Background: Amyloid-related imaging abnormalities (ARIA) have been reported with some anti-amyloid-β (Aβ) immunotherapy trials. They are detected with magnetic resonance imaging (MRI) and thought to represent transient accumulation of fluid/edema (ARIA-E) or microhemorrhages (ARIA-H). Although the clinical significance and pathophysiology are unknown, it has been proposed that anti-Aβimmunotherapy may affect blood-brain barrier (BBB) integrity. Objective: To examine vascular integrity in aged (12–16 months) PDAPP and wild type mice (WT), we performed a series of longitudinal in vivo MRI studies. Methods: Mice were treated on a weekly basis using anti-Aβimmunotherapy (3D6) and follow up was done longitudinally from 1–12 weeks after treatment. BBB-integrity was assessed using both visual assessment of T1-weighted scans and repeated T1 mapping in combination with gadolinium (Gd-DOTA). Results: A subset of 3D6 treated PDAPP mice displayed numerous BBB disruptions, whereas WT and saline-treated PDAPP mice showed intact BBB integrity under the conditions tested. In addition, the contrast induced decrease in T1 value was observed in the meningeal and midline area. BBB disruption events occurred early during treatment (between 1 and 5 weeks), were transient, and resolved quickly. Finally, BBB-leakages associated with microhemorrhages were confirmed by Perls'Prussian blue histopathological analysis. Conclusion: Our preclinical findings support the hypothesis that 3D6 leads to transient leakage from amyloid-positive vessels. The current study has provided valuable insights on the time course of vascular alterations during immunization treatment and supports further research in relation to the nature of ARIA and the utility of in vivo repeated T1 MRI as a translational tool.
Amyloid-related imaging abnormalities (ARIA) have been reported in Alzheimer disease (AD) and in cerebral amyloid angiopathy patients. Moreover, an increased risk of ARIA upon β-amyloid (Aβ) immunotherapy has been reported in both clinical and preclinical studies (Zago et al.,2013; Salloway et al.,2014). Although the underlying pathophysiological mechanisms are unknown, it has been proposed that reduced vascular integrity caused by aggressive lowering of central and vascular Aβ might be involved. In the current study we applied T 1 mapping MRI, in combination with Gadolinium (Gd, Dotarem®) injection, to probe for blood-brain-barrier (BBB) integrity applying 3D6 (the murine equivalent of bapineuzumab) immunotherapy in PDAPP mice. PDAPP mice (12 months) with established amyloid pathology were treated weekly with either 3D6 or saline. Wild type animals served as controls and were treated with saline for a period of 3 weeks. T1 weighted images were acquired weekly on a 7T small animal MRI system, before (baseline) and after intravenous administration of Gd (0.2 mmol kg-1). T1 values were quantified from cortical, hippocampal and thalamic ROIs. Gd infusion significantly decreased T 1 values (∼800ms) in the jaw muscle, whereas brain regions - due to the BBB - showed only a small reduction in T 1, which was most pronounced in the cortex (50-100ms). No differences in cortical T 1 values were observed at baseline. Two mice (out of 12) showed a considerable larger drop in T 1 values (∼300ms) after 2-3 weeks treatment with 3D6, which was not observed in the saline-treated PDAPP group. This suggests increased vascular permeability, probably induced by the clearance of vascular Aβ. In a previous study, similar drops in T 1 values were observed in 2 out of 8 animals. Moreover, in both studies the augmented permeability normalized afterwards. This study shows for the first time in vivo increased cerebral vascular permeability upon Aβ immunotherapy in a subset of PDAPP mice. In addition, the time course of these events was documented. Moreover, the methodology may be useful to assess ARIA risk in the development of other Aβ lowering compounds. Hence, our findings are important to increase our understanding of ARIA observed in AD patients upon Aβ lowering therapies.
Neurodegenerative disorders such as Parkinson's Disease (PD), PD dementia (PDD) and Dementia with Lewy bodies (DLB) are characterized by progressive accumulation of α-synuclein (α-syn) in neurons. Recent studies have proposed that neuron-to-neuron propagation of α-syn plays a role in the pathogenesis of these disorders. We have previously shown that antibodies against the C-terminus of α-syn reduce the intra-neuronal accumulation of α-syn and related deficits in transgenic models of synucleinopathy, probably by abrogating the axonal transport and accumulation of α-syn in in vivo models. Here, we assessed the effect of passive immunization against α-syn in a new mouse model of axonal transport and accumulation of α-syn. For these purpose, non-transgenic, α-syn knock-out and mThy1-α-syn tg (line 61) mice received unilateral intra-cerebral injections with a lentiviral (LV)-α-syn vector construct followed by systemic administration of the monoclonal antibody 1H7 (recognizes amino acids 91-99) or control IgG for 3 months. Cerebral α-syn accumulation and axonopathy was assessed by immunohistochemistry and effects on behavior were assessed by Morris water maze. Unilateral LV-α-syn injection resulted in axonal propagation of α-syn in the contra-lateral site with subsequent behavioral deficits and axonal degeneration. Passive immunization with 1H7 antibody reduced the axonal accumulation of α-syn in the contra-lateral side and ameliorated the behavioral deficits. Together this study supports the notion that immunotherapy might improve the deficits in models of synucleinopathy by reducing the axonal propagation and accumulation of α-syn. This represents a potential new mode of action through which α-syn immunization might work.
Melanoma cell adhesion molecule (MCAM; CD146) is expressed on the surfaces of a small population of T cells that have the capacity to produce a multitude of cytokines; further, expression of MCAM is a defining characteristic of TH17 cells. As such, MCAM is hypothesized to be central to the pathogenesis of numerous autoimmune disorders, including psoriasis and psoriatic arthritis. Laminin α4 (LAMA4), the primary MCAM ligand, promotes T-cell transmigration into sites of inflammation. Thus, inhibition of MCAM/LAMA4 binding may limit cell infiltration and subsequent pathogenic inflammation. PRX003, a monoclonal antibody designed to bind an MCAM epitope critical for LAMA4 interactions, blocks adhesion of MCAM-expressing cells to LAMA4 rather than targeting an individual cytokine, thus preventing the infiltration of cells responsible for disease pathogenesis.
Objectives
Study objectives were twofold: (1) to demonstrate preclinical proof of concept for anti-MCAM antibodies in mice overexpressing RAC1, an aggressive transgenic model of psoriasis and (2) to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of PRX003 in healthy human subjects.
Methods
The effects of anti-MCAM antibodies (murine homolog of PRX003) on the MCAM expression of circulating immune cells and on disease progression were assessed in RAC1 mice treated 3×/week with anti-MCAM antibodies (0.1 or 10 mg/kg) or isotype control from postnatal day 7 (when disease progression is evident) through postnatal day 34. In a first-in-human, randomized, double-blind, placebo-controlled, phase 1, single ascending dose-escalation study (NCT02458677), PRX003 was administered by intravenous infusion over approximately 60 minutes. Five escalating dose cohorts received 0.3, 1.0, 3.0, 10, or 30 mg/kg of PRX003 or placebo (6 subjects randomized to PRX003 and 2 to placebo per cohort) and were subsequently monitored in an inpatient unit for 24 hours and by periodic follow-up for 12 weeks. PD measurements in humans included MCAM expression on circulating T-lymphocytes and serum soluble MCAM.
Results
Anti-MCAM antibodies demonstrated target-mediated binding to circulating immune cells after administration to RAC1 transgenic mice. Furthermore, anti-MCAM antibody-treated mice experienced marked reductions in MCAM expression levels on circulating cells, resulting in diminished (by 40% to 60%) psoriatic disease progression, as evidenced by less erythema, scaling, and skin thickness than were observed in RAC1 transgenic mice treated with an isotype control antibody. PRX003 may thus have a beneficial impact on inflammatory diseases in humans. Data on the safety, tolerability, serum PRX003 PK, and immunogenicity of PRX003 in healthy human subjects, including PK/PD modeling of markers of target engagement, will be presented for the first time.
Conclusions
The results of these studies and of previously published reports support further examination of PRX003 in patients with inflammatory disease. A phase 1 multiple ascending dose trial of PRX003 in patients with plaque psoriasis (NCT02630901) is planned.
Disclosure of Interest
M. Koller Shareholder of: Prothena, Employee of: Prothena, K. Flanagan Shareholder of: Prothena, Employee of: Prothena, M. Skov Shareholder of: Prothena, Employee of: Prothena, R. Goldblum Consultant for: Prothena, Vascular BIogenics Limited, Sophiris, S. Griffith Consultant for: Prothena, R. Barbour Shareholder of: Prothena, Employee of: Prothena, N. Ehsani-Chimeh: None declared, M. Marinkovich: None declared, W. Zago Shareholder of: Prothena, Employee of: Prothena, T. Yednock Shareholder of: Prothena, Consultant for: Prothena, G. Kinney Shareholder of: Prothena, Consultant for: Janssen, Employee of: Prothena, D. Ness Employee of: Prothena
Melatonin, the pineal hormone produced during the dark phase of the light-dark cycle, modulates neuronal acetylcholine receptors located presynaptically on nerve terminals of the rat vas deferens. Recently we showed the presence of high affinity nicotine-binding sites during the light phase, and low and high affinity binding sites during the dark phase. The appearance of the low affinity binding sites was due to the nocturnal melatonin surge and could be mimicked by exposure to melatonin in vitro. The aim of the present research was to identify the receptor subtypes responsible for the functional response during the light and the dark phase. The rank order of potency of agonists was dimethylphenylpiperazinium (DMPP) = cytisine > nicotine > carbachol and DMPP = nicotine = cytisine > carbachol, during the light and dark phase, respectively, due to an increase in apparent affinity for nicotine. Mecamylamine similarly blocked the DMPP response during the light and the dark phase, while the response to nicotine was more efficiently blocked during the light phase. In contrast, methyllycaconitine inhibited the nicotine-induced response only at 21:00 h. Since a7 nicotinic acetylcholine receptors (nAChRs) have low affinity for nicotine in binding assays, we suggest that a mixed population composed of a3ß4 - plus a7-bearing nAChR subtypes is present at night. This plasticity in receptor subtypes is probably driven by melatonin since nicotine-induced contraction in organs from animals sacrificed at 15:00 h and incubated with melatonin (100 pg/ml, 4 h) is not totally blocked by mecamylamine. Thus melatonin, by acting directly on the short adrenergic neurons that innervate the rat vas deferens, induces the appearance of the low affinity binding site, probably an a7 nAChR subtype.
We characterized autonomic pilomotor and sudomotor skin function in early Parkinson's disease (PD) longitudinally.We enrolled PD patients (Hoehn and Yahr 1-2) and healthy controls from movement disorder centers in Germany, Hungary, and the United States. We evaluated axon-reflex responses in adrenergic sympathetic pilomotor nerves and in cholinergic sudomotor nerves and assessed sympathetic skin response (SSR), predominantly parasympathetic neurocardiac function via heart rate variability, and disease-related symptoms at baseline, after 2 weeks, and after 1 and 2 years.gov: NCT03043768.We included 38 participants: 26 PD (60% females, aged 62.4 ± 7.4 years, mean ± SD) and 12 controls (75% females, aged 59.5 ± 5.8 years). Pilomotor function was reduced in PD compared to controls at baseline when quantified via spatial axon-reflex spread (78 [43-143], median [interquartile range] mm2 vs. 175 [68-200] mm2 , p = 0.01) or erect hair follicle count in the axon-reflex region (8 [6-10] vs. 11 [6-16], p = 0.008) and showed reliability absent any changes from baseline to Week 2 (p = not significant [ns]). Between-group differences increased over the course of 2 years (p < 0.05), although no decline was observed within groups (p = ns). Pilomotor impairment in PD correlated with motor symptoms (rho = -0.59, p = 0.017) and was not lateralized (p = ns). Sudomotor axon-reflex and neurocardiac function did not differ between groups (p = ns), but SSR was reduced in PD (p = 0.0001).Impairment of adrenergic sympathetic pilomotor function and SSR in evolving PD is not paralleled by changes to cholinergic sudomotor function and parasympathetic neurocardiac function, suggesting a sympathetic pathophysiology. A pilomotor axon-reflex test might be useful to monitor PD-related pathology.
Cerebral amyloid angiopathy is a common neuropathological feature of Alzheimer's disease and is characterized by vascular deposition of fibrillar amyloid β-protein (VAβ). Vascular structural changes are associated with VAβ deposits, including localized loss of smooth muscle cells (SMC) and changes in extracellular matrix (ECM). Passive immunization with antibodies to the Aβ N-terminus (3D6, aa 1–5) has been shown to effectively reduce amyloid deposition in the vasculature of PDAPP transgenic mice. In the present study we characterized structural changes induced by amyloid on SMC and ECM of PDAPP mouse vessels and assessed the effects of passive immunotherapy. Mice were immunized weekly for either 3 or 9 months with 1 or 3 mg/Kg of 3D6 antibody. High-resolution, quantitative IHC analyses of vascular components (α-actin for SMC and collagen-IV for ECM) were performed on meningeal vessels from the sagittal sinus, where VAβ deposition is prominent (∼70% of vessels affected). Microhemorrhage events were monitored by hemosiderin detection or ferritin immunohistochemistry. We found that changes in the vascular wall are invariably associated with VAβ, and they included both degeneration (decreased thickness) and hyperplasia/hypertrophy (increased thickness) of SMC and ECM. These two contrasting findings were often observed in the same vessel and were not present in wild type animals or PDAPP vessels lacking amyloid. The extreme degrees of thickening and thinning of the SM resulted in a widely variable vascular phenotype in untreated PDAPP mice. Passive immunotherapy restored the pattern of vascular SMC and ECM thicknesses and reduced the phenotypic variability in a dose- and time-dependent manner, with the high dose of 3D6 reaching control levels (wild type) at 9 months. Although the incidence of microhemorrhage increased in the 3-month group, it reduced to control levels after 9 months of treatment. Our results suggest that passive immunotherapy allows the recovery of meningeal vessels from amyloid-induced structural changes. Furthermore, the treatment-related increase in microhemorrhage appears to be a transient event that resolves during VAβ clearance. Mechanisms of repair may be triggered by VAβ removal, which ultimately lead to recovery from vascular dysfunction. Experiments to assess this possibility are in progress.
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