A Strategy to Detect Emerging Non-Delta SARS-CoV-2 Variants with a Monoclonal Antibody Specific for the N501 Spike Residue.

Efforts to control SARS-CoV-2 have been challenged by the emergence of variant strains that have important implications for clinical and epidemiological decision making. Four variants of concern (VOCs) have been designated by the Centers for Disease Control and Prevention (CDC), namely, B.1.617.2 (delta), B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), although the last three have been downgraded to variants being monitored (VBMs). VOCs and VBMs have shown increased transmissibility and/or disease severity, resistance to convalescent SARS-CoV-2 immunity and antibody therapeutics, and the potential to evade diagnostic detection. Methods are needed for point-of-care (POC) testing to rapidly identify these variants, protect vulnerable populations, and improve surveillance. Antigen-detection rapid diagnostic tests (Ag-RDTs) are ideal for POC use, but Ag-RDTs that recognize specific variants have not yet been implemented. Here, we describe a mAb (2E8) that is specific for the SARS-CoV-2 spike protein N501 residue. The 2E8 mAb can distinguish the delta VOC from variants with the N501Y meta-signature, which is characterized by convergent mutations that contribute to increased virulence and evasion of host immunity. Among the N501Y-containing mutants formerly designated as VOCs (alpha, beta, and gamma), a previously described mAb, CB6, can distinguish beta from alpha and gamma. When used in a sandwich ELISA, these mAbs sort these important SARS-CoV-2 variants into three diagnostic categories, namely, (1) delta, (2) alpha or gamma, and (3) beta. As delta is currently the predominant variant globally, they will be useful for POC testing to identify N501Y meta-signature variants, protect individuals in high-risk settings, and help detect epidemiological shifts among SARS-CoV-2 variants.

Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids.

BACKGROUND: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs. PURPOSE: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer. METHODS: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions. RESULTS: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence. CONCLUSION: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.

Effects of Fluorescent Diamond Particles FDP-NV-800nm on Essential Biochemical Functions of Primary Human Umbilical Vein Cells and Human Hepatic Cell Line, HepG-2 in vitro (Part VI): Acute Biocompatibility Studies.

BACKGROUND: Recently, we reported the safety and biocompatibility of fluorescent diamond particles, FDP-NV-Z-800nm (FDP-NV) injected intravenously into rats, where no morbidity and mortality were noted over a period of 3 months. The acute effects of FDP-NV-800nm particles on cultured human endothelial and hepatic cells remain unexplored. PURPOSE: In this study, we aimed to explore select cellular and biochemical functions in cultured human umbilical endothelial cells (HUVEC) and a human hepatic cancer cell line (HepG-2) exposed to FDP-NV-800 in vitro at exposure levels within the pharmacokinetics (Cmax and the nadir) previously reported in vivo. METHODS: Diverse cellular and biochemical functions were monitored, which cumulatively can provide insights into some vital cellular functions. Cell proliferation and migration were assessed by quantitative microscopy. Mitochondrial metabolic functions were tested by the MTT assay, and cytosolic esterase activity was studied by the calcein AM assay. Chaperons (CHOP), BiP and apoptosis (caspase-3 activation) were monitored by using Western blot (WB). MAPK Erk1/2 signaling was assessed by the detection of the phosphorylated form of the protein (P-Erk 1/2) and its translocation into the cell nucleus. RESULTS: At all concentrations tested (0.001-0.1mg/mL), FDP-NV did not affect any of the biomarkers of cell integrity of HepG2 cells. In contrast, the proliferation of HUVEC was affected at the highest concentration tested (0.1mg/mL, Cmax). Exposure of HUVEC to (0.01 mg/mL) FDP-NV had a mild-moderate effect on cell proliferation as evident in the MTT assay and was absent when proliferation was assessed by direct cell counting or by using the calcein AM assays. In both cell types, exposure to the highest concentration (0.1 mg/mL) of FDP-NV did neither affect FBS-stimulated cell signaling (MAPK Erk1/2 phosphorylation) nor did it activate of Caspase 3. CONCLUSION: Our data suggest that FDP-NV-800nm are largely biocompatible with HepG-2 cells proliferation within the pharmacokinetic data reported previously. In contrast, HUVEC proliferation at the highest exposure dose (0.1 mg/mL) responded adversely with respect to several biomarkers of cell integrity. However, since the Cmax levels are very short-living, the risk for endothelial injury is likely minimal for slow rate cell proliferation such as endothelial cells.

The Use of Near-Infrared Light-Emitting Fluorescent Nanodiamond Particles to Detect Ebola Virus Glycoprotein: Technology Development and Proof of Principle.

BACKGROUND: There is a dire need for rapid diagnostic tests of high sensitivity, efficiency, and point-of-test reporting capability to mitigate lethal viral epidemic outbreaks. PURPOSE: To develop a new operating system within the lateral flow assay (LFA) format for Ebola virus (EBOV), based on fluorescent nanodiamond particles (FNDP) nitrogen vacancy (NV) emitting near-infrared (NIR) light. Specifically, we aimed to detail technical issues and the feasibility of mobilizing FNDP-NV on nitrocellulose membranes (NCM) and capturing them at test and control lines. METHODS: FNDP-NV-200nm, 400nm or 800nm were linked to anti-EBOV glycoprotein (GP) monoclonal antibodies (mAb) and tested for LFA performance by monitoring NIR emissions using an in vivo imaging system or optoelectronic device (OED). Anti-EBOV recombinant glycoprotein (GP) humanized mAb c13C6 was linked to FNDP-NV-200nm for the mobile phase; and a second anti-GP mouse mAb, 6D8, was printed on NCM at the test line. Goat anti-human IgG (GAH-IgG) served as a nonspecific antibody for conjugated FNDP-NV-200nm at the control line. RESULTS: FNDP-NV-200nm-c13C6 specifically and dose-dependently bound to recombinant EBOV GP in vitro and was effectively captured in a sandwich configuration at the test line by mAb 6D8. FNDP-NV-200nm-c13C6 was captured on the control line by GAH-IgG. The OED quantitative analysis of NIR (obtained in less than 1 minute) was further validated by an in vivo imaging system. CONCLUSION: FNDP-NV-200nm performance as a reporter for EBOV GP rapid diagnostic tests suggests an opportunity to replace contemporary visual tests for EBOV GP and other highly lethal viral pathogens. Mobile, battery-operated OED adds portability, quantitative data, rapid data collection, and point-of-test reporting capability. Further development of FNDP-NV-200nm within a LFA format is justified.

Biocompatibility studies of fluorescent diamond particles-(NV)~800nm (part V): in vitro kinetics and in vivo localization in rat liver following long-term exposure.

BACKGROUND: We recently reported on long-term comprehensive biocompatibility and biodistribution study of fluorescent nanodiamond particles (NV)-Z-average 800nm (FNDP-(NV)) in rats. FNDP-(NV) primary deposition was found in the liver, yet liver function tests remained normal. PURPOSE: The present study aimed to gain preliminary insights on discrete localization of FNDP-(NV) in liver cells of the hepatic lobule unit and venous micro-vasculature. Kinetics of FDNP-(NV) uptake into liver cells surrogates in culture was conducted along with cell cytokinesis as markers of cells' viability. METHODS: Preserved liver specimens from a pilot consisting of two animals which were stained for cytoskeletal elements (fluorescein-isothiocyanate-phalloidin) were examined for distribution of FNDP-(NV) by fluorescent microscopy (FM) and Confocal-FM (CFM) using near infra-red fluorescence (NIR). Hepatocellular carcinoma cells (HepG-2) and human umbilical vein endothelial cells (HUVEC) were cultured with FNDP-(NV) and assayed for particle uptake and location using spectrophotometric technology and microscopy. RESULTS: HepG-2 and HUVEC displayed rapid (<30 mins) onset and concentration-dependent FNDP-(NV) internalization and formation of peri-nuclear corona. FM/CFM of liver sections revealed FNDP-(NV) presence throughout the hepatic lobules structures marked by spatial distribution, venous microvascular spaces and parenchyma and non-parenchyma cells. CONCLUSION: The robust presence of FNDP-(NV) throughout the hepatic lobules including those internalized within parenchyma cells and agglomerates in the liver venous micro-circulation were not associated with macro or micro histopathological signs nor vascular lesions. Cells cultures indicated normal cytokinesis in cells containing FNDP-(NV) agglomerates. Liver parenchyma cells and the liver microcirculation remain agnostic to presence of FNDP-(NV) in the sinusoids or internalized in the hepatic cells.

Rosiglitazone treatment restores renal responsiveness to atrial natriuretic peptide in rats with congestive heart failure.

The thiazolidinedione (TZD) class of Peroxisome proliferator-activated receptor gamma agonists has restricted clinical use for diabetes mellitus due to fluid retention and potential cardiovascular risks. These side effects are attributed in part to direct salt-retaining effect of TZDs at the renal collecting duct. A recent study from our group revealed that prolonged rosiglitazone (RGZ) treatment caused no Na+/H2 O retention or up-regulation of Na+ transport-linked channels/transporters in experimental congestive heart failure (CHF) induced by surgical aorto-caval fistula (ACF). The present study examines the effects of RGZ on renal and cardiac responses to atrial natriuretic peptide (ANP), Acetylcholine (Ach) and S-Nitroso-N-acetylpenicillamine (SNAP-NO donor). Furthermore, we assessed the impact of RGZ on gene expression related to the ANP signalling pathway in animals with ACF. Rats subjected to ACF (or sham) were treated with either RGZ (30 mg/kg/day) or vehicle for 4 weeks. Cardiac chambers pressures and volumes were assessed invasively via Miller catheter. Kidney excretory and renal hemodynamic in response to ANP, Ach and SNAP were examined. Renal clearance along with cyclic guanosine monophosphate (cGMP), gene expression of renal CHF-related genes and ANP signalling in the kidney were determined. RGZ-treated CHF rats exhibited significant improvement in the natriuretic responses to ANP infusion. This 'sensitization' to ANP was not associated with increases in neither urinary cGMP nor in vitro cGMP production. However, RGZ caused down-regulation of several genes in the renal cortex (Ace, Nos3 and Npr1) and up-regulation of ACE2, Agtrla, Mme and Cftr along down-regulation of Avpr2, Npr1,2, Nos3 and Pde3 in the medulla. In conclusion, CHF+RGZ rats exhibited significant enhancement in the natriuretic responses to ANP infusion, which are known to be blunted in CHF. This 'sensitization' to ANP is independent of cGMP signalling, yet may involve post-cGMP signalling target genes such as ACE2, CFTR and V2 receptor. The possibility that TZD treatment in uncomplicated CHF may be less detrimental than thought before deserves additional investigations.

Long-term biocompatibility of fluorescent diamonds-(NV)-Z~800 nm in rats: survival, morbidity, histopathology, particle distribution and excretion studies (part IV).

BACKGROUND: Thromboembolic events are a major cause of heart attacks and strokes. However, diagnosis of the location of high risk vascular clots is hampered by lack of proper technologies for their detection. We recently reported on bio-engineered fluorescent diamond-(NV)-Z~800nm (FNDP-(NV)) conjugated with bitistatin (Bit) and proven its ability to identify iatrogenic blood clots in the rat carotid artery in vivo by Near Infra-Red (NIR) monitored by In Vivo Imaging System (IVIS). PURPOSE: The objective of the present research was to assess the in vivo biocompatibility of FNDP-(NV)-Z~800nm infused intravenously to rats. Multiple biological variables were assessed along this 12 week study commissioned in anticipation of regulatory requirements for a long-term safety assessment. METHODS: Rats were infused under anesthesia with aforementioned dose of the FNDP-(NV), while equal number of animals served as control (vehicle treated). Over the 12 week observation period rats were tested for thriving, motor, sensory and cognitive functions. At the termination of study, blood samples were obtained under anesthesia for comprehensive hematology and biochemical assays. Furthermore, 6 whole organs (liver, spleen, brain, heart, lung and kidney) were collected and examined ex vivo for FNDP-NV) via NIR monitored by IVIS and histochemical inspection. RESULTS: All animals survived, thrived (no change in body and organ growth). Neuro-behavioral functions remain intact. Hematology and biochemistry (including liver and kidney functions) were normal. Preferential FNDP-(NV) distribution identified the liver as the main long-term repository. Certified pathology reports indicated no outstanding of finding in all organs. CONCLUSION: The present study suggests outstanding biocompatibility of FNDP-(NV)-Z~800nm after long-term exposure in the rat.

Pilot study on biocompatibility of fluorescent nanodiamond-(NV)-Z~800 particles in rats: safety, pharmacokinetics, and bio-distribution (part III).

INTRODUCTION: We hereby report on studies aimed to characterize safety, pharmacokinetics, and bio-distribution of fluorescent nanodiamond particles (NV)-Z~800 (FNDP-(NV)) administered to rats by intravenous infusion in a single high dose. METHODS: Broad scale biological variables were monitored following acute (90 minutes) and subacute (5 or 14 days) exposure to FNDP-(NV). Primary endpoints included morbidity and mortality, while secondary endpoints focused on hematology and clinical biochemistry biomarkers. Particle distribution (liver, spleen, lung, heart, and kidney) was assessed by whole organ near infrared imaging using an in vivo imaging system. This was validated by the quantification of particles extracted from the same organs and visualized by fluorescent and scanning electron microscopy. FNDP-(NV)-treated rats showed no change in morbidity or mortality and preserved normal motor and sensory function, as assessed by six different tests. RESULTS: Blood cell counts and plasma biochemistry remained normal. The particles were principally distributed in the liver and spleen. The liver particle load accounted for 51%, 24%, and 18% at 90 minutes, 5 days, and 14 days, respectively. A pilot study of particle clearance from blood indicated 50% clearance 33 minutes following the end of particle infusion. CONCLUSION: We concluded that systemic exposure of rats to a single high dose of FDNP-(NV)-Z~800 (60 mg/kg) appeared to be safe and well tolerated over at least 2 weeks. These data suggest that FNDP-(NV) should proceed to preclinical development in the near future.

Vascular thrombus imaging in vivo via near-infrared fluorescent nanodiamond particles bioengineered with the disintegrin bitistatin (Part II).

The aim of this feasibility study was to test the ability of fluorescent nanodiamond particles (F-NDP) covalently conjugated with bitistatin (F-NDP-Bit) to detect vascular blood clots in vivo using extracorporeal near-infrared (NIR) imaging. Specifically, we compared NIR fluorescence properties of F-NDP with N-V (F-NDPNV) and N-V-N color centers and sizes (100-10,000 nm). Optimal NIR fluorescence and tissue penetration across biological tissues (rat skin, porcine axillary veins, and skin) was obtained for F-NDPNV with a mean diameter of 700 nm. Intravital imaging (using in vivo imaging system [IVIS]) in vitro revealed that F-NDPNV-loaded glass capillaries could be detected across 6 mm of rat red-muscle barrier and 12 mm of porcine skin, which equals the average vertical distance of a human carotid artery bifurcation from the surface of the adjacent skin (14 mm). In vivo, feasibility was demonstrated in a rat model of occlusive blood clots generated using FeCl3 in the carotid artery bifurcation. Following systemic infusions of F-NDPNV-Bit (3 or 15 mg/kg) via the external carotid artery or femoral vein (N=3), presence of the particles in the thrombi was confirmed both in situ via IVIS, and ex vivo via confocal imaging. The presence of F-NDPNV in the vascular clots was further confirmed by direct counting of fluorescent particles extracted from clots following tissue solubilization. Our data suggest that F-NDPNV-Bit associate with vascular blood clots, presumably by binding of F-NDPNV-Bit to activated platelets within the blood clot. We posit that F-NDPNV-Bit could serve as a noninvasive platform for identification of vascular thrombi using NIR energy monitored by an extracorporeal device.

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbß3 and activated platelets.

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the αIIbß3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbß3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

Pharmacology in cerebrovascular disease research: Pharmacokinetics-pharmacodynamics to the rescue.

Pharmacological tools (compounds) have an important role in validation of biological molecular targets for their role in disease processes and their prospective development for therapeutic objectives. Effective utilization of such pharmacological tools impacts on the veracity of the information by which decisions regarding target validation is reached, and investment in clinical development is committed. This commentary addresses frequent gaps in effective utilization of pharmacological principles and practices of pharmacokinetics and pharmacodynamics in experimental pre-clinical research, which if more rigorously implemented could contribute to eventual success in drug development for stroke and neurotrauma.

Potential early predictors for outcomes of experimental hemorrhagic shock induced by uncontrolled internal bleeding in rats.

Uncontrolled hemorrhage, resulting from traumatic injuries, continues to be the leading cause of death in civilian and military environments. Hemorrhagic deaths usually occur within the first 6 hours of admission to hospital; therefore, early prehospital identification of patients who are at risk for developing shock may improve survival. The aims of the current study were: 1. To establish and characterize a unique model of uncontrolled internal hemorrhage induced by massive renal injury (MRI), of different degrees (20-35% unilateral nephrectomy) in rats, 2. To identify early biomarkers those best predict the outcome of severe internal hemorrhage. For this purpose, male Sprague Dawley rats were anesthetized and cannulas were inserted into the trachea and carotid artery. After abdominal laparotomy, the lower pole of the kidney was excised. During 120 minutes, hematocrit, pO2, pCO2, base excess, potassium, lactate and glucose were measured from blood samples, and mean arterial pressure (MAP) was measured through arterial tracing. After 120 minutes, blood loss was determined. Statistical prediction models of mortality and amount of blood loss were performed. In this model, the lowest blood loss and mortality rate were observed in the group with 20% nephrectomy. Escalation of the extent of nephrectomy to 25% and 30% significantly increased blood loss and mortality rate. Two phases of hemodynamic and biochemical response to MRI were noticed: the primary phase, occurring during the first 15 minutes after injury, and the secondary phase, beginning 30 minutes after the induction of bleeding. A Significant correlation between early blood loss and mean arterial pressure (MAP) decrements and survival were noted. Our data also indicate that prediction of outcome was attainable in the very early stages of blood loss, over the first 15 minutes after the injury, and that blood loss and MAP were the strongest predictors of mortality.

Effect of PSI-697, a novel P-selectin inhibitor, on platelet-monocyte aggregate formation in humans.

BACKGROUND: Platelet activation is central to the pathogenesis of acute coronary syndromes. Surface expression of P-selectin on activated platelets induces formation of platelet-monocyte aggregates and promotes vascular inflammation and thrombosis. P-selectin antagonism may represent a novel therapeutic strategy in vascular disease. We aimed to investigate the effects of the novel P-selectin antagonist PSI-697 on platelet-monocyte aggregate formation in humans. METHODS AND RESULTS: In a double-blind, randomized, placebo-controlled crossover study, healthy smokers were randomized to receive either oral PSI-697 600 mg or matched placebo. The sequence of treatment was also randomized, with all subjects receiving both PSI-697 and placebo. Platelet-monocyte aggregates were measured by flow cytometry at 4 and 24 hours in the presence and absence of thrombin receptor-activating peptide (TRAP; 0.1 to 1.0 µm/L). The ex vivo addition of TRAP caused a concentration-dependent increase in platelet-monocyte aggregates from 8.2% to 94.8% (P<0.001). At 4 and 24 hours, plasma concentrations of PSI-697 increased to 1906 and 83 ng/mL, respectively (P<0.001). PSI-697 had no demonstrable effect on either stimulated or unstimulated platelet-monocyte aggregates at 4 or 24 hours (P>0.05). P-selectin-blocking antibody (CLB-Thromb6), but not PSI-697, inhibited both stimulated and unstimulated platelet-monocyte aggregate formation in vitro (P<0.001). CONCLUSIONS: The novel small-molecule P-selectin antagonist PSI-697 did not inhibit basal or stimulated platelet-monocyte aggregate formation in humans at the dose tested. Its clinical efficacy remains to be established. CLINICAL TRIAL REGISTRATION: URL: http://EudraCT.ema.europa.eu Unique identifier: 2007-005695-14.

In vivo and ex vivo imaging of amyloid-ß cascade aggregates with a Pronucleon™ peptide.

Accumulation of amyloid-ß (Aß) cascade aggregates is considered a hallmark of Alzheimer's disease (AD). Current dogma holds that the appearance of Aß oligomers and larger aggregates occur many years prior to plaque formation associated with the advanced and irreparable neurocognitive decline characteristic of AD. This premise is the impetus to identify these Aß precursor structures prior to advanced plaque development. The Pronucleon™ technology platform is comprised of a novel series of engineered peptides that provide a unique readout when associated with beta-rich fiber and oligomeric Aß. This technology has been applied to Ex Vivo tissue sections and In Vivo mouse models of AD to determine the potential utility of these synthetic peptides as potential imaging agents. In Ex Vivo studies, the Pronucleon™ peptide binds plaque like structures in brain sections obtained from transgenic mice overexpressing hAPP with both the human Swedish and London Aß mutations. In Vivo, Pronucleon™ peptide administered peripherally can localize to the brain and label plaques throughout the brain in transgenic mice. Taken together, the data suggest that Pronucleon™ could provide a new imaging tool for Aß cascade elements that precede advanced plaque and fibril formation, thereby advancing early diagnosis and treatment opportunities.

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.

In vitro and in vivo properties of a fully human IgG1 monoclonal antibody that combats multidrug resistant Pseudomonas aeruginosa.

The development of an anti-bacterial drug in the form of a monoclonal antibody (mAb) targeting an exposed virulence factor, represents an innovative therapeutic strategy. Consequently, a fully human IgG1 mAb (LST-007) targeting Pseudomonas aeruginosa (PA) flagellin type b was recombinantly expressed and characterized in vitro and in an infection model driven by a multidrug resistant (MDR) PA strain. LST-007 demonstrated a highly specific binding towards whole PA bacteria harboring flagellin type b and its recombinant counterpart, with a K(D) of 7.4x10(-10) M. In bioactivity assays, LST-007 or titers of Cmax sera derived from pharmacokinetic studies, markedly attenuated PA motility in an equipotent manner. In vivo, parenteral LST-007 (20 mg/kg) given as a single or double-dosing paradigm post-infection, afforded survival (up to 75% at Day 7) in a lethal model of pneumonia driven by the intratracheal (i.t.) instillation of an LD(80) of the MDR PA isolate. This protective effect was markedly superior to that of imipenem (30% survival at Day 7) and totally devoid with an irrelevant, human isotype mAb. These data lay credence that LST-007 may be a valuable adjunct to the limited list of anti-bacterials that can tackle MDR PA strains, thereby warranting its continued development for eventual clinical evaluation.

Lyophilised reconstituted human platelets increase thrombus formation in a clinical ex vivo model of deep arterial injury.

Platelets are the principal component of the innate haemostatic system that protect from traumatic bleeding. We investigated whether lyophilised human platelets (LHPs) could enhance clot formation within platelet-free and whole blood environments using an ex vivo model of deep arterial injury. Lyophilised human platelets were produced from stored human platelets and characterised using conventional, fluorescent and electron microscopic techniques. LHPs were resuspended in platelet-free plasma (PFP) obtained from citrated whole human blood to form final concentrations of 0, 20 and 200 x 109 LHPs/L. LHPs with recalcified PFP or whole blood were perfused through the chamber at low (212 s⁻¹) and high (1,690 s⁻¹) shear rates with porcine aortic tunica media as thrombogenic substrate. LHPs shared morphological characteristics with native human platelets and were incorporated into clot generated from PFP or whole blood. Histomorphometrically measured mean thrombus area increased in a dose-dependent manner following the addition of LHPs to PFP under conditions of high shear [704 µm² ± 186 µm² (mean ± SEM), 1,511 µm² ± 320 µm² and 2,378 µm² ± 315 µm², for LHPs at 0, 20 and 200 x 109 /l, respectively (p= 0.012)]. Lyophilised human platelets retain haemostatic properties when reconstituted in both PFP and whole blood, and enhance thrombus formation in a model of deep arterial injury. These data suggest that LHPs have the potential to serve as a therapeutic intervention during haemorrhage under circumstances of trauma, and platelet depletion or dysfunction.

Sputum matrix metalloproteinase-12 in patients with chronic obstructive pulmonary disease and asthma: relationship to disease severity.

BACKGROUND: Matrix metalloproteinase (MMP)-12 has been implicated in the pathogenesis of both chronic obstructive pulmonary disease (COPD) and asthma. The influence of disease severity on sputum MMP-12 concentrations and activity is not known. OBJECTIVES: We sought to examine the relationship between disease severity assessed by means of lung function and computed tomography (CT) and induced sputum MMP-12 concentrations and activity in patients with asthma and COPD. METHODS: In 208 subjects (109 asthmatic patients, smokers and never smokers, mild, moderate, and severe; 53 patients with COPD, smokers and exsmokers, mild, moderate, and severe; and 46 healthy control subjects, smokers and never smokers), we measured induced sputum MMP-12 concentrations (ELISA) and enzyme activity (fluorescence resonance energy transfer), sputum cell MMP12 mRNA expression (quantitative PCR [qPCR]), diffusing capacity for carbon monoxide (Dlco), and CT assessment of emphysema (percentage of low-attenuation areas at less -950 Hounsfield units). RESULTS: Sputum MMP-12 concentrations are greater in patients with COPD and smokers with asthma than in healthy nonsmokers (P = .003 and P = .035, respectively) but similar to those seen in healthy smokers. In patients with COPD, disease severity, when measured by means of CT-assessed emphysema, but not by means of spirometry or Dlco values, is directly associated with sputum MMP-12 concentrations and activity. In the asthma groups there is no significant association between disease severity and sputum MMP-12 concentrations or activity. CONCLUSIONS: Sputum MMP-12 concentrations and activity in patients with COPD are directly associated with the extent of emphysema measured by means of CT. This finding supports a role for MMP-12 in the pathogenesis of COPD and might suggest that blocking MMP-12 activity in patients with COPD could prevent the further development of emphysema.

Clinical validity of plasma and urinary desmosine as biomarkers for chronic obstructive pulmonary disease.

BACKGROUND: Although an increased concentration of degraded elastin products in patients with chronic obstructive pulmonary disease (COPD) has been reported for many years, its clinical validity and utility remain uncertain due to technical difficulties, small study groups and the unknown relationship between exacerbation and elastin degradation. The objectives of this study were to determine the validity of urinary and blood total desmosine/isodesmosine in patients with COPD and asthma and to evaluate their relationship to exacerbation status and lung function. METHODS: Urinary and blood desmosine levels were measured using validated isotopic dilution liquid chromatography-tandem mass spectrometry methods. RESULTS: 390 study participants were recruited from the following groups: healthy volunteers, stable asthma, stable and 'during an exacerbation' COPD. Compared with healthy non-smokers, we found increased urinary or blood desmosine levels in patients with COPD, but no differences in patients with asthma or healthy smokers. The elevation of urinary desmosine levels was associated with the exacerbation status in patients with COPD. Approximately 40% of patients with stable and 'during an exacerbation' COPD showed elevated blood desmosine levels. Blood desmosine levels were strongly associated with age and were negatively correlated with lung diffusing capacity for carbon monoxide. CONCLUSION: The results suggest that urinary desmosine levels are raised by exacerbations of COPD whereas blood desmosine levels are elevated in a subgroup of patients with stable COPD and reduced lung diffusing capacity. The authors speculate that a raised blood desmosine level may identify patients with increased elastin degradation suitable for targeted therapy. Future prospective studies are required to investigate this hypothesis.

Synergistic effect of liver X receptor activation and simvastatin on plaque regression and stabilization: an magnetic resonance imaging study in a model of advanced atherosclerosis.

AIMS: The aim of this study was to investigate the effects of liver X receptors (LXRs)-ß preferential activation by LXR-623 (WAY-252623), a novel LXRs agonist, on plaque progression/regression in a rabbit model of advanced atherosclerosis. METHODS AND RESULTS: Advanced atherosclerosis was induced in New Zealand White Rabbits (n= 41). At the end of atherosclerosis induction, animals underwent a baseline magnetic resonance imaging (MRI) and were randomized to receive LXR-623 (1.5, 5, or 15 mg/kg/day), simvastatin (5 mg/kg/day), or placebo. The combination of LXR-1.5/simvastatin was also tested. After a final MRI, animals were euthanized and their aortas processed for further analysis. Simvastatin significantly reduced lesion progression (-25%; P< 0.01) in comparison with the placebo group. A similar effect was observed in the LXR-1.5 and -5 groups. A significant regression (16.5%; P< 0.01) of existing atherosclerosis was observed in the LXR-1.5/simvastatin group. Histological and molecular analysis showed plaque stabilization in the animals treated with the LXR-1.5 and -5, and LXR-1.5/simvastatin. The effects of LXR-623 were observed in the presence of a non-significant effect on total-cholesterol, low-density lipoproteins-cholesterol, and triglyceride levels. CONCLUSION: The results of the present study show that LXR-623 significantly reduces the progression of atherosclerosis and induces plaque regression in combination with simvastatin. These observations could drive future development of novel anti-atherosclerotic therapeutic approaches.