Anti‐inflammatory effect of 4‐methylcyclopentadecanone in rats submitted to ischemic stroke

This study aimed to investigate the anti‐inflammatory effect of 4‐methylcyclopentadecanone (4‐MCPC) in rats suffering from a cerebral ischemia/reperfusion (I/R) injury. In this study, the focal cerebral ischemia in rats was induced by middle cerebral artery occlusion (MCAO) for 2 h, and the rats were treated with 4‐MCPC (8 mg/kg) just 0.5 h before reperfusion. The ischemic infarct volume was recorded 24 h after the MCAO. In addition, myeloperoxidase (MPO) activity and TNF‐α and IL‐1β levels in the ischemic cerebral cortex were determined by ELISA, while nuclear translocation of NF‐κB p65 subunit and expression of p‐IκBα were investigated by Western blotting. Our results showed that 4‐MCPC treatment decreased infarct volume significantly, compared with I/R group (16.8%±7.5% vs. 39.7%±10.9%); it reduced MPO activity (0.43 ± 0.10 vs. 1.00 ± 0.51 U/g) and expression levels of TNF‐α (18.90 ± 3.65 vs. 35.87 ± 4.87 ng/g) and IL‐1β (1.68 ± 0.23 vs. 2.67 ± 0.38 ng/g) in ischemic brain tissues of rats. Further study revealed that 4‐MCPC treatment markedly reduced nuclear translocation of NF‐κB p65 subunit and expression of p‐IκBα in ischemic cerebral cortex. Taken together, our results suggest that 4‐MCPC protects against cerebral I/R injury and displays anti‐inflammatory actions through inhibition of the NF‐κB signal pathway.     

Effects of variation in the human α2A‐ and α2C‐adrenoceptor genes on cognitive tasks and pain perception

Background: The mechanisms underlying interindividual variability in pain perception and cognitive responses are undefined but highly heritable. α_2C‐ and α_2A‐adrenergic receptors regulate noradrenergic activity and are important mediators of pain perception and analgesia. We hypothesized that common genetic variants in these genes, particularly the ADRA2C 322–325 deletion variant, affect pain perception or cognitive responses. Methods: We studied 73 healthy subjects (37 Caucasians and 36 African–Americans) aged 25.4 ± 4.6 years. Pain response to a cold pressor test was measured using a 10 cm visual analog scale and again on the next day, after three infusions of the selective α₂‐agonist dexmedetomidine. Standardized cognitive tests were administered at baseline and after each infusion. The contribution of ADRA2C deletion genotype, dexmedetomidine concentration, and other covariates to pain perception and cognitive responses was determined using multiple linear regression models. Secondary analysis examined the effects of ADRA2A and other ADRA2C variants on pain perception. Results: ADRA2C Del homozygotes had higher pain scores in response to cold at baseline (6.3 ± 1.8 cm) and after dexmedetomidine (5.6 ± 2.2 cm) than insertion allele carriers (4.6 ± 2.1 cm [baseline] and 3.8 ± 1.9 cm [after dexmedetomidine]; adjusted P‐values = 0.019 and 0.004, respectively). Cognitive responses were unrelated to ADRA2C Ins/Del genotype. None of the other ADRA2A and ADRA2C variants was significantly related to cold pain sensitivity before dexmedetomidine; after dexmedetomidine, ADRA2A rs1800038 was marginally associated (P = 0.03). Conclusion: The common ADRA2C del322–325 variant affected pain perception before and after dexmedetomidine but did not affect other cognitive responses, suggesting that it contributes to interindividual variability in pain perception.     

Flow mediated vasodilation and circulating concentrations of high sensitive C‐reactive protein,interleukin‐6 and tumor necrosis factor‐α in normal pregnancy – The Cardiovascular Risk in Young Finns Study

Background: Traditional risk factors such as hyperlipidemia induce a state of inflammation that impairs vascular function. Despite marked maternal hyperlipidemia, endothelial function improves during pregnancy. In non‐pregnant state increased circulating levels of pro‐inflammatory cytokines and high sensitive C‐reactive protein (hsCRP) lead to attenuated flow mediated vasodilation. Relation between endothelial function and pro‐inflammatory cytokines has not been studied thoroughly in pregnancy. The aim of this study was to evaluate the effect of pregnancy on hsCRP and pro‐inflammatory cytokines and their associations with vascular endothelial function. Methods: As part of population‐based, prospective cohort Cardiovascular Risk in Young Finns study conducted in Finland we measured brachial artery flow mediated dilation (FMD) and serum concentrations of hsCRP, interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) in 57 pregnant Finnish women throughout gestation and 62 control women matched for age and smoking. Results: HsCRP‐concentration was greater in pregnancy compared to non‐pregnant controls (median hsCRP 2·52 mg l^?1 versus 1·21 mg l^?1, P<0·001). IL‐6‐concentration was slightly increased in pregnancy compared with the non‐pregnant controls (median 1·66 versus 1·32 mg l^?1, non‐significant [NS]) and TNF‐α‐concentration was slightly decreased in pregnant group (2·11 versus 2·38 pg ml^?1, NS). FMD increased during pregnancy and IL‐6 had a positive correlation to the FMD in pregnancy (R = 0·288, P = 0·031). Conclusions: Improvement of FMD in normal pregnancy was not affected by increase in hsCRP concentration. We found an association with IL‐6 and FMD but we believe that improvement in endothelial function during normal pregnancy is not caused by variation in hsCRP, IL‐6 or TNF‐α.     

Inhibition of transient receptor potential vanilloid‐1 confers neuroprotection,reduces tumor necrosis factor‐alpha,and increases IL‐10 in a rat stroke model

Stroke is a major cause of mortality and long‐term disability in adults. Transient receptor potential vanilloid‐1 (TRPV1) plays a crucial role in neuroinflammation. In this study, the effects of TRPV1 agonist (capsaicin) and antagonist (AMG9810) on cerebral ischemia were investigated. Forty male Wistar rats were assigned to the following experimental groups: sham, vehicle) ischemic), AMG9810 (selective TRPV1 antagonist, 0.5 mg/kg; 3 h after stroke), and capsaicin (1 mg/kg; 3 h after stroke). Stroke was induced by permanent middle cerebral artery occlusion and neurological deficits were evaluated 1, 3, and 7 days after stroke. Then, infarct volume, brain edema, body temperature, mRNA expression of TRPV1, and serum concentrations of tumor necrosis factor‐alpha (TNF‐α) and IL‐10 were measured. Compared to the vehicle group, AMG9810 significantly decreased the infarct volume (P < 0.01). Latency for the removal of sticky labels from the forepaw and the hanging time were significantly decreased and increased, respectively, following administration of AMG9810 (P < 0.01 and P < 0.001 vs. vehicle) 3 and 7 days after stroke. Compared to the sham group, the mRNA expression of TRPV1 was significantly increased in vehicle group (P < 0.01). Administration of AMG9810 significantly increased the anti‐inflammatory cytokine IL‐10 and decreased the inflammatory cytokine TNF‐α (P < 0.05). Moreover, our results indicate that AMG9810 might a promising candidate for the hypothermic treatment of stroke. The findings also suggest a key role for AMG9810 in reducing inflammation after stroke and imply that TRPV1 could be a potential target for the treatment of ischemic stroke.     

Establishment of a cut‐point value of serum TNF‐α levels in the metabolic syndrome

Cardiovascular diseases and type 2 diabetes are the major causes of mortality in Mexico. Metabolic syndrome (MS) is a cluster of factors that increase the risk to develop such diseases. Previous studies have shown that MS is associated with high tumor necrosis factor (TNF‐α) levels. In fact, TNF‐α has been proposed to be a useful marker for clinical diagnosis of inflammation at an early stage. Therefore, we analyzed TNF‐α concentrations in Mexican individuals with or without MS and related these levels to the associated MS components. Clinical, anthropometric, and biochemical data were analyzed in 41 healthy and 39 MS individuals. Individuals were similarly grouped by age and gender.The serum TNF‐α levels measured bya highly sensitive enzyme‐linked immunosorbent assay (ELISA) kit were increased significantly in MS subjects compared with healthy individuals (P<0.001). The assay showed 78.1% sensitivity and 61.5% specificity with a cut‐point level of 1.36 pg/mL. TNF‐α levels higher than the cut‐point value were correlated with insulin resistance indices. These findings support the hypothesis that serum TNF‐α concentration could be a useful marker for early MS diagnosis. Nevertheless, we suggest the establishment of specific cut‐point values in each studied population to evaluate potential clinical applications. J. Clin. Lab. Anal. 23:51–56, 2009. © 2009 Wiley‐Liss, Inc.     

The calcification potential of human MSCs can be enhanced by interleukin‐1β in osteogenic medium

Inflammation is one of the key regulators of the repair process in bone tissues. Current data about the effect of interleukin‐1β (IL‐1β) on MSCs and osteoblasts are conflicting. We investigated the long‐term effect of IL‐1β on direct osteogenic differentiation of hMSCs in vitro. IL‐1β‐stimulated cells showed enhanced proliferation and entered maturation prior to non‐stimulated ones, as monitored by ALP activity. The process of calcification was accelerated during long‐term stimulation of hMSCs with IL‐1β. Since donor variability is a well‐known issue, we suggest a new method to illustrate global changes of a random chosen donor population through collative analysis. We further demonstrate an absorbance assay to evaluate the degree of calcification during in vitro culture of monolayer expanded hMSCs. Our findings support the importance of IL‐1β in osteogenic differentiation of hMSCs in an in vitro monolayer culture model. A new online absorbance assay is a useful method to evaluate the osteogenic differentiation of hMSCs at early stages. These findings will be helpful in optimizing predifferentiation of hMSCs in vitro for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

The profibrotic cytokine transforming growth factor‐β1 increases endothelial progenitor cell angiogenic properties

Summary. Background: Transforming growth factor‐β1 (TGF‐β1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF‐β1 modulation of EPC angiogenic properties. Methods: TGF‐β1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF‐β1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony‐forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGF‐β1 in ECFCs by using short interfering RNA. Results: Total TGF‐β1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF‐β1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF‐β1 receptors. Conclusions: TGF‐β1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF‐β1 may play a role during vascular remodeling in fibrotic disease states via EPCs.     

Granulocyte colony‐stimulating factor produces a decrease in IFNγ and increase in IL‐4 when administrated to healthy donors

Hematopoietic stem cells transplantation (HSCT) is the leading curative therapy for a variety of hematological and hereditary diseases; however, graft versus host disease (GVHD), an immunologic phenomenon that is favored by Th1 cytokines and cytotoxic cells from donors, is present frequently and is one of the most important causes of transplant related mortality. Peripheral blood HSCT is the preferred source of stem cells in almost 100% of the cases of autologous HSCT and in 70% of allogeneic transplants. The best mobilizing agent to get the stem cells out from the bone marrow is the Granulocyte‐Colony Stimulating Factor (G‐CSF). In this work, our main objective was to study a possible correlation between the graft cell dose and the patient's clinical outcome. We evaluated the immunologic changes produced by G‐CSF in the lymphocyte and cytokine profiles in allogeneic HSC donors. HSC from twelve donors were mobilized with G‐CSF at 16 μg/kg/day, for 5 days. Basal Peripheral Blood (BPB), Mobilized Peripheral Blood (MPB), and aphaeresis mononuclear cells (G‐MNC) samples were taken from all donors. Using flow cytometry, we quantified CD19⁺, CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, NK, NKT, DC1, and DC2 cells. Cytokines were determined by ELISA in culture supernatants. CD19⁺ (p = 0.001), DC1 (p < 0.002) and DC2 (p < 0.001) cells were increased in MPB with respect to BPB. An increase in Th2 cytokines such as (IL‐4) and a decrease in Th1 cytokines (IFNγ, IL‐2) were also found in MPB samples. In conclusion, Th1 and Th2 cytokines are relevant in predicting the clinical outcome after allogeneic peripheral blood HSCT. J. Clin. Apheresis 25:181–187, 2010. © 2010 Wiley‐Liss, Inc.     

Mobilization effects of G‐CSF,GM‐CSF,and darbepoetin‐α for allogeneic peripheral blood stem cell transplantation

The effects of GM‐/G‐CSF and darbepoetin‐α on stem cell mobilization were investigated. From February 2005 to March 2007, 30 allogeneic sibling donors were randomly assigned to a G‐CSF group (5 μg/kg/day for 5–7 days) or triple group (GM‐CSF 10 μg/kg/day on 1st and 2nd day, G‐CSF 5 μg/kg/day for 5–7 days, and darbepoetin‐α 40 mg on 1st day). The MNCs and CD34⁺ cells were not different between the two groups, although the doses (×10⁸/kg of recipient body weight) of CD3⁺ cells (3.64 ± 1.75 vs. 2.63 ± 1.36, P = 0.089) and CD8⁺ cells (1.07 ± 0.53 vs. 0.60 ± 0.30, P = 0.006) were lower in the triple group. The engraftments, frequency of RBC transfusions, and hemoglobin recovery were not different between the two groups. The cumulative incidence of overall and Grades II–IV aGVHD was 64.3% vs. 61.1% and 25.9% vs. 27.1% in the G‐CSF and triple regimen group, respectively, whereas the cumulative incidence of cGVHD was 20.8 ± 1.3% and 24.4 ± 1.7%, respectively. In conclusion, the triple regimen did not seem to be superior to G‐CSF alone in terms of the CD34+ cell dose, hemoglobin recovery, and GVHD. However, the CD8+ cell count was significantly lower in the triple regimen group. The role of a lower CD8+ cell count in the graft may need to be elucidated in the future. J. Clin. Apheresis, 2009. © 2009 Wiley‐Liss, Inc.     

Functional association of phosphoinositide‐3‐kinase with platelet glycoprotein Ibα, the major ligand‐binding subunit of the glycoprotein Ib–IX–V complex

Summary. Background: The adhesion receptor glycoprotein (GP)Ib–IX–V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet‐mediated tumor metastasis. The cytoplasmic C‐terminal domain of the ligand‐binding GPIbα subunit contains binding sites for filamin (residues 561–572, critically Phe568/Trp570), 14‐3‐3ζ (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide‐3‐kinase (PI3‐kinase) regulatory subunit, p85. Objectives: We previously showed that, as compared with wild‐type receptor, deleting the contiguous sequence 580–590 or 591–610, but not upstream sequences, of GPIbα expressed as a GPIb–IX complex in Chinese hamster ovary cells inhibited VWF‐dependent Akt phosphorylation, which is used as a read‐out for PI3‐kinase activity. Pulldown experiments using glutathione‐S‐transferase (GST)–p85 or GST–14‐3‐3ζ constructs, and competitive inhibitors of 14‐3‐3ζ binding, suggested an independent association of 14‐3‐3ζ and PI3‐kinase with GPIbα. The objective of this study was to analyze a further panel of GPIbα deletion mutations within residues 580–610. Results: We identified a novel deletion mutant, Δ591–595, that uniquely disrupts 14‐3‐3ζ binding but retains the functional p85/PI3‐kinase association. Deletion of other sequences within the 580–610 region were less discriminatory, and either partially affected p85/PI3‐kinase and 14‐3‐3ζ binding (Δ580–585, Δ586–590, Δ596–600, Δ601–605), or strongly inhibited binding of both proteins (Δ606–610). Conclusions: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14‐3‐3ζ in GPIb‐dependent signaling or platelet functional studies involving truncation of the C‐terminal residues in cell‐based assays and mouse models. The Δ591–595 mutation provides another strategy for determining the function of GPIbα‐associated 14‐3‐3ζ by selective disruption of 14‐3‐3ζ but not p85/PI3‐kinase binding.