Effects of ferumoxides-protamine sulfate labeling on immunomodulatory characteristics of macrophage-like THP-1 cells

Superparamagnetic Iron Oxide (SPIO) complexed with cationic transfection agent is used to label various mammalian cells. Labeled cells can then be utilized as an in vivo magnetic resonance imaging (MRI) probes. However, certain number of in vivo administered labeled cells may be cleared from tissues by the host's macrophages. For successful translation to routine clinical application of SPIO labeling method it is important that this mode of in vivo clearance of iron does not elicit any diverse immunological effects. The purpose of this study was to demonstrate that SPIO agent ferumoxides-protamine sulfate (FePro) incorporation into macrophages does not alter immunological properties of these cells with regard to differentiation, chemotaxis, and ability to respond to the activation stimuli and to modulate T cell response. We used THP-1 cell line as a model for studying macrophage cell type. THP-1 cells were magnetically labeled with FePro, differentiated with 100 nM of phorbol ester, 12-Myristate-13-acetate (TPA) and stimulated with 100 ng/ml of LPS. The results showed 1) FePro labeling had no effect on the changes in morphology and expression of cell surface proteins associated with TPA induced differentiation; 2) FePro labeled cells responded to LPS with slightly higher levels of NFkappaB pathway activation, as shown by immunobloting; TNF-alpha secretion and cell surface expression levels of CD54 and CD83 activation markers, under these conditions, were still comparable to the levels observed in non-labeled cells; 3) FePro labeling exhibited differential, chemokine dependent, effect on THP-1 chemotaxis with a decrease in cell directional migration to MCP-1; 4) FePro labeling did not affect the ability of THP-1 cells to down-regulate T cell expression of CD4 and CD8 and to induce T cell proliferation. Our study demonstrated that intracellular incorporation of FePro complexes does not alter overall immunological properties of THP-1 cells. The described experiments provide the model for studying the effects of in vivo clearance of iron particles via incorporation into the host's macrophages that may follow after in vivo application of any type of magnetically labeled mammalian cells. To better mimic the complex in vivo scenario, this model may be further exploited by introducing additional cellular and biological, immunologically relevant, components.     

FACTORS AFFECTING THE REDISTRIBUTION OF SURFACE-BOUND CONCANAVALIN A ON HUMAN POLYMORPHONUCLEAR LEUKOCYTES

Human neutrophil polymorphonuclear leukocytes (PMN) were studied to determine the influence of cellular locomotion upon the redistribution and capping of concanavalin A (Con A). Con A was detected by fluorescence (using Con A conjugated to fluorescein isothiocyanate [Con A-FITC]), or on shadow-cast replicas (using Busycon canaliculatum hemocyanin as a marker for Con A). After labeling with Con A 100 µg/ml at 4°C and warming to 37°C, locomotion occurred, and the Con A quickly aggregated into a cap at the trailing end of the cell. When locomotion was inhibited (with cytochalasin B, or by incubation in serum-free medium at 18°C) Con A rapidly formed a cap over the central region of the cell. Iodoacetamide inhibited capping. PMN labeled with FITC, a monovalent ligand, developed caps at the tail only on motile cells; FITC remained dispersed on immobilized cells. PMN exposed to Con A 100 µg/ml at 37°C bound more lectin than at 4°C, became immobilized, and showed slow central capping. The Con A soon became internalized to form a perinuclear ring. Such treatment in the presence of cytochalasin B resulted in the quick formation of persistent central caps. Colchicine (or prior cooling) protected PMN from the immobilizing effect of Con A, and tail caps were found on 30–40% of cells. Immobilization of colchicine-treated cells caused Con A to remain in dispersed clusters. Thus, capping on PMN is a temperature- and energy-dependent process that proceeds independently of cellular locomotion, provided a colchicine-sensitive system is intact and the ligand is capable of cross linking receptors. On the other hand, if the cell does move, it appears that ligands may be swept into a cap at the tail whether cross-linking occurs or not.     

EXPRESSION OF THE MITOCHONDRIAL GENOME IN HELA CELLS : XV. Effect of Inhibition of Mitochondrial Protein Synthesis on Mitochondrial Formation

The effect of selective inhibition of mitochondrial protein synthesis by chloramphenicol at 40 or 200 µg/ml on the formation of mitochondria in HeLa cells was investigated. HeLa cells, under the conditions used in the present work, grow at a decreasing rate for at least four cell generations in the presence of 40 µg/ml chloramphenicol, and for two generations in the presence of 200 µg/ml chloramphenicol. The progressive cell growth inhibition which begins after 2 days of exposure of the cells to 40 µg/ml chloramphenicol is immediately or gradually reversible, upon removal of the drug, for periods up to at least 8 days of treatment, though there is a progressive loss of cloning efficiency. In cells which have been treated for 6–7 days with 40 or 200 µg/ml of chloramphenicol, mitochondrial protein synthesis occurs at a normal or near-normal rate 1 h after removal of the drug. Mitochondria increase normally in number and show a normal size and amount of cristae in the presence of either concentration of drug. However, in 4–5% of the mitochondrial profiles the cristae appear to be arranged in unusual, circular, looped or whorled configuration.     

Cytotoxic Effects of Tetracycline Analogues (Doxycycline,Minocycline and COL-3) in Acute Myeloid Leukemia HL-60 Cells

Tetracycline analogues (TCNAs) have been shown to inhibit matrix metalloproteinases and to induce apoptosis in several cancer cell types. In the present study, the cytotoxic effects of TCNAs doxycycline (DOXY), minocycline (MINO) and chemically modified tetracycline-3 (COL-3) were investigated in the human acute myeloid leukemia HL-60 cell line. Cells were incubated with TCNAs in final concentrations of 0.5–100 µg/ml for 24 h. Viability of the leukemic cells was inhibited in a concentration-dependent manner using resazurin assay. The estimated IC_50s were 9.2 µg/ml for DOXY, 9.9 µg/ml for MINO and 1.3 µg/ml for COL-3. All three TCNAs induced potent cytotoxic effects and cell death. Apoptosis, which was assessed by morphological changes and annexin V positivity, was concentration- and time-dependent following incubation with any one of the drugs. TCNAs induced DNA double strand breaks soon after treatment commenced as detected by γH2AX and western blot. The loss of mitochondrial membrane potential (Δψm), caspase activation and cleavage of PARP and Bcl-2 were observed; however, the sequence of events differed among the drugs. Pancaspase inhibitor Z-VAD-FMK improved survival of TCNAs-treated cells and decreased TCNAs-induced apoptosis. In summary, we demonstrated that TCNAs had a cytotoxic effect on the HL-60 leukemic cell line. Apoptosis was induced via mitochondria-mediated and caspase-dependent pathways in HL-60 cells by all three TCNAs. COL-3 exerted the strongest anti-proliferative and pro-apoptotic effects in concentrations that have been achieved in human plasma in reported clinical trials. These results indicate that there is a therapeutic potential of TCNAs in leukemia.     

The Sonic Hedgehog Signaling Pathway Induces Myopic Development by Activating Matrix Metalloproteinase (MMP)-2 in Guinea Pigs

Purpose

To investigate whether the Sonic hedgehog (Shh) signaling induces myopic development by increasing the expression of matrix metalloproteinase (MMP)-2 in guinea pigs.

Methods

A translucent diffuser was glued onto the right eye to induce form-deprivation myopia (FDM) in 10 guinea pigs. Four guinea pigs were served as a control group. The other 100 guinea pigs were subdivided into 5 groups (20 per group) and received a 10 µl intravitreal injection every 2 days for 4 times. Two groups were injected with 20 or 50 µg/ml Shh amino-terminal peptide (Shh-N) into the right eye and 0.1% bovine serum albumin into the other. FDM was induced in the right eyes of the three cyclopamine-treated groups and both eyes were injected with 50, 100, or 200 µg/ml cyclopamine. Retinoscopic refraction and eye dimensions were assessed on Day 14 of treatment. MMP-2 protein expression was determined in both scleras by western blotting.

Results

Both concentrations of Shh-N stimulated myopic development and axial growth as compared with control eyes. Myopia and axial elongation were significantly greater in the 50 µg/ml than in the 20 µg/ml Shh-N group (P<0.001 and P = 0.0019, respectively). All three doses of cyclopamine significantly attenuated myopic development compared with the FDM group (P<0.0001). Cyclopamine at 100 or 200 µg/ml significantly reduced axial elongation compared with the FDM group (P = 0.044 and P = 0.001, respectively). FDM-induced myopia and axial elongation were significantly greater in the 50 µg/ml than in the 200 µg/ml cyclopamine group (P<0.0001 and P = 0.008, respectively). MMP-2 expression was significantly greater in Shh-N–treated eyes than in the control eyes, and was lower in the cyclopamine plus FDM groups than in the FDM group.

Conclusions

The Shh signaling pathway induces myopic development by activating MMP-2 in guinea pigs.     

Feasibility of Using Pseudo-Continuous Arterial Spin Labeling Perfusion in a Geriatric Population at 1.5 Tesla

Objectives

To evaluate the feasibility of using pseudo-continuous arterial spin labeling (pCASL) perfusion in a geriatric population at 1.5-Tesla.

Materials and Methods

In 17 participants (mean age 78.8±1.63 years) we assessed; 1) inter-session repeatability and reliability of resting state perfusion in 27 brain regions; 2) brain activation using finger-tapping as a means to evaluate the ability to detect flow differences; 3) reliability by comparing cerebral blood flow (CBF) with pCASL to CBF with phase contrast (PC-MR).

Results

The CBF (mean±standard deviation (SD)) for the whole brain grey matter (GM) was 40.6±8.4 and 41.4±8.7 ml/100g/min for the first and second scan respectively. The within-subject standard deviation (SDw), the repeatability index (RI) and intra-class correlation coefficient (ICC) across the 27 regions ranged from 1.1 to 7.9, 2.2 to 15.5 and 0.35 to 0.98 respectively. For whole brain GM the SDw, RI and ICC were 1.6, 3.2 and 0.96 respectively. The between-subject standard deviation (SD_B) was larger than the SDw for all regions. Comparison of CBF at rest and activation on a voxel level showed significantly higher perfusion during finger tapping in the motor- and somatosensory regions. The mean CBF for whole brain GM was 40.6±8.4 ml/100g/min at rest and 42.6±8.6 ml/100g/min during activation. Finally the reliability of pCASL against the reference standard of PC-MR was high (ICC = 0.80). The mean CBF for whole brain measured with PC-MRI was 54.3±10.1 ml/100g/min and 38.3±7.8 ml/100g/min with pCASL.

Conclusions

The results demonstrate moderate to high levels of repeatability and reliability for most brain regions, comparable to what has been reported for younger populations. The performance of pCASL at 1.5-Tesla shows that region-specific perfusion measurements with this technique are feasible in studies of a geriatric population.     

220D-F2 from Rubus ulmifolius Kills Streptococcus pneumoniae Planktonic Cells and Pneumococcal Biofilms

Streptococcus pneumoniae (pneumococcus) forms organized biofilms to persist in the human nasopharynx. This persistence allows the pneumococcus to produce severe diseases such as pneumonia, otitis media, bacteremia and meningitis that kill nearly a million children every year. While bacteremia and meningitis are mediated by planktonic pneumococci, biofilm structures are present during pneumonia and otitis media. The global emergence of S. pneumoniae strains resistant to most commonly prescribed antibiotics warrants further discovery of alternative therapeutics. The present study assessed the antimicrobial potential of a plant extract, 220D-F2, rich in ellagic acid, and ellagic acid derivatives, against S. pneumoniae planktonic cells and biofilm structures. Our studies first demonstrate that, when inoculated together with planktonic cultures, 220D-F2 inhibited the formation of pneumococcal biofilms in a dose-dependent manner. As measured by bacterial counts and a LIVE/DEAD bacterial viability assay, 100 and 200 µg/ml of 220D-F2 had significant bactericidal activity against pneumococcal planktonic cultures as early as 3 h post-inoculation. Quantitative MIC’s, whether quantified by qPCR or dilution and plating, showed that 80 µg/ml of 220D-F2 completely eradicated overnight cultures of planktonic pneumococci, including antibiotic resistant strains. When preformed pneumococcal biofilms were challenged with 220D-F2, it significantly reduced the population of biofilms 3 h post-inoculation. Minimum biofilm inhibitory concentration (MBIC)₅₀ was obtained incubating biofilms with 100 µg/ml of 220D-F2 for 3 h and 6 h of incubation. 220D-F2 also significantly reduced the population of pneumococcal biofilms formed on human pharyngeal cells. Our results demonstrate potential therapeutic applications of 220D-F2 to both kill planktonic pneumococcal cells and disrupt pneumococcal biofilms.     

In vitro cytoprotective activity of cyanidin 3-glucoside extracts from Haematocarpus validus pomace on streptozotocin induced oxidative damage in pancreatic β-cells

Cyanidin-3-glucoside (C3Ghv) compounds were purified and isolated from the anthocyanins extract of Haematocarpus validus. C3Ghv were studied for antioxidant and cytoprotective properties on pancreatic β-cells of rat insulinoma cells (RINm5F) against the oxidative stress induced by streptozotocin (STZ). The exposure of RINm5F cells to C3Ghv at concentration of 100 and 200 μg/mL for 24 h reduced 10% and 23% cell viability, respectively, as compared to control cells. The pre-treatment of RINm5F cells with C3Ghv (50 µg/mL) increased the cell viability by 29% as compared to control, on being treated with STZ (10 mM) for 24 h. The pre-treatment of RINm5F cells with C3Ghv (50 µg/mL) for 24 h followed by exposure to STZ (10 mM) for 1 h decreased the generation of reactive oxygen species (ROS) by 57%, generation of nitric oxide by 22.8%, generation of malondialdehyde (MDA) by 32%, the production of p-ERK ½ by 83%, p-JNK by 82.6%, p-MEK by 57%, and p-p38 MAPK by 64%. The C3Ghv treatment also decreased the ratio of apoptotic proteins Bax to Bcl-2 by 61%, and improved the M2 phase of cell cycle by 75% as compared to STZ treated cells. The overall results suggest that C3Ghv protects pancreatic β-cells against oxidative stress-induced apoptosis, thereby implicating the significant role of C3Ghv as an antidiabetic agent.     

Dose-Response of Superparamagnetic Iron Oxide Labeling on Mesenchymal Stem Cells Chondrogenic Differentiation: A Multi-Scale In Vitro Study

Aim

The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues.

Methods

MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity.

Results

No influence of various SPIO concentrations was noted on human bone marow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations.

Conclusions

This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5–25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.     

Selective stimulation of Hsp27 and αB-crystallin but not Hsp70 expression by p38 MAP kinase activation

The levels of Hsp27 and αB-crystallin in C6 rat glioma cells, that had been heated at 43°C for 30 min with a subsequent culture for 16 h at 37°C, were markedly increased. The exposure of the cells to a low concentration (0.1–3 µg/ml) of anisomycin for a few hours after heat stress stimulated the accumulation of the small stress proteins Hsp27 and αB-crystallin, but not that of Hsp70. The levels of mRNAs for Hsp27 and αB-crystallin but not that for Hsp70 increased in cells that had been exposed to heat and subsequently for 2 h to 0.1–3 µg/ml anisomycin. The results of a reporter assay, using an αB-crystallin promotor fused to a luciferase reporter gene, suggested that the increase in level of αB-crystallin mRNA was due to the production of new mRNA. The activation of the binding of heat shock factors to heat shock elements induced in cells that had been heat stressed was barely affected by subsequent exposure to anisomycin at 0.3 µg/ml. The stimulatory effects of anisomycin were also observed in cells that had been exposed to NaAsO₂, or CdCl₂. The active form of p38 mitogen activated protein (MAP) kinase was increased in cell that had been subjected to heat shock and subsequent exposure to 0.3 µg/ml of anisomycin. The heat-induced accumulations of Hsp27 and αB-crystallin were also stimulated by cycloheximide, another stimulator of p38 MAP kinase. SB202190, a specific inhibitor of p38 MAP kinase, suppressed the stimulation by anisomycin of the heat stress-induced expressions of Hsp27 and αB-crystallin. These results suggest that the signal transduction pathway of the stress-induced expressions of Hsp27 and αB-crystallin in C6 glioma cells includes a process that is sensitive to p38 MAP kinase.     

INHIBITION OF CELLULAR DIFFERENTIATION BY PHOSPHOLIPASE C : I. Effects of the Enzyme on Myogenesis and Chondrogenesis In Vitro

In cell culture, a partially purified commercial preparation of phospholipase C (PLC) from Clostridium welchii inhibited fusion of myoblasts at concentrations of 12–50 µg per ml. At lower concentrations, PLC-treated cultures were indistinguishable from controls, and at concentrations above 100 µg per ml, PLC-treated cells detached from their substrates. The effect was reversible and fusion resumed approximately one cell cycle time after removal of the enzyme. Neither the percent of cells in the mitotic cycle nor the duration of the different phases of the cycle were altered by PLC at concentrations which inhibited fusion. Cell motility was not reduced by the enzyme. Unfused, PLC-treated myoblasts were virtually indistinguishable in ultrastructure from untreated cells just before fusion. In the presence of PLC, mononucleated myogenic cells did not synthesize thick (150 Å) filaments. Treatment of culture medium with insolubilized commercial PLC did not abolish the capacity of the medium to support myogenesis. Chondrocytes treated with PLC divided repeatedly but failed to synthesize metachromatic matrix and failed to incorporate labeled sulfate into chondroitin sulfate. PLC was further purified by chromatography on Sephadex G-100. The resulting preparation was free of detectable protease, yielded one band on SDS-acrylamide gel electrophoresis, and displayed all of the biological activities of the less pure material.     

A Pro-Inflammatory Role of C5L2 in C5a-Primed Neutrophils for ANCA-Induced Activation

Background

The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In particular, C5a and its receptor on neutrophils, CD88, play a central role. The functional role of the second receptor of C5a, C5L2, remains unclear. In the current study, we investigated the role of C5L2 in C5a-primed neutrophils for ANCA-induced activation.

Methods

The effect of blocking C5L2 by anti-human C5L2 blocking antibody were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on membrane-bound proteinase 3 (mPR3) and concentration of myeloperoxidase (MPO) in supernatant of C5a-primed neutrophils. An antagonist for CD88 was also employed.

Results

Blocking C5L2 resulted in a significantly decreased MPO concentration in the supernatant of C5a-primed neutrophils. mPR3 expression increased from 209.0±43.0 in untreated cells to 444.3±60.8 after C5a treatment (P<0.001), and decreased to 375.8±65.44, 342.2±54.3 and 313.7±43.6 by pre-incubating blocking C5L2 antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-priming group, P<0.001, P<0.001, and P<0.001), respectively. In C5a-primed neutrophils, subsequently activating with MPO-ANCA-positive IgG, the MFI value was 425.8±160.6, which decreased to 292.8±141.2, 289.7±130.0 and 280.3±136.4 upon pre-incubation with mouse anti-human C5L2 blocking antibody at 2.5 µg/ml, 5 µg/ml or 10 µg/ml (compared with C5a-primed neutrophils, for MPO-ANCA-positive IgG-induced activation, P<0.05, P<0.05, and P<0.05), respectively. Blocking C5L2 also resulted in significantly decreased C5a-primed neutrophils for PR3-ANCA-positive IgG-induced activation. Moreover, the lactoferrin concentration in the supernant significantly decreased in pre-incubation with anti-human C5L2 blocking antibody, compared with C5a-primed neutrophils induced by PR3- or MPO-ANCA-positive IgG.

Conclusions

C5L2 may be implicated in the pro-inflammatory role in C5a-primed neutrophils for ANCA-induced activation.     

Visible Light Is a Better Co-Inducer of Apoptosis for Curcumin-Treated Human Melanoma Cells than UVA

Curcumin attracts worldwide scientific interest due to its anti-proliferative and apoptosis inducing effects on different tumor cells at concentrations ranging from 10 to 150 µM (3.7–55 µg/ml). Unfortunately, because of a low oral bioavailability, only low and pharmacologically ineffective serum levels are achievable. In this study, an alternative treatment concept consisting of low concentration curcumin (0.2–5 µg/ml) and irradiation with UVA or visible light (VL) has been tested. The experimental results show clearly that this treatment decreases the proliferation and the viability of human melanoma cells while the cell membrane integrity remains intact. We identified the onset of apoptosis characterized by typical markers such as active caspases 8, 9 and 3 as well as DNA fragmentation accompanied by the loss of cell adhesion. The mitochondrial apoptosis signaling pathway is predominant due to an early activation of caspase-9. The present data indicate a higher efficacy of a combination of curcumin and VL than curcumin and UVA. Reduced effects as a result of light absorption by heavily pigmented skin are unlikely if VL is used. These results indicate that a combination of curcumin and light irradiation may be a useful additional therapy in the treatment of malignant disease.     

Quantitative Renal Perfusion Measurements in a Rat Model of Acute Kidney Injury at 3T: Testing Inter- and Intramethodical Significance of ASL and DCE-MRI

Objectives

To establish arterial spin labelling (ASL) for quantitative renal perfusion measurements in a rat model at 3 Tesla and to test the diagnostic significance of ASL and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in a model of acute kidney injury (AKI).

Material and Methods

ASL and DCE-MRI were consecutively employed on six Lewis rats, five of which had a unilateral ischaemic AKI. All measurements in this study were performed on a 3 Tesla MR scanner using a FAIR True-FISP approach and a TWIST sequence for ASL and DCE-MRI, respectively. Perfusion maps were calculated for both methods and the cortical perfusion of healthy and diseased kidneys was inter- and intramethodically compared using a region-of-interest based analysis.

Results/Significance

Both methods produce significantly different values for the healthy and the diseased kidneys (P<0.01). The mean difference was 147±47 ml/100 g/min and 141±46 ml/100 g/min for ASL and DCE-MRI, respectively. ASL measurements yielded a mean cortical perfusion of 416±124 ml/100 g/min for the healthy and 316±102 ml/100 g/min for the diseased kidneys. The DCE-MRI values were systematically higher and the mean cortical renal blood flow (RBF) was found to be 542±85 ml/100 g/min (healthy) and 407±119 ml/100 g/min (AKI).

Conclusion

Both methods are equally able to detect abnormal perfusion in diseased (AKI) kidneys. This shows that ASL is a capable alternative to DCE-MRI regarding the detection of abnormal renal blood flow. Regarding absolute perfusion values, nontrivial differences and variations remain when comparing the two methods.     

Persistent DNA damage caused by low levels of mitomycin C induces irreversible cell senescence

Mutations of oncogenes and tumor suppressor genes which activate mTOR through several downstream signaling pathways are common to cancer. Activation of mTOR when combined with inhibition of cell cycle progression or DNA replication stress has previously been shown to promote cell senescence. In the present study, we examined the conditions under which human non-small cell lung carcinoma A549 cells can undergo senescence when treated with the DNA alkylating agent mitomycin C (MMC). While exposure of A549 cells to 0.1 or 0.5 µg/ml of MMC led to their arrest in S phase of the cell cycle and subsequent apoptosis, exposure to 0.01 or 0.02 µg/ml for 6 d resulted in induction of cell senescence and near total (0.01 µg/ml) or total (0.02 µg/ml) elimination of their reproductive potential. During exposure to these low concentrations of MMC, the cells demonstrated evidence of DNA replication stress manifested by expression of γH2AX, p21^WAF1 and a very low level of EdU incorporation into DNA. The data are consistent with the notion that enduring DNA replication stress in cells known to have activated oncogenes leads to their senescence. It is reasonable to expect that tumors having constitutive activation of oncogenes triggering mTOR signaling may be particularly predisposed to undergoing senescence following prolonged treatment with low doses of DNA damaging drugs.     

CT Perfusion with Acetazolamide Challenge in C6 Gliomas and Angiogenesis

Background

This study was performed to investigate the correlation between CT perfusion with acetazolamide challenge and angiogenesis in C6 gliomas.

Methods

Thirty-two male Sprague-Dawley rats were evaluated. The rats were divided randomly to four groups: eight rats with orthotopically implanted C6 gliomas at 10-days old (Group A), eight rats with gliomas at 14-days old (Group B), eight rats with gliomas at 18-days old (Group C), eight rats with orthotopically injected normal saline served as controls. CT perfusion was performed before and after administration of acetazolamide. Changes in perfusion parameters due to acetazolamide administration were calculated and analyzed.

Results

Elevated carbon dioxide partial pressure and decreased pH were found in all 32 rats post acetazolamide challenge (P<0.01). Cerebral blood flow_{pre-challenge} was increased in group C (95.0±2.5 ml/100g/min), as compared to group B (80.1±11.3 ml/100g/min) and group A (63.1±2.1 ml/100g/min). Cerebral blood flow percentage changes were detected with a reduction in group C (54.2±4.8%) as compared to controls (111.3±22.2%). Cerebral blood volume _{pre-challenge} was increased in group C (50.8±1.7ml/100g), as compared to group B (45.7±1.9 ml/100g) and group A (38.2±0.8 ml/100g). Cerebral blood volume percentage changes were decreased in group C (23.5±4.6%) as compared to controls (113.5±30.4%). Angiogenesis ratio = [(CD105-MVD) / (FVIII-MVD)] ×100%. Positive correlations were observed between CD105-microvessel density, angiogenesis ratio, vascular endothelial growth factor, proliferation marker and cerebral blood flow_{pre-challenge}, cerebral blood volume _{pre-challenge}. Negative correlations were observed between CD105-microvessel density and cerebral blood flow percentage changes (P<0.01, correlation coefficient r=-0.788), cerebral blood volume percentage changes (P<0.01, r=-0.703). Negative correlations were observed between angiogenesis ratio, vascular endothelial growth factor, proliferation marker and cerebral blood flow percentage changes, cerebral blood volume percentage changes.

Conclusion

Our findings suggest that CT perfusion with challenge can provide new insight into non-invasive assessment of rat C6 glioma angiogenesis.     

In vitro antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia laevis ethanolic leaf extract

Background

The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract.

Results

Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH^.), superoxide anion (O₂^.-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO⁻), singlet oxygen (¹O₂), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC₅₀ of the extract showed significant difference only in singlet oxygen (¹O₂) and iron chelating activity when compared with the controls.

Conclusions

The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.     

Tacrolimus (FK506) Suppresses TREM-1 Expression at an Early but Not at a Late Stage in a Murine Model of Fungal Keratitis

Purpose

To investigate the efficacy and mechanism of tacrolimus(FK506), which is a novel macrolide immunosuppressant, in inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) expression in a murine keratitis model induced by Aspergillus fumigatus.

Method

TREM-1 was detected in 11 fungus-infected human corneas by quantitative real-time PCR (qRT-PCR). RAW264.7 macrophages were divided into four groups, which received treatment with zymosan (100 µg/ml), zymosan (100 µg/ml) + mTREM-1/Fc protein (1 µg/ml), or zymosan (100 µg/ml) + FK506 (20 µM) or negative-control treatment. After this treatment, the expression of TREM-1, interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) was assayed using qRT-PCR and ELISA. The mouse model of fungal keratitis was created by intrastromal injection with Aspergillus fumigatus, and the mice were divided into 2 groups: group A received vehicle eye drops 4 times each day, and group B received 4 doses of FK506 eye drops each day. Corneal damage was evaluated by clinical scoring and histologic examination,and myeloperoxidase (MPO) protein levels were also detected by ELISA. The expression of TREM-1, IL-1β and TNFα was then determined at different time points using qRT-PCR and ELISA.

Results

TREM-1 expression dramatically increased in the human corneas with fungal keratitis. In contrast, FK506 reduced the expression of TREM-1, IL-1β and TNFα in RAW264.7 macrophages stimulated with zymosan. In the mouse model, at day 1 post-infection, the corneal score of the FK506-treated group was lower than that of the control, and polymorphonuclear neutrophil (PMN) infiltration was diminished. TREM-1, IL-1β and TNFα expression was significantly reduced at the same time point. However, the statistically significant differences in cytokine expression, clinical scores and infiltration disappeared at 5 days post-infection.

Conclusions

FK506 may inhibit the inflammation induced by fungi and alleviate the severity of corneal damage at an early stage of fungal keratitis by downregulating TREM-1 expression.     

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells

Background

Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10–40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied.

Methods

The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting.

Results and conclusion

In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1–2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01–16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0025-8) contains supplementary material, which is available to authorized users.