The CC chemokine CCL21 is a potent chemoattractant for lymphocytes and dendritic cells in vitro. In the murine genome there are multiple copies of CCL21 encoding two CCL21 proteins that differ from each other by one amino acid at position 65 (either a serine or leucine residue). In this report, we examine the expression pattern and biological activities of both forms of CCL21. We found that although both serine and leucine forms are expressed in most tissues examined, the former was the predominant form in lymphoid organs while the latter was predominantly expressed in nonlymphoid organs. When expressed in transgenic pancreas, both forms of CCL21 were capable of inducing the formation of lymph node-like structures composed primarily of T and B cells and a few dendritic cells. Induction of lymph node-like structures by these CCL21 proteins, however, could not be reproduced in every tissue. For instance, no lymphocyte recruitment or accumulation was observed when CCL21 was overexpressed in the skin. We conclude that both forms of CCL21 protein are biologically equivalent in promoting lymphocyte recruitment to the pancreas, and that their ability to induce the formation of lymph node-like structures is dependent on the tissues in which they are expressed.

BackgroundObesity and inflammation are highly integrated processes in the pathogenesis of insulin resistance, diabetes, dyslipidemia, and non-alcoholic fatty liver disease. Molecular mechanisms underlying inflammatory events during high fat diet-induced obesity are poorly defined in mouse models of obesity. This work investigated gene activation signals integral to the temporal development of obesity.MethodsGene expression analysis in multiple organs from obese mice was done with Taqman Low Density Array (TLDA) using a panel of 92 genes representing cell markers, cytokines, chemokines, metabolic, and activation genes. Mice were monitored for systemic changes characteristic of the disease, including hyperinsulinemia, body weight, and liver enzymes. Liver steatosis and fibrosis as well as cellular infiltrates in liver and adipose tissues were analyzed by histology and immunohistochemistry.ResultsObese C57BL/6 mice were fed with high fat and cholesterol diet (HFC) for 6, 16 and 26 weeks. Here we report that the mRNA levels of macrophage and inflammation associated genes were strongly upregulated at different time points in adipose tissues (6-16 weeks) and liver (16-26 weeks), after the start of HFC feeding. CD11b+ and CD11c+ macrophages highly infiltrated HFC liver at 16 and 26 weeks. We found clear evidence that signals for IL-1β, IL1RN, TNF-α and TGFβ-1 are present in both adipose and liver tissues and that these are linked to the development of inflammation and insulin resistance in the HFC-fed mice.ConclusionsMacrophage infiltration accompanied by severe inflammation and metabolic changes occurred in both adipose and liver tissues with a temporal shift in these signals depending upon the duration of HFC feeding. The evidences of gene expression profile, elevated serum alanine aminotransferase, and histological data support a progression towards nonalcoholic fatty liver disease and steatohepatitis in these HFC-fed mice within the time frame of 26 weeks.

We developed transgenic mice conditionally expressing the neutrophil chemoattracting chemokine KC and the β-galactosidase gene in multiple tissues. In these transgenic mice, doxycycline treatment induced a strong up-regulation in the expression of KC in several tissues, including heart, liver, kidney, skin, and skeletal muscle. Expression of KC within these tissues led to a rapid and substantial increase in the serum levels of KC (serum KC levels were higher than 200 ng/ml 24 h after treatment). Accordingly, β-galactosidase expression was also detected after injection of doxycycline and was highest in skeletal muscle, pancreas, and liver. Surprisingly, despite expression of KC in multiple tissues, no neutrophil infiltration was observed in any of the tissues examined, including skin. Doxycycline treatment of nontransgenic mice grafted with transgenic skin caused dense neutrophilic infiltration of the grafts, but not the surrounding host skin, indicating that the KC produced in transgenic tissues was biologically active. In separate experiments, neutrophil migration toward a localized source of recombinant KC was impaired in animals overexpressing KC but was normal in response to other neutrophil chemoattractants. Analysis of transgenic neutrophils revealed that high concentrations of KC in transgenic blood had no influence on L-selectin cell surface expression but caused desensitization of the receptor for KC, CXCR2. These results confirm the neutrophil chemoattractant properties of KC and provide a mechanistic explanation for the paradoxical lack of leukocyte infiltration observed in the presence of elevated concentrations of this chemokine.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

Background: The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738. Results: In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collageninduced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC 50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC 90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment.