367A.P. Nicholas and S.K. Bhattacharya (eds.), Protein Deimination in Human Health and Disease, DOI 10.1007/978-1-4614-8317-5_20, © Springer Science+Business Media New York 2014 Keywords Homocitrulline • Carbamoylation • Autoantibodies • Chemical modifi -cation • Cross-reaction • Monoclonal antibody F95 20.1 Introduction A structural homolog to citrulline, homocitrulline, can be present in proteins and peptides as a product of posttranslational modifi cation. Homocitrulline is formed nonenzymatically from lysine residues in the polypeptide chain by the action of cyanate. The latter compound is derived either from urea or, via a reaction catalyzed by the enzyme myeloperoxidase (MPO), from thiocyanate. The formation of homocitrulline is known as carbamoylation or carbamylation. In humans, this pro-cess takes place predominantly in two types of situations: uremia and infl ammation. Smoking increases the circulating thiocyanate concentration and thus enhances car-bamoylation of proteins. The similarities between citrulline and homocitrulline are relevant in two ways. First, as structural analogs, they can act as confounding factors in assays aimed at Chapter 20 Homocitrulline: An Analog and Confounder Related to Citrulline Sanna Turunen , Marja-Kaisa Koivula , Anthony P. Nicholas , Leila Risteli , and Juha Risteli S. Turunen Department of Clinical Chemistry , Institute of Diagnostics, University of Oulu , Oulu , FI-90014 , Finland M.-K. Koivula • L. Risteli • J. Risteli (*) Department of Clinical Chemistry , Institute of Diagnostics, University of Oulu , Oulu , FI-90014 , Finland Nordlab Oulu, Oulu University Hospital , Oulu , Finland e-mail: juha.risteli@oulu.fi A. P. Nicholas Department of Neurology , University of Alabama at Birmingham and the Birmingham Veterans Administration Medical Center , Birmingham , AL , USA 368specifi cally measuring just one of them. If such an assay is based on detecting ure-ido groups in proteins, it is likely that it cannot distinguish between citrulline and homocitrulline. If the presence of citrulline in tissues or in a protein is studied, it is imperative to avoid conditions that cause carbamoylation, in particular the use of high concentrations of urea. Secondly, immunization with proteins containing one of the amino acids can elicit antibodies that also recognize the other. Experimentally produced antibodies against carbamoylated proteins have been shown to bind to synthetic cyclic citrullinated peptide (CCP), an antigen used to demonstrate the presence of anti-citrulline antibodies in patients with rheumatoid arthritis (RA). Anti-homocitrulline antibodies have also been found to be present in the sera of patients with RA and can also predict joint damage. These fi ndings are particularly interesting, as smoking is the only environmental factor that has been associated with the development of RA (see Chap. 5 ) and the pathogenetic link has not been found so far. 20.2 Why Homocitrulline Is Important Citrulline and homocitrulline (ε-carbamyl lysine) are two amino acids that both contain an ureido group (Fig. 20.1 ). In addition to similar structures, the two amino acids have common features in their biosynthesis and immunogenicity. In proteins, neither exists in a primary translation products. Their posttranslational formation mechanisms are different and the conditions that favor their respective formations are not identical; however, their close structural resemblance makes them potential Fig. 20.1 Structures of citrulline and homocitrulline S. Turunen et al. 369confounders in assays designed to detect just one of them. Furthermore, these amino acids, when present in proteins, can induce antibodies that recognize them both. Citrulline is formed from polypeptide-bound arginine by PAD enzymes (Chap. 1 ), while homocitrulline is formed from lysine in proteins via a spontaneous carbamo-ylation (also known as carbamylation) reaction that involves isocyanic acid, an isomer of cyanate. The latter compound, in turn, is derived either from the spontane-ous decomposition of urea or from the oxidation of thiocyanate by the enzyme MPO (Fig. 20.2 ). Kidney failure with uremia leads to increased homocitrulline formation in tissue and plasma proteins. In addition, increased protein carbamoylation has been demonstrated in atherosclerosis and in infl ammatory situations (Jaisson et al. 2011 ; Wang et al. 2007 ). As hydrophilic, basic residues, arginine and lysine are often located on the surface of proteins. Thus, the loss of their charges caused by citrullination (also known as deimination) or carbamoylation (Kraus et al. 2001 ), respectively, distorts the protein structure (Kraus and Kraus 2001 ; Tarcsa et al. 1996 ), thus increasing its susceptibility to proteolytic degradation and can produce potential antigens (Mydel et al. 2010 ; Turunen et al. 2010 ). The antigenicity of peptide-bound citrulline is relevant in RA, as these patients have been shown to possess antibodies against deiminated proteins, even years before the clinical onset of the disease (Koivula et al. 2007 ; Nielen et al. 2004 ). The presence of such antibodies is also related to a more severe disease course (Rantapää-Dahlqvist et al. 2003 ). The antigenicity of homocitrullinated proteins is a more recent discovery, and, so far, the clinical signifi cance of this modifi cation in RA is not clear (Mydel et al. 2010 ). However, antibodies against homocitrullinated pro-teins can be induced in experimental animals (Turunen et al. 2010 ) and have also been found in the sera of RA patients, where they predict joint damage (Shi et al. 2011 ). In this chapter, we review what is presently known about homocitrulline, in the contexts that are relevant for citrulline. However, it is beyond the scope of this chapter to provide a general review on the chemistry or biological functions of homocitrulline. Fig. 20.2 Formation of homocitrulline by carbamoylation. MPO myeloperoxidase 20 Homocitrulline: An Analog and Confounder Related to Citrulline 37020.3 How Homocitrulline Is Formed in the Body Spontaneous carbamoylation is a reaction between cyanate and a free amino group of an amino acid. Carbamoylation can affect all amino groups in proteins, but some-times at different rates. In fact, the reaction for α-amino groups is 100 times faster than for the ε-amino group of lysine (Stark 1965 ). Also, the reaction is practically irreversible. Cyanate in tissues is derived from the chemical equilibrium reaction between urea, on one hand, and ammonium and cyanate ions, on the other hand (Fig. 20.2 ). Consequently, any urea-containing solution will eventually also contain cyanate. In the human body, the urea concentration in body fl uids is particularly high in uremia, which is a state of accumulation of urea and other nitrogenous waste products due to kidney failure. Because of its irreversible nature, carbamoylation leads to the accumulation of homocitrulline in proteins during aging and in various diseases (reviewed in Jaisson and Gillery 2010 ). In healthy individuals, the concentration of plasma cyanate is around 50 nmol/L, while in uremic patients, it increases by threefold (Nilsson et al. 1996 ). Even such a high cyanate concentration is signifi cantly lower than the theo-retical value, calculated from the corresponding urea concentration, and indicates that cyanate disappears by effi ciently reacting with the available amino groups. In kidney failure, the large load of functionally altered carbamoylated proteins may further aggravate renal dysfunction (Kraus et al. 2001 ). In addition to spontaneous carbamoylation, there is also another enzyme-assisted carbamoylation route. The enzyme MPO, presents in infl ammatory cells, catalyzes the oxidation of thiocyanate to cyanate in the presence of hydrogen peroxide. As a result, the cyanate then becomes available for protein carbamoylation (Wang et al. 2007 ). Phagocytic cells, at least granulocytes and monocytes, contain MPO (reviewed in Klebanoff 2005 ), which is part of a defense mechanism against outside pathogens. MPO acts in the phagosome or, if the pathogen is too large to be ingested by the phagocytic cell, it is released into the extracellular space. An antimicrobial system that consists of MPO, halides (iodide, bromide, chloride) (Klebanoff 1968 ), or thiocyanate (Klebanoff et al. 1966 ), and hydrogen peroxide (H 2 O 2 ) is effective against numerous organisms. In this reaction, the thiocyanate, which has been gained from diet or tobacco smoke, is turned to active cyanate. This reaction sequence links infl ammation and smoking to protein carbamoylation (Wang et al. 2007 ). 20.4 Homocitrulline as a Confounding Factor in Deimination (Citrulline) Assays Many methods for detecting citrulline rely on demonstrating the presence of a ureido group in the protein or tissue to be studied. Such methods should be regarded as detecting both homocitrulline and citrulline, unless higher specifi city is rigorously proved and the reaction with homocitrulline excluded. One example of such a method is the chemical modifi cation of a ureido group that involves the use of antipyrine and S. Turunen et al. 3712,3-butanedione monoxime under acidic conditions, after which the modifi ed amino acid is then detected immunologically. There is a commercially available antibody preparation designated as anti-citrulline (modifi ed) antibody (Millipore; previously Upstate). As presented in Fig. 20.3 , the antibody reacts with both citrulline and homocitrulline-containing proteins (Turunen et al. 2010 ; Shi et al. 2013 ). This is to be expected, as both amino acids contain identical ureido groups and the side chain of the homocitrulline residue is even longer than that of the citrulline residue. The results that have been obtained with this antibody should be interpreted with this possible cross-reaction in mind, paying particular attention to how probable it is that the conditions used favor the formation of homocitrulline by carbamoylation. As discussed earlier, urea in solution is in equilibrium with cyanate, the latter of which readily carbamoylates the amino groups of proteins. We have treated human albumin with potassium cyanate under different conditions (Fig. 20.3 ) and found that, even at 37 °C, the degree of carbamoylation is signifi cant. It seems that avoiding the use of urea when studying the presence of citrulline in tissues is really important. Urea is generally used for solubilizing proteins, such as from tissue samples, and it is not easy to fi nd suitable replacements for it. When high concentrations (6–8 M) of urea are needed, the generation of cyanate is often inevitable. Removal of the cya-nate by ion-exchange does not solve the problem completely, as the equilibrium between urea and cyanate is reestablished. However, the process can be controlled to some extent by using appropriate buffers and cyanate scavangers, such as Tris, glycine, ethanolamine, or diethylenediamine. In this respect, demonstrations of pepti-dyl-citrulline in tissue samples are in need of verifi cation with more specifi c methods (Klareskog et al. 2006 ; Senshu et al. 1992 ). Fig. 20.3 Immunological cross-reaction between protein-bound citrulline and homocitrulline. SDS-polyacrylamide gel electrophoresis with subsequent blotting onto nitrocellulose membrane, chemical modifi cation and immunostaining shows ( a ) protein staining (Coomassie) and ( b ) immu-noreaction with polyclonal anti (modifi ed)-citrulline antibody (Upstate Biotechnology/Millipore) (Senshu et al. 1992 ). The lanes for each reaction are molecular mass standard ( lane 1 ), native human serum albumin ( lane 2 ), PAD-treated human serum albumin ( lane 3 ), and carbamo-ylated human serum albumin ( lanes 4–6 and 8–10 ), under different conditions. Lane 7 is blank. The KCNO treatment was carried out at 37 °C ( lane 4 : 2 h, 5 : 6 h and 6 : 17 h) and at 70 °C ( lane 8 : 2 h, 9 : 6 h and 10 : 17 h) 20 Homocitrulline: An Analog and Confounder Related to Citrulline 37220.5 Detection of Modifi ed and Unmodifi ed Citrulline and Homocitrulline in Hydrolysates The chemical modifi cation mentioned above can also be used for detecting free citrulline and homocitrulline in solution. The reaction between a ureido group- containing structure with 2,3-butanedione (also known as diacetyl) creates an adduct with a yellow/orange color. Absorbance of this color has been used for quantifying ureido group-containing molecules since the 1930s (Fearon 1939 ), but it can also be used as part of other techniques. If the modifi ed protein is hydrolyzed, citrulline and homocitrulline can be separated on HPLC and detected (Turunen et al. 2010 ) as separate absorbance peaks at 464 nm (Fig. 20.4 ). The disadvantage of this method is that the sample is lost in the analysis and the location of citrulline or homocitrulline in the protein can only be determined by sample purifi cation and identifi cation before this analysis. Methods have been suggested for 2,3- butanedione-modifi ed citrulline by mass spectrometry (De Ceuleneer et al. 2011 ), using the 50 mass shift as recognition tool, but the 2,3-butanedione reaction still remains common for all substituted ureas, including both citrulline and homocitrulline. Classical methods for quantifying both citrulline and homocitrulline in proteins include amino acid analysis, either by ion-exchange chromatography or by high- performance liquid chromatography (HPLC), possibly coupled to tandem mass spec-trometry, both after hydrolysis of the protein sample. Some loss of homocitrulline takes place during the hydrolysis, resulting in reappearance of lysine. These methods require the isolation of the protein in question, with subsequent hydrolysis, and are thus generally not used for demonstrating the presence of either citrulline or homoc-itrulline in complex tissue samples. 20.6 Citrulline and Homocitrulline as Immunogens Citrullinated proteins can be used for raising antibodies in experimental animals, both in their native state and when citrulline has been chemically modifi ed. Two immunological methods have been described for the detection of citrullinated proteins, respectively: a commercially available anti-(modifi ed) citrulline rabbit antibody (Millipore; previously Upstate) (Senshu et al. 1992 ) and the monoclonal antibody F95 (Nicholas and Whitaker 2002 ). The former antibody recognizes citrulline in proteins after the amino acid has undergone a chemical modifi cation by 2,3-butanedione monoxime and antipyrine in acidic conditions. The F95 antibody is against non-modifi ed citrulline in proteins (see Chap. 14 for more details). As with any antibodies, verifying their specifi city with respect to similar, homocitrulline- containing structures is essential. As shown in Figs. 20.3b and 20.5 , both of these antibodies are able to react with carbamoylated (homocitrulline-containing) proteins, as well as with their citrulline-containing counterparts. In rabbits, homocitrulline-containing proteins can be used to elicit antibodies that also recognize citrulline in proteins (Turunen et al. 2010 ). The immunogens S. Turunen et al. 373have been albumin and type I collagen or its peptide parts, and the antibodies produced also react with the CCP-2 antigen, an artifi cial cyclic peptide that has been designed for detecting anti-citrulline antibodies in humans with RA (Turunen et al. 2010 ). Furthermore, immunization with homocitrulline-containing peptides causes a T cell response in mice, similar to that known to be critical to the induction of autoimmune arthritis (Mydel et al. 2010 ). Fig. 20.4 Separation of modifi ed citrulline and homocitrulline on HPLC. Hydrolyzed human serum albumin samples run on HPLC after chemical modifi cation with 2,3-butanedione and anti-pyrine in acidic conditions. ( a ) Native albumin; ( b ) citrullinated albumin (PAD treated), together with the structure of citrulline; ( c ) homocitrullinated albumin (KCNO treated), together with the structure of homocitrulline. X axis = time in min, Y axis = absorbance at 464 nm 20 Homocitrulline: An Analog and Confounder Related to Citrulline 37420.7 What Homocitrulline Has to Do with Citrulline In Vivo The clinical signifi cance of citrulline is largely the subject of this book. At the protein level, effects of carbamoylation are similar to those of citrullination. Lysine is often located on the surface of proteins and enhances its solubility in an aqueous environ-ment. The loss of charge, caused by carbamoylation (Kraus et al. 2001 ; Kraus and Kraus 2001 ), distorts the protein structure (Kraus and Kraus 2001 ; Tarcsa et al. 1996 ) and results in a potentially antigenic structure (Mydel et al. 2010 ; Turunen et al. 2010 ). Antigenicity of carbamoylated proteins in experimental animals has been demonstrated (Turunen et al. 2010 ), and carbamoylation-dependent activation of T cells has been recorded (Mydel et al. 2010 ). In addition, interaction of carbamo-ylated albumin and collagen with immune responsive cells has also been shown (Jaisson et al. 2006 , 2007 ). Carbamoylation is present in tissues of patients with chronic renal failure and in atherosclerosis (reviewed in Jaisson et al. 2011 ). It is a relatively new fi nding that patients with RA also have antibodies against homocitrulline, in addition to those against citrulline, and such antibodies are also able to predict joint damage (Shi et al. 2011 ). Antibodies of these two specifi cities can be separated by immunoabsorption (Shi et al. 2013 ). The major assay used for detecting anti-citrulline antibodies in RA, that for anti-CCP, does not distinguish between these two specifi cities, as antisera that are experimentally produced to car-bamoylated antigens, also readily bind to the CCP antigen (Turunen et al. 2010 ). Smoking is an environmental factor that has a role in several diseases. In RA (see Chap. 5 ), it is the only environmental factor connected to citrulline antibodies in genetically susceptible individuals (Klareskog et al. 2006 ). The mechanism of the effect of smoking on the immune response against citrullinated proteins is not yet clear. However, the fi nding that antibodies against carbamoylated proteins are also present in RA sera and correlate with the disease outcome is interesting, considering Fig. 20.5 Binding of protein-bound homocitrulline to monoclonal antibody F95 raised against a deca- citrullinated peptide (Nicholas and Whitaker 2002 ). The proteins have not been chemically modifi ed before SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting. Lanes 1–3 are human serum albumin carbamoylated with KCNO at 70 °C ( lane 1 : 2 h, 2 : 6 h and 3 : 17 h). No staining was seen with unmodifi ed albumin S. Turunen et al. 375the carbamoylation route through MPO. The serum levels of thiocyanate are elevated in smokers (Rubab and Rahman 2006 ), who are also more prone to infec-tions (reviewed in Huttunen et al. 2011 ). As a result of these factors, the contribution of carbamoylation, as it relates to that of deimination in different disease states, still needs to be further explored. 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J Biol Chem 271:30709–30716 Turunen S, Koivula MK, Risteli L, Risteli J (2010) Anticitrulline antibodies can be caused by homocirtulline-containing proteins in rabbits. Arthritis Rheum 62:3345–3352 Wang Z, Nicholls SJ, Rodriguez ER, Kummu O, Hörkkö S, Barnard J, Reynolds WF, Topol EJ, DiDonato JA, Hazen SL (2007) Protein carbamylation links infl ammation, smoking, uremia and atherogenesis. Nat Med 13:1176–1184 S. Turunen et al.Citations (6)References (31)... Homocitrulline is a product of protein carbamylation, generated non-enzymatically from lysine residues by the action of cyanate. Cyanate is itself derived from the spontaneous decomposition of urea or via the oxidation of thiocyanate by myeloperoxidase in the presence of H2O2 [22]. Finally, to gain insight into the lipoprotein status, Apo-AI, Apo-B, and also myeloperoxidase-oxidized LDLs (Mox-LDLs) were quantified in dialysis patients versus healthy volunteers. ...... Finally, the concentration of homocitrulline was quantified. Homocitrulline is a product of protein carbamylation that accumulates after the oxidation of thiocyanate by myeloperoxidase in the presence of H2O2 [22]. An increase in protein carbamylation has been observed in inflammatory diseases such as atherosclerosis [22]. ...... Homocitrulline is a product of protein carbamylation that accumulates after the oxidation of thiocyanate by myeloperoxidase in the presence of H2O2 [22]. An increase in protein carbamylation has been observed in inflammatory diseases such as atherosclerosis [22]. The plasma concentration of homocitrulline (Hcit) increased in HD patients, as compared to the controls (Figure 2e). ...M2 Monocyte Polarization in Dialyzed Patients Is Associated with Increased Levels of M-CSF and Myeloperoxidase-Associated Oxidative Stress: Preliminary ResultsArticleFull-text availableJan 2021 Valérie Pireaux Cédric Delporte Alexandre Rousseau Karim Zouaoui BoudjeltiaCardiovascular diseases represent a major issue in terms of morbidity and mortality for dialysis patients. This morbidity is due to the accelerated atherosclerosis observed in these patients. Atherosclerosis is a chronic inflammatory disease characterized by key players such as monocytes, macrophages, or oxidized LDLs. Monocytes-macrophages are classified into subsets of polarized cells, with M1 and M2 macrophages considered, respectively, as pro- and anti-inflammatory. (1) Methods: The monocyte subsets and phenotypes were analyzed by flow cytometry. These data were completed by the quantification of plasma M-CSF, IL-8, CRP, Mox-LDLs, Apo-B, Apo-AI, chloro-tyrosine, and homocitrulline concentrations. The statistical differences and associations between two continuous variables were assessed using the Mann–Whitney U test and Spearman’s correlation coefficient, respectively. (2) Results: Hemodialyzed patients showed a significant increase in their concentrations of CRP, M-CSF, and IL-8 (inflammation biomarkers), as well as chloro-tyrosine and homocitrulline (myeloperoxidase-associated oxidative stress biomarkers). Moreover, we observed a higher percentage of M2 monocytes in the plasma of hemodialysis patients as compared to the controls. (3) Conclusions: Our data suggest that oxidative stress and an inflammatory environment, which is amplified in hemodialysis patients, seems to favor an increase in the concentration of circulating M-CSF, therefore leading to an increase in M2 polarization among circulating monocytes.ViewShow abstract... Homocitrulline is a product of protein carbamylation, generated non enzymatically from lysine residues by the action of cyanate. Cyanate is itself derived from the spontaneous decomposition of urea or via the oxidation of thiocyanate by myeloperoxidase in the presence of H2O2 [22]. Finally, to have a glance on the lipoprotein status, Apo-AI, Apo-B, but also myeloperoxidase-oxidized LDLs (Mox-LDLs) were quantified in dialysis patients versus healthy volunteers. ...... The copyright holder for this preprint this version posted May 11, 2020. . https://doi.org/10.1101/2020.05.07.20094011 doi: medRxiv preprint myeloperoxidase in the presence of H2O2 [22]. An increase in protein carbamylation has been observed in inflammatory diseases such as atherosclerosis [22]. ...... https://doi.org/10.1101/2020.05.07.20094011 doi: medRxiv preprint myeloperoxidase in the presence of H2O2 [22]. An increase in protein carbamylation has been observed in inflammatory diseases such as atherosclerosis [22]. The plasma concentration of homocitrulline (Hcit) increased in HD patients, as compared to the controls (Fig. 2 C). ...M2 monocyte polarization in dialyzed patients is associated with increased levels of M-CSF and myeloperoxidase-associated oxidative stress: preliminary resultsPreprintFull-text availableMay 2020Valerie Pireaux Cédric Delporte Alexandre Rousseau Karim Zouaoui BoudjeltiaCardiovascular diseases represent a major issue in terms of morbidity and mortality for dialysis patients. This morbidity is due to the accelerated atherosclerosis observed in these patients. Atherosclerosis is a chronic inflammatory disease characterized by key players such as monocytes, macrophages or oxidized LDLs. Monocytes-macrophages are classified into subsets of polarized cells, with M1 and M2 macrophages considered respectively as pro- and anti-inflammatory.The monocyte subsets and phenotypes were analyzed by flow cytometry. These data was completed by the quantification of plasma M-CSF, IL-8, CRP, Mox-LDLs, Apo-B and Apo-AI, chloro-tyrosine and homocitrulline concentrations.The statistical differences and associations between two continuous variables were assessed using the Mann-Whitney U test and Spearman correlation coefficient, respectively.Hemodialyzed patients showed a significant increase in the concentrations of CRP, M-CSF and IL-8 (inflammation biomarkers) as well as of chloro-tyrosine and homocitrulline (myeloperoxidase-associated oxidative stress biomarkers). Moreover we observed a higher percentage of M2 monocytes in the plasma of hemodialysis patients, as compared to the controls.Our data suggests that an oxidative stress and an inflammatory environment, amplified in hemodialysis patients, seems to favor an increase in the concentration of circulating M-CSF, therefore leading to an increase of M2 polarization among circulating monocytes.ViewShow abstract... Carbamylation of lysine (Lys) residues oppositely occurs spontaneously using the urea intermediate isocyanic acid as substrate to generate the non-coded amino acid homocitrulline (HCit) [10]. Cit and HCit are almost identical residues and differ only by one carbon in their side chain, although HCit results from a non-enzymatic chemical reaction and Cit formation is enzymatic [14]. Particularly, both non-coded amino acids are considered as ureido DPMs (uDPMs); hence, they share an ureido group in the side chain [14]. ...... Cit and HCit are almost identical residues and differ only by one carbon in their side chain, although HCit results from a non-enzymatic chemical reaction and Cit formation is enzymatic [14]. Particularly, both non-coded amino acids are considered as ureido DPMs (uDPMs); hence, they share an ureido group in the side chain [14]. Citrullination is known to cause loss of positive charge in Arg residues, which in turn disturbs the structural order of the protein and makes it more prone to proteolysis [11]. ...... Though until now, very few uDPMcontaining proteins have been identified and characterized from brain-diseased proteomes [31]. This is mainly due to the chemical and structural similarities that Arg and Lys present with their respective modified counterparts, and between Cit and HCit, which by the use of classical biochemical methods result in significant cross-reactivity [14,21,32]. Recent advances in liquid chromatography mass spectrometry (LC-MS/MS)-based proteomics have made possible the unbiased identification of proteins in complex samples and characterization of isoforms expression, turnover rate, subcellular localization, PTMs, and quantification of altered abundances in disease states [33]. ...Brain ureido degenerative protein modifications are associated with neuroinflammation and proteinopathy in Alzheimer’s disease with cerebrovascular diseaseArticleFull-text availableSep 2017J NEUROINFLAMM Xavier Gallart-Palau Aida SerraBenjamin Sian Teck LeeSiu Kwan SzeBackgroundBrain degenerative protein modifications (DPMs) are associated with the apparition and progression of dementia, and at the same time, Alzheimer’s disease with cerebrovascular disease (AD + CVD) is the most prevalent form of dementia in the elder population. Thus, understanding the role(s) of brain DPMs in this dementia subtype may provide novel insight on the disease pathogenesis and may aid on the development of novel diagnostic and therapeutic tools. Two essential DPMs known to promote inflammation in several human diseases are the ureido DPMs (uDPMs) arginine citrullination and lysine carbamylation, although they have distinct enzymatic and non-enzymatic origins, respectively. Nevertheless, the implication of uDPMs in the neuropathology of dementia remains poorly understood.MethodsIn this study, we use the state-of-the-art, ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC)-coupled mass spectrometry technology to undertake a comparative characterization of uDPMs in the soluble and particulate postmortem brain fractions of subjects diagnosed with AD + CVD and age-matched controls.ResultsAn increase in the formation of uDPMs was observed in all the profiled AD + CVD brains. Citrulline-containing proteins were found more abundant in the soluble fraction of AD + CVD whereas homocitrulline-containing proteins were preferentially abundant in the particulate fraction of AD + CVD brains. Several dementia-specific citrulline residues were also identified in soluble proteins previously categorized as pro-immunogenic, which include the receptor P2X7, alpha-internexin, GFAP, CNP, MBP, and histones. Similarly, diverse dementia-specific homocitrulline residues were also observed in the particulate fractions of AD + CVD in proteins that have been vastly implicated in neuropathology. Intriguingly, we also found that the amino acids immediately flanking arginine residues may specifically influence the increase in protein citrullination.ConclusionsTaken together, these results indicate that uDPMs widely contribute to the pathophysiology of AD + CVD by promoting neuroinflammation and proteinopathy. Furthermore, the obtained results could help to identify disease-associated proteins that can act as potential targets for therapeutic intervention or as novel biomarkers of specific neuropathology.Electronic supplementary materialThe online version of this article (10.1186/s12974-017-0946-y) contains supplementary material, which is available to authorized users.ViewShow abstract... Synovial samples of knees were obtained from patients that underwent a total knee replacement. We compared the findings of four osteoarthritis (OA, patients 1-4) with seven seronegative RA (RA-, patients 5-11) and eight seropositive RA (RA+, patients [12][13][14][15][16][17][18][19] patients. All RA patients fulfilled the diagnostic criteria of the American College of Rheumatology for RA [18]. ...... The strongest myeloperoxidase staining was also detected in the precipitate fraction of the necrotic inner mass of the rheumatoid nodule. [12][13][14][15][16][17][18][19] patients. Statistically significant differences are indicated ( ** p 0.01, *** p 0.001). ...... More adipose tissue was found in the OA synovial samples, and the synovial lining layer was thicker in the RA samples. One of the seropositive RA knee synovial tissues was mostly necrosis (patient 19), and in the metatarsal joint synovial tissues of seropositive RA patient s necrosis could be found in three out of five samples (Fig. 3). ...Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissueArticleFull-text availableOct 2016ARTHRITIS RES THER Sanna Palosaari Johanna HuhtakangasTomi Nousiainen Petri Pauli LehenkariBackgroundSeropositive rheumatoid arthritis (RA) is characterized by autoantibodies binding to citrullinated and homocitrullinated proteins. We wanted to study the expression patterns of these disease-associated protein forms and if the rheumatoid nodule and synovial tissue itself contain biologically active levels of citrullinating peptidyl arginine deiminases 2, 3 and 4 and homocitrullination-facilitating neutrophil enzyme myeloperoxidase. MethodTotal of 195 synovial samples from metatarsal joints from five ACPA/RF-positive RA patients (n = 77), synovial samples from knees of eight seropositive RA (n = 60), seven seronegative RA (n = 33) and five osteoarthritis (n = 25) patients were analyzed for citrulline and homocitrulline contents using HPLC. The location of citrulline- and homocitrulline-containing proteins, PAD 2, 3, 4 and myeloperoxidase were shown by immunostaining. Myeloperoxidase and citrulline- or homocitrulline-containing proteins were stained on Western blot. ResultsOverall, necrosis was frequent in metatarsals of seropositive RA and absent in seronegative RA and osteoarthritis patients. In histological analysis, there was a significant local patterning and variation in the citrulline and homocitrulline content and it was highest in metatarsal synovial tissues of seropositive RA patients. We found peptidyl arginine deiminase 2, 3 and 4 in the lining and sublining layers of intact synovial tissue. Myeloperoxidase was found locally around necrotic areas. The tissues with necrosis contained the highest levels of citrulline and homocitrulline. ConclusionsRheumatoid nodules and synovia contain significant amount of PAD2, 3 and 4 and myeloperoxidase enzymes. These enzymes could explain the levels of citrulline and homocitrulline in seropositive RA synovial and rheumatoid nodule tissues especially around necrotic tissue.ViewShow abstract... However, a difficulty is associated with distinguishing citrullinated proteins from carbamylated proteins: not only the cross-reaction of antibodies in patient serum, but also that of commercially available antibodies for experiments. Although the antimodified citrulline (AMC)-Senshu antibody [56] and antibody against the deca-citrulline peptide (F95) [57] have been used in many studies on citrullinated antigens, both were shown to bind to carbamylated antigens [9,58,59]. These findings indicate the potential misinterpretation of carbamylated antigens as citrullinated antigens in many previous studies because citrullinated proteins and carbamylated proteins may both be produced by activated neutrophils, including NETs-forming neutrophils, as discussed above. ...Clinical and etiological meaning of anti-carbamylated protein antibodies in rheumatoid arthritisArticleFull-text availableFeb 2019 Shuichiro NakaboSeveral autoantibodies against proteins with post-translational modifications have been detected in patients with rheumatoid arthritis (RA) and are called anti-modified protein antibodies (AMPAs). Anti-carbamylated protein antibodies (Anti-CarP Ab) are the second most vigorously researched AMPAs following anti-citrullinated protein/peptide antibodies (ACPA). Anti-CarP Ab and ACPA show cross-reactivity to some extent and frequently co-exist with each other in RA, but are two distinct antibodies. Although the diagnostic efficacy of anti-CarP Ab is inferior to that of ACPA, the diagnostic specificity of RA may improve when used in combination with ACPA and rheumatoid factor. Anti-CarP Ab and ACPA are also useful for identifying patients at high risk of more severe joint destruction and cardiovascular diseases. The high prevalence of the co-existence of both antibodies suggests a common factor in their production, and this is important for the development of RA because both antibodies emerge before the onset of clinical symptoms. Neutrophils may also be crucially involved. It is important to distinguish citrullinated antigens from carbamylated antigens because the methods commonly used to detect the former are now known to be cross-reactive with the latter. Research on anti-CarP Ab will provide novel insights into the pathology and etiology of RA.ViewShow abstractDegenerative protein modifications in the aging vasculature and central nervous system: A problem shared is not always halvedArticleMay 2019AGEING RES REV Xavier Gallart-PalauLe Min Tan Aida SerraSiu Kwan SzeAging influences the pathogenesis and progression of several major diseases affecting both the cardiovascular system (CVS) and central nervous system (CNS). Defining the common molecular features that underpin these disorders in these crucial body systems will likely lead to increased quality of life and improved health-span in the global aging population. Degenerative protein modifications (DPMs) have been strongly implicated in the molecular pathogenesis of several age-related diseases affecting the CVS and CNS, including atherosclerosis, heart disease, dementia syndromes, and stroke. However, these isolated findings have yet to be integrated into a wider framework, which considers the possibility that, despite their distinct features, CVS and CNS disorders may in fact be closely related phenomena. In this work, we review the current literature describing molecular roles of the major age-associated DPMs thought to significantly impact on human health, including carbamylation, citrullination and deamidation. In particular, we focus on data indicating that specific DPMs are shared between multiple age-related diseases in both CVS and CNS settings. By contextualizing these data, we aim to assist future studies in defining the universal mechanisms that underpin both vascular and neurological manifestations of age-related protein degeneration.ViewShow abstractRecognition of citrullinated and carbamylated proteins by human antibodies: Specificity, cross-reactivity and the AMC-Senshu methodArticleFull-text availableJul 2012Ann Rheum Dis Jing Shi Annemiek Willemze George M C Janssen Rene e ToesRecently, a novel family of autoantibodies in rheumatoid arthritis (RA) patients was described: anti-carbamylated protein (Anti-CarP) antibodies, which target carbamylated (homocitrulline-containing) epitopes.1 ,2 Since citrulline and homocitrulline have a similar structure, we wished to determine to what extent human autoantibodies can differentiate between them. Unlike human antibodies, the anti-modified citrulline (AMC) antibody developed by Dr Senshu3–8 is able to recognise citrullinated epitopes irrespective of the neighbouring amino acids. Thus, we also aimed to verify whether the AMC assay could distinguish between these two amino acids.To address this, we loaded gels with citrullinated, carbamylated and non-modified forms of foetal calf s serum (FCS) and human fibrinogen (Fib). Gels loaded with equal amounts of these protein preparations (figure 1A) were used for western blotting and staining with the ‘AMC-Senshu’ method. Both the citrullinated and the carbamylated forms of the proteins tested were strongly recognised, whereas, the non-modified form did not reveal any staining (figure 1B). Staining similar western blots with selected human sera1 revealed that sera-positive …ViewShow abstractAutoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damageArticleFull-text availableOct 2011Proc Natl Acad Sci Unit States Am Jing Shi Rachel Knevel Parawee Suwannalai Leendert A TrouwAutoimmune responses against posttranslationally modified antigens are a hallmark of several autoimmune diseases. For example, antibodies against citrullinated protein antigens (ACPA) have shown their relevance for the prognosis and diagnosis of rheumatoid arthritis (RA), and have been implicated in disease pathogenesis. It is conceivable that other autoantibody systems, recognizing other posttranslationally modified proteins, are also present in RA. Here, we describe the presence of an autoantibody system that discriminates between citrulline- and homocitrulline-containing antigens in the sera of RA-patients. IgG antibodies recognizing carbamylated (homocitrulline-containing) antigens were present in sera of over 45% of RA-patients. Likewise, anticarbamylated protein (anti-CarP) IgA antibodies were observed in 43% of RA-sera. ACPA and anti-CarP antibodies are distinct autoantibodies because, in selected double-positive patients, the anti-CarP antibody binding to carbamylated antigens could be inhibited by carbamylated antigens, but not by control or citrullinated antigens. Similarly, ACPA-binding to citrullinated antigens could only be inhibited by citrullinated antigens. In line with this observation, 16% of ACPA-negative RA-patients, as measured by a standard ACPA assay, harbored IgG anti-CarP antibodies, whereas 30% of these patients tested positive for IgA anti-CarP antibodies. The presence of anti-CarP antibodies was predictive for a more severe disease course in ACPA-negative patients as measured by radiological progression. Taken together, these data show the presence of a unique autoantibody system recognizing carbamylated, but not citrullinated, protein antigens. These antibodies are predictive for a more severe clinical course in ACPA-negative RA-patients, indicating that anti-CarP antibodies are a unique and relevant serological marker for ACPA-negative RA.ViewShow abstractPreparation of a monoclonal antibody to citrullinated epitopes: Its characterization and some applications to immunohistochemistry in human brainArticleMar 2002GLIA Anthony P NicholasJohn N. WhitakerUsing 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. (C) 2002 Wiley-Liss, Inc.ViewShow abstractCarbamylation of amino acids and proteins in uremiaArticleFeb 2001Kidney Int SupplLorraine M. KrausJr. Kraus A.P.Cyanate spontaneously transformed from urea increases as renal function decreased. Acting as a potential toxin, the active form of cyanate, isocyanic acid, carbamoylates amino acids, proteins, and other molecules, changing their structure, charge, and function. The resulting in vivo carbamoylation can modify the molecular activity of enzymes, cofactors, hormones, low-density lipoproteins, antibodies, receptors, and transport proteins. Antibodies specific for ε-amino-carbamoyl-lysine (homocitrulline) located carbamoylated proteins in situ in neutrophils, monocytes, and erythrocytes. Carbamoylated proteins were found in renal tissue from uremic patients but not in normal transplanted kidneys. The irreversible reaction with cyanate converts free amino acids (F-AAs) to carbamoyl-amino acids (C-AAs). The Carbamoylation Index (CI), C-AA/F-AA, quantifies the decrease of the F-AA pool for each essential amino acid. C-AAs contribute, in part, to malnutrition of uremia. C-AAs interfered with protein synthesis to lower 14C hemoglobin synthesis in human reticulocytes and osteocalcin synthesis in rat osteosarcoma-derived tissue culture. Insulin-sensitive glucose uptake was decreased 33% in cultured rat adipocytes by α-amino-carbamoyl-asparagine. α-Amino carbamoylation occurs primarily in F-AA, while ε-amino carbamoylation of lysine in protein occurs continuously during the protein life span. Protein catabolism releases ε-amino-carbamoyl-lysine. Quantitation of α versus ε carbamoylation may yield a more sensitive measurement of protein intake versus protein catabolism, and could be useful in decisions concerning the time to initiate dialysis or subsequent changes in dialysis prescription. Carbamoylated molecules can block, enhance, or be excluded from metabolic pathways, thereby influencing the fate of noncarbamoylated molecules. Although not an all-or-none phenomenon, urea-derived cyanate and its actions are contributing causes of toxicity in uremia.ViewShow abstractReactions of Cyanate with Functional Groups of Proteins. III. Reactions with Amino and Carboxyl GroupsArticleJun 1965BIOCHEMISTRY-USGeorge R. StarkViewA new model for an etiology of rheumatoid arthritis: Smoking may trigger HLA–DR (shared epitope)–restricted immune reactions to autoantigens modified by citrullinationArticleJan 2006Arthritis RheumPhD Lars Klareskog MD Patrik Stolt Karin Lundberg Lars AlfredssonObjectiveTo investigate whether smoking and HLA–DR shared epitope (SE) genes may interact in triggering immune reactions to citrulline-modified proteins.MethodsIn a case–control study involving patients with recent-onset rheumatoid arthritis (RA), we studied interactions between a major environmental risk factor (smoking), major susceptibility genes included in the SE of HLA–DR, and the presence of the most specific autoimmunity known for RA (i.e., antibodies to proteins modified by citrullination). Immunostaining for citrullinated proteins in cells from bronchoalveolar lavage fluid was used to investigate whether smoking is associated with citrullination in the lungs.ResultsPrevious smoking was dose-dependently associated with occurrence of anticitrulline antibodies in RA patients. The presence of SE genes was a risk factor only for anticitrulline-positive RA, and not for anticitrulline-negative RA. A major gene–environment interaction between smoking and HLA–DR SE genes was evident for anticitrulline-positive RA, but not for anticitrulline-negative RA, and the combination of smoking history and the presence of double copies of HLA–DR SE genes increased the risk for RA 21-fold compared with the risk among nonsmokers carrying no SE genes. Positive immunostaining for citrullinated proteins was recorded in bronchoalveolar lavage cells from smokers but not in those from nonsmokers.ConclusionWe identified an environmental factor, smoking, that in the context of HLA–DR SE genes may trigger RA-specific immune reactions to citrullinated proteins. These data thus suggest an etiology involving a specific genotype, an environmental provocation, and the induction of specific autoimmunity, all restricted to a distinct subset of RA.ViewShow abstractCarbamylation-Derived Products: Bioactive Compounds and Potential Biomarkers in Chronic Renal Failure and AtherosclerosisArticleJul 2011Clin Chem Stéphane JaissonChristine PietrementPhilippe GilleryCarbamylation is a posttranslational modification of proteins resulting from the nonenzymatic reaction between isocyanic acid and specific free functional groups. This reaction alters protein structural and functional properties and thus contributes to molecular ageing. Many studies have shown the involvement of carbamylated proteins in diseases, especially in chronic renal failure and atherosclerosis.In this review we describe the biochemical basis of the carbamylation process and its role in protein molecular ageing. We summarize the current evidence of protein carbamylation involvement in disease, identify available biomarkers of the carbamylation process and their related analytical methods, and discuss the practical relevance of these biomarkers.Carbamylation-induced protein alterations are involved in the progression of various diseases, because carbamylation-derived products (CDPs) are bioactive compounds that trigger specific and inappropriate cellular responses. For instance, carbamylation may promote hormone and enzyme inactivation, and carbamylated proteins, as diverse as collagen or LDLs, induce characteristic biochemical events of atherosclerosis progression. CDPs are potential biomarkers to monitor diseases characterized by an increased rate of carbamylation (e.g., chronic renal failure and atherosclerosis). Different methods (e.g., liquid chromatography-tandem mass spectrometry and immunoassays) to measure specific carbamylated proteins or general markers of carbamylation, such as protein-bound homocitrulline, have been described. Their use in clinical practice must still be validated by appropriate clinical studies.ViewShow abstractModification of citrulline residues with 2,3-butanedione facilitates their detection by liquid chromatography/mass spectrometryArticleJun 2011Rapid Comm Mass Spectrom Marlies De CeuleneerVanessa De Wit Katleen Van Steendam Dieter DeforceCitrullination is a post-translational modification (PTM) that results from the deimination of the amino acid arginine into citrulline by Peptidyl Arginine Deiminase enzymes and occurs in a wide range of proteins in health and disease. This modification causes a 1 Da mass shift, which can be used to identify citrullination sites in proteins by the use of mass spectrometry. However, other PTMs, such as deamidation from asparagine to aspartic acid or from glutamine to glutamic acid, can also cause a 1 Da mass shift, making correct interpretation of the data more difficult. We developed a chemical tagging strategy which, combined with an open source search application, allowed us to selectively pinpoint citrullinated peptides in a complex mixture after liquid chromatography/mass spectrometry (LC/MS) analysis. After incubation of a peptide mixture with 2,3 butanedione, citrulline residues were covalently modified which resulted in a 50 Da shift in singly charged mass. By comparison of the peptide mass fingerprint from a modified and an unmodified version of the same sample, our in-house search application was able to identify the citrullinated peptides in the mixture. This strategy was optimized on synthetic peptides and validated on a digest of in vitro citrullinated fibrinogen, where different proteolytic enzymes were used to augment the protein coverage. This new method results in easy detection of citrullinated residues, without the need for complex mass spectrometry equipment.ViewShow abstractSmoking and outcome of infectionArticleMar 2011J Intern Med Reetta Huttunen Terho HeikkinenJaana SyrjänenHuttunen R, Heikkinen T, Syrjänen J. (Department of Internal Medicine, Tampere University Hospital, Tampere; Department of Pediatrics, Turku University Hospital, Turku; Finland) Smoking and the Outcome of Infection (Review). J Intern Med 2010; 269: 258–269.Smoking has substantial local and systemic adverse effects on the immune system, respiratory tract and skin and soft tissues. Smokers are at increased risk of invasive pneumococcal disease, pneumonia, periodontitis, surgical infections, tuberculosis, influenza and meningococcal disease. The results of several studies indicate that smokers with periodontitis or tuberculosis suffer more severe disease. Data on the impact of smoking on sepsis and pneumonia are controversial and limited, and systematic data regarding the outcome of the majority of infections in smokers are scarce. Abundant data indicate that children exposed to environmental tobacco smoke (ETS) suffer from more severe infections. However, information regarding the effects of ETS on the outcome of infections in adults is limited. Various aspects of the relation between smoking and the outcome of bacterial infection (e.g. potential dose-dependent effects and the interactions between smoking and other environmental factors that may affect the course of infectious diseases) remain to be established.ViewShow abstractAnticitrulline Antibodies Can Be Caused by Homocitrulline-Containing Proteins in RabbitsArticleNov 2010Arthritis Rheum Sanna Palosaari Marja-Kaisa Koivula Leila Risteli Juha RisteliTo clarify the possible roles of protein-bound homocitrulline in causing an antibody response to citrulline and as a confounding factor in citrulline detection assays.Native, carbamoylated, and citrullinated albumins were used for testing the specificity of commercial antibodies against modified citrulline by Western blot. Rabbits were immunized with human albumin and/or bone type I collagen, both of which were treated with cyanate to produce homocitrulline, or with citrullinated synthetic telopeptide of type I collagen. These antisera were tested for binding to cyclic citrullinated peptide 2 (CCP-2), mutated citrullinated vimentin, and type I or II collagen telopeptides containing either citrulline or homocitrulline.Commercial antibodies that had been considered to be specific for chemically modified citrulline were found to recognize both homocitrulline- and citrulline-containing albumins. The rabbits immunized with proteins containing homocitrulline produced high-affinity antibodies against CCP-2 and, to a lesser extent, against mutated citrullinated vimentin. The antisera also bound homocitrulline-containing collagen telopeptides and, less strongly, citrulline-containing telopeptides.Our findings demonstrate that homocitrulline, which is a structural analog of citrulline (longer by 1 carbon, and readily formed in the body), can be involved in raising citrulline-recognizing antibodies in experimental animals and can cause false-positive reactions in assays for citrulline.ViewShow abstractShow moreAdvertisementRecommendationsDiscover moreProjectArthritis project Johanna Huhtakangas Petri Pauli Lehenkari Sanna Palosaari[...]View projectProjectRheumatoid arthritis Marja-Kaisa Koivula Juha Risteli Leila Risteli[...]H KautiainenView projectProjectEffect of nicotine from cigarette smoke on preterm babies: A Finnish study Ashok Aspatwar Sanna PalosaariTarja Tanner[...]Petri Pauli LehenkariThe goal of the project is to study the effect of nicotine and the chemicals from cigarette smoke on preterm babies using zebrafish as a model organism. View projectProjectMOM project Sanna Palosaari Petri Pauli LehenkariTomi NousiainenMetal-on-metal hip revisions, a translational study. View projectArticlePeptidylarginine deiminases in citrullination, gene regulation, health and pathogenesisJuly 2013 · Biochimica et Biophysica ActaShu Wang Yanming WangPeptidylarginine deiminases are a family of enzymes that mediate post-translational modifications of protein arginine residues by deimination or demethylimination to produce citrulline. In vitro, the activity of PADs is dependent on calcium and reductive reagents carrying a free sulfhydryl group. The discovery that PAD4 can target both arginine and methyl-arginine for citrullination about 10years ... [Show full abstract] ago renewed our interest in studying this family of enzymes in gene regulation and their physiological functions. The deregulation of PADs is involved in the etiology of multiple human diseases, including cancers and autoimmune disorders. There is a growing effort to develop isoform specific PAD inhibitors for disease treatment. However, the regulation of the activity of PADs in vivo remains largely elusive, and we expect that much will be learned about the role of these enzymes in normal life cycle and under pathology conditions.Read moreArticleOP0148 First in Class Therapeutic Acpa Reduces Inflammation by Inhibition of NetosisJune 2016 · Annals of the Rheumatic Diseases Renato G S Chirivi Jos van RosmalenGonny Schmets[...] Jos RaatsBackgroundNeutrophils together with aberrant Neutrophil Extracellular Trap (NET) formation contribute to the induction and propagation of inflammation. A growing number of studies indicate that Peptidyl Arginine Deiminases (PADs) mediated conversion of arginine to citrulline residues in proteins are essential for NETosis, generation of auto-antigens, autoimmunity, and the breaking of tolerance ... [Show full abstract] in rheumatoid arthritis (RA). In RA patients, enhanced NETosis is observed in circulating and synovial neutrophils, and NET components are observed in blood and joints.ObjectivesOur objective is to develop a novel first in class NET-inhibiting therapeutic anti-citrullinated protein antibody (tACPA) treatment for RA and other auto-immune diseases in which aberrant NET formation adds to the severity of the disease.MethodsHuman neutrophils from blood donors have been used in order to visualize the NETosis inhibiting properties of tACPA. Furthermore, collagen antibody-induced (CAIA) as well as collagen-induced (CIA) arthritis mouse models have been used in order to test the therapeutic properties of tACPA.ResultsIn human neutrophils, NETosis induction with calcium ionophore A-23187 or physiological stimuli like human synovial fluid is strongly inhibited by tACPA treatment (40–90% NET reduction compared to non-treated cells (n 40 different donors)). This observation has been confirmed by Myeloperoxidase and Neutrophil Elastase activity as well as immunohistochemistry read-outs. In both CAIA and CIA mouse models, NET-inhibiting tACPAs are able to prevent the onset and/or exacerbation of inflammation, and prevent or strongly reduce joint damage. Histological analysis of tACPA-treated inflamed mouse joints revealed a significant decrease in neutrophil influx. We identified the epitopes recognized by tACPA to be citrullinated domains of histones 2A and 4.ConclusionsWe have identified antibodies directed against a citrullinated epitope in murine and human histones 2A and 4. In RA mouse models, we demonstrate that tACPAs strongly prevents the occurrence of swelling and joint damage. We propose that the therapeutic effect of tACPA acts through the inhibition of NET and auto-antigen formation, clearance of formed NETs and toxic histones. In RA patients extinguishing auto-antigen production offers an orthogonal approach for treating this destructive autoimmune disease, potentially without impacting systemic immunity. Citryll and ModiQuest are developing tACPAs as treatment for diseases where NETs and NETosis are driving or contributing to disease progression.Disclosure of InterestR. Chirivi Shareholder of: Citryll BV, Employee of: ModiQuest BV, J. van Rosmalen Employee of: ModiQuest BV, G. Schmets Employee of: ModiQuest BV, H. van Es Shareholder of: Citryll BV, Employee of: Citryll BV, J. Raats Shareholder of: Citryll BV and ModiQuest BVRead moreArticleAutoantibodies as predictors of diseaseJune 2004 · The LancetR Hal ScofieldContext: Many human diseases are the result of autoimmune attack, presumably related to a loss of tolerance to self. Autoimmune disease can be divided into either organ-specific illnesses, such as thyroid disease, type 1 diabetes, and mysasthenia gravis, or systemic illnesses, such as rheumatoid arthritis and systemic lupus erythematosus. The pathogenesis of autoimmune damage also segregates ... [Show full abstract] autoimmune disease in that some diseases or manifestations are mainly induced by autoantibodies. Pathogenesis may be mainly mediated by autoimmune T lymphocytes. Notwithstanding the underlying mechanism of disease, virtually all autoimmune diseases are associated with circulating autoantibodies, which bind self-protein. Furthermore, for many diseases these autoantibodies are found in serum samples many years before disease onset.Starting point: In the past several years a new autoantibody specificity has been identified in the sera of patients with rheumatoid arthritis. These autoantibodies bind citrulline, a post-translational modification of arginine. Markus Nielen and colleagues recently studied the presence of these autoantibodies and rheumatoid factor in blood donors who later developed rheumatoid arthritis (Arthritis Rheum 2004; 50: 380-86). About half the patients were positive for at least autoantibody at a median of 4.5 years before the onset of disease. The negative predictive value of these tests was high, while the positive predictive value was very high. WHERE NEXT? Autoantibodies might not be directly responsible for many of the manifestations of autoimmune disease, but they are markers of future disease in presently healthy individuals. Long-term large studies of outcome are needed to assess the use of assaying autoantibodies for prediction of disease. Such data could lead to intervention trials to prevent autoimmune disease, as are already underway in type 1 diabetes.Read moreArticleFree amino acids and related compounds in cow s milk and their changes by heat treatmentsJanuary 1968 · Nihon Chikusan GakkaihoGosei KawanishiNobuaki AbeKensuke Saito1. 東京と釧路で集乳された混合乳15試料の遊離アミノ酸(FAA)類について,イオン交換樹脂カラムクロマトグラフィーにより検索し,17種のFAA, P-Ser,GPEA, PEA, EA, Urea, Taurine, NT3, MethioninesulfoxideおよびCitrullineをクロマトグラフィー的に認め,その定量を行なった.全体ではUreaが多く64%,燐脂質構成体17%,ついでFAA 15%であった.FAAは60μmole/100mlで,その中ではGluが25μmole/100mlでもっとも多く,ついでGly, Lys,Ala, Proが比較的多かった.各試料は時期,地区を異にするにもかかわらず定量値に大きな変動はなく,FAA含量の変動係数は13%であった.2. ... [Show full abstract] 乳中のFAA類の加熱による変化を定量的に検討した.63°Cでは90分でも変化は少ないが,75°C15分～120°C10分の間ではFAAの減少とNH3の増加が認められ,120°C20分では蛋白質の加水分解によると考えられるFAAの減少の低下とNH3の急激な増加が認められた.これに比較して工業的な殺菌,減菌法では85°CHTST法,115°C,140°C UHT Plale法,150°C UHT Steam injection法のいずれとも,FAAの減少,NH3の増加は著しくないことを認め,種々の考察を行なった.Read moreDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! Please log in.Email · HintTip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleNo account? 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