L., Cook, J.A. and Di Luzio, N.R.

Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan. Science 199, 1340-1342 (1978). 2 Sherwood, E.R., Williams, D.l. and N.R. Di Luzio.

Glucan stimulates production of antitumor cytolytic cytostatic factors by macrophages. J. Biol. Resp. Mod- 6, 358-381 (1986)

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Management Of Adenoid Hypertrophy: Efficacy Of Beta -Glucan Tricarico, D; Ascione, E; Avvisati, F; Caterino, R;Varricchio, A; Imperiali, M, Italy The medical treatmem of the adenoid hypertrophy cannot put aside from an immunostimulant theraphy. We report the results of a placebo "controlled, randomized,double-blind study on the efficacy and the safety of beta-glucan. A total of 103 pediatric patients (middle age 6,5 years) were enrolled in our study between September 2000 and December 2001. The selected patients were affected from odenoid hypertrophy of 3°(68%) and 4° (32%) degree. The elegibility criteria including: 1) a documented diagnosis of adenoid hypertrophy; 2} not allergy; 3} any benefit from precedent therapies; 4) common environment conditions of life. The patients treated with beta-glucan (group A) had a reduction of adenoid hypertrophy of 62,5% with improvement of signs and symptoms,

whereas the patients that received placebo (group B) had an improvement of adenoid hypertrophy of 13,1% (p<0,05). Our study shows that the administration of beta-glucan reduces the adenoid hypertrophy and improves nasal functions. The beta-glucan, with the immunostimulant action, that is expounded with the increase of lymphocyte B and T and with the incentive to macrophages activity, improves the trophism and the immunologic action of Waldeyer's ring, particularly of the vegetations adenoids.

La terapia dell’ipertrofia delle adenoidi: efficacia del beta-glucano Tricarico D; Ascione E; Avvisati F; Caterino R; Varricchio A; Imperiali M Il trattamento medico dell’ipertrofia delle adenoidi non può prescindere da una terapia immunostimolante. Riportiamo i risultati di uno studio controllato con placebo, randomizzato e in doppio cieco riguardo l’efficacia e la sicurezza del beta-glucano. Per il nostro studio è stato arruolato un totale di 103 pazienti pediatrici (età media 6.5 anni), tra il settembre 2000 ed il dicembre 2001. I pazienti selezionati erano affetti da ipertrofia delle adenoidi di 3° (68 %) e di 4° grado (32 %). I criteri di selezione hanno considerato: 1) una diagnosi documentata di ipertrofia delle adenoidi; 2) l’assenza di allergie; 3) assenza di benefici da precedenti terapie; 4) condizioni ambientali di vita comuni. I pazienti trattati col beta-glucano (gruppo A) hanno presentato una riduzione dell’ipertrofia delle adenoidi del 62.5 % con miglioramento dei segni e dei sintomi, mentre i pazienti che hanno ricevuto il placebo (gruppo B) hanno avuto un miglioramento

dell’ipertrofia delle adenoidi del 13.1 % (p<0.05). Il nostro studio mostra che la somministrazione del beta-glucano riduce l’ipertrofia delle adenoidi e migliora la funzionalità nasale. Il beta-glucano, con la sua azione immunostimolante, che si manifesta con l’aumento di linfociti B e T e con l’incremento dell’attività macrofagica, migliora il trofismo e l’azione immunologica dell’anello di Waldeyer, particolarmente della vegetazione adenoide.

Immunostimulant oxidized beta-glucan conjugates.

Cross GG; Jennings HJ; Whitfield DM; Penney CL; Zacharie B; Gagnon L Int Immunopharmacol 2001 Mar;1(3):539-50 (ISSN: 1567-5769) National Research Council, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6. Beta-Glucans are polysaccharides that act as nonspecific immune system stimulants. However, many beta-Glucans are sparingly soluble in water. This work describes an oxidative procedure, which solubilizes the beta-Glucan from Saccharomyces cerevisiae and maintains its immunostimulatory properties. Furthermore, the carboxylates at the site of oxidation allow for the conjugation of small molecule immunostimulants. Both the parent oxidized beta-glucan and its conjugates with O-beta-alanyl-5-[6-(N,N'-dimethylamino)purin-9-yl]pentanol stimulate cytotoxic T-lymphocytes (CTLs), B cells and macrophages. In addition, they both stimulate natural killer (NK) cells, a property which the small molecule purine does not possess.

Beta-glucani coniugati ossidati immunostimolanti. Cross GG; Jennings HJ; Whitfield DM; Penney CL; Zacharie B; Gagnon L Int Immunopharmacol 2001 Mar;1(3):539-50 (ISSN: 1567-5769) National Research Council, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6. I beta-glucani sono polisaccaridi che agiscono come stimolanti non specifici del sistema immunitario. Molti beta-glucani sono moderatamente solubili in acqua. Questo lavoro descrive una procedura ossidativa che solubilizza il beta-glucano ricavato da Saccaromyces cerevisiae mantenendo le sue proprietà immunostimolatorie. Inoltre, i carbossilati presenti ai siti di ossidazione consentono la coniugazione di immunostimolanti a molecola piccola. Sia il beta-glucano ossidato precursore che i suoi coniugati con O-beta-alanil-5-[6-(N,N’-dietilamino)purin-9-yl]pentanolo, stimolano i linfociti T citotossici (CTL), le cellule B e i macrofagi. Inoltre entrambi stimolano le cellule “natural killer” (NK), proprietà che la purina a molecola piccola non possiede.

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Effect of glucan on granulopoiesis and macrophage genesis in mice C Burgaleta and DW Golde Cancer Research; 37: 1739-1742; Jun 1977 Glucan, a potent reticuloendothelial stimulant, is a glucopyranose

polysaccharide derived from zymosan. Because of glucan's potential as an immunotherapeutic agent, we performed studies in order to determine its effect on granulopoiesis and macrophage production in mice. One week after the i.p. injection of 4 mg of glucan, there was a tenfold increase in colony-forming cells in the spleen and approximately a twofold increment of cells in the bone marrow and the peritoneal cavity capable of colony formation in vitro. There was a relative and absolute increase in the number of pure macrophage colonies from bone marrow and spleen. The total

macrophage content in spleen, peritoneal cavity, and bone marrow as also increased in the treated mice. Serum from glucan-injected mice had high colony-stimulating activity levels, and the peritoneal macrophages elaborated increased colony-stimulating activity in vitro as compared to controls. Peripheral white blood cell counts were two times greater than those of control in the glucan-treated mice. These studies indicate that glucan administration results in increased granulocyte and macrophage production. The enhanced leukopoiesis is probably mediated in part by augmented release of colony-stimulating activity from macrophages. These observations suggest that the use of glucan as an immunotherapeutic agent can result in an increased number of available effector cells.

Effetto del glucano sulla granulopoiesi e la generazione di macrofagi nel topo Burgaleda C. and Golde D.W. Cancer Research; 37: 1739-1742; Jun 1977 Il glucano è un potente stimolante reticoloendoteliale, e questo è dimostrato, tra gli altri, anche attraverso questo studio che ha come obiettivo di determinare gli effetti del glucano sulla produzione di macrofagi e la granulocitopoiesi. A seguito di un’iniezione intra peritoneale giornaliera per una settimana di 4mg di glucano nel topo, è stato rilevato un aumento di circa 10 volte del numero delle cellule staminali nella milza e, un incremento di circa il doppio dei granulociti nel midollo osseo e nella cavità peritoneale. Il siero dei topi trattati con glucano ha presentato alti livelli di leucociti circolanti e peritoneali degli animali trattati erano di dimensioni doppie rispetto ai controlli.

Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan. Kokoshis PL; Williams DL; Cook JA; Di Luzio NR, Science 1978 Mar 24;199(4335):1340-2 (ISSN: 0036-8075) Glucan is a potent reticuloendothelial stimulant whose immunobiological activity is mediated, in part, by an increase in the number and function of macrophages. In studying the role of glucan as a mediator of antibacterial activity, we attempted to ascertain the ability of glucan to modify the mortality of mice with experimentally induced Gram-positive bacteremia, and to enhance antibacterial defenses in rats as denoted by serum lysozyme and phagocytic activity. After intravenous administration of glucan, serum lysozyme concentrations were increased approximately sevenfold over control concentrations. The increase in serum lysozyme appeared to parallel the glucan-induced increase in phagocytosis and induced hyperplasia of macrophages. Prior treatment of mice with glucan significantly enhanced their survival when they were challenged systemically with Staphylococcus aureus. These studies indicate that glucan confers an enhanced state of host defense against bacterial infections.

Aumento dovuto al glucano della resistenza all’infezione da Staphylococcus aureus e dell’attività del lisozima nel siero. Kokoshis PL; Williams DL; Cook JA; Di Luzio NR, Science 1978 Mar 24;199(4335):1340-2 (ISSN: 0036-8075) Il glucano è un potente stimolatore reticolo-endoteliale la cui attività immunobiologica è mediata, in parte, da un aumento nel numero e nella funzionalità dei macrofagi. Nell’ambito dello studio del ruolo del glucano come mediatore dell’attività antibatterica, abbiamo tentato di accertare la capacità del glucano di modificare la mortalità in topi con una batteremia gram-positiva indotta sperimentalmente, e di aumentare le difese antibatteriche nei ratti, come indicato dal lisozima nel siero e dall’attività fagocitica. Dopo la somministrazione endovenosa di glucano, le concentrazioni di lisozima nel siero sono state approssimativamente di sette volte maggiori rispetto a quelle del controllo. L’aumento di lisozima nel siero sembra seguire parallelamente la crescita della fagocitosi indotta dal glucano e l’iperplasia indotta nei macrofagi. Il trattamento preventivo dei topi con il glucano ha migliorato significativamente la loro sopravvivenza quando erano infettati sistemicamente con Staphylococcus aureus. Questi studi indicano che il glucano conferisce un miglioramento della difesa contro le infezioni batteriche.

Immune recognition. A new receptor for beta-glucans. Brown GD; Gordon S Sir William Dunn, School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK..Nature 2001 Sep 6;413(6851):36-7 The carbohydrate polymers known as beta-1,3-d-glucans exert potent effects on the immune system - stimulating antitumour and antimicrobial activity, for example - by binding to receptors on macrophages and other white blood cells and activating them. Although beta-glucans are known to bind to receptors, such as complement receptor 3 (ref. 1), there is evidence that another beta-glucan receptor is present on macrophages. Here we identify this unknown receptor as dectin-1 (ref. 2), a finding that provides new insights into the innate immune recognition of beta-glucans.

Riconoscimento immunitario. Un nuovo recettore per i beta-glucani. Brown GD; Gordon S Sir William Dunn, School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK..Nature 2001 Sep 6;413(6851):36-7 I polimeri carbossilati noti come beta-1,3-d-glucani esercitano potenti effetti sul sistema immunitario – ad esempio, stimolando l’attività antitumorale ed antimicrobica – legandosi a recettori sui macrofagi e su altri globuli bianchi del sangue ed attivandoli. Benchè sia noto che i beta-glucani si legano a dei recettori, come ad esempio il recettore di tipo 3 del complemento (rif. 1), vi è evidenza della presenza di un altro recettore per il beta-glucano sui macrofagi. Qui noi identifichiamo tale recettore sconosciuto come dectina-1 (rif. 2), un risultato che fornisce nuovi approfondimenti sul riconoscimento immunitario innato dei beta-glucani.

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Immunomodulatory activities of oat beta-glucan in vitro and in vivo. Estrada A; Yun CH; Van Kessel A; Li B; Hauta S; Laarveld B Animal Biotechnology Centre, Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Canada. Microbiol Immunol 1997;41(12):991-8 (ISSN: 0385-5600) Previous studies have shown that beta-glucans extracted from yeast or fungi potentiate immune responses. In the present study, the immunomodulatory activities of beta-(1-->3,1-->4)-glucan, derived from oats, were investigated. The ability of oat beta-glucan (ObetaG) to stimulate IL-1 and TNF-alpha release from murine peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of macrophages with ObetaG resulted in the production of IL-1 in a dose and time-dependent manner, whereas only small amounts of TNF-alpha could be detected in the culture supernatants. ObetaG also induced the production of IL-2, IFN-gamma and IL-4 secretion in a dose-dependent manner in cultured spleen cells. The intraperitoneal administration of ObetaG in mice resulted in the accumulation of leucocytes, predominantly macrophages, in the peritoneal cavity. Furthermore, ObetaG was tested for its ability to enhance non-specific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was enhanced by a single intraperitoneal administration of 500 microg of ObetaG 3 days prior to bacterial challenge. In conclusion, these studies demonstrated that ObetaG possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.

Attività immunomodulatorie del beta-glucano dell’avena in vitro ed in vivo. Estrada A; Yun CH; Van Kessel A; Li B; Hauta S; Laarveld B Animal Biotechnology Centre, Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Canada. Microbiol Immunol 1997;41(12):991-8 (ISSN: 0385-5600) Precedenti studi hanno mostrato che i beta-glucani estratti dai lieviti o dai funghi potenziano le risposte immunitarie. Nel presente studio sono state investigate le attività immunomodulatorie del beta-(1-->3, 1-->4)-glucano, derivato dall’avena. È stata valutata la capacità del beta-glucano dell’avena (ObetaG) di stimolare il rilascio di IL-1 e TNF-alfa da macrofagi peritoneali murini e da macrofagi della linea cellulare murina P388D1. La stimolazione in vitro dei macrofagi con ObetaG ha causato la produzione di IL-1 in modo dose- e tempo-dipendente, mentre solamente piccole quantità di TNF-alfa sono state rinvenute nei surnatanti della cultura. ObetaG ha inoltre indotto la produzione di IL-2, IFN-gamma, e la secrezione di IL-4 in modo dose-dipendente in cellule coltivate della milza. La somministrazione intraperitoneale di ObetaG nei topi ha causato l’accumulo di leucociti, prevalentemente di macrofagi, nella cavità peritoneale. Inoltre, è stata esaminata la capacità di ObetaG di migliorare la resistenza aspecifica ad un’aggressione batterica nei topi. La sopravvivenza di topi infettati con Staphylococcus aureus è stata migliorata da un’unica somministrazione intraperitoneale di 500 microgrammi di ObetaG 3 giorni prima dell’infezione. In conclusione, questi studi dimostrano che ObetaG possiede attività immunomodulatorie tali da stimolare le funzioni immunitarie sia in vitro che in vivo.

Macrophage activation in vitro by chemically cross-linked (1----3)-beta-D-glucans. Adachi Y; Ohno N; Ohsawa M; Oikawa S; Yadomae T Tokyo Chem Pharm Bull (Tokyo) 1990 Apr;38(4):988-92 College of Pharmacy, Japan. The chemical cross-linking of soluble (1----3)-beta-D-glucans having molecular weights of 21000 (CL 3 h) and 6400 (CL 6 h), and laminarin (CL LAMI), which showed negligible biological activity, by epichlorohydrin provided rigid particles. These particles showed no gel-to-sol transition upon the addition of sodium hydroxide. We compared the effects of chemical cross-linking on the biological activities of glucans. The alternative complement pathway was not activated by any of the cross-linked glucans. Glucose consumption, lysosomal enzyme release, and interleukin-1 production by mouse resident peritoneal macrophages incubated in vitro were strongly induced by CL 3 h, CL 6 h and CL LAMI. However, cross-linked dextran, Sephadex, did not exhibit any of these biological activities. These results suggested that chemical cross-linking of (1----3)-beta-D-glucans enhances macrophage activities without opsonization by complement components.

Attivazione dei macrofagi in vitro tramite (1----3)-beta-D-glucani sottoposti a crosslink chimico. Adachi Y; Ohno N; Ohsawa M; Oikawa S; Yadomae T Tokyo Chem Pharm Bull (Tokyo) 1990 Apr;38(4):988-92 College of Pharmacy, Japan. Il crosslink chimico degli (1----3)-beta-D-Glucani di peso molecolare 21000 (CL 3 h) e 6400 (CL 6 h), e della laminarina (CL LAMI), che ha mostrato un’attività biologica trascurabile, con epicloroidrina ha fornito particelle rigide. Tali particelle non hanno mostrato transizione gel-sol a seguito dell’aggiunta di idrossido di sodio. Abbiamo confrontato gli effetti del crosslink chimico sull’attività biologica dei glucani. La via alternativa del complemento non è stata attivata da nessuno dei glucani sottoposti a crosslink. CL 3 h, CL 6 h e CL LAMI hanno fortemente indotto: il consumo di glucosio, il rilascio di enzimi lisosomiali e la produzione di interleuchina-1 da parte di macrofagi residenti nel peritoneo di topi e incubati in vitro. Peraltro, il destrano sottoposto a crosslink, il Sephadex, non ha manifestato nessuna di queste attività biologiche. Questi risultati suggeriscono che il crosslink chimico degli (1----3)-beta-D-glucani accresce le attività macrofagiche senza opsonizzazione da parte di componenti complementari.

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Effects of fungal beta-glucan and interferon-gamma on the secretory functions of murine alveolar macrophages. J Leukoc Biol 1996 Jul;60(1):118-24 (ISSN: 0741-5400) Sakurai T; Ohno N; Yadomae T Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan. We investigated the effect of a fungal component, soluble beta-glucan, on secretory functions of murine alveolar macrophages (AMs) in vitro. Stimulation by beta-glucan (500 microg/mL) or interferon-gamma (IFN-gamma; 100 U/mL) alone had a slight effect on AM functions, but when AMs were incubated together with beta-glucan and IFN-gamma, the production and secretion of some immune mediators, such as nitric oxide, interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha), were markedly augmented. This combined effect of beta-glucan and IFN-gamma was based on a priming effect of IFN-gamma, because prestimulation with IFN-gamma followed by beta-glucan induced high nitric oxide production of AMs, but reversal of the sequence of treatments had only a slight effect. We also found that preincubation of AMs with IFN-gamma enhanced the binding of fluorescein-labeled beta-glucan on the AM surface, and this increased binding was abrogated to the control level by the addition of three species of soluble unlabeled (1-->3)-beta-D-glucans but not by soluble alpha-glucan. These data imply that the priming effect of IFN-gamma on the AM response to beta-glucan was dependent, at least in part, on the enhancement of beta-glucan specific binding sites on the AM surface. It was suggested that IFN-gamma is one of the principal factors controlling the pulmonary immune system against both severe fungal infection and inflammation via AM activation at the alveoli.

Effetti di beta-glucano ed interferone gamma derivato da funghi sulle funzioni secretorie dei macrofagi alveolari murini. J Leukoc Biol 1996 Jul;60(1):118-24 (ISSN:0741-5400) Sakurai T; Ohno N; Yadomae T Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan. Abbiamo investigato l’effetto di un componente fungino, il beta-glucano solubile, sulle funzioni secretorie di macrofagi alveolari murini (AM) in vitro. La stimolazione con solo beta-glucano (500 microg/mL) o solo interferone gamma (IFN-gamma, 100 U/mL) ha avuto un lieve effetto sulle funzioni degli AM, ma si è osservato un marcato aumento nella produzione e secrezione di alcuni mediatori della risposta immunitaria, come l’ossido di azoto, l’interleuchina-1 (IL-1), l’IL-6 e il fattore alfa di necrosi tumorale (TNF-alfa) quando gli AM sono stati incubati contemporaneamente con beta-glucano e IFN-gamma. Questo effetto combinato del beta-glucano e dell’IFN-gamma è basato su un effetto di innesco dell’IFN-gamma, dato che la prestimolazione con IFN-gamma seguita dal beta-glucano ha causato un’elevata produzione di ossido di azoto da parte degli AM, mentre il ribaltamento della sequenza di trattamento ha avuto solo un lieve effetto. Abbiamo inoltre scoperto che la preincubazione degli AM con IFN-gamma ha aumentato il legame sulla superficie degli AM con il beta-glucano marcato con fluorescina, e che questo aumento di legame è stato ridotto fino al livello dei controlli aggiungendo tre specie di (1-->3)-beta-D-glucani solubili non marcati, mentre non è stato ridotto aggiungendo alfa-glucani solubili. Questi dati implicano che l’effetto di innesco dell’IFN-gamma sulla risposta degli AM ai beta-glucani dipende, almeno in parte dall’aumento di siti di legame specifici per il beta-glucano sulla superficie degli AM. È stato suggerito che l’IFN-gamma sia uno dei fattori principali nel controllo del sistema immunitario polmonare nei confronti di gravi infezioni ed infiammazioni fungine tramite l’attivazione degli AM negli alveoli.

Intravenously administered (1----3)-beta-D-glucan, SSG, obtained from Sclerotinia sclerotiorum IFO 9395 augments murine peritoneal macrophage functions in vivo. Sakurai T; Ohno N; Yadomae T,Laboratory of Immunopharmacology of Microbial Products, Tokyo College of Pharmacy, Japan. Chem Pharm Bull (Tokyo) 1992 Aug;40(8):2120-4 (ISSN:0009-2363) Effect of intravenously (i.v.) or intraperitoneally (i.p.) administered (1----3)-beta-D-glucan, SSG, obtained from Sclerotinia sclerotiorum IFO 9395 on the murine peritoneal macrophage (PM) functions were examined. A single i.v. administration of SSG increased the number of PMs at a dose of 250 micrograms/mouse, and the peak appeared 4 d after administration. However, no special change was observed on peritoneal exude cell (PEC) populations. These PMs showed augmented lysosomal enzyme activity and the peaks appeared in 2 phases, on days 2 and 10. In contrast, SSG administered i.p. (250 micrograms/mouse) increased the number of PMs and enhanced the lysosomal enzyme activity of PMs from day 4, and a broad peak appeared until days 8--12. The populations of PECs were also changed by i.p. injection of SSG. Additionally, SSG administered i.v. enhanced phagocytic activity, H2O2 production and interleukin 1 (IL-1) production, and the kinetics of the activation differed depending on the activities. These data suggest that the effects of SSG on macrophage functions are different depending on administration routes, and there are some different mechanisms in the activation of macrophages in vivo by SSG.

La somministrazione endovenosa di (1----3)-beta-D-glucano, SSG, ottenuto dalla Sclerotinia sclerotiorum IFO9395 incrementa la funzionalità dei macrofagi peritoneali murini in vivo. Sakurai T; Ohno N; Yadomae T,Laboratory of Immunopharmacology of Microbial Products, Tokyo College of Pharmacy, Japan. Chem Pharm Bull (Tokyo) 1992 Aug;40(8):2120-4 (ISSN:0009-2363) Sono stati esaminati gli effetti della somministrazione endovenosa (i.v.) o intraperitoneale (i.p.) di (1----3)-beta-D-glucano, SSG, ottenuto dalla Sclerotinia sclerotiorum IFO 9395 sulla funzionalità dei macrofagi peritoneali murini (PM). Una singola somministrazione i.v. di SSG ad una dose di 250 microgrammi/topo ha accresciuto il numero di PM, ed il picco è comparso 4 gg dopo la somministrazione. Comunque, non si è osservato nessun particolare cambiamento nelle popolazioni delle cellule dell’essudato peritoneale (PEC). Questi PM hanno mostrato un incrementata attività dell’enzima lisosomiale ed i relativi picchi sono comparsi in 2 fasi, al giorno 2 ed al giorno 10. Per contro, la somministrazione i.p. di SSG (250 microgrammi/topo) ha accresciuto il numero dei PM ed incrementato l’attività dell’enzima lisosomiale dei PM a partire dal giorno 4, ed è comparso un ampio picco fino ai giorni 8--12. Anche le popolazioni di PEC sono state modificate dall’iniezione i.p. di SSG. Inoltre, l’SSG somministrato i.v. ha aumentato l’attività fagocitica, e la produzione di H202 e di interleuchina 1 (IL-1), inoltre la cinetica di attivazione è risultata differente in dipendenza dalle attività. Questi dati suggeriscono che gli effetti dell’SSG sulla funzionalità dei macrofagi differiscono in dipendenza dalle vie di somministrazione, e che esistono differenti meccanismi nell’attivazione dei macrofagi in vivo da parte dell’SSG.

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PGG-glucan, a soluble beta-(1,3)-glucan, enhances the oxidative burst response, microbicidal activity, and activates an NF-kappa B-like factor in human PMN: evidence for a glycosphingolipid beta-(1,3)-glucan receptor. Wakshull E; Brunke-reese D; Lindermuth J; Fisette L; Nathans RS; Crowley JJ; Tufts JC; Zimmermann J; Mackin W; Adams DS Department of Biology, Alpha-beta Technology, Worcester, MA 01605, USA Immunopharmacology 1999 Feb;41(2):89-107 (ISSN: 0162-3109) PGG-Glucan, a soluble beta-(1,6)-branched beta-(1,3)-linked glucose homopolymer derived from the cell wall of the yeast Saccharomyces cerevisiae, is an immunomodulator which enhances leukocyte anti-infective activity and enhances myeloid and megakaryocyte progenitor proliferation. Incubation of human whole blood with PGG-Glucan significantly enhanced the oxidative burst response of subsequently isolated blood leukocytes to both soluble and particulate activators in a dose-dependent manner, and increased leukocyte microbicidal activity. No evidence for inflammatory cytokine production was obtained under these conditions. Electrophoretic mobility shift assays demonstrated that PGG-Glucan induced the activation of an NF-kappaB-like nuclear transcription factor in purified human neutrophils. The binding of 3H-PGG-Glucan to human leukocyte membranes was specific, concentration-dependent, saturable, and high affinity (Kd approximately 6 nM). A monoclonal antibody specific to the glycosphingolipid lactosylceramide was able to inhibit activation of the NF-kappaB-like factor by PGG-Glucan, and ligand binding data, including polysaccharide specificity, suggested that the PGG-Glucan binding moiety was lactosylceramide. These results indicate that PGG-Glucan enhances neutrophil anti-microbial functions and that interaction between this beta-glucan and human neutrophils is mediated by the glycosphingolipid lactosylceramide present at the cell surface.

Il PGG-glucano, un beta-(1,3)-glucano solubile, migliora la risposta di attivazione metabolica, l’attività microbicida ed attiva un fattore NF-kappa B-simile nei PMN umani: evidenza di un recettore glicosfingolipidico per il beta-(1,3)-glucano. Wakshull E; Brunke-reese D; Lindermuth J; Fisette L; Nathans RS; Crowley JJ; Tufts JC; Zimmermann J; Mackin W; Adams DS Department of Biology, Alpha-beta Technology, Worcester, MA 01605, USA Immunopharmacology 1999 Feb;41(2):89-107 (ISSN: 0162-3109) Il PGG-glucano, un omopolimero del glucosio beta-(1,6)-ramificato e beta-(1,3)-legato derivato dalla parete cellulare del lievito Saccaromyces cerevisiae, è un immunomodulatore che incrementa l’attività anti-infettiva dei leucociti e la proliferazione dei precursori mieloidi e megacariocitici. L’incubazione di sangue intero umano con PGG-glucano ha incrementato significativamente e in modo dose-dipendente la risposta di attivazione metabolica dei leucociti del sangue successivamente isolati agli attivatori, sia solubili che particellati, ed ha accresciuto l’attività microbicida dei leucociti. In queste condizioni, non si è ottenuta alcuna evidenza della produzione di citochine infiammatorie. I test di variazione della mobilità elettroforetica hanno mostrato che il PGG-glucano induce l’attivazione di un fattore di trascrizione nucleare NF-kappaB-simile nei neutrofili umani purificati. Il legame del 3H-PGG-glucano alle membrane dei leucociti umani è risultato specifico, dipendente dalla concentrazione, saturabile e ad alta affinità (Kd approssimativamente 6 nM). Un anticorpo monoclonale specifico per il glicofosfolipide lactosilceramide è stato in grado di inibire l’attivazione del fattore NF-kappaB-simile da parte del PGG-glucano, ed i dati sul legame del ligando, che includono la specificità al polisaccaride, hanno suggerito che il gruppo funzionale di legame del PGG-glucano è il lactosilceramide. Questi risultati indicano che il PGG-glucano incrementa le funzioni anti-microbiche dei neutrofili e che l’interazione tra questo beta-glucano ed i neutrofili umani è mediata dal glicosfingolipide lactosilceramide presente sulla superficie cellulare.

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Modulation of endotoxin- and enterotoxin-induced cytokine release by in vivo treatment with beta-(1,6)-branched beta-(1,3)-glucan. Soltys J; Quinn MT; Department of Veterinary Molecular Biology, Montana State University, Bozeman 59717, USA.Infect Immun 1999 Jan;67(1):244-52 (ISSN: 0019-9567) Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, beta-(1,6)-branched beta-(1,3)-glucan (soluble beta-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble beta-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-alpha), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-gamma) and suppressed production of IL-2 and TNF-alpha compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble beta-glucan-treated mice with LPS also resulted in suppressed TNF-alpha production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-alpha production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble beta-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-alpha. Taken together, our results suggest that treatment with soluble beta-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality.

Modulazione del rilascio di citochine mediato da endotossine e enterotossine tramite trattamento in vivo con beta-(1,3)-glucano beta-(1,6)-ramificato. Soltys J; Quinn MT; Department of Veterinary Molecular Biology, Montana State University, Bozeman 59717, USA.Infect Immun 1999 Jan;67(1):244-52 (ISSN: 0019-9567) I leucociti attivati dalle endotossine o dalle enterotossine rilasciano citochine proinfiammatorie, contribuendo così alla cascata di eventi che porta allo shock settico. Negli studi qui presentati, abbiamo analizzato gli effetti della somministrazione in vivo di un immunomodulatore solubile, beta-(1,3)-glucano beta-(1,6)-ramificato (beta-glucano solubile), sulla produzione di citochine indotta dalle tossine in monociti e linfociti isolati da topi trattati. La stimolazione in vitro con lipopolisaccaride (LPS) di linfociti isolati da topi trattati col beta-glucano solubile ha provocato un aumento della produzione di interleuchina-6 (IL-6) ed una soppressione della produzione del fattore alfa di necrosi tumorale (TNF-alfa), mentre la stimolazione di queste cellule con enterotossina B di staffilococco (SEB) o con tossina 1 della sindrome da shock tossico (TSST-1) ha provocato un aumento della produzione di interferone gamma (IFN-gamma) ed una soppressione della produzione di IL-2 e TNF-alfa rispetto alle cellule isolate da topi non trattati. Anche la stimolazione in vitro con LPS di monociti isolati da topi trattati con beta-glucano solubile ha soppresso la produzione di TNF-alfa, mentre la stimolazione di queste cellule con SEB o TSST-1 ha soppresso la produzione di IL-6 e TNF-alfa rispetto alle cellule isolate dai topi non trattati. Pertanto, lo schema complessivo delle citochine leucocitarie provenienti da topi trattati con glucano riflette la soppressione della produzione di citochine proinfiammatorie, specialmente del TNF-alfa. Considerati nel complesso, i nostri risultati suggeriscono che il trattamento con beta-glucano solubile può modulare l’induzione di citochine durante la sepsi, portando ad una diminuzione della mortalità dell’ospite.

Receptor binding and internalization of a water-soluble (1-->3)-beta-D-glucan biologic response modifier in two monocyte/macrophage cell lines. Muller A; Rice PJ; Ensley HE; Coogan PS; Kalbfleish JH; Kelley JL; Love EJ; Portera CA; Ha T; Browder IW; Williams DL Department of Surgery, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614, USA. J Immunol 1996 May 1;156(9):3418-25 (ISSN: 0022-1767) Glucan phosphate, a water-soluble, chemically defined (1-->3)-beta-D-glucan biologic response modifier, has been reported to exert antisepsis activity and accelerate wound healing. In this study we describe the specific binding of glucan phosphate to human and murine monocyte/macrophage cell lines, U937 and J774A.1, respectively. At 37 degrees C, equilibrium binding was rapidly achieved, i.e., within 1 min. In U937 cells, binding occurred with an affinity (Kd) of 37 microM and a Bmax of 65 x 106 binding sites/cell at 37 degrees C. In J774A.1 cells, glucan phosphate bound with an affinity (Kd) of 24 microM and a Bmax of 53 x 106 binding sites/cell at 37 degrees C. In both cases there was insignificant nonspecific binding. We further demonstrated that bound glucan phosphate cannot be displaced by a 50-fold excess of unlabeled ligand, suggesting internalization of glucan phosphate. Transmission electron microscopy showed significantly increased cytoplasmic vacuolization and significantly decreased mitotic activity in glucan phosphate-treated U937 cells compared with that in untreated cells. Pullulan, a random coil alpha-(1-->4)-(1-->6)-linked glucose polymer that served as a control, did not compete for the same binding site as glucan phosphate in either cell line, indicating the specificity of the binding site for (1-->3)-beta-D-glucans. We conclude that water-soluble pharmaceutical grade (1-->3)-beta-D-glucan phosphate specifically binds to and is internalized by U937 and J774A.1 cells.

Legame al recettore e internalizzazione di un modificatore della risposta biologica (1-->3)-beta-D-glucano idrosolubile in due linee cellulari di monociti/macrofagi. Muller A; Rice PJ; Ensley HE; Coogan PS; Kalbfleish JH; Kelley JL; Love EJ; Portera CA; Ha T; Browder IW; Williams DL Department of Surgery, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614, USA. J Immunol 1996 May 1;156(9):3418-25 (ISSN: 0022-1767) È stato affermato che il glucano fosfato, un modificatore della risposta biologica chimicamente definito come (1-->3)-beta-D-glucano idrosolubile, presenta attività antisettica e accelera la guarigione delle ferite. Nel presente studio, descriviamo il legame specifico del glucan fosfato con linee cellulari umane e murine di monociti/macrofagi: U937 e J774A.1. Il legame di equilibrio è stato raggiunto rapidamente, cioè entro 1 min, a 37 gradi C. Nelle cellule U937, il legame si è avuto con un’affinità (Kd) di 37 microM e con un Bmax di 65 x 10(6) siti di legame/cellula a 37 gradi C. Nelle cellule J774A.1, il glucan fosfato si è legato con un’affinità (Kd) di 24 microM ed un Bmax di 53 x 10(6) siti di legame/cellula a 37 gradi C. In entrambi i casi ci sono stati legami aspecifici non significativi. Abbiamo inoltre dimostrato che che il glucan fosfato legato non può essere rimosso neppure da una quantità 50 volte in eccesso di ligando non marcato, il che suggerisce un’internalizzazione del glucan fosfato. La microscopia elettronica a trasmissione ha mostrato una crescita significativa della vacuolizzazione del citoplasma ed una riduzione significativa dell’attività mitotica nelle cellule U937 trattate con il glucan fosfato, rispetto alle medesime cellule non trattate. Il pullulano, un polimero a catena casuale del glucosio alfa-(1-->4)-(1-->6)-legato che serviva come controllo, non è risultato in competizione per i medesimi siti di legame del glucan fosfato in nessuna delle due linee cellulari, il che indica la specificità dei siti di legame per gli (1-->3)-beta-D-glucani. Ne concludiamo che l’ (1-->3)-beta-D-glucan fosfato idrosolubile per uso farmaceutico si lega specificamente e viene internalizzato dalle cellule U937 e J774A.1.

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A beta-glucan inhibitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway. Czop JK; Austen KF; J Immunol 1985 Apr;134(4):2588-93 (ISSN: 0022-1767) The ligand specificity of the human monocyte receptor that mediates phagocytosis of particulate activators of the human alternative complement pathway was defined by inhibiting the phagocytic response with glycans known to be present in zymosan. When monocytes in monolayers were preincubated with 100 micrograms/ml of beta-glucan and then incubated with 1.25 to 2.5 X 10(6) zymosan particles, the percentage of cells that exhibited phagocytosis was inhibited in a time-dependent manner; maximal inhibition occurred within 20 min of preincubation. beta-Glucan inhibited monocyte phagocytosis of zymosan and rabbit erythrocytes (Er) in a similar dose-dependent fashion and at 100 micrograms/ml reduced monocyte ingestion of 5 X 10(6)/ml zymosan and 2 X 10(8)/ml Er by 63 +/- 8% and 68 +/- 16% (mean +/- SD, n = 3), respectively. The other glycan constituent of zymosan, mannan, was less than 1% as active, and 10 mg/ml of mannan reduced the number of monocytes ingesting zymosan and Er by 56 +/- 12% and 26 +/- 11%, respectively. At concentrations as high as 500 micrograms/ml, beta-glucan had no effect on monocyte Fc, C3b, or fibronectin receptor-mediated functions. Enzymatic hydrolysis of beta-glucan and alpha-mannan with beta-glucosidase or beta-glucanase before their incubation with monocytes abrogated their inhibitory capacity, whereas hydrolysis with alpha-mannosidase or alpha-glucosidase did not. Neither of the two alpha-glucans tested (dextran T-70 and nigeran) affected monocyte ingestion of zymosan particles or sheep erythrocytes (Es) sensitized with rabbit 7S anti-Es (EsIgG) at concentrations as high as 2 mg/ml. In contrast, a number of beta-glucans were active against zymosan but not EsIgG ingestion with a 75% reduction in the number of monocytes ingesting zymosan occurring with 100 micrograms/ml laminarin, 500 micrograms/ml soluble pachyman, and 900 micrograms/ml of soluble pustulan. The galactan, agarose, either in suspensions at 2 mg/ml or in a soluble portion at 600 micrograms/ml failed to affect monocyte ingestion of zymosan particles or Er. Thus, the monocyte receptor for particulate activators that is specifically inhibited by beta-glucan at a rate compatible with a phagocytic process and that recognizes beta-glucans but not alpha-glucans, mannan, or galactan is a beta-glucan receptor.

Un recettore per il beta-glucano sui monociti umani che puo’ essere inibito: sua identità con il recettore fagocitico per le particelle attivatrici della via alternativa del complemento. Czop JK; Austen KF; J Immunol 1985 Apr;134(4):2588-93 (ISSN: 0022-1767) La specificità di ligando del recettore dei monociti umani che media la fagocitosi delle particelle attivatrici della via alternativa del complemento è stata definita inibendo la risposta fagocitica tramite dei glucani notoriamente presenti nello zimosano. Preincubando i monociti, coltivati in monostrato, con 100 microgrammi/ml di beta-glucano e successivamente incubandoli con un numero di particelle di zimosano variabile da 1.25 a 2.5 X 10(6), si è ottenuta una riduzione tempo-dipendente della percentuale di cellule che esibiscono fagocitosi; la massima inibizione si è avuta dopo 20 min di preincubazione. Il beta-glucano ha inibito in maniera simile e dose-dipendente la fagocitosi dello zimosano e degli eritrociti di coniglio (Er) e, a 100 microgrammi/ml, ha ridotto l’ingestione da parte dei monociti di 5 X 10(6) particelle/ml di zimosano e di 2 X 10(8)/ml Er, rispettivamente del 63 +/- 8% e del 68 +/- 16% (media +/- deviazione standard, n = 3). L’altro glucano tra i costituenti dello zimosano, il mannano, era meno dell’ 1% come composto attivo, e 10 mg/ml di mannano hanno causato una riduzione del numero di monociti che ingeriscono zimosano ed Er rispettivamente del 56 +/- 12% e del 26 +/- 11%. Per concentrazioni fino a 500 microgrammi/ml, il beta-glucano non ha avuto alcun effetto sulle funzioni mediate dai recettori Fc, C3b e per la fibronectina. L’idrolisi enzimatica con beta-glucosidasi o beta-glucanasi prima dell’incubazione con i monociti ha eliminato la capacità inibitoria del beta-glucano e dell’alfa-mannano, per contro l’idrolisi con alfa-mannosidasi o alfa-glucosidasi non ha avuto effetto. Nessuno dei due alfa-glucani esaminati (destrano T-70 e nigerano), fino a concentrazioni di 2 mg/ml, ha avuto effetti sull’ingestione da parte dei monociti di particelle di zimosano o di eritrociti di pecora (Es) sensibilizzati con 7S anti-Es di coniglio (EsIgG). Per contro, alcuni beta-glucani si sono dimostrati attivi nei confronti dell’ingestione di zimosano ma non dell’ingestione di EsIgG, con una riduzione del 75% del numero di monociti che ingeriscono zimosano che si verifica con 100 microgrammi/ml di laminarina, 500 microgrammi/ml di pachyman, e 900 microgrammi/ml di pustulan solubile. Il galattano, un agarosio, sia in sospensione a 2 mg/ml che in soluzione a 600 microgrammi/ml non ha avuto alcun effetto sull’ ingestione da parte dei monociti di particelle di zimosano o di Er. Pertanto, il recettore per le particelle attivatrici dei monociti che viene inibito specificamente dal beta-glucano ad un livello compatibile con un processo di fagocitosi, e che riconosce i beta-glucani ma non gli alfa-glucani, il mannano o il galattano, è un recettore per il beta-glucano.

Functional beta-glucan receptor expression by a microglial cell line Muller CD; Bocchini V; Giaimis J; Guerrieri P; Lombard Y; Poindron P,Departement d’Immunologie, Universitè Louis Pasteur de Strasbourg, Illkirch, France.Res Immunol 1994 May;145(4):267-75 (ISSN: 0923-2494) In the central nervous system, the functions of microglia appear crucial after brain damage, when phagocytes eliminate cell debris, acting as the scavengers of the brain. Diseases where an active role for microglia has been proposed recently include Alzheimer's disease, the acquired immune deficiency syndrome (AIDS) and multiple sclerosis. Only recently has it been possible to obtain a microglial cell line retaining morphological and functional aspects of these cells and their secretory products. Sugar receptors are expressed by a variety of phagocytes in primary cultures, but in contrast, are absent on the majority of the described macrophage-like cell lines. We here establish, by 4 degrees C binding experiments, that this murine cell line, called BV-2, expresses a high level (9.86 +/- 0.91 x 10(5); n = 3) of beta-glucan receptors. At 37 degrees C, BV-2 cells show high phagocytic power that can only be inhibited by the free polysugar beta-laminarin (a poly-glucose) and not by mannan (a poly-mannose) as described for macrophages. The beta-glucan receptor expressed by the microglial cell line BV-2 is fully functional in phagocytosis of unopsonized heat-killed yeast particles.

Espressione di un recettore per il beta-glucano da parte di una linea di cellule microgliali. Muller CD; Bocchini V; Giaimis J; Guerrieri P; Lombard Y; Poindron P, Departement d’Immunologie, Universitè Louis Pasteur de Strasbourg, Illkirch, France.Res Immunol 1994 May;145(4):267-75 (ISSN: 0923-2494) La funzione della microglia nel sistema nervoso centrale sembra essere cruciale a seguito di un danno cerebrale, quando i fagociti eliminano i detriti cellulari, agendo come gli “spazzini” del cervello. Le patologie per le quali è stato proposto un ruolo attivo della microglia comprendono la malattia di Alzheimer, la sindrome da immunodeficienza acquisita (AIDS) e la sclerosi multipla. Solo di recente è stato possibile ottenere una linea cellulare microgliale che mantenesse gli aspetti morfologici e funzionali di questo tipo cellulare ed i relativi prodotti di secrezione. I recettori per gli zuccheri sono espressi da una varietà di fagociti in culture primarie ma, per contro, sono assenti nella maggioranza delle linee cellulari macrofago-simili già descritte. Qui noi stabiliamo, tramite esperimenti di legame a 4 gradi C, che la linea cellulare murina, denominata BV-2, esprime un elevato livello (9.86 +/- 0.91 x 10(5); n = 3) di recettori per il beta-glucano. A 37 gradi C, le cellule BV-2 mostrano un alto potere fagocitico che può essere inibito solamente dal polisaccaride libero beta-laminarina (un poli-glucosio) e non dal mannano (un poli-mannosio), in analogia a quanto descritto per i macrofagi. I recettori per il beta-glucano espressi dalla linea cellulare microgliale BV-2 è completamente funzionale nella fagocitosi di particelle di lievito non opsonizzate uccise con calore.

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Isolation of a yeast heptaglucoside that inhibits monocyte phagocytosis of zymosan particles. Janusz MJ; Austen KF; Czop JK, Department of Medicine, Harvard Medical School, Boston, MA J Immunol 1989 Feb 1;142(3):959-65 (ISSN: 0022-1767). To isolate a unit ligand recognized by human monocyte beta-glucan receptors, acid-solubilized oligoglucosides were prepared by partial acid hydrolysis of purified yeast cell walls, gel filtered sequentially on Bio-Gel P-4 and P-2, derivatized with 2-aminopyridine, and separated by normal-phase HPLC. Ligand recognition was assessed by quantitating the effect of pretreatment with isolated materials on the capacities of adherent monocytes to phagocytose zymosan particles. Partial acid hydrolysis solubilized 23 +/- 4% (mean +/- SD; n = 7) of the cell wall glucans; at an input of 50 micrograms/ml, the solubilized products reduced the numbers of monocytes ingesting zymosan by an average of 44%. Gel filtration of acid-solubilized glucans on Bio-Gel P-4 revealed several peaks with phagocytosis-inhibiting activity, and fractions from the peak containing the smallest oligoglucosides, which accounted for 10 +/- 2% (mean +/- SD; n = 7) of the carbohydrate applied, were pooled. Further purification on Bio-Gel P-2 resolved this phagocytosis-inhibiting activity to a single peak that contained apparent heptaoses and accounted for 8 +/- 2% (mean +/- SD; n = 6) of the carbohydrate applied. At a concentration of 0.5 microgram/ml, the oligoglucosides pooled from the Bio-Gel P-4 and P-2 columns reduced the numbers of ingesting monocytes by 45 +/- 1% and 42 +/- 7% (mean +/- SD; n = 3), respectively. When derivatized with 2-aminopyridine, the oligoglucosides were resolved by HPLC to a number of peaks; a peak that eluted as an apparent heptaglucoside contained virtually all the inhibitory activity and accounted for only 6.6 +/- 0.7% (mean +/- SD, n = 7) of the carbohydrate applied. Gas chromatography analysis revealed only glucose and FAB-mass spectrometric analysis showed only heptaglucoside and no noncarbohydrate molecules. At a concentration of 1.6 ng/ml, the derivatized yeast heptaglucoside reduced the numbers of monocytes ingesting zymosan and glucan particles by 44 +/- 9% (mean +/- SD; n = 5) and 45 +/- 6% (n = 3), respectively. Thus, a heptaglucoside present in yeast cell walls is a unit ligand for human monocyte beta-glucan receptors.

Isolamento di un eptaglucoside del lievito che inibisce la fagocitosi da parte dei monociti di particelle di zimosano. Janusz MJ; Austen KF; Czop JK, Department of Medicine, Harvard Medical School, Boston, MA J Immunol 1989 Feb 1;142(3):959-65 (ISSN: 0022-1767). Per isolare un ligando riconosciuto dai recettori dei monociti per i beta-glucani, sono stati preparati degli oligoglucosidi solubilizzati con acidi tramite idrolisi acida parziale di pareti cellulari purificate di lievito, filtrate sequenzialmente con Bio-Gel P-4 e P-2, derivatizzate con 2-aminopiridine, e separate con HPLC (cromatografia liquida ad alte prestazioni) in fase normale. Il riconoscimento del ligando è stato effettuato tramite la quantificazione dell’effetto del pretrattamento con i materiali isolati sulle capacità dei monociti aderenti di fagocitare particelle di zimosano. Il 23 +/- 4% (media +/- deviazione standard; n = 7) dei glucani della parete cellulare è stato solubilizzato usando l’idrolisi acida parziale; ad un input di 50 microgrammi/ml, i prodotti di solubilizzazione hanno ridotto in media del 44% il numero di monociti che ingeriscono zimosano. La filtrazione su gel di glucani solubilizzati in acidi con Bio-Gel P-4 ha evidenziato numerosi picchi con attività inibitoria della fagocitosi, e sono state raggruppate le frazioni del picco contenenti gli oligoglucosidi più piccoli, che rappresentano il 10 +/- 2% (media +/- deviazione standard; n = 7) dei carboidrati applicati. Un’ulteriore purificazione con Bio-Gel P-2 ha permesso di risolvere tale attività di inibizione della fagocitosi ad un singolo picco contenente eptosi apparenti che rappresenta l’ 8+/- 2% (media +/- deviazione standard; n = 6) dei carboidrati applicati. Gli oligoglucosidi raccolti dalle colonne Bio-Gel P-4 e P-2 hanno ridotto il numero di monociti che ingeriscono zimosano rispettivamente del 45 +/- 1% e del 42 +/- 7% (media +/- deviazione standard; n = 3). Gli oligoglucosidi, una volta derivatizzati con 2-aminopiridina, sono stati risoluti tramite HPLC ad alcuni picchi; uno di essi, che rilasciava come un eptaglucoside apparente, conteneva sostanzialmente tutta l’attività inibitoria e rappresentava solamente il 6.6 +/- 0.7% (media +/- deviazione standard; n = 7) dei carboidrati applicati. L’analisi gas-cromatografica ha evidenziato solamente glucosio e l’analisi spettrometrica FAB-mass ha mostrato solo eptaglucoside e nessuna molecola di elementi diversi dai carboidrati. L’eptaglucoside derivatizzato dal lievito, alla concentrazione di 1.6 ng/ml, ha ridotto del 44 +/- 9% (media +/- deviazione standard; n = 5) e del 45 +/- 6% (n = 3) il numero di monociti che ingeriscono rispettivamente zimosano e particelle di glucano. Pertanto, uno degli eptaglucosidi presenti sulla parete cellulare dei lieviti è un unità ligando per i recettori del beta-glucano dei monociti umani.

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Isolation of soluble yeast beta-glucans that inhibit human monocyte phagocytosis mediated by beta-glucan receptors. Janusz MJ; Austen KF; Czop JK J Immunol 1986 Nov 15;137(10):3270-6 (ISSN: 0022-1767) The trypsin-sensitive receptor that mediates phagocytosis of unopsonized zymosan particles by human monocytes has been designated as a beta-glucan receptor because of its functional inhibition by specific algal and plant beta-glucans. Soluble ligands that are chemically and structurally identical to beta-glucan constituents of zymosan were isolated from a carbohydrate-enriched fraction of yeast extract by sequential chromatography on DE-cellulose, SP-Sephadex, and Con A-Sepharose. Preincubation of adherent human monocytes with 278, 210, and 2.5 micrograms/ml hexose equivalents in pooled chromatographic fractions from DE-cellulose, SP-Sephadex, and Con A-Sepharose, respectively, effected 50% reductions in subsequent phagocytosis of zymosan particles without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (ESIgG). The purified yeast extract-derived beta-glucans, which contained 92% glucose and 8% mannose by gas chromatographic analysis and eluted from a Sephacryl S-200 column as a broad peak with a Kav of 0.39 and estimated molecular sizes of from 20,000 to 70,000 m.w., required only 3.5 +/- 0.9 micrograms/ml (mean +/- SD, n = 6), as compared with 31.5 micrograms/ml of the algal beta-glucan laminarin to achieve 50% decreases in zymosan ingestion. Alternatively, soluble yeast beta-glucans with estimated molecular sizes of from 2 X 10(5) to 2 X 10(6) were prepared from yeast glucan particles, which contained 98% glucose and 0% mannose, by sonication and sequential centrifugation at 15,000 and 100,000 X G for 30 and 60 min, respectively. Monocyte ingestion of zymosan was reduced by 50% by pretreatment with 60 ng/ml of the soluble beta-glucans in 15,000 X G supernatants, whereas ingestion of ESIgG was unaffected by as much as 50 micrograms/ml of this material. Partial acid hydrolysis of soluble glucan-derived beta-glucans in 15,000 X G supernatants followed by gel filtration on Bio-Gel P-4 revealed two well-defined peaks within the inclusion volume of the column with phagocytosis-inhibiting activity. Oligoglucosides that eluted at a Kav of 0.46 had an estimated molecular size of 2,000 m.w. and effected a 48% reduction in zymosan ingestion at inputs of 2 to 5 micrograms/ml, and smaller oligoglucosides with a Kav of 0.82 and an estimated molecular size of 1,000 m.w. effected a 50% reduction at inputs of 25 micrograms/ml. Preincubation of monocytes for 2 min with 25 micrograms/ml of the oligoglucosides with estimated molecular size of 1,000 m.w. and with 50 ng/ml of soluble glucan-derived beta-glucans in 100,000 X G supernatants reduced zymosan ingestion by 41% +/- 4 and 44% +/- 3 (mean +/- SD, n = 3), respectively.

Isolamento di beta-glucani solubili del lievito che inibiscono la fagocitosi dei monociti umani mediata dai recettori per il beta-glucano.

Janusz MJ; Austen KF; Czop JK J Immunol 1986 Nov 15;137(10):3270-6 (ISSN: 0022-1767) Il recettore sensibile alla tripsina, che media la fagocitosi di particelle non opsonizzate di zimosano da parte dei monociti umani, è stato classificato come recettore per il beta-glucano a causa della sua inibizione funzionale da parte di specifici beta-glucani ricavati dalle alghe e dalle piante. A partire da una frazione di estratto di lievito arricchita in carboidrati, utilizzando la cromatografia sequenziale su DE-cellulosa, SP-sephadex e Con A-Sepharose, sono stati isolati dei ligandi solubili chimicamente e strutturalmente identici ai beta-glucani che costituiscono lo zimosano. La preincubazione di monociti umani aderenti con 278, 210 e 2.5 microgrammi/litro di esoso equivalenti in frazioni cromatografiche raggruppate da DE-cellulosa, SP-sephadex e Con A-Sepharose, rispettivamente, ha ridotto del 50% la successiva fagocitosi delle particelle di zimosano, senza effetto sull’ingestione mediata dagli Fc di eritrociti di pecora rivestiti con IgG (ESIgG). I beta-glucani derivati dall’estratto purificato di lievito, contenente il 92% di glucosio e l’8% di mannosio secondo l’analisi gas cromatografica ed eluito da una colonna Sephacryl S-200 come un picco ampio con una Kav di 0.39 e delle dimensioni molecolari stimate da 20000 a 70000 m.w., hanno richiesto solo 3.5 +/- 0.9 microgrammi/ml del beta-glucano algale laminarina per causare una diminuzione del 50% nell’ingestione di zimosano. In alternativa, i beta-glucani solubili del lievito con dimensioni molecolari stimate da 2 X 10(5) a 2 X 10(6) sono stati preparati da particelle di glucano del lievito, contenenti il 98% di glucosio e lo 0% di mannosio, rispettivamente per sonicazione e centrifugazione sequenziale a 15000 e 100000 X G per 30 e 60 min. L’ingestione dello zimosano da parte dei monociti è stata ridotta del 50% grazie al pretrattamento con 60 ng/ml di beta glucani solubili contenuti nei surnatanti ottenuti a 15000 X G, mentre l’ingestione di ESIgG è risultata non influenzata da quantità di questo materiale dell’ordine dei 50 microgrammi/ml. L’idrolisi acida parziale dei beta glucani derivati dai glucani solubili contenuti nei surnatanti ottenuti a 15000 X G, seguita da filtrazione di gel su Bio-Gel P-4, ha rivelato due picchi ben definiti all’interno del volume di inclusione della colonna che presenta attività di inibizione fagocitotica. Gli oligoglucosidi che rilasciano ad una Kav di 0.46 hanno una dimensione molecolare dell’ordine di 2000 m.w. ed hanno ridotto l’ingestione di zimosano del 48% per input da 2 a 5 microgrammi/ml, mentre gli oligoglucosidi più piccoli che rilasciano ad una Kav di 0.82 e hanno una dimensione molecolare dell’ordine di 1000 m.w. hanno ridotto l’ingestione di zimosano del 50% per input di 25 microgrammi/ml. La preincubazione di monociti per 2 min con 25 microgrammi/ml degli oligoglucosidi con dimensione molecolare dell’ordine di 1000 m.w. e con 50 ng/ml di beta glucani derivati da glucani solubili contenuti nei surnatanti ottenuti a 100000 X G ha ridotto l’ingestione di zimosano rispettivamente del 41% 4 e del 44%

3 (media SD, n = 3).

12

Human monocyte scavenger receptors are pattern recognition receptors for (1-->3)-beta-D-glucans. Rice PJ; Kelley JL; Kogan G; Ensley HE; Kalbfleisch JH; Browder IW; Williams DL ; Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Building 119 Room 1-29, Dogwood Lane, Johnson City, TN 37614-1708, USA. J Leukoc Biol 2002 Jul;72(1):140-6 Glucans are cell wall constituents of fungi and bacteria that bind to pattern recognition receptors and modulate innate immunity, in part, by macrophage activation. We used surface plasmon resonance to examine the binding of glucans, differing in fine structure and charge density, to scavenger receptors on membranes isolated from human monocyte U937 cells. Experiments were performed at 25 degrees C using a biosensor surface with immobilized acetylated low density lipoprotein (AcLDL). Inhibition of the binding by polyinosinic acid, but not polycytidylic acid, confirmed the interaction of scavenger receptors. Competition studies showed that there are at least two AcLDL binding sites on human U937 cells. Glucan phosphate interacts with all sites, and the CM-glucans and laminarin interact with a subset of sites. Polymer charge has a dramatic effect on the affinity of glucans with macrophage scavenger receptors. However, it is also clear that human monocyte scavenger receptors recognize the basic glucan structure independent of charge.

I recettori “scavenger” dei monociti umani sono recettori di pattern recognition per gli (1-->3)-beta-D-glucani. Rice PJ; Kelley JL; Kogan G; Ensley HE; Kalbfleisch JH; Browder IW; Williams DL ; Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Building 119 Room 1-29, Dogwood Lane, Johnson City, TN 37614-1708, USA. J Leukoc Biol 2002 Jul;72(1):140-6 I glucani sono costituenti della parete cellulare dei funghi e dei batteri che si legano a recettori di pattern recognition e modulano, in parte, l’immunità innata tramite l’attivazione dei macrofagi. Abbiamo utilizzato la risonanza plasmonica di superficie per esaminare il legame dei glucani, che differiscono nella struttura fine e nella densità di carica, ai recettori “scavenger” su membrane cellulari isolate da monociti umani U937. Gli esperimenti sono stati condotti a 25 gradi C utilizzando una superficie del biosensore dove è immobilizzata una lipoproteina acetilata a bassa densita (AcLDL). L’inibizione del legame, che avviene con l’acido polinosinico, ma non con l’acido policitidilico, ha confermato l’interazione con i recettori “scavenger”. Studi di confronto hanno mostrato che ci sono almeno due siti di legame per le AcLDL sulle cellule U937 umane. Il glucan fosfato interagisce con tutti i siti, i CM-glucani e la laminarina interagiscono con un sottoinsieme di siti. La carica del polimero ha un pesante effetto sull’affinità dei glucani per i recettori “scavenger” dei macrofagi. È comunque chiaro che i recettori “scavenger” dei monociti umani riconoscono la struttura di base del glucano indipendentemente dalla carica.

Specificity of membrane complement receptor type three (CR3) for beta-glucans. Ross GD; Cain JA; Myones BL; Newman SL; Lachmann PJ Complement 1987;4(2):61-74 (ISSN: 0253-5076) The binding of the iC3b receptor (CR3) to unopsonized zymosan was shown to result from CR3 attachment to cell wall beta-glucans. A specificity of neutrophil responses for beta-glucan was first suggested by a comparison of yeast (Saccharomyces cerevisiae) cell wall components for stimulation of a neutrophil superoxide burst. Neutrophils responded poorly to heat-killed yeast, but gave increasingly better responses to cell wall polysaccharides devoid of proteins (zymosan) and nearly pure beta-glucan particles derived from zymosan. Zymosan triggered a burst that was 29% as great as that stimulated by phorbol myristate acetate (PMA), and beta-glucan particles stimulated a burst that was 72% as great as that produced by PMA. Phagocytic responses to yeast were also inhibited by soluble glucans but not by soluble mannans. Three types of experiments demonstrated a role for CR3 in these responses. First, neutrophil ingestion of either yeast or yeast-derived beta-glucan particles was blocked by monoclonal anti-CR3, fluid-phase iC3b, or soluble beta-glucan from barley. Monocyte ingestion of beta-glucan particles was also blocked by anti-CR3, but not by anti-CR1 or anti-C3. Second, the neutrophil superoxide burst response to either zymosan or beta-glucan particles was blocked by anti-CR3 or fluid-phase iC3b, and was completely absent with neutrophils from 3 patients with an inherited deficiency of CR3. Third, CR3 was isolated from solubilized neutrophils by affinity chromatography on beta-glucan-Sepharose.

Specificità del recettore di membrana per il complemento tipo 3 (CR3) per i beta-glucani. Ross GD; Cain JA; Myones BL; Newman SL; Lachmann PJ Complement 1987;4(2):61-74 (ISSN: 0253-5076)

È stato dimostrato che il legame del recettore iC3B (CR3) con lo zimosano non opsonizzato è dovuto al legarsi di CR3 coi beta-glucani della parete cellulare. Una specificità della risposta dei neutrofili al beta-glucano è stata originariamente suggerita dal confronto tra le capacità dei componenti della parete cellulare nel lievito (Saccaromyces cerevisiae) di stimolare un “burst” del perossido (superoxide burst) nei neutrofili. I neutrofili hanno risposto scarsamente al lievito ucciso al calore, ma hanno dato risposte sempre migliori ai polisaccaridi della parete cellulare priva di proteine (zimosano) ed alle particelle quasi pure di beta-glucano derivate dallo zimosano. Lo zimosano ha indotto un “burst” del 29% maggiore rispetto a quello indotto dal forbolo miristato acetato (PMA), e le particelle di beta-glucano hanno indotto un “burst” del 72 % maggiore rispetto a quello prodotto dal PMA. Le risposte fagocitiche al lievito sono state inoltre inibite da glucani solubili, ma non da mannani solubili. Tre tipi di esperimento hanno dimostrato un ruolo del CR3 in queste risposte. Primo: l’ingestione da parte dei neutrofili sia di lievito che di particelle di beta-glucano derivate dal lievito è stata bloccata da anti-CR3 monoclonali, iC3b in fase liquida o beta-glucani solubili derivati dall’orzo. L’ingestione di particelle di beta-glucano da parte dei monociti è stata inoltre bloccata dagli anti-CR3, ma non dagli anti-CR1 o dagli anti-C3. Secondo: la risposta allo zimosano o alle particelle di beta-glucano in termini di “burst” del perossido nei neutrofili è stata bloccata dagli anti-CR3 o dagli iC3b in fase liquida, ed è stata del tutto assente nei neutrofili provenienti da 3 pazienti con una carenza ereditaria di CR3. Terzo: il CR3 è stato isolato da neutrofili solubilizzati tramite cromatografia di affinità su beta-glucan-sefarosio.

13

GGLLUUCCAANN

AAnnttiibbaacctteerriiaall AAccttiivviittyy aanndd

ssyynneerrggiissmm wwiitthh aannttiibbiioottiiccss

O

O

O

O

OH

HO

HO

OH

CH 2OCH 2COO - Na +

CH 2OHO

O

O

O

OH

HO

HO

OH

CH 2OCH 2COO - Na +

CH 2OH

14

AA pphhaassee IIII mmuullttiicceenntteerr,, ddoouubbllee--bblliinndd,, rraannddoommiizzeedd,, ppllaacceebboo--

ccoonnttrroolllleedd ssttuuddyy ooff tthhrreeee ddoossaaggeess ooff aann iimmmmuunnoommoodduullaattoorr

((PPGGGG--gglluuccaann)) iinn hhiigghh--rriisskk ssuurrggiiccaall ppaattiieennttss.. Babineau TJ; Hackford A; Kenler A; Bistrian B; Forse RA; Fairchild PG; Heard S; Keroack M; Caushaj P; Benotti P ;Department of Surgery, Deaconess Hospital, Harvard Medical School, Boston. Arch Surg 1994 Nov;129(11):1204-10 (ISSN: 0004-0010) OBJECTIVE: To examine the safety and efficacy of multiple doses of PGG-glucan (poly-[1-6]-B-D-glucopyranosyl-[1-3]-B-D-glucopyranose) in high-risk patients undergoing major thoracic or abdominal surgery. DESIGN: An interventional, multicenter, double-blind, randomized, placebo-controlled study. SETTING: Four university-affiliated medical centers. PATIENTS: Sixty-seven high-risk patients undergoing major thoracic or abdominal surgery. INTERVENTION: Patients were randomized in a 1:1:1:1 ratio to receive saline placebo or PGG-glucan at a dose of 0.1 mg/kg, 0.5 mg/kg, and 1.0 mg/kg or 2.0 mg/kg. One dose was administered before surgery and three doses were administered after surgery. MAIN OUTCOME MEASURES: To examine the safety and efficacy of PGG-glucan infusion and to identify potentially important factors for a planned phase III study. RESULTS: A dose-response trend with regard to infection incidence among patients who received PGG-glucan was observed. Serious infections occurred in four patients who received placebo and in three patients who received PGG-glucan at a dose of 0.1 mg/kg. However, only one patient who received PGG-glucan at a high dose had a serious infection. The incidence and severity of adverse events was comparable in all groups. CONCLUSIONS: PGG-glucan was generally safe and well tolerated, may decrease postoperative infection rates, and warrants further investigation in a planned phase III trial.

UUnnoo ssttuuddiioo ddii ffaassee IIII mmuullttiicceennttrriiccoo,, iinn ddooppppiioo cciieeccoo,,

ccoonnttrroollllaattoo ccoonn ppllaacceebboo ddii ttrree ddoossaaggggii ddii uunn

iimmmmuunnoommoodduullaattoorree ((PPGGGG--gglluuccaannoo)) iinn ppaazziieennttii cchhiirruurrggiiccii

aadd aallttoo rriisscchhiioo.. Babineau TJ; Hackford A; Kenler A; Bistrian B; Forse RA; Fairchild PG; Heard S; Keroack M; Caushaj P; Benotti P ; Department of Surgery, Deaconess Hospital, Harvard Medical School, Boston. Arch Surg 1994 Nov;129(11):1204-10 (ISSN: 0004-0010) OBIETTIVO: esaminare la sicurezza e l’efficacia di diversi dosaggi di PGG-glucano (poli-[1,6]-B-D-glucopiranosil-[1,3]-B-D-glucopiranosio) in pazienti ad alto rischio sottoposti a gravi interventi chirurgici toracici o addominali. PROGETTO: uno studio basato su interventi chirurgici, multicentrico, in doppio cieco, randomizzato, controllato tramite placebo. AMBIENTE: quattro centri medici affiliati ad università. PAZIENTI: sessantasette pazienti ad alto rischio sottoposti a gravi interventi chirurgici toracici o addominali. INTERVENTO: i pazienti sono stati randomizzati con un rapporto 1:1:1:1 per ricevere: soluzione salina placebo o PGG-glucano in dosi da: 0.1 mg/kg, 0.5 mg/kg, e 1.0 mg/kg o 2.0 mg/kg. Una dose è stata somministrata prima dell’intervento e tre dosi sono state somministrate dopo l’intervento. PRINCIPALI MISURE: l’esame della sicurezza e dell’efficacia delle iniezioni di PGG-glucano e l’identificazione di fattori potenzialmente importanti per uno studio pianificato di fase III. RISULTATI: la tendenza dose-risposta rispetto all’incidenza di infezioni tra i pazienti che hanno ricevuto il PGG-glucano. Infezioni gravi si sono verificate in quattro pazienti che hanno ricevuto il placebo ed in tre pazienti che hanno ricevuto il PGG-glucano alla dose di 0.1 mg/kg. Comunque, solo un paziente che ha ricevuto il PGG-glucano a dosi elevate ha presentato un infezione grave. L’incidenza e la gravità di eventi sfavorevoli è stata comparabile in tutti i gruppi. CONCLUSIONI: il PGG-glucano è risultato generalmente sicuro e ben tollerato, in grado di diminuire il tasso di infezioni post-operatorie e in grado di garantire ulteriori ricerche in uno studio pianificato di fase III.

CCoommppaarraattiivvee ttuummoorr--iinnhhiibbiittoorryy aanndd aannttii--bbaacctteerriiaall aaccttiivviittyy ooff

ssoolluubbllee aanndd ppaarrttiiccuullaattee gglluuccaann.. Di Luzio NR; Williams DL; McNamee RB; Edwards BF; Kitahama A Int J Cancer 1979 Dec 15;24(6):773-9 (ISSN: 0020-7136) A soluble fraction of particulate glucan was prepared and evaluated for its anti-tumor and anti-bacterial activity. Thin-layer chromatographic analysis indicated that the soluble preparation was composed of a variety of polyglucoses. Intravenous administration of soluble or particulate glucan resulted in significant reductions in the growth of a syngeneic anaplastic mammary carcinoma and melanoma B16. Survival data demonstrated that intravenous administration of soluble or particulate glucan prolonged survival of A/J and C57BL/6J mice with subcutaneous tumor implants. As regards to bacterial infections, soluble and particulate glucan decreased renal necrosis in S. aureus challenged mice as compared to control mice. Although the exact nature of the active soluble fraction(s) of glucan remains to be delineated, these studies demonstrate that a soluble glucan preparation exhibits significant anti-tumor and anti-staphylococcal activity. The active soluble fraction of particulate glucan may be preferable to particulate glucan in view of the inherent ease of parenteral administration.

AAttttiivviittàà iinniibbiittrriiccee ddii ttuummoorrii ee aannttiibbaatttteerriiccaa ccoommppaarraattaa ddeell

gglluuccaannoo ssoolluubbiillee ee ppaarrttiicceellllaattoo.. Di Luzio NR; Williams DL; McNamee RB; Edwards BF; Kitahama A Int J Cancer 1979 Dec 15;24(6):773-9 (ISSN: 0020-7136) È stata preparata una frazione solubile del glucano particellato e ne è stata valutata l’attività antitumorale ed antibatterica. L’analisi cromatografica in strato sottile ha indicato che la preparazione solubile era composta da un varietà di poliglucosi. La somministrazione endovenosa di glucano solubile o particellato ha prodotto una significativa inibizione della crescita di un carcinoma mammario singenico anaplastico e di un melanoma B16. I dati di sopravvivenza hanno dimostrato che la somministrazione endovenosa di glucano particellato o solubile ha prolungato la sopravvivenza di topi A/J e C57BL/6J con impianti tumorali sottocutanei. Per quanto riguarda le infezioni batteriche, il glucano particellato e solubile hanno ridotto la necrosi renale nei topi infettati con S. aureus in confronto ai controlli. Benchè l’esatta natura delle frazioni solubili attive di glucano resti da definire, questi studi dimostrano che una preparazione solubile di glucano presenta un’attività antitumorale e anti stafilococcica significativa. La frazione solubile attiva può essere preferita al glucano particellato in vista della semplicità intrinseca nella somministrazione parenterale.

15

PPrrootteeccttiivvee iimmmmuunniittyy aaggaaiinnsstt SSttrreeppttooccooccccuuss mmuuttaannss iinnffeeccttiioonn

iinn mmiiccee aafftteerr iinnttrraannaassaall iimmmmuunniizzaattiioonn wwiitthh tthhee gglluuccaann--

bbiinnddiinngg rreeggiioonn ooff SS.. mmuuttaannss gglluuccoossyyllttrraannssffeerraassee.. Jespersgaard C, Hajishengallis G, Huang Y, Russell MW, Smith DJ, Michalek SM Departments of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, SA. Infect Immun 1999 Dec;67(12):6543-9

Here we present the construction and characterization of a chimeric vaccine protein combining the glucan-binding domain (GLU) of the gtfB-encoded water-insoluble glucan-synthesizing glucosyltransferase enzyme (GTF-I) from Streptococcus mutans and thioredoxin from Escherichia coli, which increases the solubility of coexpressed recombinant proteins and stimulates proliferation of murine T cells. The protective potential of intranasal (i.n.) immunization with this chimeric immunogen was compared to that of the GLU polypeptide alone in a mouse infection model. Both immunogens were able to induce statistically significant mucosal (salivary and vaginal) and serum responses (P < 0.01) which were sustained to the end of the study (experimental day 100). Following infection with S. mutans, sham-immunized mice maintained high levels of this cariogenic organism ( approximately 60% of the total oral streptococci) for at least 5 weeks. In contrast, animals immunized with the thioredoxin-GLU chimeric protein (Thio-GLU) showed significant reduction (>85%) in S. mutans colonization after 3 weeks (P < 0.05). The animals immunized with GLU alone required 5 weeks to demonstrate significant reduction (>50%) of S. mutans infection (P < 0.05). Evaluation of dental caries activity at the end of the study showed that mice immunized with either Thio-GLU or GLU had significantly fewer carious lesions in the buccal enamel or dentinal surfaces than the sham-immunized animals (P < 0.01). The protective effects against S. mutans colonization and caries activity following i.n. immunization with GLU or Thio-GLU are attributed to the induced salivary immunoglobulin A (IgA) anti-GLU responses. Although in general Thio-GLU was not significantly better than GLU alone in stimulating salivary IgA responses and in protection against dental caries, the finding that the GLU polypeptide alone, in the absence of any immunoenhancing agents, is protective against disease offers a promising and safe strategy for the development of a vaccine against caries.

IImmmmuunniittàà pprrootteettttiivvaa ccoonnttrroo ll’’iinnffeezziioonnee ddaa SSttrreeppttooccooccccuuss

mmuuttaannss nneeii ttooppii ddooppoo iimmmmuunniizzzzaazziioonnee iinnttrraannaassaallee ccoonn iill

ssiittoo ddii lleeggaammee ddeell gglluuccaannoo ddeellllaa gglluuccoossiillttrraannssffeerraassii ddii SS..

mmuuttaannss.. Jespersgaard C, Hajishengallis G, Huang Y, Russell MW, Smith DJ, Michalek SM Departments of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, SA. Infect Immun 1999 Dec;67(12):6543-9

Presentiamo qui la preparazione e caratterizzazione di una proteina vaccino chimerica che combina il dominio di legame per il glucano (GLU) della glucosiltransferasi (GTF-I), l’enzima insolubile in acqua per la sintesi del glucano, proveniente da Streptococcus mutans, con la tioredoxina proveniente da Escherichia Coli, la quale incrementa la solubilità delle proteine ricombinanti coespresse e stimola la proliferazione di cellule T murine. Il potenziale protettivo dell’immunizzazione intranasale (i.n.) con questo immunogeno chimerico è stato confrontato con quello del solo polipeptide GLU in un modello di infezione murino. Entrambi gli immunogeni sono stati in grado di indurre risposte delle mucose (salivare e vaginale) e del siero statisticamente significative (P<0.01) che si sono mantenute fino al termine dello studio (giorno 100 dell’esperimento). Dopo l’infezione con S. mutans, i topi con falsa immunizzazione hanno mantenuto per almeno 5 settimane elevati livelli di questo microrganismo cariogeno (che rappresenta circa il 60% del totale degli streptococchi orali). Per contro, gli animali immunizzati con la proteina chimerica tioredoxina-GLU (Thio-GLU) hanno mostrato riduzioni significative (>85%) della colonizzazione da S. mutans dopo 3 settimane (P<0.05). Gli animali immunizzati con il solo GLU hanno necessitato di 5 settimane per mostrare una riduzione significativa (>50%) dell’infezione da S. mutans (P<0.05). La valutazione dell’attività delle carie dentali al termine dello studio ha mostrato che i topi immunizzati con Thio-GLU o GLU hanno presentato significativamente meno lesioni cariose nello smalto dentale o sulla superficie della dentina rispetto ai topi con falsa immunizzazione (P<0.01). Gli effetti protettivi contro la colonizzazione da S. mutans dopo l’immunizzazione i.n. con GLU o Thio-GLU si possono attribuire all’induzione della risposta dell’immunoglobulina A (IgA) anti-GLU salivare. Benchè in generale Thio-GLU non sia stata significativamente migliore del solo GLU nella stimolazione dell’IgA salivare e nella protezione contro la carie, la scoperta che il polipeptide GLU da solo, in assenza di qualsiasi agente immunostimolatore, è protettivo verso questa malattia offre una strategia promettente e sicura per lo sviluppo di un vaccino contro la carie.

Prophylaxis with the immunomodulator PGG glucan enhances antibiotic efficacy in rats infected with antibiotic-resistant bacteria. Tzianabos AO; Cisneros RL ;Channing Laboratory Brigham and Women's Hospital, Harvard Medical School Boston, Massachusetts 02115, USA. Ann N Y Acad Sci 1996 Oct 25;797:285-7 (ISSN: 0077-8923) The emergence of multiple antibiotic-resistant microorganisms has led to a search for alternatives to traditional therapeutic regimens. PGG glucan is a soluble beta-glucan immunomodulator that selectively enhances the microbicidal activities of neutrophils and macrophages without stimulating proinflammatory cytokine production. In the present studies, we examined the ability of PGG glucan to act in concert with antibiotics to decrease mortality in a rat model of intraabdominal sepsis using antibiotic-resistant bacteria as infectious inocula. Results of these studies demonstrated that prophylaxis with PGG glucan in combination with antibiotics provided enhanced protection against lethal challenge with Esherichia coli or Staphylococcus aureus as compared with the use of antibiotics alone.

La profilassi con l’immunomodulatore PGG glucano incrementa l’efficacia degli antibiotici nei ratti infettati con batteri antibiotico-resistenti. Tzianabos AO; Cisneros RL ;Channing Laboratory Brigham and Women's Hospital, Harvard Medical School Boston, Massachusetts 02115, USA. Ann N Y Acad Sci 1996 Oct 25;797:285-7 (ISSN: 0077-8923) L’emergere di molti microorganismi antibiotico-resistenti ha portato a ricercare delle alternative ai regimi terapeutici tradizionali. Il PGG glucano è un beta-glucano immunomodulatore solubile che aumenta selettivamente le attività microbicide dei neutrofili e dei macrofagi senza stimolare la produzione di citochine proinfiammatorie. Nel presente studio abbiamo esaminato la capacità del PGG glucano di agire di concerto con gli antibiotici nel decrescere la mortalità in un modello murino di sepsi intraaddominale ottenuto utilizzando batteri antibiotico-resistenti come infettanti. I risultati di questi studi hanno dimostrato che la profilassi con PGG glucano in associazione con antibiotici ha fornito un’incrementata protezione contro le infezioni letali da E. coli o da S. aureus, rispetto all’uso del solo antibiotico.

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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. Liang J; Melican D; Cafro L; Palace G; Fisette L; Armstrong R; Patchen ML;Alpha-Beta Technology, Inc., Worcester, MA 10605, USA.Int J Immunopharmacol 1998 Nov;20(11):595-614 (ISSN: 0192-0561) 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 (0.25-4 mg/kg) was administered intramuscularly to male Wistar rats 48 h, 24 h, and 4 h before and 4 h after intraperitoneal implantation of a gelatin capsule containing 10(8)S. aureus colony forming units (CFU). Blood samples were collected at various times after challenge to determine CFU levels, leukocyte counts and neutrophil oxidative burst activity; serum TNF-alpha, and IL-1beta levels were also evaluated. The 0.25 mg/kg PGG-Glucan dose had no effect on reducing blood CFU levels; however, PGG-Glucan doses of 0.5 mg/kg, 1 mg/kg, 2 mg/kg or 4 mg/kg significantly reduced blood CFU levels by 48 h after challenge. Reduced CFU levels correlated with significantly elevated absolute monocyte counts, absolute neutrophil counts, and neutrophil oxidative burst activity in the absence of any effect on TNF-alpha or on IL-1beta levels. In additional studies, effects on mortality and blood CFU levels were evaluated in rats treated with ampicillin (an antibiotic to which the S. aureus was resistant), PGG-Glucan, or both agents. Mortality and blood CFU levels were reduced most in combination-treated rats compared to saline control rats or rats treated with either ampicillin alone or PGG-Glucan alone. We conclude that in vivo (1) PGG-Glucan can enhance clearance of an antibiotic resistant S. aureus, (2) that this clearance is accompanied by an increase in monocytes and neutrophils as well as a potentiation of neutrophil oxidative microbiocidal activity without alteration of the proinflammatory cytokine response, and (3) PGG-Glucan can enhance the effectiveness of traditional antibiotic treatment.

L’aumento dell’eliminazione di uno Staphylococcus aureus con resistenza multipla agli antibiotici in ratti trattati con PGG-glucano è associata con l’aumento della conta leucocitaria e dell’attivazione metabolica dei neutrofili. Liang J; Melican D; Cafro L; Palace G; Fisette L; Armstrong R; Patchen ML;Alpha-Beta Technology, Inc., Worcester, MA 10605, USA.Int J Immunopharmacol 1998 Nov;20(11):595-614 (ISSN: 0192-0561) Il PGG-glucano [betafectina], un glucano solubile altamente purificato beta-(1-6)-ramificato beta-(1-3)-legato isolato da Saccaromyces cerevisiae, ha un’ampia attività anti-infettiva in vitro ed in vivo, non correlata all’induzione di citochine. Qui presentiamo i risultati dello studio dell’attività anti-infettiva del PGG-glucano verso uno Staphylococcus aureus con resistenza multipla agli antibiotici. Il PGG-glucano è stato somministrato (0.25-4 mg/kg) per via intramuscolare a ratti Wistar maschi 48 ore, 24 ore e 4 ore prima e 4 ore dopo l’impianto intraperitoneale di una capsula di gelatina contenente di 10(8) unità produttrici di colonie (CFU) di S. aureus. Sono stati raccolti dei campioni di sangue a vari periodi di tempo dopo l’infezione per determinare: i livelli di CFU, la conta leucocitaria e l’attivazione metabolica dei neutrofili; sono stati anche valutati i livelli di TNF-alfa nel siero e di IL-1beta. La dose da 0.25 mg/kg di glucano non ha avuto effetti sulla riduzione del livello di CFU; invece, le dosi di PGG-glucano da 0.5 mg/kg, 1 mg/kg, 2 mg/kg o 4 mg/kg hanno ridotto significativamente i livelli di CFU nel sangue 48 ore dopo l’infezione. La riduzione dei livelli ematici di CFU è risultata correlata con conte assolute dei monociti e dei neutrofili significativamente alte e con un aumento significativo dell’attivazione metabolica dei neutrofili, in assenza di effetti sui livelli di TNF-alfa e IL-1beta. Negli studi aggiuntivi sono stati valutati gli effetti sulla mortalità e sui livelli ematici di CFU in ratti trattati con ampicillina (un antibiotico cui lo S. aurus era resistente), PGG-glucano, o entrambi gli agenti. La mortalità ed i livelli ematici di CFU sono diminuiti maggiormente nei ratti trattati con la combinazione PGG-glucano-ampicillina rispetto sia ai ratti di controllo trattati con soluzione salina, sia ai ratti trattati con sola ampicillina o con solo PGG-glucano. Ne concludiamo che in vivo (1) il PGG-glucano può incrementare l’eliminazione di uno S. aureus resistente agli antibiotici, (2) che questa eliminazione è accompagnata da un aumento di monociti e neutrofili e da un potenziamento dell’attività ossidativa microbicida dei neutrofili senza alterazioni nella risposta delle citochine proinfiammatorie, e che (3) il PGG-glucano può incrementare l’efficacia dei trattamenti antibiotici tradizionali.

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Passive transfer of poly-(1-6)-beta-glucotriosyl-(1-3)-beta- glucopyranose glucan protection against lethal infection in an animal model of intra-abdominal sepsis. RL Cisneros, FC Gibson 3rd and AO Tzianabos; Channing Laboratory, Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA. Infect Immun 1996 Jun;64(6):2201-5 Previous studies have established the efficacy of soluble polymers of

poly-(1-6)-beta-glucotriosyl-(1-3)-beta-glucopyranose (PGG) glucan, a biological-response modifier, in protecting against mortality associated with experimentally induced peritonitis in a rat model. PGG glucan-treated animals showed increases in total leukocyte counts and enhanced bacterial clearance from blood. To further explore the mechanisms) by which this agent confers protection, studies were performed to examine whether protection could be transferred from PGG glucan-treated animals to naive recipients via spleen cells (SC), SC lysates, or serum. Passive-transfer experiments indicated that the responsible factor(s) was transferable by whole SC and SC lysates, as well as by peripheral leukocytes or serum from

animals treated with PGG glucan. The transferable factor(s) was resistant to pronase and trypsin digestion, was heat stable at 56 or 80 degrees C, and was not removed by NH4SO4 precipitation. The protective effect of PGG glucan was abrogated by treatment with indomethacin, a potent inhibitor of prostaglandin synthesis. Administration of a purified prostaglandin extract from the sera of PGG glucan-treated animals protected against mortality in the peritonitis model. Furthermore, treatment of rats with exogenous

synthetic prostaglandin E2 also conferred protection against mortality. These results suggest that the protective effect exhibited by PGG glucan in the rat peritonitis model is mediated, at least in part, by prostaglandins.

Trasferimento passivo della protezione da poli-(1-6)-beta-glucotriosil-(1-3)-beta-glucopiranosio glucano contro infezioni letali in un modello animale di sepsi intraaddominale. RL Cisneros, FC Gibson 3rd and AO Tzianabos; Channing Laboratory, Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA. Infect Immun 1996 Jun;64(6):2201-5 Precedenti studi hanno dimostrato l’efficacia dei polimeri solubili di poli-(1-6)-beta-glucotriosil-(1-3)-beta-glucopiranosio (PGG) glucano, un modificatore della risposta biologica, nel proteggere dalla mortalità associata alla peritonite indotta sperimentalmente in un modello murino. Gli animali trattati con PGG glucano hanno mostrato aumenti nella conta totale dei leucociti ed hanno aumentato la clearance batterica ematica. Per analizzare ulteriormente i meccanismi tramite i quali questo agente fornisce la protezione, sono stati condotti degli studi per esaminare se la protezione potesse essere trasferita da animali trattati con PGG-glucano verso riceventi non sensibilizzati, tramite le cellule della milza (SC), i lisati di SC, o il siero. Gli esperimenti di trasferimento passivo indicano che il fattore (o i fattori) responsabili sono trasferibili tramite gli SC ed i lisati di SC, così come tramite i leucociti periferici o il siero proveniente da animali trattati con PGG glucano. Il fattore (o i fattori) responsabili si sono mostrati resistenti alla digestione con pronasi e tripsina, stabili fino a 56 o 80 gradi C e non sono stati rimossi con precipitazione NHS404. L’effetto protettivo del PGG glucano è stato eliminato tramite trattamento con indometacina, un potente inibitore della sintesi della prostaglandina. La somministrazione di un estratto purificato di prostaglandine proveniente dal siero degli animali trattati con PGG glucano ha protetto contro la mortalità nel modello di peritonite. Inoltre, anche il trattamento dei topi con prostaglandina E2 sintetica esogena ha conferito protezione contro la mortalità. Questi risultati suggeriscono che l’effetto protettivo mostrato dal PGG glucano nel modello murino di peritonite è mediato, almeno in parte, dalle prostaglandine.

Synergism between poly-(1-6)-beta-d-glucopyranosyl-(1-3) -beta-d-glucopyranose glucan and cefazolin in prophylaxis of staphylococcal wound infection in a guinea pig model. Kaiser AB, Kernodle DS. Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2605, USA. Antimicrob Agents Chemother 1998 Sep; 42(9):2449-51 To determine whether the infection-preventing capability of the neutrophil-activating agent poly-(1-6)-beta-D-glucopyranosyl-(1-3)-beta-D-glucopyranose glucan (PGG-glucan) can be enhanced with antibiotic prophylaxis, we administered PGG-glucan and cefazolin, alone and in combination, to guinea pigs inoculated with isolates of staphylococci. Guinea pigs receiving both PGG-glucan and cefazolin had 50% infective doses that were 8- to 20-fold higher than those obtained with cefazolin alone and 100- to 200-fold higher than those obtained with PGG-glucan alone. PGG-glucan and cefazolin are synergistic in their ability to prevent staphylococcal wound infection.

Sinergia tra il poli-(1-6)-beta-d-glucopiranosil-(1-3)-beta-d-glucopiranosio glucano e la cefazolina nella profilassi dell’infezione stafilococcica delle ferite nella cavia. Kaiser AB, Kernodle DS. Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2605, USA. Antimicrob Agents Chemother 1998 Sep; 42(9):2449-51 Al fine di determinare se la capacità di prevenire le infezioni dell’agente attivatore di neutrofili poli-(1-6)-beta-d-glucopiranosil-(1-3)-beta-d-glucopiranosio glucano (PGG-glucano) possa o meno essere incrementata tramite profilassi antibiotica, abbiamo somministrato PGG-glucano e cefazolina, da soli o in combinazione, a cavie inoculate con isolati di staffilococchi. Le cavie che hanno ricevuto sia PGG-glucano che cefazolina hanno presentato dosi infettive al 50% da 8 a 20 volte più elevate rispetto a quelle ottenute con sola cefazolina, e da 100 a 200 volte più elevate rispetto a quelle ottenute col solo PGG-glucano. Il PGG-glucano e la cefazolina sono sinergici nel prevenire l’infezione stafilococcica delle ferite.

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Anti-infective effect of poly-beta 1-6-glucotriosyl-beta 1-3- glucopyranose glucan in vivo. AB Onderdonk, RL Cisneros, P Hinkson and G Ostroff Department of Pathology, Channing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts. Infect Immun 1992 Apr;60(4):1642-7 Mice challenged with Escherichia coli or Staphylococcus aureus were

protected against lethal peritonitis by the intravenous administration of 10 micrograms of poly-beta 1-6-glucotriosyl-beta 1-3-glucopyranose (PGG) glucan per animal 4 to 6 h prior to bacterial challenge. Subsequent studies with the rat model for intra-abdominal sepsis indicated that intramuscular doses of 10 to 100 micrograms per animal 24 and 4 h prior to surgical implantation of the bacterial inoculum reduced the early mortality associated with the peritonitis phase of this experimental disease process.

Quantitative cultures of blood obtained from challenged rats showed that significantly fewer organisms were present in the blood of PGG

glucan-treated animals than in that of untreated animals. Quantitative studies of leukocytes of rats and mice following a single injection of PGG glucan showed a modest transient increase in the total leukocyte count. The possible mechanisms by which protection occurs in the animal model system are discussed.

Effetto anti-infettivo del poli 1-6-glucotriosil-beta 1-3-glucopiranosio glucano in vivo. AB Onderdonk, RL Cisneros, P Hinkson and G Ostroff Department of Pathology, Channing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts. Infect Immun 1992 Apr;60(4):1642-7 Topi infettati con Escherichia coli o Staphylococcus aureus sono stati protetti contro la peritonite letale tramite la somministrazione endovenosa di 10 microgrammi per animale di 1-6-glucotriosil-beta 1-3-glucopiranosio (PGG) glucano da 4 a 6 ore prima dell’infezione batterica. Successivi studi con modelli animali della sepsi intra-addominale nel ratto hanno indicato che dosi intramuscolari da 10 a 100 microgrammi per animale 24 e 4 ore prima dell’impianto dell’inoculo batterico hanno ridotto la mortalità precoce associata con la fase peritonitica di questo processo patologico sperimentale. Culture quantitative di sangue ottenuto da ratti infettati hanno mostrato che erano presenti un numero significativamente minore di microrganismi nel sangue dei topi trattati con PGG-glucano rispetto agli animali non trattati. Studi quantitativi dei leucociti dei ratti e dei topi dopo una singola iniezione di PGG glucano hanno mostrato un modesto aumento transitorio della conta totale dei leucociti. I possibili meccanismi tramite i quali avviene la protezione nei modelli animali sono discussi.

Diepitopic construct of functionally and epitopically complementary peptides enhances immunogenicity, reactivity with glucosyltransferase, and protection from dental caries. Taubman MA; Holmberg CJ; Smith DJ, Department of Immunology, The Forsyth Institute, Boston, Massachusetts 02115, USA Infect Immun 2001 Jul;69(7):4210-6 (ISSN: 0019-9567) Coimmunization with peptide constructs from catalytic (CAT) and glucan-binding (GLU) domains of glucosyltransferase (GTF) of mutans streptococci has resulted in enhanced levels of antibody to the CAT construct and to GTF. We designed and synthesized a diepitopic construct (CAT-GLU) containing two copies of both CAT (B epitope only) and GLU (B and T epitope) peptides. The immunogenicity of this diepitopic construct was compared with that of individual CAT and GLU constructs by immunizing groups of Sprague-Dawley rats subcutaneously in the salivary gland vicinity with the CAT-GLU, CAT, or GLU construct or by treating rats by sham immunization. Levels of serum immunoglobulin G (IgG) antibody to GTF or CAT in the CAT-GLU group were significantly greater than in GLU- or CAT-immunized groups. Immunization with CAT-GLU was compared to coimmunization with a mixture of CAT and GLU in a second rodent experiment under a similar protocol. CAT-GLU immunization resulted in serum IgG and salivary IgA responses to GTF and CAT which were greater than after coimmunization. Immunization with the diepitopic construct and communization with CAT and GLU constructs showed proliferation of T lymphocytes to GTF. Immunization with either the CAT or GLU construct has been shown to elicit significant protection in a rodent dental caries model. Similarly in this study, the enhanced response to GTF after immunization with the CAT-GLU construct resulted in protective effects on dental caries. Therefore, the CAT-GLU diepitopic construct can be a potentially important antigen for a caries vaccine, giving rise to greater immune response than after immunization with CAT, GLU, or a mixture of the two.

Un costrutto diepitopico di peptidi funzionalmente ed epitopicamente complementari aumenta l’immunogenicità, la reattività con la glucosiltransferasi e la protezione dalle carie dentali. AB Onderdonk, RL Cisneros, P Hinkson and G Ostroff Department of Pathology, Channing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts. Infect Immun 1992 Apr;60(4):1642-7 La coimmunizzazione con costrutti peptidici composti dai domini catalitico (CAT) e di legame del glucano (GLU) della glucosiltransferasi (GTF) di streptococchi mutanti, ha generato un incremento del livello di antricorpi anti-costrutti CAT e anti-GTF. Noi abbiamo progettato e sintetizzato un costrutto diepitopico (CAT-GLU) contenente due copie ciascuno dei peptidi CAT (solo l’epitopo B) e GLU (epitopi B e T). L’immunogenicità di questo costrutto diepitopico è stata confrontata con quella dei costrutti singoli di CAT e GLU tramite l’immunizzazione sottocutanea in prossimità della ghiandola salivare di gruppi di ratti di Sprague-Dawley con i costrutti CAT-GLU, CAT oppure GLU, ovvero trattando i ratti con falsa immunizzazione. I livelli nel siero di immunoglobulina G (IgG) anticorpale verso GTF o CAT erano significativamente maggiori nei gruppi CAT-GLU rispetto ai gruppi immunizzati GLU o CAT. L’immunizzazione con CAT-GLU è stata confrontata con la coimmunizzazione con una miscela di CAT e GLU in un secondo esperimento con roditori utilizzando un protocollo simile. L’immunizzazione CAT-GLU ha prodotto risposte al GTF ed al CAT, in termini di IgG del siero e IgA salivare, che sono risultate maggiori rispetto a quelle conseguenti alla coimmunizzazione. L’immunizzazione col costrutto diepitopico e la coimmunizzazione con i costrutti CAT e GLU ha mostrato proliferazione dei linfociti T anti-GTF. L’immunizzazione con costrutti CAT o GLU si è dimostrata efficace nel fornire una protezione significativa in un modello di carie dentale nei roditori. Analogamente, in questo studio l’incrementata risposta al GTF successiva all’immunizzazione con il costrutto CAT-GLU ha avuto effetti protettivi sulle carie dentali. Pertanto, il costrutto diepitopico CAT-GLU può essere un antigene potenzialmente importante per un vaccino contro la carie, poiché da una maggior risposta immunitaria rispetto all’immunizzazione con CAT, GLU o una miscela dei due.

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Prophylaxis with the immunomodulator PGG glucan enhances antibiotic efficacy in rats infected with antibiotic-resistant bacteria. A. O. Tzianabos and R. L. Cisneros; Channing Laboratory Brigham and Women's Hospital, Harvard Medical School Boston, Massachusetts 02115, USA. Ann N Y Acad Sci. 1996 Oct 25; 797:285-7. The emergence of multiple antibiotic-resistant microorganisms has led to a search for alternatives to traditional therapeutic regimens. PGG glucan is a soluble beta-glucan immunomodulator that selectively enhances the microbicidal activities of neutrophils and macrophages without stimulating proinflammatory cytokine production. In the present studies, we examined the ability of PGG glucan to act in concert with antibiotics to decrease mortality in a rat model of intraabdominal sepsis using antibiotic-resistant bacteria as infectious inocula. Results of these studies demonstrated that prophylaxis with PGG glucan in combination with antibiotics provided enhanced protection against lethal challenge with Esherichia coli or Staphylococcus aureus as compared with the use of antibiotics alone.

La profilassi con l’immunomodulatore PGG glucano incrementa l’efficacia degli antibiotici in ratti infettati con batteri antibiotico-resistenti. A. O. Tzianabos and R. L. Cisneros; Channing Laboratory Brigham and Women's Hospital, Harvard Medical School Boston, Massachusetts 02115, USA. Ann N Y Acad Sci. 1996 Oct 25; 797:285-7. L’emergere di molti microorganismi antibiotico-resistenti ha portato a ricercare delle alternative ai regimi terapeutici tradizionali. Il PGG glucano è un beta-glucano immunomodulatore solubile che aumenta selettivamente le attività microbicide dei neutrofili e dei macrofagi senza stimolare la produzione di citochine proinfiammatorie. Nel presente studio abbiamo esaminato la capacità del PGG glucano di agire di concerto con gli antibiotici nel decrescere la mortalità in un modello animale di sepsi intraaddominale nel ratto ottenuto utilizzando batteri antibiotico-resistenti come infettanti. I risultati di questi studi hanno dimostrato che la profilassi con PGG glucano in associazione con antibiotici ha fornito un’incrementata protezione contro infezioni letali da E. coli o da S. aureus, rispetto all’uso del solo antibiotico.

Protective effect of beta-glucan against systemic Streptococcus pneumoniae infection in mice. Hetland G; Ohno N; Aaberge IS; Lovik M Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway. FEMS Immunol Med Microbiol 2000 Feb;27(2):111-6 (ISSN: 0928-8244) The antimicrobial effect of soluble beta-1,3-D-glucan from Sclerotinia sclerotiorum (SSG) was examined in mice experimentally infected intraperitoneally (i.p.) with Streptococcus pneumoniae serotypes 4 and 6B. SSG was administered i.p. either 3 days before challenge or 3-48 h after challenge. The number of bacteria in blood samples and the mouse survival rates were recorded. Pre-challenge SSG administration protected dose-dependently against both S. pneumoniae type 4 and 6B infections. SSG injected 24 h post-challenge had a curative effect against type 6B but not type 4 pneumococcal infection. The data demonstrate that SSG administered systemically protects against pneumococcal infection in mice.

Effetto protettivo del beta-glucano contro l’infezione sistemica da Streptococcus pneumoniae nei topi. Hetland G; Ohno N; Aaberge IS; Lovik M Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway. FEMS Immunol Med Microbiol 2000 Feb;27(2):111-6 (ISSN: 0928-8244) L’effetto antimicrobico del beta-1,3-D-glucano estratto da Sclerotinia sclerotiorum (SSG) è stato esminato in topi infettati sperimentalmente per via intraperitoneale (i.p.) con i sierotipi 4 e 6B di Streptococcus pneumoniae. SSG è stato somministrato i.p. o 3 giorni prima dell’infezione o 3-48 ore dopo l’infezione. Sono stati registrati il numero di batteri presenti nei campioni di sangue e i tassi di sopravvivenza dei topi. La somministrazione di SSG pre-infezione ha protetto in modo dose-dipendente sia dall’infezione col tipo 4 di S. pneumoniae, sia dall’infezione col tipo 6B. SSG iniettato 24 ore dopo l’infezione ha avuto un effetto terapeutico verso il tipo 6B ma non verso il tipo 4 di infezione pneumococcica. I dati dimostrano che SSG somministrato per via sistemica è protettivo contro le infezioni pneumococciche nei topi.

The protective effect of beta 1-3D-glucan-derivatized plastic beads against Escherichia coli infection in mice. Seljelid R; Rasmussen LT; Larm O; Hoffman J, Scand J Immunol 1987 Jan;25(1):55-60 (ISSN: 0300-9475) Pretreatment with beta-1,3-D-glucan-derivatized plastic beads conferred strong protection against Escherichia coli infection in mice. The protective effect showed a dose-response relationship to the amount of beads injected and was dependent on the time point of the injection relative to the infection with E. coli. A similar protection could be obtained in nude mice. Experiments with radioactively labelled bacteria as well as beads indicated a systemic effect of the beads. Macrophages extracted from animals treated with glucan plastic beads appeared highly stimulated. This was also true of cells that did not contain beads and presumably therefore not glucan, which seems to indicate a soluble stimulatory factor

L’effetto protettivo di granuli plastici derivatizzati con beta 1-3D-glucano verso l’infezione da Escherichia coli nel topo. Seljelid R; Rasmussen LT; Larm O; Hoffmann J, Scand J Immunol 1987 Jan;25(1):55-60 (ISSN:0300-9475) Il pretrattamento con granuli plastici derivatizzati con beta 1-3-D-glucano ha conferito una forte protezione contro l’infezione da Escherichia coli nei topi. L’effetto protettivo ha mostrato una relazione dose-risposta alla quantità di granuli iniettati ed una dipendenza dall’intervallo di tempo intercorrente tra l’iniezione e l’infezione con E. coli. Una protezione simile può essere ottenuta nei topi nudi. Gli esperimenti con batteri e granuli marcati radioattivamente hanno mostrato un effetto sistemico dei granuli. I macrofagi estratti dagli animali trattati con i granuli plastici al glucano apparivano altamente stimolati. Questo valeva anche per le cellule non contenenti granuli, e pertanto presumibilmente nemmeno glucano, il che sembra indicare la presenza di un fattore stimolatorio solubile.

20

GGLLUUCCAANN

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aaccttiivviittiieess

O

O

O

O

OH

HO

HO

OH

CH 2OCH 2COO - Na +

CH 2OHO

O

O

O

OH

HO

HO

OH

CH 2OCH 2COO - Na +

CH 2OH

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Promotion of Wound repair in Mice by Application of Glucan. S.J Leibovich and D. Danon, The section of biological ultrastructure, the weizmann institute of science, P.O.B. 26, Rehovot, Israel Journal of the Reticuloendothelial Society, Vol.27, No1,January 1980

The effects of topical application of a variety of substances (glucan, carrageenan, levan, inulin, dextran, starch and talcum powder) on the repair of skin wounds in SWR mice were examined. Of the substance tested, only glucan showed any marked beneficial effects. Glucan –treated wounds showed a higher number of macrophages in the early, inflammatory stage of repair, with fever polymorphonuclear neutrophilic leukocytes then did control wounds. Both re-epithalialization and the onset of fibroplasia commenced at an early stage in glucan treated wounds then in control wounds. Five days following wounding glucan treated wounds were generally completely rehepitelializeted, while control wounds were not the organization of fibroblats in glucan treaded wounds was more advanced at 5 and 7 days following injury, and the extent of fibroplasias was also greater. By 10 days following injury glucan treated wounds were completely re-epithelializated , as were control wounds treated with medium alone inulin dextran, starch or talc. Carrageenan and levan-treated wounds showed impaired re-epitheliliazation. Only occasional residual glucan-containg macrophages were present in the glucan-treated wounds at this stage. In glucan treated wounds, no formation of granulomas was observed up to one month following wounding.

La promozione della guarigione della ferita nei topi attraverso l’applicazione di glucano. S.J Leibovich and D. Danon, The section of biological ultrastructure, the weizmann institute of science, P.O.B. 26, Rehovot, Israel Journal of the Reticuloendothelial Society, Vol.27, No1,January 1980 Sono stati esaminati gli effetti di applicazioni topiche di una varietà di sostanze (glucano, carragenina, levan, inulina, destrano, amido e polvero di talco) sulla guarigione di ferite della pelle di topi SWR. Tra le sostanze testate, solamente il glucano ha mostrato degli effetti benefici significativi. Le ferite trattate con glucano hanno mostrato un più alto numero di macrofagi nella prima fase infiammatoria della guarigione, con più leucociti polimorfonucleati neutrofili di quanto abbiano mostrato le ferite di controllo. Sia la riepitelizzazione sia lo sviluppo di tessuto fibroso sono comparsi prima nelle ferite trattate con glucano che in quelle di controllo. Le ferite testate con glucano erano generalmente completamente riepitelizzate dopo cinque giorni dalla lesione, mentre le ferite di controllo non lo erano e l’organizzazione dei fibroblasti nelle ferite trattate con glucano era più avanzata a 5 e 7 giorni dopo la lesione, e anche l’organizzazione dei fibroblasti e la quantità di fibroplasia era maggiore. Dopo 10 giorni dalla lesione, le ferite trattate con glucano erano completamente riepitelizzate, così come le ferite di controllo trattate solamente con inulina, destrano, amido o talco. Le ferite trattate con carragenina o levan hanno mostrato una riepitelizzazione ridotta. Solo occasionali macrofagi residui contenenti glucani erano presenti nelle ferite trattate con glucano in questa fase. Nelle ferite trattate con glucano nessuna formazione di granulomi è stata osservata fino ad un mese dalla lesione.

Effect of macrophage stimulation on collagen biosynthesis in the healing wound. Portera CA; Love EJ; Memore L; Zhang L; Muller A; Browder W; Williams DL; Department of Surgery, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614-0575, USA. Am Surg 1997 Feb;63(2):125-31 (ISSN: 0003-1348) Immunomodulators that enhance macrophage function have been shown to be beneficial in a number of wound-healing models in humans and in experimental animals. The exact mechanism of this improved healing is unclear. To assess the role of collagen biosynthesis, the immunomodulator glucan phosphate was utilized in two murine models of wound healing, i.e., colon anastomosis and full-thickness skin incision. Tensile strength was evaluated using computer-assisted constant velocity tensiometry. Collagen biosynthesis was determined by assaying hydroxyproline content of wound hydrolysates by N-(9-fluorenyl)methoxycarbonyl/o-phthalaldehyde high-performance liquid chromatography. Experimental animals were treated with (1-3)-beta-D-glucan phosphate (250 mg/kg) intravenously 24 hours prior to colon anastomosis or skin incision. A second dose of glucan phosphate was given immediately postoperatively. Control animals received dextrose and water (5% w/v) intravenously. Tensile strength and hydroxyproline content were measured on postoperative Day 3. In the skin wound model, glucan phosphate treatment increased (P < 0.05) tensile strength by 42 per cent (342.5 +/- 12.2 vs 241.8 +/- 4.8 g), and hydroxyproline content was increased by 23.5 per cent (242.0 +/- 14.4 vs 196.8 +/- 10.5 pmol/microg; P < 0.05). In the glucan phosphate group, colon tensile strength was significantly (P < 0.05) increased by 34 per cent (34.2 +/- 2.3 g vs 45.8 +/- 2.1 g), and hydroxyproline content was increased by 7 per cent (47.45 +/- 3.31 vs 44.34 +/- 3.74 pmol/microg). These data indicate that macrophage modulation with glucan phosphate will increase tensile strength in experimental colon and skin wounds. In addition, we observed a positive correlation between glucan phosphate treatment, wound tensile strength, and collagen biosynthesis.

Effetto della stimolazione macrofagica sulla biosintesi del collagene durante la guarigione della ferita. Portera CA; Love EJ; Memore L; Zhang L; Muller A; Browder W; Williams DL; Department of Surgery, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614-0575, USA. Am Surg 1997 Feb;63(2):125-31 (ISSN: 0003-1348) Gli immunomodulatori che potenziano la funzione macrofagica hanno mostrato di essere benefici in numerosi modelli di guarigione della ferita nell’uomo e nell’animale da esperimento. L’esatto meccanismo di questo miglioramento della guarigione non è chiaro. Per verificare il ruolo della biosintesi del collagene, è stato utilizzato l’immunomodulatore glucanfosfato in due modelli murini di guarigione della ferita, ossia l’anastomosi del colon e l’incisione della pelle a tutto spessore. Lo resistenza di trazione è stata valutata usando la tensiometria computerizzata a velocità costante. La biosintesi del collagene è stata determinata misurando il contenuto di idrossiprolina degli idrolisati di ferite attraverso cromatografia liquida ad alte prestazioni di N-(9-fluorenil) metocsicarbonil/oftalaldeide. Gli animali da esperimento sono stati trattati con iniezione endovenosa di glucano (1-3)-beta-D fosfato (250 mg/kg) 24 ore prima dell’anastomosi del colon o dell’incisione della pelle. Una seconda dose di glucanfosfato è stata somministrata immediatamente dopo l’operazione. Gli animali di controllo hanno ricevuto destrosio e acqua (5% w/v) per via endovenosa. La forza di trazione e il contenuto di idrossiprolina sono stati misurati il terzo giorno postoperatorio. Nel modello della ferita della pelle, il trattamento di glucanfosfato ha aumentato (P < 0.05) la resistenza a trazione del 42% (342.5 +/- 12.2 vs 241.8 +/- 4.8 g), e il contenuto di idrossiprolina del 23.5% (242.0 +/- 14.4 vs 196.8 +/- 10.5 pmol/microg; P < 0.05). Nel gruppo trattato con glucanfosfato, la resistenza a trazione del colon è aumentata significativamente (P < 0.05) del 34% (34.2 +/- 2.3 g vs 45.8 +/- 2.1 g), e il contenuto di idrossiprolina del 7 % (47.45 +/- 3.31 vs 44.34 +/- 3.74 pmol/microg). Questi dati indicano che la modulazione macrofagica con glucanfosfato è in grado di aumentare la resistenza a trazione nelle ferite sperimentali del colon e della pelle. Inoltre, è stata riscontrata una correlazione positiva tra il trattamento con glucanfosfato, la resistenza a trazione della ferita e la biosintesi del collagene.

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Effect of enhanced macrophage function on early wound healing. Browder W; Williams D; Lucore P; Pretus H; Jones E; McNamee R Department of Surgery, Tulane University School of Medicine, New Orleans, LA 70112. Surgery 1988 Aug;104(2):224-30 (ISSN: 0039-6060) Although the macrophage is important to wound healing, research has focused on its relationship to fibroblast and collagen synthesis. This study was designed to assess effects of enhanced macrophage function on early wound healing, before established collagen synthesis. Sprague-Dawley rats had dorsal incisions after one of three treatment regimens: (1) saline solution, 0.5 ml administered intravenously, (2) intravenous glucan, a macrophage stimulant, 20 mg; (3) topical glucan, 20 mg. Intravenous therapy was administered 24 hours before and after incision. Breaking strength was significantly increased (p less than 0.01) by both intravenous glucan (49.8 +/- 5.5 gm) and topical glucan (59.7 +/- 5.6 gm) on the fourth day after incision, compared with controls (22.0 +/- 2.6 gm). Similar results occurred on the seventh day after incision. Although formalin fixation significantly enhanced breaking strength in fresh control wounds (22.0 +/- 2.6 vs 39.5 +/- 2.2 gm), no increase occurred in wounds treated with intravenous glucan (49.8 +/- 5.0 vs 55.3 +/- 6.4 gm), indicating maximal cross-linking of collagen. Collagen synthesis, reflected by tritiated proline uptake, was no different in control versus glucan groups. Supernatants from control or glucan-activated macrophages were injected intraperitoneally or applied topically in the rat model. Activated supernatant, both intraperitoneal and topical, resulted in increased breaking strength on the fourth day after incision. Formalin fixation did not increase breaking strength in the activated supernatant groups. We conclude that enhanced macrophage function increases early wound breaking strength. This effect appears unrelated to collagen synthesis but may be related to increased cross-linking of collagen. Similar effects are seen with activated macrophage secretory products administered intraperitoneally or topically.

Effetto del potenziamento della funzione macrofagica nella prima fase di guarigione della ferita. Browder W; Williams D; Lucore P; Pretus H; Jones E; McNamee R Department of Surgery, Tulane University School of Medicine, New Orleans, LA 70112. Surgery 1988 Aug;104(2):224-30 (ISSN: 0039-6060) Sebbene i macrofagi siano ritenuti importanti nella del collagene ma potrebbe essere collegato all’aumento di guarigione di una ferita, la ricerca si è focalizzata sui nessi dei macrofagi stessi con i fibroblasti e con la sintesi del collagene. Questo studio è stato pensato per quantificare gli effetti del potenziamento della funzione macrofagica nella prima fase della guarigione della ferita, prima che si stabilisca la sintesi del collagene. Dei ratti Sprague-Dowley hanno subito una incisione dorsale dopo uno dei seguenti tre trattamenti: (1) fisiologica, 0.5 ml somministrati per via endovenosa, (2) glucano endovenoso, uno stimolatore di macrofagi, 20 mg, (3) glucano topico, 20 mg. La terapia endovenosa è stata somministrata 24 ore prima e dopo l’incisione. La resistenza a rottura è aumentata in maniera significativa (p < 0.01) sia per il glucano endovenoso (49.5 +/- 5.5 gm) sia per il glucano topico (59.7 +/- 5.6 gm) al quarto giorno dopo l’incisione, se confrontato con il gruppo di controllo (22.0 +/- 2.6 gm). Risultati simili sono stati osservati al settimo giorno dopo l’incisione. Sebbene il fissaggio in formalina abbia aumentato significativamente la resistenza a rottura delle ferite fresche nel gruppo di controllo (22.0 +/- 2.6 gm vs 39.5 +/-2.2 gm), non è stato riscontrato un aumento nelle ferite trattate con glucano endovenoso (49.8 +/- 5.0 vs 55.3 +/- 6.4 gm), fatto che indica un massimo cross-linking del collagene. La sintesi del collagene, evidente dall’assorbimento di prolina marcata con trizio radioattivo, non era differente tra il gruppo di controllo e quelli trattati con glucano. I surnatanti dai macrofagi di controllo o dai macrofagi attivati dal glucano sono stati iniettati per via intraperitoneale o applicati per via topica nel modello di ratto. I supernatanti attivati, sia per via peritoneale che per via topica, hanno aumentato la resistenza a rottura al quarto giorno dopo l’incisione. La fissazione in formalina non ha aumentato la resistenza a rottura nel gruppo di supernatanti attivati. Concludiamo che il potenziamento della funzione macrofagica aumenta la resitenza a rottura nella prima fase di guarigione della ferita. Questo effetto appare non correlato alla sintesi cross-linking del collagene. Effetti simili sono stati osservati con prodotti di secrezione dei macrofagi attivati somministrati per via peritoneale o topica.

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Normal human fibroblasts express pattern recognition

receptors for fungal (1-->3)-beta-D-glucans. Kougias P; Wei D; Rice PJ; Ensley HE; Kalbfleisch J; Williams DL; Browder. IW Departments of Surgery, James H. Quillen College of Medicine, Johnson City, Tennessee 37614, USA. Infect Immun 2001 Jun;69(6):3933-8 (ISSN: 0019-9567) Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity. Glucans are thought to mediate their effects via interaction with membrane receptors on macrophages, neutrophils, and NK cells. There have been no reports of glucan receptors on nonimmune cells. We investigated the binding of a water-soluble glucan in primary cultures of normal human dermal fibroblasts (NHDF). Membranes from NHDF exhibited saturable binding with an apparent dissociation constant (K(D)) of 8.9 +/- 1.9 microg of protein per ml and a maximum binding of 100 +/- 8 resonance units. Competition studies demonstrated the presence of at least two glucan binding sites on NHDF. Glucan phosphate competed for all binding sites, with a K(D) of 5.6 microM (95% confidence interval [CI], 3.0 to 11 microM), while laminarin competed for 69% +/- 6% of binding sites, with a K(D) of 3.7 microM (95% CI, 1.9 to 7.3 microM). Glucan (1 microg/ml) stimulated fibroblast NF-kappaB nuclear binding activity and interleukin 6 (IL-6) gene expression in a time-dependent manner. NF-kappaB was activated at 4, 8, and 12 h, while IL-6 mRNA levels were increased by 48% at 8 h. This is the first report of pattern recognition receptors for glucan on human fibroblasts and the first demonstration of glucan binding sites on cells other than leukocytes. It also provides the first evidence that glucans can directly modulate the functional activity of NHDF. These results provide new insights into the mechanisms by which the host recognizes and responds to fungal (1-->3)-beta-D-glucans and suggests that the response to glucans may not be confined to cells of the immune system.

I fibroblasti umani normali esprimono recettori di “pattern recognition” per i gli (1-->3)-beta-D-glucani fungini.

Kougias P; Wei D; Rice PJ; Ensley HE; Kalbfleisch J; Williams DL; Browder. IW Departments of Surgery, James H. Quillen College of Medicine, Johnson City, Tennessee 37614, USA. Infect Immun 2001 Jun;69(6):3933-8 (ISSN: 0019-9567) I glucani della parete cellulare dei funghi stimolano in maniera aspecifica vari aspetti dell’immunità innata. Si ritiene che i glucani agiscano tramite l’interazione con i recettori di membrana sui macrofagi, sui neutrofili e sulle cellule NK. Non è stata mai riportata la presenza di recettori per i glucani su cellule non immunitarie. Noi abbiamo investigato il legame di un glucano idrosolubile usando culture primarie di fibroblasti normali del derma umano (NHDF). Le membrane degli NHDF hanno mostrato un legame saturabile con una costante di dissociazione apparente (K(D)) di 8.9 +/- 1.9 microgrammmi di proteina per ml ed un legame massimo di 100 +/- 8 unità di risonanza. Studi comparativi hanno mostrato la presenza di almeno due siti di legame per il glucano sugli NHDF. Il glucan fosfato competeva per tutti i siti di legame, con una K(D) di 5.6 microM (intervallo di confidenza [CI] al 95%, da 3 ad 11 microM), mentre la laminarina competeva per il 69% +/- 6% dei siti di legame, con una K(D) di 3.7 microM (CI 95%, da 1.9 a 7.3 microM). Il glucano (1 microg/ml) ha stimolato in maniera tempo-dipendente l’attività di legame nucleare NF-kappaB e l’espressione del gene per l’ intereleuchina-6 (IL-6) dei fibroblasti. NF-kappaB è stata attivata a 4, 8 e 12 ore, mentre i livelli di mRNA per IL-6 sono cresciuti del 48% ad 8 ore. Questo è il primo rapporto riguardo ai recettori di “pattern recognition” per il glucano sui fibroblasti umani e la prima dimostrazione della presenza di siti di legame per il glucano su cellule diverse dai leucociti. Inoltre, questo studio evidenzia per la prima volta che i glucani possono modulare direttamente l’attività funzionale degli NHDF. Questi risultati forniscono nuovi approfondimenti riguardo ai meccanismi tramite i quali l’organismo riconosce e risponde agli (1-->3)-beta-D-glucani fungini e suggeriscono che la risposta ai glucani può non essere limitata alle cellule del sistema immunitario.

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The beneficial effect of enhanced macrophage function on the healing of bowel anastomoses. Compton R; Williams D; Browder W , Department of Surgery, East Tennessee State University, Johnson City 37614, USA. Am Surg 1996 Jan;62(1):14-8 (ISSN: 0003-1348) Inadequate healing and subsequent leakage of bowel anastomoses are serious postoperative complications in abdominal surgery. Previous studies have demonstrated the macrophage to be a key cell in the physiology of wound healing. The current study was undertaken to evaluate the effects of enhanced macrophage function on the healing of bowel anastomoses. Sprague-Dawley rats (250 gm) underwent laparotomy and jejunojejunostomy following IV treatment with glucan (100 mg per kg), a potent macrophage stimulant, or 5 per cent dextrose 24 hours before surgery and again on the day of surgery. Animals were killed and the anastomoses underwent wound tensiometry on Day 3 using a computer-assisted constant velocity tensiometer. The glucan treated animals had a significantly greater anastomotic breaking strength (88.5 gm +/- 10.7 versus 45.45 gm +/- 5.1) (P < 0.01). Formalin fixation increased the breaking strength of the untreated anastomosis but not of the treated anastomosis (92.9 gm +/- 11.77 versus 92.3 +/- 12.44). Analysis of macrophage supernatant for the growth factors epidermal growth factor (EGF), platelet derived growth factor (PDGF), and transforming growth factor-beta (TGF-beta) was accomplished by immunoblot assay. Results indicated no difference in the presence of EGF in the stimulated and unstimulated macrophage supernatants. PDGF and TGF-beta were decreased in the stimulated macrophage supernatants. We conclude that 1) Enhanced macrophage function had a beneficial effect on the early tensile strength of bowel anastomoses. 2) Effects of the activated macrophage on bowel anastomoses may not be related to secretion of conventional growth factors. 3) Immunopharmacologic agents that enhance macrophage function may be an important adjunct to surgical therapy requiring bowel anastomosis.

Effetto benefico del potenziamento della funzione macrofagica nella guarigione dell’anastomosi dell’intestino. Compton R; Williams D; Browder W , Department of Surgery, East Tennessee State University, Johnson City 37614, USA. Am Surg 1996 Jan;62(1):14-8 (ISSN: 0003-1348) Guarigioni inadeguate e conseguenti perdite delle anastomosi dell’intestino sono serie complicazioni postoperatorie nella chirurgia addominale. Ricerche passate hanno dimostrato che i macrofagi sono le cellule-chiave nella fisiologia della guarigione delle ferite. Il presente studio è stato intrapreso per valutare gli effetti del potenziamento della funzione macrofagica sulla guarigione delle anastomosi dell’intestino. Dei ratti Sprague-Dowley (250 gm) sono stati sottoposti a laparotomia e digiunostomia dopo il trattamento IV con glucano (100 mg per kg), un potente stimolante microfagico, o con 5% di destrosio 24 ore prima e nel giorno stesso dell’operazione chirurgica. Gli animali sono stati sacrificati e le anastomosi sono state testate a trazione con un tensiometro computerizzato a velocità costante. Gli animali trattati con glucano avevano una resistenza a rottura della ferita significativamente più grande (88.5 gm +/- 10.7 vs 45.45 gm +/- 5.1) (P < 0.01). La fissazione con formalina ha aumentato la forza di rottura delle anastomosi non trattate ma non quella delle anastomosi trattate (92.9 gm +/- 11.77 vs 92.3 gm +/- 12.44). L’analisi dei surnatanti macrofagici per individuare i fattori di crescita: fattore di crescita epidermico (EFG), fattore di crescita derivato delle piastrine (PDGF) e fattore di crescita beta (TGF-beta) è stata condotta con dosaggio immunoblot*. I risultati non hanno indicato differenze nella presenza dell’EGF sia nei surnatanti di macrofagi stimolati sia in quelli non stimolati. Il PDGF ed il TGF-beta sono diminuiti nei surnatanti di macrofagi stimolati. Noi concludiamo che 1) il potenziamento della funzione macrofagica ha avuto un effetto benefico sulla resistenza a trazione dell’anastomosi dell’intestino nella prima fase di guarigione, 2) gli effetti dei macrofagi attivati sull’anastomosi dell’intestino possono essere non correlati alla secrezione di fattori di crescita convenzionali, 3) gli agenti immunofarmacologici che potenziano la funzione macrofagica possono essere un importante integrazione alla terapia chirurgica richiesta nell’anastomosi dell’intestino. * Trasferimento elettroforetico di proteine separate su un gel di poliacrilammide dal gel a una membrana di nitrocellulosa, su cui vengono immobilizzate. La presenza di una specifica proteina viene quindi evidenziata mediante reazione con il relativo anticorpo opportunamente marcato.

ANNEX A

LIST OF SOME STUDIES ON BETA GLUCAN

ABDOMINAL OR THORACIC SURGERY - HARVARD MEDICAL SCHOOL (USA) "There were no adverse drug experiences....is safe and appears to be effective in the further reduction of the morbidity and cost of major surgery."

BACTERIAL INFECTIONS - BAYLOR COLLEGE OF MEDICINE; Wyde, P., "Beta-1,3-glucan activity in mice: intraperitoneal and oral applications." " Beta glucan, through the stimulation of host defense systems, creates a more supportive environment within the body to assist the primary killing action of the conventional agent." CANCER - CHEM PHARM BULL (Japan); "Antitumor and immunomodulating activities of a beta-glucan...." CANCER, LUNG AND BREAST - NATIONAL CANCER INST (USA); "The initial 9 patients studied had malignant melanoma, adenosquamous carcinoma of the lung, or carcinoma of the breast. Control and experimental lesions were injected: subsequently biopsies were performed at varying intervals for histologic evaluation. Always when glucan or glucan and RF fraction were administered intralesionally, the size of the lesion was strikingly reduced in as short a period as 5 days. This reduction was associated with necrosis of the tumor and a monocytic infiltrate. In small lesions, resolution was complete, whereas in large lesions, resolution was partial. CANCER - MAYO CLINIC (USA); "..... beta-glucan interacts with vitronectin and stimulates tumor necrosis factor alpha release from macrophage's." CANCER - UNIVERSITY OF TROMSO (Norway); "Macrophages stimulated by an insoluble beta 1-3-D-glucan from yeast cell walls were able to destroy tumour cells as measured by the release of radioactive label from prelabelled 14C-thymidine cells. Target cells were B-16 melanoma, P-815 mastocytoma, and the L-929 cell line. A significant target cell killing by macrophages stimulated by glucan was observed after 72-96 h." CANDIDA ALBICANS - DEPARTMENT OF SURGERY, TULANE UNIVERSITY; "Protection against C. albicans was observed in the glucan-treated groups. ...These observations suggest that Biologic Response Modifiers such as glucan may be effectively employed in patients who are at risk for post-operative infections." CHOLESTEROL (LDL) - OTTAWA CIVIC HOSPITAL (Canada); "CONCLUSIONS: The main component of the soluble fiber of oats, beta-glucan, significantly reduced the total and LDL cholesterol levels of hypercholesterolemic adults without changing HDL cholesterol." CHOLESTEROL - DEPARTMENT OF AGRICULTURE (USA); "Beneficial reduction of cholesterol was obtained with modest amounts....." DIABETES - OTTAWA CIVIC HOSPITAL (Canada); "A diet rich in beta-glucan may therefore be of benefit in the regulation of plasma glucose levels in subjects with Type 2

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diabetes." DIABETES - NESTLAE RESEARCH CENTER (Switzerland); "Diabetic individuals can benefit from diets that are high in beta-glucan, .." E. COLI - TULANE UNIVERSITY (USA, La); "Glucan therapy also increased bone marrow proliferation. We conclude that (1) glucan enhances peritoneal neutrophil levels, (2) Peripheral blood neutrophils are increased following glucan and E. coli, (3) ip glucan increase bone marrow proliferation, .... Thus, the beneficial effect of glucan is mediated not only by activated macrophages, but also by the neutrophilic leukocyte." E. COLI, STAPHYLOCOCCUS - DEPARTMENT OF PATHOLOGY, BRIGHAM AND WOMEN'S HOSPITAL (USA, Mass) "Mice challenged with Escherichia coli or Staphylococcus aureus were protected against lethal peritonitis by the intravenous administration of 10 micrograms of poly-beta 1-6-glucotriosyl-beta 1-3-glucopyranose (PGG) glucan per animal 4 to 6 h prior to bacterial challenge." FUNGAL INFECTION - TULANE UNIVERSITY; "The broad spectrum of immunopharmacological activities of glucan includes not only the modification of certain bacterial, fungal, viral and parasitic infections, but also inhibition of tumor growth." HEPATITIS, VIRAL - SCIENCE (1980);"Thus glucan is capable of increasing survival, inhibiting hepatic necrosis, and maintaining an activated state of phagocytic activity in mice challenged with [mouse hepatitis virus strain] MHV-A59." HERPES SIMPLEX 1 - PLANTA MED., 62:4, 301-7. (1996); "The antiviral effect of scleroglucan seems to be related to its binding with membrane glycoproteins of HSV-1 particles which impedes the complex interactions of the virus with the cell plasma membrane." HIGH RISK SURGICAL PATIENTS - HARVARD MEDICAL SCHOOL (USA); "Patients who received PGG-glucan had significantly fewer infectious complications (3.4 infections per infected patient vs. 1.4 infections per infected patient, p = 0.05), decreased intravenous antibiotic requirement (10.3 days vs. 0.4 days, p = 0.04) and shorter intensive care unit length of stay (3.3 days vs. 0.1 days, p = 0.03). CONCLUSIONS: PGG-glucan is safe and appears to be effective in the further reduction of the morbidity and cost of major surgery. INFECTION PREVENTION - GYNECOLOGY & OBSTETRICS, 177:383-388. (1993); "The incidence of hospital pneumonia of 55% and sepsis of 35% confirms results of previous studies of patients with multitrauma. Glucan decreased pneumonia and sepsis to a significantly lower level of 9.5%....The mortality rate related to infection decreased from 30.0 to 4.8%. The lower number of instances of pneumonia and sepsis....decreased the period of time in the intensive care and the hospital, with a global reduction of 40% on hospital cost." INTERLEUKIN - INT J IMMUNOPHARMACOL, 1987, 9:3, 261-7; "The study demonstrates that; (1) glucan will enhance IL-1 and IL-2 production and (2) elevations in lymphokine production can be maintained up to 12 days post-glucan" PARASITES - TULANE UNIVERSITY (USA); "Trypanosoma cruzi, the causative agent of Chagas' disease, infects humans and animals..... Glucan significantly (P less than 0.05) increased survival rate as denoted by 60%...." PNEUMONIA - HOSPITAL ARTHUR RIBEIRO DE SABOYA (Brazil); "The mortality rate

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related to infection was 30.0 percent in patients in the control group and 4.8 percent in the group treated with glucan...." RADIATION - RADIOPROTECTIVE EFFECT; "These results suggest that early after irradiation glucan may mediate its radioprotection by enhancing resistance to microbial invasion via mechanisms not necessarily predicated on hemopoietic recovery. In addition, preliminary evidence suggests that glucan can also function as an effective free-radical scavenger." RADIATION SURVIVABILITY- ARMED FORCES RADIOBIOLOGY RESEARCH INSTITUTE (USA); "Immunomodulators, either microbial agents (e.g. glucan, TDM) or recombinant cytokines (e.g. Interleukin-1, colony-stimulating factor), can enhance hematopoietic and functional cell recovery after irradiation." RADIATION SURVIVABILITY; Abstract: "Glucan, a beta-1, 3 polyglucose, was administered to mice either 1h before or 1h after a 650 rad exposure to cobalt-60 radiation. Compared to radiation controls, glucan-treated mice consistently exhibited a more rapid recovery of pluripotent stem cells and committed granulocyte, macrophage and erythroid progenitor cells. This may partially explain the mechanism by which glucan also enhances survival in otherwise lethally irradiated mice." STAPHYLOCOCCAL WOUND INFECTION - VANDERBILT UNIVERSITY SCHOOL OF MEDICINE (USA); "We conclude that PGG Glucan reduces the risk of staphylococcal abscess formation." STRESS, PHYSICAL, OR EMOTIONAL - TOWNSEND LETTER FOR DOCTORS, (1996);"The following list includes benefits from the use of Beta 1,3-glucan supplementation: Professional and amateur athletes as well as people who work outdoors intensively. People under physical or emotional stress" TRAUMA PATIENTS - TULANE UNIVERSITY (USA); "total mortality rate was significantly less in the glucan group (0% versus 29%) (p less then 0.05), the mortality rate from sepsis was not statistically different (0% versus 17.6%). Glucan therapy significantly decreased septic morbidity (9.5% versus 49%; p less than 0.05). Serum IL-1 had a greater increase in glucan patients on day 3 after trauma (143.4 +/- 19.3% versus 78.6 +/- 11.7%; p less than 0.05),..." WOUND HEALING - EAST TENNESSEE STATE UNIVERSITY (USA); "These data indicate that macrophage modulation with glucan phosphate will increase tensile strength in experimental colon and skin wounds."

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ANNEX B

Beta Glucan Studies and Research Abel, G. & Czop, J.K.; - "Stimulation of human monocyte beta-glucan receptors by glucan particles induces production of TNF-alpha and IL-1 beta, "Int. J. Immunopharmacolol, 14: 1363-1373. 1992.* Abel, G. & Czop, J.K., - "Activation of Human Monocyte GM-CSF and TNF-alpha. Production by Particulate Yeast Glucan," International Congress for Infectious Diseases, Montreal, Canada (abstract). 1990. * Dept of Medicine, Harvard Medical School, Boston, MA. Quote: "Beta-glucans are pharmacological agents that rapidly enhance the host resistance to a variety of biologic insults through mechanisms involving macrophage activation." Adachi Y., Ohno N., Yacomae T.; - "Preparation and antigen specificity of an anti- (1-->3)-beta-D-glucan antibody," Biol Pharm bull 17: 1508-1512; 1994. * Adachi Y., Ohno N., Yacomae T.; - "Inhibitory effect of beta-glucans on Zymosan-mediated hydrogen peroxide production by murine peritoneal macrophages in vitro," Biol Pharm Bull, 16: 462-467; 1993. Adachi Y., Ohno N., Ohsawa M., Oikawa S.,Yacomae T.; - "Macrophage activation in vitro by chemically cross-linked (1--3)-beta-D-glucans," Chem Pharm Bull (Tokyo), 38:988-992 1990. Laboratory of Immunopharmacology of Microbial Products, Tokyo College of Pharmacy, Japan. * Ainsworth A.J., - "A beta-glucan inhibitable Zymosan receptor on channel catfish neutrophils," Vet Immunol Immunopathol, 41: 141-152. 1994. * Almdahl SM, Bogwald J, Hoffman J, Seljelid R; - "Treatment of experimental peritonitis in rats by transfer of peritoneal mononuclear cells from rats injected with semisoluble aminated glucan." Acta Chir Scand 153(9): 535-539, Sep 1987. Dept of Surgery, University Hospital, Tromso, Norway. * Almdahl SM, Bogwald J, Hoffman J, Seljelid R; - "The effect of splenectomy on Escherichia coli sepsis and its treatment with semisoluble aminated glucan," Scand J Gastroenterol 22(3): 261-267; Apr 1987. * Almdahl SM, Bogwald J, Hoffman J, Seljelid R Giercksky KE; - "Protection by aminated glucan in experimental endogenous peritonitis," Eur Surg Res 19(2): 78-85, 1987. * Almdahl SM, Seljelid R; - "Semisoluble animated glucan: long-term efficacy against an intraperitoneal E. coli challenge and its effect on formation of abdominal adhesions," Res Exp Med (Berlin) 187(5): 369-377, 198 . * Andaluz E., Guillen A., Larriba G.; - "Preliminary evidence for a glucan acceptor in the yeast Candida albicans," Biochem J.; 240: 495-502. 1986. Anti-free Radical Activity of Beta (1-3) glucan Molecule. Seporga Laboratories, Sophia Antipolis, France. Research Report. 1990. Aono R., Hammura M. et al; - "Isolation of extracellular 28- and 42-kilodalton beta-1-3-glucanases and comparison of three beta-1, 3-glucanases produced by Bacillus circulans IAM1165," Appl. Environ. Microbiol 61: 122-129.1995 Babineau, et al., - "A Phase II Multicenter, Double-Blind Randomized, Placebo-Controlled Study of Three Dosages of an Immunomodulator (PGG-Glucan) in High Risk Surgical Patients", Arch. Surg.; 129:1204-1210. 1994. Dept of Surgery, Deaconess Hospital, Harvard Medical School, Boston MA. * Babineau, et al., - "Randomized Phase I/II Trial of a Macrophage-Specific Immunomodulator (PGG-

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Glucan) in High Risk Surgical Patients", Annals of Surgery; 220: (5): 601-609. 1994. Dept of Surgery, Deaconess Hospital, Harvard Medical School, Boston MA. * Quote: "PGG-glucan is safe and appears to be effective in further reduction of the morbidity and cost of major surgery."* Bacon J., et al., - "The Glucan Components of the Cell Wall of Baker's Yeast (Saccharomyces cerevisiae) Considered in Relation to its Ultrastructure," Chemical Abstracts, 71:109168c. 1991. Ballou CE; - "The yeast cell wall and cell surface;" The Molecular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratories. New York. p 335; 1982. Benach J.L., et al., - "Glucan as an adjuvant for a murine Babesia microti immunization trial," Infection and Immunity, 35(3): 947-951. 1982. Quote: "These observations demonstrate that glucan is an effective adjuvant in enhancing immunity to murine babesiosis."* Beta (1-3) glucan 1.3 Glucan Activity in Mice: Intraperitoneal and Oral Applications. Baylor College of Medicine. Research Summary. 1989. Beta (1-3) glucan: "I1-1 Cytokine Release after Oral Application in Mice". Baylor College of Medicine. Research Report. 1994. Bogwald J, Johnson E, Hoffman J, Seljelid R, - "Lysosomal Glycosidase in Mouse Peritoneal Macrophages Stimulated in Vitro with Soluble and Insoluble Glucans". J. Leukocyte Biol.; 35: 357-371. 1984. * Bogwald J, Johnson E, Seljelid R; - "The Cytotoxic Effect of Mouse Macrophages Stimulated in vitro by a beta. 1,3-D-Glucan from Yeast Cell Walls". Scand. J. Immuol. 15: 297-304. 1982. Institute of Med Bio, U of Tromso, Norway. Quote: " Macrophages stimulated by an insoluble beta 1-3-D-glucan from yeast cell walls were able to destroy tumor cells as measured by the release of radioactive label from prelabelled 14C-thymidine cells. Target cells were B-16 melanoma, P-815 mastocytoma, and the L-929 cell line. A significant target cell killing by macrophages stimulated by glucan was observed after 72-96 h." Bomford and Moreno, - "Mechanisms of the Anti-Tumor Effect of Glucans and Fructosans: A Comparison with C. Parvum". Br. J. Cancer; 36:41-48. 1977. Boone C, Sdicu A, Laroche M, Bussey H; - "Isolation from Candida albicans of a functional homolog of the Saccharomyces cerevisiae KRE1 Gene, which is involved in cell wall beta-glucan synthesis," J Bacteriol 173(21); 6859-6864, Nov 1991. * Boone C., Sommer SS, Hensel A., Bussey H., - "Yeast KRE genes provide evidence for a pathway of cell wall beta-glucan assembly," J Cell Biol; 110: 1833-1843. 1990. Borriss, et al., - "Molecular cloning of a gene coding for thermostable beta-glucanase from Bacillus macerns," J. Basic Microbiol; 28:3-10. 1988. Borriss, et al., - "Expressions in Escherichia coli of a cloned beta-glucanase gene from Bacillus Amyloliquefaciens," Appl. Microbiol. Biotechnol; 22:63-71. 1985. Borriss, - Purification and characterization of an extracelluar beta-glucanase from Bacillus IMET B376 (1)), Z. Alg. Mikrobiologie; 21:7-17. 1981. Borriss, et al., - "Beta-1, 3-1,4-glucanase in sporeforming microorganisms. V. The efficiency of beta-glucanase in reducing the viscosity of wort", Zbl. Bakt II Abt. 136:324-329. 1981. Bousquet M., Escoula L. et al; "Immunopharmacologic study in mice of 2 beta-1, 3, beta-1, 6 polysaccharides (Scleroglucan and PSAT) on the activation of macrophages and T lymphocytes," Ann Rech Vet 20: 165-173. 1989. Station of Pharmacologie-Toxicologie, INRA, Toulouse, France. * Quote: "...PSAT and scleroglucan favorably affect the non-specific host defense and cellular immune

30

response in mice." Bousquet M., Escoula L., Pippy B, Besssieres MH, Chavant L, Seguela JP, "Enhancement of Resistance of mice Toxoplasma gondi by 2 polysaccharides beta (1-3) glucan 1-3, beta (1-3) glucan 1-6 (PSAT and Scleroglucan)" Ann Parasitol Hum Comp., ^63 (6): 398-409. 1988. * Bowers G., J. Patchen MLl, et al, "Glucan enhances survival in an intraabdominal infection model," J Surg Res 47(2): 183-188; Aug 1989. * Broach JR, Pringle JR and Jones EW; "The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae;" Genome Dynamics, Protein Synthesis, and Energetics; Cold Springs Harbor Laboratory Press, Cold Spring Harbor, New York. 1991. Browder W., Williams D., Pretus H., et al; Beneficial Effect of Enhanced Macrophage Function in the Trauma Patients. Ann. Surg.; Vol 211: 605-613. 1990. Dept of Surg and Physiol, Tulane U Sch of Med, LA and Istituto Di Chirurgia D'Urgenza, U of Torino, Torino, Italy. * Quote: "Previous studies have demonstrated that glucan, a beta-1, 3-linked glucopyranose polymer, isolated from the inner cell wall of Saccharomyces cerevisiae, is a potent macrophage stimulant and is beneficial in the therapy of experimental bacterial, viral, and fungal diseases. Use of glucan in a murine model of hind-limb crush injury decreased macrophage PGE2 release while stimulating bone marrow proliferation." Browder Iw, Sherwood E., Williams D., Jones E., Mcnamee R., Diluzio N., "Protective effect of glucan-enhanced macrophage function in experimental pancreatitis", Am J Surg.; 1153 (1): 25-33. 1987. Browder W., et al., "Modification of Post-Operative C. albicans Sepsis by Glucan Immunostimulation," Int. J. Immunopharmac.; 6:19-26. 1984. Dept of Surg and Physiol, Tulane U Sch of Med, LA Quote: "These observations suggest that Biologic Response Modifiers such as glucan may be effectively employed in patients who are at risk for post-operative infections."* Browder W., et al., "Protective Effect of Nonspecific Immunostimulation in Post Splenectomy Sepsis". J. Surg. Res.; 35: 474-479. 1983. Dept of Surg and Physiol, Tulane U Sch of Med, LA * Quote: "This study reports the use of glucan, a beta-1, 3-polyclucose, as a nonspecific immunostimulant for postsplenectomy pneumococcal sepsis. ...Nonspecific immunostimulation appears to have significant potential as a treatment strategy against postplenectomy infection." Brown Jl, et al; "A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-->6)-beta-glucan synthesis," Genetics 133(4) 837-849, Apr 1993. * Buddle BM, et al, "Protective effect of glucan against experimentally induced staphylococcal mastitis in ewes." Vet Microbiol 16(1): 67-76, Jan 1988. Bulone V., Fevre m.; "A 34-kilodalton polypeptide is associated with 1,3-beta-glucan synthase activity from the fungus Saprolegnia monoica," FEMS Microbiol Lett: 140: 145-150, 1996. Burgaleta C., Goide D. W.; Increased granulopoiesis and macrophage production in glucan-treated mice; Chirigos MA, ed. Immune Modulation and Control of Neoplosia by Adjuvant Therapy. New York: Raven Press, 195-219, 1978. Burgaleta C., Territo M.C., Quan C.G., Goide D.W.; Glucan activated macrophages: functional characteristics and surface morphology; J Reticuloendothel Soc 23: 195-204. 1978. Burgaleta, C. and Golde, D.W.; "Effect of Glucan on Granulopoiesis and Macrophage Genesis in Mice". Cancer Research; 37:1739-1742; Jun 1977. * Cain J.A., Newman S.L., Ross G.D., "Role of complement receptor type three and serum opsonins in the neutrophil response to yeast," Complement 4: 75-86.1987.

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Campbell I And Duffus jh; eds., "Yeast." 1988. Carrow, D.J.; "Beta-1, 3-glucan as a Primary Immune Activator," Townsend Letter; June 1996. Cerenius L., Liang Z., Duvie B., et al, "Structure and biological activity of a 1,3 beta-D-glucan-binding protein in crustacean blood," J. Biol Chem 269: 29462-29467. 1994. Cisreros RL, Gibson FC 3, Tzianabos AO; "Passive transfer of poly- (1-6)-beta-Glucotrisyl- (1-3)-beta glucopyranose glucan protection against lethal infection in an animal model of intra-abdominal sepsis," Infect Immun 64(6): 2201-2205, Jun 1996. Channing Laboratory, Brigham and Women's Hospital, Boston, MA. * Clark A.E., Stone B.A.; "Beta-glucan hydrolases from Aspergillus niger. Isolation of a beta- (1-4)-glucan hydrolase and some properties of the beta- (1-3)-glucan-hydrolase components," Bichem J 96: 793-801. 1965. Cook J. A., et al, "Protective Effect of Glucan against Visceral Leishmaniasis in Hamsters". Immun.; 37: 1261-1269. 1982. Cook J. A., et al,, "Viscereal Leishmaniasis in Mice: Protective Effect of Glucan". J. Reticuloendothel; Soc. 27: 567-573. 1980. Cross CE, Bancroft GJ, "Ingestion of acapsular Cryptococcus neoformans occurs via mannose and beta-glucan receptors, resulting in cytokine production and increased phagocytosis of the encapsulated form." Infect Immun 63:2604-2611. 1995. Dept Clin Sci, London Sch of Hyg and Trop Med, England. Czop J.K., Kay J., Isolation and Characterization of B-glucan Receptors on Human Mononuclear Phagocytes. J. Exp. Medicine; V.173: 1511-1520. 1991. (Copy available) Dept of Med, Harvard Med Sch, Boston, MA. * Quote: "...human alveolar macrophages ...possess phagocytic receptors of comparable ligand specificity for the Beta glucans commonly present in yeast and fungi. Pathogens such as Candida and Aspergilli contain "yeast" glucan, cell wall components consisting of branched homopolymers of Beta-D-glucose with 1,3 consecutive and 1,6-crosslinked chains and prototypic of Saccharomyces cerevisiae."* Czop J.K., Gurish M.F., Kadish J.l., Production and Isolation of Rabbit Anti-idiotypic Antibodies Directed Against the Human Monocyte Receptor for Yeast B-glucans. Journal of Immunology; 145:995-1001. 1990. Dept of Med, Harvard Med Sch, Boston, MA. * Quote (p1): "Beta-Glucans with 1,3 and/or 1,6 linkages are the major structural components of yeast and fungi and are pharmacological agents in animals...The cell wall glucans of S. cerevisiae consist of two structurally distinct Beta-glucans: major components comprised of consecutively, 1,3-linked glucopyranosyl residues with small numbers of 1,6-linked branches, and minor components with consecutive 1,6-linkages and 1,3-branches." Czop, J.K., Valiante N.M., Janusz M.J.; "Phagocytosis of particulate activators of the human alternative complement pathway through monocyte beta-glucan receptors," Prog Clin Biol Res 297: 287-296; 1989. Dept of Med, Harvard Med S, Boston, MA. * Quote (p1): "Animal studies indicate that beta-glucans with 1,3-and/or 1,6-linkages are active pharmacologic agents that rapidly confer protection to a normal host against a variety of biological insults. The beta-glucan receptors provide a mechanism by which a heightened state of host responsiveness is initiated." Czop J.K., Puglisi A.V., Miorandi D.Z., Austen K.F.; "Pertubation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production," J. Immunol 141: 3170-3176. 1988. * Czop, Joyce K., "The Role of Beta-Glucan Receptors on Blood and tissue Leukocytes in Phagocytosis and metabolic Activation". Pathology and Immunopathology Research; 5:286-296. 1986. *

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Czop J.K., Austen K.F., A B-glucan Inhibitable Receptor on Human Monocytes: Its Identity with the Phagocytic Receptor for Particular Activators of the Alternative Complement Pathway. Journal of Immunology 134: 1985; 2588-2593. 1985. * Czop J.K., Austen K.F.; "Properties of glycans that activate the human alternative complement pathway and interact with the human monocyte beta-glucan receptor," J Immunol 135: 3388-3393. 1985. * Czop J.K., Austen K.F.; "A beta-glucan inhibitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway," J Immunol 134(4): 2588-2593, Apr 1985. * Czop J.K., Austen K.F.; "Generation of leukotrienes by human monocytes upon stimulation of their beta-glucan receptor during phagocytosis," Proc Natl Acad Sci USA; 82: 2751-2755 1985. *

Daum T., Rohrbach M.S.; "Zymosan induces selective release of arachidonic acid from rabbit alveolar macrophages via stimulation of a beta-glucan receptor," FEBS Lett 309: 119-122; 1992. Deimann W, Fahimi HD, "The Appearance of Transition Forms Between Monocytes and Kupffer Cells in the Liver of Rats Treated with Glucan," J Exp Med, p883-897, Apr 1979. * Dept of Anat, U of Heidelberg, Germany.* Delville, et al., entitled "Le-.beta-1, 3-Glucan et Autres Immunomodulateurs dans L'Unfection lepresis Experimentale Chez La Souris". Acta Leprologica; 77/76: 273-281. 1979. Deslanders, et al., "Triple-Helical Structure (1,3)-beta-D-Glucans". Macromolecules 13: 1466-1471. 1980. Diluzio N.R., "Soluble phosphorylated glucan," U.S. Patent 487777, Issued Oct 31, 1989. Diluzio N.R. (deceased), Williams D.L., Browder I.W.; Soluble phosphorylated glucan: methods and compositions for treatment of neoplastic diseases; U.S. Patent 4818752; 1989. Diluzio N.R.; Soluble phosphorylated glucan; U.S. Patent 4739046; 1988. Diluzio N.R. and Williams D.L., " The Roll of Glucan in the Prevention and Modification of Microparasitic Diseases;" in Chemical Regulation of Immunology in Veterinary Medicine, Alan R. Liss, Inc.; pp. 443-456. 1984. Diluzio N.R.,"Immunopharmacology of glucan: a broad-spectrum enhancer of host defense mechanisms," Trends in Pharmacol. SCI., 4:344-347. 1983. Dept of Physiology, Tulane U, New Orleans, LA. * Quote: (p347) "The broad spectrum of immunopharmacological activities of glucan includes not only the modification of certain bacterial, fungal, viral and parasitic infections, but also inhibition of tumor growth." Diluzio N.R. Williams D.L. et al, "Comparative evaluation of the tumor inhibitory and antibacterial activity of solubilized and particulate glucan," Recent Results Cancer Res 75:165-172. 1980. * Quote: "Intravenous administration of soluble or particulate glucan resulted in significant reduction in the growth of a syngeneic anaplastic mammary carcinoma and melanoma B16 and enhanced survival." Diluzio NR, Williams DL; "Enhancement of host susceptibility to Staphylococcus aureus infection by chronic ethanol ingestion-modification by glucan immunostimulation," Alcohol Clin Exp Res 4(3): 254-260. Jul 1980. * Quote: "The administration of glucan significantly prolonged survival of S. Aureus infected control and chronic ethanol mice." Diluzio NR, Williams DL, et al, "Comparative tumor-inhibitory and anti-bacterial activity of soluble and particulate glucan," Int J Cancer, 24(6): 773-779. Dec 1979. * Quote: "...these studies demonstrate

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that a soluble glucan preparation exhibits significant anti-tumor and anti-staphylococcal activity." Diluzio N.R. and Williams D.L., "Glucan-Induced Modification of the Increases Susceptibility of Cyclophosphamide-Treated Mice to Staphylococcus aureus Infection". Cancer Immunol. Immunother.; 6: 73-79. 1979. Diluzio NR, Williams DL, "Protective effect of glucan against systemic Staphylococcus aureus septicemia in normal and leukemic mice," Infect Immun 20(3): 804-810. Jun 1978. * Dept of Physiology, Tulane U, New Orleans, LA. * Quote: "These data denote that glucan enhances nonspecific resistance to S. aureus sepsis, promotes survival during leukemic episodes, and increases survival time of leukemic mice with experimentally induced staphylococcal infection." Diluzio N.R., Williams D.L., Cook J.L., Hoffman E.O.; Protective effect of glucan in experimentally induced Candidiasis; J Reticuloendothel Soc 53: 479-490, 1978. Diluzio N.R., et al., "The Employment of Glucan and Glucan Activated Macrophages in the Enhancement of Host Resistance to Malignancies in Experimental Animals," in The Macrophage in Neoplasia; Academic Press, Inc. New York; pp. 181-198. 1976. DiLuzio N.R., et al., "Evaluation of the Mechanism of Glucan-Induced Stimulation of the Reticuloendothelial System". J. Reticuloendothelial Soc.; Soc.7: 731-742. 1970. Di Renzo, L., Yefenof, E., Klein E., "The Function of human NK cells is enhanced by B-Glucan, a ligand of CR3 (CD11b/CD18)". Eur. J. Immunol., 21:1755-1758. 1991. Doita M, Rasmussen LT, Seljelid R, Lipsky PE, "Effect of soluble aminated beta-1, 3-D-polyglucose on human monocytes: stimulation of cytokine and prostaglandin E2 production but not antigen-presenting function." J Leukoc Biol 49(4): 342-351. Apr 1991. * Donzis B. A.; Substantially purified beta (1,3) finely ground yeast cell wall glucan composition with dermatological and nutritional uses; U.S. Patent 5576015; 1996. Donzis B.A.; Solubilized yeast glucan; U.S. Patent 5519009; 1996. Donzis B.A.; Photoprotective composition containing yeast extract; U.S. Patent 5397773; 1995. Donzis B.A.; Method of revitalizing skin by applying topically water insoluble glucan; U.S. Patent 5223491; 1993. Duan X., Ackerly M. et al; "Evidence for involvement of beta-glucan-binding cell surface lectins."Cell Immunol 157: 393-402; 1994.* Duvic B., Soderhall K.; "Purification and partial characterization of a beta-1, 3-glucan-binding protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculus," Eur J. Biochem 207: 223-228; 1992. Enhanced Healing of Decubitus Ulcers by Topical Application of Particulate Glucan. Tulane University School of Medicine; Research Summary. 1984. Bisu et al, "Studies on the Structure of Polysaccharides (Glucans and Fructans) Produced by Cariogenic Streptococci and on an Enzyme Hydrolyzing the Insoluble Glucan I. Structural Studies of Insoluble Glucan, Soluble Glucan, and Fructans," Chemical Abstracts; vol. 38:pp. 374-381. 1976. Elstad MR, Parker C et al; "CD11b/CD18 integrin and a beta-glucan receptor act in concert to induce the synthesis of platelet-activating factor by monocytes," J Immunol 152:220-230. 1994. Dept of Med, Veterans Affairs Medical Center, Salt Lake City, UT. * Engstad RE, Robertsen B, "Recognition of yeast cell wall glucan by Atlantic salmon (Salmo salar L.)

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macrophages," Dev Comp Immunol 17:319-330. 1993. * Felippe J., Silva M., Maciel F.M., et al., Infection prevention in patients with severe multiple trauma with the immunomodulator beta (1-3) glucan 1-3 polyglucose (glucan). Surg. Gynecol Obstet., 177: 3833-388. 1993. Fleet. G. H., et al., "Isolation and Composition of an Alkali-Soluble Glucan from the Cell Walls of Saccharomyces cerevisiae," Chemical Abstracts; 85:89819z. 1976. Fleet. G. H., et al.,"Isolation and Composition of an Alkali-Soluble Glucan from the Cell Walls of Saccharomyces cerevisiae," Journal of General Microbiology; 94:180-192. 1976. Franek J, Malina J, Kratka H, "Bacterial infection modulated by glucan: a search for the host defense potentiation mechanisms," Folia Microbiol (Praha) 37(2): 146-152. 1992. * Giaimis J., Lombard Y., et al; "Both mannose and beta-glucan receptors are involved in phagocytosis of unopsonized, heat-killed Saccharomyces cerevisiae by murine macrophages," J. Leukoc Biol 54: 564-571. 1993. * Gillet et al., "Particulate beta 1-3 Glucan and Casual Prophylaxis of Mouse Malaria (Plasmodium berghei)". In advances in Exper. Med. and Biology; vol 121A, Escobar and Friedman, eds. Plenum Press, New York, pp. 307-313. 1980. Glovsky MM, et al; "Effects of particulate beta-1, 3 glucan on human, rat, and guinea pig complement activity," J. Reticuloendothel Soc. 33:401-413. 1983. * Quote: "Glucan administration is associated with the modification of a variety of experimentally induced infectious disease states as well as the inhibition of growth of implantable and spontaneous tumors." Goldman R., "Characteristics of the B-glucan Receptor of Murine Macrophages". Exp.Cel. Res.; 174:481-490; 1988. * Goldman R., "Induction of a beta-1, 3-D-Glucan Receptor in P388D1 Cells Treated with Retinoic Acid of 1,25-dihydroxyvitamin D (3)," Immunology; 63:319-324. 1988. Goto H., Yuasa K., Rylander R.; "(1-->3)-beta-D-glucan in indoor air, its measurement and in vitro activity," Am J. Ind Med 25: 81-83.1994. Hall MN and Linder P; "The Early Days of Yeast Genetics," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 1993. Hara C., et al., "A Branched (1.fwdarw.3)-beta-D-Glucan From a Water Extract of Dictyophora indusiata FISCH," Carb. Res.; 145:237-246. 1986. Harada, et al., " Agricultural Biological, Growth and beta-Glucan 10C3K Production by a Mutant of Alcaligenes faecalis var. myxogenes in Defined Medium"; vol. 30, No. 8, pp. 764-769. 1966. Hartland RP, Emerson GW, Sullivan PA, "A secreted beta-glucan-branching enzyme from Candida albicans," Pro R Soc Lond B Biol Sci, 246(1316): 155-160. Biochem Dept, U of Otago, Dunedin, New Zealand. Nov 1991 Hassid, W.Z., Joslyn, M.A., McReady, R.M., "The Molecular Constitution of an Insoluble Polysaccharide From Yeast Saccharomyces Cerevisae"; Journal of American Chemical Society, 1941; 63:295-298. 1941. Hofemeister, "The beta-glucanase gene from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis," Gene; 49:177-187. 1986. Holbrook J.A.C., Parker J.L.; Immunization against Leishmania donovani: glucan as an adjuvant with

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killed promastigotes; Am J Trop Med Hyg 30: 762-768, 1981. Holbrook T.W., et al., "Glucan-Enhanced Immunogenicity of Killed Erythrocylic Stages of Plasmodium Benghei"; Infection and Immunity, 32, 542. 1981. Honda S., et al, "Activation of the alternative pathway of complement by an antitumor (1----3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140, and its lower molecular weight and carboxymethylated derivatives,""Immunopharmacology 11:29-37. 1986. * Inai et al., "Activation of the Alternative Complement Pathway by Water-Insoluble Glucans of Streptococcus mutans: the Relation between Their Chemical Structures and Activating Potencies." J. Immunol.; 117" 1256-1260. 1976. Jamas S., Easson D., Ostroff G.R., DavidsonDd; "Method for producing soluble glucans," U.S. Patent 5633369. Issued May 27, 1997. * Jamas S., Easson D., Ostroff G.R.; "Glucan Preparation," U.S. Patent 5622939. Issued April 22, 1997. * Jamas S., Easson D., Ostroff G.R.;"Glucan drug delivery system and adjuvant," U.S.Patent 5607677. Issued March 4, 1997. * Jamas S., Easson D., Ostroff G.R.;"Use of aqueous soluble glucan preparations to stimulate platelet production." U.S. Patent 5532223. Issued July 2, 1996. * Jamas S., Easson D., Ostroff G.R.;"Use of neutral soluble glucan preparations to stimulate platelet production." U.S. Patent 5488040. Issued January 30, 1996. * Jamas S., Easson D., Ostroff G.R.;"Method for producing neutral glucans for pharmaceutical applications," U.S. Patent 5322841. Issued June 21, 1994. *< James S., Chen Y-CJ, Von Der Osten C.H., et al., "Spectral analysis of glucan produced by wild-type and mutant Saccharomyces cerevesiae". Carbohydr. Polym., 13:207-219. 1990. Janusz M.J., Austen K.F., Czop J.K.; "Isolation of a Yeast Heptaglucoside that Inhibits Monocyte Phagocytosis of Zymosan Particles". The Journal of Immunology; 142:959-965. 1989. Dept of Med, Harvard Med Sch, Boston, MA. * Quote: "Beta-Glucans with 1, 3-and 1, 6 glycosidic linkages are the major structural components of yeast and fungal cell walls and are active pharmacologic agents in host defense systems of plants and animals.... The administration of particulate glucans from S. cerevisiae to laboratory animals induces host resistance to a variety of lethal pathogens by mechanisms involving macrophage stimulation. In vitro studies reveal that bone marrow-derived mouse macrophages and human peripheral blood monocytes possess Beta-glucan receptors that mediate phagocytosis of glucan particles and induce release of proinflammatory mediators..." Janusz M.J., Austen K.F., Czop J.K.; Phagocytosis of Heat-killed Blastophores of Candida Albicans by Human Monocyte B-glucan Receptors. Immunology; 65:181-185. 1988. * Janusz M.J., Austen K.F., Czop J.K.; "Lysosomal enzyme release from human monocytes by particulate activators is mediated by beta-glucan inhibitable receptors," J. Immunol 138: 3897-3901. 1987. * Janusz M.J., et al, "Isolation of Soluble Yeast beta-Glucan that Inhibit Human Monocyte Phagocytosis Mediated by beta-Glucan Receptors," L. Immunol; 137:3270-3276. 1986. * Jiang B., Sheraton J., et al; "CWH41 encodes a novel endoplasmic reticulum membrane N-glycoprotein involved in beta 1, 6-glucan assembly," J. Bacteriol 178: 1162-1171. 1996. Jones EW, Broach JR and Pringle JR. ;"The Molecular and Cellular Biology of the Yeast

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Saccharomyces cerevisiae;" Gene Expression; Cold Springs Harbor Laboratory Press, Cold Spring Harbor, New York. 1992. Jorgensen J.B., Robertsen B.; "Yeast beta-glucan stimulates respiratory burst activity of Atlantic salmon (Salmo salar L.) macrophages," Dev Comp Immunol 19: 43-57. 1995. * Jorgensen J.B., "Quantification of high molecular weight (1-3)(1-4)-beta-D-glucan using calcofluor complex formation and flow injection analysis. I. Analytical principle and its standardization," Carlsberg Res. Commun. (1988); 53:277-285. 1988.

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Konopski Z., Seljelid R., Eskeland T.; "Interferon-gamma inhibits endocytosis of soluble animated beta-1, 3-D-glucan and neutral red in mouse peritoneal macrophages," J Interferon Cytokine Res 15(7): 597-603. Jul 1995. * Dept of Exper Path and Anat, U of Tromso, Norway. Konopski Z., Seljelid R., Eskeland T.; "IFN-gamma inhibits internalization of soluble aminated beta-1-3-D-glucan by macrophages and thereby down-regulates the glucan induced release of TNF-alpha and IL-1 beta," Scand J. Immunol 40: 57-63. 1994. * Konopski Z., Seljelid R., Eskeland T.; "A novel immunomodulator soluble aminated beta-1, 3-D-glucan: binding characteristics to mouse peritoneal macrophages," Biochem Biophys Acta 1221(1): 61-65. Mar 1994. * Konopski Z., Seljelid R., Eskeland T.; "Cytokines and PGE2 modulate the phagocytic function of the beta-glucan receptor in macrophages," Scand J. Immunol 37: 587-592. 1993. * Konopski, Z., et al., "Phagocytosis of beta-1, 3-D-Glucan-Derivatized Microbeads by Mouse Peritoneal Macrophages Involves Three Different Receptors," Scand. J. Immunol.; 33:297-306. 1991. * Kopecka M.; "Electron microscopic study of purified polysaccharide components glucans and mannan of the cell walls in the yeast Saccharomyces cerevisiae," J Basic Microbiol 25: 161-174. 1985. * Lahnborg G., Hedstrom K.G., Nord C.E.; "The Effect of Glucan - A Host Resistance Activator and Ampicillin on Experimental Intraabdominal Sepsis". Journal of Reticuloendothelial Society. 32: 347-353. 1982. * Quote: "It is concluded that glucan, in combination with Ampicillin, has a significant effect on the survival rate of rats with induced peritonitis, probably by enhancing the activities of the reticuloendothelial system, an important part of the total host resistance." Lahnborg, et al., "Glucan-Induced Enhancement of Host Resistance in Experimental Intraabdominal Sepsis". Eur. Surg. Res.; 401-408. 1982. * Larm O., Seljelid R., "Water-soluble aminated beta-1, 3-bount D-glucan and composition containing same," U.S. Patent 4707471; Issued Nov 17, 1987. Leibovich S.J., et al., "Promotion of Wound Repair in Mice by Application of Glucan". J. Reticuloendothel, Soc. 27: 1-11. 1980. Lejeune FJ., Vercammen-Granfjean A., Mendes Da Costa P., Bron D., Defleur V., "Suppressor cell induction and reticuloendothelial cells activation produced in the mouse by beta(1-3)glucan 1-3 glucan", Adv. Exp. Med. Biol. 121 (A): 235-244. 1979. * Lotzova and Gutterman, "Effect of Glucan on Natural Killer (NK) Cells: Further Comparison between NK Cell and Bone Marrow Effector Cell Activities". J. Immunol., 123: 607-611. 1979. Mahauthaman R, Howell CJ, Spur BW, Youlten LJ, Clark TJ, Lessof MH and Lee TH; "The generation and cellular distribution of leukotriene C4 in human eosinophils stimulated by unopsonized zymosan and glucan particles;" J Allergy Clin Immunol 81:696. 1988. Manners, D.J., Masson, A.J. & Patterson, J.C.: "Heterogeneity of Glucan Preparations from the Walls of Various Yeasts". J. of Gen Micro.; 411-417. 1974. Manners, D.J., et al., "The Structure of a beta- (1.fwdarw.3)-D-Glucan from Yeast Cell Walls," BiochemJ.; 135: 19-30. 1973. Mansell P.W.A., Rowden G., Hammer C.; Clinical experiences with the use of glucan. Chirigos MA, ed.; Immune Modulation and Control of Neoplasia by Adjuvant Therapy. Raven Press, New York 255-280; 1978.

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Tomos et al., "A protein-glucan intermediate during paramylon synthesis" Biochem. J.; 174:283-290. 1978. Tong, D.W., Barnetson R.S.; B-1, 3-D-glucan gel in the treatment of solar keratoses; Australasian J of Dermatology, 37: 137-138, 1996. * Dept of Dermat, Royal Prince Alfred Hosp, Camperdown, Australia. Truscheit E., Bierling R., Schlumberger H., Oettgen H.; "Process for the preparation of immunopotentiating agents from components of yeast cell wall material; " U.S. Patent 4138479; Issued Feb 6, 1979. * Tsujinaka T., Yokota M.K.; Modification of septic processes by B-glucan administration. Eur Surg Res; 22:540-546, 1990. * Tzianabos AO, Cisneros RL; "Prophylaxis with the immunomodulator PGG glucan enhances antibiotic efficacy in rats infected with antibiotic-resistant bacteria, "Ann NY Acad Sci 797: 285-287; Oct 1996. * Quote: "Results of these studies demonstrated that prophylaxis with PGG glucan in combination with antibiotics provided enhanced protection against lethal challenge with Escherichia coli or Staphylococcus aureus as compared with the use of antibiotics alone." Uchida, A.; "Method for treatment of chronic fatigue syndrome, "U.S. Patent 5424300 (A method for the treatment of chronic fatigue syndrome, comprising administering a polysaccharide which further contains a beta (1-3) glucan-1, 3/1, 6-glucoside bond). Issued June 13, 1995. Van Der Vaart J.M., et al, and "The retention mechanism of cell wall proteins in Saccharomyces cerevisiae Wall-bound Cwp2p is beta-1, 6-glucosylated," Biochim Biophys Acta, 1291(3): 206-214. Dept Molecular Cell Biol, Utrecht U., The Netherlands. Dec 1996. Van Der Vaart J.M., et al, "The beta-1,6-glucan containing side-chain of cell wall proteins of Saccharomyces cerevisiae is bound to the glucan core of the GPI moiety," FEMS Microbiol Lett 145: 401-407. 1996. Vargas-Albores F., Jimenez-Vega, Soderhall K.; "A plasma protein isolated from brown shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidase system by beta-1, 3-glucan," Dev Comp Immunol 20: 299-306. 1996. Vetvicka V., Thornton B.P., Ross G.D.; "Soluble Beta-glucan Polysaccharide Binding to the Lectin Site of Neutrophil or Natural Killer Cell Complement Receptor Type 3 (CD11b/CD18) Generates a Primed State of the Receptor Capable of Mediating Cytotoxicity of iC3b-Opsonized Target Cells," Journal Clin Invest 98: 50-61. 1996. Div of Experimental Immuno and Immunopath, Dept Path, U of Louisville, KY. * Quote: "This investigation showed that soluble CR3-specific polysaccharides such as beta-glucan induced a primed state of CR3 that could trigger killing of iC3b-target cells that were otherwise resistant to cytotoxicity." Wessels J.G.; "A beta 1,6-glucan glucanohydrolase involved in hydrolysis of cell-wall glucan in Schizophyllum commune," Biochem Biophys Acta 178: 191-193. 1969. Williams D.L. ,Mueller A., Mueller P., Swails W., et al., "Randomized phase I/II trial of a macrophage-specific immunomodulator (PGG-glucan) in high-risk surgical patients". Ann. Surg.; 220(5): 601-609. 1994. Williams D.L., et al,"Development of a Water-Soluble, Sulfated (1.fwdarw.3)-beta-D-Glucan Biological Response Modifier Derived from Saccharomyces cerevisiae," Carbohydrate Research. 247-257. 1992. Williams D.L., et al, Development, Physicochemical Characterization and Preclinical Efficacy Evaluation of a Water Soluble Glucan Sulfate Derived from Saccharomyces cerevisiae," Immunopharmacology; 22:139-156. 1991.

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Williams D.L., Mcnamee R.B., Jones E.L., et al., "A method for the solubilization of a (1-2)-B-D-glucan isolated from Saccharomyces cerevisiae." Carbohydr Res.; 219: 203-213. 1991. Williams D.L., Browder I. and Diluzio N.R., "Soluble phosphorylated glucan: methods and compositions for wound healing," U.S. Patent 4975421, Issued Dec 4, 1990. Quote: "The soluble phosphorylated glucans are useful for promoting the wound healing process. The soluble phosphorylated glucans are also useful for prophylactic and therapeutic applications against neoplastic, bacteria, viral, fungal and parasitic diseases." Williams D.L., Browder I. and Diluzio N.R., "Methods and compositions for prophylactic and therapeutic treatment of infections," U.S. Patent 4900722, Issued Feb 13, 1990. Quote: "The soluble phosphorylated glucans are also useful for stimulating macrophage cells, either in vivo or in vitro, to produce a cytotoxic/cyctostatic factor effective against cancer cells." Williams D.L., Sherwood E.R., Browder I.W., McNamee R.B., Jones E.L., Di Luzio N.R.; Pre-clinical Safety Evaluation of Soluble Glucan. International Journal Immunopharmac. 1988; 10: 405-411. 1988. Williams D.L., et al; "Pre-clinical Safety Evaluation of Soluble Glucan", Int. J. Immunophamac. Vol. 10, No. 4: 405-414. 1988. * Dept of Phys, Tulane U Sch of Med, New Orleans, LA. *Quote: "Soluble glucan, a beta-1, 3-linked glucopyranose biological response modifier, is effective in the therapy of experimental neoplasia, infectious diseases and immune suppression." Williams D.L., et al; "Therapeutic efficacy of glucan in a murine model of hepatic metastatic disease," Hepatology 5(2): 198-206. Mar 1985. *Quote: "...coincubation of particulate glucan with diverse populations of normal or tumor cells in vitro indicated that glucan exerted a direct cytostatic effect on sarcoma and melanoma cells and, in contrast, had a proliferative effect on normal spleen and bone marrow cells." Williams D., Browder IW and Diluzio N.R, "Immunotherapeutic modification of Escherichia coli-induced experimental peritonitis and bacteremia by glucan," Surgery 93(3): 448-454. Mar 1983. * Quote: "These data denote that the intraperitoneal administration of glucan significantly modifies the course of E. coli-induced peritonitis and bacteremia due, in part, to glucan-induced enhancement of macrophage function." Williams D.L. and Diluzio N.R.; "Modification of Experimental Viral Hepatitis by Glucan Induced Macrophage Activation". In the Reticuloendothelial System and Pathogenesis of Liver Disease, Liehr and Grun, eds. Elsevier/North Holland Biomedical Press; pp. 363-368. 1983. Williams D.L. and Diluzio N.R.; "Glucan-Induced Modification of murine Viral Hepatitis". Science (1980), 208: 67-69. 1980. *Quote: "Thus glucan is capable of increasing survival, inhibiting hepatic necrosis, and maintaining an activated state of phagocytic activity in mice challenged with [mouse hepatitis virus strain] MHV-A59." Williams D.L., et al; "Protective Effect of Glucan in Experimentally Induced Candidiasis". J. Reticuloendothel; Soc 23: 479-490. 1978. Williams D.L, Diluzio NR, "Glucan induced modification of experimental Staphylococcus aureus infection in normal, leukemic and immunosuppressed mice." Adv Exp Med Biol 121(A): 291-306. 1979* Williams D.L, Diluzio NR, , Reticuloendothelial System and Pathogenesis of Liver Disease; Liehr and Grun. eds. Solubilization of a (1-3_-B-D-glucan isolated from Saccharomyces cerevisiae. Carbohydr. Res. 219: 203-213. 1991. Wolk, M. and Danon, D.; "Promotion of Wound Healing by Yeast Glucan Evaluated on Single Animals"; Medical Biology; 63:73-80. 1985. *

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Wooles and Diluzio N.R.; "The Phagocytic and Proliferative Responses of the Reticuloendothelial System Following Glucan Administration". J. Reticuloendothelial..; Soc. 1: 169-169. 1964. Yoxhida M, et al, "Soluble (1-->3)-beta-D-glucan purified from Candida albicans: biologic effects and distribution in blood and organs of rabbits," J Lab Clin Med 128(1): 103-114. Jul 1996. *Dept of Lab Med, U of Cal Sch of Med, San Francisco, CA. Yoshida H., Ochiai M., Ashida M.; "Beta-1, 3-glucan receptor and peptidoglycan receptor are present as separate entities within insect prophenoloxidase activating system," Biochem Biophys Res Commun 141: 1177-1184. 1986.