Candida Albicans Extract Research Articles

Identification and production of novel potential pathogen-specific biomarkers for diagnosis of histoplasmosis.

Immunological assays based on the detection of circulating fungal biomarkers are helpful in clinical practice for diagnosing invasive fungal infections. Some of these targeting antigenic components are common to several different fungi. Histoplasmosis is a mycosis caused by the dimorphic fungus Histoplasma capsulatum, which in recent years has gained significant relevance due to the increase in the population susceptible to developing severe clinical forms, including those living with HIV/AIDS. Immunological tests that detect cell wall polysaccharide antigens are among the most used laboratory techniques for diagnosing this mycosis. However, none have shown adequate performance, and cross-reactivity with other fungal pathogens may be observed. Considering that there is a real need to improve the sensitivity and specificity of current diagnostic methods, we explored a novel strategy for the identification of H. capsulatum-specific antigens (Hc_Ags) that could be detected in clinical samples during infection based on a computational analysis model that included the application of bioinformatics tools and the analysis of experimental data from transcriptomics and proteomics. The Hc_Ags identified were expressed and purified by eukaryotic and prokaryotic systems. First, the Hc_Ags were used in an in-house immunization model in mice (BALB/c) to obtain Hc_Ag-specific polyclonal antibodies (Hc_Ag_PAb). Then, the presence of these antigens in H. capsulatum-yeast culture extracts and the specificity of Hc_Ag_PAb against culture extracts of Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Fusarium spp., and Paracoccidioides brasiliensis were confirmed. Finally, we demonstrated the immunoreactivity of these Hc_Ag-specific polyclonal antibodies with urine samples from patients previously diagnosed with histoplasmosis.IMPORTANCEHistoplasmosis is considered one of the most important mycoses due to the increasing number of individuals susceptible to develop severe clinical forms, particularly those with HIV/AIDS or receiving immunosuppressive biological therapies, the high mortality rates reported when antifungal treatment is not initiated in a timely manner, and the limitations of conventional diagnostic methods. In this context, there is a clear need to improve the capacity of diagnostic tools to specifically detect the fungal pathogen, regardless of the patient's clinical condition or the presence of other co-infections. The proposed novel pathogen-specific biomarkers have the potential to be used in immunodiagnostic platforms and antifungal treatment monitoring in histoplasmosis. In addition, the bioinformatics strategy used in this study could be applied to identify potential diagnostic biomarkers in other models of fungal infection of public health importance.

FORMULASI SEDIAAN SABUN CAIR EKSTRAK DAUN SIRIH HIJAU (Piper battle folium L.) DAN EKSTRAK BAWANG PUTIH (Allium sativum L.) SEBAGAI ANTIJAMUR Candida albicans

Islam is a religion that likes cleanliness, in case of body hygiene, clothing, and environment. One of the diseases that arise because it does not keep the body clean is white discharge. Piper bettle folium and Alium sativum are some of the natural ingredients that has the potential as an antifungal. This research aims to see the effect of antifungal Candida albicans extract of Piper bettle folium and Alium sativum after formulated as a liquid soap preparation. Liquid soaps are made by hot process methods with 3 formulations of combination of Piper bettle folium and Alium sativum extract with a ratio of 1) 25%: 75% 2) 50%: 50% 3) 75%: 25%. The evaluation of liquid soap include organoleptic examination, pH, high foam, moisture content, homogeneity, and antifungal power test against Candida albicans fungus. The results of the testing of liquid soap quality of green betel leaf extract and garlic have met the standards SNI (Indonesia’s National Standards) set for the manufacture of liquid soap.The results showed that liquid soap extract of green betel leaf and garlic have antifungal activity against Candida albicans. The result of formulation with the highest inhibition zone is formulation 3 and formulation with the lowest inhibition zone is the formulation 1. The test of this liquid soap activity is higher than fluconazole as the positive control and liquid soap of female area spread in the market.

Analysis of LC3-Associated Phagocytosis and Antigen Presentation.

The noncanonical macroautophagy pathway, LC3-associated phagocytosis (LAP) has recently emerged as an important catabolic process involved during exogenous antigen processing. It has been described that in human macrophages and dendritic cells the direct recruitment of LC3 to the phagosomal membrane is associated with its maturation impairment, allowing the stabilization of the cargo to prolong antigen presentation on major histocompatibility complex (MHC) class II molecules.In this chapter, we describe methods to monitor, manipulate, and understand the role of LAP during MHC class II presentation. We show how to enhance LAP formation resulting in antigen presentation by using zymosan or beads coated with Candida albicans extract. Then, we describe how to determine the localization of Rab7 or Lamp2 on LC3-phagosomes by confocal microscopy, a useful technique to follow phagosome maturation. Finally, we propose an assay to understand how MHC class II antigen presentation can be modulated by the LAP pathway.

The effect of immunological status, in-vitro treatment and culture time on expression of eleven candidate reference genes in bovine blood mononuclear cells.

BackgroundTechnical feasibility of RNA quantification by real time RT-PCR has led to enormous utilization of this method. However, real time PCR results need to be normalized due to the high sensitivity of the method and also to eliminate technical variation. Normalization against a reference gene that is constitutively transcribed and has minimum variation among samples is the ideal method. Nevertheless, many studies have shown that there is no general reference gene(s) with ideal characteristics and candidate reference genes should be tested before being used as a “normalizer” in each study.MethodsThe current study investigated the effects of previous exposure of the host to experimental test antigens and culturing time on the expression of 11 candidate genes when blood mononuclear cells (BMCs) were cultured and treated in-vitro by hen egg white lysozyme, Candida albicans extract and a mitogen. Mononuclear cells were isolated and cultured from 12 bovine blood samples representing 3 different immunological statuses. The expression of candidate housekeeping genes were measured by real-time RT-PCR at 4 and 24 hours post culture. The expression of candidate genes were first compared between the two time points in untreated samples. Constitutively expressed genes were further tested in linear mixed effects models to examine the effect of previous host exposure and in-vitro treatments.ResultsOur findings showed that the expression of the most common reference genes, β-actin, and Glyceraldehydes-3-phosphate dehydrogenase (GAPDH), are significantly decreased at 24 hours after culturing BMCs, even without any treatment. The effect of culturing time was also significantly influenced the expression of 18s ribosomal RNA, β2-microglobulin, Tyrosine 3-monooxygenase/tryptophan 5-monoxygenase activation protein, zeta polypeptide (YWHAZ) in BMCs. Only the expression of C-terminal binding protein 1 (CTBP1) and RAD50 among all tested genes were consistent after treatment of cultured BMCs with C. albicans whole yeast extract and Hen Egg White Lysozyme (HEWL), respectively. In addition, expressions of CTBP1, and RAD50 were independent from previous exposure of the host to the antigen.ConclusionsThe results of this study demonstrated inconsistent expression of commonly used reference genes in untreated cultured BMCs over time. As this condition applies to negative controls in real time RT-PCR study designs, normalization against these genes can largely deceive the outcome, especially in kinetic studies. Moreover, the potential effects of immunological memory on the expression of reference genes should be considered if BMCs are collected from different individuals under different environmental conditions and if these cells are treated in-vitro by an antigen.

ELISpot and ELISA analyses of human IL-21-secreting cells: Impact of blocking IL-21 interaction with cellular receptors

Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed. The selected detection mAb also neutralized IL-21-mediated activation of human cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors (n=24) were stimulated polyclonally (phytohemagglutinin; PHA) or with antigen (Candida albicans extract and tetanus toxoid). Using ELISpot, high numbers of IL-21-producing cells were detected after PHA activation; lower but positive responses to antigen were seen in approximately 50% of the donors. In contrast, the ELISA detected IL-21 in supernatants from PHA-activated cells but not from antigen-stimulated cells. When analyzing IL-17A in parallel, PHA and antigens induced detectable responses in ELISpot as well as in ELISA. Hypothesizing that the lack of detectable IL-21 levels after antigenic stimulation was due to a combination of low frequencies of IL-21-secreting cells and consumption of IL-21 by cellular receptors during cell culture, PBMCs (n=18) were stimulated in the presence of the neutralizing detection mAb. When preventing IL-21 from interacting with its receptor, increased IL-21 levels were found by ELISA after PHA activation and IL-21 could also be measured after antigen stimulation. ELISpot results were unaffected by the addition of the neutralizing mAb. In conclusion, IL-21 secreted by low frequencies of antigen-specific ex vivo-stimulated PBMC can be difficult to detect by ELISA but prevention of IL-21 interaction with its receptor leads to detectable IL-21 levels. In ELISpot, where the cytokine is captured by mAbs on a solid phase immediately upon secretion, blocking the receptor interaction does not affect the detection of IL-21-secreting cells.

Triple Cytokine FluoroSpot Analysis of Human Antigen-Specific IFN-γ, IL-17A and IL-22 Responses.

The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was developed and evaluated using human peripheral blood mononuclear cells from healthy donors incubated with tetanus toxoid, Candida albicans extract, mycobacterial purified protein derivative or medium only. Antigen stimulation yielded mainly cells secreting IFN-γ, IL-17A or IL-22 alone but lower proportions of double-secreting cells were also found; triple-secreting cells were rare. The response to C. albicans contrasted in that higher proportions of IL-17A single secreting as well as co-secreting cells, in particular IL-17A/IL-22, were found. The FluoroSpot analysis correlated well with single cytokine ELISpot assays ran in parallel and the methods displayed a comparable sensitivity. The results demonstrate the functionality of the FluoroSpot assay for simultaneous analysis of distinct Th1, Th17, Th22 as well as intermediate cell populations. The method provides a mean for a simple and rapid analysis of the involvement of these cells in immunity and disease.

Characterization of Nucleotides and Nucleotide Sugars in Candida albicans by High Performance Liquid Chromatography–Mass Spectrometry with a Porous Graphite Carbon Column

An optimized high performance liquid chromatography electrospray ionization/tandem mass spectrometry method was developed and optimized for the determination of seven nucleotides and nucleotide sugars in the extract of Candida albicans. The chromatographic separation was performed on a porous graphitic carbon column with gradient elution using acetonitrile and aqueous ammonia acetate containing 0.1% formic acid adjusted to pH 8.8. Negative-ion mode electrospray ionization mass spectrometry was applied to improve the selectivity and sensitivity of the analysis. The calibration curves showed excellent linearity over approximately three orders of magnitude with correlation coefficients exceeding 0.9946. The relative standard deviations of measurements were smaller than 4% (n = 5) and the accuracy of the intra-day and inter-day measurements were between 95.2% and 106.7%. The extraction recovery of each component was within satisfactory ranges. The validated method was applied to characterize the nucleotides metabolic profiles of the biofilm compared to the planktonic mode to evaluate changes of energy states in different growth modes of C. albicans. The levels of nucleotides and nucleotide sugar in the biofilm model treated with 5-fluoropyrimidine, baicalein, and sodium houttuyfonate were determined to investigate their effects on nucleotide metabolism and the mechanisms of antifungal agents.

Clinical-scale generation of multi-specific anti-fungal T cells targeting Candida, Aspergillus and mucormycetes

Invasive fungal infections, in particular, infections caused by Candida, Aspergillus and mucormycetes, are a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Adoptive transfer of donor-derived anti-fungal T cells shows promise to restore immunity and to offer a cure. Because T cells recognize only specific epitopes, the low rate of patients in which the causal fungal pathogen can be identified and the considerable number of patients with co-infection with several genera or species of fungi significantly limit the application of adoptive immunotherapy. Using the interferon-γ secretion assay, we isolated multi-specific human anti-fungal T cells after simultaneous stimulation with cellular extracts of Aspergillus fumigatus, Candida albicans and Rhizopus oryzae. Cells were phenotypically and functionally characterized by flow cytometry. Of a total of 1.1 × 10(9) peripheral blood mononuclear cells, a median number of 5.2 × 10(7) CD3+ CD4+ T cells was generated within 12 days. This cell population consisted of activated memory TH1 cells and reproducibly responded to a multitude of Aspergillus spp., Candida spp. and mucormycetes with interferon-γ production. On re-stimulation, the generated T cells proliferated and enhanced anti-fungal activity of phagocytes and showed reduced alloreactivity compared with the original cell fraction. Our rapid and simple method of simultaneously generating functionally active multi-specific T cells that recognize a wide variety of medically relevant fungi may form the basis for future clinical trials investigating adoptive immunotherapy in allogeneic hematopoietic stem cell transplantation recipients with invasive fungal infection.

In this issue

AbstractMALDI‐TOF “Flip‐flop” drug susceptibility testpp. 4627–4631The emergence of drug‐resistant pathogens over the last 50 years has presented/created major problems for clinical microbiologists: is the bug resistant to a given drug and, if so, how resistant? Current susceptibility tests are not as clear cut as might be wished, requiring an educated eyeball for the final determination. Marinach et al. have taken a new approach to this problem, applying the power of a MALDI‐TOF mass spectrometer to “fingerprint” organisms and extracts rapidly and inexpensively. Using an acid extract of Candida albicans, which had been exposed to various concentrations of Fluconazole, they found the MALDI‐TOF mass spectrum showed an “on–off” response: in one example there were no peak differences between low control and up to 2 μg/mL, then, clear changes were visible from 4 μg/mL and up. magnified imageProteome in motionpp. 4674–4685Pulse‐chase has been one of the most powerful techniques in molecular biology for eliciting relationships and pathways. Here it is applied to the yeast proteome to ask the “Big Bang” question: what is the proteome at the instant of synthesis and what does it become in time? Massoni et al. used [35S]‐methionine to pulse label Saccharomyces cerevisiae, sampling after various times, from 0.1 to 4 doublings. Analyzing by 2‐D electrophoresis, these researchers were able to detect more than 500 spots and determine protein half‐lives down to ∼15 min. Spots that appeared or disappeared during the chase period were undergoing post‐translational modification (PTM). The most common PTM is seen as a pI shift due to phosphorylation. A number of unusual PTMs were confirmed. Rates of PTM appearance ranged from virtually immediately upon expression to several hours. magnified imageQuick switch key for mitochondriapp. 4787–4798One of the benefits of working with Saccharomyces cerevisiae is the speed at which it can switch between aerobic and anaerobic growth. How do the mitochondria do that? The answer: they don't do a total shut‐down when subjected to anaerobic conditions. They retain the energetically expensive mitochondrial ribosomes and most of the multi‐subunit respiratory complex components. To gather this kind of information, Helbig et al. required better starting material than had been used in previously published studies, they required separately grown and labeled aerobic and anaerobic cell lysates. The final experimental design was in three parts and produced quantitative results for mitochondrial components under virtually identical conditions for aerobic and anaerobic growth. Blue native (BN)‐PAGE was used for improved coverage of membrane proteins. magnified image

Cover Picture: Proteomics 20'09

AbstractMALDI‐TOF “Flip‐flop” drug susceptibility testThe emergence of drug‐resistant pathogens over the last 50 years has presented/created major problems for clinical microbiologists: is the bug resistant to a given drug and, if so, how resistant? Current susceptibility tests are not as clear cut as might be wished, requiring an educated eyeball for the final determination. Marinach et al. have taken a new approach to this problem, applying the power of a MALDI‐TOF mass spectrometer to “fingerprint” organisms and extracts rapidly and inexpensively. Using an acid extract of Candida albicans, which had been exposed to various concentrations of Fluconazole, they found the MALDI‐TOF mass spectrum showed an “on–off” response: in one example there were no peak differences between low control and up to 2 μg/mL, then, clear changes were visible from 4 μg/mL and up.Marinach, C. et al., Proteomics 2009, 9, 4627–4631.Proteome in motionPulse‐chase has been one of the most powerful techniques in molecular biology for eliciting relationships and pathways. Here it is applied to the yeast proteome to ask the “Big Bang” question: what is the proteome at the instant of synthesis and what does it become in time? Massoni et al. used [35S]‐methionine to pulse label Saccharomyces cerevisiae, sampling after various times, from 0.1 to 4 doublings. Analyzing by 2‐D electrophoresis, these researchers were able to detect more than 500 spots and determine protein half‐lives down to ∼15 min. Spots that appeared or disappeared during the chase period were undergoing post‐translational modification (PTM). The most common PTM is seen as a pI shift due to phosphorylation. A number of unusual PTMs were confirmed. Rates of PTM appearance ranged from virtually immediately upon expression to several hours.Massoni, A. et al., Proteomics 2009, 9, 4674–4685.Quick switch key for mitochondriaOne of the benefits of working with Saccharomyces cerevisiae is the speed at which it can switch between aerobic and anaerobic growth. How do the mitochondria do that? The answer: they don't do a total shut‐down when subjected to anaerobic conditions. They retain the energetically expensive mitochondrial ribosomes and most of the multi‐subunit respiratory complex components. To gather this kind of information, Helbig et al. required better starting material than had been used in previously published studies, they required separately grown and labeled aerobic and anaerobic cell lysates. The final experimental design was in three parts and produced quantitative results for mitochondrial components under virtually identical conditions for aerobic and anaerobic growth. Blue native (BN)‐PAGE was used for improved coverage of membrane proteins.Helbig, A. O. et al., Proteomics 2009, 9, 4787–4798.

A novel monoclonal antibody recognizing β(1–3) glucans in intact cells of Candida and Cryptococcus

The cell walls of all medically important fungi contain a unique polyglucose compound, beta(1-3) glucan. In the present study, murine monoclonal antibodies were produced against linear and beta(1-6) branched beta(1-3) glucans, and their specificities were characterized for reactivity to other beta glucans, fungal cell wall fragments, and fungal cells. Their reactivity was also compared with that of rabbit polyclonal antibodies raised against the same immunogens. Two mouse monoclonal antibodies (AG and BG) recognized immunoreactive epitopes in beta(1-3)(1-6) glucan by ELISA. In an inhibition assay of the anti-beta(1-3)(1-6) activity of the monoclonals, the homologous antigen effectively inhibited the activity as expected, while beta(1-3) also inhibited the assay but to a much lesser extent. No inhibition was obtained by beta(1-3)(1-4) or beta(1-6), while a cell wall extract of Candida albicans (PPM) effectively inhibited both monoclonals. Cell wall fragments of C. albicans (CaCW) and Cryptococcus neoformans (CnCW) inhibited the anti-beta(1-3)(1-6) activity of AG, while BG was much less or not inhibited at all. Immunofluorescence confirmed the unique antibody specificity of AG by its recognition of a beta(1-3)(1-6)-associated epitope on the cell surfaces of C. albicans,C. krusei, C. glabrata, and nonencapsulated C. neoformans. The epitope for the AG antibody is suggested to be present in the branching point of beta(1-3)(1-6), or in the randomly coiled beta(1-3) polyglucan due to the presence of branches. Thus, monoclonal antibodies to beta(1-3)(1-6) glucans may have potential as tools in the laboratory diagnosis of invasive yeast infections.

Inhibition of sterol 14 α-demethylation of Candida albicans with NND-502, a novel optically active imidazole antimycotic agent

To investigate the mode of action of the newly synthesized optically active imidazole compound, NND-502, (−)-(E)-[4-(2,4-dichlorophenyl)-1,3-dithiolan-2-ylidene]-1-imidazolylacetonitrile, its effect on ergosterol biosynthesis in cell-free extracts of Candidaalbicans was examined and compared with that of the (S)-enantiomer of NND-502 in addition to lanoconazole and bifonazole, both of which are clinically used for the treatment of dermatomycoses. NND-502 was found to interfere with ergosterol biosynthesis by inhibition of sterol 14α-demethylase, while no interference due to the (S)-enantiomer of NND-502 was found, indicating that the stereochemical orientation of the 2,4-dichlorophenyl group plays an important role in the interaction with the enzyme. In terms of drug concentration exerting 50% inhibition of ergosterol biosynthesis, NND-502 was 2·5 and 28 times more effective than that of lanoconazole and bifonazole, respectively.

Generation of highly purified and functionally active human TH1 cells against Aspergillus fumigatus

AbstractInvasive aspergillosis remains a serious complication in patients undergoing allogeneic stem cell transplantation (SCT). Since it became clear that lymphocytes provide a critical secondary defense against fungi, adoptive transfer of functionally active anti-Aspergillus T cells might be an option to restore adaptive immune effector mechanisms. Using the interferon (IFN)-γ secretion assay, we isolated human activated T cells upon stimulation with a cellular extract of Aspergillus fumigatus. Culturing this cell population for 14 days, we obtained an average of 1.1 × 107 cells from a single 100-mL blood draw in 7 of 7 healthy individuals. Within another 14 days, these cells were expanded to an average number of 2.0 × 108 T-helper 1 (TH1) cells secreting IFN-γ on stimulation with Aspergillus antigens. Testing various fungal antigen extracts, similar proportions of IFN-γ-producing CD3+/CD4+ cells were obtained upon activation with antigen extracts of A fumigatus, A flavus, A niger, and Penicillium chrysogenum, whereas no significant IFN-γ production was observed upon activation with antigen extracts of Alternaria alternata and Candida albicans. In addition, generated T cells were able to induce damage to A fumigatus hyphae, and significantly increased hyphal damage induced by human neutrophils. CD4+ T-cell-mediated alloreactivity of generated anti-Aspergillus T cells was clearly reduced compared with that of the original cell population. In conclusion, we present a simple and feasible strategy for rapid generation of a high number of functional active T cells against Aspergillus from a single blood draw. Our data suggest that functionally active T cells against Aspergillus could be a promising treatment option for patients undergoing allogeneic SCT. (Blood. 2006;107: 2562-2569)

Histopathological features of murine systemic vasculitis caused by Candida albicans extract--an animal model of Kawasaki disease.

We examined the histopathological features of systemic vasculitis caused in mice by injection of a Candida albicans ( C. albicans) extract and investigated the principal genetic roles in the development of vasculitis. C. albicans extract was injected intraperitoneally for five consecutive days in the 1st and 5th weeks to CD-1, C57BL/6N, C3H/HeN, BALB/cAnN, DBA/2N and CBA/JN mice. At week 8, mice were killed, and histological examination was performed by light microscopy. Arteritis had developed in 66% of CD-1 mice. The extramural coronary arteries and aortic root close to the orifice of coronary arteries were most frequently involved. Histologically, the characteristic feature of the arteritis was proliferative and granulomatous inflammation accompanied by numerous macrophages, lymphocytes, plasma cells and neutrophils. Fibrocellular intimal thickening with destruction of the internal elastic lamina and media was also observed. Five mouse strains after injection of C. albicans extract were clearly classified into a resistant group (CBA/JN, DBA/2N and BALB/cAnN mice) and a sensitive group (C3H/HeN and C57BL/6N mice). The inbred mouse strains which showed the same histocompatibility-2 (H-2) haplotype exhibited a different susceptibility to development of vasculitis. This arteritis murine model shows unique histological features that have not been observed in other animal vasculitis models and it most closely resembles Kawasaki disease in humans. The genetic control of susceptibility to induction of vasculitis by the C. albicans extract is dependent to the mouse strains, but is not linked to the H-2 loci.

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans.

A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans.

Immune Reaction of Candida albicans Soluble Polysaccharide Fraction CAWS Developing Coronary Arteritis in Mice

Potassium hydroxide extract of Candida albicans (CADS) isolated from Kawasaki disease (KD) patient has been known to induce coronary arteritis in mice resembles to KD. Incidence of the coronary arteritis is strain dependent: C3H/HeN; susceptible, DBA/2; resistant. In addition, a water-soluble polysaccharide fraction (CAWS) released from C. albicans into synthetic medium induced coronary arteritis by i.p. administration and showed lethality resembled to the anaphylactic shock by i.v.. Lethality was strain dependent: ICR, C3H/HeN; susceptible, DBA/2; resistant. CAWS is mainly composed of a complex of mannoprotein and β-glucan and reacts with limulus factor G test, specific for 1,3-β-D-glucan. Half clearance time of CAWS in DBA/2 mice was 19 min after i.v. administration. Lethality was partially inhibited by pretreatment with yeast mannan and salbutamol. Incidence rate of coronary arteritis by CAWS was significantly higher than that of CADS. Then, various activities of CAWS were examined by i.p. administration to clarify underlying mechanisms for the development of coronary arteritis, i) CAWS induced cell infiltration into peritoneal cavity and spleen in both C3H/HeN and DBA/2. Ratio of macrophages and granulocytes in the spleen was significantly increased, ii) IL-6 and IL-10 production of the spleen cells was increased in both strains of mice, iii) INF-γ production of the spleen cells was enhanced only in DBA/2 mice, iv) myeloperoxidase (MPO) activity of the spleen cells were increased in both strains of mice, v) Anti-MPO autoantibody in sera was induced more significantly in DBA/2 mice, vi) IL-6 and MIP-2 activity of PEC were significantly reduced in both strains of mice. These results suggested that CAWS showed inflammatory activity and immuno-toxicity and IFN-γ production might be related to the susceptibility to arteritis.

IL-5 Production by Peripheral Blood Th Cells of Adult Asthma Patients in Response to Candida albicans Allergen

Cytokines produced by T cells are involved in chronic asthma. Relevant antigens for nonatopic asthma are still mostly unknown. Peripheral blood mononuclear cells (PBMCs) were obtained from adult asthmatic donors and incubated with Candida albicans extract. Proliferation response and cytokine production were analyzed. IL-2 and IL-5 were detectable in the cultures incubated with C. albicans extract, whereas IL-4 was undetectable. PBMCs obtained from some nonatopic asthma patients produced IL-5 upon stimulation of C. albicans extract. C. albicans allergen may be involved in the airway eosinophilic inflammation through the induction of IL-5 production by Th cells.

Inhibition of sterol 14 alpha-demethylation of Candida albicans with NND-502, a novel optically active imidazole antimycotic agent.

To investigate the mode of action of the newly synthesized optically active imidazole compound, NND-502, (-)-(E)-[4-(2, 4-dichlorophenyl)-1,3-dithiolan-2-ylidene]-1-imidazolylacetonit rile, its effect on ergosterol biosynthesis in cell-free extracts of Candida albicans was examined and compared with that of the (S)-enantiomer of NND-502 in addition to lanoconazole and bifonazole, both of which are clinically used for the treatment of dermatomycoses. NND-502 was found to interfere with ergosterol biosynthesis by inhibition of sterol 14alpha-demethylase, while no interference due to the (S)-enantiomer of NND-502 was found, indicating that the stereochemical orientation of the 2, 4-dichlorophenyl group plays an important role in the interaction with the enzyme. In terms of drug concentration exerting 50% inhibition of ergosterol biosynthesis, NND-502 was 2.5 and 28 times more effective than that of lanoconazole and bifonazole, respectively.

Evaluation of IgE-sensitization to fungi in HIV-positive patients with eczematous skin reactions.

Human immunodeficiency virus infection is associated with declining immune function and polyclonal B-cell activation leading to elevated IgE-levels. In selected patient categories, increased total IgE may be associated with allergic diseases. Furthermore, a significant number of patients with low CD4+ cell numbers have various skin manifestations, eg, eczema and dermatophytosis. Patients with chronic fungal infections and a tendency to produce increased levels of specific IgE may become allergic and IgE-mediated mechanism may contribute to inflammatory reactions in the skin. This study investigates IgE-sensitization of patients infected with human immunodeficiency virus to a panel of fungal extracts of Candida albicans, Fusarium moniliforme, Penicillium notatum, Pityrosporum ovale, and Trichophyton rubrum. Fifteen HIV-positive patients with eczematous skin manifestations and five non-atopic healthy controls were evaluated by basophil histamine release and skin prick test with fungal extracts. The extracts were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions and analyzed by IgE-immunoblotting with sera from the patients and controls. Thirteen of 15 patients (87%) released histamine to one or more of the fungi. Skin prick test was positive to one or more fungi in 7 (47%) patients. Patient sera revealed binding to a wide range of IgE-binding components present in the fungal extracts. The IgE response was most often directed against a 46-kD main protein in the Candida albicans extract. There was no correlation between total serum IgE, CD4+ cell counts, and frequency of IgE-sensitization to fungi. The human IgE response in HIV-infected patients appears to be polyspecific and may be directed against various fungi of which Candida albicans may be an important allergen. It is possible that the sensitization is due to frequent infections with Candida albicans in this patient population. No unspecific fungal reactions were noted among control patients. These results suggest that allergen-specific IgE-mediated mechanism may contribute to the pathogenesis of the eczematous skin reaction in HIV-infected patients.

Cellular and humoral immune responses to MUC1 mucin and tandem-repeat peptides in ovarian cancer patients and controls.

The objective of this study was to demonstrate the presence of proliferative T cell responses to human polymorphic epithelial mucin (MUC1) and its tandem-repeat peptides in peripheral blood mononuclear cells (PBMC) from ovarian cancer patients and from controls and to correlate these cellular responses to a humoral response to MUC1. PBMC were obtained from 6 healthy women, from 13 women in the third trimester of pregnancy and from 21 ovarian cancer patients. Only 1 of the 6 healthy women showed a weak primary proliferative response (stimulation index, SI <2) to a 20-mer MUC1 tandem-repeat peptide in the presence of interleukin-2 (IL-2). In PBMC from 5/13 pregnant women (38%) a weak response could be induced by the 20-mer and/or 60-mer tandem-repeat peptides (SI < or =3.0) and in PBMC from 8/15 ovarian cancer patients (53%) 20-mer and/or 60-mer MUC1 tandem-repeat peptides induced primary responses (SI < or =5.4). MUC1 mucin purified from a breast tumor cell line and/or from urine of a healthy donor had a relatively strong stimulating effect (SI < or =19) on PBMC from 4 of 16 ovarian cancer patients (25%). In contrast, in PBMC of 9 ovarian cancer patients stimulated by the addition of a Candida albicans extract, MUC1 mucin strongly inhibited proliferation. This inhibition could partially be abrogated by the addition of IL-2. MUC1 (CA 15.3 assay) and free circulating MUC1 IgG and IgM antibodies (PEM.CIg assay) were determined in the plasma of all subjects. The MUC1 and the free circulating MUC1 IgG antibody plasma levels were significantly higher in the ovarian cancer patients than in the healthy women. Although no significant correlations were found between MUC1 mucin, MUC1 Ab plasma levels and the individual proliferative responses to the MUC1 antigens, an association may exist between them, since all three are significantly higher in the ovarian cancer patients than in the healthy women.