Emily J. Gilles

In vivo targeting of antibody fragments to the nervous system for Alzheimer’s disease immunotherapy and molecular imaging of amyloid plaques

Targeting therapeutic or diagnostic proteins to the nervous system is limited by the presence of the blood–brain barrier. We report that a F(ab′)2 fragment of a monoclonal antibody against fibrillar human Aβ42 that is polyamine (p)‐modified has increased permeability at the blood–brain barrier, comparable binding to the antigen, and comparable in vitro binding to amyloid plaques in Alzheimer’s disease (AD) transgenic mouse brain sections. Intravenous injection of the pF(ab′)24.1 in the AD transgenic mouse demonstrated efficient targeting to amyloid plaques throughout the brain, whereas the unmodified fragment did not. Removal of the Fc portion of this antibody derivative will minimize the inflammatory response and cerebral hemorrhaging associated with passive immunization and provide increased therapeutic potential for treating AD. Coupling contrast agents/radioisotopes might facilitate the molecular imaging of amyloid plaques with magnetic resonance imaging/positron emission tomography. The efficient delivery of immunoglobulin G fragments may also have important applications to other neurodegenerative disorders or for the generalized targeting of nervous system antigens.

Selective Contrast Enhancement of Individual Alzheimer’s Disease Amyloid Plaques Using a Polyamine and Gd-DOTA Conjugated Antibody Fragment Against Fibrillar Aβ42 for Magnetic Resonance Molecular Imaging

ABSTRACTPurposeThe lack of an in vivo diagnostic test for AD has prompted the targeting of amyloid plaques with diagnostic imaging probes. We describe the development of a contrast agent (CA) for magnetic resonance microimaging that utilizes the F(ab′)2 fragment of a monoclonal antibody raised against fibrillar human Aβ42MethodsThis fragment is polyamine modified to enhance its BBB permeability and its ability to bind to amyloid plaques. It is also conjugated with a chelator and gadolinium for subsequent imaging of individual amyloid plaquesResultsPharmacokinetic studies demonstrated this 125I-CA has higher BBB permeability and lower accumulation in the liver and kidney than F(ab′)2 in WT mice. The CA retains its ability to bind Aβ40/42 monomers/fibrils and also binds to amyloid plaques in sections of AD mouse brain. Intravenous injection of 125I-CA into the AD mouse demonstrates targeting of amyloid plaques throughout the cortex/hippocampus as detected by emulsion autoradiography. Incubation of AD mouse brain slices in vitro with this CA resulted in selective enhancement on T1-weighted spin-echo images, which co-register with individual plaques observed on spatially matched T2-weighted spin-echo imageConclusionsDevelopment of such a molecular probe is expected to open new avenues for the diagnosis of AD.

HH Domain of Alzheimer’s Disease Aβ Provides Structural Basis for Neuronal Binding in PC12 and Mouse Cortical/Hippocampal Neurons

A key question in understanding AD is whether extracellular Aβ deposition of parenchymal amyloid plaques or intraneuronal Aβ accumulation initiates the AD process. Amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce soluble Aβ which is then released into the brain interstitial fluid. Intraneuronal Aβ accumulation is hypothesized to predominate from the neuronal uptake of this soluble extracellular Aβ rather than from ER/Golgi processing of APP. We demonstrate that substitution of the two adjacent histidine residues of Aβ40 results in a significant decrease in its binding with PC12 cells and mouse cortical/hippocampal neurons. These substitutions also result in a dramatic enhancement of both thioflavin-T positive fibril formation and binding to preformed Aβ fibrils while maintaining its plaque-binding ability in AD transgenic mice. Hence, alteration of the histidine domain of Aβ prevented neuronal binding and drove Aβ to enhanced fibril formation and subsequent amyloid plaque deposition - a potential mechanism for removing toxic species of Aβ. Substitution or even masking of these Aβ histidine residues might provide a new therapeutic direction for minimizing neuronal uptake and subsequent neuronal degeneration and maximizing targeting to amyloid plaques.

Regional differences in MRI detection of amyloid plaques in AD transgenic mouse brain

Our laboratory and others have reported the ability to detect individual Alzheimers disease (AD) amyloid plaques in transgenic mouse brain in vivo by magnetic resonance imaging (MRI). Since amyloid plaques contain iron, most MRI studies attempting to detect plaques in AD transgenic mouse brain have employed techniques that exploit the paramagnetic effect of iron and have had mixed results. In the present study, using five-way anatomic spatial coregistration of MR images with three different histological techniques, properties of amyloid plaques in AD transgenic mouse brain were revealed that may explain their variable visibility in gradient- and spin-echo MR images. The results demonstrate differences in the visibility of plaques in the cortex and hippocampus, compared to plaques in the thalamus, by the different MRI sequences. All plaques were equally detectable by T(2)SE, while only thalamic plaques were reliably detectable by T(2)*GE pulse sequences. Histology revealed that cortical/hippocampal plaques have low levels of iron while thalamic plaques have very high levels. However, the paramagnetic effect of iron does not appear to be the sole factor leading to the rapid decay of transverse magnetization (short T(2)) in cortical/hippocampal plaques. Accordingly, MRI methods that rely less on iron magnetic susceptibility effect may be more successful for eventual human AD plaque MR imaging, particularly since human AD plaques more closely resemble the cortical and hippocampal plaques of AD transgenic mice than thalamic plaques.

Pharmacokinetics and Amyloid Plaque Targeting Ability of a Novel Peptide-Based Magnetic Resonance Contrast Agent in Wild-Type and Alzheimer's Disease Transgenic Mice

A novel magnetic resonance (MR) imaging contrast agent based on a derivative of human amyloid β (Aβ) peptide, Gd[N-4ab/Q-4ab]Aβ 30, was previously shown to cross the blood-brain barrier (BBB) and bind to amyloid plaques in Alzheimers disease (AD) transgenic mouse (APP/PS1) brain. We now report extensive plasma and brain pharmacokinetics of this contrast agent in wild-type (WT) and in APP/PS1 mice along with a quantitative summary of various physiological factors that govern its efficacy. Upon i.v. bolus administration, 125I-Gd[N-4ab/Q-4ab]Aβ 30 was rapidly eliminated from the plasma following a three-exponential disposition, which is saturable at higher concentrations. Nevertheless, the contrast agent exhibited rapid and nonsaturable absorption at the BBB. The brain pharmacokinetic profile of 125I-Gd[N-4ab/Q-4ab]Aβ 30 showed a rapid absorption phase followed by a slower elimination phase. No significant differences were observed in the plasma or brain kinetics of WT and APP/PS1 animals. Emulsion autoradiography studies conducted on WT and APP/PS1 mouse brain after an i.v. bolus administration of 125I-Gd[N-4ab/Q-4ab]Aβ 30 in vivo confirmed the brain pharmacokinetic data and also demonstrated the preferential localization of the contrast agent on the plaques for an extended period of time. These attributes of the contrast agent are extremely useful in providing an excellent signal/noise ratio during longer MR scans, which may be essential for obtaining a high resolution image. In conclusion, this study documents the successful plaque targeting of Gd[N-4ab/Q-4ab]Aβ 30 and provides crucial pharmacokinetic information to determine the dose, mode of administration, and scan times for future in vivo MR imaging of amyloid plaques in AD transgenic mice.

Transvenous Stimulation of the Renal Sympathetic Nerves Increases Systemic Blood Pressure: A Potential New Treatment Option for Neurocardiogenic Syncope

Neurocardiogenic syncope (NCS) is a common and sometimes debilitating disorder, with no consistently effective treatment. NCS is due to a combination of bradycardia and vasodilation leading to syncope. Although pacemaker devices have been tried in treating the bradycardic aspect of NCS, no device‐based therapy exists to treat the coexistent vasodilation that occurs. The renal sympathetic innervation has been the target of denervation to treat hypertension. We hypothesized that stimulation of the renal sympathetic nerves can increase blood pressure and counteract vasodilation in NCS.

Physiological and Biophysical Factors That Influence Alzheimer's Disease Amyloid Plaque Targeting of Native and Putrescine Modified Human Amyloid β40

Amyloid β40 (Aβ40) and its derivatives are being developed as probes for the ante-mortem diagnosis of Alzheimers disease. Putrescine-Aβ40 (PUT-Aβ40) showed better plaque targeting than the native Aβ40, which was not solely explained by the differences in their blood-brain-barrier (BBB) permeabilities. The objective of this study was to elucidate the physiological and biophysical factors influencing the differential targeting of Aβ40 and PUT-Aβ40. Despite better plaque-targeting ability 125I-PUT-Aβ40 was more rapidly cleared from the systemic circulation than amyloid β40 labeled with 125I (125I-Aβ40) after i.v. administration in mice. The BBB permeability of both compounds was inhibited by circulating peripheral Aβ40 levels. 125I-Aβ40 but not 125I-PUT-Aβ40 was actively taken up by the mouse brain slices in vitro. Only fluorescein-Aβ40, not fluorescein-PUT-Aβ40, was localized in the brain parenchymal cells in vitro. The metabolism of 125I-Aβ40 in the brain slices was twice as great as 125I-PUT-Aβ40. 125I-Aβ40 efflux from the brain slices was saturable and found to be 5 times greater than that of 125I-PUT-Aβ40. Thioflavin-T fibrillogenesis assay demonstrated that PUT-Aβ40 has a greater propensity to form insoluble fibrils compared with Aβ40, most likely due to the ability of PUT-Aβ40 to form β sheet structure more readily than Aβ40. These results demonstrate that the inadequate plaque targeting of Aβ40 is due to cellular uptake, metabolism, and efflux from the brain parenchyma. Despite better plaque targeting of PUTAβ40, its propensity to form fibrils may render it less suitable for human use and thus allow increased focus on the development of novel derivatives of Aβ with improved characteristics.

Novel balloon catheter device with pacing, ablating, electroporation, and drug-eluting capabilities for atrial fibrillation treatment--preliminary efficacy and safety studies in a canine model.

Pulmonary vein isolation is an established therapeutic procedure for symptomatic atrial fibrillation (AF). This approach involves ablation of atrial tissue just outside the pulmonary veins. However, patient outcomes are limited because of a high rate of arrhythmia recurrence. Ablation of electrically active tissue inside the pulmonary vein may improve procedural success, but is currently avoided because of the complication of postablation stenosis. An innovative device that can ablate inside pulmonary veins and prevent stenosis is a viable strategy to increase long-term efficacy. We have developed a prototypical balloon catheter device capable of nonthermal pulmonary vein ablation along with elution of an antifibrotic agent intended to eliminate arrhythmogenic substrate without the risk of stenosis and have demonstrated its functionality in 4 acute canine experiments. Further optimization of this device may provide an innovative means to simultaneously ablate and prevent pulmonary vein stenosis for improved AF treatment in humans.

Novel Bloodless Potassium Determination Using a Signal‐Processed Single‐Lead ECG

Background Hyper‐ and hypokalemia are clinically silent, common in patients with renal or cardiac disease, and are life threatening. A noninvasive, unobtrusive, blood‐free method for tracking potassium would be an important clinical advance. Methods and Results Two groups of hemodialysis patients (development group, n=26; validation group, n=19) underwent high‐resolution digital ECG recordings and had 2 to 3 blood tests during dialysis. Using advanced signal processing, we developed a personalized regression model for each patient to noninvasively calculate potassium values during the second and third dialysis sessions using only the processed single‐channel ECG. In addition, by analyzing the entire development groups first‐visit data, we created a global model for all patients that was validated against subsequent sessions in the development group and in a separate validation group. This global model sought to predict potassium, based on the T wave characteristics, with no blood tests required. For the personalized model, we successfully calculated potassium values with an absolute error of 0.36±0.34 mmol/L (or 10% of the measured blood potassium). For the global model, potassium prediction was also accurate, with an absolute error of 0.44±0.47 mmol/L for the training group (or 11% of the measured blood potassium) and 0.5±0.42 for the validation set (or 12% of the measured blood potassium). Conclusions The signal‐processed ECG derived from a single lead can be used to calculate potassium values with clinically meaningful resolution using a strategy that requires no blood tests. This enables a cost‐effective, noninvasive, unobtrusive strategy for potassium assessment that can be used during remote monitoring.

Direct pulmonary vein ablation with stenosis prevention therapy

The dominant location of electrical triggers for initiating atrial fibrillation (AF) originates from the muscle sleeves inside pulmonary veins (PVs). Currently, radiofrequency ablation (RFA) is performed outside of the PVs to isolate, rather than directly ablate these tissues, due to the risk of intraluminal PV stenosis.