Ml Of RPMI Research Articles

In vitro Effect of Arnebia densiflora and Artemisia annua Plant Extracts against Theileria annulata Schizonts

Background: This study investigated the in vitro activities of naphthoquinones and artemisinin contained in the extracts obtained from Arnebia densiflora and Artemisia annua plants against peripheral blood mononuclear cells infected with Theileria annulata schizonts. For this purpose, the n-hexane extract of A. densiflora and the petroleum ether and methanol extracts of A. annua were obtained. Methods: Non-infected and infected cells were cultivated and 2×106 cells were seeded in each well in 0.5 ml of RPMI 1640 medium. The n-hexane extract of A. densiflora was tested six times at concentrations of 125, 62.5, 15.625, 7.8125 and 3.9062 mg/ml. A. annua petroleum ether and methanol extracts were also tested six times at 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, and 1.9531 mg/ml concentrations on PBMCs. A Mann-Whitney U test was used to determine differences between control and experimental groups. Result: The A. densiflora extract killed both non-infected and infected cells at significant levels (P less than 0.05) compared to the control group at all tested concentrations. A. annua methanol extract had no adverse effect on non-infected cells and the effect on infected cells was insufficient, with the only observed significant effect at a concentration of 125 µg/ml. The A. annua petroleum ether extract had no adverse effect on non-infected cells and killed infected cells at significant levels (P less than 0.05) at concentrations of 15.625 µg/ml and above at 24 hours. At 48 hours, all infected cells were killed at a concentration of 62.5 µg/ml. In conclusion, A. annua petroleum ether extract can be used in future in vivo studies for Theileriosis at these effective concentrations.

UJI AKTIVITAS BUAH MAHKOTA DEWA (PHALERIA MACROCARPA) SEBAGAI ANTIMALARIA PADA MENCIT YANG DIINFEKSI PLASMODIUM BERGHEI

Malaria is a parasitic infection caused by Plasmodium sp. Malaria’s medication and prevention are known, but there is a problem of increasing Plasmodium resistance to antimalarial drugs such as drugs from the chloroquine class, to overcome this problem traditional medicine is chosen because it is safer and cheaper. God’s crown (Phaleria macrocarpa) is one of the plants commonly used for traditional medicine which contains several active substances, namely saponins, polyphenols, flavonoids, tannins, sterols, alkaloids and terpenoids which have potential as antimalarials which can be proven by observing the degree of parasitemia in the mice blood. This study was conducted in vivo using the Randomized Post Test Only Controlled Group method using 24 samples of mice which were divided into 4 groups. The negative control group was only 0.2 ml of RPMI as a treatment control and the treatment group was treated with God’s crown extract at doses of 1, 10, and 100 mg/kgBW, then the degree of parasitemia was examined in each mouse. The data analysis technique used is One Way ANOVA and Post Hoc test. The results obtained mean the degree of parasitemia in the control group, doses of 1 mg/kgBW, 10 mg/kgBW and 100 mg/kgBW respectively 41.70; 37.96; 31.55; and 37.0. The results based on the analysis using One Way ANOVA posttest, it was found that there were significant differences between the control group and treatment groups 1,2, and 3 with p value of 0.000 (p < 0.05). The results showed that the extract of the fruit of the God’s crown (Phaleria macrocarpa) could inhibit the growth of Plasmodium berghei, the best dose was found at a dose of 10 mg/kgBW.

Longitudinal Evaluation of Ethyl Cyanoacrylate Adhesives on Candida albicans Biofilm

The aim of this study is to evaluate the effect of two ethyl cyanoacrylate-based adhesives on the growth of Candida albicans biofilms on a heat-polymerized resin, after 7, 14, and 30 days of exposure. Ninety circular (10 x 2 mm) heat-polymerized resin specimens were equally divided into three groups: control, conventional ethyl cyanoacrylate (ECAc), and ethyl cyanoacrylate gel (ECAg). Two layers of 50 µL of each material were applied to the respective groups. C. albicans SC5314 strain was activated and standardized to 107 cells/mL-1. Specimens were immersed in 1 mL of artificial saliva and deposited in 1 mL fungal suspension, washed, and immersed in 1 mL of RPMI for 7, 14, and 30 days. The medium was changed at 48-hour intervals. The final suspension was diluted (10 -1 to 10-4) and deposited on Sabouraud dextrose agar for 48 h at 37 °C. After this period, the colonies were quantified using the CFU/mL calculation. Data were evaluated using one- way ANOVA and Tukey’s test for post-hoc analysis (P=0.05). It was observed that both adhesives significantly reduced (P<0.05) biofilm formation compared to the control at all evaluated periods. In conclusion, an immediate and long-term inhibitory effect on C. albicans biofilm formation was observed.

Importance of Mitochondrial Metabolism As a Selective Therapeutic Approach in Chronic Lymphocytic Leukemia Patients

Objective: Chronic lymphocytic leukemia (CLL) is the most common blood cancer among adults in Western countries. Impairments in mitochondrial physiology play a role in CLL. Selectively targeting mitochondrial bioenergetics in leukemia cells may be an effective therapeutic approach for CLL. However, mitochondrial metabolism in CLL remains relatively unexplored. Nicotinamidephospho-robosyltransferase (NAMPT) is a key enzyme in the nitoinamide adenine dinucleotide (NAD) salvage pathway. FK866, a chemical inhibitor of NAMPT depletes cellular NAD and ATP levels and triggers apoptosis, suggesting NAMPT contributes to the prolonged survival of CLL cells. Idelalisib, an inhibitor of phosphatidylinositol-3-kinase-delta (PI3Kδ) is an approved therapy for B-cell malignancies including CLL. Herein, bioenergetics profile in 20 CLL patients have been defined and the effects of these drugs on this profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of mitochondrial complexes and pPI3Kδ were analyzed. We hypothesize that both inhibitors will alter CLL mitochondrial bioenergetics profiles and function, cell viability, ROS levels and enable lower doses and profound effects for combination therapies rather than single agent therapy.Methods: A high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria), a Clarke-type oxygen electrode is used to measure mitochondrial respiration rates CLL cells at 370C. Freshly isolated CLL cells (5-10 mil./ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates (BR), the following chemicals were added: oligomycin (2μM), FCCP: carbonyl cyanide p-trifluoromethoxy phenyhydrazone (2.5-12.5 μM), and antimycin A (2 μM). Oxygen consumption rate is expressed in pmol oxygen/s/mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by Novocyte flow cytometer.Results: Bioenergetics profile (BR: 14.45 ± 3.40; FCCP-induced MR: 57.48 ± 14.94; reserve respiratory capacity, RRC: 43.03 ± 12.80; respiratory control ratio, RCR: 9.36 ± 3.99; proton leak: 6.77 ± 2.45 and residual respiration: 6.03 ± 1.49; Mean ± SD) of 20 CLL patients was individually illustrated and summarized. Our data show that CLL cells have a high mitochondrial RRC and require a higher concentration of uncoupler, namely FCCP to achieve MR than commonly used. BR was not significantly decreased by 24 hours of treatment with FK866 at 1 nM (FK1) nor idelalisib at 6.25 μM (ID6.25) or the combination. After 48 hours, basal respiration of ID6.25 or ID6.25 + FK1 - treated cells was significantly decreased compared to DMSO vehicle control. However, after 24 or 48 hours MR, RRC, and RCR were significantly decreased by each individual drug or its combination compared to DMSO vehicle control. After 24 hours, combination therapy did not significantly decrease BR, RRC, and RCR compared to the treatment of FK1 (except MR), but MR, RRC, and RCR were decreased significantly compared to ID6.25 (except BR). The combination treatment of these two drugs significantly decreased all bioenergetics parameters compared to each individual drug after 48 hours. Preliminary data with another NAMPT-inhibitor, GMX-1778 at 1nM also showed similar effect to FK866 and in combination with ID6.25. MMP was not affected by these drug treatments, cell viability corresponded to the effect of mitochondrial bioenergetics profile, however, ROS levels were increased. Protein expression of certain subunits of mitochondrial complexes I, III and V and pPI3Kδ were decreased after the combined treatment.Conclusion:Bioenergetics profile can be directly evaluated by high resolution Oroboros oxygraph in CLL cells without any excess manipulation. CLL cells have immense mitochondrial RRC and highly sensitive to an uncoupler. Both FK866 and idelalisib affected bioenergetics profile in CLL cells at doses as low as 1 nM or 6.25 µM, respectively. The combined effect of these drugs is more profound than each agent alone on mitochondrial bioenergetics profile, cell viability, ROS and protein levels of mitochondrial complexes and pPI3Kδ. These novel data derived from the combined treatment with lower doses targeting mitochondrial metabolism provide rational therapeutic combination that may lead to reduce toxicity and increased drug efficacy in CLL. DisclosuresNo relevant conflicts of interest to declare.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5mg/kg) for 14days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1×106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500µL ip, POD 1, 5). Banff grade III or 120days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28days in CTRL (n=7), 34 in ASC (n=6), and 27.5 in ASC-CYP (n=4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

Ibrutinib in Combination with Venetoclax Decreases Mitochondrial Bioenergetics through the Impaired BTK, AKT and AMPK/SIRT/PGC-1α Signaling Pathway in CLL

Objective: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Altered mitochondrial metabolism has been shown to be involved in the pathogenesis of CLL. Ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK), and venetoclax, an inhibitor of B-cell lymphoma 2 (Bcl-2) protein are approved therapies for CLL. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling axis senses the metabolic demands of cells and regulates mitochondrial function. Silent information regulator T1 (SIRT1) is a cytoplasmic enzyme that mediates NAD+-dependent deacetylation of target substrates. The importance of BTK, AKT, and AMPK/SIRT/PGC-1α signaling pathway have to be explored yet in CLL. Ibrutinib and venetoclax may trigger a switching off of AMPK and /or SIRT1 signaling leading to impaired PGC-1α expression/activity and diminished mitochondrial activity. The effects of these drugs on mitochondrial bioenergetics profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of p-BTK, p-AKT, Bcl-2, p-AMPK, SIRT1, and PGC-1α were analyzed. We hypothesize that ibrutinib, along with venetoclax, will alter CLL mitochondrial bioenergetics profiles and function, cell viability, and ROS levels through the involvement of BTK, AKT and AMPK/SIRT/PGC-1α signaling pathway and enable lower doses and profound effects for combination therapies rather than single agent therapy. Methods: The high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Austria), a Clarke-type oxygen electrode, is used to measure mitochondrial respiration rates of CLL cells at 370C. Freshly isolated CLL cells (10 mil. cells/ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates, the following chemicals were added: oligomycin (2uM), FCCP: carbonylcyanide p-trifluoromethoxyphenylhydrazone (2-12 uM), and antimycin A (2 uM). Oxygen consumption rate is expressed in pmol oxygen/s /mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by the flow cytometer (NovoCyte). Results: CLL cells obtained ex vivo from ibrutinib treated patients had decreased mitochondrial bioenergetics compared to pre-treatment samples. Primary CLL cells treated in vitro with sub-lethal doses of ibrutinib for 24 or 48h showed significantly decreased basal respiration rates, maximal respiration rates, and spare respiratory capacity compared to DMSO vehicle control. These parameters were also significantly affected by low doses (0.5 - 2 nM) of venetoclax. The combination treatment of ibrutinib with venetoclax for 24 or 48h significantly decreased these bioenergetics parameters compared to each individual drug alone except for the spare respiratory capacity vs venetoclax alone at 24 h. MMP was significantly decreased by these drug treatments and its combinations compared to DMSO. Cell viability was used to confirm a sub-lethal phenotype. ROS levels were significantly increased by venetoclax compared to DMSO, and in combination with ibrutinib vs ibrutinib alone. However, ROS levels by ibrutinib were not significantly changed compared to DMSO or venetoclax. Protein levels of p-BTK, p-AKT, p-AMPK, SIRT1, and PGC-1α were decreased by ibrutinib or venetoclax alone compared to DMSO and further decreased by the combination of these two drugs compared to single agent. Conclusion: CLL cells from ibrutinib treated patients, a clinically approved BTK inhibitor, demonstrated decreased bioenergetics similar to normal B-lymphocytes suggesting ibrutinib treatment normalizes the mitochondrial bioenergetics in CLL. Ibrutinib and venetoclax affected bioenergetics profiles in CLL cells at low doses. The combined effect of these drugs on the mitochondrial bioenergetics profiles and cell viability is more profound than each BTK or Bcl-2 inhibitor agent alone. Protein levels of p-BTK, p-AKT, p-AMPK, SIRT1, and PGC-1α in ibrutinib and venetoclax treated samples were reduced compared to DMSO and each single agent inhibitor. These novel data suggest the involvement of BTK, AKT and AMPK/SIRT/PGC-1α signaling pathway to target mitochondrial metabolism and provide rational therapeutic combinations that may lead to reduced toxicity and increased drug efficacy in CLL. Disclosures Johnston: Janssen: Research Funding. Banerji:Janssen: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Licensing fee, Research Funding; Abbvie: Consultancy, Honoraria; CIHR: Research Funding; LLSC: Research Funding; Research Manitoba: Research Funding; Astra-Zeneca: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Dana-Farber Cancer Institute: Other: Licencing fee; CCMF: Research Funding; CancerCare Manitoba/University of Manitoba: Employment; CAPhO: Honoraria; BIOGEN: Other: Licensing fee.

Venetoclax in Combination with NAMPT Inhibitors Decreases Mitochondrial Bioenergetics through the Impaired AMPK/SIRT/PGC1α Signaling Pathway in CLL

Objective: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Altered mitochondrial metabolism has been shown to be involved in the pathogenesis of CLL. Nicotinamidephospho-ribosyltransferase (NAMPT) is a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage pathway. FK866 and GMX1778, chemical inhibitors of NAMPT, deplete cellular NAD and ATP levels and trigger apoptosis in CLL cells, suggesting NAMPT contributes to the prolonged survival of these cells. Venetoclax is an approved therapy for CLL and blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, leading to programmed cell death of CLL cells. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling axis senses the metabolic demands of cells and regulates mitochondrial function. Silent information regulator T1 (SIRT1) is a cytoplasmic enzyme that mediates NAD+-dependent deacetylation of target substrates. The importance of the AMPK/SIRT/PGC1α signaling pathway has to be explored yet in CLL. Venetoclax may trigger a switching off of AMPK and /or SIRT1 signaling leading to impaired PGC-1α expression/activity and diminished mitochondrial activity. The effects of these drugs on mitochondrial bioenergetics profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of Bcl-2, AMPK, SIRT1, and PGC1α were analyzed. We hypothesize that venetoclax, along with FK866 and GMX1778, will alter CLL mitochondrial bioenergetics profiles and function, cell viability, and ROS levels through the involvement of the AMPK/SIRT/PGC1α signaling pathway and enable lower doses and profound effects for combination therapies rather than single agent therapy.Methods: A high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria), a Clarke-type oxygen electrode is used to measure mitochondrial respiration rates of CLL cells at 370C. Freshly isolated CLL cells (10 mil./ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates, the following chemicals were added: oligomycin (2uM), FCCP: carbonyl cyanide p-trifluoromethoxy phenyhydrazone (2.5-12.5 uM), and antimycin A (2 uM). Oxygen consumption rate is expressed in pmol oxygen/s/mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by Novocyte flow cytometer.Results: Primary CLL cells treated with 1.25 nM of venetoclax for 24 or 48h showed significantly decreased basal respiration rates, maximal respiration rates, and spare respiratory capacity compared to DMSO vehicle control. These parameters were also significantly affected by 1nM of FK866 or 1 nM of GMX1778 except basal respiration rates. The combination treatment of venetoclax with FK866 or GMX1778 for 24 or 48h significantly decreased all these bioenergetics parameters compared to each individual drug. MMP was not affected by these drug treatments and cell viability corresponded to the effect of the mitochondrial bioenergetics profile. ROS levels after 24 hours in venetoclax treated CLL cells were significantly increased compared to DMSO, any single agent or in combination. However, after 48 hours of treatment, increased ROS levels were not statistically significant compared to DMSO. Protein levels of Bcl-2, P-AMPK, AMPK, SIRT1, and PGC1α were decreased by venetoclax alone or in combination with FK866 or GMX1778.Conclusion: Venetoclax, FK866 and GMX1778 affected bioenergetics profiles in CLL cells at doses as low as 1.25 nM, 1nM, and 1 nM, respectively. The combined effect of these drugs on the mitochondrial bioenergetics profiles and cell viability is more profound than each NAMPT inhibitor agent alone. Protein levels of Bcl-2, AMPK, SIRT1, and PGC1α in venetoclax treated samples were reduced compared to DMSO and each single agent NAMPT inhibitor. These novel data suggest the involvement of the AMPK/SIRT/PGC1α signaling pathway to target mitochondrial metabolism and provide rational therapeutic combinations that may lead to reduced toxicity and increased drug efficacy in CLL. DisclosuresBanerji:Roche: Other: Unrestricted grant received in the past; Janssen: Other: Unrestricted grant received in the past; Gilead: Other: Unrestricted grant received in the past; Abbvie: Other: Unrestricted grant received in the past; Teva: Other: Unrestricted grant received in the past.

Resistance to Azacitidine Is Determined at Cellular Level By Lower Expression of Nucleoside Metabolizing Enzymes

Introduction: Azacitidine is at present the standard of treatment of myelodysplastic syndromes (MDS). We demonstrated that MDS patients responsive to azacitidine have significantly higher expression of the azacitidine-activating enzyme uridine-cytidine kinase-1 (UCK1) in bone marrow mononuclear cells (Valencia et al. Leukemia 2014). Correlation of the expression of nucleoside transporter, activating and disactivating enzymes with clinical and in vitro response to hypomethylating drugs azacitidine and decitabine has been suggested by several authors. Yet, the crucial role of these enzymes has to be ascertained, as well as their possible different importance in determining resistance.Objectives: To confirm that the cellular expression of nucleoside metabolizing enzymes plays a major role in cellular resistance and significantly impacts on clinical response to azacitidine.Methods: Two cell lines, SKM1 sensitive (SKM1-S) and SKM1 resistant (SKM1-R) to azacitidine (Cluzeau et al. Oncogene 2014) were analyzed for expression of UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 by quantitative PCR, using an ABI GeneAmp® 5700. The specific oligonucleotides and TaqMan® probes were acquired among the Assay-on-Demand® Gene Expression Products (Applied Biosystems). Corresponding proteins were quantitated by western blotting in both cell lines. The expression of UCK1 and UCK2 was blunted by siRNAs in order to determine their role in in vitro sensitivity to azacitidine.For UCK2 silencing in SKM1-S, specific siRNAs were used (OriGene Technologies, Inc., Rockville, MD, USA). SKM1-S cells were cultured at a density of 1x106 cells/ml in 9 ml of RPMI 1940 medium. After 72 h of transfection, cells were treated for further 48h with azacitidine at the concentrations of 0,1 and 1 μM. After assessment of effective gene silencing, apoptosis and DNA methylation were evaluated by Annexin V test, MSP and pyrosequencing in cells exposed to azacitidine.In addition, the expression of nucleoside metabolizing enzymes was evaluated prospectively in IPSS high risk MDS patients treated with azacitidine 75mg/m2/7 days every 28 days.Results: SKM1-R cells did not express UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2. Corresponding proteins were also not expressed. In order to clarify the specific role of UCK2 protein in the mechanisms of resistance to azacitidine, we knocked-down UCK2. A reduction of apoptosis was observed in UCK2-silenced SKM-1 S after azacitidine 0.1 μM treatment: 31%±0,85% Annexin V-positive cells versus 21%±0,35% in non-silenced control SKM1-S cultures (P= 0.054). Hypomethylation induced by in vitro azacitidine treatment was also hampered by absence of expression of UCK2.Quite surprisingly, in MDS cases, gene expression of UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 in primary cellsdid not predict different clinical response to azacitidine.Conclusions: We demonstrated that UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 and the corresponding proteins are absent in an in vitro generated azacitidine-resistant cell line SKM1-R. These abnormalities could be major determinants of the induced resistance to the nucleoside analogue. Supporting the importance of these enzymes in resistance to nucleosides, and the major role played by UCK2, its silencing induced by synthetic siRNAs significantly decreased azacitidine effects. Prospective evaluation of the predictive role of cellular expression of genes involved in azacitidine metabolism is ongoing in a larger cohort of MDS patients.Establishing the mechanisms of resistance to azacitidine could lead us to optimize treatment of MDS patients whose scarce susceptibility could be predictable. DisclosuresSantini:Celgene: Consultancy, Honoraria, Research Funding; Amgen: Other: advisory board; Novartis: Consultancy, Honoraria; Onconova: Other: advisory board; Janssen: Consultancy, Honoraria; Astex: Other: advisory board.

P47phox Directs Murine Macrophage Cell Fate Decisions

Macrophage differentiation and function are pivotal for cell survival from infection and involve the processing of microenvironmental signals that determine macrophage cell fate decisions to establish appropriate inflammatory balance. NADPH oxidase 2 (Nox2)-deficient chronic granulomatous disease (CGD) mice that lack the gp91(phox) (gp91(phox-/-)) catalytic subunit show high mortality rates compared with wild-type mice when challenged by infection with Listeria monocytogenes (Lm), whereas p47(phox)-deficient (p47(phox-/-)) CGD mice show survival rates that are similar to those of wild-type mice. We demonstrate that such survival results from a skewed macrophage differentiation program in p47(phox-/-) mice that favors the production of higher levels of alternatively activated macrophages (AAMacs) compared with levels of either wild-type or gp91(phox-/-) mice. Furthermore, the adoptive transfer of AAMacs from p47(phox-/-) mice can rescue gp91(phox-/-) mice during primary Lm infection. Key features of the protective function provided by p47(phox-/-) AAMacs against Lm infection are enhanced production of IL-1α and killing of Lm. Molecular analysis of this process indicates that p47(phox-/-) macrophages are hyperresponsive to IL-4 and show higher Stat6 phosphorylation levels and signaling coupled to downstream activation of AAMac transcripts in response to IL-4 stimulation. Notably, restoring p47(phox) protein expression levels reverts the p47(phox)-dependent AAMac phenotype. Our results indicate that p47(phox) is a previously unrecognized regulator for IL-4 signaling pathways that are important for macrophage cell fate choice.

Inhibition of IL-17 Release by the Novel Anti-Inflammatory Drug Vidofludimus Involves Attenuation of STAT3 and NF-kappa B Signaling Pathways in Murine Splenocytes and Hapten-Induced Colitis

IL-17 Secretion: Murine Splenocytes Vidofludimus Inhibits the Nuclear Binding of STAT3 in a Dose Dependent Fashion: Murine Splenocytes Vidofludimus Does Not Affect the Expression of IκB-α: Murine Splenocytes Vidofludimus Attenuates Phospho-p65 Staining: Murine TNBS Colitis The STAT3 and NF-kappa B (NF-κB) pathways play a prominent role in IL-17 production and intestinal inflammation. We have shown that a novel drug named Vidofludimus (Vido, 4SC-101, SC12267) inhibited colonic IL-17 and murine colitis [Inflammatory Bowel Diseases, 2010]. Aims: The aims of our study were: 1) to determine if Vido inhibited IL-17 production by murine splenocytes, 2) to evaluate if Vido inhibited key components of the STAT3 and NF-κB signaling pathways in splenocytes and 3) to determine if Vido inhibited phosphorylated (phospho) STAT3 and NF-κB in the colons of mice with hapten (TNBS)-induced colitis. Methods: Splenocytes were collected from BALB/c mice. These cells were stimulated with IL-23 or IL-1β (10 ng/ml) alone, or in combination. Vido (25 to 100 μM), or vehicle, were added along with the cytokines. IL-17 secretion was determined by ELISA at Vidofludimus (4SC-101, SC12267) is a proprietary compound of 4SC AG Vidofludimus Inhibits Cytokine-Induced IL-17 Secretion: Murine Splenocytes Synergistic activation by IL-23 + IL-1β Vidofludimus Attenuates Phospho-AKT1 Expression: Murine Splenocytes Effects of Vidofludimus on Signaling Pathways: Murine Splenocytes 48 hours. Cytosolic extracts from splenocytes were used for western blots of phospho JAK2, STAT3 and AKT1 (STAT3 pathway proteins). Phospho I-κB and I-κB degradation were evaluated as indices of NF-κB pathway activation. Extracts were also collected from stimulated splenocytes for the evaluation of STAT3 and NF-κB nuclear binding by ELISA. Immunohistochemistry for phospho STAT3 and p65 was done on samples from BALB/c mice that underwent colitis induction with TNBS ± oral treatment for six days with Vido (50-200 mg/kg). Using coded slides, the staining for these proteins in the mucosa/submucosa was graded on a six point intensity scale. Results: IL-23 and IL-1β alone did not induce significant IL-17 production (< 30 pg/ml), but combined cytokine treatment induced a synergistic stimulation of IL-17 secretion (630 pg/ml). IL-17 production was blocked by Vido in a dose dependent fashion (IC50 = 58.1 μM). At 10 to 60 minutes after cytokine stimulation, Vido reduced the expression of phospho proteins (JAK2, STAT3, and AKT1) involved in STAT3 signaling, and also inhibited the nuclear binding of STAT3 (IC50 = 53.9 μM). Vido did not reduce the phosphorylation or degradation of I-κB, but at 100 μM inhibited the nuclear binding of p65 by 47%. Evidence of increased phospho STAT3 and p65 immunostaining was found in leukocytes within the colons of TNBS-treated mice. The staining was attenuated in mice treated with Vido. Phospho STAT3 staining scores (2-3 mice per group) were: 1.3 ± 0.8 (no TNBS), 4.3 ± 0.9 (Vehicle/TNBS), 1.1 ± 0.3 (Vido-50 mg/kg), 1.2 ± 0.7 (Vido-100 mg/kg) and 0.3 ± 0 (Vido-200 mg/kg/TNBS). Similar METHODS immuno-staining scores were found for phospho p65. Summary: Vidofludimus inhibited STAT3 and NF-κB signaling pathways in murine splenocytes. It also normalized the enhanced phospho STAT3 and p65 immunostaining associated with murine colitis. Conclusion: The inhibition of STAT3 and NF-κB pathways by Vidofludmus reduces IL-17 production and contributes to the established anti-colitis activity of this drug. Splenocyte Cultures: Spleens were rapidly harvested from BALB/c mice. Splenocytes were collected by standard techniques, and erythrocytes were removed by using RBC lysis buffer. For the IL-17 secretion studies, we plated 2 x 106 cells/ml in 4 ml of RPMI 1640 medium containing 10% FBS and 5 x 105 β-mercaptoethanol. Therefore, there were 8 x 106 cells per well in the standard six-well plates. We utilized two wells (1.6 x 107 cells) per treatment condition in these studies. Splenocytes were cultured for 48 hours at 37 degrees C. Then, the culture media was collected for the measurement of IL-17 by ELISA (R&D systems, Minneapolis, MN). In subsequent studies (western blots, transcription factor binding assays), splenocytes were plated at a density of 1 x 107 cells/ml in 4 ml of RPMI 1640 medium. Therefore,

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.

Rapid and Highly Sensitive Detection of Malaria-Infected Erythrocytes Using a Cell Microarray Chip

BackgroundMalaria is one of the major human infectious diseases in many endemic countries. For prevention of the spread of malaria, it is necessary to develop an early, sensitive, accurate and conventional diagnosis system.Methods and FindingsA cell microarray chip was used to detect for malaria-infected erythrocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene, and the formation of monolayers of erythrocytes in the microchambers was observed. Cultured Plasmodium falciparum strain 3D7 was used to examine the potential of the cell microarray chip for malaria diagnosis. An erythrocyte suspension in a nuclear staining dye, SYTO 59, was dispersed on the chip surface, followed by 10 min standing to allow the erythrocytes to settle down into the microchambers. About 130 erythrocytes were accommodated in each microchamber, there being over 2,700,000 erythrocytes in total on a chip. A microarray scanner was employed to detect any fluorescence-positive erythrocytes within 5 min, and 0.0001% parasitemia could be detected. To examine the contamination by leukocytes of purified erythrocytes from human blood, 20 µl of whole blood was mixed with 10 ml of RPMI 1640, and the mixture was passed through a leukocyte isolation filter. The eluted portion was centrifuged at 1,000×g for 2 min, and the pellet was dispersed in 1.0 ml of medium. SYTO 59 was added to the erythrocyte suspension, followed by analysis on a cell microarray chip. Similar accommodation of cells in the microchambers was observed. The number of contaminating leukocytes was less than 1 on a cell microarray chip.ConclusionThe potential of the cell microarray chip for the detection of malaria-infected erythrocytes was shown, it offering 10–100 times higher sensitivity than that of conventional light microscopy and easy operation in 15 min with purified erythrocytes.

CAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression

CD39 is a transmembrane enzyme that inhibits platelet reactivity and inflammation by phosphohydrolyzing ATP and ADP to AMP. Cyclic AMP (cAMP), an essential second messenger, is particularly important in regulating genes controlling vascular homeostasis. These experiments test the hypothesis that cAMP might positively regulate the expression of CD39 and thereby modulate important vascular homeostatic properties. Cd39 mRNA was induced by 13.8- fold in RAW cells treated with a membrane-permeant cAMP analogue (8-bromo-cyclic AMP; 8-Br-cAMP), stimulation of adenylate cyclase, or prostanoids known to drive cAMP response. Fluorescence-activated cell sorting, immunofluorescence, and TLC assays demonstrated that both CD39 protein expression and enzymatic activity were increased in cells treated with 8-Br-cAMP but not in cells transfected with short hairpin RNA against CD39. This analogue drove a significant increase in transcriptional activity at the Cd39 promoter although not when the promoter's cAMP-response element sites were mutated. Pretreatment with cAMP-dependent protein kinase (PKA), phosphoinositide 3-kinase (PI3K), or ERK inhibitors nearly obliterated the cAMP-driven increase in Cd39 mRNA, protein expression, and promoter activity. 8-Br-cAMP greatly increased the phosphorylation of CREB1 (Ser(133)) and ATF2 (Thr(71)) in a PKA-, PI3K-, and ERK-dependent fashion. Chromatin immunoprecipitation assays demonstrated that binding of phosphorylated CREB1 and ATF2 to cAMP-response element-like sites was significantly increased with 8-Br-cAMP treatment and that binding was reduced with PKA, PI3K, and ERK inhibition, whereas transfection of Creb1 and Atf2 overexpression constructs enhanced cAMP-driven Cd39 mRNA expression. Transfection of RAW cells with mutated Creb1 (S133A) reduced cAMP-driven Cd39 mRNA expression. Furthermore, the cAMP-mediated induction of Cd39 mRNA, protein, and phosphohydrolytic activity was replicated in primary peritoneal macrophages. These data identify cAMP as a crucial regulator of macrophage CD39 expression and demonstrate that cAMP acts through the PKA/CREB, PKA/PI3K/ATF2, and PKA/ERK/ATF2 pathways to control a key vascular homeostatic mediator.

LANCL2 Is Necessary for Abscisic Acid Binding and Signaling in Human Granulocytes and in Rat Insulinoma Cells

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic beta cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38(+)/LANCL2(+) HeLa transfected with a chimeric G-protein (G alpha(q/i)) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G(i). Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish S-Adenosylmethionine Synthesis

We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of methionine adenosyltransferase-II (MAT-II), which catalyzes SAMe synthesis from ATP and l-Met. Specifically, we silenced the expression of the regulatory MAT-IIbeta subunit in Jurkat cells and accordingly shifted the K(m L-Met) of the enzyme 10-15-fold above the physiologic levels of l-Met, thereby reducing enzyme activity and SAMe pools, inducing excessive apoptosis and diminishing leukemic cell growth in vitro and in vivo. These effects were reversed at unphysiologically high l-Met (>50 microm), indicating that diminished leukemic cell growth at physiologic l-Met levels was a direct result of the increase in MAT-II K(m L-Met) due to MAT-IIbeta ablation and the consequent reduction in SAMe synthesis. In our NOD/Scid IL-2Rgamma(null) humanized mouse model of leukemia, control shRNA-transduced Jurkat cells exhibited heightened engraftment, whereas cells lacking MAT-IIbeta failed to engraft for up to 5 weeks post-transplant. These stark differences in malignant cell survival, effected by MAT-IIbeta ablation, suggest that it may be possible to use this approach to disadvantage leukemic cell survival in vivo with little to no harm to normal cells.

CCL2 Protects Prostate Cancer PC3 Cells from Autophagic Death via Phosphatidylinositol 3-Kinase/AKT-dependent Survivin Up-regulation

Resistance to cell death is a hallmark of cancer. Autophagy is a survival mechanism activated in response to nutrient deprivation; however, excessive autophagy will ultimately induce cell death in a nonapoptotic manner. The present study demonstrates that CCL2 protects prostate cancer PC3 cells from autophagic death, allowing prolonged survival in serum-free conditions. Upon serum starvation, CCL2 induced survivin up-regulation in PC3, DU 145, and C4-2B prostate cancer cells. Both cell survival and survivin expression were stunted in CCL2-stimulated PC3 cells when treated either with the phosphatidylinositol 3-kinase inhibitor LY294002 (2 microm) or the Akt-specific inhibitor-X (Akti-X; 2.5 microm). Furthermore, CCL2 significantly reduced light chain 3-II (LC3-II) in serum-starved PC3; in contrast, treatment with LY294002 or Akti-X reversed the effect of CCL2 on LC3-II levels, suggesting that CCL2 signaling limits autophagy in these cells. Upon serum deprivation, the analysis of LC3 localization by immunofluorescence revealed a remarkable reduction in LC3 punctate after CCL2 stimulation. CCL2 treatment also resulted in a higher sustained mTORC1 activity as measured by an increase in phospho-p70S6 kinase (Thr389). Rapamycin, an inducer of autophagy, both down-regulated survivin and decreased PC3 cell viability in serum-deprived conditions. Treatment with CCL2, however, allowed cells to partially resist rapamycin-induced death, which correlated with survivin protein levels. In two stable transfectants expressing survivin-specific short hairpin RNA, generated from PC3, survivin protein levels controlled both cell viability and LC3 localization in response to CCL2 treatment. Altogether, these findings indicate that CCL2 protects prostate cancer PC3 cells from autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway and reveal survivin as a critical molecule in this survival mechanism.

Identification of Novel Endogenous Cytochrome P450 Arachidonate Metabolites with High Affinity for Cannabinoid Receptors

Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Phospholipase C-δ1 Expression Is Linked to Proliferation, DNA Synthesis, and Cyclin E Levels

Phospholipase C-δ1 Expression Is Linked to Proliferation, DNA Synthesis, and Cyclin E Levels

Heterogeneity of Human Macrophages in Culture and in Atherosclerotic Plaques

Research suggests that monocytes differentiate into unique lineage-determined macrophage subpopulations in response to the local cytokine environment. The present study evaluated the atherogenic potential of two divergent lineage-determined human monocyte-derived macrophage subpopulations. Monocytes were differentiated for 7 days in the presence of alternative macrophage development cytokines: granulocyte-macrophage colony-stimulating factor to produce granulocyte-macrophage-CSF macrophages (GM-Mac), or macrophage colony-stimulating factor (M-CSF) to produce M-Mac. Gene chip analyses of three monocyte donors demonstrated differential expression of inflammatory and cholesterol homeostasis genes in the macrophage subpopulations. Quantitative PCR confirmed a fivefold elevation in the expression of genes that promote reverse cholesterol transport (PPAR-gamma, LXR-alpha, and ABCG1) and macrophage emigration from lesions (CCR7) in GM-Mac compared to that in M-Mac. Immunocytochemistry confirmed enhanced expression of the proinflammatory marker CD14 in M-Mac relative to GM-Mac. M-Mac spontaneously accumulated cholesterol when incubated with unmodified low-density lipoprotein whereas GM-Mac only accumulated similar levels of cholesterol after protein kinase C activation. Immunostained human coronary arteries showed that macrophages with similar antigen expression to that of M-Mac (CD68(+)/CD14(+)) were predominant within atherosclerotic lesions whereas macrophages with antigen expression similar to GM-Mac (CD68(+)/CD14(-)) were predominant in areas devoid of disease. The identification of macrophage subpopulations with different gene expression patterns and, thus, different potentials for promoting atherosclerosis has important experimental and clinical implications and could prove to be a valuable finding in developing therapeutic interventions in diseases dependent on macrophage function.