Small Heat Shock Proteins, Amyloid Fibrils, and Nicotine Stimulate a Common Immune Suppressive Pathway with Implications for Future Therapies.

The α7 nicotinic acetylcholine receptor (α7nAChR) is central to the anti-inflammatory function of the vagus nerve in a physiological mechanism termed the inflammatory reflex. Studies on the inflammatory reflex have been instrumental for the current development of the field of bioelectronic medicine. An independent investigation of the biological role of αB-crystallin (HspB5), the most abundant gene transcript present in active multiple sclerosis lesions in human brains, also led to α7nAChR.

Identification of a common immune regulatory pathway induced by small heat shock proteins, amyloid fibrils, and nicotine.

Although certain dogma portrays amyloid fibrils as drivers of neurodegenerative disease and neuroinflammation, we have found, paradoxically, that amyloid fibrils and small heat shock proteins (sHsps) are therapeutic in experimental autoimmune encephalomyelitis (EAE). They reduce clinical paralysis and induce immunosuppressive pathways, diminishing inflammation. A key question was the identification of the target for these molecules.

Genetic background modulates outcome of therapeutic amyloid peptides in treatment of neuroinflammation.

Amyloid hexapeptide molecules are effective in the treatment of the murine model of neuroinflammation, known as experimental autoimmune encephalomyelitis (EAE). Efficacy however differs between two inbred mouse strains, C57BL/6J (B6) and C57BL/10SnJ (B10). Amyloid hexapeptide treatments improved the clinical outcomes of B6, but not B10 mice, indicating that genetic background influences therapeutic efficacy.

Amyloid fibrils activate B-1a lymphocytes to ameliorate inflammatory brain disease.

Amyloid fibrils composed of peptides as short as six amino acids are therapeutic in experimental autoimmune encephalomyelitis (EAE), reducing paralysis and inflammation, while inducing several pathways of immune suppression. Intraperitoneal injection of fibrils selectively activates B-1a lymphocytes and two populations of resident macrophages (MΦs), increasing IL-10 production, and triggering their exodus from the peritoneum. The importance of IL-10-producing B-1a cells in this effective therapy was established in loss-of-function experiments where neither B-cell-deficient (μMT) nor IL10(-/-) mice with EAE responded to the fibrils.

Self-Assembling Peptides Form Immune Suppressive Amyloid Fibrils Effective in Autoimmune Encephalomyelitis.

Amyloidogenic proteins have long been linked to neurodegenerative diseases. However, amyloid fibrils composed of six amino acids are protective in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). The reduction of pro-inflammatory cytokines, decrease in the number of inflammatory foci in the parenchyma and meninges of the brain and spinal cord, and amelioration of the neurological signs of EAE when amyloid fibril-forming hexapeptides are administered reveal that some fibrils provide benefit.

Mechanisms of action of therapeutic amyloidogenic hexapeptides in amelioration of inflammatory brain disease.

Amyloid fibrils composed of peptides as short as six amino acids are effective therapeutics for experimental autoimmune encephalomyelitis (EAE). Immunosuppression arises from at least two pathways: (1) expression of type 1 IFN by pDCs, which were induced by neutrophil extracellular traps arising from the endocytosis of the fibrils; and (2) the reduced expression of IFN-γ, TNF, and IL-6. The two independent pathways stimulated by the fibrils can act in concert to be immunosuppressive in Th1 indications, or in opposition, resulting in inflammation when Th17 T lymphocytes are predominant.

Janus faces of amyloid proteins in neuroinflammation.

Amyloid forming molecules are generally considered harmful. In Alzheimer's Disease two amyloid molecules Aβ A4 and tau vie for consideration as the main pathogenic culprit. But molecules obey the laws of chemistry and defy the way we categorize them as humans with our well-known proclivities to bias in our reasoning.

CRYAB modulates the activation of CD4+ T cells from relapsing-remitting multiple sclerosis patients.

Background: Suppression of activation of pathogenic CD4(+) T cells is a potential therapeutic intervention in multiple sclerosis (MS). We previously showed that a small heat shock protein, CRYAB, reduced T cell proliferation, pro-inflammatory cytokine production and clinical signs of experimental allergic encephalomyelitis, a model of MS.

Objective: We assessed whether the ability of CRYAB to reduce the activation of T cells translated to the human disease.

Amyloid fibrils composed of hexameric peptides attenuate neuroinflammation.

The amyloid-forming proteins tau, αB crystallin, and amyloid P protein are all found in lesions of multiple sclerosis (MS). Our previous work established that amyloidogenic peptides from the small heat shock protein αB crystallin (HspB5) and from amyloid β fibrils, characteristic of Alzheimer's disease, were therapeutic in experimental autoimmune encephalomyelitis (EAE), reflecting aspects of the pathology of MS. To understand the molecular basis for the therapeutic effect, we showed a set of amyloidogenic peptides composed of six amino acids, including those from tau, amyloid β A4, major prion protein (PrP), HspB5, amylin, serum amyloid P, and insulin B chain, to be anti-inflammatory and capable of reducing serological levels of interleukin-6 and attenuating paralysis in EAE.

Piet Mondrian's trees and the evolution in understanding multiple sclerosis, Charcot Prize Lecture 2011.

Four questions were posed about multiple sclerosis (MS) at the 2011 Charcot Lecture, Oct. 22, 2011. 1.

Chaperone activity of small heat shock proteins underlies therapeutic efficacy in experimental autoimmune encephalomyelitis.

To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis.

Therapeutic effects of systemic administration of chaperone αB-crystallin associated with binding proinflammatory plasma proteins.

The therapeutic benefit of the small heat shock protein αB-crystallin (HspB5) in animal models of multiple sclerosis and ischemia is proposed to arise from its increased capacity to bind proinflammatory proteins at the elevated temperatures within inflammatory foci. By mass spectral analysis, a common set of ∼70 ligands was precipitated by HspB5 from plasma from patients with multiple sclerosis, rheumatoid arthritis, and amyloidosis and mice with experimental allergic encephalomyelitis. These proteins were distinguished from other precipitated molecules because they were enriched in the precipitate as compared with their plasma concentrations, and they exhibited temperature-dependent binding.

Systemic augmentation of alphaB-crystallin provides therapeutic benefit twelve hours post-stroke onset via immune modulation.

Tissue plasminogen activator is the only treatment option for stroke victims; however, it has to be administered within 4.5 h after symptom onset, making its use very limited. This report describes a unique target for effective treatment of stroke, even 12 h after onset, by the administration of αB-crystallin (Cryab), an endogenous immunomodulatory neuroprotectant.

Chaperone activity of α B-crystallin is responsible for its incorrect assignment as an autoantigen in multiple sclerosis.

For 15 y, α B-crystallin (heat shock protein [Hsp] B5) has been labeled an autoantigen in multiple sclerosis (MS) based on humoral and cellular responses found in humans and animal models. However, there have been several scientific inconsistencies with this assignment, ranging from studies demonstrating small differences in anticrystallin responses between patients and healthy individuals to the inability of crystallin-specific T cells to induce symptoms of experimental allergic encephalomyelitis in animal models. Experiments in this article demonstrate that the putative anti-HspB5 Abs from 23 MS patients cross-react with 7 other members of the human small Hsp family and were equally present in normal plasma.

Quantitation of cellular and topical uptake of luciferin-oligoarginine conjugates.

A major challenge confronting the further advancement of using molecular transporters conjugated to small molecular weight therapeutics in the clinic is the development of linkers that would allow for the controllable release of a free drug/probe only after cell entry. Development of assays that would allow for the rapid real-time quantification of transporter conjugate uptake and cargo release in cells and animals would greatly help in their development. In this chapter, we describe a imaging method that quantitatively measures transporter conjugate uptake and cargo release in real-time in both cell culture and animal models.

Binding of biotinylated peptides to MHC class II proteins on cell surfaces.

This unit describes a simple, reproducible, and semiquantitative assay for measuring ligand binding to cell surface receptors. This approach is useful as a quick screen for peptide binding to MHC class II proteins that avoids the need to purify the MHC class II molecules and that uses small numbers of cells. The basic protocol describes procedures for incubating cells expressing the MHC molecule of interest with a biotinylated peptide, washing off excess peptide, and detecting the bound peptides by staining with fluorescently labeled avidin.

Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of guanidinium-rich transporters into cells.

A mechanistic hypothesis is presented for how water-soluble guanidinium-rich transporters attached to small cargoes (MW ca. <3000) can migrate across the non-polar lipid membrane of a cell and enter the cytosol. Positively charged and water-soluble, arginine oligomers can associate with negatively charged, bidentate hydrogen bond acceptor groups of endogenous membrane constituents, leading to the formation of membrane-soluble ion pair complexes.

Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells.

The results described herein support a mechanistic hypothesis for how guanidine-rich transporters attached to small cargos (MW ca. <3000) can migrate across the lipid membrane of a cell and directly enter the cytosol. Arginine oligomers are found to partition almost completely into the aqueous layer of a water-octanol bilayer.

L-Arginine polymers enhance coronary flow and reduce oxidative stress following cardiac transplantation in rats.

Background: Hearts treated with l-arginine polymers have demonstrated upregulated production of nitric oxide. The current study examined whether these polymers improved coronary flow and reduced myocardial oxidative stress after rat heart transplantation.

Methods: PVG donor hearts were incubated ex vivo with either 100 mumol/L l-arginine polymers 9 amino acids in length (R9) (n = 7) or phosphate-buffered saline (n = 7) for 30 minutes after arrest and then transplanted heterotopically into the abdomen of ACI recipient rats.

Arginine-based molecular transporters: the synthesis and chemical evaluation of releasable taxol-transporter conjugates.

[structure: see text] A flexible and efficient procedure has been developed for the conjugation of taxol to various arginine-based molecular transporters via the taxol C2' O-chloroacetyl derivative. The resultant taxol-transporter conjugates are highly water soluble and release free taxol with half-lives of minutes to hours depending on the pH and the linker structure.

Guanidinium rich peptide transporters and drug delivery.

The use of peptide or peptidomimetic transporters to enable or enhance the uptake of drugs or probe molecules into cells and tissues has received increasing research attention and clinical interest over the past 10 years. This review summarizes a class of transporters that have been studied and focuses on the variation and use of guanidinium based transporters to facilitate the uptake of various types of molecules into cells and tissues. Lead conjugates in this area are currently in clinical trials.