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1.
Oncologist ; 29(6): 493-503, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38330461

RESUMO

BACKGROUND: One of the most common sporadic homozygous deletions in cancers is 9p21 loss, which includes the genes methylthioadenosine phosphorylase (MTAP), CDKN2A, and CDKN2B, and has been correlated with worsened outcomes and immunotherapy resistance. MTAP-loss is a developing drug target through synthetic lethality with MAT2A and PMRT5 inhibitors. The purpose of this study is to investigate the prevalence and genomic landscape of MTAP-loss in advanced gastrointestinal (GI) tumors and investigate its role as a prognostic biomarker. MATERIALS AND METHODS: We performed next-generation sequencing and comparative genomic and clinical analysis on an extensive cohort of 64 860 tumors comprising 5 GI cancers. We compared the clinical outcomes of patients with GI cancer harboring MTAP-loss and MTAP-intact tumors in a retrospective study. RESULTS: The prevalence of MTAP-loss in GI cancers is 8.30%. MTAP-loss was most prevalent in pancreatic ductal adenocarcinoma (PDAC) at 21.7% and least in colorectal carcinoma (CRC) at 1.1%. MTAP-loss tumors were more prevalent in East Asian patients with PDAC (4.4% vs 3.2%, P = .005) or intrahepatic cholangiocarcinoma (IHCC; 6.4% vs 4.3%, P = .036). Significant differences in the prevalence of potentially targetable genomic alterations (ATM, BRAF, BRCA2, ERBB2, IDH1, PIK3CA, and PTEN) were observed in MTAP-loss tumors and varied according to tumor type. MTAP-loss PDAC, IHCC, and CRC had a lower prevalence of microsatellite instability or elevated tumor mutational burden. Positive PD-L1 tumor cell expression was less frequent among MTAP-loss versus MTAP-intact IHCC tumors (23.2% vs 31.2%, P = .017). CONCLUSION: In GI cancers, MTAP-loss occurs as part of 9p21 loss and has an overall prevalence of 8%. MTAP-loss occurs in 22% of PDAC, 15% of IHCC, 8.7% of gastroesophageal adenocarcinoma, 2.4% of hepatocellular carcinoma, and 1.1% of CRC and is not mutually exclusive with other targetable mutations.


Assuntos
Neoplasias Gastrointestinais , Purina-Núcleosídeo Fosforilase , Humanos , Purina-Núcleosídeo Fosforilase/genética , Masculino , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Pessoa de Meia-Idade , Idoso , Estudos Retrospectivos , Biomarcadores Tumorais/genética , Adulto , Prognóstico , Genômica/métodos
2.
Am J Respir Crit Care Med ; 207(2): 138-149, 2023 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-35972987

RESUMO

Rationale: High circulating galectin-3 is associated with poor outcomes in patients with coronavirus disease (COVID-19). We hypothesized that GB0139, a potent inhaled thiodigalactoside galectin-3 inhibitor with antiinflammatory and antifibrotic actions, would be safely and effectively delivered in COVID-19 pneumonitis. Objectives: Primary outcomes were safety and tolerability of inhaled GB0139 as an add-on therapy for patients hospitalized with COVID-19 pneumonitis. Methods: We present the findings of two arms of a phase Ib/IIa randomized controlled platform trial in hospitalized patients with confirmed COVID-19 pneumonitis. Patients received standard of care (SoC) or SoC plus 10 mg inhaled GB0139 twice daily for 48 hours, then once daily for up to 14 days or discharge. Measurements and Main Results: Data are reported from 41 patients, 20 of which were assigned randomly to receive GB0139. Primary outcomes: the GB0139 group experienced no treatment-related serious adverse events. Incidences of adverse events were similar between treatment arms (40 with GB0139 + SoC vs. 35 with SoC). Secondary outcomes: plasma GB0139 was measurable in all patients after inhaled exposure and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc analysis of covariance [ANCOVA] over days 2-7; P = 0.0099 vs. SoC). Plasma biomarkers associated with inflammation, fibrosis, coagulopathy, and major organ function were evaluated. Conclusions: In COVID-19 pneumonitis, inhaled GB0139 was well-tolerated and achieved clinically relevant plasma concentrations with target engagement. The data support larger clinical trials to determine clinical efficacy. Clinical trial registered with ClinicalTrials.gov (NCT04473053) and EudraCT (2020-002230-32).


Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Galectina 3 , Inflamação , Resultado do Tratamento
3.
Int Arch Occup Environ Health ; 95(3): 645-664, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34628523

RESUMO

OBJECTIVE: Workplace mental health is relevant to public safety organizations due to the exposure that many public safety personnel (PSP) have to psychological trauma in the course of their daily work. While the importance of attending to PSP mental health has been established, the implementation of workplace mental health interventions is not as well understood. This scoping review describes workplace mental health interventions and their implementation in public safety organizations. METHODS: English published primary studies with any publication date up to July 3, 2020 were considered. JBI methodology and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews was followed. RESULTS: 89 citations met inclusion criteria out of the 62,299 found. Articles and reports found were largely published within the last decade, most frequently from Western nations, and most often applied to police, followed by firefighters. The focus of interventions was commonly stress management and resilience, and a frequent implementation strategy was multi-session group training. Comprehensive quality improvement initiatives, a focus on supervisors and managers, and interventions across primary, secondary, and tertiary prevention, were infrequent. CONCLUSION: Public safety organizations are frequently reporting on stress management and resilience interventions for police and firefighters, implemented through multi-session group training. A focus across a range of PSP, including paramedics, corrections officers, and emergency dispatchers, using implementation strategies beyond group training, is suggested. This area of research is currently expanding, with many studies published within the past decade; ongoing evaluation of the quality of interventions and implementation strategies is recommended.


Assuntos
Bombeiros , Saúde Mental , Pessoal Técnico de Saúde , Humanos , Psicoterapia , Local de Trabalho
4.
Eur J Nucl Med Mol Imaging ; 48(3): 800-807, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32915268

RESUMO

PURPOSE: The relentless rise in antimicrobial resistance is a major societal challenge and requires, as part of its solution, a better understanding of bacterial colonization and infection. To facilitate this, we developed a highly efficient no-wash red optical molecular imaging agent that enables the rapid, selective, and specific visualization of Gram-positive bacteria through a bespoke optical fiber-based delivery/imaging endoscopic device. METHODS: We rationally designed a no-wash, red, Gram-positive-specific molecular imaging agent (Merocy-Van) based on vancomycin and an environmental merocyanine dye. We demonstrated the specificity and utility of the imaging agent in escalating in vitro and ex vivo whole human lung models (n = 3), utilizing a bespoke fiber-based delivery and imaging device, coupled to a wide-field, two-color endomicroscopy system. RESULTS: The imaging agent (Merocy-Van) was specific to Gram-positive bacteria and enabled no-wash imaging of S. aureus within the alveolar space of whole ex vivo human lungs within 60 s of delivery into the field-of-view, using the novel imaging/delivery endomicroscopy device. CONCLUSION: This platform enables the rapid and specific detection of Gram-positive bacteria in the human lung.


Assuntos
Fibras Ópticas , Staphylococcus aureus , Endoscópios , Bactérias Gram-Positivas , Humanos , Pulmão/diagnóstico por imagem
5.
Crit Care Med ; 46(9): e937-e944, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29957711

RESUMO

OBJECTIVES: Mild traumatic brain injury in the form of concussion is extremely common, and the potential effects on pulmonary priming have been underestimated. The aim of this study was to characterize the pulmonary response following mild traumatic brain injury and assess the pulmonary susceptibility to lung injury after a subsequent innocuous pulmonary insult. DESIGN: Experimental in vivo study. SETTING: University research laboratory. SUBJECTS: Male CD1 mice. INTERVENTIONS: We developed a model of concussive traumatic brain injury in mice followed by pulmonary acid microaspiration. To assess the dependent role of neutrophils in mediating pulmonary injury, we specifically depleted neutrophils. MEASUREMENTS AND MAIN RESULTS: Lateral fluid percussion to the brain resulted in neuronal damage and neutrophil infiltration as well as extensive pulmonary interstitial neutrophil accumulation but no alveolar injury. Following subsequent innocuous acid microaspiration, augmented alveolar neutrophil influx led to the development of pulmonary hemorrhage that was reduced following neutrophil depletion. CONCLUSIONS: This model shows for the first time that innocuous acid microaspiration is sufficient to induce neutrophil-mediated lung injury following mild concussion and that the extracranial effects of mild traumatic brain injury have been underestimated.


Assuntos
Concussão Encefálica/complicações , Lesão Pulmonar/etiologia , Infiltração de Neutrófilos , Animais , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos
6.
Thorax ; 72(10): 928-936, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28469031

RESUMO

BACKGROUND: Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. METHODS: Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography-tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. RESULTS: Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1-/- mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. CONCLUSIONS: We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS.


Assuntos
Receptores de Formil Peptídeo/imunologia , Síndrome do Desconforto Respiratório/imunologia , Animais , Líquido da Lavagem Broncoalveolar/química , Quimiotaxia de Leucócito/imunologia , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Camundongos , Mitocôndrias/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Espectrometria de Massas em Tandem
7.
Am J Respir Crit Care Med ; 186(6): 514-24, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22822022

RESUMO

RATIONALE: Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood. OBJECTIVES: To determine the influence of peripheral blood monocytes (PBMs) in established ALI. METHODS: In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2(hi) monocytes. MEASUREMENTS AND MAIN RESULTS: PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1(hi) and Gr1(lo) PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes. CONCLUSIONS: These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI.


Assuntos
Lesão Pulmonar Aguda/patologia , Movimento Celular/imunologia , Macrófagos/citologia , Monócitos/citologia , Neutrófilos/citologia , Lesão Pulmonar Aguda/fisiopatologia , Análise de Variância , Animais , Líquido da Lavagem Broncoalveolar/citologia , Movimento Celular/fisiologia , Ácido Clodrônico/farmacologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/imunologia , Neutrófilos/fisiologia , Distribuição Aleatória , Estatísticas não Paramétricas
9.
Artigo em Inglês | MEDLINE | ID: mdl-36360872

RESUMO

Public safety personnel (PSP), including correctional officers, firefighters, paramedics, and police officers, have higher rates of mental health conditions than other types of workers. This scoping review maps the impact of organizational factors on PSP mental health, reviewing applicable English language primary studies from 2000-2021. JBI methodology for scoping reviews was followed. After screening, 97 primary studies remained for analysis. Police officers (n = 48) were the most frequent population studied. Correctional officers (n = 27) and paramedics (n = 27) were the second most frequently identified population, followed by career firefighters (n = 20). Lack of supervisor support was the most frequently cited negative organizational factor (n = 23), followed by negative workplace culture (n = 21), and lack of co-worker support (n = 14). Co-worker support (n = 10) was the most frequently identified positive organizational factor, followed by supervisor support (n = 8) and positive workplace culture (n = 5). This scoping review is the first to map organizational factors and their impact on PSP mental health across public safety organizations. The results of this review can inform discussions related to organizational factors, and their relationship to operational and personal factors, to assist in considering which factors are the most impactful on mental health, and which are most amenable to change.


Assuntos
Bombeiros , Transtornos Mentais , Humanos , Saúde Mental , Polícia , Local de Trabalho
10.
JBI Evid Synth ; 20(1): 229-237, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34137740

RESUMO

OBJECTIVE: This scoping review aims to map workplace mental health implementation strategies in public safety organizations and describe the characteristics, participants, and contexts of these strategies. INTRODUCTION: Workplace mental health implementation strategies are relevant to public safety organizations due to the exposure that many public safety personnel, such as firefighters, paramedics, and police officers, have to psychological trauma in the course of their daily work. While the importance of attending to public safety personnel's mental health has been established, workplace mental health implementation strategies have historically varied in public safety organizations. INCLUSION CRITERIA: This scoping review will address workplace mental health implementation strategies used in public safety organizations. It will exclude studies that do not focus on workplace mental health, do not report on the implementation strategies used, or do not take place in a public safety context. METHODS: Primary studies published in English with any publication date up to the present will be considered. JBI methodology and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) will be followed. The search will be carried out in five databases and reference lists will also be searched for additional studies. Duplicates will be removed, and two independent reviewers will screen the titles, abstracts, and full text of the selected studies. Data collection will be performed using a tool developed by the researchers, based on JBI's model instrument for extracting study details, characteristics, and results. A summary of the results will be presented in diagrams, narratives, and tables.


Assuntos
Saúde Mental , Local de Trabalho , Pessoal Técnico de Saúde , Atenção à Saúde , Humanos , Organizações , Literatura de Revisão como Assunto , Revisões Sistemáticas como Assunto
11.
EBioMedicine ; 76: 103856, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35152152

RESUMO

BACKGROUND: Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials in COVID19 without characterisation of Pharmacokinetics /Pharmacodynamics including safety data. One such drug is nafamostat mesylate. METHODS: We present the findings of a phase Ib/IIa open label, platform randomised controlled trial of intravenous nafamostat in hospitalised patients with confirmed COVID-19 pneumonitis. Patients were assigned randomly to standard of care (SoC), nafamostat or an alternative therapy. Nafamostat was administered as an intravenous infusion at a dose of 0.2 mg/kg/h for a maximum of seven days. The analysis population included those who received any dose of the trial drug and all patients randomised to SoC. The primary outcomes of our trial were the safety and tolerability of intravenous nafamostat as an add on therapy for patients hospitalised with COVID-19 pneumonitis. FINDINGS: Data is reported from 42 patients, 21 of which were randomly assigned to receive intravenous nafamostat. 86% of nafamostat-treated patients experienced at least one AE compared to 57% of the SoC group. The nafamostat group were significantly more likely to experience at least one AE (posterior mean odds ratio 5.17, 95% credible interval (CI) 1.10 - 26.05) and developed significantly higher plasma creatinine levels (posterior mean difference 10.57 micromol/L, 95% CI 2.43-18.92). An average longer hospital stay was observed in nafamostat patients, alongside a lower rate of oxygen free days (rate ratio 0.55-95% CI 0.31-0.99, respectively). There were no other statistically significant differences in endpoints between nafamostat and SoC. PK data demonstrated that intravenous nafamostat was rapidly broken down to inactive metabolites. We observed no significant anticoagulant effects in thromboelastometry. INTERPRETATION: In hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous nafamostat, and there were additional adverse events. FUNDING: DEFINE was funded by LifeArc (an independent medical research charity) under the STOPCOVID award to the University of Edinburgh. We also thank the Oxford University COVID-19 Research Response Fund (BRD00230).


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Benzamidinas/uso terapêutico , Tratamento Farmacológico da COVID-19 , Guanidinas/uso terapêutico , Administração Intravenosa , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios não Esteroides/farmacocinética , Benzamidinas/efeitos adversos , Benzamidinas/farmacocinética , Biomarcadores/sangue , Biomarcadores/metabolismo , COVID-19/mortalidade , COVID-19/virologia , Esquema de Medicação , Feminino , Guanidinas/efeitos adversos , Guanidinas/farmacocinética , Meia-Vida , Humanos , Imunofenotipagem , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Resultado do Tratamento , Carga Viral
12.
BME Front ; 2021(2021): 9834163, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-37851586

RESUMO

Objective and Impact Statement. There is a need to develop platforms delineating inflammatory biology of the distal human lung. We describe a platform technology approach to detect in situ enzyme activity and observe drug inhibition in the distal human lung using a combination of matrix metalloproteinase (MMP) optical reporters, fibered confocal fluorescence microscopy (FCFM), and a bespoke delivery device. Introduction. The development of new therapeutic agents is hindered by the lack of in vivo in situ experimental methodologies that can rapidly evaluate the biological activity or drug-target engagement in patients. Methods. We optimised a novel highly quenched optical molecular reporter of enzyme activity (FIB One) and developed a translational pathway for in-human assessment. Results. We demonstrate the specificity for matrix metalloproteases (MMPs) 2, 9, and 13 and probe dequenching within physiological levels of MMPs and feasibility of imaging within whole lung models in preclinical settings. Subsequently, in a first-in-human exploratory experimental medicine study of patients with fibroproliferative lung disease, we demonstrate, through FCFM, the MMP activity in the alveolar space measured through FIB One fluorescence increase (with pharmacological inhibition). Conclusion. This translational in situ approach enables a new methodology to demonstrate active drug target effects of the distal lung and consequently may inform therapeutic drug development pathways.

13.
Sci Rep ; 11(1): 976, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441792

RESUMO

Neutrophil activation is an integral process to acute inflammation and is associated with adverse clinical sequelae. Identification of neutrophil activation in real time in the lungs of patients may permit biological stratification of patients in otherwise heterogenous cohorts typically defined by clinical criteria. No methods for identifying neutrophil activation in real time in the lungs of patients currently exist. We developed a bespoke molecular imaging probe targeting three characteristic signatures of neutrophil activation: pinocytosis, phagosomal alkalinisation, and human neutrophil elastase (HNE) activity. The probe functioned as designed in vitro and ex vivo. We evaluated optical endomicroscopy imaging of neutrophil activity using the probe in real-time at the bedside of healthy volunteers, patients with bronchiectasis, and critically unwell mechanically ventilated patients. We detected a range of imaging responses in vivo reflecting heterogeneity of condition and severity. We corroborated optical signal was due to probe function and neutrophil activation.


Assuntos
Pulmão/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Animais , Bronquiectasia/imunologia , Humanos , Inflamação/imunologia , Masculino , Elastase Pancreática/imunologia , Pinocitose/imunologia , Espectrometria de Fluorescência/métodos
14.
Br J Haematol ; 146(2): 185-92, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19438469

RESUMO

Circadian (c. 24 h) rhythms of physiology are entrained to either the environmental light-dark cycle or the timing of food intake. In the current work the hypothesis that rhythms of platelet turnover in mammals are circadian and entrained by food intake was explored in mice. Mice were entrained to 12 h light-dark cycles and given either ad libitum (AL) or restricted access (RF) to food during the light phase. Blood and megakaryocytes were then collected from mice every 4 h for 24 h. It was found that total and reticulated platelet numbers, plasma thrombopoietin (TPO) concentration and the mean size of mature megakaryocytes were circadian but not entrained by food intake. In contrast, a circadian rhythm in the expression of Arnt1 in megakaryocytes was entrained by food. Although not circadian, the expression in megakaryocytes of Nfe2, Gata1, Itga2b and Tubb1 expression was downregulated by RF, whereas Ccnd1 was not significantly affected by the feeding protocol. It is concluded that circadian rhythms of total platelet number, reticulated platelet number and plasma TPO concentration are entrained by the light-dark cycle rather than the timing of food intake. These findings imply that circadian clock gene expression regulates platelet turnover in mammals.


Assuntos
Plaquetas/fisiologia , Ritmo Circadiano/fisiologia , Comportamento Alimentar/fisiologia , Megacariócitos/fisiologia , Estimulação Luminosa , Trombopoetina/metabolismo , Análise de Variância , Animais , Proteínas de Transporte/metabolismo , Ciclina D1/metabolismo , Proteínas Fetais/metabolismo , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos , Contagem de Plaquetas , Trombopoese/fisiologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismo
15.
Sci Rep ; 9(1): 8422, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182770

RESUMO

Rapid in situ detection of pathogens coupled with high resolution imaging in the distal human lung has the potential to provide new insights and diagnostic utility in patients in whom pneumonia is suspected. We have previously described an antimicrobial peptide (AMP) Ubiquicidin (fragment UBI29-41) labelled with an environmentally sensitive fluorophore that optically detected bacteria in vitro but not ex vivo. Here, we describe further chemical development of this compound and demonstrate that altering the secondary structure of the AMP to generate a tri-branched dendrimeric scaffold provides enhanced signal in vitro and ex vivo and consequently allows the rapid detection of pathogens in situ in an explanted human lung. This compound (NBD-UBIdend) demonstrates bacterial labelling specificity for a broad panel of pathogenic bacteria and Aspergillus fumigatus. NBD-UBIdend demonstrated high signal-to-noise fluorescence amplification upon target engagement, did not label host mammalian cells and was non-toxic and chemically robust within the inflamed biological environment. Intrapulmonary delivery of NBD-UBIdend, coupled with optical endomicroscopy demonstrated real-time, in situ detection of bacteria in explanted whole human Cystic Fibrosis lungs.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Corantes Fluorescentes/metabolismo , Pulmão/microbiologia , Modelos Biológicos , Animais , Bactérias/metabolismo , Células Cultivadas , Fibrose Cística/microbiologia , Modelos Animais de Doenças , Fungos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Inflamação/patologia , Pulmão/patologia , Oxidiazóis/metabolismo , Pneumonia/microbiologia , Ovinos , Razão Sinal-Ruído
16.
Biosens Bioelectron ; 119: 209-214, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30138864

RESUMO

Human neutrophil elastase (HNE) is a serine protease, produced by polymorphonuclear neutrophils (PMNs), whose uncontrolled production has been associated with various inflammatory disease states as well as tumour proliferation and metastasis. Here we report the development and characterisation of an electrochemical peptide-based biosensor, which enables the detection of clinically relevant levels of HNE. The sensing platform was characterised in terms of its analytical performance, enzymatic cleavage kinetics and cross-reactivity and applied to the quantitative detection of protease activity from PMNs from human blood.


Assuntos
Técnicas Biossensoriais/métodos , Análise Química do Sangue/métodos , Técnicas Eletroquímicas , Elastase de Leucócito/metabolismo , Humanos , Neutrófilos/enzimologia , Peptídeos/química , Proteólise
17.
Sci Rep ; 8(1): 13490, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30201982

RESUMO

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.


Assuntos
Microscopia Intravital/métodos , Sondas Moleculares/química , Neutrófilos/metabolismo , Sistemas Automatizados de Assistência Junto ao Leito , Serina Proteases/metabolismo , Células Cultivadas , Citometria de Fluxo/métodos , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Voluntários Saudáveis , Humanos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/metabolismo , Cultura Primária de Células
18.
J Biomed Opt ; 23(7): 1-12, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29992799

RESUMO

A highly sensitive, modular three-color fluorescence endomicroscopy imaging platform spanning the visible to near-infrared (NIR) range is demonstrated. Light-emitting diodes (LEDs) were sequentially pulsed along with the camera acquisition to provide up to 20 frames per second (fps) three-color imaging performance or 60 fps single color imaging. The system was characterized for bacterial and cellular molecular imaging in ex vivo human lung tissue and for bacterial and indocyanine green imaging in ex vivo perfused sheep lungs. A practical method to reduce background tissue autofluorescence is also proposed. The platform was clinically translated into six patients with pulmonary disease to delineate healthy, cancerous, and fibrotic tissue autofluorescent structures. The instrument is the most broadband clinical endomicroscopy system developed to date (covering visible to the NIR, 500 to 900 nm) and demonstrates significant potential for future clinical utility due to its low cost and modular capability to suit a wide variety of molecular imaging applications.


Assuntos
Endoscopia , Microscopia de Fluorescência , Imagem Molecular , Idoso , Animais , Broncoscopia , Ensaios Clínicos como Assunto , Endoscopia/economia , Endoscopia/instrumentação , Endoscopia/métodos , Desenho de Equipamento , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Limite de Detecção , Pulmão/diagnóstico por imagem , Masculino , Microscopia de Fluorescência/economia , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Pessoa de Meia-Idade , Imagem Molecular/economia , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Ovinos
19.
Sci Transl Med ; 10(464)2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30355797

RESUMO

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.


Assuntos
Corantes Fluorescentes/metabolismo , Bactérias Gram-Negativas/isolamento & purificação , Lipídeo A/metabolismo , Pulmão/microbiologia , Peptídeos/metabolismo , Animais , Bronquiectasia/microbiologia , Bronquiectasia/patologia , Humanos , Unidades de Terapia Intensiva , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Polimixinas/farmacologia , Ovinos , Razão Sinal-Ruído , Relação Estrutura-Atividade
20.
J Vis Exp ; (129)2017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-29286374

RESUMO

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.


Assuntos
Pulmão/microbiologia , Microscopia Confocal/métodos , Imagem Óptica/métodos , Humanos
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