Phagocytosis and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages

Aspergillus fumigatus is the most prevalent airborne fungal pathogen responsible for fatal invasive aspergillosis in immunocompromised patients. Upon arrival in the lung alveolus, conidia of A. fumigatus are phagocytosed by alveolar macrophages, the major phagocytic cells of the lung. Engulfment and intracellular trafficking of A. fumigatus conidia in alveolar macrophages of two different origins, the murine cell line MH-S and human pulmonary alveolar macrophages, were analyzed by electron microscopy and immunofluorescence. Phagocytosis of A. fumigatus conidia required actin polymerization and phosphatidylinositol 3-kinase activity. Fusion of A. fumigatus phagosomes with early and late endosomes was shown by immunolabeling with specific markers for the transferrin receptor, early endosome antigen, and Rab7. Maturation of A. fumigatus phagolysosomes was monitored by using a fixable acidotropic probe, LysoTracker Red DND-99, and an anti-cathepsin D antibody. Bafilomycin A-induced inhibition of lysosomal acidification abolished the conidial killing by the macrophages. These data suggest that the maturation of A. fumigatus phagosomes results from fusion with the compartments of the endocytic pathway and that the killing of conidia depends on phagolysosome acidification. A model for the phagocytosis of A. fumigatus conidia by alveolar macrophages is proposed on the basis of these results.     

In vivo autofluorescence imaging of early cancers in the human tracheobronchial tree with a spectrally optimized system

The changes in the autofluorescence characteristics of the bronchial tissue is of crucial interest as a cancer diagnostic tool. Evidence exists that this native fluorescence or autofluorescence of bronchial tissues changes when they turn dysplastic and to carcinoma in situ. There is good agreement that the lesions display a decrease of autofluorescence in the green region of the spectrum under illumination with violet-light, and a relative increase in the red region of the spectrum is often reported. Imaging devices rely on this principle to detect early cancerous lesions in the bronchi. Based on a spectroscopic study, an industrial imaging prototype is developed to detect early cancerous lesions in collaboration with the firm Richard Wolf Endoskope GmbH, Germany. A preliminary clinical trial involving 20 patients with this spectrally optimized system shows that the autofluorescence can help to detect most lesions that would otherwise have remained invisible to an experienced endoscopist under white light illumination. A systematic off line analysis of the autofluorescence images pointed out that real-time decisional functions can be defined to reduce the number of false positive results. Using this method, a positive predictive value (PPV) of 75% is reached using autofluorescence only. Moreover, a PPV of 100% is obtained, when combining the white light (WL) mode and the autofluorescence (AF) mode, at the applied conditions. Furthermore, the sensitivity is estimated to be twice higher in the AF mode than in WL mode.     

Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.     

Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells

Exosomes are small vesicles (60-100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen-presenting cells (APC), such as B lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC-derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of specific T cells by MHC class II-bearing exosomes in vitro. The rat mast cell line, RBL-2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide [HLA-DR1-hemagglutinin (HA)]. These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross-linking. Exosomes bearing DR1-HA(306-318) complexes activated HA/DR1-specific T cells only weakly, whereas the cross-linking of such exosomes to latex beads increased stimulation of specific T cells. By contrast, the incubation of free exosomes with DC resulted in the highly efficient stimulation of specific T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for efficient T cell activation.     

Neutralizing antibodies in gene-defective hosts

In a host with a normal immune system and a complete gene defect, the nondefective gene product will be immunogenic. Consequently, neutralizing antibodies against the respective protein can arise either 'spontaneously' or after immunization, as shown in patients and in animal models, such as knockout mice. Accordingly, patients with X-linked or homozygous autosomal gene defects are at risk of developing neutralizing antibodies, in particular after protein substitution or gene therapy. This Review compares and exemplifies the various genetic and immunological contexts that lead to 'neutralizing and generated by gene defect' or 'nagged' antibodies, and outlines implications and solutions for therapeutic strategies.     

The UTX gene escapes X inactivation in mice and humans

We recently have identified a ubiquitously transcribed mouse Y chromosome gene, Uty , which encodes a tetratricopeptide repeat (TPR) protein. A peptide derived from the UTY protein confers H-Y antigenicity on male cells. Here we report the characterization of a widely transcribed X-linked homologue of Uty , called Utx , which maps to the proximal region of the mouse X chromosome and which detects a human X-linked homologue at Xp11.2. Given that Uty is ubiquitously transcribed, we assayed for Utx expression from the inactive X chromosome (Xi) in mice and found that Utx escapes X chromosome inactivation. Only Smcx and the pseudoautosomal Sts gene on the mouse X chromosome have been reported previously to escape inactivation. The human UTX gene was also found to be expressed from Xi. We discuss the significance of these data for our understanding of dosage compensation of X-Y homologous genes in humans and mice.      

Maternal immunoglobulins have no effect on the rate of maturation of the B cell compartment of the offspring

To elucidate a general role of maternal immunoglobulins (Ig) on the kinetics of B cell development of the offspring, we studied non-genetic influences of maternal Ig on the developing immune system of B cell-competent mice. These animals were the offsprings of either B cell-deprived μMT or of normal C57BL/6 females. In these mice, we have compared the kinetics of Ig production, the numbers of B cell progenitors, the expression of surface markers specific of the B lineage and the progression of Ig variable gene expression. We show that the absence of maternal Ig has no detectable effect on the kinetics of IgM and IgG production by the offspring's immune system. The number of B cell precursors, the kinetics of generation of B cells and their pattern of surface markers expression is identical in both types of mice. The acquisition of diversity in the B cell repertoire and the changes in the ratios of variable gene family expression are also indistinguishable. We conclude that maternally derived Ig has no influence on the rate of development and maturation of the B cell compartment of the offspring.