anti-CarP Antibodies Research Articles

A1.55 Development of antibodies specific for carbamylated protein precedes disease onset in MICE with collagen-induced arthritis

BackgroundAntibodies against citrullinated proteins are a characteristic of rheumatoid arthritis (RA). Recently, we demonstrated that autoantibodies recognizing homocitrulline containing proteins are present in sera of RA patients and predict joint...

Anti-carbamylated protein (anti-CarP) antibodies precede the onset of rheumatoid arthritis

ObjectiveThe presence of anti-citrullinated protein antibodies (ACPA) and IgM-rheumatoid factor (IgM-RF) years before the clinical diagnosis of rheumatoid arthritis (RA) suggests they are possibly involved in the pathogenic process underlying...

THU0031 Anti-Carbamylated Protein Antibodies (Anti-CarP) Precede the Onset of Rheumatoid Arthritis

BackgroundAnti-citrullinated protein antibodies (ACPA) and IgM-Rheumatoid Factor (IgM-RF) are auto-antibodies that can be detected many years before the clinical diagnosis of Rheumatoid Arthritis (RA) [1]. Since ACPA and IgM-RF are...

AB1187 Autoantibodies recognizing carbamylated proteins (anti-carp) in sera of patients with JIA

BackgroundAnti-citrullinated protein antibodies (ACPA) have a low prevalence in Juvenile Idiopathic Arthritis (JIA). Autoantibodies recognizing carbamylated protein (anti-CarP) are a promising new serological marker for ACPA negative Rheumatoid Arthritis and...

Autoimmunity in rheumatoid arthritis: different antigens—common principles

Since the identification of antibodies directed against citrullinated protein antibodies (ACPA) as specific serological markers for rheumatoid arthritis (RA) the insight into the pathogenesis of RA has made significant progress....

The ‘Senshu’ method often used to detect citrullinated proteins does not discriminate between citrullination and carbamylation

Backgroundand objectivesAnticitrullinated protein antibodies (ACPA) play an important role in the diagnosis and prognosis of rheumatoid arthritis (RA). ACPA have been implicated in disease pathogenesis partially based on the argument...

Autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage

Autoimmune 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.

Hybrid ELISAs for vitellogenins of the endangered copper redhorse Moxostoma hubbsi and the shorthead redhorse Moxostoma macrolepidotum (Cypriniformes, catostomidae)

The shorthead redhorse (sr) Moxostoma macrolepidotum is endemic in North America, while in Canada the copper redhorse (cr) Moxostoma hubbsi is classified as endangered. A commercial heterologous carp vitellogenin (VTG) sandwich ELISA underestimates VTGs in these species. Hybrid ELISAs, using anti-carp VTG antibodies and purified sr- and cr-VTGs as standards, were validated to measure VTGs in plasma and surface mucus of redhorse species. The hybrid ELISAs showed good precision, sensitivity and accuracy. Following induction with β-estradiol 3-benzoate (E2B), levels of plasma VTG (mean±SEM) in male sr increased from 2.7±1.5 μg/ml to 16.5±2.2 mg/ml. In immature cr, plasma VTG increased to 70.1±13.8 mg/ml after induction, from an initial value of 37.7±51.0 μg/ml. Levels of VTG in mucus from E2B-injected fish were highly correlated ( p<0.0001) with levels in plasma, but not in a non-induced mature female. Hybrid assays are a good compromise between homologous and heterologous assays, especially for small-bodied or endangered fish species. The performance of the hybrid assays strongly suggests they could be used to determine exposure of these species to estrogenic contaminants and contribute to restoration efforts of the copper redhorse.

Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme‐linked immunoassay in the fathead minnow (Pimephales promelas)

The present study compared three different methods for measuring plasma vitellogenin (VTG) in fathead minnow (FHM; Pimephales promelas): A procedure using liquid chromatography with electrospray ionization combined with tandem mass spectrometry (LC/ESI-MS/MS), and two commercial enzyme-linked immunoassay (ELISA) kits using either anti-carp or anti-FHM antibodies. The influence on plasma VTG measurements of using the protease-inhibitor aprotinin during blood sampling and of submitting the plasma samples to a freeze-thaw cycle before analysis also was evaluated. The addition of aprotinin to the blood during sampling significantly reduced the plasma VTG concentrations measured by ELISA, whereas the VTG values measured after plasma samples were submitted to a freeze-thaw cycle were significantly higher than those measured before freezing. This inflating effect of freezing on VTG measurements made by ELISA could be prevented if plasma samples were frozen diluted in citrate buffer containing 16 mg/ml of polyethylene glycol (PEG). In contrast, measurements of VTG made by LC/ESI-MS/MS were unaffected by freezing and, conceptually, are independent from enzymatic degradation. Although the use of aprotinin and PEG effectively reduced the influence of enzymatic and physical degradation caused by freezing and thawing on VTG measurements made by ELISA, it did not improve agreement between the three analytical techniques evaluated. More information is needed regarding the molecular structure and the existence of possible multiple forms of VTG before this protein can be measured adequately in FHM.

CARP, a Cardiac Ankyrin Repeat Protein, Is Up-Regulated during Wound Healing and Induces Angiogenesis in Experimental Granulation Tissue

Cardiac ankyrin repeat protein (CARP) was identified by subtractive hybridization as one of a group of genes that are rapidly modulated by acute wounding of mouse skin. Quantitative RT-PCR showed that CARP was strongly induced during the first day after wounding (157.1-fold), and the high level persisted for up to 14 days. Immunohistochemistry and in situ hybridization revealed that CARP was expressed in skeletal muscle, vessel wall, hair follicle, inflammatory cells, and epidermis in the wound area. To examine the effects of CARP on wound healing, we developed an adenoviral CARP vector to treat subcutaneously implanted sponges in either rats or Flk-1(LacZ) knock-in mice. Four days after infection, CARP-infected sponges in rats showed a remarkable increase in the vascular component in granulation tissue as compared to Ad-LacZ controls. This result was confirmed by CD34 immunostaining. By 7 days post-infection of sponge implants in Flk-1(LacZ) knock-in mice, granulation tissue showed many more LacZ-positive cells in Ad-CARP-infected sponges than in virus controls. Ad-CARP treatment also induced neovascularization and increased blood perfusion in rabbit excisional wounds in and ischemic rat wounds. These findings indicate that CARP could play a unique role in therapeutic angiogenesis during wound healing.

Isolation and characterisation of pentraxin-like serum proteins from the common carp Cyprinus carpio

There is increasing economic and ecological interest in the development of assays for the early detection of infection, disease activity and environmental stress in marine and freshwater animals. In humans the serum pentraxin C-reactive protein (CRP) is universally used as a clinical indicator of inflammation and underlying infection. As a first step towards assessing the potential of an immunoassay for CRP in fish, we have isolated and characterised common carp Cyprinus carpio CRP and a highly specific and sensitive anti-carp CRP polyclonal antibody has been raised. The results show levels of CRP in healthy fish similar to those found in healthy humans. A protein of unknown function, which displays the characteristic calcium-dependent phosphate monoester binding exhibited by CRP and some similarity to the known fish pentraxin sequences, has also been identified.

Cyclic AMP-receptor proteins in human salivary glands.

In mammalian species, cyclic AMP receptor proteins (cARP) are the regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), the cellular effector of cyclic AMP-mediated signal transduction. An isoform of the PKA type II R subunit (RII), cARP, is a polyfunctional protein, present in most tissues and cells. It is expressed in salivary and other glands of rodents, and secreted into the saliva of rats and Man. The aim of the present study was to determine the expression of cARP in human salivary glands using immunoelectron microscopy. Thin sections of normal salivary glands embedded in LR Gold resin were labeled with anti-cARP primary antibody, then with gold-conjugated secondary antibody. Labeling was present in the secretory granules and cytoplasm of parotid, submandibular (SMG) and sublingual gland serous cells. Quantitative analysis showed considerable variability in granule labeling from sample to sample, indicating shifts in expression and cellular location of cARP. Unlike rodent salivary glands, the granules of intercalated and striated duct cells also were labeled. The cytoplasm and granules of mucous cells of the SMG and sublingual glands were unlabeled, while the Golgi complex and filamentous bodies in these cells showed moderate reactivity. Mitochondria and nuclei of both serous and mucous cells were unlabeled. Labeling also was present in the connective tissue adjacent to the epithelial cells. The results indicate that serous cells of the parotid and SMG are the major source of salivary cARP. They also reveal significant species differences in the glandular distribution of RII. RII binds to cytoskeletal and nuclear proteins, and may function to regulate extracellular cyclic AMP levels. Thus, the tissue and cellular distribution of RII may serve as an index of regulation of gene expression and cell differentiation.

Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio).

To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. 22 carp. Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.

Products of proteolytic cleavage of transferrin induce nitric oxide response of goldfish macrophages

Enzymatic cleavage product of transferrin induced the production of nitric oxide (NO) by LPS-stimulated goldfish macrophages. A NO-inducing factor was purified from the supernatants of mitogen-stimulated goldfish kidney leukocytes using fast performance liquid chromatography (FPLC) and the purified proteins analyzed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry. The proteins were identified as truncated forms of transferrin, having approximate molecular weights (MW) of 33, 35, and 37 kDa (kilodaltons). The precursor form (i.e. full-length) of transferrin did not enhance NO production by LPS-stimulated goldfish macrophages, but enzymatic cleavage of this precursor form correlated with enhanced production of NO by goldfish macrophages. Enzymatic cleavage of transferrin was dependent on the presence of stimulated kidney leukocytes and was shown to occur in response to both mixed lymphocyte reactions (MLR) and the mitogenic stimulation of goldfish kidney leukocytes. Time course analysis revealed that 24 h after kidney leukocyte MLR or mitogen stimulation, cleaved transferrin products appeared in the supernatants of cultured cells, which was related to the on-set of NO-inducing activity of these preparations. To confirm these findings, bovine transferrin was digested in vitro using protease XXVII. The resulting cleavage products had approximate MW of 33, 35, and 37 kDa. When these peptides were subjected to the purification protocols used to purify a NO-inducing factor from goldfish leukocyte supernatants, they were shown to elute to identical fractions. To examine the potential role of fish transferrin in mediating goldfish NO production, carp transferrin was purified from serum and following protease-digestion and purification by FPLC, the truncated proteins were found to elute to similar fractions as bovine transferrin. Furthermore, mitogen-stimulated leukocyte supernatants prepared in the absence of bovine serum (carp serum only) retained NO-inducing activity, indicating that this response was not an artifact of bovine serum components (i.e. bovine transferrin). Anti-bovine and anti-carp transferrin polyclonal antibodies identified the presence of truncated forms of transferrin in the active fractions of FPLC-separated mitogen-stimulated leukocyte supernatants prepared in the presence of bovine or carp serum, respectively. Thus, our results suggest a novel role for fish transferrin as one of the factors that mediates teleost macrophage antimicrobial functions.

Immunolabelling of fish host molecules on the tegumental surface of Ligula intestinalis (Cestoda: Ppseudophyllidea)

Immunoblotting, SDS-PAGE and western biotting procedures were used to demonstrate cross-reactivity of a polyclonal anti-carp IgM antibody with components of roach serum. The polyclonal antibody labelled 2 major bands in both immune and normal roach sera corresponding to molecular mannes of approximately 90 and 65 kDa. One of these bands (65 kDa) was considered to be the heavy chain of fish immunoglobulin whilst the identity of the other remains uncertain. This cross-reaction has been exploited in immunofluorescence and immunogold labelling studies to localize fish host molecules on the tegumental membrane of Ligula intestinalis freshly removed from roach fry. Immunogold studies revealed a low level of host molecules to be associated mainly with the microthrix spines of the tegament with less gold labelling being observed on the microthrix shafts.

A time-study analysis to evaluate albumin carbamylation in vitro

Carbamylation is a non-enzymatic post-translational modification in which cyanate binds to molecules containing primary amine or thiol groups and forms carbamyl groups. Cyanate is in equilibrium with urea in body fluid and increased carbamylation was first reported in patients with increased urea levels such as patients suffering renal diseases. Next, increased carbamylation related to inflammation has also been described in other conditions such as cardiovascular disease.Recently, a new consequence of carbamylation has been observed: induction of an autoantibody response. We identified anti-carbamylated protein (anti-CarP) antibodies in rheumatoid arthritis (RA) patients and in patients having ‘pre-RA’ symptoms, arthralgia. The presence of anti-CarP antibodies in arthralgia patients is associated with an increased risk of developing RA. The presence of anti-CarP antibodies in RA patients is associated with more severe joint damage in RA patients who do not have anti-citrullinated protein antibodies. It is currently unknown to what extent carbamylation and/or the formation of anti-CarP antibodies contributes to the disease processes of chronic diseases such as renal diseases, cardiovascular diseases and RA. This review summarizes the current knowledge on carbamylation and the formation of anti-CarP antibodies and discusses their possibly important implications.

Cutaneous tumor of northern pike, [formula omitted][formula omitted] L. II. Expression of surface and cytoplasmic immunoglobulins and alpha-naphthyl acetate esterase by mononuclear cells from lymphoid tissues and tumor

Rabbit anti-pike IgM antibodies showed that up to 95% of mononuclear (MN) cells isolated on Ficoll-Isopaque gradients from peripheral blood, spleen, and head kidney of northern pikes carrying cutaneous tumors were surface (SIg) and cytoplasmic (CIg) immunoglobulin positive by indirect immunofluorescence. On the contrary, rabbit anti-winter flounder IgM antibodies gave totally negative results. Monoclonal anti-carp IgM antibodies stained approximately 40–56% of peripheral blood, splenic, and head kidney MN cells in assays for SIg and CIg. Monoclonal anti-rainbow trout IgM antibodies, however, did not cross-react with pike Ig. A maximum of 5% of MN cells from the tumor were SIg and CIg positive by anti-pike IgM as well as 6% by anti-carp IgM antibodies, and were negative by the other two antibodies even at low dilutions. A majority of cells in the tumor exhibited an intense diffuse staining pattern for alpha-naphthyl acetate esterase (ANAE) which was inhibited by sodium fluoride and was reminiscent of that noted in human monocytes. These findings show that the Åland pike neoplasm is not a B cell tumor or plasmacytoma, but more representative markers are needed to demonstrate whether it is of T lymphocytic or monocytic origin.

Comparative studies on the structure of biliary immunoglobulins of some avian species II. Antigenic properties of the biliary immunoglobulins of chicken, turkey, duck and goose

Studies on the immunological cross-reactivity of the Igs + from the bile of chicken and turkey (order Galliformes ) as well as duck and goose (order Anseriformes ) to Igs with antigenic known properties carried out by a series of RIA. Various class-specific anti-Ig sera produced in rabbits, guinea-pigs and mice were used. The following results were obtained: (1) A high extent of antigenic relationship between the chicken and turkey biliary Igs was demonstrated by a radiobinding system consisting of 125I-turkey biliary Ig and class-specific rabbit anti-turkey biliary Ig antibodies. The biliary Igs of duck and goose did not inhibit this antigen binding system. (2) The duck and goose biliary Igs were able to inhibit the binding of 125 I-carp IgM to Fc-fragment-specific rabbit anti-carp IgM antibodies and of 125I-human IgM to Fc-fragment-specific guinea-pig anti-human IgM antibodies, respectively. (3) None of the biliary Igs show cross-reactive properties common with human and porcine IgA. That was detected by inhibition of the radio-binding system from 125I-human IgA and α chain-specific mouse anti-human IgA antibodies. Therefore, the conclusion was made that in anseriform birds IgM-like Igs are the secretory Igs, while in galliform birds biliary Igs with special antigenic properties occur.

The differentiation of germ cells and gonads during development of carp (Cyprinus carpio L.). A study with anti-carp sperm monoclonal antibodies

Gonadal development, germ cell differentiation and the appearance of membrane antigenic determinants, specific for male and female germ cells during gonadogenesis, was studied in larval and juvenile carp (Cyprinus carpio L.) until 25 weeks after fertilization. Indirect immunofluorescence studies with four monoclonal antibodies raised against carp spermatozoa revealed that monoclonal antibody WCS 29 stained the outer membranes of primordial germ cells in larvae from 3 days after fertilization. The monoclonal antibodies WCS 3 and 17 reacted with the outer membranes of germ cells from 7 weeks after fertilization onwards, simultaneously with the onset of germ cell proliferation. With monoclonal antibody WCS 28 germ cell membranes were clearly stained from 18 weeks after fertilization. Similar reactions were observed in both sexes, however, female germ cells reacted at an earlier developmental stage with the monoclonal antibody WCS 28 than male germ cells. In the developing testis the monoclonal antibodies stained all types of spermatogenic cells. In the ovary, however, only oogonia and early prophase oocytes showed a positive reaction with the four monoclonal antibodies. The results indicate that germline-specific antigens are present on the outer membranes of primordial germ cells and their male and female descendants, with the exception of elderly oögenic stages. It is assumed that the appearance and disappearance of these membrane antigens reflect differentiation steps of germ cells during gonadogenesis.

Ontogeny of the immune system in carp ( [formula omitted]) the appearance of antigenic determinants on lymphoid cells detected by mouse anti-carp thymocyte monoclonal antibodies

Mouse anti-carp thymocyte monoclonal antibodies were used to investigate the appearance of lymphoid cell determinants in young carp. Determinants detected by clone WCT 4 were present on thymocytes as soon as the thymus was visible histologically, day 4 after fertilization (a.f.) at 21°C, but WCT 4-positive lymphocytes were not present in the pronephros until day 7 a.f.. In contrast, determinants detected by monoclonal antibodies from clones WCT 8 and WCT 10 appeared on thymocytes from day 7 a.f. at 21°C, and WCT 8-positive and WCT 10-positive lymphocytes were not detected in the pronephros until days 16 and 12 a.f., respectively. These results are discussed in relation to the appearance of determinants detected by anti-carp serum immunoglobulin antisera.