Elevated plasma fibronectin levels associated with venous thromboembolism

Elevated plasma fibronectin levels occur in various clinical states including arterial disease. Increasing evidence suggests that atherothrombosis and venous thromboembolism (VTE) share common risk factors. To assess the hypothesis that high plasma fibronectin levels are associated with VTE, we compared plasma fibronectin levels in the Scripps Venous Thrombosis Registry for 113 VTE cases vs. age and sex matched controls. VTE cases had significantly higher mean fibronectin concentration compared to controls (127% vs. 103%, p<0.0001); the difference was greater for idiopathic VTE cases compared to secondary VTE cases (133% vs. 120%, respectively). Using a cut-off of >90% of the control values, the odds ratio (OR) for association of VTE for fibronectin plasma levels above the 90th percentile were 9.37 (95% CI 2.73-32.2; p<0.001) and this OR remained significant after adjustment for sex, age, body mass index (BMI), factor V Leiden and prothrombin nt20210A (OR 7.60, 95% CI 2.14-27.0; p=0.002). In particular, the OR was statistically significant for idiopathic VTE before and after these statistical adjustments. For the total male cohort, the OR was significant before and after statistical adjustments and was not significant for the total female cohort. In summary, our results suggest that elevated plasma fibronectin levels are associated with VTE especially in males, and extend the potential association between biomarkers and risk factors for arterial atherothrombosis and VTE. © 2008 Schattauer GmbH.

Study of particulate matter removal mechanism by using non-thermal plasma technology

Numbers of diesel engines in both stationary and mobile applications are increasing nowadays. Diesel engines emit lower Hydrocarbon (HC) and Carbon monoxide (CO) than gasoline engines. However, they can produce more nitrogen oxides (NOx) and have higher particulate matter (PM). On the other hand, emissions standards are getting stringent day by day due to considerable concerns about unregulated pollutants and particularly ultrafine particles deleterious effect on human health. Non-thermal plasma (NTP) treatment of exhaust gas is known as a promising technology for both NOx and PM reduction by introducing plasma inside the exhaust gas. Vehicle exhaust gases undergo chemical changes when exposed to plasma. In this study, the PM removal mechanism using NTP by applying high voltage pulses of up to 20 kVpp with a repetition rate of 10 kHz are investigated. It is found that, voltage increase not necessarily has a positive effect on PM removal in diesel engine emissions.

Investigating epidermogenesis in a human skin equivalent model

Skin is the largest, and arguably, the most important organ of the body. It is a complex and multi-dimensional tissue, thus making it essentially impossible to fully model in vitro in conventional 2-dimensional culture systems. In view of this, rodents or pigs are utilised to study wound healing therapeutics or to investigate the biological effects of treatments on skin. However, there are many differences between the wound healing processes in rodents compared to humans (contraction vs. re-epithelialisation) and there are also ethical issues associated with animal testing for scientific research. Therefore, the development of skin equivalent (HSE) models from surgical discard human skin has become an important area of research. The studies in this thesis compare, for the first time, native human skin and the epidermogenesis process in a HSE model. The HSE was reported to be a comparable model for human skin in terms of expression and localisation of key epidermal cell markers. This validated HSE model was utilised to study the potential wound healing therapeutic, hyperbaric oxygen (HBO) therapy. There is a significant body of evidence suggesting that lack of cutaneous oxygen results in and potentiates the chronic, non-healing wound environment. Although the evidence is anecdotal, HBO therapy has displayed positive effects on re-oxygenation of chronic wounds and the clinical outcomes suggest that HBO treatment may be beneficial. Therefore, the HSE was subjected to a daily clinical HBO regime and assessed in terms of keratinocyte migration, proliferation, differentiation and epidermal thickening. HBO treatment was observed to increase epidermal thickness, in particular stratum corneum thickening, but it did not alter the expression or localisation of standard epidermal cell markers. In order to elucidate the mechanistic changes occurring in response to HBO treatment in the HSE model, gene microarrays were performed, followed by qRT-PCR of select genes which were differentially regulated in response to HBO treatment. The biological diversity of the HSEs created from individual skin donors, however, overrode the differences in gene expression between treatment groups. Network analysis of functional changes in the HSE model revealed general trends consistent with normal skin growth and maturation. As a more robust and longer term study of these molecular changes, protein localisation and expression was investigated in sections from the HSEs undergoing epidermogenesis in response to HBO treatment. These proteins were CDCP1, Metallothionein, Kallikrein (KLK) 1 and KLK7 and early growth response 1. While the protein expression within the HSE models exposed to HBO treatment were not consistent in all HSEs derived from all skin donors, this is the first study to detect and compare both KLK1 and CDCP1 protein expression in both a HSE model and native human skin. Furthermore, this is the first study to provide such an in depth analysis of the effect of HBO treatment on a HSE model. The data presented in this thesis, demonstrates high levels of variation between individuals and their response to HBO treatment, consistent with the clinical variation that is currently observed.

Examination of the role of nitric oxide synthase and renal kallikrein as candidate genes for essential hypertension

Nitric oxide synthase and renal kallikrein are both involved in blood pressure regulation. Genes for these enzymes may, therefore, be considered candidates for hypertension pathogenesis. 2. In the present study, genotypes for nitric oxide synthase and renal kallikrein microsatellite markers were determined in a cross-sectional association analysis of hypertensive patients and normotensive control subjects. 3. Results from this study did not indicate an association of either of the candidate gene polymorphisms with essential hypertension. Hence, findings for this study do not support a role for these genes in human hypertension.

Molecular genetic analyses of RFLPs for PCR-amplified growth hormone gene, renal kallikrein gene and atrial natriuretic factor gene in essential hypertension

The present study examined polymorphisms of genes that might be involved in the onset of essential hypertension (HT). These included the (i) growth hormone gene (GH1), whose locus has recently been linked to elevated blood pressure (BP) in the stroke-prone SHR, although recent sib-pair analysis of a polymorphism near the human chorionic somatomammotropin gene (a member of the GH cluster) was unable to show linkage with HT; (ii) renal kallikrein gene (KLK1); and (iii) atrial natriuretic factor gene (ANF), where a primary defect in production or activity of kallikrein or ANF could cause NaCl retention and vasoconstriction. Association analyses were conducted to compare restriction fragment length polymorphisms (RFLPs) of each gene in 85 HT and 95 normotensive (NT) Caucasian subjects whose parents had a similar BP status at age ≥50 years. The frequency of the minor allele of (i) a RsaI RFLP in the promoter of GH1, amplified from leukocyte DNA by the polymerase chain reaction, was 0.15 in the HT group and 0.14 in the NT group (χ1=0.34, P=0.55); (ii) a TaqI RFLP for KLK1 was 0.035 in the HT group and 0.015 in the NT group (χ2=1.5, P=0.21); and (iii) a XhoI RFLP for ANF was 0.50 in HTs and 0.46 in NTs (χ2=0.20, P=0.65). Studies of HT pedigrees found one family in which the ANF locus and HT were not linked, owing to an obligate recombinant. The present data thus provide no evidence for involvement of the growth hormone, renal kallikrein, nor ANF gene in the causation of essential hypertension.

Metastasis of ovarian cancer is mediated by kallikrein related peptidases

Ovarian cancer, in particular epithelial ovarian cancer (EOC), is commonly diagnosed when the tumor has metastasized into the abdominal cavity with an accumulation of ascites fluid. Combining histopathology and genetic variations, EOC can be sub-grouped into Type-I and Type-II tumors, of which the latter are more aggressive and metastatic. Metastasis and chemoresistance are the key events associated with the tumor microenvironment that lead to a poor patient outcome. Kallikrein-related peptidases (KLKs) are aberrantly expressed in EOC, in particular, in the more metastatic Type-II tumors. KLKs are a family of 15 serine proteases that are expressed in diverse human tissues and involved in various patho-physiological processes. As extracellular enzymes, KLKs function in the hydrolysis of growth factors, proteases, cell membrane bound receptors, adhesion proteins, and cytokines initiating intracellular signaling pathways and their downstream events. High KLK levels are differentially associated with the prognosis of ovarian cancer patients, suggesting that they not only have application as biomarkers but also function in disease progression, and therefore are potential therapeutic targets. Recent studies have demonstrated the function of these proteases in promoting and/or suppressing the invasive behavior of ovarian cancer cells in metastasis in vitro and in vivo. Both conventional cell culture methods and three-dimensional platforms have been applied to mimic the ovarian cancer microenvironment of patients, such as the solid stromal matrix and ascites fluid. Here we summarize published studies to provide an overview of our understanding of the role of KLKs in EOC, and to lay the foundation for future research directions.

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell lines in vitro and in vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-rasH, v-rasKi, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determined in vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection with ras, despite similar ras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-rasH + SV40T and v -rasH + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

Plasma-induced death of HepG2 cancer cells : intracellular effects of reactive species

Reports show that cold atmospheric-pressure plasmas can induce death of cancer cells in several minutes. However, very little is presently known about the mechanism of the plasma-induced death of cancer cells. In this paper, an atmospheric-pressure plasma plume is used to treat HepG2 cells. The experimental results show that the plasma can effectively control the intracellular concentrations of ROS, NO and lipid peroxide. It is shown that these concentrations are directly related to the mechanism of the HepG2 death, which involves several stages. First, the plasma generates NO species, which increases the NO concentration in the extracellular medium. Second, the intracellular NO concentration is increased due to the NO diffusion from the medium. Third, an increase in the intracellular NO concentration leads to the increase of the intracellular ROS concentration. Fourth, the increased oxidative stress results in more effective lipid peroxidation and consequently, cell injury. The combined action of NO, ROS and lipid peroxide species eventually results in the HepG2 cell death. The mechanism of death of human hepatocellular carcinoma cells (HepG2) induced by atmospheric-pressure room-temperature plasma, related to the plasma-controlled intracellular concentrations of reactive oxygen species (ROS), nitric oxide (NO) and lipid peroxide is revealed. Only 34.75 s are required to reduce the number of the viable HepG2 cells by 50%.

Open-air direct current plasma jet : scaling up, uniformity, and cellular control

Atmospheric-pressure plasma jets are commonly used in many fields from medicine to nanotechnology, yet the issue of scaling the discharges up to larger areas without compromising the plasma uniformity remains a major challenge. In this paper, we demonstrate a homogenous cold air plasmaglow with a large cross-section generated by a direct current power supply. There is no risk of glow-to-arc transitions, and the plasmaglow appears uniform regardless of the gap between the nozzle and the surface being processed. Detailed studies show that both the position of the quartz tube and the gas flow rate can be used to control the plasma properties. Further investigation indicates that the residual charges trapped on the inner surface of the quartz tube may be responsible for the generation of the air plasma plume with a large cross-section. The spatially resolved optical emission spectroscopy reveals that the air plasma plume is uniform as it propagates out of the nozzle. The remarkable improvement of the plasma uniformity is used to improve the bio-compatibility of a glass coverslip over a reasonably large area. This improvement is demonstrated by a much more uniform and effective attachment and proliferation of human embryonic kidney 293 (HEK 293) cells on the plasma-treated surface.

Room-temperature, atmospheric plasma needle reduces adenovirus gene expression in HEK 293A host cells

Room-temperature, atmospheric-pressure plasma needle treatment is used to effectively minimize the adenovirus (AdV) infectivity as quantified by the dramatic reduction of its gene expression in HEK 293A primary human embryonic kidney cells studied by green fluorescent protein imaging. The AdV titer is reduced by two orders of magnitude within only 8 min of the plasma exposure. This effect is due to longer lifetimes and higher interaction efficacy of the plasma-generated reactive species in confined space exposed to the plasma rather than thermal effects commonly utilized in pathogen inactivation. This generic approach is promising for the next-generation anti-viral treatments and imunotherapies.

Control of energy and matter at nanoscales : challenges and opportunities for plasma nanoscience in a sustainability age

Plasma nanoscience is an emerging multidisciplinary research field at the cutting edge of a large number of disciplines including but not limited to physics and chemistry of plasmas and gas discharges, materials science, surface science, nanoscience and nanotechnology, solid-state physics, space physics and astrophysics, photonics, optics, plasmonics, spintronics, quantum information, physical chemistry, biomedical sciences and related engineering subjects. This paper examines the origin, progress and future perspectives of this research field driven by the global scientific and societal challenges. The future potential of plasma nanoscience to remain a highly topical area in the global research and technological agenda in the age of fundamental-level control for a sustainable future is assessed using a framework of the five Grand Challenges for Basic Energy Sciences recently mapped by the US Department of Energy. It is concluded that the ongoing research is very relevant and is expected to substantially expand to competitively contribute to the solution of all of these Grand Challenges. The approach to controlling energy and matter at nano- and subnanoscales is based on identifying the prevailing carriers and transfer mechanisms of the energy and matter at the spatial and temporal scales that are most relevant to any particular nanofabrication process. Strong accent is made on the competitive edge of the plasma-based nanotechnology in applications related to the major socio-economic issues (energy, food, water, health and environment) that are crucial for a sustainable development of humankind. Several important emerging topics, opportunities and multidisciplinary synergies for plasma nanoscience are highlighted. The main nanosafety issues are also discussed and the environment- and human health-friendly features of plasma-based nanotech are emphasized.

Investigation on bioactivity of titanium dioxide thin films fabricated by electron cyclotron resonance plasma aided pulsed laser deposition

Titanium dioxide thin films with a rutile crystallinite size around 20 nm were fabricated by pulsed laser deposition (PLD) aided with an electron cyclotron resonance (ECR) plasma. With annealing treatment, the crystal size of the rutile crystallinite increased to 100 nm. The apatite-forming ability of the films as deposited and after annealing was investigated in a kind of simulated body fluid with ion concentrations nearly equal to those of human blood plasma. The results indicate that ECR aided PLD is an effective way both to fabricate bioactive titanium dioxide thin films and to optimize the bioactivity of titanium dioxide, with both crystal size and defects of the film taken into account.

Distribution of methylene chloride in human blood

Methylene chloride (dichloromethane) is widely used as a solvent for stripping of paint, as industrial cleaning agent, for coating of pills in the pharmaceutical industry, and in the decaffeination of coffee. There is “sufficient evidence for the carcinogenicity” of methylene chloride in animals and “inadequate evidence for its carcinogenity in humans”, according to IARC (IARC 1987; CEC 1990).