Candida albicans-secreted aspartic proteinases modify the epithelial cytokine response in an in vitro model of vaginal candidiasis.

Secreted aspartyl proteinases (Saps) are important virulence factors of Candida albicans during mucosal and disseminated infections and may also contribute to the induction of an inflammatory host immune response. We used a model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) to study the epithelial cytokine response induced by C. albicans. In order to study the impact of the overall proteolytic activity and of distinct Sap isoenzymes, we studied the effect of the proteinase inhibitor pepstatin A on the immune response and compared the cytokine expression pattern induced by the wild-type strain SC5314 with the pattern induced by Sap-deficient mutants. Infection of RHVE with the C. albicans wild-type strain induced strong interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha responses in comparison with cytokine expression in noninfected tissue. Addition of the aspartyl proteinase inhibitor pepstatin A strongly reduced the cytokine response of RHVE. Furthermore, SAP-null mutants lacking either SAP1 or SAP2 caused reduced tissue damage and had a significantly reduced potential to stimulate cytokine expression. In contrast, the vaginopathic and cytokine-inducing potential of mutants lacking SAP4 to SAP6 was similar to that of the wild-type strain. These data show that the potential of specific Saps to cause tissue damage correlates with an epithelium-induced proinflammatory cytokine response, which may be crucial in controlling and managing C. albicans infections at the vaginal mucosa in vivo.

Buonocore Memorial Lecture. Review of the clinical survival of direct and indirect restorations in posterior teeth of the permanent dentition.

This review provides a survey on the longevity of restorations in stress-bearing posterior cavities and assesses possible reasons for clinical failure. The dental literature, predominantly since 1990, was reviewed for longitudinal, controlled clinical studies and retrospective cross-sectional studies of posterior restorations. Only studies investigating the clinical performance of restorations in permanent teeth were included. Longevity and annual failure rates of amalgam, direct composite restorations, compomers, glass ionomers and derivative products, composite and ceramic inlays and cast gold restorations were determined for Class I and II cavities. Mean (SD) annual failure rates in posterior stress-bearing cavities are: 3.0% (1.9) for amalgam restorations, 2.2% (2.0) for direct composites, 3.6% (4.2) for direct composites with inserts, 1.1% (1.2) for compomer restorations, 7.2% (5.6) for regular glass ionomer restorations, 7.1% (2.8) for tunnel glass ionomers, 6.0% (4.6) for ART glass ionomers, 2.9% (2.6) for composite inlays, 1.9% (1.8) for ceramic restorations, 1.7% (1.6) for CAD/CAM ceramic restorations and 1.4% (1.4) for cast gold inlays and onlays. Publications from 1990 forward showed better results. Indirect restorations exhibited a significantly lower mean annual failure rate than direct techniques (p=0.0031). Longevity of dental restorations is dependent upon many different factors, including material, patient- and dentist-related. Principal reasons for failure were secondary caries, fracture, marginal deficiencies, wear and postoperative sensitivity. We need to learn to distinguish between reasons that cause early failures and those that are responsible for restoration loss after several years of service.

Polymorphonuclear leukocytes (PMNs) induce protective Th1-type cytokine epithelial responses in an in vitro model of oral candidosis.

The immune response and the anticandidal activity of keratinocytes and polymorphonuclear leukocytes (PMNs) play a key role in host defence against localized Candida albicans infection. An established model of oral candidosis based on reconstituted human oral epithelium (RHE) was supplemented with PMNs to study the effect of these immune cells during experimental oral candidosis. Infection of RHE with C. albicans induced a strong expression of the chemokine interleukin-8 (IL-8) and the cytokine granulocyte-macrophages colony-stimulating factor (GM-CSF), and a moderate stimulation of interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) by keratinocytes. This immune response was associated with chemoattraction of PMNs to the site of infection, whereas uninfected RHE failed to induce cytokine expression or to attract PMNs. Growth of the pathogen and tissue damage of C. albicans-infected RHE were significantly reduced when PMNs were applied to the apical epithelial surface or when PMNs migrated through a perforated basal polycarbonate filter of the model. Notably, protection against epithelial tissue damage was also observed when PMNs were placed on the basal side of non-perforated filters, which prevented PMN migration into the RHE. Addition of PMNs enhanced a Th1-type immune response (IFN-gamma, TNF-alpha), down-regulated the expression of the Th2-type cytokine interleukin-10 (IL-10), and was associated with protection against Candida-induced tissue damage. This PMN-supplemented model of oral candidosis mimics the in vivo situation, and provides a promising tool for studying the immunological interactions between keratinocytes and C. albicans, as well as the influence of PMNs on C. albicans pathogenesis.

Reduced expression of the hyphal-independent Candida albicans proteinase genes SAP1 and SAP3 in the efg1 mutant is associated with attenuated virulence during infection of oral epithelium.

The transition of Candida albicans from a yeast to a hyphal form is controlled by several transcriptional factors, including the key regulators Cph1 and Efg1, and is considered an important virulence attribute. These factors, especially Efg1, regulate the expression of hyphal-associated genes e.g. SAP4-SAP6. In order to investigate the relevance of these transcriptional regulators for hyphal-independent SAP genes, recently constructed cph1 and efg1 single mutants and a cph1/efg1 double mutant lacking these factors were tested during interaction with oral epithelium and polymorphonuclear neutrophils. In contrast to the parental wild-type strain and the cph1 mutant, the efg1 and the cph1/efg1 mutants did not produce hyphal forms in all experiments and were less capable of damaging epithelial cells and neutrophil granulocytes. The attenuated epithelial lesions of these mutants were correlated not only with reduced expression of the hyphal-associated gene SAP4, but also with the lack of SAP1 and SAP3 expression previously shown to be important for oral infections. An efg1 mutant strain carrying a plasmid-borne copy of the EFG1 gene regained hyphal growth, damage of keratinocytes, granulocytes and the expression of SAP1 and SAP3. Although efg1 and cph1/efg1 mutants did not produce germ tubes during infection, expression of the hyphal-associated genes SAP5 and SAP6 was not completely abolished. A reduced capacity to stimulate an epithelial immune response manifested by a delayed onset of IL-1beta, IL-8 and TNF expression was only observed in the cph1/efg1-infected tissue. These results provide further evidence for a combined regulation of different virulence factors, such as dimorphism and expression of SAP genes. Furthermore, it could be demonstrated that the lack of Efg1 also caused reduced expression of hyphal-independent SAP genes. Both the EFG1 and the CPH1 gene products are necessary for adequate induction of an immune response.

The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on reconstituted human vaginal epithelium.

Secreted aspartyl proteinases (Saps) contribute to the ability of Candida albicans to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these C. albicans proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by C. albicans SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of SAP2, SAP9, and SAP10 was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of SAP1, SAP4, and SAP5. Additional transcripts of SAP6 and SAP7 were detected at a later stage of the artificial infection (24 h). Similar SAP expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by C. albicans was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by C. albicans cells. Furthermore, SAP null mutants lacking either SAP1 or SAP2 had a drastically reduced potential to cause tissue damage even though SAP3, SAP4, and SAP7 were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking SAP3 or SAP4 to SAP6 was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in C. albicans vaginal infections.

Effect of antimycotic agents on the activity of aspartyl proteinases secreted by Candida albicans.

The inhibitory effect of human immunodeficiency virus (HIV) proteinase inhibitors amprenavir and saquinavir and antifungal agents terbinafine, ketoconazole, amphotericin B and ciclopiroxolamine on aspartyl proteinases (Saps) secreted by Candida albicans was tested in an in vitro spectophotometric assay. As expected, both HIV proteinase inhibitors showed a significant inhibitory effect on Sap activity, which was comparable to that of the classical aspartyl proteinase inhibitor pepstatin A (P < 0.001). Antifungal drugs such as ketoconazole, terbinafine and amphotericin B had no, or only minor, inhibitory effects on proteolytic activity. In contrast, a significant reduction in Sap activity could be demonstrated during treatment with the antifungal agent ciclopiroxolamine (P < 0.001). These results point to a multiple effect of this antimycotic agent and might explain the reduced adherence of C. albicans to human epithelial cells at subinhibitory doses.