Xiulong Xu

A Novel Polymorphic CAAT/Enhancer-Binding Protein β Element in the FasL Gene Promoter Alters Fas Ligand Expression: A Candidate Background Gene in African American Systemic Lupus Erythematosus Patients

A single-nucleotide polymorphism (SNP), identified at nucleotide position −844 in the 5′ promoter of the FasL gene, lies within a putative binding motif for CAAT/enhancer-binding protein β (C/EBPβ). Electrophoretic mobility shift and supershift assays confirmed that this element binds specifically to C/EBPβ and demonstrated that the two alleles of this element have different affinities for C/EBPβ. In luciferase reporter assays, the −844C genotype had twice the basal activity of the −844T construct, and basal expression of Fas ligand (FasL) on peripheral blood fibrocytes was also significantly higher in −844C than in −844T homozygous donors. FasL is located on human chromosome 1q23, a region that shows linkage to the systemic lupus autoimmune phenotype. Analysis of 211 African American systemic lupus erythematosus patients revealed enrichment of the −844C homozygous genotype in these systemic lupus erythematosus patients compared with 150 ethnically matched normal controls (p = 0.024). The −844C homozygous genotype may lead to the increased expression of FasL, to altered FasL-mediated signaling in lymphocytes, and to enhanced risk for autoimmunity. This functionally significant SNP demonstrates the potential importance of SNPs in regulatory regions and suggests that differences in the regulation of FasL expression may contribute to the development of the autoimmune phenotype.

Two activities of the immunosuppressive metabolite of leflunomide, A77 1726: Inhibition of pyrimidine nucleotide synthesis and protein tyrosine phosphorylation☆

Previous studies have demonstrated that the active metabolite of leflunomide, A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], is capable of inhibiting the activities of tyrosine kinases and dihydroorotate dehydrogenase (DHO-DHase). In the present study, we define the relative contribution of these activities to the ability of A77 1726 to inhibit proliferation of the murine leukemia cell line LSTRA. A77 1726 inhibited LSTRA cell growth and proliferation (IC50 = 10-30 microM); this inhibition, however, could be reversed by the addition of exogenous uridine, suggesting that the anti-proliferative activity of A77 1726 may be due to inhibition of de novo pyrimidine nucleotide synthesis. Quantitation of nucleotide levels revealed that A77 1726, at an IC50 of about 10 microM, selectively inhibited pyrimidine nucleotide but not purine nucleotide synthesis. In vitro enzyme assays confirmed that A77 1726 directly inhibited the activity of DHO-DHase, the fourth enzyme in the de novo pathway of pyrimidine nucleotide synthesis (IC50 = 220 nM). LSTRA cells overexpress p56lck and have elevated levels of tyrosine phosphorylated intracellular proteins. A77 1726 reduced the intracellular levels of tyrosine phosphorylated proteins with relatively high IC50 values ranging from 50 to 100 microM. A77 1726 also inhibited p56lck activity in LSTRA membrane preparation and immunoprecipitates; the IC50 values for inhibition of immunoprecipitated p56lck autophosphorylation and exogenous substrate histone 2B were 80 and 40 microM, respectively. The anti-tyrosine phosphorylation activity of A77 1726 was not affected by uridine. These studies therefore demonstrate the two activities of A77 1726: inhibition of pyrimidine nucleotide synthesis and interference with tyrosine phosphorylation.

In Vivo Activity Of Leflunomide: Pharmacokinetic analyses and mechanism of immunosuppression

BACKGROUND Leflunomide is an experimental drug with demonstrated ability to prevent and reverse acute allograft and xenograft rejection. The two biochemical activities reported for the active metabolite of leflunomide, A77 1726, are inhibition of tyrosine phosphorylation and inhibition of dihydroorotate dehydrogenase, an enzyme necessary for de novo pyrimidine synthesis. These activities can be distinctly separated in vitro by the use of uridine, which reverses the anti-proliferative effects of A77 1726 caused by inhibition of de novo pyrimidine synthesis. We report the effect of uridine on the in vivo immunosuppressive activities of leflunomide. METHODS We first quantified the serum levels of A77 1726, the active metabolite of leflunomide, after a single treatment of leflunomide (5, 15, and 35 mg/kg). Additionally, we quantified the levels of serum uridine and of nucleotide triphosphates in the liver, spleen, and lymph nodes of Lewis rats after the administration of a single dose of uridine (500 mg/kg; i.p.). Lewis rats heterotopically transplanted with brown Norway or Golden Syrian hamster hearts were treated for 50 or 75 days with leflunomide (5, 15, and 35 mg/kg/day; gavage) alone or in combination with uridine (500 mg/ kg/day; i.p.). Hematocrits were determined and the levels of alloreactive or xenoreactive immunoglobulin (Ig)M and IgG were determined by flow cytometric analysis. The allograft and xenografts, small bowel, liver, kidney, and spleen were subjected to pathological examination. RESULTS A linear relationship was observed between the serum A77 1726 concentrations in Lewis rats and the dose of leflunomide administered. Peak A77 1726 concentrations were 20.9, 71.8 and 129.3 mg/l (77.5, 266.1 and 478.8 microM) for the 5, 15, and 35 mg/kg doses of leflunomide, respectively. The concentration of uridine in the serum of normal Lewis rats is 6.5 microM; after i.p. administration of 500 mg/kg uridine, the serum uridine concentrations peaked at 384.1 microM in 15-30 min. The rapid elimination of uridine was not reflected in the lymphoid compartments, and the pharmacokinetics of pyrimidine nucleotides in the spleen resembled that of A77 1726. This dose of uridine, when administered daily (500 mg/kg/day, i.p.), weakly antagonized the immunosuppressive activities of leflunomide (5, 15, and 35 mg/kg/day) in the allotransplantation model. In contrast, in the xenotransplantation model, the same concentration of uridine completely antagonized the immunosuppressive activities of low-dose leflunomide (15 mg/kg/day) and partially antagonized the immunosuppressive activities of high-dose leflunomide (35 mg/kg/day). Toxicities associated with high-dose leflunomide (35 mg/kg/day) were anemia, diarrhea, and pathological changes in the small bowel and liver. These toxicities were significantly reduced by uridine co-administration. CONCLUSION These studies reveal that the blood levels of A77 1726 in Lewis rats satisfy in vitro requirements for both inhibition of de novo pyrimidine synthesis and protein tyrosine kinase activity. Our data also illustrate that the in vivo mechanism of immunosuppression by leflunomide is complex and is affected by at least the following four factors: type and vigor of the immune response, availability of uridine for salvage by proliferating lymphocytes, species being investigated, and concentration of serum A77 1726.

Effects of leflunomide and other immunosuppressive agents on T cell proliferation in vitro.

Leflunomide and its active metabolite, A771726, are structurally unrelated to immunosuppressive agents currently under investigation. Previous in vitro studies have revealed that leflunomide primarily inhibits interleukin-2-stimulated T cell proliferation. In the current study, we have extended our previous work and demonstrate that leflunomide prevents T cell progression induced by phytohemagglutinin into the S phase of the cell cycle. To discriminate further the action on T cells of leflunomide from other immunosuppressive agents, we performed kinetic studies where leflunomide was added either after the initiation of mixed lymphocyte cultures (MLC) or after interleukin-2 stimulation of CTLL-4 cell proliferation. These studies revealed that leflunomide acted comparably to rapamycin, but was distinct from brequinar sodium in the MLC, and from cyclosporine and mycophenolic acid in both MLC and CTLL-4. Although previous biochemical studies indicated that leflunomide can inhibit src-family tyrosine kinase activity, more recent studies have suggested that leflunomide can also inhibit pyrimidine synthesis. Our data demonstrate that the ability of leflunomide (25-100 microM) to inhibit MLC and CTLL-4 cell proliferation is partially antagonized by uridine (25-100 microM), and support the hypothesis that leflunomide inhibits pyrimidine synthesis in T cells. Unique molecular mechanisms of immunosuppression suggest that drug combinations may result in synergistic immunosuppression. Our in vitro studies revealed synergistic inhibition of T cell proliferation with the combinations of leflunomide with cyclosporine or with rapamycin. We have extended those studies to quantitate inhibition of MLC by the combinations of leflunomide and brequinar sodium or mycophenolic acid.

In vitro and in vivo antitumor activity of a novel immunomodulatory drug, leflunomide : Mechanisms of action

Leflunomide, a novel immunomodulatory drug, has two biochemical activities: inhibition of tyrosine phosphorylation and inhibition of pyrimidine nucleotide synthesis. In the present study, we first showed that A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], the active metabolite of leflunomide, was more effective at inhibiting the tyrosine kinase activity of platelet-derived growth factor (PDGF) receptor than that of epidermal growth factor (EGF) receptor, and had no effect on the tyrosine kinase activity of the fibroblast growth factor receptor. In the presence of exogenous uridine, A77 1726 was more effective at inhibiting the PDGF-stimulated proliferation of PDGF receptor-overexpressing C6 glioma than the EGF-stimulated proliferation of EGF receptor-overexpressing A431 cells. In vivo studies demonstrated that leflunomide treatment strongly inhibited the growth of the C6 glioma but had only a modest effect on the growth of the A431 tumor. Uridine co-administered with leflunomide did not reverse the antitumor activity of leflunomide on C6 and A431 tumors significantly. Quantitation of nucleotide levels in the tumor tissue revealed that leflunomide treatment significantly reduced pyrimidine nucleotide levels in the fast-growing C6 glioma but had no effect on the relatively slow-growing A431 tumor. Whereas uridine co-administration normalized pyrimidine nucleotide levels, it had minimal effects on the antitumor activity of leflunomide in both tumor models. Immunohistochemical analysis revealed that leflunomide treatment significantly reduced the number of proliferating cell nuclear antigen-positive cells in C6 glioma, and that uridine only partially reversed this inhibition. These results collectively suggest that the in vivo antitumor effect of leflunomide is largely independent of its inhibitory effect on pyrimidine nucleotide synthesis. The possibility that leflunomide exerts its antitumor activity by inhibition of tyrosine phosphorylation or by a yet unidentified mode of action is discussed.

Abnormal Glomerular Permeability Characteristics in Diabetic Nephropathy Implications for the therapeutic use of low–molecular weight heparin

The physicochemical characteristics of the glomerular capillary filtration membrane restrict the passage of macromolecules on the basis of molecular weight, charge, and shape. The proposed ionic charge permselectivity characteristics of the glomerular basement membrane (GBM) are determined by its chemical composition, primarily the highly sulfated glycosaminoglycan heparan. In diabetic nephropathy, the heparan sulfate content of the GBM is diminished. It has been proposed that decreased GBM heparan sulfate content causes decreased permselectivity to negatively charged macromolecules such as albumin, allowing this protein to leak into the urinary space. One possible explanation for decreased GBM heparan sulfate content in diabetic nephropathy is the observation that heparanase, an enzyme capable of degrading heparan sulfate, is upregulated in the glomerular epithelial cell (GEC) in response to increased glucose. Increased GEC heparanase activity has been demonstrated in glomeruli in diabetic kidneys, and increased urine heparanase has been observed in diabetic nephropathy. In vitro studies have shown that GEC heparanase activity depends on the glucose concentration of the culture medium. GEC heparanase activity can be inhibited by heparin compounds. Sulodexide, an orally active low–molecular weight heparin, has been shown to lower urine albumin excretion. The working hypothesis that has emerged is that sulodexide may be an in vivo heparanase inhibitor that reaches the glomerular capillary wall and prevents heparan sulfate degradation, thus allowing reconstruction of heparan sulfate content and restoration of GBM ionic permselectivity. Two clinical trials are currently being carried out to determine whether sulodexide is renoprotective in diabetic nephropathy.

Human Serum from Patients with Septic Shock Activates Transcription Factors STAT1, IRF1, and NF-κB and Induces Apoptosis in Human Cardiac Myocytes

Proinflammatory cytokines have been linked to depression of myocardial contractility in vivo in patients with acute septic shock and in vitro models employing isolated myocytes exposed to serum from such patients. The key pathways involved in mediating this septic organ dysfunction (cell adhesion molecule expression, inducible nitric-oxide synthase induction, and apoptosis) are known to be regulated by transcription factors STAT1, IRF1, and NF-κB. Utilizing a model that mimics human disease, we have demonstrated activation of the transcription factors STAT1, IRF1, and NF-κB in human fetal myocytes exposed to human septic serum. Both reporter and electrophoretic mobility shift assays demonstrated a 5–19-fold increase in activation of transcription factors STAT1, IRF1, and NF-κB in response to incubation with human septic serum. The addition of human septic serum to human fetal myocytes induced apoptosis in human fetal myocytes and activation of the mitogen-activated protein kinase c-Jun NH -terminal kinase and caspase 1 as measured by Western blot. These data suggest that transcription factor activation and early myocyte apoptosis play a mechanistic role in septic myocardial depression and sepsis-induced organ dysfunction.

Major histocompatibility complex class I-related chain A/B (MICA/B) expression in tumor tissue and serum of pancreatic cancer: Role of uric acid accumulation in gemcitabine-induced MICA/B expression

BackgroundMajor histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind the immunoreceptor NKG2D and play an important role in mediating the cyotoxicity of NK and T cells. In this study, we sought to study MICA/B expression in pancreatic cancer and to determine whether and how genotoxic drugs such as gemcitabine can affect MICA/B expression and natural killer cytotoxity.MethodsSeven pancreatic cancer cell lines were analyzed for MICA/B expression by flow cytometry and for their sensitivity to NK-92 cell killing by a 51Cr release assay. MICA/B expression in tumor tissues and sera of pancreatic cancer was analyzed by immunohistochemical staining (IHC) and ELISA, respectively.ResultsTwo MICA/B-positive cell lines were sensitive to the cytotoxic activity of NK-92 cells. Other two MICA/B-positive cell lines and three MICA/B-negative cell lines were resistant to NK-92 cell killing. MICA/B expression was positive in 17 of 25 (68%) pancreatic ductal adenocarcinomas but not in normal pancreatic ductal epithelial cells. Serum MICA/B levels were significantly elevated in patients with pancreatic adenocarcinomas but did not correlate with the stage of pancreatic cancer and patient survival. Gemcitabine therapy led to increased serum MICA levels in 6 of 10 patients with detectable serum MICA. Allopurinol, an inhibitor of xanthine oxidoreductase that converts xanthine to uric acid, blocked uric acid production, MICA/B expression, and sensitivity to NK-92 cell killing toward a PANC-1 cancer cell line exposed to radiation and two genotoxic drugs, gemcitabine and 5-fluorouracil.ConclusionsThe levels of MICA/B expression in serum and tissue of pancreatic cancer are elevated. DNA damage-induced MICA/B expression is mediated through increased uric acid production.

Elevated serum heparanase‐1 levels in patients with pancreatic carcinoma are associated with poor survival

It has previously been shown that heparanase‐1 (HPR1), an endoglycosidase, is up‐regulated in pancreatic carcinoma. The purpose of this study was to test whether serum HPR1 levels in pancreatic carcinoma patients are elevated, and whether higher serum HPR1 levels are associated with a shortened survival.

The sonic hedgehog signaling pathway maintains the cancer stem cell self-renewal of anaplastic thyroid cancer by inducing snail expression.

CONTEXT Cancer stem cells (CSCs) have been recently identified in thyroid neoplasm. Anaplastic thyroid cancer (ATC) contains a higher percentage of CSCs than well-differentiated thyroid cancer. The signaling pathways and the transcription factors that regulate thyroid CSC self-renewal remain poorly understood. OBJECTIVE The objective of this study is to use two ATC cell lines (KAT-18 and SW1736) as a model to study the role of the sonic hedgehog (Shh) pathway in maintaining thyroid CSC self-renewal and to understand its underlying molecular mechanisms. DESIGN The expression and activity of aldehyde dehydrogenase (ALDH), a marker for thyroid CSCs, was analyzed by Western blot and ALDEFLUOR assay, respectively. The effect of three Shh pathway inhibitors (cyclopamine, HhAntag, GANT61), Shh, Gli1, Snail knockdown, and Gli1 overexpression on thyroid CSC self-renewal was analyzed by ALDEFLUOR assay and thyrosphere formation. The sensitivity of transfected KAT-18 cells to radiation was evaluated by a colony survival assay. RESULTS Western blot analysis revealed that ALDH protein levels in five thyroid cancer cell lines (WRO82, a follicular thyroid cancer cell line; BCPAP and TPC1, two papillary thyroid cancer cell lines; KAT-18 and SW1736, two ATC cell lines) correlated with the percentage of the ALDH(High) cells as well as Gli1 and Snail expression. The Shh pathway inhibitors, Shh and Gli1 knockdown, in KAT-18 cells decreased thyroid CSC self-renewal and increased radiation sensitivity. In contrast, Gli1 overexpression led to increased thyrosphere formation, an increased percentage of ALDH(High) cells, and increased radiation resistance in KAT-18 cells. Inhibition of the Shh pathway by three specific inhibitors led to decreased Snail expression and a decreased number of ALDH(High) cells in KAT-18 and SW1736. Snail gene knockdown decreased the number of ALDH(High) cells in KAT-18 and SW1736 cells. CONCLUSIONS The Shh pathway promotes the CSC self-renewal in ATC cell lines by Gli1-induced Snail expression.