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Identification of individuals at high risk for rapid progression of motor and cognitive signs in Parkinson disease (PD) is clinically significant. Postural instability and gait dysfunction (PIGD) are associated with greater motor and cognitive deterioration. We examined the relationship between baseline clinical factors and the development of postural instability using 5-year longitudinal de-novo idiopathic data (n = 301) from the Parkinson's Progressive Markers Initiative (PPMI). Logistic regression analysis revealed baseline features associated with future postural instability, and we designated this cohort the emerging postural instability (ePI) phenotype. We evaluated the resulting ePI phenotype rating scale validity in two held-out populations which showed a significantly higher risk of postural instability. Emerging PI phenotype was identified before onset of postural instability in 289 of 301 paired comparisons, with a median progression time of 972 days. Baseline cognitive performance was similar but declined more rapidly in ePI phenotype. We provide an ePI phenotype rating scale (ePIRS) for evaluation of individual risk at baseline for progression to postural instability.

Publisher's version (útgefin grein), Pentraxins arefluid phase pattern recognition molecules that form an important part of the innate immunedefence and are conserved betweenfish and human. In Atlantic cod (Gadus morhuaL.), two pentraxin-likeproteins have been described, CRP-I and CRP-II. Here we show for thefirst time that these two CRP forms arepost-translationally deiminated (an irreversible conversion of arginine to citrulline) and differ with respect totissue specific localisation in cod ontogeny from 3 to 84 days post hatching. While both forms are expressed inliver, albeit at temporally differing levels, CRP-I shows a strong association with nervous tissue while CRP-II isstrongly associated to mucosal tissues of gut and skin. This indicates differing roles for the two pentraxin types inimmune responses and tissue remodelling, also elucidating novel roles for CRP-I in the nervous system. Thepresence of deimination positive bands for cod CRPs varied somewhat between mucus and serum, possiblyfacilitating CRP protein moonlighting, allowing the same protein to exhibit a range of biological functions andthus meeting different functional requirements in different tissues. The presentedfindings may further currentunderstanding of the diverse roles of pentraxins in teleost immune defences and tissue remodelling, as well as invarious human pathologies, including autoimmune diseases, amyloidosis and cancer., Thanks are due to Matthías Oddgeirsson, Agnar Steinarsson and other staff the staff at the Marine Institute's Mariculture Laboratory, Staður Grindavík, Iceland for providing sampling facilities and the fish. The authors also thank Margrét Jónsdóttir, Keldur, Institute for Experimental Pathology University of Iceland, for cod larvae sample preparation. This work was partly supported by The Icelandic Research Council (RANNIS), EC grant Fishaid QLK2-CT-2000-01076 and a University of Westminster start-up grant to SL. The authors declare no competing interest.

BACKGROUND Monoamine oxidase type B (MAO-B) inhibitors exhibit neuroprotective effects in preclinical models of PD but clinical trials have failed to convincingly demonstrate disease modifying benefits in PD patients. OBJECTIVE To perform a secondary analysis of NET-PD LS1 to determine if longer duration of MAO-B inhibitor exposure was associated with less clinical decline. METHODS The primary outcome measure was the Global Outcome (GO), comprised of 5 measures: change from baseline in the Schwab and England (ADL) scale, the 39-item Parkinson's Disease Questionnaire (PDQ-39), the UPDRS Ambulatory Capacity Scale, the Symbol Digit Modalities Test, and the most recent Modified Rankin Scale. A linear mixed model was used to explore the association between the cumulative duration of MAO-B inhibitor exposure and the GO, adjusting for necessary factors and confounders. Associations between MAO-B inhibitor exposure and each of the five GO components were then studied individually. RESULTS 1616 participants comprised the analytic sample. Mean observation was 4.1 (SD = 1.4) years, and 784 (48.5%) participants received an MAO-B inhibitor. The regression coefficient of cumulative duration of MAO-B inhibitor exposure (in years) on the GO was - 0.0064 (SE = 0.002, p = 0.001). Significant associations between duration of MAO-B inhibitor exposure and less progression were observed for ADL (p

Outer membrane and membrane vesicles (OMV/MV) are released from bacteria and participate in cell communication, biofilm formation and host-pathogen interactions. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes that catalyze post-translational deimination/citrullination of proteins, causing structural and functional changes in target proteins. PADs also play major roles in the regulation of eukaryotic extracellular vesicle release. Here we show phylogenetically conserved pathways of PAD-mediated OMV/MV release in bacteria and describe deiminated/citrullinated proteins in E. coli and their derived OMV/MVs. Furthermore, we show that PAD inhibitors can be used to effectively reduce OMV/MV release, both in Gram-negative and Gram-positive bacteria. Importantly, this resulted in enhanced antibiotic sensitivity of both E. coli and S. aureus to a range of antibiotics tested. Our findings reveal novel strategies for applying pharmacological OMV/MV-inhibition to reduce antibiotic resistance.

Publisher's version (útgefin grein)., Peptidylarginine deiminases (PADs) are calcium dependent enzymes with physiological and pathophysiologicalroles conserved throughout phylogeny. PADs promote post-translational deimination of protein arginine to ci-trulline, altering the structure and function of target proteins. Deiminated proteins were detected in the earlydevelopmental stages of cod from 11 days post fertilisation to 70 days post hatching. Deiminated proteins werepresent in mucosal surfaces and in liver, pancreas, spleen, gut, muscle, brain and eye during early cod larvaldevelopment. Deiminated protein targets identified in skin mucosa included nuclear histones; cytoskeletalproteins such as tubulin and beta-actin; metabolic and immune related proteins such as galectin, mannan-binding lectin, toll-like receptor, kininogen, Beta2-microglobulin, aldehyde dehydrogenase, bloodthirsty andpreproapolipoprotein A-I. Deiminated histone H3, a marker for anti-pathogenic neutrophil extracellular traps,was particularly elevated in mucosal tissues in immunostimulated cod larvae. PAD-mediated protein deimina-tion may facilitate protein moonlighting, allowing the same protein to exhibit a range of biological functions, intissue remodelling and mucosal immune defences in teleost ontogeny., The authors wish to thank the staffat Staður Grindavík, Iceland forproviding thefish and sampling facilities. Thanks are due to MargrétJónsdóttir, Keldur, Institute for Experimental Pathology University ofIceland, for preparation of cod larvae samples and tissue sections. Thiswork was supported in parts by the EC grant Fishaid QLK2-CT-2000-01076, The Icelandic Research Council (RANNIS), and a University ofWestminster start-up grant to SL. The authors declare no competinginterest.

Publisher's version (útgefin grein), Post-translational protein deimination is mediated by peptidylarginine deiminases (PADs), which are calcium dependent enzymes conserved throughout phylogeny with physiological and pathophysiological roles. Protein deimination occurs via the conversion of protein arginine into citrulline, leading to structural and functional changes in target proteins. In a continuous series of early halibut development from 37 to 1050° d, PAD, total deiminated proteins and deiminated histone H3 showed variation in temporal and spatial detection in various organs including yolksac, muscle, skin, liver, brain, eye, spinal cord, chondrocytes, heart, intestines, kidney and pancreas throughout early ontogeny. For the first time in any species, deimination of complement components C3 and C4 is shown in halibut serum, indicating a novel mechanism of complement regulation in immune responses and homeostasis. Proteomic analysis of deiminated target proteins in halibut serum further identified complement components C5, C7, C8 C9 and C1 inhibitor, as well as various other immunogenic, metabolic, cytoskeletal and nuclear proteins. Post-translational deimination may facilitate protein moonlighting, an evolutionary conserved phenomenon, allowing one polypeptide chain to carry out various functions to meet functional requirements for diverse roles in immune defences and tissue remodelling., The authors wish to thank Birgir Kristjánsson and the staff at Fiskeldi Eyjafjardar, þorlákshöfn, Iceland, and the staff at Fiskey hf, Hjalteyri, Iceland for providing the fish and sampling facilities. Thanks are due to Sigurður Helgason, Gísli Jónsson and Margrét Jónsdóttir, Keldur, Institute for Experimental Pathology University of Iceland, and to Paul Cook, FRS Marine Laboratory, Aberdeen, Scotland, for preparation of larval samples. Thanks also to Antony Willis, MRC Immunochemistry Unit, Department of Biochemistry, Oxford, for N-terminal amino acid sequence analysis and Michael Deery at the Cambridge Centre for Proteomics. This work was supported in parts by the EC grant Fishaid QLK2-CT-2000-01076 , The Icelandic Ministry of Fisheries , The Icelandic Research Council (RANNIS) , the European Molecular Biology Organisation (EMBO) and a University of Westminster start-up grant to SL. The authors declare no competing interest.

Background A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA. Methods Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann–Whitney and Fisher’s exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test. Results Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes. Conclusion Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin. Electronic supplementary material The online version of this article (10.1186/s12967-018-1588-2) contains supplementary material, which is available to authorized users.

Objective A C-to-T single-nucleotide polymorphism (SNP) located at position 1858 of human protein tyrosine phosphatase PTPN22 complementary DNA carries the highest risk of rheumatoid arthritis (RA) among all non-HLA genetic variants. This C1858T SNP converts an arginine (R620) to a tryptophan (W620), but it is unclear why it has such a strong impact on RA, a disease characterized by anti–citrullinated protein antibodies. The aim of this study was to test the hypothesis that PTPN22 regulates protein citrullination. Methods The level of citrullinated proteins in immune cells was quantified by Western blotting. The physical interaction between PTPN22 and peptidyl arginine deiminase type 4 (PAD-4), which is one of the enzymes that catalyzes protein citrullination, was examined by coimmunoprecipitation. Neutrophils were collected from healthy donors carrying the C1858T SNP and healthy donors not carrying this SNP. The formation of neutrophil extracellular traps (NETs) was examined by immunocytochemistry. Results PTPN22 physically interacted with PAD-4, and a deficiency in PTPN22 enhanced protein citrullination. This abnormality was reversed by exogenous wild-type PTPN22 or catalytically dead mutant PTPN22. The R-to-W conversion rendered PTPN22 unable to interact with PAD-4 and suppress citrullination. The C1858T SNP was associated with hypercitrullination in peripheral blood mononuclear cells and a heightened propensity for spontaneous formation of NETs, which is a PAD-4–dependent process. Conclusion PTPN22 is an inhibitor of PAD-4 and protein citrullination. This function of PTPN22 is independent of its phosphatase activity but requires R620. Our data not only establish a molecular link between PTPN22 and PAD-4, but also suggest that the C1858T SNP increases the risk of RA by enhancing protein citrullination and spontaneous formation of NETs.

Background Citrullination is a presently under-recognized posttranslational protein modification catalyzed by PAD enzymes. Immune responses to citrullinated neo-epitopes are identified in a growing number of inflammatory and autoimmune diseases. However, the involvement of hypercitrullination in the pathogenesis of bronchial asthma is still unknown. Methods As main experimental tool, we examined the effect of 2-chloroacetamidine (2CA), a PAD enzyme inhibitor, on OVA-immunized and airway-challenged BALB/c mice; a commonly used model of allergic airway inflammation. We also measured the effect of 2CA on ex vivo lymphocytes and cell lines. Results In vivo, 2CA dramatically suppressed lung tissue hypercitrullination, inflammatory cell recruitment, and airway-Th2 cytokine secretion. 2CA also suppressed systemic OVA-specific and total IgE production dramatically, effectively preventing de novo and diminishing established disease without measurably impacting general immunocompetence. In vitro, 2CA markedly inhibited the proliferation of mouse and human T cells with cell cycle block and apoptosis during a limited, postactivation phase. Conclusions 2CA acts as narrow-spectrum immunosuppressant that selectively targets lymphocyte populations involved in active inflammatory tissue lesions. If hypercitrullination is generated in patients with asthma, 2CA may represent a novel disease modulator for human asthmatics/allergic diseases.

Objective Malondialdehyde-acetaldehyde (MAA) adducts are a product of oxidative stress associated with tolerance loss in several disease states. This study was undertaken to investigate the presence of MAA adducts and circulating anti-MAA antibodies in patients with rheumatoid arthritis (RA). Methods Synovial tissue from patients with RA and patients with osteoarthritis (OA) were examined for the presence of MAA-modified and citrullinated proteins. Anti-MAA antibody isotypes were measured in RA patients (n = 1,720) and healthy controls (n = 80) by enzyme-linked immunosorbent assay. Antigen-specific anti–citrullinated protein antibodies (ACPAs) were measured in RA patients using a multiplex antigen array. Anti-MAA isotype concentrations were compared in a subset of RA patients (n = 80) and matched healthy controls (n = 80). Associations of anti-MAA antibody isotypes with disease characteristics, including ACPA positivity, were examined in all RA patients. Results Expression of MAA adducts was increased in RA synovial tissue compared to OA synovial tissue, and colocalization with citrullinated proteins was found. Increased levels of anti-MAA antibody isotypes were observed in RA patients compared to controls (P < 0.001). Among RA patients, anti-MAA antibody isotypes were associated with seropositivity for ACPAs and rheumatoid factor (P < 0.001) in addition to select measures of disease activity. Higher anti-MAA antibody concentrations were associated with a greater number of positive antigen-specific ACPA analytes (expressed at high titer) (P < 0.001) and a higher ACPA score (P < 0.001), independent of other covariates. Conclusion MAA adduct formation is increased in RA and appears to result in robust antibody responses that are strongly associated with ACPAs. These results support speculation that MAA formation may be a cofactor that drives tolerance loss, resulting in the autoimmune responses characteristic of RA.

Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease, considered to be autoimmune in origin. Post-translational modification of central nervous system proteins, including glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP), through citrullination of arginine residues, may lead to exposure of neoepitopes, triggering autoimmunity. Here we investigated the expression of citrullinated proteins in active MS lesions, MS normal appearing white matter and control brain white matter. We demonstrate increased citrullinated GFAP and MBP by immunohistochemistry and western blotting in areas of ongoing demyelination, suggesting a pivotal role for deimination of GFAP and MBP in MS pathogenesis MS.

Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post-translational modification caused by Ca(+2) -regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue-specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan-PAD inhibitor Cl-amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS-treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl-amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug-directed intervention in neurotrauma. Hypoxic Ischaemic Insult (HI) results in activation of peptidylarginine deiminases (PADs) because of calcium dysregulation. Target proteins undergo irreversible changes of protein bound arginine to citrulline, resulting in protein misfolding. Infection in synergy with HI causes up-regulation of TNFα, nuclear translocation of PAD4 and change in gene regulation as a result of histone deimination. Pharmacological PAD inhibition significantly reduced HI brain damage.

Background The effects of dopaminergic therapy in parkinson's disease (PD) can vary depending on the class of medication selected. Objective The aim of this post hoc study was to determine if the class of dopaminergic therapy correlated with disease severity in persons with early, treated PD. Methods A non-parametric global statistical test (GST) was used to assess the status of participants treated with dopamine agonist (DA) monotherapy, levodopa (LD) monotherapy or combined LD and DA therapy on multiple PD outcomes encompassing motor, cognitive, psychiatric and autonomic function, as well as disability and quality of life. Results The outcomes measured at the beginning of the study showed lower disease burden for participants on initial DA monotherapy compared to those taking combined LD and DA therapy after controlling for age, education, taking cog-meds and amantadine. Conclusion This observation suggests that clinicians treating early PD patients favor combined LD and DA therapy in patients with more disabling features over DA monotherapy. As such, studies of PD progression in treated PD patients may be affected by the class of symptomatic dopaminergic therapy.

Nonmotor symptoms (NMS) of Parkinson's disease (PD) are critical to identify and treat because of their impact on quality of life. Despite growing evidence of the importance of NMS on patients' quality of life, gaps remain in their recognition and treatment. The result is a need for increased information and understanding of specific NMS and the clinical approaches for their assessment and management in the context of PD as a whole. This article discusses the NMS of PD, their relationship to the pathologic basis of PD, and how NMS can be best managed.

Numerous scales assess dyskinesia in Parkinson's disease (PD), variably focusing on anatomi- cal distribution, phenomenology, time, severity, and dis- ability. No study has compared these scales and their rela- tive ability to detect change related to an established treatment. We conducted a randomized placebo-con- trolled trial of amantadine, assessing dyskinesia at base- line and at 4 and 8 weeks using the following scales: Uni- fied Dyskinesia Rating Scale (UDysRS), Lang-Fahn Activities of Daily Living Dyskinesia Rating Scale (LF), 26- Item Parkinson's Disease Dyskinesia scale (PDD-26), patient diaries, modified Abnormal Involuntary Movements Scale (AIMS), Rush Dyskinesia Rating Scale (RDRS), dys- kinesia items from the Movement Disorder Society-spon- sored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS), and Clinical Global Impression (se- verity and change: CGI-S, CGI-C). Scale order was randomized at each visit, but raters were aware of each scale as it was administered. Sensitivity to treatment was assessed using effect size. Sixty-one randomized dyski- netic PD subjects (31 amantadine, 30 placebo) completed the study. Four of the 8 scales (CGI-C, LF, PDD-26, and UDysRS) detected a significant treatment. The UDysRS Total Score showed the highest effect size (h 2 5 0.138) for detecting treatment-related change, with all other scales having effect sizes < 0.1. No scale was resistant to pla- cebo effects. This study resolves 2 major issues useful for future testing of new antidyskinesia treatments: among tested scales, the UDysRS, having both subjective and objective dyskinesia ratings, is superior for detecting treat- ment effects; and the magnitude of the UDysRS effect size from amantadine sets a clear standard for compari- son for new agents. V C 2013 Movement Disorder Society

Multiple sclerosis (MS) is a chronic, autoimmune, demyelinating, and degenerative disease that affects the central nervous system. The majority of patients are diagnosed between the ages of 20 and 40, and it is a leading cause of disability among young adults (Hauser and Goodin 2015; Lavery et al. 2014). MS typically presents with acute attacks of neurologic symptoms such as weakness, imbalance, vision loss, or numbness that are called “relapses,” and these occur early in the course of the most common form, historically known as relapsing-remitting MS (RRMS). Over time, increasing numbers of relapses contribute to disability accumulation (Fig. 18.1). At 19 years of disease duration, 75% of RRMS patients will have developed a more progressive course, whereby their worsening occurs in a steady decline and with fewer relapses (Confavreux and Vukusic 2006), a disease process historically known as secondary progressive MS (SPMS). In contrast, about 15% of patients will have progressive neurologic decline from the start, and this has been referred to as primary progressive MS (PPMS) (Lublin and Reingold 1996). In a 2013 effort to align the nomenclature with the underlying pathological changes, these diagnoses were renamed simply relapsing MS (RMS) and progressive MS (PMS) along with other, more subtle changes in diagnostic terminology that are beyond the scope of this chapter (Lublin et al. 2014).

A primary impetus for interest in a role for deiminated proteins in neurodegenerative diseases has been suggested that deimination of myelin basic protein (MBP) is a primary step in the development of multiple sclerosis (MS) (reviewed in Chap. 19). In brief, it is proposed that an unknown precipitating event causes increased peptidylarginine deiminase (PAD) activity, leading to deimination of MBP. This in turn results in disruption of the myelin structure, increased susceptibility of MBP to proteolysis, and release of MBP peptides that precipitate the autoimmune response that results in clinical symptoms. It was surprising, therefore, that a survey of proteins deiminated in MS, as well as in other CNS disorders, suggested that glial fibrillary acidic protein (GFAP) is the more predominant target. This finding raises the possibility that deimination of GFAP could have a general role in neurodegenerative disorders. This chapter explores this premise, with special emphasis on Alexander disease (AxD) as a likely candidate for an effect, since this disease is caused by dominant mutations in the GFAP gene. We begin with a description of GFAP and AxD, and then review evidence that astrocytes are a major repository for PAD in the CNS, and finally that GFAP is a primary target of its activity in both normal conditions and in several neurodegenerative disorders. This is followed by a discussion of the possible physiological effects of GFAP deimination and how these might influence disease states.

Objective To characterize the expression of malondialdehdye-acetaldehyde (MAA) adducts and anti-MAA antibody in articular tissues and serum of patients with RA. Methods Paired sera and SF were examined from 29 RA and 13 OA patients. Anti-MAA antibody, RF, ACPA and total immunoglobulin were quantified. SF-serum measures were compared within and between disease groups. The presence and co-localization of MAA, citrulline and select leukocyte antigens in RA and OA synovial tissues were examined using immunohistochemistry. Results Circulating and SF anti-MAA antibody concentrations were higher in RA vs OA by 1.5- to 5-fold. IgG (P < 0.001), IgM (P = 0.006) and IgA (P = 0.036) anti-MAA antibodies were higher in paired RA SF than serum, differences not observed for total immunoglobulin, RF or ACPA. In RA synovial tissues, co-localization of MAA with citrulline and CD19+ or CD27+ B cells was demonstrated and was much higher in magnitude than MAA or citrulline co-localization with T cells, monocytes, macrophages or dendritic cells (P < 0.01). Conclusion Anti-MAA antibodies are present in higher concentrations in the RA joint compared with sera, a finding not observed for other disease-related autoantibodies. Co-localization of MAA and citrulline with mature B cells, coupled with the local enrichment of anti-MAA immune responses, implicates MAA-adduct formation in local autoantibody production.

Background: The efficacy of rotigotine has been demonstrated in studies of patients with early (i.e. not receiving levodopa) and advanced (i.e. not adequately controlled on levodopa; average 2.5 h/day in ‘off’ state) Parkinson’s disease (PD). Objective: To further investigate the efficacy of rotigotine transdermal patch across different stages of PD symptom severity and functional disability, according to baseline Hoehn and Yahr (HY) staging. Methods: Post hoc analysis of six placebo-controlled studies of rotigotine in patients with early PD (SP506, SP512, SP513; rotigotine ≤8 mg/24 h) or advanced-PD (CLEOPATRA-PD, PREFER, SP921; rotigotine ≤16 mg/24 h). Data were pooled and analyzed according to baseline HY stage (1, 2, 3 or 4) for change from baseline to end of maintenance in Unified Parkinson’s Disease Rating Scale (UPDRS) II (activities of daily living), UPDRS III (motor) and UPDRS II+III; statistical tests are exploratory. Results: Data were available for 2057 patients (HY 1 : 262; HY 2 : 1230; HY 3 : 524; HY 4 : 41). Patients at higher HY stages were older, had a longer time since PD diagnosis and higher baseline UPDRS II+III scores vs patients at lower HY stages. Rotigotine improved UPDRS II+III versus placebo for each individual HY stage (p

Background The relationship between lung and joint inflammation in rheumatoid arthritis is poorly understood. Lung inflammation with resultant protein citrullination may trigger anti-citrullinated protein antibodies, inflammation, and arthritis. Alternatively, lung and joint inflammation may be two manifestations of a single underlying pathology. The lung has increased citrullination and TNF-α levels are high in rheumatoid arthritis; however, it is unknown if TNF-α can induce lung protein citrullination. The citrullinating enzyme peptidylarginine deiminase 4 (PAD4) exacerbates TNF-α-induced arthritis, but a role for PAD4 in lung citrullination and TNF-α-induced lung inflammation has not been explored. Our aim was to use TNF-α-overexpressing mice to clarify the intersection of TNF-α, citrullination, PAD4, arthritis, and lung inflammation. Methods Lung protein citrullination in wild-type mice, mice that overexpress TNF-α systemically (TNF+), TNF+PAD4+/+, and TNF+PAD4-/- mice was quantified by both gel electrophoresis using a citrulline probe and western blot. Hematoxylin and eosin (H&E)-stained lung sections from TNF+PAD4+/+ and TNF+PAD4-/- mice were scored for lung inflammation. H&E-stained ankle joint sections from mice that overexpress TNF-α only in the lungs were assessed for arthritis. Results TNF+ mice have increased lung protein citrullination. TNF+PAD4-/- mice do not have significantly reduced lung protein citrullination, but do have decreased lung inflammation compared to TNF+PAD4+/+ mice. Mice that overexpress TNF-α only in the lungs do not develop arthritis. Conclusions PAD4 exacerbates lung inflammation downstream of TNF-α without having a major role in generalized protein citrullination in inflamed lungs. Also, TNF-α-induced lung inflammation is not sufficient to drive murine arthritis. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1068-0) contains supplementary material, which is available to authorized users.

Purpose A hallmark of retinal gliosis is the increased detection and modification of the type III intermediate filament (IF) proteins vimentin and glial fibrillary acidic protein (GFAP). Here, we investigated vimentin and GFAP in Müller glia in a mouse model of alkali injury, focusing on the posttranslational modification of citrullination. Methods Mice were injured by corneal exposure to 1.0 N NaOH, and eyes were enucleated at different time points following injury. The levels of soluble and cytoskeletal forms of IF proteins and citrullination were measured using western blot analysis. Citrullinated GFAP was identified by immunoprecipitation followed by two-dimensional (2D) isoelectric focusing–polyacrylamide gel electrophoresis (IEF-PAGE) western blotting using a specific antibody that recognizes citrullinated GFAP. Vimentin, GFAP, and citrullinated proteins were localized in the retina by immunohistochemistry (IHC). Drug treatments were investigated in retinal explant cultures of posterior eyecups obtained from mouse eyes that were injured in vivo. Results Detection of GFAP in injured retinas increased over a period of 1 to 7 days, showing increased levels in both soluble and cytoskeletal forms of this IF protein. The global level of citrullinated proteins was also induced over this period, with low-salt buffer extraction showing the most abundant early changes in citrullination. Using IHC, we found that GFAP filaments assembled at Müller glial end feet, growing in size with time through the inner layers of the retina at 1–3 h postinjury. Interestingly, over this early time period, levels of soluble citrullinated proteins also increased within the retina, as detected by western blotting, coincident with the localization of the citrullinated epitopes on growing GFAP filaments and existing vimentin filaments by 3 h after injury. Taking advantage of the in vivo injury model to promote a robust gliotic response, posterior eyecups from 7-day postinjured eyes were treated in explant cultures with the peptidyl arginine deiminase inhibitor Cl−amidine, which was found to reduce global citrullination. Surprisingly, the detection of injury-induced high-molecular-weight GFAP species containing citrullinated epitopes was also reduced by Cl−amidine treatment. Using a low dose of the potent type III IF drug withaferin A (WFA), we showed that Cl−amidine treatment in combination with WFA reduced global protein citrullination further, suggesting that GFAP may be a key component of pathological citrullinated targets. Conclusions Our findings illuminate citrullination as a potential novel target for trauma-induced retinal gliosis. We also propose that strategies for combining drugs targeting type III IFs and citrullination may potentiate tissue repair, which is an idea that needs to be validated in vivo.

Objective: Fipamezole, a selective α 2 -adrenergic receptor antagonist, reduced levodopa-induced dyskinesias (LID) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys. In 10 dyskinetic subjects with Parkinson disease (PD), a proof-of-concept study showed beneficial effects at single doses of 60 and 90 mg. The primary study objective was to assess suppression of LID by fipamezole at day 28, as measured by the Levodopa-Induced Dyskinesia Scale (LIDS), a modification of the Abnormal Involuntary Movement Scale. Methods: This was a double-blind, randomized, placebo-controlled, dose-escalating 28-day study in levodopa-treated patients with PD experiencing LID, conducted at 25 centers in the United States (115 subjects) and 7 centers in India (64 subjects). Dyskinesias were evaluated 3 times after subjects became “on” from levodopa. Outcome assessment was performed with analysis of variance, which evaluated fipamezole dose-effects in a hierarchical stepwise manner and the Jonckheere test for dose responsiveness. Results: The total study population showed no statistically significant primary endpoint difference. However, because of inhomogeneity recognized between US and Indian study populations, a prespecified subgroup analysis of US subjects was conducted, showing fipamezole at 90 mg reduced LID (mean, 95% CI, LID rating improvement vs placebo −1.9 [0.0 to −3.8; p = 0.047]). Dose responsiveness was demonstrated ( p = 0.014 for placebo, 30, 60, and 90 mg fipamezole). Fipamezole induced mild, transient blood pressure elevation and was associated with an acceptable profile of adverse effects. Conclusions: The evidence of efficacy in the US subgroup suggested that fipamezole at 90 mg TID may be useful to treat LID in PD without exacerbating parkinsonism. Classification of evidence: This study provides Class III evidence that fipamezole is well-tolerated and, in the US subpopulation, lessens LID at 90 mg TID.

Deimination is a post-translational modification of proteins in which selected arginine amino acids are enzymatically converted to citrullines. Using dual-color immunofluorescence and an established monoclonal antibody (F95) against peptidyl-citrulline moieties, the present study is the first to compare immunohistochemical staining patterns for deiminated proteins in human substantia nigra (SN) from patients with Parkinson disease (PD) versus similar control specimens supplied by the Harvard Brain Bank. In control SN sections, many tyrosine hydroxylase (TH)-immunoreactive dopamine neurons were seen surrounded either by small fibers immunoreactive for deiminated proteins, or large reactive astrocytes, co-localized with glial fibrillary acidic protein (GFAP). However, in SN specimens from PD patients, immunoreactivity for deiminated proteins was also demonstrated within the cytoplasm of many surviving dopamine neurons that were also immunoreactive for TH, but this staining was not specifically restricted to Lewy bodies. Although the identity of neuronal deiminated proteins in these SN dopamine neurons is unknown, the present study provides evidence that the anatomical expression of these proteins in PD is altered and thus suggests that deimination may be involved in the pathophysiology of this disease.

Glioblastoma multiforme (GBM) is the most aggressive form of adult primary malignant brain tumour with poor prognosis. Extracellular vesicles (EVs) are a key-mediator through which GBM cells promote a pro-oncogenic microenvironment. Peptidylarginine deiminases (PADs), which catalyze the post-translational protein deimination of target proteins, are implicated in cancer, including via EV modulation. Pan-PAD inhibitor Cl-amidine affected EV release from GBM cells, and EV related microRNA cargo, with reduced pro-oncogenic microRNA21 and increased anti-oncogenic microRNA126, also in combinatory treatment with the chemotherapeutic agent temozolomide (TMZ). The GBM cell lines under study, LN18 and LN229, differed in PAD2, PAD3 and PAD4 isozyme expression. Various cytoskeletal, nuclear and mitochondrial proteins were identified to be deiminated in GBM, including prohibitin (PHB), a key protein in mitochondrial integrity and also involved in chemo-resistance. Post-translational deimination of PHB, and PHB protein levels, were reduced after 1 h treatment with pan-PAD inhibitor Cl-amidine in GBM cells. Histone H3 deimination was also reduced following Cl-amidine treatment. Multifaceted roles for PADs on EV-mediated pathways, as well as deimination of mitochondrial, nuclear and invadopodia related proteins, highlight PADs as novel targets for modulating GBM tumour communication.

Purpose In this article, the authors report a case of acquired stuttering associated with Parkinson’s disease (PD) that was responsive to unilateral subthalamic nucleus deep-brain stimulation (STN DBS) in the language-dominant hemisphere. Method A single-subject, masked, multiple baseline design was used to evaluate the effects of unilateral left STN DBS on stuttering associated with PD. The patient underwent 3 formal speech assessments of spontaneous speech and the reading of passages with DBS off and on. Speech samples were videotaped and placed in random order, and 2 independent speech-language pathologists calculated the percentage of stuttered syllables and classified individual stuttering events. Results Stuttering improved significantly in the DBS-on condition. In total, 10% of syllables were affected by stuttering events with DBS off, and less than 1% of syllables were affected by stuttering events with DBS on ( n = 2,281 syllables, p < .00001, in a χ 2 test). The effect of unilateral STN DBS on stuttering was relatively independent of whether the patient was on or off dopaminergic medications. Conclusion This article emphasizes the important role of the subthalamic region in the motor control of speech and language.

OBJECTIVES: To investigate financial capacity in patients with mild cognitive impairment (MCI) and Alzheimer’s disease (AD) using a clinician interview approach. DESIGN: Cross-sectional. SETTING: Tertiary care medical center. PARTICIPANTS: Healthy older adults (n 575) and patients with amnestic MCI (n 558), mild AD (n 597), and moderate AD (n 531). MEASUREMENTS: The investigators and five study physicians developed a conceptually based, semistructured clinical interview for evaluating seven core financial domains and overall financial capacity (Semi-Structured Clinical Interview for Financial Capacity; SCIFC). For each participant, a physician made capacity judgments (capable, marginally capable, or incapable) for each financial domain and for overall capacity. RESULTS: Study physicians made more than 11,000 capacity judgments across the study sample (N 5261). Very good interrater agreement was obtained for the SCIFC judgments. Increasing proportions of marginal and incapable judgment ratings were associated with increasing disease severity across the four study groups. For overall financial capacity, 95% of physician judgments for older controls were rated as capable, compared with 82% for patients with MCI, 26% for patients with mild AD, and 4% for patients with moderate AD. CONCLUSION: Physicians and other clinicians can reliably evaluate financial capacity in cognitively impaired older adults using a relatively brief, semistructured clinical interview. Patients with MCI have mild impairment in financial capacity, those with mild AD have emerging global impairment, and those with moderate AD have advanced global impairment. Patients with MCI and their families should proactively engage in financial and legal planning, given these patients’ risk of developing AD and accelerated loss of financial abilities. J Am Geriatr Soc 57:806–814, 2009.

Little is currently known about the higher order functional skills of patients with Parkinson disease and cognitive impairment. Medical decision-making capacity (MDC) was assessed in patients with Parkinson's disease (PD) with cognitive impairment and dementia. Participants were 16 patients with PD and cognitive impairment without dementia (PD-CIND), 16 patients with PD dementia (PDD), and 22 healthy older adults. All participants were administered the Capacity to Consent to Treatment Instrument (CCTI), a standardized capacity instrument assessing MDC under five different consent standards. Parametric and non-parametric statistical analyses were utilized to examine capacity performance on the consent standards. In addition, capacity outcomes (capable, marginally capable, or incapable outcomes) on the standards were identified for the two patient groups. Relative to controls, PD-CIND patients demonstrated significant impairment on the understanding treatment consent standard, clinically the most stringent CCTI standard. Relative to controls and PD-CIND patients, patients with PDD patients were impaired on the three clinical standards of understanding, reasoning, and appreciation. The findings suggest that impairment in decisional capacity is already present in cognitively impaired PD patients without dementia, and increases as these patients develop dementia. Clinicians and researchers should carefully assess decisional capacity in all PD patients with cognitive impairment.

Despite recent advances in the treatment of Parkinson disease (PD), levodopa remains the most effective and widely used therapy. A major limitation to the use of levodopa is the development of abnormal involuntary movements, termed levodopa-induced dyskinesia (LDID), following chronic levodopa treatment. Since recent studies have suggested that modifications of chromatin structure may be responsible for many long-lasting changes in brain function, we have examined post-translational modifications of striatal histones in two models of LDID: an acute murine model and a chronic macaque monkey model, both exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In the primate model, which closely resembles human LDID, we observed that chronic levodopa and the appearance of LDID was associated with marked deacetylation of histone H4, hyperacetylation and dephosphorylation of histone H3, and enhancement of the phosphorylation of extracellular signal-regulated kinase (ERK). In the murine model of acutely rather than chronically induced LDID, dopamine depletion and levodopa treatment also induced deacetylation of histone H4 and phosphorylation of ERK, but histone H3 exhibited decreased trimethylation and reduced rather than enhanced acetylation. These data demonstrate striking changes in striatal histones associated with the induction of LDID in both animal models. The pattern of changes observed, as well as the behavioral features, differed in the two models. However, both models exhibit marked deacetylation of histone H4, suggesting that inhibitors of H4 deacetylation may be useful in preventing or reversing LDID.

The peptidylarginine deiminase (PAD) family of enzymes are responsible for conversion of protein-bound arginine to citrulline in most tissues of the body and are garnering increased interest for their physiological and pathological roles. Although it has been shown that oligodendrocytes of the CNS express the PAD isoenzyme type 2, nothing is presently known about PAD expression in Schwann cells, the myelinating cells of the PNS. To evaluate PAD expression in the PNS, cultivated rat and human Schwann cells and slices of fetal, juvenile, and normal and regenerated adult sciatic nerves were examined with RT-PCR, Western blot, and immunohistochemical analysis. Samples from cerebellar cultures and skin served as positive controls. One of the principle findings was that cultivated Schwann cells expressed significant levels of mRNA and protein for the PAD isoenzymes 2 and 3. PAD1 and PAD4, however, were not expressed in any types of Schwann cells. Using double immunofluorescence, the majority of PAD2 staining was localized in immature cell stages. Moreover, increased amounts of PAD2, PAD3, and peptidyl-citrulline were also found in human fetal and rat juvenile and regenerated sciatic nerves as compared to similar normal adult specimens. Neuronal and inducible nitric oxide synthases, enzymes that convert free arginine to citrulline, were also expressed in Schwann cells; however, their massive induction by LPS/K+, was not reflected in an enhanced peptidyl-citrulline immunosignal. These data suggest that, similar to the CNS, citrullination of proteins may also exert a specific role in thecourse of PNS development and repair. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2008

Objective: To define the contribution of citrullination on GFAP and vimentin in a traumatic eye injury model. Methods: An ocular alkali injury model of retinal gliosis was employed in mice. Retinas from injured mice were examined at different times by western blot or immunochemistry for expression of GFAP, vimentin and citrullinated proteins. Posterior eye-cups from 7-day injured mice were placed into explant culture to investigate effects of peptidyl arginine deiminase (PAD) inhibitor Cl-amidine. Results: GFAP expression increases initially in the soluble extracts, starting as early as 1 hour after injury and continues over the 7-day post-injury period, followed by increased abundance of polymeric filaments in insoluble extracts. GFAP filament growth begins at the Muller glial end-feet and advances towards the outer retina, showing punctate co-staining for citrullinated precursors that become incorporated into vimentin and GFAP filaments from soluble pools. At later time points, these citrullinated prote...

Malondialdehyde-acetaldehyde (MAA) adducts are a product of oxidative stress associated with tolerance loss in several disease states. This study was undertaken to investigate the presence of MAA adducts and circulating anti-MAA antibodies in patients with rheumatoid arthritis (RA).Synovial tissue from patients with RA and patients with osteoarthritis (OA) were examined for the presence of MAA-modified and citrullinated proteins. Anti-MAA antibody isotypes were measured in RA patients (n = 1,720) and healthy controls (n = 80) by enzyme-linked immunosorbent assay. Antigen-specific anti-citrullinated protein antibodies (ACPAs) were measured in RA patients using a multiplex antigen array. Anti-MAA isotype concentrations were compared in a subset of RA patients (n = 80) and matched healthy controls (n = 80). Associations of anti-MAA antibody isotypes with disease characteristics, including ACPA positivity, were examined in all RA patients.Expression of MAA adducts was increased in RA synovial tissue compared to OA synovial tissue, and colocalization with citrullinated proteins was found. Increased levels of anti-MAA antibody isotypes were observed in RA patients compared to controls (P0.001). Among RA patients, anti-MAA antibody isotypes were associated with seropositivity for ACPAs and rheumatoid factor (P0.001) in addition to select measures of disease activity. Higher anti-MAA antibody concentrations were associated with a greater number of positive antigen-specific ACPA analytes (expressed at high titer) (P0.001) and a higher ACPA score (P0.001), independent of other covariates.MAA adduct formation is increased in RA and appears to result in robust antibody responses that are strongly associated with ACPAs. These results support speculation that MAA formation may be a cofactor that drives tolerance loss, resulting in the autoimmune responses characteristic of RA.

We describe the case of an adult female with Hallervorden-Spatz disease (HSD), "eye-of-the-tiger" sign on cranial magnetic resonance imaging scan, and two mu- tations in the pantothenate kinase 2 (PANK2) gene. Symp- tomatic presentation included stuttering dysarthria, dys- tonic posturing, increased limb and axial muscle tone, choreoathetosis, stereotyped motor behaviors, and obses- sive- compulsive symptomatology since adolescence. Exten- sive neuropsychological testing at 40 and 44 years of age revealed a relatively normal IQ and stable cognitive pattern overall. This case demonstrates that HSD patients who survive into middle age should not be assumed to have a progressive dementia. In such cases, atypical behavioral problems such as persistent obsessions and compulsions may be present instead. © 2005 Movement Disorder Society

In this study, we demonstrate for the first time the immunohistochemical expression of citrullinated proteins in the central nervous system (CNS) of mice with myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). By using an established monoclonal antibody (F95) against natural and synthetic citrullinated proteins (Nicholas and Whitaker [2002] Glia 37:328-336), numerous, small, previously unrecognized "patches" of citrullinated proteins were discovered throughout EAE brains, whereas EAE spinal cords showed similar but much larger lesions. On dual color immunofluorescence, these lesions were found to contain citrullinated myelin basic protein (MBP) and were surrounded by astrocytes immunoreactive for both glial fibrillary acidic protein (GFAP) and F95. These lesions became evident about the time when EAE mice became symptomatic and increased in size and number with increasing disease severity. In some sections of spinal cord but not brains of severely debilitated EAE mice, a widespread gliotic response was seen, with astrocytes containing citrullinated GFAP spread throughout the gray and white matter. Western blot analysis of acidic proteins from the brains and spinal cords of EAE mice had higher levels of multiple citrullinated GFAP isoforms compared with controls, with more F95-positive bands in the EAE brains vs. spinal cords. These results raise the possibility that citrullination of both GFAP and MBP may contribute to the pathophysiology of EAE and that the brains of EAE mice may contain more pathology than previously realized.

Objective To address the ongoing debate concerning the specificity of synovial citrullinated proteins for rheumatoid arthritis (RA) and to analyze their pathophysiologic relevance to the induction or perpetuation of the RA-specific anti–citrullinated protein antibodies (ACPAs). Methods Synovium of 19 RA patients and 19 non-RA controls was immunostained for the presence of citrullinated proteins with a mouse monoclonal antibody (F95), for the citrullinating enzyme peptidyl arginine deiminase type 2 (PAD-2), and for the free citrulline–producing enzyme inducible nitric oxide synthase (iNOS). Extending the RA cohort to 61 patients, the findings of anticitrulline staining in synovium were related to serum and synovial fluid ACPA levels, as measured by enzyme-linked immunosorbent assay. Results F95 staining indicated the presence of synovial intracellular citrullinated proteins in 53% of RA samples versus 5% of control samples, whereas extracellular staining was not RA specific. Immunoblotting and inhibition experiments confirmed that the antibody recognized citrullinated proteins but not free citrulline. Accordingly, iNOS was equally found in RA and control synovium and in intracellular citrullinated protein–positive and intracellular citrullinated protein–negative samples. In contrast, intracellular citrullinated proteins colocalized with PAD-2, which was found in 59% of RA samples versus 17% of control samples. Independent of local disease activity, the presence of the RA-specific synovial intracellular citrullinated proteins was associated with significantly higher systemic and local ACPA levels and with local ACPA production in the joint. Conclusion These data confirm the presence of RA-specific intracellular citrullinated proteins in synovium. The link with PAD-2 and local and systemic ACPA levels emphasizes their pathophysiologic relevance for RA-specific humoral autoimmunity.

The most common form of autosomal dominant hereditary spastic paraparesis (HSP), SPG4, is caused by mu- tations in the spastin gene on chromosome 2p. This disease is characterized by intra- and interfamilial phenotypic variation. To determine the predictive values of clinical signs and symp- toms in SPG4, we examined 43 members of a large pedigree with autosomal dominant HSP. We then identified the genetic etiology of the disorder in this family, a novel nonsense muta- tion in exon 1 of spastin, carried by 24 of the examined family members. The best clinical predictors of positive gene status were the presence of hyperreflexia in the lower extremities, 2 beats of ankle clonus, pes cavus, bladder symptoms and in- creased tone in the legs. The mean age of onset was 32.2 7.4 years, but the age of onset was earlier in children from 10 of 12 child-parent gene-positive pairs, with a mean difference of 10.8 3.3 years. The finding of leg weakness was especially common in older-onset affected family member with leg hy- perreflexia. These results suggest that specific clinical signs and symptoms may be of value in differentiating individuals af- fected with SPG4 from family members with nonspecific neu- rological findings. © 2004 Movement Disorder Society

By using hybridoma technology, an IgM monoclonal antibody (F95) against multiple citrullinated synthetic and natural peptides was recently developed and used to stain immunohistochemically subsets of astrocytes and myelin basic protein (MBP) from selected regions of human brain (Nicholas and Whitaker [2002] Glia 37:328-336). With this antibody, the present study provides a more detailed localization of citrullinated epitopes in the central nervous system (CNS) by examining immunohistochemical staining patterns for F95 in the normal adult rat brain. Thus, immunohistochemical labeling for citrullinated epitopes was seen in white matter areas consistent with myelin staining; however, in general, it was more prominent and uniform in the caudal CNS (spinal cord, medulla oblongata, pons, and cerebellum) than in more rostral areas. F95 staining was also seen in cells and fibers often intimately associated with blood vessels and/or ventricular surfaces. By using dual-color immunofluorescence, the vast majority of this latter staining was colocalized within a subset of astrocytes also immunoreactive for glial fibrillary acidic protein (GFAP). By using Western blot analysis of rat brain proteins, multiple GFAP- and MBP-immunoreactive proteins and peptide fragments were seen, and many of them were also reactive with the F95 antibody. Thus, the present study not only demonstrates that citrullinated epitopes in normal rat brain are most concentrated in subsets of myelin and astrocytes but also provides evidence that GFAP, like MBP, may be present as multiple citrullinated isoforms.

A C-to-T single-nucleotide polymorphism (SNP) located at position 1858 of human protein tyrosine phosphatase PTPN22 complementary DNA carries the highest risk of rheumatoid arthritis (RA) among all non-HLA genetic variants. This C1858T SNP converts an arginine (R620) to a tryptophan (W620), but it is unclear why it has such a strong impact on RA, a disease characterized by anti-citrullinated protein antibodies. The aim of this study was to test the hypothesis that PTPN22 regulates protein citrullination.The level of citrullinated proteins in immune cells was quantified by Western blotting. The physical interaction between PTPN22 and peptidyl arginine deiminase type 4 (PAD-4), which is one of the enzymes that catalyzes protein citrullination, was examined by coimmunoprecipitation. Neutrophils were collected from healthy donors carrying the C1858T SNP and healthy donors not carrying this SNP. The formation of neutrophil extracellular traps (NETs) was examined by immunocytochemistry.PTPN22 physically interacted with PAD-4, and a deficiency in PTPN22 enhanced protein citrullination. This abnormality was reversed by exogenous wild-type PTPN22 or catalytically dead mutant PTPN22. The R-to-W conversion rendered PTPN22 unable to interact with PAD-4 and suppress citrullination. The C1858T SNP was associated with hypercitrullination in peripheral blood mononuclear cells and a heightened propensity for spontaneous formation of NETs, which is a PAD-4-dependent process.PTPN22 is an inhibitor of PAD-4 and protein citrullination. This function of PTPN22 is independent of its phosphatase activity but requires R620. Our data not only establish a molecular link between PTPN22 and PAD-4, but also suggest that the C1858T SNP increases the risk of RA by enhancing protein citrullination and spontaneous formation of NETs.

Using hybridoma technology, an IgM monoclonal antibody (mAb), designated as F95, was developed against a deca-citrullinated peptide (DCP) consisting of 10 citrulline residues and a carboxyl Gly-Gly-Cys through which DCP was covalently linked to an activated carrier protein, keyhole limpet hemocyanin (KLH). Clones were selected on the basis of not reacting with human unmodified and noncitrullinated myelin basic protein (MBP), MBP-C1, but reacting well with human citrullinated MBP (MBP-C8). When tested by ELISA, this mAb demonstrated minimal reactivity with human MBP-C1, varying reactivity with the C2-C5 isomers of human MBP, moderate binding with guinea pig MBP-C8, and strong reactivity with human MBP-C8. By ELISA, mAb F95 was directed predominantly against citrulline, not MBP, as revealed by its binding to DCP linked with activated KLH, bovine serum albumin (BSA), or ovalbumin (OA), but not with KLH, BSA, or OA alone. Immunohistochemistry of normal human brain demonstrated that F95 stained central nervous system myelin and a subset of astrocytes. Given the citrulline-directed features of mAb F95, this immunohistochemical pattern suggests that certain astroglial filaments expressing glial fibrillary acidic protein also contain citrulline-bearing components. These potentially implicate citrullinated proteins, notably in astroglial filaments, in a variety of normal and pathological neurobiological processes.

Background: Previous phase III studies in patients with advanced Parkinson’s disease (PD) not adequately controlled on levodopa demonstrated significant reduction of ‘off’ time with rotigotine transdermal system up to 16 mg/24 h. However, the minimal effective dose has not been established. Objective: This international, randomized, double-blind, placebo-controlled study (SP921; NCT00522379) investigated rotigotine dose response up to 8 mg/24 h. Methods: Patients with advanced idiopathic PD (≥2.5 h of daily ‘off’ time on stable doses of levodopa) were randomized 1:1:1:1:1 to receive rotigotine 2, 4, 6, or 8 mg/24 h or placebo, titrated over 4 weeks and maintained for 12 weeks. The primary efficacy variable was change from baseline to end of maintenance in absolute time spent ‘off’. Results: 409/514 (80%) randomized patients completed maintenance. Mean (±SD) baseline daily ‘off’ times (h/day) were placebo: 6.4 (±2.5), rotigotine 2–8 mg/24 h: 6.4 (±2.6). Rotigotine 8 mg/24 h was the minimal dose to significantly reduce ‘off’ time versus placebo. LS mean (±SE) absolute change in daily ‘off’ time (h/day) from baseline was −2.4 (±0.28) with rotigotine 8 mg/24 h, and −1.5 (±0.26) with placebo; absolute change in ‘off’ time in the 8 mg/24 h group compared with placebo was −0.85 h/day (95% CI −1.59, −0.11; p = 0.024). There was an apparent dose-dependent trend. Adverse events (AEs) reported at a higher incidence in the rotigotine 8 mg/24 h group versus placebo included application site reactions, nausea, dry mouth, and dyskinesia; there was no worsening of insomnia, somnolence, orthostatic hypotension, confusional state or hallucinations, even in patients ≥75 years of age. Conclusions: The minimal statistically significant effective dose of rotigotine to reduce absolute ‘off’ time was 8 mg/24 h. The AE profile was similar to previous studies.