Amelioration of hydrolyzed guar gum on high-fat diet-induced obesity: Integrated hepatic transcriptome and metabolome.

Hydrolyzed guar gum has gained attention as an anti-obesity agent; however, few studies have focused on its role in amelioration of hepatic-associated metabolic processes. Here, the anti-obesity effect of low molecular weight hydrolyzed guar gum (GMLP, 1-10 kDa) on high-fat diet (HFD)-fed C57BL/6 J mice was investigated via transcriptome and metabolome in liver. GMLP reduced body weight gain and hepatic lipid accumulation dose-dependently, regulated blood lipid levels, and improved liver damage in HFD-fed mice. Integrated transcriptome and metabolome indicated that GMLP mainly altered lipid metabolism pathways (glycerophospholipid metabolism, glycerolipid metabolism, and fatty acid degradation), reduced disease biomarkers of ethyl glucuronide and neopterin, and increased levels of choline, flavin adenine dinucleotide, and pantetheine metabolites. Real-time quantitative PCR showed that GMLP downregulated key genes involved in de novo lipogenesis and triacylglycerol synthesis, while promoting fatty acid oxidation and choline synthesis. This study provides a theoretical basis for GMLP treatment in future clinical applications.

Clinical comparative study assessing the effect of ivabradine on neopterin and NT-Pro BNP against standard treatment in chronic heart failure patients.

PURPOSE: Heart rate reduction (HR) is a cornerstone in heart failure therapy as it improves patient outcomes. The aim of this study is to evaluate short-term effect of ivabradine on NT-Pro BNP and neopterin in heart failure patients and assess the association between HR and these biomarkers. METHODS: Sixty patients on standard heart failure therapy were randomly allocated into ivabradine group (n = 30) and non-ivabradine group (n = 30). Ivabradine 5 mg twice daily was given for 3 months. Lipid profile and kidney functions were performed and blood samples for NT-Pro BNP and neopterin were analysed at baseline and after 3 months of intervention in both groups. RESULTS: There was a significant improvement in NYHA class in ivabradine group (p < 0.001). Ejection fraction was improved in ivabradine and non-ivabradine groups after intervention (p < 0.001), with a greater improvement in ivabradine group (p = 0.026). Heart rate was reduced in ivabradine group (p < 0.001) and non-ivabradine group (p < 0.001) yet greater reduction was seen in ivabradine group (p < 0.001). Serum creatinine and blood urea nitrogen were reduced in ivabradine group (Scr: p = 0.001, BUN: p = 0.001). NT-Pro BNP and neopterin levels significantly decreased in ivabradine group (NT-Pro BNP: p < 0.001, neopterin p < 0.001). Significant positive correlation was found between HR and biomarker levels after intervention (NT-Pro BNP: r = 0.475, p < 0.001, neopterin: r = 0.384, p = 0.002). CONCLUSION: Ivabradine therapy reduced levels of both biomarkers which correlated well with HR. Biomarker levels might provide a tool for assessing ivabradine effectiveness in HF. Trial registration Date: June 26, 2020. Identifier: NCT04448899. Link: Ivabradine in Patients with Congestive Heart Failure-Full Text View-ClinicalTrials.gov.

Foam cell formation but not oxLDL cytotoxicity is inhibited by CD36 down regulation by the macrophage antioxidant 7,8-dihydroneopterin.

BACKGROUND AND AIMS: Cluster of differentiation 36 (CD36) is a key scavenger receptor in the control of macrophage uptake of oxidised low-density lipoproteins (oxLDL). CD36 expression levels are not down regulated by intracellular cholesterol but are upregulated by oxidised low density lipoprotein (oxLDL) leading to the formation of lipid loaded foam cells, a major constituent of atherosclerotic plaques. We have previous shown that CD36 is down regulated by 7,8-dihydroneopterin, an antioxidant generated by γ-interferon activated macrophages. How CD36 down regulation affects oxLDL induced cytotoxicity, CD36 oxLDL upregulation and foam cell formation is examined using human monocyte like U937 cell line as a model system of human macrophages. METHODS: Low density lipoprotein (LDL) was prepared by ultracentrifugation from human plasma and oxidised in copper chloride. CD36 levels in U937 cells were measured by western blot analysis. and lipid accumulation was measured by oil red-O staining and 7-ketocholesterol accumulation by high performance liquid chromatography. Cell viability was measured by flow cytometry analysis after propidium iodide staining. RESULTS: 7,8-dihydroneopterin concentrations above 100 µM caused a concentration and time dependent decrease in cellular CD36 levels to 20 % of the untreated cells after 24 h. Upregulation of CD36 by oxLDL was inhibited by 7,8-dihydroneopterin treatment. The CD36 down regulation was associated with decrease in foam cell formation but not a reduction on oxLDL cytotoxicity. CONCLUSIONS: 7,8-dihydroneopterin down regulated CD36 in U937 cells, inhibiting foam cell formation but not oxLDL mediated cell death. 7,8-dihydroneopterin may modulate foam cell formation in atherosclerotic plaques.

Exercise-induced immune system response: Anti-inflammatory status on peripheral and central organs.

A wide array of molecular pathways has been investigated during the past decade in order to understand the mechanisms by which the practice of physical exercise promotes neuroprotection and reduces the risk of developing communicable and non-communicable chronic diseases. While a single session of physical exercise may represent a challenge for cell homeostasis, repeated physical exercise sessions will improve immunosurveillance and immunocompetence. Additionally, immune cells from the central nervous system will acquire an anti-inflammatory phenotype, protecting central functions from age-induced cognitive decline. This review highlights the exercise-induced anti-inflammatory effect on the prevention or treatment of common chronic clinical and experimental settings. It also suggests the use of pterins in biological fluids as sensitive biomarkers to follow the anti-inflammatory effect of physical exercise.

Nucleoside transporters are critical to the uptake and antioxidant activity of 7,8-dihydroneopterin in monocytic cells.

7,8-Dihydroneopterin protects cells intracellularly from oxidative stress-induced death, but its mode of transport across the cell membrane is unknown. Nucleosides, such as guanosine, are transported via nucleoside transporters of the equilibrative and concentrative forms. Therefore, the objective of this study was to identify which membrane transporters are responsible for 7,8-dihydroneopterin transport in cells and whether this is necessary for protection against oxidative stress. Monocytic cell lines U937, THP-1 and human monocytes were incubated with varying concentrations of 7,8-dihydroneopterin with or without nucleoside transporter inhibitors nitrobenzylthioinosine (NBMPR; ENT1), dipyridamole (DP; ENT1 and ENT2) or indomethacin (INDO; CNT). Only DP inhibited 7,8-dihydroneopterin uptake in U937 cells, while NBMPR and DP inhibited 7,8-dihydroneopterin uptake in THP-1 cells. All three inhibitors limited 7,8-dihydroneopterin uptake in human monocytes at short time points only. When the cells were incubated with 10 mM of the peroxyl radical generator 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) a 50-80% loss of cell viability was measured. 7,8-dihydroneopterin protected all cell lines against AAPH-induced cell death, which was prevented with DP in U937 cells, NBMPR in THP-1 cells and a combination of all three nucleoside inhibitors in human monocytes. These data indicate 7,8-dihydroneopterin is transported across the cell membrane of monocytic cells via equilibrative and concentrative nucleoside transporters in a cell lineage-dependent manner. The data also indicate protection from peroxyl radical-generated cell death with 7,8-dihydroneopterin is intracellular and facilitated through nucleoside transporters in monocytic cells.

Soluble CD163, a unique biomarker to evaluate the disease activity, exhibits macrophage activation in systemic juvenile idiopathic arthritis.

This study aims to investigate the clinical significance of serum soluble CD163 (sCD163) levels as a predictor of the disease activity of systemic juvenile idiopathic arthritis (s-JIA). In this study, we examined 63 patients with s-JIA, four with Epstein-Barr virus-induced hemophagocytic lymphohistiocytosis (EBV-HLH), and seven with Kawasaki disease (KD), along with 14 healthy controls. We quantified serum cytokine levels (sCD163, neopterin, IL-18, IL-6) by enzyme-linked immunosorbent assay and compared the results with the clinical features of s-JIA. Serum sCD163 levels were significantly elevated in patients with s-JIA associated macrophage activation syndrome (MAS) and EBV-HLH compared to those in patients with acute-phase s-JIA and KD. In addition, serum sCD163 levels profoundly increased with the progress of MAS and correlated positively with the disease activity of s-JIA, even in patients receiving tocilizumab. Furthermore, serum sCD163 levels significantly decreased in the inactive phase compared to those in the active phase and normalized in remission. The correlation between macrophage activation and serum sCD163 levels might be a unique indicator of the disease activity and a potential diagnostic laboratory criterion for clinical remission in patients with s-JIA, including those receiving tocilizumab.

Effect of 7,8-dihydroneopterin mediated CD36 down regulation and oxidant scavenging on oxidised low-density lipoprotein induced cell death in human macrophages.

The role of CD36 in oxidised low-density lipoprotein (oxLDL) mediated cell death was examined by down regulating the receptor level with the macrophage generated antioxidant 7,8-dihydroneopterin. Down regulation of CD36 protein levels in human monocyte derived macrophages by 7,8-dihydroneopterin corresponded to a decrease in CD36-mRNA. The oxidation products of 7,8-dihydroneopterin, dihydroxanthopterin and neopterin did not significantly down regulate CD36. The CD36 down regulation resulted in a decrease in oxLDL uptake measured as 7-ketocholesterol accumulation. Though less oxLDL was taken up by the macrophages as a result of the 7,8-dihydroneopterin induced down regulation in CD36 levels, the cytotoxicity of the oxLDL was not decreased. Addition of 7,8-dihydroneopterin to oxLDL treated macrophages decreased the concentration of intracellular oxidants. In the presence of oxLDL, 7,8-dihydroneopterin was oxidised to neopterin showing that the 7,8-dihydroneopterin was scavenging intracellular oxidants generated in response to the oxLDL. The results show CD36 down regulation does not protect human macrophages form oxLDL cytotoxicity but 7,8-dihydroneopterin intracellular oxidant scavenging is protective.

Deletion of nudB Causes Increased Susceptibility to Antifolates in Escherichia coli and Salmonella enterica.

Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes (pabB and nudB) involved in the folate biosynthesis of Escherichia coli were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudB resulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of nudB also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudB affects the bacterial growth and susceptibility to antifolates, 7,8-dihydroneopterin and 7,8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on susceptibilities to the antifolates. Reverse mutants of the nudB deletion strain were isolated to further study the mechanism of how the deletion of nudB affects susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the nudB deletion. Meanwhile, overexpression of folM could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudB resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudB also resulted in increased susceptibility to both SMX and TMP in Salmonella enterica Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be interesting to design new compounds targeting dihydroneopterin triphosphate hydrolase, which may inhibit bacterial growth and simultaneously potentiate the antimicrobial activities of antifolates targeting other components of folate biosynthesis.

Neopterin acts as an endogenous cognitive enhancer.

Neopterin is found at increased levels in biological fluids from individuals with inflammatory disorders. The biological role of this pteridine remains undefined; however, due to its capacity to increase hemeoxygenase-1 content, it has been proposed as a protective agent during cellular stress. Therefore, we investigated the effects of neopterin on motor, emotional and memory functions. To address this question, neopterin (0.4 and/or 4pmol) was injected intracerebroventricularly before or after the training sessions of step-down inhibitory avoidance and fear conditioning tasks, respectively. Memory-related behaviors were assessed in Swiss and C57BL/6 mice, as well as in Wistar rats. Moreover, the putative effects of neopterin on motor and anxiety-related parameters were addressed in the open field and elevated plus-maze tasks. The effects of neopterin on cognitive performance were also investigated after intraperitoneal lipopolysaccharide (LPS) administration (0.33mg/kg) in interleukin-10 knockout mice (IL-10(-/-)). It was consistently observed across rodent species that neopterin facilitated aversive memory acquisition by increasing the latency to step-down in the inhibitory avoidance task. This effect was related to a reduced threshold to generate the hippocampal long-term potentiation (LTP) process, and reduced IL-6 brain levels after the LPS challenge. However, neopterin administration after acquisition did not alter the consolidation of fear memories, neither motor nor anxiety-related parameters. Altogether, neopterin facilitated cognitive processes, probably by inducing an antioxidant/anti-inflammatory state, and by facilitating LTP generation. To our knowledge, this is the first evidence showing the cognitive enhancer property of neopterin.

Oxidised low density lipoprotein causes human macrophage cell death through oxidant generation and inhibition of key catabolic enzymes.

Oxidised low density lipoprotein (oxLDL) is thought to be a significant contributor to the death of macrophage cells observed in advanced atherosclerotic plaques. Using human-derived U937 cells we have examined the effect of cytotoxic oxLDL on oxidative stress and cellular catabolism. Within 3h of the addition of oxLDL, there was a rapid, concentration dependent rise in cellular reactive oxygen species followed by the loss of cellular GSH, and the enzyme activity of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aconitase. The loss of these catabolic enzymes was accompanied by the loss of cellular ATP and lower lactate generation. Addition of the macrophage antioxidant 7,8-dihydroneopterin inhibited the ROS generation, glutathione loss and catabolic inactivation. NOX was shown to be activated by oxLDL addition while apocynin inhibited the loss of GSH and cell viability. The data suggests that oxLDL triggers an excess of ROS production through NOX activation, and catabolic failure through thiol oxidation resulting in cell death.

Intracellular glutathione protects human monocyte-derived macrophages from hypochlorite damage.

AIMS: Macrophages must function in an inflammatory environment of high oxidative stress due to the production of various oxidants. Hypochlorous acid (HOCl) is a potent cytotoxic agent generated by neutrophils and macrophages within inflammatory sites. This study determines whether glutathione is the key factors governing macrophage resistance to HOCl. MAIN METHODS: Human monocyte derived macrophages (HMDM) were differentiated from human monocytes prepared from human blood. The HMDM cells were exposed to micromolar concentrations of HOCl and the timing of the cell viability loss was measured. Cellular oxidative damage was measured by loss of glutathione, cellular ATP, tyrosine oxidation, and inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). KEY FINDINGS: HOCl causes a rapid loss in HMDM cell viability above threshold concentrations. The cell death occurred within 10 min of treatment with the morphological characteristics of necrosis. The HOCl caused the extensive cellular protein oxidation with the loss of tyrosine residue and inactivation of GAPDH, which was accompanied with the loss of cellular ATP. This cellular damage was only observed after the loss of intracellular GSH from the cell. Removal of intracellular GSH with diethyl maleate (DEM) increased the cells' sensitivity to HOCl damage while protecting the intracellular GSH pool with the antioxidant 7,8-dihydroneopterin prevented the HOCl mediated viability loss. Variations in the HOCl LD(50) for inducing cell death were strongly correlated with initial intracellular GSH levels. SIGNIFICANCE: In HMDM cells scavenging of HOCl by intracellular glutathione is sufficient to protect against oxidative loss of key metabolic functions within the cells.

Neopterin, inflammation-associated product, prolongs erythropoiesis suppression in aged SAMP1 mice due to senescent stromal-cell impairment.

Anemia induced by inflammation is well known to be more serious in the elderly than in non-elderly adults; however, the reason why this is so remains unclear. Neopterin produced by monocytes during inflammation promotes myelopoiesis but suppresses B-lymphopoiesis and erythropoiesis, by activating stromal cells in mice. Here, age-related changes in the erythropoietic response to neopterin were determined using senescence accelerated mice (SAMP1) with senescence stromal-cell impairment. Intravenous injection of neopterin into young mice (8-12 weeks old) resulted in a decrease in erythroid progenitor cell number in the bone marrow (BM), concomitant with an increase in myeloid progenitor cell number over one week. Intravenous injection of neopterin into aged mice (30-36 weeks old) resulted in a prolonged decrease in erythroid progenitor cell number in the BM over three weeks and a limited increase in myeloid progenitor cell number over one day. Neopterin treatment induced a decrease in serum erythropoietin concentrations in young mice but not in aged mice. The gene expression of tumor necrosis factor α (TNF-α), a negative regulator of erythropoiesis, was up-regulated in the BM of both young and aged mice, and the degree of TNF-α up-regulation was the same in both groups. The gene expression of interleukin (IL)-11, a positive regulator of erythropoiesis, was also up-regulated over one day in both young and aged mice. However, IL-11 gene expression remained up-regulated thereafter in young mice, whereas it was rapidly down-regulated in aged mice. These data suggest that prolonged suppression of erythropoiesis in aged mice may be due to a decrease in the production of positive regulators rather than to an increase in the production of negative regulators. Our combined data suggest that age-related impairment of stromal cells induces serious anemia in the elderly during inflammation.

Oxidant production, oxLDL uptake, and CD36 levels in human monocyte­derived macrophages are downregulated by the macrophage-generated antioxidant 7,8-dihydroneopterin.

The severity of atheroma burden in patients strongly correlates to increasing levels of plasma neopterin, the oxidation product of 7,8-dihydroneopterin. Interferon-γ stimulation of macrophages causes the synthesis of 7,8-dihydroneopterin, a potent antioxidant that inhibits oxidative damage to cells, and the cytotoxicity of oxidized low-density lipoprotein (oxLDL) to monocyte-like U937 cells but not THP-1 cells. With human monocyte-derived macrophages (HMDMs), oxLDL triggered a large oxidative stress, causing the rapid loss of cellular glutathione, glyceradehyde-3-phosphate dehydrogenase (GAPDH) inhibition, and eventual loss of viability without caspase-3 activation. Inhibition of oxLDL cytotoxicity to HMDMs occurred at 7,8-dihydroneopterin concentrations >100 µM. The oxLDL-mediated glutathione loss and GAPDH inactivation was inhibited by 7,8-dihydroneopterin. 7,8-Dihydroneopterin rapidly entered the HMDMs, suggesting that much of the protective effect was scavenging of intracellular oxidants generated in response to oxLDL. OxLDL uptake by HMDMs was reduced by 30% by 7,8-dihydroneopterin. Immunoblot analysis suggests that this decrease in oxLDL uptake was due to a significant downregulation in the levels of CD36. These results imply that 7,8-dihydroneopterin protects human macrophages both by scavenging oxidants generated in response to oxLDL and by decreasing CD36-mediated uptake of oxLDL.

Inflammatory biomarker, neopterin, predominantly enhances myelopoiesis, which suppresses erythropoiesis via activated stromal cells.

Neopterin is produced by monocytes and is a useful biomarker for inflammation. We found previously that neopterin enhanced myelopoiesis but suppressed B-lymphopoiesis triggered by the positive and negative regulations of cytokines produced by stromal cells in mice. The effects of neopterin on erythropoiesis during the enhancement of myelopoiesis were determined in the present study using C57BL/6J mice. The intravenous injection of neopterin into mice resulted in a prolonged decrease in the number of femoral erythroid progenitor cells (BFU-Es and CFU-Es), whereas the number of femoral myeloid progenitor cells (CFU-GMs) was increased. Interestingly, the oscillatory changes in the number of erythroid progenitor cells were reciprocal to those of myeloid progenitor cells. The expression of Cdc42, a regulator of the balance between erythropoiesis and myelopoiesis, was down-regulated, implying that the suppression of erythropoiesis is due to myelopoietic predominance. Furthermore, the expression of SDF-1 in stromal cells, a negative regulator of erythropoiesis, was up-regulated. These results suggest that neopterin facilitates myelopoiesis in the bone marrow by suppressing erythropoiesis, thereby contributing to the potential up-regulation of inflammatory process.

[Pteridine-dependent oxygen activation in neutrophils].

We investigated the influence ofneopterin and 7, 8-dihydroneopterin on the activity and secretory degranulation of myeloperoxidase in neutrophils and the ability of pteridines to interact with the main substrate of this enzyme, hydrogen peroxide, and with the intermediate product of halogenation cycle--hypochlorous acid. It was shown that neopterin and 7, 8-dihydroneopterin while being a redox-pair regulated the process of oxygen activation in neutrophils by functioning of myeloperoxidase. Depending on concentration, pteridines can influence the secretion of myeloperoxidase into intracellular medium and decrease the level of hydrogen peroxide and hypochlorous acid that are a substrate and an intermediate product of the enzyme respectively. It was shown that 7, 8-dihydroneopterin in micromolar concentration appeared to be noncompetitive inhibitor of myeloperoxidase. We suppose that myeloperoxidase assists 7, 8-dihydroneopterin oxidation by hypochlorous acid that leads to neopterin concentration increase. These changes modify the concentration of reactive oxygen species in intracellular and extracellular media.

Macrophage mediated protein hydroperoxide formation and lipid oxidation in low density lipoprotein are inhibited by the inflammation marker 7,8-dihydroneopterin.

The formation of oxidised low density lipoprotein (LDL) within the atherosclerotic plaque appears to be a factor in the development of advanced atherosclerotic plaques. LDL oxidation is dependent on the balance of oxidants and antioxidants within the intima. In addition to producing various oxidants, human macrophages release 7,8-dihydroneopterin which in vivo is oxidised to the inflammation marker neopterin. Using macrophage-like THP-1 cells and human monocyte-derived macrophages, we demonstrate that 7,8-dihydroneopterin is a potent inhibitor of cell-mediated LDL oxidation. 7,8-Dihydroneopterin scavenges the chain propagating lipid peroxyl radical, inhibiting both lipid and protein hydroperoxide formation. A significant amount of the hydroperoxide formed during cell-mediated LDL oxidation was protein hydroperoxide. 7,8-Dihydroneopterin oxidation to 7,8-dihydroxanthopterin was only observed in the presence of both cells and LDL, showing that 7,8-dihydroneopterin had no effect on initiating oxidant generation by the cells. 7,8-Dihydroneopterin did not regenerate alpha-tocopherol but competed with it for the lipid peroxyl radical. Although stimulation of both cell types with gamma-interferon failed to produce sufficient 7,8-dihydroneopterin to inhibit LDL oxidation in tissue culture, analysis of advanced atherosclerotic plaque removed from patients showed that total neopterin levels could reach low micromolar concentrations. This suggests that 7,8-dihydroneopterin synthesis by macrophages could play a significant role in the development of atherosclerotic plaques.

In vivo bioactivity of interferon-beta in multiple sclerosis patients with neutralising antibodies is titre-dependent.

BACKGROUND: Development of neutralising antibodies (NAbs) against recombinant interferon-beta (IFNbeta) is a significant clinical problem in the treatment of multiple sclerosis (MS). Several methods are available to assess NAbs, but there is a lack of consensus on how the different NAb titre levels interfere with the efficacy of the drug, especially in the individual patient. METHODS: NAb titres were measured with an in vitro MxA induction assay and the in vivo IFNbeta response was assessed by measuring MxA mRNA expression using real-time PCR. RESULTS: We identified titre levels of NAbs at which the IFNbeta biological activity was reduced or abrogated. Patients with NAb titres of up to 150 TRU/ml (ten times reduction units per ml) still had retained IFNbeta bioactivity, whereas greatly reduced levels of IFNbeta bioactivity were found in patients with NAbs of 150-600 TRU/ml. Titres above 600 TRU/ml were associated with loss of IFNbeta bioactivity. Similar results were obtained when TRAIL mRNA was used as a marker of the in vivo response to IFNbeta. CONCLUSION: There is a stepwise loss of IFNbeta bioactivity with increasing NAb titres and it is possible to identify functionally critical NAb titre levels that are useful to support treatment decisions at the individual patient level.

Mechanism of apoptosis induced by apigenin in HepG2 human hepatoma cells: involvement of reactive oxygen species generated by NADPH oxidase.

Although plant-derived flavonoids have been reported to have anti-cancer activities, the exact mechanism of these actions is not completely understood. In this study we investigated the role for reactive oxygen species (ROS) as a mediator of the apoptosis induced by apigenin, a widespread flavonoid in plant, in HepG2 human hepatoma cells. Apigenin reduced cell viability, and induced apoptotic cell death in a dose-dependent manner. In addition, it evoked a dose-related elevation of intracellular ROS level. Treatment with various inhibitors of the NADPH oxidase (diphenylene iodonium, apocynin, neopterine) significantly blunted both the generation of ROS and induction of apoptosis induced by apigenin. These results suggest that ROS generated through the activation of the NADPH oxidase may play an essential role in the apoptosis induced by apigenin in HepG2 cells. These results further suggest that apigenin may be valuable for the therapeutic management of human hepatomas.

Inflammatory biomarker, neopterin, suppresses B lymphopoiesis for possible facilitation of granulocyte responses, which is severely altered in age-related stromal-cell-impaired mice, SCI/SAM.

Neopterin is produced by monocytes and is a useful biomarker of inflammatory activation. We found that neopterin enhanced in vivo and in vitro granulopoiesis triggered by the stromal-cell production of cytokines in mice. The effects of neopterin on B lymphopoiesis during the enhancement of granulopoiesis were determined using the mouse model of senescent stromal-cell impairment (SCI), a subline of senescence-accelerated mice (SAM). In non-SCI mice (a less senescent stage of SCI mice), treatment with neopterin decreased the number of colonies, on a semisolid medium, of colony-forming units of pre-B-cell progenitors (CFU-preB) from unfractionated bone marrow (BM) cells, but not that from a population rich in pro-B and pre-B cells without stromal cells. Neopterin upregulated the expression of genes for the negative regulators of B lymphopoiesis such as tumor necrosis factor-alpha (TNF-alpha ), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta) in cultured stromal cells, implying that neopterin suppressed the CFU-preB colony formation by inducing negative regulators from stromal cells. The intraperitoneal injection of neopterin into non-SCI mice resulted in a marked decrease in the number of femoral CFU-preB within 1 day, along with increases in TNF-alpha and IL-6 expression levels. However, in SCI mice, in vivo and in vitro responses to B lymphopoiesis and the upregulation of cytokines after neopterin treatment were less marked than those in non-SCI mice. These results suggest that neopterin predominantly suppressed lymphopoiesis by inducing the production of negative regulators of B lymphopoiesis by stromal cells, resulting in the selective suppression of in vivo B lymphopoiesis. These results also suggest that neopterin facilitated granulopoiesis in BM by suppressing B lymphopoiesis, thereby contributing to the potentiation of the inflammatory process; interestingly, such neopterin function became impaired during senescence because of attenuated stromal-cell function, resulting in the downmodulation of the host-defense mechanism in the aged.

Influence of neopterin and 7,8-dihydroneopterin on the replication of Coxsackie type B5 and influenza A viruses.

Pteridine derivatives neopterin and 7,8-dihydroneopterin are produced by human macrophages and dendritic cells upon stimulation with interferon-gamma (IFN-gamma) and therefore become detectable in increased amounts in humans during cell-mediated (Th1-type) immune response. Compounds produced upon influence of cytokine IFN-gamma often exert antiproliferative and antiviral activity. The aim of this study was to investigate the effect of neopterin and 7,8-dihydroneopterin on the replication of Coxsackie type B5 and influenza A viruses. The changes in the replication of these viruses were evaluated by the degree of cytopathic effect and their ability to form plaques in Coxsackie B5-infected human larynx carcinoma epithelial (Hep-2) cells and in influenza A-infected canine kidney epithelial cells (MDCK). Potential toxicity of neopterin and 7,8-dihydroneopterin was estimated by the incorporation of (3)H-thymidine and (3)H-uridine into Hep-2 and MDCK cells. Whereas 30 nmol/l neopterin delayed the development of the cytopathic effect of Coxsackie B5 virus in Hep-2 cells (P < 0.01), 7,8-dihydroneopterin did not have any essential influence at any of the concentrations tested between 10 nmol/l and 1,000 micromol/l. However, 100-1,500 micromol/l 7,8-dihydroneopterin significantly suppressed the propagation of influenza A virus. Neopterin and 7,8-dihydroneopterin were practically nontoxic for Hep-2 and MDCK cells even at high microM concentration. Results suggest that the increased production of neopterin derivatives by activated macrophages and dendritic cells may represent part of the antiviral armature induced by IFN-gamma. The mechanisms of the inhibitory effects of neopterin and 7,8-dihydroneopterin on virus replication apparently are different.