THP-1 Viability Research Articles

Dendrobium officinale polysaccharides attenuate uropathogenic Escherichia coli (UPEC)-induced pyroptosis in macrophage cells

Urinary tract infections (UTI) are recognized as one of the most common infectious diseases worldwide, and uropathogenic Escherichia coli (UPEC) is the main causative agent of UTI. Dendrobium officinale polysaccharides (DOPs), the main effective ingredient in Dendrobium officinale, have been reported to possess an anti-inflammatory role. Whether DOPs can attenuate the inflammatory injury (pyroptosis) induced by UPEC remains unknown. The present study aimed to assess the protective effect and potential mechanism of DOPs in UPEC-induced pyroptosis. Cell viability of THP-1 differentiated macrophage cells with DOPs was determined using MTT assay. Pyroptosis by UPEC in macrophage cells with or not DOPs pre-treatment was evaluated with flow cytometry analysis, lactate dehydrogenase (LDH) assay, and proinflammatory cytokines secretion. Expression level of key proteins in the NLRP3/Caspase-1/GSDMD pyroptotic pathway was analyzed with western blot. Furthermore the effect of DOPs on ROS activation was investigated. Results indicated that DOPs attenuated UPEC-induced cell damage in macrophage cells, inhibited the activation of NLRP3 mediated inflammasome, subsequently decreased induction and activation of caspase-1/GSDMD, and reduced the secretion of pro-inflammatory cytokine (IL-1β et al.). Moreover, pretreatment with DOPs significantly reduces ROS production, an important/putative pyroptosis stimulus signal. These results suggested that DOPs successfully mitigate UPEC-promoted pyroptosis in macrophage cells. The protective effects of DOPs are associated with the inhibition of the NLRP3/Caspase-1/GSDMD pathway and ROS signal activation.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC). Results: Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors.The high toxicity forH929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC). Results: Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for (thus forming: The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.) H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Andrographolide, isolated from Andrographis paniculata , induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC). Results: Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. Results: Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. The apoptotic activities of Andro were largely suppressed by N-acetyl-L-cysteine (NAC), an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Furthermore, whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. Notably, the high sensitivity of H929 cells is encouraging for further studies on the use of Andro against multiple myeloma.

Impact of BCR-ABL Tyrosine Kinase Inhibitors on Atherosclerosis Plaque Rupture

BackgroundBCR-ABL tyrosine kinase inhibitors (TKIs) are successful treatments extensively used to treat chronic myeloid leukemia (CML). Recent investigations demonstrate that new generation TKIs (dasatinib, nilotinib, bosutinib and ponatinib) increase the risk of thromboembolism at all arterial location in CML patients compared with the first generation (imatinib). To date, the pathophysiology of these events is poorly understood but clinical data suggest that new generation TKIs might accelerate atherosclerosis and induce plaque rupture. The stability of atherosclerotic plaque correlates to the thickness of its fibrous cap. A major mechanism inducing fibrous cap thickening is the production of matrix metalloproteinases (MMPs) by macrophages that degrade components of the fibrous cap. Until now, the impact of BCR-ABL TKIs on monocytes and macrophages have been little studied (Table 1).AimsThe aim of this research is to determine using in vitro model, the effect of BCR-ABL TKIs on MMP production.MethodsThe 5 BCR-ABL TKIs were tested at 3 clinically relevant concentrations. The THP-1 cell line was used to assess the effect of TKIs on human macrophages. THP-1 cells were differentiated to macrophage using PMA at 100ng/mL during 24h. Macrophage viability after 24h and 48h of treatment with TKI was assessed using MTS and LDH assays following standard protocols. Production and activity of 2 MMPs (MMP-2 and MMP-9) were assessed by gelatin zymography. Differentiated THP-1 were treated with TKIs during 24h and 48h. Media from these cultures were then loaded in SDS-PAGE gels in which gelatin was copolymerized. After electrophoresis, proteins were renatured and gels were incubated in developing buffer to induce gelatin lysis. Gels were finally colored by Coomassie blue to reveal lytic bands.ResultsThe 5 BCR-ABL TKIs possess few impact on THP-1 viability. MTS assay demonstrate decreased metabolic activity with nilotinib at high concentration (2µM), oppositely to increased cell metabolism with high-dose bosutinib (2µM). LDH assay indicate increased cell death with high-dose (2µM) nilotinib, and decreased cell death with dasatinib (0.5µM).MMP-2 and MMP-9 are 2 MMPs highly involved in atherosclerotic plaque rupture by degrading collagen, a major component of the extracellular matrix. Gelatin zymography is a common method for examining MMP-2 and MMP-9 in media samples. Results demonstrate no difference between TKI in MMP-2 and MMP-9 production and activity after 24h of treatment. After 48h of treatment, imatinib at the highest dosage (5µM) increases production of the active form of MMP-9 after 48h of treatment. Nilotinib at 2µM induces decreased production of activated MMP-9, which might be the result of decreased cell viability. Other BCR-ABL TKIs do not affect the production and activity of MMP-2 and MMP-9 by THP-1.ConclusionsNew generation BCR-ABL TKIs have a few impact on macrophage survival as well as on MMP production and activity. These results do not suggest high impact of BCR-ABL TKIs on macrophages and vascular occlusive events are probably not mediated by impact on macrophages. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cytotoxicity and proinflammatory effects of titanium and zirconia particles

BackgroundTo assess the effects of differently sized titanium (Ti) and zirconia (Zr) particles on (1) the metabolic activity of osteosarcoma-derived osteoblasts (SaOs-2) and human gingival fibroblasts (HGF) and (2) the cytokine expression of monocytes (THP-1)MethodsTi (60–80 nm and 100 nm) and Zr (2 μm and 75 μm) particles were incubated with SaOs-2, HGF, and THP-1 cells. At days 0, 2, 4, and 7 and 0, 1, 2, and 4 (THP-1), the mitochondrial activity was assessed and enzyme-linked immunosorbent assays were used to determine interleukin (IL)-1 beta and IL-6 concentrations of stimulated THP-1 at day 1.ResultsTi60–80, Ti100, Zr2, and Zr75 particles were associated with gradual and significant within-group decreases in the viability of SaOs-2 and HGF cells. These effects were less pronounced in the Zr group. Similar to control cells, THP-1 did not reveal any significant increases in IL-1 beta and IL-6 concentrations. Viability of THP-1 was merely impaired in the presence of Ti100.ConclusionsTi and Zr particles had a detrimental effect on the viability of SaOs-2 and HGF, but no proinflammatory effect on THP-1.

Withania somnifera modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC\u2019s)

BackgroundCancer and inflammation are associated with cachexia. Withania somnifera (W. somnifera) possesses antioxidant and anti-inflammatory potential. We investigated the potential of an aqueous extract of the root of W. somnifera (WRE) to modulate cytokines, antioxidants and apoptosis in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC’s).MethodsCytotoxcity of WRE was determined at 24 and 72 h (h). Oxidant scavenging activity of WRE was evaluated (2, 2-diphenyl-1 picrylhydrazyl assay). Glutathione (GSH) levels, caspase (− 8, − 9, − 3/7) activities and adenosine triphosphate (ATP) levels (Luminometry) were thereafter assayed. Tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 levels were also assessed using enzyme-linked immunosorbant assay.ResultsAt 24 h, WRE (0.2–0.4 mg/ml) decreased PBMC viability between 20 and 25%, whereas it increased THP-1 viability between 15 and 23% (p < 0.001). At 72 h, WRE increased PBMC viability by 27–39% (0.05, 0.4 mg/ml WRE) whereas decreased THP-1 viability between 9 and 16% (0.05–0.4 mg/ml WRE) (p < 0.001). Oxidant scavenging activity was increased by WRE (0.05–0.4 mg/ml, p < 0.0001). PBMC TNF-α and IL-10 levels were decreased by 0.2–0.4 mg/ml WRE, whereas IL-1β levels were increased by 0.05–0.4 mg/ml WRE (p < 0.0001). In THP-1 cells, WRE (0.05–0.4 mg/ml) decreased TNF-α, IL-1β and IL-6 levels (p < 0.0001). At 24 h, GSH levels were decreased in PBMC’s, whilst increased in THP-1 cells by 0.2–0.4 mg/ml WRE (p < 0.0001). At 72 h, WRE (0.1–0.4 mg/ml) decreased GSH levels in both cell lines (p < 0.0001). At 24 h, WRE (0.2–0.4 mg/ml) increased PBMC caspase (-8, -3/7) activities whereas WRE (0.05, 0.1, 0.4 mg/ml) increased THP-1 caspase (-9, -3/7) activities (p < 0.0001). At 72 h, PBMC caspase (-8, -9, -3/7) activities were increased at 0.05–0.1 mg/ml WRE (p < 0.0001). In THP-1 cells, caspase (-8, -9, -3/7) activities and ATP levels were increased by 0.1–0.2 mg/ml WRE, whereas decreased by 0.05 and 0.4 mg/ml WRE (72 h, p < 0.0001).ConclusionIn PBMC’s and THP-1 cells, WRE proved to effectively modulate antioxidant activity, inflammatory cytokines and cell death. In THP-1 cells, WRE decreased pro-inflammatory cytokine levels, which may alleviate cancer cachexia and excessive leukaemic cell growth.

In vitro co-culture models to evaluate acute cytotoxicity of individual and combined mycotoxin exposures on Caco-2, THP-1 and HepaRG human cell lines

Deoxynivalenol (DON) and zearalenone (ZEA) are mycotoxins primarily produced by Fusarium species and commonly co-occur in European grains. Some in vitro studies reported synergistic combined effects on cell viability reduction for these two natural food contaminants. However, most of these studies were carried out on conventional cell culture systems involving only one cell type and thus did not include cell-cell communication that is closer to in vivo conditions. In this context, we developed easy bi- and tri-culture systems using the Caco-2 (intestinal epithelial cells), THP-1 (monocytes) and HepaRG (hepatic cells) human cell lines in a proliferating state. Individual and combined cytotoxic effects of DON and ZEA were then assessed using co-cultures during 48 h. In bi-culture systems, results showed that only the highest tested dose of ZEA (IC30) induced a significant reduction in THP-1 viability with both Caco-2 and HepaRG cells cultured in transwells above. On the contrary, only the highest tested dose of DON (IC30) significantly affected HepaRG cell viability located under the Caco-2 cell monolayer. In addition, the DON + ZEA combination seemed to induce higher cytotoxicity than each toxin alone. Mycotoxin quantification in the abluminal compartment by Q-TOF LC-MS suggested uptake of both mycotoxins by the different cell lines. According to the co-culturing cell type, possible cell-cell interactions were also observed. Finally, in the tri-culture system, no cytotoxic effects were observed, regardless of the treatment. These findings highlighted the importance of the proposed models to better decipher toxicological impacts of mycotoxins on more complex cellular systems.

The influence of sweet pepper pectin structural characteristics on cytokine secretion by THP-1 macrophages

Pectins can modulate the biological responses interacting directly with immune cells. The observed responses can strongly be affected by polysaccharide structural features. We analyzed the intrinsic activation capacity of native and modified sweet pepper pectin on cytokine secretion by THP-1 macrophages as well as compare their effects in the presence of lipopolysaccharide. Modified pectin was obtained by partial acid hydrolysis which promoted the removal of side chains as well as the reduction of molecular weight and the degree of methyl esterification of native pectin. The results showed that both fractions had no effect on THP-1 viability. Native pectin at 300μg/mL increased TNF-α, IL-1β and IL-10 cytokine secretion by THP-1 macrophages. However, in the presence of lipopolysaccharide, it can attenuate the inflammatory response by reducing the production of the pro-inflammatory cytokines TNF-α and IL-1β and increasing the anti-inflammatory cytokine IL-10, as well as decreasing the TNF-α/IL-10 and IL-1β/IL-10 ratios. The structural modifications caused by acid hydrolysis affected the intrinsic activation capacity of native pectin to modulate the cytokines secretion. These results indicate that degree of methyl esterification, molecular weight and presence of side chains are important structural features of pectins involved in the modulation of cytokine secretion by THP-1 macrophages.

Centella asiatica modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC\u2019s)

BackgroundCancer cachexia is associated with increased pro-inflammatory cytokine levels. Centella asiatica (C. asiatica) possesses antioxidant, anti-inflammatory and anti-tumour potential. We investigated the modulation of antioxidants, cytokines and cell death by C. asiatica ethanolic leaf extract (CLE) in leukaemic THP-1 cells and normal peripheral blood mononuclear cells (PBMC’s).MethodsCytotoxcity of CLE was determined at 24 and 72 h (h). Oxidant scavenging activity of CLE was evaluated using the 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. Glutathione (GSH) levels, caspase (−8, −9, −3/7) activities and adenosine triphosphate (ATP) levels (Luminometry) were then assayed. The levels of tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 were also assessed using enzyme-linked immunosorbant assay.ResultsCLE decreased PBMC viability between 33.25–74.55% (24 h: 0.2–0.8 mg/ml CLE and 72 h: 0.4–0.8 mg/ml CLE) and THP-1 viability by 28.404% (72 h: 0.8 mg/ml CLE) (p < 0.0001). Oxidant scavenging activity was increased by CLE (0.05–0.8 mg/ml) (p < 0.0001). PBMC TNF-α and IL-10 levels were decreased by CLE (0.05–0.8 mg/ml) (p < 0.0001). However, PBMC IL-6 and IL-1β concentrations were increased at 0.05–0.2 mg/ml CLE but decreased at 0.4 mg/ml CLE (p < 0.0001). In THP-1 cells, CLE (0.2–0.8 mg/ml) decreased IL-1β and IL-6 whereas increased IL-10 levels (p < 0.0001). In both cell lines, CLE (0.05–0.2 mg/ml, 24 and 72 h) increased GSH concentrations (p < 0.0001). At 24 h, caspase (−9, −3/7) activities was increased by CLE (0.05–0.8 mg/ml) in PBMC’s whereas decreased by CLE (0.2–0.4 mg/ml) in THP-1 cells (p < 0.0001). At 72 h, CLE (0.05–0.8 mg/ml) decreased caspase (−9, −3/7) activities and ATP levels in both cell lines (p < 0.0001).ConclusionIn PBMC’s and THP-1 cells, CLE proved to effectively modulate antioxidant activity, inflammatory cytokines and cell death. In THP-1 cells, CLE decreased pro-inflammatory cytokine levels whereas it increased anti-inflammatory cytokine levels which may alleviate cancer cachexia.

Sugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-\u03baB Pathway in a Myeloid Cell Line

PurposeFourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors.MethodsTo estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4.ResultsWe found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 μM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25–100 μM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected.ConclusionWe conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines).

In vitro immunotoxicity assessment of culture-derived extracellular vesicles in human monocytes

The potential to engineer extracellular vesicles (EV) that target specific cells and deliver a therapeutic payload has propelled a growing interest in their development as promising therapeutics. These EV are often produced from cultured cells. Very little is known about the interaction of cell culture-derived EV with cells of the immune system and their potential immunomodulatory effects. The present study evaluated potential immunotoxic effects of HEK293T-derived EV on the human monocytic cell lines THP-1 and U937. Incubation of cells with different doses of EV for 16–24 h was followed by assessment of cytotoxicity and cell function by flow cytometry. Changes in cell functionality were evaluated by the capacity of cells to phagocytize fluorescent microspheres. In addition, the internalization of labeled EV in THP-1 and U937 cells was evaluated. Exposure to EV did not affect the viability of THP-1 or U937 cells. Although lower doses of the EV increased phagocytic capacity in both cell lines, phagocytic efficiency of individual cells was not affected by EV exposure at any of the doses evaluated. This study also demonstrated that THP-1 and U937 monocytic cells are highly permissive to EV entry in a dose-response manner. These results suggest that, although HEK293T-derived EV are efficiently internalized by human monocytic cells, they do not exert a cytotoxic effect or alter phagocytic efficiency on the cell lines evaluated.

The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl

The effects of an equimolar mixture of l-arginine and l-glutamate (Arg·Glu) on cell viability and cellular stress using in vitro cell culture systems are examined with reference to NaCl, in the context of monoclonal antibody formulation. Cells relevant to subcutaneous administration were selected: the human monocyte cell line THP-1, grown as a single cell suspension, and adherent human primary fibroblasts. For THP-1 cells, the mechanism of cell death caused by relatively high salt concentrations was investigated and effects on cell activation/stress assessed as a function of changes in membrane marker and cytokine (interleukin-8) expression. These studies demonstrated that Arg·Glu does not have any further detrimental effects on THP-1 viability in comparison to NaCl at equivalent osmolalities, and that both salts at higher concentrations cause cell death by apoptosis; there was no significant effect on measures of THP-1 cellular stress/activation. For adherent fibroblasts, both salts caused significant toxicity at ~400mOsm/kg, although Arg·Glu caused a more precipitous subsequent decline in viability than did NaCl. These data indicate that Arg·Glu is of equivalent toxicity to NaCl and that the mechanism of toxicity is such that cell death is unlikely to trigger inflammation upon subcutaneous injection in vivo.

Increased Proinflammatory Cytokine Production and Decreased Cholesterol Efflux Due to Downregulation of ABCG1 in Macrophages Exposed to Indoxyl Sulfate.

One of the possible causes of enhanced atherosclerosis in patients with chronic kidney disease (CKD) is the accumulation of uremic toxins. Since macrophage foam cell formation is a hallmark of atherosclerosis, we examined the direct effect of indoxyl sulfate (IS), a representative uremic toxin, on macrophage function. Macrophages differentiated from THP-1 cells were exposed to IS in vitro. IS decreased the cell viability of THP-1 derived macrophages but promoted the production of inflammatory cytokines (IL-1β, IS 1.0 mM: 101.8 ± 21.8 pg/mL vs. 0 mM: 7.0 ± 0.3 pg/mL, TNF-α, IS 1.0 mM: 96.6 ± 11.0 pg/mL vs. 0 mM: 15.1 ± 3.1 pg/mL) and reactive oxygen species. IS reduced macrophage cholesterol efflux (IS 0.5 mM: 30.3% ± 7.3% vs. 0 mM: 43.5% ± 1.6%) and decreased ATP-binding cassette transporter G1 expression. However, lipid uptake into cells was not enhanced. A liver X receptor (LXR) agonist, T0901317, improved IS-induced production of inflammatory cytokines as well as reduced cholesterol efflux. In conclusion, IS induced inflammatory reactions and reduced cholesterol efflux in macrophages. Both effects of IS were improved with activation of LXR. Direct interactions of uremic toxins with macrophages may be a major cause of atherosclerosis acceleration in patients with CKD.

Inhibition of protein phosphatase-1 and -2A decreases the chemosensitivity of leukemic cells to chemotherapeutic drugs

The phosphorylation of key proteins balanced by protein kinases and phosphatases are implicated in the regulation of cell cycle and apoptosis of malignant cells and influences anticancer drug actions. The efficacy of daunorubicin (DNR) in suppression of leukemic cell survival was investigated in the presence of tautomycin (TM) and calyculin A (CLA), specific membrane permeable inhibitors of protein phosphatase-1 (PP1) and -2A (PP2A), respectively. CLA (50nM) or TM (1μM) suppressed viability of THP-1 and KG-1 myeloid leukemia cell lines to moderate extents; however, they significantly increased survival upon DNR-induced cell death. CLA increased the phosphorylation level of Erk1/2 and PKB/Akt kinases, the retinoblastoma protein (pRb), decreased caspase-3 activation by DNR and increased the phosphorylation level of the inhibitory sites (Thr696 and Thr853) in the myosin phosphatase (MP) target subunit (MYPT1) as well as in a 25kDa kinase-enhanced phosphatase inhibitor (KEPI)-like protein. TM induced enhanced phosphorylation of pRb only, suggesting that this event may be a common factor upon CLA-induced PP2A and TM-induced PP1 inhibitory influences on cell survival. Silencing PP1 by siRNA in HeLa cells, or overexpression of Flag-KEPI in MCF-7 cells coupled with inducing its phosphorylation by PMA or CLA, resulted in increased phosphorylation of pRb. Our results indicate that PP1 directly dephosphorylates pRb, while PP2A might have an indirect influence via mediating the phosphorylation level of PP1 inhibitory proteins. These data imply the importance of PP1 inhibitory proteins in controlling the phosphorylation state of key proteins and regulating drug sensitivity and apoptosis in leukemic cells.

Cell-penetrating fusion peptides OD1 and OD2 interact with Bcr–Abl and influence the growth and apoptosis of K562 cells

The Bcr-Abl oncoprotein is the cause of chronic myelogenous leukemia (CML). Crystal structure analysis suggests that Bcr30-63 is the core of the Bcr-Abl oligomerization interface for aberrant kinase activity; however, the precise role of other residues of Bcr1-72 excluding Bcr30-63 have not been evaluated. In this study, Bcr30-63 was named OD2 and other residues of Bcr1-72 were named OD1. Cytoplasmic transduction peptide (CTP) was used to carry molecules into cytoplasm. CTP-OD1 and CTP-OD2 fusion peptides were expressed from a cold-inducible expression system. Our results demonstrated that both fusion peptides could localize into the cytoplasm, specifically interact with the Bcr-Abl protein and further inhibit growth, induce apoptosis, and decrease the phosphorylation of Bcr-Abl in K562 cell lines. However, the viability of THP-1, a Bcr-Abl negative cell line, was unaffected. These results suggested that CTP-OD1 and CTP-OD2 may be an attractive therapeutic option to inhibit the activation of Bcr-Abl kinase in CML.

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

SONODYNAMIC EFFECT OF BERBERINE ON MACROPHAGES

ObjectivesAtherosclerosis (AS) is the major contributing factor that results in acute cardiovascular events, which leading to the high mortality of cardiovascular diseases, which. The pathophysiological mechanism of atherosclerosis remains unknown....

Both leukotoxin and poly- N-acetylglucosamine surface polysaccharide protect Aggregatibacter actinomycetemcomitans cells from macrophage killing

Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly- N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48 h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately twofold in wells infected with the wild-type strain, but decreased by approximately 70–90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans.