Anne Burdess

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 1–5×109 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 108 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 109 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.

Platelet activation in patients with peripheral vascular disease: Reproducibility and comparability of platelet markers ☆

BACKGROUND Many markers of platelet activation have been described but their reproducibility and comparability in patient populations are poorly defined. OBJECTIVES We sought to compare markers of platelet and monocyte activation with platelet-monocyte aggregates, a proposed gold standard of in vivo platelet activation, and assess their reproducibility in patients with peripheral arterial disease: a population with substantial platelet activation, inflammation and risk of thrombotic events. PATIENTS/METHODS Thirty patients with peripheral vascular disease attended on two occasions to permit within-day and between-day comparisons. In vivo platelet and monocyte activation were determined by flow-cytometric quantification of platelet-monocyte aggregation, platelet surface expression of P-selectin and CD40L, platelet-derived microparticles, and monocyte surface expression of CD40 and CD11b. Plasma concentrations of platelet-derived microparticles, soluble P-selectin and CD40L were measured by enzyme-linked immunosorbant assays. RESULTS Platelet-monocyte aggregation (36.7±7.86%), and platelet surface expression of P-selectin (5.8±1.65%) and CD40L (3.3±1.45%) demonstrated comparable within-day (mean difference±co-efficient of reproducibility; 0.9±15.4%, 0.21±1.65% and 0.2±2.8% respectively) and between-day reproducibility (2.0±12.4%, 0.10±2.25% and 0.9±6.4% respectively). Platelet-monocyte aggregates correlated well with other platelet (r=0.30-0.50, P<0.02) and monocyte (r=0.27-0.47, P<0.03) activation markers. Flow cytometric and assay quantified platelet-derived microparticles showed poorer reproducibility (co-efficient of reproducibility >40). CONCLUSIONS In patients with peripheral arterial disease, measurements of platelet-monocyte aggregates have good reproducibility and consistently reflect other markers of platelet and monocyte activation.

Randomized controlled trial of dual antiplatelet therapy in patients undergoing surgery for critical limb ischemia

Background and Objective:Patients with critical limb ischemia have a perioperative cardiovascular morbidity comparable to patients with acute coronary syndromes. We hypothesized that perioperative dual antiplatelet therapy would improve biomarkers of atherothrombosis without causing unacceptable bleeding in patients undergoing surgery for critical limb ischemia. Methods:In a double-blind randomized controlled trial, 108 patients undergoing infrainguinal revascularization or amputation for critical limb ischemia were maintained on aspirin (75 mg daily) and randomized to clopidogrel (600 mg prior to surgery, and 75 mg daily for 3 days; n = 50) or matched placebo (n = 58). Platelet activation and myocardial injury were assessed by flow cytometry and plasma troponin concentrations, respectively. Results:Clopidogrel reduced platelet-monocyte aggregation before surgery (38%–30%; P = 0.007). This was sustained in the postoperative period (P = 0.0019). There were 18 troponin-positive events (8 [16.0%] clopidogrel vs. 10 [17.2%] placebo; relative risk [RR]: 0.93, 95% confidence interval [CI]: 0.39–2.17; P = 0.86). Half of troponin-positive events occurred preoperatively with clopidogrel causing a greater decline in troponin concentrations (P < 0.001). There was no increase in major life-threatening bleeding (7 [14%] vs. 6 [10%]; RR: 1.4, 95% CI: 0.49–3.76; P = 0.56) or minor bleeding (17 [34%] vs. 12 [21%]; RR 1.64, 95% CI: 0.87–3.1; P = 0.12), although blood transfusions were increased (28% vs. 12.6%, RR: 2.3, 95% CI: 1.0–5.29; P = 0.037). Conclusions:In patients with critical limb ischemia, perioperative dual antiplatelet therapy reduces biomarkers of atherothrombosis without causing unacceptable bleeding. Large-scale randomized controlled trials are needed to establish whether dual antiplatelet therapy improves clinical outcome in high-risk patients undergoing vascular surgery.

Perioperative platelet and monocyte activation in patients with critical limb ischemia.

BACKGROUND Patients with critical limb ischemia (CLI) have a high rate of adverse cardiovascular events, particularly when undergoing surgery. We sought to determine the effect of surgery and vascular disease on platelet and monocyte activation in vivo in patients with CLI. METHODS An observational, cross-sectional study was performed at a tertiary referral hospital in the southeast of Scotland. Platelet and monocyte activation were measured in whole blood in patients with CLI scheduled for infrainguinal bypass and compared with matched healthy controls, patients with chronic intermittent claudication, patients with acute myocardial infarction, and those undergoing arthroplasty (n = 30 per group). Platelet and monocyte activation were quantified using flow cytometric assessment of platelet-monocyte aggregation, platelet P-selectin expression, platelet-derived microparticles, and monocyte CD40 and CD11b expression. RESULTS Compared with those with intermittent claudication, subjects with CLI had increased platelet-monocyte aggregates (41.7% +/- 12.2% vs 32.6% +/- 8.5%, respectively), platelet microparticles (178.7 +/- 106.9 vs 116.9 +/- 53.4), and monocyte CD40 expression (70.0% +/- 12.2% vs 52.4% +/- 15.2%; P < .001 for all). Indeed, these levels were equivalent (P-selectin, 4.4% +/- 2.0% vs 4.9% +/- 2.2%; P > .05) or higher (platelet-monocyte aggregation, 41.7% +/- 12.2% vs 33.6% +/- 7.0%; P < .05; platelet microparticles, 178.7 +/- 106.9 vs 114.4 +/- 55.0/microL; P < .05) than in patients with acute myocardial infarction. All platelet and monocyte activation markers remained elevated throughout the perioperative period in patients with CLI (P < .01) but not those undergoing arthroplasty. CONCLUSIONS Patients undergoing surgery for CLI have the highest level of in vivo platelet and monocyte activation, and these persist throughout the perioperative period. Additional antiplatelet therapy may be of benefit in protecting vascular patients with more severe disease during this period of increased risk.

Harnessing the preconditioning phenomenon: does remote organ ischaemia provide the answer?

Despite progress in defining the cellular mechanisms of the ischaemic preconditioning phenomenon, its conversion into convenient clinical practice has been slow. The possibility that an innate mechanism of tissue resistance to ischaemia could be harnessed as a clinical tool is an attractive and enticing prospect

In Vivo Mononuclear Cell Tracking Using Superparamagnetic Particles of Iron Oxide

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 1–5×109 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 108 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 109 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.

Reply to Letter: "Randomized Controlled Trial of Dual Antiplatelet Therapy in Patients Undergoing Surgery for Critical Limb Ischemia"

W e thank Dr. Zhang for his recent comments regarding our trial of dual antiplatelet therapy in patients undergoing surgery for critical limb ischemia.1 Dr. Zhang begins by suggesting that the nonsignificant increases in minor and life threatening bleeding might be attributable to the relatively small study size. He goes on to state that the study failed to demonstrate any benefit of dual antiplatelet therapy in reducing adverse cardiovascular events. This was a proof of concept study and was powered to examine the effect of combination antiplatelet therapy on markers of platelet activation— surrogate markers of clinical risk—not clinical end points. Clinical trials often require several thousands of patients to demonstrate a clinical benefit from antiplatelet regimes.2 The potential for adverse bleeding would seem greater for patients undergoing open surgery under antiplatelet therapy. Consequently, any large-scale trial powered to examine clinical end points should be justified by “pilot” data suggesting that some cardiovascular benefit could be achieved without excessive bleeding. Existing reports of antiplatelet therapy during the perioperative period have been largely anecdotal. Our study provides the first attempt to examine objectively the benefits and risks of such regimes in high-risk patients with vascular disease. We demonstrate that increased bleeding is a side effect of dual antiplatelet therapy and found an increase in nonlife threatening bleeding (transfusion of 2 or less units of red cells) and wound leak. However, we believe that this was not an excessive or prohibitive level of increased bleeding. Indeed, our results are consistent with those reported by the CURE trial2 where those patients who continued clopidogrel therapy within 5 days of coronary artery bypass had a 2-fold increased relative risk of major bleeding. In patients with acute coronary syndromes, additional clopidogrel therapy results

In Vivo Mononuclear Cell Tracking Using Superparamagnetic Particles of Iron OxideClinical Perspective

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 1–5×109 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 108 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 109 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.

In Vivo Mononuclear Cell Tracking Using Superparamagnetic Particles of Iron OxideClinical Perspective: 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 1–5×109 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 108 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 109 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.

074 In vivo cell tracking of superparamagnetic iron oxide-labelled mononuclear cells in humans

Introduction Stem cell and other cell-based therapies have emerged as promising novel treatment options for acute myocardial infarction and heart failure. Ascertaining whether cells reach and remain in the target site is essential to monitor the success of these therapeutic strategies. We have developed a Good Manufacturing Practice (GMP)-compliant protocol for labelling monocytes with an MRI contrast agent (Endorem, Guerbet) containing superparamagnetic particles of iron oxide (SPIO), and evaluated the potential for its use for human cell tracking studies. Method Up to 109 human peripheral blood mononuclear cells were labelled with SPIO using protamine sulphate as a transfection agent. In vitro labelling efficiency, viability and migratory capacity were evaluated. Six healthy volunteers each received intramuscular thigh injections of labelled cells (107), unlabelled cells (107) and SPIO alone, and underwent T2-weighted (T2W) MRI (1.5T) immediately and 7 days later. Safety of intravenous infusion was determined using a phased-dosing protocol in which two volunteers each received six doses of cells (104–109cells). Six further volunteers received 5×107 SPIO-labelled cells intravenously, and underwent serial T2 and multiecho (TE 4.1–22.1 ms) T2*-weighted imaging (3 T) before and serially after administration of cells. Imaging focused on the liver and spleen since this was where cells were expected to accumulate in healthy volunteers in the absence of an inflammatory focus. Results An optimised labelling protocol maximised SPIO uptake without affecting viability or migratory capacity. SPIO-labelled cells were visualised on T2W imaging following intramuscular administration (Abstract 74 Figure 1). Intravenous administration of up to 109 labelled cells was well tolerated with no effect on coagulation parameters. Reduction in signal intensity was seen in the liver and spleen on T2W imaging (Abstract 74 Figure 2) and a significant reduction in T2* value (Abstract 74 Figure 3) was observed in the liver (p<0.01) and spleen (p<0.001) following intravenous administration of cells, reflecting accumulation of SPIO-labelled cells at these sites. Abstract 74 Figure 1 T2-weighted imaging of the thigh following intra-musclar administration. A signal deficit is seen in relation to injection of Endorem alone (A) and Endorem-labelled cells (B) but not unlabelled cells (C). Abstract 74 Figure 2 T2-weighted transaxial slice through the upper abdomen before (A) and after (B) intravenous administration of Endorem-labelled cells. Signal intensity is reduced in the liver (L) and spleen (S) following administration of cells. Abstract 74 Figure 3 Change in T2* value following administration of Endorem-labelled cells. A significant reduction in T2* value is seen in the spleen (2–48 hrs, P<0.001) and liver (2–24 hrs, P<0.01) but not skeletal muscle (control). Conclusions We have shown that up to 109 human mononuclear cells can be labelled with SPIO under GMP-compliant conditions without affecting cell viability or migratory capacity. Intramuscular and intravenous administration of up to 109 SPIO-labelled cells is safe, and we have demonstrated for the first time in humans that SPIO-labelled cells can be imaged at a target site following local or systemic administration. Key to the success of cell-based studies, we have demonstrated the operational capacity of our institution to deliver a study of this nature. This pilot work enables us to progress to clinical cell tracking studies in patients with advanced atherosclerotic disease.