雌激素对阴道上皮细胞抗念珠菌作用的影响

  目的：研究阴道上皮细胞对白色念珠菌的生长抑制作用及雌激素对此作用的影响。 方法： 用酶消化法分别分离雌激素处理组、动情间期组和去势组小鼠阴道上皮细胞作为效应细胞，白色念珠菌芽生孢子作为靶细胞，将效靶细胞共同孵育9 h，测定白色念珠菌生长抑制率，观察其生长密度及透射电镜超微结构的改变。 结果： 白色念珠菌与阴道上皮细胞共同孵育后，生长受到抑制，生长密度降低，超微结构表现为与阴道上皮细胞靠近的念珠菌细胞壁崩解，细胞膜连续性破坏，甚至细胞器肿胀溶解。雌激素处理组与去势组和动情间期组比较，阴道上皮细胞对念珠菌的生长抑制率下降（P<0.05）。 结论： 小鼠阴道上皮细胞具有天然的抗白色念珠菌作用，雌激素降低此抑制作用。     

Potential role for a carbohydrate moiety in anti-Candida activity of human oral epithelial cells

Candida albicans is both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candida host defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of several Candida species in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candida activity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candida activity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candida activity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence of C. albicans to oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose- and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candida activity occurs exclusively with viable epithelial cells through contact with C. albicans by an as-yet-undefined carbohydrate moiety.     

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.     

Growth inhibition of Candida albicans by vaginal cells from na?ve mice.

Recurrent vulvovaginal candidiasis (RVVC) is a common idiopathic mucosal infection caused by Candida albicans. Current data suggests that local immunity is more important than that in the peripheral circulation for protection against infection. In the present study, anti-Candida innate resistance at the vaginal mucosa was investigated using a murine model. For this, splenic and vaginal cells were assessed for in vitro growth inhibition (GI) of C. albicans and cytotoxicity of natural killer (NK) cell-sensitive tumour targets (YAC-1). As expected, significant GI of C. albicans by splenic cells was mediated predominantly by polymorphonuclear leucocytes (PMNL) at effector to target (E:T) ratios of 100 and 50:1. From the vaginal mucosa, na?ve unfractionated, but not nylon wool non-adherent (NWN), cells extracted from whole vaginal tissue showed significant GI of C. albicans at E:T ratios as low as 1:1, but only modest killing of YAC-1 targets at all E:T ratios. Subsequent experiments showed significant GI of C. albicans by vaginal epithelioid-enriched cells and with several epithelial cell lines, but not in supernatants collected from the co-cultures. In contrast, lymphoid cell lines had no anti-Candida activity. These results suggest that anti-Candida activity is present at the vaginal mucosa, but unlike that from the spleen, the vaginal activity appears to be predominantly mediated by epithelial cells.     

Adherence of Candida albicans to a fibrin-platelet matrix formed in vitro.

The adherence of Candida albicans to a fibrin-platelet matrix formed in vitro was studied. Platelet-rich plasma obtained from rabbits was incubated with thrombin and CaCl2 to form a clot in tissue culture dishes. Such clots were then infected with 3 x 10(7) C. albicans cells per 0.3 ml prelabeled with [U14C]-glucose, and the percent adherence was measured after 30 min of incubation by counting the radioactivity in saline washes of the clot as well as a streptokinase-streptodornase digest of the corresponding clot. Heat- and formaldehyde-killed cells did not adhere as well as viable cells. Pretreatment of C. albicans with trypsin, chymotrypsin, and pronase reduced adherence to the clots. Normal rabbit serum and anti-Candida antiserum also inhibited adherence 40 and 100%, respectively, Diethylaminoethyl-purified anti-Candida gamma globulin (1:8) completely inhibited adherence, whereas purified normal serum gamma globulin did not. Several Candida spp. and Saccharomyces cerevisiae showed differences in their ability to adhere to clots. C. albicans and C. stellatoidea presented the highest adherence, whereas C. krusei, C. guilliermondii, and S. cerevisiae adhered less readily. Other species were intermediate in their ability to adhere.     

Vaginal epithelial cell anti-Candida albicans activity is associated with protection against symptomatic vaginal candidiasis

Vaginal epithelial cell (VEC) anti-Candida albicans activity, despite being measured in vitro, is considered an innate host defense mechanism. This was supported further by the fact that women protected from symptomatic infection following a live intravaginal Candida challenge had increased VEC anti-Candida activity compared to those who acquired a symptomatic infection.     

Augmentation of GG2EE macrophage cell line-mediated anti-Candida activity by gamma interferon, tumor necrosis factor, and interleukin-1.

The expression of anti-Candida activity in the GG2EE macrophage cell line, generated by immortalization of fresh bone marrow with v-raf and v-myc oncogenes, was studied. GG2EE cells spontaneously inhibited the growth of an agerminative mutant of Candida albicans in vitro. The anti-Candida activity was maximal after 8 h of coculture and was proportional to the effector-to-target ratio. Gamma interferon (IFN-gamma), interleukin-1 (IL-1), and tumor necrosis factor (TNF) all significantly enhanced the anti-Candida activity of GG2EE cells. In contrast, IL-3, IL-4, and colony-stimulating factor 1 were ineffective. The augmentation of anti-Candida activity was not always concomitant with enhancement of phagocytosis, since IFN-gamma and colony-stimulating factor 1, but not IL-1 or TNF, augmented the phagocytic ability of GG2EE cells. Furthermore, the augmentation of anti-Candida activity in GG2EE cells did not correlate with the acquisition of antitumor activity. In fact, none of the cytokines alone were able to induce antitumor activity in GG2EE cells, which, however, could be activated to a tumoricidal stage by IFN-gamma plus heat-killed Listeria monocytogenes. These findings demonstrate that GG2EE cells exhibit spontaneous anti-Candida activity and that such activity is enhanced by TNF, IL-1, and IFN-gamma.     

Lactoferrin release and interleukin-1, interleukin-6, and tumor necrosis factor production by human polymorphonuclear cells stimulated by various lipopolysaccharides: relationship to growth inhibition of Candida albicans.

Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and Salmonella typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro. Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells. LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C. albicans in vitro. Antibodies against lactoferrin effectively and specifically reduced the anti-C. albicans activity of both LPS-stimulated and unstimulated PMN. Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS. The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide. These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN. These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C. albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN. Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.     

Oral epithelial cell antifungal activity: approaches to evaluate a broad range of clinical conditions.

Anti-Candida activity by oral epithelial cells is considered one of several innate mucosal defense mechanisms against oropharyngeal candidiasis (OPC). OPC is the most common fungal infection in HIV disease. Previously we reported that oral epithelial cell anti-Candida activity is reduced in those with OPC, potentially representing a contributing factor to OPC. However, testing clinical epithelial cells possessing high levels of Candida has been limiting due to high background in the assay controls. HIV+ smokers often develop OPC sooner than non-smokers during progression to AIDS, suggesting additional immune aberrations. The purpose of this study was to design a means to reduce Candida associated with epithelial cells collected from saliva without affecting their in vitro growth inhibitory activity, and to employ that approach to evaluate antifungal activity in HIV+ smokers. To do so, oral epithelial cells with and without known levels of Candida were subjected to various treatments including azole, polyene, or echinocandin antifungal drugs or fixation followed by the standard growth inhibition (GI) assay. The results indicated that antifungal drugs, while effectively reducing cell-associated Candida, also affected epithelial cell function. In contrast, fixation with paraformaldehyde eliminated cell-associated Candida and had minimal effects on epithelial cell anti-Candida activity. Employing the fixation design that allowed a broad range of patients to be evaluated showed no difference in oral epithelial anti-Candida activity between HIV+ smokers and non-smokers. Therefore, oral epithelial cell antifungal activity does not appear compromised in those who smoke, reducing it as a contributing factor in susceptibility to premature OPC.     

Role of L3T4+ lymphocytes in protective immunity to systemic Candida albicans infection in mice.

Protective immunity to lethal Candida albicans challenge in vivo and activation of splenic macrophages with highly candidacidal activity in vitro were detected in mice infected with low-virulence agerminative yeast cells of the variant strain PCA-2, at a time when a strong delayed-type hypersensitivity (DTH) reaction to C. albicans occurred in the footpads of PCA-2-treated mice. The DTH reaction was transferable with spleen cell populations from these animals, and enrichment of splenic lymphocytes in L3T4+ cells significantly increased the footpad swelling. The reactivity transferred by L3T4+ cells was a radiosensitive (2,500 rads in vitro) phenomenon that required collaboration with radioresistant, silica-sensitive syngeneic cells in the host and was inhibited by treatment of recipient mice with antibodies to the L3T4 antigen or murine gamma interferon. In vitro, the PCA-2-immune L3T4+ cells produced various lymphokine activities upon incubation with C. albicans, including gamma interferon and granulocyte-macrophage colony-stimulating factor. Anti-L3T4 monoclonal antibody treatment of PCA-2-infected mice significantly impaired their footpad reaction and resistance to C. albicans, as shown by increased recovery of yeast cells from the kidneys of anti-L3T4-treated mice. These results suggested that the mechanisms of anti-Candida resistance induced by PCA-2 may involve specific induction of a DTH response mediated by inflammatory L3T4+ T cells and lymphokine-activated phagocytic effectors. However, the survival rate of the PCA-2-immune mice challenged with C. albicans was not significantly modified by administration of the anti-L3T4 antibody, thus allowing for the conclusion that compensatory mechanisms lead to considerable anti-Candida resistance when the activity of L3T4+ cells is deficient.     

Growth inhibition of Candida albicans by interleukin-2-activated splenocytes.

Murine splenocytes, Percoll-enriched low-density lymphocytes, and interleukin-2 (IL-2)-activated lymphocytes were assessed for the capacity to limit the growth of the hyphal form of Candida albicans. No fungal-growth-inhibitory activity was exhibited for C. albicans by either splenocytes or Percoll-enriched lymphocytes. These cells were capable of cytotoxic activity for a natural killer cell-sensitive cell line. However, when cultured for several days with IL-2, splenocytes acquired the capacity to inhibit the growth of the fungus. The appearance of the antifungal activity coincided with the development of cytotoxic activity for the natural killer cell-insensitive cell line. Anti-C. albicans and antitumor activities of IL-2-activated lymphocytes were competitively and reciprocally inhibited by C. albicans and the natural killer cell-sensitive and -insensitive cell lines. The antifungal activity of the IL-2-activated lymphocytes was exhibited against a number of clinical isolates of C. albicans and related fungal species. IL-2-activated human peripheral blood lymphocytes also acquired the capacity to inhibit the growth of C. albicans. These data show that in vitro growth inhibition can be mediated by IL-2-stimulated lymphocytes which are neither fungal strain nor mammalian species restricted in their biological activity.     

Enhanced killing of Candida albicans by human macrophages adherent to type 1 collagen matrices via induction of phagolysosomal fusion

Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candida activity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes in C. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candida activity and that C. albicans pathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.     

Interspecies variation in Candida biofilm formation studied using the Calgary biofilm device

An in vitro assay to study multiple Candida biofilms, in parallel, has been carried out using the Calgary biofilm device (CBD). We here report: i) standardization of the CBD for Candida albicans biofilm formation, ii) kinetics of C. albicans biofilm formation, iii) biofilm formation by five Candida species, and iv) effect of dietary carbohydrates on biofilm formation. The biofilm metabolic activity on all CBD pegs was similar (p=0.6693) and C. albicans biofilm formation revealed slow growth up to 36 h and significantly higher growth up to 48 h (p<0.001). Significant differences in total biofilm metabolic activity were seen for glucose, fructose and lactose grown C. albicans compared with sucrose and maltose grown yeasts. Candida krusei developed the largest biofilm mass (p<0.05) relative to C. albicans, C. glabrata, C. dubliniensis and C. tropicalis. Scanning electron microscopy revealed that C. krusei produced a thick multilayered biofilm of pseudohyphal forms embedded within the polymer matrix, whereas C. albicans, C. dubliniensis and C. tropicalis biofilms consisted of clusters or chains of cells with sparse extracellular matrix material. We conclude that CBD is a useful, simple, low cost miniature device for parallel study of Candida biofilms and factors modulating this phenomenon.     

Co-stimulation of murine CD4 T cell growth: cooperation between B7 and heat-stable antigen.

The B cell activation antigen B7/BB1 has been shown to co-stimulate growth of human T cells by binding the T cell molecule CD28. In mice, the heat-stable antigen (HSA) has also been shown to act as a co-stimulator for T cell growth. In this study, we have evaluated the contributions of B7 and HSA to the co-stimulatory activity of antigen-presenting cells (APC). Mouse B7 provides co-stimulatory activity for murine CD4 T cells in anti-CD3-induced proliferation. Human CTLA4Ig, a chimeric molecule comprising the extracellular region of CTLA-4 fused to an immunoglobulin C gamma fragment, binds to murine B7. We, therefore, use human CTLA4Ig and the hamster anti-HSA monoclonal antibody 20C9 to analyze the relative contributions of B7 and HSA to the co-stimulatory activity of murine spleen APC. Our data reveal that both murine B7 and HSA are expressed by dendritic cells and by low-density spleen B cells. Either CTLA4Ig alone or anti-HSA alone inhibited CD4 T cell proliferation to anti-CD3 by > 90%, while CTLA4Ig and anti-HSA together were far more efficient in inhibiting clonal expansion of CD4 T cells. These results demonstrate that functionally defined co-stimulation involves at least B7 and HSA and suggest that signals delivered by B7 and HSA synergize in promoting T cell growth.     

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.     

Heterogeneous activity of immature and mature cells of the murine monocyte-macrophage lineage derived from different anatomical districts against yeast-phase Candida albicans.

Mature mononuclear phagocytes have been receiving much attention as effectors of spontaneous candidacidal activity, although with controversial results due to differences in the effector populations and the methods used in different laboratories. We here systematically compare the fungistatic activity of immature and mature cells of the murine macrophage series. The results show that nonadherent, nonphagocytic precursor cells (isolated either [90% purity] from bone marrow liquid cultures or from the organs of mice in which inflammatory conditions had been elicited in vivo) exerted a strong extracellular candidastatic activity. In contrast, mature macrophages, either obtained from different anatomical areas (spleen, liver, lung, peritoneal cavity) or matured in vitro from the precursor populations, displayed striking heterogeneity in their ability to inhibit the growth of Candida albicans, depending on the anatomical site they were derived from. Lymphokine activation did not alter the fungistatic pattern of the untreated cells. The different macrophage populations behaved very differently also in the production of reactive oxygen intermediates (ROI) in response to phagocytosis of C. albicans. The amounts of ROI generated, however, showed no correlation with candidastatic ability. Low levels of candidastatic activity exerted by resident peritoneal macrophages (good ROI producers) were inhibited by catalase, whereas high levels of growth inhibition by Kupffer cells (poor ROI producers) after 8 h of assay were hardly influenced by the enzyme. Our data suggest the existence of two different effector mechanisms in macrophage-mediated C. albicans growth inhibition, a rather inefficient ROI-dependent one, and a second, very efficient oxygen-independent mechanism. The implications of these findings are discussed.     

Characterisation of yeasts implicated in vulvovaginal candidosis in Irish women

High-vaginal swabs were taken from 98 women attending a genitourinary clinic in Dublin city who presented with symptoms of vaginitis. Twenty-eight (28.6%) proved culture-positive for yeasts. Candida albicans was isolated from 26 of the yeast-positive patients and Candida glabrata and Candida tropicalis were isolated from a further two patients. Two patients were colonized by two yeasts simultaneously (C. albicans and Candida lucitaniae, and C. albicans and Candida krusei). Yeasts were characterised on the basis of their adherence ability, extracellular enzyme production and resistance to antifungal agents. All C. albicans isolates demonstrated high adherence abilities to buccal epithelial cells, but produced relatively low levels of phospholipase and acid proteinase. The non-C. albicans isolates demonstrated low levels of adherence, but no extracellular enzyme production was detected. Isolates were most sensitive to the polyenes amphotericin B and nystatin but a large proportion (50%) of C. albicans isolates were resistant to clotrimazole, which is an important agent in the treatment of vulvovaginal candidosis.     

抗白念珠菌芽管单克隆抗体的制备及特性鉴定

目的制备抗白念珠菌芽管的单克隆抗体mAb03.2C1-C2,并对其特性进行分析,以探讨其应用于实验室检测的可行性。方法利用杂交瘤技术制备分泌抗白念珠菌芽管胞壁外膜抗原mAb的杂交瘤细胞株,对mAb的Ig亚类进行鉴定。用间接免疫荧光(IIF)法对mAb的抗体活性及特异性进行测定。在白念珠菌芽管形成条件下,加入mAb,观察其对白念珠菌芽管诱导的抑制及白念珠菌对上皮细胞、内皮细胞黏附的抑制。复苏冻存的杂交瘤细胞株,分析其持续分泌mAb的能力。结果获得1株mAb03.2C1-C2,其Ig亚类为IgG1。该mAb仅与白念珠菌芽管特异性结合,并能显著降低白念珠菌芽管的生成率以及在芽管形成条件下对上皮细胞、内皮细胞的黏附,其抑制作用与该mAb的浓度呈正比。结论制备的mAb03.2C1-C2抗体效价高,在体外能抑制白念珠菌芽管的形成,降低白念珠菌的侵袭力。而且其对白念珠菌芽管具有高度的特异性,可用于白念珠菌和都柏林念珠菌的快速鉴别。     

Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants.

Strains of Candida albicans, selected on the basis of their reduced agglutination with a polyclonal anti-Candida antiserum, were tested for their adherence to human buccal epithelial cells (BEC). Of four strains, one (A9V2) had reduced binding to BEC in vitro. Adherence of wild type (wt) yeast cells (A9), as measured by the percentage of BEC with adhering Candida cells, was 73.4% +/- 3.8% compared with 49.3% +/- 3.1% for A9V2 (P less than 0.01). From yeast cells of A9 and A9V2, whole-cell extracts and dithiothreitol-, Zymolyase-, or beta-mercaptoethanol-solubilized cell extracts were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). From dithiothreitol-solubilized cell extracts, proteins with molecular masses of 55 to 60, 80 to 84, 115, and 165 kDa were observed from wt (A9) cells but were highly reduced in amount or absent from A9V2 cells. Western blot profiles of Zymolyase-solubilized extracts from both A9 and A9V2 were similar in appearance, while 55-, 80- to 84-, 115-, and 165-kDa proteins were observed only in A9 cells extracted with beta-mercaptoethanol. Strain A9V4, also selected by reduced agglutination but which adhered as well as strain A9, lacked the 80- to 84-kDa and 115-kDa proteins but otherwise was similar to strain A9. These results indicate that the 55- to 60- and 165-kDa proteins may be related to an adhesin function in C. albicans. The differences observed in the protein profiles of the wt adhering strain and its derived nonadhering mutant are similar to those described for another matched pair of C. albicans strains.     

Redundant role of TLR9 for anti-Candida host defense

The role of Toll-like receptor-9 (TLR9) in the recognition of Candida albicans and anti-Candida host defense was investigated in a murine model of disseminated candidiasis and in human peripheral blood mononuclear cells (PBMC). Blocking TLR9 by a specific inhibitor of human TLR9 or stimulation of cells isolated from TLR9-deficient (TLR9-/-) mice resulted in a 20-30% reduction in cytokine production induced by C. albicans. However, this defect was not accompanied by differences in mortality and organ fungal growth between TLR9-/- and TLR9+/+ mice. In conclusion, TLR9 is a pathogen-recognition receptor for C. albicans, and TLR9 is involved in the induction of cytokines in response to C. albicans. However, the cytokine defect in TLR9-/- mice is compensated by alternative pathways, and the TLR9-dependent pathway seems to be redundant in the disseminated candidiasis model in mice.