Effect of early tranexamic acid administration on mortality, hysterectomy, and other morbidities in women with post-partum haemorrhage (WOMAN): an international, randomised, double-blind, placebo-controlled trial

Background Post-partum haemorrhage is the leading cause of maternal death worldwide. Early administration of tranexamic acid reduces deaths due to bleeding in trauma patients. We aimed to assess the effects of early administration of tranexamic acid on death, hysterectomy, and other relevant outcomes in women with post-partum haemorrhage. Methods In this randomised, double-blind, placebo-controlled trial, we recruited women aged 16 years and older with a clinical diagnosis of post-partum haemorrhage after a vaginal birth or caesarean section from 193 hospitals in 21 countries. We randomly assigned women to receive either 1 g intravenous tranexamic acid or matching placebo in addition to usual care. If bleeding continued after 30 min, or stopped and restarted within 24 h of the first dose, a second dose of 1 g of tranexamic acid or placebo could be given. Patients were assigned by selection of a numbered treatment pack from a box containing eight numbered packs that were identical apart from the pack number. Participants, care givers, and those assessing outcomes were masked to allocation. We originally planned to enrol 15 000 women with a composite primary endpoint of death from all-causes or hysterectomy within 42 days of giving birth. However, during the trial it became apparent that the decision to conduct a hysterectomy was often made at the same time as randomisation. Although tranexamic acid could influence the risk of death in these cases, it could not affect the risk of hysterectomy. We therefore increased the sample size from 15 000 to 20 000 women in order to estimate the effect of tranexamic acid on the risk of death from post-partum haemorrhage. All analyses were done on an intention-to-treat basis. This trial is registered with ISRCTN76912190 (Dec 8, 2008); ClinicalTrials.gov, number NCT00872469; and PACTR201007000192283. Findings Between March, 2010, and April, 2016, 20 060 women were enrolled and randomly assigned to receive tranexamic acid (n=10 051) or placebo (n=10 009), of whom 10 036 and 9985, respectively, were included in the analysis. Death due to bleeding was significantly reduced in women given tranexamic acid (155 [1·5%] of 10 036 patients vs 191 [1·9%] of 9985 in the placebo group, risk ratio [RR] 0·81, 95% CI 0·65–1·00; p=0·045), especially in women given treatment within 3 h of giving birth (89 [1·2%] in the tranexamic acid group vs 127 [1·7%] in the placebo group, RR 0·69, 95% CI 0·52–0·91; p=0·008). All other causes of death did not differ significantly by group. Hysterectomy was not reduced with tranexamic acid (358 [3·6%] patients in the tranexamic acid group vs 351 [3·5%] in the placebo group, RR 1·02, 95% CI 0·88–1·07; p=0·84). The composite primary endpoint of death from all causes or hysterectomy was not reduced with tranexamic acid (534 [5·3%] deaths or hysterectomies in the tranexamic acid group vs 546 [5·5%] in the placebo group, RR 0·97, 95% CI 0·87-1·09; p=0·65). Adverse events (including thromboembolic events) did not differ significantly in the tranexamic acid versus placebo group. Interpretation Tranexamic acid reduces death due to bleeding in women with post-partum haemorrhage with no adverse effects. When used as a treatment for postpartum haemorrhage, tranexamic acid should be given as soon as possible after bleeding onset. Funding London School of Hygiene & Tropical Medicine, Pfizer, UK Department of Health, Wellcome Trust, and Bill & Melinda Gates Foundation

Epithelial tight junction function in a co-culture model of the human bronchial epithelium and endothelium

Epithelial cells line the conducting airways of the bronchial tract, with endothelial-lined capillaries in the submucosa, forming an integrated system and acting as a barrier to the environment.  When the epithelial surface is challenged, the epithelial and endothelial response is locally co-ordinated to allow infiltrating cells to cross the endothelium and target the site of challenge on the luminal surface.  This thesis reports the establishment of a co-culture model reflecting this co-ordinated system, to enable epithelial-endothelial cell interactions to be investigated. 16HBE 14o-, SV40-transformed bronchial epithelial cells were grown on the underside of insert membranes, and HUVEC (human umbilical vein endothelial cells) were grown on the upper side.  Immunofluorescent staining with HEA-125 (human epithelial antigen-125) provided an epithelial-surface marker and vWF (von Willebrand factor) an intracellular endothelial-specific marker.  Using these markers in confocal microscopy, showed that confluent epithelial and endothelial cell layers were growing on opposite sides of the membrane.  This was confirmed by scanning and transmission electron microscopy.  Transepithelial electrical resistance (TER) was used as a functional measurement reflecting tight junction formation.  The final model improves on existing models of the conducting airways, allowing the effects of cell-to-cell interaction on tight junction function to be investigated, better reflecting the in vivo situation in which epithelial and endothelial cells coexist. The bilayer had an increased TER (1547Ω.cm2, S.E. ± 59) in comparison to the epithelial monolayer (792Ω.cm2, S.D. ± 84), and this increase only occurred when 16HBE 14o- cells were grown with endothelial cells.  Furthermore, this same increase was seen when HUVEC conditioned medium was added to the basolateral side of 16HBE 14o- cells, confirming that the increase in TER was occurring in the 16HBE 14o- cells.  These results revealed that functional differences exist between epithelial cells grown as a monolayer and epithelial cells grown as a bilayer with endothelial cells.</p

Epithelial tight junction function in a co-culture model of the human bronchial epithelium and endothelium

Epithelial cells line the conducting airways of the bronchial tract, with endothelial-lined capillaries in the submucosa, forming an integrated system and acting as a barrier to the environment.  When the epithelial surface is challenged, the epithelial and endothelial response is locally co-ordinated to allow infiltrating cells to cross the endothelium and target the site of challenge on the luminal surface.  This thesis reports the establishment of a co-culture model reflecting this co-ordinated system, to enable epithelial-endothelial cell interactions to be investigated. 16HBE 14o-, SV40-transformed bronchial epithelial cells were grown on the underside of insert membranes, and HUVEC (human umbilical vein endothelial cells) were grown on the upper side.  Immunofluorescent staining with HEA-125 (human epithelial antigen-125) provided an epithelial-surface marker and vWF (von Willebrand factor) an intracellular endothelial-specific marker.  Using these markers in confocal microscopy, showed that confluent epithelial and endothelial cell layers were growing on opposite sides of the membrane.  This was confirmed by scanning and transmission electron microscopy.  Transepithelial electrical resistance (TER) was used as a functional measurement reflecting tight junction formation.  The final model improves on existing models of the conducting airways, allowing the effects of cell-to-cell interaction on tight junction function to be investigated, better reflecting the in vivo situation in which epithelial and endothelial cells coexist. The bilayer had an increased TER (1547Ω.cm2, S.E. ± 59) in comparison to the epithelial monolayer (792Ω.cm2, S.D. ± 84), and this increase only occurred when 16HBE 14o- cells were grown with endothelial cells.  Furthermore, this same increase was seen when HUVEC conditioned medium was added to the basolateral side of 16HBE 14o- cells, confirming that the increase in TER was occurring in the 16HBE 14o- cells.  These results revealed that functional differences exist between epithelial cells grown as a monolayer and epithelial cells grown as a bilayer with endothelial cells.</p

From research to regulated: challenges in transferring methods

The current decade has seen an evolution in biomarker research, with a breakthrough from traditional single analyte studies to simultaneous multiple analyte technologies, aided by the progressive development of research tools and the discovery of many novel biomarkers. It is foreseeable that the application of such technologies will have an integral role in clinical studies for establishing biomarker profiles of disease status and prognosis. However, the transfer of such complex procedures to a regulated environment presents many obstacles. Here, we discuss some of these applied technologies and the validation approaches we have taken as an academic unit to prove their suitability and appropriateness for clinical application. We discuss the advantages and limitations for such end point assays in early Phase clinical trial

Validation and comparison of two multiplex technologies, Luminex® and Mesoscale Discovery, for human cytokine profiling

Biomarker research has rapidly expanded over recent years aided by the progressive development of research tools, in particular the different multiplex technologies allowing simultaneous measurement of multiple analytes. It is foreseeable that such technology will have an integral role in clinical studies for establishing biomarker profiles of disease status, but validation of the tools is essential to confirm the reliability of their application. More comparable studies between multiplex platforms are required to enable users to determine which of these are best for a particular clinical study, as different platforms will have varying levels of performance for the validation parameters. Comparison of two multiplex platforms, the Luminex® and the Mesoscale Discovery, has been performed to determine their performance for the validation parameters of sensitivity, precision and accuracy for the cytokines IL-2, IL-4, IL-8, IL-10, IL-12, IFN? and TNF?. When measuring high concentrations both platforms show good accuracy (within +/? 25% recovery) with all cytokines except IL-12 for the MSD. At low concentrations, +/? 25% recovery was seen with all cytokines except IL-2 and IL-8 for the Luminex and IL-2 and IL-12 for the MSD. Although quantitative differences are found, relative differences are comparable, and consequently both platforms have been shown to be suitable for analyzing trends in multiple cytokine profiles, with the Luminex having better precision and the Mesoscale Discovery having greater sensitivity.<br/

Ex vivo assays of dendritic cell activation and cytokine profiles as predictors of in vivo effects in an anti-human CD40 monoclonal antibody ChiLob 7/4 phase I trial

Immunostimulatory antibodies entering the clinic create challenge in terms of not only pharmacodynamics for monitoring anticipated mechanisms but also predetermining cytotoxicity. We show the use of ex vivo whole-blood samples to predict the activation requirements, cytokine signature, and adverse events of an anti-human-CD40 chimeric IgG1 antibody, ChiLob 7/4. Assessments were initially undertaken on human myeloid (mDC1) and plasmacytoid (pDC) dendritic cells, in which an absolute need for cross-linking was shown through the upregulation of activation markers CD83 and CCR7. Subsequent cytokine secretion evaluations of ex vivo whole blood showed the cross-linked antibody-induced increases in MIP1?, interleukin (IL)-8, IL-12, TNF?, and IL-6. This cytokine signature compared favorably with the Toll-like receptor (TLR) ligand lipopolysaccharide (LPS), in which levels of TNF? and IL-6 were significantly higher, suggesting a less intense proinflammatory response and possible modified cytokine release syndrome when used in human trials. Following first-in-human use of this agent within a dose escalation study, in vivo evaluations of dendritic cell activation and secreted cytokines closely matched the predetermined immunomonitoring endpoints. Patients showed a comparable pattern of MIP1?, IL-8, and IL-12 secretion, but no TNF? and IL-6 were identified. Mild symptoms relating to a cytokine release syndrome were seen at an equivalent dosage to that observed for dendritic cell activation and cytokine release. In summary, ChiLob 7/4 induces a distinctive pattern of dendritic cell activation and cytokine secretion in ex vivo assays that can be predictive of in vivo responses. Such preclinical approaches to monoclonal antibody evaluation may inform both the starting dosages and the anticipated cytokine release events that could occur, providing a valuable adjunct for future first-in-human assessments of immunostimulatory antibodies

Fit for purpose? A case study: validation of immunological endpoint assays for the detection of cellular and humoral responses to anti-tumour DNA fusion vaccines.

Clinical trials are governed by an increasingly stringent regulatory framework, which applies to all levels of trial conduct. Study critical immunological endpoints, which define success or failure in early phase clinical immunological trials, require formal pre-trial validation. In this case study, we describe the assay validation process, during which the sensitivity, and precision of immunological endpoint assays were defined. The purpose was the evaluation of two multicentre phase I/II clinical trials from our unit in Southampton, UK, which assess the effects of DNA fusion vaccines on immune responses in HLA-A2+ patients with carcinoembryonic antigen (CEA)-expressing malignancies and prostate cancer. Validated immunomonitoring is being performed using ELISA and IFNgamma ELISPOTs to assess humoral and cellular responses to the vaccines over time. The validated primary endpoint assay, a peptide-specific CD8+ IFNgamma ELISPOT, was tested in a pre-trial study and found to be suitable for the detection of low frequency naturally occurring CEA- and prostate-derived tumour-antigen-specific T cells in patients with CEA-expressing malignancies and prostate cancer