Activity In U937 Cells Research Articles

Rs2564978(T) allele associated with severe influenza a disrupts binding site for myeloid differentiation factor PU.1 and reduces <i>CD55/DAF</i> gene promoter activity in macrophages

An inhibitor of the complement system CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity during influenza A infection, as well as with vascular complications in pathologies involving excessive activation of the complement system. Using a luciferase reporter system, we performed functional analysis of the single nucleotide polymorphism rs2564978 located in the promoter of the CD55 gene in human pro-monocytic cell line U937. We have shown a decreased activity in activated U937 cells of the CD55 gene promoter carrying minor rs2564978(T) allele associated with the severe course of influenza A(H1N1)pdm09. Using bioinformatic resources, we determined that transcription factor PU.1 can potentially bind to the CD55 promoter region containing rs2564978 in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was determined by genetic knockdown of PU.1 using small interfering RNAs under specific monocyte activation conditions.

The rs2564978(T) Allele Associated with Severe Influenza A Disrupts the Binding Site for Myeloid Differentiation Factor PU.1 and Reduces CD55/DAF Gene Promoter Activity in Macrophages

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

The rs2564978(T) Allele Associated with Severe Influenza A Disrupts the Binding Site for Myeloid Differentiation Factor PU.1 and Reduces CD55/DAF Gene Promoter Activity in Macrophages

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

In Vitro Effects of Low Doses of β-Caryophyllene, Ascorbic Acid and d-Glucosamine on Human Chondrocyte Viability and Inflammation

β-caryophyllene (BCP), a plant-derived sesquiterpene, has been reported to have anti-inflammatory and antioxidant effects. The purpose of this study is to evaluate the effects of BCP in combination with ascorbic acid (AA) and d-glucosamine (GlcN) against macrophage-mediated inflammation on in vitro primary human chondrocytes. Changes in cell viability, intracellular ROS generation, gene expression of pro-inflammatory mediators, metalloproteinases (MMPs), collagen type II and aggrecan were analyzed in primary human chondrocytes exposed to the conditioned medium (CM) of activated U937 monocytes and subsequently treated with BCP alone or in combination with AA and GlcN. The CM-induced chondrocyte cytotoxicity was reduced by the presence of low doses of BCP alone or in combination with AA and GlcN. The exposure of cells to CM significantly increased IL-1β, NF-κB1 and MMP-13 expression, but when BCP was added to the inflamed cells, alone or in combination with AA and GlcN, gene transcription for all these molecules was restored to near baseline values. Moreover, chondrocytes increased the expression of collagen type II and aggrecan when stimulated with AA and GlcN alone or in combination with BCP. This study showed the synergistic anti-inflammatory and antioxidative effects of BCP, AA and GlcN at low doses on human chondrocyte cultures treated with the CM of activated U937 cells. Moreover, the combination of the three molecules was able to promote the expression of collagen type II and aggrecan. All together, these data could suggest that BCP, AA and GlcN exert a chondro-protective action.

Complexes between C-Reactive Protein and Very Low Density Lipoprotein Delay Bacterial Clearance in Sepsis.

C-reactive protein (CRP) can increase up to 1000-fold in blood and form complexes with very low density lipoproteins (VLDL). These complexes are associated with worse outcomes for septic patients, and this suggests a potential pathological role in sepsis. Complex formation is heightened when CRP is over 200 mg/l and levels are associated with the severity of sepsis and blood bacterial culture positivity. Using a mouse bacteremia model, blood bacterial clearance can be delayed by i.v. injection of CRP-VLDL complexes. Complexes are more efficiently taken up by activated U937 cells in vitro and Kupffer cells in vivo than VLDL alone. Both in vitro-generated and naturally occurring CRP-VLDL complexes reduce phagocytosis of bacteria by activated U937 cells. Fcγ and scavenger receptors are involved and a competitive mechanism for clearance of CRP-VLDL complexes and bacteria is demonstrated. Interaction of phosphocholine groups on VLDL with CRP is the major driver for complex formation and phosphocholine can disrupt the complexes to reverse their inhibitory effects on phagocytosis and bacterial clearance. Increased formation of CRP-VLDL complexes is therefore harmful and could be a novel target for therapy in sepsis.

Proliferation, migration and apoptosis of acute myeloid leukemia cells regulated by mir-23a-3p targeting SMC1A and the mechanism

Objective: To investigate the effects of miR-23a-3p on proliferation, migration and apoptosis on human acute myeloid leukemia (AML) cells by targeting SMC1A. Methods: Microarray analysis was used to screen differentially expressed microRNAs and mRNAs in human AML cells. Real-time fluorescence quantitative PCR (RT-qRCR) was used to detect the expressions of miR-23a-3p and SMCA in human AML cell line U937. TargetScan database was used to analyze the correlation between miR-23a-3p and SMC1A. Double luciferase reporter gene was used to detect the interaction between miR-23a-3p and SMC1A. The effect of miR-23a-3p expression on the proliferation of U937 cells was detected by clonal assay. The migration, apoptosis, cell cycle and caspase-3 activity of U937 cells regulated by miR-23a-3p were detected by cell scratch assay and flow cytometry, respectively. Western blot was used to detect the expressions of Bax and Bcl-2 in U937 cells. Results: Compared with human normal monocyte SC group (1.00), the expression of miR-23a-3p in U937 cells was up-regulated (2.56±0.78) (P<0.01), while the expression of SMC1A was down-regulated (0.48±0.56, P<0.01). miR-23a-3p specifically bond to SMC1A 3'UTR and regulated the expression activity of SMC1A. Overexpression of miR-23a-3p promoted the proliferation and migration of U937 cells and inhibited the apoptosis of U937 cells, while up-regulation of SMC1A inhibited the proliferation and migration of U937 cells and promoted the apoptosis of U937 cells. The percentages of G(0)/G1 phase, G(2)/M phase and S phase cells in the negative control group were (37.48±0.21)%, (16.78±0.18)% and (45.74±0.15)% respectively, and those in the miR-23a-3p mimics group were (19.96±0.11)%, (41.69±0.24)% and (38.24±0.34)%, respectively. The difference was statistically significant (all P<0.05). The proportions of G(0)/G(1) phase, G(2)/M phase and S phase cells in the group of miR-23a-3p mimics+ pcDNA3.1-SMC1A were (36.88±0.21)%, (30.44±0.33)% and (32.88±0.16)%, respectively, without significant difference when compared with those of the miR-23a-3p mimics group (P>0.05). The relative expression levels of Bax and Bcl-2 protein in the negative control group were 0.55±0.45 and 0.31±0.54, respectively. Overexpression of miR-23a-3p inhibited the expression of Bax protein in U937 cells (0.23±0.13, P<0.001), promoted the expression of Bcl-2 protein (0.50±0.23, P<0.01), while SMC1A increased the expression of Bax protein in U937 cells (0.40±0.11, P<0.01), and inhibited the expression of Bcl-2 protein (0.37±0.15). In the negative control group, caspase-3 activity was (25.82±0.89)%. Overexpression of miR-23a-3p inhibited caspase-3 activity in U937 cells (3.64±0.56)%, P<0.01, while up-regulation of SMC1A promoted caspase-3 activity in U937 cells (15.29±0.85)%, P<0.01. Conclusion: miR-23a-3p can inhibit the proliferation and migration and promote apoptosis of human AML cells by targeting SMC1A.

The Biological Effects of Interleukin-17A on Adhesion Molecules Expression and Foam Cell Formation in Atherosclerotic Lesions.

Interleukin-17A (IL-17A), a major effector cytokine secreted by T helper 17 (Th17) cells, is elevated in atherosclerosis lesions. The purpose of this study was to assess the role of IL-17A in the pathogenesis of atherosclerosis. To measure the expression of adhesion molecules, human umbilical vein endothelial cells (HUVECs) and U937 cells were stimulated with IL-17A. Western blot and real-time polymerase chain reaction analyses revealed that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in HUVECs, and very late antigen-4 (VLA-4), lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (MAC-1) expression in U937 cells was upregulated by IL-17A. Furthermore, IL-17A stimulation resulted in mRNA and protein expression of scavenger receptor (LOX-1) in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. Oil Red O also demonstrated that IL-17A enhanced foam cell formation by PMA-activated U937 cells induced by oxidized low-density lipoprotein (ox-LDL), and this enhancement of ox-LDL-induced foam cell formation in IL-17A-treated U937 cells was downregulated by transfection of LOX-1 siRNA. These results indicated that IL-17A induced the expression of adhesion molecules, promoted the adherence of monocytes to vascular endothelial cells. IL-17A also stimulated ox-LDL-induced foam cell formation via upregulation of LOX-1 in activated macrophages. IL-17A may be responsible for the pathogenesis of atherosclerosis by inducing the adhesion of leukocytes to vascular endothelium and foam cell formation.

Pushing the limits of detection for proteins secreted from single cells using quantum dots.

Single cell analysis methods are increasingly being utilized to investigate how individual cells process information and respond to diverse stimuli. Soluble proteins play a critical role in controlling cell populations and tissues, but directly monitoring secretion is technically challenging. Microfabricated well arrays have been developed to assess secretion at the single cell level, but these systems are limited by low detection sensitivity. Semiconductor quantum dots (QD) exhibit remarkably bright and photostable luminescence signal, but to date they have not been evaluated in single cell secretion studies using microfabricated well arrays. Here, we used QDs in a sandwich immunoassay to detect secretion of the soluble cytokine tumor necrosis factor-α (TNF-α) from single cells. To enhance detection sensitivity, we employed two different strategies. First, we used a unique single QD imaging approach, which provided a detection threshold (180 attomolar) that was >100-fold lower than previously reported results using QDs. We also amplified QD binding to each captured TNF-α molecule using the bioorthogonal cycloaddition reaction between trans-cyclooctene and tetrazine, which further lowered detection threshold to 60 attomolar. This is 6 orders of magnitude more sensitive than organic fluorophores that have been used for single cell secretion studies, and far surpasses single molecule resolution within sub-picoliter microwells that are used to assess single cell secretion. Finally, single cell secretion studies were performed using phorbol 12-myristate 13-acetate (PMA) differentiated and lipopolysaccharide (LPS) activated U-937 cells. TNF-α secretion was detected from 3-fold more single cells using the QD-based method in comparison to rhodamine, which was accomplished by extending sensitivity into the range of ∼2 to 10 000 molecules captured per microwell. In future work, we will apply this technique to assess immune cell secretion dynamics under diverse stimuli and disease settings. We will also incorporate multiplexing capabilities to evaluate the secretome at the resolution of single molecules.

Design and synthesis of novel 1,3,5-triphenyl pyrazolines as potential anti-inflammatory agents through allosteric inhibition of protein kinase Czeta (PKCζ).

Much light has been shed on the vital role of protein kinase Czeta (PKCζ) in NF-κB activation and the potential use of PKCζ inhibitors as anti-inflammatory agents. We previously reported a series of 1,3,5-trisubstituted pyrazolines as potent and selective allosteric inhibitors of PKCζ; in that series of compounds, the phenolic OH at the 5-phenyl was essential for binding to the PKCζ PIF pocket. In the present study, we surprisingly found that replacing it by a halogen and at the same time moving the OH to the 3-phenyl still resulted in active compounds. An extension of this class of compounds with a new focused library is presented herein, where the phenolic OH at the 5-phenyl, which was reported to be an irreplaceable feature for activity, was moved to the 3-phenyl and replaced by halogen. The new set of compounds maintained the same level of potency against PKCζ and selectivity against PKC isoforms, and showed reduced potency against the PIF pocket mutant PKCζ[Val297Leu]. Of note, the repositioning of the key functional groups resulted in a marked enhancement of cellular potency. One of the most potent new PKCζ inhibitors, 2h, was able to suppress NO production in RAW 264.7 macrophage cells with 8 times higher efficacy than the previous series, and inhibited the NF-κB transcriptional activity in U937 cells with a sub-micromolar IC50.

Leukemia-associated gene MLAA-34 reduces arsenic trioxide-induced apoptosis in HeLa cells via activation of the Wnt/β-catenin signaling pathway.

Our laboratory previously used the SEREX method in U937 cells and identified a novel leukemia-associated gene MLAA-34, a novel splice variant of CAB39L associated with acute monocytic leukemia, that exhibited anti-apoptotic activities in U937 cells. Whether MLAA-34 has an anti-apoptotic role in other tumor cells has not yet been reported. We explored whether MLAA-34 exhibited anti-apoptotic effects in HeLa cervical cancer cells and the possible mechanism of action. We generated a HeLa cell line stably expressing MLAA-34 and found that MLAA-34 overexpression had no effect on the growth, apoptosis and cell cycle of HeLa cells. However, upon treatment with arsenic trioxide (ATO) to induce apoptosis, the cell viability and colony formation ability of ATO-treated MLAA-34 stable HeLa cells were significantly higher than that of ATO-treated controls, and the apoptosis rate and proportion of G2/M cells also decreased. We found that ATO treatment of HeLa cells resulted in significant decreases in the expression of β-catenin mRNA and protein and the downstream target factors c-Myc, cyclin B1, and cyclin D1 in the Wnt signaling pathway. Notably, ATO-treated MLAA-34 stable HeLa cells showed a significant reduction in the ATO-mediated downregulation of these factors. In addition, MLAA-34 overexpression significantly increased the expression of nuclear β-catenin protein in ATO-treated cells compared with HeLa cells treated only with ATO. Thus, here we have found that the Wnt/β-catenin signaling pathway is involved in ATO-induced apoptosis in HeLa cells. MLAA-34 reduces ATO-induced apoptosis and G2/M arrest, and the anti-apoptotic effect may be achieved by activating the Wnt/β-catenin signaling pathway in HeLa cells.

Differential effect of p38 and MK2 kinase inhibitors on the inflammatory and toxicity biomarkers in vitro.

Many inflammatory responses including chemotaxis, production of nitric oxide, and modulation of pro-inflammatory cytokines in immunological cells are mediated by p38MAPK. Due to its pivotal role, p38MAPK has been extensively explored as a molecular target for inhibition of chronic inflammation; however, it has not been successful so far due to serious toxicity issues. Among several downstream substrates of p38, mitogen-activated protein kinase-activated protein kinase 2 (MK2) has been reported to be a direct and essential downstream component in regulation of innate immune and inflammatory responses. Thus, in this study, we aimed to understand relative molecular differences between p38 and MK2 kinase inhibition in terms of a comparative anti-inflammatory potential along with molecular regulation of toxicity biomarkers such as Phospho c-Jun N-Terminal Kinase (pJNK), caspase-3, and hepatic enzyme levels in relevant human cells in vitro. Both p38 and MK2 inhibitors attenuated lipopolysaccharide-induced pro-inflammatory biomarkers expression. In addition, both these kinase inhibitors inhibited release of Th1 and Th17 cytokines in phytohemagglutinin-induced cells with MK2 inhibitor showing a better potency for inhibition of Th1 cytokine release, interferon-γ. In the mechanistic differentiation studies, p38 inhibitors displayed an increase in pJNK and caspase-3 activity in U937 cells and elevation in aspartate transaminase enzyme in HepG2 cells, whereas MK2 inhibitor did not show such adverse toxic effects. Taken together, inhibition of MK2 kinase can be a relatively preferred strategy as an anti-inflammatory therapy over direct inhibition of p38 kinase in p38MAPK pathway.

Synthesis and evaluation of the anti-proliferative activity of diaryl-3-pyrrolin-2-ones and fused analogs

Analogs containing a central 3-pyrrolin-2-one core with different methoxyphenyl and/or indole substituents were prepared and tested for anti-proliferative activity in U-937 cells. The most efficacious analogs were non-rigid, (non-fused) contained methoxyaryl groups located at the 4-position, and contained either methoxyaryl or indole groups located at the 3-position. Both the number of methoxy groups contained in the substituents and the particular location of the indole rings with respect to the lactam carbonyl had significant affects on anti-proliferative activity. This work provides a framework to better understand structure-activity relationships for inducing anti-proliferative activity in diaryl heterocyclic scaffolds.

Staurosporine enhances ATRA-induced granulocytic differentiation in human leukemia U937 cells via the MEK/ERK signaling pathway.

Although all-trans retinoic acid (ATRA) is regarded as a prominent example of differentiation therapy, it is not effective for the treatment of other subtypes of acute myeloid leukemia(AML) beyond acute promyelocytic leukemia (APL). Therefore, new strategies need to be explored to extend the efficacy of ATRA-based therapy to non-APL AML patients. In the present study, staurosporine, a protein kinaseC(PKC) pan-inhibitor, exhibited synergism with ATRA to promote granulocytic differentiation in poorly ATRA-sensitive U937 cells but not in ATRA unresponsive K562 and Kasumi cells. Staurosporine or the combined treatment did not affect PKC activity in U937 cells. Moreover, other selective PKC inhibitors, UCN-01, Go6976 or rottlerin failed to enhance ATRA‑induced granulocytic differentiation in U937 cells. Therefore, staurosporine-enhanced ATRA-induced granulocytic differentiation in U937 cells may be independent of PKC. Staurosporine activated mitogen‑activated protein kinase kinase(MEK) and extracellular signal‑regulated kinase(ERK). Meanwhile, staurosporine also enhanced ATRA-promoted upregulation of the protein level of CCAAT/enhancer‑binding proteinβ(C/EBPβ) and C/EBPε in U937 cells. Furthermore, blockade of MEK activation suppressed staurosporine‑enhanced differentiation as well as the elevated protein level of C/EBPs. Taken together, we concluded that staurosporine enhanced ATRA‑induced granulocytic differentiation in U937 cells via MEK/ERK-mediated modulation of the protein level of C/EBPs.

CARD15 gene overexpression reduces effect of etanercept, infliximab, and adalimumab on cytokine secretion from PMA activated U937 cells

Crohn's disease (CD), a subcategory of inflammatory bowel disease, is an immune-related disorder characterized by inflammation of the gastrointestinal mucosa, which can take place in any region along the alimentary tract. The most important gene involved in the etiology of CD is NOD2/CARD15 located on chromosome 16. It has been shown that CARD15 is overexpressed in monocytes of CD patients. The common treatment for the disease is anti-TNF-alpha drugs, the most hopeful of which are probably infliximab and etanercept. Infliximab rapidly reduces signs and symptoms of active Crohn’s disease. In contrast, etanercept shows no such effect.In the present study, we evaluated the effects of the CARD15 gene overexpression in monocytic cell line U937 in the production of anti-inflammatory cytokine, IL-10, and proinflammatory cytokine, Il-1 beta, produced after incubation with infliximab, adalimumab, and etanercept separately.Our results show that infliximab and adalimumab significantly decreased IL-10 and IL-1beta secretion levels. However, etanercept inhibition of secretion was less compared with infliximab or adalimumab.In all three cases, suppression of cytokine production is reduced by CARD15 overexpression.

Role of diallyl disulfide-mediated cleavage of c-Myc and Sp-1 in the regulation of telomerase activity in human lymphoma cell line U937

ObjectiveGarlic (Allium sativum) has been considered a wonder herb for years with a reputation of disease prevention. Telomerase, a ribonucleoprotein enzyme responsible for telomere integrity, is strongly up-regulated in different types of cancers. The aim of this study was to reveal the role of diallyl disulfide (DADS), an organosulfur component of garlic, on telomerase activity in human lymphoma with an emphasis on key transcription factors c-Myc and Sp-1. MethodsHuman lymphoma cell line U937 was used as model cell line. Telomerase activity was measured by telomerase repeat amplification protocol assay, levels of related proteins and mRNAs were measured by Western blot and reverse transcriptase polymerase chain reaction, respectively. Moreover, in vitro binding assay was performed using radiolabeled double-stranded DNA having specific sequences to detect involvement of transcription factors in DADS-dependent modulation of telomerase activity. ResultsThe present study demonstrated DADS-mediated decrease in telomerase activity in U937 cells with concomitant transcriptional down-regulation of human telomerase reverse transcriptase (hTERT) that is caused by reduced binding of c-Myc and Sp-1 to their respective binding sites on hTERT promoter. Lowering of DNA-binding activity of c-Myc and Sp-1 due to DADS treatment is caused by the deactivation of these transcription factors due to cleavage. Additionally, Mad1—the repressor protein of hTERT expression—is also overexpressed in DADS-treated U937 cells. ConclusionsThese findings strongly suggest that DADS down-regulate telomerase activity through c-Myc-, Sp-1-, and Mad1-dependent transcriptional down-regulation of hTERT.

Delivery of lipid micelles into infarcted myocardium using a lipid-linked matrix metalloproteinase targeting peptide.

There is a great need for delivery strategies capable of efficiently localizing drugs to the damaged myocardium that do not require direct intramyocardial injection of therapeutic molecules. In the work discussed here, we exploited the myocardium-specific upregulation of matrix metalloproteinases (MMPs) that occurs during myocardium remodeling by designing a micellar vehicle containing an MMP-targeting peptide (MMP-TP). The binding of MMP-TP to MMP was evaluated with purified MMP-2 protein and U-937 cells induced to overexpress MMP. Inhibition of MMP-2 activity was not observed in the presence of unmodified micelles but was pronounced at a 5 mol % MMP-TP ligand density. In a FACS analysis, MMP-TP micelles containing 5 mol % of the MMP-targeting peptide showed ∼10-fold higher binding to activated U937 cells than plain micelles and micelles containing a control peptide with two amino acid replacements. MMP-TP-micelles and plain micelles were injected intravenously into C57BL/6 mice 1, 3, and 7 days after the induction of a myocardial infarction (MI). Immunohistochemistry performed on heart tissue sections revealed that MMP-TP-micelles colocalize with both MMP and infiltrating macrophages. MMP-TP micelles showed significantly enhanced accumulation to the necrotic area of the heart after MI on days 3 and 7 when compared to plain micelles and negative control peptide micelles. This is coincident with the measured temporal profile of MMP gene expression in the heart after MI. These results suggest that MMP-TP micelles are candidates for the development of targeted regenerative heart therapeutics because of their ability to target the infarcted myocardium in a MMP dependent manner.

Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.

BackgroundLPS-binding protein (LBP) and its ligand CD14 are located upstream of the signaling pathway for LPS-induced inflammation. Blocking LBP and CD14 binding might prevent LPS-induced inflammation. In previous studies, we obtained a peptide analog (MP12) for the LBP/CD14 binding site and showed that this peptide analog had anti-endotoxin activity. In this study, we used in vitro directed evolution for this peptide analog to improve its in vivo and in vitro anti-endotoxin activity.MethodsWe used error-prone PCR (ep-PCR) and induced mutations in the C-terminus of LBP and attached the PCR products to T7 phages to establish a mutant phage display library. The positive clones that competed with LBP for CD14 binding was obtained by screening. We used both in vivo and in vitro experiments to compare the anti-endotoxin activities of a polypeptide designated P1 contained in a positive clone and MP12.Results11 positive clones were obtained from among target phages. Sequencing showed that 9 positive clones had a threonine (T) to methionine (M) mutation in amino acid 287 of LBP. Compared to polypeptide MP12, polypeptide P1 significantly inhibited LPS-induced TNF-α expression and NF-κB activity in U937 cells (P<0.05). Compared to MP12, P1 significantly improved arterial oxygen pressure, an oxygenation index, and lung pathology scores in LPS-induced ARDS rats (P<0.05).ConclusionBy in vitro directed evolution of peptide analogs for the LBP/CD14 binding site, we established a new polypeptide (P1) with a threonine (T)-to-methionine (M) mutation in amino acid 287 of LBP. This polypeptide had high anti-endotoxin activity in vitro and in vivo, which suggested that amino acid 287 in the C-terminus of LBP may play an important role in LBP binding with CD14.

MIF inhibits monocytic movement through a non-canonical receptor and disruption of temporal Rho GTPase activities in U-937 cells

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that was initially identified by its ability to inhibit the movement of macrophages. Cell migration is a highly complex process involving changes to the cytoskeleton and cell adhesion molecules, and is regulated by the Rho GTPases. A simple model using human monocytic U-937 cells to elicit the classic MIF response was implemented to examine the mechanism of MIF-induced migration inhibition. Our results demonstrate that MIF inhibits migration of these U-937 cells through a non-canonical receptor, CXCR4, in the absence of the putative primary MIF receptor CD74. Migration inhibition is dependent upon a series of temporal perturbations of the activities of the Rho GTPases: initial activation followed by subsequent inactivation of RhoA, inactivation of Rac1, and cyclic activation of Cdc42. MIF-mediated changes in the activities of the Rho GTPases jointly contributed to migration inhibition in these cells. Collectively, these data suggest that the MIF-mediated migration inhibition is mediated by the outcome of G-protein signaling, and in less adherent cells such as those of the monocyte/macrophage lineage, RhoA directly affects net translocation through its ability to induce cell body contraction. These findings demonstrate that CXCR4 can mediate MIF signaling in the absence of CD74 in addition to serving as a MIF co-receptor along with CD74. These results correlate MIF activity to specific and sequential Rho GTPase activity perturbations, and given that CXCR4 functions in numerous processes, suggests potential roles for the modulation of cell movement in those events including development, cell survival and viral infection.

Low-level laser therapy suppresses the oxidative stress-induced glucocorticoids resistance in U937 cells: Relevance to cytokine secretion and histone deacetylase in alveolar macrophages

Oxidative stress is present in severe asthma and contributes to the low response to corticoids through the downregulation of histone deacetylase (HDAC) and the increase of cytokines. Low-level laser therapy (LLLT) has been proven to be an anti-inflammatory. Thus, we investigated the laser effect on lipopolysaccharide (LPS)-induced cytokine secretion and HDAC activity in U937 cells under oxidative stress. U937 cells activated with oxidative stress were treated with dexamethasone (dexa) or laser. Cytokines and phosphoinositide 3-kinase (PI3K) were measured by ELISA whilst the HDAC was detected through colorimetric assay. LPS activated- U937 cells cytokines secretion increased with H2O2 (hydrogen peroxide) as well as with TSA (trichostatin). The HDAC activity in activated U937 cells was decreased. LLLT and dexa inhibited the LPS-stimulated U937 cells cytokines, but dexa effect disappeared with H2O2. With TSA, the LLLT was less effective on H2O2/LPS stimulated- U937 cells cytokines. Dexa failed on H2O2/LPS- induced HDAC, while LLLT restored the HDAC and the dexa effect. LLLT plus prostaglandin E2 (PGE2) increased cyclic adenosine monophosphate (cAMP) and potentiated the laser action on oxidative stress-induced cytokine. LLLT reduced the PI3K and its effects on cytokine and HDAC was suppressed with LY294002. In situations of corticoid resistance, LLLT acts decreasing the cytokines and HDAC through the activation of the protein kinase A via the inhibition of PI3K.

Experimental and Computational Studies on Newly Synthesized Resveratrol Derivative: A New Method for Cancer Chemoprevention and Therapeutics?

Nature has been a provenance of medicinal agents for thousands of years. Resveratrol (RESL) is a naturally occurring polyphenolic compound in food stuffs such as peanuts, seeds, berries, grapes, and beverages (red wine). RESL has received significant attention due to a plethora of in vitro and in vivo reports on its cancer chemopreventive and therapeutic properties. In the present study, diacetate RESL derivative (RESL43) was synthesized. The RESL43 displayed potent cytotoxicity and triggered apoptosis in U937 cells as evidenced by poly (ADP-ribose) polymerase (PARP) cleavage, DNA fragmentation, morphological changes, and activation of FasR and FasL genes. The electrophoretic mobility shift assay revealed the suppression NFkB activity in U937 cells after treatment with RESL43 in corroboration with the deactivation of NFkB dependent genes such as IL-8, TNFR, and TNFα. Furthermore, molecular docking and dynamics studies have shown that RESL and RESL43 might exert their inhibitory activity on NFkB by altering the intramolecular binding abilities between DNA and NFkB. Taken together, RESL43 can have greater putative activity than parental RESL in a context of cancer chemoprevention and therapeutics. We suggest that the diacetate resveratrol derivative RESL43 warrants further evaluation in preclinical and clinical bridging studies in the near future.