RATIONALEFew noninvasive biomarkers for pulmonary inflammation are currently available that can assess the lung-specific response to antiinflammatory treatments. Positron emission tomography with [(18)F]fluorodeoxyglucose (FDG-PET) is a promising new method that can be used to quantify pulmonary neutrophilic inflammation.OBJECTIVESTo evaluate the ability of FDG-PET to measure the pulmonary antiinflammatory effects of hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and recombinant human activated protein C (rhAPC) in a human model of experimentally-induced lung inflammation.METHODSEighteen healthy volunteers were randomized to receive placebo, lovastatin, or rhAPC before intrabronchial segmental endotoxin challenge. FDG-PET imaging was performed before and after endotoxin instillation. The rate of [(18)F]FDG uptake was calculated as the influx constant K(i) by Patlak graphical analysis. Bronchoalveolar lavage (BAL) was performed to determine leukocyte concentrations for correlation with the PET imaging results.MEASUREMENTS AND MAIN RESULTSThere was a statistically significant decrease in K(i) in the lovastatin-treated group that was not seen in the placebo-treated group, suggesting attenuation of inflammation by lovastatin treatment despite a small decrease in BAL total leukocyte and neutrophil counts that was not statistically significant. No significant decrease in K(i) was observed in the rhAPC-treated group, correlating with a lack of change in BAL parameters and indicating no significant antiinflammatory effect with rhAPC.CONCLUSIONSFDG-PET imaging is a sensitive method for quantifying the lung-specific response to antiinflammatory therapies and may serve as an attractive platform for assessing the efficacy of novel antiinflammatory therapies at early phases in the drug development process. Clinical trial registered with www.clinicaltrials.gov (NCT00741013).

Objective:Acute respiratory distress syndrome is an abrupt inflammatory illness that involves damage from reactive oxygen species. We examined the efficacy and safety of oxothiazolidine-4-carboxylic acid (OTZ), a free radical scavenger, in treating acute respiratory distress syndrome. Design:Double-blind, placebo-controlled trial. Setting:Multicentered study. Patients:Patients with a Pao2/Fio2 ≤200 and bilateral infiltrates on chest radiograph, and requiring mechanical ventilation. Interventions:We randomized 215 patients to receive OTZ, 210 mg/kg per day every 8 hrs for 14 days or placebo. Measurements and Main Results:Ventilator-free days (the number of days alive and free from ventilator requirement) during the first 30 days of study were 8.3 vs. 13.5 days for the OTZ and placebo groups, respectively (p < .001). Mortality was 30/101 (29.7%) in the OTZ group and 18/114 (15.8%) in the placebo group during the 30-day study period (p = .014). This study was terminated prematurely for safety reasons after 215 of the planned 352 patients were enrolled. Conclusions:OTZ does not improve survival or reduce ventilator time in patients with acute respiratory distress syndrome and may worsen outcome, although mortality in the OTZ group was similar or lower than most similar trials. Alternatively, our results may be best explained by the unusually excellent outcome in the placebo group.

Biopharmaceuticals, such as proteins and DNA, have demonstrated their potential to prevent and cure diseases. The success of such therapeutic agents hinges upon their ability to cross complex barriers in the body and reach their target intact. In order to reap the full benefits of these therapeutic agents, a delivery vehicle capable of delivering cargo to all cell types, both phagocytic and non-phagocytic, is needed. This article presents the synthesis and evaluation of a microparticle delivery vehicle capable of cell penetration and sub-cellular triggered release of an encapsulated payload. pH-sensitive polyacrylamide particles functionalized with a polyarginine cell-penetrating peptide (CPP) were synthesized. The incorporation of a CPP into the microparticles led to efficient uptake by non-phagocytic cells in culture. In addition, the CPP-modified particles showed no cytotoxic effects at concentrations used in this study. The results suggest that these particles may provide a vehicle for the successful delivery of therapeutic agents to various cell types.

This article reviews the potential use of positron emission tomography (PET), alone and in combination with computed tomography, for evaluating the severity of disease in cystic fibrosis. PET scanning using injected 18F-fluorodeoxyglucose provides visual and quantitative information for the rate at which glucose is taken up by the lung, a process that should relate to the presence of inflammation and reflect the extent of the disease. The computed tomography scan gives highly accurate density and anatomic information to locate areas of inflammation seen on the PET scan, increasing the accuracy of the interpretation. Until recently, the scanners have been single systems, often located in separate hospital departments. Combined systems are now commercially available, with major advantages for patients and in the quality of analytical information obtained for interpretation by the physician. The use of 18F-fluorodeoxyglucose uptake and PET scanning has been suggested as a biomarker of progressive pulmonary inflammation in cystic fibrosis. Although promising, the data so far are limited. Further studies will be needed to validate this measurement for this purpose.

Positron emission tomography (PET) provides three-dimensional images of the distributions of radionuclides that have been inhaled or injected into the lungs. By using radionuclides with short half-lives, the radiation exposure of the subject can be kept small. By following the evolution of the distributions of radionuclides in gases or compounds that participate in lung function, information about such diverse lung functions as regional ventilation, perfusion, shunt, gas fraction, capillary permeability, inflammation, and gene expression can be inferred. Thus PET has the potential to provide information about the links between cellular function and whole lung function in vivo. In this paper, recent advancements in PET methodology and techniques and information about lung function that have been obtained with these techniques are reviewed.

Current methodologies for quantifying radiolabeled nucleoside monophosphates and nucleoside analogues result in high retention of unphosphorylated guanosine nucleosides in the case of lanthanum chloride precipitation or inconsistent retention of nucleotides in the case of DEAE cellulose filter papers. This study describes an innovative method for quantifying thymidine kinase (TK) activity that is compatible with both purine and pyrimidine nucleoside analogues by using lanthanum phosphate coprecipitation at pH 4.0. This methodology maintains quantitative precipitation of nucleoside monophosphates and yields minimal background binding from a variety of nucleoside analogues. In addition, use of PCR thermocyclers enhances the temporal precision of TK assays. This method was shown to be useful for assaying TK activity in a broad range of biochemically relevant systems, including purified enzymes, stable cell lines, and virally infected cells. Use of this methodology should aid researchers in the evaluation of novel nucleoside analogues and TK enzymes while decreasing radioactive waste, minimizing assay time, increasing accuracy, and enhancing dynamic range.

The pathogenesis of many lung diseases involves neutrophilic inflammation. Neutrophil functions, in turn, are critically dependent on glucose uptake and glycolysis to supply the necessary energy to meet these functions. In this study, we determined the effects of p38 mitogen-activated protein kinase and hypoxia-inducible factor (HIF)-1, as well as their potential interaction, on the expression of membrane glucose transporters and on glucose uptake in murine neutrophils. Neutrophils were harvested and purified from C57BL/6 mice and stimulated with lipopolysaccharide (LPS) in the presence or absence of specific p38 and HIF-1 inhibitors. Glucose uptake was measured as the rate of [3H]deoxyglucose (DG) uptake. We identified GLUT-1 in mouse neutrophils, but neither GLUT-3 nor GLUT-4 were detected using Western blot analysis, even after LPS stimulation. LPS stimulation did not increase GLUT-1 protein levels but did cause translocation of GLUT-1 from the cell interior to the cell surface, together with a dose-dependent increase in [3H]DG uptake, indicating that glucose uptake is regulated in these cells. LPS also activated both p38 and the HIF-1 pathway. Inhibitors of p38 and HIF-1 blocked GLUT-1 translocation and [3H]DG uptake. These data suggest that LPS-induced increases in neutrophil glucose uptake are mediated by GLUT-1 translocation to the cell surface in response to sequential activation of neutrophil p38 and HIF-1alpha in neutrophils. Given that neutrophil function and glucose metabolism are closely linked, control of the latter may represent a new target to ameliorate the deleterious effects of neutrophils on the lungs.

Nanoparticles (NPs) targeting the intercellular adhesion molecule 1 (ICAM-1) hold promise as a mean of delivering therapeutics to the pulmonary endothelium in patients with acute and chronic respiratory diseases. As these new materials become available, strategies are needed to understand their behavior in vivo. We have evaluated the use of Cu-64 and PET to noninvasively image the lung uptake and distribution of NPs coated with an anti-ICAM antibody. Methods: Model fluorescent NPs were coated with a mixture of an anti-ICAM antibody (or nonspecific IgG) and Cu-64-DOTA-IgG (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Biodistribution and small-animal PET and CT studies were performed in healthy mice and in mice pre-treated with lipopolysaccharides (LPSs). Metabolism studies were also performed to evaluate the stability of (CU)-C-64-labeled NPs in lungs in vivo. Results: The lungs of mice administered anti-ICAM NPs labeled with Cu-64 were clearly imaged by small-animal PET 1, 4, and 24 h after administration. Both biodistribution and small-animal imaging showed a 3- to 4-fold higher uptake in the lungs of mice injected with ICAM-targeted NPs relative to that of the control group. Lung uptake was further enhanced by pretreating the mice with LPS, presumably because of ICAM-1 upregulation. However, an approximately 2-fold decrease in lung signal was observed in each experimental group over 24 h. Metabolism studies in lung tissues harvested from mice injected with Cu-64-labeled anti-ICAM NPs showed considerable release of a small Cu-64-radiometabolite from the NPs beginning as early as 1 h after injection. A decrease in lung fluorescence was also observed, most likely reflecting partial release of NPs from the lungs in vivo. Conclusion: The use of small-animal PET to track Cu-64-labeled nanostructures in vivo shows potential as a strategy for the preclinical screening of new NP drug delivery agents targeting the lung endothelium and other tissues. Future design optimization to prolong the stability of the radiolabel in vivo will further improve this promising approach.

Recent advances in imaging offer exciting opportunities to develop and validate lung-specific biomarkers as valuable adjuncts to diagnosis, tests of treatment efficacy, and/or treatment monitoring. State-of-the-art structural, functional, and molecular imaging methods allow the lungs to be visualized noninvasively in vivo at submillimeter and subsecond spatial and temporal scales. However, the development and validation of imaging biomarkers present some special challenges, including the following: equipment evaluation, procedure standardization, data regarding reproducibility and replication, interrater variability, the production and measurement of reference standards, sensitivity to interventions or disease progression, intersubject variance, choice of image reconstruction and segmentation algorithms, automated versus observer-dependent image analysis, data acquisition during conditions of standardized lung volume, whether a reliable association can be demonstrated between the imaging biomarker and a clinical endpoint, and whether its use will have a favorable cost-effective impact on drug development or disease management. Establishing such performance characteristics, especially for single investigators at single institutions, can be daunting if not impossible for costly biomarkers such as imaging. Therefore, to take full advantage of the opportunities presented by state-of-the-art imaging methods, new approaches to analytic and clinical validation must be developed in collaboration with industry, foundation, and federal funding agencies.

Recent studies indicate that a focal, limited, inflammatory response can be safely elicited after direct bronchial instillation of small doses of endotoxin into a single lung segment. Because the radiotracer [18F]fluorodeoxyglucose ([18F]FDG) is taken up at accelerated rates within inflamed tissues, we hypothesized that we could detect and quantify this regional inflammatory response with positron emission tomography (PET). We imaged 18 normal volunteers in a dose-escalation study with 3 endotoxin dosing groups (n = 6 in each group): 1 ng/kg, 2 ng/kg, and 4 ng/kg. Endotoxin was instilled by bronchoscopy into a segment of the right middle lobe, with imaging performed approximately 24 h later, followed by bronchoalveolar lavage (BAL). A "subtraction imaging analysis" was performed in the highest dose cohort to identify the area of inflammation, using the preendotoxin scan as a baseline. BAL neutrophil counts were significantly higher in the highest dose group compared with the other two groups (1,413 +/- 625 vs. 511 +/- 396 and 395 +/- 400 cells/mm3; P < 0.05). Autoradiography performed on cells harvested by BAL showed specific [3H]deoxyglucose ([3H]DG) uptake limited to neutrophils. In vitro [3H]DG uptake in BAL neutrophils in the 4 ng/kg dose group (but not in the 2 ng/kg group) was statistically greater than in peripheral blood neutrophils obtained before endotoxin instillation. The rate of [18F]FDG uptake was greatest in the 4 ng/kg group, with a consistent, statistically significant increase in the rate of uptake after endotoxin instillation compared with baseline. We conclude that the inflammatory response to low-dose endotoxin in a single lung segment can be visualized and quantified by imaging with FDG-PET.

Methods currently used to assess lung and airway inflammation are often poorly quantitative, invasive, nonspecific, or insensitive. Positron emission tomography (PET) with [18F]fluorodeoxyglucose [18F]FDG), on the other hand, is a noninvasive, highly sensitive imaging technique that can be used to quantify pulmonary inflammation. [18F]FDG, an analogue of glucose, is taken up by the same transporters that take up glucose into the cell; therefore, [18F]FDG uptake tracks cellular glucose transport, which is highly correlated to the rate of cellular glucose metabolism. Recent studies in animal models of neutrophilic lung inflammation, as well as in patients with inflammatory lung disease, indicate that increased [18F]FDG uptake by the lungs correlates with the number of activated neutrophils recovered from the lungs. Therefore, the in vivo measurement of pulmonary glucose metabolism is a measure of neutrophil burden within the lungs. We propose that FDG-PET imaging can be used as a measurable biomarker in the development of drug therapies targeting lung inflammation.

RATIONALE Although infection contributes to morbidity in patients with cystic fibrosis (CF), the host inflammatory response is also an important cause of progressive pulmonary function deterioration. Quantifying the inflammatory burden in these patients is challenging and often requires invasive procedures. Positron emission tomographic imaging with [18F]fluorodeoxyglucose ([18FDG]) could be used as a noninvasive alternative to quantify lung inflammation. OBJECTIVE To determine the relationships among lung [18F]FDG uptake, bronchoalveolar lavage (BAL) neutrophil concentrations, and pulmonary function in patients with CF. METHODS Twenty patients and seven healthy volunteers were studied. A subset of seven patients also consented to undergo BAL. The uptake of [18F]FDG by the lungs was measured as the net influx rate constant Ki. Patients were stratified by rate of decline in pulmonary function into stable, intermediate, and rapidly declining groups. Ki was compared among groups and was correlated against neutrophil concentrations in BAL fluid. RESULTS Ki was significantly elevated (p<0.05) among patients with CF as a whole compared with healthy control subjects (0.0015+/-0.0009 versus 0.0007+/-0.0002 ml blood/ml lung/min) but especially in patients with rapidly declining pulmonary function (0.0022+/-0.0011 ml blood/ml lung/min). Ki correlated positively with the number of neutrophils present in BAL fluid. CONCLUSION Imaging with [18F]fluorodeoxyglucose and positron emission tomography can be used to assess inflammatory burden in patients with CF. Elevations in Ki may be able to identify patients with more aggressive disease and may be useful in monitoring changes in inflammatory burden in response to novel treatments.

Washington University School of Medicine, St. Louis, MO The author has not disclosed any potential conflicts of interest.