RESUMO
Our aim was to observe whether normal human T-cells respond to mitogenic stimulation with large whole-cell inward currents (composed of identifiable single-channel contributions) when [Ca2+]i is not markedly lowered but instead kept normal or moderately low, as has been reported in human leukaemic Jurkat T-cell line and T-cell clones [Kuno et al. (1986) Nature 323, 269-73; Kuno and Gardner (1987) Nature 326, 301-304; Gardner (1990) Annu. Rev. Immunol. 8, 231-252]. Whole-cell patch recordings showed no such currents in cells otherwise normally responding to depolarisation with the macroscopic IK described in T-lymphocytes and thus deemed viable, in agreement with the notion that Ca2+ influx in normal T-cells enterily depends on depletion of internal stores [Putney (1986) Cell Calcium 7, 1-12; Putney (1990) Cell Calcium 11, 611-624].
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Ativação Linfocitária , Linfócitos T/fisiologia , Potenciais Evocados , Humanos , Técnicas In Vitro , Cinética , Potenciais da Membrana , Técnicas de Patch-Clamp , Fito-Hemaglutininas , Linfócitos T/imunologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Changes in the thymus are often encountered in myasthenia gravis (MG), together with neuromuscular pathology. In the present study, the cell population of 28 thymuses from patients with MG were analyzed by immunofluorescent and 49 thymuses by immunoperoxidase techniques. We were able to show the presence of sIg receptor positive, SpA reactive medium and large B lymphocytes of light specific density in the majority of thymuses, and to demonstrate a characteristic pattern of distribution in the gland. PAP analysis showed that the infiltrating B cells appeared in the interlobular septa, then reached the medulla, occasionally the cortex which mostly revealed signs of atrophy. These findings are consistent with an autoimmune reaction occurring against a thymic antigen; such an antigen could be acetylcholine receptor (AchR) of the thymic structures, since anti-AchR antibody or serum from patients with MG will react with 60% thymocytes and some epithelial cells and Hassall's corpuscles.
Assuntos
Miastenia Gravis/imunologia , Timo/imunologia , Acetilcolina/imunologia , Adolescente , Adulto , Autoanticorpos/imunologia , Linfócitos B/ultraestrutura , Feminino , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoglobulinas/imunologia , Masculino , Pessoa de Meia-Idade , Receptores Colinérgicos/imunologia , Timo/ultraestruturaRESUMO
The effect of human myasthenia gravis (MG) sera and complement on isolated adult rat muscle fibres was investigated. Heat-inactivated MG sera reduced the frequency of single acetylcholine receptor (AChR)-channel activity. One of the MG sera tested had a stronger effect on the extrajunctional type of AChRs than on the junctional type. The simultaneous addition of normal human serum (NHS), as source of complement, and MG serum to freshly dissociated muscle fibres caused contraction restricted to the endplate area and progressive depolarization of the muscle membrane, followed by contracture. An MG antibody-dependent complement-mediated damage of the muscle fibres is suggested.
Assuntos
Proteínas do Sistema Complemento/farmacologia , Músculos/efeitos dos fármacos , Miastenia Gravis/sangue , Animais , Técnicas de Cultura , Eletrofisiologia , Humanos , Masculino , Músculos/fisiologia , Ratos , Ratos WistarRESUMO
Qualitative and/or quantitative alterations in the expression of the MHC class II molecules affect the onset and maintenance of the immune response and may be the basis of a wide variety of disease states, such as autoimmunity and immunodeficiency.CIITA is a major physiological regulator of the expression of MHC class II genes. The availability of CIITA ap- pears generally essential for MHC class II gene expression, and hence its own transcriptional regulatory mechanisms result of fundamental importance for a correct homeostasis of the immune response. Therefore, it is possible to hypothesize that variability at the CIITA-encoding locus, AIR-1, could constitute an additional source of susceptible traits to autoimmune diseases. Mutations at AIR-1/CIITA promoters could modulate expression of CIITA. Variations in CIITA expression could influence the qualitative and quantitative expression of MHC class II molecules at cell surface. We have analyzed sequence variation at AIR-1/CIITA promoters by PCR-SSCP in 23 IDDM and 30 RA patients compared to a sample of 19 unaffected normal controls and 16 unaffected IDDM family members, for a total of 88 Caucasian subjects from the Northeast of Italy. No sequence difference was found at the four AIR-1/CIITA promoters between autoimmune patients and normal controls. Moreover, the promoters resulted invariant within the entire group of 88 subjects analyzed, comprising patients and controls. This finding suggests a possible selective advantage in maintaining CIITA upstream regulatory sequences invariant.
Assuntos
Artrite Reumatoide/genética , Diabetes Mellitus Tipo 1/genética , Genes MHC da Classe II , Proteínas Nucleares , Transativadores/genética , Artrite Reumatoide/imunologia , DNA/análise , Diabetes Mellitus Tipo 1/imunologia , Variação Genética , Humanos , Itália , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Regiões Promotoras GenéticasRESUMO
Productive infection by the LAV strain has been demonstrated in T cell precursors at different stages of intrathymic development, while viral replication in thymic epithelial cells is still controversial. In this article we show that epithelial cell cultures derived from the medullary component of normal thymus are infectable by HTLV-IIIB virus through cell-free and lymphoid-mediated transmission. Free virus inoculum results in the integration of proviral copies undergoing poor replication, whereas lymphoid-mediated transmission leads to substantial viral expression and the production of viral progeny able to secondary infect lymphoid cells. Interleukin 6 production and phenotype changes (increased expression of MHC class I and ICAM-1) were induced in TE cells by contact with free virus or by adhesion to infected lymphoid cells. By contrast, NF-kappa B-binding activity on the HIV-1 LTR kappa B enhancer element was upregulated only by contact with infected lymphoid cells, but not with virus. The viral replication observed in TE cells after lymphoid-mediated transmission correlates with the upregulation of NF-kappa B-binding activity. Interleukin 6 increased production and phenotype changes and increased NF-kappa B-binding activity were also induced by adhesion to uninfected lymphoid cells, demonstrating that lymphoepithelial cell contacts can activate TE cells. These results demonstrate that thymic epithelial cells are permissive to HIV infection and that viral replication in this cell lineage can be modulated by intracellular signals delivered by adhesive contacts.
Assuntos
HIV-1/metabolismo , NF-kappa B/metabolismo , Timo/metabolismo , Adesão Celular , Linhagem Celular , Pré-Escolar , Células Epiteliais , Epitélio/metabolismo , Humanos , Interleucina-6/metabolismo , Fenótipo , Timo/citologia , Timo/virologiaRESUMO
Optimal conditions for in vitro histamine release mediated either by Con A (3 micrograms/ml) or by Ionophore A23187 (1 microgram/ml), were established for mouse peritoneal mast cells and human normal basophils. In both systems N-5' exerts potent inhibiting effects on histamine release after short term (1-5 minutes) in vitro preincubation. At concentrations of 1mM for mouse mast cells and 3mM for normal human basophils, N-5' inhibits up to 95% Con A-induced histamine release and more than 50% ionophore-induced histamine release. Such in vitro effects are more potent than DSCG-mediated inhibition, under similar experimental conditions, and are resistant to challenge with exceeding doses of the two releasing agents, particularly in the Con A system. Interestingly, basophils with apparent normal morphology, from a CML patient, were resistant to both challenges.
Assuntos
Basófilos/efeitos dos fármacos , Liberação de Histamina/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , ortoaminobenzoatos/farmacologia , Animais , Calcimicina/farmacologia , Células Cultivadas , Concanavalina A/farmacologia , Humanos , CamundongosRESUMO
Carbohydrate-deficient transferrin (CDT) is a reliable marker of chronic or repeated alcohol abuse. It indicates a group of isoforms of human transferrin (Tf), the main iron transport serum protein, deficient in sialic acid residues (asialo-, monosialo- and disialo-Tf) in comparison to the main isotransferrin which contains four sialic acid groups (tetrasialo-Tf). The aim of the present work was to develop a capillary electrophoretic method suitable for rapid determination of CDT components in serum. Serum samples (0.1 ml) were saturated with iron by incubation with 10 mM FeCl3 (2 microl) and 500 mM NaHCO3 (3 microl) for 30 min, then diluted 1:10 in water and injected by positive pressure (0.5 p.s.i. for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (57 cm x 20 microm I.D.) and a buffer composed of 100 mM sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 mM diaminobutane. Applied voltage was 20 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were baseline separated. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-Tf, and 0.5% of trisialo-Tf, expressed as percentages of the terasialo-Tf peak area. Day-to-day RSDs of relative migration times were < or = 0.2%. Quantitation showed day-to-day RDSs < or = 6.9% and < or = 10.9% for disialo- and trisialo-Tf, respectively. The results from 79 control subjects, including social drinkers, and 23 alcoholics showed disialo- and trisialo-Tf significantly increased in patients (P<0.0001 and <0.01, respectively). A clear interference from trisialo-Tf in an immunoassay for CDT was demonstrated. The present method is suitable for confirmation of CDT immunoassays by independent technique.
Assuntos
Eletroforese Capilar/métodos , Transferrina/análogos & derivados , Humanos , Imunoensaio/métodos , Controle de Qualidade , Reprodutibilidade dos Testes , Transferrina/análiseRESUMO
The involvement of immunological reactivity to ranitidine base (R-b) and ranitidine hydrochloride (R-HCl) in the development of occupationally related symptomatology was analyzed in 40 subjects employed in a pharmaceutical plant producing ranitidine and in 33 nonexposed controls, using a specific dose-response lymphocyte proliferative test (lymphocyte transformation test: LTT). Of the 40 workers, 11 (28%) gave positive reactions to LTT: 3/11 to R-b, 4/11 to R-HCl, and 4/11 to both compounds. None of the controls gave positive reactions. Cutaneous, oculonasal, or respiratory work-related symptoms were cited by 23 of the 40 (58%) subjects; ten of these 23 subjects (43%) were LTT positive. One asymptomatic case was LTT positive. The present results indicate that specific immune reactivity to ranitidine, analyzed by LTT, is associated with the presence of occupational symptomatology; R-HCl and R-b seem to share some antigenic determinants, because of the partial cross-reactivity shown by the examined compounds. Nonimmunological, probably irritative, mechanisms are also present in some of the symptomatic subjects.
Assuntos
Ativação Linfocitária/efeitos dos fármacos , Doenças Profissionais/induzido quimicamente , Ranitidina/efeitos adversos , Oftalmopatias/induzido quimicamente , Feminino , Humanos , Masculino , Doenças Respiratórias/induzido quimicamente , Fatores de TempoRESUMO
In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represented by both the Burkitt lymphoma line Raji and normal tonsil- derived B cells. However, some peculiar differences were observed between the TEC, either IFN-gamma-induced or not, and the constitutive B cells. These results suggest that both common mechanisms, such as the one mediated by the CIITA trans-activator, and distinct tissue-specific constraints contribute to the transcriptional control of constitutive and IFN-gamma-induced MHC class II gene expression.
Assuntos
Genes MHC da Classe II , Antígenos HLA-DR/genética , Proteínas Nucleares , Regiões Promotoras Genéticas , Transativadores/biossíntese , Linfócitos B/imunologia , Sequência de Bases , Células Cultivadas , Primers do DNA/genética , Células Epiteliais , Epitélio/imunologia , Regulação da Expressão Gênica , Humanos , Interferon gama/farmacologia , Dados de Sequência Molecular , Proteínas Recombinantes , Linfócitos T/imunologia , Timo/citologia , Timo/imunologiaRESUMO
Two monoclonal antibodies (1H6.2 and 45.30) were raised against MBP purified from human brain under experimental conditions that allowed MBP to retain binding to surrounding myelin lipids (human lipid-bound MBP (hLB-MBP)). 1H6.2 and 45.30 MoAbs were selected on the basis of their different binding properties to: hLB-MBP, human lipid-free-MBP (hLF-MBP) and bovine lipid-free-MBP (bLF-MBP). Although the isotype of both MoAbs was IgM, their specificity, as tested in ELISA assays against chemical haptens and unrelated protein antigens, was restricted to MBP. 1H6.2 and 45.30 MoAbs stained MBP from human brain white matter tissue extracts, as well as bLF-MBP, in Western blot assays. Both MoAbs stained oligodendrocytes and myelin in immunohistochemical analysis of white matter from human brain. Tissue sections from human peripheral nerves were labelled by 1H6.2 only, however, demonstrating that the MoAbs recognize two different epitopes. Epitopes recognized by 1H6.2 and 45.30 MoAbs were also expressed by a wide array of human non-neural cells of either normal or pathological origin, as evidenced by cytofluorimetric assays. In particular, MBP epitopes (MEs) were expressed by lymphoid cells as well as by cells which play a pivotal role in immune homeostasis and in the immune response, such as thymic epithelial cells and professional antigen-presenting cells. Both MoAbs were efficiently internalized by cells from a human B cell line, suggesting trafficking of MEs along the endocytic pathways. These findings support hypotheses regarding the role of MEs expressed by non-neural cells in establishing self-tolerance and/or in triggering the immune response against MBP antigen.