Cytokines in the oral mucosa of mice infected with Candida albicans

Oropharyngeal candidiasis is associated with defects in cell-mediated immunity, and is commonly seen in immunocompromised patients. We have previously shown that T-cell-deficient BALB/c nude (nu/nu) mice are extremely susceptible to oropharyngeal candidiasis, and that recovery from a chronic infection is dependent on CD4 T lymphocytes. In this study we describe the local tissue cytokine profile in lymphocyte-reconstituted immunodeficient mice and their euthymic counterparts. Mice were infected orally with 108 cells of the yeast Candida albicans, and oral tissues sampled on days 0, 4, 8, and 14. Nude mice were reconstituted with 3 x 107 na?ve lymphocytes following oral inoculation. Interleukin (IL)-6, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were identified in the oral tissues of infected euthymic mice recovering from oral infection, as well as na?ve controls. TNF-alpha was identified in nude oral tissue on days 4 and 8, but only after lymphocyte reconstitution. No IL-2, IL-4 or IL-10 was detected in either euthymic or athymic mice at any time-point throughout the experiment. This study confirms the functional activity of T lymphocytes in reconstituted nude mice, and suggests that TNF-alpha may be an important mediator in the recovery from oropharyngeal candidiasis.     

Monoclonal antibody-mediated inhibition of adhesion of Candida albicans and Candida dubliniensis to human epithelial cells

The aim of this study was to assess the role of whole saliva, four saliva-derived preparations, and six monoclonal antibodies (mAbs), directed against components of the cell wall of Candida albicans , on the adhesion of C. albicans and Candida dubliniensis to human epithelial cells (HEC). C. albicans serotype A NCPF 3153 and C. albicans serotype B ATCC 90028 showed higher adhesion to HEC than C. dubliniensis NCPF 3949. Pooled whole saliva was more efficient than salivary secretory immunoglobulin A, partially purified by chromatography, at inhibiting the adhesion of C. albicans serotype A NCPF 3153 to HEC. Monoclonal antibodies C7, 14-8, and 26G7 were the most potent inhibitors of adhesion. Our results show that mAbs can mimic the inhibition of adhesion of C. albicans to HEC that is mediated by human saliva.     

Systemic fluconazole therapy and in vitro adhesion of Candida albicans to human buccal epithelial cells

The adhesion of Candida albicans to buccal epithelial cells (BEC) from four healthy dentate men was studied in vitro 1 wk before, during and after a course of systemic fluconazole (50 mg once daily for 1 wk). After 1 wk of fluconazole intake candidal adhesion to BEC was significantly reduced and this reduction persisted for a week following completion of therapy. Fluconazole, therefore, in addition to being an effective antifungal agent may also have a sustained action on reducing adherence of candidal species to the oral mucosa.     

Induction of apoptosis in oral epithelial cells by Candida albicans

During infection, interactions between Candida albicans and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of oral mucosal ulcerations generally associated with discomfort. In vitro studies have shown that C. albicans induces early apoptotic alterations in oral epithelial cells; however, these studies have also shown that treatment of infected cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C. albicans stimulates oral epithelial signaling pathways that promote apoptotic cell death. Activation of specific death pathways in response to microbial organisms plays an essential role in modulating the pathogenesis of a variety of infectious diseases. The aim of this study was to (i) characterize C. albicans‐induced apoptotic morphological alterations in oral epithelial cells, and (ii) investigate the activation of apoptotic signaling pathways and expression of apoptotic genes during infection. Candida albicans induced early apoptotic changes in over 50% of oral epithelial cells. However, only 15% of those showed mid‐late apoptotic alterations. At the molecular level, C. albicans caused a loss of the mitochondrial transmembrane potential and translocation of mitochondrial cytochrome c. Caspase‐3/9 activities increased only during the first hours of infection. Moreover, poly[ADP ribose] polymerase 1 was cleaved into apoptotic and necrotic‐like fragments. Finally, five anti‐apoptotic genes were significantly upregulated and two pro‐apoptotic genes were downregulated during infection. Altogether, these findings indicate that epithelial apoptotic pathways are activated in response to C. albicans, but fail to progress and promote apoptotic cell death.     

Oral and vaginal epithelial cell anti‐Candida activity is acid labile and does not require live epithelial cells

Background: Candida albicans is the causative agent of oral and vaginal candidiasis. Innate host defenses against C. albicans are important against each infection. Among these are oral and vaginal epithelial cells that have anti‐Candida activity. The mechanism of action includes a requirement for cell contact with no role for soluble factors, and a putative role for carbohydrates based on the sensitivity of the activity to periodic acid. Methods: Periodic acid treatment of epithelial cells as well as the property of partial resistance of antifungal activity to fixation was used to further dissect the mechanism of action. Results: The results herein effectively now challenge a role for carbohydrates alone. Firstly, the putative carbohydrate(s) released into supernatants of periodic acid‐treated epithelial cells could not compete with fresh epithelial cells for activity, and equivalent abrogation of activity was observed by periodic acid‐treated cells irrespective of the amount of carbohydrate released. Instead, the similar abrogation of activity following treatment with other acids or when cocultured under acidic conditions suggests that the activity is acid‐labile. Finally, while activity requires intact epithelial cells, it does not require live cells; activity was minimally affected by fixing epithelial cells prior to coculture where the majority of cells remained impermeable to Trypan blue but were defined as non–viable by positive nuclear staining with propidium iodide. Conclusion: These results suggest that antifungal activity is dependent on contact by intact, but not necessarily live, epithelial cells through an acid‐labile mechanism.     

Immunoglobulin A antibodies to mutans streptococci in human saliva and serum comparing fresh and subcultivated strains and activity in repeated saliva samples

The aims of this study were i) to characterize and compare the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns of reference Streptococcus mutans and Streptococcus sobrinus strains, subcultivated for years, with fresh isolates of the same serotype; ii) to study possible differences between the human salivary immunoglobulin A (IgA) activity to reference strains and to fresh bacterial isolates of the saliva donors; iii) to examine for potential differences in the salivary IgA activity to the streptococcal antigens during 1 week; and iv) to map, in the same individuals, the serum IgA activity against the selected bacteria. S. mutans reference and fresh isolated strains showed a similar protein pattern with few exceptions. The immunoblot also revealed similarity in saliva IgA response, with only one subject's saliva displaying clearly one band's difference. For S. sobrinus a larger discrepancy was seen. The antibody activity during the one week interval was essentially unchanged. When incubated with serum, a different immunoblot profile was seen compared with saliva, although most bands revealed by saliva were also displayed by serum.     

Asymptomatic oral Candida albicans carriage in HIV-infection: frequency and predisposing factors

Many studies have focused on the epidemiology and pathogenesis of oral candidiasis in HIV infection. Little is known on the incidence and predisposing factors of asymptomatic oral Candida carriage in this setting, obviously an important issue in view of prophylaxis. To address this question. 261 consecutive HIV-infected individuals without clinical evidence of candidiasis were investigated. C. albicans was isolated from cultured oral cavity swabs of 63 subjects (24%). Colonization was significantly more frequent in IV drug users. CDC groups IV. and in subjects with lymphocytopenia. CD4+ cell depletion, or elevated beta-2 microglobulin. These data further suggest that oral candidiasis occurs in HIV infection as a result of C. albicans overgrowth and raise the question of primary antifungal prophylaxis in subjects with low CD4 counts and asymptomatic oral Candida carriage.     

Active and passive immunization against oral Candida albicans infection in a murine model

BACKGROUND/AIMS: Clinical and laboratory studies are consistent with a major role for cell-mediated immunity in recovery from oral infection with Candida albicans, but the role of humoral immunity remains controversial. The purpose of this study was to establish the relative contributions of cellular and humoral immunity to protection against oral candidiasis in a murine model, and to determine whether host responses could be enhanced by different immunization strategies. RESULTS: Active oral immunization was protective in BALB/c and CBA/CaH mice, reducing both fungal burden and duration of infection after secondary challenge, whereas systemic immunization failed to protect against subsequent oral challenge. Candida-specific IgM was the predominant antibody detected in serum following both primary and secondary oral challenge; however, Candida-specific salivary IgA was not detectable. Immunization by passive transfer of either lymphocytes or immune serum did not confer any significant protection against oral infection in either susceptible or resistant mouse strain. CONCLUSION: The data demonstrate a possible role for mucosa-associated immunity following active immunization by the oral route, most likely exerted by local T lymphocytes resident in the oral mucosa, but there was no evidence to support a role for humoral immunity in protection against oral candidiasis.     

Saliva promotes Candida albicans adherence to human epithelial cells

Adhesion of Candida cells to oral surfaces is an initial event in pathogenesis. Since specific immobilized salivary components mediate the binding of Candida albicans to hydroxyapatite, we hypothesized that saliva may also promote adherence to oral epithelia via a similar mechanism. In an in vitro model, C. albicans ATCC 10261 yeast cells adhered in a saturable manner to monolayers of three cultured human epithelial cell lines (A549, HEp-2, and HET-1A). The addition of whole saliva to the assay promoted the binding of C. albicans to all cell lines in a dose-dependent manner, but pre-incubation of the epithelial cells with pooled whole saliva had no effect on subsequent adherence. Pre-incubation of the yeast cells with pooled whole saliva, however, significantly enhanced (by up to 120%, P < 0.05) binding to epithelial cell monolayers, and pooled saliva that had been pre-incubated with C. albicans yeast cells was defective in promoting yeast adherence. There was a negative correlation (r = 0.68, P < 0.005) between specific IgA titers against whole cells of C. albicans and adherence-promoting activities for individual saliva samples. The adhesion-inhibitory effect of specific anti-C. albicans IgA was reversed by depletion of IgA from saliva by affinity chromatography. Factors in whole saliva, therefore, bound to the yeast cells, counter the C. albicans-specific salivary IgA inhibitory effect on adhesion and promote the adherence of C. albicans yeast cells to cultured epithelial cells.     

Quantitative evaluation of tissue invasion by wild type, hyphal and SAP mutants of Candida albicans, and non-albicans Candida species in reconstituted human oral epithelium

BACKGROUND: Oral candidiasis is a common problem in compromised patients. Although several non-albicans Candida species have emerged as pathogens the majority of candidal infections are caused by Candida albicans. Morphogenesis from the blastospore to filamentous phase, and production of secretory aspartyl proteinases (SAP) are two major virulence attributes of these opportunistic yeast. Histopathology of oral candidiasis is characterized by fungal invasion of the superficial epithelium although the invasive potentials of different Candida species vary. Computerized image analysis systems (IAS) utilizing immunohistochemistry have been successfully employed for quantification of such histopathological features. The purpose of this study was to evaluate quantitatively the in vitro invasive potential of C. albicans and its hyphal and SAP mutants, and five other non-albicans Candida species using a computerized IAS. METHODS: In vitro human oral candidiasis was produced using five wild type and one reference C. albicans isolates, hyphal and SAP mutants of C. albicans SC 5314, and one wild type and one reference isolate each of C. tropicalis, C. dubliniensis, C. glabrata, C. parapsilosis and C. krusei in a reconstituted human oral epithelium (RHOE) model. The infected tissues were examined histologically at 12, 24 and 48 h. Invading fungal elements were visualized by periodic acid-Schiff (PAS) staining and quantitatively evaluated as a percentage of total tissue invasive area, using a computerized IAS. RESULTS: All C. albicans isolates including hyphal mutant cph1/cph1 and SAP mutants; sap 1-3, sap 4-6 produced hyphae and differentially (P < 0.05) invaded the tissue over 48 h. The invasive potential of hyphal mutant cph1/cph1 and SAP mutants (sap 1-3, sap 4-6) were similar to the parent wild-type isolate at 12 h although after 24 h their invasion was dissimilar (P < 0.05). Non-albicans Candida species and hyphal mutants; efg1/efg1, efg1/efg1 cph1/cph1 were all non-invasive. CONCLUSIONS: RHOE model in combination with computerized image analysis permits for the first time, the assessment of invasive potential of Candida species in a quantitative manner. The differential tissue invasive patterns of various C. albicans isolates, their mutants and other Candida species are also described.     

口腔扁平苔藓患者白色念珠菌分离株的颊细胞粘附力研究

的　研究健康人和口腔扁平苔藓患者口腔白色念珠菌分离株的颊细胞粘附力。方法　应用颊细胞粘 附实验法,比较来自于健康人(26株)、糜烂型口腔扁平苔藓患者(62株)以及非糜烂型口腔扁平苔藓患者(24株)共 112株白色念珠菌对人颊细胞粘附力的大小。结果　糜烂型口腔扁平苔藓患者组的白色念珠菌分离株的平均粘附 数较健康对照组高(P<0·05),说明糜烂型口腔扁平苔藓患者组白色念珠菌分离株的颊细胞粘附力高于健康对照 组。结论　糜烂型口腔扁平苔藓患者组的白色念珠菌分离株与健康对照组相比,具有不同的毒性特征,白色念珠 菌与糜烂型口腔扁平苔藓发生发展可能相关。     

Acquired resistance and persistence of Candida albicans following oral candidiasis in the mouse: a model of the carrier state in humans

In our experimental model of oral candiasis in the CD1 mouse, the primary infection showed reproducible Candida overgrowth kinetics with a peak level on day 5 of the infection. After day 7, the population stabilized at about 300 colony-forming units per excised mucosal tissue. The primary infection triggered an inflammatory response that resolved in under 8 days. At this point, the histological pattern of the mucosa reached a new equilibrium between recruited and resident mononuclear cells. The primary infection also rapidly stimulated cellular immunity, as measured from day 4 by a delayed-type hypersensitivity footpad reaction. Following a second topical challenge with Candida 30 days after the primary infection, the infection was barely delectable and a typical local delayed-type hypersensitivity reaction occurred between 24–72 h. It is proposed that acquired resistance, in conjunction with low-level persistence of Candida in our model, mimics the carrier stale in sensitized humans.     

The effect of subinhibitory concentrations of gentian violet on the germ tube formation by Candida albicans and its adherence to oral epithelial cells

ObjectiveThis study investigated the effect of subinhibitory concentrations of gentian violet on the germ tube formation by Candida albicans and its adherence ability to oral epithelial cells.MethodsThirty strains of C. albicans isolated from denture wearers, normal healthy individuals and HIV positive patients were used in the study. The antifungal property (Minimum Fungicidal Concentration) of gentian violet was determined at various time intervals using a microdilution technique. The effect of subinhibitory concentrations of gentian violet on the adherence ability (0.000244%) and on germ tube formation ((0.000244%, 0.000122%, 0.000061% and 0.000031%) was determined. In both experiments, water was used as a control. The test results were compared using the Kruskal-Wallis test.ResultsAt 60 min a high concentration (0.0078%) of gentian violet was required to completely kill C. albicans. Subinhibitory concentrations of gentian violet significantly reduced the adherence ability of C. albicans by 57% (p < 0.01) and equally inhibited germ tube formation (p < 0.01) compared with the controls. The inhibition was concentration dependent, with up to 98% reduction at a concentration of 0.000244%. Germ tube reduction was significantly higher in the isolates from the HIV positive patients than in the isolates from denture wearers.ConclusionAt high concentrations, gentian violet killed C. albicans, whereas at subinhibitory concentrations it reduced its virulence by preventing the adherence ability and germ tube formation. This suggests that the beneficial effects of gentian violet would last beyond the fungicidal concentrations in the treatment of candidiasis.     

Studies on the binding of Treponema pectinovorum to HEp-2 epithelial cells

We developed a radioassay to assess the adherence of the oral treponemes Treponema denticola and Treponema pectinovorum to live HEp-2 epithelial cells. T. pectinovorum bound firmly to the epithelial cell monolayer in a concentration-dependent manner. The results indicated that a subpopulation of T. pectinovorum appeared to bind and that the binding could be influenced by environmental factors. Increasing concentrations of fetal bovine serum inhibited binding, whereas T. pectinovorum membrane vesicles and co-incubation with T. denticola ATCC 35404 increased the number of cells bound to the monolayer. Treatment of T. pectinovorum with periodic acid, but not trypsin or proteinase K, decreased the binding suggesting that a cell surface carbohydrate, such as the O-antigenic component of the lipopolysaccharide, mediates attachment of the bacteria to the epithelial cells. Co-infection of the HEp-2 cells with both T. denticola and T. pectinovorum did not interfere with each other in attachment to the epithelial cell suggesting that they do not compete for the same cellular receptor on the host cell surface. This study demonstrates that T. pectinovorum is capable, in vitro, of forming a tight association with host cells and that this binding could represent an initial step in the pathogenesis of T. pectinovorum.     

Facial Candida albicans cellulitis occurring in a patient with oral submucous fibrosis and unknown diabetes mellitus after local corticosteroid injection treatment

Facial cellulitis caused by odontogenic bacterial infection is frequently encountered; however, facial cellulitis caused by Candida albicans infection is rarely found. A patient with oral submucous fibrosis (OSF) and unknown diabetes mellitus (DM) was treated in our out-patient dental clinic by biweekly submucosal injection of 40 mg triamcinolone acetonide into bilateral buccal mucosae plus forced mouth opening performed by the two hands of the clinician. The interincisal distance of the patient improved from 28 to 48 mm after four times of steroid injection. The symptoms and signs of OSF also improved markedly. Unfortunately, facial candidal cellulitis occurred 2 months after the last time of steroid injection treatment. The infection was cured by incision and drainage, intravenous administration of amphotericin B (100 mg once a day for a week), and an appropriate medical control of DM. No recurrence of facial cellulitis was found during the follow-up period of 18 months. To prevent the occurrence of facial cellulitis after a high-dose steroid therapy, some prophylactic procedures should be taken before the initiation of the steroid treatment.     

口腔放线菌对白假丝酵母菌拮抗作用的体外实验研究

目的观测3种口腔放线菌对白假丝酵母菌的生长有无抑制作用。方法采用直接点种法观察抑菌圈,分别采用反点种菌落计数法和液体共培养法,观察在口腔放线菌作用下白假丝酵母菌菌落数量的变化。结果1）各实验组均未观察到清晰的抑菌圈;2）在黏性放线菌软琼脂上的白假丝酵母菌菌落数较对照组明显减少（P<0.05）;在溶牙放线菌和内氏放线菌软琼脂上无明显白假丝酵母菌生长,与对照组相比差异有统计学意义（P<0.01）;黏性放线菌组与溶牙放线菌组、内氏放线菌组相比,白假丝酵母菌生长差异有统计学意义（P<0.01）;3）分别与3种放线菌共培养的白假丝酵母菌菌落数量减少,与对照组相比差异有统计学意义（P<0.05）;3个实验组间差异无统计学意义（P＞0.05）。结论口腔黏性放线菌、溶牙放线菌和内氏放线菌对白假丝酵母菌的体外生长有抑制作用。