Immunochemical studies on the mannans of Candida albicans species, with special reference to the presence and function of β-1, 2-linked mannopyranose units have been reviewed in the following order: 1. Structural investigation of C. albicans mannans in earlier stage; 2. Detection of β-1, 2 linkage in C. albicans mannans; 3. Characterization of β-1, 2-linked mannopyranose units in mannans as integral part of epitopes; 4. Chemical structures of C. albicans mannans and those of antigenic factors containing β-1, 2-linked mannopyranose units; 5. Conclusion and future objectives.

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We have engaged in the extraction and purification of the epidermal extract with fungcidal nature, I will introduce the progress of the study to date, and also the basic polypeptides exist in various tissues are also discussed together with the possibility of the same kind of substance on the self-defense in the kingdom of animal other than humans.

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Immunopharmacological activities of fungal β-glucans are well recognized to be useful for the medical treatment of cancer and infectious diseases. These (1-3)-β-D-glucans possess several conformers, such as sol (random coil), gel (single and triple helixes), and insoluble. In this review, structure-activity relationships of these conformers were summarized. The glucans having appropriate branches showed significant antitumor activity in all conformers tested. However, activation of Mφ (in vivo and in vitro) and of complement by gel and insoluble conformers were significantly stronger than sol conformer. In the gel conformer, the triple helix conformer was a stronger activator of alternative pathway of complement and the single helix conformer was a stronger activator of classical pathway of complement. Activation of limulus lysates was also dependent on the molecular weight, branch and conformation. Considering these facts, it is suggested that β-glucans would be recognized by several distinct ways in the host immune system.

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We have established an antigenic formula for two serotypes and three subtypes of Saccharomyces cerevisiae by agglutination system with adsorption experiments. This antigenic formulation identifies five distinct antigenic factors or determinants that are found on various serotypes of S. cerevisiae. Serotype Ia and Ib have antigenic factors 1, 10, 18 and 18a and 1, 10, 18 and p, respectively. Serotype Iab has both antigenic factors 18a and p in addition to the common antigenic factors 1, 10 and 18. Serotype II has only the common antigenic factors 1 and 10. ¹H-n. m. r. spectrum (H-1 region) of the mannan of serotype Ia, almost free from phosphate, was characterized by the signal at δ5.127 which corresponded to the H-1 of 3-O-substituted α-D-Manp residue adjacent to terminal α (1-3) linkage of mannopentaose side chain, for the specific determinant of antigenic factor 18a. In contrast, phosphodiester mannan from serotype Ib released mannose and α-(1-3)-linked mannobiose by mild acid hydrolysis. The signals at δ5.422-5.461 and 5.284 disappeared, and the signal at δ5.160 was diminished in phosphomonoester mannan treated by mild acid hydrolysis. An immunochemical characterization of the antigenic factor p using agglutination inhibition test between factor p serum and serotype Ib cell ssuggests that the α-(1-3)-linked mannobiosyl-1-phosphate group is a part of the antigenic determinant for the factor p.

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The Saccharomyces cerevisiae och1 mutant shows a deficiency in the mannose outer chain elongation at the non-permissive temperature. We have cloned the OCH1 gene by complementation of temperature sensitive (ts) phenotype for growth. The OCH1 gene sequence predictsa 55kDa protein consisting of 480 amino acids. It contains four potential asparagine-linked (N-linked) glycosylation sites and three distinct regions; a short NH₂-terminal domain lacking a signal sequence, a single transmembrane domain and a long hydrophilic COOH-terminal domain. In vitro translation/translocation analysis revealed that the large COOH-terminal domain of the OCH1 protein is located at the luminal side of microsomal membranes with some sugar modification. The OCH1 protein was detected in yeast membrane fractions as four forms of 58-66kDa, which correspond to the size of a glycoprotein containing four N-linked sugar chains, the length of which is almost the same or slightly larger than the inner core (Man₈GlcNAc₂) formed in the endoplasmic reticulum (ER). Finally, the OCH1 gene was found to encode a novel mannosyltransferase which specifically transfers GDP-[¹⁴C]-mannose to the unique acceptor, the core like oligosaccharide of cell wall mannan accumulated in the och1 disruptant.

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Mannan and N-acetylchitohexaose from the cell well of bakers' yeast manifested antitumor activity against several mouse transplantable tumors and protective effects against microbial infections in mice. In order to clarify the underlying mechanism of immunoenhancing effect, we examined the differentiation of thymocyte, splenocyte and lymphocyte in mice administered with WNM or N-acetylchitohexaose by immunofluorescence assays. The ratio of L3T4/Lyt-2 double negative cells was decreased and L3T4 or Lyt-2 single positive cells was increased in thymus and the ratio of double negative cells was increased in lymphnode and spleen in the same mannan-treated mice. The ratio of L3T4⁺ and Lyt-2⁺ cells was increased in N-acetylchitohexaose-administered Meth A tumor-bearing mice. On the other hand, two conjugates of mitomycin C and the mannan or N-acetylchitohexaose exhibited a higher growth-inhibition ratio against several mouse syngeneic tumors. These conjugates were also found to kill the same tumor cells in vitro. Because a dextran-mitomycin C conjugate did not manifest any antitumor effect against tumors, it was postulated that interaction of the mannan and N-acetylchitohexaose moiety in the mannan-mitomycin C and N-acetylchitohexaose-mitomycin C conjugates with the mannose/N-acetylchitohexaose receptor locating the cell surface of immunocytes, and/or these tumor cells participated in the common initiating action steps in antitumor effects. The results of binding assay of ¹⁴C-labeled mannan-mitomycin C conjugate to tumor cells substantiate this concept.

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The prophylactic effect of NND-318 in polyethylene glycol 300 (PEG 300) solution and cream preparation was examined on experimental dermatophytosis in comparison with that of clotrimazole and bifonazole. Each agent was applied to the skin on the back of guinea pigs which were subsequently inoculated with Trichophyton mentagrophytes.
When animals were treated once with 1% NND-318 in PEG 300 solution 1, 2, 3 and 4 days before the inoculation, the number of infected loci with clinical symptoms was 0/12, 1/12, 0/12 and 2/12, respectively, and fungus-positive rates were 3, 7, 8 and 21%, respectively. Both parameters were significantly lower than those in the groups treated with 1% clotrimazole and 1% bifonazole in PEG 300 solution.
When animals were treated once with 0.5% and 1% cream preparations of NND-318 1-4 days before the inoculation, the number of infected loci with clinical symptoms was 1/12-8/12 and 0/12, respectively, with increasing time intervals between drug-pretreatment and fungal challenge, and fungus-positive rates were 26-59% and 0-13%, respectively. Both parameters were significantly or tended to be lower than those in the groups treated with 1% clotrimazole and 1% bifonazole creams, which formulations are already available on the market.
Since the prophylactic effect of NND-318 in both PEG 300 solution and cream preparation was better than that of reference agents, it is suggested that a sufficient amount of the antimycotic is retained in the skin tissue in an active form long enough to exert an antifungal effect.

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The in vitro antifungal activity of NND-318, a new topical imidazole, was studied in comparison with other imidazole antimycotics.
NND-318 exhibited potent activity against a wide variety of pathogenic fungi including yeasts, dermatophytes, dematiaceous fungi, dimorphic fungi, Aspergillus spp. and Penicillium spp. Especially, it was 1-64 times more potent against filamentous fungi including dermatophytes and dimorphic fungi than was clotrimazole. The in vitro activity of NND-318, like that of clotrimazole and bifonazole, became less potent with increase in inoculum size and the addition of serum. The activity of NND-318 was not much affected by change in pH, and became more potent with the addition of urea.

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MJR-1762, a gel formulation of miconazole newly developed for oral mycosis, was mycologically and histopathologically evaluated for experimental oral Candidosis in rats.
Sprague-Dawley rats receiving tetracycline in drinking water (TC-receiving rats) or rats of the same strain receiving subcutaneous cortisone acetate concomitant with tetracycline in drinking water (immunosuppressed rats) were orally infected with Candida albicans. Daily oral treatments with MJR-1762 containing 2% miconazole significantly reduced the average numbers of C. albicans (p<0.01) in the oral cavities of both TC-receiving and immunosuppressed rats. MJR-1762 at the same content of miconazole also reduced the average number of C. albicans (p<0.05) in the feces of TC-receiving rats after 1-week therapy. Mycelial penetration of C. albicans and inflammatory cell infiltration in the tongue epithelium were observed only in the immunosuppressed rats, and MJR-1762 containing 2% miconazole markedly prevented the penetration. Furthermore, miconazole was found to inhibit the germ tube formation of C. albicans enhanced in the presence of human saliva in vitro, and this was thought to be one of the therapeutic mechanisms of MJR-1762.

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Effect of NND-318 on the morphology of growing hyphae of Trichophyton mentagrophytes was observed by scanning and transmission electron microscopy. Microconidia of T. mentagrophytes were inoculated on a slide-culture and incubated at 27°C for 60 hours. In the control culture grown without the drug, the hyphae showed a smooth surface, straight extensions and consistent width. In the culture grown with NND-318 at concentrations ranging from 0.08 to 0.4ng/ml, scanning electron microscopy revealed that hyphae were wavy, curled and bent with wrinkled surface. In ultrathin sections, thickening of the cell wall, accumulation of electron denes granular structures formed within the cell wall, and the number of cytoplasmic lipid droplets were observed to increase with drug concentration. At concentrations ranging from 0.8 to 1.6ng/ml, hyphae were short in length and the surface was heavily wrinkled. At concentrations above 4ng/ml, germination of inoculated conidia was not observed. Similar morphological changes were seen in T. mentagrophytes treated with higher concentrations of bifonazole.
These results suggest that NND-318, at concentrations considerably lower than those of other imidazoles, is able to inhibit hyphal growth of T. mentagrophytes with thickening of cell wall, accumulation of electron dense granules formed in the cell wall and increased number of cytoplasmic lipid droplets.

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To clarify the mode of antifungal action of NND-318, a new imidazole antimycotic, the effect of the agent on ergosterol synthesis in Trichophyton mentagrophytes and Candida albicans was examined, and compared to that of bifonazole.
NND-318 was found to interfere with ergosterol biosynthesis by inhibition of sterol C-14 demethylation. In terms of drug concentrations exerting 50% inhibition of ergosterol biosynthesis, NND-318 was ≥25 times stronger than bifonazole.

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The in vitro postantifungal effect (PAFE) following exposure of Candia albicans to cispentacin and pradimicin (BMY-28864) for short periods of time was investigated. A turbidometric method was used to measure cell regrowth following exposure to different concentrations (0.313 to 20μ/ml) of the two drugs for 0.5, 1 and 2hr at 30°C and 37°C. The PAFE was determined by the difference in time (hr) required for growth of the control and test cultures to reach the 0.5 absorbance level after removal of the drug by dilution. The length of the PAFE was dependent upon the drug concentration, exposure time and temperature and the strain of C. albicans being assayed. Optimal PAFEs ranged from 3.7 to >17.0hr with cispentacin and from 1.7 to >21.0hr with pradimicin (BMY-28864).

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In vitro antifungal activity of NND-318, a new imidazole antimycotic, against clinical isolates obtained from patients with cutaneous mycosis was examined in comparison with that of bifonazole (BFZ). The clinical isolates tested were 112 isolates of dermatophytes including Trichophyton mentagrophytes (62 isolates), Trichophyton rubrum (47 isolates), Microsporum canis (2 isolates), Epidermophyton floccosum (1 isolate), and 40 isolates of Candida albicans.
MIC values of NND-318 for 112 isolates of dermatophytes distributed in a range of ≤0.0012-0.04μg/ml indicating that NND-318 had more potent in vitro activity than BFZ. MIC values of NND-318 for the isolates of C. albicans distributed in a wide range of 0.63-20μg/ml which were similar to those of BFZ (1.25-20μg/ml).
These results showed that in vitro antifungal activity of NND-318 against clinical isolates of dermatophytes and C. albicans was greater than or similar to that of BFZ.

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The administration of antileukemic drugs results in remarkable reduction in leucocytes, so that the opportunistic infection (especially Candida infection) are frequently observed in these cases. Recently the usefulness of rhG-CSF (G-CSF) administration has been reported to stimulate recovery from leucopenia. In this study, the effect of G-CSF administration on Candida antigen titer in serum and number of Candida in feces was examined in patients with leucopenia induced by antileukemic therapy. Relation to respiratory Candida infection was also investigated.
Candida antigen titer in serum and number of Candida in feces significantly (p<0.02) decreased, while leucocytes and neutrocytes significantly (p<0.01) increased following G-CSF administration. Respiratory Candida infection was reversed in proportion to the increases of leucocytes and neutrocytes, as well as by the lower number of Candida in feces. Thus, not only the counting of leucocytes and/or neutrocytes but also the determination of Candida antigen in serum or number of Candida in feces were useful in evaluating the effect of G-CSF administration in leukemic patients with leucopenia.

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The pathogenicity of two strains of Entomophthora exitialis (ATCC 32890 and its artificial mutant No. 1), one strain of Conidiobolus heterosporus (TACC 12941) and two strains of Basidiobolus haptosporus (MMC 68 and MMC 69) was examined experimentally using normal and diabetic BALB/c male mice (5-6 weeks, 20-25g).
Both normal and diabetic mice were inoculated intravenously with each fungus suspension consisting of conidia and zygospores. The mice were then sacrificed at predetermined intervals. Portions of each organ were inoculated on potato dextrose agar plates supplemented with chloramphenicol and incubated for 7 days at 27°C. The remaining organs were fixed in a 10% formalin solution, and histological sections were prepared by conventional procedures. They were stained by H & E or PAS.
Spores of E. exitialis ATCC 32890 and the mutant No. 1, and B. haptosporus MMC 68 and MMC 69 were recovered from the lungs of both groups of mice, while from other organs isolation was very rare.
Spores of C. heterosporus ATCC 12941 were not recovered from any of the organs examined.
Histopathologically, in the lungs of mice inoculated with E. exitialis ATCC 32890 and the mutant No. 1, a few zygospores were observed until the 21st day after inoculation. In general, these spores existed discretely in the alveolar tissues and their cellular reactions were very weak or entirely lacking. In the lungs of mice inoculated with C. heterosporus, a few conidia with or without cell infiltrates were observed in the sections 3 days after inoculation. In the lungs of mice inoculated with B. haptosporus MMC 68 and MMC 69, a few conidia and zygospores with or without cell infiltrates existed until the 14th day. Spores did not germinate during this experiment.
These results indicate that the pathogenicity of these fungi is very feeble.

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A 61-year-old Japanese woman was admitted to the hospital on December 13, 1988, because of severe brain contusion sustained in a traffic accident. She was unconscious, febrile, and sweating profusely. She was administered intravenous cefazolin sodium and prednisolone daily, and on the 10th day of hospitalization, clusters of pustules and crusts were observed on her back. Potassium hydroxide preparations of the crust specimens revealed numerous nonseptate branching mucoraceous hyphae. Biopsy specimens from the pustules showed abscess formed around partially destroyed hair follicles and numerous hyphal cross sections within the abscess. The causative organism was isolated from the pustules in pure cultures and identified as Rhizopus arrhizus Fischer. She died of respiratory failure on the 16th day; no autopsy was performed.
The cutaneous lesions in this patient were apparently primary cutaneous mucormycosis. Thirteen cases of cutaneous mucormycosis reported to date in Japan are reviewed.

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A turbidometric method was used to evaluate Candida albicans yeast cell growth and to quantitate the in vitro postantifungal effect (PAFE) after exposure to various concentrations of cispentacin and pradimicin (BMY-28864), alone and in combination with flucytosine, for 2hr at 30°C and 37°C. Initial MIC and FIC (fractional inhibitory concentration) assays were made to determine approximate drug concentrations to use in the PAFE studies. The PAFE was calculated by the difference in time (hr) required for growth of the control and test cultures to reach the 0.5 absorbance level following removal of the drug by dilution. A synergistic PAFE was evidenced with both cispentacin and pradimicin (BMY-28864) when combined with flucytosine. Optimal PAFEs ranging from 5.7 to 18.5hr were induced by concentrations of cispentacin (0.078 to 0.313μ/ml) and flucytosine (0.049-0.098μg/ml) and from 3.3 to 11.7hr with pradimicin (BMY-28864) (0.078 to 0.313μg/ml) and flucytosine (0.049-0.098μg/ml). The PAFEs persisted for 1.9 to 4.8hr (cispentacin+flucytosine) and for 0.3 to 2.9hr (pradimicin-BMY-28864+flucytosine) longer than those achieved with either agent alone.

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