Clinical Significance of Antibodies to DFS70 in Immunoinflammatory Rheumatic Diseases.

The relevance of the problem of immunoinflammatory rheumatic diseases (IIRD) for modern medicine is determined by their high prevalence in the population, the difficulty of early diagnosis, the rapid development of disability and poor life prognosis. Recent data on the significance of anti-DFS70 have opened up new possibilities for optimizing the step-by-step diagnosis of IIRD. The detection of these antibodies can help in the interpretation of a positive result for antinuclear antibodies (ANA) by indirect immunofluorescence assay on HEp-2 cells (IIFA-HEp-2) in the absence of autoantibodies specific for IIRD. Detection of anti-DFS70 in antinuclear factor (ANF) seropositive patients without clinical and/or serological markers characteristic of a certain disease from the IIRD group can be considered as a potential marker that excludes this group of diseases.

A Rare Post-Infectious Autoimmune Manifestation of COVID-19.

The incidence of post-infectious autoimmune diseases has been on the rise following the COVID-19 pandemic. Recently, an autistic patient was admitted to the hospital presenting with a mild upper respiratory system COVID-19 infection. Months after recovery and polymerase chain reaction negativity, the patient developed HEp-2 cell positivity and presented with relapsing polychondritis (RP), a rare autoimmune disease. The mechanism of this autoimmune invasion is ultimately caused by activating a myriad of immune reactions. Lymphocytopenia almost always accompanies various clinical forms of COVID-19; however, it may drive the lymphocytopenia-induced proliferation of autoreactive T cells via the activation of interleukin-6 (IL-6). Moreover, high levels of neutrophils during infection promote autoimmune disease by releasing cytokine and chemokine cascades that accompany inflammation, and neutrophil extracellular traps regulating immune responses through cell-cell interactions. Furthermore, autism spectrum disorder patients display an altered immune system that includes an augmented inflammatory cytokine milieu leading to an increased pro-inflammatory Th1/Th2 ratio. In addition, the pathophysiology of RP is majorly associated with a cell-mediated immune reaction; thus, the predisposing exaggerated immune system of such patients must also be considered as a predisposing factor to the development of post-infectious autoimmune diseases. LEARNING POINTS: COVID-19 infection is a potential trigger for relapsing polychondritis, an autoimmune disease affecting cartilage, and must be considered as a rare post-COVID complication.The hyperactive immune system in autism spectrum disorder (ASD) is an important predisposing factor to the induction of more autoimmune diseases after the occurrence of post-infectious dysregulation.Lymphocytopenia-induced proliferation possibly initiates the post-infection immune dysregulation.

Citalopram, an antipsychotic agent, induces G1/G0 phase cell cycle arrest and promotes apoptosis in human laryngeal carcinoma HEP-2 cells.

Human laryngeal squamous carcinoma (LSCC) is a common malignant tumor in the head and neck. Despite the recently developed therapies for the treatment of LSCC, patients' overall survival rate still did not enhance remarkably; this highlights the need to formulate alternative strategies to develop novel treatments. The antitumor effects of antidepressant drugs such as citalopram have been reported on several cancer cells; however, they have yet to be investigated against LSCC. The current study was directed to explore the possible antitumor effects of citalopram on human laryngeal carcinoma cell lines (HEP-2). HEP-2 cells were cultured and treated with different doses of citalopram (50-400 µM) for 24, 48, and 72 h. The effects of citalopram on the viability of cancer cells were determined by the MTT assay. In addition, apoptosis and cell cycle analysis were performed by flow cytometry. Moreover, evaluation of the expression of proapoptotic and apoptotic proteins, such as cytochrome c, cleaved caspases 3 and 9, Bcl-2, and BAX, was performed by western blotting analysis. Our results revealed that citalopram significantly suppressed the proliferation of HEP-2 cells through the upregulation of p21 expression, resulting in the subsequent arrest of the cell cycle at the G0/G1 phase. Furthermore, citalopram treatment-induced HEP-2 cell apoptosis; this was indicated by the significant increase of cytochrome c, cleaved caspases 3 and 9, and BAX protein expression. On the contrary, Bcl-2 protein expression was significantly downregulated following treatment with citalopram. The ultrastructure studies were in accordance with the protein expression findings and showed clear signs of apoptosis with ring chromatin condensation upon treatment with citalopram. These findings suggest that citalopram's anti-tumor activities on HEP-2 cells entailed stimulation of the intrinsic apoptotic pathway, which was mediated via Bcl-2 suppression.

Complex patterns on HEp-2 indirect immunofluorescence assay in a large sample referred for anti-cell autoantibodies detection.

Introduction: The combination of patterns is a frequent and challenging situation in the daily laboratory routine of autoantibodies testing using HEp-2 cells indirect immunofluorescence assay (HEp-2-IFA). Recently, the Brazilian Consensus on Autoantibodies (BCA) named these combinations as complex patterns (CPs) and organized them into 3 subtypes: multiple, mixed, and composite. This study aimed to describe the most frequent combinations of HEp-2-IIF patterns according to this new nomenclature. Methods: Routine HEp-2-IFA results reported in January and June 2017 were reviewed using the new BCA classification. Visual pattern recognition was performed by experts on HEp-2-IFA readings, using the International Consensus on Antinuclear Antibodies (ANA) Patterns (ICAP) and BCA recommendations. Results: 54,990 serum samples from different patients were tested for ANA-HEp-2, and 11,478 (20.9%) were positive at a titer ≥ 1/80. Among these positive samples, 1,111 (9.7%) displayed CPs, divided into 95 different combinations. A higher proportion of CPs was observed in the pediatric age group. Multiple, mixed, and composite patterns were present in 85.3, 5.4, and 9.5% of the samples, respectively. In the multiple/mixed pattern group (n=1,005), double, triple, and quadruple combinations (ICAP/BCA codes) were observed in 97.7%, 2.2%, and 0.1%, respectively. The double nuclear pattern was the most prevalent combination observed (67.6%). The most common CPs registered were AC-4 (nuclear fine speckled) + AC-6,7 (nuclear discrete dots) (n=264); AC-2 (nuclear dense fine speckled) + AC-6,7 (n=201); AC-4+AC-8,9,10 (nucleolar) (n=129); and AC-3 (centromere)+AC-4 (n=124). All of these combinations were in the multiple subgroup. Conclusion: Almost 10% of positive results in the HEp-2 procedure displayed CPs. Among the 3 subtypes of CPs proposed, the multiple pattern was the most prevalent, especially in the pediatric population. The AC-4, AC-2, and AC-6,7 were the most prevalent single patterns observed in the combinations described in this study. There was a significant association between age and the prevalence of most combined patterns. The AC-4+AC-6,7 combination was the most prevalent complex pattern detected regardless of the age group. The AC-2+AC-6,7 was more prevalent in younger individuals. The concepts involved in the CPs definition should add value to the reading and interpretation of the HEp-2-IIF assay.

The influence of demography and referral medical specialty on the detection of autoantibodies to HEP-2 cells in a large sample of patients

Abstract Background: The prevalence of anti-cell autoantibodies detected by indirect immunofluorescence assay on HEp-2 cells (HEp-2-IIFA) increases with age and is higher in female sex. The number of medical specialties that use HEp-2-IIFA in the investigation of autoimmune diseases has increased lately. This study aimed to determine the prevalence and patterns of autoantibodies on HEp-2-IIFA according to demographics variables and referring medical specialties. Methods: A retrospective analysis of the HEp-2-IIFA carried out between January and June of 2017 was performed. The International Consensus on Antinuclear Antibodies Patterns (ICAP) and the Brazilian Consensus on Autoantibodies were used for patterns definition on visual reading of the slides. Anti-cell (AC) codes from ICAP and Brazilian AC codes (BAC) were used for patterns classification. Results: From 54,990 samples referred for HEp-2-IIF testing, 20.9% were positive at titer ≥ 1/80. HEp-2-IIFA positivity in females and males was 24% and 12%, respectively ( p < 0.0001). The proportion of positive results in the 4 age groups analyzed: 0-19, 20-39, 40-59, and ≥ 60 years was 23.3, 20.2, 20.1, and 22.8%, respectively ( p < 0.0001). Considering all positive sera (n = 11,478), AC-4 nuclear fine speckled (37.7%), AC-2 nuclear dense fine speckled (21.3%), BAC-3 nuclear quasi -homogeneous (10%) and mixed/composite patterns (8.8%) were the most prevalent patterns. The specialties that most requested HEp-2-IIFA were general practitioner (20.1%), dermatology (15%), gynecology (9.9%), rheumatology (8.5%), and cardiology (5.8%). HEp-2-IIFA positivity was higher in patients referred by rheumatologists (35.7% vs. 19.6%) ( p < 0.0001). Moderate (46.4%) and high (10.8%) titers were more observed in patients referred by rheumatologists ( p < 0.0001). We observed a high proportion of mixed and cytoplasmic patterns in samples referred by oncologists and a high proportion of BAC-3 (nuclear quasi -homogeneous) pattern in samples referred by pneumologists. Conclusions: One-fifth of the patients studied were HEp-2-IIFA-positive. The age groups with more positive results were 0-19 and ≥ 60 years. AC-4, AC-2, BAC-3 and mixed/composite patterns were the most frequent patterns observed. Rheumatologists requested only 8.5% of HEp-2-IIFA. Positive results and moderate to high titers of autoanti-bodies were more frequent in patients referred by rheumatologists.

Diagnosis and treatment of HEp-2 cells contaminated with mycoplasma / Diagnóstico e tratamento de células HEp-2 contaminadas por micoplasma

Abstract Contamination of primary and cell cultures by mycoplasmas is one of the main economic and biological pitfalls in basic research, diagnosis and manufacture of biotechnological products. It is a common issue which may be difficult to conduct surveillance on. Mycoplasma presence may affect several physiological parameters of the cell, besides being considered an important source of inaccurate and/or non-reproducible scientific results. Each cell type presents characteristical symptoms, mainly morphological, that indicate a contamination by mycoplasma. HEp-2 cells originate from carcinoma of the larynx and are, therefore, part of the respiratory tract, which is one of mycoplasma habitats. Despite the importance these cells in several biological research (evaluation of cell proliferation and migration, apoptosis, antiviral and antitumor compounds), the alterations induced by mycoplasma contamination in HEp-2 cells have not yet been described. Here, we describe the progressive morphological alterations in culture of HEp-2 cells infected with mycoplasma, as well as the-diagnosis of the infection and its treatment. Mycoplasma contamination described within this work led to cytoplasm elongation, cell-to-cell spacing, thin plasma membrane projections, cytoplasmic vacuoles, fusion with neighboring cells, and, finally, cell death. Contamination was detected by fluorescence imaging (DAPI) and PCR reactions. The cultures were treated with BM-Cyclin antibiotic to eliminate contamination. The data presented here will be of relevance to researchers whose investigations involve cell culture, especially respiratory and HEp-2 cells.

Resumo A contaminação de culturas primárias e celulares por micoplasmas é uma das principais armadilhas econômicas e biológicas da pesquisa básica, diagnóstico e fabricação de produtos biotecnológicos. Trata-se de uma contaminação rotineira, mas de difícil acompanhamento. A presença de micoplasma pode afetar vários parâmetros fisiológicos da célula, além de ser considerada uma importante fonte de resultados científicos imprecisos e/ou não reprodutíveis. Cada tipo de célula apresenta sintomas característicos, principalmente morfológicos, que indicam uma contaminação por micoplasma. As células HEp-2 são originárias do carcinoma da laringe e, portanto, fazem parte do trato respiratório, um dos habitats do micoplasma. Apesar da importância destas células em diversas pesquisas biológicas (avaliação da proliferação e migração celular, apoptose, compostos antivirais e antitumorais), as alterações decorrentes da contaminação por micoplasma nestas células ainda não foi descrita. Aqui, descrevemos as alterações morfológicas progressivas na cultura de células HEp-2 infectadas por micoplasma, bem como o diagnóstico da infecção e seu tratamento. A contaminação por micoplasma descrita neste trabalho resultou em alongamento citoplasmático, espaçamento entre células, projeções delgadas da membrana plasmática, vacúolos citoplasmáticos, fusão de células vizinhas e, finalmente, morte celular. A contaminação foi detectada por imagens de fluorescência (DAPI) e reações de PCR. As culturas foram tratadas com antibiótico BM-Cyclin para eliminar a contaminação. Os dados aqui apresentados serão de relevância para pesquisadores cujas investigações envolvem cultura celular, principalmente células respiratórias e HEp-2.

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

Abstract 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.

Diagnosis and treatment of HEp-2 cells contaminated with mycoplasma

Abstract Contamination of primary and cell cultures by mycoplasmas is one of the main economic and biological pitfalls in basic research, diagnosis and manufacture of biotechnological products. It is a common issue which may be difficult to conduct surveillance on. Mycoplasma presence may affect several physiological parameters of the cell, besides being considered an important source of inaccurate and/or non-reproducible scientific results. Each cell type presents characteristical symptoms, mainly morphological, that indicate a contamination by mycoplasma. HEp-2 cells originate from carcinoma of the larynx and are, therefore, part of the respiratory tract, which is one of mycoplasma habitats. Despite the importance these cells in several biological research (evaluation of cell proliferation and migration, apoptosis, antiviral and antitumor compounds), the alterations induced by mycoplasma contamination in HEp-2 cells have not yet been described. Here, we describe the progressive morphological alterations in culture of HEp-2 cells infected with mycoplasma, as well as the-diagnosis of the infection and its treatment. Mycoplasma contamination described within this work led to cytoplasm elongation, cell-to-cell spacing, thin plasma membrane projections, cytoplasmic vacuoles, fusion with neighboring cells, and, finally, cell death. Contamination was detected by fluorescence imaging (DAPI) and PCR reactions. The cultures were treated with BM-Cyclin antibiotic to eliminate contamination. The data presented here will be of relevance to researchers whose investigations involve cell culture, especially respiratory and HEp-2 cells.

Resumo A contaminação de culturas primárias e celulares por micoplasmas é uma das principais armadilhas econômicas e biológicas da pesquisa básica, diagnóstico e fabricação de produtos biotecnológicos. Trata-se de uma contaminação rotineira, mas de difícil acompanhamento. A presença de micoplasma pode afetar vários parâmetros fisiológicos da célula, além de ser considerada uma importante fonte de resultados científicos imprecisos e/ou não reprodutíveis. Cada tipo de célula apresenta sintomas característicos, principalmente morfológicos, que indicam uma contaminação por micoplasma. As células HEp-2 são originárias do carcinoma da laringe e, portanto, fazem parte do trato respiratório, um dos habitats do micoplasma. Apesar da importância destas células em diversas pesquisas biológicas (avaliação da proliferação e migração celular, apoptose, compostos antivirais e antitumorais), as alterações decorrentes da contaminação por micoplasma nestas células ainda não foi descrita. Aqui, descrevemos as alterações morfológicas progressivas na cultura de células HEp-2 infectadas por micoplasma, bem como o diagnóstico da infecção e seu tratamento. A contaminação por micoplasma descrita neste trabalho resultou em alongamento citoplasmático, espaçamento entre células, projeções delgadas da membrana plasmática, vacúolos citoplasmáticos, fusão de células vizinhas e, finalmente, morte celular. A contaminação foi detectada por imagens de fluorescência (DAPI) e reações de PCR. As culturas foram tratadas com antibiótico BM-Cyclin para eliminar a contaminação. Os dados aqui apresentados serão de relevância para pesquisadores cujas investigações envolvem cultura celular, principalmente células respiratórias e HEp-2.

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO3^2-) is toxic to most microorganisms, TeO3^2--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO3^2--resistance (Te^R) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative Te^R determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO3^2- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegansand the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the Te^R and pathogenic potential of C. diphtheriae.

Comparación del cultivo celular de HeLa y HEp-2: Perspectivas de estudios con Chlamydia trachomatis / Comparison of cell culture HeLa and HEp-2: Studies prospects Chlamydia trachomatis

Objetivo. Estandarizar el cultivo de células HeLa en diferentes condiciones, con el fin de utilizarlo en protocolos de infección con Chlamydia trachomatis serovar L2. Materiales y métodos. Este estudio se llevó a cabo en cuatro fases principales que son: 1.Viabilidad celular por la técnica de azul de tripán y su posterior observación, 2. Estandarización del cultivo de células HeLa, 3. Coloración de Giemsa, 4. Cultivo de células HEp-2.Resultados. Se determinó que la línea celular HeLa debe ser cultivada en medio DMEM, 0,1% de L- glutamina, 10% de SFB. Así mismo, que la coloración de Giemsa es mejor realizarla en un tiempo de 40 minutos por que se evidencia una clara definición de núcleo y citoplasma. Frente a la comparación de las dos líneas celulares se obtuvo que la línea HeLa desde el primer día muestra un crecimiento adecuado y alcanza rápidamente la confluencia esperada, en contraposición la línea HEp-2 presenta un crecimiento más lento pero alcanzando la confluencia deseada al último día.

Objective. The goal of this study was to standardize the cultivation of HeLa cells under different conditions, to be used in Chlamydia trachomatis serovar L2 infection's protocols. Materials and Methods. This study was conducted in four phases that are: 1.Cell Viabilidad by trypan blue technique and his subsequent remark, 2. Standardization HeLa cell culture, 3. Giemsa, 4. Cultivation of Hep-2 cells. Results. As a result of standardization it is determined that the HeLa cell line should be cultured in DMEM, 0.1% L-glutamine, 10% FBS.It was determined that the Giemsa perform better over time of 40 minutes that a clear definition of nucleus and cytoplasm is evident. Comparing against both cell lines HeLa was obtained that the line from day growing well and quickly reaches the expected confluence, the opposed line HEp-2 has a slower growth but achieving the desired confluence the last day.

Apoptosis in HEp-2 cells infected with Ureaplasma diversum

BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location ofUreaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversuminvasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

IV Consenso Brasileiro para pesquisa de autoanticorpos em celulas HEp-2 / IV Brazilian Guidelines for autoantibodies on HEp-2 cells

Objetivo: O IV Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 (FAN) realizado em Vitória (ES), no dia 18 de setembro de 2012, objetivou discutir estratégias e recomendações relacionadas ao procedimento técnico, à padronização e à interpretação dos resultados da pesquisa de autoanticorpos em células HEp-2. Métodos: Participaram do evento 23 pesquisadores e especialistas de Universidades e laboratórios brasileiros. Foram abordados diferentes tópicos, discutidos amplamente a fim de se estabelecer recomendações específicas. Resultados e conclusão: O IV Consenso integrou à árvore de decisão o padrão citoplasmático em Anéis e Bastões, o padrão nuclear pontilhado Quasi-homogêneo (QH) e o padrão misto CENP-F. Discutiu-se ainda a necessidade de atenção para a classificação do padrão misto relacionado à presença de anticorpos anti-DNA topoisomerase I (Scl70), compreendendo os componentes nuclear pontilhado fino, nucleolar homogêneo, NOR na placa metafásica e citoplasmático pontilhado fino. Foram sugeridas diretrizes para o controle de qualidade do teste, diluição de triagem e diluição de esgotamento, e foi emitido alerta quanto à necessidade de atenção em relação à heterogeneidade de substratos disponíveis no mercado e a utilização de metodologias automatizadas para detecção de autoanticorpos. .

Objective: The Fourth Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells (ANA) was held in Vitória, Espírito Santo, and aimed to discuss strategies and recommendations about the technique, standardization, interpretation and quality control of the indirect immunofluorescence reaction on HEp-2 cells. Methods: Twenty three ANA experts from university centers and private laboratories in different areas from Brazil discussed and agreed upon recommendations for the fourth edition of the Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells. Results and conclusion: The 4th ANA Consensus included three novel patterns into the existing algorithm (cytoplasmic Rods and Rings, nuclear Quasi-homogeneous, and CENP-F). Emphasis was given to the need of attention in describing the peculiar mixed pattern elicited by anti-DNA topoisomerase I (Scl-70) autoantibodies, comprising nuclear fine specked, nucleolar homogeneous pattern, NOR staining in metaphase plates, and cytoplasmic fine speckled patterns. The group also emphasized the need for continuous quality control in indirect immunofluorescence assays, the establishment of screening dilutions, as well as conjugate titration. An alert was made regarding the heterogeneity of commercial kits in defining patterns and the use of solid phase methodologies to determine the presence of autoantibodies. .

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

Frecuencia de los patrones de anticuerpos anti-nucleares en pacientes con sospecha clínica de LES / Frequency of antinuclear antibody patterns in patients with SLE clinical suspicion

Las enfermedades autoinmunes son alteraciones de los mecanismos de tolerancia inmunológica, con producción de anticuerpos contra antígenos propios, como los anticuerpos antinucleares (ANA), que detectados por inmunofluorescencia indirecta,permiten reconocer varios patrones nucleares y citoplasmáticos, como en el Lupus Eritematoso Sistémico (LES). El objetivo del estudio fue describir los patrones de tinciónde ANA en sueros de pacientes con sospecha clínica de LES, que concurrieron al Instituto de Investigaciones en Ciencias de la Salud, durante el año 2008. En este estudio observacional descriptivo, se analizaron 150 sueros positivos para ANA, codificados,respetando la confidencialidad de los mismos. Para la determinación de ANA se utilizaron láminas con células HEp-2 (BIO-RAD-USA), y el conjugado fue anti human IgG de Sigma(USA); la técnica fue inmunofluorescencia indirecta. Del total de 150 pacientes estudiados, 134 (89%) fueron mujeres y 16 (11%) hombres, cuyas edades estaban comprendidas entre 11 y 87 años. De los sueros positivos analizados para ANA, 44 (29%)correspondieron al patrón periférico, 18(12%) puntillado grueso, 39(26%) puntillado fino,42(28%) homogéneo, 5(3%) nucleolar y 2(1.3%) citoplásmico puntillado fino. En este estudio se encontró en mayor porcentaje el patrón periférico, seguido del homogéneo y puntillado fino, los cuales se hallan asociados a pacientes con LES, indicando un pronóstico poco favorable para estos pacientes.

Autoimmune diseases are alterations of the immunological tolerance mechanisms, with production of antibodies against self-antigens, such as antinuclear antibodies (ANA), which are detected by indirect immunofluorescence, and allow the recognition of several nuclear and cytoplasmic patterns, as shown in the systemic lupus erythematosus (SLE).The objective of this study was to describe the ANA staining patterns in sera of patients with clinical suspicion of SLE that attended the Instituto de Investigaciones en Ciencias de la Salud during 2008. In this descriptive observational study, 150 coded positive ANA sera were analyzed, respecting the confidentiality of patients. ANA determination was made by indirect immunofluorescence using slides with HEp-2 cells (BIO-RAD-USA) and aantihuman-IgG-fluorescein conjugate from Sigma (USA). Of the 150 studied patients, 134 (89%) were females and 16 (11%) males with ages between 11 and 87 years. Of the ANA positive sera analyzed, 44 (29%) showed peripheral patterns, 18 (12%) coarse speckled patterns, 39 (26%) fine speckled patterns, 42 (28%) homogeneous patterns, 5 (3%) nucleolar patterns and 2 (1.3%) fine speckled cytoplasmic patterns. In this study, the peripheral pattern was found in greater percentage, followed by homogeneous and fine speckled patterns, which are associated to patients with SLE, indicating an unfavorable prognosis for these patients.

Avaliação clínico-laboratorial de pacientes com síndrome antifosfolípide primária segundo a frequência de anticorpos antinucleares (FAN Hep-2) / Clinical and laboratory evaluation of patients with primary antiphospholipid syndrome according to the frequency of antinuclear antibodies (ANA Hep-2)

OBJETIVO: Avaliar a frequência de manifestações clínicas e laboratoriais em pacientes com síndrome antifosfolípide primária (SAFP) com anticorpos antinucleares positivos (FAN Hep-2+), comparados àqueles com esses anticorpos negativos (FAN Hep-2 -). PACIENTES E MÉTODOS: Estudo transversal em 58 pacientes (82,8 por cento mulheres) com SAFP. Foram avaliados os dados demográficos, clínicos, comorbidades, medicações e anticorpos antifosfolípides. RESULTADOS: Dos 58 pacientes incluídos no estudo, vinte (34,5 por cento) apresentaram presença de FAN Hep-2. Comparando-se o grupo de pacientes FAN Hep-2+ com aqueles FAN Hep-2 -, verificou-se que ambos os grupos de pacientes com SAFP não diferiram estatisticamente em relação aos dados demográficos, bem como em relação ao tempo de doença. Em relação às manifestações clínicas e laboratoriais, o grupo com FAN Hep-2 + apresentou maior frequência de trombose venosa profunda (85 versus 52,6 por cento, P = 0,04), uma frequência estatística e significativamente maior de anticardiolipina IgG (85 versus 52,6 por cento, P = 0,02) e uma tendência para anticardiolipina IgM (80 por cento versus 52,6 por cento, P = 0,05), bem como maiores medianas desses anticorpos [33 (0-128) versus 20 (0-120) GPL, P = 0,008] e [33 (0-120) versus 18,5 (0-120) MPL, P = 0,009]. Tal diferença não foi observada no que se refere a outras manifestações da SAF, presença de comorbidades, estilo de vida e uso de medicações. CONCLUSÃO: Pacientes com SAFP que apresentam FAN Hep-2+ têm maior frequência de trombose venosa profunda e anticardiolipinas IgG e IgM.

OBJECTIVE: To evaluate the frequency of clinical and laboratory manifestations in patients with primary antiphospholipid syndrome (PAPS) with positive antinuclear antibodies (ANA Hep-2+) compared to those in whom this antibody is negative (ANA Hep-2-). PATIENTS AND METHODS: This is a transversal study with 58 patients (82.8 percent females) with PAPS. Demographic and clinical data, comorbidities, medications, and antiphospholipid antibodies were evaluated. RESULTS: Twenty (34.5 percent) out of 58 patients were positive for ANA Hep-2. Comparing the group of patients ANA Hep-2+ with those that were ANA Hep-2-, it was observed that both groups of patients with APS did not show statistically significant differences regarding demographic data, as well as the duration of the disease. As for clinical and laboratorial manifestations, the ANA Hep-2+ group showed higher frequency of deep venous thrombosis (85 versus 52.6 percent, P = 0.04), a statistically higher frequency of anticardiolipin IgG (85 versus 52.6 percent, P = 0.02), and a tendency for anticardiolipin IgM (80 percent versus 52.6 percent, P = 0.05), as well as greater medians of those antibodies [33 (0-128) versus 20 (0-120) GPL, P = 0.008] and [33 (0-120) versus 18,5 (0-120) MPL, P = 0.009]. Such difference was not observed regarding other manifestations of APS, presence of comorbidities, lifestyle, and medications used. CONCLUSIONS: Patients with PAPS with ANA Hep-2+ have a higher frequency of deep venous thrombosis and anticardiolipin IgG and IgM.

3º Consenso Brasileiro para pesquisa de autoanticorpos em células HEp-2 (FAN): recomendações para padronização do ensaio de pesquisa de autoanticorpos em células HEp-2, controle de qualidade e associações clínicas / Third Brazilian Consensus for autoantibodies screening in HEp-2 cells (ANA): recommendations for standardization of autoantibodies screening trial in HEp-2 cells, quality control and clinical associations

OBJETIVO: O 3º Consenso Brasileiro para pesquisa de autoanticorpos em Células HEp-2 (FAN) teve como propósito avaliar as dificuldades de implantação do 2º Consenso ocorrido no ano de 2002, discutir estratégias para controlar a qualidade do ensaio e promover a atualização das associações clínicas dos diversos padrões. MÉTODOS: Participaram do encontro em Goiânia nos dias 13 e 14 de abril de 2008 pesquisadores e especialistas de diversos centros universitários e laboratórios clínicos de diferentes regiões do Brasil, com o propósito de discutir e aprovar as recomendações que visam à melhor padronização, interpretação e utilização do ensaio pelos clínicos. Representantes comerciais de diferentes empresas produtoras de insumos para realização do teste de FAN foram convidados como ouvintes. RESULTADOS E CONCLUSÕES: O 3º Consenso enfatizou a necessidade do controle de qualidade em imunofluorescência dada a heterogeneidade de microscópios e reagentes disponíveis no mercado, promoveu adequações na terminologia utilizada para classificar os diferentes padrões e, finalmente, atualizou as associações clínicas com finalidade de facilitar cada vez mais o melhor uso do ensaio pelos clínicos.

OBJECTIVE: The Third Brazilian Consensus for autoantibodies Screening in HEp-2 cells had as purpose the evaluation of difficulties in the accomplishment of the 2nd Consensus recommendations that took place in the year of 2002, the discussion of strategies for quality control of the assay and the promotion of an update of the clinical associations of the several immunofluorescent patterns. METHODS: Several ANA experts from university centers and private laboratories in different areas in Brazil joined the workshop in Goiânia on 2008 April 13 and 14 with the purpose of discussing and approving the recommendations for standardization, interpretation and use of the test by physicians. Commercial representatives of different ANA slide brands were also invited as listeners to the workshop. RESULTS AND CONCLUSIONS: The 3rd Consensus emphasized the need for quality control in indirect immunofluorescent since there is a considerable heterogeneity of available microscopes and reagents. It also promoted adaptations in the previously approved terminology used to classify the different patterns and finally updated the clinical associations of the several patterns with the purpose of providing guidance for interpretation of the assay by clinical pathologists and assistant physicians.

III Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2: perspectiva histórica, controle de qualidade e associações clínicas / Third Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells: historical perspective, quality control and clinical associations

OBJETIVO: O III Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 (FAN) objetivou discutir estratégias para controlar a qualidade do ensaio, promover a atualização das associações clínicas dos diversos padrões e avaliar as dificuldades de implantação do II Consenso ocorrido no ano de 2002. MÉTODOS: Nos dias 13 e 14 de abril de 2007 participaram do encontro em Goiânia pesquisadores e especialistas de diversos centros universitários e laboratórios clínicos de diferentes regiões do Brasil, com o propósito de discutir e aprovar as recomendações que visam a melhores padronização, interpretação e utilização do ensaio pelos clínicos. Foram convidados como ouvintes representantes comerciais de diferentes empresas produtoras de insumos para realização do teste de FAN. RESULTADOS E CONCLUSÃO: Dada a heterogeneidade de microscópios e reagentes disponíveis no mercado, o III Consenso enfatizou a necessidade do controle de qualidade em ensaios de imunofluorescência indireta. Foram também feitas algumas adequações na terminologia utilizada para classificar os diferentes padrões. Finalmente, foi realizada uma atualização das associações clínicas com finalidade de facilitar cada vez mais o melhor uso do ensaio pelos clínicos.

OBJECTIVE: The Third Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells (ANA) had as purpose the evaluation of difficulties in the accomplishment of the 2nd Consensus recommendations that took place in the year of 2002, the discussion of strategies for quality control of the assay and the discussion of an update of the clinical associations of the several immunofluorescent patterns. METHODS: Several ANA experts from university centers and private laboratories in different areas in Brazil joined the workshop in Goiânia on 2007 April 13 and 14 with the purpose of discussing and approving the recommendations for standardization, interpretation and use of the test by physicians. Commercial representatives of different ANA slide brands were also invited as listeners to the workshop. RESULTS AND CONCLUSION: The 3rd ANA Consensus emphasized the need for quality control in indirect immunofluorescent assays since there is a considerable heterogeneity of available microscopes and reagents. It also promoted adaptations in the previously approved terminology used to classify the different patterns and finally updated the clinical associations of the several patterns with the purpose of providing guidance for interpretation of the assay by clinical pathologists and assistant physicians.

Experiência da adoção do I e II Consensos Brasileiros de Fator Antinuclear por Imunofluorescência Indireta em Células HEp-2 em um hospital universitário / Experience of an university hospital on the implementation of I and II Brazilian Consensuses for Standardization of ANA in HEp-2 Cells

OBJETIVO: Analisar a prevalência, padrões e títulos do Fator Antinuclear (FAN) por imunofluorescência indireta (IFI) em células HEp-2 em um hospital universitário após a adoção do I e II Consensos Nacional para Padronização dos Laudos de FAN em Células HEp-2. MÉTODO: Foi realizado um estudo transversal, em que foram revisados os laudos de FAN por IFI originários de solicitações encaminhadas ao Serviço de Patologia Clínica do Hospital de Clínicas de Porto Alegre (SPC/HCPA) entre 2002 e 2005. RESULTADOS: Foram analisados 12.095 testes de FAN no período entre 2002 e 2005. As solicitações com resultado reagente foram de 2.577 (21,30 por cento), com média anual de 644±233). Houve um aumento significativo na proporção de resultados reagentes posterior à adoção dos Consensos (p < 0,001). A Reumatologia foi a especialidade que mais solicitou exames por paciente atendido, mas houve um declínio nesse número nos anos posteriores à adoção do Consenso, ocorrido em 2004 (p < 0,001). O padrão de imunofluorescência de FAN mais frequentemente encontrado foi o padrão nuclear pontilhado fino, presente em 52,3 por cento dos resultados reagentes (453/866), e os títulos mais encontrados foram 1/80 e 1/160 (27,8 por cento e 29,4 por cento, respectivamente). CONCLUSÃO: Após a adoção do Consenso Nacional de Padronização de Laudos de FAN, observou-se um aumento de exames com resultados reagentes, na sua maioria, com títulos baixos e padrão nuclear pontilhado fino. Na Reumatologia, observou-se uma diminuição no número de solicitações desse exame. As potenciais causas para essas observações são discutidas, mas seu real impacto sobre a situação clínica do paciente e seu tratamento merece ser mais bem estudado.

OBJECTIVE: To evaluate the prevalence of patterns and titers of antinuclear antibodies (ANA) detected by indirect immunofluorescence (IIF) technique on HEp-2 cells in a university hospital following the introduction of I and II Brazilian Consensuses for Standardization of ANA in HEp-2 Cells. METHODS:A transversal study was performed between 2002 and 2005 during which all ANA orders to Serviço de Hospital de Clínicas de Porto Alegre (SPC/HCPA) and cognate results were reviewed. RESULTS:12.095 tests of ANA were revised. The number of positive results during this period was 2.577 (21.30 percent), annual mean 644 (SD: 233). A marked increase in the number of positive results was observed following the introduction of the Consensuses (p < 0.001). Rheumatology was the medical specialty which requested the highest number of ANA testing per patient although a significant decrease of these numbers was observed after the introduction of the Consensus in 2004 (p < 0,001). Nuclear fine speckled immunofluorescence labeling was the most frequently ANA pattern observed, 52.3 percent (453/866), and low ANA titers (1/80 and 1/160) more commonly detected (27.8 percent and 29.4 percent, respectively). CONCLUSION: Following the introduction of the Brazilian Consensus for standardization of ANA in HEp-2 cells an increased number of positive results was observed, mostly in low titers and with nuclear fine speckled immunofluorescence pattern. Moreover, there were decreasing numbers of ANA orders by rheumatologists in the same period. Potential causes for these observations are discussed but the real impact in the clinical condition of the patient and therapy deserves to be better studied.

Potential pathogenic role of aggregative-adhering Corynebacterium diphtheriae of different clonal groups in endocarditis

Invasive diseases caused by Corynebacterium diphtheriae have been described increasingly. Several reports indicate the destructive feature of endocarditis attributable to nontoxigenic strains. However, few reports have dealt with the pathogenicity of invasive strains. The present investigation demonstrates a phenotypic trait that may be used to identify potentially invasive strains. The study also draws attention to clinical and microbiological aspects observed in 5 cases of endocarditis due to C. diphtheriae that occurred outside Europe. Four cases occurred in female school-age children (7-14 years) treated at different hospitals in Rio de Janeiro, Brazil. All patients developed other complications including septicemia, renal failure and/or arthritis. Surgical treatment was performed on 2 patients for valve replacement. Lethality was observed in 40 percent of the cases. Microorganisms isolated from 5 blood samples and identified as C. diphtheriae subsp mitis (N = 4) and C. diphtheriae subsp gravis (N = 1) displayed an aggregative adherence pattern to HEp-2 cells and identical one-dimensional SDS-PAGE protein profiles. Aggregative-adhering invasive strains of C. diphtheriae showed 5 distinct RAPD profiles. Despite the clonal diversity, all 5 C. diphtheriae invasive isolates seemed to display special bacterial adhesive properties that may favor blood-barrier disruption and systemic dissemination of bacteria. In conclusion, blood isolates from patients with endocarditis exhibited a unique adhering pattern, suggesting a pathogenic role of aggregative-adhering C. diphtheriae of different clones in endocarditis. Accordingly, the aggregative-adherence pattern may be used as an indication of some invasive potential of C. diphtheriae strains.