Hearing impairment as an early sign of alpha‐mannosidosis in children with a mild phenotype: Report of seven new cases

Alpha‐mannosidosis (AM) is a very rare (prevalence: 1/500000 births) autosomal recessive lysosomal storage disorder. It is characterized by multi‐systemic involvement associated with progressive intellectual disability, hearing loss, skeletal anomalies, and coarse facial features. The spectrum is wide, from very severe and lethal to a milder phenotype that usually progresses slowly. AM is caused by a deficiency of lysosomal alpha‐mannosidase. A diagnosis can be established by measuring the activity of lysosomal alpha‐mannosidase in leucocytes and screening for abnormal urinary excretion of mannose‐rich oligosaccharides. Genetic confirmation is obtained with the identification of MAN2B1 mutations. Enzyme replacement therapy (LAMZEDE^R) was approved for use in Europe in August 2018. Here, we describe seven individuals from four families, diagnosed at 3–23 years of age, and who were referred to a clinical geneticist for etiologic exploration of syndromic hearing loss, associated with moderate learning disabilities. Exome sequencing had been used to establish the molecular diagnosis in five cases, including a two‐sibling pair. In the remaining two patients, the diagnosis was obtained with screening of urinary oligosaccharides excretion and the association of deafness and hypotonia. These observations emphasize that the clinical diagnosis of AM can be challenging, and that it is likely an underdiagnosed rare cause of syndromic hearing loss. Exome sequencing can contribute significantly to the early diagnosis of these nonspecific mild phenotypes, with advantages for treatment and management.     

Detection of apple chlorotic leaf spot virus using a 5' nuclease assay with a fluorescent 3' minor groove binder-DNA probe

The development of a real-time 5' nuclease RT-PCR assay for the detection of apple chlorotic leaf spot virus (ACLSV) from infected plant material is described. A short fluorogenic 3' minor groove binder-DNA hydrolysis probe was used to circumvent genome variability between isolates and target a short conserved sequence. The covalent attachment of the minor groove binder moiety at the 3' end of the probe increased the probe/target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The method is rapid, sensitive and takes place within a single tube without post-PCR handling of the amplification products.     

Pituitary corticotroph adenoma with crooke’s hyalinization

The diagnosis of pituitary corticotroph adenoma relies on the demonstration of a loss of the normal feedback control of adrenocorticotropic hormone (ACTH) biosynthesis by cortisol. The marked variability in the degree of ACTH suppression by glucocorticoids in these tumors, however, greatly enhances the difficulty in distinguishing Cushing’s disease from other syndromes of glucocorticoid excess. To illustrate this variability, we describe the clinical, biochemical, and morphological characteristics of a pituitary corticotroph adenoma in a 63-year-old woman, who presented with symptoms of a sellar mass but did not initially have florid Cushing’s disease. Light and electron microscopy of the pituitary tumor showed a corticotroph adenoma with Crooke’s hyalinization of the tumor cells, characterized by the accumulation of keratin immunoreactive microfilaments similar to those observed in normal corticotrophs in the presence of excess glucocorticoids. This case illustrates an unusual clinical presentation that may be associated with pituitary corticotroph adenoma showing Crooke’s hyalinization.     

Congenital muscular dystrophy associated with lethal arthrogryposis multiplex congenita

Summary Two unrelated patients with severe arthrogryposis multiplex congenita (AMC) who died perinatally, are presented. In both, postmortem examination revealed an intact nervous system and striking dystrophic muscle changes, consistent with congenital muscular dystrophy (CMD). Few similar cases have been reported before, but since the condition is not well known, it seems probable that in the past many have been labeled as mere multiple malformations. The possibility of an underlying muscular disorder, either primary myopathic or neurogenic should be considered in any patient with early lethal AMC. Our findings confirm that the fetal akinesia-arthrogryposis sequence is a nonspecific clinical syndrome resulting from various causes of muscular inactivity in utero. The main objective of this report is to provide reasonable guidelines on how to approach the problem of classification. We favor a pathogenetic approach, depending upon careful sampling of the central nervous system and skeletal muscles at autopsy.     

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.