RESUMO
AIM: To investigate the retreatability of two calcium silicate-based materials (BioRoot RCS, Septodont, Saint-Maur-des-Fossés, France and GuttaFlow Bioseal, Colténe/Whaledent AG, Langenau, Germany) using rotary instrumentation combined with supplementary irrigant agitation techniques using extracted teeth in a laboratory setting. METHODOLOGY: The root canals of extracted single-rooted mandibular premolars were prepared to size 40, .04 taper and randomly divided into two experimental groups (n = 36) depending on the root filling material. Root canals were filled with gutta-percha and GuttaFlow Bioseal (GB, group 1) or BioRoot RCS (BR, group 2), scanned using a micro-CT scanner and stored in phosphate-buffered saline for 4 months. Removal of root filling was performed with rotary instruments, and specimens were randomly allocated to one of the subgroups for supplementary irrigant agitation (n = 12): subgroup A, syringe irrigation (control); subgroup B, Tornado Brush (M.I.B, Suresnes, France) and subgroup C, ultrasonically activated irrigation. Specimens were re-scanned with micro-CT to calculate the volume of remnant root filling material. Data were analysed statistically by two-way ANOVA and post hoc Tukey's tests (P = 0.05). RESULTS: Specimens filled with GuttaFlow Bioseal were associated with a significantly smaller volume of root filling remnants compared with BioRoot RCS (P < 0.05). There was no significant difference between the supplementary irrigant agitation subgroups in the removal of GB (P > 0.05). In group 2 (BioRoot RCS), subgroups B (Tornado Brush) and C (ultrasonically activated irrigation) were associated with a significantly smaller volume of root filling remnants compared with subgroup A (syringe irrigation) (P < 0.05). There was no significant difference between subgroups B and C (P > 0.05). CONCLUSIONS: Significantly smaller volumes of root filling remnants of GuttaFlow Bioseal, than BioRoot RCS, were present after their removal with rotary instruments and irrigation. Supplementary irrigant agitation techniques were associated with smaller volumes of remnants during the removal of BioRoot RCS but not that of GuttaFlow Bioseal.
Assuntos
Cavidade Pulpar , Materiais Restauradores do Canal Radicular , Cálcio , Compostos de Cálcio , Alemanha , Guta-Percha , Preparo de Canal Radicular , SilicatosRESUMO
Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (â¼2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.
Assuntos
Proteínas Relacionadas a Receptor de LDL/imunologia , Miastenia Gravis/epidemiologia , Miastenia Gravis/imunologia , Testes Sorológicos/métodos , Timo/patologia , Adolescente , Adulto , Idade de Início , Idoso , Autoanticorpos/sangue , Criança , Pré-Escolar , Progressão da Doença , Feminino , Células HEK293 , Humanos , Hiperplasia , Imunoglobulina G/sangue , Lactente , Recém-Nascido , Cooperação Internacional , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/diagnóstico , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Fatores Sexuais , Adulto JovemRESUMO
We fine mapped two primary biliary cirrhosis (PBC) risk loci, CLEC16A (C-type lectin domain family 16 member A)-suppressor of cytokine signaling 1 (SOCS1) and Spi-B protein (SPIB) and sequenced a locus, sialic acid acetylesterase (SIAE), proposed to harbor autoimmunity-associated mutations. In all, 1450 PBC cases and 2957 healthy controls were genotyped for 84 single-nucleotide polymorphisms (SNPs) across the CLEC16A-SOCS1 and SPIB loci. All 10 exons of the SIAE gene were resequenced in 381 cases and point substitutions of unknown significance assayed for activity and secretion. Fine mapping identified 26 SNPs across the CLEC16A-SOCS1 and 11 SNPs across the SPIB locus with significant association to PBC, the strongest signals at the CLEC16A-SOCS1 locus emanating from a SOCS1 intergenic SNP (rs243325; P=9.91 × 10(-9)) and at the SPIB locus from a SPIB intronic SNP (rs34944112; P=3.65 × 10(-9)). Among the associated SNPs at the CLEC16A-SOCS1 locus, two within the CLEC16A gene as well as one SOCS1 SNP (rs243325) remained significant after conditional logistic regression and contributed independently to risk. Sequencing of the SIAE gene and functional assays of newly identified variants revealed six patients with functional non-synonymous SIAE mutations (Fisher's P=9 × 10(-4) vs controls) We demonstrate independent effects on risk of PBC for CLEC16A, SOCS1 and SPIB variants, while identifying functionally defective SIAE variants as potential factors in risk for PBC.
Assuntos
Acetilesterase/genética , Proteínas de Ligação a DNA/genética , Lectinas Tipo C/genética , Cirrose Hepática Biliar/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores de Transcrição/genética , Acetilesterase/metabolismo , Alelos , Estudos de Casos e Controles , Mapeamento Cromossômico/métodos , Proteínas de Ligação a DNA/metabolismo , Ensaios Enzimáticos , Loci Gênicos , Predisposição Genética para Doença , Haplótipos , Humanos , Lectinas Tipo C/metabolismo , Cirrose Hepática Biliar/imunologia , Cirrose Hepática Biliar/metabolismo , Modelos Logísticos , Proteínas de Transporte de Monossacarídeos/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [3H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na+-dependent transcellular transport of [3H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na+-dependent uptake of [3H]taurocholate, with apparent Km and Vmax values of 209+/-45 microM and 1.23+/-0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na+-dependent taurocholate-cotransporting polypeptide and rat ileal apical Na+-dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids.
Assuntos
Ácidos e Sais Biliares/metabolismo , Ductos Biliares/metabolismo , Proteínas de Transporte/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio , Simportadores , Animais , Ductos Biliares/citologia , Transporte Biológico , Proteínas de Transporte/genética , Células Cultivadas , Masculino , RNA , Ratos , Ratos Endogâmicos F344 , Ácido Taurocólico/metabolismoRESUMO
Pharmacogenomics, studying genetic variation related to drug response, was established decades ago. Today, performing clinical pharmacogenomics testing has increased, creating great potential to improve patient care. Yet widespread implementation of pharmacogenomics in practice is currently limited, resulting in the "therapeutic odyssey" of patients. Preemptive clinical pharmacogenomics testing prior to the time of prescribing is now emerging as an option that could tailor the pharmacotherapy of patients by increasing drug effectiveness while reducing adverse drug reaction risk.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Variação Genética , Preparações Farmacêuticas/administração & dosagem , Farmacogenética/métodos , Testes Genéticos/métodos , Humanos , Assistência ao Paciente/normas , Resultado do TratamentoRESUMO
Primary sclerosing cholangitis (PSC) is strongly associated with several human leukocyte antigen (HLA) haplotypes. Due to extensive linkage disequilibrium and multiple polymorphic candidate genes in the HLA complex, identifying the alleles responsible for these associations has proven difficult. We aimed to evaluate whether studying populations of admixed or non-European descent could help in defining the causative HLA alleles. When assessing haplotypes carrying HLA-DRB1*13:01 (hypothesized to specifically increase the susceptibility to chronic cholangitis), we observed that every haplotype in the Scandinavian PSC population carried HLA-DQB1*06:03. In contrast, only 65% of HLA-DRB1*13:01 haplotypes in an admixed/non-European PSC population carried this allele, suggesting that further assessments of the PSC-associated haplotype HLA-DRB1*13:01-DQA1*01:03-DQB1*06:03 in admixed or multi-ethnic populations could aid in identifying the causative allele.
Assuntos
Colangite Esclerosante/genética , Predisposição Genética para Doença , Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/genética , Haplótipos , Alelos , Colangite Esclerosante/etnologia , Colangite Esclerosante/imunologia , Etnicidade , Expressão Gênica , Frequência do Gene , Cadeias beta de HLA-DQ/classificação , Cadeias beta de HLA-DQ/imunologia , Cadeias HLA-DRB1/classificação , Cadeias HLA-DRB1/imunologia , Humanos , Desequilíbrio de Ligação , Países Escandinavos e Nórdicos , População BrancaRESUMO
Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction of skeletal muscles. Triple-seronegative MG (tSN-MG, without detectable AChR, MuSK and LRP4 antibodies), which accounts for ~10% of MG patients, presents a serious gap in MG diagnosis and complicates differential diagnosis of similar disorders. Several AChR antibody positive patients (AChR-MG) also have antibodies against titin, usually detected by ELISA. We have developed a very sensitive radioimmunoprecipitation assay (RIPA) for titin antibodies, by which many previously negative samples were found positive, including several from tSN-MG patients. The validity of the RIPA results was confirmed by western blots. Using this RIPA we screened 667 MG sera from 13 countries; as expected, AChR-MG patients had the highest frequency of titin antibodies (40.9%), while MuSK-MG and LRP4-MG patients were positive in 14.6% and 16.4% respectively. Most importantly, 13.4% (50/372) of the tSN-MG patients were also titin antibody positive. None of the 121 healthy controls or the 90 myopathy patients, and only 3.6% (7/193) of other neurological disease patients were positive. We thus propose that the present titin antibody RIPA is a useful tool for serological MG diagnosis of tSN patients.
Assuntos
Autoanticorpos/sangue , Conectina/imunologia , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Cooperação Internacional , Proteínas Relacionadas a Receptor de LDL/imunologia , Masculino , Miastenia Gravis/epidemiologia , Ensaio de Radioimunoprecipitação , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologiaRESUMO
Myasthenia gravis (MG) is usually caused by antibodies against the muscle acetylcholine receptor (AChR). Plasmapheresis and immunoadsorption are often used to treat non-responsive patients. Antigen-specific immunoadsorption would remove only the pathogenic autoantibodies reducing side-effects. We expressed AChR extracellular domain mutants for use as specific adsorbents, and characterized them. Antigenicity and capacity for autoantibody binding were improved compared to the wild-type proteins. Adsorption appeared to be fast, as high plasma flow-rates could be applied. The bound autoantibodies were eluted repeatedly, allowing column reuse, without compromise in efficiency. Overall, the adsorbents were specific, efficient and suitable for use in therapy.
Assuntos
Remoção de Componentes Sanguíneos , Espaço Extracelular/metabolismo , Proteínas Mutantes/imunologia , Miastenia Gravis/sangue , Receptores Nicotínicos/genética , Receptores Nicotínicos/imunologia , Especificidade de Anticorpos , Autoanticorpos/sangue , Espaço Extracelular/imunologia , Feminino , Humanos , Imunoadsorventes , Masculino , Miastenia Gravis/imunologia , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fatores de TempoRESUMO
BACKGROUND: The relationships between primary sclerosing cholangitis (PSC) and the environment are largely unknown. AIM: To validate associations reported in previous studies and to identify novel environmental exposures among PSC patients. METHODS: We performed a multicenter, case-control analysis utilising self-administered questionnaires. Responses between cases (n = 1000) and controls (n = 663) were compared using multivariable logistic regression adjusted for age and gender. The model was further stratified based on inflammatory bowel disease (IBD) status (with IBD n = 741 without IBD n = 259). RESULTS: Smoking was associated with PSC only when IBD was present (OR, 0.5; 95% CI 0.4-0.7) but not among those PSC patients without IBD (OR, 0.9; 95% CI 0.7-1.2). Compared to controls, women with PSC (irrespective of the presence of IBD) were less likely to have received hormone replacement therapy (HRT; OR, 0.5; 95% CI 0.4-0.7) and were more likely to have recurrent urinary tract infections (OR, 1.6; 95% CI 1.2-2.3). PSC patients regardless of gender or IBD status were less likely to eat fish (OR, 0.4; 95% CI 0.3-0.6) and grilled/barbecued meat (OR, 0.8; 95% CI 0.7-0.9). In contrast, PSC patients with and without IBD were more likely to consume steak/burgers that were more well done (OR, 1.3; 95% CI 1.2-1.5). CONCLUSIONS: IBD (rather than PSC) is associated with smoking. Women with PSC are more likely to have recurrent urinary tract infections and less likely to receive HRT. Dietary intake and methods of food preparation differ in PSC patients when compared to controls.
Assuntos
Colangite Esclerosante/epidemiologia , Exposição Ambiental/efeitos adversos , Doenças Inflamatórias Intestinais/epidemiologia , Fumar/efeitos adversos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Canadá/epidemiologia , Estudos de Casos e Controles , Criança , Colangite Esclerosante/etiologia , Feminino , Humanos , Doenças Inflamatórias Intestinais/etiologia , Masculino , Pessoa de Meia-Idade , Fumar/epidemiologia , Inquéritos e Questionários , Estados Unidos/epidemiologia , Adulto JovemRESUMO
Seronegative myasthenia gravis (MG) presents a serious gap in MG diagnosis and understanding. We applied a cell based assay (CBA) for the detection of muscle specific kinase (MuSK) antibodies undetectable by radioimmunoassay. We tested 633 triple-seronegative MG patients' sera from 13 countries, detecting 13% as positive. MuSK antibodies were found, at significantly lower frequencies, in 1.9% of healthy controls and 5.1% of other neuroimmune disease patients, including multiple sclerosis and neuromyelitis optica. The clinical data of the newly diagnosed MuSK-MG patients are presented. 27% of ocular seronegative patients were MuSK antibody positive. Moreover, 23% had thymic hyperplasia suggesting that thymic abnormalities are more common than believed.
Assuntos
Autoanticorpos/sangue , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , Receptores Proteína Tirosina Quinases/imunologia , Adulto , Idoso , Feminino , Citometria de Fluxo , Humanos , Cooperação Internacional , Proteínas Relacionadas a Receptor de LDL/imunologia , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/patologia , Neuromielite Óptica/diagnóstico , Radioimunoensaio , Receptores Colinérgicos/imunologia , Timo/patologia , Hiperplasia do Timo/diagnósticoRESUMO
Cytokines play a major role in tissue destruction caused by autoimmune dysregulation. In Sjögren's syndrome (SS) patients, salivary glands are the target organs for autoimmune tissue damage. In the present study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to look for cytokine mRNA expressed in SS salivary glands. Focus score was used to determine the severity of the lesions. Cytokine production in supernatants of the salivary gland cell culture was measured by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining was used to identify the local presence of transforming growth factor beta (TGF-beta). Interleukin (IL)-2, IL-6, and IL-10 mRNA were expressed in moderate to severe SS salivary gland lesions. TGF-beta mRNA was constitutively expressed in normal and SS salivary glands. In SS salivary gland cell cultures, IL-6 and IL-10 proteins were produced. TGF-beta production was reduced in high focus score SS glands. Normal and minimally involved SS salivary gland ductal epithelium and acinar cells were found to produce TGF-beta by immunostaining. In conclusion, an excess production of IL-2, IL-6, and IL-10 and a reduced production of the immunosuppressive cytokine, TGF-beta, may be responsible for the progression of the salivary gland lesion in SS. Specific immunotherapy can now be designed based on mechanisms to correct this cytokine imbalance and benefit patients with autoimmune diseases, such as SS.
Assuntos
Citocinas/fisiologia , Doenças das Glândulas Salivares/metabolismo , Síndrome de Sjogren/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Adulto , Idoso , Sequência de Bases , Células Cultivadas , Citocinas/análise , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/biossínteseRESUMO
Hepatic hydrothorax is defined as a pleural effusion in a patient with cirrhosis of the liver and no cardiopulmonary disease. The estimated prevalence of this often debilitating complication in patients with liver cirrhosis is 4% to 10%. Its pathophysiology involves movement of ascitic fluid from the peritoneal cavity into the pleural space through diaphragmatic defects. As a result patients are at increased risk of respiratory infection. Initial management consists of sodium restriction, diuretics, and thoracentesis. A transjugular intrahepatic portosystemic shunt may be required. Because most patients with hepatic hydrothorax have end-stage liver disease, a liver transplant should be considered if these options fail.
Assuntos
Hidrotórax/diagnóstico , Hidrotórax/terapia , Cirrose Hepática/complicações , Derrame Pleural/complicações , Algoritmos , Diagnóstico Diferencial , Diafragma/anormalidades , Diafragma/cirurgia , Diuréticos/uso terapêutico , Humanos , Hidrotórax/tratamento farmacológico , Hidrotórax/etiologia , Hidrotórax/cirurgia , Derivação Peritoneovenosa , Derrame Pleural/etiologia , Pleurodese , Derivação Portossistêmica Transjugular Intra-Hepática , Toracoscopia , ToracostomiaRESUMO
The aim of this study was to assess the value of the early exercise test (ET) in patients with acute myocardial infarction (AMI) treated with IV streptokinase (SK). The authors studied 70 patients with first AMI; 31 were treated with SK and 39 were not. Before discharge everyone was given early exercise up to 5-6 METs and catheterized within 22.9 +/- 7.2 days. There was no significant difference in the number of positive ETs between the two groups (11/31 and 14/39 respectively). There was significant difference in favor of: (1) the recanalization of the infarct-related artery in the SK group, (2) the negative ET in patients with recanalized vessels in both groups, (3) the positive ET in patients with multi-vessel coronary disease. It is concluded that the results of early ET in patients with AMI are related to the recanalization of the infarct-related artery and the coexistence of multi-vessel coronary artery disease, regardless of SK treatment. Patients with successful thrombolysis have negative ET more frequently.
Assuntos
Teste de Esforço , Infarto do Miocárdio/tratamento farmacológico , Estreptoquinase/uso terapêutico , Terapia Trombolítica , Cateterismo Cardíaco , Estudos de Avaliação como Assunto , Humanos , Infarto do Miocárdio/diagnóstico , Fatores de TempoRESUMO
Three common methods of isolating amplified DNA were evaluated for their effects on cloning efficiency in a phage vector designed to express human kappa chain. The "glass milk" technique gave higher cloning efficiency and protein expression than phenol-chloroform extraction or microfiltration. This shows that the quality of amplified cDNA should be considered when studying the human antibody repertoire using this vector system.
Assuntos
DNA/isolamento & purificação , Genes de Imunoglobulinas , Cadeias kappa de Imunoglobulina/genética , Reação em Cadeia da Polimerase , Bacteriófago lambda/genética , Sequência de Bases , Clonagem Molecular , Vetores Genéticos , Humanos , Dados de Sequência MolecularRESUMO
Primary biliary cirrhosis (PBC) is one of the most common chronic cholestatic liver diseases affecting the adult population. The clinical presentation of PBC can be diverse, ranging from the presymptomatic individual to the patient with advanced liver disease. Initial evaluation to establish diagnosis and appropriate follow-up are very important in the life-long management of these patients. The primary medical treatment in PBC should focus on reducing the rate of disease progression. To this extent, treatment with ursodeoxycholic acid has been extensively evaluated and has been shown to improve liver biochemistries and survival in patients with PBC. Secondary medical management in PBC should address the treatment of complications of chronic cholestasis, hepatic cirrhosis, and liver failure. Liver transplantation remains the only established therapeutic approach in treatment of patients with end-stage PBC and the associated complications.
Assuntos
Colagogos e Coleréticos/uso terapêutico , Cirrose Hepática Biliar/terapia , Falência Hepática/terapia , Ácido Ursodesoxicólico/uso terapêutico , Adulto , Doenças Ósseas Metabólicas/etiologia , Estrogênios/uso terapêutico , Humanos , Estilo de Vida , Cirrose Hepática Biliar/complicações , Cirrose Hepática Biliar/diagnóstico , Transplante de Fígado , Qualidade de Vida , Análise de SobrevidaRESUMO
UDCA exerts its beneficial effect in liver diseases through a diverse, probably, complementary array of mechanisms. The clinical use and efficacy of UDCA in PBC have been evident. UDCA may also have a place in the management of PSC, ICP, cystic fibrosis, PFIC and GVHD involving the liver, although, more studies are needed to further determine its therapeutic potential in these diseases and in other hepatobiliary disorders such as liver allograft rejection, drug and TPN-induced cholestasis, NASH, and alcoholic liver disease.
Assuntos
Doenças dos Ductos Biliares/tratamento farmacológico , Doenças dos Ductos Biliares/fisiopatologia , Hepatopatias/tratamento farmacológico , Hepatopatias/fisiopatologia , Ácido Ursodesoxicólico/fisiologia , Animais , Fenômenos Químicos , Físico-Química , Humanos , Ácido Ursodesoxicólico/química , Ácido Ursodesoxicólico/farmacologia , Ácido Ursodesoxicólico/uso terapêuticoRESUMO
A human mAb designated 4B4 with anti-Sm activity was derived from a patient with systemic lupus erythematosus. This antibody expressed a lupus-associated cross-reactive Id, partially related to the monoclonal murine anti-Sm (Y2) from MRL/lpr mice. Studies were performed to investigate the ability of 4B4 to induce lupus in nonautoimmune-prone mice. BALB/c mice immunized with 4B4 produced antibodies to dsDNA, ssDNA, Sm ribonucleoprotein, and mouse Fc fragment. There was no antibody activity against SSA/Ro, SSB/La, and hen egg lysozyme. Ag inhibition studies show that the autoantibodies were not polyreactive. Mice were also immunized with r4B4 polypeptides representing the H/L heterodimer, H chain and L chain. Autoantibodies were induced in mice immunized against the H/L and H polypeptides. No autoantibodies were induced in mice immunized with recombinant L chain. Furthermore, from 20 to 68% of antibody activity to Sm or dsDNA could be inhibited with anti-Id antiserum (either anti-4B4 or Y2). The autoantibody was initially IgM and then underwent an isotype switch to IgG. These results show that lupus-associated autoantibodies can be induced by immunization with 4B4 and that the 4B4 VH region is important in this induction process. The finding of murine IgG autoantibody expressing a cross-reactive Id similar to the immunizing 4B4 suggests a role for anti-idiotypic Th cells in this autoimmune response.
Assuntos
Autoanticorpos/biossíntese , Idiótipos de Imunoglobulinas , Lúpus Eritematoso Sistêmico/imunologia , Ribonucleoproteínas Nucleares Pequenas , Animais , Anticorpos Antinucleares/biossíntese , Anticorpos Monoclonais , Autoantígenos , Reações Cruzadas , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteínas Recombinantes/imunologia , Ribonucleoproteínas/imunologia , Proteínas Centrais de snRNPRESUMO
Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.
Assuntos
Regiões 5' não Traduzidas/química , Picornaviridae/genética , Biossíntese de Proteínas , Ribossomos/química , Regiões 5' não Traduzidas/fisiologia , Animais , Drosophila/genética , Triticum/genéticaRESUMO
While previous work has demonstrated that monosaccharides can be absorbed from bile, studies of sugar transport by the biliary, epithelia (i.e., cholangiocytes) are lacking. Using a novel model of polarized rat cholangiocytes in primary culture, designated normal rat cholangiocytes (NRC), we examined directly the uptake and transcellular transport of a nonmetabolizable monosaccharide, methyl alpha-D-glucopyranoside (AMG). When the apical or basolateral domain of cholangiocytes was exposed to radiolabeled AMG or sucrose (control), only apical absorption of AMG was evident. This apical uptake was time dependent, saturable, and significantly inhibited (> or = 90%) by removal of Na+ or in the presence of phlorizin (0.1 mM), a competitive inhibitor of the Na(+)-glucose cotransporter. The transcellular flux of AMG was also polar (i.e., apical to basolateral). Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of the transcript for the specific Na(+)-glucose cotransporter SGLT1 in NRC and in freshly isolated cholangiocytes but not in purified hepatocytes; in contrast, the transcript for SGLT2 was absent in all liver samples. In situ RT-PCR on frozen sections of normal rat liver showed that SGLT1 was expressed exclusively in cholangiocytes. Immunoblot analysis using a specific polyclonal antibody for the facilitative glucose transporter GLUT1 demonstrated it to be present in vesicles derived from NRC enriched in basolateral plasma membrane domains. Our data are consistent with the concept that SGLT1 is present on the apical domain of biliary epithelia and, in conjunction with GLUT1 on the basolateral domain, accounts for glucose absorption from bile.