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Monocytes play a major role in the cellular defence against Aspergillus fumigatus in immunocompromised patients. To obtain a better understanding of the mechanisms involved in this interaction, phagocytosis and gene expression profiling of human monocytes was carried out after incubation with A. fumigatus resting, swollen and germinating conidia and hyphae (for 3, 6 and 9 h). The majority of monocytes phagocytosed up to three conidia during the first 3 h of incubation. Microarray analysis showed an increased expression level of immune-relevant genes, which was dependent on the germination state of the fungus and the incubation period. Among these genes, those encoding interleukin-8, macrophage inflammatory protein 3-α (CCL20) and monocyte chemotactic protein-1 (CCL2) were found to be potential key regulators involved in the A. fumigatus-induced immune response. In addition, A. fumigatus was found to be an inducer of the genes encoding urokinase type plasminogen activator (uPA), urokinase type plasminogen activator receptor (uPAR),plasminogen activator inhibitor (PAI), pentraxin-3 (PTX3) and intercellular adhesion molecule-1 (ICAM-1), which, in combination, may contribute to thrombosis and local lung tissue injury.

Among all candidates tested, RPL13 was the best housekeeper for qRT-PCR studies in autopsy brain tissue samples from controls and AD cases. RNA quality should be assessed and data normalized on this index as well.

The glial transporter excitatory amino acid transporter-2 (EAAT2) is the main mediator of glutamate clearance in brain. The wild-type transporter (EAAT2wt) forms trimeric membrane complexes in which each protomer functions autonomously. Several EAAT2 variants are found in control and Alzheimer-diseased human brains; their expression increases with pathological severity. These variants might alter EAAT2wt-mediated transport by abrogating membrane trafficking, or by changing the configuration or functionality of the assembled transporter complex. HEK293 cells were transfected with EAAT2wt; EAAT2b, a C-terminal variant; or either of two exonskipping variants: alone or in combination. Surface biotinylation studies showed that only the exon-7 deletion variant was not trafficked to the membrane when transfected alone, and that all variants could reach the membrane when co-transfected with EAAT2wt. Fluorescence resonance energy transfer (FRET) studies showed that co-transfected EAAT2wt and EAAT2 splice variants were expressed in close proximity. Glutamate transporter function was measured using a whole cell patch clamp technique, or by changes in membrane potential indexed by a voltage-sensitive fluorescent dye (FMP assay): the two methods gave comparable results. Cells transfected with EAAT2wt or EAAT2b showed glutamate-dependent membrane potential changes consistent with functional expression. Cells transfected with EAAT2 exon-skipping variants alone gave no response to glutamate. Co-transfection of EAAT2wt (or EAAT2b) and splice variants in various ratios significantly raised glutamate EC 50 and decreased Hill coefficients. We conclude that exon-skipping variants form heteromeric complexes with EAAT2wt or EAAT2b that traffic to the membrane but show reduced glutamate-dependent activity. This could allow glutamate to accumulate extracellularly and promote excitotoxicity. Excitatory amino acid transporters (EAATs)6 are high-affinity, sodium-dependent glutamate carriers from solute carrier family 1 (SLC1). Five EAATs have been cloned from animal and human tissue. EAAT1 (SLC1A3; GLAST in rodents) and EAAT2 (SLC1A2; GLT1) are primarily expressed in astroglia (1). Overall, EAAT2 is responsible for most of the glutamate transport in the adult brain (1, 2).The amino acid sequence of an aspartate transporter from Pyrococcus horikoshii (Glt Ph ) is 36% homologous to EAAT2; many residues implicated in glutamate binding and transport are highly conserved (up to 90%) between all EAATs (3, 4). The crystal structure of Glt Ph is a trimer in which each protomer comprises eight ␣-helical transmembrane domains and two helical hairpins (5). It has been proposed that transmembrane domains, TM3, -6, -7, and -8, together with the two helical hairpins, HP1 and HP2, are essential for substrate translocation from the extracellular side into the cell. The first transmembrane segments, TM1, -2, -4, and -5, form the trimerization domain and provide stability to counterbalance the movements of the transport domains (3, 6, 7).In addition to EAAT...

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