Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate

The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.

Follicular helper T cell in immunity and autoimmunity

The traditional concept that effector T helper (Th) responses are mediated by Th1/Th2 cell subtypes has been broadened by the recent demonstration of two new effector T helper cells, the IL-17 producing cells (Th17) and the follicular helper T cells (Tfh). These new subsets have many features in common, such as the ability to produce IL-21 and to express the IL-23 receptor (IL23R), the inducible co-stimulatory molecule ICOS, and the transcription factor c-Maf, all of them essential for expansion and establishment of the final pool of both subsets. Tfh cells differ from Th17 by their ability to home to B cell areas in secondary lymphoid tissue through interactions mediated by the chemokine receptor CXCR5 and its ligand CXCL13. These CXCR5+ CD4+ T cells are considered an effector T cell type specialized in B cell help, with a transcriptional profile distinct from Th1 and Th2 cells. The role of Tfh cells and its primary product, IL-21, on B-cell activation and differentiation is essential for humoral immunity against infectious agents. However, when deregulated, Tfh cells could represent an important mechanism contributing to exacerbated humoral response and autoantibody production in autoimmune diseases. This review highlights the importance of Tfh cells by focusing on their biology and differentiation processes in the context of normal immune response to infectious microorganisms and their role in the pathogenesis of autoimmune diseases.

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25+/highCD127Ø/lowFoxP3+, and effector T cells were defined as CD25+CD127+FoxP3Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4+TREG and CD28+TREG cells and an increased frequency of CD40L+TREG cells. There was a decrease in the TREG/effector-T ratio for GITR+, HLA-DR+, OX40+, and CD45RO+ cells, and an increased ratio of TREG/effector-T CD40L+ cells in patients with SLE. In addition, CD40L+TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.

Autoimmune diseases in the TH17 era: [review]

A new subtype of CD4+ T lymphocytes characterized by the production of interleukin 17, i.e., TH17 cells, has been recently described. This novel T cell subset is distinct from type 1 and type 2 T helper cells. The major feature of this subpopulation is to generate significant amounts of pro-inflammatory cytokines, therefore appearing to be critically involved in protection against infection caused by extracellular microorganisms, and in the pathogenesis of autoimmune diseases and allergy. The dynamic balance among subsets of T cells is important for the modulation of several steps of the immune response. Disturbances in this balance may cause a shift from normal immunologic physiology to the development of immune-mediated disorders. In autoimmune diseases, the fine balance between the proportion and degree of activation of the various T lymphocyte subsets can contribute to persistent undesirable inflammatory responses and tissue replacement by fibrosis. This review highlights the importance of TH17 cells in this process by providing an update on the biology of these cells and focusing on their biology and differentiation processes in the context of immune-mediated chronic inflammatory diseases.

Antibodies to citrullinated peptides are not associated with the rate of joint destruction in patients with a well-established diagnosis of rheumatoid arthritis

Antibodies to citrullinated peptides are highly specific for rheumatoid arthritis (RA) and represent a significant risk factor for undifferentiated polyarthritis. This prognostic ability may be related to the very diagnostic performance of these autoantibodies, since RA is a more erosive disease than other forms of arthritis. The present study evaluated an association of antibodies to citrullinated peptides and the rate of joint destruction in patients with a well-established diagnosis of RA. Seventy-one patients with RA were evaluated in 1994 and again in 2002 (functional class, joint count, Health Assessment Questionnaire score, hands X-ray). Autoantibodies (rheumatoid factor (RF), anti-perinuclear factor, anti-cyclic citrullinated peptide (CCP) antibodies) and Sharp's index were analyzed blindly. Delta Sharp was calculated as the difference in Sharp's index obtained in 1994 and 2002. During the follow-up the Health Assessment Questionnaire score increased from 0.91 ± 0.74 to 1.39 ± 0.72 (P < 0.001). Similarly, the number of swollen joints increased from 4.6 ± 5.71 to 6.4 ± 4.1 (P = 0.002). The frequency of autoantibodies and anti-CCP titer remained stable; however, serum RF concentration increased from 202.8 ± 357.6 to 416.6 ± 636.5 IU/mL (P = 0.003). Sharp's index increased from 56.7 ± 62.1 to 92.4 ± 80.9 (P < 0.001). No correlation was observed between Delta Sharp and the presence of RF, anti-perinuclear factor, and anti-CCP antibodies at baseline. Antibodies to citrullinated epitopes are specific and early markers for the diagnosis of RA but do not seem to be associated with the rate of joint destruction in patients with a well-established diagnosis of RA.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis

E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 ± 39.94). There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P < 0.035). This correlation was even stronger within the first 48 months of diagnosis (r = 0.63, P < 0.048). On the other hand, no association was observed between selectin and ectasia. Patients with diffuse disease presented higher serum E-selectin levels than patients with limited disease, although the difference was not statistically significant (96.44 ± 48.04 vs 63.56 ± 21.77 ng/dl; P = 0.08). The present study is the first showing a correlation between soluble serum E-selectin levels and alterations in capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Long-term thalidomide use in refractory cutaneous lesions of systemic lupus erythematosus

Objective. To evaluate the efficacy of long-term thalidomide treatment in cutaneous lesions os systemic lupus erythematosus (SLE), not responsive to conventional therapy. Patients and Methods. Were selected 18 SLE patients (ACR criteria) with active cutaneous lesions not responsive to chloroquine, photoprotectors and low doses prednisone and who presented good response to thalidomide but relapsed after withdrawal of the drug. All female patients had no risk of pregnancy. Thalidomide was reintroduced and maintained at low dose (25-100 mg/day) for a minimum of 6 months. Results. Eighteen patients (16 females) with mean age of 34.2yo (16-57y.o.) received thalidomide for 6-21 months (mean 8.5m). The mean dose of prednisone at beginning of study was 38.3 mg/d and at the end was 9.7 mg/d (p<0.05). Complete remission of cutaneous lesions was observed in thirteen patients (72 per cent) and partial remission in five (28 per cent). Side effects observed were: drowsiness in eight patients, intestinal constipation in 5, transient oliguria in 1, paresthesia of hand with normal electromyography in another one. All side effects disappeared with reduction of thalidomide dose and no patient needed to stop treatment owing to side effect. Conclusion. Thalidomide is a good alternative therapy to SLE patients with refractory cutaneous lesions and without any risk of pregnancy.

Osteoartropatia hipertrófica associada a polipose colônica esquistossomótica / Hypertrophic osteoarthropathy associated with schistosomal colonic polyposis

A possibilidade de que doenças parasitárias intestinais ocasionem manifestações reumáticas está documentada por relatos de casos referentes a giardíase, teníase, amebíase e estrongilodíase. Também a esquistossomose parece poder associar-se a manifestações reumáticas. Descrevemos caso de paciente com forma pseudotumoral de esquistossomose mansônica, caracterizada por polipose colônica, que desenvolveu quadro de osteoartropatia hipertrófica. A inexistência de alterações pulmonares neste caso sugere que a osteoartropatia tenha sido secundária às alterações intestinais. Outro aspecto interessante foi a presença de anticorpos anticitoplasma de neutrófilos (ANCA).

Determinaçäo dos níveis séricos de ácido úrico em uma amostra populacional da regiäo de Marília-SP e suas correlaçöes com algumas variáveis demográficas e bioquímicas / Serum uric acid levels in a populational sample in Marília county - Säo Paulo State and correlation with some biochemical and demographic parameters

Este trabalho é um estudo epidemiológico que procura estabelecer correlaçöes entre a concentraçäo sérica de ácido úrico e outros fatores, como localizaçäo geográfica, etnia, idade, sexo e obesidade. Foram estudados 958 indivíduos da regiäo da Alta Paulista, no Estado de Säo Paulo, Brasil, submetidos a anamnese, exames clínicos, reumatológico e laboratoriais. Encontraram-se níveis de ácido úrico sérico significantemente mais elevados na populaçäo urbana quando comparados com a populaçäo rural. Nas duas populaçöes, tanto urbana quanto rural, os níveis séricos de ácido úrico foram significantemente mais elevados no grupo masculino que no feminino. Näo houve correlaçäo entre níveis séricos de ácido úrico e idade, embora entre as mulheres se observasse aumento significante da urecemia a partir dos 40 anos. Näo houve diferença quanto à urecemia entre as raças branca e näo branca. Indivíduos obesos apresentaram níveis maiores de urecemia em relaçäo aos näo obesos, embora essa diferença tenha sido estatisticamente significante apenas na amostra de origem urbana. Näo houve correlaçäo entre os níveis séricos de ácido úrico e trigliceridemia, colesterolemia e glicemia

Significado clínico dos anticorpos antinucleares na artrite reumatóide juvenil / Clinical significance of antinuclear antibodies in juvenile rheumatoid arthritis

Os anticorpos antinucleares (AAN) têm sido demonstrados em freqüência extremamente variável na artrite reumatóide juvenil (ARJ), dependendo do substrato empregado e da seleçäo dos pacientes. Além disso, a associaçÝo entre estes anticorpos e a atividade ou a gravidade da doença näo está bem estabelecida. Objetivo: Determinar a freqüência dos AAN e possível associaçöes com diferentes parâmetros clínicos da ARJ, como duraçäo, atividade e gravidade da doença. Material e métodos: 86 pacientes com ARJ foram estudados quanto à presença de AAN (pesquisados pelo método de imunofluorescência indireta em células HEp-2); 32 pacientes com lúpus eritematoso sistêmico juvenil e 52 crianças saudáveis constituíram os grupos-controles. A atividade da doença foi definida segundo uma escala de dois pontos, enquanto a gravidade foi avaliada pelo grau de capacidade funcional. Resultados: AAN foram encontrados em 36 (42 por cento) pacientes com ARJ, sendo mais prevalentes no tipo de início oligoarticular do que no sistêmico (p<0,05). Uma associaçäo com a duraçäo, atividade ou a gravidade da doença näo foi evidenciada (p>0,05), embora uma tendência tenha sido observada em pacientes do tipo de início poliarticular, em atividade de doença (P=0,054). O padräo pontilhado fino foi o mais freqëntemente observado (64 por cento), conquanto outros padröes, como o centrossômico e o do corpo intermediário, também tivessem sido notados. Conclusäo: Anticorpos antinucleares foram encontrados em menor freqüência que aquela descrita na maioria dos trabalhos da literatura para a ARJ, nÝo apresentando associaçäo com o sexo, duraçäo, atividade ou gravidade de doença. Embora o padräo de fluorescência mais freqüente tenha sido o pontilhado, a presença de padröes inusitados de fluorescência nuclear sugere que AAN na ARJ possam apresentar especificidades ainda näo definidas, que poderäo futuramente ser melhor caracterizadas

Epidermal dendritic cells in murine lupus

O lúpus eritematoso sistêmico (LES) é uma síndrome auto-imune multissistêmica. O acometimento cutâneo é freqüente e constitui manifestaçao clínica importante. O polimorfismo das lesoes cutâneas presume o envolvimento de diversos fatores etiopatogênicos. As células de Langerhans epidérmicas (CLs) têm funçoes imunorregulatórias importantes e participam de um modo decisivo na fisiopatologia de várias doenças dermatológicas inflamatórias, existindo evidência de seu envolvimento da patogênese da lesao cutânea lúpica. O camundongo F1 (NZB x NZW) um dos mais completos modelos experimentais de LES, nao desenvolve lesoes de pele. A ausência de dados na literatura referentes as CLs no camundongo F1 (NZB x NZW) motivou-lhes a realizar o presente estudo, que demonstrou, de forma original, a presença de CLs na epiderme do modelo F1, tanto por técnica citoquímica quanto por microscopia eletrônica de transmissao. Utilizando a técnica citoquímica da ATPase, estudamos as CLs no camundongo F1 (NZB x NZW) antes e após irradiaçao com luz ultravioleta B (UVB). As CLs no modelo F1 apresentaram-se em maior número e com alteraçoes morfológicas (dendritos curtos e menos ramificados) quando comparadas com os animais dos grupos-controles (BALB/c e C57BL/6). A irradiaçao UVB induziu alteraçoes morfológicas e reduçao no número das CLs do camundongo F1 em extensao semelhante ao observado no camundongo BALC/c. Nossos resultados sugerem possível alteraçao funcional nas CLs do camundongo F1 (NZB x NZW) e fornecem evidências de que estas células nao apresentam maior sensibilidade à luz UVB em comparaçao às de outras raças de camundongos. Estudos adicionais sao necessários para se testar possível associaçao entre essas alteraçoes fenotípicas das CLs e a ausência de manifestaçoes cutâneas nos camundongos F1 (NZB x NZW)

Development of an enzyme linked immunosorbent assay (ELISA) for detection of anti-native DNA antibodies using DNA from "Crithidia luciliae" as substrate

DNA de cinetoplasto de Crithidia luciliae (kDNA) foi isolado e purificado para desenvolvimento de ensaio imunoenzimático para detecçao de anticorpos anti-DNA nativo. O kDNA foi obtido por lise das células com dodecil sulfato de sódio (SDS) e pronase E e purificado por ultracentrifugaçao sobre soluçao de sacarose a 20 por cento e extraçao protéica. A pureza do kDNA foi verificada pela razao de densidades ópticas a 260 e 280nm e por eletroforese em gel de agarose, após digestao com as enzimas de restriçao HindIII e HaeIII. DNA de timo de vitelo, DNA plasmidial bacteriano e DNA cromossômico de Micrococcus lysodeikticus foram também empregados como substrato para ELISA. Foram testados 158 soros de pacientes atendidos no ambulatório da Disciplina de Reumatologia da Escola Paulista de Medicina, UNIFESP, sendo 65 com lúpus eritematoso sistêmico (LES), 30 com artrite reumatóide, 27 com esclerose sistêmica e 36 com outras doenças reumáticas auto-imunes. Foram também testados 105 soros obtidos de pacientes que estavam sendo investigados quanto a apresentarem LES e 30 soros de controles sadios. Nossos resultados mostraram que o kDNA pode ser purificado e utilizado com sucesso como substrato alternativo em ELISA. Entretanto, o mesmo nao ocorre com DNA cromossômico obtido de M. lysodeikticus, mostrando a necessidade de avaliaçao cuidadosa da fonte de DNA para detecçao de anticorpos anti-DNA, em testes imunoenzimáticos