Label-free electrochemical impedimetric immunosensor for sensitive detection of IgM rheumatoid factor in human serum.

The authors report a label-free and direct detection of rheumatoid factor- Immunoglobulin M (IgM-RF) based on an impedimetric-interdigitated wave type microelectrode array (IDWµE). IDWµE was functionalized with a self-assembled monolayer (SAM) of thioctic acid for antigen immobilization. The SAM functionalized IDWµE were characterized by atomic force microscopy, Energy Dispersive X-Ray Spectroscopy, and X-ray photoelectron spectroscopy. The covalent immobilization of IgG-Fc onto the SAM modified electrode arrays was characterized morphologically via AFM and electrochemically via cyclic voltammetry and electrochemical impedance spectroscopy. Impedimetric measurements in the presence of redox probe (Potassium ferrocyanide and potassium ferricyanide) showed a significant change in both the impedance spectrum and charge transfer resistance upon IgM-RF binding. Impedance measurements were target specific and linear with an increase in IgM-RF concentrations between 1 and 200 IU mL-1 in redox probe and human serum, respectively. The sensor showed detection limits of 0.6 IU mL-1 in the presence of redox probe and 0.22 IU mL-1 in the human serum. The biosensor exhibited good reproducibility (relative standard deviation (RSD), 4.96%) and repeatability (RSD, 2.31%) with an acceptable selectivity towards IgM-RF detection. The sensor also showed a good stability for 3 weeks at 4 °C in 1X PBS. Therefore, the sensitive and stable immunosensor exhibiting IDWµE features can be integrated with a miniaturized potentiostat to develop a sensing system that detects IgM-RF for point-of-care applications.

IgM and IgA rheumatoid factors purified from rheumatoid arthritis sera boost the Fc receptor- and complement-dependent effector functions of the disease-specific anti-citrullinated protein autoantibodies.

Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1ß ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.

False decrease of HBsAg S/CO values in serum with high-concentration rheumatoid factors.

OBJECTIVES: To investigate whether high-concentration RFs cause false decrease of S/CO values of serum HBsAg. DESIGN AND METHODS: Serum HBsAg was determined in 100 RF-positive sera using one-step ELISA. Twenty-three HBsAg-negative sera with high-concentration RFs were selected randomly to perform dilution with mixed normal sera. Serum models consisting of HBsAg and high-concentration RFs were made by blending high-concentration RFs and HBsAg-positive sera at the ratio of 9:1. RESULTS: In the 23 samples, one-step ELISA showed that HBsAg-positive rate was 69.57% at dilution of 1:2, 60.87% at dilution of 1:4 and 40.00% at dilution of 1:8, whereas two-step ELISA showed that it was 100% without any dilution. Thirty serum models were made and divided into six groups. Median S/CO value of HBsAg was 3.00 in control, whereas it ranged from 0.13 to 1.78 in the six groups. CONCLUSIONS: High-concentration RFs cause false decrease of S/CO values of HBsAg using one-step ELISA.

Dietary fish and fish oil and the risk of rheumatoid arthritis.

BACKGROUND: Intake of fish oil and oily fish has been reported to improve clinical symptoms in people who have rheumatoid arthritis. Whether the intake of oily fish and fish oil might also protect against the development of rheumatoid arthritis is not known. OBJECTIVE: We investigated the association between intake of oily fish and fish oil supplements and the risk of rheumatoid arthritis in a population-based case-control study. METHODS: The study comprised 1889 incident cases of rheumatoid arthritis and 2145 randomly selected controls recruited from a geographically defined area of Sweden during 1996-2005. Data on the consumption of oily fish and fish oil supplements 5 years preceding enrollment had been obtained through a questionnaire. We calculated odds ratios (ORs) for the development of rheumatoid arthritis, using logistic regression to adjust for age, residential area, body mass index, smoking, and alcohol consumption. RESULTS: Compared with subjects who never or seldom consumed oily fish, the OR for developing rheumatoid arthritis was 0.8 (95% confidence interval = 0.6-1.0) for subjects who consumed oily fish 1-7 times a week. The results did not change notably when stratifying the cases for rheumatoid factor or for antibodies to citrullinated peptide antigens. Similar results were seen for subjects consuming oily fish 1-3 times a month. Cases and controls did not differ in their consumption of fish oil supplements. CONCLUSION: Intake of oily fish was associated with a modestly decreased risk of developing rheumatoid arthritis.

Removal of autoantibodies by 4-mercaptoethylpyridine-based adsorbent.

Extracorporeal immunoadsorption (ECI) therapy using Staphylococcal Protein A columns has proven effective for removing autoantibodies and circulating immune complexes from patients selectively, providing a promising treatment for autoimmune diseases. However, due to the drawbacks of Protein A in terms of cost and stability, the widespread use of Protein A based ECI is limited. In this study, we investigated the feasibility of 4-mercaptoethylpyridine (MEP, MW 139 Da), a simple and inexpensive synthetic compound, as an alternative to Protein A for autoantibody removal therapy. MEP-based adsorbents were prepared by coupling MEP to Sepharose CL-6B. We found that ligand density was an adjustable parameter for the synthesis of adsorbents aiming at different pathogenic factors, depending on the class of antibody. MEP-Sepharose with a ligand density of 98.8 micromol/ml could remove 80% of the anti-double-stranded DNA antibodies from human serum, whereas a ligand density of 64.5 micromol/ml was enough to remove 96% of the rheumatoid factor (RF) in the serum. Moreover, MEP-based adsorbents showed a lower degree of individual differences compared to Protein A-Sepharose. RF removal of 90% was achieved for all 12 serum samples from different individuals. Among the 14 serum samples derived from systemic lupus erythematosus patients, 11 samples had markedly reduced antinuclear antibody titers. In addition, non-specific adsorption of plasma components to MEP-Sepharose was limited, and the binding capacity of the absorbent for IgG was still about 20 mg/ml of gel after 10 cycles. The results indicated that MEP-based adsorbent could offer a new type of adsorber for the treatment of autoimmune diseases.

Molecular studies of rheumatoid factor using pseudobioaffinity membrane chromatography.

Using pseudobioaffinity ligand L-histidine immobilized to poly(ethylene vinyl alcohol) hollow fiber membrane is an interesting approach for the purification of total IgG and its subclasses from untreated serum in a single step. Gentle adsorption and elution conditions of this chromatography system allow efficient recovery of the protein in its native form. This approach was employed for the recovery and molecular study of rheumatoid factor (RF), an anti-IgG autoantibody (AAb) that form immune complexes with autologous IgG Abs in the sera. The purity of the recovered molecule was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), revealed a 150-kDa IgG band and an additional approximately 300 kDa band which may be RF bound IgG complex. Since RF is an AAb, the purified protein was studied for its catalytic functions like peptide, DNA, and RNA hydrolyzing activities. The substrate Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) hydrolyzing activity by total IgG from different patient sera was found to be greater than healthy controls. In an effort to identify the subclass specificity for the proteolytic function, the pre-purified total IgG fractions from rheumatoid arthritis (RA) sera were subjected to rechromatography using a discriminating buffer. In this experiment, the activity was found in the non-retained fractions suggesting IgG2 specificity for the catalytic function. A comparative study between different catalytic functions was performed for IgG separated from individual patient.

Rheumatoid factors induce signaling from B cells, leading to Epstein-Barr virus and B-cell activation.

B-cell antigen receptor signaling is initiated upon binding of the antigen to membrane-bound immunoblobulin (Ig), and the anti-Ig antibody (Ab) mimics this signaling. In B cells latently infected with Epstein-Barr virus (EBV), the same signals induce virus activation. We examine here whether rheumatoid factors (RFs), autoantibodies directed against the Fc portion of IgG, induce EBV and B-cell activation. As a source of RFs, RF-producing lymphoblastoid cell line (LCL) clones were isolated from peripheral blood mononuclear cells (PBMC) and synovial cells from patients with rheumatoid arthritis (RA) by EBV transformation. Burkitt's lymphoma-derived Akata cells, which are highly responsive to EBV activation by anti-Ig Abs, were used for the assay of EBV activation. Akata cells expressed IgG3 as membrane-bound Ig. RFs from a synovium-derived LCL were directed to IgG3 and induced EBV activation in 16 to 18% of Akata cells, whereas RFs from another synovium-derived LCL were directed to IgG1 and did not induce EBV activation. Pretreatment of RFs with the purified Fc fragment of human IgG completely abolished EBV activation. Furthermore, B-cell activation was assessed by incorporation of [3H]thymidine. RFs from synovium-derived LCLs efficiently induced B-cell activation, and the addition of CD40 ligand had a synergistic effect. On the other hand, RFs from PBMC-derived LCLs were polyreactive, had a lower affinity to IgG, and did not induce EBV and B-cell activation. The present findings imply a possible role for RFs as EBV and B-cell activators.

Interference by rheumatoid factor activity in the detection of antiavian antibodies in pigeon breeders disease.

The assessment of antiavian antibodies is relevant for the study of pigeon breeder's disease; nevertheless, different factors may hamper their accurate detection. The objective of this study was to determine whether an endogenous interfering effect in pigeon breeder's disease might explain the simultaneous presence of IgM, IgG, and IgA antiavian antibodies in high titers as assessed by ELISA. Fifty-nine patients with pigeon breeder's disease, 80 healthy controls, and 47 asymptomatic breeders were studied. To assess possible interfering effects by endogenous immunoglobulins, serum IgG was separated through protein A-Sepharose CL-4B chromatography. Antiavian antibodies were measured in whole and separated samples by ELISA. Since a decline of IgM antiavian antibodies following IgG removal was consistent with a false-positive effect, the causes were studied. High values of IgM, IgG, and IgA antiavian antibodies were found in 47.4% [corrected] of patients with pigeon breeder's disease. An IgM rheumatoid factor activity against IgG was found through ELISA in sera with false-positive IgM antiavian antibodies. Rheumatoid factor binding was confirmed by Western blot. Experimental addition of purified rheumatoid factor to sera with IgG antiavian antibodies replicated the interfering effect. A control group of rheumatoid arthritis with high rheumatoid factor values did not show positive antiavian antibodies tests. No IgG with anti-IgM or anti-IgA activity was found, and the detection of IgA against IgM and IgG was negative. In conclusion, the study of antiavian antibodies might be affected by different immunoassay conditions. An endogenous rheumatoid factor activity produced false-positive IgM results. Other similar interferences warrant a careful evaluation during the serological assessment of pigeon breeder's disease.

Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation.

The occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RF activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.

Laboratory aspects of rheumatologic disease.

Assessment of laboratory values is an important function of nursing practice. Rheumatologic laboratory assessment, in particular, can be complex because few findings are actually pathognomonic. This article provides a perspective on an interpretive approach to laboratory assessment of rheumatologic disease. In conjunction with the patient's clinical status, these values can provide helpful information for monitoring or predicting the course of disease.

Rheumatoid factors in primary Sjögren's syndrome (pSS) use diverse VH region genes, the majority of which show no evidence of somatic hypermutation.

Rheumatoid factor (RF) is the most common autoantibody found in patients with Sjögren's syndrome (SS). To study the genetic origin and the mechanisms acting behind its generation we have characterized and sequenced the immunoglobulin VH genes used by 10 IgM RF MoAbs derived from peripheral blood of six female patients with pSS. We compared the structure of the RF immunoglobulin VH genes with those obtained previously from rheumatoid arthritis (RA) patients and healthy immunized donors (HID). VH1 and VH4 were each used by four RF clones, one clone was encoded by VH3 family gene and one by VH2 family gene. This distribution frequency was different from that observed in RA, where VH3 was the dominant family, followed by VH1. Eight different germ-line (GL) genes encoded the clones and all of these genes were seen previously in RA and/or HID RF. Five clones rearranged to JH6, four rearranged to JH4 and one to JH5, in contrast to RF from RA and HID, where JH4 was most frequently used. D segment use and CDR3 structure were diverse. Interestingly, three out of four VH4 clones used the GL gene DP-79 that was seen frequently in RA RF. The degree of somatic mutation in the pSS RF was very much lower than seen in RA and HID RF. All the pSS RF clones except three were in or very close to GL configuration. This indicates that there is little role for somatic hypermutation and a germinal centre reaction in the generation of RF from peripheral blood in pSS.

Heat-aggregated human IgG as immunosorbent for rheumatoid arthritis.

In order to selectively remove pathogenic rheumatoid factors (RF) from plasma of rheumatoid arthritis (RA) patients, a new immunosorbent was developed by immobilizing heat-aggregated human IgG (HAH IgG) on epichlorohydrin-activated agar gel. Different parameters influencing ligand coupling were studied, i.e., ligand concentration, reaction time, temperature and pH. The maximum amount of HAH IgG immobilized on the carrier was 2.7 mg/ml. Adsorption capacity was evaluated by ELISA technique. After incubation of 3.0 ml of RA plasma with 1.0 ml of the adsorbent, the RF IgM, IgG, and IgA were removed by 88%, 74%, and 50%, respectively. The decrease of total immunoglobulins and albumin were less than 7% and 9%. In vitro, perfusion of 10.0 ml plasma at 37 degrees C through 3.0 ml of adsorbent, at a flow rate of 5.0 ml/min for 60 min, resulted in the removal of 49% of RF IgM, 72% of RF IgG and 58% of RF IgA. No leakage of ligand and no decline in adsorption capacity and mechanical strength were observed after steam-sterilization.

Efectos de la bromocriptina en pacientes con artritis reumatoide activa / Effects of bromocriptine in patients with active rheumatoid arthritis

Background: Neuroendocrine factors play an important role in the expression of autoimmune disease. Proclatin (PRL) can induce T-cell proliferation and macrophage activation. Elevated PRL levels have been described in patients with rheumatoid arthritis and (RA). Aim and Methods: We studiend immunological and clinical effects of PRL suppression in 9 RA patients with active disease, treated for 3 months with bromocriptiner (BRC), an inihibitor of PRL secretion. Results: BRC induced a significant depression of the peripheral blood mononuclear cells response to antigen (p=0.008) and mitogen (p=0.008) which was significantly correlated with improvements in the HAQ disability index (r=0.68; p=0.04) and grip strength (r=0.7; p=0.02). Also, the in-vitro production of IL-2, nitric oxide and poliamines -that are critical for the proliferative response of lymphoid cells- decreased significantly. The group experienced significant improvement of grip strength (p=0.028) and the HAQ disability index (p=0.025), whereas 4 individuals archieved clinical improvement according to the American College of Rheumatology preliminary definition. We conclude that BRC treatment induces a significant depression of in-vitro immune function in RA patients and these changes are related to parameters of disease activity. The effects of BRC on immune function and disease activity in RA patients warrant further investigation

IgG rheumatoid factors isolated by the surface-displaying phage library technique.

Our analysis of IgG rheumatoid factors (RFs) from three patients with rheumatoid arthritis (RA) revealed that most contained significant numbers of skewed mutations per V region, suggesting that these RFs arose from antigen-driven responses. To further study IgG RFs in RA, we used pComb3 vector to construct an IgG1,lambda combinatorial antibody library from a synovial fluid sample. After panning against human IgG, Fab fragments from 71/96 phage clones bound to Fc-coated wells. Sequence analysis of 20 randomly chosen Fc-binders showed that 17 (85%) clones had identical heavy (H) and light (L) chain V regions, represented by Humha311 and Humla211, respectively. Of the remaining three clones, two had the same Humla211 L chain, but each with a different H chain V region. All the putative germline V genes for these RFs also encode RF in RA patients. However, none of these RF V regions are similar to those of the two IgG RFs derived by the hybridoma technique from the same synovial fluid. The Humha311 H chain has two frameshifts: a one-base insertion upstream of the JH region and a four-base deletion near the end of the CH1 region, resulting in a mainly unconventional amino acid sequence in the CH1 region. In the future, it will be important to study the presence of IgG molecules with such unconventional CH1 amino acid sequences, and the effects of these amino acid sequences on the structures and immunological properties of the IgG molecules.

Acidosis tubular renal distal y litiasis renal en tres casos de síndrome de Sjögren primario / Distal renal tubular acidosis and renal lithiasis in 3 cases of primary Sjögren's syndrome

Tubulo interstitial nephritis, the main manifestation of renal involvement in Sjögren syndrome, may lead to a tubular dysfunction that is usually subclinical. We report 3 women, aged 32, 35 and 35 years old, with a primary Sjögren's syndrome and symptomatic type I or distal tubular acidosis. Two patients had nephrolithiasis and one a nephrocalcinosis. Two had a basal hyperchloremic metabolic acidosis. The ammonium chloride acidification test was abnormal in all, demonstrating a distal tubular defect. None had proximal tubular dysfunction. All had an urinary pH over 6.5 and hypocitraturia and none had hypercalciuria. Renal calculi were composed of calcium oxalate and calcium phosphate in 2 patients and calcium phosphate and ammonium phosphate in the other. All women had positive antinuclear antibodies with mottled pattern, 2 had anti Ro antibodies and positive rheumatoid factor and 1 had hypergammaglobulinemia. None had anti La antibodies, crioglobulinemia or monoclonal proteins