Monocytic U937 Research Articles

Gain-of-function Tibetan PHD2D4E;C127S variant suppresses monocyte function: A lesson in inflammatory response to inspired hypoxia.

BackgroundWe have previously described an evolutionarily selected Tibetan prolyl hydroxylase-2 (PHD2D4E;C127S) variant that degrades the hypoxia-inducible factor (HIFα) more efficiently and protects these highlanders from hypoxia-triggered elevation in haemoglobin concentration. High altitude is known to cause acute mountain sickness (AMS) and high-altitude pulmonary edema (HAPE) in a section of rapidly ascending non-acclimatised lowlanders. These morbidities are often accompanied by inflammatory response and exposure to hypobaric hypoxia is presumed to be the principal causative agent. We have investigated whether PHD2D4E;C127S variant is associated with prevention of hypoxia-mediated inflammatory milieu in Tibetan highlanders and therefore identify a potential target to regulate inflammation.MethodsWe genotyped the Tibetans using DNA isolated from whole blood. Thereafter immunophenotying was performed on PBMCs from homozygous PHD2D4E;C127S and PHD2WT individuals using flow cytometry. RNA isolated from these individuals was used to evaluate the peripheral level of important transcripts associated with immune as well as hypoxia response employing the nCounter technology. The ex-vivo findings were validated by generating monocytic cell lines (U937 cell line) expressing PHD2D4E;C127S and PHD2WT variants post depletion of endogenous PHD2. We had also collected whole blood samples from healthy travellers and travellers afflicted with AMS and HAPE to evaluate the significance of our ex-vivo and in vitro findings. Hereafter, we also attempted to resolve hypoxia-triggered inflammation in vitro as well as in vivo by augmenting the function of PHD2 using alpha-ketoglutarate (αKG), a co-factor of PHD2.FindingsWe report that homozygous PHD2D4E;C127S highlanders harbour less inflammatory and patrolling monocytes in circulation as compared to Tibetan PHD2WT highlanders. In response to in vitro hypoxia, secretion of IL6 and IL1β from PHD2D4E;C127S monocytes, and their chemotactic response compared to the PHD2WT are compromised, corresponding to the down-modulated expression of related signalling molecules RELA, JUN, STAT1, ATF2 and CXCR4. We verified these functional outcomes in monocytic U937 cell line engineered to express PHD2D4E;C127S and confirmed the down-modulation of the signalling molecules at protein level under hypoxia. In contrast, non-Tibetan sojourners with AMS and HAPE at high altitude (3,600 m above sea level) displayed significant increase in these inflammatory parameters. Our data henceforth underline the role of gain-of-function of PHD2 as the rate limiting factor to harness hyper-activation of monocytes in hypoxic environment. Therefore upon pre-treatment with αKG, we observed diminished inflammatory response of monocytes in vitro and reduction in leukocyte infiltration to the lungs in mice exposed to normobaric hypoxia.InterpretationOur report suggests that gain-of-function PHD2 D4E;C127S variant can therefore protect against inflammation elicited by hypobaric hypoxia. Augmentation of PHD2 activity therefore may be an important method to alleviate inflammatory response to inspired hypoxia.FundingThis study is supported by the Department of Biotechnology, Government of India.

Porphyromonas gingivalis lipopolysaccharide and gingival fibroblast augment MMP-9 expression of monocytic U937 cells through cyclophilin A.

Intercellular cross-talking was suggested in matrix metalloproteinase (MMP)-9 expression with unknown mechanisms. Studies showed cyclophilin A (CypA) playing an important role in regulating MMP-9 expression in varied diseases. The aim of the study was to examine the CyPA on the MMP-9 augmentation in monocytic U937 cells after Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) treatment and human gingival fibroblast (hGF) co-culture. In independent culture or co-culture of hGF and U937 cell, quantitative real-time polymerase chain reaction (qPCR) and zymography were selected to examine the mRNA and protein activity of MMP-9, respectively. The CyPA expression was determined by qPCR. LPS could enhance MMP-9 mRNA expression and enzyme activity in U937 cell. However, the enhancements were not observed in hGF. Similarly, LPS enhanced CyPA mRNA in U937, but not in hGF. After co-cultured with hGF, however, MMP-9 and CyPA in U937 increased regardless of the presence/absence of LPS. In U937 cells, the extra-supplied CyPA increased MMP-9 mRNA and enzyme activity, whereas the CyPA inhibitor, cyclosporine A, suppressed the LPS- and co-culture-enhanced MMP-9. Moreover, the inhibitors for MAP kinase, including PD98059 (ERK) and SP600125 (JNK), suppressed the CyPA-enhanced MMP-9 in U937. Through the CyPA pathway, the LPS and the hGF could augment the MMP-9 expression in the U937 cells.

Fomitopsis cajanderi extracts have differential tumoricidal actions on human MCF7 and U-937 cancer cells and inhibit TNF secretion by U-937 monocytes

Abstract This study aimed to determine if hot ethanol and aqueous extracts from the Fomitopsis cajanderi mushroom species are cytotoxic to human cancer cell lines and modulate TNF secretion by human monocytes. The hot ethanol extract was made via a 2 hour Soxhlet extraction of F. cajanderi fruiting body in ethanol using a cold finger condenser. The hot aqueous extract was made via a 2 hour decoction in water at 100°C. Human MCF7 mammary adenocarcinoma and U-937 histiocytic lymphoma cells were treated with increasing concentrations of extract or solvent controls and were assessed for cytotoxicity by XTT colorimetric assay. Effects of the aqueous extract on TNF secretion by PMA-differentiated, LPS-stimulated U-937 cells were determined by TNF ELISA. Glycosyl content and linkage analysis of the aqueous extract were assessed by the Complex Carbohydrate Research Center (Athens, Georgia, USA). The hot ethanol extract induced concentration-dependent cytotoxicity of MCF7 and U-937 cells, with stronger U-937 tumoricidal effects. The hot aqueous extract was not cytotoxic to MCF7 cells, but had cytotoxic effects on undifferentiated U-937 cells at high concentrations. The aqueous extract, found to contain beta glucans, inhibited TNF secretion by LPS-stimulated U-937 cells in a concentration-dependent manner. Hot ethanol and aqueous F. cajanderi extracts showed differential tumoricidal actions on human cancer cells. The ethanol extract was cytotoxic to the human cancer cells tested. The aqueous extract contained beta glucans reported to be immunomodulatory, and inhibited U-937 TNF secretion. These results warrant further research of F. cajanderi extracts as novel adjunctive anti-cancer and anti-inflammatory agents.

Anti-Inflammatory Activity of Benzyl Glycosides from the Flowers of Lilium Asiatic Hybrids

Repeated column chromatography of solvent fractions extracted from the flowers of Lilium Asiatic hybrids ensured purification of three benzyl glycosides I – III, which were reliably identified based on the analysis of various spectroscopic data. Compounds I – III (20 μg/mL) reduced nuclear factor-kappa B (NF-kB) (p-p65/p-65) by 9.64 ± 0.66%, 30.98 ± 1.08%, and 54.04 ± 1.12%, respectively. Compounds II and III markedly inhibited the expression of vascular cell adhesion molecule 1 (VCAM-1) in tumor necrosis factor (TNF)-α-stimulated human aortic smooth muscle cells (HASMCs) by 38.68 ± 2.52% and 37.9 ± 2.76%, respectively. Compound III significantly suppressed U937 monocytes attachment to the TNF-α-stimulated HASMCs.

Amyloid beta peptide (Aβ 1–42 ) antagonizes nicotinic acetylcholine receptors of monocytes and enables ATP‐mediated release of interleukin‐1β

Amyloid-β peptide (Aβ1-42) plays a pathogenic role in Alzheimer´s disease. Its physiological functions are, however, still debated. Aβ1-42 can induce the secretion of the pro-inflammatory cytokine intereukin-1β (IL-1β) by immune cells. Known interaction partners of Aβ1-42 are α7 nicotinic acetylcholine receptors (nAChRs). Recently, we identified a cholinergic mechanism that inhibits the ATP-associated release of IL-1β by human monocytes via nAChRs containing α7, α9 and/or α10 subunits. Moreover, we discovered novel nAChR agonists that efficiently inhibit monocytic IL-1β release via a metabotropic mechanism. The objective of this study was to test if Aβ1-42 can modulate this cholinergic mechanism. Human monocytic U937 cells and freshly isolated human peripheral blood mononuclear cells (approved by the ethics committee of the medical faculty Giessen, Germany, and performed in accordance with the Helsinki Declaration) were primed with lipopolysaccharide and ATP-induced IL-1β release in the presence or absence nAChR agonists and Aβ1-42 was measured by enzyme-linked immunosorbent assay. We found that Aβ1-42 dose-dependently (IC50= 2.5 µM) antagonized the inhibitory effect of canonical and novel non-canonical nicotinic agonists like phosphocholine (100 µM), C-reactive protein (5 µg/mL) and glycerophosphocholine (10 µM), indicating a novel pro-inflammatory Aβ1-42 function that enables monocytic IL-1β release in the presence of nAChR agonists. In whole-cell patch-clamp and calcium imaging experiments on human P2X7 receptor overexpressing HEK293 cells, no direct effect of Aβ1-42 on P2X7 receptor activity was observed. In blood plasma samples from major surgery patients, IL-1β was mostly detected together with increased Aβ1-42 levels, which is in line with our hypothesis. Taken together, our results suggest that Aβ1-42 can antagonize the cholinergic control of ATP-mediated monocytic IL-1β-release and, thus, provide evidence for a novel function of Aβ1-42 that also might be considered in situations of systemic inflammation.

Stealthiness and Hematocompatibility of Gold Nanoparticles with Pre-Formed Protein Corona.

Studies have established that a serum protein corona pre-formed around gold nanorods (NRs) could be exploited for loading photosensitizers and chemotherapeutics to result in efficient cell kill in vitro with an extremely low dose. In this study, we further demonstrated that pre-forming a serum protein corona (PC) around citrate-capped NRs (NR-Cit) to form NR-PC conferred them stealth property and high hematocompatibility similar to the common strategy of PEGylating NRs, which would otherwise not be able to evade the immune system. Specifically, the NR-PC caused minimal complement activation with significantly lower formation of the terminal complement complex SC5b-9 measured in human serum containing NR-PC, and this resulted in low uptake by phagocytic U937 monocytes of 5.9% of the initial gold dose compared to 55.8% of NR-Cit. In addition, NR-PC exhibited very low hemolytic activity of less than 0.2% hemolysis with no observable effect on RBC morphology as opposed to 0.6% for NR-Cit at the same concentration of 1 nM NRs. Furthermore, we showed that the high hematocompatibility and stealth property of NR-PC were maintained even after the loading of small molecules, photosensitizer Chlorine e6 (Ce6), into the protein corona, thus further establishing the potential clinical relevance of exploiting the inevitably formed serum protein corona on nanoparticles as an effective delivery vector for small molecular therapeutics.

LCZ696 ameliorates lipopolysaccharide-induced endothelial injury.

Lipopolysaccharide (LPS)-induced endothelial dysfunction plays an important role in the pathogenesis of cardiovascular diseases. LCZ696, the dual-acting angiotensin receptor blocker, and neprilysin inhibitor has been used for the treatment of heart failure with reduced ejection fraction. Recent work suggests that LCZ696 therapy might have an anti-inflammatory effect in cardiovascular tissue. In the current study, we show that LCZ696 attenuates LPS-induced oxidative stress by reducing the production of intracellular reactive oxygen species (ROS) and the measurements of malonyl dialdehyde (MDA) level in human umbilical vascular endothelial cells (HUVECs). LCZ696 inhibits LPS-induced expressions and secretions of the pro-inflammatory cytokines, interleukin-6 (IL-6), interleukin-1α (IL-1α), and tumor necrosis factor β (TNF-β) as well as the chemokines, monocyte chemotactic protein 1 (MCP-1), and chemokine (C-X-C motif) ligand 1 protein (CXCL1). Additionally, we found that LCZ696 reduces LPS-induced expressions of vascular cell adhesion molecule 1 (VCAM-1) and P-selectin and the attachment of U937 monocytes to HUVECs. Mechanistically, LCZ696 prevents LPS-induced activation of the TLR4/Myd88 pathway and nuclear translocation of nuclear factor kappa-B (NF-κB) p65 factor. Based on these findings, we conclude that LCZ696 is capable of ameliorating LPS-induced endothelial dysfunction via anti-inflammatory properties.

Single cell morphological metrics and cytoskeletal alignment regulate VCAM-1 protein expression

In the initial stages of atherosclerosis, vascular adhesion molecule-1 (VCAM-1) is a surface protein that mediates leukocyte adhesion to the endothelium’s luminal surface. VCAM-1 expression is upregulated on endothelial cells (ECs) under pro-inflammatory conditions and is known to be modulated by fluid shear stress (FSS). High, pulsatile FSS induces endothelial elongation and cytoskeletal alignment and downregulates pro-inflammatory induced VCAM-1 expression, which is associated with an athero-protective EC phenotype. In contrast, athero-prone ECs under low, oscillatory FSS fail to elongate and maintain a cobblestone morphology with random cytoskeletal alignment, while VCAM-1 expression is upregulated. Whether EC shape and cytoskeletal alignment play a role in the regulation of VCAM-1 protein expression independent of FSS has not been previously determined. The goal of this study was to determine the effect of EC morphology, specifically cell elongation and alignment, and cytoskeletal alignment on VCAM-1 protein expression using topographical micropatterning of an endothelial monolayer and single cell image analysis techniques. Elongated ECs with an aligned cytoskeleton significantly downregulated VCAM-1 protein expression in the absence of FSS compared to planar controls. In addition, linear correlations between morphological metrics and protein expression showed that actin alignment had a significantly stronger effect on VCAM-1 expression than cell elongation. Functionally, monocytic U937 cells statically adhered less on micropatterns compared to planar substrates, in a VCAM-1 dependent manner. Therefore, endothelial cellular elongation and alignment as well as cytoskeletal alignment regulate VCAM-1 protein expression and immunogenic functions to produce a less inflammatory phenotype in the absence of hemodynamic effects.

Candida albicans Activation of Human Monocytes Toward M2 Profile is Reversed by Amphotericin B and Fluconazole

Phagocytes, including monocytes/macrophages, play an important role in the host defense during Candida albicans infections. In the L-arginine metabolism, the balance between the activation of two enzymes, inducible Nitric Oxide Synthase (iNOS) and arginase, promotes in the macrophages two alternative metabolic states, while M1 profile is related with host protection, M2 favored the fungal growth and evasion. Our aim was to evaluate the effect of Amphotericin B (AMB) and Fluconazole (FLC) on polarization of human monocytes to M2 profile induced by C. albicans. The human monocytic (Mo) cell line U937 was co-cultured with viable yeast of C. albicans, or Lipopolysaccharides (LPS) or Phorbol-12-myristate-13-acetate (PMA). Nitric Oxide (NO), cytokines production and arginase activity were evaluated. The effect of AMB or FLC on these metabolic pathways in immune cells and on fungus intrinsic arginase activity was studied. C. albicans inhibits NO production in human-monocyte and induces strong host arginase activity (p<0.0001). AMB and FLC inhibited C. albicansinduced arginase activity in immune cells (p<0.001), reaching a percentage of inhibition of 90% for AMB and 78% for FLC. Arginase intrinsic activity of the fungus was blocked by nor-NOHA (arginase inhibitor) and AMB (p<0.05). These results show that C. albicans drives human Mo toward M2 profile and that both antifungal drugs evaluated have the ability to revert C. albicans-induced M2 profile. In a relevant manner, it also provides data about additional effect of AMB as inhibitor of C. albicans endogenous arginase activity. Here in we provide new evidence for the effect of these drugs over the immune cells and the yeast.

Commercial α1-antitrypsin preparations markedly differ in their potential to inhibit the ATP-induced release of monocytic interleukin-1β

The acute phase protein α1-antitrypsin (AAT) inhibits numerous proteases, specifically neutrophil elastase. Patients with an AAT deficiency due to mutations frequently develop early onset emphysema. The commercial preparations of human plasma AAT are clinically used as biopharmaceuticals to protect the lung tissue of AAT-deficient patients from damage caused by neutrophil elastase. Accordingly, preparations of AAT are validated for their anti-elastase activity. However, several anti-inflammatory effects of AAT were described, some of them being independent from its anti-protease function. We recently demonstrated that AAT isolated from the blood of healthy persons efficiently inhibits the ATP-induced release of interleukin-1β by human monocytes. This finding is of therapeutic relevance, because IL-1β plays an important role in numerous debilitating and life-threatening inflammatory diseases. As anti-inflammatory functions of AAT are of increasing clinical interest, we compared the potential of two widely used AAT preparations, Prolastin® and Respreeza®, to inhibit the ATP-induced release of IL-1β using human monocytic U937 cells. We detected marked functional differences between both medicaments. The AAT preparation Respreeza® is less active compared to Prolastin® regarding the inhibition of the ATP-induced release of monocytic IL-1β. Chemical oxidation of Respreeza® restored this anti-inflammatory activity, while destroying its anti-protease function. Our data suggest that the anti-inflammatory potential and the anti-protease function of AAT can be fully uncoupled. In the light of the increasing clinical interest in anti-inflammatory functions of AAT, commercial AAT preparations should be carefully reinvestigated and optimized to preserve the dual anti-protease and anti-inflammatory activity of native AAT.

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

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

N6-methyladenosine modification of HIV-1 RNA suppresses type-I interferon induction in differentiated monocytic cells and primary macrophages.

N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in CD4+ T cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m6A levels of RNA by manipulating the expression of the m6A erasers (FTO and ALKBH5) or pharmacological inhibition of m6A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro. HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies indicated that m6A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m6A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells.

5-((7-Chloro-6-fluoro-1h-indol-3-yl) methyl)-3-methylimidazolidine-2,4-dione as a RIP1 inhibitor protects LPS/D-galactosamine-induced liver failure

AimsNecroptosis, an inflammatory form of regulated necrosis mediated by receptor-interacting kinase 1 (RIP1), RIP3, and pseudokinase mixed lineage kinase domain-like protein (MLKL) is extensively implicated in liver inflammatory disease. Thus identification small-molecule inhibitor of necroptosis has emerged as a potential therapeutic strategy to prevent liver damage. In this study, we identified 5-((7-chloro-6-fluoro-1 h-indol-3-yl) methyl)-3-methylimidazolidine-2,4-dione (F-nec) as a novel potent necroptosis inhibitor. Main methodsTo find out the potent chemical inhibitors of necroptosis, human monocytic U937 cells were treated with a combination of tumor necrosis factor alpha (TNFα) and a pan-caspase inhibitor z-VAD-fmk. LPS and D-galactosamine (LPS/GalN) were further employed to simulate acute liver failure to explore therapeutic potency of F-nec in vivo. In addition, a specific inhibitor of c-Jun NH (2)-terminal kinases (JNK) SP600125 and its activator anisomycin are used to elucidate its mechanisms in acute liver failure therapy. Necroptosis pathway related proteins were tested by western blot. Key findingsIn this study, we identified F-nec as a novel potent RIP1 inhibitor which efficiently blocked TNFα-induced necroptosis in human and mice cells. Furthermore, pre-treatment of F-nec could prevent hepatic necrosis by reducing RIP1-mediated necroptosis also effectively ameliorated LPS/GalN induced acute liver failure by attenuating cell death signaling-stimulated JNK pathway activation and then suppressing JNK-triggered inflammation. SignificanceAltogether, this study demonstrates that F-nec is a potent inhibitor of RIP1 and highlights its great potential for use in the treatment of RIP1-driven inflammatory liver diseases.

L-Cysteine Stimulates the Effect of Vitamin D on Inhibition of Oxidative Stress, IL-8, and MCP-1 Secretion in High Glucose Treated Monocytes

Objective: Vitamin D deficiency is common in the general population and diabetic patients, and supplementation with vitamin D is widely used to help lower oxidative stress and inflammation. The cytokine storm in SARS-CoV2 infection has been linked with both diabetes and Vitamin D deficiency. This study examined the hypothesis that supplementation with vitamin D, in combination with l-cysteine (LC), is better at reducing oxidative stress and thereby, more effective, at inhibiting the secretion of the pro-inflammatory cytokines, Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in U937 monocytes exposed to high glucose concentrations. Methods: U937 monocytes were pretreated with 1,25 (OH)2 vitamin D (VD, 10 nM) or LC (250 µM) or VD + LC for 24 h and then exposed to control or high glucose (HG, 25 mM) for another 24 h. Results: There were significantly greater reactive oxygen species (ROS) levels in monocytes treated with HG than those in controls. Combined supplementation with VD and LC showed a more significant reduction in ROS (46%) in comparison with treatment with LC (19%) or VD (26%) alone in monocytes exposed to HG. Similarly, VD supplementation, together with LC, caused a more significant inhibition in the secretion of IL-8 (36% versus 16%) and MCP-1 (46% versus 26%) in comparison with that of VD (10 nM) alone in high-glucose treated monocytes. Conclusions: These results suggest that combined supplementation with vitamin D and LC has the potential to be more effective than either VD or LC alone in lowering the risk of oxidative stress and inflammation associated with type 2 diabetes or COVID-19 infection. Further, this combined vitamin D with LC/N-acetylcysteine may be a potent alternative therapy for SARS-CoV2 infected subjects. This approach can prevent cellular damage due to cytokine storm in comorbid systemic inflammatory conditions, such as diabetes, obesity, and hypertension.

Cationic peptidomimetics derived from CAP-18 cathelicidin shown activity in vitro against mycobacterium tuberculosis H37RA and synergy combined with isoniazid

Aims & objectives: Tuberculosis (TB) is the infectious disease that results in the largest number of deaths around the world caused by a bacterial infectious agent. According to the World Health Organization (WHO), there were 10 million new cases and 1.2 million deaths caused by TB in 2018. The problem increases due to drug resistance and HIV/AIDS coinfection, which is why it is necessary to develop new anti-TB compounds with different mechanisms of action. Antimicrobial peptides are molecules gener42ated in the innate immune response of the host with an effect on pathogens. This is the case of the cathelicidins present in mammals involved in the formation of channels in the bacterial cell membrane that leads to cell lysis. Methods: In this study, the antimycobacterial activity and the possible mechanism of action of peptides derived from CAP-18 with unnatural amino acids were evaluated. Using bioinformatic tools, the epitope of the native sequence with the best characteristics of helical structure, amphipathicity and antibacterial value was determined. The corresponding peptides, CAP18-B and CAP18-C, had changes from L-Lys to D-Lys and from L-Gly to N-methyl-Gly in the sequence, generating the peptides CAP18-BD, CAP18-CD and the peptoid CAP18-BP. Peptides and peptoids were synthesized using the Fmoc strategy, purified by RP-HPLC and characterized by ESI-MS, circular dichroism and H1 -NMR. The in vitro assesament of the designed sequences was carried out in Mycobacterium smegmatis mc2 155 and Results: Mycobacterium tuberculosis H37Ra, obtaining a minimum inhibitory concentration increase from 2 to 6 times in the ranges from 1200 to 60 μg/mL, less than 10% hemolytic activity and no cytotoxic activity on U937 monocytes with LC50 values at least 10 times higher than the MIC and synergy combined with isoniazid. Confocal microscopy showed the insertion of peptides and peptoids in Mycobacterium smegmatis mc2 155 and transmission electron microscopy showed alterations in the cell membrane of Mycobacterium smegmatis mc2 155 evidenced in the formation of blisters, microspheres, channels and cell lysis. Conclusions: The results suggest that analogous sequences derived from CAP-18 can be considered as potential antimycobacterial compounds.

Pro-Inflammatory Effect of Gliadins and Glutenins Extracted from Different Wheat Cultivars on an In Vitro 3D Intestinal Epithelium Model.

There is a need to assess the relationship between improved rheological properties and the immunogenic potential of wheat proteins. The present study aimed to investigate the in vitro effects of total protein extracts from three modern and two landrace Triticum aestivum commercial flour mixes, with significant differences in gluten strength (GS), on cell lines. Cytotoxicity and innate immune responses induced by wheat proteins were investigated using Caco-2 monocultures, two dimensional (2D) Caco-2/U937 co-cultures, and three dimensional (3D) co-cultures simulating the intestinal mucosa with Caco-2 epithelial cells situated above an extra-cellular matrix containing U937 monocytes and L929 fibroblasts. Modern wheat proteins, with increased GS, significantly reduced Caco-2 cell proliferation and vitality in monoculture and 2D co-cultures than landrace proteins. Modern wheat proteins also augmented Caco-2 monolayer disruption and tight junction protein, occludin, redistribution in 3D co-cultures. Release of interleukin-8 into the cell medium and increased U937 monocyte migration in both 2D and 3D co-cultures were similarly apparent. Immuno-activation of migrating U937 cells was evidenced from cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. The modern wheat proteins, with gluten polymorphism relatedness and increased GS, were shown to be more cytotoxic and immunogenic than the landrace wheat proteins.

Effect of Calomelanone, a Dihydrochalcone Analogue, on Human Cancer Apoptosis/Regulated Cell Death in an In Vitro Model.

Calomelanone, 2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, possesses anticancer activities. This study was conducted to investigate the cytotoxic effect of calomelanone, a dihydrochalcone analogue, on human cancer cells and its associated mechanisms. The cytotoxic effect of calomelanone was measured by MTT assay. Annexin V-FITC/propidium iodide and DiOC6 staining that employed flow cytometry were used to determine the mode of cell death and reduction of mitochondrial transmembrane potential (MTP), respectively. Caspase activities were measured using specific substrates and colorimetric analysis. The expression levels of Bcl-2 family proteins were determined by immunoblotting. Reactive oxygen species were also measured using 2′,7′-dihydrodichlorofluorescein diacetate and dihydroethidium (fluorescence dyes). Calomelanone was found to be toxic towards various human cancer cells, including acute promyelocytic HL-60 and monocytic leukemic U937 cells, in a dose-dependent manner at 24 h and human hepatocellular HepG2 cells at 48 h. However, the proliferation of HepG2 cells increased at 24 h. Calomelanone was found to induce apoptosis in HL-60 and U937 at 24 h and HepG2 apoptosis at 48 h via the intrinsic pathway by inducing MTP disruption. This compound also induced caspase-3, caspase-8, and caspase-9 activities. Calomelanone upregulated proapoptotic Bax and Bak and downregulated antiapoptotic Bcl-xL proteins in HepG2 cells. Moreover, signaling was also associated with oxidative stress in HepG2 cells. Calomelanone induced autophagy at 24 h of treatment, which was evidenced by staining with monodansylcadaverine (MDC) to represent autophagic flux. This was associated with a decrease of Akt (survival pathway) and an upregulation of Atg5 (the marker of autophagy). Thus, calomelanone induced apoptosis/regulated cell death in HL-60, U937, and HepG2 cells. However, it also induced autophagy in HepG2 depending on duration, dose, and type of cells. Thus, calomelanone could be used as a potential anticancer agent for cancer treatment. Nevertheless, acute and chronic toxicity should be further investigated in animals before conducting investigations in human patients.

MLL and CALM Are Fused to AF10 in Morphologically Distinct Subsets of Acute Leukemia With Translocation t(10;11): Both Rearrangements Are Associated With a Poor Prognosis

AbstractThe translocation t(10;11)(p13;q14) has been observed in acute lymphoblastic leukemia (ALL) as well as acute myeloid leukemia (AML). A recent study showed a MLL/AF10fusion in all cases of AML with t(10;11) and various breakpoints on chromosome 11 ranging from q13 to q23. We recently cloned CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene), the fusion partner of AF10 at 11q14 in the monocytic cell line U937. To further define the role of these genes in acute leukemias, 10 cases (9 AML and 1 ALL) with cytogenetically proven t(10;11)(p12-14;q13-21) and well-characterized morphology, immunophenotype, and clinical course were analyzed. Interphase fluorescence in situ hybridization (FISH) was performed with 2 YACs flanking the CALM region, a YAC contig of the MLLregion, and a YAC spanning the AF10 breakpoint. Rearrangement of at least one of these genes was detected in all cases with balanced t(10;11). In 4 cases, including 3 AML with immature morphology (1 AML-M0 and 2 AML-M1) and 1 ALL, the signals of the CALM YACS were separated in interphase cells, indicating a translocation breakpoint within the CALM region. MLL was rearranged in 3 AML with myelomonocytic differentiation (2 AML-M2 and 1 AML-M5), including 1 secondary AML. In all 3 cases, a characteristic immunophenotype was identified (CD4+, CD13−, CD33+, CD65s+).AF-10 was involved in 5 of 6 evaluable cases, including 1 case without detectable CALM or MLL rearrangement. In 2 complex translocations, none of the three genes was rearranged. All cases had a remarkably poor prognosis, with a mean survival of 9.6 ± 6.6 months. For the 7 AML cases that were uniformly treated according to the AMLCG86/92 protocols, disease-free and overall survival was significantly worse than for the overall study group (P = .03 and P = .01, respectively). We conclude that the t(10;11)(p13;q14) indicates CALM and MLL rearrangements in morphologically distinct subsets of acute leukemia and may be associated with a poor prognosis.

Effect of Peganum Harmala Seeds Extract on Nitric Oxide in U937 Monocytes and Macrophages

Background and Aims: Nitric oxide (NO) has an essential role in inflammation and has been related to pathogenesis and the progress of numerous inflammatory-based diseases, including some cancers. Peganum harmala (P. harmala) is a medicinal plant used for the treatment of numerous diseases such as several infections. Also, anti-inflammatory effects of P. harmala extracts and its derivatives (harmaline and harmine) by suppressing myeloperoxidase, NO, and other mediators have been demonstrated in vivo. In this study, the effect of P. harmala seeds aqueous extract on NO production in U937 monocytic cells and peritoneal macrophages has been evaluated in vitro. Materials and Methods: U937 and mice peritoneal macrophages were cultured in Roswell Park Memorial institute-1640 with 10% fetal calf serum. Then, the cells at the logarithmic growth phase were incubated with different concentrations of aqueous extract of P. harmala seeds (0.1-1 mg/ml) for 24 hours. Next, NO production was assessed by the Griess method in the culture medium. Results: P. harmala seeds aqueous extract did not significantly affect lipopolysaccharide-induced NO production in U937 cells and peritoneal macrophages after 24 hours incubation time compared with untreated control cells. Conclusion: These results suggest that the anti-inflammatory effects of P. harmala may be mediated through NO-independent mechanism(s). However, further studies are warranted to define the P. harmala aqueous extract impact on NO expression in other related normal and cancerous cells.

Methods to Validate Binding and Kinetics of "Proximity-Inducing" Covalent Immune-Recruiting Molecules.

The emergence of covalent inhibitors and chemoproteomic probes in translational chemical biology research requires the development of robust biophysical and analytical methods to characterize their complex interactions with target biomolecules. Importantly, these methods must efficiently assess target selectivity and accurately discern noncovalent binding from the formation of resultant covalent adducts. One recently reported covalent chemical tool used in tumor immune oncology, covalent immune recruiters (CIRs), increases the proximity of immune cells and cancer cells, promoting immune recognition and response. Herein we describe biolayer interferometry (BLI) biosensor, flow cytometry, and solution fluorescence-based assay approaches to characterize CIR:antibody binding and CIR-antibody covalent-labeling kinetics. BLI technology, akin to surface plasmon resonance, provides the unique opportunity to investigate molecular binding and labeling kinetics both on a solid surface (Basic Protocol 1) and in solution (Alternate Protocol 1). Here, recruitment of mass-containing proteins to the BLI probe via CIR is measured with high sensitivity and is used as a readout of CIR labeling activity. Further, CIR technology is used to label antibodies with a fluorescent handle. In this system, labeling is monitored via SDS-PAGE with a fluorescence gel imager, where increased fluorescence intensity of a sample reflects increased labeling (Basic Protocol 2). Analysis of CIR:antibody target-specific immune activation is demonstrated with a flow cytometry-based antibody-dependent cellular phagocytosis (ADCP) assay (Basic Protocol 3). This ADCP protocol may be further used to discern CIR:antibody binding from covalent adduct formation (Alternate Protocol 3). For the protocols described, each method may be used to analyze characteristics of any covalent-tagging or antibody-recruiting small molecule or protein-based technology. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Determining "on-probe" reaction kinetics of CIR1/CIR4 via biolayer interferometry with Octet RED96 Alternate Protocol 1: Determining "in-solution" reaction kinetics of prostate-specific membrane antigen targeting CIR (CIR3) via biolayer interferometry with Octet RED96 Basic Protocol 2: Reaction kinetics of covalently labeled antibodies via fluorescence SDS-PAGE Basic Protocol 3: Small molecule-directed antibody-dependent cellular phagocytosis on live human cells measured via flow cytometry Alternate Protocol 2: Kinetic analysis of CIR3:antibody labeling via antibody-dependent cellular phagocytosis on flow cytometry Support Protocol 1: Activation of U937 monocytes with interferon γ Support Protocol 2: Labeling streptavidin beads with biotinylated prostate-specific membrane antigen receptor.