Surface hydrophobin prevents immune recognition of airborne fungal spores.

The air we breathe is filled with thousands of fungal spores (conidia) per cubic metre, which in certain composting environments can easily exceed 10(9) per cubic metre. They originate from more than a hundred fungal species belonging mainly to the genera Cladosporium, Penicillium, Alternaria and Aspergillus. Although these conidia contain many antigens and allergens, it is not known why airborne fungal microflora do not activate the host innate immune cells continuously and do not induce detrimental inflammatory responses following their inhalation. Here we show that the surface layer on the dormant conidia masks their recognition by the immune system and hence prevents immune response. To explore this, we used several fungal members of the airborne microflora, including the human opportunistic fungal pathogen Aspergillus fumigatus, in in vitro assays with dendritic cells and alveolar macrophages and in in vivo murine experiments. In A. fumigatus, this surface 'rodlet layer' is composed of hydrophobic RodA protein covalently bound to the conidial cell wall through glycosylphosphatidylinositol-remnants. RodA extracted from conidia of A. fumigatus was immunologically inert and did not induce dendritic cell or alveolar macrophage maturation and activation, and failed to activate helper T-cell immune responses in vivo. The removal of this surface 'rodlet/hydrophobin layer' either chemically (using hydrofluoric acid), genetically (DeltarodA mutant) or biologically (germination) resulted in conidial morphotypes inducing immune activation. All these observations show that the hydrophobic rodlet layer on the conidial cell surface immunologically silences airborne moulds.

PE_PGRS antigens of Mycobacterium tuberculosis induce maturation and activation of human dendritic cells.

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosis and host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M. tuberculosis and, in particular, the proline-glutamic acid_polymorphic guanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappaB signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis.

Modulation of human dendritic cell maturation and function by natural IgG antibodies.

Dendritic cells (DCs) are professional antigen-presenting cells, which have a central role in the initiation of primary immune responses and in maintaining immune tolerance. The functions of DCs can be regulated both by environmental signals as well as signals delivered by endogenous molecules. Recently we have examined regulation of human DCs by B cells via natural IgG antibodies. Natural antibodies (NAbs) are defined as antibodies that circulate in normal individuals in the absence of deliberate immunization or microbial aggression. We demonstrate that the differentiation of DCs is severely impaired in primary immunodeficient patients such as X-linked agammaglobulinemia (XLA) and common variable immunodeficiency (CVID) at least in part due to the deficiency of circulating NAbs. Further, we show that NAbs are able to restore normal phenotypes of DCs from patients with XLA and CVID. Our results suggest that B cells promote bystander DC development through NAbs and the interaction between NAbs and DCs may play a role in steady-state migration of DCs.

Induction of maturation and activation of human dendritic cells: a mechanism underlying the beneficial effect of Viscum album as complimentary therapy in cancer.

BACKGROUND: Viscum album (VA) preparations have been used as a complimentary therapy in cancer. In addition to their cytotoxic properties, they have also been shown to have immunostimulatory properties. In the present study, we examine the hypothesis that the VA preparations induce activation of human DC that facilitates effective tumor regression. METHODS: Four day old monocyte-derived immature DCs were treated with VA Qu Spez at 5, 10 and 15 microg/ml for 48 hrs. The expression of surface molecules was analyzed by flow cytometry. The ability of Qu Spez-educated DC to stimulate T cells was analyzed by allogeneic mixed lymphocyte reaction and activation of Melan-A/MART-1-specific M77-80 CD8+T cells. Cytokines in cell free culture supernatant was analyzed by cytokine bead array assay. RESULTS: VA Qu Spez stimulated DCs presented with increased expression of antigen presenting molecule HLA-DR and of co-stimulatory molecules CD40, CD80 and CD86. The VA Qu Spez also induced the secretion of inflammatory cytokines IL-6 and IL-8. Further, Qu Spez-educated DC stimulated CD4+T cells in a allogeneic mixed lymphocyte reaction and activated melanoma antigen Melan-A/MART-1-specific M77-80 CD8+T cells as evidenced by increased secretion of TNF-alpha and IFNgamma. CONCLUSION: The VA preparations stimulate the maturation and activation of human DCs, which may facilitate anti-tumoral immune responses. These results should assist in understanding the immunostimulatory properties of VA preparations and improving the therapeutic strategies.

The protective role of immunoglobulins in fungal infections and inflammation.

Increased incidence of fungal infections in the immunocompromised individuals and fungi-mediated allergy and inflammatory conditions in immunocompetent individuals is a cause of concern. Consequently, there is a need for efficient therapeutic alternatives to treat fungal infections and inflammation. Several studies have demonstrated that antibodies or immunoglobulins have a role in restricting the fungal burden and their clearance. However, based on the data from monoclonal antibodies, it is now evident that the efficacy of antibodies in fungal infections is dependent on epitope specificity, abundance of protective antibodies, and their isotype. Antibodies confer protection against fungal infections by multiple mechanisms that include direct neutralization of fungi and their antigens, inhibition of growth of fungi, modification of gene expression, signaling and lipid metabolism, causing iron starvation, inhibition of polysaccharide release, and biofilm formation. Antibodies promote opsonization of fungi and their phagocytosis, complement activation, and antibody-dependent cell toxicity. Passive administration of specific protective monoclonal antibodies could also prove to be beneficial in drug resistance cases, to reduce the dosage and associated toxic symptoms of anti-fungal drugs. The longer half-life of the antibodies and flexibilities to modify their structure/forms are additional advantages. The clinical data obtained with two monoclonal antibodies should incite interests in translating pre-clinical success into the clinics. The anti-inflammatory and immunoregulatory role of antibodies in fungal inflammation could be exploited by intravenous immunoglobulin or IVIg.

Regulation of human dendritic cell functions by natural anti-CD40 antibodies.

Dendritic cells (DCs) are professional antigen presenting cells that play a pivotal role in the initiation of immune responses. DCs ingest antigens and then present these antigens to T cells to initiate T cell activation and polarization. DCs receive signals both from environment and from endogenous molecules. DCs in the immune system constantly interact with immunoglobulins (or antibodies) and a substantial amount of these immunoglobulins are natural. We found that natural antibodies have a key role in regulating the DC functions and that CD40-reactive natural antibodies constitute one of the endogenous molecules that provide maturation-associated signals to DCs in physiology. In this chapter, we describe the isolation of anti-CD40 natural antibodies from pooled normal immunoglobulin preparations (intravenous immunoglobulin, IVIg) and their biological effects on human DC maturation and functions.

Antiangiogenic properties of viscum album extracts are associated with endothelial cytotoxicity.

BACKGROUND: Viscum album (VA) extracts are widely used in cancer therapy and are known to be cytotoxic to tumors and endothelial cells. Angiogenesis plays an important role in the growth, sustenance and metastasis of tumors. Inhibition of angiogenesis is now being explored as a new therapeutic avenue for cancer. MATERIALS AND METHODS: The cytotoxicity of VA extracts was analyzed by Annexin V labeling and propidium iodide uptake in EA-hy926 endothelial cells. The antiangiogenic effect was studied in vitro by treating the EA-hy926 cells in matrigel and subsequent analysis of vascular formation. Computer-assisted image analysis of vascular formation was analyzed to quantify the in vitro data. In vivo studies were performed by implanting matrigel +/- VA extracts in Balb/C mice that had been subjected to IP treatment with VA extracts. RESULTS: The combination of systemic and intra- matrigel treatment with the VA Qu Spez extract caused significant inhibition of angiogenesis. The VA P extract treatment showed insignificant change in vessel formation. CONCLUSION: These results may provide novel guidelines towards improved strategies using VA extracts based on the inhibition of angiogenesis of tumors.