For evaluation of fungal and host factors in pathogenesis of tinea pedis, Trichophyton mentagrophytes zoophilic and anthropophilic strains were compared for invasiveness and induced of tissue response and cell-mediated immunity in experimental tinea pedis in guinea pigs. In vitro comparison of keratinolytic and albuminolytic activities was also made. T. mentagrophytes zoophilic strain SM-110 quickly penetrated the horny layer of an inoculated foot within 24 hours after inoculation, invading the whole horny layer just above the granular layer, provoking a strong inflammatory response and clinical manifestations. Anthropophilic strain NTM-105, in contrast, invaded the upper two-thirds of the horny layer slowly and induced no inflammatory response. Zoophilic strains, SM-110, VUT-85001, VUT-85002 and VUT-85003 showed deep perforations in a hair perforation test and demonstrated clear bovine serum albuminolytic activities, while anthropophilic strains NTM-105 and SM-8500 male only infrequent shallow perforations and showed weak albuminolytic activities. These results indicate that the invasiveness of infecting fungi depends on the protelytic activities of the fungi and relates to the strength of tissue response in tinea pedis. Zoophilic strain SM-110 infections showed clearer response to dermatophyte antigen in intradermal skin test and a lymphocyte stimulation test than did anthropophilic strain NTM-105 infections. In the experimental tinea pedis described herein, it was possible to analyze the strength of tissue reactions, cell-mediated immunity and invasiveness of the infecting fungi.
Although the pathogenic factors and mechanisms in fungal infection are not yet fully understood, many investigators have pointed out the important role of fungus in producing extracellular proteinases. The present study demonstrated the characteristics of Sporothrix schenckii in producing proteinases and identified the roles of proteinases in the pathogenesis of sporothrichosis. (1) S. schenckii produced extracellular proteinases to catalyze albumin, collagen, elastin, keratin and so forth. (2) Proteinases were produced in culture media containing these substrates, but were not produced in Sabouraud's medium. (3) Two proteinases (proteinase I and II) were purified. Proteinase I was Mr 36, 500, pH optima 6.0 and serine proteinase inhibited by chymostatin. Proteinase II was Mr 39, 000, pH optima 3.5 and carboxyl proteinase inhibited by pepstatin. (4) Neither proteinase I nor II was produced nor were cells grown in culture when pepstatin or chymostatin was added. (5) Cell growth was not inhibited in culture by addition of either pepstatin or chymostatin. (6) S. schenckii infection was suppressed in vivo (mouse model) by the application of both inhibitors. These results conclude that the 2 proteinases produced by S. schenckii work cooperatively and compensatively in S. schenckii infection.
Relationships between immunity and tissue reaction in mice infected with Candida albicans or Cryptococcus neoformans have been investigated. When mice immunized sublethally with live C. albicans were lethally infected with the same yeast, small inflammatory lesions were restricted in the renal glomerulus, while several large lesions were developed in the pyelic and urethric regions in control mice. When mice immunized with Cr. neoformans were infected with the same yeast, granulomatous lesions were predominant in the lung and kidney, while many cystic lesions were developed in control mice. On the other hand, in mice treated with interferon-γ (IFN-γ) before and after infection with C. albicans, a reduction in viable C. albicans cells in kidney were seen compared with that in control mice, although no significant difference in tissue response was found between treated and control mice. Likewise, in IFN-γ-treated mice, the viable cells in the lung and kidney decreased after Cr. neoformans infection compared with control mice without any change in tissue reactions from cystic to granulomatous lesions. These results suggest that although IFN-γ was able to activate phagocytes for killing, chemotactic lymphokine(s) other than IFN-γ is responsible for inducing changes of tissue reaction in immunized mice. Moreover, although Cr. neoformans cell walls activate the complement via the alternative pathway in vitro, chemotaxis of phagocytes was not demonstrated in fresh serum activated by Cr. neoformans whole cells, suggesting that the released chemotactic factor(s) for the phagocytes is consumed by adsorption with capsular polysaccharide.
The experimental results of the relationship between iron and a fungal infection were obtained and tissue reactin for deep-seated mycosis was evaluated. 1) Increased susceptibility to infection in iron overload: When experimental candidiasis was induced in ICR mice with iron overload, the mice had renal abscesses containing candidial pseudohyphae in the eraly stage after the inoculation. By contrast, in mice with Candida albicans and without iron overload, no fungal elements were detected in renal abscesses in the same early stage. The excess iron thus promoted the growth of Candida; however, there was no significant histological difference when iron was loaded or not. The same results were gained when leukemic mice were employed. 2) Resistance to infection due to decreased serum iron: The decreased serum iron induced by lipopolysaccharides or muramyl dipeptide had a deterrent effect on the growth of Aspergillus, so that production of the fungal lesion occurred later. 3) Increased susceptibility to infection by decreased UIBC (unbound iron binding capacity): Decreased UIBC produced by diabetic ketoacidosis enhanced the growth of Rhizopus oryzae. 4) Increased susceptibility to infection due to decreased amount of transferrin: A decreased transferrin level in D(+) galactosamine-induced liver injury accelearted the growth of Candida. From these results, the following was concluded; there was no significant difference in tissue reaction for deep-seated mycosis between the group with deranged iron metabolism and the control group; however, the deranged iron metabolism influenced the growth speed of the fungi and the extent of the fungal lesion.
Tissue reactions against Coccidioides immitis infection were investigated. Congenitally athymic nude mice (nu/nu), their heterozygous littermates (nu/+) and ddY mice were used as experimental animals. Each mouse was inoculated intravenously with 3.3×105 or 530 of arthroconidia and was sacrificed at determined intervals for histological examination. The virulence of C. immitis used in this experiment was as follows: When 3.3×105 arthroconidia were inoculated intravenously, all of 6 nu/nu or 6 nu/+ mice died 5 or 6 days after inoculation, respectively. When 530 arthroconidia were inoculated, all of 6 nu/nu or 6 nu/+ mice died 14 or 13 days after inoculation, respectively. The parasitic cycle of the fungus in the mouse organs was completed within 5 days. As the C. immitis had predilection for the liver, spleen and lung, we focussed on the liver to investigate the tissue reactions. Without distinction among mouse strains, cell reactions to the arthroconidia were very weak. A few polymorphonuclear leucocytes (PMN), monocytes or a mixture of the two accumulated at the arthroconidia. This pattern of cell infiltration continued until the completion of spherules; it took about 4 days. When part of the cell wall of a spherule was broken down and numerous endospores were release into the tissue, PMN accumulated immediately and remarkably at the endospores and formed relatively large pyogenic lesions 4 days after inoculation, in which most of the released endospores were destroyed by the PMN. On the 7th day, lesions changed to granulomatous ones, and surviving endospores began to be destroyed in the granulomatous lesions. In the nu/nu mice, granulomatous lesions were formed 7 days after inoculation. However, different from the granulomatous lesions formed in the nu. + and ddY mice, endospores released from spherules were not destroyed in the granulomatous lesions and continued to develop into mature spherules.
The efficacy of FK-936, PY(1)+CTZ(2), -an external liquid preparation-was evaluated in guinea pigs with Trichophyton spp. infection and the following results were obtained: 1) FK-936 was significantly superior to CTZ and PY preparations in therapeutic activity against experimentally induced infection with T. mentagrophytes Junten. The mean negative rates of the culture specimens treated with FK-936, CTZ and PY preparations were 85, 76 and 61%, respectively. 2) The efficacy of FK-936 on experimental infection with clinically isolated T. rubrum No. 68 was also significantly superior to that of CTZ and PY preparations. The mean negative rates of the culture specimens treated with of FK-936, CTZ and PY preparations were 90, 69 and 60%, respectively. 3) The excellent efficacy of FK-936 against experimental Trichophytosis was confirmed by the MIC and fungicidal activity in vitro.
The separation of plasma components capable of binding to Asp-hemolysin by affinity chromatography using a column of Sepharose 4B coupled covalently with this hemolytic toxin has been investigated. IgG, IgM and α2-macroglobulin were isolated from normal human blood plasma by this chromatography, and IgG which was eluted from the column with 1M NaCl was found to be the major component. It was also shown that Fab'fragment was able to bind to Asp-hemolysin more strongly than did the pFc'fragment and no difference was observed between the binding abilities of all IgG subclasses in vitro. Neither IgG nor its fragment influenced the hemolytic activity of Asp-hemolysin in vitro. However, the addition of α2-macroglobulin in vitro caused a marked inhibition, indicating that α2-macroglobulin is one of the plasma components inhibitory to the hemolytic activity of Asp-hemolysin.
A new method utilizing nitroblue tetrazolium (NBT) reduction was to designed to evaluate the superoxide (O-2) production of human polymorphonuclear leukocytes (PMN) at phagocytosis of C. albicans. The most ideal experimental condition was set up as follows: 0.2ml each of PMN (2.5×106/ml) and Candida (2.5×107/ml) suspensions with the addition of 0.1ml of 50% autoserum were put into a polyethylene tube for 30 minutes of incubation in a CO2 incubator followed the measurement of phagocytosis (NBT-phagocytosis) and NBT reduction. The same experimental procedure but without the addition of NBT was performed simultaneously to determine the phagocytosis (PBS-phagocytosis) and candidacidal activity of PMN after 30 and 180 minutes of incubation, respectively. Killing activity was estimated by counting the rate of killed Candida (killing rate) from the culture of the incubated suspension on Sabourud destrose agar. In studies of normal control, positive correlations existed between PBS- and NBT-phagocytic rates (p<0.001) and between NBT reduction and killing rates (p<0.05). The mew method was applied to clinical patients with pulmonary diseases to verify its usefulness, and some were revealed to have various degrees of impaired PMN functions. These experimental results thus confirmed the value of the new method in the analysis of human PMN functions.
We report the first case of cutaneous mucormycosis caused by Rhizopus microsporus var. rhizopodiformis in Japan. A 56-year-old woman affected with acute myeloblastic leukemia noted a small black papular lesion on her left forearm under the adhesive bandage. The ulcer rapidly enlarged to more than 5cm in diameter by the 5th day thereafter. Histological sections of the skin lesion showed numerous non-septated hyphae spreading throughout the dermis and invading the walls of blood vessels. R. microsporus var. rhizopodiformis was isolated form the skin lesion. The patient died on the 17th day after that on which the initial skin lesion was noted.