Autoantibody and metalloproteinase activity in early arthritis.

OBJECTIVES: The aim of the study was to evaluate the frequency of anti-mutated citrullinated vimentin antibodies (a-Sa), anti-citrullinated α-enolase peptide 1 antibodies (a-CEP-1), anti-filaggrin antibodies (AFAs), heterogeneous nuclear ribonucleoprotein compies/anti-RA33-antibodies (a-hnRNP/RA33), anti-carbamylated protein antibodies (a-CarP), and metalloproteinase (MMPs) activity in patients with early inflammatory arthritis (EIA). METHODS: Seventy-four patients with EIA: 51 diagnosed with RA (rheumatoid arthritis) and 23 with UA (undifferentiated arthritis), and 20 healthy volunteers were enrolled to the study. Inflammatory markers, rheumatoid factor (RF), and antibodies mentioned above were assessed in all patients. RESULTS: In the EIA group, we observed significantly higher concentration of a-CEP-1 (65.8 ± 111.6 RU/mL) than in controls (2.0 ± 0.0 RU/mL). In RF(+) RA patients, we observed higher concentration of a-Sa and a-CEP-1 than in other groups. A-Sa were positive in 69% of RF(+) RA, 37% of RF(-) RA, 26% of UA patients and in 10% of controls. A-CEP-1 were positive in 77% of RF(+) RA patients, in 56% of RF(-) RA patients, in 8.7% of UA patients, but they were negative in controls. In patients with RF(+) RA, positive a-CarP were present statistically significantly more often than in RF (-) RA patients. No statistically significant difference in frequency of a-hnRNP/RA33 and AFA between RF(+) RA, RF(-) RA, and UA was observed. CONCLUSIONS: Our results suggest that a-CEP-1 may help in differentiation between RF(-) RA and UA. a-CEP-1 and a-Sa may be useful while diagnosing EIA. a-CarP may be used in differentiation of RA RF(-) and UA. However, a follow-up study is needed to evaluate the prognostic value of analyzed antibodies.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

The effect of targeted rheumatoid arthritis therapies on anti-citrullinated protein autoantibody levels and B cell responses.

Rheumatoid arthritis (RA) is a complex inflammatory disorder associated with synovitis and joint destruction that affects an estimated 1·3 million Americans and causes significant morbidity, a reduced life-span and lost work productivity. The use of biological therapies for the treatment of RA is costly, and the selection of therapies is still largely empirical and not guided by the underlying biological features of the disease in individual patients. The synovitis associated with RA is characterized by an influx of B and T cells, macrophages and neutrophils and the expansion of fibroblast-like synoviocytes, which form pannus and lead to cartilage and bone destruction. RA is associated with synovial production of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) and with the production of inflammatory cytokines, including interleukin (IL)-1, IL-6, IL-17 and tumour necrosis factor (TNF)-α, which are targets for RA therapeutics. Recent ideas about the pathogenesis of RA emphasize a genetic predisposition to develop RA, a preclinical phase of disease that is associated with the production of ACPA and the development of symptomatic disease following inflammatory initiating events that are associated with expression of citrullinated epitopes in the joints of patients. However, we still have a limited understanding of the cytokine and intracellular pathways that regulate ACPA levels. In humans, therapy with biological agents affords a unique opportunity to better understand the cytokine and signalling pathways regulating ACPA levels and the impact of ACPA level changes on disease activity. In this study we summarize the effect of RA therapies on ACPA levels and B cell responses.

[Effects of acupuncture at "Zusanli" (ST 36) on cerebral proinflammatory cytokine and plasma neuron specific enolase in septic rats].

OBJECTIVE: To explore protective effects of acupuncture at "Zusanli" (ST 36) on cerebral tissue in rats with sepsis. METHODS: Cecal ligation and puncture (CLP) was applied to duplicate the rat model of sepsis. According to random number table, thirty SD rats were divided into a sepsis model group (group A), a sepsis model plus electroacupuncture (EA) group (group B), and a sepsis model plus non-acupoint EA group (group C), ten rats in each one. EA with the same frequency and intensity at "Zusanli" (ST 36) and non-acupoint (0.5 cm laterally to "Zusanli") for 30 min was applied in the group B and group C, respectively. No treatment was given in the goup A. 6 hours after CLP, blood was acquired from abdominal aorta to measure the levels of neuron specific enolase (NSE). Then the rats were sacrificed by abdominal aorta exsanguination to take their cerebral tissue for measuring the levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). RESULTS: Six hours after CLP, the level of NSE was (3.51 +/- 0.39) ng/mL in group B, which was significantly lower than (7.72 +/- 0.64) ng/mL in group A (P<0.05). The level of NSE was (8.02 +/- 0.72) ng/mL in the group C, which had no statistical significance with group A (P>0.05). The levels of TNF-alpha, IL-6 in cerebral tissue in group B were significantly lower than that of group A and C (all P>0.05). CONCLUSION: EA at "Zusanli" (ST 36) has certain protective effect on septic rat's brain, which has some relationship with decreasing levels of cerebral tissue proinflammatory cytokine and plasma NSE. EA at non-acupoint has no the same action.

Peptidylarginine deiminase modulates the physiological roles of enolase via citrullination: links between altered multifunction of enolase and neurodegenerative diseases.

The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. In the present study, we report that citrullination diversely regulates the biological functions of ENO1 (α-enolase) and NSE (neuron-specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (i) citrullination of Arg9 of ENO1 [ENO1Cit9; anti-CE1 (citrullinated enolase 1) antibody]; (ii) citrullination of Arg9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2 antibody); and (iii) citrullination of Arg429 of NSE (NSECit429; anti-CE2 antibody). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer's disease compared with controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by Ca2+-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen-binding affinity, which was blocked by the lysine analogue ϵ-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases.

Ex vivo soft-laser treatment inhibits the synovial expression of vimentin and α-enolase, potential autoantigens in rheumatoid arthritis.

BACKGROUND: Soft-laser therapy has been used to treat rheumatic diseases for decades. The major effects of laser treatment may be dependent not on thermal mechanisms but rather on cellular, photochemical mechanisms. However, the exact cellular and molecular mechanisms of action have not been elucidated. OBJECTIVE: The aim of this study was to investigate the ex vivo effects of low-level laser treatment (with physical parameters similar to those applied previously) on protein expression in the synovial membrane in rheumatoid arthritis (RA). DESIGN: Synovial tissues were laser irradiated, and protein expression was analyzed. METHODS: Synovial membrane samples obtained from 5 people who had RA and were undergoing knee surgery were irradiated with a near-infrared diode laser at a dose of 25 J/cm(2) (a dose used in clinical practice). Untreated synovial membrane samples obtained from the same people served as controls. Synovial protein expression was assessed with 2-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry. RESULTS: The expression of 12 proteins after laser irradiation was different from that in untreated controls. Laser treatment resulted in the decreased expression of α-enolase in 2 samples and of vimentin and precursors of haptoglobin and complement component 3 in 4 samples. The expression of other proteins, including 70-kDa heat shock protein, 96-kDa heat shock protein, lumican, osteoglycin, and ferritin, increased after laser therapy. LIMITATIONS: The relatively small sample size was a limitation of the study. CONCLUSIONS: Laser irradiation (with physical parameters similar to those used previously) resulted in decreases in both α-enolase and vimentin expression in the synovial membrane in RA. Both proteins have been considered to be important autoantigens that are readily citrullinated and drive autoimmunity in RA. Other proteins that are expressed differently also may be implicated in the pathogenesis of RA. Our results raise the possibility that low-level laser treatment of joints affected with RA may be effective, at least in part, by suppressing the expression of autoantigens. Further studies are needed.

Proteomics: advances in biomarker discovery.

The challenges encountered by proteomic researchers seeking diagnostic, prognostic and mechanistic markers were the subject of the 1-day meeting, Proteomics: Advances in Biomarker Discovery hosted by EuroSciCon. The speakers had a broad range of clinical and basic science interests, and presented data using a number of proteomic platforms to search for discriminant biomarkers of disease in easily accessible bodily fluids including serum and urine. Several potential pitfalls for proteomic researchers were mentioned and the potential of collaborative networks between research institutions to increase the size and power of clinical studies was discussed. Overall, the meeting highlighted the exciting opportunities that proteomic techniques offer for discovering not only diagnostic but also prognostic and mechanistic markers of a number of clinically important diseases.

Decoding serological response to Candida cell wall immunome into novel diagnostic, prognostic, and therapeutic candidates for systemic candidiasis by proteomic and bioinformatic analyses.

In an effort to bring novel diagnostic and prognostic biomarkers or even potential targets for vaccine design for systemic candidiasis (SC) into the open, a systematic proteomic approach coupled with bioinformatic analysis was used to decode the serological response to Candida wall immunome in SC patients. Serum levels of IgG antibodies against Candida wall-associated proteins (proteins secreted from protoplasts in active wall regeneration, separated by two-dimensional gel electrophoresis, and identified by mass spectrometry) were measured in 45 SC patients, 57 non-SC patients, and 61 healthy subjects by Western blotting. Two-way hierarchical clustering and principal component analysis of their serum anti-Candida wall antibody expression patterns discriminated SC patients from controls and highlighted the heterogeneity of their expression profiles. Multivariate logistic regression models demonstrated that high levels of antibodies against glucan 1,3-beta-glucosidase (Bgl2p) and the anti-wall phosphoglycerate kinase antibody seropositivity were the only independent predictors of SC. Receiver operating characteristic curve analysis revealed no difference between their combined evaluation and measurement of anti-Bgl2p antibodies alone. In a logistic regression model adjusted for known prognostic factors for mortality, SC patients with high anti-Bgl2p antibody levels or a positive anti-wall enolase antibody status, which correlated with each other, had a reduced 2-month risk of death. After controlling for each other, only the seropositivity for anti-wall enolase antibodies was an independent predictor of a lower risk of fatality, supporting that these mediated the protective effect. No association between serum anti-cytoplasmic enolase antibody levels and outcomes was established, suggesting a specific mechanism of enolase processing during wall biogenesis. We conclude that serum anti-Bgl2p antibodies are a novel accurate diagnostic biomarker for SC and that, at high levels, they may provide protection by modulating the anti-wall enolase antibody response. Furthermore serum anti-wall enolase antibodies are a new prognostic indicator for SC and confer protection against it. Bgl2p and wall-associated enolase could be valuable candidates for future vaccine development.

Clinical significance of serum antibody against neuron-specific enolase in glaucoma patients.

PURPOSE: In a recent study, we found the presence of serum autoantibody against neuron-specific enolase (NSE) in glaucoma patients. The purpose of the present paper is to investigate further the clinical significance of the presence of the serum antibody against NSE in glaucoma patients. METHODS: Serum autoantibody against NSE was examined by Western blot analysis in 143 patients with glaucoma (normal tension glaucoma [NTG], 45 cases; primary open angle glaucoma [POAG] 98 cases). Clinical characteristics including visual acuity, visual field, intraocular pressure (IOP), and optic disc features were compared between the serum autoantibody-positive and the serum autoantibody-negative patients. RESULTS: Maximum IOP in the serum anti-NSE antibody-positive patients was significantly lower than that in the negative patients (P <.05). However, no statistical differences were observed in visual field loss, disc cupping, or other clinical factors. During the clinical course, rates of the presence of anti-NSE antibody were significantly higher in the early stages of POAG (P <.0001) with visual field deterioration than without it. Although it was not statistically significant, the positive rates of serum anti-NSE antibody were relatively higher in the later stages of POAG and NTG with visual field deterioration than without it. CONCLUSION: The present observations suggest that the presence of serum autoantibody against NSE may be clinically useful for predicting the progression of visual field loss in POAG patients.

Skin prick test response to enzyme enolase of the baker's yeast (Saccharomyces cerevisiae) in diagnosis of respiratory allergy.

BACKGROUND: The aim of the study is to prove that Saccharomyces cerevisiae enolase, the major allergen of the baker's yeast, induces allergic immediate response in patients with inhalant allergy sensitized to Candida albicans extract. MATERIAL AND METHODS: The study was performed in three groups of patients: I. 20 atopic patients with respiratory allergy sensitized to Candida albicans and inhalant allergens (mite, feather, pollens) II. 30 patients with respiratory allergy, positive skin tests to inhalant allergens but negative skin tests to Candida albicans and other fungi; III. 20 nonatopic, healthy individuals. Skin prick test of purified enolase from Saccharomyces cerevisiae (bakers yeast) at concentration 1 and 10 mg/ml was performed in all groups. The results were documented planimetrically. RESULTS: 95% of patients sensitized to Candida albicans extract showed positive skin reactions to Saccharomyces cerevisiae enolase, 10% of patients of group II and none of the patients of the control group had positive skin responses to enolase. The mean wheal size (mm2) in skin prick test to Candida albicans, Saccharomyces cerevisiae enolase at concentration 10 mg/ml was x = 15.17 +/- 11.08, 15.76 +/- 19.67 and at concentration 1 mg/ml 10.02 +/- 10.49, respectively. CONCLUSIONS: 1. Saccharomyces cerevisiae enolase induces an immediate allergic reaction in skin in subjects with respiratory allergy and positive skin prick test results to Candida albicans and other fungi. 2. Enolase can be an important allergenic component of the Candida albicans extract.

Retinal ganglion cells recognized by serum autoantibody against gamma-enolase found in glaucoma patients.

PURPOSE: To study pathologic roles of the presence of serum autoantibodies against retinal ganglion cells in patients with glaucoma. METHODS: Serum autoantibody reactions were detected by Western blot analysis using retinal soluble fractions in 79 patients with glaucoma (normal-tension glaucoma [NTG], 23 cases; primary open-angle glaucoma [POAG], 56 cases) and 60 age-matched healthy subjects. Clinical characteristics including visual acuity, visual field, intraocular pressure (IOP), and optic disc features were compared between the serum autoantibody-positive and -negative patients. The retinal autoantigen recognized by patients' sera was identified by a combination of in-gel digestion and Edman sequencing. RESULTS: Western blot analysis revealed that serum autoantibody against retinal 50-kDa antigen was recognized in 20 out of 79 glaucoma patients (25.3%; 14 POAG and 6 NTG patients) and 60 age-matched control subjects (11.7%), respectively. Immunocytochemistry revealed that labeling of the ganglion cell layer (GCL) by IgG from glaucoma patients (POAG: 13/56, 23.2%; NTG: 6/23, 26%) existed at a significantly higher rate than that by IgG from control subjects (2/60, 3.3%; P < 0.05). In POAG, maximum IOP in the serum antibody positive-patients was significantly lower than that in the antibody-negative patients (P < 0.05). However, no statistical differences were observed in visual field loss, disc cupping, and other clinical factors between the antibody-positive and -negative groups in POAG and NTG. In-gel digestion of the 50-kDa band in two-dimensional polyacrylamide gels and Edman sequence analysis of the high-performance liquid chromatography-purified peptides identified the 50-kDa protein as gamma-enolase. Injection of the 50-kDa IgG from glaucoma patients or anti-gamma-enolase serum into the vitreous cavity of Lewis rats caused reduction of the b-wave of the electroretinogram and TdT-dUTP terminal nick-end labeling (TUNEL)-positive staining within the GCL. CONCLUSIONS: In the current study, serum autoantibody against 50-kDa protein identified as gamma-enolase in 25% of glaucoma patients.