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Fibroblast growth factor (FGF) 13, a member of the FGF11 subfamily, is a kind of intracrine protein similar to other family members including FGF11, FGF12, and FGF14. Unlike classical FGF, FGF13 exerts its bioactivities independent of fibroblast growth factor receptors (FGFRs). However, the effect of exogenous administration of FGF13 still remains further investigated. In the present study, we established an Escherichia coli expression system for the large-scale production of FGF13 and then obtained two isoform proteins including recombinant human FGF13A (rhFGF13A) and rhFGF13B with a purity greater than 90% by column chromatography, respectively. Otherwise, soluble analysis indicated that both rhFGF13A and rhFGF13B expressed in E. coli BL21 (DE3) pLysS were soluble. Furthermore, cellular-based experiments demonstrated that rhFGF13A, rather than rhFGF13B, could promote the proliferation of NIH3T3 cells in the presence of heparin. Mechanistically, the mitogenic effect of FGF13 was mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), but not p38. Moreover, blockage of FGFRs also significantly attenuated the mitogenic effects of rhFGF13A, implying that FGFRs are still related to FGF13. Thus, our research shows that exogenous FGF13 can act as secreted FGF to participate in cell signal transmission and heparin is still required as an ancillary cofactor for the mitogenic effects of FGF13, which may help people to discover more potential functions of FGF13 in cell life activities.

Amino acid racemases catalyze the stereoinversion of the chiral Cα to produce the D-enantiomers that participate in biological processes, such as cell wall construction in prokaryotes. Within this large protein family, bacterial proline racemases have been extensively studied as a model of enzymes acting with a pyridoxalphosphate-independent mechanism. Here we report the crystal structure of the proline racemase from the human parasite Trypanosoma cruzi (TcPRACA), a secreted enzyme that triggers host B cell polyclonal activation, which prevents specific humoral immune responses and is crucial for parasite evasion and fate. The enzyme is a homodimer, with each monomer folded in two symmetric α/α subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism. Mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein. In contrast, inhibitor binding promotes the closure of the interdomain crevice and completely abrogates B cell proliferation, suggesting that the mitogenic properties of TcPRACA depend on the exposure of transient epitopes in the ligand-free enzyme.

In Chronic Kidney Disease (CKD), immune cells are affected by uremic retention toxins. Given this effect, we analyzed lymphocyte proliferative response and immune modulators production following in vitro stimulation. Whole blood was drawn from healthy controls, patients with eGFR <20 ml/min/1.73 m(2) (Pre-dialysis, CKD stages 4 and 5) and hemodialysis patients (stage 5D). Peripheral cells were incubated for six days with pokeweed mitogen, concanavalin A, Staphylococcus enterotoxin A or influenza A vaccine. Peripheral lymphocyte proliferation was then analyzed by the \"Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood\" (FASCIA) method, and cytokine profile in the cell supernatants was analyzed by the Milliplex multi-array method. The absolute number of lymphoblasts in response to mitogenic stimulation and the number of cells in each CD4+ and CD8+ subpopulation were similar comparing the three groups, except for a single decline in number of lymphoblasts after stimulation with Staphylococcus enterotoxin A, comparing dialysis patients with healthy controls. Levels of interleukin (IL)-2 (p=0.026), -10 (p=0.019) and -15 (p=0.027) in the Staphylococcus enterotoxin A-stimulated supernatant were lower in hemodialysis patients compared to healthy controls. Levels of IL-15 (p=0.017) from pre-dialysis patients and levels of IL-5 (p=0.019) from hemodialysis patients in influenza A vaccine-stimulated supernatants were also lower compared to controls. In pokeweed mitogen-stimulated supernatant, IL-2 levels (p=0.013) were lower in hemodialysis patients compared to pre-dialysis patients. TNF-α, IL-10, IL-12, IL-15, IL-8, MCP-1, IP-10, IFN-α2, IL-1α and eotaxin levels were all significantly higher in plasma obtained from CKD patients. Our results suggest that T-cells from CKD patients have similar proliferative response to stimulation compared with healthy individuals. Moreover, however the immune cells show inability to produce selected cytokines, most likely due to the uremic milieu or dialysis procedure.

As a phenotypically plastic cellular population, macrophages change their physiology in response to environmental signals. Emerging evidence suggests that macrophages are capable of tightly coordinating their metabolic programs to adjust their immunological and bioenergetic functional properties, as needed. Upon mitogenic stimulation, quiescent macrophages enter the cell cycle, increasing their bioenergetic and biosynthetic activity to meet the demands of cell growth. Proinflammatory stimulation, however, suppresses cell proliferation, while maintaining a heightened metabolic activity imposed by the production of bactericidal factors. Here, we report that the mitogenic stimulus, colony-stimulating factor 1 (CSF-1), engages a myelocytomatosis viral oncogen (Myc)-dependent transcriptional program that is responsible for cell cycle entry and the up-regulation of glucose and glutamine catabolism in bone marrow-derived macrophages (BMDMs). However, the proinflammatory stimulus, lipopolysaccharide (LPS), suppresses Myc expression and cell proliferation and engages a hypoxia-inducible factor alpha (HIF1α)-dependent transcriptional program that is responsible for heightened glycolysis. The acute deletion of Myc or HIF1α selectively impaired the CSF-1– or LPS-driven metabolic activities in BMDM, respectively. Finally, inhibition of glycolysis by 2-deoxyglucose (2-DG) or genetic deletion of HIF1α suppressed LPS-induced inflammation in vivo. Our studies indicate that a switch from a Myc-dependent to a HIF1α-dependent transcriptional program may regulate the robust bioenergetic support for an inflammatory response, while sparing Myc-dependent proliferation.

Vitamin E is divided into two subgroups; tocopherols and tocotrienols. Both have protective roles in biological systems. The present study was conducted to compare the effect of short-term supplementation at 200 mg/d of either α-tocopherol or a tocotrienol-rich fraction (TRF) from palm oil on immune modulation and plasma vitamin E levels in normal healthy Asian volunteers. In a randomised, double-blind placebo-controlled trial conducted, fifty-three healthy volunteers aged 20-50 years were recruited based on the study's inclusion and exclusion criteria. They were randomly assigned into three groups, i.e. two experimental groups that received daily supplementation at 200 mg of either α-tocopherol or the TRF, and the control group that received a placebo. Blood was drawn on days 0, 28 and 56 for several laboratory analyses. Differences in the production of IL-4 or interferon-γ by concanavalin A-stimulated lymphocytes isolated from these volunteers were not significant (P>0·05). There were no significant differences observed in immune parameters between the healthy volunteers who received daily supplementation with either α-tocopherol or the TRF. As these observations were made in the absence of any immunogenic challenge, we feel it would be of benefit to study if there would be any differences observed when an immunogenic challenge such as vaccination were introduced.

Ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, is the major component of many plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes and has been reported to possess antioxidant and anti-tumor properties. These properties of UA have been attributed to its ability to suppress NF-κB (nuclear factor kappa B) activation. Since NF-κB, in co-ordination with NF-AT (nuclear factor of activated T cells) and AP-1(activator protein-1), is known to regulate inflammatory genes, we hypothesized that UA might exhibit potent anti-inflammatory effects. The anti-inflammatory effects of UA were assessed in activated T cells, B cells and macrophages. Effects of UA on ERK, JNK, NF-κB, AP-1 and NF-AT were studied to elucidate its mechanism of action. In vivo efficacy of UA was studied using mouse model of graft-versus-host disease. UA inhibited activation, proliferation and cytokine secretion in T cells, B cells and macrophages. UA inhibited mitogen-induced up-regulation of activation markers and co-stimulatory molecules in T and B cells. It inhibited mitogen-induced phosphorylation of ERK and JNK and suppressed the activation of immunoregulatory transcription factors NF-κB, NF-AT and AP-1 in lymphocytes. Treatment of cells with UA prior to allogenic transplantation significantly delayed induction of acute graft-versus-host disease in mice and also significantly reduced the serum levels of pro-inflammatory cytokines IL-6 and IFN-γ. UA treatment inhibited T cell activation even when added post-mitogenic stimulation demonstrating its therapeutic utility as an anti-inflammatory agent. The present study describes the detailed mechanism of anti-inflammatory activity of UA. Further, UA may find application in the treatment of inflammatory disorders.

The aim of this study was to investigate the influence of 2 established anesthetic techniques: total intravenous anesthesia and balanced inhalation anesthesia (BAL) on the perioperative-induced changes of peripheral blood mononuclear cells (PBMCs), changes in lymphocyte subsets, and the balance of proinflammatory and anti-inflammatory cytokines. This is a prospective, randomized, clinical comparison study. This study was set at a university hospital. This study involved 50 patients with American Society of Anesthesiologists physical status I who were scheduled for elective minimal invasive partial diskectomy. There was no intervention involved in this study. Changes in differential counts, lymphocyte subsets, and proliferation rates were determined before surgery and in the early postoperative period. Plasma concentrations of proinflammatory cytokines (IL-2, IL-6, IL-12, interferon γ) and anti-inflammatory cytokines (IL-10, IL-1RA, transforming growth factor β), and plasma concentrations of cortisol, epinephrine, and norepinephrine were measured before, during, and after surgery. Absolute number of CD3 +, CD4 +, and CD8 +, and expression of HLA-DR and activation marker CD25 +, CD26 +, and CD69 + decreased more in response to surgery after BAL. Changes in distribution of T-lymphocyte cells seem to be in part related to severe postoperative pain. Plasma concentration of IL-6 significantly increased during and after surgery with BAL without relation to pain. Anesthetic management may have varying influences on the postoperative immune response. Surgery-induced inflammatory response and alteration in cell-mediated immunity seem to be more pronounced after BAL. These effects were attributed to the enhanced stress response after BAL.

Global transcriptional and epigenomic analyses in diverse cell types reveal that the primary action of Myc is to up- and downregulate transcription of distinct groups of genes, rather than to amplify transcription of all active genes; general RNA amplification, when observed, is better explained as an indirect consequence of Myc’s action on cellular physiology. Selective gene regulation by Myc The mammalian Myc oncoprotein is a transcription factor that binds to thousands of promoters. Two current models for Myc function propose that it is either a gene-specific regulator of transcription, or a global amplifier of all active genes. Two groups reporting in this issue of Nature present evidence in support of the idea that Myc regulates specific genes. Arianna Sabò et al . analyse Myc genomic distribution and RNA expression profiles during B-cell lymphomagenesis in mice and Susanne Walz et al . compare normal cells and Myc-transformed tumour cells. Although both groups find that Myc overexpression can result in a general increase in gene expression, the effect is an indirect one. Modulated by various other transcription factors, Myc seems to act primarily by regulating specific groups of genes. The c- myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci 1 . Recent work suggested that rather than up- and downregulating selected groups of genes 1 , 2 , 3 , Myc targets all active promoters and enhancers in the genome (a phenomenon termed ‘invasion’) and acts as a general amplifier of transcription 4 , 5 . However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations 6 , we detected general increases in total RNA or messenger RNA copies per cell (hereby termed ‘amplification’) 4 , 5 when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) 7 , 8 or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome 4 , 5 , 9 , 10 , 11 , Myc does not directly act as a global transcriptional amplifier 4 , 5 . Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.

Pharmacological fibroblast growth factor 1 (FGF1) normalizes blood glucose in diabetic mice by means of an FGF receptor signalling pathway that is independent of its mitogenic activity. Glucose-lowering activity of 'endocrine' FGF1 As a non-endocrine member of the fibroblast growth factor (FGF) family, FGF1 is known as a classic growth factor with mitogenic and angiogenic activity. This study identifies FGF1 as a powerful metabolic regulator. Injection of recombinant FGF1 (rFGF1) results in potent, insulin-dependent glucose lowering in diabetic mice, but does not lead to hypoglycaemia. Chronic pharmacological treatment with rFGF1 increases insulin-dependent glucose uptake in skeletal muscle and suppresses hepatic glucose production to achieve whole-body insulin sensitization. This work raises the possibility that FGF1 could have therapeutic potential for the treatment of insulin resistance and type 2 diabetes. Fibroblast growth factor 1 (FGF1) is an autocrine/paracrine regulator whose binding to heparan sulphate proteoglycans effectively precludes its circulation 1 , 2 . Although FGF1 is known as a mitogenic factor, FGF1 knockout mice develop insulin resistance when stressed by a high-fat diet, suggesting a potential role in nutrient homeostasis 3 , 4 . Here we show that parenteral delivery of a single dose of recombinant FGF1 (rFGF1) results in potent, insulin-dependent lowering of glucose levels in diabetic mice that is dose-dependent but does not lead to hypoglycaemia. Chronic pharmacological treatment with rFGF1 increases insulin-dependent glucose uptake in skeletal muscle and suppresses the hepatic production of glucose to achieve whole-body insulin sensitization. The sustained glucose lowering and insulin sensitization attributed to rFGF1 are not accompanied by the side effects of weight gain, liver steatosis and bone loss associated with current insulin-sensitizing therapies. We also show that the glucose-lowering activity of FGF1 can be dissociated from its mitogenic activity and is mediated predominantly via FGF receptor 1 signalling. Thus we have uncovered an unexpected, neomorphic insulin-sensitizing action for exogenous non-mitogenic human FGF1 with therapeutic potential for the treatment of insulin resistance and type 2 diabetes.

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.