Cytotoxic and cytokine-inducing properties of Candida glabrata in single and mixed oral infection models

Oral candidiasis is a common opportunistic infection, with Candida albicans being the most prevalent etiologic agent and Candida glabrata emerging as an important pathogen. C. glabrata is frequently co-isolated with C. albicans from oral lesions. Although C. albicans has been shown to trigger significant cytokine responses and cell damage, C. glabrata has not been systematically studied yet. The purpose of this study was to characterize the ability of C. glabrata to induce proinflammatory cytokine responses and host damage as a single infecting organism and in combination with C. albicans, using in vitro models of the oral mucosa. In monolayer oral epithelial cell cultures, C. glabrata failed to induce a significant interleukin-1alpha and interleukin-8 cytokine response and showed lower cytotoxicity, compared to C. albicans. However, C. glabrata triggered a significantly higher granulocyte macrophage colony stimulating factor response than C. albicans. C. glabrata strains showed a strain-dependent tissue damaging ability and a superficial invasion of the mucosal compartment in a three-dimensional (3-D) in vitro model of the human oral mucosa and submucosa. In the 3-D system, co-infection failed to promote host damage beyond the levels of infection with C. albicans alone. These studies indicate that C. glabrata induces cytokines in human oral epithelium in a strain-specific manner, but its tissue/cell damaging ability, compared to C. albicans, is low. Synergy between C. glabrata and C. albicans in cytokine induction and host damage was not observed with the strains tested.     

Immunopathogenesis of oropharyngeal candidiasis in human immunodeficiency virus infection

Oropharyngeal and esophageal candidiases remain significant causes of morbidity in human immunodeficiency virus (HIV)-infected patients, despite the dramatic ability of antiretroviral therapy to reconstitute immunity. Notable advances have been achieved in understanding, at the molecular level, the relationships between the progression of HIV infection, the acquisition, maintenance, and clonality of oral candidal populations, and the emergence of antifungal resistance. However, the critical immunological defects which are responsible for the onset and maintenance of mucosal candidiasis in patients with HIV infection have not been elucidated. The devastating impact of HIV infection on mucosal Langerhans' cell and CD4(+) cell populations is most probably central to the pathogenesis of mucosal candidiasis in HIV-infected patients. However, these defects may be partly compensated by preserved host defense mechanisms (calprotectin, keratinocytes, CD8(+) T cells, and phagocytes) which, individually or together, may limit Candida albicans proliferation to the superficial mucosa. The availability of CD4C/HIV transgenic mice expressing HIV-1 in immune cells has provided the opportunity to devise a novel model of mucosal candidiasis that closely mimics the clinical and pathological features of candidal infection in human HIV infection. These transgenic mice allow, for the first time, a precise cause-and-effect analysis of the immunopathogenesis of mucosal candidiasis in HIV infection under controlled conditions in a small laboratory animal.     

Development of a novel three-dimensional in vitro model of oral Candida infection

Oropharyngeal candidiasis is emerging as a serious health problem in the elderly as well as other chronically immunosuppressed patient populations. Several approaches have been used to study the interactions between Candida and the oral mucosa in vitro. Recently, three-dimensional organotypic systems of the oral mucosa have been developed, which provide an organizational complexity that is between the culture of single cell types and organ cultures in vivo. In this report we describe the development of a novel three-dimensional system of the human oral mucosa based on an immortalized oral keratinocyte cell line. Unlike the commercially available cell line systems, this system also contains a connective tissue cell component, which ensures the quality and resemblance of the tissue model to the human oral masticatory mucosa and submucosa. Using a panel of Candida albicans strains with variable virulence in vivo, we showed that the extent of tissue damage, fungal invasion and host inflammatory response in this system was proportional to the well-documented in vivo virulence potential of these strains. Therefore, this investigation has added another useful tool in the study of host pathogen interactions in oral candidiasis.     

Widespread geographic distribution of oral Candida dubliniensis strains in human immunodeficiency virus-infected individuals.

Candida dubliniensis is a recently identified chlamydospore-positive yeast species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected (HIV+) patients and is closely related to Candida albicans. Several recent reports have described atypical oral Candida isolates with phenotypic and genetic properties similar to those of C. dubliniensis. In this study 10 atypical chlamydospore-positive oral isolates from HIV+ patients in Switzerland, the United Kingdom, and Argentina and 1 isolate from an HIV-negative Irish subject were compared to reference strains of C. albicans and Candida stellatoidea and reference strains of C. dubliniensis recovered from Irish and Australian HIV+ individuals. All 11 isolates were phenotypically and genetically similar to and phylogenetically identical to C. dubliniensis. These findings demonstrate that the geographical distribution of C. dubliniensis is widespread, and it is likely that it is a significant constituent of the normal oral flora with the potential to cause oral candidiasis, particularly in immunocompromised patients.     

Bioactive interleukin-1alpha is cytolytically released from Candida albicans-infected oral epithelial cells.

Oral epithelial cells are primary targets of Candida albicans in the oropharynx and may regulate the inflammatory host response to this pathogen. This investigation studied the mechanisms underlying interleukin-1alpha (IL-1alpha) release by oral epithelial cells and the role of IL-1alpha in regulating the mucosal inflammatory response to C. albicans. Infected oral epithelial cells released processed IL-1alpha protein in culture supernatants. The IL-1alpha generated was stored intracellularly and was released upon cell lysis. This was further supported by the fact that different C. albicans strains induced variable IL-1alpha release, depending on their cytolytic activity. IL-1alpha from C. albicans-infected oral epithelial cells upregulated proinflammatory cytokine secretion (IL-8 and GM-CSF) in uninfected oral epithelial or stromal cells. Our studies suggest that production of IL-1alpha, IL-8 and GM-CSF may take place in the oral mucosa in response to lytic infection of epithelial cells with C. albicans. This process can act as an early innate immune surveillance system and may contribute to the clinicopathologic signs of infection in the oral mucosa.     

Salivary secretory leukocyte protease inhibitor and oral candidiasis in human immunodeficiency virus type 1-infected persons

Oropharyngeal candidiasis, typically caused by Candida albicans, is the most common oral disease associated with human immunodeficiency virus type 1 (HIV-1) infection. Secretory leukocyte protease inhibitor (SLPI), a 12-kDa antiprotease, suppresses the growth of C. albicans in vitro. To determine whether the mucosal protein plays a role in protecting oral tissues against fungal infection, we conducted a cross-sectional study investigating the oral and systemic health and salivary SLPI levels in 91 dentate HIV-1-infected adults receiving medical care in the southeastern United States. Participants with a self-reported history of clinical oropharyngeal candidiasis during the previous 2 years constituted the test group (n = 52), while the comparison group (n = 39) had no oropharyngeal candidiasis during that period. Data collected from medical records, oral examination, and SLPI enzyme-linked immunosorbent assay quantitation of whole saliva were analyzed by t test, analysis of variance, linear regression, and unconditional logistic regression. The test group had a significantly higher mean salivary SLPI level than the comparison group (1.9 microg/ml versus 1.1 microg/ml, P < 0.05). Linear regression modeling identified CD4 cell count and history of oropharyngeal candidiasis as key predictors of salivary SLPI and revealed a significant interaction (P < 0.05) between immunosuppression (CD4 cell count below 200 cells/ microl) and positive history of oropharyngeal candidiasis in predicting salivary SLPI level. By logistic regression modeling, a salivary SLPI level exceeding 2.1 microg/ml, low CD4 count, antiretroviral monotherapy, and smoking were key predictors of oropharyngeal candidiasis. These data support a key role for SLPI in the oral mucosal defense against C. albicans. The antimicrobial mucosal protein may serve as an indicator of previous oropharyngeal candidiasis infection among immunosuppressed persons.     

Candida albicans-infected oral epithelial cells augment the anti-fungal activity of human neutrophils in vitro.

Oropharyngeal candidiasis (OPC) is the most common opportunistic infection in immunosuppressed patients. In OPC, Candida albicans persists intraepithelially triggering inflammatory events, without generally causing invasive infection. Since neutrophils play an important role in preventing invasive infection and since they establish contact with the microorganisms only within the epithelial cell layer, we examined the ability of Candida-infected oral epithelial cells to augment neutrophil-mediated hyphal damage in vitro. We found that challenge of neutrophils with hyphal organisms in the presence of C. albicans-infected oral epithelial cell supernatants resulted in a significantly greater suppression of hyphal cell metabolic activity compared to basal neutrophil anti-fungal function. Anti-hyphal activity in response to these supernatants was partly inhibited by neutralizing anti-IL-1alpha antibody and IL-1 receptor antagonist. Control supernatants from uninfected oral epithelial cells, as well as C. albicans conditioned-medium had a much less pronounced effect on neutrophil anti-fungal activity, which was not inhibited by these cytokine antagonists. We conclude that oral epithelial cells can act as activators of neutrophil anti-hyphal function, an effect that can be partly attributed to the generation of immunomodulatory cytokines during the interaction of oral mucosal cells with the pathogen.     

Oral candidiasis: clinical features and control

Candidiasis is the most commonly encountered fungal infection, and oral candidiasis is often observed as a local opportunistic infection. Oral candidiasis is clinically divided into three types: acute forms, chronic forms, and Candida-associated lesions. Candida adhesion and multiplication are largely regulated by the local and systemic factors of the host. The local factors include impairment of the oral mucosal integrity, which is usually impaired by hyposalivation, anticancer drugs/radiation for head and neck cancers, denture wearing, a decrease in the oral bacterial population, and poor oral hygiene. Among Candida species, oral candidiasis is mostly caused by Candida albicans (C. albicans), C. glabrata, or C. tropicalis. Oral Candida induces a variety of symptoms, such as oral mucosal inflammation manifesting as an uncomfortable feeling, pain, erythema, erosion, taste abnormalities, and hyperplasia of the oral mucosa. Candida overgrowth in the oral cavity may disseminate to distant organs. Therefore, in order to avoid the sequelae of systemic candidiasis, oral candidiasis should be rapidly controlled. Oral candidiasis is usually treated by the local application of antifungal drugs. However, oral candidiasis occasionally escapes the control of such local treatment due to the development of multi-drug resistant Candida strains and species or due to the suppression of salivation or cellular immune activity. When drug-resistant strains are suspected as the pathogens and when the host is generally compromised, the oral administration of combinations of antifungal drugs, enhancement of cellular immune activity, and improvement of the nutritional condition are recommended.     

Nitric oxide-enhanced resistance to oral candidiasis

A murine model of oral candidiasis was used to show that nitric oxide (NO) is involved in host resistance to infection with Candida albicans in infection-'resistant' BALB/c and infection-'prone' DBA/2 mice. Following infection, increased NO production was detected in saliva. Postinfection samples of saliva inhibited the growth of yeast in vitro. Treatment with NG-monomethyl-L-arginine (MMLA), an inhibitor of NO synthesis, led to reduced NO production, which correlated with an increase in C. albicans growth. Reduction in NO production following MMLA treatment correlated with an abrogation of interleukin-4 (IL-4), but not interferon-gamma (IFN-gamma), mRNA gene expression in regional lymph node cells. Down-regulation of IL-4 production was accompanied with an increase in IFN-gamma production in infection-'prone' DBA/2 mice. There was a functional relationship between IL-4 and NO production in that mice treated with anti-IL-4 monoclonal antibody showed a marked inhibition of NO production in saliva and in culture of cervical lymph node cells stimulated with C. albicans antigen. The results support previous conclusions that IL-4 is associated with resistance to oral candidiasis and suggest that NO is involved in controlling colonization of the oral mucosal surface with C. albicans.     

Local Th1/Th2 cytokine production during experimental vaginal candidiasis: potential importance of transforming growth factor-beta.

Host defense mechanisms against vaginal Candida albicans infections are poorly understood. Despite the protective role of T helper (Th)1-type cell-mediated immunity (CMI) against mucosal C. albicans infections, studies using an estrogen-dependent murine model of vaginal candidiasis have shown a lack of effect of systemic Th1-type CMI against a vaginal C. albicans infection, and a lack of changes in local T cells during infection. In the present study, the local Thl- (interleukin [IL]-2, interferon [IFN]-gamma and IL-12) and Th2- (IL-4, IL-10 and transforming growth factor [TGF]-beta1) type cytokines were evaluated in vaginal tissue during an experimental C. albicans infection. Results showed constitutive expression of TGF-beta1 in vaginal tissue of naive mice that was two-fold higher than the levels of the other cytokines examined. These high levels of TGF-beta1 were further increased as a result of pseudoestrus and/or infection, and were corroborated at the messenger RNA level. Furthermore, the levels of TGF-beta in naive or infected mice were significantly higher in the vagina compared to other areas of the genital tract. Finally, TGF-beta1 predominated as well in the draining, but not non-draining, lymph nodes during infection. These results suggest that TGF-beta1, a potent immunoregulatory cytokine, may be important in the lack of demonstrable CMI at the vaginal mucosa against C. albicans.     

Vaginal and oral epithelial cell anti-Candida activity

Candida albicans is the causative agent of acute and recurrent vulvovaginal candidiasis (VVC), a common mucosal infection affecting significant numbers of women in their reproductive years. While any murine host protective role for cell-mediated immunity (CMI), humoral immunity, and innate resistance by neutrophils against the vaginal infection appear negligible, significant in vitro growth inhibition of Candida species by vaginal and oral epithelial cell-enriched cells has been observed. Both oral and vaginal epithelial cell anti-Candida activity has a strict requirement for cell contact to C. albicans with no role for soluble factors, and oral epithelial cells inhibit C. albicans through a cell surface carbohydrate moiety. The present study further evaluated the inhibitory mechanisms by murine vaginal epithelial cells and the fate of C. albicans by oral and vaginal epithelial cells. Similar to human oral cells, anti-Candida activity produced by murine vaginal epithelial cells is unaffected by enzymatic cleavage of cell surface proteins and lipids but sensitive to periodic acid cleavage of surface carbohydrates. Analysis of specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose and mannose-containing carbohydrates, also similar to oral cells. Staining for live and dead Candida in the coculture with fluorescein diacetate (FDA) and propidium iodide (PI), respectively, showed a clear predominance of live organisms, suggesting a static rather than cidal action. Together, the results suggest that oral and vaginal epithelial cells retard or arrest the growth rather than kill C. albicans through an as-yet-unidentified carbohydrate moiety in a noninflammatory manner.     

Synergistic interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model

Candida albicans is a commensal colonizer of the gastrointestinal tract of humans, where it coexists with highly diverse bacterial communities. It is not clear whether this interaction limits or promotes the potential of C. albicans to become an opportunistic pathogen. Here we investigate the interaction between C. albicans and three species of streptococci from the viridans group, which are ubiquitous and abundant oral commensal bacteria. The ability of C. albicans to form biofilms with Streptococcus oralis, Streptococcus sanguinis, or Streptococcus gordonii was investigated using flow cell devices that allow abiotic biofilm formation under salivary flow. In addition, we designed a novel flow cell system that allows mucosal biofilm formation under conditions that mimic the environment in the oral and esophageal mucosae. It was observed that C. albicans and streptococci formed a synergistic partnership where C. albicans promoted the ability of streptococci to form biofilms on abiotic surfaces or on the surface of an oral mucosa analogue. The increased ability of streptococci to form biofilms in the presence of C. albicans could not be explained by a growth-stimulatory effect since the streptococci were unaffected in their growth in planktonic coculture with C. albicans. Conversely, the presence of streptococci increased the ability of C. albicans to invade organotypic models of the oral and esophageal mucosae under conditions of salivary flow. Moreover, characterization of mucosal invasion by the biofilm microorganisms suggested that the esophageal mucosa is more permissive to invasion than the oral mucosa. In summary, C. albicans and commensal oral streptococci display a synergistic interaction with implications for the pathogenic potential of C. albicans in the upper gastrointestinal tract.     

Base-metal dental casting alloy biocompatibility assessment using a human-derived three-dimensional oral mucosal model

Nickel-chromium (Ni-Cr) alloys used in fixed prosthodontics have been associated with type IV Ni-induced hypersensitivity. We hypothesised that the full-thickness human-derived oral mucosa model employed for biocompatibility testing of base-metal dental alloys would provide insights into the mechanisms of Ni-induced toxicity. Primary oral keratinocytes and gingival fibroblasts were seeded onto Alloderm™ and maintained until full thickness was achieved prior to Ni-Cr and cobalt-chromium (Co-Cr) alloy disc exposure (2-72 h). Biocompatibility assessment involved histological analyses with cell viability measurements, oxidative stress responses, inflammatory cytokine expression and cellular toxicity analyses. Inductively coupled plasma mass spectrometry analysis determined elemental ion release levels. We detected adverse morphology with significant reductions in cell viability, significant increases in oxidative stress, inflammatory cytokine expression and cellular toxicity for the Ni-Cr alloy-treated oral mucosal models compared with untreated oral mucosal models, and adverse effects were increased for the Ni-Cr alloy that leached the most Ni. Co-Cr demonstrated significantly enhanced biocompatibility compared with Ni-Cr alloy-treated oral mucosal models. The human-derived full-thickness oral mucosal model discriminated between dental alloys and provided insights into the mechanisms of Ni-induced toxicity, highlighting potential clinical relevance.     

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.     

Tissue engineering of lips and muco-cutaneous junctions: in vitro development of tissue engineered constructs of oral mucosa and skin for lip reconstruction

We report for the first time the fabrication of a three-dimensional tissue structure containing, in a continuous layer, the morphological features of a lip: epidermal skin, vermillion, and oral mucosa. This tissue engineered muco-cutaneous (M/C) equivalent was manufactured using human oral and skin keratinocytes grown on an acellular, nonimmunogenic dermal equivalent (AlloDerm(?)) to produce a tissue equivalent with similar anatomic and handling properties as native human lips. Confirmation of the structural composition of the construct was performed using routine histology and immunohistochemistry by identification of epithelial markers that are differentially expressed in separate anatomic areas of the lips. These full-thickness human lip skin equivalents can be used in surgical lip reconstruction in individuals suffering from lip loss from cancer, congenital deformations, and injuries after accidents. We propose this technique can be used as a general basis for tissue engineering of M/C junctions in other parts of the body, such as anus and vagina.     

The protective immune response against vaginal candidiasis: lessons learned from clinical studies and animal models

Recurrent vulvovaginal candidiasis (RVVC) is a significant problem in women of childbearing ages and is caused by Candida albicans, a commensal organism of the intestinal and reproductive tracts. As a result of this commensalism, most healthy individuals have demonstrable Candida-specific adaptive immunity that is considered protective. In women with RVVC, a deficiency/dysfunction of this protective immunity is postulated to affect susceptibility to infection. Although cell-mediated immunity (CMI) is considered important for protection against mucosal candidal infections, little is understood about specific host defenses that are important at the vaginal mucosa. Studies to date suggest that a compartmentalized local, rather than systemic, immunity is important for defense against vaginitis. This review will summarize the current state of knowledge regarding protective host defense mechanisms against vaginal C. albicans infections both from clinical studies and animal models. From these data, hypotheses are presented for what host defense mechanisms appear important for resistance/susceptibility to vaginal infection.     

THE PROTECTIVE IMMUNE RESPONSE AGAINST VAGINAL CANDIDIASIS: LESSONS LEARNED FROM CLINICAL STUDIES AND ANIMAL MODELS

Recurrent vulvovaginal candidiasis (RVVC) is a significant problem in women of childbearing ages and is caused by Candida albicans, a commensal organism of the intestinal and reproductive tracts. As a result of this commensalism, most healthy individuals have demonstrable Candida -specific adaptive immunity that is considered protective. In women with RVVC, a deficiency/dysfunction of this protective immunity is postulated to affect susceptibility to infection. Although cell-mediated immunity (CMI) is considered important for protection against mucosal candidal infections, little is understood about specific host defenses that are important at the vaginal mucosa. Studies to date suggest that a compartmentalized local, rather than systemic, immunity is important for defense against vaginitis. This review will summarize the current state of knowledge regarding protective host defense mechanisms against vaginal C. albicans infections both from clinical studies and animal models. From these data, hypotheses are presented for what host defense mechanisms appear important for resistance/susceptibility to vaginal infection.     

Local Production of Chemokines during Experimental Vaginal Candidiasis

Recurrent vulvovaginal candidiasis, caused by Candida albicans, is a significant problem in women of childbearing age. Although cell-mediated immunity (CMI) due to T cells and cytokines is the predominant host defense mechanism against C. albicans at mucosal tissue sites, host defense mechanisms against C. albicans at the vaginal mucosa are poorly understood. Based on an estrogen-dependent murine model of vaginal candidiasis, our data suggest that systemic CMI is ineffective against C. albicans vaginal infections. Thus, we have postulated that local immune mechanisms are critical for protection against infection. In the present study, the kinetic production of chemokines normally associated with the chemotaxis of T cells, macrophages (RANTES, MIP-1alpha, MCP-1), and polymorphonuclear neutrophils (MIP-2) was examined following intravaginal inoculation of C. albicans in estrogen-treated or untreated mice. Results showed significant increases in MCP-1 protein and mRNA in vaginal tissue of infected mice as early as 2 and 4 days postinoculation, respectively, that continued through a 21-day observation period, irrespective of estrogen status. No significant changes were observed with RANTES, MIP-1alpha, or MIP-2, although relatively high constitutive levels of RANTES mRNA and MIP-2 protein were observed. Furthermore, intravaginal immunoneutralization of MCP-1 with anti-MCP-1 antibodies resulted in a significant increase in vaginal fungal burden early during infection, suggesting that MCP-1 plays some role in reducing the fungal burden during vaginal infection. However, the lack of changes in leukocyte profiles in vaginal lavage fluids collected from infected versus uninfected mice suggests that MCP-1 functions to control vaginal C. albicans titers in a manner independent of cellular chemotactic activity.     

Microsatellite-based genotyping of Candida albicans isolated from patients with superficial candidiasis

This study aimed to examine the genotype distribution of Candida albicans and the major genotypes involved in superficial candidiasis. The genotypes of C. albicans isolated from the infection sites of patients with superficial candidiasis (referred to as infection isolates) were analyzed by fragment analysis using 4 microsatellite markers (HIS3, CDC3, CAI and CAIII). Genotypes of the infection isolates were compared with those of C. albicans isolated from oral mucosa of non-candidiasis patients (referred to as oral isolates). Isolates of C. albicans showed 4 major genotypes for HIS3/CAI (" a " for 148 : 148 / 23 : 23," b " for 148 : 160 / 33 : 41," c " for 148 : 164 / 32 : 41 and " d " for 152 : 152 / 18 : 27). The genotypes " a "," b " and " d " were commonly found in oral (4.7, 8.8 and 7.6%, respectively) and infection (6.6, 9.2 and 15.4%, respectively) isolates. No isolates of genotype " c " were isolated from infection sites. The genotype " a " was found in the isolates from patients with genitalia candidiasis. Genotyping of multiple isolates from an individual patient showed that C. albicans from infection sites was genetically homogenous as compared with that of oral isolates, even in the same patient with candidiasis.