Candida albicans does not invade carious human dentine.

AIM: Candida albicans has been proposed to be a caries pathogen, but the evidence for its specific role is lacking. To be considered significant in caries progression, a marked amount of yeasts should be present in a lesion. The aim of the study was to investigate the presence of C. albicans in dentinal caries lesions. MATERIALS AND METHODS: To demonstrate the extension of caries and to identify the bacteria in a lesion, sections of 10 carious human teeth were stained with Gram and Giemsa stains. C. albicans was detected with periodic acid-Schiff (PAS) staining and by immunohistochemistry using a C. albicans-specific antibody 3H8. Thirty sections were used for each staining (in total 120 sections). RESULTS: Extensive bacterial invasion and intensive staining by PAS occurred in all samples. However, with the C. albicans-specific antibody, only 30 (3.3%) sections stained weakly positive, with a few stained cells on the lesion surface. However, the positive identification of C. albicans, based on the morphology of the cells, was not possible. CONCLUSIONS: The results do not support the previous suggestion that C. albicans is important in the dentine caries pathology. In addition, because of its unspecific nature, PAS turned out to be an unsuitable method for detecting yeasts in carious tooth samples.

Expression of interleukin-8 and its receptor IL-8RA in chronic hyperplastic candidosis.

INTRODUCTION: Neutrophils are the main opponents of Candida albicans in chronic hyperplastic candidosis. They migrate from the circulation to the epithelium where they form microabscesses. We therefore hypothesized that the neutrophil chemokine interleukin-8 (IL-8) might play a role in the neutrophil-Candida interaction. METHODS: Biopsies from patients with chronic hyperplastic candidosis (n = 10) were stained using the avidin-biotin-peroxidase complex protocol for IL-8 and IL-8 receptor A and were compared to healthy control mucosa (n = 3). A set of C. albicans agar sections was similarly analysed. RESULTS: In chronic hyperplastic candidosis lesions IL-8 was strongly expressed in both vascular endothelium and mucosal epithelium. Many resident and immigrant inflammatory cells, including intraepithelial neutrophils, were IL-8 receptor A positive. In addition, IL-8 (or an analogue) was found in the candidal mother cell in chronic hyperplastic candidosis and in agar, whereas the tips of the hyphae expressed IL-8 receptor A (or an analogue). CONCLUSION: IL-8 may play a role in the recruitment of neutrophils from the vascular compartment to the epithelial microabscesses. C. albicans may have developed an ability to sense IL-8. The IL-8 ligand-receptor interaction may help to direct the growth of the IL-8-receptor-containing tips of the hyphae away from the IL-8-producing candidal cell body (a centrifugal growth pattern to facilitate host tissue penetration). Later, this ability might help to keep the vulnerable hyphal tips away from areas with high concentrations of host IL-8 and candidacidal neutrophils. We suggest that this phenomenon, in contrast to chemotropism, is named chemophobia.

Specificity of the monoclonal antibody 3H8 in the immunohistochemical identification of Candida species.

Candida albicans has been shown to be involved in the pathogenesis of adult periodontitis (AP). The diagnosis of Candida-associated AP depends largely on the identification of yeast and pseudomycelial forms in gingival tissue samples by using periodic acid-Schiff and Gomori methenamine silver stains. However, these stains are non-specific and also reveal confusing artifacts seemingly rather difficult to distinguish from yeasts. With the recent development and availability of monoclonal antibodies (Mabs) to various epitopes of C. albicans, for example Mab 3H8 which recognizes a mannoprotein, it is now possible to identify Candida in human tissue biopsies. To explore further the usefulness of this Mab in detecting Candida in periodontal disease the antibody was tested against a wide range of yeast species and strains and various morphological forms, grown in agar blocks at various temperatures and for various time periods. Furthermore, considering the location of the 3H8 epitope on the external cell wall of certain C. albicans strains, it seemed reasonable to determine whether the epitope could be expressed into the surrounding environment, further aiding the recognition of the organism in tissue. The 3H8 epitope appeared to be located at the external surface and on the septum between the mother cell and germ tube of some C. albicans strains but it was partially cryptic in the cell wall of other strains. Both yeast blastoconidia and pseudohyphae were labeled by the 3H8 antibody. Candida lusitaniae, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis did not posses the epitope. The epitope was expressed extracellularly by both blastoconidia and pseudohyphae of C. albicans. This Mab appears to be suitable for the identification of C. albicans in periodontal tissue and may provide further insight into the role of C. albicans in the pathogenesis and diagnosis of periodontal diseases.

Intracellular localization of Porphyromonas gingivalis thiol proteinase in periodontal tissues of chronic periodontitis patients.

OBJECTIVES: Porphyromonas gingivalis is a significant periodontal pathogen that has been shown in vitro to be able to invade gingival epithelial cells and grow intracellularly. The aim of the present study was to detect P. gingivalis in gingival tissues from chronic periodontitis (CP) patients. MATERIALS AND METHODS: Monoclonal antibodies specific to a cell membrane-bound thiol proteinase of P. gingivalis were used to detect the microbe in gingival tissues of CP patients (n = 13) by immunohistochemistry. The presence of P. gingivalis was also analysed by polymerase chain reaction (PCR). RESULTS: Immunohistochemical analysis of the periodontal tissues revealed positive staining for P. gingivalis thiol proteinase in 11 of the 13 patients. Positive staining was mainly located intracellularly in the perinuclear region of the cytoplasm in the periodontal epithelial cells and it could be detected throughout the whole depth of both pocket and oral epithelium. The sensitivity of immunohistochemistry was found to be comparable with that of PCR. CONCLUSIONS: Our results provide in vivo evidence of the ability of P. gingivalis to enter human gingival epithelial cells. Intracellular localization of P. gingivalis contributes to its evasion of the host immune surveillance and eventually increases its resistance to conventional treatments of periodontal diseases.

Candida yeasts in chronic periodontitis tissues and subgingival microbial biofilms in vivo.

The frequency of Candida infection in periodontal tissues of chronic periodontitis (CP) patients and the extent of candidal penetration into gingival tissues was studied. Tissue specimens collected from 25 CP patients during periodontal flap operations of initial periodontal therapy were examined by immunohistochemistry using Candida albicans-specific antibodies. Sections were also stained with periodic acid-Schiff (PAS) and subgingival plaque samples from 17 patients were cultured. Immunoreactivity for Candida was present in four of the 25 CP specimens (16%). Only one yeast-positive specimen was found when PAS-staining was used (4%) and two yeast-positive specimens were found with plaque culture (8%). Hyphal formation seemed essential and candidal hyphae were found to extend into the periodontal connective tissue. The degree and type of inflammation adjacent to the hyphae varied from a site and a patient to another. The sensitivity of specific antibodies was superior to PAS-stain or plaque culture in detection of Candida in tissues.

Examination on Candida spp. in refractory periapical granulomas.

AIM: To examine the occurrence of Candida spp. in refractory periapical granulomas. METHODOLOGY: One hundred and three surgically removed periapical granulomas were subjected to molecular analysis for the occurrence of Candida albicans. DNA was extracted from the samples using a modified phenol/chloroform/isoamyl alcohol method and was subjected to polymerase chain reaction (PCR) with OPA-03 and repetitive sequence (GACA)4 primers. The PCR products were separated in agarose gel electrophoresis, stained with ethidium bromide, visualized using UV light and the sequences were analysed. Samples indicating possible occurrence of Candida were further investigated by histological and immunohistological methods. Periodic acid-Shiff staining (PAS) was used to detect yeast cells and hyphae, and specific monoclonal antibodies to recognize high molecular mass mannoproteins present in the C. albicans cell wall. DNA extraction was controlled by running PCR using beta-actin primers (a housekeeping gene). C. albicans CCUG19915, C. tropicalis ATCC750, C. krusei ATCC6258, C. guilliermondii ATCC6260 and C. glabrata CCUG32725 served as positive controls in PCR. A tissue preparation of chronic atrophic candidosis in oral buccal mucosa served as a positive control for histological and immunohistological examinations. RESULTS: Polymerase chain reaction with beta-actin primers indicated successful DNA extraction in 68 out of 103 samples. The majority of the samples (50) were negative whereas 18 of the samples showed PCR products indicating possible occurrence of Candida spp. PAS-staining and immunohistological examination of these samples were, however, negative. Further analysis of the PCR products revealed sequences not typical for Candida spp. CONCLUSIONS: Candida spp. do not seem to occur in periapical granuloma.