Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

Fc N-glycosylation of autoreactive Aß antibodies as a blood-based biomarker for Alzheimer's disease.

INTRODUCTION: Naturally occurring autoantibodies (nAbs) against the pathologic isoform of amyloid beta (Aß42 ) were found in body fluids and indicate a systemic B cell response that may prevent Alzheimer's disease (AD) onset. N-glycans attached to immunoglobulin G-Fab/Fc fragments are features that influence their mechanism of action. The aim was to study the role of N-glycans in nAbs-Aß42 . METHODS: nAbs-Aß42 were isolated from AD patients and age-/sex-matched controls (n = 40) and immunoglobulin preparations. Glycosylated/deglycosylated nAbs-Aß42 were analyzed for their effect on Aß42 's aggregation, toxicity, and phagocytosis. Glycan structure was analyzed using matrix assisted laser desorption ionization time of flight mass spectrometry. RESULTS: Deglycosylation of nAbs-Aß42 had a major impact on Aß42 's aggregation/toxicity/phagocytosis. The glycan structure showed considerable differences between AD and controls. We were able to predict disease status with a sensitivity/specificity of 95% (confidence interval [CI]: 76.4-99.7%)/100% (CI: 83.9-100%). DISCUSSION: N-glycosylation has been identified as a critical attribute maintaining the beneficial effects of autoreactive Aß antibodies. These data have consequences for the development of monocloncal Aß antibodies and may open new avenues for diagnostics.

Repetitive head injuries in German American football players do not change blood-based biomarker candidates for CTE during a single season.

BACKGROUND: Repetitive traumatic brain injuries in American football players (AFPs) can lead to the neurodegenerative disease chronic traumatic encephalopathy (CTE). Clinical symptoms of CTE range from mood and behavioral changes to cognitive impairment, depression, and suicidality. So far, CTE cannot be diagnosed in vivo and thus specific diagnostic parameters for CTE need to be found, to observe and treat exposed athletes as early as possible. Promising blood-based biomarkers for CTE include total tau (tTau), hyperphosphorylated tau (pTau), neurofilament light protein (NF-L), glial fibrillary acidic protein (GFAP), amyloid-ß40 (Aß40), amyloid-ß42 (Aß42) and calcium-binding protein B (S100-B). Previous studies have found elevated levels of these biomarkers in subjects exposed to TBIs, whereas cerebrospinal fluid (CSF) levels of Aß40 and Aß42 were decreased in CTE subjects. Here, we investigated whether young AFPs already exhibit changes of these biomarker candidates during the course of a single active season. METHODS: Blood samples were drawn from n = 18 American Football Players before and after a full season and n = 18 male age-matched control subjects. The plasma titers of tTau, pTau, NF-L, GFAP, Aß40, Aß42 and S100-B were determined. Additionally, Apathy, Depression, and Health status as well as the concussion history and medical care were assessed and analyzed for correlations. RESULTS: Here we show, that the selected biomarker candidates for CTE do not change significantly during the seven-month period of a single active season of American Football in blood samples of AFPs compared to healthy controls. But interestingly, they exhibit generally elevated pTau titers. Furthermore, we found correlations of depression, quality-of-life, career length, training participation and training continuation with headache after concussion with various titers. CONCLUSION: Our data indicates, that changes of CTE marker candidates either occur slowly over several active seasons of American Football or are exclusively found in CSF. Nevertheless, our results underline the importance of a long-term assessment of these biomarker candidates, which might be possible through repeated blood biomarker monitoring in exposed athletes in the future.

The Avidity of Autoreactive Alpha-Synuclein Antibodies in Leucine-Rich Repeat Kinase 2 Mutation Carriers Is Not Altered Compared to Healthy Controls or Patients with Parkinson's Disease.

The accumulation and aggregation of alpha-synuclein (α-Syn) are pathological processes associated with Parkinson's disease, indicating that the regulation of protein is a crucial etiopathological mechanism. Interestingly, human serum and cerebrospinal fluid contain autoantibodies that recognize α-Syn. This potentially demonstrates an already existing, naturally decomposing, and protective system. Thus, quantitative or qualitative alterations, such as the modified antigen binding of so-called naturally occurring autoantibodies against α-Syn (nAbs-α-Syn), may induce disease onset and/or progression. We investigated the serum titers and binding characteristics of nAbs-α-Syn in patients suffering from sporadic Parkinson's disease (n = 38), LRRK2 mutation carriers (n = 25), and healthy controls (n = 22). METHODS: Titers of nAbs-α-Syn were assessed with ELISA and binding affinities and kinetics with SPR. Within the patient cohort, we discriminated between idiopathic and genetic (LRRK2-mutated) variants. RESULTS: ELISA experiments revealed no significant differences in nAbs-α-Syn serum titers among the three cohorts. Moreover, the α-Syn avidity of nAbs-α-Syn was also unchanged. CONCLUSIONS: Our findings indicate that nAbs-α-Syn concentrations or affinities in healthy and diseased persons do not differ, independent of mutations in LRRK2.

Alpha-synuclein Immunization Strategies for Synucleinopathies in Clinical Studies: A Biological Perspective.

The therapeutic strategies currently available for neurodegenerative diseases such as Parkinson's disease target only the symptoms of the disease. Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy can be summarized as synucleinopathies, as they are all characterized by the aggregation and accumulation of alpha-synuclein (α-syn) in the brain. Targeting α-syn by its formation and progression opens a new and promising disease-modifying therapeutic strategy. Thus, several distinct immunotherapeutic approaches are currently being evaluated in clinical trials. The objective of this article is to review, from a biological perspective, the most important properties of these passive and active immunotherapies to point out their relevance and suitability for the treatment of synucleinopathies.

Effects of a Multimerized Recombinant Autoantibody Against Amyloid-ß.

Alzheimer's disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved. As promising approach passive immunization strategies have thereby come forth. In this study, we focused on naturally occurring autoantibodies against the AD-associated peptide amyloid-ß. These antibodies have already reported to show beneficial functions in vitro and in mouse models of AD. However, their availability is limited due to their low abundance in peripheral blood. In a recent study, we were able to generate four recombinant antibodies against amyloid-ß. In the present study, we tested these antibodies in ELISA and SPR assays for their binding behavior and by aggregation- and phagocytosis assays as functional evidences to characterize their amyloid-ß-related neutralizing and clearance abilities. Further ex vivo assay on organotypic hippocampal slice cultures gave first evidence of microglial activation and inflammatory features. The tested recombinant antibodies in IgG format showed, in comparison to naturally occurring autoantibodies against amyloid-ß, insufficient binding capacities and -affinities. However, after conversion of one antibody into a single chain format multimerization of the scFv-Fc construct, the investigated binding capacity and -affinity showed improvements. Further functional assays predict a protective effect of this antibody. Although, all four recombinant antibodies showed binding to amyloid-ß, promising features were only detectable after conversion into a multimeric format. The multimeric scFv-Fc antibody exhibited thereby strong impact on amyloid-ß clearance and inhibition of oligomerization.

Encoding the Sequence of Specific Autoantibodies Against beta-Amyloid and alpha-Synuclein in Neurodegenerative Diseases.

There is no effective disease-modifying therapy for Alzheimer's or Parkinson's disease. As pathological hallmarks, the specific peptide amyloid-ß and the specific protein α-Synuclein aggregate and deposit in and destabilize neurons, which lead to their degeneration. Within the context of a potential immunization strategy for these diseases, naturally occurring autoantibodies could play a crucial role in treatment due to their ability to inhibit peptide/protein aggregation and mediate their phagocytosis. We developed a procedure to extract the genetic information of such amyloid-ß- and α-Synuclein- specific naturally occurring autoantibodies for future passive immunization strategies. We performed FACS-based single-cell sorting on whole blood donated from healthy individuals and performed single-cell RT-PCR analysis to amplify the coding sequences of antigen-binding regions of each antibody-secreting B1 cell. Sequences were further analyzed to determine CDR sequences and germline expression. Therefore, only low percentages of B1 cells obtained were amyloid-ß+/α-Synuclein+. After cell sorting, the variable regions of full IgGs were sequenced, demonstrating preferred usage of IGVH3 and IGKV1. The study we present herein describes an approaching for extracting and amplifying the sequence information of autoantibodies based on single-cell analysis of donated blood and producing a recombinant antibody pool for potential passive immunization against neurodegenerative diseases. We sorted a small pool of CD20+ CD27+ CD43+ CD69- IgG+ and Aß+/α-Syn+ B cells.

Extending the functional characteristics of naturally occurring autoantibodies against ß-Amyloid, Prion Protein and α-Synuclein.

BACKGROUND: Abnormal aggregation of proteins induces neuronal cell loss in neurodegenerative disorders such as Alzheimer's Disease, Creutzfeldt-Jakob Disease and Parkinson's Disease. Specific stimuli initialize conformational changes in physiological proteins, causing intra- or extracellular protein aggregation. We and other groups have identified naturally occurring autoantibodies (nAbs) as part of the human antibody pool that are able to prevent peptide fibrillation. These nAbs show a rescue effect following exposure of toxic aggregates on neurons, and they support microglial uptake of aggregated peptides. OBJECTIVE: Identification of a putative common epitope among the relevant proteins ß-Amyloid, α-Synuclein and Prion Protein for the respective nAbs. MATERIAL AND METHODS: Binding affinity between the aforementioned proteins and nAbs was tested by Dot Blot, ELISA and SPR-technology. Furthermore, the functionality of the protein-nAbs-complexes was studied in Thioflavin-T assays and microglial uptake experiments to study dependent inhibition of protein aggregation and enhancement of Fcγ mediated uptake by microglial cells. RESULTS: ß-Amyloid and Prion Protein fragment showed considerable binding affinity and functional efficacy for all applied nAbs. Thereby, no significant difference within the different nAbs was detected. In contrast, α-Synuclein was bound exclusively by nAbs-α-Synuclein, which was reproduced in all binding studies. Surprisingly, functional assays with α-Synuclein revealed no significant effect of nAbs in comparison to IVIg treatment. However, all applied nAbs as well as IVIg show a minimal functionality on the microglial uptake of α-Synuclein. CONCLUSION: nAbs-Aß, nAbs-PrP possibly display comparable affinity to the same structural epitope within Aß and PrP106-126 A117V whereas the epitope recognized by nAbs-α-Syn is only present in α-Syn. The structural similarity of Aß and PrP fragment promotes the outline for an efficient antibody for the treatment of several neurodegenerative disorders and extend the functional characteristics of the investigated nAbs.

Naturally Occurring Autoantibodies against Tau Protein Are Reduced in Parkinson's Disease Dementia.

BACKGROUND AND OBJECTIVE: Altered levels of naturally occurring autoantibodies (nAbs) against disease-associated neuronal proteins have been reported for neurodegenerative diseases, such as Alzheimer's (AD) and Parkinson's disease (PD). Recent histopathologic studies suggest a contribution of both Lewy body- and AD-related pathology to Parkinson's disease dementia (PDD). Therefore, we explored nAbs against alpha-synuclein (αS), tau and ß-amyloid (Aß) in PDD compared to cognitively normal PD patients. MATERIALS AND METHODS: We established three different ELISAs to quantify the nAbs-tau, nAbs-αS, and nAbs-Aß levels and avidity towards their specific antigen in serum samples of 18 non-demented (PDND) and 18 demented PD patients (PDD), which were taken from an ongoing multi-center cohort study (DEMPARK/LANDSCAPE). RESULTS: PDD patients had significantly decreased nAbs-tau serum levels compared to PDND patients (p = 0.007), whereas the serum titers of nAbs-αS and nAbs-Aß were unchanged. For all three nAbs, no significant differences in avidity were found between PDD and PDND cohorts. However, within both patient groups, nAbs-tau showed lowest avidity to their antigen, followed by nAbs-αS, and nAbs-Aß. Though, due to a high interassay coefficient of variability and the exclusion of many samples below the limit of detection, conclusions for nAbs-Aß are only conditionally possible. CONCLUSION: We detected a significantly decreased nAbs-tau serum level in PDD patients, indicating a potential linkage between nAbs-tau serum titer and cognitive deficits in PD. Thus, further investigation in larger samples is justified to confirm our findings.

Diacylglycerol mediates regulation of TASK potassium channels by Gq-coupled receptors.

The two-pore domain potassium (K2P) channels TASK-1 (KCNK3) and TASK-3 (KCNK9) are important determinants of background K(+) conductance and membrane potential. TASK-1/3 activity is regulated by hormones and transmitters that act through G protein-coupled receptors (GPCR) signalling via G proteins of the Gαq/11 subclass. How the receptors inhibit channel activity has remained unclear. Here, we show that TASK-1 and -3 channels are gated by diacylglycerol (DAG). Receptor-initiated inhibition of TASK required the activity of phospholipase C, but neither depletion of the PLC substrate PI(4,5)P2 nor release of the downstream messengers IP3 and Ca(2+). Attenuation of cellular DAG transients by DAG kinase or lipase suppressed receptor-dependent inhibition, showing that the increase in cellular DAG-but not in downstream lipid metabolites-mediates channel inhibition. The findings identify DAG as the signal regulating TASK channels downstream of GPCRs and define a novel role for DAG that directly links cellular DAG dynamics to excitability.