Regulation of Inflammation by IL-17A and IL-17F Modulates Non-Alcoholic Fatty Liver Disease Pathogenesis

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. While it is well-accepted that inflammation is central to NAFLD pathogenesis, the immune pathway(s) orchestrating disease progression are poorly defined. Notably, IL-17RA signaling, via IL-17A, plays an important role in obesity-driven NAFLD pathogenesis. However, the role of the IL-17F, another IL-17RA ligand, in NAFLD pathogenesis has not been examined. Further, the cell types expressing IL-17RA and producing IL-17RA ligands in the pathogenesis of NAFLD have not been defined. Here, IL-17RA^-/-, IL-17A^-/-, IL-17F^-/- and wild-type (WT) mice were fed either standard chow diet or methionine and choline deficient diet (MCDD)—a diet known to induce steatosis and hepatic inflammation through beta-oxidation dysfunction—and hepatic inflammation and NAFLD progression were subsequently quantified. MCDD feeding augmented hepatic IL-17RA expression and significantly increased hepatic infiltration of macrophages and IL-17A and IL-17F producing CD4⁺ and CD8⁺ T cells in WT mice. In contrast, IL-17RA^-/-, IL-17A^-/-, and IL-17F^-/- mice, despite increased steatosis, exhibited significant protection from hepatocellular damage compared to WT controls. Protection from hepatocellular damage correlated with decreased levels of hepatic T-cell and macrophage infiltration and decreased expression of inflammatory mediators associated with NAFLD. In sum, our results indicate that the IL-17 axis also plays a role in a MCDD-induced model of NAFLD pathogenesis. Further, we show for the first time that IL-17F, and not only IL-17A, plays an important role in NAFLD driven inflammation.     

Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily.

The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes l-arginine to l-ornithine and urea. Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents. The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H. pylori infection. Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production. The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid-borne rocF abolished arginase activity. A translationally fused His(6)-RocF was purified from E. coli under nondenaturing conditions and had catalytic activity. Remarkably, the purified enzyme had an acidic pH optimum of 6.1. Both purified arginase and arginase-containing H. pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine. Viable H. pylori or E. coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal. His(6)-RocF arginase activity was inhibited by low concentrations of reducing agents. Antibodies raised to purified His(6)-RocF reacted with both H. pylori and E. coli extracts containing arginase, but not with extracts from rocF mutants of H. pylori or E. coli lacking the rocF gene. The results indicate that H. pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism.     

Poly (8-bromo-2′-deoxyadenylic acid): The syn/anti relationship

The synthesis of a high-molecular-weight, putatively all-syn DNA analogue, poly(8-bromo-2′-deoxyadenylic acid), is described. The syn → anti transition was shown to be both salt and temperature dependent. Conditions were found which favored ‘normal’ Watson-Crick pairing and duplex formation with poly(dT).     

Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells

Using a flow cytometry-based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.     

Fluid flow induced PGE2 release by bone cells is reduced by glycocalyx degradation whereas calcium signals are not

It has been hypothesized that bone cells have a hyaluronic acid (HA) rich glycocalyx (cell coat or pericellular matrix) and that this contributes to bone cell mechanotransduction via fluid flow. The glycocalyx of bone cells of the MC3T3-E1 osteoblastic cell line and the MLO-Y4 osteocytic cell line were characterized. Alcian blue staining and lectin binding experiments suggested that these cells have a glycocalyx rich in HA. Sulphated proteoglycans were not detected. Staining with hyaluronic acid binding protein and degradation by hyaluronidase confirmed that HA was a major component of the glycocalyx. We subjected cells, with and without hyaluronidase treatment, to oscillating fluid flow under standardized in vitro conditions. There was no effect of glycocalyx degradation on the intracellular calcium signal, in either cell type, in terms of the percentage of cells responding (40-80%) or the magnitude of the response (2-5 times baseline). However, a 4-fold fluid flow induced increase in PGE2 was eliminated by hyaluronidase pre-treatment in MLO-Y4 cells. We conclude that under these conditions the calcium and PGE2 responses occur via different pathways. An intact glycocalyx is not necessary in order to initiate a calcium signal in response to oscillating fluid flow. However, in osteocyte-like cells the PGE2 pathway is more dependent on mechanical signals transmitted through the glycocalyx.     

Cold Shock Lethality and Injury in Clostridium perfringens

Several observations have been made in regard to cold shock lethality of Clostridium perfringens: (i) loss of viability was not consequence of exposure of the cells to air; (ii) stationary-phase cells were much more resistant to cold shock at 4 C than exponential-phase cells; (iii) at 4 C 96% of an initial population of exponential-phase cells was killed upon cold shock and 95% of the remaining population was killed within 90 min of continued exposure at 4 C; (iv) the minimal temperature differential for detectable cold shock lethality was between 17 and 23 C, and the maximum beyond which lethality was not appreciably increased was between 28 and 33 C. Up to 75% of viable cold-shocked cells were injured, as demonstrated by cold shocking late exponential-phase cells at 10 C and using differential plating procedure for recovery. Repair of injury was temperature dependent, and occurred in a complex medium and 0.1% peptone but not water. Nalidixic acid, chloramphenicol, and rifampin did not inhibit repair of injury.