Increased Generation of TRAP Expressing Multinucleated Giant Cells in Patients with Granulomatosis with Polyangiitis

Background

Tissue-infiltrating multinucleated giant cells (MNGs) within geographic necrosis are pathologic hallmarks of granulomatosis with polyangiitis (GPA). However, the origin, phenotype, and function of these cells in GPA remain undefined.

Methodology/Principal Findings

MNG phenotype in GPA lung tissue was examined by immunohistochemistry using antibody directed against cathepsin K and calcitonin-receptor. Tartrate-resistant-acid-phosphatase (TRAP) expression was assessed using enzymatic color reaction. Peripheral blood mononuclear cells (PBMCs) from 13 GPA patients (5 with localized and 8 with systemic disease) and 11 healthy controls were cultured in the presence of RANKL and M-CSF for 9 days, and TRAP+ MNGs containing 3 or more nuclei were identified. GPA lung granulomata contained numerous MNGs that expressed osteoclastic TRAP and cathepsin K but not calcitonin receptors. In the presence of RANKL and M-CSF, PBMCs of GPA patients formed significantly more MNGs than healthy controls (114±29 MNG/well vs. 22±9 MNG/well, P = 0.02). In a subgroup analysis, patients with systemic disease generated significantly more MNGs than patients with localized disease (161±35 MNG/well vs. 39±27 MNG/well, P<0.01) or healthy controls (P<0.01). MNG production did not differ between localized GPA and control subjects (P = 0.96).

Conclusions/Significance

MNGs in granulomata in the GPA lung express osteoclastic enzymes TRAP and cathepsin K. GPA patients have a higher propensity to form TRAP+ MNGs from peripheral blood than healthy controls. These data suggest that (i) the tendency to form MNGs is a component of the GPA phenotype itself, and (ii) that lesional MNGs might participate in the destructive process through their proteolytic enzymes.     

Regulation of monocyte functional heterogeneity by miR-146a and Relb

Monocytes serve as a central defense system against infection and injury but can also promote pathological inflammatory responses. Considering the evidence that monocytes exist in at least two subsets committed to divergent functions, we investigated whether distinct factors regulate the balance between monocyte subset responses in vivo. We identified a microRNA (miRNA), miR-146a, which is differentially regulated both in mouse (Ly-6C(hi)/Ly-6C(lo)) and human (CD14(hi)/CD14(lo)CD16(+)) monocyte subsets. The single miRNA controlled the amplitude of the Ly-6C(hi) monocyte response during inflammatory challenge whereas it did not affect Ly-6C(lo) cells. miR-146a-mediated regulation was cell-intrinsic and depended on Relb, a member of the noncanonical NF-κB/Rel family, which we identified as a direct miR-146a target. These observations not only provide mechanistic insights into the molecular events that regulate responses mediated by committed monocyte precursor populations but also identify targets for manipulating Ly-6C(hi) monocyte responses while sparing Ly-6Clo monocyte activity.     

Conservation of Gene Cassettes among Diverse Viruses of the Human Gut

Viruses are a crucial component of the human microbiome, but large population sizes, high sequence diversity, and high frequencies of novel genes have hindered genomic analysis by high-throughput sequencing. Here we investigate approaches to metagenomic assembly to probe genome structure in a sample of 5.6 Gb of gut viral DNA sequence from six individuals. Tests showed that a new pipeline based on DeBruijn graph assembly yielded longer contigs that were able to recruit more reads than the equivalent non-optimized, single-pass approach. To characterize gene content, the database of viral RefSeq proteins was compared to the assembled viral contigs, generating a bipartite graph with functional cassettes linking together viral contigs, which revealed a high degree of connectivity between diverse genomes involving multiple genes of the same functional class. In a second step, open reading frames were grouped by their co-occurrence on contigs in a database-independent manner, revealing conserved cassettes of co-oriented ORFs. These methods reveal that free-living bacteriophages, while usually dissimilar at the nucleotide level, often have significant similarity at the level of encoded amino acid motifs, gene order, and gene orientation. These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/.     

The Aldosterone-Mineralocorticoid Receptor Pathway Exerts Anti-Inflammatory Effects in Endotoxin-Induced Uveitis

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.     

Actin- and dynamin-dependent maturation of bulk endocytosis restores neurotransmission following synaptic depletion

Bulk endocytosis contributes to the maintenance of neurotransmission at the amphibian neuromuscular junction by regenerating synaptic vesicles. How nerve terminals internalize adequate portions of the presynaptic membrane when bulk endocytosis is initiated before the end of a sustained stimulation is unknown. A maturation process, occurring at the end of the stimulation, is hypothesised to precisely restore the pools of synaptic vesicles. Using confocal time-lapse microscopy of FM1-43-labeled nerve terminals at the amphibian neuromuscular junction, we confirm that bulk endocytosis is initiated during a sustained tetanic stimulation and reveal that shortly after the end of the stimulation, nerve terminals undergo a maturation process. This includes a transient bulging of the plasma membrane, followed by the development of large intraterminal FM1-43-positive donut-like structures comprising large bulk membrane cisternae surrounded by recycling vesicles. The degree of bulging increased with stimulation frequency and the plasmalemma surface retrieved following the transient bulging correlated with the surface membrane internalized in bulk cisternae and recycling vesicles. Dyngo-4a, a potent dynamin inhibitor, did not block the initiation, but prevented the maturation of bulk endocytosis. In contrast, cytochalasin D, an inhibitor of actin polymerization, hindered both the initiation and maturation processes. Both inhibitors hampered the functional recovery of neurotransmission after synaptic depletion. Our data confirm that initiation of bulk endocytosis occurs during stimulation and demonstrates that a delayed maturation process controlled by actin and dynamin underpins the coupling between exocytosis and bulk endocytosis.     

Candida albicans-epithelial interactions: dissecting the roles of active penetration, induced endocytosis and host factors on the infection process

Candida albicans frequently causes superficial infections by invading and damaging epithelial cells, but may also cause systemic infections by penetrating through epithelial barriers. C. albicans is a remarkable pathogen because it can invade epithelial cells via two distinct mechanisms: induced endocytosis, analogous to facultative intracellular enteropathogenic bacteria, and active penetration, similar to plant pathogenic fungi. Here we investigated the contributions of the two invasion routes of C. albicans to epithelial invasion. Using selective cellular inhibition approaches and differential fluorescence microscopy, we demonstrate that induced endocytosis contributes considerably to the early time points of invasion, while active penetration represents the dominant epithelial invasion route. Although induced endocytosis depends mainly on Als3-E-cadherin interactions, we observed E-cadherin independent induced endocytosis. Finally, we provide evidence of a protective role for serum factors in oral infection: human serum strongly inhibited C. albicans adhesion to, invasion and damage of oral epithelial cells.     

HRP-2 determines HIV-1 integration site selection in LEDGF/p75 depleted cells

Background

The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here.

Findings

Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs).

Conclusions

These findings add TBC1D20 to the network of host factors regulating HIV replication cycle.     

A novel heart valve stent design: mechanical interaction with the aortic root

Percutaneous aortic valve implantation has become an alternative technique to surgical valve replacement in patients with high risk for open chest surgery. Vascular stents clinically used today for non-invasive aortic valve replacement tend, however, to impede the dimension changes of the compliant aortic root over the cardiac cycle. The purpose of the present work is to assess the influence of a novel heart valve stent, designed specifically to limit the traumatism in tissue, on the compliance of the aortic root. A theoretical approach is adopted to model the mechanical behaviour of the different stent parts and assess the compliance modification induced by the stent. The validity of the model is then tested experimentally. Both approaches show that the specific geometry of the stent makes it possible to keep the compliance of the aortic root close to the native root values.