Fleury Medicine and Health Laboratories

São Paulo, Brazil

Fleury Medicine and Health Laboratories

São Paulo, Brazil

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Pereira K.M.C.,University of Sao Paulo | Pereira K.M.C.,Fleury Medicine and Health Laboratories | Dellavance A.,University of Sao Paulo | Dellavance A.,Fleury Medicine and Health Laboratories | And 2 more authors.
Clinica Chimica Acta | Year: 2014

Background: Autoantibodies to extractable nuclear antigens (ENA) are good biomarkers for systemic autoimmune rheumatic diseases (SARD), but no one assay for the detection of these antibodies provides satisfactory sensitivity and positive predictive value (PPV). Here we evaluate current assays and propose novel strategies to detect anti-ENA antibodies. Methods: Diagnostic performance of double immunodiffusion (DID) and several enzyme immunoassays (EIA) for the detection of anti-ENA autoantibodies was determined using samples from 144 patients with a previous clinical diagnosis of SARD and 121 non-autoimmune individuals. A 2-step assay combining EIA and DID was developed and tested on 16,458 serum samples. Results: EIA was more sensitive than DID for all anti-ENA antibodies, but yielded lower PPV (mean = 66%) than DID (mean = 96%) and a higher percentage of unexpected positive results. ROC-curve guided cut-off adjustments improved PPV for most EIA kits. Using the 2-step assay, over 80% of the samples were screened out by the first step (EIA), with results available within 24. h, leaving only about 20% to be confirmed by DID. 2.9% of the 16,485 samples were found to be positive. Conclusions: A 2-step assay combining the speed and potential for automation of EIA with the high specificity and PPV of DID allows efficient and reliable detection of anti-ENA antibodies. Alternatively, improved PPV can be achieved by adjusting cut-off values for EIA assay results. © 2014 Elsevier B.V.


Keppeke G.D.,University of Sao Paulo | John Calise S.,University of Florida | Chan E.K.L.,University of Florida | Andrade L.E.C.,University of Sao Paulo | Andrade L.E.C.,Fleury Medicine and Health Laboratories
World Journal of Gastroenterology | Year: 2016

Chronic inflammation associated with hepatitis C virus (HCV) infection can lead to disabling liver diseases with progression to liver cirrhosis and hepatocellular carcinoma. Despite the recent availability of more effective and less toxic therapeutic options, in most parts of the world the standard treatment consists of a weekly injection of pegylated interferon α (IFN-α) together with a daily dose of ribavirin. HCV patients frequently present circulating non-organ-specific autoantibodies demonstrating a variety of staining patterns in the indirect immunofluorescence assay for antinuclear antibodies (ANA). Between 20% to 40% of HCV patients treated with IFN-α and ribavirin develop autoantibodies showing a peculiar ANA pattern characterized as rods and rings (RR) structures. The aim of this article is to review the recent reports regarding RR structures and anti-rods/rings (anti-RR) autoantibody production by HCV patients after IFN-α/ribavirin treatment. Anti-RR autoantibodies first appear around the sixth month of treatment and reach a plateau around the twelfth month. After treatment completion, anti-RR titers decrease/disappear in half the patients and remain steady in the other half. Some studies have observed a higher frequency of anti-RR antibodies in relapsers, i.e. , patients in which circulating virus reappears after initially successful therapy. The main target of anti-RR autoantibodies in HCV patients is inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme involved in the guanosine triphosphate biosynthesis pathway. Ribavirin is a direct IMPDH2 inhibitor and is able to induce the formation of RR structures in vitro and in vivo . In conclusion, these observations led to the hypothesis that anti-RR autoantibody production is a human model of immunologic tolerance breakdown that allows us to explore the humoral autoimmune response from the beginning of the putative triggering event: exposure to ribavirin and interferon. © The Author(s) 2016.


Damoiseaux J.,Maastricht University | Andrade L.E.,University of Sao Paulo | Andrade L.E.,Fleury Medicine and Health Laboratories | Fritzler M.J.,University of Calgary | Shoenfeld Y.,The Zabludowicz Center for Autoimmune Diseases
Autoimmunity Reviews | Year: 2015

At the 12th International Workshop on Autoantibodies and Autoimmunity (IWAA), organized in August 2014 in Sao Paulo, Brazil, more than 300 autoimmunologists gathered to discuss the status of many novel autoantibodies in clinical practice, and to envisage additional value of autoantibodies in terms of prediction, prognosis and prevention of autoimmune diseases. Two separate workshops were dedicated to standardization and harmonization of autoantibody testing and nomenclature: International Autoantibody Standardization (IAS) and International Consensus on ANA Patterns (ICAP). It was apparent to all in attendance that the discovery and elucidation of novel autoantibodies did not slow down, but that multiple challenges lay ahead of us in order to apply these discoveries to effective and efficient clinical practice. Importantly, this requires optimal bidirectional communication between clinicians and laboratory specialists, as well as close collaboration with the diagnostic industry. This paper is a report on the 12th IWAA in combination with a review of the recent developments in the field of autoantibodies. © 2015 Elsevier B.V.


John Calise S.,University of Florida | Keppeke G.D.,University of Sao Paulo | Andrade L.E.C.,University of Sao Paulo | Andrade L.E.C.,Fleury Medicine and Health Laboratories | Chan E.K.L.,University of Florida
Frontiers in Immunology | Year: 2015

In recent years, autoantibodies targeting subcellular structures described as the rods and rings pattern in HEp-2 ANA have been presented as a unique case of autoantibody generation. These rod and ring structures (RR) are at least partially composed of inosine monophosphate dehydrogenase type 2 (IMPDH2), and their formation can be induced in vitro by several small-molecule inhibitors, including some IMPDH2 inhibitors. Autoantibodies targeting these relatively unknown structures have been almost exclusively observed in hepatitis C virus (HCV) patients who have undergone treatment with pegylated interferon-α/ribavirin (IFN/RBV) combination therapy. To date, anti-RR antibodies have not been found in treatment-naïve HCV patients or in patients from any other disease groups, with few reported exceptions. Here, we describe recent advances in characterizing the RR structure and the strong association between anti-RR antibody response and HCV patients treated with IFN/RBV, detailing why anti-RR can be considered a human model of drug-induced autoantibody generation. © 2015 Calise, Keppeke, Andrade and Chan.


Keppeke G.D.,University of Sao Paulo | Nunes E.,University of Sao Paulo | Ferraz M.L.G.,University of Sao Paulo | Silva E.A.B.,University of Sao Paulo | And 5 more authors.
PLoS ONE | Year: 2012

Background: A novel pattern in the indirect immunofluorescence antinuclear antibody assay on HEp-2 cells (IIF-HEp-2) characterized by cytoplasmic rods and rings (RR) was reported in HCV patients, but stringent disease specificity studies and longitudinal analysis are lacking. We investigated the clinical significance of anti-RR in an HCV cohort with up to a 12-month treatment follow up. Methodology/Results: 597 patients (342 HCV, 55 HCV/HIV, 200 non-HCV) were screened and titered for anti-RR. Serial samples were available from 78 of 176 treated and 27 of 166 untreated patients. Anti-RR was detected in 14.1% of 342 HCV patients, 9.1% of 55 HCV/HIV, 3.4% of 29 Hepatitis B, and none of 171 non-HCV (p<0.0001; HCV versus non-HCV). Anti-RR was present in 38% of 108 patients receiving interferon-α/ribavirin, but none in 26 receiving either interferon-α or ribavirin, or 166 untreated patients (p<0.0001). Other IIF-HEp-2 patterns were more frequently associated with interferon-α treatment alone (52.2%) as compared to interferon-α/ribavirin (25%), ribavirin alone (33.3%), and no therapy (26.5%). Anti-RR frequency was not associated with sex, age, ethnicity, HCV genotype or viral load. Anti-RR occurred only after initiation of treatment, beginning as early as 1 month (6%), but by the sixth month >47% tested positive for anti-RR. The anti-RR titer generally increased with sustained treatment and remained high in 53% of patients. After treatment, anti-RR titer was negative in 41%. Non-responders to HCV therapy were 77% in anti-RR-positive versus 64% in anti-RR-negative patients. Response to treatment was not associated with anti-RR titer or the dynamics of anti-RR reactivity during and after treatment. Conclusions: The exquisite association of anti-RR reactivity with combined interferon-α/ribavirin therapy in HCV patients represents a unique model for drug-induced autoantibody generation in humans as demonstrated by the fact that a significant fraction of patients who have anti-RR during therapy becomes anti-RR-negative after completion of therapy. © 2012 Keppeke et al.


Keppeke G.D.,University of Sao Paulo | Keppeke G.D.,University of Florida | Satoh M.,Health Science University | Satoh M.,University of Florida | And 4 more authors.
Immunologic Research | Year: 2014

Autoantibodies to inosine monophosphate dehydrogenase-2 (IMPDH2), an enzyme involved in de novo biosynthesis of guanine nucleotides, are observed in a subset of hepatitis C virus (HCV) patients receiving interferon alpha (IFN-α) plus ribavirin. Anti-IMPDH2 antibodies display a peculiar cytoplasmic “rod/ring” (RR) pattern in IIF-HEp-2. We examined the dynamics of anti-RR autoimmune response with respect to immunoglobulin isotypes, titer, avidity, and protein targets in 80 sequential samples from 15 HCV patients (plus 12 randomly selected anti-RR-positive, totalizing 92 samples) collected over an 18-month period, including samples collected before, during, and after IFN-α + ribavirin treatment. Immunoprecipitation showed reactivity with the 55 kDa IMPDH2 protein in 12/15 patients (80 %) and 11/15 (73 %) reacted with IMPDH2 in a sandwich ELISA. During treatment, anti-IMPDH2 autoantibodies hit their highest levels after 6–12 months of treatment and decreased post-treatment, while anti-HCV antibodies levels were stable over time. Anti-IMPDH2 IgM levels increased up until the sixth month of treatment and remained stable thereafter, while IgG levels increased steadily up to the twelfth month. Both IgG and IgM decreased during the post-treatment period. IgG avidity increased steadily up to the twelfth month of treatment. In conclusion, this study showed that the temporal kinetics of IFN-α + ribavirin-induced humoral autoimmune response to IMPDH2 exhibited a considerably delayed pace of increase in antibody levels and avidity as well as in isotype class switch in comparison with a conventional humoral response to infectious agents. These unique findings uncover intriguing differences between the autoimmune response and the immune response to exogenous agents in humans. © 2014, Springer Science+Business Media New York.


Keppeke G.D.,Federal University of São Paulo | Keppeke G.D.,University of Florida | Calise S.J.,University of Florida | Chan E.K.L.,University of Florida | And 2 more authors.
Journal of Genetics and Genomics | Year: 2015

Inhibition of guanosine triphosphate (GTP) and cytidine triphosphate (CTP) biosynthetic pathways induces cells to assemble rod/ring (RR) structures, also named cytoophidia, which consist of the enzymes cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures. HeLa and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine (DON), ribavirin, or mycophenolic acid (MPA). Over 90% of DON-treated cells presented RR structures. In HeLa cells, 35% of the RR structures were positive for IMPDH2 alone, 26% were CTPS alone, and 31% were IMPDH2/CTPS mixed, while in COS-7 cells, 42% of RR were IMPDH2 alone, 41% were CTPS alone, and 10% were IMPDH2/CTPS mixed. Ribavirin and MPA treatments induced only IMPDH2-based RR. Cells were also transfected with an N-terminal hemagglutinin (NHA)-tagged CTPS1 construct. Over 95% of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100% presented CTPS1-based RR; when treated with ribavirin, over 94% of transfected cells presented IMPDH2-based RR and 37% presented CTPS1-based RR, whereas 2% of untreated transfected cells presented IMPDH2-based RR and 28% presented CTPS1-based RR. These results may help in understanding the relationship between CTP and GTP biosynthetic pathways, especially concerning the formation of filamentous RR structures. © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China.


Perazzio S.F.,UNIFESP EPM | Salomao R.,Immunology and Virology Laboratory | Silva N.P.,UNIFESP EPM | Andrade L.E.C.,UNIFESP EPM | Andrade L.E.C.,Fleury Medicine and Health Laboratories
Lupus | Year: 2012

Introduction: There is increased frequency of discoid lesions (2.7%) and SLE (0.5%) in patients with chronic granulomatosus disease, but the literature is still controversial about phagocyte oxidative burst in SLE patients. Materials and methods: 300 SLE patients and 301 blood donors were evaluated for quantitation of the oxidative burst in phagocytes by flow cytometry based on the oxidation of 2,7-dichlorofluorescein-diacetate after stimuli with Staphylococcus aureus and Pseudomonas aeruginosa. Results: Neutrophils from SLE patients displayed higher basal reactive oxygen species (ROS) production than healthy controls [Mean of fluorescence intensity (MFI) = 53.77 ± 11.38 vs 15.08 ± 2.63, p < 0.001] and after stimulation with S. aureus (MFI = 355.46 ± 58.55 vs 151.92 ± 28.25, p < 0.001) or P. aeruginosa (MFI = 82.53 ± 10.1 vs 48.99 ± 6.74, p < 0.001). There was stronger neutrophil response after bacterial stimuli (ΔMFI) in SLE patients than in healthy controls (S. aureus = 301.69 ± 54.42 vs 118.38 ± 26.03, p < 0.001; P. aeruginosa = 28.76 ± 12.3 vs 15.45 ± 5.15, p < 0.001), but no difference with respect to the oxidative burst profile according to disease activity (SLEDAI ≥ 6) or severity (SLICC-DI ≥2). Patients with kidney involvement presented higher basal and stimulated ROS production in neutrophils. Discussion: The present findings corroborate the important role of innate immunity in SLE and implicate neutrophils in the pathophysiology of the disease. © 2012 The Author(s).


Dellavance A.,Fleury Medicine and Health Laboratories | Coelho Andrade L.E.,Federal University of São Paulo
Lupus | Year: 2014

Autoantibodies are valuable markers for the recognition of autoimmune diseases. Over the last 25 years, several investigators have consistently shown that autoantibodies precede the clinical onset of cognate diseases by years or decades. This phenomenon, regularly observed in the natural history of autoimmune diseases, indicates that autoimmunity develops through successive stages across a variable period of time until the characteristic manifestations of disease are clinically apparent. Recent evidence indicates that the pre-clinical stages of autoimmune diseases involve a series of immunologic derangements and that this process is dynamic and progressive. During the years preceding clinical disease onset, there is progressive intensification in the humoral autoimmune response, characterized by increases in autoantibody titer, avidity, number of immunoglobulin isotypes, and spread of epitopes and of autoantigens targeted. This scenario is reminiscent of cancer processes that develop slowly by means of progressive stages, and may be interrupted by early detection and therapeutic intervention. Therefore, it might be reasoned that early intervention may be more effective in reverting the less firmly established autoimmune abnormalities at the pre-clinical stage of autoimmunity. With the continuous progress in novel immunologic therapeutic strategies, one can envision the possibility that early intervention at pre-clinical stages may lead to prevention of overt disease development and even cure of the autoimmune disorder. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.


Sheldon J.,St Georges Hospital | Dellavance A.,Fleury Medicine and Health Laboratories
Frontiers in Immunology | Year: 2015

Producing robust, certified, traceable reference material for autoantibody testing is a vital element in maintaining the validity of results that are generated in the daily clinical laboratory routine. This is a huge challenge because of the high number of variables involved in the detection and measurement of the autoantibodies. The production of such materials is time consuming and needs rigorous attention to detail; this is best achieved by an overarching independent body who will oversee the process in a "not for profit" manner. Much effort has been made to build international standards for quantitative and qualitative assays based on monoclonal antibodies, obtained from affinity purification and plasmapheresis. The big challenge is to respect individual differences in immune response to the same antigen. A promising ongoing initiative is the construction of pools with monospecific samples from different individuals. © 2015 Sheldon and Dellavance.

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