Neutrophils enhance the clearance of systemic amyloid deposits in a murine amyloidoma model.

Introduction: Amyloid-specific antibodies have been shown to opsonize and enhance amyloid clearance in systemic amyloidosis mouse models. However, the immunological mechanisms by which amyloid is removed have not been clearly defined. Previous reports from preclinical studies suggest polymorphonuclear cells (, neutrophils) can affect amyloid removal.

Cardiac Amyloid Detection by PET/CT Imaging of Iodine (I) Evuzamitide (I-p5+14): A Phase 1/2 Study.

Background: The noninvasive detection of cardiac amyloid, as well as deposits in other vital organs, is critical for early diagnosis and quantitative disease monitoring. Positron emission tomography is an intrinsically quantitative imaging modality suitable for high-resolution amyloid detection.

Objectives: This study sought to evaluate the safety and efficacy of a novel amyloid-reactive peptide, designated p5+14, labeled with iodine-124 (I), in patients with diverse types of systemic amyloidosis.

Development and characterization of a prototypic pan-amyloid clearing agent - a novel murine peptide-immunoglobulin fusion.

Introduction: Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition.

Collagen inhibits phagocytosis of amyloid and and may act as a 'don't eat me' signal.

Background: Systemic amyloidosis refers to a group of protein misfolding disorders characterized by the extracellular deposition of amyloid fibrils in organs and tissues. For reasons heretofore unknown, amyloid deposits are not recognized by the immune system, and progressive deposition leads to organ dysfunction.

Methods: and phagocytosis assays were performed to elucidate the impact of collagen and other amyloid associated proteins (eg serum amyloid p component and apolipoprotein E) had on amyloid phagocytosis.

First in Human Evaluation and Dosimetry Calculations for Peptide I-p5+14-a Novel Radiotracer for the Detection of Systemic Amyloidosis Using PET/CT Imaging.

Purpose: Accurate diagnosis of amyloidosis remains a significant clinical challenge and unmet need for patients. The amyloid-reactive peptide p5+14 radiolabeled with iodine-124 has been developed for the detection of amyloid by PET/CT imaging. In a first-in-human evaluation, the dosimetry and tissue distribution of I-p5+14 peptide in patients with systemic amyloidosis.

Anticytomegalovirus Peptides Point to New Insights for CMV Entry Mechanisms and the Limitations of Screenings.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that can cause severe disease following exposure, during primary infection, or latent virus reactivation in immunocompromised populations. These complications lead to a 1- to 2-billion-dollar economic burden, making vaccine development and/or alternative treatments a high priority. Current treatments for HCMV include nucleoside analogues such as ganciclovir (GCV), foscarnet, and cidofovir.

Macrophage-Mediated Phagocytosis and Dissolution of Amyloid-Like Fibrils in Mice, Monitored by Optical Imaging.

Light chain-associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore.

Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy.

Amyloidosis is a malignant pathology associated with the formation of proteinaceous amyloid fibrils that deposit in organs and tissues, leading to dysfunction and severe morbidity. More than 25 proteins have been identified as components of amyloid, but the most common form of systemic amyloidosis is associated with the deposition of amyloid composed of Ig light chains (AL). Clinical management of amyloidosis focuses on reducing synthesis of the amyloid precursor protein.

A functional assay to identify amyloidogenic light chains.

Introduction: Multiple myeloma (MM) and light chain monoclonal gammopathy of undetermined significance (LCMGUS) are plasma cell disorders associated with the secretion of monoclonal free light-chain (LC) proteins. Due to the high concentrations of LC in circulation, both of these populations are at risk for developing LC-associated amyloidosis (AL) - a protein misfolding disease characterized by the deposition of LC protein fibrils in organs and tissues, leading to dysfunction and significant morbidity. At present, accurate identification of subjects at risk for developing amyloidosis is not possible, but with the advent of novel, amyloid-targeted therapies, identification of pre-symptomatic individuals is of clinical import.

A Peptide-Fc Opsonin with Pan-Amyloid Reactivity.

There is a continuing need for therapeutic interventions for patients with the protein misfolding disorders that result in systemic amyloidosis. Recently, specific antibodies have been employed to treat AL amyloidosis by opsonizing tissue amyloid deposits thereby inducing cell-mediated dissolution and organ improvement. To develop a pan-amyloid therapeutic agent, we have produced an Fc-fusion product incorporating a peptide, p5, which binds many if not all forms of amyloid.

IgG Conformer's Binding to Amyloidogenic Aggregates.

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aβ and transthyretin (TTR) than the monomeric antibody.

Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential.

Abeta(1-40) forms five distinct amyloid structures whose beta-sheet contents and fibril stabilities are correlated.

The ability of a single polypeptide sequence to grow into multiple stable amyloid fibrils sets these aggregates apart from most native globular proteins. The existence of multiple amyloid forms is the basis for strain effects in yeast prion biology, and might contribute to variations in Alzheimer's disease pathology. However, the structural basis for amyloid polymorphism is poorly understood.

Anti-amyloidogenic activity of IgGs contained in normal plasma.

Introduction: We have previously shown that a subpopulation of naturally occurring human IgGs has therapeutic potential for the amyloid-associated disorders. These molecules cross-react with conformational epitopes on amyloidogenic assemblies, including amyloid beta (Abeta) protein fibrils that are a pathological hallmark of Alzheimer's disease.

Materials And Methods: Using our europium-linked immunosorbant assay, we established that approximately 95% of 260 screened donor plasma samples had amyloid fibril-reactive IgGs and Abeta conformer-reactive IgGs with minimal binding to Abeta monomers.

Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease.

Plasticity of amyloid fibrils.

In experiments designed to characterize the basis of amyloid fibril stability through mutational analysis of the Abeta (1-40) molecule, fibrils exhibit consistent, significant structural malleability. In these results, and in other properties, amyloid fibrils appear to more resemble plastic materials generated from synthetic polymers than globular proteins. Thus, like synthetic polymers and plastics, amyloid fibrils exhibit both polymorphism, the ability of one polypeptide to form aggregates of different morphologies, and isomorphism, the ability of different polypeptides to grow into a fibrillar amyloid morphology.

Kinetics and thermodynamics of amyloid assembly using a high-performance liquid chromatography-based sedimentation assay.

Nonnative protein aggregation has been classically treated as an amorphous process occurring by colloidal coagulation kinetics and proceeding to an essentially irreversible endpoint often ascribed to a chaotic tangle of unfolded chains. However, some nonnative aggregates, particularly amyloid fibrils, exhibit ordered structures that appear to assemble according to ordered mechanisms. Some of these fibrils, as illustrated here with the Alzheimer's plaque peptide amyloid beta, assemble to an endpoint that is a dynamic equilibrium between monomers and fibrils exhibiting a characteristic equilibrium constant with an associated free energy of formation.

Distinct thymidine kinases encoded by cowpox virus and herpes simplex virus contribute significantly to the differential antiviral activity of nucleoside analogs.

Orthopoxviruses and herpesviruses are both large enveloped DNA viruses, yet these virus families exhibit very different susceptibilities to antiviral drugs. We investigated the activation of nucleoside analogs by the types I and II thymidine kinase (TK) homologs expressed by herpes simplex virus type 1 (HSV-1) and cowpox virus (CV). Antiviral activity against TK(-) and TK(+) strains of HSV-1 and CV was determined, and the ratio of the EC(50) values was used as a measurement of TK dependence.

Alanine scanning mutagenesis of Abeta(1-40) amyloid fibril stability.

We describe here an alanine scanning mutational analysis of the Abeta(1-40) amyloid fibril monitored by fibril elongation thermodynamics derived from critical concentration values for fibril growth. Alanine replacement of most residues in the amyloid core region, residues 15-36, leads to destabilization of the elongation step, compared to wild-type, by about 1kcal/mol, consistent with a major role for hydrophobic packing in Abeta(1-40) fibril assembly. Where comparisons are possible, the destabilizing effects of Ala replacements are generally in very good agreement with the effects of Ala replacements of the same amino acid residues in an element of parallel beta-sheet in the small, globular protein Gbeta1.

Oligoproline effects on polyglutamine conformation and aggregation.

There are nine known expanded CAG repeat neurological diseases, including Huntington's disease (HD), each involving the repeat expansion of polyglutamine (polyGln) in a different protein. Similar conditions can be induced in animal models by expression of the polyGln sequence alone or in other protein contexts. Besides the polyGln sequence, the cellular context of the disease protein, and the sequence context of the polyGln within the disease protein, are both likely to contribute to polyGln physical behavior and to pathology.

Structural properties of Abeta protofibrils stabilized by a small molecule.

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of Abeta protofibrils. Library screening yielded several molecules that stimulate Abeta aggregation.

Hydrogen-deuterium (H/D) exchange mapping of Abeta 1-40 amyloid fibril secondary structure using nuclear magnetic resonance spectroscopy.

We describe here details of the hydrogen-deuterium (H/D) exchange behavior of the Alzheimer's peptide Abeta(1)(-)(40), while it is a resident in the amyloid fibril, as determined by high-resolution solution NMR. Kinetics of H/D exchange in Abeta(1)(-)(40) fibrils show that about half the backbone amide protons exchange during the first 25 h, while the other half remain unexchanged because of solvent inaccessibility and/or hydrogen-bonded structure. After such a treatment for 25 h with D(2)O, fibrils of (15)N-enriched Abeta were dissolved in a mixture of 95% dimethyl sulfoxide (DMSO) and 5% dichloroacetic acid (DCA) and successive heteronuclear (1)H-(15)N HSQC spectra were collected to identify the backbone amides that did not exchange in the fibril.

Kinetic analysis of beta-amyloid fibril elongation.

We used surface plasmon resonance biosensors to evaluate the kinetics associated with the initial events of beta-amyloid (Abeta) fibril elongation. Fibrils were immobilized on the sensor chip surface and extended by exposure to soluble Abeta(1-40) peptide. The fibril surfaces bound Congo red, a marker for beta sheet structures, and exhibited a slow linear background decay that is consistent with fibril depolymerization.

Seeding specificity in amyloid growth induced by heterologous fibrils.

Over residues 15-36, which comprise the H-bonded core of the amyloid fibrils it forms, the Alzheimer's disease plaque peptide amyloid beta (Abeta) possesses a very similar sequence to that of another short, amyloidogenic peptide, islet amyloid polypeptide (IAPP). Using elongation rates to quantify seeding efficiency, we inquired into the relationship between primary sequence similarity and seeding efficiency between Abeta-(1-40) and amyloid fibrils produced from IAPP as well as other proteins. In both a solution phase and a microtiter plate elongation assay, IAPP fibrils are poor seeds for Abeta-(1-40) elongation, exhibiting weight-normalized efficiencies of only 1-2% compared with Abeta-(1-40) fibrils.

Mapping abeta amyloid fibril secondary structure using scanning proline mutagenesis.

Although the amyloid fibrils formed from the Alzheimer's disease amyloid peptide Abeta are rich in cross-beta sheet, the peptide likely also exhibits turn and unstructured regions when it becomes incorporated into amyloid. We generated a series of single-proline replacement mutants of Abeta(1-40) and determined the thermodynamic stabilities of amyloid fibrils formed from these mutants to characterize the susceptibility of different residue positions of the Abeta sequence to proline substitution. The results suggest that the Abeta peptide, when engaged in the amyloid fibril, folds into a conformation containing three highly structured segments, consisting of contiguous sequence elements 15-21, 24-28, and 31-36, that are sensitive to proline replacement and likely to include the beta-sheet portions of the fibrils.