Anti-beta-glucan antibodies in healthy human subjects.

Previous data by our group demonstrated the antifungal efficacy of a vaccine consisting of laminarin (beta-(1,3)-glucan), conjugated with diphtheria toxoid, which generated protective anti-laminarin antibodies in mice. In this paper, we sought for the presence, isotype and subclass composition of natural anti-laminarin antibodies in an unselected population of human healthy subjects, in a comparison with antibodies directed against beta-(1,6)-glucan (pustulan) and branched beta-(1,3/1,6)-glucan (Pool 1) and mannan from Candida albicans. Almost all subjects showed detectable levels of anti-beta-glucan antibodies, with IgG largely prevailing on IgM, little, if any, IgA and no IgE. However, the titer of anti-beta-glucan antibodies was overall about 1log lower than that of anti-mannan antibodies of the corresponding isotype. In particular, the level of anti-laminarin IgG was the lowest one, its geometrical mean titer (95% confidence interval, CI) being 1838 (1245-2714) as compared to 8157 (6067-10,931) and 3940 (2911-5332) for pustulan and Pool 1 fungal glucan, respectively. Analysis of IgG subclass composition showed that IgG2 was the prevalent subclass against any antigen, and again the concentration of anti-laminarin IgG2 was the lowest one, its geometrical mean concentration being 0.13 (0.07-0.24)microg/ml as compared to anti-pustulan and anti-Pool 1 glucan and mannan IgG2 levels, which were 0.33 (0.2-0.5), 1.35 (0.9-2.0), and 36.1 (25.2-51.3)microg/ml, respectively. These data show that anti-laminarin antibodies are present at low levels in humans as compared to other anti-beta-glucan and, mostly, anti-mannan antibodies, and suggest that a protective antifungal vaccination in humans should attempt to tip the balance of antifungal antibodies in favour of the anti-laminarin ones.

The interaction of human dendritic cells with yeast and germ-tube forms of Candida albicans leads to efficient fungal processing, dendritic cell maturation, and acquisition of a Th1 response-promoting function.

T helper cell type 1 (Th1) cell-mediated immunity plays a critical role in protection against the opportunistic pathogen Candida albicans. Virulence of the fungus is closely associated with its ability to form germ-tubes (GT), the early phase of the dimorphic transition from the commensal yeast (Y) to the more invasive hyphal (H) form. In this study, we examined the functional outcome of the interaction of Y or GT forms with human dendritic cells (DCs), professional antigen-presenting cells, which are pivotal for initiation and modulation of T cell responses. DCs phagocytosed and killed Y and GT cells with a comparable efficiency, becoming able to trigger strong proliferative responses by Candida-specific, autologous T cell clones. Both fungal forms induced DC maturation, as indicated by up-regulation of CD83, CD80, CD86, CD40, and major histocompatibility complex classes I and II surface antigens. Chemokine receptors were also modulated in Candida-DCs, which showed increased CCR7/CXCR4 and decreased CCR5 expression. Y- and GT-activated DCs differed in the pattern of cytokine expression. In particular, GT cells, in common with fully differentiated H cells, induced significantly more elevated levels of interleukin (IL)-10 than Y cells. Nevertheless, Y-, GT-, or H-pulsed DCs secreted comparable amounts of IL-12p70. In addition, irrespective of the fungal form triggering DC activation, Candida-DCs acquired the ability to prime naive T lymphocytes with a defined Th1 phenotype. Overall, our findings highlight the induction of substantially similar functional patterns in human DCs encountering the different forms of growth of C. albicans, both seemingly activating the Th1-type immunity which is characteristic of the healthy human subjects, naturally immunized and protected against the fungus.

Differential chemokine response of human monocytes to yeast and hyphal forms of Candida albicans and its relation to the beta-1,6 glucan of the fungal cell wall.

Hyphae formation from yeast cells is a virulence trait enabling the human opportunistic pathogen Candida albicans to invade host tissues. Hyphal cells proved to be much less efficient than yeast cells in stimulating production of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, interleukin-8 (IL-8), and particularly, monocyte chemotactic protein-1 (MCP-1) by human monocyte. This different stimulation did not depend on the monocyte inability to ingest the hyphae nor did it imply hyphal resistance to the extracellular killing by the monocytes. Purified hyphal and yeast cell walls reproduced the differences shown by the intact cells, and chemical-enzymatic dissection of cell wall components suggested that cell wall beta-1,6 rather than beta-1,3 glucan was the main chemokine inducer. Coherently, immunofluorescence studies with an anti beta-1,6 glucan serum showed that the surface expression of this polysaccharide was much lower on hyphae than on yeast cells. By minimizing chemokine induction, the formation of hyphal filaments might facilitate C. albicans escaping from host immunity.

Defective induction of interleukin-12 in human monocytes by germ-tube forms of Candida albicans.

Yeast (Y) to germ-tube (GT) transition of Candida albicans is considered a putative virulence trait. On the other hand, interleukin-12 (IL-12) is a key promoter of T-helper type 1 protective immunity against this human opportunistic pathogen. We studied IL-12 production by human monocytes cocultured in vitro with Y or GT forms of C. albicans. Following stimulation by Y cells, monocytes produced appreciable levels of IL-12, which, upon addition of gamma interferon (IFN-gamma), compared to those achievable by lipopolysaccharide (100 ng/ml) stimulation (140 +/- 65 and 185 +/- 80 pg/ml, respectively [mean +/- standard deviation in four independent experiments]). In contrast, IL-12 production by GT cell-stimulated monocytes was much lower or absent (<5 pg/ml) and could not be brought to the level induced by Y cells by the addition of IFN-gamma (30 +/- 10 pg/ml in the four independent experiments above). Besides being observed as actual cytokine production, this lower response was also observed as specific IL-12 p40 mRNA transcript and was not associated with hyperproduction of the IL-12-competing cytokine IL-10. Phagocytosis and killing experiments in the presence of cytochalasin D showed that IL-12 production by Y cell-stimulated monocytes was phagocytosis dependent and that GT cells of C. albicans were not phagocytized by the human monocytes. Importantly, however, Y and GT cells were equally killed by the monocytes. Thus, the virulence trait attributed to the Y-GT transition of C. albicans might also be related to the lack of induction by GT cells of a protective anticandidal immunity through defective IL-12 production.

Fusarium infections in patients with severe aplastic anemia: review and implications for management.

BACKGROUND AND OBJECTIVE: The prognosis of severe fungal infections, such as fusarium infections, in patients with aplastic anemia is directly related to the recovery of bone marrow functions. In this study, in vitro anti-Fusarium activity of granulocytes was investigated, the case of disseminated infection in a child with very severe aplastic anemia is reported, and implications for management of such infective complications are discussed. DESIGN AND METHODS: The in vitro efficiency of PMNL from three untreated, normal blood donors and from two G-CSF-treated WBC donors in contrasting the growth of the Fusarium sp strain isolated from the patient we present was measured by a 3H-glucose uptake inhibition assay and confirmed by microscopic examination. RESULTS: Basic growth inhibitory activity of unstimulated PMNL on Fusarium cells was significantly enhanced in the presence of GM-CSF in all three blood donors tested. In one of the two G-CSF-treated donors, in vitro efficiency of PMNL in contrasting the growth of the fungus increased notably after G-CSF treatment. We report the case of a 3-year-old girl with very severe aplastic anemia unresponsive to conventional immunosuppressant therapy who developed a disseminated fusarium infection. The child initially responded to liposomal amphotericin B and granulocyte transfusions from G-CSF stimulated donors. Subsequently she was given a cord blood stem cell transplantation but died of disseminated infection. INTERPRETATION AND CONCLUSIONS: Including the present case, there are only ten reports of invasive infections caused by the genus Fusarium in aplastic anemia patients and only two of the patients survived. In vitro data seem to suggest that in vivo treatment with rh-G-CSF could have a stimulatory effect on the anti-Fusarium activity of neutrophils. Despite the efficacy of granulocyte transfusions by G-CSF-stimulated donors in the temporary control of fusarium infection, treatment of the underlying hematologic disease is required to cure the infection in patients with severe aplastic anemia. Granulocyte transfusions by G-CSF-stimulated donors while awaiting bone marrow recovery following the blood stem cell transplant should be considered.

Suckling CD1 mice as an animal model for studies of Legionella pneumophila virulence.

On the assumption that specific host defences are lower in newborn and infant animals, the susceptibility of CD1 suckling mice to Legionella pneumophila was studied with the hypothesis that this model could detect consistent differences in virulence among Legionella isolates from various clinical and environmental sources. Mice 3-14 days old were indeed markedly susceptible to intraperitoneal challenge with fresh clinical isolates, but not to serially subcultured or type collection strains of L. pneumophila. For example, intraperitoneal inoculation of 10(7) cells of a fresh clinical isolate of L. pneumophila (strain Monza 3) caused 60% mortality of suckling mice in 1 day whereas the same number of cells of a culture-attenuated derivative (strain Monza 3p50) caused <10% mortality in >15 days. Lethal infection by the 'virulent' Monza 3 strain was strictly dependent on mouse age (no death was observed in mice >26 days old), required the inoculation of viable cells and was not related to endotoxin production. The 'virulent' L. pneumophila strain was cleared from mouse lungs less rapidly, while adhering to, and being internalised into the peritoneal exudate cells (PEC) of suckling mice to a greater extent, than the avirulent derivative, as shown by immunofluorescence and confocal microscopy. The Monza 3 strain also induced the production by PEC in vivo of 5-to-10 times more tumour necrosis factor-alpha (TNF-alpha) mRNA than the Monza 3p50 strain. Overall, suckling CD1 mice appear to provide a promising, easily handled, highly reproducible and relatively inexpensive animal model for studies of the virulence of L. pneumophila, and possibly, of the role of pro-inflammatory cytokine production in this phenomenon.

Possible participation of polymorphonuclear cells stimulated by microbial immunomodulators in the dysregulated cytokine patterns of AIDS patients.

Macrophages and polymorphonuclear cells (PMN) play a major role as cells primarily responsive to microbial biological response modifiers (BRM). Although much attention has been given to macrophages, PMN have been relatively underinvestigated. We have recently studied the responses of PMN from HIV- and HIV+ subjects after stimulation with a powerful immunomodulatory fraction from the cell wall of Candida albicans (MP-F2) and compared this to bacterial lipopolysaccharide (LPS). Both cytokine patterns and PMN anticandidal activity were investigated. MP-F2, like LPS, was an active inducer of interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), IL-6, and IL-1 beta production by PMN and monocytes from all subjects. IL-12 was also produced by MP-F2-stimulated PMN in the presence of interferon-gamma (IFN-gamma). PMN from HIV+ subjects showed increased in vitro expression of TNF-alpha and IL-6 genes as determined by semiquantitative reverse transcriptase-polymerase chain reaction. In all subjects, cytokine gene expression was strongly stimulated by MP-F2 or LPS and inhibited by IL-10. Production of IL-6 and TNF-alpha protein (measured by ELISA) was higher in PMN from HIV+ subjects in at least one of the conditions tested (unstimulated or stimulated by LPS or MP-F2). However, the amount of the C-X-C chemokine IL-8 was equal in PMN from HIV- and HIV+ subjects. PMN from HIV+ subjects were at least as active in inhibiting candide growth as PMN from HIV- controls. In both groups PMN were equally stimulated by MP-F2 and LPS. Only in severely neutropenic subjects was there some reduction in the anticandidal activity but not in cytokine responses. When appropriately stimulated by microbial BRM, PMN are active producers of pro-inflammatory and immunomodulatory cytokines. This production is not only totally preserved in HIV+ subjects but may be higher than in PMN from HIV- subjects and may be coupled with an efficient anticandida activity. We suggest that during common bacterial or fungal infections PMN may contribute to the dysregulated production of inflammatory cytokines in AIDS patients.

Responsiveness of human polymorphonuclear cells (PMNL) to stimulation by a mannoprotein fraction (MP-F2) of Candida albicans; enhanced production of IL-6 and tumour necrosis factor-alpha (TNF-alpha) by MP-F2-stimulated PMNL from HIV-infected subjects.

IL-1beta, IL-6, IL-8 and TNF-alpha production by PMNL from 21 HIV-infected (HIV+), including 11 full-blown AIDS, and 20 HIV-uninfected (HIV-) subjects (matched for age and sex to HIV+ ones) was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA. PMNL from both categories of subjects were strongly stimulated in their actual cytokine production by a mannoprotein fraction (MP-F2) of Candida albicans, as well as by the bacterial lipopolysaccharide (LPS). These stimulatory effects were apparently due to increased cytokine gene expression and were substantially reversed by the physiological inhibitor IL-10. However, PMNL from HIV+ subjects showed increased IL-6 and TNF-alpha gene expression and produced more IL-6 and TNF-alpha than PMNL from HIV- controls, under similar stimulation conditions. This difference could not be attributed to a given stage of HIV infection, any associated medication, or to a generalized increase of gene expression, as quantitatively similar beta-actin and IL-1beta transcripts were detected. Moreover, no significant difference in IL-8 production by the PMNL from HIV+ and HIV- subjects was observed. Our studies suggest that PMNL from HIV+ subjects might add to other cellular sources of IL-6 and TNF-alpha (e.g. monocytes-macrophages) in contributing to the cytokine-dysregulated pattern typical of the HIV+ patient.

Elevated aspartic proteinase secretion and experimental pathogenicity of Candida albicans isolates from oral cavities of subjects infected with human immunodeficiency virus.

Isolates of Candida albicans from the oral cavities of subjects at different stages of human immunodeficiency virus (HIV) infection or uninfected controls were examined for (i) production of aspartic proteinase(s), a putative virulence-associated factor(s); (ii) the presence in the fungal genome of two major genes (SAP1 and SAP2) of the aspartic proteinase family; and (iii) experimental pathogenicity in a murine model of systemic infection. It was found that the fungal isolates from symptomatic patients secreted, on average, up to eightfold more proteinase than the isolates from uninfected or HIV-infected but asymptomatic subjects. This differential property was stably expressed by the strains even after years of maintenance in stock cultures. Moreover, representative high-proteinase isolates were significantly more pathogenic for mice than low-proteinase isolates of C. albicans. The characters high proteinase and increased virulence were not associated with a single molecular type or category identifiable through DNA fingerprinting or pulsed-field electrophoretic karyotype, and both SAP1 and SAP2 genes were present in both categories of isolates, on the same respective chromosomes. In conclusion, our data suggest that during HIV infection more-virulent strains or biotypes of C. albicans which are identifiable by direct analysis of virulence determinants are selected. It also appears that the biotype switch to increased aspartic proteinase and virulence properties occurs before the HIV-infected subject enters the symptomatic stage and overt AIDS.