Tolerogenic nanoparticles suppress central nervous system inflammation.

Therapeutic approaches for the induction of immune tolerance remain an unmet clinical need for the treatment of autoimmune diseases, including multiple sclerosis (MS). Based on its role in the control of the immune response, the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is a candidate target for novel immunotherapies. Here, we report the development of AhR-activating nanoliposomes (NLPs) to induce antigen-specific tolerance. NLPs loaded with the AhR agonist ITE and a T cell epitope from myelin oligodendrocyte glycoprotein (MOG)35-55 induced tolerogenic dendritic cells and suppressed the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, in preventive and therapeutic setups. EAE suppression was associated with the expansion of MOG35-55-specific FoxP3+ regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells), concomitant with a reduction in central nervous system-infiltrating effector T cells (Teff cells). Notably, NLPs induced bystander suppression in the EAE model established in C57BL/6 × SJL F1 mice. Moreover, NLPs ameliorated chronic progressive EAE in nonobese diabetic mice, a model which resembles some aspects of secondary progressive MS. In summary, these studies describe a platform for the therapeutic induction of antigen-specific tolerance in autoimmune diseases.

Atypical Pediatric Demyelinating Diseases of the Central Nervous System.

PURPOSE OF REVIEW: Pediatric central nervous system demyelinating diseases include multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), and acute disseminated encephalomyelitis (ADEM). As diagnostic criteria become more inclusive, the risk of misdiagnosis of atypical demyelinating diseases of rheumatologic, infectious, and autoimmune etiology increases. RECENT FINDINGS: We review mimics of multiple sclerosis, neuromyelitis optica spectrum disorder, and acute disseminated encephalomyelitis, including rheumatologic diseases: systemic lupus erythematosus and neuro-Behçet disease; infectious diseases: human immunodeficiency virus, progressive multifocal leukoencephalopathy, and subacute sclerosis panencephalitis; and autoimmune diseases including X-linked Charcot-Marie-Tooth disease, chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) and autoimmune glial fibrillary acidic protein (GFAP) encephalopathy. Atypical demyelinating disease may mimic classic neuroinflammatory diseases of the central nervous system. Imaging may meet criteria for a diagnosis of multiple sclerosis, or patients may present with optic neuritis and transverse myelitis consistent with neuromyelitis optica spectrum or myelin oligodendrocyte glycoprotein (MOG) antibody disorders. Through careful history-taking and review of atypical MRI findings, we may avoid misdiagnosis and mistreatment.

Overcoming Hurdles in Nanoparticle Clinical Translation: The Influence of Experimental Design and Surface Modification.

Nanoparticles are becoming an increasingly popular tool for biomedical imaging and drug delivery. While the prevalence of nanoparticle drug-delivery systems reported in the literature increases yearly, relatively little translation from the bench to the bedside has occurred. It is crucial for the scientific community to recognize this shortcoming and re-evaluate standard practices in the field, to increase clinical translatability. Currently, nanoparticle drug-delivery systems are designed to increase circulation, target disease states, enhance retention in diseased tissues, and provide targeted payload release. To manage these demands, the surface of the particle is often modified with a variety of chemical and biological moieties, including PEG, tumor targeting peptides, and environmentally responsive linkers. Regardless of the surface modifications, the nano-bio interface, which is mediated by opsonization and the protein corona, often remains problematic. While fabrication and assessment techniques for nanoparticles have seen continued advances, a thorough evaluation of the particle's interaction with the immune system has lagged behind, seemingly taking a backseat to particle characterization. This review explores current limitations in the evaluation of surface-modified nanoparticle biocompatibility and in vivo model selection, suggesting a promising standardized pathway to clinical translation.

Conjugation of Transforming Growth Factor Beta to Antigen-Loaded Poly(lactide- co-glycolide) Nanoparticles Enhances Efficiency of Antigen-Specific Tolerance.

Current strategies for treating autoimmunity involve the administration of broad-acting immunosuppressive agents that impair healthy immunity. Intravenous (i.v.) administration of poly(lactide- co-glycolide) nanoparticles (NPs) containing disease-relevant antigens (Ag-NPs) have demonstrated antigen (Ag)-specific immune tolerance in models of autoimmunity. However, subcutaneous (s.c.) delivery of Ag-NPs has not been effective. This investigation tested the hypothesis that codelivery of the immunomodulatory cytokine, transforming growth factor beta 1 (TGF-ß), on Ag-NPs would modulate the immune response to Ag-NPs and improve the efficiency of tolerance induction. TGF-ß was coupled to the surface of Ag-NPs such that the loadings of Ag and TGF-ß were independently tunable. The particles demonstrated bioactive delivery of Ag and TGF-ß in vitro by reducing the inflammatory phenotype of bone marrow-derived dendritic cells and inducing regulatory T cells in a coculture system. Using an in vivo mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, TGF-ß codelivery on Ag-NPs resulted in improved efficacy at lower doses by i.v. administration and significantly reduced disease severity by s.c. administration. This study demonstrates that the codelivery of immunomodulatory cytokines on Ag-NPs may enhance the efficacy of Ag-specific tolerance therapies by programming Ag presenting cells for more efficient tolerance induction.

Conditioned Medium from the Stem Cells of Human Exfoliated Deciduous Teeth Ameliorates Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a major neuroinflammatory demyelinating disease of the CNS. Current MS treatments, including immunomodulators and immunosuppressants, do not result in complete remission. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells derived from dental pulp. Both SHED and SHED-conditioned medium (SHED-CM) exhibit immunomodulatory and regenerative activities and have the potential to treat various diseases. In this study, we investigated the efficacy of SHED-CM in treating experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE mice treated with a single injection of SHED-CM exhibited significantly improved disease scores, reduced demyelination and axonal injury, and reduced inflammatory cell infiltration and proinflammatory cytokine expression in the spinal cord, which was associated with a shift in the microglia/macrophage phenotype from M1 to M2. SHED-CM also inhibited the proliferation of myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, as well as their production of proinflammatory cytokines in vitro. Treatment of EAE mice with the secreted ectodomain of sialic acid-binding Ig-like lectin-9, a major component of SHED-CM, recapitulated the effects of SHED-CM treatment. Our data suggest that SHED-CM and secreted ectodomain of sialic acid-binding Ig-like lectin-9 may be novel therapeutic treatments for autoimmune diseases, such as MS.

Monodisperse magnetic poly(glycidyl methacrylate) microspheres for isolation of autoantibodies with affinity for the 46 kDa form of unconventional Myo1C present in autoimmune patients.

Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-Fe2O3) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 µm in diameter, contain 0.9 mM · g-1 of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.

Design of Chemical Conjugate for Targeted Therapy of Multiple Sclerosis Based of Constant Fragment of Human Antibody Heavy Chain and Peptoid Analog of Autoantigen MOG35-55.

Elimination of B cells producing autoantibodies to neuroantigens is considered as beneficial in the treatment of multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG) is a significant autoantigen in multiple sclerosis. It was shown that MOG-like peptoid AMogP3 can bind autoantibodies produced by pathological lymphocytes. We propose a structure of an innovative drug for targeted elimination of the pool of autoreactive B cells responsible for multiple sclerosis pathogenesis; this compound is a complex of peptoid AMogP3 with Fc fragment of human immunoglobulin. The obtained Fc-PEG-AMogP3 conjugate effectively interact with autoreactive antibodies, which attests to their high therapeutic potential.

[Oral health in multiple sclerosis patients]. / Choroby jamy ustnej u chorych na stwardnienie rozsiane.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system occuring in young adults, mainly female. MS dominates in Caucasians living in regions far away from the equator. The coexistence of genetic and environmental factors is considered in its etiopathogenesis. MS mostly occurs in the form of relapses and remissions, leading to the physical disability and cognitive decline. The diagnosis is based on MRI images and cerebrospinal fluid testing. The current guidelines for therapy recommend immunosuppression (steroids during relapses) and immunomodulation. Symptomatic treatment of pain or muscle rigidity is used additionally. The epidemiological data draw attention to the geographical distribution of indicators related to the increased prevalence of MS and dental caries. The role of D3 vitamin is discussed in the development of both diseases, but the role of amalgam filling in the development of MS is rejected. The demyelinating process in MS and applied treatment predispose to the neurological pain in the facial area or the temporomandibular joints. The increasing disability and used treatment enhance the susceptibility to mucosal inflammation and xerostomia, and activate viral and fungal infections. Patients with MS require regular dental control often in conditions arranged for disable people.

Multiple sclerosis and oral health: an update.

Multiple sclerosis is a chronic, neurodegenerative disease seen in 69.1 per 100,000 person-years in the world. As multiple sclerosis and periodontal disease both have an inflammatory origin, dental professionals should be aware of the link between these two diseases. In patients unable to carry out effective oral hygiene, dental treatment should be done by dental hygienists and/or dentists to prevent dental caries and periodontal disease. It is hoped that by identifying multiple sclerosis patients in dental clinics, the required support and treatment could be provided to these patients to improve their quality of life and that dental professionals would feel comfortable treating patients with multiple sclerosis.

Spinal fluid IgG antibodies from patients with demyelinating diseases bind multiple sclerosis-associated bacteria.

A panel of 10 IgG enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of anti-microbial immune responses in the cerebrospinal fluid (CSF) of patients with demyelinating diseases (DD). The anti-microbial ELISA assays follow on prior human brain tissue RNA sequencing studies that established multiple sclerosis (MS) microbial candidates. Lysates included in the ELISA panel were derived from Akkermansia muciniphila, Atopobium vaginae, Bacteroides fragilis, Lactobacillus paracasei, Odoribacter splanchnicus, Pseudomonas aeruginosa, Cutibacterium (Propionibacterium) acnes, Fusobacterium necrophorum, Porphyromonas gingivalis, and Streptococcus mutans. CSF responses from patients with demyelinating diseases (DD, N = 14) were compared to those with other neurological diseases (OND, N = 8) and controls (N = 13). Commercial positive and negative control CSF specimens were run with each assay. ELISA index values were derived for each specimen against each of the 10 bacterial lysates. CSF reactivity was significantly higher in the DD group compared to the controls against Akkermansia, Atopobium, Bacteroides, Lactobacillus, Odoribacter, and Fusobacterium. Four of the 11 tested DD group subjects had elevated antibody indexes against at least one of the 10 bacterial species, suggesting intrathecal antibody production. This CSF serological study supports the hypothesis that several of the previously identified MS candidate microbes contribute to demyelination in some patients. KEY MESSAGES: A panel of 10 IgG enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of anti-microbial immune responses in the cerebrospinal fluid (CSF) of patients with demyelinating diseases, including multiple sclerosis and acute disseminated encephalomyelitis. CSF reactivity was significantly higher in the demyelination group compared to the controls against the bacteria Akkermansia, Atopobium, Bacteroides, Lactobacillus, Odoribacter, and Fusobacterium. Several of the demyelination subjects had elevated antibody indexes against at least one of the 10 antigens, suggesting at least limited intrathecal production of anti-bacterial antibodies. This CSF serological study supports the hypothesis that several of the previously identified MS candidate microbes contribute to demyelination in some patients.

Engineering release kinetics with polyelectrolyte multilayers to modulate TLR signaling and promote immune tolerance.

Autoimmune disorders, such as multiple sclerosis and type 1 diabetes, occur when immune cells fail to recognize "self" molecules. Recently, studies have revealed aberrant inflammatory signaling through pathogen sensing pathways, such as toll-like receptors (TLRs), during autoimmune disease. Therapeutic inhibition of these pathways might attenuate disease development, skewing disease-causing inflammatory cells towards cell types that promote tolerance. Delivering antagonistic ligands to a TLR upstream of an inflammatory signaling cascade, TLR9, has demonstrated exciting potential in a mouse model of MS; however, strategies that enable sustained delivery could reduce the need for repeated administration or enhance therapeutic efficacy. We hypothesized that GpG - an oligonucleotide TLR9 antagonist - which is inherently anionic, could be self-assembled into polyelectrolyte multilayers (PEMs) with a cationic, degradable poly(ß-amino ester) (Poly1). We hypothesized that degradable Poly1/GpG PEMs could promote sustained release of GpG and modulate inflammatory immune cell functions. Here we demonstrate layer-by-layer assembly of degradable PEMs, as well as subsequent degradation and release of GpG. Following assembly and release, GpG maintains the ability to reduce dendritic cell activation and inflammatory cytokine secretion, restrain TLR9 signaling, and polarize myelin specific T cells towards regulatory phenotypes and functions in primarily immune cells. These results indicate that degradable PEMs may be able to promote tolerogenic immune function in the context of autoimmunity.

Interaction of PEGylated interferon-beta with antibodies to recombinant interferon-beta.

Because PEGylated molecules exhibit different physicochemical properties from those of the parent molecules, PEGylated interferonß-1a (pegIFNß-1a) may be able to be used with retained bioactivity in Multiple Sclerosis (MS) patients who have previously developed neutralizing antibodies (NABs) to recombinant interferonß (rIFNß). Hence, the objective of the present study was to test whether pegIFNß-1a is less antigenic for NABs in vitro than rIFNß. Two in vitro assays were used to quantitate NABs in 115 sera obtained from MS patients included in the INSIGHT study: the cytopathic effect (CPE) assay, and the MxA protein induction assay. NABs cross-reactivity was assessed by comparing dilutions of serum with fixed doses of rIFNß-1a Avonex® and pegIFNß-1a Plegridy®. NABs were shown to cross-react in both assays. The y-intercept (c), the slope of the line of agreement (b), the Pearson coefficients as well as the Bland-Altman analysis, indicated that there is good level of agreement between NAB titers against the two IFNß-1a formulations, with both the CPE (c = 0.1044 ±â€¯0.1305; b = 0.8438 ±â€¯0.06654; r2 = 0.587; bias index ±â€¯SD = -0.01702 ±â€¯0.6334), and the MxA protein induction (c = 0.08246 ±â€¯0.1229; b = 0.8878 ±â€¯0.06613; r2 = 0.615; bias index ±â€¯SD = -0.09965 ±â€¯0.6467) assays. Until further in vivo evidence is established, clinicians should consider the current in vitro data demonstrating NAB cross-reactivity between pegIFNß-1a and rIFNß when discussing new treatment options with MS patients.

Autoimmune disease and the female patient.

This article focuses on the pathogenesis of the gender gap of autoimmune disease. Specifically, the discussion characterizes the role of sex hormones in the immune response and a female predilection for the common diseases seen in daily practice (ie, lupus erythematosus, myasthenia gravis, and other autoimmune diseases). A comparison between the sexes, with respect to autoimmune disease mechanisms, is presented to give oral and maxillofacial surgeons a better insight as to the role of sex and successful surgical treatment outcomes in this population of patients.

Epstein-Barr virus and multiple sclerosis: Updating Pender's hypothesis.

Substantial epidemiological evidence supports the involvement of the Epstein-Barr virus (EBV) in multiple sclerosis (MS). Mechanisms through which EBV may increase MS risk are reviewed here. Most individuals contract EBV in early childhood yet only develop MS in early adulthood, by which time EBV has been latent for decades. When latent, EBV is confined to a minute subset of memory B cells: about 1000 cells in peripheral blood and 500,000 cells in the lymphoid system, mainly in the mouth. Reactivation of EBV in the central nervous system (CNS) has been proposed as a cause of MS. Alternatively, EBV may enable the recognition of "forbidden" antigens by memory B cells through its presence in this leukocyte type, as first proposed by Pender. Though the requirement for B cells in MS supports both hypotheses, EBV has not been consistently found in MS lesions, as would be expected. EBV episome replication during B cell division is now known to be inefficient, resulting in some descendant B cells becoming EBV-free after a few dozen divisions. EBV-free memory B cells in the CNS may thus have descended from a memory B cell which matured while containing EBV episomes, enabling its B cell receptor to recognize "forbidden" MS-causing antigens in the CNS, even if EBV is absent from this site.

Liposome-based immunotherapy against autoimmune diseases: therapeutic effect on multiple sclerosis.

AIM: Based on the ability of apoptosis to induce immunological tolerance, liposomes were generated mimicking apoptotic cells, and they arrest autoimmunity in Type 1 diabetes. Our aim was to validate the immunotherapy in other autoimmune disease: multiple sclerosis. MATERIALS & METHODS: Phosphatidylserine-rich liposomes were loaded with disease-specific autoantigen. Therapeutic capability of liposomes was assessed in vitro and in vivo. RESULTS: Liposomes induced a tolerogenic phenotype in dendritic cells, and arrested autoimmunity, thus decreasing the incidence, delaying the onset and reducing the severity of experimental disease, correlating with an increase in a probably regulatory CD25+ FoxP3- CD4+ T-cell subset. CONCLUSION: This is the first work that confirms phosphatidylserine-liposomes as a powerful tool to arrest multiple sclerosis, demonstrating its relevance for clinical application.

Intravenous Injection of Myelin Oligodendrocyte Glycoprotein-coated PLGA Microparticles Have Tolerogenic Effects in Experimental Autoimmune Encephalomyelitis.

The abnormal function of the T lymphocytes causes a range of autoimmune diseases, particularly multiple sclerosis; hence, several methods have been used to treat these disorders through the induction of antigen-specific tolerance in T cells. The present study aims to use a simple and low-cost method to produce poly (lactic-co-glycolic acid) (PLGA) nanoparticles for carrying antigens and inducing antigen-specific tolerance. In this study, PLGA nanoparticles were produced using the water/oil/water (W/O/W) method. The myelin oligodendrocyte glycoprotein (MOG) peptide and ovalbumin peptide(OVA) were covalently bound to the synthetic PLGA nanoparticles in the presence of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) and were injected to six groups of C57BL/6 mice one week before the induction of the experimental autoimmune encephalomyelitis (EAE) intravenously or subcutaneously; one group was considered as control; finally, immunologic responses including delayed-type hypersensitivity (DTH) response and lymphocyte proliferation were investigated. The results showed that the intravenous injection of microparticles containing MOG peptides before the development of the EAE model, not only could delay the incidence of syndrome, but also increase the antigen-specific tolerance. Moreover, a reduced delayed-type hypersensitivity response was observed in the mice primed with microparticles containing MOG peptides. In addition, a reduced spleen lymphocyte proliferation was found in the same mice when challenged with antigens. The present study proposes a simple, inexpensive, effective and safe method for preparing MOG-conjugated PLGA microparticles with immune tolerance properties that can be used in the treatment or reducing clinical syndromes of EAE model.

Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of monospecific anti-p46 kDa/Myo1C antibodies for early diagnosis of multiple sclerosis patients.

The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing ∼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.

An antigen-specific semi-therapeutic treatment with local delivery of tolerogenic factors through a dual-sized microparticle system blocks experimental autoimmune encephalomyelitis.

Antigen-specific treatments are highly desirable for autoimmune diseases in contrast to treatments which induce systemic immunosuppression. A novel antigen-specific therapy has been developed which, when administered semi-therapeutically, is highly efficacious in the treatment of the mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). The treatment uses dual-sized, polymeric microparticles (dMPs) loaded with specific antigen and tolerizing factors for intra- and extra-cellular delivery, designed to recruit and modulate dendritic cells toward a tolerogenic phenotype without systemic release. This approach demonstrated robust efficacy and provided complete protection against disease. Therapeutic efficacy required encapsulation of the factors in controlled-release microparticles and was antigen-specific. Disease blocking was associated with a reduction of infiltrating CD4+ T cells, inflammatory cytokine-producing pathogenic CD4+ T cells, and activated macrophages and microglia in the central nervous system. Furthermore, CD4+ T cells isolated from dMP-treated mice were anergic in response to disease-specific, antigen-loaded splenocytes. Additionally, the frequency of CD86hiMHCIIhi dendritic cells in draining lymph nodes of EAE mice treated with Ag-specific dMPs was reduced. Our findings highlight the efficacy of microparticle-based drug delivery platform to mediate antigen-specific tolerance, and suggest that such a multi-factor combinatorial approach can act to block autoimmunity.

A practical approach to immunomodulatory therapy for multiple sclerosis.

This article provides a current overview of the definition and pathogenesis of the disease, the different types of MS, a new diagnostic criteria, the rationale for early therapy, a review of the approved MS therapies, the strategies to evaluate ongoing treatment efficacy,the management of suboptimal treatment responders, and the prospects for future therapies. The article focuses on relapsing remitting MS because most of the therapeutic data deal with this type of MS. The role of mitoxantrone as a "rescue therapy" for suboptimal responders to IMTs is discussed.

Immunogenicity evaluation strategy for a second-generation therapeutic, PEG-IFN-ß-1a.

AIMS: Neutralizing antibodies can diminish clinical efficacy of IFN-ß in multiple sclerosis patients. Therefore, monitoring immunogenicity was considered critical during clinical development of a second-generation, pegylated IFN-ß product, PEG-IFN-ß-1a. MATERIALS & METHODS: Assays previously used to evaluate immunogenicity of IFN-ß-1a were used to assess PEG-IFN-ß-1a immunogenicity, with modifications to apply current best bioanalytical practices. A separate testing paradigm was used to monitor antibodies to polyethylene glycol. RESULTS & CONCLUSION: Final assay cut points and relevant titer levels were established in-study. Immunogenicity evaluation strategies for second-generation therapeutics should take into consideration current best bioanalytical practices while retaining consistency with legacy assays to facilitate data comparison and interpretation. This study illustrates challenges in assessing immunogenicity of second-generation therapeutics.