Heterogeneity in Surface Sensing Suggests a Division of Labor in Pseudomonas aeruginosa Populations

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration

The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity

The primary aim of this clinical trial was to determine the feasibility of delivering first-generation CAR T cell therapy to patients with advanced, CEACAM5(+) malignancy. Secondary aims were to assess clinical efficacy, immune effector function and optimal dose of CAR T cells. Three cohorts of patients received increasing doses of CEACAM5(+)-specific CAR T cells after fludarabine pre-conditioning plus systemic IL2 support post T cell infusion. Patients in cohort 4 received increased intensity pre-conditioning (cyclophosphamide and fludarabine), systemic IL2 support and CAR T cells. No objective clinical responses were observed. CAR T cell engraftment in patients within cohort 4 was significantly higher. However, engraftment was short-lived with a rapid decline of systemic CAR T cells within 14 days. Patients in cohort 4 had transient, acute respiratory toxicity which, in combination with lack of prolonged CAR T cell persistence, resulted in the premature closure of the trial. Elevated levels of systemic IFNγ and IL-6 implied that the CEACAM5-specific T cells had undergone immune activation in vivo but only in patients receiving high-intensity pre-conditioning. Expression of CEACAM5 on lung epithelium may have resulted in this transient toxicity. Raised levels of serum cytokines including IL-6 in these patients implicate cytokine release as one of several potential factors exacerbating the observed respiratory toxicity. Whilst improved CAR designs and T cell production methods could improve the systemic persistence and activity, methods to control CAR T 'on-target, off-tissue' toxicity are required to enable a clinical impact of this approach in solid malignancies

Past and current asbestos exposure and future mesothelioma risks in Britain: The Inhaled Particles Study (TIPS)

BACKGROUND: Occupational and environmental airborne asbestos concentrations are too low and variable for lifetime exposures to be estimated reliably, and building workers and occupants may suffer higher exposure when asbestos in older buildings is disturbed or removed. Mesothelioma risks from current asbestos exposures are therefore not known. METHODS: We interviewed and measured asbestos levels in lung samples from 257 patients treated for pneumothorax and 262 with resected lung cancer, recruited in England and Wales. Average lung burdens in British birth cohorts from 1940 to 1992 were estimated for asbestos-exposed workers and the general population. RESULTS: Regression analysis of British mesothelioma death rates and average lung burdens in birth cohorts born before 1965 suggests a lifetime mesothelioma risk of approximately 0.01% per fibre/mg of amphiboles in the lung. In those born since 1965, the average lung burden is ∼1 fibre/mg among those with no occupational exposure. CONCLUSIONS: The average lifetime mesothelioma risk caused by recent environmental asbestos exposure in Britain will be about 1 in 10 000. The risk is an order of magnitude higher in a subgroup of exposed workers and probably in occupants in the most contaminated buildings. Further data are needed to discover whether asbestos still present in buildings, particularly schools, is a persistent or decreasing hazard to workers who disturb it and to the general population, and whether environmental exposure occurs predominantly in childhood or after beginning work. Similar studies are needed in other countries to estimate continuing environmental and occupational mesothelioma hazards worldwide, including the contribution from chrysotile

Expression of the 5T4 oncofoetal antigen in renal cell carcinoma: a potential target for T-cell-based immunotherapy

The 5T4 oncofoetal antigen is a heavily glycosylated cell surface protein found on human placental trophoblast and on diverse types of human cancer but is not expressed at significant levels on adult human tissues in health. It therefore satisfies the criteria for a tumour-associated antigen and is an ideal target for the immunotherapy of cancer. We report here that 5T4 is strongly expressed on the majority of renal cell carcinomas and therefore this population of patients is suitable for trials of 5T4-targeted therapies. In particular, we have shown that T cells from renal cell carcinoma patients can be genetically modified to kill 5T4 expressing renal cancer cell lines by introduction of a chimeric-signalling protein. This protein consists of a single chain antibody fragment capable of binding antigen directly at the cell surface and then activating the T cell by virtue of a CD3ζ-signalling domain. This is a powerful tool that bypasses a number of mechanisms that allow tumours to escape T-cell killing and can be readily scaled up for clinical use

Engineering T cells for cancer therapy

It is generally accepted that the immune system plays an important role in controlling tumour development. However, the interplay between tumour and immune system is complex, as demonstrated by the fact that tumours can successfully establish and develop despite the presence of T cells in tumour. An improved understanding of how tumours evade T-cell surveillance, coupled with technical developments allowing the culture and manipulation of T cells, has driven the exploration of therapeutic strategies based on the adoptive transfer of tumour-specific T cells. The isolation, expansion and re-infusion of large numbers of tumour-specific T cells generated from tumour biopsies has been shown to be feasible. Indeed, impressive clinical responses have been documented in melanoma patients treated with these T cells. These studies and others demonstrate the potential of T cells for the adoptive therapy of cancer. However, the significant technical issues relating to the production of natural tumour-specific T cells suggest that the application of this approach is likely to be limited at the moment. With the advent of retroviral gene transfer technology, it has become possible to efficiently endow T cells with antigen-specific receptors. Using this strategy, it is potentially possible to generate large numbers of tumour reactive T cells rapidly. This review summarises the current gene therapy approaches in relation to the development of adoptive T-cell-based cancer treatments, as these methods now head towards testing in the clinical trial setting

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Overcoming Target Driven Fratricide for T Cell Therapy

Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells