Relationship between solubility of grifolan, a fungal 1,3-beta-D-glucan, and production of tumor necrosis factor by macrophages in vitro.

Grifolan, GRN, is a fungal antitumor beta-glucan isolated from Grifola frondosa. Various studies suggested that the underlying mechanism of the antitumor activity of GRN is strongly related to immune modulation. In the previous publication (Adachi et al., 1994; Okazaki et al., 1995), we have shown that GRN activates macrophages to produce tumor necrosis factor (TNF) in vitro. In this study, the structural unit essential to produce TNF was examined by chemical modifications of GRN. GRN suspended in distilled water was treated at 150 degrees C for up to 3 h. Addition of the resulting turbid solution to the RAW 264.7 macrophage-like cell line produced TNF, and the relative activity was diminished in relation to the heat treatment period. The fractions with a heating period longer than 15 min did not show any activity. After centrifugation of the resulting solution, significant activity was shown by precipitate fractions, suggesting that the insoluble form of GRN is important for TNF production. Interestingly, the precipitate fraction obtained from 9 min of treatment also had significant activity. In addition, admixing the soluble fraction with the particles significantly inhibited the TNF production. In contrast to these observations, the high-molecular-mass subfraction of the soluble fraction prepared by ultrafiltration produced significant amounts of TNF. Similar phenomena were shown with sodium hydroxide treatment and dimethylsulfoxide treatment. These facts strongly suggested that insoluble as well as a high molecular mass soluble form of GRN are required for TNF production by macrophages.

Association of immunological disorders in lethal side effect of NSAIDs on beta-glucan-administered mice.

(1-->3)-beta-D-Glucan (beta-glucan) is a biological response modifier that regulates host immune response. We have found that the combination of a beta-glucan and a non-steroidal anti-inflammatory drug (NSAID), indomethacin (IND), induced lethal toxicity in mice [Yoshioka et al. (1998) FEMS Immunol. Med. Microbiol., 21, 171-179]. This study was undertaken to analyze the mechanism of the lethal side effect. Combination of a beta-glucan and IND increased the number of leukocytes, especially macrophages and neutrophils, in various organs and these cells were activated. The activated state of these cells was supported by the enhanced production of interferon-gamma in the presence of IND in vitro culture of the peritoneal exudate cells. Intestinal bacterial flora was translocated into the peritoneal cavity in these mice to cause peritonitis. Comparing the toxicity of various NSAIDs, nabumetone, a partially cyclooxygenase-2-selective NSAID with weaker toxicity to the gastrointestinal tract, did not exhibit a lethal side effect. These facts strongly suggested that gastrointestinal damage by NSAIDs was more severe in beta-glucan-administered mice, resulting in peritonitis by enteric bacteria and leading to death.

Th1/Th2-Balancing immunomodulating activity of gel-forming (1-->3)-beta-glucans from fungi.

The immunomodulating effects of various gel-forming (1-->3)-beta-glucans, grifolan (GRN), SSG, sonifilan (SPG) and alkaline-treated SPG (SPG-OH), on balancing helper T cell activity were examined in a murine model. Plasma from mice that were injected with GRN or SPG-OH and trinitrophenyl ovalbumin (TNP-OVA) contained TNP-specific antibodies of both IgG1 and IgG2a isotypes. Administration of SSG and TNP-OVA significantly augmented the synthesis of IgG2a antibodies, while the synthesis of IgG1 was reduced. However, SPG did not enhance the antibody response. In the culture supernatants of splenocytes obtained from GRN- or SPG-OH-administered mice, high levels of IgGI and low levels of IgG2a and IFN gamma were detected. In contrast, high levels of IgG2a and IFN gamma and low levels of IgGI were detected in the case of administration of SSG. Furthermore, it was shown by intracellular cytokine staining that the proportion of IFN gamma+CD4+ double-positive cells among the CD4+ cells from mice administered SSG was most strongly increased by addition of PMA and A23187. On the other hand, the expression of IL-12 p40 mRNA was more markedly elevated in splenocytes after combined administration of TNP-OVA plus SSG than after administration of TNP-OVA alone. The highest IFN gamma production was observed when adherent cells of mice administered TNP-OVA and SSG were cultured with TNP-primed lymphocytes. This effect of administration of SSG on IFN-y production was completely inhibited by addition of anti-IL-12 mAb. In conclusion, our study showed that beta-glucans have various effects on the Th1 or Th2-dependent antibody subclasses, in particular, SSG induces the development of Th1 cells via the IL-12 pathway.

Antitumor beta glucan from the cultured fruit body of Agaricus blazei.

Agaricus blazei is a medically important mushroom widely eaten and prescribed in Japan. Polysaccharide fractions were prepared from cultured A. blazei by repeated extraction with hot water (AgHWE), cold NaOH (AgCA), and then hot NaOH (AgHA). By chemical, enzymic, and NMR analyses, the primary structures of AgHWE, AgCA, and AgHA were mainly composed of 1,6-beta-glucan. Among these fractions, the NaOH extracts showed antitumor activity against the solid form of Sarcoma 180 in ICR mice. To demonstrate the active component in these fractions, several chemical and enzymic treatments were applied. These fractions were found to be i) neutral beta-glucan passing DEAE-Sephadex A-25, ii) resistant to periodate oxidation (I/B) and subsequent partial acid hydrolysis (I/B/H), iii) resistant to a 1,3-beta-glucanase, zymolyase, before I/B, but sensitive after I/B/H. In addition, after I/B/H treatment of the neutral fraction of AgCAE, a signal around 86 ppm attributable to 1,3-beta glucosidic linkage was detectable in the 13C-NMR spectrum. These facts strongly suggest that a highly branched 1,3-beta-glucan segment forms the active center of the antitumor activity.

Role of plasma, lipopolysaccharide-binding protein, and CD14 in response of mouse peritoneal exudate macrophages to endotoxin.

Plasma lipopolysaccharide (LPS)-binding protein (LBP) and membrane CD14 function to enhance the responses of monocytes to low concentrations of endotoxin. Surprisingly, recent reports have suggested that LBP or CD14 may be dispensable for macrophage responses to low concentrations of LPS or may even exert an inhibitory effect in the case of LBP. We therefore investigated whether LBP and CD14 participated in the response of mouse peritoneal exudate macrophages (PEM) to LPS stimulation. In the presence of a low amount of plasma (<1%) or of recombinant mouse or human LBP, PEM were found to respond to low concentrations of LPS (<5 to 10 ng/ml) in an LBP- and CD14-dependent manner. However, tumor necrosis factor production (not interleukin-6 production) by LPS-stimulated PEM was reduced when cells were stimulated in the presence of higher concentrations of plasma or serum (5 or 10%). Yet, the inhibitory effect of plasma or serum was not mediated by LBP. Taken together with previous results obtained with LBP and CD14 knockout mice in models of experimental endotoxemia, the present data confirm a critical part for LBP and CD14 in innate immune responses of both blood monocytes and tissue macrophages to endotoxins.

Measurement of blood clearance time by Limulus G test of Candida-water soluble polysaccharide fraction, CAWS, in mice.

The Limulus G test, responsive to beta-1,3-D-glucan, is a well-established method for the detection of invasive fungal infection. We have recently found that Candida albicans released a water-soluble polysaccharide fraction (CAWS) into synthetic medium (Uchiyama et al., FEMS Immunol. Med. Microbiol. 24 (1999) 411-420). CAWS was composed of a mannoprotein-beta-glucan complex and activated Limulus factor G, and thus would be similar to the Limulus active substance in patient's blood. In a preliminary investigation, we have found that CAWS is lethal when administered intravenously in a murine system. In this study, we examined the toxicity and then the fate of CAWS in mice. The lethal toxicity was strain-dependent and strain DBA/2 was the most resistant. The toxicity was, at least in part, reduced by salbutamol sulfate and prednisolone treatment in the sensitive strains. On intravenous administration, the half clearance time (t1/2) was approximately 40 min in mice (DBA/2). On intraperitoneal administration, CAWS appeared in the blood with a peak concentration at 1 h. In order to establish a treatment plan, it is important to demonstrate the onset and the termination of deep-seated mycosis. The Limulus G test is suitable for the above purpose; however, it is necessary to fully understand the fate of beta-1,3-D-glucan in patients' blood.

Antitumor 1,3-beta-glucan from cultured fruit body of Sparassis crispa.

Sparassis crispa is an edible mushroom recently cultivable in Japan. Polysaccharide fractions were prepared from the cultured S. crispa by repeated extraction with hot water (SCHWE), cold NaOH (SCCA), and then hot NaOH (SCHA). HWE was further separated by 1 volume (SCHWE1v) or 4 volumes (SCHWE4v) of ethanol-precipitable fractions. By chemical, enzymic, and NMR analyses, the primary structures of SCHWE1v, SCCA, and SCHA were 6-branched 1,3-beta-glucan, having one branch in approximately every third mainchain unit. All of these fractions showed antitumor activity to the solid form of Sarcoma 180 in ICR mice with strong vascular dilation and hemorrhage reaction. These fractions also showed enhanced hematopoietic response to cyclophosphamide induced leukopenic mice following intraperitoneal or peroral administration.

Enhancement of IL-8 production from human monocytic and granulocytic cell lines, THP-1 and HL-60, stimulated with Malassezia furfur.

Previously, we reported that Malassezia furfur, causing systemic fungal infection, was taken up into human monocytic cell line, THP-1, in a concentration-dependent manner. This fact suggested that M. furfur could activate phagocytes, such as monocyte and polymorphonuclear leukocyte. Thus we examined cytokine mRNA expression from human monocytic and granulocytic cell line, THP-1 and HL-60, stimulated with M. furfur by using reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA. We chose IL-1alpha, IL-6, IL-8, IL-12 and TNF-alpha as primers for THP-1, and IL-1alpha, IL-6 and IL-8 for HL-60. M. furfur induced the expression of IL-8 mRNA from THP-1 and HL-60 following incubation for 3 h, and also induced IL-1alpha mRNA from HL-60, although this induction was weaker than that of IL-8 mRNA. Furthermore, opsonized M. furfur induced stronger expression of IL-8 mRNA in comparison with intact M. furfur. IL-8 production from THP-1 and HL-60 was enhanced in a concentration- and incubation time-dependent manner. These facts strongly suggested that M. furfur could activate phagocytes, and could induce inflammatory responses in systemic infection.

Solubilized cell wall beta-glucan, CSBG, is an epitope of Candida immune mice.

Antibody to beta-glucan is generally difficult to produce in mice. We have recently developed a protocol to obtain a soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction. CSBG is composed mainly of beta-(1-->3) and beta-(1-->6)-glucosidic linkages with a small amount of branch. In this paper, mice were immunized with Candida albicans and the specificity of the resulting sera to CSBG was examined by ELISA. Using CSBG coated plate, sera of the Candida immune mice showed higher reactivity than non-immune, normal mice and the reactivity was neutralized by adding soluble CSBG as a competitor. However, the reactivity could not be neutralized by a beta-(1-->6) branched beta-(1-->3)-glucan, grifolan. Similar specificity of the sera was obtained by commercially available beta-glucan particle, zymosan or zymocel, immune mice. These facts strongly suggested that CSBG included epitopes of the specific antibody in Candida immune mice.

[Structure and biological activities of fungal beta-1,3-glucans].

This paper summarizes the structure, biological activities, signaling, and metabolic degradation of fungal beta-1,3-glucans. Fungal beta-glucans exist both soluble and particulate forms. Conformation of the soluble beta-glucan was classified into three groups: triple helix, single helix and random coil. Fungal beta-1,3-glucans exhibit a variety of biological and immuno-pharmacological activities, and the significance of these activities is dependent on the structure, such as solubility in water, molecular weight, degree of branching, and conformation. Many of the activities, such as nitrogen oxide synthesis of macrophage and limulus factor G activation, were dependent on the conformation, but some of the others were independent, such as interferon-gamma and colony stimulating factor syntheses. These activities are beneficial and pharmacologically useful, while some strongly related to allergic and inflammatory adverse reactions. It should be noted that the beta-glucans, once administered into the body, remain mainly in the liver and spleen for a very long period of time. The activity, at least in part, is maintained during these periods. The biochemical mechanisms of the beta-glucan, especially in soluble form, mediating biological activity was still not clearly demonstrated.

Modulation of calcium-induced clot formation of human plasma by Malassezia furfur.

The effect of Malassezia furfur on clot formation by human plasma was examined. The clotting time in the presence of M. furfur or Candida albicans was measured. M. furfur shortened the clotting time of calcium-induced clots by human plasma in a concentration-dependent manner. However, the protein content of the clots formed were not significantly different between the M. furfur-treated and the control group. The clotting time of clots triggered by thrombin or by placing plasma in glass tubes, which artificially activate the blood coagulation systems, were not affected by treatment with M. furfur. Moreover, acetone-treated M. furfur also shortens the calcium-induced clot formation time, while treatment with zymolyase, which causes decomposition of beta-glucan, did not shorten it. These results suggest that M. furfur activates the blood coagulation systems, and the beta-glucan portion of M. furfur plays a key role in shortening calcium-induced clot formation time.

Failure in antitumor activity by overdose of an immunomodulating beta-glucan preparation, sonifilan.

Schizophyllan (SPG, Sonifilan) is a soluble (1-->3)-beta-D-glucan, used as a biological response modifier (BRM) with radiation therapy for cancer treatment in Japan. The mechanism of SPG mediated antitumor activity is thought to be via immune stimulation, which includes cytokine production, hematopoietic response, and so on. In this paper, we found that the activity of SPG was quite long-lived and an overdose significantly failed to display the antitumor activity. To demonstrate the mechanism several parameters were examined using a high dose of SPG administration as follows: i) the effect on vascular permeability in vivo, ii) the priming effect on tumor necrosis factor (TNF-alpha) production in vivo, iii) the effect on macrophage adherence to plastic plate in vitro, and iv) anti-Sarcoma 180 antibody production in vivo. It was evident that vascular permeability and anti-Sarcoma 180 antibody production remained unchanged, but TNF-alpha production and adherence to a plastic plate was significantly reduced by a high dose of SPG. These facts strongly suggested that modulation of the cytokine syntheses and the leukocyte traffic would be the causative mechanisms of the failure of antitumor activity by an overdose of SPG.

Immunopharmacological and immunotoxicological activities of a water-soluble (1-->3)-beta-D-glucan, CSBG from Candida spp.

We have established a convenient, two-step procedure to solubilize the yeast cell wall (1-->3)-beta-D-glucan using the combination of NaClO oxidation and DMSO extraction. Candida soluble beta-D-glucan (CSBG) was mainly composed of a linear beta-1,3 glucan with a linear beta-1,6-glucan moiety. In this study, we screened for several immunopharmacological activities of CSBG and found the following activities: (1) interleukin-6 synthesis of macrophages in vitro; (2) antagonistic effect for zymosan mediated-tumor necrosis factor synthesis of macrophages; (3) augmentation for lipopolysaccharide mediated tumor necrosis factor and nitrogen oxide syntheses of macrophages; (4) activation of alternative pathway of complement; (5) hematopoietic response on cyclophosphamide induced leukopenia; (6) the antitumor effect on ascites form tumor; (7) Enhanced vascular permeability; (8) priming effect on lipopolysaccharide triggered TNF-alpha synthesis; and (9) adjuvant effect on antibody production. These results strongly suggested that CSBG possessed various immunopharmacological activity.

Amplitude and frequency modulation of metabolic signals in leukocytes: synergistic role of IFN-gamma in IL-6- and IL-2-mediated cell activation.

Many stimuli cause intracellular concentration oscillations of second messengers or metabolites, which, in turn, may encode information in their amplitudes and frequencies. We now test the hypothesis that synergistic cellular responses to dual cytokine exposure correlate with cross-talk between metabolic signaling pathways of leukocytes. Polarized RAW264.7 macrophages and human neutrophils and monocytes exhibited NAD(P)H autofluorescence oscillation periods of congruent with20 s. IFN-gamma tripled the NAD(P)H oscillatory amplitude for these cells. Although IL-6 had no effect, incubation of cells with IFN-gamma and IL-6 increased both oscillatory amplitude and frequency. Parallel changes were noted after treatment with IFN-gamma and IL-2. However, IL-1beta and TNF-alpha did not display frequency doubling with or without IFN-gamma exposure. To determine whether frequency doubling required complete IFN-gamma signaling or simply metabolic amplitude modulation, an electric field was applied to cells at NAD(P)H troughs, which has been shown to enhance NAD(P)H amplitudes. Electric field application led to frequency doubling in the presence of IL-6 or IL-2 alone, suggesting that amplitude modulation is crucial to synergism. Because NADPH participates in electron trafficking to NO, we tested NO production during cytokine exposure. Although IL-6 and IL-2 alone had no effect, IFN-gamma plus IL-6 and IFN-gamma plus IL-2 enhanced NO release in comparison to IFN-gamma treatment alone. When NO production was examined for single cells, it incrementally increased with the same phase and period as NAD(P)H. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy and entrain NO production.

Chemical and immunochemical characterization of limulus factor G-activating substance of Candida spp.

The limulus test is a well-established method for the diagnosis of both gram (-) sepsis and invasive fungal infection. To diagnose deep-seated fungal infections, a (1-->3)-beta-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. It is suggested that the limulus reactive substance was released from the fungi to the blood, however, its chemical properties were not precisely examined in detail because of the limited quantity available. In this study, we used chemically defined liquid medium to culture Candida spp. and collected the water soluble fraction, CAWS. The yield of CAWS was circa 100 mg/l, independent of the strain of Candida. CAWS reacted with limulus factor G (Fungitec G test MK) at concentrations as low as 100 ng/ml. Limulus factor G reactivity of CAWS was sensitive to (1-->3)-beta-glucanase, zymolyase and was, at least in part, bound to ConA-agarose. The ConA-bound fraction also reacted with anti-beta-glucan antibody. CAWS is mainly composed of mannan and (1-->6)-beta-glucan, in addition to protein, assessed by 1H-NMR spectroscopy. CAWS also reacted with typing sera of Candida spp., specific for cell wall mannan. Chemical, immunochemical and biochemical analyses of CAWS strongly suggested that the limulus factor G-activating substance was a mannan-beta-glucan complex, present within the architecture of the yeast cell wall.

Solubilization of yeast cell-wall beta-(1-->3)-D-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction.

The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a beta-(1-->3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The beta-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of beta-(1-->3)- and beta-(1-->6)-D-glucosidic linkages. The specific activity of the beta-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.

Antigen-specific response of murine immune system toward a yeast beta-glucan preparation, zymosan.

Zymosan, a particulate beta-glucan preparation from Saccharomyces cerevisiae, shows various biological activities, including anti-tumor activity. We have previously shown that soluble beta-glucan initiated anti-tumor activity was long-lived and was effective even by prophylactic treatment at 1 month prior to tumor challenge. However, the activity by zymosan was relatively short-lived. Antigen-specific responses of mice to zymosan might be a causative mechanism. In this paper, mice were immunized with zymosan and antibody production and antigen-specific responses of lymphocytes to zymosan were analyzed. Sera of zymosan immune mice contained zymosan-specific IgG assessed by enzyme-linked immunosorbent assay and FACS. Spleen and bone marrow cells of zymosan-immune mice showed higher cytokine production in response to zymosan. Specificity of zymosan-specific responses were also analyzed using various derivatives prepared from zymosan. These facts strongly suggested that mice recognize zymosan as antigen in addition to non-specific immune stimulant.

Inflammatory and immunopharmacological activities of meta-periodate oxidized zymosan.

Zymosan (ZYM), a strong complement-activating yeast cell preparation composed mainly of mannan and beta-glucan moieties, is a potent inflammatory substance with immunopharmacological activity. We previously analyzed the metabolism of ZYM in mice and found that it was deposited in liver and spleen for at least several months and then gradually oxidatively degraded. In this paper, we prepared oxidized ZYM by sodium metaperiodate oxidation (NaIO4) and borohydride reduction (I/B-ZYM) and/or limited hydrolysis of oxidized moieties (I/B/H-ZYM). Activities of the resulting products were assessed by (i) vascular permeability in mice, (ii) H2O2 synthesis by macrophages, (iii) TNF-alpha synthesis by macrophages, and (iv) reactivity to anti-ZYM sera. As a general trend, NaIO4, oxidation products exhibited reduced, but still significant, activity. Interestingly, the H2O2 production induced by I/B/H-ZYM was significantly reduced after extensive sonication. Antagonist(s) for H2O2 synthesis were concomitantly solubilized by sonication of I/B/H-ZYM. On the contrary, TNF-alpha production induced by I/B/H-ZYM was comparable with that of ZYM. These facts strongly suggest that highly branched 1,3-beta- and 1,6-beta-glucosidic linkages resistant to NaIO4 oxidation are important for biological activity of ZYM. Further, the minimal structure in ZYM necessary for biological activity may depend on the activity tested.

Increase of hematopoietic responses by triple or single helical conformer of an antitumor (1-->3)-beta-D-glucan preparation, Sonifilan, in cyclophosphamide-induced leukopenic mice.

It has been suggested that the immunopharmacological activity of soluble (1-->3)-beta-D-glucan depends on its conformation in mice. In this study, we examined the relationship between the conformation of Sonifilan (SPG) and hematopietic responses in cyclophosphamide (Cy)-induced leukopenic mice. SPG, a high molecular weight (1-->3)-beta-D-glucan, has a triple helical conformation in water, and it was changed by treatment with aqueous sodium hydroxide to the single helical conformer (SPG-OH). The effects of SPG or SPG-OH on hematopoietic responses in cyclophosphamide induced leukopenic mice were investigated by monitoring i) gene expression of cytokines by RT-PCR, ii) protein synthesis of interleukin 6 (IL-6) by ELISA and iii) colony formation of bone marrow cells (BMC). The mice administered Cy and SPG or SPG-OH expressed and produced higher levels of IL-6 mRNA and protein than the mice administered only Cy. Gene expression of NK1.1 was also induced by Cy/SPG (or SPG-OH) treatment. Induced gene expression of stem cell factor (SCF) and macrophage-colony stimulating factor (M-CSF) by SPG/SPG-OH were also found in in vitro culture of BMC from Cy treated mice. These results strongly suggested that conformation of the glucans, single and triple helix, are independent of the hematopietic response.

Cytokine synthesis of human monocytes stimulated by triple or single helical conformer of an antitumour (1-->3)-beta-D-glucan preparation, sonifilan.

It has been suggested that the immunopharmacological activity of soluble (1-->3)-beta-D-glucan depends on it's conformation in mice. In this study, we examined the relationship between the conformation of Schizophyllan (SPG), a high molecular weight (1-->3)-beta-D-glucan, and cytokine productivity in an in vitro human system. Monocyte-like human cell lines, THP-1 and U-937, and peripheral blood mononuclear cells (PBMC) were used. THP-1 and U-937 cells were differentiated by phorbol myristate acetate (PMA) before use. SPG usually has a triple helical conformation in water, but it was modified by treatment with aqueous sodium hydroxide to become a single helical conformer (SPG-OH). SPG or SPG-OH was added to the macrophage cell culture and gene expression and translation of several cytokines was analyzed by RT-PCR, ELISA, or bioassays. Differentiated THP-1 expressed high levels of cytokine genes, such as IL-8, in response to SPG-OH. High levels of IL-12 p70 were detected from THP-1 cells stimulated with SPG-OH. U-937 cells expressed high levels of IL-8 and TNF-alpha after SPG-OH treatment. Furthermore, PBMC isolated from healthy donors also strongly reacted with SPG-OH but not with SPG. High concentrations of TNF-alpha were detected in SPG-OH-stimulated PBMC cultures. These data suggest that the biological activities of SPG are strongly associated with its conformation in humans.