Introduction Ubiquitous naturally occurring autoantibodies (nAbs) against alpha-synuclein (α-syn) may play important roles in the pathogenesis of Multiple System Atrophy (MSA) and Parkinson's disease (PD). Recently, we reported reduced high-affinity/avidity anti-α-syn nAbs levels in plasma from MSA and PD patients, along with distinct inter-group immunoglobulin (Ig)G subclass distributions. The extent to which these observations in plasma may reflect corresponding levels in the cerebrospinal fluid (CSF) is unknown. Methods Using competitive and indirect ELISAs, we investigated the affinity/avidity of CSF anti-α-syn nAbs as well as the CSF and plasma distribution of IgG subclasses and IgM nAbs in a cross-sectional cohort of MSA and PD patients. Results Repertoires of high-affinity/avidity anti-α-syn IgG nAbs were reduced in CSF samples from MSA and PD patients compared to controls. Furthermore, anti-α-syn IgM nAb levels were relatively lower in CSF and plasma from MSA patients but were reduced only in plasma from PD patients. Interestingly, anti-α-syn IgG subclasses presented disease-specific profiles both in CSF and plasma. Anti-α-syn IgG1, IgG2 and IgG3 levels were relatively increased in CSF of MSA patients, whereas PD patients showed increased anti-α-syn IgG2 and reduced anti-α-syn IgG4 levels. Conclusions Differences in the plasma/CSF distribution of anti-α-syn nAbs seem to be a common feature of synucleinopathies. Our data add further support to the notion that MSA and PD patients may have compromised immune reactivity towards α-syn. The differing α-syn-specific systemic immunological responses may reflect their specific disease pathophysiologies. These results are encouraging for further investigation of these immunological mechanisms in neurodegenerative diseases.

Aggregation of alpha-synuclein (α-syn) is considered to be the major pathological hallmark and driving force of Multiple System Atrophy (MSA) and Parkinson's disease (PD). Immune dysfunctions have been associated with both MSA and PD and recently we reported that the levels of natural occurring autoantibodies (NAbs) with high-affinity/avidity toward α-synuclein are reduced in MSA and PD patients. Here, we aimed to evaluate the plasma immunoglobulin (Ig) composition binding α-syn and other amyloidogenic neuropathological proteins, and to correlate them with disease severity and duration in MSA and PD patients. All participants were recruited from a single neurological unit and the plasma samples were stored for later research at the Bispebjerg Movement Disorder Biobank. All patients were diagnosed according to current consensus criteria. Using multiple variable linear regression analyses, we observed higher levels of anti-α-syn IgG1 and IgG3 NAbs in MSA vs. PD, higher levels of anti-α-syn IgG2 NAbs in PD compared to controls, whereas anti-α-syn IgG4 NAbs were reduced in PD compared to MSA and controls. Anti-α-syn IgM levels were decreased in both MSA and PD. Further our data supported that MSA patients' immune system was affected with reduced IgG1 and IgM global levels compared to PD and controls, with further reduced global IgG2 levels compared to PD. These results suggest distinct autoimmune patterns in MSA and PD. These findings suggest a specific autoimmune physiological mechanism involving responses toward α-syn, differing in neurodegenerative disease with overlapping α-syn pathology.

The present invention is based on antibodies that are both highly specific to tau p-s396 hyperphosphorylated pathogenic and highly specific for the sections labeled human retina, as well as methods of using these antibodies and their binding of tau fragments in the treatment of amyloidosis retinoid-related macular degeneration (ARMD) and glaucoma.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer’s disease (AD). However, the mechanisms that link disruption of the blood–brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377–395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases. Fibrin deposition occurs after the blood–brain barrier is breached. Akassoglou and colleagues generate a therapeutic monoclonal antibody that targets a cryptic fibrin epitope to suppress activation of innate immune responses in the CNS and diminish neuroinflammation.

ObjectiveCADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), caused by dominant mutations in the NOTCH3 receptor, is the most aggressive small vessel disease of the brain. A key feature of its pathogenesis is accumulation of the extracellular domain of NOTCH3 receptor (Notch3(ECD)) in small vessels, with formation of characteristic extracellular deposits termed granular osmiophilic material (GOM). Here, we investigated the therapeutic potential of a mouse monoclonal antibody (5E1) that specifically recognizes Notch3(ECD). MethodsThe binding affinity of 5E1 toward purified NOTCH3 was assessed using Octet analysis. The ability of 5E1 to bind Notch3(ECD) deposits in brain vessels and its effects on disease-related phenotypes were evaluated in the CADASIL mouse model, which overexpresses a mutant rat NOTCH3. Notch3(ECD) and GOM deposition, white matter lesions, and cerebral blood flow deficits were assessed at treatment initiation (10 weeks) and study completion (30 weeks) using quantitative immunohistochemistry, electron microscopy, and laser-Doppler flowmetry. Results5E1 antibody bound recombinant rat NOTCH3 with an average affinity of 317nM. A single peripheral injection of 5E1 robustly decorated Notch3(ECD) deposits in the brain vasculature. Chronic administration of 5E1 did not attenuate Notch3(ECD) or GOM deposition and was not associated with perivascular microglial activation. It also failed to halt the development of white matter lesions. Despite this, 5E1 treatment markedly protected against impaired cerebral blood flow responses to neural activity and topical application of vasodilators and normalized myogenic responses of cerebral arteries. InterpretationThis study establishes immunotherapy targeting Notch3(ECD) as a new avenue for disease-modifying treatment in CADASIL that warrants further development. Ann Neurol 2018;84:246-259

The 140-residue natively disordered protein α-synuclein (aSN) is a central component in the development of a family of neurodegenerative diseases termed synucleinopathies. This is attributed to its ability to form cytotoxic aggregates such as oligomers and amyloid fibrils. Consequently there have been intense efforts to avoid aggregation or reroute the aggregation pathway using pharmaceutical agents such as small molecules, chaperones and antibodies. aSN's lack of persistent structure in the monomeric state, as well as the multitude of different oligomeric and even different fibrillar states, makes it difficult to raise antibodies that would be efficacious in neutralizing all conformations of aSN. However, the C-terminal 20-40 residues of aSN are a promising epitope for antibody development. It is primarily disordered in both monomeric and aggregated forms, and an anti-C-terminal antibody will therefore be able to bind all forms. Furthermore, it might not interfere with the folding of aSN into membranes, which could be important for its physiological role. Here we report a screen of a series of monoclonal antibodies, which all target the C-terminal of aSN. According to dot blot analyses, different antibodies bound different forms of aSN with different preferences and showed reduced binding to monomeric compared to aggregated (oligomeric and fibrillary) aSN. Consequently they have different effects on aSN's ability to fibrillate and permeabilize membranes. Generally, the antibodies with strongest binding to aggregated aSN in dot blot, also inhibited fibrillation and membrane permeabilization the most, and promoted formation of amorphous aggregates surrounded by small and thin fibers. This suggests that the development of antibodies that targets the C-terminus, exposed in the aggregated forms of aSN, may be beneficial for improved immunotherapy against PD.

Background Parkinson’s’ disease (PD) and Multiple System Atrophy (MSA) are progressive brain disorders characterized by intracellular accumulations of α-synuclein and nerve cell loss in specific brain areas. This loss causes problems with movement, balance and/or autonomic functions. Naturally occurring autoantibodies (NAbs) play potentially an important role in clearing or/and blocking circulating pathological proteins. Little is known about the functional properties of anti-α-synuclein NAbs in PD and MSA, and there have been opposing reports regarding their plasma concentrations in these disorders. Methods We have investigated the apparent affinity of anti-α-synuclein NAbs in plasma samples from 46 PD patients, 18 MSA patients and 41 controls using competitive enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD) set-ups. Results We found that the occurrence of high affinity anti-α-synuclein NAbs in plasma from PD patients is reduced compared to healthy controls, and nearly absent in plasma from MSA patients. Also, levels of α-synuclein/NAbs immunocomplexes is substantially reduced in plasma from both patient groups. Further, cross binding of anti-α-synuclein NAbs with β- and γ-synuclein monomers suggest, the high affinity anti-α-synuclein plasma component, seen in healthy individuals, is directed mainly against C-terminal epitopes. Furthermore, we also observed reduced occurrence of high affinity anti-phosphorylated-α-synuclein NAbs in plasma from PD and MSA patients. Conclusions One interpretation implies that these patients may have impaired ability to clear and/or block the effects of pathological α-synuclein due to insufficient/absent concentration of NAbs and as such provides a rationale for testing immune-based therapeutic strategies directed against pathological α-synuclein. Following this interpretation, we can hypothesize that high affinity autoantibodies efficiently bind and clear potentially pathological species of α-synuclein in healthy brain, and that this mechanism is impaired or absent in PD and MSA patients.

Aged companion dogs with canine cognitive dysfunction (CCD) spontaneously develop varying degrees of progressive cognitive decline and particular neuropathological features correspondent to the changes associated with Alzheimer’s disease (AD) in humans. The aim of the present study was to character ize certain aspects of neuropathology and inflammatory markers related to aging and CCD in dogs in comparison with human AD. Fifteen brains from aged dogs with normal cognitive function, mild cognitive impairment, or CCD were investigated and compared with two control brains from young dogs and brain sections from human AD subjects. The neuropathological investigations included evaluation of amyloid-β (Aβ) plaque deposition (N-terminally truncated and pyroglutamyl-modified Aβ included), tau pathology, and inflammatory markers in prefrontal cortex. Cortical Aβ deposition was found to be only of the diffuse subtype as no dense-core or neuritic plaques were found. The Aβ deposition followed a progressive pattern in four maturation stages. Accumulation of the Aβ peptide was also observed in the vessel walls. Both immunohistochemically and biochemically measured levels of Aβ pathology in prefrontal cortex showed a consistent positive correlation to age but not to cognitive deficit severity. No evidence of neurofibrillary tau pathology was found. The level of pro-inflammatory cytokines was generally low and showed no significant association to cognitive status. The findings of the present study support the senescent dog with spontaneous cognitive dysfunction as a valuable non-transgenic model for further investigations of the molecular events involved in the neurodegenerative processes associated with aging and early stage AD, especially the Aβ-related pathology.

Immunotherapeutic approaches to treating Alzheimer's disease (AD) using vaccination strategies must overcome the obstacle of achieving adequate responses to vaccination in the elderly. Here we demonstrate for the first time that application of the Escherichia coli heat-labile enterotoxin adjuvant-laden immunostimulatory patches (LT-IS) dramatically enhances the onset and magnitude of immune responses to DNA- and protein-based vaccines for Alzheimer's disease following intradermal immunization via gene gun and conventional needles, respectively. Our studies suggest that the immune activation mediated by LT-IS offers improved potency for generating AD-specific vaccination responses that should be investigated as an adjuvant in the clinical arena.

Tissue-type plasminogen activator (t-PA) is the only thrombolytic treatment available for patients with acute ischaemic stroke. However, t-PA can increase permeability of the blood-brain barrier (BBB). Desmoteplase is a plasminogen activator derived from the common vampire bat, currently under clinical development for ischaemic stroke. We compared how t-PA and desmoteplase influenced BBB permeability using a human in vitro model where primary brain endothelial cells (BEC) and astrocytes are co-cultured on the opposite sides of a porous membrane. Permeability changes were evaluated 6 or 24h post-stimulation by passage of fluorescent albumin across the membrane. Under normoxic conditions, t-PA, but not desmoteplase, increased BBB permeability. Surprisingly, the ability of t-PA to affect the barrier was lost under conditions of oxygen-glucose deprivation (OGD). Addition of plasminogen re-sensitised the BBB to the action of t-PA under both normoxia and OGD, but did not affect the inert behaviour of desmoteplase, even when digested fibrinogen was added to ensure optimal plasmin generation. These observations coincided with plasmin-dependent changes in astrocyte and BEC morphology and disruption of tight junction proteins in BECs, specifically initiated by t-PA but not by desmoteplase. Finally, inhibition of plasmin post-stimulation with t-PA and plasminogen, especially within 2h, protected the BBB against t-PA-mediated barrier opening. Hence t-PA, but not desmoteplase, increases BBB permeability under both normoxic and OGD conditions in a reversible, plasmin-dependent process. The inability of desmoteplase to increase permeability despite its capacity to generate plasmin provides further support for its use as thrombolytic in patients with ischaemic stroke.

The Alzheimer's disease (AD) process is understood to involve the accumulation of amyloid plaques and tau tangles in the brain. However, attempts at targeting the main culprits, neurotoxic Aβ peptides, have thus far proven unsuccessful for improving cognitive function. Recent clinical trials with passively administrated anti-Aβ antibodies failed to slow cognitive decline in mild to moderate AD patients, but suggest that an immunotherapeutic approach could be effective in patients with mild AD. Using an AD mouse model (Tg2576), we tested the immunogenicity (cellular and humoral immune responses) and efficacy (AD-like pathology) of clinical grade Lu AF20513 vaccine. We found that Lu AF20513 induces robust "non-self" T-cell responses and the production of anti-Aβ antibodies that reduce AD-like pathology in the brains of Tg2576 mice without inducing microglial activation and enhancing astrocytosis or cerebral amyloid angiopathy. A single immunization with Lu AF20513 induced strong humoral immunity in mice with preexisting memory T-helper cells. In addition, Lu AF20513 induced strong humoral responses in guinea pigs and monkeys. These data support the translation of Lu AF20513 to the clinical setting with the aims of: (1) inducing therapeutically potent anti-Aβ antibody responses in patients with mild AD, particularly if they have memory T-helper cells generated after immunizations with conventional tetanus toxoid vaccine, and (2) preventing pathological autoreactive T-cell responses.

INTRODUCTION:As we found no recent published reports on the amount and kind of research published from Danish hospitals without university affiliation, we have found it relevant to conduct a bibliometric survey disclosing these research activities.MATERIAL AND METHODS:We retrieved all scientific papers published in the period 2000-2009 emanating from all seven Danish non-university hospitals in two regions, comprising 1.8 million inhabitants, and which were registered in a minimum of one of the three databases: PubMed MEDLINE, Thomson Reuters Web of Science and Elsevier's Scopus.RESULTS:In 878 of 1,252 papers, the first and/or last author was affiliated to a non-university hospital. Original papers made up 69% of these publications versus 86% of publications with university affiliation on first or last place. Case reports and reviews most frequently had authors from regional hospitals as first and/or last authors. The total number of publications from regional hospitals increased by 48% over the 10-year period. Publications were cited more often if the first or last author was from a university hospital and even more so if they were affiliated to foreign institutions. Cardiology, gynaecology and obstetrics, and environmental medicine were the three specialities with the largest number of regional hospital publications.CONCLUSION:A substantial number of scientific publications originate from non-university hospitals. Almost two thirds of the publications were original research published in international journals. Variations between specialities may reflect local conditions.FUNDING:not relevant.TRIAL REGISTRATION:not relevant.

Purpose: Sentinel lymph node (SLN) biopsy is an established method for the identification of early breast cancer metastases. SLN lymphoscintigraphy is pivotal in many cases, but the diagnostic value of early dynamic versus late static imaging procedures remains unclear. The objective of this study was to evaluate the diagnostic value of early dynamic versus late static imaging in the identification of SLN in breast cancer patients.Materials and Methods: A total of 104 consecutive female patients who were referred for SLN scintigraphy prior to breast cancer surgery were included in this retrospective study. All patients underwent both a 20-minute dynamic lymphoscintigraphy and a 2-hour static acquisition. The images were independently evaluated for the presence, location, and numbers of SLN and echelon nodes in a blinded and random manner by 2 trained observers. Any discrepancy was solved by a third party arbitrator.Results: SLN was identified in 101 of 104 (97%) patients on the static images versus 41 of 104 (39%) of the patients based on the dynamic acquisition. The combined use of dynamic and static images did not increase the diagnostic performance versus late lymphoscintigraphy alone.Conclusions: Identification of SLN by lymphoscintigraphy in breast cancer can be performed using a 2-hour static image alone.

Tissue viability imaging (TIVI) is a novel polarization spectroscopy method for assessing dermal vascular viability. The purpose of the present study was to compare TIVI with laser Doppler flowmetry (LDF) for assessment of pharmacologically induced vasodilation and vasoconstriction in human skin. Eight individual skin sites on the backs of seven healthy volunteers were randomized to receive an intradermal injection of prostaglandin E2 (PGE2, 10−6 to 10−9M), norepinephrine (NE, 10−5 to 10−7M), or vehicle. Vascular responses were measured by TIVI and LDF at the injection sites at 1-min intervals starting 2min before and ending 15min after the skin challenge. TIVI and LDF demonstrated significant dose-dependent and time-related vasodilator responses to PGE2 and vasoconstrictor responses to NE, respectively (p<0.001). The time course and dose–response functions for LDF and TIVI showed notable differences. Dose–response data showed a significant reduction in TIVI signal with NE 10-7M (10−6 NE with LDF) whereas PGE2 10−6M was required to elicit a significant increase in TIVI signal (10−8M PGE2 with LDF). TIVI demonstrated relative vascular response changes of 0.79 to 1.63 of baseline values at 15min with NE 10−5M or PGE2 10−6M compared to values of 0.59 to 8.38 with LDF. There was a modest though significant correlation between relative changes in vascular responses measured by the two methods (p<0.0001, r2=0.521). A Bland–Altman difference plot demonstrated significant underestimation of relative increase versus baseline measured by TIVI (r2=0.99, p<0.0001). We conclude that TIVI polarization spectroscopy is a sensitive method for measurement of NE-induced vascular responses but that it is less sensitive than LDF for measurement of the PGE2-induced reactions.

INTRODUCTION:Painful bone metastases are common in advanced prostate cancer. We report the clinical outcome after administration of Samarium-153 ((153)Sm), an emitter of beta-particles that concentrates in the areas of enhanced osteoblastic activity.METHODS:Twenty-two patients (median age 73 years) with metastatic, hormone-refractory prostate cancer received a single bolus infusion of (153)Sm (37 MBq/kg). All patients had painful bone metastases to more than one anatomical region, and most had inadequate pain relief to narcotic analgesics. Bone specific pain, analgesic score according to WHO, ECOG performance status, and blood count were evaluated before and up to 28 weeks after treatment.RESULTS:Median follow-up was six weeks (mean 14 weeks). Eleven patients died within the 28 week observation period (ten from terminal disease), and four patients had their observation period truncated. Median pain score was 56.3%, 50.0%, and 50.0% of baseline values at week 4 (n = 20), 16 (n = 10), and 28 (n = 7), respectively. A reduction of baseline pain score by 50% or more was observed in 50%, 70% and 71% of patients at week 4, 16, and 28, respectively. Hematological toxicity was mild and reversible in most cases.CONCLUSION:Administration of (153)Sm to prostate cancer patients with painful bone metastases offered clinical relevant pain relief with tolerable hematological toxicity.

Animal models have shown histamine to be released from the skin during the acute phase of a burn injury. The role of histamine during the early phase of thermal injuries in humans remains unclear.

Cancer patients in general have a high risk of venous thromboembolic events (VTE) driven not only by patient-related risk factors, but also risk factors related to the disease and anti-cancer therapies. Cancer patients with documented VTE have a notably worse outcome than non-cancer VTE patients. Since VTE is a highly preventable condition, it is striking that large surveys have shown significant underuse of VTE prophylaxis in surgical cancer patients and in medical cancer patients in particular. Recently, guidelines have been issued from European and American medical oncology societies and organizations for identification of cancer patients at risk, and the guidelines give recommendations for treatment of individual groups of cancer patients. This review summarizes the recommendations for VTE prophylaxis and treatment from the recent guidelines and reviews some outstanding issues in VTE prophylaxis and treatment of cancer patients.

Alzheimer's disease (AD) is hypothesized to result from elevated brain levels of beta-amyloid peptide (A beta) which is the main component of plaques found in AD brains and which cause memory impairment in mice. Therefore, there has been a major focus on the development of inhibitors of the A beta producing enzymes gamma-secretase and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1). In this study, we investigated the A beta-lowering effects of the BACE1 inhibitor LY2434074 in vitro and in vivo, comparing it to the well characterized gamma-secretase inhibitor LY450139. We sampled interstitial fluid A beta from awake APPswe/PS1dE9 AD mice by in vivo A beta microdialysis. In addition, we measured levels of endogenous brain A beta extracted from wildtype C57BL/6 mice. In our in vitro assays both compounds showed similar A beta-lowering effects. However, while systemic administration of LY450139 resulted in transient reduction of A beta in both in vivo models, we were unable to show any A beta-lowering effect by systemic administration of the BACE1 inhibitor LY2434074 despite brain exposure exceeding the in vitro IC50 value several fold. In contrast, significant reduction of 40-50% of interstitial fluid A beta and wildtype cortical A beta was observed when infusing LY2434074 directly into the brain by means of reverse microdialysis or by dosing the BACE1 inhibitor to p-glycoprotein (p-gp) mutant mice. The effects seen in p-gp mutant mice and subsequent data from our cell-based p-gp transport assay suggested that LY2434074 is a p-gp substrate. This may partly explain why BACE1 inhibition by LY2434074 has lower in vivo efficacy, with respect to decreased A beta 40 levels, compared with gamma-secretase inhibition by LY450139.

The primary factor driving pathogenesis in Alzheimers' disease (AD) according to the amyloid hypothesis is the accumulation of amyloid β-peptide (Aβ) in the brain. Aβ is generated by sequential processing of amyloid precursor protein (APP) by β- and γ-secretases. BACE-1 (≤-site A PP C leaving Enzyme 1) has been identified as the enzyme responsible for the β-secretase activity and is the key rate-limiting enzyme that initiates the formation of Aβ. BACE-1 appears as promising therapeutic target for amyloid-lowering strategies in AD. We established neuronal cultures from wildtype mice and characterized cell-permeable BACE-1 inhibitors by detection of full-length APP, sAPPβ (N-terminal product of APP following BACE-1 cleavage), sAPPα (N-terminal product of APP following α-secretase cleavage) and secreted Aβ1–40 from primary neurons and from brain homogenates. Cell-permeable BACE-1 inhibitors displayed a dose-dependent reduction of secreted Aβ1–40 with an IC50 in the nM range. Furthermore, we observed reduction in sAPPβ and accumulation of full-length APP. In contrast to γ-secretase inhibitors, BACE-1 inhibitors increased the amount of sAPPα. Additionally, we validated these neuronal models genetically by knock-down of BACE-1 expression by siRNA and observed inhibition of secreted Aβ1–40 in concert with reduction of sAPPβ. Finally, in vivo efficacy of a CNS active BACE-1 inhibitor was tested in wildtype mice. Reduction of soluble Aβ1–40 levels in brain homogenates was observed within 2 hours in concert with reduction of sAPPβ and elevation of sAPPα levels. Our characterization showed that in vitro data combined with data on brain exposure were predictive of in vivo activity. These studies confirm pharmacologically, that BACE-1 plays the predominant role in the β-site cleavage of APP and that inhibition of β-secretase activity could potentially redirect APP-processing via the non-amyloidogenic α-secretase pathway.