Paclitaxel resistance and multicellular spheroid formation are induced by kallikrein-related peptidase 4 in serous ovarian cancer cells in an ascites mimicking microenvironment

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.

Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

New particle formation and growth at a remote, sub-tropical coastal location

A month-long intensive measurement campaign was conducted in March/April 2007 at Agnes Water, a remote coastal site just south of the Great Barrier Reef on the east coast of Australia. Particle and ion size distributions were continuously measured during the campaign. Coastal nucleation events were observed in clean, marine air masses coming from the south-east on 65% of the days. The events usually began at ~10:00 local time and lasted for 1-4 hrs. They were characterised by the appearance of a nucleation mode with a peak diameter of ~10 nm. The freshly nucleated particles grew within 1-4 hrs up to sizes of 20-50 nm. The events occurred when solar intensity was high (~1000 W m-2) and RH was low (~60%). Interestingly, the events were not related to tide height. The volatile and hygroscopic properties of freshly nucleated particles (17-22.5 nm), simultaneously measured with a volatility-hygroscopicity-tandem differential mobility analyser (VH-TDMA), were used to infer chemical composition. The majority of the volume of these particles was attributed to internally mixed sulphate and organic components. After ruling out coagulation as a source of significant particle growth, we conclude that the condensation of sulphate and/or organic vapours was most likely responsible for driving particle growth during the nucleation events. We cannot make any direct conclusions regarding the chemical species that participated in the initial particle nucleation. However, we suggest that nucleation may have resulted from the photo-oxidation products of unknown sulphur or organic vapours emitted from the waters of Hervey Bay, or from the formation of DMS-derived sulphate clusters over the open ocean that were activated to observable particles by condensable vapours emitted from the nutrient rich waters around Fraser Island or Hervey Bay. Furthermore, a unique and particularly strong nucleation event was observed during northerly wind. The event began early one morning (08:00) and lasted almost the entire day resulting in the production of a large number of ~80 nm particles (average modal concentration during the event was 3200 cm-3). The Great Barrier Reef was the most likely source of precursor vapours responsible for this event.

Long-term trends in fine particle number concentrations in the urban atmosphere of Brisbane : the relevance of traffic emissions and new particle formation

The measurement of submicrometre (< 1.0 m) and ultrafine particles (diameter < 0.1 m) number concentration have attracted attention since the last decade because the potential health impacts associated with exposure to these particles can be more significant than those due to exposure to larger particles. At present, ultrafine particles are not regularly monitored and they are yet to be incorporated into air quality monitoring programs. As a result, very few studies have analysed their long-term and spatial variations in ultrafine particle concentration, and none have been in Australia. To address this gap in scientific knowledge, the aim of this research was to investigate the long-term trends and seasonal variations in particle number concentrations in Brisbane, Australia. Data collected over a five-year period were analysed using weighted regression models. Monthly mean concentrations in the morning (6:00-10:00) and the afternoon (16:00-19:00) were plotted against time in months, using the monthly variance as the weights. During the five-year period, submicrometre and ultrafine particle concentrations increased in the morning by 105.7% and 81.5% respectively whereas in the afternoon there was no significant trend. The morning concentrations were associated with fresh traffic emissions and the afternoon concentrations with the background. The statistical tests applied to the seasonal models, on the other hand, indicated that there was no seasonal component. The spatial variation in size distribution in a large urban area was investigated using particle number size distribution data collected at nine different locations during different campaigns. The size distributions were represented by the modal structures and cumulative size distributions. Particle number peaked at around 30 nm, except at an isolated site dominated by diesel trucks, where the particle number peaked at around 60 nm. It was found that ultrafine particles contributed to 82%-90% of the total particle number. At the sites dominated by petrol vehicles, nanoparticles (< 50 nm) contributed 60%-70% of the total particle number, and at the site dominated by diesel trucks they contributed 50%. Although the sampling campaigns took place during different seasons and were of varying duration these variations did not have an effect on the particle size distributions. The results suggested that the distributions were rather affected by differences in traffic composition and distance to the road. To investigate the occurrence of nucleation events, that is, secondary particle formation from gaseous precursors, particle size distribution data collected over a 13 month period during 5 different campaigns were analysed. The study area was a complex urban environment influenced by anthropogenic and natural sources. The study introduced a new application of time series differencing for the identification of nucleation events. To evaluate the conditions favourable to nucleation, the meteorological conditions and gaseous concentrations prior to and during nucleation events were recorded. Gaseous concentrations did not exhibit a clear pattern of change in concentration. It was also found that nucleation was associated with sea breeze and long-range transport. The implications of this finding are that whilst vehicles are the most important source of ultrafine particles, sea breeze and aged gaseous emissions play a more important role in secondary particle formation in the study area.

The mechanism of breath aerosol formation

Background: Aerosol production during normal breathing is often attributed to turbulence in the respiratory tract. That mechanism is not consistent with a high degree of asymmetry between aerosol production during inhalation and exhalation. The objective was to investigate production symmetry during breathing. Methods: The aerosol size distribution in exhaled breath was examined for different breathing patterns including normal breathing, varied breath holding periods and contrasting inhalation and exhalation rates. The aerosol droplet size distribution measured in the exhaled breath was examined in real time using an aerodynamic particle sizer. Results and Conclusions: The dependence of the particle concentration decay rate on diameter during breath holding was consistent with gravitational settling in the alveolar spaces. Also, deep exhalation resulted in a 4 to 6 fold increase in concentration and rapid inhalation produced a further 2 to 3 fold increase in concentration. In contrast rapid exhalation had little effect on the measured concentration. A positive correlation of the breath aerosol concentration with subject age was observed. The results were consistent with the breath aerosol being produced through fluid film rupture in the respiratory bronchioles in the early stages of inhalation and the resulting aerosol being drawn into the alveoli and held before exhalation. The observed asymmetry of production in the breathing cycle with very little aerosol being produced during exhalation, is inconsistent with the widely assumed turbulence induced aerosolization mechanism.