Candida albicans derived fungal PAMPS, CAWS, water soluble mannoprotein-beta-glucan complex shows similar immunotoxicological activity with bacterial endotoxin from Escherichia coli O9

Candida albicans water soluble fraction (CAWS), water soluble fraction of Candida albicans mainly composed of mannoprotein-beta-glucan complex, has various biological effects, such as anaphylactoid shock and coronary arteritis. These toxicological effects fit CAWS as one of PAMPs, pathogen-associated molecular patterns. Acute anaphylactoid reaction is known to be induced by lipopolysaccharide from Escherichia coli O9 (O9 LPS), which possesses the mannose homopolysaccharide as the O-antigen region. In the present study, we compared immunotoxicological and immunochemical similarity between CAWS and O9 LPS. CAWS strongly reacted with Candida serum factors, and the reactivity was found to be partially competed with O9 LPS. CAWS induced lethal toxicity was inhibited by pretreatment of mice with i.v. injection of CAWS. The lethality was found to be inhibited by i.v. injection of O9 LPS. Vice versa, O9 LPS induced acute lethal toxicity was also inhibited by pretreatment of mice with CAWS. These results suggested that CAWS, fungal PAMPs, and O9 LPS from Gram-negative bacteria share, at least in part, immunochemical and immunotoxicological activities.     

Involvement of platelet activating factor, histamine and serotonin in acute lethal shock induced by Candida albicans water-soluble extracellular polysaccharide fraction (CAWS) in mice

CAWS (Candida albicans water-soluble extracellular polysaccharide fraction) is a water-soluble extracellular mannoprotein-beta-glucan complex obtained from the culture supernatant following the culture of pathogenic Candida albicans in a completely synthetic medium. CAWS administered intraperitoneally induces vasculitis in mice, however, administered intravenously, it causes lethal shock. The acute lethal reaction to CAWS occurs within 1 h of intravenous administration, with the mice demonstrating anaphylactic shock-like symptoms including convulsion, diarrhea, and collapse. In this study, we analyzed the factors involved in this lethal effect. We examined physiologically active substances believed to be involved in anaphylactic shock, and found that the lethal effect of CAWS could be inhibited by blocking histamine, serotonin, and platelet activating factor (PAF) simultaneously, but by blocking only one. This finding strongly suggests that the acute lethal reaction to CAWS is a result of the simultaneous production of several physiologically active substances.     

Histopathological examination and analysis of mortality in DBA/2 mouse vasculitis induced with CAWS, a water-soluble extracellular polysaccharide fraction obtained from Candida albicans

CAWS, a water-soluble extracellular polysaccharide fraction obtained from the culture supernatant of Candida albicans, is one of the fungal pathogen-associated molecular patterns (PAMPs). It has been reported to show potent activity inducing arteritis and coronaritis in mice. Especially, CAWS-induced arteritis has a 100% incidence and severe mortality in the DBA/2 mouse strain. This artificial vasculitis was reported to provide a good murine model of Kawasaki disease and other inflammatory vascular disease. However, severe mortality was observed only in DBA/2 mice, which is a CAWS-sensitive strain. In this study, to clarify the mechanisms of CAWS-induced arteritis and mortality, we investigated microscopic histopathological changes in cardiovascular tissues in DBA/2 mice. Severe inflammatory infiltration was observed from the external elastic lamina in the aorta and proximal coronary arteries within 1 week after CAWS administration. Severe stenosis of the aorta and coronary arteries was observed more than 3 weeks after CAWS administration. Fibrinoid necrosis was observed in these vessel walls. All CAWS-treated mice died between the fifth and twelfth week after administration. Severe inflammatory change with aortic valve transformation suggested that CAWS-treated mice died of valvular endocarditis or cardiac dysfunction. Based on the simple induction method and complete incidence, these data suggest that CAWS-induced arteritis is a good model of not only Kawasaki disease but also other cardiovascular diseases such as valvular endocarditis.     

Effect of CAWS,a mannoprotein-beta-glucan complex of Candida albicans,on leukocyte,endothelial cell,and platelet functions in vitro

Candida albicans is a medically important fungus which induces a disseminated candidasis and candidemia in immunocompromised hosts, and releases a polysaccharide fraction into the blood. We recently found that C. albicans released a water-soluble polysaccharide fraction (CAWS) into synthetic medium and demonstrated that CAWS was mainly composed of a complex of mannan and beta-glucan. In the murine system, CAWS showed a lethality resembling anaphylactic shock when administered i.v., and induced coronary arteritis similar to Kawasaki Disease (KD) when given i.p. In the present study, we examined the biological activity of CAWS in the cell culture and found the following: i) CAWS slightly induced production of IFN-gamma and IL-6 by splenocytes at lower dose (ca. 10 micro g/ml), but at a higher dose strongly inhibited the proliferation of splenocytes induced by a B cell mitogen, lipopolysaccharide (LPS) and a T cell mitogen, concanavalin A. ii) The viability of these splenocytes monitored by propidium iodide staining was significantly reduced. iii) The addition of CAWS to a culture of monophage RAW264.7 cells significantly reduced cellular growth rate dose dependently. iv) The LPS-mediated synthesis of cytokines by RAW264.7 cells was significantly inhibited by CAWS. v) CAWS induced an aggregation of platelets in human platelet-rich plasma, and vi) CAWS inhibited the production of thrombomodulin by human umbilical endothelial cells and acted synergistically with TNF-alpha. Thus, CAWS strongly inhibited the cellular functions of leukocytes in vitro, partly through direct cytotoxicity. The enhanced production in injured cells of the vascular endothelium would be related to the local inflammatory response in the coronary artery.     

A murine model of vasculitis induced by fungal polysaccharide

CAWS is a mannoprotein-beta-glucan complex obtained from the culture supernatant of the fungal pathogen Candida albicans. CAWS exhibits various biological activities, and induces prominent vasculitis of the aortic valve and the coronary arteries in mouse. A significant difference was noted in the susceptibility to and the degree of vasculitis induction among mouse lines. The difference in cytokine production among mouse lines may be strongly related to that difference, namely, IL-6, IFN-gamma and TNF-alpha presumably act as positive factors, and IL-10, as a negative regulator. On the other hand, as a structural component of the inducing substance, the presence or absence of beta-1,2-mannose residues was suggested to be closely related to the activity. An understanding of the molecular mechanisms underlying this model could lead to the conquest of many modern diseases. This model is also expected to be useful for the development of new therapeutic drugs for vasculitis and cardiovascular diseases.     

复方镰形棘豆凝胶剂的皮肤安全性评价

目的:制备一种外用复方镰形棘豆凝胶剂,并对其皮肤用药的安全性进行评价。方法:选用壳聚糖为凝胶基质制备复方镰形棘豆凝胶剂;用复方镰形棘豆凝胶剂考察豚鼠皮肤致敏性、家兔皮肤刺激试验、急性毒性试验和30 d 长期毒性试验。结果:复方镰形棘豆凝胶剂对豚鼠无致敏性,对家兔完整皮肤及破损皮肤无刺激;急性毒性试验中家兔体质量变化无差异,未见死亡及中毒反应发生;家兔连续给药30 d 后凝胶剂低中高剂量组对家兔的一般情况、体质量、脏器系数及病理组织、血液学指标、血液生化学指标均无明显影响,恢复期亦无延迟性毒性反应。结论:制备的复方镰形棘豆凝胶剂是一种安全性较高的外用制剂。     

Antibacterial effect of the culture fluid of Lentinus edodes mycelium grown in submerged liquid culture

The antimicrobial activity of the culture fluid of Lentinus edodes mycelium grown in submerged liquid culture was tested against some common bacterial species and Candida albicans. The mycelium-free culture fluid was bacteriostatic against Streptococcus pyogenes, Staphylococcus aureus and Bacillus megaterium. The substance responsible for the activity was heat-stable, could be extracted with chloroform and had a molecular weight under 10000. These characteristics suggested that the component might be lenthionine, an antibacterial and antifungal sulphur-containing compound. The culture fluid was less toxic to human tissue culture cells than to microbes. The antibacterial activity and toxicity could not be attributed to the same component.     

亚抑菌量多粘菌素B在大肠埃希氏菌和白色念珠菌混合感染小鼠中的作用

探讨亚抑菌量多粘菌素B对大肠埃希氏菌和白色念珠菌混合感染小鼠病情的影响。建立小鼠大肠埃希氏菌、白色念珠菌单独及混合感染模型，以亚抑菌量多粘菌素B拮抗内毒素活性，观察各组小鼠死亡率、血浆内毒素水平，肺组织中白色念珠菌菌落数及中性粒细胞杀菌能力。结果：大肠埃希氏菌和白色念珠菌混合感染组小鼠死亡率明显高于各自单独感染组（P＜0．05）；大肠埃希氏菌和白色念珠菌混合感染组24h后血浆内毒素水平明显高于大肠埃希氏菌单独感染组（P＜0．01）；大肠埃希氏菌和白色念珠菌混合感染组24h和48h后白色念珠菌菌落数明显高于白色念珠菌单独感染组（P＜0．01）；大肠埃希氏菌和白色念珠菌混合感染组小鼠中性粒细胞杀菌能力明显低于两者单独感染组（P＜0．01）；亚抑菌量多粘菌素B能明显降低混合感染组小鼠死亡率、血浆内毒素水平及肺组织中菌落数，并能增强混合感染组小鼠中性粒细胞杀菌功能（P＜0．05）。结论：亚抑菌量多粘菌量B可改善大肠埃希氏菌和白色念珠菌混合感染小鼠病情。     

Late acute,delayed neurotoxic and cholinergic effects of S,S,S-tributyl phosphorotrithioite (merphos) in hens

This study reports three toxicologic effects of S,S,S-tributyl phosphorotrithioite (merphos) in hens: acute cholinergic, late acute, or delayed neurotoxic. These effects can be differentiated by route of administration, dosage required, severity and reversibility of clinical signs, and production of pathologic lesions of the nervous system. Oral administration of a single dose (200 to 2000 mg/kg) of merphos caused weak cholinergic effects, which were rapidly relieved by atropine sulfate treatment. Four days after start of administration of large daily doses (80 mg/kg/day) a late acute effect was observed. The clinical signs of the late acute effects were identical to those produced by n-butyl mercaptan (nBM), a hydrolytic product of merphos, and were not relieved by atropine sulfate. The late acute effect overlapped with the clinical signs of delayed neurotoxicity. Late acute toxicity was not seen with topical application of single or daily doses of merphos; rather, delayed neurotoxicity was consistently produced. Degeneration of the anterior columns in the spinal cord was identical to that found in tri-o-cresyl phosphate-treated hens and was the most consistent histopathologic change. Topical administration of merphos caused a more prolonged inhibition of plasma butyrylcholinesterase activity than the orally administered compound. Orally administered merphos was rapidly metabolized in the gastrointestinal tract directly to nBM or following its oxidation to S,S,S-tributyl phosphorotrithioate. nBM apparently caused the late acute toxic effect. Topically administered merphos, which was not metabolized in the gastrointestinal tract, caused delayed neurotoxicity but did not produce a late acute effect. The present results demonstrate that evaluation of the neurotoxic effect of organophosphorus esters should include investigation with both oral and topical application.     

Characterization of the arteritis induced by infusion of rats with UK-61,260, an inodilator,for 24 h

 Administration of fenoldopam mesylate (FM), a dopaminergic agonist, or of cyclic cAMP phosphodiesterase inhibitors (PDE III), for example theophylline and caffeine, induces arteritis in the rat. In this study we characterized the arteritis induced by UK-61,260, an investigational inotropic agent with vasodilatory properties which displays an inhibitory action on cyclic AMP phosphodiesterase, in comparison with lesions induced by FM. The compounds were administered to Sprague-Dawley rats by intravenous infusion over 24 h (FM and UK-61,260), orally or subcutaneously (UK-61,260); the rats were killed and necropsied for pathological examination at various times between 0 h and 28 days post-infusion. Infusion of UK-61,260 at doses of 100, 300 or 400 mg/kg produced arteritis mainly in the mesenteric arteries and occasionally in the renal, pancreatic, gastric and coronary arteries. There were no arterial lesions after infusion of 30 mg/kg, or after administration of 30, 100 or 200 mg/kg per day subcutaneously for 7 days, or after acute administration of 100, 300, 400 or 600 mg/kg orally. Infusion of rats with 72 or 144 mg/kg FM produced arteritis over a wider range of tissues than did UK-61,260. However, the arterial lesions produced by infusion of either drug have the same initial aspect and a similar evolution with time. Immediately after the end of the infusion, minimal necrosis and haemorrhage occurred in the media only, without involvement of the endothelium or the perivascular space. This indicates that the media of the artery is the primary site of injury. The lesions seen 1 and 3 days post-infusion were characterized by severe medial necrosis and haemorrhage with perivascular acute inflammation and appeared macroscopically as haemorrhagic spots on the vessels. On days 7, 14 and 28 post-infusion, no medial necrosis or haemorrhage were present, while perivascular chronic inflammation and moderate smooth muscle hyperplasia were seen. It appeared, therefore, that the lesions underwent repair in 28 days, but footprints of the damage were still present 28 days post-infusion. The similarity between arteritis induced in rats by fenoldopam or by UK-61,260, at doses inducing PDE III inhibition, is consistent with the view that they have a similar pathogenesis. In our view it is probable that these pharmacologically and chemically distinct drugs trigger an increase in intracellular levels of cAMP which in turn triggers vascular damage. The arterial changes observed in the current study after acute administration may explain the increased incidence of polyarteritis nodosa occurring in long term toxicity studies with FM or PDE III inhibitors. Received: 21 October 1994 / Accepted: 13 March 1995     

磷酸川芎嗪气雾剂急性毒性及局部刺激性试验研究

目的观察磷酸川芎嗪气雾剂的急性毒性、黏膜刺激性。方法采用最大给药量试验观察鼻腔及腹腔给药后SD大白鼠短期内出现的急性毒性反应及其程度。观察一般情况与解剖显微镜下检查呼吸道黏膜局部刺激性反应。结果鼻腔及腹腔给药后SD大鼠的最大给药量为1%磷酸川芎嗪气雾剂1 mL,短期内未出现急性毒性反应及死亡情况;局部刺激性试验中大鼠一般情况良好,未发生毒副反应,显微镜下观察鼻、喉、气管等呼吸道黏膜未见损伤性变化。结论磷酸川芎嗪气雾剂鼻腔用药无明显毒性及刺激性,可供临床使用。     

Regulation of Candida albicans morphogenesis by tumor necrosis factor-alpha and potential for treatment of oral candidiasis

BACKGROUND: Endogenous tumor necrosis factor-alpha (TNF-alpha) has a beneficial effect as an activation mediator of host defense against infection by the fungus Candida albicans (C. albicans). However, it is unclear whether exogenous TNF-alpha has a beneficial or detrimental effect against Candida. MATERIALS AND METHODS: The direct effect of TNF-alpha on CO2-induced morphological transformation of C. albicans blastoconidia was examined in vitro and the effect of TNF-alpha was determined in a mouse model of oral candidiasis. RESULTS: TNF-alpha suppressed hyphal formation from C. albicans blastoconidia directly and dose-dependently, whereas it did not affect the fungal budding rate at concentrations ranging from 0.01 to 10 microg/ml. In vivo, the oral administration of TNF-alpha significantly reduced the C. albicans CFU in tongue tissues of treated mice. Histopathologically, there was a decrease in the number and size of C. albicans fungi in the tongue tissues. CONCLUSION: Since orally administered TNF-alpha suppressed fungal burden in the tongue tissue without significant detrimental effects, TNF-alpha has potential as a therapeutic agent against Candida.     

Fungal diseases of vulva and vagina caused by Candida species

Fungal diseases of vulva and vagina attributed to Candida species (vulvovaginal candidosis) are the most frequent mycoses of women. They show acute or chronic courses and different disease patterns which can strongly affect the quality of life of the women who are concerned. In general, the most common cause of acute vulvovaginal candidosis is Candida albicans, followed by C. glabrata. In chronic recurrent vulvovaginal candidosis, C. albicans and C. glabrata are often equally distributed. In several cases, treatment requires an antimycotic therapy which refers to the severity and main form of disease as well as to the aetiological agent. Most vulvovaginal candidoses are accessible to the treatment with local and systemic antimycotic agents. Generally, in Germany azoles such as clotrimazole, fluconazole and itraconazole, the polyens nystatin and Amphotericin B and the hydroxypyridone derivative ciclopirox are available for antimycotic therapy of vulvovaginal candidoses. Significance of non-conventional and adjuvant therapeutic approaches is considered to be generally low.     

Antimycotic 2-alkyldithio, 2-aralkyldithio, 2-aryldithiobenzamides

Some 2-alkyldithio, 2-aralkyldithio, 2-aryldithio benzoic acids, their methyl esters and N-monosubstituted amides were prepared and tested in vitro against Candida albicans and Trichophyton mentagrophytes. Some N-monosubstituted amides displayed activities similar to those of clotrimazole and pyrrolnitrin. Against Candida albicans, N-monosubstituted amides exhibited a generally higher activity than the corresponding N-monosubstituted amides of 2,2'-dicarboxydiphenyldisulfide.     

Lactobacillus helveticus HY7801 ameliorates vulvovaginal candidiasis in mice by inhibiting fungal growth and NF-κB activation

The anti-inflammatory effects of hydrogen peroxide-producing lactic acid bacteria (LAB) against Candida albicans-induced vulvovaginal candidiasis in β-estradiol-immunosuppressed mice were examined. Oral and intravaginal treatment with these LABs significantly decreased the level of viable C. albicans within the vaginal cavity as well as the quantitated myeloperoxidase activity in the vaginal tissues when compared with control untreated mice. Out of all of the LABs tested, Lactobacillus helveticus HY7801 (LH) most potently inhibited vulvovaginal candidiasis. LH also inhibited the expression of the pro-inflammatory cytokines including TNF-α, IL-1β and IL-6, and inflammatory enzymes, COX-2 and iNOS, as well as the activation of NF-κB. However, the addition of LH led to an increase in IL-10 cytokine expression in the vaginal tissues. In addition, the decrease of Lactobacillaceae and the increase of Pasteurellaceae caused by treatment with C. albicans were reversed with oral and intravaginal administration of LH, suggesting a potential shift in the vaginal microflora present. Addition of LH was toxic to C. albicans in vitro when cultured with HeLa cells. Oral administration of LH inhibited lipopolysaccharide (LPS)-induced TNF-α and IL-1β expressions in β-estradiol-immunosuppressed mice but reversed the expression of anti-inflammatory cytokine IL-10 in comparison to levels observed in the normal control group. LH also inhibited the expression of the pro-inflammatory cytokines, TNF-α and IL-1β, and the activation of NF-κB in LPS-stimulated peritoneal macrophages. Based on these findings, LH may ameliorate vulvovaginal candidiasis by suppressing the NF-κB pathway, as well as through inhibition of the growth of C. albicans.     

BE-31405, a new antifungal antibiotic produced by Penicillium minioluteum. I. Description of producing organism, fermentation, isolation, physico-chemical and biological properties

A new antifungal antibiotic, BE-31405, was isolated from the culture broth of a fungal strain, Penicillium minioluteum F31405. BE-31405 was isolated by adsorption on high porous polymer resin (Diaion HP-20), followed by solvent extraction, precipitation and crystallization. BE-31405 showed potent growth inhibitory activity against pathogenic fungal strains such as Candida albicans, Candida glabrata and Cryptococcus neoformans, but did not show cytotoxic activity against mammalian cells such as P388 mouse leukemia. The mechanism studies indicated that BE-31405 inhibited the protein synthesis of C. albicans but not of mammalian cells.     

Relationship between the physical properties of Candida albicans cell well beta-glucan and activation of leukocytes in vitro

We previously reported that the fungal particle 1,3-beta-D-glucan derived from Candida albicans, a pathogenic fungus, was obtained by oxidation of the cell wall with sodium hypochlorite (NaClO). It could be solubilized by treatment with dimethylsulfoxide (DMSO). In the present study, we prepared Candida 1,3-beta-D-glucan having different physical properties, and examined the relationship between leukocyte activation and the physicochemical properties. Beta-glucan activated leukocytes significantly more effectively in a particulate than solubilized form in terms of TNF-alpha production by RAW 264.7 cells, hydrogen peroxide production by murine PEC and IL-8 production by human PBMC. Furthermore, we compared the biological activity of the glucan particles oxidized under various conditions. Interestingly, inactive and antagonistic particles were obtained under strong oxidation conditions. However, the inactive particles showed significant agonistic activity on dissolution in DMSO and following lyophilization. These facts strongly suggested that the solubility and assembly of the components influence the immunopharmacological activities of 1,3-beta-D-glucans.     

New beauvericins, potentiators of antifungal miconazole activity, Produced by Beauveria sp. FKI-1366. I. Taxonomy, fermentation, isolation and biological properties

Three new beauvericins, designated beauvericins D, E and F, were isolated along with known beauvericin and beauvericin A, from the culture of Beauveria sp. FKI-1366 by solvent extraction, ODS column chromatography and HPLC. These compounds potentiate miconazole activity against not only wild Candida albicans but also fluconazole resistant C. albicans. Beauvericins D and E decreased the IC50 value of miconazole against fluconazole resistant C. albicans from 1.3 microM to 0.25 and 0.31 microM, respectively.     

Antifungal mechanism of hinokitiol against Candida albicans

The growth of Candida albicans was dose-dependently inhibited by addition of hinokitiol. The sensitivity of C. albicans to hinokitiol under aerobic conditions was higher than that under anaerobic conditions. Amount of ATP in C. albicans was not inhibited by hinokitiol under both conditions. The expression of mRNAs related to the growth signal, CYR1 and RAS1, was inhibited by hinokitiol. These findings suggested that the growth inhibition of C. albicans by hinokitiol was due to the interruption of RAS-signal transmission, such as the cAMP pathway.     

Antifungal properties of 2-n-alkyn-1-ols

Fourteen 2-n-alkynols (C3-C14, C16, and C18) were tested against Aspergillus oryzae, Aspergillus niger, Trichoderma viride, and Myrothecium verrucaria in Sabouraud dextrose agar at pH 5.6 and 7.0. Toxicity to Candida albicans, Candida tropicalis, Trichophyton mentagrophytes, and Mucor mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. Fungitoxicity was strongly influenced by chain length, slightly by the pH of the medium, and significantly by the presence of beef serum. 2-n-Undecyn-1-ol was the most active member of the series, and there was marked synergism between it and ketoconazole.