Nitroblue Tetrazolium Reduction Research Articles

Effect of Human Umbilical Cord-derived Mesenchymal Stem Cells on Proliferation and Differentiation of Leukemia Cells

To investigate the effect of human umbilical cord-derived mesenchymal stem cells(HUC-MSC) on the proliferation and differentiation of NB4 treated with all-trans retinoid acid (ATRA) and its underlining mechanisms . Human umbilical cord mesenchymal stem cells were isolated from umbilical cord of newborns. Co-culture system was established by HUC-MSC and NB4 in vitro. The experiment was divided into 4 groups: NB4 group (NB4 cells alone) , NM group (NB4 cells co-cultured with HUC-MSC) , NA group (NB4 cells treated with ATRA) , NMA group (NB4 cells co-cultured with HUC-MSC and treated with ATRA) . NB4 cells were counted by a microscopy, NB4 proliferation was monitored by CCK-8 assay, NB4 differentiation was assessed by Wright ' s staining and nitroblue tetrazolium reduction test. IL-6 levels in the culture supernatant of different groups were tested by ELISA kit. Quantitative PCR was used to detect the transcription level of CDKN1A, CCND1 and Survivin. NB4 and HUC-MSC in the co-culturing systems were in good condition with a slight repression of NB4 proliferation by HUC-MSC. HUC-MSC could collaborate with ATRA to induce significant NB4 differentiation. Consistent with this finding, IL-6 expression levels of co-cultured groups were remarkably higher than that in any other groups or the group of HUC-MSC alone. The quantitative PCR analysis showed that the levels of CDKN1A and CCND1 mRNA expression were increased or decreased respectively in the co-cultured groups. HUC-MSC co-culture can reduce proliferation but promote the differentiation of NB4 cells, suggesting that this effect may be closely related with the secretion of IL-6 which can affect the expression of some factors in vitro.

Killing of Staphylococcus aureus by allylpyrocatechol is potentiated by induction of intracellular oxidative stress and inhibition of catalase activity

ObjectiveThis study investigated the effects of allylpyrocatechol (APC), the major component in ethanolic extract of Piper betle, on key oxidative stress resistance enzymes important for the survival of Staphylococcus aureus, a major pathogen in the human host. MethodsEffects of APC on expressions of genes encoding catalase (katA), superoxide dismutases (SODs), including sodA and sodM, and alkyl hydroperoxide reductase (ahpC) in S. aureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species (ROS) levels were determined using the nitroblue tetrazolium (NBT) reduction assay. ResultsAPC-treated S. aureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL (P<0.05). Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells (P<0.05). Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 (A310nm) compared to untreated cells which was 0.394 (A310nm). In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 × 10−4 (U/L or μmol/(min·L)) compared to untreated cells, which was 4.8 × 10−4 U/L (P<0.05). Absorbance readings (A575nm) for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated S. aureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively (P<0.05). ConclusionAPC induced expressions of both sodA and sodM, resulting in increased total SOD activity in S. aureus. Higher sodA expression indicated stress induced intracellularly involving O2−, presumably leading to higher intracellular pools of H2O2. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternate pathway involving ahpC to reduce stress invoked by O2− and H2O2. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.

Synthesis and biological evaluation of PEGylated CuO nanoparticles

There is a growing field of research into the physicochemical properties of metal oxide nanoparticles (NPs) and their potential use against tumor formation, development and progression. Coated NPs with biocompatible surfactants can be incorporated into the natural metabolic pathway of the body and specifically favor delivery to the targeted cancerous cells versus normal cells. Polyethylene glycol (PEG) is an FDA approved, biocompatible synthetic polymer and PEGylated NPs are regarded as “stealth” nanoparticles, which are not recognized by the immune system. Herein, PEGylated cupric oxide nanoparticles (CuO NPs) with either PEG 1000 or PEG 8000 were hydrothermally prepared upon properly adjusting the reaction conditions. Depending on the reaction time CuO NPs in the range of core sizes 11–20nm were formed, while hydrodynamic sizes substantially varied (330–1120nm) with improved colloidal stability in PBS. The anticancer activity of the NPs was evaluated on human cervical carcinoma HeLa cells by using human immortalized embryonic kidney 293 FT cells as a control. Viability assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) revealed that CuO NPs could selectively reduce viability of tumor cells (IC50 values 11.91–25.78μg/mL). Reactive oxygen species (ROS) production, cell membrane damage and apoptotic DNA laddering were also evident by nitroblue tetrazolium (NBT) reduction, lactate dehydrogenase (LDH) release assays and DNA electrophoresis, respectively. CuO NPs strongly inhibited lipoxygenase (LOX) enzymatic activity with IC50 values 4–5.9μg/mL, highlighting in that manner their anti-inflammatory activity.

Effect of recombinant malarial antigen on monocyte functionality.

Recombinant proteins and vaccine candidates of Plasmodium vivax have met with limited success. One of the reasons could be their effect on monocytes which are important in malaria pathogenesis. Our aim was therefore to investigate the effect of selected recombinant malarial proteins on monocytes functions. Phagocytosis rate and respiratory burst of healthy individuals' monocytes treated with antigens were examined. The homing capacity of monocytes was studied by examining the mRNA level of chemokine receptors from patients and healthy individuals. Phagocytosis rate was reduced in antigen treated monocytes whereas nitroblue tetrazolium (NBT) reduction was more in apical membrane antigen-1 (AMA-1) and merozoite surface protein-7 (MSP7) treated than in untreated and von Willebrand factor A domain-related protein (WARP) treated monocytes. Patient monocytes showed higher mRNA expression for CCR2 and CX3CR1 and reduced levels for CCR7 and CXCR4. AMA-1 and WARP treated monocytes showed lower expression for CCR2, CX3CR1 and CXCR4, but unchanged for CCR7. However, with MSP7, all the receptor levels were reduced. CX3CR1 in monocytes from activated PBMCs was either unchanged (AMA-1) or increased (MSP7, WARP) while remaining receptors were reduced. These antigens may modulate the monocyte functionality and hence may not have desired therapeutic effect.

Abstract 1186: Inhibition of topoisomerase 2β sensitizes acute myeloid leukemia cells to ATRA induced apoptosis independent of the MAPK/ERK pathway

Abstract Pharmacologic inhibition or molecular down-regulation of topoisomerase 2β (TOP2β) potentiates all trans retinoic acid (ATRA)-induced differentiation and apoptosis in acute myeloid leukemia (AML) cells. Since ATRA-induced myeloid differentiation involves activation of the MAPK/ERK signaling pathway and aberrant activation of MAPK/ERK is frequently observed in AML we tested the role of this pathway in modulating the cellular effects of ATRA when TOP2β is inhibited with the bisdioxopiperazines, ICRF 187 (dexrazoxane, Zinecard™) or ICRF193. Treatment of HL-60 AML (M2) cells with ATRA increased the percentage of differentiated cells, as assessed by the nitroblue tetrazolium reduction assay, and enhanced levels of phosphorylated ERK. Paradoxically, while combination treatment with ATRA/ICRF187 enhanced differentiation (P&amp;lt;0.05), ERK phosphorylation was attenuated compared to treatment with ATRA alone. Moreover, pretreatment with the MEK inhibitor PD98059, which completely blocks ATRA-induced differentiation and ERK phosphorylation, only partially blocked (46% reduction) differentiation induced by ATRA/ICRF187 (P&amp;lt;0.01) and had no effect on ATRA/ICRF187-induced apoptosis. In agreement with this observation, combination treatment with ATRA/ICRF187 induced apoptosis (P&amp;lt;0.05) in KG-1 (M1) AML cells and primary AML cells from patients, that do not phosphorylate ERK or differentiate in response to ATRA treatment. In summary, our data suggest that inhibition of TOP2β sensitizes AML cells to ATRA induced differentiation and apoptosis primarily via an ERK-independent mechanism. Further, combination treatment ATRA and ICRF187 may be clinically relevant to overcome ATRA resistance in AML patients with a deregulated MAPK/ERK signaling pathway. Citation Format: Eric J. Norris, Amy Dorszynski, Aaron Lucander, Darla Destephanis, Ram Ganapathi, Mahrukh Ganapathi. Inhibition of topoisomerase 2β sensitizes acute myeloid leukemia cells to ATRA induced apoptosis independent of the MAPK/ERK pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1186.

Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells.

Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, calreticulin (CRT) was found to be downregulated in differentiated HL-60 cells treated with DADS. The present study investigated the role of CRT proteins in DADS-induced proliferation, invasion and differentiation in HL-60 cells. The present study demonstrated that DADS treatment significantly changed the morphology of HL-60 cells and caused the significant time-dependent downregulation of CRT. Small interfering RNA (siRNA)-mediated knockdown of CRT expression significantly inhibited proliferation, decreased invasion ability, increased the expression of cluster of differentiation (CD)11b and reduced the expression of CD33 in DADS-treated HL-60 cells. DADS also significantly affected cell proliferation, invasion and differentiation in CRT-overexpressed HL-60 cells. Nitroblue tetrazolium (NBT) reduction assays showed decreased NBT reduction activity in the CRT overexpression group and increased NBT reduction in the CRT siRNA group. Following treatment with DADS, the NBT reduction abilities in all groups were increased. In conclusion, the present study clearly demonstrates the downregulation of CRT during DADS-induced differentiation in HL-60 cells and indicates that CRT is involved in cell proliferation, invasion and differentiation in DADS-treated HL-60 cells.

Impact of the Fenton process in meat digestion as assessed using an in vitro gastro-intestinal model

The production of oxygen free radicals catalysed by non-haem iron was investigated in an in vitro mimetic model of the digestive tract using specific chemical traps. Superoxide radicals (O2∗−) and their protonated form (hydroperoxyl radicals, HO2∗) were detected by the reduction of nitroblue tetrazolium into formazan, and hydroxyl radicals (OH∗) were detected by the hydroxylation of terephthalate. Under gastric conditions, O2∗−/HO2∗ were detected in higher quantity than OH∗. Increasing the pH from 3.5 to 6.5 poorly affected the kinetics of free radical production. The oxidations generated by these free radicals were estimated on myofibrils prepared from pork rectus femoris muscle. Myofibrillar lipid and protein oxidation increased with time and oxidant concentration, with a negative impact on the digestibility of myofibrillar proteins. Plant food antioxidants considerably decreased free radical production and lipid oxidation but not protein oxidation.

Human Umbilical Cord-derived Mesenchymal Stem Cells Secrete Interleukin-6 to Influence Differentiation of Leukemic Cells

To investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells. The co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b. UC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells. UC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.

Innate Cellular Immunity in Newly Diagnosed Pulmonary Tuberculosis Patients and During Chemotherapy

BackgroundLeukocyte migration (LM) and intracellular killing aspects of the innate immune response play important roles in protection against and containment and cure of Mycobacterium tuberculosis infection, and thus may be exploited as immunotherapeutic targets to improve the management and treatment outcomes of patients with tuberculosis (TB). ObjectivesThe aim of this study was to assess LM and mediators of intracellular killing in patients with TB at the time of diagnosis and during anti-TB chemotherapy and compare them with apparently healthy controls. MethodsWe recruited 24 patients who were newly diagnosed with pulmonary TB and 20 apparently healthy individuals. Blood was drawn from patients with TB at the time of diagnosis, and after 2, 4, and 6 months of anti-TB chemotherapy and control. In vitro percentage LM (%LM) upon stimulation with Bacillus Calmette-Guérin vaccine, percentage nitroblue tetrazolium (%NBT) reduction, plasma concentrations of hydrogen peroxide (H2O2), and nitric oxide (NO) were assessed in both groups. FindingsPercentage NBT was significantly reduced in patients with TB at 2 months of anti-TB chemotherapy compared with patients at diagnosis and in healthy controls, whereas %LM was significantly increased in patients at 4 months of anti-TB chemotherapy compared with patients at diagnosis and controls. Mean plasma H2O2 and NO were significantly reduced in patients at diagnosis and throughout the period of anti-TB chemotherapy compared with the control group. Significant decreases were demonstrated in mean plasma H2O2 and NO in patients at 2 and 4 months of anti-TB chemotherapy, respectively, compared with patients at diagnosis. There was significant positive correlation between %NBT with plasma H2O2 and NO, but %LM was negatively correlated with plasma H2O2 in this group. ConclusionThe intracellular killing aspect of innate cellular immunity is deficient in patients with TB, especially 2 to 4 months after commencement of treatment. Therefore, measures (eg, arginine supplementation) to improve intracellular killing in these patients is advocated. Moreover, %LM assay with Bacillus Calmette-Guérin vaccine as an antigen may be used to differentiate those newly diagnosed patients from those on anti-TB chemotherapy.

Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD+ adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD+ adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages’ cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV–Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD+. Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV–Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD+. This adduct was confirmed to be isonicotinyl-NAD+ using LC–MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity.

The Effects of Purified Artemia Extract Proteins on Proliferation, Differentiation and Apoptosis of Human Leukemic HL-60 Cells

There has been an increment in the number of studies focused on marine bioactive materials. Many peptides and other biomaterials with anticancer potential have been extracted from various marine animals. Artemia extracts have found uses in sun-light protection cosmetics and anti-aging products. However, contents of biochemical compounds in Artemia spp. and molecular mechanisms of have not been clearly studied in leukemic cells in vitro. In this work, we isolated and purified proteins of Artemia Urmiana. Six clear fractions (A-F) observed on DEAE-cellulose chromatography were assayed for effects on cell growth, differentiation and apoptosis using the human leukemic HL-60 cell line. Cell proliferation analysis by MTT and BrdU assays indicated that did not affect cells, growth. Cells treated with crude extract and fractions A, B and C, but not E and F (up to 100 µg/mL), exhibited increase of cell growth in a dose dependent manner. Stimulatory effects of fraction D were observed at concentrations of 10 µg/ml and above. In nitro blue tetrazolium (NBT) reduction assays, treatment with 100 µg/mL of fraction E or F for 96 hr increased the fraction of differentiated cells up to 14.8 ± 3.56% and 16.5 ± 2.08% respectively. Combination of those fractions with retinoic acid had significant synergistic effects on the differentiation of cells (56.8 ± 3.7% and 67.4 ± 4.2%, p≤0.01). Annexin-V FITC staining for apoptosis and flow cytometric assays indicated induction of apoptosis by fractions E and F up to 23.8 and 31.8% of cells.

Standardization and regulation of the rate of the superoxide-generating adrenaline autoxidation reaction used for evaluation of pro/antioxidant properties of various materials

The superoxide-generating reaction of adrenaline autoxidation is widely used for determination of the activity of superoxide dismutase and pro/antioxidant properties of various materials. There are two variants of the spectrophotometric registration of the products of this reaction. The first is based on registration of adrenochrome, as adrenaline autooxidation product at 347 nm; the second employs nitro blue tetrazolium (NBT) and registration of diformazan, a product of NBT reduction at 560 nm. In the present work, recommendations for the standardization of the reaction rate in both variants have been proposed. The main approach consists in the use of the pharmaceutical form of 0.1% adrenaline hydrochloride solution. Although each of two adrenaline preparations available in the Russian market has some features in kinetic behavior of its autooxidation; they are applicable in the superoxide generating system based on adrenaline autooxidation. Performing measurements at 560 nm, the reaction rate can be regulated by lowering the concentration of added adrenaline, whereas during spectrophotometric registration at 347 nm, this cannot be done. These features of adrenaline autoxidation may be due to the fact that the intrinsic multistage process of the conversion of adrenaline to adrenochrome, which is recorded at 347 nm, is coupled with the transition of electrons from adrenaline and intermediate products of its oxidation to oxygen, carbon dioxide, and carbonate bicarbonate ions, which is detected in the presence of added NBT.

A comparative evaluation of whole blood total antioxidant capacity using nitroblue tetrazolium reduction test in patients with oral lichen planus and healthy subjects

Background and Objectives: Oral Lichen Planus is an inflammatory disease of unknown etiology. Recently, reactive oxygen species have been implicated in the pathogenesis of this disease. The investigation of disease associated oxidant-antioxidant imbalance is hard because constrained availability of specific biomarkers of oxidative stress, and the fact that measurement of individual antioxidant may give a deceptive image due to the fact that antioxidants work in concert through chain breaking reactions. Therefore, analysis of total antioxidant capacity may be the maximum applicable investigation. As blood is continuously exposed to oxidative stress, the aim of this study was to analyze overall blood antioxidant potential in healthy and oral lichen planus patients through the use of Nitroblue Tetrazolium reduction test. Methods: The study included 30 individuals which were selected from outpatients department with age ranging between 30 and 60 years, 15 patients in oral lichen planus group and 15 in healthy group (control), respectively. Blood samples were collected early morning and tested using Nitroblue Tetrazolium Reduction test (NBT). The test was performed and gave us the clear picture of whole blood total antioxidant capacity. Results: Results were obtained, analyzed and results showed that the total antioxidant capacity in whole blood of patients with oral lichen planus was significantly decreased as compared with healthy group (P

Antihypergluco-lipidemic and Antioxidant activities in Aqueous methanol Extract of Some Vegetables Peel: An in vitro Analysis

Objective: To examine phytochemical constituents, antihyperglycemic, antihyperlipidemic and free radical scavenging activities in 50% methanol extract of peels of Cucumis melo var. chito (CM), Cucumis pubescens (CP), Lagenaria siceraria (LS), Luffa acutangula (LA), and Momordica charantia (MC) applying in vitro methods. Material and Methods: Total polyphenol (gallic acid equivalent; GAE), flavonoid (rutin equivalent; RE) and anthocy anin (cyanidine-3-glucoside equivalent; CGE) content were estimated. The 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2-azinobis(3-ethylbenzthiazoline6-sulphonic acid) (ABTS .+ ); nitric oxide (NO) scavenging; nitro blue tetrazolium (NBT) reduction; advanced glycation end-products (AGEs), intestinal α-glucosidase, pancreatic lipase and liver Protein-tyrosine phosphatase 1β (PTP 1β) inhibitory activities were examined in vitro. Results: Peel of CM (1190 μgGAE/g) and MC (923 μgGAE/g) was rich in polyphenol and flavo noids (68.8, 145.1 μgRE/g respectively). Anthocyanin content was more in MC (167 mg CGE/100 g) than others. LS potently scavenged ABTS .+ (SC50, 99 μg) and DPPH (SC50 , 0.6 mg) radicals. Inhibition of vesperlysinelike AGEs (71.5%) was significant (p<0.006) than pentosidine-like AGEs (14.8%) by LA peel. Inhibition of pentosidine-like AGEs was significant (p<0.009) by CM than CP peel. MC reduced (84.5 %) only vesperlysine-like AGEs. Intestinal α-glucosidase was inhibited (23.02 %) by MC peel only. Pancreatic lipase was inhibited by CM (74.5%) and CP (58.6 %) peels. The LS reduced PTP 1β (32.1 %) more potently than others. Conclusion: Vegetable peels present variable degree of antioxidant, antihyperglycemic and antihyperlipidemic activities.

Antioxidant activity of Curculigoside, a phenolic glucoside from Curculigo orchioides Gaertn

Background: Curculigoside is phenolic glucoside derived from the plant Curculigo orchioides Gaertn, a well known immune stimulatory and rejuvenating tuberous plant. A systematic analysis of the antioxidant potential of curculigoside is not reported as far. Methods: In the present study antioxidant activity of curculigoside was evaluated both in the in vivo and in vitro models. Tube test were performed to analyse 1, 1-diphenyl-2-picrylhydrazyl (DPPH), super oxide (Nitro blue tetrazolium reduction) and hydroxyl radical (Fenton reaction) scavenging effects. Besides, effect of the compound on lipid peroxidation and nitric oxide scavenging was also tested. In vivo antioxidant activity was checked by PMA (Phorbol-12-myristate-13-acetate) induced superoxide radical formation by mouse peritoneal macrophages. Result: Curculigoside was found to scavenge the hydroxyl radicals generated by Fenton reaction and also inhibited the in vitro lipid peroxidation. The compound was also found to be an effective scavenger of superoxide, DPPH and nitric oxide (NO) radicals. Curculigoside showed a significant scavenging effect on the NO release by cultured macrophages. Besides, oral administration of curculigoside inhibited superoxide generation in macrophages in vivo . Conclusion: Curculigoside was found to exhibit good free radical scavenging effects which is comparable to known standards, both in the in vitro as well as in vivo systems.

The effects of phenolic microbial metabolites on the activities of mitochondrial enzymes

The effects of microbial phenolic metabolites on the activities of enzymes of the tricarboxylic acid cycle were investigated in isolated mitochondria. The detection of metabolites of the tricarboxylic acid cycle in the blood of patients with sepsis as potential biomarkers of mitochondrial dysfunction was investigated. We found that microbial phenolic metabolites possess an inhibitory effect on the activity of dehydrogenases, as determined by the reduction of dichlorophenolindophenol and nitroblue tetrazolium in liver mitochondria and liver homogenates. The effect was more pronounced in the case of the oxidation of NAD-dependent substrates than succinate oxidation, as well as at lower concentrations of microbial metabolites than in the case of inhibition of respiration. Using gas chromatography coupled with mass spectrometry it was shown that the content of the tricarboxylic acid cycle metabolites is lower in the blood of patients with sepsis as compared to healthy donors. Our data demonstrate that microbial phenolic acids can significantly contribute to mitochondrial dysfunction and to metabolic suppression, both of which are characteristic of these pathologies.

Immunomodulatory Effect of Gymnema sylvestre (R.Br.) Leaf Extract: An In Vitro Study in Rat Model.

Gymnema sylvestre Wild R.Br (family: Asclepidaceae) is a valuable medicinal plant used in folk medicine to treat diabetes, obesity, asthma etc. in India for antiquity. Diabetes mellitus is a syndrome characterized immunologically by lymphocyte apoptosis and reduced cell-mediated and humoral immunity. Modulation of immune responses to alleviate diseases has been of interest, and traditional herbal medicines may play an important role in this regard. In this study, we aim to evaluate the immunomodulatory potential of methanolic extract of G. sylvestre leaf using rat model. HPLC analysis of leaf extract was carried out for gymnemic acid. The method involves the initial hydrolysis of gymnemic acids, the active ingredients, to a common aglycone followed by the quantitative estimation of gymnemagenin, using gymnemagenin as reference standard. Gymnemic acid content was 2.40% (w/w) in G. sylvestre leaf extract. In vitro immunomodulatory activity of the methanolic extract of G. sylvestre leaf (1–200μg/ml) was evaluated by gauging its effects on nitroblue tetrazolium reduction and nitrite release in rat peritoneal macrophages and on mitogen (ConA, PHA and LPS) induced splenic lymphocyte proliferation. G. sylvestre leaf extract showed significant (<0.05) enhancement in NO and ROS generation in macrophages and in proliferation of lymphocytes in dose dependent manner. EC50 value was 3.10, 3.75 and 2.68μg/ml for NBT reduction, nitrite release and lymphoproliferation, respectively. Potential effect was observed at 100 μg/ml in NO and ROS generation in macrophages and 20 μg/ml in lymphocyte proliferation. G. sylvestre leaf extract stimulates macrophage reactivity, increasing the level of activity even higher when combined with PMA or LPS. These findings suggest the presence of active compounds, gymnemic acid, in methanolic extract of G. sylvestre leaf that stimulates both myeloid and lymphoid components of immune system, and therefore can restore the innate immune function. Through this study, the traditional knowledge of anti-diabetic property of G. sylvestre is scientifically supplemented with its immunomodulatory properties.

Reactive Oxygen Intermediate (ROI) in Dog Macrophage Infected with Mycobacterium tuberculosis

experiment used 24 healthy dogs, aged between 1 and 2 years, both male and female which were divided into twodifferent groups consisting of 12 dogs each. The first group was the treatment group, that is they were infected with Mtuberculosis and the second one was the control group. The activity of macrophages ROI secretion were measured at1st, 2nd, 12th, and 24th after infection using nitroblue tetrazolium (NBT) reduction assay. Three cats were used to measure themacrophage activity in each period, using triplicate sample for each cat. The results of the experiment showed thatROI secretion increased in infected group compared with the control group, and this activity reached to the plateaulevel at 2 weeks after infection. Although these enhanced activities were gradually diminished thereafter, higherlevels of these activities were consistently observed until the end of experiment compared with control group. Theresults of the experiment indicated that ROI played an important role to against M.tuberculosis infection in dogs.Keyword: macrophage, ROI, M.tuberculosis, dogs

Internalization of titanium dioxide nanoparticles by glial cells is given at short times and is mainly mediated by actin reorganization-dependent endocytosis

Many nanoparticles (NPs) have toxic effects on multiple cell lines. This toxicity is assumed to be related to their accumulation within cells. However, the process of internalization of NPs has not yet been fully characterized. In this study, the cellular uptake, accumulation, and localization of titanium dioxide nanoparticles (TiO2 NPs) in rat (C6) and human (U373) glial cells were analyzed using time-lapse microscopy (TLM) and transmission electron microscopy (TEM). Cytochalasin D (Cyt-D) was used to evaluate whether the internalization process depends of actin reorganization. To determine whether the NP uptake is mediated by phagocytosis or macropinocytosis, nitroblue tetrazolium (NBT) reduction was measured and the 5-(N-ethyl-N-isopropyl)-amiloride was used. Expression of proteins involved with endocytosis and exocytosis such as caveolin-1 (Cav-1) and cysteine string proteins (CSPs) was also determined using flow cytometry.TiO2 NPs were taken up by both cell types, were bound to cellular membranes and were internalized at very short times after exposure (C6, 30min; U373, 2h). During the uptake process, the formation of pseudopodia and intracellular vesicles was observed, indicating that this process was mediated by endocytosis. No specific localization of TiO2 NPs into particular organelles was found: in contrast, they were primarily localized into large vesicles in the cytoplasm. Internalization of TiO2 NPs was strongly inhibited by Cyt-D in both cells and by amiloride in U373 cells; besides, the observed endocytosis was not associated with NBT reduction in either cell type, indicating that macropinocytosis is the main process of internalization in U373 cells. In addition, increases in the expression of Cav-1 protein and CSPs were observed.In conclusion, glial cells are able to internalize TiO2 NPs by a constitutive endocytic mechanism which may be associated with their strong cytotoxic effect in these cells; therefore, TiO2 NPs internalization and their accumulation in brain cells could be dangerous to human health.

Abstract 5347: SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells

Abstract Although differentiation-inducing agents have significantly improved the management of acute promyelocytic leukemia, no significant progress has been made in the treatment of other acute myeloid leukemias (AML). Numerous proteins involved in tumor development have so-called allosteric disulfide bonds amenable to modifications affecting protein structure and function. We have developed SK053, a small molecule and mechanism-selective inhibitor of enzymes involved in allosteric disulfide bonds formation such as thioredoxin, thioredoxin reductase and protein disulfide isomerase (PDI). The aim of our studies was to evaluate anti-leukemic activity of SK053 in human AML cells. To validate if SK053 targets PDI, a binding assay and an insulin turbidimetric activity assay were used. Cytostatic/cytotoxic effects in HL60, NB4, KG-1 and MOLM14 cells as well as in primary AML cells were assessed with trypan blue exclusion. Differentiation of AML cells was studied with May-Grünwald-Giemsa staining, nitro blue tetrazolium reduction assay and flow cytometry analysis of CD11b, CD14 and CD15 levels and by RNA sequencing, qRT-PCR and western blotting (WB). We observed covalent binding of SK053 to PDI and inhibition of its enzymatic activity with IC50 of 10 μM. Since PDI blocks translation of CCAAT enhancer binding protein alpha (CEBPA), a transcription factor crucial for neutrophils maturation, we evaluated the potential of SK053 to induce differentiation and cytostatic/cytotoxic effects in human AML cells. SK053 exerts significant cytostatic/cytotoxic activity in human AML cells (HL60, NB4, KG-1 and MOLM14), and induces differentiation of AML blasts into more mature myeloid cells. Incubation of AML cells with SK053 induced expression of CEBPA and hexokinase 3 mRNA in quantitative RT-PCR and increased amount of CEBPA protein in nuclear fraction measured in WB. Finally, SK053 induces differentiation of primary leukemic cells freshly isolated from AML patients. RNA-seq analysis revealed that incubation of HL60 cells with SK053 down-regulates mRNA for MYC and ID1 oncogenes as well as for histone proteins. Expression of other genes of mature myeloid lineage such as adhesion molecules (collagen type XV, fibronectin I, MAC-1), hydrolytic enzymes (carboxypeptidase, proteinase 3, CA12 anhydrase, ADAM19 metalloprotease), proteoglycan 2 (core of eosinophilic granules) and PGLYRP3 (peptidoglycan recognition protein 3) was significantly up-regulated. The GeneOntology analysis done with the RNAseq results revealed enrichment of gene transcripts regulating myeloid cells differentiation. SK053 exerts potent anti-leukemic activity and induces differentiation of numerous types of human AML cells. Targeting allosteric disulfide bonds with small molecule inhibitors presents a promising therapeutic strategy in AML. Citation Format: Dominika Nowis, Justyna Chlebowska, Pawel Gaj, Michal Lazniewski, Malgorzata Firczuk, Karolina Furs, Radoslaw Sadowski, Pawel Leszczynski, Piotr Stawinski, Szymon Klossowski, Ryszard Ostaszewski, Krzysztof Giannopoulos, Rafal Ploski, Dariusz Plewczynski, Jakub Golab. SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2015-5347