FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.     

Structural elucidation of the O-antigenic polysaccharide from enterohemorrhagic (EHEC) Escherichia coli O48:H21

The structure of the antigenic O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) produced by the enterohemorrhagic strain of Escherichia coli O48:H21 (EHEC) has been elucidated. The O-PS obtained by mild acid hydrolysis of the LPS had [alpha]D +95 (water) and was composed of L-rhamnose (L-Rha), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN), and D-galacturonic acid (D-GalA) (1:1:1:1:1). From the results of methylation analysis, mass spectrometry, 2D NMR, and DOC-PAGE, the O-PS was shown to be a high molecular mass polymer of a repeating pentasaccharide unit having the structure: [structure: see text]. The D-Gal pA non-reducing end groups in the O-PS were partially O-acetylated (approximately 30%) at the O-2 and O-3 positions and the degree of acetylation was variable from batch to batch cell production.     

Nematode and macrofaunal diversity in central Arctic Ocean benthos

Deep-sea diversity studies have revealed intriguing patterns on both local and regional scales, but there is insufficient evidence with which to evaluate these trends in the Arctic Ocean basin. We collected data on the diversity of benthic macrofauna and meiofaunal nematodes along two transects from the shelf margin to the North Pole. Contrary to prevailing paradigms, there was no change in diversity with depth between 1000 and 4273 m. There was a trend, however, toward reduced taxonomic richness for both macrofauna and nematodes with increasing latitude. Regional (β-) diversity differences were not observed for nematodes, but significant contrasts in Bray-Curtis similarity-based community structure of macrofauna were seen between the Eurasian and Amerasian Basins, as well as between the Lomonosov and Mendeleev Ridges. Since fauna within the deep Arctic Ocean appear to represent a single species pool, we suggest that both local (α-) and β-diversity may be determined by ecological processes in the Arctic, and are not the consequence of historical or evolutionary processes. Furthermore, insights gained from studies of benthic-pelagic coupling, known to play a significant role in determining benthic community structure and function at high latitudes, may also be useful in investigations of Arctic biodiversity. This model may be valuable in designing future studies of biodiversity, and for predicting potential impacts of climate change on diversity patterns.     

Similarity in Several Properties of Psychrophilic Bacteria Grown at Low and Moderate Temperatures

Several properties of psychrophilic pseudomonads were studied with cells grown in batch culture in nutrient broth at 2 and 30 C. No differences were observed in the size, catalase activity, deoxyribonucleic acid, ribonucleic acid, or protein content of cells grown at either temperature. The importance of comparing physiologically similar cells is discussed.     

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales,Rhodophyta) from East Asia

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.     

Autophagy Modulates Articular Cartilage Vesicle Formation in Primary Articular Chondrocytes

Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair.