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1.
Pediatr Rheumatol Online J ; 19(1): 37, 2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33743728

RESUMO

BACKGROUND: Systemic sclerosis (SSc; scleroderma) is an autoimmune connective tissue disease that affects the skin and subcutaneous tissue, in addition to the internal organs of the whole body. Onset in childhood is uncommon; however, both patients with childhood-onset and adult-onset SSc are positive for anti-nuclear antibodies (ANAs).Detection of SSc-related anti-nuclear antibodies is often useful for predicting clinical features, disease course, and outcomes. CASE PRESENTATION: A 5-year-old Japanese female manifested gradually progressive abnormal gait disturbance, regression of motor development, Raynaud's phenomenon, and the shiny appearance of the skin of the face and extremities at age 2. On admission, she presented a mask-like appearance, loss of wrinkles and skin folds, puffy fingers, moderate diffuse scleroderma (18/51 of the modified Rodnan total skin thickness score), and contracture in the ankle and proximal interphalangeal joints. Grossly visible capillary hemorrhage on nail fold and severe abnormal capillaroscopy findings including bleeding, giant loop and disappearance of capillaryconsistent with the late phase in SSc. A skin biopsy showed fibrous thickening of the dermis, entrapment of an eccrine sweat glands, and thickened fiber. Chest high-resolution computed tomographic scanning demonstrated patchy areas of ill-defined air-space opacity and consolidation predominantly involving the posterior basilar aspects of the lower lobes presenting withinterstitial lung disease. Positive ANA (1:160 nucleolar and homogeneous nuclear staining by indirect fluorescent antibody technique) and double-seropositive for anti-Th/To and anti-PM-Scl antibodies were identified. She was diagnosed with diffuse cutaneous SSc based on the Pediatric Rheumatology European Society/American College of Rheumatology/European League Against Rheumatism Provisional Classification Criteria for Juvenile Systemic Sclerosis and was successfully treated with immunosuppressive agents, including methylprednisolone pulses and intravenous cyclophosphamide. CONCLUSIONS: We experienced the first case of juvenile SSc with anti-PM-Scl and anti-Th/To antibodies. ILD was identified as a typical feature of patients with these autoantibodies; however, diffuse cutaneous SSc and joint contraction were uncharacteristically associated. The case showed unexpected clinical findings though the existence of SSc-related autoantibodies aids in determining possible organ involvement and to estimate the children's outcome.


Assuntos
Autoanticorpos/sangue , Nucléolo Celular/imunologia , Contratura/sangue , Contratura/tratamento farmacológico , Imunossupressores/uso terapêutico , Escleroderma Sistêmico/sangue , Pré-Escolar , Contratura/etiologia , Feminino , Humanos , Escleroderma Sistêmico/complicações , Resultado do Tratamento
2.
Dokl Biochem Biophys ; 494(1): 261-265, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33119830

RESUMO

Proliferation of mammalian cells is often accompanied by an increase in the content of the nucleolar proteins, which allows researchers to consider such proteins as potential activation markers. To test this assumption experimentally, we examined the expression pattern of the nucleolar rRNA processing factor SURF6 in normal (resting) peripheral blood lymphocytes, lymphocytes activated for proliferation in vitro, and in blood samples from patients with lymphoproliferative diseases. Using two methods (immunofluorescence and immunoblotting), we for the first time showed that the SURF6 protein is not detected in normal lymphocytes but can easily be visualized in lymphocytes after PHA activation and in lymphocytes of lymphocytic leukemia patients. The level of SURF6 expression in patients correlated with the aggressiveness of the disease development determined by the content of Ki-67-positive lymphocytes. These results allow the SURF6 nucleolar protein to be considered as a putative activation marker of lymphocytes in human blood disorders.


Assuntos
Linfócitos/imunologia , Transtornos Linfoproliferativos/metabolismo , Proteínas Nucleares/biossíntese , Biomarcadores/metabolismo , Estudos de Casos e Controles , Nucléolo Celular/imunologia , Humanos , Ativação Linfocitária , Linfócitos/citologia , Transtornos Linfoproliferativos/patologia , Proteínas Nucleares/imunologia , RNA Ribossômico/metabolismo
3.
PLoS One ; 15(8): e0237109, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804939

RESUMO

INTRODUCTION: There is no evidence on the role of Human Anti Nucleolus Antibody (ANCAb) in type 2 diabetes mellitus (T2DM). We compared prevalence and concentration of ANCAb between age and a gender-matched sample of T2DM with and without diabetes-related complications. METHODS: In this study, the reaction to ANCAb was compared quantitatively between 38 T2DM patients complicated with microvascular conditions and 43 T2DM without complications as controls. RESULTS: The patients in complicated and non-complicated groups were comparable in diabetes duration (9.0 vs. 5.0 years; P = 0.065), respectively. The study found that 27 cases (71.1%) of the complicated group reacted to ANCAb test compared to 25 (58.1%) in non-complicated patients (P = 0.226; 3.53 vs. 2.72 ng/mL; P = 0.413). The reaction response to ANCAb in patients with neuropathy and cardiovascular complications was 80.0%, 76.2% in patients with neuropathy compared to 58.1% in the control group (P = 0.398). The reaction response to ANCAb in patients with mono-complication was 72.7% compared 68.8% in patients with multi-complication (P = 0.466). Similarly, 76.2% of patients with T2DM and complicated with neuropathy (n = 21 patients) reacted to ANCAb compared to 58.1% in control patients with (P = 0.158). CONCLUSIONS: Reaction to ANCAb was not statistically different between the T2DM patients with and without complications.


Assuntos
Autoanticorpos/sangue , Nucléolo Celular/imunologia , Diabetes Mellitus Tipo 2/sangue , Angiopatias Diabéticas/sangue , Neuropatias Diabéticas/sangue , Adulto , Autoanticorpos/imunologia , Biomarcadores/sangue , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Pessoa de Meia-Idade
4.
Genes (Basel) ; 11(6)2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32585969

RESUMO

The view of the nucleolus as a mere ribosomal factory has been recently expanded, highlighting its essential role in immune and stress-related signalling and orchestrating. It has been shown that the nucleolus structure, formed around nucleolus organiser regions (NORs) and attributed Cajal bodies, is prone to disassembly and reassembly correlated to various physiological and pathological stimuli. To evaluate the effect of parasite stimulus on the structure of the leukocyte nucleolus, we exposed rat peripheral blood mononuclear cells (PBMC) to the crude extract of the nematode A. pegreffii (Anisakidae), and compared the observed changes to the effect of control (RPMI-1640 media), immunosuppressive (MPA) and immunostimulant treatment (bacterial lipopolysaccharide (LPS) and viral analogue polyinosinic:polycytidylic acid (poly I:C)) by confocal microscopy. Poly I:C triggered the most accentuated changes such as nucleolar fragmentation and structural unravelling, LPS induced nucleolus thickening reminiscent of cell activation, while MPA induced disassembly of dense fibrillar and granular components. A. pegreffii crude extract triggered nucleolar segregation, expectedly more enhanced in treatment with a higher dose. This is the first evidence that leukocyte nucleoli already undergo structural changes 12 h post-parasitic stimuli, although these are likely to subside after successful cell activation.


Assuntos
Anisaquíase/imunologia , Anisakis/imunologia , Nucléolo Celular/imunologia , Região Organizadora do Nucléolo/imunologia , Animais , Anisaquíase/genética , Anisaquíase/patologia , Anisakis/patogenicidade , Nucléolo Celular/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Células Intersticiais de Cajal/efeitos dos fármacos , Células Intersticiais de Cajal/imunologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/farmacologia , Região Organizadora do Nucléolo/efeitos dos fármacos , Região Organizadora do Nucléolo/genética , Poli I-C/farmacologia
5.
Immunol Res ; 66(6): 790-799, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30515730

RESUMO

The "X chromosome-nucleolus nexus" hypothesis provides a comprehensive explanation of how autoantibodies can develop following cellular stress. The hypothesis connects autoimmune diseases with the impact of environmental factors, such as viruses, through epigenetic disruption. The inactive X chromosome, a major epigenetic structure in the female cell's nucleus, is a key component of the hypothesis. The inactive X is vulnerable to disruption due to the following: (1) its heavy requirements for methylation to suppress gene expression, (2) its peripheral location at the nuclear envelope, (3) its late replication timing, and (4) its frequently observed close association with the nucleolus. The dynamic nucleolus can expand dramatically in response to cellular stress and this could disrupt the neighboring inactive X, particularly during replication, leading to expression from previously suppressed chromatin. Especially vulnerable at the surface of the inactive X chromosome would be genes and elements from Xp22 to the terminus of the short arm of the X. Expression of these genes and elements could interfere with nucleolar integrity, nucleolar efficiency, and future nucleolar stress response, and even lead to fragmentation of the nucleolus. Ribonucleoprotein complexes assembled in the nucleolus could be left in incomplete states and inappropriate conformations, and/or contain viral components when the nucleolus is disrupted and these abnormal complexes could initiate an autoimmune response when exposed to the immune system. Epitope spreading could then lead to an autoimmune reaction to the more abundant normal complexes. Many autoantigens reported in lupus and other autoimmune diseases are, at least transiently, nucleolar components.


Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Nucléolo Celular/imunologia , Cromossomo X/imunologia , Animais , Autoantígenos/imunologia , Humanos
7.
J Immunol ; 199(12): 3981-3990, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29070672

RESUMO

Anti-nuclear autoantibodies, which frequently target the nucleoli, are pathogenic hallmarks of systemic lupus erythematosus (SLE). Although the causes of these Abs remain broad and ill-defined, a genetic deficiency in C1 complex (C1qC1r2C1s2) or C4 is able to induce these Abs. Considering a recent finding that, in dead cells, nucleoli were targeted by C1q and two nucleolar autoantigens were degraded by C1r/C1s proteases, we considered that C1 could help protect against antinuclear autoimmunity by broadly degrading nucleolar proteins or autoantigens. Nucleoli were isolated to homogeneity and structurally defined. After C1 treatment, cleaved nucleolar proteins were identified by proteomic two-dimensional fluorescence difference gel electrophoresis and mass spectrometry, and further verified by Western blotting using specific Abs. The extent of nucleolar autoantigen degradation upon C1 treatment was estimated using SLE patient autoantibodies. The isolated nucleoli were broadly reactive with SLE patient autoantibodies. These nucleoli lacked significant autoproteolysis, but many nucleolar proteins and autoantigens were degraded by C1 proteases; >20 nucleolar proteins were identified as C1 cleavable. These were further validated by Western blotting using specific Abs. The broad autoantigenicity of the nucleoli may attribute to their poor autoproteolysis, causing autologous immune stimulation upon necrotic exposure. However, C1q targets at these nucleoli to cause C1 protease activation and the cleavage of many nucleolar proteins or autoantigens. This may represent one important surveillance mechanism against antinuclear autoimmunity because C1 genetic deficiency causes anti-nuclear autoantibodies and SLE disease.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/metabolismo , Nucléolo Celular/imunologia , Complemento C1r/metabolismo , Complemento C1s/metabolismo , Imunoglobulina G/imunologia , Proteínas Nucleares/metabolismo , Autoanticorpos/sangue , Nucléolo Celular/ultraestrutura , Células HeLa , Humanos , Imunoglobulina G/sangue , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Proteólise , Proteômica , Especificidade por Substrato
8.
Oncotarget ; 8(25): 40079-40089, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28445143

RESUMO

The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus limiting the extent of its potentially deleterious off-target effects.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Nucléolo Celular/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Células Cultivadas , Citidina Desaminase/imunologia , Citidina Desaminase/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Hibridização in Situ Fluorescente/métodos , Ativação Linfocitária/imunologia , Microscopia Confocal , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia
9.
Lupus ; 25(9): 980-7, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26846693

RESUMO

Nucleolar staining of antinuclear antibodies (ANAs) is not exclusive to patients suffering systemic sclerosis (SSc) since it can occur in other autoimmune diseases, such as systemic lupus erythematosus (SLE). The nucleolar ANA pattern presents a low incidence in patients with SLE, with less than 9% reported in some studies. The significance of nucleolar staining and antinucleolar antibodies (ANoA) in SLE is still unknown, as is its association with clinical manifestations. To address these issues, a case-control study was carried out. Twenty-eight cases of SLE with nucleolar staining were enrolled, as well as 73 controls with no nucleolar staining and different ANA patterns (homogeneous, speckled, and combined homogeneous and speckled). The homogeneous nucleolar pattern was the most frequent (27 out of 28), and in 75% was combined with other ANA patterns. The anti-double stranded DNA antibodies showed no differences between the two groups of patients, nor the auto-antibodies detected by line immunoassay (LIA). However, we have found an increased frequency of anti-PM-Scl antibodies with respect to the controls (p = 0.02), in addition to the association between Raynaud's phenomenon (RP) and anti-PM-Scl antibodies (OR = 20.72, 95% CI 1.33-323.19, p = 0.03). Moreover, the cases of SLE showed a 7.78-fold increase in the risk of developing cancer (95%, CI 1.85-32.75, p = 0.005) with respect to the control group. Taken together these findings suggest that nucleolar staining represents a comorbidity factor in patients with SLE, although its significance must still be determined.


Assuntos
Anticorpos Antinucleares/metabolismo , Autoanticorpos/metabolismo , Nucléolo Celular/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Neoplasias/diagnóstico , Adulto , Anticorpos Antinucleares/sangue , Autoanticorpos/sangue , Estudos de Casos e Controles , Comorbidade , Feminino , Células Hep G2 , Humanos , Lúpus Eritematoso Sistêmico/sangue , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/imunologia , Fatores de Risco , Espanha
10.
Toxicol Ind Health ; 32(9): 1663-74, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25765285

RESUMO

The growth of the influence of anthropogenic factors aimed on the improvement of human life has its side effect, for example, living organisms receive increasing exposure to toxic mercuric compounds. Experimental data show that mercury (Hg) salts are able to induce systemic autoimmunity in rodents. This Hg-induced autoimmune process (HgIA) is characterized by T cell-dependent polyclonal activation of B lymphocytes, increased level of serum immunoglobulin G1 (IgG1) and immunoglobulin E (IgE), production of antinucleolar autoantibodies (ANoA), and immune complex deposition in multiple organs. HgIA in mice is used as a model of human systemic autoimmune disorders. However, the dose of mercuric chloride (HgCl2) usually used in laboratory mice to induce HgIA is above the allowable limit for everyday levels of Hg exposure in humans. So, we decided to determine the lowest dose of HgCl2 that is able to trigger autoimmunity in outbred Carworth Farms Swiss Webster (CFW) mice not genetically prone to HgIA development. The lowest dose (50 µg/kg body weight (b.w.)/week) was chosen to match the World Health Organization provisional weekly tolerable intake of total Hg for humans. We also tested HgCl2 at 500 and 1500 µg/kg b.w./week (6.5- and 2-fold less than usually used for induction of HgIA in mice). We found that even the lowest dose of Hg resulted in a statistically significant increase in serum level of IgG1 after 8 weeks of treatment. HgCl2 in doses 500 and 1500 µg/kg b.w./week resulted in a significant increase in serum level of IgG1 after 4 weeks of treatment, followed by ANoA production. Sera of HgCl2-treated mice stained the regions in which the major autoantigen in HgIA, fibrillarin, was revealed. These results suggest that low doses of Hg are able to induce the main features of HgIA in genetically heterozygous mice, and that humans chronically exposed to low doses of Hg may be at risk of autoimmunity induction regardless of their genetic background.


Assuntos
Antígenos Nucleares/metabolismo , Doenças Autoimunes/etiologia , Autoimunidade/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Cloreto de Mercúrio/toxicidade , Intoxicação por Mercúrio/fisiopatologia , Animais , Animais não Endogâmicos , Autoanticorpos/análise , Autoanticorpos/biossíntese , Autoantígenos/metabolismo , Nucléolo Celular/imunologia , Nucléolo Celular/metabolismo , Nucléolo Celular/patologia , Proteínas Cromossômicas não Histona/metabolismo , Relação Dose-Resposta a Droga , Poluentes Ambientais/administração & dosagem , Feminino , Imunoglobulina G/análise , Injeções Subcutâneas , Cloreto de Mercúrio/administração & dosagem , Intoxicação por Mercúrio/sangue , Intoxicação por Mercúrio/imunologia , Intoxicação por Mercúrio/patologia , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Baço/patologia
11.
BMC Cancer ; 15: 407, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25975273

RESUMO

BACKGROUND: The objective of this work was to demonstrate that autoantibodies in breast cancer sera are not epiphenomena, and exhibit unique immunologic features resembling the rheumatic autoimmune diseases. METHODS: We performed a comprehensive study of autoantibodies on a collection of sera from women with breast cancer or benign breast disease, undergoing annual screening mammography. All women in this study had suspicious mammography assessment and underwent a breast biopsy. We used indirect immunofluorescence, the crithidia assay for anti-dsDNA antibodies, and multiple ELISAs for extractable nuclear antigens. RESULTS: Autoantibodies were detected in virtually all patients with breast cancer, predominantly of the IgG1 and IgG3 isotypes. The profile detected in breast cancer sera showed distinctive features, such as antibodies targeting mitochondria, centrosomes, centromeres, nucleoli, cytoskeleton, and multiple nuclear dots. The majority of sera showing anti-mitochondrial antibodies did not react with the M2 component of pyruvate dehydrogenase, characteristic of primary biliary cirrhosis. Anti-centromere antibodies were mainly anti-CENP-B. ELISAs for extractable nuclear antigens and the assays for dsDNA were negative. CONCLUSIONS: The distinctive autoantibody profile detected in BC sera is the expression of tumor immunogenicity. Although some of these features resemble those in the rheumatic autoimmune diseases and primary biliary cirrhosis, the data suggest the involvement of an entirely different set of epithelial antigens in breast cancer. High titer autoantibodies targeting centrosomes, centromeres, and mitochondria were detected in a small group of healthy women with suspicious mammography assessment and no cancer by biopsy; this suggests that the process triggering autoantibody formation starts in the pre-malignant phase and that future studies using validated autoantibody panels may allow detection of breast cancer risk in asymptomatic women. Autoantibodies developing in breast cancer are not epiphenomena, but likely reflect an antigen-driven autoimmune response triggered by epitopes developing in the mammary gland during breast carcinogenesis. Our results support the validity of the multiple studies reporting association of autoantibodies with breast cancer. Results further suggest significant promise for the development of panels of breast cancer-specific, premalignant-phase autoantibodies, as well as studies on the autoantibody response to tumor associated antigens in the pathogenesis of cancer.


Assuntos
Anticorpos Antinucleares/sangue , Neoplasias da Mama/imunologia , Carcinogênese/imunologia , Carcinoma in Situ/imunologia , Carcinoma Ductal de Mama/imunologia , Imunoglobulina G/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Nucleares , Doenças Mamárias/imunologia , Nucléolo Celular/imunologia , Centrômero/imunologia , Proteína B de Centrômero/imunologia , Centrossomo/imunologia , Feminino , Humanos , Pessoa de Meia-Idade , Mitocôndrias/imunologia
13.
Acta Pharmacol Sin ; 33(7): 873-8, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22659623

RESUMO

AIM: To explore the signalling pathways involved in aldosterone-induced inflammation and fibrosis in rat vascular smooth muscle cells (VSMCs). METHODS: Using Western blotting and real-time RT-PCR, we investigated the effects of aldosterone on the expression of cyclooxygenase-2 (Cox-2) and IL-6, two important proinflammatory factors, and TGFß1, a critical profibrotic factor, in VSMCs. RESULTS: Aldosterone treatment significantly increased the expression of Cox-2 and IL-6 and activation of p38MAPK and NF-κB. The expression of both Cox-2 and IL-6 could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone and the p38MAPK inhibitor SB203580. Also, the rapid phosphorylation of p38MAPK could be suppressed by SB203580 but not by spironolactone, implicating in nongenomic effects of aldosterone. Similar to SB203580 and spironolactone, the NF-κB inhibitor α-p-tosyl-L-lysine chloromethyl ketone (TLCK) markedly attenuated expression of Cox-2, indicating that MR, p38MAPK and NF-κB are associated with aldosterone-induced inflammatory responses. Furthermore, aldosterone enhanced expression of TGFß1 in rat VSMCs. This result may be related to activation of the MR/ERK-Sp1 signalling pathway because PD98059, an ERK1/2 inhibitor, significantly blocked the rapid phosphorylation of ERK1/2 and function of Sp1 and led to reduced expression of TGFß1. Spironolactone was also shown to significantly inhibit TGFß1 and Sp1 expression but not ERK1/2 phosphorylation. CONCLUSION: These results suggest that aldosterone-induced inflammatory responses and fibrotic responses may be mediated by the MR/p38MAPK-NF-κB pathways and the MR/ERK-Sp1 pathways in VSMCs, respectively.


Assuntos
Aldosterona/imunologia , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos de Músculo Liso/imunologia , NF-kappa B/imunologia , Proteínas Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Linhagem Celular , Nucléolo Celular/imunologia , Nucléolo Celular/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/citologia , Fosforilação , Proteínas Quinases/genética , RNA Mensageiro/genética , Ratos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Regulação para Cima
14.
Arthritis Res Ther ; 14(3): R128, 2012 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-22643159

RESUMO

INTRODUCTION: Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies. METHODS: Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard. RESULTS: By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP. CONCLUSIONS: Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.


Assuntos
Anticorpos Antinucleares/análise , Imunoprecipitação/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Escleroderma Sistêmico/diagnóstico , Autoantígenos/imunologia , Nucléolo Celular/imunologia , Humanos , Células K562 , Ribonucleoproteínas/imunologia , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/imunologia , Sensibilidade e Especificidade
15.
J Gastrointestin Liver Dis ; 20(3): 267-70, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21961094

RESUMO

BACKGROUND AND AIMS: Immunosuppressive drugs have been used to prevent graft rejection in most allo-liver recipients and the immune state of these patients differs greatly before and after transplant operation. This study aims at evaluating the immune state of liver transplant patients treated with tacrolimus by investigating the production of anti-nuclear antibodies (ANA). METHODS: A hundred and eighty-eight serum samples from 94 allo-liver recipients treated with tacrolimus and from 94 patients with matched liver diseases were tested for ANA by indirect immunofluorescence assay with HEp-2 cells as substrate. RESULTS: ANA were detected as positive in 20.2% of the liver transplant patients treated with tacrolimus, and in 12.8% of disease-matched control patients, but this difference was not statistically significant (P=0.17). However, the frequency of nucleolar ANA pattern in ANA-positive cases was significantly higher in the liver transplant patients (63.2%) than in the control group (16.7%) (P=0.01). CONCLUSION: Tacrolimus may contribute to producing nucleolar ANA in liver transplant patients. The autoimmune disease susceptibility of allo-liver recipients treated with tacrolimus requires further studying.


Assuntos
Anticorpos Antinucleares/biossíntese , Nucléolo Celular/imunologia , Imunossupressores/efeitos adversos , Transplante de Fígado/efeitos adversos , Tacrolimo/efeitos adversos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tacrolimo/sangue
16.
Mol Immunol ; 49(3): 441-52, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22014684

RESUMO

The nucleolus, site of ribosome biogenesis, is a dynamic subnuclear organelle involved in diverse cellular functions. The size, number and organisation of nucleoli are cell-specific and while it remains to be established, the nucleolar protein composition would be expected to reflect lineage-specific transcriptional regulation of rDNA genes and have cell-type functional components. Here, we describe the first characterisation of the human T-cell nucleolar proteome. Using the Jurkat T-cell line and a reproducible organellar proteomic approach, we identified 872 nucleolar proteins. In addition to ribosome biogenesis and RNA processing networks, network modeling and topological analysis of nucleolar proteome revealed distinct macromolecular complexes known to orchestrate chromatin structure and to contribute to the regulation of gene expression, replication, recombination and repair, and chromosome segregation. Furthermore, among our dataset, we identified proteins known to functionally participate in T-cell biology, including RUNX1, ILF3, ILF2, STAT3, LSH, TCF-1, SATB1, CTCF, HMGB3, BCLAF1, FX4L1, ZAP70, TIAM1, RAC2, THEMIS, LCP1, RPL22, TOPK, RETN, IFI-16, MCT-1, ISG15, and 14-3-3τ, which support cell-specific composition of the Jurkat nucleolus. Subsequently, the nucleolar localisation of RUNX1, ILF3, STAT3, ZAP70 and RAC2 was further validated by Western Blot analysis and immunofluorescence microscopy. Overall, our T-cell nucleolar proteome dataset not only further expands the existing repertoire of the human nucleolar proteome but support a cell type-specific composition of the nucleolus in T cell and highlights the potential roles of the nucleoli in lymphocyte biology.


Assuntos
Nucléolo Celular/química , Proteínas Nucleares/análise , Linfócitos T/química , Nucléolo Celular/imunologia , Humanos , Células Jurkat , Proteínas Nucleares/imunologia , Proteômica , Linfócitos T/imunologia
17.
Arthritis Res Ther ; 13(4): R119, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21781293

RESUMO

INTRODUCTION: Anti-RNA polymerase III (RNAP III) antibodies are highly specific markers of scleroderma (systemic sclerosis, SSc) and associated with a rapidly progressing subset of SSc. The clinical presentation of anti-RNAP III positive patients, onset of Raynaud's phenomenon (RP) and SSc in unselected patients in a rheumatology clinic were evaluated. METHODS: Autoantibodies in sera from 1,966 unselected patients (including 434 systemic lupus erythematosus (SLE), 119 SSc, 85 polymyositis/dermatomyositis (PM/DM)) in a rheumatology clinic were screened by radioimmunoprecipitation. Anti-RNAP III positive sera were also tested by immunofluorescence antinuclear antibodies and anti-RNAP III ELISA. Medical records of anti-RNAP III positive patients were reviewed. RESULTS: Among 21 anti-RNAP III positive patients, 16 met the American College of Rheumatology (ACR) SSc criteria at the initial visit but 5 did not; diagnoses were vasculitis, early polyarthritis, renal failure with RP, interstitial lung disease, and Sjögren's syndrome. The first two patients developed rapidly progressive diffuse SSc. An additional case presented with diffuse scleroderma without RP and RP developed two years later. Anti-RNAP III antibodies in these 6 cases of atypical clinical presentation were compared with those in 15 cases of typical (SSc with RP) cases. Anti-RNAP III levels by ELISA were lower in the former group (P = 0.04 by Mann-Whitney test) and 3 of 6 were negative versus only 1 of 15 negative in the latter (P < 0.05 by Fisher's exact test). Three cases of non-SSc anti-RNAP III positive patients had predominant reactivity with RNAP I with weak RNAP III reactivity and had a strong nucleolar staining. Three anti-RNAP III patients, who did not have RP at the initial visit, developed RP months later. Scleroderma developed prior to RP in 5 out of 16 (31%) in the anti-RNAP III group, but this was rare in patients with other autoantibodies. The interval between the onset of RP to scleroderma was short in anti-RNAP III positive patients. CONCLUSIONS: Anti-RNAP III antibodies are highly specific for SSc; however, a subset of anti-RNAP III positive patients do not present as typical SSc. The interval between RP and scleroderma in this group is short, and 31% of patients developed scleroderma prior to RP in this group. Anti-RNAP III positive patients may not present as typical SSc and detecting anti-RNAP III may have predictive value.


Assuntos
Autoanticorpos/sangue , RNA Polimerase III/imunologia , RNA Polimerase I/imunologia , Doença de Raynaud/imunologia , Escleroderma Sistêmico/imunologia , Adulto , Idoso , Autoanticorpos/imunologia , Autoantígenos/imunologia , Nucléolo Celular/imunologia , Nucléolo Celular/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Pessoa de Meia-Idade , Ensaio de Radioimunoprecipitação , Doença de Raynaud/diagnóstico , Doença de Raynaud/metabolismo , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/metabolismo
18.
Clin Rheumatol ; 30(10): 1333-9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21523365

RESUMO

Autoantibodies to topoisomerase I (topo I), RNA polymerase III (RNAPIII), centromere, U3RNP/fibrillarin, Th, PM-Scl, and U1RNP found in scleroderma (SSc) are associated with unique clinical subsets. The effects of race and gender on autoantibody prevalence and clinical manifestations were examined. Autoantibodies in sera from 105 SSc (include 75 Caucasian, 24 African-American, 6 others; 89 females and 16 males) were analyzed by immunofluorescence and immunoprecipitation. Clinical information was from database. SSc-related autoantibodies seldom coexist except for anti-topo I and anti-U1RNP. Anti-topo I (35% vs 15%), anti-U3RNP (30% vs 3%, p = 0.0005), and anti-U1RNP (30% vs 13%) were more common in African-Americans vs Caucasians. Anti-centromere (17%) and anti-PM-Scl (only in 8% of female) were found only in Caucasians. In race/gender combination, all three African-American males had anti-topo I (p = 0.04). Anti-U3RNP (35% vs 3%, p = 0.0005) and anti-U1RNP were common in African-American females. In African-American, all nucleolar dominant staining sera had anti-U3RNP; nuclear pattern was topo I (50%), U1RNP (19%), and RNAPIII (13%). In Caucasian, nucleolar was anti-Th (43%) and PM-Scl (29%); nuclear pattern was RNAPIII (29%), topo I (24%), and U1RNP (18%). Anti-topo I, anti-RNAPIII, and anti-U3RNP were associated with diffuse SSc while anti-centromere, anti-Th, and anti-U1 with limited disease. Proximal scleroderma was less common in African-American with anti-topo I (38% vs 91% in Caucasian, p = 0.04). The production of SSc-related autoantibodies is gender and race dependent, and this can be highly relevant in understanding their clinical significance.


Assuntos
Anticorpos Antinucleares/sangue , Escleroderma Sistêmico/etnologia , Escleroderma Sistêmico/imunologia , Adulto , Negro ou Afro-Americano/etnologia , Anticorpos Antinucleares/imunologia , Nucléolo Celular/imunologia , Centrômero/imunologia , DNA Topoisomerases Tipo I/imunologia , Feminino , Florida/etnologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Polimerase III/imunologia , Ribonucleoproteínas Nucleolares Pequenas/imunologia , Escleroderma Sistêmico/sangue , Fatores Sexuais , População Branca/etnologia
20.
J Rheumatol ; 37(10): 2071-5, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20682663

RESUMO

OBJECTIVE: Patients with scleroderma (systemic sclerosis; SSc) can be classified into subsets based on autoantibody profile and clinical features. Specificities such as anti-Th/To and anti-fibrillarin (U3RNP) are detectable mainly by immunoprecipitation (IP), which is not widely used in clinical laboratories. We examined the autoantibody profiles and clinical manifestations in a cohort of Italian patients with SSc, focusing on anti-Th/To and anticentromere (ACA) antibodies, associated with limited cutaneous SSc (lcSSc). METHODS: Sera from 216 consecutive patients with SSc were tested for ACA (by indirect immunofluorescence), antitopoisomerase I (topo I; by counterimmunoelectrophoresis), and anti-RNA polymerase III (RNAPIII; by ELISA). Forty-one sera negative for these specificities were tested by IP analysis of proteins ((35)S-methionine labeled K562 cell extract) and RNA (silver staining). RESULTS: Among 216 SSc patients analyzed, anti-topo I, ACA, and anti-RNAPIII were detected in 38% (81/216), 31% (67/216) and 7% (15/216), respectively. Among 41 sera negative for ACA, anti-topo I, and anti-RNAPIII and which were tested by IP, 14 were nucleolar stain-positive. Eight out of 14 (57%) showed anti-Th/To reactivity, but no anti-U3RNP was found. In comparison with ACA-positive patients, anti-Th/To-positive patients were younger (p = 0.0046) and more commonly were male (p = 0.0006). All 8 anti-Th/To-positive and all but one ACA-positive patients had lcSSc. Interstitial lung disease (ILD) and pericarditis were more frequent in anti-Th/To-positive patients. CONCLUSION: Anti-Th/To are common in antinucleolar antibody-positive Italian patients with SSc. Anti-Th/To and ACA patients had lcSSc, with excellent prognosis. The anti-Th/To group had frequent pericarditis and ILD, although impairment of pulmonary function appeared mild.


Assuntos
Anticorpos Antinucleares/sangue , Autoanticorpos/sangue , Nucléolo Celular/imunologia , Escleroderma Sistêmico , Adulto , Idoso , Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/imunologia
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