The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The cell wall-associated mycolactone polyketide synthases are necessary but not sufficient for mycolactone biosynthesis

Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules

Hyperexpression of alpha-hemolysin explains enhanced virulence of sequence type 93 community-associated methicillin-resistant Staphylococcus aureus

BACKGROUND: The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined. RESULTS: Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus. CONCLUSIONS: These data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA

'A Divided Soul'? the Cold War odyssey of O. John Rogge

In 1948 O. John Rogge, a prominent American liberal, was a contender for the Progressive Party's vice-presidential nomination. He was then a man of the Left: an activist in the international peace movement, a champion of radical causes and a defender of organizations deemed subversive by the Department of Justice. In 1951 he persuaded his\ud client to turn government witness in the Rosenberg espionage trial and was converted into 'Rogge the Rat' by his former allies. In tracing this transformation, this paper will argue that Rogge was neither a typical Cold War apostate nor a typical anti-Stalinist intellectual. Instead, his political trajectory was the outcome of a failed attempt to steer global politics away from Cold War dichotomies. The paper will therefore throw new light\ud both on the movement to find a 'third way' between East and West, and on the phenomenon of non-communist Left activism during the early Cold War

Expression of mycolactone accessory enzymes encoded by mup045 and mup038.

<p>(A) RT-PCR of mup045, mup038 and <i>crtI</i> on RNA extracted from <i>M. marinum</i> M containing plasmids expressing LM-M8 and mup045-mup038 under control of the P<i>_ermE</i> promoter. Western immunoblots showing: (B) presence of Mup038 in the cell wall fraction of <i>M. marinum</i> TPS8334; (C) presence of Mup045 in whole cell lysates from <i>M. marinum</i> M expressing LM-M8 and mup045-mup038 (TPS8334) as well as <i>M. ulcerans</i> 06-3844 harbouring pTPS331 (TPS8164) or pTPS333 (TPS8162); (D) Localization of Mup045 to the cell wall in <i>M. ulcerans</i> 06-3844 wild type and <i>M. marinum</i> TPS8334 cell fractions, showing reactivity against Mup045 in all strains, and localized to the cell wall fraction in both strains. Positive controls are purified, recombinant Mup038 and Mup045. (D).</p

Stability of pTPS629 in <i>M. marinum</i> M cultured in the absence of apramycin antibiotic selection.

<p>A late log-phase culture of <i>M. marinum</i> harbouring pTPS629 (and <i>mlsA2</i> on pTPS334) grown in the presence of apramycin and hygromycin, was shifted to media without apramycin and then monitored at successive time points by determining the cfu/ml on media with the antibiotic (squares) and calculating the percentage of <i>M. marinum</i> cells retaining apramycin resistance (right hand Y-axis, triangles). These results depict the mean and standard deviation of at least biological triplicates.</p

SDS-PAGE and western immunoblot analysis of whole cell lysates and cell fractions of <i>M. marinum</i> M harbouring plasmids expressing LM-M8 and MlsA2_His.

<p>(A) SDS-PAGE separation and Coomassie-stained protein gel of 10 μg of whole cell lysate of <i>M. marinum</i> M with LM-M8 and <i>mlsA2</i> (TPS8313) and empty vector control (TPS8256); (B) Western immunoblot of (A) using an anti-AT domain antibody; (C) Western immunoblot of (A) using an anti-His antibody; (D) Western immunoblot of cell fractions from <i>M. marinum</i> M harbouring plasmids expressing LM-M8 and MlsA2_His using an anti-His antibody, showing MlsA2 is present only in the cell wall (P27) fraction. The reactivity of the anti-His antibody to a protein with a mass ∼65 kDa in panels (C) and (D) is the known cross-reactivity with the polyhistidines of mycobacterial GroEL (Hsp65) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070520#pone.0070520-Noens1" target="_blank">[37]</a>.</p