Production of anti-Candida antibodies in mice with gut colonization of Candida albicans

BACKGROUND: Production of antibodies that are specific for allergens is an important pathological process in inflammatory allergic diseases. These contain the antibodies against antigens of Candida albicans, one of the normal microbial flora in an intestinal tract. We studied the effects of the prednisolone administration on the production of anti-Candida antibodies in the gastrointestinally C. albicans-colonized mice. METHODS AND MATERIALS: BALB/c mice, treated with antibacterial antibiotics to decontaminate indigenous intestinal bacterial flora, were inoculated intragastrically with C. albicans. The mice, in which C. albicans grows intestinally, were administered prednisolone to induce temporary immunosuppression. The Candida growth in their intestinal tract and their antibody response to Candida were examined. RESULTS: Antibiotic treatment allowed establishment of C. albicans gastrointestinal colonization, but did not cause subsequent systemic dissemination of C. albicans in all the animals. When these animals received an additional treatment with prednisolone, they showed a significantly higher population of C. albicans in their feces than those of animals treated with antibiotics alone, and the organisms were recovered even from their kidney. This systemic dissemination by C. albicans appeared to be temporal, because all the mice survived without any symptoms for more than 2 months. Examination of the serum titers of total immunoglobulin (Ig)E antibodies and specific IgE and IgG antibodies against Candida antigens demonstrated that titers of total IgE increased, partially by day 14 and clearly at day 27, in prednisolone-treated Candida-colonized mice. Without prednisolone treatment, an increment of the serum titer was scarcely observed. By day 27, corresponding to the increase of total IgE, the anti-Candida IgE and IgG titer increased in mice of the prednisolone-treated group. CONCLUSION: Administration of prednisolone to Candida-colonized mice can induce production of the IgG, IgE antibodies against Candida antigens, perhaps through temporal systemic dissemination of Candida from the intestinal tract.     

Detection of specificity of a new antigen in Candida tropicalis and its evaluation by taxonomic DNA analyses

Monospecific factor serum for identifying Candida tropicalis was obtained either from rabbit antiserum to heated cells of C. tropicalis M 1519 (S 96) or from antiserum to C. tropicalis IFO 1400, by adsorption with heated cells of Candida albicans serotype A, or C. albicans (A) and Candida krusei, respectively. We designated this adsorbed serum factor t serum. The monospecific factor serum reacted with 31 out of 32 strains of C. tropicalis, only when tested on heat-treated cell antigens, whereas it did not react with any of 72 strains of the six other medically important species of Candida. The morphological and physiological characteristics of the one strain of C. tropicalis that did not react with the factor t serum, designated the t- -strain, were shown to be similar to those of the type strain of C. tropicalis by most of the methods employed for identifying Candida. Therefore, cell wall mannan from the t- -strain was compared with that from several typical strains of C. tropicalis for its specificity by the precipitation reaction and also for its 1H-nuclear magnetic resonance spectrum. The results showed that these mannans are similar to each other serologically and physicochemically, suggesting that the new antigen t is not mannan. Taxonomic characterization of the t-- and several typical strains of C. tropicalis was carried out by determining the mol% G+C of their DNA and also their DNA homology. Although the mol% G+C values of four typical strains of C. tropicalis were fairly similar (35.2 to 36.2 mol% by the Tm method and 35.5 to 36.4 mol% by the HPLC method), the t- -strain had a G+C content of 44.1 (Tm) and 43.3 (HPLC) mol%. Furthermore, the DNAs of the t- -strain and the type strain of C. tropicalis showed only 18.2% relatedness. These results suggest that the antigen corresponding to serum factor t exists only in the cell wall of C. tropicalis strains, not in those of the other medically important Candida, and that the t- -strain should not be classified as C. tropicalis. In conclusion, the taxonomic value and usefulness of factor t serum is primarily for differentiating C. tropicalis from C. albicans serotype A serologically.     

Effective inhibition of Candida albicans growth by the combination of murine peritoneal neutrophils and activated macrophages.

The effect of leukocytes on the anti-Candida activity of neutrophils was examined. Murine neutrophils which were purified from casein-induced peritoneal cells inhibited the mycelial growth of Candida albicans. This anti-Candida activity of neutrophils was augmented by the addition of spleen cells prepared from mice pretreated with bacterial lipopolysaccharide 3 hr before, but not from non-treated mice. The population in the spleen cells, which enhanced the anti-Candida activity of neutrophils, was plastic-plate adherent, nylon-fiber columns adherent and anti-Mac-1 antigen-positive. These immunological profiles suggested that the enhancing cells are classified to splenic macrophages. Peritoneal-exudated macrophages from mice treated with lipopolysaccharide also augmented the anti-Candida activity of neutrophils. These results suggest that the anti-Candida activity of neutrophils may be upregulated by activated macrophages.     

Granulocyte colony-stimulating factor potentiates anti-Candida albicans growth inhibitory activity of polymorphonuclear cells

Abstract Granulocyte colony-stimulating factor (G-CSF) stimulates a subset of granulocyte colony forming cells and when administered to neutropenic individuals results in recovery of blood neutrophil numbers to normal levels. Therefore, G-CSF may be a useful therapeutic agent for infections in immunocompromised hosts. However, to date there has been only limited information that G-CSF activates the antimicrobial activity of neutrophils. In the present study, we found that recombinant G-CSF promotes the anti- Candida albicans activity of normal human blood polymorphonuclear (PMN) cells in vitro using both a ³H-glucose uptake procedure and a Candida colony counting assay. As little as 0.1 ng/ml G-CSF induced significant anti-Candida activity in the PMN cultures. G-CSF treatment also enhanced superoxide anion production by the PMNs in response to f-MLP as determined by the superoxide dismutase inhibitable cytochrome C reduction method. Such results show that G-CSF can promote the antimicrobial activity of peripheral blood PMNs against C. albicans .     

Regulation of human polymorphonuclear neutrophil (PMN) activity against Candida albicans by large granular lymphocytes via release of a PMN-activating factor

Using a 24-hr radiolabel microassay developed in our laboratory that measures [3H]glucose uptake in residual Candida, we have identified the effector cells responsible for in vitro inhibition of Candida albicans growth as mainly polymorphonuclear neutrophils (PMN) and monocytes within the human peripheral blood cells. Highly purified T cells and large granular lymphocytes (LGL) that mediate natural killer activity which were obtained by Percoll density gradient centrifugation were found to have no innate activity against C. albicans. The LGL could not be activated by interferon-alpha, interferon-gamma or interleukin 2 to inhibit Candida growth although their K562 tumor cytotoxic activity was readily enhanced by these cytokines. Stimulation with heat-killed C. albicans also did not activate fungal growth inhibitory function in LGL and the supernatant of these activated LGL had no direct fungicidal activity. However, the activated LGL supernatant had the capability to enhance PMN function against C. albicans growth. Addition of recombinant human tumor necrosis factor, affinity-purified interferon-alpha, or interferon-gamma to PMN caused increased antifungal activity in PMN. However, antibodies to these cytokines had only a partial adverse effect on the ability of the activated LGL supernatant to stimulate PMN anti-Candida function. Therefore, the activated LGL supernatant appeared to contain a potent stimulator of PMN function which is as yet unidentified. These data indicate that LGL did not directly mediate anti-Candida activity but could indirectly influence C. albicans growth by activating PMN against the fungi through the release of a specific PMN-activating factor. Our findings therefore add another role to LGL which is the regulation of PMN function, the consequence of which is regulation of fungal immunity.     

Antimicrobial activity of Wedelia trilobata crude extracts.

A biological screening of activity against Gram-positive and Gram-negative bacteria, yeasts, and fungi of crude extracts from Wedelia trilobata is reported. The n-hexane extract showed antibacterial activity against Bacillus subtilis, Mycobacterium smegmatis, Staphylococcus aureus, and Staphylococcus epidermidis (Gram-positive bacteria); along with Proteus vulgaris, Pseudomonas aeruginosa, Salmonella group C, Salmonella paratyphi, and Shigella sonnei (Gram-negative bacteria). The ethyl acetate extract was active only against Salmonella group C; and the aqueous extract was inactive against the tested bacteria. None of the tested extracts showed biological activity against the yeasts (Candida albicans, Candida tropicalis, Rhodotorula rubra) or the fungi (Aspergillus flavus, Aspergillus niger, Mucor sp., Trichophyton rubrum).     

Anti-Candida albicans properties of novel benzoxazine analogues

We previously reported that azole 1,4-benzothiazine derivatives have appreciable anti-Candida activity. In this study, we synthesized 1,4-benzoxazine analogues and examined their possible antifungal activity to further analyze the structure-activity relationships. Results of in vitro and in vivo experiments showed that 1,4-benzoxazine analogues show appreciable antifungal activity. In particular, they have significant capability to cure mice systemically infected with a lethal challenge of Candida albicans, as indicated by increased survival time paralleling reduction of colony forming units. Moreover, 1,4-benzoxazine derivatives also showed immunomodulating activity, as indicated by a significant increase of interleukin-12 and interferon-gamma production by splenocytes and reinforcement of a T helper type 1 protective immune response to C. albicans. In conclusion, the results demonstrate that replacement of sulfur by oxygen may improve immune response against C. albicans infection.     

Antifungal and antitumor activities of a monoclonal antibody directed against a stress mannoprotein of Candida albicans

Immunization of mice with a stress mannoprotein of >200 kDa from the cell wall of Candida albicans led to the production of monoclonal antibody (Mab) C7. The immunogen is a major target of secretory IgA and its expression is regulated by different environmental conditions including temperature, pH, glucose concentration and ammonium sulphate in the culture medium. Mab C7 reacted with a peptide epitope present in the >200 kDa antigen as well as in a number of antigens from the blastoconidium and germ tube cell wall, including enolase. In addition to its reactivity with C. albicans, Mab C7 also reacted with antigens present in C. krusei, C, tropicalis, C. glabrata, C. dubliniensis and C. lusitaniae, as well as in Cryptococcus neoformans, Scedosporium prolificans and Aspergillus fumigatus. Mab C7 exhibited four important biological activities, namely inhibition of adhesion of C. albicans to a variety of surfaces, inhibition of germination of C. albicans, direct candidacidal activity and direct tumoricidal activity. In tumor cells, Mab C7 reacted with nucleoporin Nup88, a reactivity that can be utilized for diagnostic and prognostic purposes.     

抗系统性白念珠菌感染的先天性免疫

白念珠菌是引起浅部、深部真菌感染常见的病原菌.先天免疫反应在宿主抗系统性白念珠菌感染中起主导作用.介导宿主抗念珠菌感染的先天性免疫包括一系列真菌识别受体及免疫效应细胞.宿主对系统性白念珠菌感染的免疫反应是决定患者预后的关键.本文就宿主抗系统性白念珠菌感染的先天性免疫机制进行综述.     

Horizontal transmission of Candida albicans and evidence of a vaccine response in mice colonized with the fungus

Disseminated candidiasis is the third leading nosocomial blood stream infection in the United States and is often fatal. We previously showed that disseminated candidiasis was preventable in normal mice by immunization with either a glycopeptide or a peptide synthetic vaccine, both of which were Candida albicans cell wall derived. A weakness of these studies is that, unlike humans, mice do not have a C. albicans GI flora and they lack Candida serum antibodies. We examined the influence of C. albicans GI tract colonization and serum antibodies on mouse vaccination responses to the peptide, Fba, derived from fructose bisphosphate aldolase which has cytosolic and cell wall distributions in the fungus. We evaluated the effect of live C. albicans in drinking water and antimicrobial agents on establishment of Candida colonization of the mouse GI tract. Body mass, C. albicans in feces, and fungal-specific serum antibodies were monitored longitudinally. Unexpectedly, C. albicans colonization occurred in mice that received only antibiotics in their drinking water, provided that the mice were housed in the same room as intentionally colonized mice. The fungal strain in unintentionally colonized mice appeared identical to the strain used for intentional GI-tract colonization. This is the first report of horizontal transmission and spontaneous C. albicans colonization in mice. Importantly, many Candida-colonized mice developed serum fungal-specific antibodies. Despite the GI-tract colonization and presence of serum antibodies, the animals made antibodies in response to the Fba immunogen. This mouse model has potential for elucidating C. albicans horizontal transmission and for exploring factors that induce host defense against disseminated candidiasis. Furthermore, a combined protracted GI-tract colonization with Candida and the possibility of serum antibody responses to the presence of the fungus makes this an attractive mouse model for testing the efficacy of vaccines designed to prevent human disseminated candidiasis.     

In Vitro Inhibition of Oral Candida albicans by Chicken Egg Yolk Antibody (IgY)

This study was conducted to measure Candida albicans-specific chicken egg yolk antibody (IgY) inhibition of fluconazole-sensitive and resistant strains of C. albicans in order to assess potential use in the prevention and treatment of oral candidiasis. In this study, laying hens were immunized, and IgY was extracted by water dilution. The Minimal Inhibitory Concentrations (MICs) of IgY for inhibiting C. albicans growth were determined using the broth microdilution method from the CLSI M27-A2 protocol. Fluconazole (FLC) was used as the control. The results were analyzed with the chi(2) test. The anti-Candida titer of anti-C. albicans IgY was 1:12,000. The concentration of the IgY extract that effectively inhibited the growth of C. albicans was between 1.25 g/l and 5.0 g/l, and the efficacy rate was 82.98% during the observed 24-48 h time period. No correlation was recorded between the drug resistance of FLC and growth inhibition by IgY. It was concluded that anti-C. albicans IgY inhibited the growth of C. albicans in vitro and there was no correlation between the drug resistance of FLC and the growth inhibition by IgY (P > 0.99).     

Biochemical characterization of an anti-Candida factor produced by Enterococcus faecalis

ABSTRACT: BACKGROUND: Because Candida albicans is resistant to several antifungal antibiotics, there is a need to identify other less toxic natural products, particularly antimicrobial proteins, peptides or bacteriocin like inhibitory substances. An attempt has been made to purify and characterise an anti-Candida compound produced by Enterococcus faecalis. RESULTS: An anti-Candida protein (ACP) produced by E. faecalis active against 8 C. albicans strains was characterised and partially purified. The ACP showed a broad-spectrum activity against multidrug resistant C. albicans MTCC 183, MTCC 7315, MTCC 3958, NCIM 3557, NCIM 3471 and DI. It was completely inactivated by treatment with proteinase K and partially by pronase E. The ACP retained biological stability after heat-treatment at 90 degreesC for 20 min, maintained activity over a pH range 6-10, and remained active after treatment with alpha-amylase, lipase, organic solvents, and detergents. The antimicrobial activity of the E. faecalis strain was found exclusively in the extracellular filtrate produced in the late logarithmic growth phase. The highest activity (1600 AU mL-1) against C. albicans MTCC 183 was recorded at 48 h of incubation, and activity decreased thereafter. The peptide showed very low haemagglutination and hemolytic activities against human red blood cells. The antimicrobial substance was purified by salt-fractionation and chromatography. Partially purified ACP had a molecular weight of approximately 43 KDa in tricine-PAGE analysis. The 12 amino acid N terminal sequence was obtained by Edman degradation. The peptide was de novo sequenced by ESI-MS, and the deduced combined sequence when compared to other bacteriocins and antimicrobial peptide had no significant sequence similarity. CONCLUSIONS: The inhibitory activity of the test strain is due to the synthesis of an antimicrobial protein. To our knowledge, this is the first report on the isolation of a promising non-haemolytic anti- Candida protein from E. faecalis that might be used to treat candidiasis especially in immunocompromised patients.     

Proteomics-based identification of novel Candida albicans antigens for diagnosis of systemic candidiasis in patients with underlying hematological malignancies

Systemic candidiasis remains a major cause of disease and death, particularly among patients suffering from hematological malignancies. In an attempt to contribute to the discovery of useful biomarkers for its diagnosis and therapeutic monitoring, we embarked on a mapping of Candida albicans immunogenic proteins specifically recognized by antibodies produced during the natural course of systemic Candida infection in this high-risk population. About 85 immunoreactive protein species were detected with systemic candidiasis patients' serum specimens by using immunoproteomics (i.e., two-dimensional electrophoresis followed by Western blotting), and identified through a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), de novo peptide sequencing using nano-electrospray ionization-ion trap (ESI-IT) MS, and genomic database searches. This proteomic approach has led to the characterization of 42 different housekeeping enzymes as C. albicans antigens. Their biological significance is also discussed. Furthermore, this study is the first to report that 26 of them exhibit antigenic properties in C. albicans, and 35 of them become targets of the human antibody response to systemic candidiasis. Our findings suggest that the production of antibodies to C. albicans phosphoglycerate kinase and alcohol dehydrogenase during systemic candidiasis could be associated with a differentiation of the human immune response. We also highlight the relationship between changes in maintenance of circulating levels of specific anti-Candida antibodies and patients' outcome. Some of these variations, especially the rise of high anti-enolase antibody concentrations, appear to be related to recovery from systemic candidiasis in these patients, which might serve as markers for predicting their outcome. This approach could therefore provide new challenges for diagnosis and clinical follow-up of these fungal infections, and even for antifungal drug or vaccine design.     

Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.     

Bioactivity studies of extracts from Tridax procumbens.

An updated review on the biological activity of Tridax procumbens is presented. A detailed biological screening comprised of gram-positive and gram-negative bacteria, yeasts and fungi using crude extracts of this plant was undertaken. The n-hexane extract of the flowers showed activity against Escherichia coli. The same extract of the whole aerial parts was active against Mycobacterium smegmatis, Escherichia coli, Salmonella group C and Salmonella paratyphi. The ethyl-acetate extract of the flowers was active against Bacillus cereus and Klebsiella sp. The aerial parts extract also showed activity only against Mycobacterium smegmatis and Staphylococcus aureus, while the aqueous extract showed no antimicrobial activity. None of the tested extracts was active against the yeasts, Candida albicans, Candida tropicalis and Rhodotorula rubra; or the fungi: Aspergillus flavus, Aspergillus niger, Mucor sp. and Trichophyton rubrum.     

Comparison of a Newly Developed Latex Agglutination Test and an Immunodiffusion Test in the Diagnosis of Systemic Candidiasis

A newly developed latex agglutination (LA) test and a modified immunodiffusion (ID) test were evaluated. The antigen used was a homogenate of Candida albicans. A total of 167 antisera were employed in the evaluation. They included 36 sera from clinically well persons; 78 from patients with various clinical forms of candidiasis; 52 from patients with proven cases of aspergillosis, blastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, nocardiosis, paracoccidioidomycosis, sporotrichosis, and tuberculosis; and one serum from a patient with toruloposis. Use of the LA test in conjunction with the ID test permitted the detection of more than 90% of 43 proven candidiasis cases. Of all the heterologous cases and normal human sera tested, LA reactions were noted with 3 of 10 cryptococcosis case specimens, 1 of 9 tuberculosis case specimens, and with the torulopsemia case serum. In contrast, the only heterologous serum reactive in the ID test was that from the patient with torulopsemia. Torulopsis glabrata and C. albicans antisera gave identical reactions in LA and ID tests with T. glabrata or C. albicans antigens. ID tests with selected antigens, however, permitted differentiation of rabbit and human T. glabrata antibody from that of C. albicans antibody. Six different precipitins were recognized with the C. albicans antigens. The occurrence of multiple precipitin lines and high LA titers was suggestive of severe candidiasis. The LA test, in contrast to the ID test, appeared to have prognostic value. Together, the LA and ID tests provided a simple, rapid, and accurate means of detecting and monitoring infections by species of Candida.     

Lymphoproliferative and cytotoxic responses of human peripheral blood mononuclear cells to mannoprotein constituents of Candida albicans.

Two major proteoglycan constituents (designated F1 and F2) of the cell wall of Candida albicans were separated by ion-exchange chromatography from a crude carbohydrate-rich extract (GMP), and investigated for their chemical and molecular composition, antigenicity and immunomodulatory properties in cultures of human peripheral blood mononuclear cells (PBMC). Both fractions consisted predominantly of Periodic acid-Schiff (PAS) and concanavalin A (Con A)-reactive material consisting of greater than 90% mannose, 3-5% protein and small amounts of phosphorus; each was recognized by an anti-Candida rabbit serum as well as by a monoclonal antibody (mAb AF1) directed against an oligosaccharide epitope present on the fungal cell surface. When F1 and F2 were subjected to SDS-PAGE, transblotted and stained with enzyme-conjugated mAb AF1 or Con A, most of the antibody or lectin bound to high molecular mass (greater than 200 kDa) polydisperse material, some of which was present in F2 (as in the starting GMP extract) but absent in F1. This difference was also observed in PAS-stained gels of the two fractions. The F2, but not the F1, constituent was as active as the unfractionated GMP extract in inducing lymphoproliferation, production of the cytokines interleukin-2 and interferon-gamma, and generation of cytotoxicity against a natural-killer-sensitive target cell line (K562). These immunomodulatory properties were, like those possessed by GMP, protease-sensitive and heat-stable. Treatment of PMBC cultures with a modulatory anti-T-cell receptor antibody abolished the lymphoproliferation induced by GMP and F2 but not that induced by phytohaemagglutinin, showing that the mannoprotein materials of C. albicans acted through interaction with the antigen receptor complex.     

Secretion of inducible proteinase by pathogenic Candida species

The ability of three isolates each of seven pathogenic Candida species to grow in a liquid medium containing bovine serum albumin (BSA) as a nitrogen source was determined. All three strains of C. albicans, two strains of C. guilliermondii and one strain of C. tropicalis grew well. At any time proteinase activity was detected in the culture filtrates of only the most virulent species--C. albicans, C. tropicalis and C. parapsilosis and this observation was related to complete hydrolysis of BSA. Serologically, cross reactions were demonstrated between anti-proteinase antiserum and C. albicans and C. tropicalis culture filtrates. These results further emphasise the role of the inducible proteinase of Candida in the pathogenesis of candidosis.     

Toxicity to Candida albicans mediated by human serum and peripheral blood mononuclear cells

This study evaluates the conditions in which peripheral blood mononuclear cells mediate toxicity to Candida albicans opsonized with heat-inactivated human serum. Serum concentrations as low as 1% resulted in 50% inhibition of C. albicans metabolic activity after incubation with peripheral blood mononuclear cells at an effector to target ratio of 8. Measurable inhibition was also achieved at lower effector to target ratios and lower serum concentrations, and at least a portion of the metabolic inhibition reflected fungal cell death. Depletion of C. albicans-specific antibody decreased the toxic effect while opsonization with purified human IgG restored toxicity, and cell-cell contact between peripheral blood mononuclear cells and fungus was required. Depletion of or enrichment for monocytes from the peripheral blood mononuclear cells preparation diminished the toxic effect and the monocytic cell line, THP-1, was likewise incapable of toxicity. These studies provide evidence that antibody augments antifungal host defense and underscore the complex interrelationship between humoral and cellular immunity in these infections.