Time-Resolved Spectroscopy of Fluorescence Quenching in Optical Fibre-Based pH Sensors.

Numerous optodes, with fluorophores as the chemical sensing element and optical fibres for light delivery and collection, have been fabricated for minimally invasive endoscopic measurements of key physiological parameters such as pH. These flexible miniaturised optodes have typically attempted to maximize signal-to-noise through the application of high concentrations of fluorophores. We show that high-density attachment of carboxyfluorescein onto silica microspheres, the sensing elements, results in fluorescence energy transfer, manifesting as reduced fluorescence intensity and lifetime in addition to spectral changes. We demonstrate that the change in fluorescence intensity of carboxyfluorescein with pH in this "high-density" regime is opposite to that normally observed, with complex variations in fluorescent lifetime across the emission spectra of coupled fluorophores. Improved understanding of such highly loaded sensor beads is important because it leads to large increases in photostability and will aid the development of compact fibre probes, suitable for clinical applications. The time-resolved spectral measurement techniques presented here can be further applied to similar studies of other optodes.

Rapid Polymer Conjugation Strategies for the Generation of pH-Responsive, Cancer Targeting, Polymeric Nanoparticles.

The combination of controlled living polymerization in association with rapid and highly efficient macromolecule conjugation strategies provides a powerful tool for the synthesis of novel polymeric materials. Here functional block copolymers were rapidly and quantitatively conjugated using an efficient reaction between polymers containing a phenolic group and the 4-phenyl-3 H-1,2,4-triazole-3,5(4 H)-dione (PTAD) moiety and used to generate nanoparticles that encapsulated drugs. pH responsive amphiphilic block copolymers, which self-assemble into nanoparticles, were fabricated using our novel polymer conjugation strategy with the resulting system designed to promote drug release within the acidic milieu of the cancer microenvironment. The conjugation strategy also enabled the direct tagging of the nanoparticles with a range of fluorophores, targeting assets, or both with cargo release demonstrated in cancer cells.

C5a-mediated neutrophil dysfunction is RhoA-dependent and predicts infection in critically ill patients.

Critically ill patients are at heightened risk for nosocomial infections. The anaphylatoxin C5a impairs phagocytosis by neutrophils. However, the mechanisms by which this occurs and the relevance for acquisition of nosocomial infection remain undetermined. We aimed to characterize mechanisms by which C5a inhibits phagocytosis in vitro and in critically ill patients, and to define the relationship between C5a-mediated dysfunction and acquisition of nosocomial infection. In healthy human neutrophils, C5a significantly inhibited RhoA activation, preventing actin polymerization and phagocytosis. RhoA inhibition was mediated by PI3Kδ. The effects on RhoA, actin, and phagocytosis were fully reversed by GM-CSF. Parallel observations were made in neutrophils from critically ill patients, that is, impaired phagocytosis was associated with inhibition of RhoA and actin polymerization, and reversed by GM-CSF. Among a cohort of 60 critically ill patients, C5a-mediated neutrophil dysfunction (as determined by reduced CD88 expression) was a strong predictor for subsequent acquisition of nosocomial infection (relative risk, 5.8; 95% confidence interval, 1.5-22; P = .0007), and remained independent of time effects as assessed by survival analysis (hazard ratio, 5.0; 95% confidence interval, 1.3-8.3; P = .01). In conclusion, this study provides new insight into the mechanisms underlying immunocompromise in critical illness and suggests novel avenues for therapy and prevention of nosocomial infection.

A novel subpopulation of monocyte-like cells in the human lung after lipopolysaccharide inhalation.

The co-ordinated recruitment of monocyte subpopulations, neutrophils and regulatory T-cells (Tregs) during the early stages of human acute lung inflammation remains poorly understood. We therefore performed a detailed characterisation of these lineages in the blood and lungs in a model of human acute lung inflammation. Healthy volunteers inhaled lipopolysaccharide (LPS) or saline (n=6 for each group). Blood was collected at 0, 2, 4, 6 and 8 h and bronchoscopy with bronchoalveolar lavage (BAL) performed at 8 h. Multiparameter flow cytometry was used to characterise monocyte subpopulations, neutrophils and Tregs in the blood and lung. Inhalation of LPS was associated with significant blood and BAL fluid neutrophilia. Blood populations of monocyte subpopulations and Tregs were unaltered by LPS. In contrast, LPS induced an accumulation of a pulmonary monocyte-like cell (PMLC) population, which was further subdivided into "inducible" CD14(++)CD16(-) and "resident" CD14(++)CD16(+) subsets. Inducible PMLCs were significantly increased following LPS inhalation (p=0.0046), whereas resident PMLCs were unchanged. In addition, we noted a significant decrease in Tregs in BAL fluid with LPS inhalation (p=0.027). The early stages of LPS-induced inflammation in humans is characterised by pulmonary accumulation of a novel inducible monocyte-like subpopulation, accompanied by significant changes in both neutrophil and Treg numbers.

Full spectrum fluorescence lifetime imaging with 0.5 nm spectral and 50 ps temporal resolution.

The use of optical techniques to interrogate wide ranging samples from semiconductors to biological tissue for rapid analysis and diagnostics has gained wide adoption over the past decades. The desire to collect ever more spatially, spectrally and temporally detailed optical signatures for sample characterization has specifically driven a sharp rise in new optical microscopy technologies. Here we present a high-speed optical scanning microscope capable of capturing time resolved images across 512 spectral and 32 time channels in a single acquisition with the potential for ~0.2 frames per second (256 × 256 image pixels). Each pixel in the resulting images contains a detailed data cube for the study of diverse time resolved light driven phenomena. This is enabled by integration of system control electronics and on-chip processing which overcomes the challenges presented by high data volume and low imaging speed, often bottlenecks in previous systems.

Developing Novel Host-Based Therapies Targeting Microbicidal Responses in Macrophages and Neutrophils to Combat Bacterial Antimicrobial Resistance.

Antimicrobial therapy has provided the main component of chemotherapy against bacterial pathogens. The effectiveness of this strategy has, however, been increasingly challenged by the emergence of antimicrobial resistance which now threatens the sustained utility of this approach. Humans and animals are constantly exposed to bacteria and have developed effective strategies to control pathogens involving innate and adaptive immune responses. Impaired pathogen handling by the innate immune system is a key determinant of susceptibility to bacterial infection. However, the essential components of this response, specifically those which are amenable to re-calibration to improve host defense, remain elusive despite extensive research. We provide a mini-review focusing on therapeutic targeting of microbicidal responses in macrophages and neutrophils to de-stress reliance on antimicrobial therapy. We highlight pre-clinical and clinical data pointing toward potential targets and therapies. We suggest that developing focused host-directed therapeutic strategies to enhance "pauci-inflammatory" microbial killing in myeloid phagocytes that maximizes pathogen clearance while minimizing the harmful consequences of the inflammatory response merits particular attention. We also suggest the importance of One Health approaches in developing host-based approaches through model development and comparative medicine in informing our understanding of how to deliver this strategy.

Functional characterisation of human pulmonary monocyte-like cells in lipopolysaccharide-mediated acute lung inflammation.

BACKGROUND: We have previously reported the presence of novel subpopulations of pulmonary monocyte-like cells (PMLC) in the human lung; resident PMLC (rPMLC, HLA-DR(+)CD14(++)CD16(+)cells) and inducible PMLC (iPMLC, HLA-DR(+)CD14(++)CD16(-) cells). iPMLC are significantly increased in bronchoalveolar lavage (BAL) fluid following inhalation of lipopolysaccharide (LPS). We have carried out the first functional evaluation of PMLC subpopulations in the inflamed lung, following the isolation of these cells, and other lineages, from BAL fluid using novel and complex protocols. METHODS: iPMLC, rPMLC, alveolar macrophages (AM), neutrophils, and regulatory T cells were quantified in BAL fluid of healthy subjects at 9 hours post-LPS inhalation (n = 15). Cell surface antigen expression by iPMLC, rPMLC and AM and the ability of each lineage to proliferate and to undergo phagocytosis were investigated using flow cytometry. Basal cytokine production by iPMLC compared to AM following their isolation from BAL fluid and the responsiveness of both cell types following in vitro treatment with the synthetic corticosteroid dexamethasone were assessed. RESULTS: rPMLC have a significantly increased expression of mature macrophage markers and of the proliferation antigen Ki67, compared to iPMLC. Our cytokine data revealed a pro-inflammatory, corticosteroid-resistant phenotype of iPMLC in this model. CONCLUSIONS: These data emphasise the presence of functionally distinct subpopulations of the monocyte/macrophage lineage in the human lung in experimental acute lung inflammation.

The use of a portable digital thoracic suction Thopaz drainage system for the management of a persistent spontaneous secondary pneumothorax in a patient with underlying interstitial lung disease.

We present the case of a 68-year-old woman who presented in extremis with a secondary pneumothorax with a past history of severe idiopathic pulmonary fibrosis. Following insertion of a 32F intercostal drain, she developed a persistent broncho-pleural fistula and became dependent on negative-pressure wall-mounted suction to prevent respiratory compromise. She declined definitive surgical intervention and was therefore managed conservatively. After adhering to the wall-mounted suction method for 49 days, we obtained for use a portable digital thoracic drainage system previously used only in the cardiothoracic postoperative patient. This electronically delivered, negative-pressure drainage system induced radiographic improvement within 24 h, and allowed the patient to mobilise for the first time since admission. The patient was discharged home with the Thopaz drain in situ 8 weeks after placing it, and the drain was removed successfully with a resolved pneumothorax 20 weeks after her initial presentation.

In vivo mononuclear cell tracking using superparamagnetic particles of iron oxide: feasibility and safety in humans.

BACKGROUND: Cell therapy is an emerging and exciting novel treatment option for cardiovascular disease that relies on the delivery of functional cells to their target site. Monitoring and tracking cells to ensure tissue delivery and engraftment is a critical step in establishing clinical and therapeutic efficacy. The study aims were (1) to develop a Good Manufacturing Practice-compliant method of labeling competent peripheral blood mononuclear cells with superparamagnetic particles of iron oxide (SPIO), and (2) to evaluate its potential for magnetic resonance cell tracking in humans. METHODS AND RESULTS: Peripheral blood mononuclear cells 1-5 × 10(9) were labeled with SPIO. SPIO-labeled cells had similar in vitro viability, migratory capacity, and pattern of cytokine release to unlabeled cells. After intramuscular administration, up to 10(8) SPIO-labeled cells were readily identifiable in vivo for at least 7 days using magnetic resonance imaging scanning. Using a phased-dosing study, we demonstrated that systemic delivery of up to 10(9) SPIO-labeled cells in humans is safe, and cells accumulating in the reticuloendothelial system were detectable on clinical magnetic resonance imaging. In a healthy volunteer model, a focus of cutaneous inflammation was induced in the thigh by intradermal injection of tuberculin. Intravenously delivered SPIO-labeled cells tracked to the inflamed skin and were detectable on magnetic resonance imaging. Prussian blue staining of skin biopsies confirmed iron-laden cells in the inflamed skin. CONCLUSIONS: Human peripheral blood mononuclear cells can be labeled with SPIO without affecting their viability or function. SPIO labeling for magnetic resonance cell tracking is a safe and feasible technique that has major potential for a range of cardiovascular applications including monitoring of cell therapies and tracking of inflammatory cells. Clinical Trial Registration- URL: http://www.clinicaltrials.gov; Unique identifier: NCT00972946, NCT01169935.