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Localized Merkel cell carcinoma treatment considerations: a response to the forty-year experience at the Peter MacCallum cancer centre
Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine tumour of the skin with poor prognosis and rising global incidence. A recently published article in BMC Cancer, titled “Merkel cell carcinoma: a forty-year experience at the Peter MacCallum Cancer Centre” (Wang et al.), provides a contemporary analysis of locoregional disease outcomes in Australia which highlights the comparative effectiveness of radiotherapy for excisions with involved margins versus wide local excision. There is a persistent lack of clear, well-defined guidelines to manage MCC in Australia despite experiencing the highest rates globally. The advanced age at onset also provides inherent challenges for optimal management and often, a case-by-case approach is necessary based on patient preferences, baseline function and fitness for surgery. This paper responds to the recently published article by Wang et al. and will expand the discourse regarding management of localized MCC. Specifically, we will discuss the surgical excision approaches; alternative treatment options for MCC including radiotherapy, Mohs micrographic surgery and novel immunotherapy agents being investigated through several clinical trials.
Journal Article
Reproducible isolation of bovine mammary macrophages for analysis of host pathogen interactions
Background
Macrophages residing in milk are vital during intramammary infections. This study sought to develop a method enabling the investigation of macrophage responses to pathogens.
Streptococcus uberis
is the predominant cause of bovine mastitis UK-wide and its pathogenesis is unusual compared to other intramammary pathogens. Previous studies utilise macrophage cell lines, isolated bovine blood derived monocytes, or macrophages from raw milk through complex or inconsistent strategies such as fluorescence activated cell sorting (FACS), centrifugation and selective adherence, and CD14 antibody-microbeads. The centrifuge steps required in the initial stages often damage cells. Thus, the aim of this study was to develop a reliable, reproducible, and cost-effective method for isolating mammary macrophages from milk in a way that allows their culture, challenge with bacteria, and measurement of their response
ex-vivo
.
Results
This method achieves an average yield of 1.27 × 10
7
cells per litre of milk. Whole milk with somatic cell range of 45–65 cells/µL produced excellent yields, with efficient isolations accomplished with up to 150 cells/µL. This strategy uses milk diluted in PAE buffer to enable low-speed centrifugation steps followed by seeding on tissue-culture-treated plastic. Seeding 1,000,000 milk-extracted cells onto tissue culture plates was sufficient to obtain 50,000 macrophage. Isolated macrophage remained responsive to challenge, with the highest concentration of IL-1β measured by ELISA at 20 h after challenge with
S. uberis
. In this model, the optimal multiplicity of infection was found to be 50:1 bacteria:macrophage. No difference in IL-1β production was found between macrophages challenged with live or heat-killed
S. uberis
. Standardisation of the production of IL-1β to that obtained following macrophage stimulation with LPS allowed for comparisons between preparations.
Conclusions
A cost-effective method, utilising low-speed centrifugation followed by adherence to plastic, was established to isolate bovine mammary macrophages from raw milk. This method was shown to be appropriate for bacterial challenge, therefore providing a cost-effective,
ex-vivo
, and non-invasive model of macrophage-pathogen interactions. The optimal multiplicity of infection for
S. uberis
challenge was demonstrated and a method for standardisation against LPS described which removes sample variation. This robust method enables, reproducible and reliable interrogation of critical pathogen-host interactions which occur in the mammary gland.
Journal Article
Deface the face
\"Long ago, Harvey Dent wasn't just a good man--he was the best man in all of Gotham City. As the town's District Attorney, the city's so-called golden boy put away murderers, thieves and mob bosses by the dozens. But there's one organization he couldn't put away. And now they're back. A desparate Dark Knight needs help and there's only one man he can turn to--unfortunately, that one man isn't the person he used to be. In fact... he's two.\"-- Provided by publisher.
Book
Priming from within: TLR2 dependent but receptor independent activation of the mammary macrophage inflammasome by Streptococcus uberis
is a member of the pyogenic cluster of
commonly associated with intramammary infection and mastitis in dairy cattle. It is a poorly controlled globally endemic pathogen responsible for a significant cause of the disease worldwide. The ruminant mammary gland provides an atypical body niche in which immune cell surveillance occurs on both sides of the epithelial tissue.
does not cause disease in non-ruminant species and is an asymptomatic commensal in other body niches.
exploits the unusual niche of the mammary gland to initiate an innate response from bovine mammary macrophage (BMMO) present in the secretion (milk) in which it can resist the host immune responses. As a result - and unexpectedly - the host inflammatory response is a key step in the pathogenesis of
, without which colonisation is impaired. In contrast to other bacteria pathogenic to the bovine mammary gland,
does not elicit innate responses from epithelial tissues; initial recognition of infection is via macrophages within milk.
We dissected the role of the bacterial protein SUB1154 in the inflammasome pathway using
bovine mammary macrophages isolated from milk, recombinant protein expression, and a panel of inhibitors, agonists, and antagonists. We combine this with reverse-transcription quantitative real-time PCR to investigate the mechanisms underlying SUB1154-mediated priming of the immune response.
Here, we show that SUB1154 is responsible for priming the NLRP3 inflammasome in macrophages found in the mammary gland. Without SUB1154, IL-1β is not produced, and we were able to restore IL-1β responses to a sub1154 deletion
mutant using recombinant SUB1154. Surprisingly, only by blocking internalisation, or the cytoplasmic TIR domain of TLR2 were we able to block SUB1154-mediated priming.
Together, our data unifies several contrasting past studies and provides new mechanistic understanding of potential early interactions between pyogenic streptococci and the host.
Journal Article
Hopes and fears
\"Superman is faced with an impossible decision when Deathstroke comes to Metropolis--let the love of his life die or become a killer himself. In the wake of BLACK DAWN's barage of terror and horror, the super family decides to take a much needed vacation. What the Kents don't realize is that this may not be the relaxing family time they had initially planned. When Superman is thrust into the anti-matter universe of Qward, his only hope is Sinestro, the former greatest of the Green Lanterns. Meanwhile, Lois Lane profiles Deathstroke the Terminator for the Daily Planet and it could cost her her life! This leads Deathstroke to Metropolis and he forces Superman to make an impossible choice--let the love of his life die or become a killer himself.\"-- Provided by publisher.
Book
Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages
Streptococcus uberis
is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards
S. uberis
strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by
Escherichia coli
. Neither heat inactivated nor live
S. uberis
induced the expression of 10 key immune genes (including
TNF
,
IL1B
,
IL6
). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (
hasA
) or biofilm formation (
sub0538
and
sub0539
)
. Streptococcus uberis
failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that
S. uberis
particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact
S. uberis
such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same
S. uberis
preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of
S. uberis
mastitis.
Journal Article
Brake : seconds to live : a nation to save
Jeremy Reins wakes up in a cramped space with the only light coming from the digital numbers ticking away above his head. Confused and disoriented with no one answering his cries for help, he suddenly hears an engine rev and his predicament becomes clear: he's trapped in the trunk of a moving car. As his captors reveal themselves, Jeremy realizes he won't be set free until he gives up the whereabouts of a secret location where the U.S. President is taken in the event of a terrorist attack.
DVD
Defining the ABC of gene essentiality in streptococci
Background
Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for
Streptococcus equi
subsp.
equi
, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C
Streptococcus
was compared to that of group A and B streptococci.
Results
Six barcoded variants of pGh9:IS
S1
were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the
S. equi
genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species.
Conclusions
The use of barcoded pGh9:IS
S1
to generate mutant libraries provides a highly useful tool for the assignment of gene function in
S. equi
and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.
Journal Article