Thomas H. Craven

Endoscopic sensing of alveolar pH

Previously unobtainable measurements of alveolar pH were obtained using an endoscope-deployable optrode. The pH sensing was achieved using functionalized gold nanoshell sensors and surface enhanced Raman spectroscopy (SERS). The optrode consisted of an asymmetric dual-core optical fiber designed for spatially separating the optical pump delivery and signal collection, in order to circumvent the unwanted Raman signal generated within the fiber. Using this approach, we demonstrate a ~100-fold increase in SERS signal-to-fiber background ratio, and demonstrate multiple site pH sensing with a measurement accuracy of ± 0.07 pH units in the respiratory acini of an ex vivo ovine lung model. We also demonstrate that alveolar pH changes in response to ventilation.

The cyclin-dependent kinase inhibitor AT7519 accelerates neutrophil apoptosis in sepsis-related acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disorder with no effective pharmacological therapies. While the cyclin-dependent kinase inhibitor AT7519 induces neutrophil apoptosis to promote inflammation resolution in preclinical models of lung inflammation, its potential efficacy in ARDS has not been examined. Untreated peripheral blood sepsis-related ARDS neutrophils demonstrated prolonged survival after 20 hours in vitro culture. AT7519 was able to override this phenotype to induce apoptosis in ARDS neutrophils with reduced expression of the pro-survival protein Mcl-1. We demonstrate the first pharmacological compound to induce neutrophil apoptosis in sepsis-related ARDS, highlighting cyclin-dependent kinase inhibitors as potential novel therapeutic agents.

Substantial Metabolic Activity of Human Brown Adipose Tissue during Warm Conditions and Cold-Induced Lipolysis of Local Triglycerides

Summary Current understanding of in vivo human brown adipose tissue (BAT) physiology is limited by a reliance on positron emission tomography (PET)/computed tomography (CT) scanning, which has measured exogenous glucose and fatty acid uptake but not quantified endogenous substrate utilization by BAT. Six lean, healthy men underwent 18fluorodeoxyglucose-PET/CT scanning to localize BAT so microdialysis catheters could be inserted in supraclavicular BAT under CT guidance and in abdominal subcutaneous white adipose tissue (WAT). Arterial and dialysate samples were collected during warm (∼25°C) and cold exposure (∼17°C), and blood flow was measured by 133xenon washout. During warm conditions, there was increased glucose uptake and lactate release and decreased glycerol release by BAT compared with WAT. Cold exposure increased blood flow, glycerol release, and glucose and glutamate uptake only by BAT. This novel use of microdialysis reveals that human BAT is metabolically active during warm conditions. BAT activation substantially increases local lipolysis but also utilization of other substrates such as glutamate.

Structural modifications of the antimicrobial peptide ubiquicidin for pulmonary imaging of bacteria in the alveolar space

Abstract Background The direct visualisation of bacteria in the distal lung would increase the spatiotemporal understanding of pulmonary infection and be a powerful tool to stratify patients with suspected pneumonia. In critically ill patients, the diagnostic dilemma of pulmonary opacities leads to overprescribing of antimicrobial agents while waiting for culture results from bronchoalveolar lavage. Ubiquicidin (UBI) is an innate cytosolic antimicrobial peptide with a twelve aminoacid portion (UBI 29–41 ) that specifically binds bacteria. We aimed to modify the chemical structure of UBI 29–41 so that in-situ bacterial imaging with optical endomicroscopy (OEM) could be achieved. Methods UBI 29-41 compounds were labelled with the environmentally sensitive fluorophore NBD (NBD-UBI), with incorporation of synthetic aminoacids (NBD-UBI nma ) and alteration of the secondary structure of the native peptide on a dendrimeric scaffold (NBD-UBI dend ). These compounds were assessed in vitro and delivered endobronchially in an ex-vivo sheep lung model and then OEM applied to allow alveolar imaging. The NBD-UBI dend –OEM platform was also evaluated in explanted whole cystic fibrosis lungs. Findings NBD-UBI selectively labelled bacteria over mammalian cells but remained susceptible to proteolytic degradation and poor affinity. NBD-UBI nma improved stability but not affinity. NBD-UBI dend remained structurally stable and exhibited high affinity for bacteria in vitro. It retained bacterial selectivity over mononuclear cells (p=0·0015), neutrophils (p=0·0034), bronchoalveolar lavage macrophages (p=0·0169), and labelled Escherichia coli (p=0·0035), Klebsiella pneumoniae (p=0·0003), Pseudomonas aeruginosa (p=0·0009), and meticillin-sensitive Staphylococcus aureus (p 5 colony-forming units per mL on lavage. NBD-UBI dend also detected bacteria in situ in ex-vivo explanted human cystic fibrosis lungs (p=0·0027 compared with peptide and fluorophore control segments). Interpretation We describe an OEM strategy that can immediately detect bacteria in size-relevant preclinical models, with crucial requirements for pulmonary molecular imaging of peptide stability and affinity. This method has the potential to stratify pulmonary opacities in the intensive care unit when pneumonia is suspected and offers the substantial advantage of real-time detection, therefore allowing immediate decision making about antimicrobial treatment. This imaging strategy is now undergoing first-in-man translation. Funding Wellcome Trust, Department of Health, Engineering and Physical Sciences Research Council.

Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils

Serine proteases are released by neutrophils to act primarily as antimicrobial proteins but excessive and unbalanced serine protease activity results in serious host tissue damage. Here the synthesis of a novel chemical sensor based on a multi-branched fluorescence quencher is reported. It is super-silent, exhibiting no fluorescence until de-quenched by the exemplar serine protease human neutrophil elastase, rapidly enters human neutrophils, and is inhibited by serine protease inhibitors. This sensor allows live imaging of intracellular serine protease activity within human neutrophils and demonstrates that the unique combination of a multivalent scaffold combined with a FRET peptide represents a novel and efficient strategy to generate super-silent sensors that permit the visualisation of intracellular proteases and may enable point of care whole blood profiling of neutrophils.

Ballistic and snake photon imaging for locating optical endomicroscopy fibres

We demonstrate determination of the location of the distal-end of a fibre-optic device deep in tissue through the imaging of ballistic and snake photons using a time resolved single-photon detector array. The fibre was imaged with centimetre resolution, within clinically relevant settings and models. This technique can overcome the limitations imposed by tissue scattering in optically determining the in vivo location of fibre-optic medical instruments.

In-situ imaging of neutrophil activation in the human alveolar space with neutrophil activation probe and pulmonary optical endomicroscopy

Abstract Background Acute respiratory distress syndrome is a severe and heterogeneous condition. Clinical diagnostic criteria lack specificity and fail to differentiate the various inflammatory phenotypes. Diagnosis and stratification can be improved by profiling the neutrophil and one of its enzymes, neutrophil elastase (NE), within the alveolar space. We aimed to combine fibre-based optical endomicroscopy (OEM) with a bespoke imaging probe to deliver a new strategy to elucidate pathobiological processes in the lung. Methods We designed and synthesised an optical imaging probe termed neutrophil activation probe to fluoresce as it enters the alkaline neutrophilic vacuole and through NE-specific cleavage. Validation was performed with spectrophotometry, confocal microscopy, and flow cytometry. The probe was tested in a perfused large sheep lung model in conjunction with bronchoscopic OEM. Neutrophil activation probe was synthesised to GMP, and a phase 1 study is underway in healthy volunteers and patients in the intensive care unit. The phase 1 study is registered with EudraCT, number 2011-066167-17, and with ClinicalTrials.gov, number NCT01532024. Findings Neutrophil activation probe was neutrophil specific, specific to NE among other serine proteinases, and demonstrated dequenching in alkaline pH. Intracellular fluorescence signal determined by flow cytometry increased in chemically activated neutrophils (mean fluorescence index signal increased from 24 144 (SE 6175) to 1·218 × 10 6 (59 325), p Interpretation We have demonstrated the potential utility of this bedside molecular imaging strategy, from bench to patient: the probe functions in a predictable manner, in keeping with its design. A two-site international phase 2 clinical study is planned, which will assess the test characteristics of this novel technique and its ability to predict and stratify clinically important outcomes in a heterogeneous ventilated population in the intensive care unit. Funding Medical Research Council.

In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A

A topically administered fluorescently labeled peptide targeting lipid A permits rapid, real-time visualization of bacteria in the distal human lung. Lung infection in real time Lung infections are frequent causes of complications in mechanically ventilated and immunosuppressed patients. However, the diagnosis is challenging, requires risky procedures, and is time consuming. Now, Akram et al. have developed an imaging method that is able to detect Gram-negative bacteria in real time in the distal part of the human lung. Using a fluorescent probe binding to lipid A, a molecule expressed on Gram-negative bacterial membranes, in combination with an optical endomicroscope, the researchers rapidly detected Gram-negative infections in distal airways in hospitalized individuals. The results suggest that the approach could accelerate the diagnosis of bacterial lung infection and facilitate the evaluation of antibiotic treatment efficacy. Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.

Early arriving photon imaging for locating optical endomicroscopy fibres and medical devices (Conference Presentation)

Optical fibre based endoscopes are increasingly used for imaging and sensing within the human body without navigational guidance of the miniaturised fibre probe. Meanwhile, other medical device placement is a standard procedure in clinic. We demonstrate successful imaging of optical device location with centimetre resolution in clinically relevant models, in a realistically lit environment, achieved through the detection of early arriving photons with a time resolved single photon detector array. This prototype has been developed within the UK-EPSRC Proteus project, moving advanced research technologies towards clinical implementation. Short (~100ps) laser pulses are transmitted from the tip of the endoscope at 785nm in the “optical window” where attenuation is less severe in clinical scenarios. Most of the photons that pass through tissue undergo much scattering from the disordered tissue structures providing only low accuracy determination of the location of the light source. However, some photons probabilistically undergo less scattering, travelling through the medium in an almost straight line without a much extended path. Such photons exit the body sooner than the highly scattered light. A camera based upon a 32 × 32 array of Single Photon Avalanche Diodes (SPADs) made with CMOS technology is used to image the small number photons exiting the tissue. The time resolution capabilities of such a single photon detector (50ps time bin resolution, 200ps jitter) allow observation of the photon arrival times simultaneously for all 1024 pixels of the imaging array. Photon arrival statistics distinguish the early arriving photons from the highly scattered light, revealing the endoscope location. Scattered photon arrivals peak at delays of multiple nanoseconds due to the thick tissue samples. The progression of light through complex scattering structures can be observed. Normal fluorescent room lighting has distinct emission peaks. Appropriate choice of operating wavelength between these spectral features, combined with aggressive filtering, allows operation in normal fluorescent lighting. This compact packaged system is demonstrated in a normally lit room to determine optical endomicroscope location in a whole ventilated ovine lung as well as tissue models including bone structure. At the limit of capabilities of this prototype, demonstration through an entire human torso is shown to be possible. System improvements and the potential of the next generation prototype in development will be discussed. This offers the potential for real time (sub second) imaging of device location with a portable system for application in standard medical procedures, such as catheter insertion. The avoidance of the need to confirm device placement with X-ray imaging has potential to decrease disruption to procedures throughout clinical practice.

Endoscopic sensing of pH in the distal lung (Conference Presentation)

In healthy humans, the physiological state in the distal lung alveolar acinar units is tightly regulated by normal homeostatic mechanisms. Pulmonary abnormalities such as chronic obstructive pulmonary disease, that are characterized by recurrent cycles of inflammation and infection involving dense infiltration by myeloid derived peripheral blood cells, may result in significant perturbation of the homeostatic baselines of physiology in addition to host tissue damage. Therefore, the ability to quantify and monitor physiology (e.g. pH, glucose level, oxygen tension) within the alveolar acinar units would provide a key biomarker of distal lung innate defence. Although in vitro modeling of fundamental biological processes show remarkable sensitivity to physiological aberrations, little is known about the physiological state of the distal lung due to the inability to concurrently access the alveolar sacs and perform real-time sensing. Here we report on previously unobtainable measurements of alveolar pH using a fiber-optic optrode and surface enhanced Raman spectroscopy (SERS) and show that alveolar pH changes in response to ventilation. The endoscope-deployable optrode consisted of para-mercaptobenzoic acid functionalized 150 nm gold nanoshells located at the distal end, and an asymmetric dual-core optical fiber designed for spatially separated optical pump delivery and SERS signal collection in order to circumvent the unwanted Raman signal originating from the fiber itself. We demonstrate a ~ 100-fold increase in SERS signal-to-fiber background ratio and pH sensing at multiple sites in the respiratory acinar units of a whole ex vivo ovine lung model with a measurement accuracy of ± 0.07 pH units.