Serological Electrodetection of Rheumatoid Arthritis Using Mimetic Peptide.

BACKGROUND: Rheumatoid arthritis is the most common inflammatory autoimmune disease in the world. Recently new targets for its detection were developed as alternatives to classic biomarkers, including the M-12 peptide, that mimics carbonic anhydrase III. Thus, the application of this peptide for the development of new detection devices is attractive. OBJECTIVE: Our goal was to construct a modified electrode for immobilization of M-12 peptide and detection of a rheumatoid arthritis biomarker in serum of patients. METHODS: 3-Hydroxybenzoic acid was electropolymerized onto graphite electrodes, and M-12 peptide was immobilized by adsorption. Negative and positive serum samples for rheumatoid arthritis were diluted and applied onto the electrode. Detection was carried in potassium ferrocyanide/ ferricyanide solution by differential pulse voltammetry. Atomic force microscopy and scanning electron microscopy were used to evaluate electrode surfaces. RESULTS: Cyclic voltammograms indicated the poly(3-hydroxybenzoic acid) formation and increase of electroactive area. Immobilization of M-12 probe increased current by 1.2 times, and negative serum addition caused no suitable difference. However, positive serum showed expressive decrease in the current signal of about 2.2 times, possibly due to steric hindrance when the anti-CA3 antibody interacts with the M-12 peptide, decreasing the electron transfer. Microscopies images corroborated with the electrochemical detection, showing evident changes in the morphology of the electrode surfaces. CONCLUSION: The bioelectrode was able to discriminate positive and negative serum samples of rheumatoid arthritis by a considerable decrease in the current signal value. Morphological analyses supported the electrochemical results. Thus, the constructed bioelectrode offers a new platform for detection of rheumatoid arthritis.

Assessment of the mutagenic, recombinogenic and carcinogenic potential of fipronil insecticide in somatic cells of Drosophila melanogaster.

Fipronil (FP) is an insecticide that belongs to the phenylpyrazole chemical family and is used to control pests by blocking GABA receptor at the entrance channel of the chlorine neurons. The aim of this study was to evaluate the mutagenic, recombinogenic and carcinogenic potential of FP. The mutagenic and recombinogenic effects were evaluated using the somatic mutation and recombination test (SMART) on wing cells of Drosophila melanogaster. Third instar larvae from standard (ST) and high bioactivation (HB) crosses were treated with different concentrations of FP (0.3, 0.7, 1.5 or 3.0 × 10-5 mM). The results showed mutagenic effects at all concentrations tested in the HB cross; and all concentrations tested in the ST cross, except at concentration of 0.7 × 10-5 mM. The carcinogenic effect of FP was assayed through the test for detection of epithelial tumor (warts) in D. melanogaster. Third instar larvae from wts/TM3 virgin females mated to mwh/mwh males were treated with different concentrations of FP (0.3, 0.7, 1.5 or 3.0 × 10-5 mM). All these concentrations induced a statistically significant increase in tumor frequency. In conclusion, FP proved to be mutagenic, recombinogenic and carcinogenic in somatic cells of D. melanogaster.

Association of vitamin D receptor variants with clinical parameters in prostate cancer.

PURPOSE: Prostate Cancer (PCa) is one of the most common cancers in men and its early detection can provide a high chance of cure. The detection of Vitamin D Receptor (VDR) gene polymorphisms may be useful as a molecular indicator of clinical outcome, once VDR is implicated in a wide variety of biological processes including modulation of the immune response and inhibition of cancer cell growth, angiogenesis and metastasis. In this study we explored the Single Nucleotide Polymorphisms (SNPs) FokI, BsmI, ApaI and TaqI, to evaluate the susceptibility locus for PCa and verify its correlation with clinical parameters. METHODS: VDR polymorphisms were detected by PCR followed by Restriction Fragment Length Polymorphism (PCR-RFLP). DNA samples were extracted from peripheral blood of 342 patients: 132 PCa, 41 Benign Prostatic Hyperplasia and 169 young healthy volunteers. RESULTS: Statistical analysis showed a noteworthy correlation among SNPs and clinical pathological features. CC genotype (TaqI) was correlated with the age at diagnosis (>58 years old), and GG (BsmI) was associated to lower Prostate-Specific Antigen (PSA) levels (<10 ng/mL). Moreover, when PCa patients were subgrouped, G allele (BsmI) significantly increased the estimated chance for PSA < 10 ng/mL, and GG/GG genotype (BsmI/ApaI) provided a 9.75 fold increased chance of patients with PCa to present lower PSA levels. CONCLUSIONS: The polymorphisms of VDR gene showed a genotype-phenotype association and presented new correlations with different parameters as age and PSA levels.

Assessment of water quality and genotoxic impact by toxic metals in Geophagus brasiliensis.

This study used the pearl cichlid Geophagus brasiliensis as a bioindicator to survey the health of the aquatic environment on four sites (P1, P2, P3 and P4) of the Mumbuca stream located at Monte Carmelo/MG, Brazil. The selection of different sites was made with reference to the gradient of urban activity and via physicochemical and biological evaluation of water quality and genotoxicity. The water quality index was classified as 'good' for P1 and P4, regular in P2 and 'poor' for P3. The micronuclei (MN) frequency obtained from blood analysis was in agreement with the water quality, such that the higher values of MN were detected in sites evaluated as poor. Water degradation conditions worsen according to the flow of the stream over the sites P1, P2 and P3, but for site P4, located after the Monte Carmelo Sewage Treatment Plant, improvements in the micronuclei frequency are detected. Our results showed high levels of potentially toxic metals (chromium, lead, aluminum and nickel) in specific stream sites (P2 and P3). We suggest that the micronuclei induction in G. brasiliensis could be due to the presence of these compounds.

Improved serological detection of rheumatoid arthritis: a highly antigenic mimotope of carbonic anhydrase III selected in a murine model by phage display.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1% of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis. METHODS: Sera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope. RESULTS: After three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases. CONCLUSION: M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.