A total of 7, 283 autopsies on patients with malignant tumors were performed at the National Cancer Center Hospital from 1962 to December 1990. Among the malignancies, patients with opportunistic infections totaled 3, 179 (43.6%). The list was topped by bacterial pneumonia, which accounted for 77.2% (2, 490 cases) of all opportunistic infections and was followed by fungal infections (12.1% or 388 cases). Candidiasis accounted for 71.4% of fungi, aspergillosis for 19.1%, cryptococcosis for 6.9%, and mucormycosis for 2.6%.
The organs affected varied with the type of fungus. With candidiasis, the esophagus, stomach, lungs, small intestine, and oral cavity were prone to involvement. Systemic infection was also not uncommon. Other fungal infections were found most frequently in the lungs. The malignant tumors predisposing to fungal infections varied, but hemopoietic tumors generally accounted for the large group, constituting 32.5% of all patients with candidiasis. Predisposing factors included radiotherapy, chemotherapy, and a decrease in immunological competence due to general deterioration.

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[in Japanese]

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Serum mannan-binding protein (MBP) is a calcium dependent lectin specific for mannose and N-acetylglucosamine and was first isolated by our group from rabbits and subsequently from human and rats. Analysis of the human serum-MBP and its cDNA clones reveals that the protein is divided into four domains: a cyctein-rich NH2-terminal domain that cross-link the subunits each other, the second collagen like domain which consisted of twenty repeats of a Gly-X-Y sequence, and a third neck domain located between the second collagen like domain and a fourth COOH-terminal carbohydrate-recognition domain, the sequence homology of which is markedly high among calcium dependent mammalian lectins. The lectin has an apparent molecular mass of 600, 000 daltons consisting of multimers of a single subunit of around 31, 000 daltons. MBP has a role in host defense; this is suggested first by our finding that the lectin can activate the complement system through the classical pathway, and by the demonstration by Ezekowitz's group that the lectin inhibits in vitro infection by the human immunodeficiency virus and stimulates phagocytosis of bacterium with mannose residues on their surfaces by neutrophiles and macrophages. The physiological importance of MBP is confirmed by the finding that low serum MBP concentrations are associated with a common opsonic defect, which is found in 5-7% of the general population.

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Experimental dermatophytoses, which mimic the skin manifestations of human dermatophytoses may be good animal models to elucidate the histopathogenesis of the characteristic features of the human dermatophytoses such as “athlete's foot” and “ring worm”. In the experimental models it is always required that animals constantly form skin lesions with similar clinical appearance and course to those of human dermatophytoses after the inoculation of fungi. Experimental dermatophytoses on the backs of hairy guinea pigs generated by zoophilic strains of Trichophyton mentagrophytes have been used as first choice models, but they are quite different from human dermatophytoses, clinically, histologically and especially in their selfhealing courses. Zoophilic strains of T. mentagrophytes can infect guinea pig skin after 24 hours occlusion. However, anthropophilic strains may require a longer occlusion periods for infection.
In the present study, experimentally induced lesions on the plantar parts of guinea pigs with T. mentagrophytes zoophilic strain SM-110 closely resembled human tinea pedis clinically and histologically. On the other hand, the SM-110 generated lesions on the backs of hairless guinea pigs also resmbled human tinea corporis. In the histopathogenesis of dermatophytoses, skin structures of the infected or inoculated site seem to be important as well as strains of the dermatophytes.

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In order to elucidate the three-dimensional relationship between the corneocytes and the hyphae, we performed scanning and transmission electron microscopic observation of specimens from patients with tinea glabrosa due to T. rubrum. The specimens were stripped by the cyanoacrylate adhesive strip method. Under the scanning electron microscope, the hyphae of T. rubrum were seen to be mostly embedded in the corneocytes and appeared to be sharply elevated, cylinder-like protrusions. In the transmission electron microscopy, most of the hyphae of T. rubrum were seen to be penetrating the corneocytes, with little space between the cytoplasm and the hypha. The frequency of intracellular hyphae in cross section was 86% for T. rubrum. These results suggest that the hyphae of T. rubrum grow mainly in the corneocytes with minimal destruction of them.

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Yu, et al and Day, et al reported of the isolation and purification of keratinase from Trichophyton mentagrophytes in 1968. Release of proteolytic enzyme from the dermatophytes is assumed to be essential for their persistence, and thus has been the subject of many investigations. We also have reported isolation and purification of extracellular keratinase (e-kase) from Microsporum gypseum and Microsporum canis. In this report, we summarize our previous studies and, in addition, report new data on the quantification of e-kase extracted from lesional skin.
1. E-kase produced by M. canis was serine protease. The enzyme was induced by the addition of human hair instead of glucose and pepton in the culture medium. Its molecular weight is 32, 500 determined by SDS PAGE with a pH optimun of 7.48.
2. We produced both polyclonal and monoclonal anti-e-kase IgG. Four proteases from M. canis, M. gypseum, T. mentagrophytes, Trichophyton rubrum were produced and purified. Immunological identity between these four e-kase was confirmed by immunodiffusion and western blot analysis.
3. Localization of e-kase in clinical lesion was examined immunoenzymatically with light and electron microscopy. The results were as follows: 1) protease reactions surrounded fungal elements in the horny layer, 2) digestion of both tonofilament and marginal band of horny cells was also observed.
4. Protease was extracted and detected with EIA method from the lesional skin. Protease detected was 346ng/g of horny layer cells. This is the first successful report of quantification of e-kase in skin lesion.

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Keratinolysis by fungal proteinases has been considered a part of fungal pathogenicity. Previously reported proteinases from dermatophytes have had enzyme activities in neutral or alkaline pH. However, healthy skin has a weakly acidic pH. We isolated a new keratinolytic proteinase with enzyme activity at acidic pH from culture filtrates of Trichophyton mentagrophytes, a major pathogenic fungus of dermatophytosis. The proteinase was induced from a nitrogen-restricted medium supplemented with human plantar stratum corneum as a sole nitrogen source. The molecular weight of the purified proteinase was estimated to be 41kD with the isoelectric point at 3.9. The proteinase had a pH optimum of 4.5 for keratin and 5.5 for hemoglobin. This enzyme hydrolyzed the synthetic chymotrypsin substrate, Suc-Ala-Ala-Pro-Phe-MCA (Km, 0.59mM), and its activity was strongly inhibited by chymostatin. The purified proteinase which has an optimal activity at acidic pH and hydrolyzes skin constituents could be an important virulence factor in dermatophytosis.

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[in Japanese]

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Mycoses are caused by weak virulenced vectors and in this regard they prepare the good models to study the mechanisms of self-defense. We extracted candidacidal substance from guinea pig epidermis in 1981 and since then fractionation, purification and characterization of the substance have been carried out. Epidermal extract is consisted of many substances and the fractionation is very difficult to study. Recently we succeeded in the characterization, that is, the candidacidal activity is conducted by both basic epidermal protein and H1 histone, further characterization is now on the way.
Oxidative, chelating, enzymatic and cationic systems engaging in the self-defense are also discussed.

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We immunohistochemically examined six patients with ringworm which exhibited a well-marked actively involved edge and central resolution. To obtain a panoramic view of the immunopathological features of various evolutionary stages of skin lesion, we took a radial slice biopsy specimen extending from normal-appearing skin to central resolution. The actively involved edge had a moderate amount of dermal infiltrate of mononuclear cells which mostly consisted of CD3+ T cells. The ratio of CD4+ T helper/inducer cells to CD8+ T suppressor/cytotoxic cells was 3.4. At the central resolution site, the dermal infiltrate was mild, and CD4/CD8 ratio was 3.0. There was binding of many CD8+ T suppressor/cytotoxic cells to epidermal keratinocytes in actively involved edge and central resolution site. At the actively involved edge, a tendency towards a gathering of CD1a+ Langerhans cells and fungal elements was found and attachments of CD1a+ cells to lymphocytes in the dermis was often observed. The actively involved edge also showed a decrease of CD1a+ Langerhans cells in the epidermis, while many CD1a+ cells were present in the upper dermis. At the central resolution site, however, CD1a+ cells increased in number in the acanthotic epidermis.

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In order to justify the previous speculation proposed by Takahashi and Kakutani, which acanthotic and hyperkeratotic changes in the epidermis of experimental chronic dermatophytosis (ECD) produced in human skin grafted onto nude mouse may be a defensive reaction against dermatophyte infection in an individual with cell-mediated Immunodeficiency, we studied on such proliferative epidermis light- and electron microscopically and immunohistochemically. [Result] In ECD remarkable acanthosis, and many BrdU-positive keratinocytes not only in basal layer but lower spinous layer just above basal layer, were observed. Involucrin-positive keratinocytes were demonstrated in a wide range from granular layer to middle or lower portion of spinous layer in 19 case out of 20 ECD. On the contrary, almost all cases of control were negative for involucrin staining. Human Langerhans's cell in the epidermis were specifically detected in only 5 cases of ECD. IL-1, TNF, β-IFN and OKM-5 monoclonal staining showed to be negative in all ECD as well as controls. Electron microscopic examination of the epidermis of ECD revealed to appear many Odland bodies in keratinocytes from granular layer to middle or lower spinous layer, and to persist a residual of unit membrane of them in intercellular space between corneocytes. [Conclusion] Increase of S-phase keratinocytes represents proliferative changes of the epidermis, and early production of involucrin causes thickening of cell membrane of corneocytes, and early and many appearance of Odland bodies leads to the acceleration of desquamation. It may be conceivable that these morphological and biological changes of the epidermis cooperate for defensive mechanism against dermatophyte infection in cell-mediated immunodeficient condition.

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Well-demarcated and up to palm-sized scaly erythematous lesions with central healing were observed on the face of a 71-year-old female. A similar, thumb-sized lesion was seen on the left lower leg of a 3-year-old female, and erythematous lesions in the occipital region of a 58-year-old female, a grandmother of the second case, but this was without central healing. Hair in the lesions of the last patient was intact. T. violaceum was isolated on Sabouraud's dextrose agar from the lesional scales of all cases. All 3 had been inappropriately treated with corticosteroid ointment and were successfully treated with antifungal cream. Twenty nine cases of tinea corporis by T. violaceum (glabrum), including these 3, were reported in the Japanese literature from 1969 through 1989; those accompanied by other forms of tinea, such as tinea capitis, are not included in this number. The ratio of females/males was 25/4. Half of the cases were under 12-years-old, while the others were from their 20s to 70s. Nineteen cases (70.4%) had familial history of T. violaceum (glabrum) infection, including tinea capitis. Twelve cases (80%) had been inappropriately treated with corticosteroid ointment. The distribution of the lesions showed a predilection for exposed sites, with the highest frequency on the face, and lesions were usually up to the size of a hen's egg. There were 15 cases of solitary lesion and 9 of multiple lesions. A clinical feature of tinea corporis by T. violaceum (glabrum) may be the solitary occurrence on an exposed site of a female; as stated, though patients were of various ages, 50% were under the age of 12.

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A new antimycotic susceptibility test was devised. The peripheral area of a dermatophyte colony grown on 0.02% peptone agar medium was composed entirly of substrate hyphae. Cylindrical pieces of the agar punched out from the area were used as an inoculum. Each piece contained a constant amount of growth-phase hyphae without conidia. In vitro antifungal activities (minimum growth-inhibitory concentrations: MICs) of clotrimazole against 10 strains of Trichophyton rubrum, 10 strains of T. mentagrophytes, 3 strains of Microsporum canis and 3 strains of Epidermophyton floccosum were determined using an agar dilution technique with casitone agar medium in a 24-well plate. Each experiments was repeated four times. After 7 days, only one of the 26 strains of dermatophytes showed two similar MIC values, while the other 25 showed the same values in the same strains all four times. MIC values determined after 14 days were constant everytime in each of the 26 dermatophytes. The technique of the present antimycotic susceptibility test is simple and the results are reproducible and closely similar to the MIC values determined with the commonly used method.

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To elucidate discrepancies in the results obtained by direct microscopic examination and subsequent culture, the viability of dermatophytes in the scales was evaluated by neutral red staining. When the results of the staining of fungal elements in skin scales taken from 110 tinea patients were compared with those of subsequent culture on Sabouraud's dextrose agar, a strong positive correlation was found between the two sets of results. Autoradiographic study using ³H-thymidine showed that grain-positive cells (living cells) were stained with neutral red, whereas negative cells (dead cells) were not stained.
These findings suggest that neutral red staining is a useful method for evaluation of the viability of dermatophytes in human skin scales.

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Inhibitive effects of estrogen and progesterone on the yeast form growth of Paracoccidioides brasiliensis were examined using brain heart infusion broth with different doses of these hormones. The yeast form growth of P. brasiliensis was inhibited by estrogen within the range of physiological concentrations; this inhibition was dose dependent. Progesterone weakly inhibited the growth. The phenomenon that estrogen inhibited the yeast form growth of the fungus might be one of the important reasons for the sexual difference in susceptibility in paracoccidioidomycosis.

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The in vitro anti-Malassezia activity of NND-318, a new imidazole antimycotic, was examined with clotrimazole and bifonazole as reference drugs using Malassezia furfur-susceptibility testing medium supplemented with lipids. The MIC values of NND-318 against M. furfur were 0.63∼2.5μg/ml, and were smaller than those of clotrimazole and bifonazole. M. pachydermatis was more susceptible to NND-318 than M. furfur. Antifungal activity of NND-318 against M. furfur is thus definitely potent, and this suggests its therapeutic effectiveness on tinea versicolor.

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