Electro-orientation of Schizosaccharomyces pombe in high conductivity media

Polymorphonuclear neutrophils (PMN) and mononuclear phagocytes represent an important first line and effector function in the control of Candida infections. Their relative contribution to host defence is frequently assessed by means of microbiological assays. However, reported results are divergent and might well be associated with study design-related issues. In the present study, we compared frequently used microbiological candidacidal assays, with the purpose of determining the most adequate method for assessment of phagocytosis and intracellular killing. We concluded that microbiological assays using yeast-phagocyte suspensions are inappropriate for the assessment of intracellular killing of Candida blastoconidia by murine macrophages, due to adherence or clumping of cells. In contrast, an adherent monolayer of phagocytes can be applied as a single microbiological assay to independently study the process of phagocytosis and intracellular killing, by exudate peritoneal macrophages as well as exudate peritoneal PMN.     

Human dendritic cells are less potent at killing Candida albicans than both monocytes and macrophages

Dendritic cells (DC) function as professional phagocytes to kill Candida albicans and subsequently present it to the adaptive immune system. Monocytes, macrophages and DC were generated from five individual donors and their Candida-killing capacity and cytokine release were assessed. Compared to monocytes and macrophages, DC from healthy volunteers were significantly less effective in C. albicans--stimulated cytokine release, killing of C. albicans blastoconidia and damaging of C. albicans hyphae. In conclusion, while important as antigen-presenting cells and initiators of the adaptive immune system, DC are poor in both intracellular killing and damaging of C. albicans hyphae. Effective handling of large numbers of C. albicans is the prime task of the innate immune system consisting of large numbers of neutrophils and monocytes.     

Protective effect of acidic mannan fraction of bakers' yeast on experimental candidiasis in mice

An acidic fraction of bakers' yeast mannan, WAM025, showed a significant protective effect against Candida albicans infection in mice, but a neutral fraction of the same bakers' yeast mannan, WNM, did not exhibit this effect. Moreover, pretreatment with WAM025 resulted in a marked reduction of proliferation of C. albicans cells in the organs of the infected mice. We investigated the stimulative effect of these mannan fractions on the function of mouse peritoneal phagocytes, and found that mice administered WAM025 showed a greater increase in the number of peritoneal exudate cells, macrophages and polymorphonuclear leucocytes (PMN), than the mice treated with WNM, especially in the proportion of PMN. Peritoneal phagocytes, PMN and macrophages obtained from WAM025-treated mice showed marked candidacidal activity. Of the phagocytes, PMN were responsible for the larger part of the candidacidal activity. The myeloperoxidase activities of PMN and macrophages in WAM025-treated PEC were greater than in untreated macrophages. The myeloperoxidase activity of WAM025-treated PMN was significantly greater than that of WAM025-treated macrophages. This activity paralleled the active oxygen-releasing activity of the phagocytes. On the other hand, the phagocytic activity of phagocytes from mice administered WNM or WAM025 for C. albicans cells was identical to that of untreated phagocytes. WAM025 seems to cause enhance elimination of the pathogen from mice, by increasing the number and candidacidal activity of phagocytic cells.     

Studies towards the potential of poliovirus as a vector for the expression of HPV 16 virus-like-particles

In this study we tested the hypothesis that after administration of a single intraperitoneal dose of concanavalin A (Con-A) to mice, the proportion of neutrophils and macrophages in the peritoneal exudate and their phagocytic and candidacidal activities should change with time. The number of neutrophils in the peritoneal exudate was greatly increased 6 h after administration of Con-A, and those cells were able to kill both intracellular and extracellular yeast and germ tube forms of Candida albicans. Addition of catalase to the culture medium reduced the killing of C. albicans, suggesting that the candidacidal activity depended on the myeloperoxidase system. The survival of mice pretreated with Con-A and submitted to an inoculum of C. albicans 6 h afterwards was twice higher than that of controls, which suggests that neutrophils were able to clear the experimental infection. One day after the treatment, the population of neutrophils in the exudate was about 45%, but after 2 days it was reduced to only 5% and the candidacidal activity was also reduced. After 4 days the exudate contained over 95% of macrophages, the candidacidal activity reached a maximum, and the phagocytosis mediated by both complement receptors and mannose receptors was increased. Uptake of FITC-mannose-BSA by macrophages was maximal on about the 4th day and was inhibited by mannan, suggesting that treatment with Con-A increased the activity of mannose receptors. These results support the hypothesis that activation of cellular immunity by Con-A occurred in two phases, one dominated by neutrophils, and the other by macrophages expressing increased activity of mannose receptors.     

Candida-host cell receptor-ligand interactions

The interaction of Candida species with their cognate host receptors is a key factor in the pathogenesis of different types of candidiasis. The recognition of different forms of Candida albicans by Toll-like receptors 2 and 4 on mononuclear leukocytes has recently been discovered to determine the function and activity of regulatory T-cells, determine the balance of Type 1 and Type 2 cytokines and, thereby, influence the antifungal activity of both the innate and adaptive immune response. Different forms of C. albicans are also recognized by different lectins that are expressed on the surface macrophages. C. albicans and Candida glabrata express the ALS (agglutinin-like sequence) and EPA (epithelial adhesin) families of adhesins, respectively. A key difference between C. glabrata and C. albicans is that EPA expression in C. glabrata is governed by sub-telomeric silencing, whereas ALS expression in C. albicans is regulated by other mechanisms.     

Interaction of Candida albicans with Human Leukocytes and Serum

A quantitative assay of candidacidal activity based on differential staining of non-viable Candida albicans by methylene blue was developed and applied to studies of leukocytes from normal individuals and patients with fungal and other infections. Serum factors were necessary for optimal phagocytosis of C. albicans but lacked direct candidacidal activity. Normal human neutrophils (38 studies) killed 29.0 +/- 7.4% of ingested C. albicans in 1 hr. Eosinophils and monocytes killed a smaller percentage. Neutrophil candidacidal activity did not require protein or ribonucleic acid synthesis by the leukocyte but was inhibited by anaerobic conditions, potassium cyanide, and colchicine. Leukocytes of a patient with hereditary myeloperoxidase deficiency and of three children with chronic granulomatous disease phagocytized C. albicans normally, yet failed to kill them. Our data suggest that the neutrophil can play an important role in resistance to Candida infection and that the lysosomal enzyme myeloperoxidase and its oxidant substrate hydrogen peroxide are the major participants in neutrophil candidacidal activity.     

Influence of aging on murine neutrophil and macrophage function against Candida albicans

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.     

Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.     

Microbiological and immunological features of oral candidiasis

Candida albicans(C. albicans) is the major infectious agent of oral candidiasis, and both innate immunity and cell-mediated immune response participate in the control of the fungal infections. The aim of this study was to correlate the clinical forms of oral candidiasis with the number of colony forming units (CFU) of C. albicans in saliva and to characterize T cell response in patients with oral candidiasis. Participants included 75 subjects: 36 with lesions of candidiasis and 39 without lesions of oral candidiasis. A 2-ml sample of saliva was collected from all subjects for microbiological analysis. Cytokine levels were determined by ELISA in supernatants of peripheral blood mononuclear cells of 25 patients with oral candidiasis, after in vitro stimulation with C. albicans antigens. In 48% of patients, no association was observed with denture use. C. albicans was detected in the saliva of 91.7% of patients with oral candidiasis, and there was an association between the number of CFU and the presence of oral lesions. A type Th1 immune response was observed in supernatants of peripheral blood mononuclear cells stimulated with C. albicans antigens. In contrast, IL-5 and IL-10 levels were very low or undetectable. Together, this study shows an association between clinical forms of oral candidiasis and the number of colonies of C. albicans in saliva, and that a systemic immune response characterized by the production of TNF-alpha and IFN-gamma is observed in patients with oral candidiasis.     

Increased susceptibility of TNF-alpha lymphotoxin-alpha double knockout mice to systemic candidiasis through impaired recruitment of neutrophils and phagocytosis of Candida albicans.

TNF-alpha and lymphotoxin-alpha (LT) are members of the TNF family, and these cytokines play crucial roles in the defense against infection with Candida albicans. The aim of the present study was to investigate the role of endogenous TNF and LT during disseminated candidiasis in TNF-/-LT-/- knockout mice. The TNF- and LT-deficient animals had a significantly increased mortality following C. albicans infection compared with control mice, and this was due to a 10- to 1000-fold increased outgrowth of the yeast in their organs. No differences between TNF-/-LT-/- mice and TNF+/+LT+/+ were observed when mice were rendered neutropenic, suggesting that activation of neutrophils mediates the beneficial effects of endogenous TNF and LT. Histopathology of the organs, combined with neutrophil recruitment experiments, showed a dramatic delay in the neutrophil recruitment at the sites of Candida infection in the TNF-/-LT-/- mice. Moreover, the neutrophils of deficient animals were less potent to phagocytize Candida blastospores than control neutrophils. In contrast, the killing of Candida and the oxygen radical production did not differ between neutrophils of TNF-/-LT-/- and TNF+/+LT+/+ mice. Peak circulating IL-6 was significantly higher in TNF-/-LT-/- mice during infection. Peritoneal macrophages of TNF-/-LT-/- mice did not produce TNF, and synthesized significantly lower amounts of IL-1alpha, IL-1beta, IL-6, and macrophage-inflammatory protein-1alpha than macrophages of TNF+/+LT+/+ animals did. In conclusion, endogenous TNF and/or LT contribute to host resistance to disseminated candidiasis, and their absence in TNF-/-LT-/- mice renders the animals susceptible through impaired recruitment of neutrophils and impaired phagocytosis of C. albicans.     

Effect of iron on fluconazole activity against Candida albicans in presence of human serum or monocyte-derived macrophages

Human serum, transferrin, and apotransferrin are known to profoundly inhibit the growth of Candida albicans by iron deprivation. On the other hand, iron overload (iron saturated transferrin) is a serious risk factor for candidiasis in newborn and in leukemic patients. We tested the efficacy of fluconazole and the previously demonstrated synergy of fluconazole and effector cells against C. albicans under iron overload conditions where efficacy might be diminished. We confirm that exogenous iron completely reversed the inhibitory effect of human serum and report that the efficacy of fluconazole against C. albicans was not significantly compromised in a 24 h assay system. Although exogenous iron inhibited fungistatic activity of monocyte-derived macrophages, it did not interfere with the synergistic candidacidal activity of fluconazole and monocyte-derived macrophages. In 72 h assays, where fluconazole had candidacidal activity, exogenous iron did not compromise efficacy of fluconazole, and fluconazole activity was often increased. These in vitro results suggest that effectiveness of fluconazole therapy would not be compromised in iron overload situations in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitric oxide and nitrosative stress tolerance in yeast

The opportunistic human fungal pathogen Candida albicans encounters diverse environmental stresses when it is in contact with its host. When colonizing and invading human tissues, C. albicans is exposed to ROS (reactive oxygen species) and RNIs (reactive nitrogen intermediates). ROS and RNIs are generated in the first line of host defence by phagocytic cells such as macrophages and neutrophils. In order to escape these host-induced oxidative and nitrosative stresses, C. albicans has developed various detoxification mechanisms. One such mechanism is the detoxification of NO (nitric oxide) to nitrate by the flavohaemoglobin enzyme CaYhb1. Members of the haemoglobin superfamily are highly conserved and are found in archaea, eukaryotes and bacteria. Flavohaemoglobins have a dioxygenase activity [NOD (NO dioxygenase domain)] and contain three domains: a globin domain, an FAD-binding domain and an NAD(P)-binding domain. In the present paper, we examine the nitrosative stress response in three fungal models: the pathogenic yeast C. albicans, the benign budding yeast Saccharomyces cerevisiae and the benign fission yeast Schizosaccharomyces pombe. We compare their enzymatic and non-enzymatic NO and RNI detoxification mechanisms and summarize fungal responses to nitrosative stress.     

In-vitro killing of Candida species by murine immunoeffectors and its relationship to the experimental pathogenicity

Killing of yeast cells of several species of Candida by murine phagocytic cells was assessed in vitro by a radiolabel release microassay and measurement of colony forming units. The most effective candidacidal phagocytes, i.e. polymorphonuclear and bone marrow cells, were able to kill equally well cells of any species or isolate tested, given sufficient time (4 h) and an appropriate effector: target ratio. However, C. guilliermondii, C. krusei and C. parapsilosis were killed by polymorphonuclear and bone marrow cells much more promptly (1 h) and at a significantly lower effector:target ratio than C. albicans, C. tropicalis and C. viswanathii. Moreover, there were immune effectors such as peritoneal resident macrophages and, mostly, spleen cells which were practically ineffective against C. albicans and C. tropicalis but showed significant activity against C. guilliermondii, C. krusei and C. parapsilosis, even in mice immuno-depressed with cyclophosphamide. Three isolates of C. albicans, differing in the capacity to form germ tubes, also differed in mouse virulence: the germ-tube forming isolate was the most virulent. However, they showed an identical pattern of susceptibility to killing by mouse immunoeffectors, suggesting that virulence is probably not due to the resistance of hyphal cell to phagocytosis.     

Lysophospholipid regulation of mononuclear phagocytes

Blood monocytes and tissue macrophages derived from monocyte differentiation in tissues are central elements of innate immunity in host defense against numerous pathogens and other challenges. These mononuclear phagocytes also participate in wound healing and normal tissue remodeling in development and growth. Pathological perversion of their physiological roles leads to participation of mononuclear phagocytes in fibrosing diseases including granulomatous disorders, chronic inflammation typical of arthritis, and atherosclerosis. Lysophospholipids, including lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), are platelet-derived lipid growth factors considered to participate in leukocyte differentiation and activation. This section summarizes our recent observations of the effects of lysophospholipids on mononuclear phagocytes.     

Candida albicans isolates with different genomic backgrounds display a differential response to macrophage infection

Few human pathogens possess the ability exhibited by Candida albicans to colonize and cause symptomatic infections at different body sites. The host immune system is the major factor determining whether this opportunistic yeast behaves as a commensal or as a pathogen, since C. albicans strains appear capable of expressing similar virulence factors in response to specific body-district cues. This report provides evidence showing that C. albicans isolates with diverse genomic backgrounds (b and c karyotypes) differently modulate their pathogenic potential when assayed in cocultures with human monocytic derived macrophages (THP-1 cells). Striking differences were observed in the ability to undergo bud-hypha transition, a relevant C. albicans virulence factor, between b and c karyotypes (P<0.0001) upon their internalization by macrophages. All c types were able to develop hyphal forms, resist intracellular killing, replicate, and escape from macrophages. The b type isolates, which were shown to be more efficiently ingested by THP-1 cells than the c type strains (P=0.013), were susceptible to intracellular killing and predominantly found as blastoconidia inside macrophages. Despite their different intracellular disposition, both b and c type isolates were equally able to undergo morphogenesis and to express NRG1 and HWP1 genes, markers of the bud-hypha transition program, during in vitro propagation. Since macrophages play a critical role in the host resistance to C. albicans, the different response of b and c isolates to macrophage infection suggests that the c type strains are better suited to behave as a more virulent strain cluster.     

Phagocytosis of Candida albicans by rabbit alveolar macrophages and guinea pig neutrophils.

Studies of host-parasite relationships at the cellular level, using Candida albicans and rabbit alveolar macrophages or guinea pig neutrophils are presented. Guinea pig neutrophils killed the intracellular candida cells presumed by myeloperoxidase-halide-hydrogen peroxide system. In contrast, rabbit alveolar macrophages did not kill the intracellular candida cells although their phagocytic rate was almost comparable to that of neutrophils. Phagocytizing macrophages were eventually destroyed by the intracellular proliferation of candida cells and formation of germ tubes and pseudomycelia. No significant improvement of candidacidal activity was observed with macrophages from normal and immunized rabbits in immune serum. The mode of phagocytosis by macrophages and neutrophils were also studied under the scanning electron microscope.     

First human model of in vitro Candida albicans persistence within granuloma for the reliable study of host-fungi interactions

BACKGOUND: The balance between human innate immune system and Candida albicans virulence signaling mechanisms ultimately dictates the outcome of fungal invasiveness and its pathology. To better understand the pathophysiology and to identify fungal virulence-associated factors in the context of persistence in humans, complex models are indispensable. Although fungal virulence factors have been extensively studied in vitro and in vivo using different immune cell subsets and cell lines, it is unclear how C. albicans survives inside complex tissue granulomas. METHODOLOGY/PRINCIPAL FINDING: We developed an original model of in vitro human granuloma, reproducing the natural granulomatous response to C. albicans. Persistent granulomas were obtained when the ratio of phagocytes to fungi was high. This in vitro fungal granuloma mimics natural granulomas, with infected macrophages surrounded by helper and cytotoxic T lymphocytes. A small proportion of granulomas exhibited C. albicans hyphae. Histological and time-lapse analysis showed that C. albicans blastoconidia were located within the granulomas before hyphae formation. Using staining techniques, fungal load calculations, as well as confocal and scanning electron microscopy, we describe the kinetics of fungal granuloma formation. We provide the first direct evidence that C. albicans are not eliminated by immunocompetent cells inside in vitro human granulomas. In fact, after an initial candicidal period, the remaining yeast proliferate and persist under very complex immune responses. CONCLUSIONS/SIGNIFICANCE: Using an original in vitro model of human fungal granuloma, we herein present the evidence that C. albicans persist and grow into immunocompetent granulomatous structures. These results will guide us towards a better understanding of fungal invasiveness and, henceforth, will also help in the development of better strategies for its control in human physiological conditions.     

Candida albicans infection inhibits macrophage cell division and proliferation

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to inhibit effective destruction by host phagocytes. Using live cell video microscopy, we show here for the first time that C. albicans inhibits cell division in macrophages undergoing mitosis. Inhibition of macrophage cell division is dependent on the ability of C. albicans to form hyphae, as it is rarely observed following phagocytosis of UV-killed or morphogenesis-defective mutant Candida. Interestingly, failed cell division following phagocytosis of hyphal C. albicans is surprisingly common, and leads to the formation of large multinuclear macrophages. This raises question as to whether inhibition of macrophage cell division is another virulence attribute of C. albicans or enables host macrophages to contain the pathogen.     

Killing of Escherichia coli by mononuclear phagocytes and neutrophils stimulated in vitro with beta-1,3-D-polyglucose derivatives.

Human monocytes, human peritoneal macrophages, mouse peritoneal macrophages and human peripheral neutrophils pretreated with beta-1,3-D-polyglucose derivatives showed pronounced bactericidal capacity to Escherichia coli compared to control cells. The increased bactericidal capacity was detectable in mononuclear phagocytes over a wide range of concentrations of bacteria. Granulocytes, however, showed bactericidal capacity only at low concentrations of bacteria. The pretreated mononuclear phagocytes released significant amounts of IL-1 and PGE2. However, there was no significant release of tumor necrosis factor (TNF). By incubating unstimulated cells with purified IL-1 and TNF, the bactericidal activity of neutrophils and mononuclear phagocytes was enhanced. Our data indicate that the inability of neutrophils stimulated with beta-1,3-D-polyglucose derivatives to kill large numbers of bacteria could be overcome by a combined treatment with purified IL-1 or TNF in addition to beta-1,3-D-polyglucose derivatives. By incubating unstimulated cells with medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, the bactericidal activity of the cells was enhanced to the same extent as cells pretreated with purified TNF and IL-1. Cells incubated with IL-1-depleted medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, showed reduced bactericidal activity compared to cells incubated with undepleted medium. These studies demonstrate that beta-1,3-D-polyglucose-treated mononuclear phagocytes and neutrophils show enhanced bactericidal activity. The enhanced activity is partly caused by stimulation of the cells with IL-1 released from mononuclear phagocytes and partly by other unknown effects of beta-1,3-D-polyglucose derivatives on both mononuclear phagocytes and neutrophils.     

Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.