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Efficient response to Aspergillusfumigatus requires different mechanisms. Polymorphonuclear neutrophils (PMNs) are the predominant immune cells in the acute stage of most fungal infections and play a crucial role in determining the type of pathology associated with fungal infections in different clinical settings. Dendritic cells (DC) are able to decode the fungus-associated information and translate it into different T helper (Th) and regulatory (Treg) cell responses. Functionally distinct Treg cells are activated after exposure to Aspergillus conidia. Early in infection, inflammation/Th1 reactivity is controlled by Treg cells suppressing PMNs and the immunogenic program of DC. The levels of IFN-γ produced in this phase set the subsequent adaptive stage by conditioning the indoleamine 2, 3-dioxygenase (IDO)-dependent tolerogenic program of DC and the subsequent activation of tolerogenic Treg cells, which inhibit Th2 cells and prevent allergy to the fungus. Knowledge of the immunopathogenesis of Aspergillus infections may pave the way to promising strategies for immunotherapy.

During inflammation, host- and microbial-derived proteases trigger the activation of protease-activated receptors (PARs), a family of G-protein-coupled receptors. We report here that activation of Toll-like receptors (TLRs) by fungi unmasks an essential and divergent role for PAR(1) and PAR(2) in downstream signaling and inflammation. TLRs activated PARs and triggered distinct signal transduction pathways involved in inflammation and immunity to Candida albicans and Aspergillus fumigatus. Inflammation was promoted by PAR(1) and PAR(2) activation in response to Candida and by PAR(2) inhibition in response to Aspergillus. This occurred by TLR regulation of PAR signaling, with TLR2 promoting PAR(1) activity, and TLR4 suppressing PAR(2) activity. Thus, tissue injury and pathogens induce signals that are integrated at the level of distinct TLR/PAR-dependent pathways, the exploitation or subversion of which contributes to divergence in microbial promotion of inflammatory response.

The long pentraxin (PTX) 3 is produced by macrophages and myeloid dendritic cells in response to Toll-like receptor agonists and represents a nonredundant component of humoral innate immunity against selected pathogens. We report that, unexpectedly, PTX3 is stored in specific granules and undergoes release in response to microbial recognition and inflammatory signals. Released PTX3 can partially localize in neutrophil extracellular traps formed by extruded DNA. Eosinophils and basophils do not contain preformed PTX3. PTX3-deficient neutrophils have defective microbial recognition and phagocytosis, and PTX3 is nonredundant for neutrophil-mediated resistance against Aspergillus fumigatus. Thus, neutrophils serve as a reservoir, ready for rapid release, of the long PTX3, a key component of humoral innate immunity with opsonic activity.

Reactivation of latent human cytomegalovirus (HCMV) following allogeneic transplantation is a major cause of morbidity and mortality and predisposes to severe complications, including superinfection by Aspergillus species (spp). Antimicrobial polypeptides, including defensins and mannan-binding lectin, are known to block viral fusion by cross-linking sugars on cell surface. Pentraxin 3 (PTX3), a member of the long pentraxin family, successfully restored antifungal immunity in experimental hematopoietic transplantation. We assessed here whether PTX3 binds HCMV and murine virus (MCMV) and the impact on viral infectivity and superinfection in vivo. We found that PTX3 bound both viruses, reduced viral entry and infectivity in vitro, and protected from MCMV primary infection and reactivation as well as Aspergillus superinfection. This occurred through the activation of interferon (IFN) regulatory factor 3 (IRF3) in dendritic cells via the TLR9/MyD88-independent viral recognition sensing and the promotion of the interleukin-12 (IL-12)/IFN-γ–dependent effector pathway.

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defense without necessarily eliminating the fungus or causing unacceptable levels of host damage. In this study, we show that a division of labor occurs between functionally distinct regulatory T cells (Treg) that are coordinately activated by a CD28/B-7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation is controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of IL-10 and CTLA-4 on indoleamine 2,3-dioxygenase. The levels of IFN-γ produced in this early phase set the subsequent adaptive stage by conditioning the indoleamine 2,3-dioxygenase-dependent tolerogenic program of dendritic cells and the subsequent activation and expansion of tolerogenic Treg, which produce IL-10 and TGF-β, inhibit Th2 cells, and prevent allergy to the fungus. The coordinate activation of Treg may, however, be subverted by the fungus, as germinating conidia are capable of interfering with anti-inflammatory and tolerogenic Treg programs. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.

Immunological features of GM-490 cells, a new blood cell line from a patient with acute lymphoblastic leukemia, included lack of CD34, CD38, CD45, CD14, HLA-DR, and lymphoid and myeloid markers and expression of CD29, CD36, CD44, CD54, CD71, CD105, and CD133. Molecular analysis indicated CD45 gene expression was absent but CD34 mRNA was present. GM-490 cells constitutively produced fibronectin (FN), type III and traces of type I collagen, collagenases, glycosaminoglycans (GAG) and biglycan and betaglycan proteoglycans (PG) as well as FGF2 and TGFβ1. When FGF2 and/or TGFβ1 were added to cells in vitro, they stimulated cell proliferation and differently modulated matrix production and growth factor receptor expression. Reverse transcription-polymerase chain reaction (RT-PCR) detection of transcripts encoding for osteocalcin and RUNX2 suggests GM-490 cells differentiate towards the osteoblast pathway. GM-490 cells expressed the low affinity nerve growth factor receptor (p75LNGFR), a somatic stem cell marker that is not detected in hematopoietic cells, leading to the hypothesis that GM-490 has mesenchymal stem cell properties. The reciprocal modulating effects of FGF2 and TGFβ1 on each other's receptors make the GM-490 cell line a new model for investigating the relationship between these growth factors and their receptors in autocrine loops which are believed to sustain the malignant clone in hematological diseases. © 2005 Wiley-Liss, Inc.

Polymorphonuclear neutrophils (PMNs) are essential in initiation and execution of the acute inflammatory response and subsequent resolution of fungal infection. PMNs, however, may act as double-edged swords, as the excessive release of oxidants and proteases may be responsible for injury to organs and fungal sepsis. To identify regulatory mechanisms that may balance PMN-dependent protection and immunopathology in fungal infections, the involvement of different TLR-activation pathways was evaluated on human PMNs exposed to the fungus Aspergillus fumigatus. Recognition of Aspergillus and activation of PMNs occurred through the involvement of distinct members of the TLR family, each likely activating specialized antifungal effector functions. By affecting the balance between fungicidal oxidative and nonoxidative mechanisms, pro- and anti-inflammatory cytokine production, and apoptosis vs necrosis, the different TLRs ultimately impacted on the quality of microbicidal activity and inflammatory pathology. Signaling through TLR2 promoted the fungicidal activity of PMNs through oxidative pathways involving extracellular release of gelatinases and proinflammatory cytokines while TLR4 favored the oxidative pathways through the participation of azurophil, myeloperoxidase-positive, granules and IL-10. This translated in vivo in the occurrence of different patterns of fungal clearance and inflammatory pathology. Both pathways were variably affected by signaling through TLR3, TLR5, TLR6, TLR7, TLR8, and TLR9. The ability of selected individual TLRs to restore antifungal functions in defective PMNs suggests that the coordinated outputs of activation of multiple TLRs may contribute to PMN function in aspergillosis.

As a fungal etiology has been proposed to underlie severe nasal polyposis, the present study was undertaken to assess local antifungal immune reactivity in nasal polyposis. For this purpose, microbial colonization, along with the pattern of T helper 1 (Th1)/Th2 cytokine production and Toll-like receptor (TLR) expression, was evaluated in patients with nasal symptoms and with and without polyposis and in healthy subjects. The results show that Th2 reactivity was a common finding for patients with nasal polyposis regardless of the presence of microbes. The production of interleukin-10 was elevated in patients with bacterial and, particularly, fungal colonization, while both TLR2 expression and TLR4 expression were locally impaired in microbe-colonized patients. Eosinophils and neutrophils, highly recruited in nasal polyposis, were found to exert potent antifungal effector activities toward conidia and hyphae of the fungus and to be positively regulated by TLR2 or TLR4 stimulation. Therefore, a local imbalance between activating and deactivating signals to effector cells may likely contribute to fungal pathogenicity and the expression of local immune reactivity in nasal polyposis.

Background Silicosis is mediated by macrophages, their soluble mediators, and extracellular matrix molecules. In this study, we investigated the effects of silica and/or hyaluronate (HA) on several alveolar macrophage responses. Methods We evaluated glycosaminoglycan (GAG) production by radiolabeled precursors, nitric oxide (NO) release by its oxidation product, phagocytic activity by Candida albicans internalization, and the secretion of two fibrogenic cytokines, tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β, by specific assays. Results Silica significantly reduced GAG secretion, particularly HA secretion. Alone, it decreased Candida uptake; associated with HA, it enhanced the reduction. Silica and Candida reduced NO release, which was not significantly affected when silica- or Candida-exposed cells were also treated with HA. TNF-α and TGF-β activities were stimulated by silica but reduced by HA. Conclusions The results suggest that silica and HA modify alveolar macrophage functional differentiation. Silica- and HA-induced modifications of the microenvironment could determine whether the response proceeds toward healing and repair or toward lung chronic pathology.

In the Crouzon's syndrome the cranial morphogenic processes are altered due to the early fusion of cranial sutures. We analysed the phenotype of cultured fibroblasts from normal subjects and from Crouzon patients with a specific fibroblast growth factor receptor 2 mutation resulting in a Cys 342 Tyr substitution within the third immunoglobulin domain. Crouzon fibroblasts differed from normal fibroblasts in their extracellular matrix macromolecule accumulation. In Crouzon fibroblasts glycosaminoglycans and fibronectin were decreased and type I collagen increased. As transforming growth factors beta (TGF beta) and basic fibroblasts growth factor (bFGF) together regulate extracellular matrix deposition, we evaluated TGF beta(1), TGF beta(3) and bFGF production by Crouzon and normal fibroblasts. TGF beta(1), TGFb(3) and bFGF levels were lower while TGF beta(1) mRNA transcripts were higher in Crouzon cells. As the increased TGF beta(1) gene expression did not translate into a parallel increase of secreted TGF beta(1), control of TGF beta(1) secretion may be mainly post-transcriptional. Furthermore, adding bFGF increased TGF beta(1) and TGF beta(3) secretion, suggesting the drop may be due to the altered signal transduction of bFGF. These innovative data suggest the in vitro differences between normal and Crouzon fibroblasts may be due to an imbalance in TGF beta and bFGF levels which alters the microenvironment where morphogenesis takes place.

The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.

The present study provides the first evidence that fibroblasts obtained from patients affected by Crouzon syndrome, a rare craniosynostosis, despite mutations in the high-affinity bFGF receptor retain their capacity to respond to bFGF. The growth factor reduces IL-1 secretion, downregulates biglycan and procollagen α1(I), and increases betaglycan expression. Since betaglycan is a co-receptor for bFGF signalling, an alternative signal transduction pathway is suggested in Crouzon fibroblasts, to explain the documented changes in ECM macromolecule production.

The cytotoxicity of the most common alloys used in orthodontic appliances was determined by cell culture testing. Human gingival fibroblasts were cultured on 304 and 316 stainless steel, on brazing alloy composed of palladium (Pd), copper (Cu), and silver (Ag), and on plastic substrate (control). Studies were carried out with SEM and radiolabeled precursor incorporation. Cells were cultured in MEM without serum but with the addition of 3H-thymidine to evaluate cell proliferation and 3H-glucosamine to evaluate glycosaminoglycan (GAG) synthesis and secretion in the culture medium. Moreover, gingival fibroblasts were cultured in the presence of some metal ions generally released by orthodontic appliances to evaluate the cytotoxic effects of single ions. Morphologic observations with SEM and radiolabeled incorporation studies showed that 304 and 316 stainless steel were more biocompatible than the brazing alloy. Among the metal ions tested, Ag and Pd, constituents of the brazing alloy, showed the highest cytotoxicity. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 51, 561–568, 2000.

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defence without necessarily eliminating the fungus or causing unacceptable levels of host damage. Efficient responses to the fungus require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it into qualitatively different T helper (Th) and regulatory (Treg) cell responses. A division of labour occurred between functionally distinct Treg that were coordinately activated by a CD28/B.7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation was controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of interleukin (IL10) and cytotoxic T lymphocyte antigen 4 (CTLA4) on indoleamine 2,3-dioxygenase (IDO). The levels of IFNgamma produced in this early phase set the subsequent adaptive stage by conditioning the IDO-dependent tolerogenic program of DCs and the subsequent activation and expansion of tolerogenic Treg, which produced IL10 and transforming growth factor (TGF)beta, inhibited Th2 cells, and prevented allergy to the fungus. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defence without necessarily eliminating the fungus or causing unacceptable levels of host damage. Efficient responses to the fungus require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it into qualitatively different T helper (Th) and regulatory (Treg) cell responses. A division of labour occurred between functionally distinct Treg that were coordinately activated by a CD28/B.7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation was controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of interleukin (IL10) and cytotoxic T lymphocyte antigen 4 (CTLA4) on indoleamine 2,3-dioxygenase (IDO). The levels of IFNgamma produced in this early phase set the subsequent adaptive stage by conditioning the IDO-dependent tolerogenic program of DCs and the subsequent activation and expansion of tolerogenic Treg, which produced IL10 and transforming growth factor (TGF)beta, inhibited Th2 cells, and prevented allergy to the fungus. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.

In the last years, the clinical relevance of fungal diseases has gained importance because of an increasing population of immunocompromised hosts, such as patients who have undergone transplants, patients with various types of leukemia, and people infected with HIV. Although some virulence factors are of obvious importance, pathogenicity cannot be considered an inherent characteristic of fungi.1 Fungi seem to have a complex relationship with the vertebrate immune system, mainly due to some prominent features: among these, the ability of dimorphic fungi to exist in different forms and to reversibly switch from one to the other in infection. Although association between morphogenesis and virulence has long been presumed for fungi that are human pathogens2, no molecular data unambiguously establish a role for fungal morphogenesis as a virulence factor. What fungal morphogenesis implicates through antigenic variability, phenotypic switching, and dimorphic transition is the existence of a multitude of recognition and effector mechanisms to oppose fungal infectivity at the different body sites.

Efficient responses to fungi require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it into qualitatively different T helper (Th) immune responses. Murine and human DCs phagocytose conidia and hyphae of Aspergillus fumigatus through distinct recognition receptors. The engagement of distinct receptors translates into disparate downstream signaling events, ultimately affecting cytokine production and co-stimulation. Adoptive transfer of different types of DCs activates protective and non-protective Th cells as well as regulatory T cells, ultimately affecting the outcome of the infection in mice with invasive aspergillosis. The infusion of fungus-pulsed or RNA-transfected DCs also accelerates recovery of functional antifungal Th 1 responses in mice with allogeneic hematopoietic stem cell transplantation. Patients receiving T cell-depleted allogeneic hematopoietic stem cell transplantation are unable to develop antigen-specific T cell responses soon after transplant due to defective DC functions. Our results suggest that the adoptive transfer of DCs may restore immunocompetence in hematopoietic stem cell transplantation by contributing to the educational program of T cells. Thus, the remarkable furictional plasticity of DCs can be exploited for the deliberate targeting of cells and pathways of cell-mediated immunity in response to the fungus.

DAP12 is an immunoreceptor tyrosine-based activation motif-bearing membrane adapter molecule expressed by different cell types. Although several receptors associate with DAP12 in murine dendritic cells (DC), the function of these receptors is as yet unknown. Here we report that splenic mature DC with DAP12 overexpression are characterized by an impaired tolerogenic potential. In contrast, inhibition of DAP12 function results in enhanced tolerogenesis and constitutive expression of immunosuppressive tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO). Increased resistance to experimental encephalomyelitis is observed in DAP12 knockin mice, which is dependent on IDO expression. Therefore, DAP12-related receptors act as negative regulators of IDO-mediated tolerance in vivo.

The collectin pentraxin 3 (PTX3) is an essential component of host resistance to pulmonary aspergillosis. Here we examined the protective effects of administration of PTX3 alone or together with deoxycholate amphotericin B (Fungizone) or liposomal amphotericin B (AmBisome) against invasive aspergillosis in a murine model of allogeneic bone marrow transplantation. PTX3, alone or in combination with the polyenes, was given intranasally or parenterally either before, in concomitance with, or after the intranasal infection with Aspergillus fumigatus conidia. Mice were monitored for resistance to infection and parameters of innate and adaptive T-helper immunity. The results showed the following: (i) complete resistance to infection and reinfection was observed in mice treated with PTX3 alone; (ii) the protective effect of PTX3 was similar or superior to that observed with liposomal amphotericin B or deoxycholate amphotericin B, respectively; (iii) protection was associated with accelerated recovery of lung phagocytic cells and T-helper-1 lymphocytes and concomitant decrease of inflammatory pathology; and (iv) PTX3 potentiated the therapeutic efficacy of suboptimal doses of either antimycotic drug. Together, these data suggest the potential therapeutic use of PTX3 either alone or as an adjunctive therapy in A. fumigatus infections.

Dendritic cells (DCs) are uniquely able to initiate and control the immune response to fungi. DCs function at three levels in the manipulation of the immune response to these pathogens: 1) they mount an immediate or innate response to them, for example by producing inflammatory mediators upon capture and phagocytosis; 2) through these preceding innate functions, they decode the fungus-associated information and translate it in qualitatively different Th responses; 3) and they are key in containing and dampening inflammatory responses by tolerization through the induction of regulatory T cells (Treg). We demonstrated that both murine and human DCs sense fungi in a morphotype-specific manner, through the engagement of distinct recognition receptors ultimately affecting cytokine production and costimulation (Romani et al. Int Immunol. 2003). In this study we analyzed both myeloid and plasmacytoid murine and human DCs and we observed that they phagocytose fungi and undergo functional maturation in response to them. However, their activation program for cytokine production was different, being IL-12 mainly produced by myeloid DCs and IL-12, IL-10 and IFN-gamma mainly produced by plasmacytoid DCs. This resulted in a distinct ability for T-cell priming, being Th1, Th2 and Treg differently activated by the different DC subsets. The ability of fungus-pulsed DCs to prime for Th1, and Th2 cell activation upon adoptive transfer in vivo correlated with the occurrence of resistance and susceptibility to the infections, respectively. Antifungal protective immunity was also induced upon adoptive transfer of DCs transfected with fungal RNA (Bacci et al. J Immunol. 2002; Bozza et al. Blood. 2003). The efficacy was restricted to DCs transfected with RNA from yeasts or conidia but not with RNA from fungal hyphae. The effect was fungus-specific, as no cross-protection was observed upon adoptive transfer of DCs pulsed with either fungal species. The infusion of fungus-pulsed or RNA-transfected DCs accelerated the recovery of functional antifungal Th1 responses in mice with allogeneic hematopoietic stem cell transplantation (HSCT) without triggering graft-versus-host disease (GvHD) and affected the outcome of the infections. As the ability to respond to fungi was defective in peripheral DCs from patients with HSCT in terms of phagocytosis, cytokine production, and donor T-cell priming, our findings suggest that the adoptive transfer of DCs may restore immunocompetence in HSCT by contributing to the educational program of T-cells. Thus, the remarkable functional plasticity of DCs in response to fungi can be exploited for the deliberate targeting of cells and pathways of cell-mediated immunity in response to fungal vaccines.

Efficient responses to the different forms of fungi require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it in qualitatively different T helper (Th) immune responses, in vitro and in vivo. DCs sense fungi in a morphotype-specific manner, through the engagement of distinct recognition receptors ultimately affecting cytokine production and costimulation. Adoptive transfer of different types of DCs activates protective and non-protective Th cells as well as regulatory T cells and affects the outcome of the infections. DCs transfected with fungal RNA also restore antifungal resistance in hematopoietic transplantation. Thus, the remarkable functional plasticity of DCs in response to fungi can be exploited for the deliberate targeting of cells and pathways of cell-mediated immunity in response to fungal vaccines.

In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.

Dendritic cells (DCs) show a remarkable functional plasticity in the recognition of Aspergillus fumigatus and orchestrate the antifungal immune resistance in the lungs. Here, we show that thymosin α 1, a naturally occurring thymic peptide, induces functional maturation and interleukin-12 production by fungus-pulsed DCs through the p38 mitogen-activated protein kinase/nuclear factor (NF)-κB-dependent pathway. This occurs by signaling through the myeloid differentiation factor 88-dependent pathway, involving distinct Toll-like receptors. In vivo, the synthetic peptide activates T-helper (Th) cell 1-dependent antifungal immunity, accelerates myeloid cell recovery, and protects highly susceptible mice that received hematopoietic transplants from aspergillosis. By revealing the unexpected activity of an old molecule, our finding provides the rationale for its therapeutic utility and qualify the synthetic peptide as a candidate adjuvant promoting the coordinated activation of the innate and adaptive Th immunity to the fungus. (Blood. 2004;103: 4232-4239)

Dendritic cells (DCs) have a remarkable functional plasticity in response to conidia and hyphae of the fungus Aspergillus fumigatus. In the present study we sought to assess the capacity of DCs activated by live fungi or fungal RNA to generate antifungal immunity in vivo. We found that both human and murine DCs pulsed with live fungi or transfected with fungal RNA underwent functional maturation, as revealed by the up-regulated expression of histocompatibility class II antigen and costimulatory molecules and the production of interleukin 12 (IL-12) in response to conidia or conidial RNA and of IL-4/IL-10 in response to hyphae or hyphal RNA. DCs pulsed with conidia or transfected with conidial RNA activated antigen-specific, interferon γ (IFN-γ)-producing T lymphocytes in vitro and in vivo on adoptive transfer in mice otherwise susceptible to aspergillosis. TH1-dependent antifungal resistance could also be induced in mice receiving allogeneic bone marrow transplants and was associated with an accelerated recovery of myeloid and lymphoid cells. Because the efficacy of the infusion of DCs was superior to that obtained on the adoptive transfer of Aspergillus-specific T cells, these results indicate the vaccinating potential of DCs pulsed with Aspergillus conidia or conidial RNA in hematopoietic transplantation. (Blood. 2003;102:3807-3814)

BACKGROUND: We focused on the phenotype of non-mineralizing MG 63 and mineralizing TE 85 human osteosarcoma cells and investigated the role of bFGF in modulating their differentiative responses. Basic FGF expression and bFGF effects on osteocalcin, runt-related transcription factor-2 (RUNX2), matrix molecular production and bFGF receptors, were evaluated. MATERIALS AND METHODS: Osteocalcin and RUNX2 gene expression were studied by RT-PCR analysis. We evaluated cell proliferation by DNA content and performed differentiation studies on glycosaminoglican (GAG), collagen and proteoglican (PG) synthesis by using radiolabelled precursors and Northern blotting. BFGF receptors were quantified by bFGF receptor binding assay. RESULTS: Osteocalcin is expressed in MG63 and TE65. RUNX2 RNA is differentially spliced in the two cell lines. BFGF elicits the effects of differentially splicing RUNX2. Proliferation, GAG synthesis, bFGF and proteoglycan mRNA expression, high and low affinity bFGF receptors, were more marked in MG 63 and differently affected by bFGF. Procollagen expression and alkaline phosphatase activity were significantly reduced. BFGF increased TE 85 cell proliferation and reduced TE 85 procollagen and osteocalcin production. CONCLUSIONS: The different splice variants in RUNX2 gene in the two cell lines might be related to their different phenotypes. The less differentiated stage of MG63 could also be related to bFGF over-production and more bFGF receptors. The consequent increase in bFGF-bFGF receptor binding could explain the bFGF differentiative effects on MG 63. We suggest an autocrine role of bFGF endogenous release in controlling the different osteosarcoma phenotypes.

To determine how toremifene, an anti-oestrogen triphenylethylene derivate, reduces tumour mass, we investigated its modulation of TGF-beta1 and TNF-alpha in fibroma fibroblasts. Normal and fibroma fibroblasts, isolated from patients affected by Gardner's syndrome without or with fibroma manifestation, were cultured in vitro. Secretion of GAG, collagen and TGF-beta1 was increased in fibroma fibroblasts compared to healthy cells. The increase in TGF-beta1 secretion into the medium was associated with a parallel increase in TGF-beta1 gene expression and receptor number. Receptor cross-linking studies using radiolabelled TGF-beta1 revealed more receptors, particularly types I and II, in fibroma fibroblasts than in normal cells. Normal and fibroma fibroblasts did not synthesise TNF-alpha, but they had TNF-alpha membrane receptors, as shown by TNF-alpha assay. TNF-alpha secreted by human monocytes, which may be present in the peritumoral area, increased cell proliferation and GAG accumulation and was, in turn, enhanced by TGF-beta1 treatment. Both growth factors increased angiogenesis, as shown by the CAM assay. Toremifene reduced TGF-beta1 secretion by fibroma fibroblasts and TNF-alpha secretion by monocytes, thus downregulating cell proliferation, ECM macromolecule accumulation and angiogenic progression. We hypothesise that increased TGF-beta1 gene expression and TGF-beta1 secretion in fibroma fibroblasts as well as the subsequent rise in TNF-alpha production by monocytes may facilitate fibroma growth and that toremifene inhibits autocrine and paracrine growth factor production.

The present study provides evidence that the in vitro cultured fibroblast cell line from desmoid tumors differs from normal fibrobasts in its extracellular matrix (ECM) macromolecule composition and is modulated by treatment with toremifene, an antiestrogen that reduces tumor mass by an unknown mechanism. The results showed increased transforming growth factor-beta 1 (TGF-beta1) production, TGF-beta1 mRNA expression, and TGF-beta1 receptor number in desmoid fibroblasts compared with normal cells. As desmoid fibroblasts did not produce tumor necrosis factor-alpha (TNF-alpha) but were sensitive to it, which enhanced glycosaminoglycans (GAG) accumulation, we assessed the TGF-beta1 effects on TNF-alpha production by human monocytes. Our results showed TGF-beta1 significantly increased TNF-alpha secretion by monocytes. Toremifene mediated its effects in desmoid fibroblasts via an estrogen receptor-independent pathway. It inhibited GAG accumulation and the secretion of both latent and active forms of TGF-beta1 and had an inhibitory effect on TNF-alpha production by monocytes. Our results suggest that in reducing TGF-beta1 production by desmoid fibroblasts and TNF-alpha production by monocytes, toremifene may restore the balance between the two growth factors.

The objective of this study was to evaluate the effects of an orthodontic appliance and of its components (brackets, bands, and arch wires) on some cell functions. Fibroblasts were cultured either in the presence of one unwashed orthodontic appliance, or one orthodontic appliance immersed in MEM for 28 days before use (washed appliance), or in the presence of MEM in which the appliances had been immersed. At the end of in vitro maintenance, morphological studies were carried out with SEM and TEM. Cell proliferation and GAG synthesis and secretion by radio-labeled precursors were assessed. The data indicated that unwashed appliances were more cytotoxic than washed ones. Moreover, the arch wire was the most biocompatible component of the orthodontic appliance, and the bracket was the least biocompatible. A comparative study into the effects on cell proliferation of the most common metal ions released by the appliances was also carried out. At the concentration released by one orthodontic appliance immersed for 28 days, the highest reduction in DNA synthesis was observed in the presence of Cu++. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 53: 560–567, 2000

Glycosaminoglycans (GAG), are extracellular matrix macromolecules that affect the phagocytic properties of macrophages. In order to assess whether the interaction between macrophages and Candida albicans (iCa) provokes changes in the phenotype, we analyzed the GAG profiles in two macrophage lines, ANA-1 (from murine bone-marrow) and BV-2 (from murine brain). We also investigated GAG modulation by interleukin-1α (IL-1α) and interleukin-6 (IL-6). During iCa treatment and even after the addition of ILs, ANA-1 accumulated less total GAG compared to controls. IL-1 treatment, combined with iCa exposure, induced a decrease in heparan sulfate and chondroitin sulfate chains, and an increase in the hyaluronic acid percentage. IL-6 treatment, with or without iCa, decreased the hyaluronic acid/sulfated GAG ratio. The GAG pattern in BV-2 appears to be different to ANA-1 and iCa exposure does not induce any difference in total GAG. The inhibitory effect induced by ILs on GAG synthesis is less than that observed in ANA-1 and the GAG elution profile is modulated to a lesser extent by treatment with ILs and/or iCa compared to the ANA-1. We suggest that the observed changes in the expression of the individual GAG classes may be responsible for the macrophage functional heterogeneity. J. Leukoc. Biol. 64: 650–656; 1998.

By mediating tryptophan catabolism, the enzyme indoleamine 2,3-dioxygenase (IDO) has a complex role in immunoregulation in infection, pregnancy, autoimmunity, transplantation, and neoplasia. We hypothesized that IDO might affect the outcome of the infection in mice infected with Candida albicans by virtue of its potent regulatory effects on inflammatory and T cell responses. IDO expression was examined in mice challenged with the fungus along with the consequences of its blockade by in vivo treatment with an enzyme inhibitor. We found that IDO activity was induced at sites of infection as well as in dendritic cells and effector neutrophils via IFN-γ- and CTLA-4-dependent mechanisms. IDO inhibition greatly exacerbated infection and associated inflammatory pathology as a result of deregulated innate and adaptive/regulatory immune responses. However, a role for tryptophan catabolism was also demonstrated in a fungus-autonomous fashion; its blockade in vitro promoted yeast-to-hyphal transition. These results provide novel mechanistic insights into complex events that, occurring at the fungus/pathogen interface, relate to the dynamics of host adaptation to the fungus. The production of IFN-γ may be squarely placed at this interface, where IDO activation probably exerts a fine control over fungal morphology as well as inflammatory and adaptive antifungal responses.

BACKGROUND: Previous studies show that macrophages, lung fibroblasts, and their soluble mediators are responsible for the onset and development of pulmonary fibrosis. This study was conducted to determine whether airway epithelial cells are also directly involved in response to fibrogenic agents and consequently in the pathogenesis of lung fibrosis. To verify the hypothesis, we determined whether silica acts directly on human bronchial epithelial cells by stimulating cytokine and growth factor release and by modifying matrix production. MATERIALS AND METHODS: An SV40 large T antigen-transformed human airway epithelial cell line, 16HBE14o (16HBE), was used. The expression profile of some proinflammatory interleukins (ILs), such as IL-1alpha, IL-1beta and IL-6 and their modulation by silica, were evaluated by polymerase chain reaction (PCR) analysis. Transforming growth factor beta (TGFbeta) and basic fibroblast growth factor (bFGF) mRNA levels were tested by Northern blotting in the presence and in the absence of silica. The silica- and/or bFGF-induced effects on matrix components (total proteins, collagen, and fibronectin) were also evaluated using radio-labeled precursors. RESULTS: The results demonstrated 16HBE internalized silica particles. Silica induced a little IL-6 secretion, without affecting IL-1 and TGFbeta isoform production and strongly stimulated bFGF mRNA level and bFGF protein secretion. Silica also induced changes in 16HBE production of total proteins, collagen, and fibronectin production. When added in combination with the growth factor, it strengthened bFGF stimulation of matrix component secretion. CONCLUSIONS: These results support the hypothesis that the changes in matrix components are due to a direct effect of silica on bronchial epithelial cells. Silica-induced over-secretion of bFGF suggests that autocrine and paracrine differentiation loops for bFGF may also be operative and that these mechanisms may be involved in the pathogenesis of pulmonary fibrosis. In the future, cytokine-directed therapeutic strategies might find a place in clinical practice.

Background Silicosis, a pneumoconiosis marked by interstitial pulmonary fibrosis, is caused by inhalation of free crystalline silica particles. When silica particles are injected into the lower lung, they are translocated across the epithelium into the interstitial space, where macrophage-derived growth factors affect lung fibroblast proliferation and collagen deposition. We hypothesized that silica may act directly on pulmonary fibroblasts modifying extracellular matrix (ECM) synthesis and that the effects of silica may be mediated by transforming growth factor-β (TGFβ) overproduction. Methods To test this hypothesis, we studied a human lung fibroblast cell line (WI-1003) exposed to silica in vitro. We investigated cell morphology by electron microscopic procedure, cell growth, collagen production, and glycosaminoglycans (GAG) composition by radiolabeled precursors. Cytokine and growth factor synthesis were evaluated by specific enzyme-linked immunoadsorbent assay kits and Northern blotting analysis. Results Pulmonary fibroblasts internalized silica particles without detectable cell damage. Silica directly stimulated collagen synthesis and decreased the amount of 3H-glucosamine-labeled GAG. Silica-treated fibroblasts secreted less TGFβ than untreated controls, antagonized the stimulatory effect of TGFβ on ECM synthesis, and reversed TGFβ-induced inhibition of cell proliferation. Northern blotting analysis showed increased interleukin-1α (IL-1α) mRNA after silica treatment. IL-1α had no influence on collagen synthesis but increased the number of WI-1003 fibroblasts. Conclusions These results support our hypothesis that lung fibroblasts are direct silica targets. However, contradicting our hypothesis, silica antagonized TGFβ activities through a TGFβ downregulation and an IL-1α upregulation. The complex pattern of TGFβ and IL-1α regulation in pulmonary fibroblasts is imbalanced by silica exposure and might play a key role in silica-mediated pulmonary fibrosis.