Cytokine response to inactivated Candida albicans in mice

Inactivated Candida albicans (CA) cells induce strong activation of natural cytotoxic effectors in mice. In the present study we examined the expression of cytokine genes involved in the immune response to CA. It has been reported that differential cytokine production by natural immune cells is important for regulating the development of specific TH response. Northern blot analysis was performed on peritoneal exudate cells (PEC) recovered from CD2F1 mice injected ip with five doses of CA (CA-5d, on Days -14, -10, -7, -3, 0 with respect to the in vitro assays at 2, 24, and 72 hr) or from mice injected ip with four doses of CA (CA-4d, on Days -14, -10, -7, -3 with respect to the in vitro assay on Day 0). On Day 0, before the fifth CA injection, PEC expressed a high level of IL-2 and a low level of IL-1 beta mRNAs while genes coding for IL-4, IL-5, IL-6, IL-10, IL-12, TNF alpha, and IFN gamma were not expressed and there was a high level of NK activity. Two hours after CA-5d a high level of IFN gamma and a low level of IL-10 mRNAs were already evident, while IL-2 and much more IL-1 beta had greatly increased. IL-6, TNF alpha, and IL-2R alpha chain mRNAs were also detectable, whereas IL-4, IL-5, and IL-12 were not expressed. IL-12 mRNA was also absent in earlier stages of the CA sensitization. Both cellularity and NK activity of peritoneal exudate had increased with respect to Day 0. At 24 hr whereas IL-2 mRNA remained high, both IL-1 beta and IFN gamma mRNAs expression had decreased. Expression of other cytokines was no longer detectable but NK activity remained high and a significant LAK activity was also induced. After 72 hr, while the IL-2 mRNA level and NK activity were still high the IL-1 beta mRNA expression had further decreased. These results indicate that CA induces a predominant production of IFN gamma and IL-2, cytokines involved in the development of TH1 response but it is unable to induce IL-12. This secondary pathway, without IL-12 involvement in the development of TH1 response, is probably the result of the ability of IL-2, IL-1 beta, and TNF alpha to synergize in inducing IFN gamma synthesis by NK cells.     

Lack of effect of Candida albicans mannan on development of protective immune responses in experimental murine candidiasis

Candida albicans mannoprotein (MAN) administered to mice before or during immunization with viable C. albicans downregulates MAN-specific delayed hypersensitivity. In the experiments reported here we determined the effect of MAN downregulation on protective immunity in minimally immunized mice, i.e., mice exposed to C. albicans either intradermally or intragastrically, and in maximally immunized mice, i.e., mice immunized by a combination of intradermal and intragastric exposure, in experimental systemic candidiasis. MAN suppression did not induce statistically significant alterations in the protective responses in experimental candidiasis, although 8 of 12 groups of mice treated with MAN had fewer CFU of C. albicans in their kidneys than their non-MAN-treated counterparts. The results emphasize the lack of correlation of delayed hypersensitivity with protection in candidiasis and suggest that MAN may contain epitopes involved in the protective response.     

Cytokine levels affected by gamma-linolenic acid

The adrenergic receptors (ARs) belong to the superfamily of membrane-bound G protein coupled receptors (GPCRs). Our investigation has focused on the structure-function relationship of the alpha 1b-AR subtype used as the model system for other GPCRs. Site-directed mutagenesis studies have elucidated the structural domains of the alpha 1b-AR involved in ligand binding, G protein coupling or desensitization. In addition, a combined approach using site-directed mutagenesis and molecular dynamics analysis of the alpha 1b-AR has provided information about the potential mechanisms underlying the activation process of the receptor, i.e. its transition from the 'inactive' to the 'active' conformation.     

Chronic neutrophilic meningitis caused by Candida albicans

Meningitis caused by Candida albicans is a very infrequent entity. We present 3 intravenous drug users with chronic neutrophilic meningitis caused by Candida albicans. The clinical, microbiological and radiological features of the 3 patients are reviewed. The interval between the onset of the disease and the diagnosis was long (from 4 to 12 months). Candida albicans was cultured from cerebrospinal fluid (CSF) in the 3 patients. All of them developed hydrocephalus, meanwhile arachnoiditis was disclosed in two. The therapy with amphotericyn, 5-flucytosine and fluconazole produced clinical improvement and the sterilisation of the CSF in all the 3 cases. Clinicians should be aware of this entity because of diagnosis may be delayed in several months.     

Incidence of mother-child contamination by Candida albicans during labor

Two different studies assessed that materno-fetal contamination at time of delivery is not the usual way of colonization of newborns with Candida albicans. In a first study, occurence of Candida albicans in neonate units disappeared owing to prophylactic measures, such as strict care in hospital hygiene. In a second study, detailed herein and performed in an obstetric ward, it was found that despite a rate of vaginal Candida carrier of 19 %, candidiasis in newborns was exceptional.     

Development of a method to detect secretory mucinolytic activity from Candida albicans

Ultrastructural examinations of sites where Candida albicans invaded the bowel wall after oral intragastric inoculation of infant mice suggested that blastoconidia are capable of progressive extracellular digestion of the intestinal mucus barrier. Microplate assay methods, based on biotin or digoxigenin-labelling systems, were therefore devised for quantitation of protease and glycosidase activities against the glycoprotein mucin. Labelled mucin was adsorbed on microplate wells, incubated with sample to be assayed for enzyme activity, and the remaining labelled mucin was quantitated by spectrophotometry. Proteolytic activity against mucin was demonstrated using concentrated culture filtrate of C. albicans strain LAM-1, grown in yeast nitrogen base medium containing mucin as sole nitrogen source. The activity was inhibited by boiling for 10 min or by incubation with the aspartyl proteinase inhibitor pepstatin A.     

The involvement of nitric oxide in the anti-Candida albicans activity of rat neutrophils

Autoantibodies to intracellular constituents often occur naturally. This would be difficult to understand were they unable to penetrate live cells, as was once generally accepted; however, they can and in so doing may alter cell functions, cause damage and even kill cells by apoptosis. Different autoantibodies have different effects and in this paper, further to our previous report on the penetration of anti-DNA, the penetration of anti-RNP, which may be a possible cause of apoptosis, is demonstrated. Penetration of lymphocytes by autoantibodies may play a role in the causation of autoimmune disease, influencing immune regulation and causing cell damage either directly or through nucleosomal DNA release as a result of apoptosis. This, in turn, could also further promote antigen-driven production of anti-DNA. In addition, by causing apoptosis of autoreactive cell clones, natural autoantibodies could influence tolerance during development and also later in life, thus, paradoxically, helping prevent autoimmune disease.     

Colorimetric MTT assessment of antifungal activity of D0870 against fluconazole-resistant Candida albicans

The in vitro antifungal activity of D0870 against eight isolates of fluconazole-resistant Candida albicans was compared with that of itraconazole, ketoconazole and miconazole. The colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] assay was used to assess the antifungal activities. The 50% minimum inhibitory concentration (MIC50) of D0870 was below 0.031 microgram ml-1 for seven isolates and 0.25 microgram ml-1 for one isolate. The activity of D0870 was superior to that of the other azoles. Ketoconazole was the most effective azole next to D0870. Therefore, the new bis-triazole, D0870, is expected to be promising for the therapy of fluconazole-resistant candidosis. The present data also confirmed that the MTT assay may be useful for evaluation of resistance and detection of resistant C. albicans.     

The formation of hyphae of Candida albicans induced by cyclodextrins

Hyphal growth of Candida albicans was observed when yeast was cultured at 27 degrees C in liquid media containing 1% Tripcasine and 1.8% cyclodextrins (alpha, beta, and gamma respectively). Tripcasine as the sole nitrogen source did not induce the formation of hyphae of C. albicans, but cyclodextrins, especially CD-beta, were able to induce yeast-mycelial transition. In the TCD-beta media 25-30% septate hyphae form was observed. This study indicates the existence of an uptake system for CDs in C. albicans, provided these compounds are linearised in the medium. The CDs are inducers of hyphae and they may enter the C. albicans cells as linear oligosaccharids.     

Isotachophoretic analysis of short-chain carboxylic acids produced by Candida albicans

Short-chain carboxylic acids produced by Candida albicans in glucose supplemented batch cultures and in human denture plaque has been qualitatively and quantitatively analysed using isotachophoresis. This rapid, simple and relatively new technique which has advantages over other conventional methods, such as gas liquid chromatography, could be a valuable tool in the analysis of carboxylic acids produced by other yeasts of clinical and industrial importance.     

Inhibition of 2,3-oxidosqualene-lanosterol cyclase in Candida albicans by pyridinium ion-based inhibitors

The N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpyridinium and N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpicolinium cations were evaluated for their ability to inhibit 2,3-oxidosqualene-lanosterol cyclase activity in Candida albicans. Both compounds inhibited fungal growth, were fungicidal, and resulted in the accumulation of squalene epoxide concurrent with a decrease in ergosterol, monomethyl sterols, and lanosterol, as was expected for the specific inhibition of 2,3-oxidosqualene-lanosterol cyclase activity. These compounds are electron-poor aromatic mimics of a monocyclized transition state or high-energy intermediate formed from oxidosqualene, which may explain their selective action.     

Comparison by ELISA of serum anti-Candida albicans mannan IgG levels of a normal population and in diseased patients

A method is described for estimating the degree of genetic variability between parents and offspring for polygenic traits. Dermatoglyphic pattern elements on fingertips palms, and soles illustrate the method and the quantitative contribution of each sex was determined.     

Interferon-gamma activates the oxidative killing of Candida albicans by human granulocytes

The sequence proline-proline-glycine-proline is highly conserved in cytochrome P450 families 1 and 2, and similar proline rich sequences are found in other cytochromes P450. Since this sequence immediately follows the NH2-terminal hydrophobic membrane insertion signal, it potentially could function as a signal either for retention of cytochrome P450 in the endoplasmic reticulum or for its correct orientation in the membrane. To test this possibility, DNA sequence coding for this tetrapeptide was deleted from cytochrome P450 2C2 cDNA. Translation of the mutated mRNA in a reticulocyte cell-free system containing canine microsomal membranes resulted in the insertion of the protein into the membrane with a topology indistinguishable from that of normal cytochrome P450 2C2. The mutated protein was expressed in COS1 cells and its distribution, assayed by immunofluorescence, was similar to that of cytochrome P450 2C2. Furthermore, if a short peptide containing a potential glycosylation site was fused to the N-terminus of the mutant protein, the new hybrid protein was glycosylated in COS1 cells and the carbohydrate moiety remained sensitive to cleavage by endoglycosidase H. These results indicate that the protein was inserted and retained in the endoplasmic reticulum membrane. Pulse-chase studies showed that the mutated protein was degraded about four times as fast as cytochrome P450 2C2. In contrast to cytochrome P450 2C2, no (omega-1) hydroxylase activity was detected in COS1 cells expressing the mutated protein at similar steady-state levels as the wild-type protein. These results indicate that, although the conserved PPGP tetrapeptide is not required for cellular localization of cytochrome P450 in the endoplasmic reticulum membrane, its deletion decreases the stability of the protein and abolishes enzymatic activity.     

Endothelial cell injury caused by Candida albicans is dependent on iron

MRL-lpr/lpr mice spontaneously develop manifestations of autoimmunity including arthritis, vasculitis, and glomerulonephritis. The paramagnetic molecule nitric oxide has been implicated as an effector molecule in initiation and propagation of these inflammatory conditions. In this study, we utilized electron paramagnetic resonance spectroscopy to directly detect nitrosylated protein complexes as products of nitric oxide in whole blood and in kidneys of MRL-lpr/lpr mice. Electron paramagnetic resonance spectra of blood samples from MRL-lpr/lpr mice showed nitrosyl hemoglobin species. Amounts of blood nitrosyl hemoglobin in MRL-lpr/lpr mice were significantly increased as compared to age-matched control mice. Electron paramagnetic resonance spectra of MRL-lpr/lpr kidney tissue exhibited a signal characteristic of a dinitrosyl-iron-dithiolate complex at g approximately 2.04. Formation of nitrosylated nonheme protein in diseased kidneys is associated with development of glomerulonephritis in the autoimmune mice. The presence of nitrosylated nonheme protein indicates the formation of nitric oxide within the kidneys of the diseased mice signifying in situ renal nitric oxide formation.     

Antifungal susceptibility of clinically isolated Candida albicans by broth microdilution method

In this study we investigated the antifungal susceptibility of 285 strains of Candida albicans isolates at Kinki University Hospital from March 1995 to December 1996. The antifungal agents tested were fluconazole, miconazole, intraconazole, amphotericin B and flucytosine. The susceptibility testing were performed according to the broth microdilution method standardized by National Committee for Clinical Laboratory Standards (M27-T). Most isolates of C. albicans showed relatively a low MIC value and the MIC90S were calculated at 1 microgram/ml; fluconazole, 0.125 microgram/mg; miconazole, 0.06 microgram/ml; itraconazole, 1 microgram/ml; amphotericin B, 0.25 microgram/ml; flucytosine. There was only one strain that showed high resistance against fluconazole and it showed cross-resistance against miconazole and itraconazole. There were two flucytosine resistant strains. The MICs of amphotericin B were tightly clustered and resistant strain were not observed.     

Utility of Albicans ID plate for rapid identification of Candida albicans in clinical samples. Rapid identification of Candida albicans

A clinical study was carried out in an attempt to assess the efficacy of a newly designed electric toothbrush compared to a conventional manual toothbrush using the American Dental Association's protocol for evaluating toothbrushes. An Oral-B 35 manual toothbrush, which served as the control, was compared to the Plaq & White125 electric toothbrush. Examinations were performed by two calibrated examiners at baseline, day 15 and day 30. Examinations included the gingival index, plaque index and bleeding index. Mean indices were calculated and compared between the two brushes using the repeated measures multiple analysis of variance. No statistically significant differences between the mean indices on the three examination days were observed following the use of the manual or the electric toothbrushes. The results of this study demonstrate that the electric toothbrush was numerically more effective than the manual toothbrush in reducing supragingival plaque levels, either before or after brushing, at each examination date compared to baseline plaque values. However, this difference was not statistically significant. This and other findings concluded that the Plaq & White toothbrush is comparable to the control ADA-accepted toothbrush.     

Interleukin-15 augments superoxide production and microbicidal activity of human monocytes against Candida albicans

Overexpression of EGFR and c-erbB2 frequently occurs in human breast cancers, correlating with poor prognosis. Here we show that overexpression of EGFR and c-erbB2 in cell lines increases cell migration, an important step in metastasis formation. The effect of EGFR on migration is dependent on the addition of EGF to the cells. In contrast, c-erbB2 seems to act independently of its ligand in these assays. Overexpression of this receptor is sufficient to induce cell migration. In addition, we investigated the involvement of a number of signal transduction pathways known to be activated by the EGFR. We found that inactivation of MAPKK results in a decreased migration, while inactivation of PI3K increases migration.     

Alterations in frequency of interleukin-2 (IL-2)-, gamma interferon-, or IL-4-secreting splenocytes induced by Candida albicans mannan and/or monophosphoryl lipid A

We have shown previously that intravenous injection of Candida albicans mannan (MAN) into naive mice induced CD8+ effector downregulatory cells and that such cells were not produced if mice were deficient in CD4+ or I-A+ cells during the early interval (< or =30 h) following the introduction of MAN. Moreover, the nonspecific biological response modifier monophosphoryl lipid A (MPL), given in vivo or incubated with cells in vitro, can abrogate the MAN-specific immunomodulatory activity. The mechanism by which the abrogation is mediated is unknown, but it is hypothesized to involve cytokines. Therefore, we measured the number of cytokine-secreting cells for the Thl cytokine interleukin-2 (IL-2) and the Th2 cytokine IL-4, as well as for gamma interferon (IFN-gamma), in splenocyte populations from MAN and/or MPL-treated mice, using an enzyme-linked immunospot assay designed to detect individual cytokine-secreting cells (spot-forming cells [SFC]). Cytokine-secreting cells were demonstrated in cell suspensions enriched for CD4+ cells, but no SFC could be demonstrated in populations enriched for CD8+ cells. Both MAN and MPL, when administered to separate groups of animals, stimulated the production of increased numbers of cytokine-producing cells for each of the three cytokines tested. The response with respect to IL-4-secreting cells, however, was the most striking. Despite the fact that MAN and MPL independently caused increases in SFC to all three cytokines, when both MAN and MPL were administered to the same animal, all increases were reversed, and the numbers of SFC detected were at or below those detected in saline control animals. These data support the hypothesis that IL-4 is involved in MAN-specific immunoregulatory activities. The data also emphasize the fact that two immunomodulators, i.e., MAN and MPL, having similar effects when given in vivo independently, may be antagonistic when administered sequentially to the same animal.     

Chronic Candida albicans otitis media in children with immunodeficiency

Biliary peritonitis is a rare but serious complication of percutaneous renal access and surgery. We present such a case and review the literature.