Downloaded By: [University of Colorado Health Sciences Center] At: 20:28 10 April 2008 REVIEW ARTICLE informa B-Glucans, History, and the Present: Immunomodulatory Aspects and Mechanisms of Action |. Novak Insitute of Chemical Technology, Prague, C V. Vetvieka Department of Pathology, University of Louiuille, Louisville, Ken ch Republic ‘The present paper represents a comprehensive up-lodate review of Seulucans, thelr chemical and biological properties, And their role in immunological reactions. 3-D-Glucans belong io a group of physiologically ative compounds called biological response modifiers and represent highly conserved structural ‘components of ell walls in yeast, fungi or seaweed, Despite almost 180 years of research, the exact mechanisms oftheir action remain unclear The present review starts with the history of glucans. ‘Next attention is focused on sources and structure, comparing the effects of physicochemical properties, and sources on biological effects. As glucans belong to natural products useful in preventing ‘various diseases, they have been highly sought after throughout hhuman history. Based on extensive recent research, this paper ‘explains the various mechanisms of effets and the ways glucans mediate thelr effects on defense reactions against. infections Despite the fact that predominately pharmacological effets of lucans are positive their unfavorable and potentially toxie side effects were not overlooked. In addition, attention as focused fon the future research, possible alternatives such as synthetic ‘ligosuecharides, and on clinical applications. Keywords defense, glucan, history, immunity 1, INTRODUCTION Generally, @-plucan isa chemical name of a polymer of slucose. There ate more of such polymers, differing in glycosidic ‘bond position. Cellulose, (1>4)-f-D-glucan, isan example, The following data refer w homopolymers of glucose having a linear molecule with (=»3)-f-o-glycosidic linkages or a branched ‘one, with side chains bound by (1—+6)-fp-glycosiic linkages, Alttiough chemically beterogencous, chose polysaccharides are usually termed by common name "B-glucans.”ILis necessary to luke into account that these compounds, which otherwise causal Received 12 September 2007: accepted 7 October 2007 Address comespondcnce to Professor acl Vetvcka, Univer: sigy of Louisville, Department of Pathology, SIS. Floyd, MDR. ‘Bldg. Ro.224, Louisville, KY 40202, USAse mail: Viclvetvicka@ louie eda ro] ntucky, USA similar or nearly identical immune reactions in macroorganisms, cean differ in origin as well as in their primary, secondary, or tertiary structures or their solubility in water or alkalies. Due to these factors, numerous discrepancies can be found in the Tterature. Some sources of f-glucans ar listed in Table | {A~Glucans show notable physiological effects; ths is their ‘most important quality and the reason why so much atten: tion has been devoted to them. They belong (0 a group of physiologically active compounds, collectively termed biolog- ical response modifiers (BRM). According 10 their effect, BRM can be classified into two groups ~ eytokines, respon sible for communication between immune system cells and regulation of the system, and immuno-modulators. The im- ‘munomodulators are able to positively (i.e., immunopotentia- tion) or negatively (ie., immunosuppression) manipulate the immune system. Different known immunomodulators can be classified into three groups (Wemer, 1987): 1) Intact microbes (2, Bacille Calmette-Guérin) and components of microbi cells (cg. endotoxin of Gram-negative microorganisms (LPS), ‘muramyldipepxide (MDP), Fungal polysaccharides (2ymosan,f ¢glucans), polynucleotides, bestatine); 2) Natural components of ‘normal immune system (e.., thymic hormones, lymphokines, ‘monokines); and, 3) Synthetic compounds (eg, levamisole, iso- prinosine, diethyldithiocarbamate), ‘Thus far, among many known and (ested immunomodulators of the first group, polysaccharides isolated from different mit- erworganisms and plants are the most important. large number of such polysaccharides, which act only as immunpotentistors, isknown (Whistleretal., 1976). As mentioned earlier, B-glucans belong to this group. 2. HISTORY AL the beginning of the 18 Century it was already known ‘hat certain infectious diseases showed a therapeutic effect, ‘on malignant processes. Purposeful use of such therapy dates approximately {rom the middle of the 19" Century, when Bush (Bush, 1850) performed experiments in curing sarcoma,Downloaded By: (University of Colorado Health Sciences Center] At: 20:28 10 April 2008 8 [NOVAK AND VETVICKA TABLE! In time, other polysaccharide immunomodultors were Some A-glucans with immunomodulatory effects sought and among them were f-plueans Tei investigation be- = gan inthe 1960s and 1970s, Two lines can be traced in -lucan , Character history, each based on different starting points but gradually ud eon of polymer converging. The first took place chiefly in the United States and Europe, the second in Asia, primarily in Japan, Research on Curaan ‘Atcatigencs faecalis ——_‘tinear ope, primarily in Jp tartar tama teat feats inthe Buo-Ameian miley wae based on Ha pachymaran Poriacovos linear edge of immunomodulatory effects of zymosan, a mixture of feminan Lentinusedodes branched polysaccharides isolated from the cell walls of Saccharomyces Pleuran(HA-glucan) Pleurots ostreatus branched cerevisiae, Zymosan was for te fist sme prepared and invest schizophyllan Schicophyllam commune branched gated by Pillemer and Ecker (1941) inthe 1940s and since that seleroinan (SSG) Sclerotinia sclerotiorum branched ie it has been used in many physiofogical and immunologi- Seleroglucan Scleroium ghicanicim, branched 4 studies, Zymosan is potent stimulator especialy of alveolar Sols macrophages and, among others, itinduces the elease of aseries srifolan Grifotafrondosa branched ofe)tokines, mainly interleukin (IL-8), fom human neuropil, EAN-TSN ——Dictyophoraindusiaa branched ——-AlTROUgH zymosan was able vo stimulate non-specific in sean glucan Saccharomyces cerevisiae branched SU Fesponse, st the onset it was not clear what component by infecting patients with erysipelas, ie. with f-hemolytic streptococci of the Group A. These therapeutical procedures were repeated toward the end of the 19® Century by Coley (Coley, 1894), who applied less dangerous extracts of microbial teulures, inthis ease from cultures of Bacillus prodigiosus (aow Serrutia marcescens), This extract was at these times known as ‘Coley’s toxin,” and was lately determined as interleukin IL-12 (Tsung and Norton, 2006) However, Coley does not have priority in this regard. At the time, it was already known that ceriain components of mi «roorganisms eaused in maeroorganisms, especially mammalian ‘ones, erocious reactions comparable to pathophysiological con- sitions during infection by intact microbes. It i likely thatthe first investigated substance with these properties was LPS of Gram-negative bacteria (e.g, Escherichia, Salmonella, Shige Pseudomonas, Neisseria, and Haemophilus spp. a paper de scribing the endotoxin was published in 1865 (Billrth, 1868). In both humans and experimental animals, LPS induces in- creased phagocytosis with a potential protective effect for host However, its toxic effects (such as fever, diarthea, hypotensive shock, intravascular coagulation, multiple ofgan dysfunctions) completely predominate. A toxie principle of LPS is its lipid moiety, while the savcharidic moiety, with prevailing glucose, xzalactose and mannose content (Nowotny, 1969), is non-toxic “and bears immunomodulatng activity as well Itis apparent that ‘even polysaccharides themselves ean act as immunomodulators, ‘while their toxicity is negligible Properhistory of polysaccharides as immunomodulatorspoes back tothe 1940s when Shear and coworkers (Shearct al, 1943) described a substance, again rom Serratia marcescens cultures, that caused necrosis of tumors. Subsequently, this substance (so-called Shear’s polysaccharide) was identified as a mixture of three polysaccharides with the main chain consisting of o ‘glucose and o-mannose units connected by (I->3) glycosidic linkages (Srivastava and Adams, 1994). ‘of ths rather erade composition is responsible fr that activity ‘When zymosan was examined in dotal, glucan was idenifiod asthe componentof primary effect. was subsequently isolated and its immunological effects wer investigated. This esearch was pioneered by Nicholas R. DiLuzio fie see DiLuzio and Riggi, 1970, Williams eal, 1980) a Tulane University in New Orleans. Tho arrival of A-glucan in Japan was different. In Asian medieine, consumption of different medicinal mushrooms (8, Shiitake, maitake, reishi, et.) has along tradition, In detailed studies ofthe biological effects of these mushrooms, especially the anticancer actions, B-glucans were agin found w be a main cause of non-specific immunomodulation. The source of tis investigation is associated with Goro Chiara from Teikye Uni- ‘versity in Kawasaki, who isolated glucan, namedby him lenti- tan, ftom mushroom shitake(Lentnus edodes, now Lemna éeldes)(Chibaract a, 1969), Ail uflicenly-puifid polysaccharide immunomodulators distinguish themselves by very low toxicity (eg. lentnan has an LDayonase) > 1600 mark (Chibara eta, 1982), Time in- corporation of A-glucans ia the history of immunomodulation is shown in Table 2. The history of the investigation of the chemical composi tion of glucans is rater long and not straightforward. Com- position of the call walls of different microbial producers of fslucan, in particular yeasts, was previously investigated in the 19" Century. Van Wisselingh (1898) published an opin ion that in the fungal eel wal, ether chitin or cellulose could prevail -Glucan from te eel wall of Saccharomyces cerevisiae can serve as an example of how dificult un exact determination of chemical structure was inthe frst half ofthe last century Tn the 1950s nd 1960s, glucan isolated from theell walls cere sige was subjected to investigation by the technique of sugar analysis, partial hydrolysis, methylation analysis, periodate ‘oxidation, Smith degradation (periodate oxidation eduction by NaBH, and subsequent panial hydrolysis), ete. However, theDownloaded By: [University of Colorado Health Sciences Center] At: 20:28 10 April 2008 PROPERTIES OF GLUCANS 0 TABLE? History of immunomodulators 1865 Biro: endotoxin (LPS) 1894 W. B. Coley: erysipel toxins, B. prodigiosus 1936, J Freund: mykobakl J. Freund: mycobacterial adjuvants (BCG) 1941 LL Piliemer: zymosan 1943 M. J, Shear: polysaccharides from S. marcescens 1963 R, Prevot, B. Halpern, G. Biozzi: Corynebacterium parvum 1964-1971 A. White, A. Goldstein, LF. Bach etal: thyme hormones 1967 1H, Okamoto: piibanil (OK-432) 1968, NN. DiLazio: yeast glucan 1909 G. Chiara: ientinan 1967 W. Braun: double-stranded polynucleotides 1970 IN. Najjar euftsin 1971 G. Renoux: levamisol 1975 E, Lederer, L. Chedid : MDP results obtained were rather diserepant (Bell and Northcote, 1950; Manners and Patterson, 1966). Only afer finding that in the yeast cell wall several types of B-glucans exist (Bacon and Farmer, 1968), detailed fractionation of cell wall components their characterization was made (Manners eta, 1973). I is believed now thatthe main component of f-glucan from the _yeast cll wall isa slightly branched, high-molecular (1->3)-B- ‘glucan (DP about 1500, molecular weight ca. 240 kDa), with bout 3% of (16) branching Considerable heterogeneity of all natural B-glucans, not only from saccharomycetes but also from other sources, abviously \was and continues fo be the cause ofa series of mutually con- tradicting conclusions, Recently, an altempt was made to solve this problem using semisynthetic and synthetic probes, suitable for accurate immunological research (Jamois et al, 2005), 3. SOURCES AND STRUCTURE OF 9-GLUCAN ‘There are various natural sources of f-plucans: however, they are most frequently prepared from fungal cell walls. In addi- tion tothe Fungal cell walls, B-glucan is also isolated from sea- weed (laminaran from Laminaria sp, linear B(1—3)-O-glvcan [Black etal, 1951],commercially sold as Phycarine [Vetvicka and Yvin, 2005), Is also produced by bacteria (curdlan from Alcaligenes faecalis) (Harada eta, 1968) and ineluded in e=- reals. The composition of the cereal A-glucan is somewhat dif- ferent; it contains, in addition, (14) bound glucose ‘Taxonomic conception of kingdom Fungi went through a certain progression; many microorganisms earlier classified as fangi are now categorized into other kingdoms. In the follow- ing text, the name fungi willbe written in quotation marks to istinguish between taxonomic and practical definition. Exam- ples of composition of cell wall polyssecharides are presented in Table 3. The table makes evident that the most important producers of f-glucans are ascomycetes (where yeasts and cet= tain filamentous moulds pertain) and basidiomycetes. To basid- iomycetes belong edible or non-edible mushrooms, ier found in nature or ariticially cultivated ‘The cell wall composes an appreciable part of fungal cell sass; in yeasts it represents between 15% and 25% of woul cell TABLE3 Composition of cell wal polysaccharides of some “fungi Examples Prevailing wall polysaccharides Kingdom Class ‘Onder Genus and Species at vegetative cells Protista——Acrasiomycetes Dictyostelates Dictyostelium discoidewn cellulose - glycogen Chromista — Oomycetes Perenosporales Plasmopara viticola cellulose - glucan Phytophihora infestans Hyphochytridiomyceres — Hyphochytriales Rhizidiomyces parasiticus cellulose - chitin Fungi Zgomycetes Mucorales ‘Mucor mucedo cellulose - chitin Chytridiomycetes Chytridiates Blastocladiella emersonii chitin ~ -glucan Ascomycetes Eurotiales Aspergillus niger Homobasidiomycetes __Agaricales Agaricus bisporus Lentinula edodes Plewrotus ostreatus Stereales Grifola frondosa Schicophyllum commune Hemiascomycetes Saccharomycetates Saccharomyces cerevisiae mannan B-glucan Hypomycetes Sporobolomycetaceae Schizosacch, actosporus Sporobolomyces roseus ‘mannan - chitinDownloaded By: [University of Colorado Health Sciences Center] At: 20:28 10 April 2008 50 [NOVAK AND VETVICKA, ‘mass. The cell wall is not an inert structure but vital organelle that performs a range of functions ~ mechanical protection, os ‘moti¢ stabilizavion, compounds binding, mutual cellular adhe- sion, etc. The cell wall also serves as enzyme support and a selectively permeuble barrier. Research on the cell wall of different “fungal” species does ‘ot lead toa straightforward model ots structure, and concepts, ofits organization underwent certain development. According to Stratford (Stratford, 1994), the yeast cell wall resembles re= inforced concrete. An armature, representing about 35% of wall ‘mass and formed by fibrils of alkali insoluble A(1>3)-glucan, Js dipped into mannoproteins (about 25-35% of wall mass) and bound to the armature through amorphous glucan and chitin ‘The cell wall of other “Tungi” is constructed in a similar way. Essentially, the same model ofthe fungal cell wall was published by Selitrennikot? 2001). Recently, Grin (2001) published another model ofthe cell wall of ascomycetes and basidiomycetes. According to this ‘model, the cell wall of most “fungi” contains five main com= ponents; (1+3}-A-b-glucan, (I->6)-A-0-glucan, (1=r3)-a-. slucan, chitin, and glycoproteins. On the other hand, (1+3)- 1a-0-Glucan is not present in yeasts (e.g, in. cerevisiae and Candida lbicans). However, in many other species of the As- comycetes and Basidiomycetes, it forms 946% of cell wall mass. Chitin at saccharomycetes is found only in bud sears ‘Though different models ofthe fungal cell wall differ some= ‘what, they agree that f-glucan isnot located on the surface of the wall but is more or less immersed inthe wall material, With regard to both immunological research and pharmaceutical uti lization of f-glucans, an important conclusion can be reached. Ia ‘macroorganisins, -glucans act first ofall as markers of Fungal invasion so that avtivity of f-glucan preparations will increase with the degree of denudation of glucan fibrils. A general def- nition of glucans of any origin is given in Table 4 ‘Uniil recently, biologically efficient f-glucans were sup- posed to have similar structure - the main chain of C13) bound o-glucopyranose moieties to which some o- ilucopyranoses are randomly connected by A(1—>6) linkages (Figure 1). The degree of branching (DB) of some A-glucans iy presented in Table 5. However, the detailed structure of p -lucans from dissimilar sources differs as well as their biologi- cal activity (Rees and Scott, 1971; Wagner etal, 1988; Adachi et al, 1989; Jamas etal, 1991; Kraus and Franz, 1992), In na- tive B-glucans, their fibrils are composed from organized parts in which the main chain is coiled to triple helix. These regions are combined with single or double filaments of A(1—»3)-D- ‘luco-pyranoses (Saito et al, 1987, Ohno ct al, 1988). The {riple helix, formed by three H-bonds in C-2 position and stabi- lized by side chains, is probably present only in high-molecular ‘-glucans with molecular weight over 90 kDa (Chuah et al., 1983; Ohno et al, 1988). The H-bonds of triple helices can be interrupted by increased temperature, high pH, or certain, solvents Diverse data on the comparison of structure, molecular size, and biological effect can be found inthe literature. For example, anti-tumor activity of sehizophyllan is supposedly conditioned by tiple helix presence and a molecular weight higher than 100 kDa (Kojima et al., 1986). However, the triple helix structure ‘most likely should not be a solely effective form of f-glucan be- cause alkaline treatment, used in most of isolation procedures, destroys this structure (Young and Jacobs, 1998; Saito et al., 1991; Miura et al, 1995). In addition, the most recent opinions do not confirm established ideas of the necessity of high molec ular mass and branching of biologically active -glucans. Thirty ‘years ago, Kabat (1976) found that for antigen polysaccharidic {determinants the size ofthe binding site on an antibody corre= sponds to six or seven monosaccharide units Size ofthe binding site for 8-glucan ~in this case on a receptor of an immunocom= petent cell, eg. the macrophage ~ seems also to correspond to this numberof glucose residues, 4. GLUCAN AND IMMUNOLOGICAL REACTIONS Natural products useful in preventing or treating disease have been highly sought after throughout human history. A major problem in characterizing many natural products is that they represent a complex mixture of ingredients, each one of which ‘may contribute to their bioactivity. 6-Glucans from fungi, yeast, and seaweed are well-known biologic response modifiers that function a immunostimulants against infectious diseases and ‘cancer (Borchers et al, 1999; Brown and Gordan, 2003), Un- like most other natural products purified glucans retain their TABLE 4 General division of f-glucans of any origin ‘Type Gol-forming A-glucans (“solubl Types High-moecular branched f-glucans (eg. grfolan, schizophyllan,seleroglucen) Linear f-glucans (eg, laminaran from brown sea algae), Chemically modified particular luc phosphory-lated P-glucans). Particular insoluble") A-glucans Yeast glucan 8 (cB carboxymethylated, sulfonated or -Glacans from the fst group are usally soluble in alkalies. More ecenly a premise was ened that insolubiliy of f-ghacan in alkalies driven ony bythe extent of Binding to chitin (Brose el, 1999,Downloaded By: (University of Colorado Health Sciences Center] At: 20:28 10 April 2008 PROPERTIES OF GLUCANS 31 OH OH FIG. 1. Gane sacar of lacs bioactivity, which permits the characterization of how f-glucans ‘work on a cellular and molecular level #-Glucan has been used as an immunoadjuyant therapy for ‘cancer since 1980, primarily in Japan (Takeshita et al, 1991; imura et al., 1994; Matsuoka et al., 1997; Yan etal, 1999), Another activity demonstrated with f-glucan in the mid+1980s ‘was its ability (o stimulate hematopoiesis in an analogous man ‘ner a granulocyte monocyte-colony stimulating factor (Patchen etal, 1983), Both particulate and soluble f-glucans, all of whieh ‘were administered intravenously, caused significantly enhanced recovery of blood cell counts afler gamma radiation (Patchen et al, 1985), Others showed that -glucan could reverse the myelo- suppression produced with chemotherapeutic drugs (Wagnerova, etal, 1993), In addition to the effect in treatment of cancer, A-glucans have been demonstrated to protect against infection with both bacteria and protozoa in several experimental models and to enhance antibiotic eflicacy in infections with resistant bacteria. The protective effect of B-glucans ‘was shown in experimental infection with Leishmania major (Al Tuvsaiji et al, 1987) and L donovani (Cook and Hol- brook, 1984), C: albicans (Bacon and Farmer, 1968), Toxo- plasma gondii Bousquet etal, 1988), Sireptococeus suis (Drivz TABLES Degree of branching (DB) of different glucans P-Glucan Source DB achymaran — Poria covos (0015-0020 yeast glucan Saccharomyces cerevisiae ,03-0.20 lentinan Lensinula edodes 023-033 pleuran Pleurotus ostreatus 025 srifolan Grijola frondosa 031-036 seleroglucun — Seferorium glueanicum 030) schizophyllan Schizophyllum commune 0.38 S8G Sclerotinia scleroiorumn 0.50 etal, 1995), Plasmodium berghei (Kumar and Ahmad, 1985), Staphylococcus aureus (Liang et aly 1998), Escherichia coli (Rasmussen and Seljelid, 1990), Mesocestoides corti (White ct al., 1988), Trypanosoma cruzi (Williams e al, 1989), Eimeria vermiformis (Yun et al, 1998), and anthrax infection (Vetvicka et al, 2042). Most published studies described effects of injected f-glucans (either intraperitoneal (IP), intravenous {TV}, oF subcutaneous (SC). It is however nec- essary, for possible clinical practice, to evaluate the possi- bility of oral delivery. These experiments are summarized in Table 6, ‘Yeast A-glucan is able to absorb mycotoxins (such as zear- tenon, affatoxin B1, deoxynivalenol, ochratoxin A, and pat- ulin), probably through hydrogen bonds and van der Waals forces; this -glican effect is important particularly for livestock. ‘The influence of certain cereals (barley, oats) and edible ‘mushrooms (eg.. Grijola frondosa, L edodes and Flammulina velutipes (Fukushima et al, 2001), Pleuronus ostreanus [Bobek et al., 1996], oF Agaricus bisporus (Fukushima et al., 20001) ‘on decreasing levels of serum cholesterol and liver low-density lipoprotein, leading to lowering of arteriosclerosis and heart disease hazards, is also mediated by B-glucan. I is known that cereals, mushrooms and yeast facilitate bowel motility and can bbe used in amelioration of intestinal problems, particularly ob- stipation Batilana eta, 2001; Dongowski eta, 2002). Non- igestible f-glucans, forming a remarkable portion of these materials, are also able to modulate mucosal immunity of the intestinal tract (Tsukada et al.,2003). In the central nervous sys= n, A-glucans activate microglial cells (Muller et l., 1994), ‘These ells act as scavengers ofthe brain cell debris and play a positive role in Alzheimer's disease, AIDS, and multiple scle- rosis (Haga etal, 1989) Possible effects of glucans on macroorganisms are thus very diverse and impinge upon not only the immune system, but probably in most ofthe described activites, are in some form, ‘more or fess connected with that system,Downloaded By: [University of Colorado Health Sciences Center] At: 20:26 10 April 2008 2 NOVAK AND VETVICKA, TABLE 6 Oral effects of glucan Soure Indication Species Results Reference Yeast Cancer Human Inhibition ‘Ueno, 2000 Cancer Mouse Inhibition Hong etal, 2004 Lentinan Cancer ‘Mouse Reduetion Kurashige etal, 1997 Schizophytlan Antiviral Mouse jereased Ab Hota et al, 1993 Cancer Mouse No effects Miura et al, 1995 ssc Immunity Mouse Increase ‘Sakurai eta, 1992 Cancer Mouse Inhibition ‘Suzuki et al. 1991 Maitaki Cholesterol Rat Decrease of Lipids Kubo and Hanba, 1996 Cancer Human Reduction ‘Nanba, 1995 PSK Cancer Mouse Inhibition Nio etal, 1988 Cane Human Survival Increased Kaibara etal, 1976 Agpicwsb Cancer Mouse Enhanced clearance Bina and Fujimiya, 1997 Sparassiscrispa Cancer Mouse Inhibition Ohno etal. 2000 Seaweed Cancer Mouse Inhibition Vetvicka et al., 2007 5, MECHANISMS OF ACTION ‘The most pronounced effect of A-glucans consists of aug- ‘mentation of phagocytosis and proliferative activities of profes- sional phagocytes-granulocytes, monocytes, macrophages, and dendritic celts. n this tegard, macrophages (Chiaract al, 1982; ‘Quinn, 1990; Vetvickaetal., 1996), considered the basic effector cells in host defense against bacteria, viruses, multicellular par- sites, tumor cells. and erroneous clones of own somatic cells, play the most important role. ‘Macrophages are constituents of the non-specific (innate, ‘on-aduptive), evolutionary older, immune system, which be- yond phagocytes is comprised of a complicated family of serum. ‘proteins called complement anda numberof other soluble recog nizing and effector molecules. This innate immunity is based on ‘non-clonal receptors (pattem recognition receptors, PRRs) that recognize certain molecules onthe surface of invading microor- ganisins and are collectively termed as pathogen-associated ‘molecular patterns (PAMPS), Regardless oftheir name, PAMPs are not unigue for pathogens only, but are fundamental for the survival and pathogenicity of a given microorganism, The PAMPs differ from host molecules, are not subjected to variabil- ity, and are evolutionary highly conserved. Different biopoly- ‘mers, including glucans, belong to the PAMPS, Macrophages detect PAMPs through a numberof different re- ceptors, For glucan recognition the macrophages keep several, receptors at their disposal: TLR-2 (oll-like receptor 2), dectin-1, ‘CRS (complement receptor 3), lactosylceramide and probably ‘even other. Generally, the receptors are not too specific and usu ally detect several different PAMPS, ‘Toll-like receptors (TLRs) were not discovered until quite re- cently. although they possibly represent the most important re ‘ceptor molecules of the non-adaptive component ofthe immune system. They are typical PRRs, which when bound together with PAMPS facilitate activation of the adaptive immune system in vertebrates, The name of these receptors is derived from se- uential homology with a protein coded by the Toll gene. This gene occurs in Drosophilla flies where it plays a role in em- bryogenesis and, in mature flies, helps in defense against fungal infection (Lemaitre etal, 1996; Bilak otal, 2003). TLRs are ‘transmembrane proteins with extracellular repetitive sequences, ‘ch in leucine, Thus far, approximately eleven TLRs are known, £8-Glucan (and also zymosan, intact yeast cells, and/or LPS) is initially bound to TLR-2 (Underhill al, 1999). Dectin-1 isa lectin located on the macrophage surface and has special involvement in the detection and phagocytosis of fun- ‘gal pathogens. In certain cases, it cooperates with the TLR-2, iis also a transmembrane protein with many particular func- tions, eg. binding of a fungal PAMP, uptake and killing of invading cells, and induction of the production of cytokines and ‘chemokines. ‘The complement receptor 3 (CR3, also denoted as integrin CDIIBICDIS, (M,2 end MACI)is one ofthe most promiscuous pattern-recognition receptors. In addition to comple-ment com= Ponents, it recognizes a large number of other ligands, among. them f-glucan, The CR3 receptor is ereated from several do: ‘mains; for saccharide recognition, itis a lectin domain (Ross etal, 1999) (Figure 2). When a fragment iC3b of complement is simoltancously bound, binding of -piucan triggers phagocy- tosis and degranulation, Lactosylceramide (also CDw17 or Gal4Glefl Cer) is gly- ccosphingolipid PRR located in the plasmatic membrane. 6 Giucan recognition mediated by lactosyleeramide causes dif- ferent cellular responses, including production of cytokines and respiratory burst (Brown, 2006). Examples of macrophage recepiors and the comesponding PAMPs are given in Table 7Downloaded By: (University of Colorado Health Sciences Center] At: 20:28 10 April 2008 ‘PROPERTIES OF GLUCANS 3 metal ion-dependent adhesion site B-propeller Divalent cation-binding) repeats sequence _———— Hdomain N-terminal region (part of B-propelter) C-terminal region eeseazs ee ES Bee GS PPR FIG. 2 Schmaticepresttion of CR showing is interned two-shanstucture an the major domain of CDIIb From Ross C.D ea mop rnaolgy, 2,61, 199. With permis, Binding of f-glucan toa receptor activates macrophages. The activation consist of several interconnected processes including imereased chemokinesis, chemotaxis, migration of macrophages toparticies tobe phagocyted, degranulation leading to increased expression of adhesive molecules on the macrophage surface, adhesion to the endothelium, and migration of macrophages 10 tissues. In addition, #-glucan binding also triggers intracellular processes, characterized by the respiratory burs after phagocy tosis of invading cells (formation of reactive oxygen species and Iree radicals - hydrogen peroxide, superoxide radical, NO, HOC! {hypocttorous acid), HOT [hypoiodous aid] ec), inereasing of ‘content and activity of hydrolytic and metabolic enzymes, and signaling processes leading to activation of other phagocytes ang secretion of eytokines and other substances initiating in flammation reactions (e.g, 1L-1, IL-9, tumor necrosis factor-a (TNFa), For an excellent review regarding the interaction of slucans with macrophages, see Schepetkin and Quinn (2006) Itisimportant forthe pharmacological effet of -glucan that activated macrophages do not act only agains the activator but, also against any present antigen, microorganism or tumor eel Due to the fact that mammals lack f-glucanases in thei en- zzyme equipment, macrophages represent what is probably the ‘only tool for liguidation of glucan in che body. Within the macrophages, phayocytized f-glucan is degraded by an oxida tive pathway (Nono etal, 1991) 6, SIDE EFFECTS While the predominant pharmacological effects of -glucans| may be postive, their unfavorable side effects cannot be TABLE? Examples of macrophage receptors and corresponding PAMPS Character Receptor transmembrane proveins CRS Primary PAMP iC3b opsonized particles, LPS, f-glucan, Candida albicans, Mycobacterium ‘uberculosis, Cryplococeus neoformans dectin-1 Saccharomyces cerevisiae, B-glucan, C albicans, Pneumocystis carinit Aspergillus fumigatus TLR2 lactosyleeramide scavenger receptors _slycosphingolipid glycoproteins bacterial lipoproteins, LPS, zymosan, B-glucan ‘glucan, C, neoformans, C. albicans, Helicobacter pylori Escherichia coli, Staphylococcus aureus, Listeria monocytogenes IM. tuberculasis, Neisseria meningitidis, Streptococcus spp., H. pylork LPS, bacterial DNA: a = 8 3 : : eB i e 3 ; i 4 NOVAK AND VETVICKA, overlooked. At this time, few adverse effects have been de seribed. Lean be presumed, however, that with improved know]- edge ofthe effects of B-glucans, this area will broaden, TIntramuscularly administered -glucan induces an inflam- matory reaction and granuloma formation atthe puncture sie This isa painful method of application. The fact that -glucan can be the cause of the inflammatory reaction itself represents 4 certain risk, Physiological inflammation occurs atthe extent and rate corresponding with an inducing noxa, and also to the ‘Arglucan present. If the noxious impact persists, a pathologi= cal inflammation ean take place, manifested by excessive tissue damage subsequently ending in immune disorders and devel- ‘opment of imimunopathological (e.g, autoimmune) processes In mos serious eases, generalization of inflammatory processes can ovcur with shock development and subsequent fatal mul liple organ dysfunction syndrome (MODS) (Tanriverdi et al., 2005), ‘The function of nitric oxide is double-edged. Nitric oxide, the "Molecule of Year, 1992” for which the discoverers of is physiological effects earned the Nobel Prize in Physiology and ‘Medicine in 1998, is produced in macrophages by inducible i= tic oxide synthase (NOS). Synthesis of this enzyme is triggered by binding of f-glucan to a PRR on the macrophage surface Formed nitric oxide induces a cytotoxic effect upon tumor cells (Hibbs et al, 1984; Stuehr and Nathan, 1989) and shows dis linet impact on many pathogens (Grangeer et al., 1988; James. and Claven, 1989).On the other hand, itean also damage tissues, and DNA (Billa, 1995) and high concentrations can cause septic shock, The sustained action of the activator induces expression, ‘of NOS, and increased formation of NO results in vaseditation ‘of veins. The later, in turn, brings about an intense drop of ve- nous resistance and blood pressure (Vincent et al, 2000). As of now; such an effect of glucan has not been described, yeti is quite conceivable. Inhalation of intact cells or cellular detritus of fungi or yeasts + ingredients of home dust (Beijer et al, 2002) oF different agricultural and industrial dusts (Rylander et al., 1999) ~ ine «duces so-called syndrome of toxic organic dust (STOD) whichis ‘characterized by lung reactions that include pneumonia, cough, chronic bronchitis (Sato and Sano, 2003), rhinitis, headache, and invitation of the eyes and throat (Allis etal, 1999). The cause of these complaints is B-glucan which, through activa: tion of macrophages, monoeytes, and leukocytes, causes in- creased secretion of inllammatory components (ie, TNFa and IL). Due to the fact that B-glucan administration brings about inflammatory processes inthe human body, its possible to hy pothesize competitive interaction with anti-inflammatory drugs Induced lethal ticity elicited by acombination of f-glucan and nonsteroidal ant-inflammatory drug (indomethacin), was de- scribed in mice (Yoshioka et al, 1998). Results strongly suggest that such a combination induces lethality by compromising the {eytokine network dha leads to systemic inflammation response syndrome (SIRS) and death, 7. POSSIBLE ALTERNATIVES OF FURTHER RESEARCH Different natural sources, polymer character, methods of iso- lation, insolubility oF limited solubility of preparations, (and thus unrealistic fractionation), al resulted in the fact that each preparation of f-glucan, especially the pariculate one, is nec- essarly heterogeneous. Even ifin some cases the heterogeneity of B-glucan (from the point of view of molecular size, branch- ing, and crystalline of amorphous structure) does not principally ‘change the in vivo activities, for trustworthy pharmacological re- search as well a for regulatory authorities (such asthe USFDA) it represents substantial complications It follows then that most preparations containing A-glucan ~ with the aim of avoiding ‘complications —are classified as healthy food or nutsitional sup- plements, Asa result, the markets saturated with many products containing greater or lesser amounts of f-glucan that are often of| questionable quality, with uncerain effects, and recommended by delusory advertisement Reliable research techniques allow the problems of hetero. {geneity and nonexistent standards of various natural A-glucans to be volved, A frst possibility is 10 improve isolation tech- niques to obtain products with closely defined chemical compo- sition and to use up-to-date physicochemical methods for infal- Iible identification and analyses of these produets. Chemically ‘modified glucans, which du to their solubility can be easily fractionated, represents. second possibility, Unfortunately, bio- logical activity of some ofthese products is decreased. Recently, ‘an attempt has been made to solve this problem by construction of semi-synthetic or synthetic probes that are suitable for ex= act immunological research, A general solution is the binding of shor oligomers of glucose containing f(1—3) and (+6) linkages to a polymer carrier of defined size and structure. A. reasonable assumption is that such “synthetic f-glucans will interact with receptors of immunocompetenteelisandelictanal- ‘ogous reactions as natural -glucan (Descroix etal, 2006), From the immunopharmacological point of view, such probes would eventually replace the natural -glucan, 8. CONCLUSION ‘Among the many thus far known and tested immunomod- tulators, polysaccharides isolated from various natural sources ‘occupy & prominent position. An important group of these polysaccharides is represented by homopolymers of f-glucose, Prglucans. These contain either linear chains with (I—»3)-B- Duglycusidie Tinkages or branched ones containing additional (1-+6)-B-o-glycosidic linkages. Due to their very low-to-negligible toxicity, there was a time when glucans were considered merely a matter of fashion, This cannot be said about many other immuno-modulators, EEtfeets of @-glucans on a variety of diseases, such as infec tions, iradiation diseases, and foremost on neoplastic growth, Were investigated. A flood of various food additives and " ternative remedies” usually offered by faintly enlightened non specialists, is @ holdover from these pioneer yeurs. After some