RESUMEN
Ultrafast multiphoton ionization of Xe in strong extreme ultraviolet free-electron laser (FEL) fields (91 eV, 30 fs, 1.6×10^{12} W/cm^{2}) has been investigated by multielectron-ion coincidence spectroscopy. The electron spectra recorded in coincidence with Xe^{4+} show characteristic features associated with two-photon absorption to the 4d^{-2} double core-hole (DCH) states and subsequent Auger decay. It is found that the pathway via the DCH states, which has eluded clear identification in previous studies, makes a large contribution to the multiple ionization, despite the long FEL pulse duration compared with the lifetime of the 4d core-hole states.
RESUMEN
AIMS: Urinary tract infection (UTI) is a common clinical problem in diabetic patients; however, the relationship between UTI and glucosuria remains uncertain. To investigate the relationship, we examined the effect of glucosuria induced by sodium glucose cotransporter 2 (SGLT2) inhibitors on the progression of UTI in mice. METHODS: From 1 day before transurethral inoculation with Candida albicans, female mice were treated orally once a day with an SGLT2 inhibitor in different treatment regimens: (i) dapagliflozin at 10 mg/kg for 2, 3 or 7 days, (ii) dapagliflozin at 0.1, 1 or 10 mg/kg for 3 days and (iii) dapagliflozin, canagliflozin or tofogliflozin at 10 mg/kg for 3 days. To evaluate the ascending UTI, the kidneys were removed 6 days after the inoculation, and the number of viable C. albicans cells in kidney was measured as colony-forming units (CFU). RESULTS: In mice treated with dapagliflozin, the number of C. albicans CFU in kidney increased in accordance with both treatment duration and dose. The number of CFU significantly increased when mice were treated with 10 mg/kg dapagliflozin or canagliflozin but not tofogliflozin. With dapagliflozin and canagliflozin, urine glucose concentration (UGC) significantly increased up to 24 h after drug administration; with tofogliflozin, UGC significantly increased only up to 12 h after drug administration. CONCLUSIONS: Our data indicate that increased susceptibility to UTI is associated with a persistent increase in UGC.
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Compuestos de Bencidrilo/farmacología , Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Glucósidos/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Tiofenos/farmacología , Infecciones Urinarias/tratamiento farmacológico , Animales , Canagliflozina , Progresión de la Enfermedad , Femenino , Glucosuria/microbiología , Riñón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Infecciones Urinarias/microbiologíaRESUMEN
The cellular basis of the in vitro and in vivo T cell responses to Staphylococcus enterotoxin B (SEB) has been investigated. The proliferation and cytotoxicity of V beta 8.1,2+,CD4+ and CD8+ T cells were observed in in vitro response to SEB. In primary cytotoxicity assays, CD4+ T cells from control spleens were more active than their CD8+ counterparts, however, in cells derived from SEB-primed mice, CD8+ T cells were dominant in SEB-specific cytotoxicity. In vivo priming with SEB abrogated the response of V beta 8.1,2+,CD4+ T cells despite the fact that these cells exist in significant number. This SEB-specific anergy occurred only in V beta 8.1,2+,CD4+ T cells but not in CD8+ T cells. These findings indicate that the requirement for the induction of antigen-specific anergy is different between CD4+ and CD8+ T cells in post-thymic tolerance, and the existence of coanergic signals for the induction of T cell anergy is suggested.
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Linfocitos T CD4-Positivos/inmunología , Enterotoxinas/farmacología , Receptores de Antígenos de Linfocitos T/análisis , Staphylococcus aureus , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Citotoxicidad Inmunológica , Interleucina-2/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Bazo/inmunologíaRESUMEN
We prepared several monoclonal antibodies (mAbs) specific for the NH2- and COOH-terminal regions of the DNA helicase (WRN helicase) responsible for Werner's syndrome known as a premature aging disease. With these antibodies, we detected by immunoblot analysis the endogenous WRN helicase of a relative mass of 180 kD in several lines of cultured cells, but not in patient cells with a defined mutation. Immunocytochemical staining of proliferating fibroblasts and tumor cells showed that the major part of WRN helicase is in the nucleoplasm and not in the nucleolus. Similar experiments with a rat mAb specific to the mouse homologue of human WRN helicase yielded an identical conclusion. Although this nucleoplasmic staining was evident in cells in interphase, the condensed chromatin structure in metaphase was not stained by the same mAbs, suggesting that WRN helicases exist perhaps in a soluble form or bound to the unfolded chromatin structure. From quantitative immunoblot analysis, higher levels of WRN helicase were observed in all transformed cells and tumor cells examined than those of normal cells. The expression of WRN helicase was enhanced consistently in fibroblasts and B-lymphoblastoid cells by transformation with SV-40 and Epstein-Barr virus, respectively, suggesting that rapidly proliferating cells require a high copy numbers of WRN helicase.
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Transformación Celular Viral/fisiología , ADN Helicasas/inmunología , Epítopos de Linfocito B/inmunología , Regulación hacia Arriba , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Transformada , Células Cultivadas , ADN Helicasas/genética , Mapeo Epitopo , Epítopos de Linfocito B/genética , Fibroblastos/citología , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Wistar , Células Tumorales Cultivadas , Helicasa del Síndrome de WernerRESUMEN
Essentials Emicizumab (Emi) affects the APTT-based assays of factor (F)VIII activity and inhibitor titer. A mixture of two anti-Emi monoclonal antibodies (mAb) effectively neutralized the Emi activity. Anti-Emi mAbs completely eliminated the influence of Emi on FVIII activity and inhibitor titer. The inclusion of anti-Emi mAbs in routine FVIII assays would be useful for Emi-treated patients. SUMMARY: Background Emicizumab is an anti-factor (F)IXa/X bispecific monoclonal antibody (mAb), mimicking the factor (F)VIIIa cofactor activity. Emicizumab does not require activation by thrombin and its shortening effect on the activated partial prothrombin time (APTT) is more pronounced than that of factor (F)VIII. APTT-based FVIII activity (FVIII:C) and FVIII inhibiter titer measurements are influenced by the presence of emicizumab. Aim To establish a reliable APTT-based assay to measure FVIII in the presence of emicizumab. Methods Plasmas from hemophilia A (HA) patients without or with inhibitors were studied using one-stage FVIII:C and Bethesda inhibitor assays. Two recombinant anti-idiotype mAbs to emicizumab (anti-emicizumab mAbs) were prepared, rcAQ8 to anti-FIXa-Fab and rcAJ540 to anti-FX-Fab. Results The combined anti-idiotype mAbs (2000 nm each) eliminated the effects of emicizumab on APTTs of HA plasmas without or with inhibitor by competitive inhibition of antibody binding to FIX(a)/FX(a). Measurements of FVIII coagulation activity in HA plasmas without inhibitor were overestimated in the presence of emicizumab (1 µm = ~150 µg mL-1 ) at all reference levels of FVIII. The addition of anti-emicizumab mAbs to the assay mixtures completely neutralized the emicizumab and facilitated accurate determination of FVIII:C. Anti-FVIII inhibitor titers were undetectable in the presence of emicizumab in HA plasmas with inhibitor or normal plasmas mixed with anti-FVIII neutralizing antibodies. These effects of emicizumab were completely counteracted by the addition of the anti-idiotype mAbs, allowing accurate assessment of inhibitor titers. Conclusion The in vitro inclusion of anti-emicizumab mAbs in the standard one-stage coagulation assays prevented interference by emicizumab and enabled accurate measurements of FVIII:C and inhibitor titers.
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Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Neutralizantes/sangre , Coagulación Sanguínea/efectos de los fármacos , Coagulantes/farmacología , Factor VIII/análisis , Hemofilia A/sangre , Tiempo de Tromboplastina Parcial , Anticuerpos Biespecíficos/sangre , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales Humanizados/sangre , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Neutralizantes/inmunología , Unión Competitiva , Coagulantes/sangre , Coagulantes/inmunología , Relación Dosis-Respuesta a Droga , Factor IXa/inmunología , Factor IXa/metabolismo , Factor VIII/inmunología , Factor Xa/inmunología , Factor Xa/metabolismo , Hemofilia A/diagnóstico , Hemofilia A/inmunología , Humanos , Valor Predictivo de las Pruebas , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
FK506 is an immunosuppressive drug that inhibits T cell receptor-mediated signal transduction. This drug can induce immunological tolerance in allograft recipients. In this study, we investigated the in vivo effects of FK506 on T cell receptor-mediated apoptosis induction. Injection of anti-CD3 antibody (Ab) in mice resulted in the elimination of CD4+ CD8+ thymocytes by DNA fragmentation. FK506 treatment significantly augmented thymic apoptosis induced by in vivo anti-CD3 Ab administration. Increased thymic apoptosis resulted in the disappearance of CD4+ CD8+ thymocytes after anti-CD3 Ab/FK506 treatment. DNA fragmentation triggered by FK506 was induced exclusively in antigen-stimulated T cells, since enhanced DNA fragmentation induced by in vivo staphylococcal enterotoxin B (SEB) injection was confirmed in SEB-reactive V beta 8+ thymocytes but not in SEB-nonreactive V beta 6+ thymocytes. In addition to thymocytes, mature peripheral T cells also die by activation-induced programmed cell death. A similar effect of FK506 on activation-induced programmed cell death was observed in SEB-activated peripheral spleen T cells. In contrast, cyclosporin A treatment did not enhance activation-induced programmed cell death of thymocytes and peripheral T cells. Apoptosis is required for the generation and maintenance of self-tolerance in the immune system. Our findings suggest that FK506-triggered apoptosis after elimination of antigen-activated T cells may represent a potential mechanism of the immunological tolerance achieved by FK506 treatment.
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Apoptosis/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Activación de Linfocitos/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Subgrupos de Linfocitos T/efectos de los fármacos , Tacrolimus/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Complejo CD3/inmunología , Ciclosporina/farmacología , Daño del ADN , Enterotoxinas/inmunología , Enterotoxinas/farmacología , Ratones , Ratones Endogámicos BALB C , Superantígenos/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The present study was performed to clarify the relationship between human T cell lymphotropic virus type I (HTLV-I) infection and chronic inflammatory arthropathy. To determine the ability of HTLV-I to infect synovial cells and the effect on synovial cell proliferation, synovial cells were cocultured with the HTLV-I-producing T cell lines (MT-2 or HCT-1). After coculture with HTLV-I-infected T cells, the synovial cells expressed HTLV-I-specific core antigens, and HTLV-I proviral DNA was detected from the synovial cells by polymerase chain reaction. These cocultured synovial cells with HTLV-I-infected T cells proliferated more actively than the synovial cells cocultured with uninfected T cells. This stimulatory effect of HTLV-I-infected T cells on synovial cell proliferation seems necessary to contact each other. After being cocultured with MT-2 cells, synovial cells proliferated more actively than control cells even after several passages. Furthermore, HTLV-I-infected synovial cells produced significant amounts of granulocyte/macrophage colony-stimulating factor. These results suggest that HTLV-I can infect synovial cells, resulting their active proliferation and may be involved in the pathogenesis of proliferative synovitis similar to that found in rheumatoid arthritis.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Virus Linfotrópico T Tipo 1 Humano/fisiología , Membrana Sinovial/citología , Membrana Sinovial/metabolismo , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , División Celular , Línea Celular , Células Cultivadas , Medios de Cultivo Condicionados , ADN Viral/análisis , Técnica del Anticuerpo Fluorescente , Productos del Gen gag/análisis , Productos del Gen gag/biosíntesis , Humanos , Técnicas para Inmunoenzimas , Inmunohistoquímica , Mitomicina/farmacología , Reacción en Cadena de la Polimerasa/métodos , Provirus/fisiología , Membrana Sinovial/efectos de los fármacos , Linfocitos T , Replicación ViralRESUMEN
OBJECTIVE: To evaluate the efficacy and safety of leukocytapheresis (LCAP) in patients with rheumatoid arthritis (RA) that is refractory to disease modifying antirheumatic drugs (DMARDs), we conducted a prospective, multicenter, open-label clinical trial. METHODS: We enrolled 38 active RA patients, including 32 patients who showed an inadequate response to > or = 2 DMARDs and 6 patients with rapidly progressive RA. All patients continued drug therapy and were treated with 5 LCAP sessions conducted at 1-week intervals. The clinical response was evaluated at baseline before starting LCAP and at 4 weeks after the completion of all the LCAP sessions using the American College of Rheumatology (ACR) criteria and the 28-joint disease activity score (DAS28) of the European League Against Rheumatism (EULAR). RESULTS: Of the 35 patients who fulfilled the study's eligibility criteria, 24 (69%), 10 (29%), and 23 (66%) patients achieved 20% (ACR20), 50% (ACR50), and DAS28-C-reactive protein (CRP) EULAR improvement, respectively. The mean DAS28-CRP score of the 35 patients decreased significantly from 5.99 +/- 0.92 at baseline to 4.54 +/- 1.39 after treatment. Comparison analysis of the ACR20 responders and non-responders to LCAP revealed that 22 of 24 responders (92%) concomitantly received methotrexate, whereas significantly fewer, that is, 6 of 11 non-responders (55%) received methotrexate. Less frequent and transient mild-to-moderate adverse events, including nausea and headache, were seen in 12 of 189 LCAP sessions (6.3%). CONCLUSION: These results demonstrate the usefulness of LCAP in combination with DMARDs, particularly methotrexate, as an effective and safe treatment for refractory RA.
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Artritis Reumatoide/terapia , Leucaféresis , Adulto , Anciano , Antirreumáticos/uso terapéutico , Resistencia a Medicamentos , Femenino , Humanos , Leucaféresis/métodos , Masculino , Metotrexato/uso terapéutico , Persona de Mediana Edad , Estudios Prospectivos , Resultado del TratamientoRESUMEN
To clarify the role of the sterol regulatory element-binding protein-2 (SREBP-2), we established cell lines in which human SREBP-2(1-481) could be induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The range of IPTG-induced changes in SREBP-2(1-481) levels in '23-11' cells, one of these cell lines, was almost the same as that of sterol-induced changes in the levels of mature SREBP-2, indicating that IPTG was able to regulate the expression of SREBP-2(1-481) within the normal physiological range in this cell line. Sterols regulate the expression of the LDL receptor, HMG-CoA reductase, squalene synthase and fatty acid synthase in 23-11 cells as they also do in the parental cell line HeLa S3. IPTG increased mRNA levels of the LDL receptor and HMG-CoA reductase but not squalene synthase both in the presence or absence of excess sterols. Fatty acid synthase mRNA was increased 2 h after the IPTG addition in the absence of excess sterol (10% FBS), but was slightly increased 6 h after the IPTG addition in the presence of excess sterols. In the absence of excess sterols, both SREBP-2(1-481) and endogenous mature SREBP-2 exist in the nucleus. This suggests that an increased amount of SREBP-2 over the normal physiological range is required for the regulation of fatty acid synthase. IPTG increased both the surface binding of 125I-LDL and cholesterol biosynthesis from [14C]acetate significantly in a similar time course. In contrast, fatty acid biosynthesis from [14C]acetate was almost unchanged by IPTG during the same incubation period. These results suggest that physiological amounts of SREBP-2 play a key role in the regulation of cholesterol but not fatty acid metabolism.
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Proteínas de Unión al ADN/fisiología , Factores de Transcripción/fisiología , Línea Celular , Colesterol/metabolismo , Proteínas de Unión al ADN/genética , Farnesil Difosfato Farnesil Transferasa/genética , Ácido Graso Sintasas/genética , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Isopropil Tiogalactósido/farmacología , Metabolismo de los Lípidos , Lipoproteínas LDL/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Esteroles/farmacología , Factores de Transcripción/genética , TransfecciónRESUMEN
The mechanisms of TSH-induced growth stimulation of thyrocytes in vivo have yet to be elucidated. We examined the antiapoptotic effect of TSH toward Fas antigen-mediated apoptosis of thyrocytes. Fas antigen was expressed on approximately 40% of unstimulated thyrocytes, and the expression was significantly inhibited by the addition of TSH in a dose-dependent manner. Treatment of thyrocytes with 8-bromo-cAMP mimicked the effect of TSH, suggesting that the inhibitory effect of TSH on Fas antigen expression was mediated by activating protein kinase A. In contrast, treatment of thyrocytes with either interleukin-1 beta (IL-1 beta) or interferon- gamma (IFN gamma) markedly increased Fas antigen expression on thyrocytes, and these effects were inhibited in the presence of TSH. The expression of the protooncogene product Bcl-2 did not change after the addition of TSH, 8-bromo-cAMP, IL-1 beta, IFN gamma, or a combination of TSH and IL-1 beta or IFN gamma. When thyrocytes stimulated with either IL-1 beta or IFN gamma were treated with anti-Fas IgM mAb, the cells were committed to apoptosis, whereas this apoptotic process was significantly inhibited by the addition of TSH. These results indicate that the Fas antigen is functionally expressed on the surface of thyrocytes, and TSH inhibits Fas antigen-mediated apoptosis of thyrocytes through the inhibitory effect of Fas antigen expression, resulting in the promotion of growth of the thyroid gland.
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Apoptosis/efectos de los fármacos , Glándula Tiroides/fisiología , Tirotropina/farmacología , Receptor fas/fisiología , Anticuerpos Monoclonales/inmunología , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Glándula Tiroides/citología , Receptor fas/inmunologíaRESUMEN
This study was undertaken to investigate the natural killer (NK) and natural killer-like (NK-like) cell cytotoxic activity toward autologous thyrocytes of peripheral blood mononuclear cells (PB-MNC) and thyroid gland mononuclear cells (TG-MNC) from previously hyperthyroid patients with Graves' disease, and the effects of recombinant interleukin-2 on such cytotoxic activity. The average cytotoxicities of PB-MNC from Graves' patients toward K562 cells (NK-sensitive cells), Raji cells (NK-resistant cells), and autologous thyrocytes were 23.9 +/- 10.8 (+/- SD) lytic units (LU), 7.4 +/- 3.8 LU, and 11.7 +/- 4.4 LU, respectively. There were no differences in the NK and NK-like cell activity of PB-MNC between Graves' disease patients and normal subjects. In contrast to PB-MNC from patients with Graves' disease, NK and NK-like cell activity was markedly decreased in TG-MNC (NK cell activity, 2.1 +/- 2.3 LU; NK-like cell activity, 1.5 +/- 1.5 LU). TG-MNC from Graves' patients had no cytotoxic activity against autologous thyrocytes. Using the monoclonal anti-Leu 7 and anti-CD16 antibodies and a two-color immunofluorescence method, the NK cell subsets were examined in PB-MNC and TG-MNC from Graves' patients. The percentage of CD16+ cells was significantly decreased in TG-MNC compared to PB-MNC, whereas there was no significant difference in the percentage of Leu 7+ cells between PB-MNC and TG-MNC. Incubation of TG-MNC with medium only did not increase the NK and NK-like cell activity of these cells. Furthermore, incubation of autologous PB-MNC with supernatants of minced thyroid tissues did not alter their NK and NK-like cell activity. The decreased NK and NK-like cell activity of TG-MNC was augmented when these cells were incubated with recombinant interleukin-2. These results suggest that the reduction of NK cell activity in TG-MNC may allow perpetuation of B-cell activation and lead to excessive production of autoantibody in thyroid tissue.
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Citotoxicidad Inmunológica , Enfermedad de Graves/inmunología , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/inmunología , Glándula Tiroides/citología , Adolescente , Adulto , Femenino , Enfermedad de Graves/sangre , Humanos , Interleucina-2/farmacología , Recuento de Leucocitos , MasculinoRESUMEN
The expression of phenotypic markers and Concanavalin-A-induced suppressor activity was compared among mononuclear cells isolated from thyroid glands and peripheral blood of thionamide-treated patients with hyperthyroid Graves' disease and peripheral blood from normal subjects. Intrathyroidal lymphocytes were obtained by two different methods (TG-1 and TG-2 cells), gradient centrifugation of supernatants of minced thyroid tissue and overnight culture of thyroid debris after mechanical disaggregation and enzymatic digestion, respectively. The percentages of CD3+ cells (all mature T cells) among peripheral blood and TG-1 and TG-2 cells from Graves' patients were similar, but the percentages of B1+ cells (pan B cells) among the TG-1 and TG-2 cells were markedly increased compared to that in peripheral blood. The percentages of CD4+ cells among the TG-1 and TG-2 cells were significantly less than that in peripheral blood. The percentages of CD4+2H4+ cells among CD4+ cells in TG-1 and TG-2 cells also were significantly less than that in peripheral blood. The percentage of CD4+4B4+ cells among CD4+ cells in thyroid glands was markedly higher than that in peripheral blood. The percentages of CD8+ cells and CD8+CD11b- cells (cytotoxic T cells) in thyroid glands were significantly higher than those in peripheral blood from Graves' patients and peripheral blood from normal subjects. The CD8+CD11b+ cells were subdivided into two subpopulations on the basis of CD8 antigen density. The percentage of dull CD8+CD11b+ cells (natural killer cells) among TG-2 cells was lower than that in peripheral blood, but there was no significant difference in bright CD8+CD11b+ cells (suppressor-effector T cells) between thyroid glands and peripheral blood. The percent suppression induced by Concanavalin-A in both TG-1 and TG-2 cells was significantly decreased compared with that in peripheral blood. These results suggest that impairment of suppressor cell activity and an increased number of B cells exist in thyroid glands of patients with Graves' disease compared to those in peripheral blood. It, thus, appears likely that both B cell hyperactivity and suppressor T cell dysfunction may induce excess production of autoantibodies in the thyroid glands of such patients.
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Enfermedad de Graves/inmunología , Linfocitos T Reguladores/fisiología , Glándula Tiroides/inmunología , Adolescente , Adulto , Anticuerpos Monoclonales , Concanavalina A/farmacología , Femenino , Citometría de Flujo , Humanos , Masculino , Fenotipo , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
This study was undertaken to determine the effects of interleukin-1 (IL-1) on human thyroid epithelial cells (thyrocytes) and whether thyrocytes produce IL-1. The supernatants of cultured peripheral blood monocytes stimulated with lipopolysaccharide (LPS) increased [3H]thymidine incorporation into thyrocytes from normal subjects and patients with Grave's disease. The IL-1 levels of cultured supernatants of monocytes were measured by a thymocyte costimulation assay and a solid phase sandwich immunoenzymometric assay. The supernatants of monocyte cultures stimulated with LPS contained significant amounts of IL-1 bioactivity and IL-1 alpha and IL-1 beta immunoactivity. Recombinant IL-1 beta (rIL-1 beta) also stimulated [3H]thymidine incorporation into thyrocytes from normal subjects and patients with Graves' disease, and it increased the proportion of thyrocytes in the S phase of the cell cycle. Furthermore, thyrocytes stimulated with rIL-1 beta for 24 h produced significant amounts of prostaglandin E2. Indomethacin inhibited completely the rIL-1 beta-stimulated prostaglandin E2 production and increased markedly [3H]thymidine incorporation. IL-1-like activity also was detected in the cultured supernatants of lipopolysaccharide (LPS)-stimulated thyrocytes from Graves' and normal thyroid glands, but the amount of IL-1-like activity secreted by thyrocytes was significantly less than that secreted by circulating monocytes. The kinetics of the release of IL-1-like activity by thyrocytes were similar to those of its production by circulating monocytes. Pretreatment of thyrocytes with interferon-gamma failed to enhance the release of IL-1-like activity. Moreover, IL-1 alpha or IL-1 beta immunoreactivity could not be detected in the supernatants of LPS-stimulated thyrocytes, despite the presence of IL-1-like bioactivity. No IL-1 alpha mRNA was detected in unstimulated thyrocytes or thyrocytes stimulated with LPS and phorbol myristate acid. These findings demonstrate that thyrocytes produce an IL-1-like substance(s), but not IL-1, when stimulated by LPS. We conclude that IL-1 may regulate the proliferation of thyrocytes and that local production of IL-1 by infiltrating monocytes may contribute to the development of goiter in patients with autoimmune thyroid diseases.
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Interleucina-1/biosíntesis , Glándula Tiroides/metabolismo , Disponibilidad Biológica , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Dinoprostona/biosíntesis , Enfermedad de Graves/metabolismo , Humanos , Técnicas para Inmunoenzimas , Indometacina/farmacología , Interleucina-1/farmacocinética , Interleucina-1/fisiología , Lipopolisacáridos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Timidina/metabolismo , Glándula Tiroides/efectos de los fármacosRESUMEN
The expression of surface markers associated with activation and characterization was compared among T cells in thyroid glands and peripheral blood of 10 patients with Graves' hyperthyroidism receiving chronic antithyroid drug therapy, in peripheral blood of 15 patients with untreated hyperthyroid Graves' disease, and in peripheral blood of 21 normal subjects using two-color flow cytometry. In the chronically treated Graves' disease patients, the percentage of activated T cells (HLA-DR+ T cells) among total T cells was significantly higher in thyroid tissue than in peripheral blood, and the increase in percent activated T cells was also significant among both helper/inducer T cell (CD4+ cell) and suppressor/cytotoxic T cell (CD8+ cell) subsets. The percentage of activated T cells in peripheral blood was not significantly different between chronically treated hyperthyroid Graves' patients and normal subjects, whereas the percentage of activated T cells in the peripheral blood from untreated hyperthyroid Graves' disease patients was significantly higher than that in normal subjects or chronically treated hyperthyroid Graves' patients. The percentages of CD4+ cells and CD8+ cells among total T cells were not different between thyroid tissues and peripheral blood in patients with chronically treated hyperthyroid Graves' disease. When CD4+ were further divided into helper T cells (CD4+2H4- cells) and suppressor-inducer T cells (CD4+2H4+ cells) using two-color flow cytometry, the percentage of helper T cells among CD4+ cells was significantly higher in thyroid tissue than in peripheral blood, resulting in an increased ratio of CD4+2H4- cells to CD4+2H4+ cells. The percentage of CD4+2H4+ cells in peripheral blood, however, was not significantly different among untreated and chronically treated Graves' disease patients and normal subjects. From the findings of abnormalities in intrathyroidal T cell subsets, we suggest that the decrease in the function of suppressor T cells within the thyroids of Graves' disease patients may be due to a decrease in CD4+2H4+ cells within thyroid tissue.
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Enfermedad de Graves/inmunología , Linfocitos T Colaboradores-Inductores , Linfocitos T Reguladores , Glándula Tiroides/inmunología , Adolescente , Adulto , Anticuerpos Monoclonales , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Recuento de LeucocitosRESUMEN
To investigate the suppressor function of intrathyroidal (TG) T cells in Graves' disease, the percentage of suppressor T cell subsets and the suppressor function of TG and peripheral blood (PB) lymphocytes in Graves' disease were compared by determining the in vitro production of immunoglobulin G (IgG) in reconstituted mixtures of separated B, CD4+ (helper/suppressor-inducer T), and CD8+ (suppressor/cytotoxic T) cells. TG lymphocytes were obtained by gradient centrifugation of the supernatants of minced thyroid tissues. T Cells were separated by E-rosette formation, and CD4+ and CD8+ cell-rich populations were separated by a panning method using monoclonal anti-CD8 antibody. Mixtures of 5 X 10(4) B (PB1 or TG) cells, 2 X 10(4) CD4+ (PB or TG) cells, and 5 X 10(3) macrophages (PB or TG) were cultured with various numbers of CD8+ (PB) or CD8+ (TG) cells for 7 days with pokeweed mitogen, and IgG synthesis was determined by solid phase RIA. T cell subpopulations were quantitated by a direct immunofluorescence method using monoclonal anti-CD3, anti-CD4, and anti-CD8 antibodies. There was no difference in the percentages of CD8+ cells among total T cells between thyroid glands and peripheral blood from Graves' disease patients [mean PB, 39.8 +/- 9.8% (+/- SD); TG, 42.5 +/- 13.8%; n = 10]. IgG production by mixtures of B and CD4+ cells isolated from peripheral blood was not different from that by cells isolated from thyroid glands [mean PB, 1635 +/- 248 (+/- SE) ng/mL; TG, 1081 +/- 128 ng/mL; n = 19; P = NS]. The nonspecific suppressor activity of thyroid gland CD8+ cells was less than that of CD8+ (PB) cells [percent suppression of IgG production by mixtures of B (PB) and CD4+ (PB) cells, 12.5% vs. 57.0% (P less than 0.01); by mixtures of B (TG) and CD4+ (TG) cells, 5.8% vs. 38.9% (P less than 0.01)]. The suppressor-inducer function of CD4+ (TG) cells was also decreased compared with that of CD4+ (PB) cells. These results suggest that the impairment of suppressor cell activity may lead to excessive production of autoantibody in thyroid glands from patients with Graves' disease.
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Enfermedad de Graves/fisiopatología , Linfocitos T Reguladores/fisiología , Glándula Tiroides/fisiopatología , Adulto , Linfocitos B/metabolismo , Linfocitos B/fisiología , Células Sanguíneas/metabolismo , Células Sanguíneas/fisiología , Femenino , Enfermedad de Graves/metabolismo , Enfermedad de Graves/patología , Humanos , Inmunoglobulina G/biosíntesis , Masculino , Mitógenos de Phytolacca americana/farmacología , Linfocitos T/clasificación , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/fisiología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Colaboradores-Inductores/fisiología , Linfocitos T Reguladores/metabolismo , Glándula Tiroides/metabolismo , Glándula Tiroides/patologíaRESUMEN
Thyroid-infiltrating B lymphocytes from patients with Graves' disease were investigated in regard to their phenotypic profiles, cell size, cell cycle status, proliferative response to Staphylococcus aureus Cowan 1 (SAC), and spontaneous production of immunoglobulin G (IgG) and antithyroidal autoantibodies. Thyroid tissues and peripheral blood were obtained at the time of subtotal thyroidectomy of 27 Graves' patients who had been treated with thionamide drugs and iodide before operation. Two intrathyroidal mononuclear cell populations were obtained from these thyroid tissues. One cell population was isolated from the supernatants after mechanical disaggregation of the tissues and was defined as TG-1 cells. Another cell population, defined as TG-2 cells, was isolated from the supernatants of overnight cultures of the thyroid debris after enzymatic digestion. The percentages of B lymphocytes bearing activated markers and plasma cells (CD20+CD21-, IgM+IgD-, CD20+ transferrin receptor+, PCA-1+) were significantly higher in the TG-1 and TG-2 cell populations than in peripheral blood from Graves' disease patients and normal subjects. These phenotypic changes were accompanied by increased thyroid gland B lymphocyte cell size from patients with Graves' disease. The proliferative response of B lymphocytes to SAC was markedly lower in TG-1 and TG-2 cell populations than in peripheral blood cells from Graves' disease patients and normal subjects. B lymphocytes isolated from thyroid glands secreted significantly more IgG and antithyroidal autoantibodies than those from peripheral blood. Based on the findings of abnormalities in thyroid-infiltrating B lymphocytes, we suggest that activated B lymphocytes may induce the excessive production of antithyroidal autoantibodies in thyroid glands from patients with Graves' disease.
Asunto(s)
Linfocitos B/inmunología , Enfermedad de Graves/inmunología , Glándula Tiroides/inmunología , Adulto , Autoanticuerpos/biosíntesis , Ciclo Celular , Células Cultivadas , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
The fruiting body of Polysphondylium pallidum is composed of whorls of branches along the axis of a central stalk. In the course of fruiting body formation, the interval between neighboring whorls, and the number and the spacing of branches in a whorl are highly regulated. In this study, using the REMI (restriction-enzyme-mediated integration) insertional mutagenesis method, we obtained a mutant (strain M2323) with longer branches than those of the wild-type strain PN500. The sequence analyses revealed the presence of an ORF of 206 aa residues (23 kDa) near the vector insertion site. Disruption of the gene, lbrA (long branch A), by homologous recombination causes the same phenotype as that of M2323. A lbrA transcript is expressed maximally at the early aggregation stage in the parental strain, but is not detectable in the REMI mutant. A homology search showed that LbrA is a member of the p24 family proteins, which have been proposed to function as receptors for cargo proteins that are transported by COP I- (coat protein I) and/or COP II-coated vesicles between the endoplasmic reticulum and the Golgi complex. As far as we know, this is the first paper to show that a p24 family member is implicated in morphogenesis.
Asunto(s)
Dictyosteliida/genética , Proteínas de la Membrana/genética , Orgánulos/metabolismo , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Enzimas de Restricción del ADN/metabolismo , ADN Protozoario/química , ADN Protozoario/genética , Dictyosteliida/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Genes Protozoarios/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Morfogénesis , Mutagénesis Insercional , Mutación , Proteínas Protozoarias/fisiología , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: FK506 and glucocorticoids are used for allograft rejection, graft-versus-host disease, and autoimmune diseases. MATERIALS: We investigated the combined effect of FK506 and glucocorticoids on T-cell apoptosis. RESULTS: Dexamethasone injection in mice reduced the number of CD4+CD8+ thymocytes by increasing DNA fragmentation. Pretreatment with FK506 significantly augmented thymocyte DNA fragmentation induced by dexamethasone injection. Increased thymic apoptosis resulted in the disappearance of CD4+CD8+ thymocytes after FK506/dexamethasone injection. In addition to thymocytes, mature human peripheral blood T cells undergo apoptosis by exposure to dexamethasone in vitro. FK506 synergistically enhanced dexamethasone-mediated apoptosis of human peripheral blood T cells. CONCLUSIONS: Thus, our results showed that FK506 enhanced dexamethasone-induced apoptosis of T cells in vivo and in vitro. This interaction may enhance the therapeutic immunosuppression achieved by these two drugs.
Asunto(s)
Apoptosis/efectos de los fármacos , Dexametasona/farmacología , Inmunosupresores/farmacología , Linfocitos T/efectos de los fármacos , Tacrolimus/farmacología , Adulto , Animales , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Ratones , Ratones Endogámicos BALB C , Linfocitos T/citologíaRESUMEN
BACKGROUND: FK506 is a clinically effective immunosuppressive agent and promoter of immunologic tolerance. However, limited information is available about the mechanism of FK506-induced immunosuppression. METHODS: In the present study, we investigated the molecular mechanism of FK506-mediated enhancement of apoptosis using in vivo activated T lymphocytes. We examined the effects of FK506 on apoptosis-related proteins in superantigen-stimulated peripheral T cells. RESULTS: Injection of staphylococcal enterotoxin B (SEB) into BALB/c mice resulted in a selective apoptosis of splenic Vbeta8-positive T cells after 48 hr. Injection of FK506 within 36 hr of SEB injection resulted in a marked enhancement of DNA fragmentation of splenic Vbeta8+ T cells. FK506 did not affect the expression of Fas antigen on SEB-activated Vbeta8+ T cells. As Bcl-2-related proteins are involved in apoptotic process, we also evaluated their role by examining the expression of Bcl-2, Bcl-X(L), and Bax on SEB-FK506-treated murine splenic T cells. Although SEB injection slightly increased the expressions of Bcl-2 and Bax on V138+ T cells, FK506 did not modulate Bcl-2 or Bax expression in these cells. In contrast, the expression of Bcl-x(L) on Vgamma8+ T cells, which was markedly induced by SEB, was abrogated by FK506. CONCLUSIONS: Our findings indicate FK506-induced enhancement of apoptosis of activated T cells is mediated by down-regulation of Bcl-X(L) expression on these cells. Our results also suggest that Bcl-x(L) is a critical determinant of apoptosis of activated T cell and may represent a potential target for new therapies designed to achieve immunological tolerance.
Asunto(s)
Apoptosis/efectos de los fármacos , Enterotoxinas/farmacología , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Superantígenos/farmacología , Linfocitos T/efectos de los fármacos , Tacrolimus/farmacología , Animales , Apoptosis/fisiología , Fragmentación del ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Ratones , Ratones Endogámicos BALB C , Staphylococcus aureus/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Proteína bcl-XRESUMEN
High-dose steroid pulse therapy is effective in transplant rejection and severe autoimmune diseases. Our goal was to identify the mechanism by which high-dose steroid exerts specific immunosuppressive actions. In this study, we investigated the in vivo effects of high-dose (1 g) methylprednisolone infusion on peripheral blood T lymphocyte apoptosis induction in 15 patients with severe autoimmune diseases. DNA fragmentation was detected in peripheral blood T cells isolated from these patients after 2 and 4 hr of steroid infusion. In contrast, T cells isolated from the same patients before or 8 or more hours after infusion did not show DNA fragmentation. DNA fragmentation was more significant in CD4+ than CD8+ T cells. The susceptibility of CD4+ T cells to apoptosis was associated with a lower expression of Bcl-2 in these cells compared with that on CD8+ T cells. To support the T-cell apoptosis induction by pulse therapy, peripheral blood T cells from normal subjects underwent DNA fragmentation after in vitro exposure to 2.5-10 microg/ml of methylprednisolone for 30 min. Our results indicate that induction of peripheral blood T-cell apoptosis is an important mechanism contributing to the immunosuppression observed after high-dose steroid therapy.