Porphyromonas gingivalis lipopolysaccharide alters atherosclerotic-related gene expression in oxidized low-density-lipoprotein-induced macrophages and foam cells

The molecular mechanism between atherosclerosis formation and periodontal pathogens is not clear although positive correlation between periodontal infections and cardiovascular diseases has been reported. Objective: To determine if atherosclerosis related genes were affected in foam cells during and after its formation by P. gingivalis lipopolysaccharide (LPS) stimulation. Methods: Macrophages from human THP-1 monocytes were treated with oxidized low density lipoprotein (oxLDL) to induce the formation of foam cells. P. gingivalis LPS was added to cultures of either oxLDL-induced macrophages or foam cells. The expression of atherosclerosis related genes was assayed by quantitative real time PCR and the protein production of granulocyte-macrophage colony-stimulating factor（GM-CSF）, monocyte chemotactic protein-1 (MCP-1), IL-1β, IL-10 and IL-12 was determined by ELISA. Nuclear translocation of NF-κB P65 was detected by immunocytochemistry and western blot was used to evaluate IKB-α degradation to confirm the NF-κB pathway activation. Results: P. gingivalis LPS stimulated atherosclerosis related gene expression in foam cells and increased oxLDL induced expression of chemokines, adhesion molecules, growth factors, apoptotic genes, and nuclear receptors in macrophages. Transcription of the pro-inflammatory cytokines IL-1β and IL-12 was elevated in response to LPS in both macrophages and foam cells, whereas the anti-inflammatory cytokine IL-10 was not affected. Increased NF-κB pathway activation was also observed in LPS and oxLDL co-stimulated macrophages. Conclusion: P. gingivalis LPS appears to be an important factor in the development of atherosclerosis by stimulation of atherosclerosis related gene expression in both macrophages and foam cells via activation of the NF-κB pathway.

Differential expression of chemokine and matrix re-modelling genes is associated with contrasting schistosome-induced hepatopathology in murine models

The pathological outcomes of schistosomiasis are largely dependent on the molecular and cellular mechanisms of the host immune response. In this study, we investigated the contribution of variations in host gene expression to the contrasting hepatic pathology observed between two inbred mouse strains following Schistosoma japonicum infection. Whole genome microarray analysis was employed in conjunction with histological and immunohistochemical analysis to define and compare the hepatic gene expression profiles and cellular composition associated with the hepatopathology observed in S. japonicum-infected BALB/c and CBA mice. We show that the transcriptional profiles differ significantly between the two mouse strains with high statistical confidence. We identified specific genes correlating with the more severe pathology associated with CBA mice, as well as genes which may confer the milder degree of pathology associated with BALB/c mice. In BALB/c mice, neutrophil genes exhibited striking increases in expression, which coincided with the significantly greater accumulation of neutrophils at granulomatous regions seen in histological sections of hepatic tissue. In contrast, up-regulated expression of the eosinophil chemokine CCL24 in CBA mice paralleled the cellular influx of eosinophils to the hepatic granulomas. Additionally, there was greater down-regulation of genes involved in metabolic processes in CBA mice, reflecting the more pronounced hepatic damage in these mice. Profibrotic genes showed similar levels of expression in both mouse strains, as did genes associated with Th1 and Th2 responses. However, imbalances in expression of matrix metalloproteinases (e.g. MMP12, MMP13) and tissue inhibitors of metalloproteinases (TIMP1) may contribute to the contrasting pathology observed in the two strains. Overall, these results provide a more complete picture of the molecular and cellular mechanisms which govern the pathological outcome of hepatic schistosomiasis. This improved understanding of the immunopathogenesis in the murine model schistosomiasis provides the basis for a better appreciation of the complexities associated with chronic human schistosomiasis.

Effect of caffeine on adenosine-induced reversible perfusion defects assessed by automated analysis

Objectives This prospective study investigated the effects of caffeine ingestion on the extent of adenosine-induced perfusion abnormalities during myocardial perfusion imaging (MPI). Methods Thirty patients with inducible perfusion abnormalities on standard (caffeine-abstinent) adenosine MPI underwent repeat testing with supplementary coffee intake. Baseline and test MPIs were assessed for stress percent defect, rest percent defect, and percent defect reversibility. Plasma levels of caffeine and metabolites were assessed on both occasions and correlated with MPI findings. Results Despite significant increases in caffeine [mean difference 3,106 μg/L (95% CI 2,460 to 3,752 μg/L; P < .001)] and metabolite concentrations over a wide range, there was no statistically significant change in stress percent defect and percent defect reversibility between the baseline and test scans. The increase in caffeine concentration between the baseline and the test phases did not affect percent defect reversibility (average change −0.003 for every 100 μg/L increase; 95% CI −0.17 to 0.16; P = .97). Conclusion There was no significant relationship between the extent of adenosine-induced coronary flow heterogeneity and the serum concentration of caffeine or its principal metabolites. Hence, the stringent requirements for prolonged abstinence from caffeine before adenosine MPI—based on limited studies—appear ill-founded.

Modulation of LPS-induced chorioamnionitis by ureaplasma parvum in sheep chorioamnionitis in sheep

ABSTRACT Objective: Ureaplasma parvum colonization in the setting of polymicrobial flora is common in women with chorioamnionitis, and is a risk factor for preterm delivery and neonatal morbidity. We hypothesized that ureaplasma colonization of amniotic fluid will modulate chorioamnionitis induced by E.coli lipopolysaccharide (LPS). Methods: Sheep received intra-amniotic (IA) injections of media (control) or live ureaplasma either 7 or 70d before delivery. Another group received IA LPS 2d before delivery. To test for interactions, U.parvum exposed animals were challenged with IA LPS, and delivered 2d later. All animals were delivered preterm at 125±1 day gestation. Results: Both IA ureaplasmas and LPS induced leukocyte infiltration of chorioamnion. LPS greatly increased the expression of pro-inflammatory cytokines and myeloperoxidase in leukocytes, while ureaplasmas alone caused modest responses. Interestingly, 7d but not 70d ureaplasma exposure significantly downregulated LPS induced pro-inflammatory cytokines and myeloperoxidase expression in the chorioamnion. Conclusion: U.parvum can suppress LPS induced experimental chorioamnionitis.

Neutrophil activation, antioxidant supplements and exercise-induced oxidative stress

Neutrophils produce free radicals known as reactive oxygen species (ROS), which assist in the clearance of damaged host tissue. Tissue damage may occur during exercise due to muscle damage, thermal stress and ischaemia/reperfusion. When produced in excess, neutrophil-derived ROS may overwhelm the body's endogenous antioxidant defence mechanisms, and this can lead to oxidative stress. There is increasing evidence for links between oxidative stress and a variety of pathological disorders such as cardiovascular diseases, cancer, chronic inflammatory diseases and post-ischaemic organ injury. A small number of studies have investigated whether there is a link between neutrophil activation and oxidative stress during exercise. In this review, we have summarised the findings of these studies. Exercise promotes the release of neutrophils into the circulation, and some evidence suggests that neutrophils mobilised after exercise have an enhanced capacity to generate some forms of ROS when stimulated in vitro. Neutrophil activation during exercise may challenge endogenous antioxidant defence mechanisms, but does not appear to increase lipid markers of oxidative stress to any significant degree, at least in the circulation. Antioxidant supplements such as N-acetylcysteine are effective at attenuating increases in the capacity of neutrophils to generate ROS when stimulated in vitro, whereas vitamin E reduces tissue infiltration of neutrophils during exercise. Free radicals generated during intense exercise may lead to DNA damage in leukocytes, but it is unknown if this damage is the result of neutrophil activation. Exercise enhances the expression of inducible haem (heme)-oxygenase (HO-1) in neutrophils after exercise, however, it is uncertain whether oxidative stress is the stimulus for this response.

Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS

The promise of metabonomics, a new "omics" technique, to validate Chinese medicines and the compatibility of Chinese formulas has been appreciated. The present study was undertaken to explore the excretion pattern of low molecular mass metabolites in the male Wistar-derived rat model of kidney yin deficiency induced with thyroxine and reserpine as well as the therapeutic effect of Liu Wei Di Huang Wan (LW) and its separated prescriptions, a classic traditional Chinese medicine formula for treating kidney yin deficiency in China. The study utilized ultra-performance liquid chromatography/electrospray ionization synapt high definition mass spectrometry (UPLC/ESI-SYNAPT-HDMS) in both negative and positive electrospray ionization (ESI). At the same time, blood biochemistry was examined to identify specific changes in the kidney yin deficiency. Distinct changes in the pattern of metabolites, as a result of daily administration of thyroxine and reserpine, were observed by UPLC-HDMS combined with a principal component analysis (PCA). The changes in metabolic profiling were restored to their baseline values after treatment with LW according to the PCA score plots. Altogether, the current metabonomic approach based on UPLC-HDMS and orthogonal projection to latent structures discriminate analysis (OPLS-DA) indicated 20 ions (14 in the negative mode, 8 in the positive mode, and 2 in both) as "differentiating metabolites".

In vitro functional characterisation of IGF-I : VN-induced breast cancer progression

Members of the insulin-like growth factor (IGF) family have been shown to play critical roles in normal growth and development, as well as in tumour biology. The IGF system is complex and the biological effects of the IGFs are determined by their diverse interactions between many molecules, including their interactions with extracellular matrix (ECM) proteins. Recent studies have demonstrated that IGFs associate with the ECM protein vitronectin (VN) through IGF-binding proteins (IGFBP) and that this interaction modulates IGF-stimulated biological functions, namely cell migration and cell survival through the cooperative involvement of the type-I IGF receptor (IGF-1R) and VN-binding integrins. Since IGFs play important roles in the transformation and progression of breast cancer and VN has been found to be over-expressed at the leading edge of breast tumours, this project aimed to describe the effects of IGF-I:VN interactions on breast cell function. This was undertaken to dissect the molecular mechanisms underlying IGF-I:VN-induced responses and to design inhibitors to block the effects of such interactions. The studies described herein demonstrate that the increase in migration of MCF-7 breast cancer cells in response to the IGF-I:IGFBP-5:VN complex is accompanied by differential expression of genes known to be involved in migration, invasion and/or survival, including Tissue-factor (TF), Stratifin (SFN), Ephrin-B2, Sharp-2 and PAI-1. This „migration gene signature‟ was confirmed using real-time PCR analysis. Substitution of the native IGF-I within the IGF-I:IGFBP:VN complex with the IGF-I analogue, \[L24]\[A31]-IGF-I, which has a reduced affinity for the IGF-1R, failed to stimulate cell migration and interestingly, also failed to induce the differential gene expression. This supports the involvement of the IGF-1R in mediating these changes in gene expression. Furthermore, lentiviral shRNA-mediated stable knockdown of TF and SFN completely abrogated the increased cell migration induced by IGF-I:IGFBP:VN complexes in MCF-7 cells. Indeed, when these cells were grown in 3D Matrigel™ cultures a decrease in the overall size of the 3D spheroids in response to the IGF-I:IGFBP:VN complexes was observed compared to the parental MCF-7 cells. This suggests that TF and SFN have a role in complex-stimulated cell survival. Moreover, signalling studies performed on cells with the reduced expression of either TF or SFN had a decreased IGF-1R activation, suggesting the involvement of signalling pathways downstream of IGF-1R in TF- and/or SFN-mediated cell migration and cell survival. Taken together, these studies provide evidence for a common mechanism activated downstream of the IGF-1R that induces the expression of the „migration gene signature‟ in response to the IGF-I:IGFBP:VN complex that confers breast cancer cells the propensity to migrate and survive. Given the functional significance of the interdependence of ECM and growth factor (GF) interactions in stimulating processes key to breast cancer progression, this project aimed at developing strategies to prevent such growth factor:ECM interactions in an effort to inhibit the downstream functional effects. This may result in the reduction in the levels of ECM-bound IGF-I present in close proximity to the cells, thereby leading to a reduction in the stimulation of IGF-1R present on the cell surface. Indeed, the inhibition of IGF-I-mediated effects through the disruption of its association with ECM would not alter the physiological levels of IGF-I and potentially only exert effects in situations where abnormal over expression of ECM proteins are found; namely carcinomas and hyperproliferative diseases. In summary, this PhD project has identified novel, innovative and realistic strategies that can be used in vitro to inhibit the functions exerted by the IGF-I:IGFBP:VN multiprotein complexes critical for cancer progression, with a potential to be translated into in vivo investigations. Furthermore, TF and SFN were found to mediate IGF-I:IGFBP:VN-induced effects, thereby revealing their potential to be used as therapeutic targets or as predictive biomarkers for the efficacy of IGF-1R targeting therapies in breast cancer patients. In addition to its therapeutic and clinical scope, this PhD project has significantly contributed to the understanding of the role of the IGF system in breast tumour biology by providing valuable new information on the mechanistic events underpinning IGF-I:VN-mediated effects on breast cell functions. Furthermore, this is the first instance where favourable binding sites for IGF-II, IGFBP-3 and IGFBP-5 on VN have been identified. Taken together, this study has functionally characterised the interactions between IGF-I and VN and through innovative strategies has provided a platform for the development of novel therapies targeting these interactions and their downstream effects.

Effect of induced airway inflammation in asthma : the expression of trefoil factors, secretoglobins and genes involved in histone acetylation

Asthma is an incapacitating disease of the respiratory system, which causes extensive morbidity and mortality worldwide. Asthma affects more than 300 million people globally(Masoli et al. 2004). In Australia, it affects 10.2% of the population (Masoli et al. 2004) and causes 60,000 people to be hospitalised annually. Health care expenditure due to asthma in Australia was $606 million in 2004–2005. There are four primary biological factors that function in the initiation and exacerbation of asthma. Airway inflammation is important as it is often the first response to an airway insult, initiating the three other components: bronchoconstriction, mucus hyper-secretion and hyper-reactivity. The mediators involved in asthma are still not well understood, and current anti-inflammatory corticosteroid treatments are not effective with all asthmatics. As there is currently no cure for asthma, and airway inflammation is the primary component of the disease, it is important that we understand and investigate the mediators of airway inflammation to look for a potential cure and to produce better therapeutics to treat the inflammation. Trefoil factors (TFFs) and secretoglobins (SCGBs) are small secreted proteins involved in the mediation of inflammation and epithelial restitution. TFFs are pro-inflammatory and SCGBs anti-inflammatory by nature. The hypothesis of this study is that in response to induced acute airway inflammation, the expression of TFF1 and TFF3 will increase and expression of SCGB1A1 and SCGB3A2 will decrease in non-asthmatics (N-A), asthmatics medicating with bronchodilators (A-BD) and asthmatics medicating with corticosteroids (A-ST). When comparing the three groups, we expect to see higher expression of the TFFs in the A-BD group compared to the N-A and A-ST groups, indicating that inflammation is mediated by TFFs in asthma and that corticosteroid medication controls their expression as part of the control of inflammation. We expect to see the opposite with SCGBs, with a greater decrease in the A-BD group compared to the other two groups, suggesting that the A-BD group has the least anti-inflammatory activity in response to inflammatory insult. Epigenetic modification plays a role in the regulation of genes that initiate disease states such as inflammatory conditions and cancers. Histone acetylation is one such modification, which involves the acetylation of histones in chromatin by histone acetyltransferases (HATs). This increases the transcription of genes involved with inflammation or enrols histone deacetylases (HDACs) to down-regulate the transcription of inflammatory genes. These HATs and HDACs work in a homeostatic fashion; however, in the event of inflammation, increased HAT activity can stimulate further inflammation, which is believed to be the mechanism involved in some inflammatory diseases. This study hypothesises that in response to inflammation, the expression of HDACs (HDAC1-5) will decrease and the expression of HATs (NCOA1-3, HAT-1 and CREBBP) will increase in all groups. When comparing the expression between the groups, it was expected that a greater decrease in HDACs and a greater increase in HATs will be seen in the A-BD group compared to the other two groups. This would identify histone acetylation as a mechanism involved in the inflammatory condition of asthma and indicate that corticosteroids may treat the inflammation in asthma at least in part by controlling histone acetylation. The aim of the project was to compare the expression of inflammatory genes TFF1, TFF3, SCGB1A1 and SCGB3A2, as well as to compare the gene expression of HDAC1-5, NCOA1-3, HAT-1 and CREBBP within and between N-A (n=15), A-BD (n=15) and A-ST (n=15) groups in response to inflammation. This was performed by collecting airway cells and proteins by sputum induction in three sessions. The sessions were coordinated into an initial baseline collection (SI-1), followed by a second session at least one week later (SI-2) and a third session, six hours after SI-2 to collect a sample containing the resultant acute inflammation caused in SI-2 (SI-3). Analysis of the SI-1 and SI-2 samples in all three groups had high amounts of variability between samples. The samples were taken at least one weak apart and the environmental stimuli on each participant outside of the testing sessions could not be controlled. For this reason, the SI-1 samples were not used for analysis; instead SI-2 and SI-3 samples were compared as they were same-day collections, reducing the probability of differences being due to anything other than the sputum induction. The gene expressions of the TFFs, SCGBs, HDACs and HATs were analysed using real-time PCR. Western blot analysis was performed to analyse the protein concentrations of the TFFs and SCGBs in secreted fractions of the sputum collection. Both the secreted and intracellular protein fractions collected from the sputum inductions for pre- and post-inflammation (SI-2, SI-3) samples of the N-A and A-BD groups were analysed using a proteomic method called iTRAQ. This allowed the comparison of the change in protein expression as a result of airway inflammation in each group. This technique was used as a discovery method to identify novel proteins that are modulated by induced acute airway inflammation. Any proteins of interest would then be further validated and used for future research. Inflammation was achieved in the SI-3 samples of the N-A group with a 21% unit increase in % neutrophils compared to SI-2 (p=0.01). The N-A group had a marked 5.5-fold decrease in HDAC1 gene expression in SI-3 compared to SI-2 (p=0.03). No differences were seen in any of the TFFs, SCGBs or any of the rest of the HDACs and HATs. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. However, non-significant analysis of the data displayed increases in TFF1 and TFF3, and decreases in SCGB1A1 and SCGB3A2 for the majority of SI-3 samples compared to SI-2. The A-BD group also presented a marked increase in neutrophils in the SI-3 samples compared to SI-2 (27% unit increase, p=0.04). The A-BD group had a significant increase in TFF3 and SCGB1A1 gene expression concomitant with induced acute airway inflammation. A 7.3-fold increase in TFF3 (p=0.05) in SI-3 indicated that TFF3 is linked to inflammation in asthmatics. A 2.8-fold increase in SCGB1A1 (p=0.03) indicated that this gene is also up-regulated, suggesting that this SCGB is expressed to try to combat induced acute airway inflammation. No significant changes were seen in any of the other genes analysed. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. However, non-significant analysis of the data displayed an increase in TFF1 and TFF3, and a decrease in SCGB1A1 and SCGB3A2 in SI-3, similar to that seen in the N-A group. The A-ST group was different from the A-BD group, characterised by the use of inhaled corticosteroid medication to treat asthma symptoms. Inhaled corticosteroids are known to treat asthma symptoms through the control of inflammation. Therefore, it was expected that corticosteroid medication would also control the expression of TFFs, SCGBs, HATs and HDACs. Gene expression results only identified a 7.6-fold decrease in HDAC2 expression in SI-3 (p=0.001), which is proposed to be due to the up-regulation of HDAC2 protein that is known to be a function of corticosteroid use. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. The gene expression in SI-2 and SI-3 in each group was compared. When comparing the A-BD group to the N-A group, a 9-fold increase in TFF3 (p=0.008) and a 34-fold increase in SCGB1A1 (p=0.03) were seen in the SI-3 samples. Comparisons of the A-ST group to the N-A group had an increased expression in SI-2 samples for HDAC5 (3.6-fold, p=0.04), NCOA2 (8.5-fold, p=0.04), NCOA3 (17-fold, p=0.01), HAT-1 (36-fold, p=0.003) and CREBBP (13-fold, p=0.001). The SI-3 samples in the A-ST group compared to the N-A group had increased expression for HDAC1 (6.4-fold, p=0.04), HDAC5 (5.2-fold, p=0.008), NCOA2 (9.6-fold, p=0.03), NCOA3 (16-fold, p=0.06), HAT-1 (41-fold, p<0.001) and CREBBP (31-fold, p=0.001). Comparisons of the A-ST group to the A-BD group had SI-2 increases in HDAC1 (3.8-fold, p=0.03), NCOA3 (4.5-fold, p=0.03), HAT-1 (5.3-fold, p=0.01) and CREBBP (23-fold, p=0.001), while SI-3 comparisons saw a decrease in HDAC2 (41-fold, p=0.008) and increases in HAT-1 (4.3-fold, p=0.003) and CREBBP (40-fold, p=0.001). Results showed that TFF3 and SCGB1A1 expression is higher in asthmatics than non-asthmatics and that histone acetylation is more active in the A-ST group than either the N-A or A-BD group, which suggests that histone acetylation activity may be positively correlated with asthma severity. The iTRAQ proteomic analysis of the secreted protein samples identified the SCGB1A1 protein and found it to be decreased in both the N-A and A-BD groups post-inflammation, but significantly so only in the A-BD group. Although no significant results were obtained from the western blot data, both groups displayed a decrease in SCGB1A1 concentration in SI-3 samples, suggesting a correlation with the proteomic data. Only 31 peptides were identified from the secreted samples. The intracellular iTRAQ analysis successfully identified 664 peptides, eight of which had differential expression in association with induced acute airway inflammation. Significant increases were seen in the A-BD group in SI-3 compared to SI-2 than in the N-A group in chloride intracellular channel protein 1, keratin-19, eosinophil cationic protein, calnexin, peroxiredoxin-5, and ATP-synthase delta subunit, while decreases were seen in cystatin-A and mucin-5AC. The iTRAQ analysis was only a discovery measure and further validation must be performed. In summary, the expression of TFFs and SCGBs differed between non-asthmatics and asthmatics. It is clear that TFF3 is active in the airway inflammation associated with asthma as indicated by an increase associated with inflammation in the A-BD group compared to the N-A group. Results for HDAC and HAT genes showed high HAT expression in the A-ST group compared to the N-A and A-BD groups, suggesting that histone acetyltransferases may be responsible for the characteristic unregulated inflammatory symptoms of asthmatics taking corticosteroids. Interestingly, corticosteroid medication did not seem to silence the expression of the analysed HAT genes, which indicates that corticosteroids may not control inflammation by direct regulation of HATs, but instead by competition, most probably with HDAC2 protein. As a discovery tool, iTRAQ is a potent method to both identify and compare the concentration of proteins between samples. The method is a powerful first step into the identification of novel proteins that are regulated in response to different treatments.

Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride

OBJECTIVE: To optimize the animal model of liver injury that can properly represent the pathological characteristics of dampness-heat jaundice syndrome of traditional Chinese medicine. METHODS: The liver injury in the model rat was induced by alpha-naphthylisothiocyanate (ANIT) and carbon tetrachloride (CCl(4) ) respectively, and the effects of Yinchenhao Decoction (, YCHD), a proved effective Chinese medical formula for treating the dampness-heat jaundice syndrome in clinic, on the two liver injury models were evaluated by analyzing the serum level of alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP), malondialchehyche (MDA), total bilirubin (T-BIL), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) as well as the ratio of liver weight to body weight. The experimental data were analyzed by principal component analytical method of pattern recognition. RESULTS: The ratio of liver weight to body weight was significantly elevated in the ANIT and CCl(4) groups when compared with that in the normal control (P<0.01). The contents of ALT and T-BIL were significantly higher in the ANIT group than in the normal control (P<0.05,P<0.01), and the levels of AST, ALT and ALP were significantly elevated in CCl(4) group relative to those in the normal control P<0.01). In the YCHD group, the increase in AST, ALT and ALP levels was significantly reduced (P<0.05, P<0.01), but with no significant increase in serum T-BIL. In the CCl(4) intoxicated group, the MDA content was significantly increased and SOD, GSH-PX activities decreased significantly compared with those in the normal control group, respectively (P<0.01). The increase in MDA induced by CCl(4) was significantly reduced by YCHD P<0.05). CONCLUSION: YCHD showed significant effects on preventing liver injury progression induced by CCl(4), and the closest or most suitable animal model for damp-heat jaundice syndrome may be the one induced by CCl(4).

Oxidative stress induced lung cancer and COPD : opportunities for epigenetic therapy

Reactive oxygen species (ROS) form as a natural by-product of the normal metabolism of oxygen and play important roles within the cell. Under normal circumstances the cell is able to maintain an adequate homeostasis between the formation of ROS and its removal through particular enzymatic pathways or via antioxidants. If however, this balance is disturbed a situation called oxidative stress occurs. Critically, oxidative stress plays important roles in the pathogenesis of many diseases, including cancer. Epigenetics is a process where gene expression is regulated by heritable mechanisms that do not cause any direct changes to the DNA sequence itself, and disruption of epigenetic mechanisms has important implications in disease. Evidence is emerging that histone deacetylases (HDACs) play decisive roles in regulating important cellular oxidative stress pathways including those involved with sensing oxidative stress and those involved with regulating the cellular response to oxidative stress. In particular aberrant regulation of these pathways by HDACs may play critical roles in cancer progression. In this review we discuss the current evidence linking epigenetics and oxidative stress and cancer, using chronic obstructive pulmonary disease and non-small cell lung cancer to illustrate the importance of epigenetics on these pathways within these disease settings. © 2009 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation

An essential step for therapeutic and research applications of stem cells is their ability to differentiate into specific cell types. Neuronal cells are of great interest for medical treatment of neurodegenerative diseases and traumatic injuries of central nervous system (CNS), but efforts to produce these cells have been met with only modest success. In an attempt of finding new approaches, atmospheric-pressure room-temperature microplasma jets (MPJs) are shown to effectively direct in vitro differentiation of neural stem cells (NSCs) predominantly into neuronal lineage. Murine neural stem cells (C17.2-NSCs) treated with MPJs exhibit rapid proliferation and differentiation with longer neurites and cell bodies eventually forming neuronal networks. MPJs regulate ~. 75% of NSCs to differentiate into neurons, which is a higher efficiency compared to common protein- and growth factors-based differentiation. NSCs exposure to quantized and transient (~. 150. ns) micro-plasma bullets up-regulates expression of different cell lineage markers as β-Tubulin III (for neurons) and O4 (for oligodendrocytes), while the expression of GFAP (for astrocytes) remains unchanged, as evidenced by quantitative PCR, immunofluorescence microscopy and Western Blot assay. It is shown that the plasma-increased nitric oxide (NO) production is a factor in the fate choice and differentiation of NSCs followed by axonal growth. The differentiated NSC cells matured and produced mostly cholinergic and motor neuronal progeny. It is also demonstrated that exposure of primary rat NSCs to the microplasma leads to quite similar differentiation effects. This suggests that the observed effect may potentially be generic and applicable to other types of neural progenitor cells. The application of this new in vitro strategy to selectively differentiate NSCs into neurons represents a step towards reproducible and efficient production of the desired NSC derivatives. © 2013.

Artemisinin induced dormancy in Plasmodium falciparum : duration, recovery rates and implications in treatment failure

Background Despite the remarkable activity of artemisinin and its derivatives, monotherapy with these agents has been associated with high rates of recrudescence. The temporary arrest of the growth of ring-stage parasites (dormancy) after exposure to artemisinin drugs provides a plausible explanation for this phenomenon. Methods Ring-stage parasites of several Plasmodium falciparum lines were exposed to different doses of dihydroartemisinin (DHA) alone or in combination with mefloquine. For each regime, the proportion of recovering parasites was determined daily for 20 days. Results Parasite development was abruptly arrested after a single exposure to DHA, with some parasites being dormant for up to 20 days. Approximately 50% of dormant parasites recovered to resume growth within the first 9 days. The overall proportion of parasites recovering was dose dependent, with recovery rates ranging from 0.044% to 1.313%. Repeated treatment with DHA or with DHA in combination with mefloquine led to a delay in recovery and an ∼10-fold reduction in total recovery. Strains with different genetic backgrounds appeared to vary in their capacity to recover. Conclusions These results imply that artemisinin-induced arrest of growth occurs readily in laboratory-treated parasites and may be a key factor in P. falciparum malaria treatment failure.

Mitochondrial membrane potential in a small subset of artemisinin-induced dormant plasmodium falciparum parasites in vitro

Artemisinin induced dormancy is a proposed mechanism for failures of mono-therapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that following dihydroartemisinin (DHA) treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential (MMP) marker, and persisted to recovery. FACS sorted RH-positive parasites resumed growth at 10,000/well while RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was only detected in RH-positive dormant parasites. Importantly, after treating dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage.

Cell type-dependent, infection-induced, aberrant DNA methylation in gastric cancer

Epigenetic changes correspond to heritable modifications of the chromatin structure, which do not involve any alteration of the DNA sequence but nonetheless affect gene expression. These mechanisms play an important role in cell differentiation, but aberrant occurrences are also associated with a number of diseases, including cancer and neural development disorders. In particular, aberrant DNA methylation induced by H. Pylori has been found to be a significant risk factor in gastric cancer. To investigate the sensitivity of different genes and cell types to this infection, a computational model of methylation in gastric crypts is developed. In this article, we review existing results from physical experiments and outline their limitations, before presenting the computational model and investigating the influence of its parameters.