Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel

Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.     

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.     

IL-28 elicits antitumor responses against murine fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-gamma production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-gamma and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-gamma production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-gamma. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.     

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.     

IL-1α/IL-1R1 expression in chronic obstructive pulmonary disease and mechanistic relevance to smoke-induced neutrophilia in mice

Background

Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood.

Methodology and Principal Findings

The objective of this study was to assess IL-1 α and β expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and β. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and β levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1β- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation.

Conclusions and Significance

This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD.     

Local IL-17 Production Exerts a Protective Role in Murine Experimental Glomerulonephritis

IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of glomerulonephritis and IL-17 deficient mice are protected from nephrotoxic nephritis. However, a regulatory role for IL-17 has recently emerged. We describe a novel protective function for IL-17 in the kidney. Bone marrow chimeras were created using wild-type and IL-17 deficient mice and nephrotoxic nephritis was induced. IL-17 deficient hosts transplanted with wild-type bone marrow had worse disease by all indices compared to wild-type to wild-type bone marrow transplants (serum urea p<0.05; glomerular thrombosis p<0.05; tubular damage p<0.01), suggesting that in wild-type mice, IL-17 production by renal cells resistant to radiation is protective. IL-17 deficient mice transplanted with wild-type bone marrow also had a comparatively altered renal phenotype, with significant differences in renal cytokines (IL-10 p<0.01; IL-1β p<0.001; IL-23 p<0.01), and macrophage phenotype (expression of mannose receptor p<0.05; inducible nitric oxide synthase p<0.001). Finally we show that renal mast cells are resistant to radiation and produce IL-17, suggesting they are potential local mediators of disease protection. This is a novel role for intrinsic cells in the kidney that are radio-resistant and produce IL-17 to mediate protection in nephrotoxic nephritis. This has clinical significance as IL-17 blockade is being trialled as a therapeutic strategy in some autoimmune diseases.     

A novel phosphatidylcholine which contains pentadecanoic acid at sn-1 and docosahexaenoic acid at sn-2 in Schizochytrium sp. F26-b

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."