IKKß deletion from CNS macrophages increases neuronal excitability and accelerates the onset of EAE, while from peripheral macrophages reduces disease severity.

BACKGROUND: Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. METHODS: We used constitutive MφIKKßΚΟ mice, in which depletion of IKKß, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚßKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. RESULTS: Global depletion of IKKß from myeloid cells in MφIKKßΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKß only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKß, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKß resulted in enhanced late long-term potentiation in EAE. CONCLUSIONS: IKKß-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKß deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKß on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS.

Competitive ELISA for the identification of 35-55 myelin oligodendrocyte glycoprotein immunodominant epitope conjugated with mannan.

Analogs of immunodominant myelin peptides involved in multiple sclerosis (MS: the most common autoimmune disease) have been extensively used to modify the immune response over the progression of the disease. The immunodominant 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG35-55 ) is an autoantigen appearing in MS and stimulates the encephalitogenic T cells, whereas mannan polysaccharide (Saccharomyces cerevisiae) is a carrier toward the mannose receptor of dendritic cells and macrophages. The conjugate of mannan-MOG35-55 has been extensively studied for the inhibition of chronic experimental autoimmune encephalomyelitis (EAE: an animal model of MS) by inducing antigen-specific immune tolerance against the clinical symptoms of EAE in mice. Moreover, it presents a promising approach for the immunotherapy of MS under clinical investigation. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the MOG35-55 peptide that is conjugated to mannan. Intra- and inter-day assay experiments proved that the proposed ELISA methodology is accurate and reliable and could be used in the following applications: (i) to identify the peptide (antigen) while it is conjugated to mannan and (ii) to adequately address the alterations that the MOG35-55 peptide may undergo when it is bound to mannan during production and stability studies.

Rational Design, Synthesis and Binding Affinity Studies of Anthraquinone Derivatives Conjugated to Gonadotropin-Releasing Hormone (GnRH) Analogues towards Selective Immunosuppression of Hormone-Dependent Cancer.

Gonadotropin-releasing hormone (GnRH) is pivotal in regulating human reproduction and fertility through its specific receptors. Among these, gonadotropin-releasing hormone receptor type I (GnRHR I), which is a member of the G-protein-coupled receptor family, is expressed on the surface of both healthy and malignant cells. Its presence in cancer cells has positioned this receptor as a primary target for the development of novel anti-cancer agents. Moreover, the extensive regulatory functions of GnRH have underscored decapeptide as a prominent vehicle for targeted drug delivery, which is accomplished through the design of appropriate conjugates. On this basis, a rationally designed series of anthraquinone/mitoxantrone-GnRH conjugates (con1-con8) has been synthesized herein. Their in vitro binding affinities range from 0.06 to 3.42 nM, with six of them (con2-con7) demonstrating higher affinities for GnRH than the established drug leuprolide (0.64 nM). Among the mitoxantrone based GnRH conjugates, con3 and con7 show the highest affinities at 0.07 and 0.06 nM, respectively, while the disulfide bond present in the conjugates is found to be readily reduced by the thioredoxin (Trx) system. These findings are promising for further pharmacological evaluation of the synthesized conjugates with the prospect of performing future clinical studies.

The Influence of Single Nucleotide Polymorphisms on Vitamin D Receptor Protein Levels and Function in Chronic Liver Disease.

Single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene have been associated with chronic liver disease. We investigated the role of VDR SNPs on VDR protein levels and function in patients with chronic liver disease. VDR expression levels were determined in peripheral T lymphocytes (CD3+VDR+), monocytes (CD14+VDR+), and plasma from patients (n = 66) and healthy controls (n = 38). Genotyping of SNPs and the determination of expression of VDR/vitamin D-related genes were performed by using qPCR. The effect of FokI SNP on vitamin D-binding to VDR was investigated by molecular dynamics simulations. CD14+VDR+ cells were correlated with the MELD score. The ApaI SNP was associated with decreased CD3+VDR+ levels in cirrhotic patients and with higher liver stiffness in HCV patients. The BsmI and TaqI SNPs were associated with increased VDR plasma concentrations in cirrhotic patients and decreased CD14+VDR+ levels in HCV patients. The FokI SNP was associated with increased CD3+VDR+ levels in cirrhotic patients and controls. VDR polymorphisms were significantly related to the expression of genes critical for normal hepatocyte function and immune homeostasis. VDR expression levels were related to the clinical severity of liver disease. VDR SNPs may be related to the progression of chronic liver disease by affecting VDR expression levels.

Development of PLGA Nanoparticles with a Glycosylated Myelin Oligodendrocyte Glycoprotein Epitope (MOG35-55) against Experimental Autoimmune Encephalomyelitis (EAE).

Multiple sclerosis (MS) is one of the most common neurodegenerative diseases in young adults, with early clinical symptoms seen in the central nervous system (CNS) myelin sheaths due to an attack caused by the patient's immune system. Activation of the immune system is mediated by the induction of an antigen-specific immune response involving the interaction of multiple T-cell types with antigen-presenting cells (APCs), such as dendritic cells (DCs). Antigen-specific therapeutic approaches focus on immune cells and autoantigens involved in the onset of disease symptoms, which are the main components of myelin proteins. The ability of such therapeutics to bind strongly to DCs could lead to immune system tolerance to the disease. Many modern approaches are based on peptide-based research, as, in recent years, they have been of particular interest in the development of new pharmaceuticals. The characteristics of peptides, such as short lifespan in the body and rapid hydrolysis, can be overcome by their entrapment in nanospheres, providing better pharmacokinetics and bioavailability. The present study describes the development of polymeric nanoparticles with encapsulated myelin peptide analogues involved in the development of MS, along with their biological evaluation as inhibitors of MS development and progression. In particular, particles of poly(lactic-co-glycolic) acid (PLGA) loaded with peptides based on mouse/rat (rMOG) epitope 35-55 of myelin oligodendrocyte glycoprotein (MOG) conjugated with saccharide residues were developed. More specifically, the MOG35-55 peptide was conjugated with glucosamine to promote the interaction with mannose receptors (MRs) expressed by DCs. In addition, a study of slow release (dissolution) and quantification on both initially encapsulated peptide and daily release in saline in vitro was performed, followed by an evaluation of in vivo activity of the formulation on mouse experimental autoimmune encephalomyelitis (EAE), an animal model of MS, using both prophylactic and therapeutic protocols. Our results showed that the therapeutic protocol was effective in reducing EAE clinical scores and inflammation of the central nervous system and could be an alternative and promising approach against MS inducing tolerance against the disease.

Pleiotrophin selectively binds to vascular endothelial growth factor receptor 2 and inhibits or stimulates cell migration depending on ανß3 integrin expression.

Pleiotrophin (PTN) has a moderate stimulatory effect on endothelial cell migration through ανß3 integrin, while it decreases the stimulatory effect of vascular endothelial growth factor A (VEGFA) and inhibits cell migration in the absence of ανß3 through unknown mechanism(s). In the present work, by using a multitude of experimental approaches, we show that PTN binds to VEGF receptor type 2 (VEGFR2) with a KD of 11.6 nM. Molecular dynamics approach suggests that PTN binds to the same VEGFR2 region with VEGFA through its N-terminal domain. PTN inhibits phosphorylation of VEGFR2 at Tyr1175 and still stimulates endothelial cell migration in the presence of a selective VEGFR2 tyrosine kinase inhibitor. VEGFR2 downregulation by siRNA or an anti-VEGFR2 antibody that binds to the ligand-binding VEGFR2 domain also induce endothelial cell migration, which is abolished by a function-blocking antibody against ανß3 or the peptide PTN112-136 that binds ανß3 and inhibits PTN binding. In cells that do not express ανß3, PTN decreases both VEGFR2 Tyr1175 phosphorylation and cell migration in a VEGFR2-dependent manner. Collectively, our data identify VEGFR2 as a novel PTN receptor involved in the regulation of cell migration by PTN and contribute to the elucidation of the mechanism of activation of endothelial cell migration through the interplay between VEGFR2 and ανß3.

Combined Computational and Structural Approach into Understanding the Role of Peptide Binding and Activation of Melanocortin Receptor 4.

Melanocortin receptor 4 (MC4R) is expressed predominantly in the central nervous system and regulates food intake and sexual function and is also thought to be responsible for effects on mood and cognition. It belongs to the melanocortin receptor subfamily of G protein-coupled receptors (GPCRs). Here, we have synthesized and structurally characterized three peptides that bind to MC4R, producing different signaling events. AgRP is a naturally occurring antagonist, HLWNRS is the minimal sequence of the N-terminal with partial agonist activity, and aMSH is a full agonistic peptide. By implementing molecular dynamics simulations on the different peptide-receptor complexes, we propose their molecular basis of binding to investigate their differential molecular properties regarding the activation states of the receptor. Our analysis shows that the agonist and partial agonist may induce rotation in transmembrane helix 3, which is known to be involved in the key events occurring during GPCR activation, and this movement is impacted by certain aromatic residues and their positioning in the orthosteric binding site of the receptor.

Characterization of Asparagine Deamidation in Immunodominant Myelin Oligodendrocyte Glycoprotein Peptide Potential Immunotherapy for the Treatment of Multiple Sclerosis.

Mannan (polysaccharide) conjugated with a myelin oligodendrocyte glycoprotein (MOG) peptide, namely (KG)5MOG35-55, represents a potent and promising new approach for the immunotherapy of Multiple Sclerosis (MS). The MOG35-55 epitope conjugated with the oxidized form of mannan (poly-mannose) via a (KG)5 linker was found to inhibit the symptoms of MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) in mice using prophylactic and therapeutic vaccinated protocols. Deamidation is a common modification in peptide and protein sequences, especially for Gln and Asn residues. In this study, the structural solution motif of deaminated peptides and their functional effects in an animal model for MS were explored. Several peptides based on the MOG35-55 epitope have been synthesized in which the Asn53 was replaced with Ala, Asp, or isoAsp. Our results demonstrate that the synthesized MOG peptides were formed to the deaminated products in basic conditions, and the Asn53 was mainly modified to Asp. Moreover, both peptides (wild type and deaminated derivative) conjugated with mannan (from Saccharomyces cerevisiae) independently inhibited the development of neurological symptoms and inflammatory demyelinating spinal cord lesions in MOG35-55-induced EAE. To conclude, mannan conjugated with a deamidated product did not affect the efficacy of the parent peptide.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72-85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala96]MBP87-99, cyclo(87-99)[Ala91,96]MBP87-99 and cyclo(87-99)[Arg91, Ala96]MBP87-99, but not wild-type linear MBP87-99, strongly inhibited MBP72-85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg91, Ala96]MBP87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

Recovery and quantification of a myelin oligodendrocyte glycoprotein peptide from rat plasma after protein precipitation.

The recovery of high molecular weight peptides from complex biological samples is a challenging task. Herein, a reliable, cost effective and rapid methodology was developed for the recovery and quantification of a myelin oligodendrocyte glycoprotein epitope namely (LysGly)5MOG35-55, from rat plasma. Removal of plasma proteins before quantification of the peptide was achieved after precipitation by an acetonitrile/water/formic acid solution. Using the developed protocol, average recoveries of the peptide from plasma ranged between 83.3 and 90.3%.

Biologically relevant conformational features of linear and cyclic proteolipid protein (PLP) peptide analogues obtained by high-resolution nuclear magnetic resonance and molecular dynamics.

Proteolipid protein (PLP) is one of the main proteins of myelin sheath that are destroyed during the progress of multiple sclerosis (MS). The immunodominant PLP139-151 epitope is known to induce experimental autoimmune encephalomyelitis (EAE, animal model of MS), wherein residues 144 and 147 are recognized by T cell receptor (TCR) during the formation of trimolecular complex with peptide-antigen and major histocompability complex. The conformational behavior of linear and cyclic peptide analogues of PLP, namely PLP139-151 and cyclic (139-151) (L144, R147) PLP139-151, have been studied in solution by means of nuclear magnetic resonance (NMR) methods in combination with unrestrained molecular dynamics simulations. The results indicate that the side chains of mutated amino acids in the cyclic analogue have different spatial orientation compared with the corresponding side chains of the linear analogue, which can lead to reduced affinity to TCR. NMR experiments combined with theoretical calculations pave the way for the design and synthesis of potent restricted peptides of immunodominant PLP139-151 epitope as well as non peptide mimetics that rises as an ultimate goal.

Cyclic citrullinated MBP87-99 peptide stimulates T cell responses: Implications in triggering disease.

Amino acid mutations to agonist peptide epitopes of myelin proteins have been used to modulate immune responses and experimental autoimmune encephalomyelitis (EAE, animal model of multiple sclerosis). Such amino acid alteration are termed, altered peptide ligands (APL). We have shown that the agonist myelin basic protein (MBP) 87-99 epitope (MBP87-99) with crucial T cell receptor (TCR) substitutions at positions 91 and 96 (K91,P96 (TCR contact residues) to R91,A96; [R91,A96]MBP87-99) results in altered T cell responses and inhibits EAE symptoms. In this study, the role of citrullination of arginines in [R91,A96]MBP87-99 peptide analog was determined using in vivo experiments in combination with computational studies. The immunogenicity of linear [Cit91,A96,Cit97]MBP87-99 and its cyclic analog - cyclo(87-99)[Cit91,A96,Cit97]MBP87-99 when conjugated to the carrier mannan (polysaccharide) were studied in SJL/J mice. It was found that mannosylated cyclo(87-99)[Cit91,A96,Cit97]MBP87-99 peptide induced strong T cell proliferative responses and IFN-gamma cytokine secretion compared with the linear one. Moreover, the interaction of linear and cyclic peptide analogs with the major histocompatibility complex (MHC II, H2-IAs) and TCR was analyzed using molecular dynamics simulations at the receptor level, in order to gain a better understanding of the molecular recognition mechanisms that underly the different immunological profiles of citrullinated peptides compared to its agonist native counterpart MBP87-99 epitope. The results demonstrate that the citrullination of arginine in combination with the backbone conformation of mutated linear and cyclic analogs are significant elements for the immune response triggering the induction of pro-inflammatory cytokines.

Design and Synthesis of Non-Peptide Mimetics Mapping the Immunodominant Myelin Basic Protein (MBP83-96) Epitope to Function as T-Cell Receptor Antagonists.

Encephalitogenic T cells are heavily implicated in the pathogenesis of multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system. Their stimulation is triggered by the formation of a trimolecular complex between the human leukocyte antigen (HLA), an immunodominant myelin basic protein (MBP) epitope, and the T cell receptor (TCR). We detail herein our studies directed towards the rational design and synthesis of non-peptide mimetic molecules, based on the immunodominant MBP83-96 epitope that is recognized by the TCR in complex with HLA. We focused our attention on the inhibition of the trimolecular complex formation and consequently the inhibition of proliferation of activated T cells. A structure-based pharmacophore model was generated, in view of the interactions between the TCR and the HLA-MBP83-96 complex. As a result, new candidate molecules were designed based on lead compounds obtained through the ZINC database. Moreover, semi-empirical and density functional theory methods were applied for the prediction of the binding energy between the proposed non-peptide mimetics and the TCR. We synthesized six molecules that were further evaluated in vitro as TCR antagonists. Analogues 15 and 16 were able to inhibit to some extent the stimulation of T cells by the immunodominant MBP83-99 peptide from immunized mice. Inhibition was followed to a lesser degree by analogues 17 and 18 and then by analogue 19. These studies show that lead compounds 15 and 16 may be used for immunotherapy against MS.

A novel synthetic luteinizing hormone-releasing hormone (LHRH) analogue coupled with modified ß-cyclodextrin: insight into its intramolecular interactions.

BACKGROUND: Cyclodextrins (CDs) in combination with therapeutic proteins and other bioactive compounds have been proposed as candidates that show enhanced chemical and enzymatic stability, better absorption, slower plasma clearance and improved dose-response curves or immunogenicity. As a result, an important number of therapeutic complexes between cyclodextrins and bioactive compounds capable to control several diseases have been developed. RESULTS: In this article, the synthesis and the structural study of a conjugate between a luteinizing hormone-releasing hormone (LHRH) analogue, related to the treatment of hormone dependent cancer and fertility, and modified ß-cyclodextrin residue are presented. The results show that both the phenyl group of tyrosine (Tyr) as well as the indole group of tryptophan (Trp) can be encapsulated inside the cyclodextrin cavity. Solution NMR experiments provide evidence that these interactions take place intramolecularly and not intermolecularly. CONCLUSIONS: The study of a LHRH analogue conjugated with modified ß-cyclodextrin via high field NMR and MD experiments revealed the existence of intramolecular interactions that could lead to an improved drug delivery. GENERAL SIGNIFICANCE: NMR in combination with MD simulation is of great value for a successful rational design of peptide-cyclodextrin conjugates showing stability against enzymatic proteolysis and a better pharmacological profile.

The application of solid-state NMR spectroscopy to study candesartan cilexetil (TCV-116) membrane interactions. Comparative study with the AT1R antagonist drug olmesartan.

ΑΤ1 receptor (AT1R) antagonists exert their antihypertensive effects by preventing the vasoconstrictive hormone AngII to bind to the AT1 receptor. It has been proposed that these biological effects are mediated through a two-step mechanism reaction. In the first step, they are incorporated in the core of the lipid bilayers and in the second step they reach the active site of the receptor through lateral diffusion. In this model, drug/membrane interactions are key elements for the drugs achieving inhibition at the AT1 receptor. In this work, the interactions of the prodrug candesartan cilexetil (TCV-116) with lipid bilayers are studied at molecular detail. Solid-state (13)C-CP/MAS, 2D (1)H-(1)H NOESY NMR spectroscopy and in silico calculations are used. TCV-116 and olmesartan, another drug which acts as an AT1R antagonist are compared for their dynamic effects in lipid bilayers using solid-state (2)H-NMR. We find a similar localization of TCV-116 compared to other AT1 antagonists in the intermediate polar region. In addition, we can identify specific local interactions. These interactions may be associated in part with the discrete pharmacological profiles observed for different antagonists.

Conjugation of a peptide to mannan and its confirmation by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The conjugation of polysaccharides to peptides is essential for antigen delivery and vaccine development. Herein, we show that tricine SDS-PAGE in combination with Coomassie Blue staining was adequate to determine the conjugation efficacy of a peptide (epitope 35-55 of myelin oligodendrocyte glycoprotein) to mannan. In addition, tricine SDS-PAGE and periodic acid-Schiff stains were able to monitor the redox state of mannan. Using the described protocol, more than 99.9% of a peptide containing five lysines at its N-terminus was confirmed conjugated to mannan.

Review cyclic peptides on a merry-go-round; towards drug design.

Peptides and proteins are attractive initial leads for the rational design of bioactive molecules. Several natural cyclic peptides have recently emerged as templates for drug design due to their resistance to chemical or enzymatic hydrolysis and high selectivity to receptors. The development of practical protocols that mimic the power of nature's strategies remains paramount for the advancement of novel peptide-based drugs. The de novo design of peptide mimetics (nonpeptide molecules or cyclic peptides) for the synthesis of linear or cyclic peptides has enhanced the progress of therapeutics and diverse areas of science and technology. In the case of metabolically unstable peptide ligands, the rational design and synthesis of cyclic peptide analogues has turned into an alternative approach for improved biological activity.

Use of the 2-chlorotrityl chloride resin for microwave-assisted solid phase peptide synthesis.

A fast and efficient microwave (MW)-assisted solid-phase peptide synthesis protocol using the 2-chlorotrityl chloride resin and the Fmoc/tBu methodology, has been developed. The established protocol combines the advantages of MW irradiation and the acid labile 2-chlorotrityl chloride resin. The effect of temperature during the MW irradiation, the degree of resin substitution during the coupling of the first amino acids and the rate of racemization for each amino acid were evaluated. The suggested solid phase methodology is applicable for orthogonal peptide synthesis and for the synthesis of cyclic peptides.

Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.

Spontaneous human CD8 T cell and autoimmune encephalomyelitis-induced CD4/CD8 T cell lesions in the brain and spinal cord of HLA-DRB1*15-positive multiple sclerosis humanized immune system mice.

Autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) are only partially represented in current experimental models and the development of humanized immune mice is crucial for better understanding of immunopathogenesis and testing of therapeutics. We describe a humanized mouse model with several key features of MS. Severely immunodeficient B2m-NOG mice were transplanted with peripheral blood mononuclear cells (PBMCs) from HLA-DRB1-typed MS and healthy (HI) donors and showed rapid engraftment by human T and B lymphocytes. Mice receiving cells from MS patients with recent/ongoing Epstein-Barr virus reactivation showed high B cell engraftment capacity. Both HLA-DRB1*15 (DR15) MS and DR15 HI mice, not HLA-DRB1*13 MS mice, developed human T cell infiltration of CNS borders and parenchyma. DR15 MS mice uniquely developed inflammatory lesions in brain and spinal cord gray matter, with spontaneous, hCD8 T cell lesions, and mixed hCD8/hCD4 T cell lesions in EAE immunized mice, with variation in localization and severity between different patient donors. Main limitations of this model for further development are poor monocyte engraftment and lack of demyelination, lymph node organization, and IgG responses. These results show that PBMC humanized mice represent promising research tools for investigating MS immunopathology in a patient-specific approach.