Publications by authors named "Luis Eduardo Coelho Andrade"

57 Publications

CTPS forms the cytoophidium in zebrafish.

Exp Cell Res 2021 Aug 12;405(2):112684. Epub 2021 Jun 12.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom. Electronic address:

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.
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http://dx.doi.org/10.1016/j.yexcr.2021.112684DOI Listing
August 2021

IMPDH forms the cytoophidium in zebrafish.

Dev Biol 2021 May 25;478:89-101. Epub 2021 May 25.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.
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http://dx.doi.org/10.1016/j.ydbio.2021.05.017DOI Listing
May 2021

Antineutrophil cytoplasmic antibody profiles differ according to type of primary sclerosing cholangitis and autoimmune hepatitis.

Clinics (Sao Paulo) 2021 5;76:e2228. Epub 2021 Feb 5.

Departamento de Gastroenterologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR.

Objectives: To determine the frequency of the antineutrophil cytoplasmic antibodies (ANCA), antiproteinase-3 and antimyeloperoxidase, in primary sclerosing cholangitis (PSC) with or without inflammatory bowel disease (IBD+ or IBD-) and in different types of autoimmune hepatitis (AIH). Additionally, to verify the agreement between ANCA patterns by indirect immunofluorescence and their antigenic specificities by ELISA.

Methods: For this study, 249 patients were enrolled (42 PSC/IBD+; 33 PSC/IBD-; 31 AIH type-1; 30 AIH type-2; 31 AIH type-3; 52 primary biliary cirrhosis; 30 healthy controls) whose serum samples were tested for ANCA autoantibodies.

Results: There were fewer female subjects in the PSC/IBD- group (p=0.034). Atypical perinuclear-ANCA was detected more frequently in PSC/IBD+ patients than in PSC/IBD- patients (p=0.005), and was significantly more frequent in type-1 (p<0.001) and type-3 AIH (p=0.012) than in type-2 AIH. Proteinase-3-ANCA was detected in 25 samples (only one with cytoplasmic-ANCA pattern), and more frequently in PSC/IBD+ than in PSC/IBD- patients (p=0.025). Myeloperoxidase-ANCA was identified in eight samples (none with the perinuclear-ANCA pattern). Among the 62 reactive samples for atypical perinuclear-ANCA, 13 had antigenic specific reactions for proteinase-3 and myeloperoxidase.

Conclusions: PSC/IBD+ differed from PSC/IBD- in terms of sex and proteinase 3-ANCA and atypical perinuclear-ANCA reactivity, the latter of which was more frequently detected in type-1 and type-3 AIH than in type-2 AIH. There was no agreement between ANCA patterns and antigenic specificities in IBD and autoimmune liver diseases, which reinforces the need for proteinase-3 and myeloperoxidase antibody testing.
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http://dx.doi.org/10.6061/clinics/2021/e2228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847259PMC
April 2021

Selective decrease in complement C2 hemolytic activity is a sensitive marker for cryoglobulinemia and active disease in hepatitis C patients.

Dig Liver Dis 2021 Jul 18;53(7):860-865. Epub 2021 Jan 18.

Rheumatology Division, Medicine Department, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Electronic address:

Background: Some HCV patients present low/non-detected C2 hemolytic activity (C2h) without apparent consumption of other Complement components (selective low/non-detected C2h).

Aim: Characterization of the immunologic/clinical basis of this phenomenon.

Methods: C2h, HCV-viral load, cryoglobulinemia and Complement components were determined in 726 HCV patients, with sequential C2h determination in 189 patients.

Results: C2h was non-detected in 15.9%, low in 16.9% and normal in 67.2% subjects and showed temporal oscillation in 30.7% of patients. Samples with selective non-detected C2h presented lower C3/C4 than those with normal C2h, but still within the normal C3/C4 range. Selective non-detected C2h was associated with higher aspartate aminotransferase (AST) (p<0.001), alanine transferase (ALT) (p = 0.03) and APRI (Aspartate aminotransferase-to-Platelet Ratio Index) (p<0.001), lower serum albumin (p = 0.01) and platelet count (p = 0.012), more individuals at pre-treatment stage, with detectable HCV-RNA p<0.001), cryoglobulinemia (p<0.001) and with HCV genotype 3 (p = 0.003). Elevated ALT, HCV genotype 3, active disease and viral load were independent predictors of low/non-detected C2h. In vitro exposure of normal serum to exogenous HCV cryoglobulins caused dose-dependent decrease in C2h.

Conclusions: Selective C2h decrease is a sensitive marker of Complement activation in HCV patients and is associated with cryoglobulinemia, active disease, elevated ALT, higher viral load, and HCV genotype 3.
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http://dx.doi.org/10.1016/j.dld.2020.12.124DOI Listing
July 2021

Diagnostic power and clinical impact of exome sequencing in a cohort of 500 patients with rare diseases.

Am J Med Genet C Semin Med Genet 2020 12 30;184(4):955-964. Epub 2020 Nov 30.

Fleury Medicina e Saúde, Grupo Fleury, São Paulo, SP, Brazil.

Rare diseases comprise a diverse group of conditions, most of which involve genetic causes. We describe the variable spectrum of findings and clinical impacts of exome sequencing (ES) in a cohort of 500 patients with rare diseases. In total, 164 primary findings were reported in 158 patients, representing an overall diagnostic yield of 31.6%. Most of the findings (61.6%) corresponded to autosomal dominant conditions, followed by autosomal recessive (25.6%) and X-linked (12.8%) conditions. These patients harbored 195 variants, among which 43.6% are novel in the literature. The rate of molecular diagnosis was considerably higher for prenatal samples (67%; 4/6), younger children (44%; 24/55), consanguinity (50%; 3/6), gastrointestinal/liver disease (44%; 16/36) and syndromic/malformative conditions (41%; 72/175). For 15.6% of the cohort patients, we observed a direct potential for the redirection of care with targeted therapy, tumor screening, medication adjustment and monitoring for disease-specific complications. Secondary findings were reported in 37 patients (7.4%). Based on cost-effectiveness studies in the literature, we speculate that the reports of secondary findings may influence an increase of 123.2 years in the life expectancy for our cohort, or 0.246 years/cohort patient. ES is a powerful method to identify the molecular bases of monogenic disorders and redirect clinical care.
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http://dx.doi.org/10.1002/ajmg.c.31860DOI Listing
December 2020

Autoantibodies against phospholipase A2 receptor in Brazilian patients with glomerular diseases.

Int Urol Nephrol 2021 Apr 31;53(4):733-738. Epub 2020 Oct 31.

Division of Nephrology, Department of Medicine of the Federal University of São Paulo (UNIFESP), São Paulo, Brazil.

Background: Recently, great progress has been made in understanding the pathogenesis of membranous nephropathy (MN) with the discovery of autoantibodies (Abs) to M-type phospholipase A2 receptor (PLA2R) in serum and in immunocomplexes deposited in glomerulus in most adult patients with primary MN.

Objective: To evaluate the diagnostic performance of anti-PLA2R in Brazilian patients with MN, as well as to verify the possible association of anti-PLA2R serum levels with disease activity.

Methods: 117 patients with glomerular diseases confirmed by renal biopsy underwent routinely clinical and laboratory evaluation (serum creatinine and albumin, 24-h proteinuria, urinalysis, tests for etiological investigation) and determination of serum anti-PLA2R by ELISA.

Results: 67.5% of the patients had MN, 9.4% focal segmental glomerulosclerosis, 7.7% lupus nephritis class V and 15.4%, other proteinuric glomerular diseases. The mean level of glomerular filtration rate (estimated by the CKD-EPI formula) was 79.43 mL/min (12.00-151.20 mL/min), 24 h proteinuria of 2.89 g (0-14.90 g), serum albumin of 3.79 g/dL (1.20-4.80 g/dL). Anti-PLA2R was detected in 27 patients, all with active MN, being 26 primary and 1 secondary MN. Sensitivity and specificity rates for the test were 60.5-94.7%, and positive (PPV) and negative (NPV) predictive values were 92.9 and 67.9%, respectively.

Conclusions: Anti-PLA2R showed high specificity and PPV for the diagnosis of primary MN in Brazilian patients. There was a strong correlation between disease activity and positive anti-PLA2R. This biomarker represents an important diagnostic tool for primary MN and may contribute to the monitoring of disease activity in such patients.
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http://dx.doi.org/10.1007/s11255-020-02682-wDOI Listing
April 2021

HLA-B*51 and its main subtypes in Brazilian patients with Behçet's disease.

Clin Exp Rheumatol 2020 Sep-Oct;38 Suppl 127(5):53-59. Epub 2020 Oct 7.

Rheumatology Division, Department of Medicine, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, and Fleury Group, Research and Development, São Paulo, Brazil.

Objectives: This study aimed to evaluate the frequency of HLA-B*51 and its subtypes in Brazilian patients with Behçet's disease (BD) and healthy controls (HC) and to assess possible associations with disease manifestations.

Methods: A cross-sectional study with sequential BD patients and HC. HLAB*51 presence was determined by sequence-specific polymerase chain reaction (SSP-PCR) and HLA-B*51 subtypes by the Sanger sequencing method.

Results: Eighty-three BD patients and 258 HC were evaluated. HLA-B*51 was found in 30.1% of DB patients and in 15.5% of HC (p=0.003). The most prevalent subtypes in DB patients were HLA-B*51:01 (60.0%), HLA-B*51:08 (20.0%), HLA-B*51:22 (8.0%), HLAB* 51:29 (8.0%) and HLA-B*51:02 (4.0%), while HLA-B*51:01 (77.5%) and HLA-B*51:55 (7.5%) were the most prevalent in HC. HLA-B*51 was less frequently found in patients with neurologic involvement (8.0% vs. 29.3%; p=0.034) while HLAB*51:01 was more observed in patients with ocular involvement (93.3% vs. 60.3%; p=0.014). No BD patient with neurologic or vascular involvement presented HLA-B*51:01. HLAB*51:08 was more frequent in patients with vascular manifestations (60.0% vs. 15.4%; p=0.012). In multivariate analysis, HLA-B*51 was an independent risk factor for BD (OR=2.410; 95%CI: 1.332-4.361; p=0.004) and HLA-B*51:08 had an independent association with vascular manifestations of BD (OR = 14.843; 95%CI: 1.550 - 142.115; p=0.019).

Conclusions: The prevalence of HLAB*51 is higher in Brazilian BD patients compared to HC, and it is a risk factor for BD. The HLA-B*51:08 subtype was independently associated with vascular manifestations of BD.
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December 2020

Rationale and design of the "Tocilizumab in patients with moderate to severe COVID-19: an open-label multicentre randomized controlled" trial (TOCIBRAS).

Rev Bras Ter Intensiva 2020 Jul-Sep;32(3):337-347

BP - A Beneficência Portuguesa de São Paulo - São Paulo (SP), Brasil.

Introduction: Pro-inflammatory markers play a significant role in the disease severity of patients with COVID-19. Thus, anti-inflammatory therapies are attractive agents for potentially combating the uncontrolled inflammatory cascade in these patients. We designed a trial testing tocilizumab versus standard of care intending to improve the outcomes by inhibiting interleukin-6, an important inflammatory mediator in COVID-19.

Methods And Analysis: This open-label multicentre randomized controlled trial will compare clinical outcomes of tocilizumab plus standard of care versus standard of care alone in patients with moderate to severe COVID-19. Two of the following four criteria are required for protocol enrolment: D-dimer > 1,000ng/mL; C reactive protein > 5mg/dL, ferritin > 300mg/dL, and lactate dehydrogenase > upper limit of normal. The primary objective will be to compare the clinical status on day 15, as measured by a 7-point ordinal scale applied in COVID-19 trials worldwide. The primary endpoint will be assessed by an ordinal logistic regression assuming proportional odds ratios adjusted for stratification variables (age and sex).

Ethics And Dissemination: The TOCIBRAS protocol was approved by local and central (national) ethical committees in Brazil following current national and international guidelines/directives. Each participating center had the study protocol approved by their institutional review boards before initiating protocol enrolment. The data derived from this trial will be published regardless of the results. If proven active, this strategy could alleviate the consequences of the inflammatory response in COVID-19 patients and improve their clinical outcomes.
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http://dx.doi.org/10.5935/0103-507X.20200060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595725PMC
October 2020

Changes in the result of antinuclear antibody immunofluorescence assay on HEp-2 cells reflect disease activity status in systemic lupus erythematosus.

Clin Chem Lab Med 2020 07;58(8):1271-1281

Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.

Background The objective of the study was to determine whether the staining pattern and titer of indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) are associated with systemic lupus erythematosus (SLE) disease activity. Methods A total of 269 consecutive patients meeting the ACR and SLICC criteria for SLE were classified into three groups according to the SLE Disease Activity Index 2000 (SLEDAI2K): Remission (SLEDAI2K = 0; n = 47); Intermediate (SLEDAI2K = 1-5; n = 111); Active (SLEDAI2K ≥ 6; n = 111). All subjects were assessed for HEp-2 IFA titer and staining pattern and nine traditional parameters of SLE disease activity. After a 6 to 12-month interval, 101 of the 269 patients were reassessed. Results HEp-2 IFA homogeneous nuclear pattern (AC-1) occurred more frequently in the Active Group compared to the Remission Group (p < 0.001). Fine speckled nuclear pattern (AC-4) tended to occur more frequently in the Remission Group compared to the Active Group (p = 0.054). Subjects with AC-1 pattern had higher SLEDAI (8.8 ± 7.6) than those with AC-4 (4.8 ± 5.2) (p < 0.001). HEp-2 IFA titer and anti-nuclear antibody by enzyme-linked immunosorbent assay (ANA-ELISA) values were lower in the Remission Group compared to the other two groups (p < 0.001). Multivariate analyses identified only ELISA anti-dsDNA as an independent variable associated with disease activity. In follow-up analysis, HEp-2 IFA titer decreased significantly in the 33 subjects with decreased disease activity (p = 0.002). Receiver operator characteristic (ROC) curve analysis for determination of disease activity showed equivalent areas under the curve (AUC) for HEp-2 IFA titer and traditional disease activity parameters. Conclusions HEp-2 IFA pattern and titer can reflect SLE disease activity and may be considered in conjunction with other laboratory and clinical parameters in the assessment of SLE disease activity.
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http://dx.doi.org/10.1515/cclm-2019-0638DOI Listing
July 2020

IMPDH-Based Cytoophidium Structures as Potential Theranostics in Cancer.

Mol Ther 2020 07 11;28(7):1557-1558. Epub 2020 Jun 11.

Department of Pathology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.

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http://dx.doi.org/10.1016/j.ymthe.2020.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335744PMC
July 2020

Correction to: V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells.

Adv Rheumatol 2020 01 6;60(1). Epub 2020 Jan 6.

Pontifícia Universidade Católica de Goiás (PUC Goiás), Escola de Ciências Médicas, Farmacêuticas e Biomédicas, Avenida Universitária 1.440, Setor Universitário, Goiânia, GO, CEP 74605-010, Brazil.

After publication of the original article [1], we were notified that there is a mistake in Fig. 2.
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http://dx.doi.org/10.1186/s42358-019-0103-7DOI Listing
January 2020

Impact of C4, C4A and C4B gene copy number variation in the susceptibility, phenotype and progression of systemic lupus erythematosus.

Adv Rheumatol 2019 08 6;59(1):36. Epub 2019 Aug 6.

Disciplina de Reumatologia, Universidade Federal de São Paulo, Rua Botucatu 740, 3o andar, São Paulo, SP, ZIP: 04023-062, Brazil.

Background: Complement component 4 (C4) gene copy number (GCN) affects the susceptibility to systemic lupus erythematosus (SLE) in different populations, however the possible phenotype significance remains to be determined. This study aimed to associate C4A, C4B and total C4 GCN and SLE, focusing on the clinical phenotype and disease progression.

Methods: C4, C4A and C4B GCN were determined by real-time PCR in 427 SLE patients and 301 healthy controls, which underwent a detailed clinical evaluation according to a pre-established protocol.

Results: The risk of developing SLE was 2.62 times higher in subjects with low total C4 GCN (< 4 copies, OR = 2.62, CI = 1.77 to 3.87, p < 0.001) and 3.59 times higher in subjects with low C4A GCN (< 2 copies; OR = 3.59, CI = 2.15 to 5.99, p < 0.001) compared to those subjects with normal or high GCN of total C4 (≥4) and C4A (≥2), respectively. An increased risk was also observed regarding low C4B GCN, albeit to a lesser degree (OR = 1.46, CI = 1.03 to 2.08, p = 0.03). Furthermore, subjects with low C4A GCN had higher permanent disease damage as assessed by the Systemic Lupus International Collaborating Clinics - Damage Index (SLICC-DI; median = 1.5, 95% CI = 1.2-1.9) than patients with normal or high copy number of C4A (median = 1.0, 95% CI = 0.8-1.1; p = 0.004). There was a negative association between low C4A GCN and serositis (p = 0.02) as well as between low C4B GCN and arthritis (p = 0.02).

Conclusions: This study confirms the association between low C4 GCN and SLE susceptibility, and originally demonstrates an association between low C4A GCN and disease severity.
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http://dx.doi.org/10.1186/s42358-019-0076-6DOI Listing
August 2019

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells.

Adv Rheumatol 2019 07 3;59(1):28. Epub 2019 Jul 3.

Pontifícia Universidade Católica de Goiás (PUC Goiás), Escola de Ciências Médicas, Farmacêuticas e Biomédicas, Avenida Universitária 1.440, Setor Universitário, Goiânia, GO, CEP, 74605-010, Brazil.

Background: The V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations ( www.anapatterns.org ). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses.

Mainbody: Twenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized.

Conclusion: The V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.
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http://dx.doi.org/10.1186/s42358-019-0069-5DOI Listing
July 2019

T regulatory cell-based therapy and renal injury repair: restoring homeostatic balance of the immune response in lupus nephritis.

Rheumatology (Oxford) 2019 11;58(11):1896-1897

Rheumatology Division, Department of Medicine, Escola Paulista de Medicina - Universidade Federal de São Paulo, São Paulo, Brazil.

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http://dx.doi.org/10.1093/rheumatology/kez210DOI Listing
November 2019

Response to 'Decision making value of nuclear dense fine speckled pattern in systemic autoimmune rheumatic disease: trick or treat?' by Deng .

Ann Rheum Dis 2020 08 15;79(8):e93. Epub 2019 May 15.

Department of Oral Biology, University of Florida, Gainesville, Florida, USA.

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http://dx.doi.org/10.1136/annrheumdis-2019-215640DOI Listing
August 2020

Response to: 'Antinuclear antibodies: mitotic patterns and their clinical associations' by Betancur and Gómez-Puerta.

Ann Rheum Dis 2020 06 29;79(6):e64. Epub 2019 Apr 29.

Rheumatology Division, Universidade Federal de Sao Paulo, Sao Paulo, Brazil.

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http://dx.doi.org/10.1136/annrheumdis-2019-215439DOI Listing
June 2020

Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective.

Ann Rheum Dis 2019 07 12;78(7):879-889. Epub 2019 Mar 12.

Department of Oral Biology, University of Florida, Gainesville, Florida, USA.

The indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive, is integrated in diagnostic and classification criteria for several systemic autoimmune diseases. However, the HEp-2 IIFA test has much more to offer: besides the titre or fluorescence intensity, it also provides fluorescence pattern(s). The latter include the nucleus and the cytoplasm of interphase cells as well as patterns associated with mitotic cells. The International Consensus on ANA Patterns (ICAP) initiative has previously reached consensus on the nomenclature and definitions of HEp-2 IIFA patterns. In the current paper, the ICAP consensus is presented on the clinical relevance of the 29 distinct HEp-2 IIFA patterns. This clinical relevance is primarily defined within the context of the suspected disease and includes recommendations for follow-up testing. The discussion includes how this information may benefit the clinicians in daily practice and how the knowledge can be used to further improve diagnostic and classification criteria.
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http://dx.doi.org/10.1136/annrheumdis-2018-214436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585284PMC
July 2019

Clinical and pathophysiologic relevance of autoantibodies in rheumatoid arthritis.

Adv Rheumatol 2019 01 17;59(1). Epub 2019 Jan 17.

Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo, Disciplina de Reumatologia, Rua Botucatu 740, 3o andar, São Paulo, SP, ZIP:04023-062, Brazil.

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disease affecting 0.5 to 1% of adults worldwide and frequently leads to joint destruction and disability. Early diagnosis and early and effective therapy may prevent joint damage and lead to better long-term results. Therefore, reliable biomarkers and outcome measures are needed. Refinement of the understanding of molecular pathways involved in disease pathogenesis have been achieved by combining knowledge on RA-associated genes, environmental factors and the presence of serological elements. The presence of autoantibodies is a distinctive feature of RA. Rheumatoid Factor and Anti-Citrullinated Protein Antibodies are the two most remarkable autoantibodies in RA and provide different clinical and pathophysiological information. They precede the onset of disease symptoms and predict a more severe disease course, indicating a pathogenetic role in RA. Therefore, they promote a more accurate prognosis and contribute for a better disease management. Several RA-associated autoantibody systems have been identified: Anti-Carbamylated Antibodies, Anti-BRAF, Anti-Acetylated, Anti-PAD4 antibodies and others. Hopefully, the characterization of a comprehensive array of novel autoantibody systems in RA will provide unique pathogenic insights of relevance for the development of diagnostic and prognostic approaches compatible with an effective personalized medicine.
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http://dx.doi.org/10.1186/s42358-018-0042-8DOI Listing
January 2019

Detection of Autoantibodies by Indirect Immunofluorescence Cytochemistry on Hep-2 Cells.

Methods Mol Biol 2019 ;1901:19-46

Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.

Indirect immunofluorescence assay (IFA) has been used for detection of autoantibodies against cellular antigens for more than 50 years. Originally using rodent tissue as substrate, the method was optimized by using the human immortal HEp-2 cell line derived from a larynx epidermal carcinoma. The HEp-2/IFA platform allows for optimal visualization of several cellular domains recognized by autoantibodies in the samples being tested. Serial dilution allows for the estimation of the concentration (titer) of the autoantibodies in the sample. Judicious analysis of the topographic distribution of the immunofluorescence (pattern) provides useful hints on the most plausible autoantigens being recognized, vis-à-vis the cognate autoantibodies. The importance of the HEp-2/IFA pattern has been recently emphasized by the International Consensus on ANA Patterns (ICAP), an initiative that established a comprehensive classification of the most relevant and prevalent HEp-2/IFA patterns (designated anti-cell (AC) patterns) and harmonized its nomenclature. The former designation "antinuclear antibody test" has been progressively replaced by the term "anti-cell antibody test," due to the recognition that the HEp-2/IFA method in fact allows the detection of autoantibodies to several cellular domains, such as the cytoplasm and mitotic apparatus.The performance of the HEp-2/IFA test is strongly influenced by several technical details, including cell culture conditions, cell fixation and permeabilization methods, choice and titration of fluorochrome-conjugated secondary antibody, use and choice of blocking solutions, washing buffers, and antifading mounting medium. The several steps of the procedure must be carefully performed in order to avoid the formation of false positive fluorescent artifacts. The quality control of the assay involves the use of serum standards for negative, low positive and strongly positive reaction in each run of the assay. In addition, every new lot or new brand of HEp-2 slides should be evaluated by using a panel of standard sera yielding the most relevant AC patterns. Special attention should be dedicated to the training of personnel for the analysis of the slides at the microscope. These should be able to identify possible artifacts, recognize all relevant AC patterns, and formulate possible reflex tests according to the observed AC patterns.
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http://dx.doi.org/10.1007/978-1-4939-8949-2_3DOI Listing
June 2019

IMP/GTP balance modulates cytoophidium assembly and IMPDH activity.

Cell Div 2018 15;13. Epub 2018 Jun 15.

1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT UK.

Background: Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis, plays an important role in cell metabolism and proliferation. It has been demonstrated that IMPDH can aggregate into a macrostructure, termed the cytoophidium, in mammalian cells under a variety of conditions. However, the regulation and function of the cytoophidium are still elusive.

Results: In this study, we report that spontaneous filamentation of IMPDH is correlated with rapid cell proliferation. Intracellular IMP accumulation promoted cytoophidium assembly, whereas elevated GTP level triggered disassociation of aggregates. By using IMPDH2 CBS domain mutant cell models, which are unable to form the cytoophidium, we have determined that the cytoophidium is of the utmost importance for maintaining the GTP pool and normal cell proliferation in the condition that higher IMPDH activity is required.

Conclusions: Together, our results suggest a novel mechanism whereby cytoophidium assembly upregulates IMPDH activity and mediates guanine nucleotide homeostasis.
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http://dx.doi.org/10.1186/s13008-018-0038-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004095PMC
June 2018

Unending story of the indirect immunofluorescence assay on HEp-2 cells: old problems and new solutions?

Ann Rheum Dis 2019 06 17;78(6):e46. Epub 2018 Apr 17.

Department of Medicine and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.

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http://dx.doi.org/10.1136/annrheumdis-2018-213440DOI Listing
June 2019

Soluble CD40L is associated with increased oxidative burst and neutrophil extracellular trap release in Behçet's disease.

Arthritis Res Ther 2017 Oct 19;19(1):235. Epub 2017 Oct 19.

Division of Rheumatology, Escola Paulista de Medicina, Federal University of Sao Paulo, Rua Botucatu 740, 3° Andar, 04023-062, Sao Paulo, SP, Brazil.

Background: Studies have suggested that soluble factors in plasma from patients with active (aBD) and inactive (iBD) Behçet's disease (BD) stimulate neutrophil function. Soluble CD40 ligand (sCD40L) is an important mediator of inflammation in BD. Its expression and effect on neutrophil oxidative burst and neutrophil extracellular trap (NET) release have not been characterized. In this study, we sought to investigate the role of plasma and the CD40L pathway on NET release and the oxidative burst profile in patients with aBD and iBD.

Methods: Neutrophils and peripheral blood mononuclear cells (PBMCs) were obtained from patients with aBD (n = 30), patients with iBD (n = 31), and healthy control subjects (HCs; n = 30). sCD40L plasma concentration was determined in individual samples. A pool of plasma for each group was created. In some experiments, plasma pools were treated with recombinant CD40 (rhCD40-muIg) for sCD40L blockade. NET release and HO/O production were determined after stimulation with phorbol 12-myristate 13-acetate, sCD40L, or plasma pool. Flow cytometric analysis was performed to evaluate the expression of (1) CD40, Mac-1, and phosphorylated NF-κB p65 on neutrophils and monocytes and (2) CD40L on activated T cells and platelets. CD40L gene expression in PBMCs was determined by qRT-PCR.

Results: sCD40L plasma levels were significantly higher in patients with iBD (median 17,234, range 2346-19,279 pg/ml) and patients with aBD (median 18,289, range 413-19,883 pg/ml) than in HCs (median 47.5, range 33.7-26.7 pg/ml; p < 0.001). NET release was constitutively increased in BD compared with HC. NET release and HO/O were higher after stimulation with sCD40L or BD plasma and decreased after sCD40L blockade. Mac-1 expression was constitutively increased in neutrophils of patients with aBD (88.7 ± 13.2% of cells) and patients with iBD (89.2 ± 20.1% of cells) compared with HC (27.1 ± 18.8% of cells; p < 0.01). CD40 expression on phagocytes and CD40L expression on platelets were similar in the three groups. PBMCs as well as nonactivated and activated CD4 T cells from patients with BD showed higher CD40L expression.

Conclusions: Plasma from patients with aBD exerts a stimulus on NET release and oxidative burst, probably induced by sCD40L.
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http://dx.doi.org/10.1186/s13075-017-1443-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649058PMC
October 2017

Foundations, facts, photos and Facebook.

Rev Bras Reumatol Engl Ed 2017;57 Suppl 2:414-420. Epub 2017 Aug 10.

Sociedade Brasileira de Reumatologia, São Paulo, SP, Brazil.

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http://dx.doi.org/10.1016/j.rbre.2017.07.005DOI Listing
August 2019

Biological anti-TNF drugs: immunogenicity underlying treatment failure and adverse events.

Expert Opin Drug Metab Toxicol 2017 Sep 14;13(9):985-995. Epub 2017 Aug 14.

a Rheumatology Division, Escola Paulista de Medicina , Universidade Federal de São Paulo , Sao Paulo , Brazil.

Introduction: Genetically engineered monoclonal antibodies and fusion proteins directed against cytokines or their receptors represent a breakthrough in the treatment of various chronic immune-inflammatory diseases. Areas covered: Studies show high remission rates in several diseases, but clinical practice shows a significant percentage of individuals who do not exhibit the desired response. Loss of therapeutic benefit after initial successful response is designated secondary failure. Immune-biological agents are not self-antigens and are therefore potentially immunogenic. Secondary failure is frequently caused by antibodies against immune-biologicals, known as anti-drug antibodies (ADA). ADA that neutralize circulating immune-biologicals and/or promote their clearance can reduce treatment efficacy. Furthermore, ADA can induce adverse events by diverse immunological mechanisms. This review provides a comprehensive overview of ADA in rheumatoid arthritis patients treated with anti-TNF immune-biologicals, and explores the concept of therapeutic drug monitoring (TDM) as an effective strategy to improve therapeutic management. Expert opinion: Monitoring circulating ADA and therapeutic immune-biological drugs is helpful when evaluating patients with secondary failure. However, immunological tests for ADA vary considerably regarding their ability to detect different types of ADA. Several assays are not designed to determine ADA-induced drug neutralizing capacity, and they may report clinically non-relevant data, especially if drug is present in test samples.
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http://dx.doi.org/10.1080/17425255.2017.1360280DOI Listing
September 2017

25-Hydroxivitamin D Serum Concentration, Not Free and Bioavailable Vitamin D, Is Associated with Disease Activity in Systemic Lupus Erythematosus Patients.

PLoS One 2017 13;12(1):e0170323. Epub 2017 Jan 13.

Rheumatology Division, Universidade Federal de São Paulo/Escola Paulista de Medicina (Unifesp/ EPM), São Paulo-Brazil.

We aim to evaluate the prevalence of vitamin D deficiency in patients with systemic lupus erythematosus (SLE) and investigate the association between total, free and bioavailable vitamin D serum concentrations and disease activity. Patients with SLE (ACR 1997) consecutively seen at UNIFESP's outpatient's clinics had disease activity measured after clinical and laboratory evaluation using SLEDAI (Systemic Lupus Erythematosus Disease Activity Index). 25-hydroxyvitamin D (25(OH)D) serum concentrations measured by chemiluminescence and vitamin D binding protein (DBP) measured by ELISA were used to calculate free and bioavailable vitamin D. Healthy blood donors were used as controls. A total of 142 patients (71.4%) had 25(OH)D serum concentrations below 30 ng/mL. Total 25(OH)D serum concentration was associated with disease activity categorized in 5 continuous groups of SLEDAI. 25(OH)D serum concentrations were higher among patients with SLEDAI 1-5 and lower in those with severe activity (SLEDAI≥20) (p <0.05). On the other hand, no statistically significant difference was observed for DBP, free and bioavailable vitamin D measurements in the disease activity subgroups evaluated. Vitamin D deficiency is highly prevalent among patients with SLE and was associated with higher disease activity. DBP serum level and calculation of free and bioavailable vitamin D were not associated with SLE disease activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170323PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5234837PMC
August 2017

Fc gamma receptor IIIa polymorphism is not associated with susceptibility to systemic lupus erythematosus in Brazilian patients.

Rev Bras Reumatol Engl Ed 2016 Nov - Dec;56(6):515-520. Epub 2016 Oct 25.

Universidade Federal de São Paulo (UNIFESP), Departamento de Medicina, Disciplina de Reumatologia, São Paulo, SP, Brazil. Electronic address:

We evaluated the possible association between FCGR3A V/F (158) polymorphism and SLE susceptibility and clinical phenotype in 305 sequentially retrieved SLE patients and 300 healthy controls from the southeastern part of Brazil by allele-specific polymerase chain reaction. Our results showed no association between FCGR3A 158V/F alleles and susceptibility to SLE in this series of patients albeit the heterozygous genotype was strongly associated with the disease.
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http://dx.doi.org/10.1016/j.rbre.2016.07.013DOI Listing
October 2018

Differential capacity of therapeutic drugs to induce Rods/Rings structures in vitro and in vivo and generation of anti-Rods/Rings autoantibodies.

Clin Immunol 2016 Dec 13;173:149-156. Epub 2016 Oct 13.

Rheumatology Division, Federal University of Sao Paulo, Sao Paulo, SP 04023-062, Brazil; Immunology Division, Fleury Medicine and Health Laboratories, Sao Paulo SP 04102-050, Brazil. Electronic address:

Some HCV patients using ribavirin and interferon alpha (IFN-α) develop anti-rods and rings (RR) autoantibodies, the main target of which is inosine monophosphate dehydrogenase (IMPDH), the rate-determining enzyme in de novo GTP biosynthesis. In vitro inhibition of IMPDH by ribavirin induces RR formation. Here we investigate whether other commonly used drugs that interfere with GTP biosynthesis can induce RR structures in vitro and vivo and elicit generation of autoantibodies. HEp-2 cells treated for 24h with ribavirin, mycophenolic acid (MPA), azathioprine, methotrexate or acyclovir were positive for RR structures. However, adefovir, entecavir, tenofovir and lamivudine did not induce RR structures in these cells. Structures induced by ribavirin in HEp-2 cells are stable after 24h drug-washout, while structures induced by other drugs are relatively labile, disappearing within 2h. Looking at patients treated with these drugs, HCV patients treated with ribavirin (n=17) showed higher average percentage of RR-positive peripheral mononuclear cells than autoimmune patients treated with RR-inducing immunosuppressant drugs (n=21). Serum from 173 autoimmune patients who had been treated with MPA, azathioprine or methotrexate was tested for presence of anti-RR autoantibodies, and only one sample was found to be positive. Conversely, of 48 anti-RR autoantibody positive samples identified at Fleury Laboratories over 30months, 94% were from HCV patients treated with ribavirin plus IFN-α. These data indicate that RR structures can be induced by a variety of drugs in vitro and in vivo, but anti-RR autoantibody production is mostly restricted to HCV patients under ribavirin+IFN-α treatment.
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http://dx.doi.org/10.1016/j.clim.2016.10.004DOI Listing
December 2016

Mild and moderate Mannose Binding Lectin deficiency are associated with systemic lupus erythematosus and lupus nephritis in Brazilian patients.

Rev Bras Reumatol Engl Ed 2016 May-Jun;56(3):220-7. Epub 2016 Mar 21.

Division of Rheumatology, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil; Grupo Fleury, São Paulo, SP, Brazil. Electronic address:

Objective: The potential association of mannose binding lectin (MBL) deficiency and systemic lupus erythematosus (SLE) has been investigated in several studies, but results have been mixed. One explanation for the conflicting results could be differences in ethnic background of study subjects. In this study we investigated the association of MBL deficiency and SLE in a large cohort of Brazilian SLE patients and controls.

Methods: Serum MBL and Complement levels were determined for 286 Brazilian adult SLE patients and 301 healthy Brazilian adults as controls. MBL deficiency was classified as mild (<1000 and ≥500μg/L), moderate (<500 and ≥100μg/L) or severe (<100μg/L).

Results: SLE patients presented higher frequency of mild and moderate MBL deficiency compared to controls. SLE patients with MBL deficiency presented higher frequency of lupus nephritis compared to those without MBL deficiency. MBL deficiency was not associated with any other clinical manifestation, use of immunosuppressant therapy, disease activity, disease severity serum or Complement levels.

Conclusion: This study shows that an association between MBL deficiency and SLE does exist in the Brazilian population. We also found an association between MBL deficiency and lupus nephritis. These findings support the hypothesis that MBL deficiency contributes to the development of SLE and lupus nephritis.
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http://dx.doi.org/10.1016/j.rbre.2016.01.002DOI Listing
October 2018
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