Schmidt-Westhausen, Andrea Maria: Experimentelle Untersuchungen zur Pathogenese und Therapie der oralen Candidiasis bei Immundefizienz

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Kapitel 6. Summary

In recent years, the number of immunocompromised patients has risen due both to infection and to the use of immunosuppressive drugs and chemotherapy. Since the first reports on HIV, numerous publications have described a strong correlation between oropharyngeal candidiasis and progression of the disease to AIDS. It is a safe assumption that every HIV patient will experience at least one episode of oral candidiasis during the course of his or her disease. In both humans and animals, congenital or acquired immunodeficiencies increase susceptibility to infection with extracellular pathogens.

Previous studies have shown that a direct association between mucosal changes and a high number of colony forming units (CFU/ml) in either pharyngeal lavage or swab test results does not exist. Persons with a healthy immune response and no symptoms have shown up to 103 CFU/ml in swab samples, whereas some patients infected with HIV have shown as little as 50 CFU/ml in cases where clinical symptoms were present.

Treatment of oropharyngeal candidiasis consists primarily of topical or systemic application of fungistatic or fungicide substances. Factors such as recurrent infection, resistance and tachyphylaxis complicate therapy and the choice of antimycotic.

This study applied a candidiasis model to immunocompetent inbred mice (Balb/c) (n=27) and mice with combined B- and T-cell defects (SCID) (n=30) to address the following questions

1. Does a correlation exist between the inoculated pathogen load and the emergence of a C. albicans infection (dose/effect relationship).

To investigate this, Balb/c mice (n=3) were orally inoculated with a pathogen load of 105 C. albicans cells/10µl (Strain DSM 3454), n=3 with 106, n=6 with 107 and n=6 with 108 C. albicans cells/10µl, respectively. SCID mice (n=2) were also inoculated orally, with a pathogen load of 104, n=6 with 105, n=3 with 106 and n=6 with 107 C. albicans cells/10µl. One week following the inoculation, the animals were sacrificed, and one half of the tongue tissue was histochemically examined with the Periodic Acid Schiff Method for displaying morphological structures of C. albicans. Macroscopically, neither group‘s tissue showed clinical signs of mycosis, such as the erythematous or pseudomembranous changes associated with candidiasis.

2. Which cellular immune response takes place on the oral mucosa following inoculation with defined pathogen loads.


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For this purpose, the other half of the tongue tissue was examined with frozen section methods and immunocompetent cells were examined by immune peroxidase methods using antibodies specific for CD4, CD13, CD54 (ICAM-1), CD62E (E-Selectin), CD74, CD80, CD86 and CD103.

3. Is it possible achieve to a protective effect by inhibiting C. albicans adhesion to murine epithelium cells through the local application of mucine (glycopeptides) metabolites to the oral mucosa, and so to prevent infection?

A possible alternative therapy consisting of a blockade of the pathogen‘s adhesion to tissues was tested. Balb/c mice (n=8) were inoculated with 108 C. albicans cells in combination with glycopeptides and SCID mice (n=8) were inoculated with 105 C. albicans cells, also in combination with glycopeptides. Previous in vitro studies have shown that a standardisation of temperature dependent processes in the laboratory is necessary to attain reproducible results.

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Following complete histochemical preparation and examination of the tongue tissue, the Balb/c mouse specimens showed no invasion by hyphae up to an inoculation load of 107 pathogens. With a load of 108, 5/6 of the animals and 26/51 slices (51%) displayed an invasion of the stratum corneum. In the case of the SCID mice, hyphae invasion was present in 4 of 6 of the animals with the initial inoculation load of 105 pathogens, and 33 of 108 slices (33.6%) showed PAS positive structures. The minimal inoculation dose leading to infection of the tongue mucosa in the case of Balb/c mice was 108 pathogens. In the case of SCID mice, a load of 105 pathogens had this effect.

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Despite the lack of macroscopic signs of candidiasis, an immunologic reaction took place in the tongue mucosa of both groups of test animals. The extent of this reaction depended both on the inoculation dose given to the animals and on their immune status. Following inoculation with the greatest pathogen load, CD4+ cells were observed infiltrating the lower stratum spinosum in Balb/c mice, whereas in the tissues of SCID mice, these cells could be demonstrated only sporadically in subepithelial regions. The morphology of the cells expressing CD4 antigen supported the conclusion that these were dendritic cells, not T-lymphocytes. The Balb/c mice differed greatly from the SCID mice with respect to the number of CD54+ cells: whereas an inoculation of 108 C. albicans cells was required to illicit a strong expression in the


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subepithelial region and changes in the endothelium in the Balb/c mice, an inoculation with a pathogen load of only 105 brought about a strong expression of the CD54 antigen in the cells of the SCID mice. Following hyphae invasion of the the tongue epithelium, a distinct expression of CD74 antigen occurred in subepithelial regions and in the lower stratum spinosum in the Balb/c mice. The SCID group showed a much lower number of CD74+ cells in the same cell layers. The expression of CD80 antigen in the Balb/c mice following stimulation with foreign antigen was less than that of CD86. In immune deficient mice, the latter could be demonstrated only in small amounts following inoculation with the minimal infective dose. Both species showed a high number of CD103+ cells following this dose, which suggests the development of CD8+ lymphocytes.

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The results of an inoculation of 108 C. albicans cells in combination with glycopeptides showed a hyphae invasion of the tongue epithelium in 2/8 Balb/c mice. In the tongue epithelium of SCID mice, hyphae invasion could be demonstrated in 0/8 cases following an inoculation of 105 C. albicans cells in combination with glycopeptides. Despite the fact that the number of animals was limited and that statistical analysis served only to guide in orientation, the results indicate that when inoculation of C. albicans cells is combined with the application of glycopeptides, fewer infections occur than in cases where the same pathogen load is given without glycopeptides. A decisive factor for the differences in inhibition in Balb/c and SCID mice appears to be the pathogen load in relation to the glycopeptide dose. The results of immune-histochemical studies showed that the host‘s reaction to combined glycopeptide pathogen inoculation corresponds to the reaction without inoculation with respect to distribution within epithelial layers and the number of expressing cells in SCID and Balb/c mice.

As no side effects have been documented for the oral application of mucines, their use or the use of their metabolites as complementary therapy for patients at an increased risk for oral candidiasis should be considered.


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