Peripheral blood mononuclear cells are hypomethylated in active rheumatoid arthritis and methylation correlates with disease activity.

OBJECTIVE: Epigenetic modifications are dynamic and influence cellular disease activity. The aim of this study was to investigate global DNA methylation in peripheral blood mononuclear cells (PBMCs) of RA patients to clarify whether global DNA methylation pattern testing might be useful in monitoring disease activity as well as the response to therapeutics. METHODS: Flow cytometric measurement of 5-methyl-cytosine (5'-mC) was established using the cell line U937. In the subsequent prospective study, 62 blood samples were investigated, including 17 healthy donors and 45 RA patients at baseline and after 3 months of treatment with methotrexate, the IL-6 receptor inhibitor sarilumab, and Janus kinase inhibitors. Methylation status was assessed with an anti-5'-mC antibody and analysed in PBMCs and CD4+, CD8+, CD14+ and CD19+ subsets. Signal intensities of 5'-mC were correlated with 28-joint DASs with ESR and CRP (DAS28-ESR and DAS28-CRP). RESULTS: Compared with healthy individuals, PBMCs of RA patients showed a significant global DNA hypomethylation. Signal intensities of 5'-mC correlated with transcription levels of DNMT1, DNMT3B and MTR genes involved in methylation processes. Using flow cytometry, significant good correlations and linear regression values were achieved in RA patients between global methylation levels and DAS28-ESR values for PBMCs (r = -0.55, P = 0.002), lymphocytes (r = -0.57, P = 0.001), CD4+ (r = -0.57, P = 0.001), CD8+ (r = -0.54, P = 0.001), CD14+ (r = -0.49, P = 0.008) and CD19+ (r = -0.52, P = 0.004) cells. CONCLUSIONS: The degree of global DNA methylation was found to be associated with disease activity. Based on this novel approach, the degree of global methylation is a promising biomarker for therapy monitoring and the prediction of therapy outcome in inflammatory diseases.

Rapamycin Re-Directs Lysosome Network, Stimulates ER-Remodeling, Involving Membrane CD317 and Affecting Exocytosis, in Campylobacter Jejuni-Lysate-Infected U937 Cells.

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain-Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER-lysosome and ER-mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells.

MDM2 inhibition-mediated autophagy contributes to the pro-apoptotic effect of berberine in p53-null leukemic cells.

AIMS: Berberine (BBR) is reported to induce apoptosis and inhibit migration of leukemic cells, but the underlying pharmacological mechanisms have not been fully revealed. This study aims to investigate the possible mechanisms from the perspective of autophagy. MAIN METHODS: P-53-null leukemic cell lines Jurkat and U937 were used for the in vitro study. MDC staining was used for observation of autophagy in leukemic cells, and Western blot analysis was for determination of the expression levels of autophagy-associated proteins. Apoptosis of the leukemic cells was detected by flow cytometry. Cellular location of MDM2 was observed with immunofluorescence staining. Ubiquitination of MDM2 was assessed by immunoprecipitation. Male 6-8-week-old NOD/SCID mice were used for evaluating the effect of BBR on chemotherapy sensitivity in vivo. KEY FINDINGS: BBR induced autophagy in p53-null leukemic cells, which was inhibited by autophagy inhibitors 3-methyladenine. 3-methyladenine also inhibited BBR-induced apoptosis in leukemic cells. In addition, BBR not only decreased MDM2 mRNA expression, but also enhanced MDM2 self-ubiquitination in leukemic cells. Forced overexpression of MDM2 reversed the effect of BBR on autophagy and apoptosis. Furthermore, BBR promoted doxorubicin-induced autophagy and cell death in the leukemic cells and overexpression of MDM2 suppressed these effects. In vivo, BBR combined with doxorubicin achieved better therapeutic effect than doxorubicin alone. SIGNIFICANCE: MDM2 inhibits autophagy and apoptosis in leukemic cells in a p53-independent manner. BBR induces autophagy in p53-null leukemic cells through downregulating MDM2 expression at both transcriptional and post-transcriptional levels, which may contribute to the anti-cancer effect of BBR in leukemia.

Propofol attenuates monocyte-endothelial adhesion via modulating connexin43 expression in monocytes.

AIMS: Monocyte-endothelial adhesion is considered to be the primary initiator of inflammatory vascular diseases, such as atherosclerosis. Connexin 43 (Cx43) has been reported to play an important part in this process, however, the underlying mechanisms are not fully understood. Intravenous anesthetics, propofol is commonly used in the perioperative period and in the intensive care unit, and considered to have good anti-inflammatory and antioxidant effects. Thus, we speculate that propofol could influence monocyte-endothelial adhesion, and explore whether its possible mechanism is relative with Cx43 expression in U937 monocytes influencing cell adhesion of U937 monocytes to human umbilical vein endothelial cells (HUVEC). MAIN METHODS: Cx43-siRNAs or pc-DNA-Cx43 were used to alter Cx43 expression in U937 monocytes. Propofol was given as pretreatments to U937 monocytes. Then, cell adhesion, ZO-1, LFA-1, VLA-4, COX and MCP-1 were determined. PI3K/AKT/NF-κB signaling pathway was explored to clarify the possible mechanism. KEY FINDINGS: Alternation of Cx43 expression affects cell adhesion and adhesion molecules significantly, such as ZO-1, LFA-1, VLA-4, COX-2 and MCP-1, the mechanism of which is relative with Cx43 influencing the activation of PI3K/AKT/NF-κB signaling pathway. Preconditioning with propofol at its clinically relevant anesthesia concentration attenuates cell adhesion. Propofol not only decreases Cx43 expression in U937 monocytes, but also depresses the activation of PI3K/AKT/NF-κB signaling pathway. SIGNIFICANCE: Modulation Cx43 expression in U937 monocytes could affect cell adhesion via regulating the activation of PI3K/AKT/NF-κB signaling pathway. Propofol attenuates cell adhesion via inhibiting Cx43 and its downstream signaling pathway of PI3K/AKT/NF-κB.

The In Vitro Activity of Angelica archangelica L. Essential Oil on Inflammation.

The use of herbs with medicinal value and biomedical effects has increased tremendously in the last years. However, inadequate basic knowledge of their mode of action is the main issue related to phytotherapy, although they have shown promising potential. To provide insights into these important issues, we tested here on appropriate in vitro models the efficacy of Angelica archangelica essential oil (Aa-EO) for anti-inflammatory properties. The results demonstrated that Aa-EO induced significant apoptosis and necrosis at high doses in U937 cells. We used nontoxic concentrations to treat for anti-inflammatory capacity. The results also demonstrated a decreased proinflammatory cytokine interleukin-6 level in human umbilical vein endothelial cells, as senescence in vitro model, when cells are challenged with lipopolysaccharide (LPS), one of the most powerful proinflammatory inducer in the presence of Aa-EO. In addition, down expression of miR-126 and miR-146a (inflammamirs) produced by LPS stimulation was reverted by Aa-EO simultaneous treatment. These results provide noteworthy basis for the development/formulation of new drugs for future clinical uses and new food products or dietary supplements for contrasting inflammation.

Low-dose ionizing radiation exposure represses the cell cycle and protein synthesis pathways in in vitro human primary keratinocytes and U937 cell lines.

The effects of the high-dose ionizing radiation used in radiotherapy have been thoroughly demonstrated in vitro and in vivo. However, the effects of low-dose ionizing radiation (LDIR) such as computed tomography-guided biopsies and X-ray fluoroscopy on skin cells remain controversial. This study investigated the molecular effects of LDIR on the human primary keratinocytes (HPKs) and U937 cells, monocytes-like cell lines. These cells were exposed to 0.1 Gray (Gy) X-ray as LDIR. The modulation of transcription was assessed using a cDNA array, and the protein expression after LDIR exposure was investigated using isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis at 24 hours. These effects were confirmed by immunoblotting analysis. The direct effects of LDIR on the U937 cells and HPKs and the bystander effects of irradiated HPKs on U937 cells were also investigated. LDIR downregulated c-Myc in both U937 cells and HPKs, and upregulated the p21WAF1/CIP1 protein expression in U937 cells along with the activation of TGFß and protein phosphatase 2A (PP2A). In HPKs, LDIR downregulated the mTOR signaling with repression of S6 and 4EBP1 activation. Similar changes were observed as bystander effects of LDIR. Our findings suggest that LDIR inhibits protein synthesis and induces the cytokines activation associated with inflammation via direct and bystander effects, which might recapitulate the effects of LDIR in inflammated skin structures.

Hydroquinone-induced FOXP3-ADAM17-Lyn-Akt-p21 signaling axis promotes malignant progression of human leukemia U937 cells.

Hydroquinone (1,4-benzenediol; HQ), a major marrow metabolite of the leukemogen benzene, has been proven to evoke benzene-related hematological disorders and myelotoxicity in vitro and in vivo. The goal of the present study was to explore the role of FOXP3 in HQ-induced malignant progression of U937 human leukemia cells. U937 cells were treated with 5 µM HQ for 24 h, and the cells were re-suspended in serum-containing medium without HQ for 2 days. The same procedure was repeated three times, and the resulting U937/HQ cells were maintained in cultured medium containing 5 µM HQ. Proliferation and colony formation of U937/HQ cells were notably higher than those of U937 cells. Ten-eleven translocation methylcytosine dioxygenase-mediated demethylation of the Treg-specific demethylated region in FOXP3 gene resulted in higher FOXP3 expression in U937/HQ cells than in U937 cells. FOXP3-induced miR-183 expression reduced ß-TrCP mRNA stability and suppressed ß-TrCP-mediated Sp1 degradation, leading to up-regulation of Sp1 expression in U937/HQ cells. Sp1 up-regulation further increased ADAM17 and Lyn expression, and ADAM17 up-regulation stimulated Lyn activation in U937/HQ cells. Moreover, U937/HQ cells showed higher Lyn-mediated Akt activation and cytoplasmic p21 expression than U937 cells did. Abolishment of Akt activation decreased cytoplasmic p21 expression in U937/HQ cells. Suppression of FOXP3, ADAM17, and Lyn expression, as well as Akt inactivation, repressed proliferation and clonogenicity of U937/HQ cells. Together with the finding that cytoplasmic p21 shows anti-apoptotic and oncogenic activities in cancer cells, the present data suggest a role of FOXP3/ADAM17/Lyn/Akt/p21 signaling axis in HQ-induced hematological disorders.

SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights.

SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed.

The Daiokanzoto (TJ-84) Kampo Formulation Reduces Virulence Factor Gene Expression in Porphyromonas gingivalis and Possesses Anti-Inflammatory and Anti-Protease Activities.

Kampo formulations used in Japan to treat a wide variety of diseases and to promote health are composed of mixtures of crude extracts from the roots, bark, leaves, and rhizomes of a number of herbs. The present study was aimed at identifying the beneficial biological properties of Daiokanzoto (TJ-84), a Kampo formulation composed of crude extracts of Rhubarb rhizomes and Glycyrrhiza roots, with a view to using it as a potential treatment for periodontal disease. Daiokanzoto dose-dependently inhibited the expression of major Porphyromonas gingivalis virulence factors involved in host colonization and tissue destruction. More specifically, Daiokanzoto reduced the expression of the fimA, hagA, rgpA, and rgpB genes, as determined by quantitative real-time PCR. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to evaluate the anti-inflammatory properties of Daiokanzoto. Daiokanzoto attenuated the P. gingivalis-mediated activation of the NF-κB signaling pathway. It also reduced the secretion of pro-inflammatory cytokines (IL-6 and CXCL8) by lipopolysaccharide-stimulated oral epithelial cells and gingival fibroblasts. Lastly, Daiokanzoto, dose-dependently inhibited the catalytic activity of matrix metalloproteinases (-1 and -9). In conclusion, the present study provided evidence that Daiokanzoto shows potential for treating and/or preventing periodontal disease. The ability of this Kampo formulation to act on both bacterial pathogens and the host inflammatory response, the two etiological components of periodontal disease, is of high therapeutic interest.

Polymeric nanoparticles containing combination antiretroviral drugs for HIV type 1 treatment.

The use of combination antiretroviral nanoparticles (cART NPs) was investigated as a novel treatment approach for the inhibition of HIV-1 replication. We developed nanoparticles of biodegradable polymer, poly-(dl-lactide-co-glycolic acid; PLGA) containing efavirenz (EFV) and boosted lopinavir (lopinavir/ritonavir; LPV/r) by a high-pressure homogenization method. The method resulted in >79% drug entrapment efficiency for each of the three drugs. The average size of cART NPs was 138.3±55.4 nm as measured by dynamic light scanning, confirmed by scanning electron microscopy (SEM) with an average surface charge of -13.7±4.5. Lissamine-rhodamine-labeled fluorescent PLGA NPs exhibited efficient uptake in nonimmune (HeLa cells) and immune (H9 T cells) cells as measured by confocal microscopy. Cells treated with cART NPs resulted in minimal loss of cell viability over 28 days. Subcellular fractionation studies demonstrated that HIV-1-infected H9 monocytic cells treated with cART NPs contained significantly (p<0.05) higher nuclear, cytoskeleton, and membrane antiretroviral drug levels compared to cells treated with drug solutions alone. Finally, cART NPs efficiently inhibited HIV-1 infection and transduction. The IC50 for each of the three drugs in the cART NPs was <31 nM. These experiments demonstrate the efficacy of a novel PLGA NPs formulation for the delivery of cART to inhibit HIV-1 replication.

[Flow cytometry model for the detection of neutralizing antibodies against of IFN-ß]. / Modelo de detección de anticuerpos neutralizantes contra IFN-ß mediante citometría de flujo.

INTRODUCTION: Interferon beta (IFN-ß) is a treatment for relapsing remitting multiple sclerosis. However, the therapeutic use of recombinant proteins induces a humoral immunologic response resulting in the induction of binding (BAb) or neutralizing (NAb) antibodies against the biological product. The presence of neutralizing antibodies has been associated with decreased IFN-ß treatment efficacy. MATERIALS AND METHODS: Two tumor cell lines (K562 and U937) were cultivated with human recombinant IFN-ß1a at different concentrations and lengths of time in order to measure the expression of intracellular ISG15, an inducible molecule in the IFN-ß1a signaling cascade. Blood was obtained from non-immunized and IFN-ß1a immunized (100,000 IU) New Zealand rabbits. The presence of BAb was evaluated by ELISA. For NAb detection, sera 1:20 dilution were added to the IFN-ß1a-stimulated cell lines, and ISG15 expression was evaluated by flow cytometry. RESULTS: K562 cells provided the better cell line for the assay, stimulated with a dose of 1,000 IU of IFN-ß1a, and a 1:100 dilution for the primary antibody and a 1:200 dilution for the secondary antibody. ISG15 expression was compared between cells alone or cultivated with IFN-ß1a. Mean fluorescence intensity (MFI) for ISG-15 expression median was 198 arbitrary units (AU) with interquartile ranges of 173-231 AU for non-stimulated cells and 430 AU with interquartile ranges of 316-611.5 AU for IFN-ß1a stimulated cells (p<0.01). Immunized rabbit sera decreased the expression of ISG-15 in K562 cells stimulated with IFN-ß1a, whereas non-immunized rabbit sera did not. CONCLUSIONS: This rabbit model demonstrates that ISG15 expression evaluated with flow cytometry can be used as a detection assay for NAb.

Opisthorchis viverrini-antigen induces expression of MARCKS during inflammation-associated cholangiocarcinogenesis.

Myristoylated alanine rich C kinase substrate (MARCKS) has been implicated in PKC-mediated membrane-cytoskeleton alterations that underlie lipopolysaccharide (LPS)-induced macrophage responses. MARCKS is postulated to be involved in inflammation-associated CCA based on its overexpression in cholangiocarcinoma (CCA) and inflammatory cells. The aims of this study were to investigate localization patterns of MARCKS in hamster and human tissue during cholangiocarcinogenesis and to examine the involvement of MARCKS in inflammation. MARCKS protein expression was found prominently in inflammatory cells of Opisthorchis viverrini-treated as well as O. viverrini plus N-nitrosodimethylamine (NDMA)-treated hamsters from week 2 to week 3 of treatment. The positive signal decreased during week 4 to week 12, then increased again at week 26 when CCA developed. At the last time point the expression of MARCKS was observed in both cancer and inflammatory cells. MARCKS protein expression was also found in inflammatory cells, including macrophages in human CCA tissues. O. viverrini excretory/secretory products or worm antigen induced MARCKS mRNA and protein expression in a dose- and time-dependent manner in the human U937 macrophage cell line. The relative mRNA expression of MARCKS in white blood cells of O. viverrini-infected patients was significantly higher than in healthy subjects (P = 0.02). Thus, MARCKS is significantly expressed in macrophages and plays a role in inflammation-related CCA induced by O. viverrini.

Hibiscus sabdariffa extractivities on cadmium-mediated alterations of human U937 cell viability and activation.

OBJECTIVE: To investigate the effect of the anthocyanin-rich extract of Hibiscus sabdariffa (H. sabdariffa) calyx on the viability of cadmium-treated U937 cells and cadmium-mediated activation of U937-derived macrophages. METHODS: The macrophage cell line U937 was treated with cadmium (0.1 µ mol/L) and later incubated with the anthocyanin-rich extract and cell viability was assessed via trypan blue staining. In the other experiment, the U937 cells were transformed to the macrophage form by treatment with phorbol 12, myristate 13, and acetate and incubated with cadmium (10 µ mol/L). The anthocyanin-rich extract was added to the cells later and subsequently, the supernatant of each cell culture was analysed for the production of tumour necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), nitric oxide, and catalase activity as indices for the activation of macrophages. RESULTS: It revealed that the anthocynanin-rich extract significantly (P < 0.05) increased the viability of the cells which was suppressed by cadmium when compared to quercetin dihydrate. The extract also reduced the cadmium-mediated production of the markers of macrophage-activation when compared to quercetin dihydrate. In both experiments, the activity of the extract was concentration-dependent (P < 0.05). CONCLUSIONS: The findings show that H. sabdariffa possesses significant immunoprotective effect. These corroborate the immense reported antioxidant and medicinal potential of the calyces of the plant which could be exploited for pharmacological and neutraceutical advantages.

Impact of multidrug resistance on experimental empyema by Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a cause of infections of the lower respiratory tract among patients with chronic lung disorders. It is questionable whether virulence of this species may be influenced by multidrug resistance (MDR). OBJECTIVES: To define the impact of MDR in experimental lung infection. METHODS: Experimental empyema was induced in rabbits by MDR (group A, n = 16) and by susceptible isolates (group B, n = 10). Pleural fluid was sampled for quantitative culture and estimation of cell apoptosis and of tumor necrosis factor-alpha (TNFα) and malondialdehyde (MDA). Survival was recorded. Cytokine production was stimulated in U937 monocytes by samples of pleural fluid. Whole blood of rabbits was incubated with the isolates; induction of apoptosis was assessed. RESULTS: Survival of group A was prolonged compared to group B. This was accompanied by lower bacterial counts of the inoculated pathogens in pleural fluid and in the lungs of group A compared with group B. Early apoptosis of neutrophils of pleural fluid of group A was lower compared with group B. Pleural fluid concentrations of TNFα and MDA did not differ between the groups. Cytokine production by U937 monocytes after stimulation with pleural fluid was greater in group B than in group A. The susceptible isolate induced apoptosis of neutrophils in vitro at a greater rate than the MDR isolate. CONCLUSIONS: Experimental empyema by susceptible P. aeruginosa is accompanied by greater mortality compared with MDR P. aeruginosa. This phenomenon may be attributed to the different growth pattern of the pathogens or to their interaction with the innate immune system.

Comparative EPR study of different macrophage types stimulated for superoxide and nitric oxide production.

Despite the major impact of ROS on human health, their quantification remains difficult and requires an analytical approach, such as the EPR spin trap technique. In this study, a comparative EPR analysis of different macrophage types stimulated for superoxide and nitric oxide production was performed. U937 monocytes, J774A.1, RAW 264.7 and primary mouse (PMM) macrophages were included. In contrast to the U937 cells, all macrophages produced significant EPR signals after stimulation. The use of PMA as stimulator and CM-H as spin probe led to the highest response in EPR signals for detection of O(2)(.-) as nitroxide radical. A combination of LPS and IFN-gamma and the spin trap [Fe(DETC)(2)] turned out to be the best combination for the production and detection of intracellular NO spin adducts. In conclusion, this study established practical experimental conditions for the EPR analysis of O(2)(.-) and NO produced by different types of activated macrophages.

The DNA demethylating agent 5-aza-2'-deoxycytidine induces expression of NY-ESO-1 and other cancer/testis antigens in myeloid leukemia cells.

Azanucleoside DNA-hypomethylating agents have remarkable clinical activity in myelodysplastic syndromes and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs) encoding immunogenic proteins are not expressed in almost all normal tissues and many tumor types, but are consistently derepressed by epigenetically active agents in various cancer cell lines. Since the expression of CTA genes is usually very low or absent in myeloid leukemias, we treated various AML cell lines with 5-aza-2'-deoxycytidine (DAC) and quantified mRNA expression of the CTAs NY-ESO-1, MAGEA1, MAGEA3 and MAGEB2. Consistent time- and dose-dependent reactivation of all 4 CTA genes was observed, with maximum mRNA levels 72-144h after treatment start. As determined by RNA microarray analyses, numerous other CTA genes (all located on the X-chromosome) were also derepressed in a time-dependent fashion by DAC. NY-ESO-1 derepression was confirmed at the protein level. By Elispot and chromium release assays we showed that the de novo expressed NY-ESO-1 protein was naturally processed and presented in a time- and dose-dependent fashion up to 8 days after the start of DAC treatment, and converted the cell lines susceptible to antigen-specific recognition by CD8+ T-cell clones. In conclusion, NY-ESO-1 and numerous other CTAs localized on the X-chromosome are readily and transiently derepressed in AML cell lines treated with DAC. The susceptibility of DAC-treated AML cell lines to antigen-specific T-cell recognition has clear implications for future clinical trials combining DAC and specific immunotherapy in AML.

Molecular signaling operated by a diet-compatible mixture of oxysterols in up-regulating CD36 receptor in CD68 positive cells.

Oxidation of dietary cholesterol during food storage and processing, and/or that of endogenous cholesterol in the presence of increased steady-state levels of reactive oxygen species, leads to the production of derivatives, termed oxysterols. Among the biochemical effects exerted by an oxysterol mixture, it has recently been observed that marked up-regulation of CD36 scavenger receptor on macrophage cells plays a primary role in foam cell formation. This article reports evidence of a significant co-localization of CD36 receptor with cells of the macrophage lineage, i.e. CD68 positive cells, LDL apoprotein B100 and lipids in human advanced atherosclerotic lesions. In addition, it provides a comprehensive analysis of the molecular signaling operated by a nutritionally relevant mixture of oxysterols in overexpressing CD36 receptor in cells of the macrophage lineage. The involvement of a G protein, Src, phospholipase C cascade and peroxisome proliferator-activated receptor gamma in oxysterol-mediated signaling was demonstrated by using selective inhibitors, while the central role of the downstream protein kinase Cdelta and extracellular signal-regulated kinase pathways in oxysterol-induced enhancement of CD36 was conclusively proved by means of small interfering RNA (siRNA) technology.

Identification of two distinct cell binding sequences in the vitamin D binding protein.

The vitamin D binding protein (DBP) is a multifunctional, albumin-like plasma protein that often requires cell surface binding to mediate some of its diverse functions. DBP binds to several different molecules on the external face of the plasma membrane indicating that it may possess distinct cell binding sequences. In this report, surface plasmon resonance was utilized to evaluate the relative binding of the human myeloid cell line U937 to immobilized recombinant expressed DBP in order to identify cell localization sequences. U937 cells showed robust binding to immobilized native DBP, but essentially no interaction when sensor chips were coated with beta(2)-microglobulin or BSA. The cell-DBP interaction was completely eliminated if cells were pretreated with soluble DBP. Recombinant DBP domains and truncated domains were next evaluated to determine the location of cell binding regions. Domains I (amino acids 1-191) and III (379-458), but not domain II (192-378), could support cell binding. Further evaluation of domain I, using truncated proteins and overlapping peptides, demonstrated that a single amino acid sequence, residues 150-172 (NYGQAPLSLLVSYTKSYLSMVGS), mediated cell binding. The domain III cell binding region was investigated using truncated versions of domain III fused to full-length domain II that served as a scaffold. These experiments indicated that the cell binding sequence is located in the first portion of that domain (379-402: ELSSFIDKGQELCADYSENTFTEY). Overlapping peptides spanning this sequence could partially block cell binding only when used in combination. We conclude that DBP contains two cell localization sequences that may be required for some of the multiple functions of this protein.

Investigation of the binding of Cr(III) complexes to bovine and human serum proteins: a proteomic approach.

Chromium (Cr) compounds are widely used in alloys manufacturing and forming processes. One of the main concerns in the use of cobalt-chromium (Co-Cr) alloy-based implants is the long-term fate of Co and Cr ions in the blood, organs, and urine of patients. Our previous studies have shown that Cr(III) forms complexes in different cell culture media, whereas Cr(VI) does not form any detectable structure under the same conditions. Because Cr(VI) is known to be more toxic than Cr(III), we hypothesized that the presence of serum proteins in the molecular structure of Cr(III) may be responsible for the difference in toxicity. We investigated the interaction of the Cr(III) complexes with serum proteins and their internalization by U937 macrophage-like cells. By using a proteomic approach, we showed that in the presence of fetal bovine serum, Cr(III) complexes interacted only with albumin, whereas they interacted mainly with albumin, transferrin, and immunoglobulins (Ig) in the presence of human serum (HS). Cr(III) complexes were more easily engulfed by U937 macrophage-like cells when they were formed with HS. To the best of our knowledge, this is the first report on the formation of Cr(III) complexes in the presence of serum proteins and the interaction of these complexes with U937 macrophage-like cells. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Downregulation of c-FLIP, XIAP and Mcl-1 protein as well as depletion of reduced glutathione contribute to the apoptosis induction of glycyrrhetinic acid derivatives in leukemia cells.

The antiproliferative effects and apoptosis inducing abilities of four 18beta-glycyrrhetinic acid (GA) derivatives, methyl 2-cyano-3,11-dioxooleana-1,12-dien-30-oate (CDODO-Me-11), methyl 2-cyano-3,12-dioxooleana-1,12-dien-30-oate (CDODO-Me-12) and their non-esters were investigated in human leukemia cells. Methyl esterification and switching a keto group from position C(11) to C(12) significantly increased the antiproliferative effects. CDODO-Me-11 and CDODO-Me-12 were 10-fold more potent than their non-esters, respectively. CDODO-Me-12 was 10-fold more effective than CDODO-Me-11 in inducing apoptosis which was correlated with the activation of caspase-8 and caspase-9. Western blot analyses revealed that CDODO-Me-12 and CDODO-Me-11 downregulated the levels of anti-apoptosis proteins, c-FLIP, XIAP and Mcl-1, without altering the protein levels of Bcl-2 and the death receptors DR4 and DR5. Both agents decreased the levels of the mitochondrial membrane potential without altering the intracellular H(2)O(2) levels. Jurkat cells without expression of caspase-8 were not sensitive to CDODO-Me-12, but were somewhat responsive to CDODO-Me-11. K562 cells with higher intracellular reduced glutathione (GSH ) levels were less responsive to CDODO-Me-12 apoptosis induction than U937 cells even though both cell lines were equally sensitive to CDODO-Me-11 apoptosis induction. Both agents depleted intracellular GSH levels and exogenous GSH reversed apoptosis induction by either agent in HL-60 cells. N-acetylcysteine (NAC) significantly attenuated apoptosis induction by CDODO-Me-12, but only weakly, that by CDODO-Me-11. UV spectrophotometric analysis revealed that both agents interacted with GSH while only CDODO-Me-12 had high reactivity with NAC. These data suggest that both agents induce apoptosis requiring to bind to functional proteins with thiol groups and that GSH may play a protective role by forming inactive adducts with them.