Antibodies to calnexin and mutated calreticulin are common in human sera.

PURPOSE OF THE STUDY: Calreticulin is an endoplasmic reticulum chaperone protein, which is involved in protein folding and in peptide loading of major histocompatibility complex class I molecules together with its homolog calnexin. Mutated calreticulin is associated with a group of hemopoietic disorders, especially myeloproliferative neoplasms. Currently only the cellular immune response to mutated calreticulin has been described, although preliminary findings have indicated that antibodies to mutated calreticulin are not specific for myeloproliferative disorders. These findings have prompted us to characterize the humoral immune response to mutated calreticulin and its chaperone homologue calnexin. PATIENTS AND METHODS: We analyzed sera from myeloproliferative neoplasm patients, healthy donors and relapsing-remitting multiple sclerosis patients for the occurrence of autoantibodies to wild type and mutated calreticulin forms and to calnexin by enzyme-linked immunosorbent assay. RESULTS: Antibodies to mutated calreticulin and calnexin were present at similar levels in serum samples of myeloproliferative neoplasm and multiple sclerosis patients as well as healthy donors. Moreover, a high correlation between antibodies to mutated calreticulin and calnexin was seen for all patient and control groups. Epitope binding studies indicated that cross-reactive antibodies bound to a three-dimensional epitope encompassing a short linear sequence in the C-terminal of mutated calreticulin and calnexin. CONCLUSION: Collectively, these findings indicate that calreticulin mutations may be common and not necessarily lead to onset of myeloproliferative neoplasm, possibly due to elimination of cells with mutations. This, in turn, may suggest that additional molecular changes may be required for development of myeloproliferative neoplasm.

Virus-specific antibody indices may supplement the total IgG index in diagnostics of multiple sclerosis.

Intrathecal antibody synthesis to viruses is associated with multiple sclerosis (MS). Here, IgG levels to Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1), EBV nuclear antigen 1 (EBNA1), mumps virus (MuV) nucleoprotein (NuP), measles virus (MeV) NuP and rubella virus (RuV) capsid protein (CaP) were found to be elevated in serum and cerebrospinal fluid (CSF) of MS patients compared to healthy controls (HCs), whereas the opposite was found for cytomegalovirus (CMV) pp52. Strong correlations between serum and CSF IgG were seen for MeV, CMV and RuV in both MS patients and HCs. The antigen panel obtained high sensitivity (81%) and specificity (86%), demonstrating that antigen panels may supplement the total IgG index used in MS diagnosis.

Incidence of neuromyelitis optica spectrum disorder in the Central Denmark Region.

OBJECTIVES: Neuromyelitis optica (NMO)/NMO spectrum disorder (NMOSD) may be misdiagnosed as multiple sclerosis. The aim of this study was to (i) to measure AQP4-IgG in patients who fulfilled the clinical and radiological criteria of NMOSD in the Central Denmark Region and (ii) to estimate the incidence of NMOSD in the region, according to both the 2006 Wingerchuk criteria and the 2015 International Panel for NMO Diagnosis criteria. MATERIALS AND METHODS: Medical records of all patients diagnosed with a demyelinating disorder in the region from 1 January 2012 to 31 December 2013 were reviewed. Patients were classified as having (i) "NMO" if the 2006 criteria were met, (ii) "NMOSD with AQP4-IgG" or (iii) "NMOSD without/unknown AQP-IgG" if the new 2015 NMOSD criteria were met. Patients with core symptoms were invited to provide a blood sample for AQP4-IgG analysis with an enzyme-linked immunosorbent assay and a cell-based indirect immunofluorescence assay. RESULTS: In 191 patients with core symptoms, one met the 2015 NMOSD with AQP4-IgG criteria. Two patients met the 2006 NMO and 2015 NMOSD without/unknown AQP4-IgG criteria. Among 108 patients providing a blood sample, all were seronegative. The estimated incidence of NMO (2006 criteria) and NMOSD (2015 criteria) was 0.08 and 0.12 per 100 000 person-years, respectively. CONCLUSION: NMO/NMOSD is a rare disease in the Central Denmark Region, with a considerably lower incidence rate than previously estimated in a neighbouring region.

A fluorescence sedimentation assay for dsDNA antibodies.

The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other assays but does also present some disadvantages as it utilizes radioactive-labelled dsDNA and requires high levels of technical expertise for safe handling. Here, a new precipitation assay, 'Fluoro-Farr' assay, is described. This assay maintains a high sensitivity and specificity for SLE but is based on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic efficiency of the assay was evaluated by testing 57 sera from SLE patients and 60 healthy controls. The Fluoro-Farr assay revealed a diagnostic sensitivity of 68% at a diagnostic specificity of 95% (ROC AUC 0.91). Furthermore, the new Fluoro-Farr assay was compared to the RIA-Farr assay, and showed a correlation of the outcomes from the two assays, but the Fluoro-Farr assay did not outperform the RIA-Farr assay due to its outstanding clinical diagnostic efficiency (ROC AUC 0.99). In conclusion, the Fluoro-Farr assay presents a viable alternative to the traditional RIA-Farr assay; especially in laboratories without facilities to perform assays with radioactivity-based read-out. As the RIA-Farr assay, the Fluoro-Farr assay has the advantage of being a precipitation assay allowing antibody:dsDNA interaction in solution using native dsDNA.

Humoral markers of active Epstein-Barr virus infection associate with anti-extractable nuclear antigen autoantibodies and plasma galectin-3 binding protein in systemic lupus erythematosus.

We investigated if signs of active Epstein-Barr virus and cytomegalovirus infections associate with certain autoantibodies and a marker of type I interferon activity in patients with systemic lupus erythematosus. IgM and IgG plasma levels against Epstein-Barr virus early antigen diffuse and cytomegalovirus pp52 were applied as humoral markers of ongoing/recently active Epstein-Barr virus and cytomegalovirus infections, respectively. Plasma galectin-3 binding protein served as a surrogate marker of type I interferon activity. The measurements were conducted in 57 systemic lupus erythematosus patients and 29 healthy controls using ELISAs. Regression analyses and univariate comparisons were performed for associative evaluation between virus serology, plasma galectin-3 binding protein and autoantibodies, along with other clinical and demographic parameters. Plasma galectin-3 binding protein concentrations were significantly higher in systemic lupus erythematosus patients (P = 0.009) and associated positively with Epstein-Barr virus early antigen diffuse-directed antibodies and the presence of autoantibodies against extractable nuclear antigens in adjusted linear regressions (B = 2.02 and 2.02, P = 0.02 and P = 0.002, respectively). Furthermore, systemic lupus erythematosus patients with anti-extractable nuclear antigens had significantly higher antibody levels against Epstein-Barr virus early antigen diffuse (P = 0.02). Our study supports a link between active Epstein-Barr virus infections, positivity for anti-extractable nuclear antigens and increased plasma galectin-3 binding protein concentrations/type I interferon activity in systemic lupus erythematosus patients.

Antibodies to early EBV, CMV, and HHV6 antigens in systemic lupus erythematosus patients.

OBJECTIVES: We investigated the antibody levels against early antigens of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV6) in systemic lupus erythematosus (SLE) patients and healthy controls, and further correlated these antibodies to haematology/biochemistry, serology, and disease activity measures. METHOD: Immunoglobulin (Ig)M, IgG, and IgA levels against the DNA polymerase processivity factors of EBV, CMV, and HHV6, termed early antigen diffuse (EA/D), pp52, and p41, respectively, were determined in plasma samples from 77 SLE patients and 29 healthy controls by using enzyme-linked immunosorbent assays (ELISAs). RESULTS: IgM, IgG, and IgA levels against EBV EA/D, and IgG and IgA levels against CMV pp52, were significantly higher in SLE patients compared with healthy controls. Furthermore, EBV EA/D- and CMV pp52-directed IgG levels were inversely and positively associated, respectively, with lymphocyte counts in SLE patients. None of the findings seemed to be associated with use of immunosuppressive medication. CONCLUSIONS: Our results suggest strong, but opposite, associations of lytic EBV and CMV infections with SLE. The amplified humoral responses to EBV EA/D and CMV pp52 in our SLE patient cohort probably reflect aberrant control of EBV and CMV reactivation. However, reactivation of EBV appeared to correlate with lymphopenic manifestations in SLE patients whereas CMV reactivation seemed to correlate with increments in lymphocyte levels.

Chemical and thermal unfolding of calreticulin.

Calreticulin is a soluble endoplasmic reticulum chaperone, which has a relatively low melting point due to its remarkable structure with a relatively high content of flexible structural elements. Using far ultraviolet circular dichroism (CD) spectroscopy and a fluorescent dye binding thermal shift assay, we have investigated the chemical and thermal stability of calreticulin. When the chemical stability of calreticulin was assessed, a midpoint for calreticulin unfolding was calculated to 3.0M urea using CD data at 222 nm. Using the fluorescent dye binding thermal shift assay, calreticulin was found to obtain a molten structure in urea concentrations between 1-1.5 M urea, and to unfold/aggregate at high and low pH values. The results demonstrated that the fluorescent dye binding assay could measure the thermal stability of calreticulin in aqueous buffers with results comparable to melting points obtained by other techniques.

Epstein-Barr virus early antigen diffuse (EBV-EA/D)-directed immunoglobulin A antibodies in systemic lupus erythematosus patients.

OBJECTIVE: We sought to determine whether the serological response towards lytic cycle antigens of Epstein-Barr virus (EBV) is altered in systemic lupus erythematosus (SLE) patients. METHOD: We used enzyme-linked immunosorbent assay (ELISA) to investigate the prevalence of EBV early antigen diffuse (EBV-EA/D) antibodies in sera from 60 patients with SLE, 40 with scleroderma (SSc), 20 with primary Sjögren's syndrome (pSS), 20 with rheumatoid arthritis (RA), 20 healthy controls, and also subjects with various circulating autoantibodies. Samples from patients were obtained from clinics specialized within the diseases in Denmark and Sweden and samples from healthy controls were obtained from volunteers. RESULTS: A significant elevated titre of immunoglobulin (Ig)A, IgG, and IgM EBV-EA/D antibodies was found in SLE patients compared to healthy controls, a finding not explained by immunosuppressive treatment or disease activity. The largest difference was observed for IgA EBV-EA/D antibodies (p = 0.0013) with a seropositive rate of 58% in SLE patients and 0% in healthy controls. RA and SSc patients and individuals seropositive for anti-Scl-70 were additionally found to have elevated titres of IgA EBV-EA/D antibodies (40%, p = 0.014; 60%, p = 0.015; and 38.5%, p = 0.045, respectively). However, the titres were generally lower than in SLE patients. CONCLUSION: Our findings support an association between EBV and SLE. The elevated titre of EBV-EA/D-directed IgA antibodies found in SLE patients could suggest reactivation of EBV in epithelial cells or reinfection of epithelial cells after reactivation in B cells, indicating lack of control of the latent infection.

Production and characterization of peptide antibodies.

Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody purification, the antibodies are characterized based on their affinity or specificity. An efficient approach for characterization of peptide antibodies is epitope mapping using peptide based assays. This review describes standard solid-phase approaches for generation of peptide antibodies with special emphasis on peptide selection, generation of peptide conjugates for immunization and characterization of the resulting peptide antibodies.

Synthesis and anti-angiogenic effect of conjugates between serum albumin and non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit tumor growth and angiogenesis. Covalent linkage of naproxen to human serum albumin (HSA) has been shown to target it efficiently to the liver and this may potentially be exploited for liver-selective inhibition of angiogenesis. With the aim of investigating the anti-angiogenic efficiency of NSAID-HSA conjugates in vitro, three NSAIDs, aspirin, ibuprofen, and naproxen were conjugated to HSA using different concentrations of their N-hydroxysuccinimide esters. Conjugation ratios from 10 to 50 were achieved and the conjugates retained a growth inhibitory effect on endothelial cells at or above the level of the non-conjugated NSAIDs in an in vitro angiogenesis assay.

Interaction of the chaperone calreticulin with proteins and peptides of different structural classes.

The interaction of calreticulin with native and denatured forms and polypeptides in proteolytic digests of proteins representing structural classes of all-alpha-helix (hemoglobin, serum albumin), all-beta-sheet (IgG) and alpha-helix + beta-sheets (lysozyme, ovalbumin) was investigated. The binding of calreticulin to denatured proteins was found to depend on conformation and structural class of the protein. No interaction was observed with the native proteins, whereas binding was seen for the denatured proteins, the order of interaction being lysozyme = IgG > ovalbumin >> hemoglobin = serum albumin. Moreover, the interaction between calreticulin and the heat-denatured proteins depended on the temperature and time used for denaturation and the degree of proteolytic fragmentation. Calreticulin bound well to peptides in proteolytic digests from protease K or chymotrypsin treatment of lysozyme, IgG and ovalbumin but weakly or not at all to peptides in proteolytic digests of hemoglobin and serum albumin. Synthetic peptides from lysozyme and ovalbumin confirmed binding to hydrophobic peptides from these proteins. These results show that calreticulin has the ability to interact with denatured and fragmented forms of proteins with a preference for beta-strand structure and hydrophobicity.

Interaction of calreticulin with amyloid beta peptide 1-42.

The interaction of calreticulin with amyloid beta (Abeta) was investigated using solid phase and solution binding assays. Calreticulin bound Abeta 1-42 in a time and concentration dependent fashion. The binding was optimal at pH 5 and was stimulated by Ca2+ and inhibited by Zn2+ at pH 7. Interaction took place through the hydrophobic C-terminus of Abeta 1-42 and the polypeptide binding site of calreticulin. The results are discussed in the light of a reported role of calreticulin as a cell surface scavenger receptor.

Interaction of calreticulin with CD40 ligand, TRAIL and Fas ligand.

The molecular chaperone calreticulin has been shown to bind C1q and mannan-binding lectin (MBL), which are constituents of the innate immune defence system. C1q and MBL do not share a large sequence identity but have a similar overall molecular architecture: an N-terminal triple-helical collagen-like domain and a C-terminal globular domain with ligand-binding properties. C1q is a hetero-trimer, while MBL is a homo-trimer, but due to the presence of N-terminal cysteines they both form higher order oligomers of trimers, which are the mature functional molecules. The same molecular architecture is utilized by many other functionally diverse molecules and in this work the interaction of calreticulin with C1q and structurally similar molecules was investigated. In addition to C1q and MBL, CD40 ligand (CD40L), tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) were found to bind calreticulin strongly. A low level or no binding was observed for adiponectin, tumour necrosis factor-alpha (TNF-alpha), CD30L, surfactant protein-A and -D and collagen VIII. The interaction with calreticulin required a conformational change in CD40L, TRAIL and FasL and showed the same characteristics as calreticulin's interaction with C1q and MBL: a time-dependent saturable binding to immobilized protein, which was initially sensitive to salt but gradually developed into a salt-insensitive interaction. Thus, the interaction requires a structural change in the interaction partner and leads to a conformational change in calreticulin itself. The implications of these results are that calreticulin may function as a general response modifier for a whole group of immunologically important proteins.

Autoantibodies to the proteasome in monosymptomatic optic neuritis may predict progression to multiple sclerosis.

OBJECTIVE: Proteasome autoantibodies (PAB) have been found in multiple sclerosis (MS) patient sera and cerebrospinal fluid (CSF). Presence of PAB could thus be a possible diagnostic marker for MS. We investigated whether PAB serum status in acute monosymptomatic optic neuritis (ON) and MS differed significantly from that of healthy controls, and whether or not PAB status is predictive of later MS development in patients with ON. MATERIAL AND METHODS: Sera from ON patients, MS patients and healthy donors were analysed retrospectively using ELISA. Subsequently, a small group of PAB-positive samples were subjected to SDS-PAGE, immunoblotting and 2-D PAGE. RESULTS: We found that 20 % (6/30) ON patients, 47 % (22/47) MS patients and 9 % (7/81) controls tested PAB positive using ELISA analysis. High PAB levels were found in 2 (4 %) MS patients, 1 (3 %) ON patient and 2 (3 %) controls. PAB positivity in ELISA was confirmed by immunoblotting. Separation of proteasome subunits by 2D PAGE followed by immunoblotting revealed no particular PAB subunit preference. CONCLUSIONS: A retrospective search in available patient files revealed that 6 of 6 (100.0 %) PAB-positive ON patients developed MS over time. Eight of 24 (33 %) PAB-negative ON patients developed MS over time and 47 % (14/30) of all ON patients developed MS. A series of patient CSF was analysed by ELISA to assess the possible correlation between PAB status of concurrent serum and CSF samples, but no correlation was found. However, the results from the six PAB-positive ON patients could potentially be of prognostic value.

Comparison of rocket and crossed immuno-electrophoresis assays for determination of the level of actin complexing of Gc globulin.

OBJECTIVE: Gc globulin (vitamin D-binding protein) is a component of the extracellular actin scavenger system. The level of Gc globulin is reduced in patients with fulminant hepatic failure, septic shock and trauma. Furthermore, low levels of Gc globulin in patients with fulminant hepatic failure and multiple trauma have been found to correlate with the morbidity and mortality of patients. Owing to a large increase in the turnover of Gc globulin upon complex formation with actin, it may be important to determine both the total Gc globulin concentration and the degree of complexing with actin for estimating the clinical prognosis of a patient. For this reason, we have compared a crossed immuno-electrophoresis method (CIE), suitable for visualizing the degree of complexing with actin, with a rocket immuno-electrophoresis method (RIE), previously used for determination of the complex degree. MATERIAL AND METHODS: Sera from healthy donors and from patients with acetaminophen-induced liver disease or trauma were investigated using CIE, RIE and enzyme-linked immunosorbent assay (ELISA). RESULTS: Using the CIE, no Gc globulin-actin complexes were detected among healthy donors. Complexes were present in 21 of 39 patients with liver disease and 3 of 37 trauma patients. High complex ratios (> 20 %) were found in 6 of 7 patients with hepatic encephalopathy. Using the RIE, complexes were detected in most samples. CONCLUSION: The results show that the CIE method may be used for determining the degree of actin complexing in conjunction with ELISA or RIE in determining the levels of total Gc globulin.

Second-generation nanofiltered plasma-derived mannan-binding lectin product: process and characteristics.

BACKGROUND AND OBJECTIVES: Mannan-binding lectin (MBL) is an important component of the innate immune defence; it binds to carbohydrate structures on pathogenic micro-organisms resulting in complement activation and opsonization. Individuals with low MBL levels are at risk of recurrent and severe infections. Substitution therapy with plasma-derived MBL is a promising treatment of diseases associated with MBL deficiency. A first-generation MBL product has been shown to be safe and well tolerated, and patients have benefited from MBL treatment. Following is a description of the development of a nanofiltered second-generation MBL product from Cohn fraction III, with the use of a new affinity matrix for MBL purification and the characteristics of this improved product. MATERIALS AND METHODS: Carbohydrate-based gels were comparatively screened as affinity matrices. MBL was extracted from fraction III, and affinity purified on a Superdex 200 pg column. The eluted material underwent two virus reduction steps: filtration through Planova 20N and solvent/detergent treatment. It was further purified by anion-exchange and gel-filtration chromatography. The affinity eluate and the final MBL fraction were characterized by protein chemical, immunological, and functional assays. RESULTS: In production scale, Superdex 200 pg was found to be superior to other carbohydrate-based matrices, and MBL was affinity purified from fraction III with a yield of 70%. The viral safety was increased by performing a nanofiltration of the affinity eluate through Planova 20N with a minimal loss of MBL. The purity of the final MBL fraction was 53% excluding the MBL-associated serine proteases (MASP). The product consisted of high-oligomeric MBL, with two dominating forms, and with MASP-1, -2, -3 and 19 kDa MBL-associated protein (MAp19). Only a few protein impurities were present, the major being alpha2-macroglobulin. MBL formed complexes with alpha2-macroglobulin bridged by MASP-1 covalently attached to the latter. The functional activity, assessed by mannan-binding activity and opsonic function, was intact, whereas half of the C4 activating capacity was lost during the production process. CONCLUSION: A second-generation MBL process was developed with an average yield of 50%. It was possible to nanofilter the MBL-MASP complexes through Planova 20N with only a minor loss resulting in an increased safety profile of this MBL product.

Absence of high-affinity calreticulin autoantibodies in patients with systemic rheumatic diseases and coeliac disease.

Calreticulin has been reported to be an autoantigen in various autoimmune connective tissue diseases and in coeliac disease. Previous studies have used incubation buffers with low salt and low detergent concentrations (low stringency conditions) with serum albumin or other proteins as a blocking agent. Using these conditions we found a relatively high level of non-specific binding in many sera. Antibodies to proteins that are used as blocking reagents in ELISA (bovine serum albumin (BSA), ovalbumin, skimmed milk powder) are frequently present in sera, and these may cause false-positive results. Moreover, the low isoelectric point of calreticulin and its chaperone properties may give rise to false-positive results under low stringency conditions. We report that the use of a simple buffer without protein (50 mM Tris, pH 7.5, 1% Tween 20, 0.3 M NaCl) removes most of the problems with unwanted binding (high stringency conditions). Using the high stringency conditions, we screened sera from 107 patients with systemic lupus erythematosus, sera from patients with other systemic autoimmune diseases and from children with coeliac disease for the presence of high-affinity calreticulin autoantibodies by immunoblotting and ELISA. None of the sera contained high-affinity calreticulin antibodies. It is concluded that calreticulin is not a common autoantigen in patients with autoimmune connective tissue diseases or coeliac disease.

Interaction of C1q with the receptor calreticulin requires a conformational change in C1q.

The interaction between C1q and the chaperone calreticulin was studied under various conditions. When both proteins were present in equal amounts in solution, no interaction could be demonstrated. However, C1q immobilized on a hydrophobic surface, exposed to heat-treatment or bound to immunoglobulins (Igs) showed a strong, rapid and specific binding of calreticulin. The interaction appeared to be a two-step process, and the initial phase of interaction was sensitive to high concentrations of salt but not to a physiological salt concentration. The following strong binding was insensitive to salt and extremes of pH but sensitive to strongly denaturing agents (urea and guanidine). The sensitivity to salt during the initial phase of interaction was practically identical to that observed when calreticulin was bound to type V collagen. Binding between C1q and calreticulin could be inhibited by serum amyloid P component and by proteinase K-digested ovalbumin, and the binding of calreticulin to proteinase K-digested ovalbumin was shown to be inhibited by C1q. The data indicate that C1q binds stably to the peptide-binding site of calreticulin and that the initial binding of calreticulin to C1q involves the collagen-like domain of the C1q molecule. In conclusion, our results suggest calreticulin as a potential receptor for an altered conformation of C1q as occurs during binding to Igs. Thus, the chaperone and protein-scavenging function of calreticulin may extend from the endoplasmic reticulum to the topologically equivalent cell surface, where it may contribute to the elimination of immune complexes and apoptotic cells.