RESUMEN
BACKGROUND AND OBJECTIVES: Anti-CD38 monoclonal antibodies, including daratumumab and isatuximab, often interfere with pretransfusion testing. Dithiothreitol (DTT) treatment of red blood cells (RBCs) negates this interference. However, the optimum DTT concentration and treatment time have not been well defined. Here, we quantified CD38 on RBCs before and after DTT treatment using a flow cytometric antibody binding assay (FABA) to specify the optimum conditions for CD38 inactivation. MATERIALS AND METHODS: For FABA, untreated or DTT-treated RBCs were incubated with fluorescein isothiocyanate-labelled anti-CD38 antibody, in the presence or absence of 100-fold or more excess of unlabelled anti-CD38 antibody, and then analysed by flow cytometry (FCM). Dissociation of CD38-positive and control histograms was determined from the D-value using the Kolmogorov-Smirnov test. The results from FABA were compared with those from conventional FCM, indirect antiglobulin test (IAT) and Western blotting. RESULTS: The results from FABA were more consistent than those from conventional FCM. The D-value was found to be reliable in the analysis of difference between CD38 before and after DTT treatment. Our data showed that 0·0075 mol/l DTT for 30 min is sufficient to inactivate CD38 on RBCs. These results were stable and consistent with the findings from IAT. CONCLUSION: Flow cytometric antibody binding assay is an objective way of evaluating the efficacy of DTT treatment for CD38 on RBCs. This approach allows the detection of a small number of cell surface antigens and will be useful for assessing the various chemical treatments to denature RBC antigens.
Asunto(s)
Ditiotreitol , Eritrocitos , Mieloma Múltiple , ADP-Ribosil Ciclasa 1 , Transfusión Sanguínea , Prueba de Coombs , Ditiotreitol/farmacología , Recuento de Eritrocitos , Citometría de Flujo , HumanosRESUMEN
BACKGROUND AND OBJECTIVES: It is sometimes difficult to obtain antigen-negative red blood cells (RBCs) for patients with antibodies against RBCs. However, the frequency and severity of the adverse reactions have not been well elucidated. Here, we conducted a multi-institutional collaborative study to clarify the background, frequency and clinical significance of antigen-positive RBC transfusions to patients with the respective antibodies. MATERIALS AND METHODS: The survey included the background of patients, antigens on RBCs transfused, total amount of antigen-positive RBCs transfused, results from antibody screen and direct antiglobulin tests, specificity of antibodies, adverse reactions and efficacies. All antibodies were surveyed regardless of their clinical significance. RESULTS: In all, 826 cases containing 878 antibodies were registered from 45 institutions. The main reasons for antigen-positive RBC transfusions included 'negative by indirect antiglobulin test' (39%) and 'detection of warm autoantibodies' (25%). In 23 cases (3% of total), some adverse reactions were observed after antigen-positive RBC transfusion, and 25 antibodies (9 of 119 clinically significant and 16 of 646 insignificant antibodies) were detected. Non-specific warm autoantibodies were detected in 9 cases, anti-E in 5 cases, 2 cases each of anti-Lea , anti-Jra or cold alloantibodies, and 1 case each of anti-Dib , anti-Leb or anti-P1. Other antibodies were detected in 2 further cases. Five (22%) of these 23 cases, who had anti-E (3 cases) or anti-Jra (2 cases), experienced clinically apparent haemolysis. CONCLUSIONS: Adverse reactions, especially haemolysis, were more frequently observed in cases with clinically significant antibodies than those with clinically insignificant antibodies (P < 0·001).
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Antígenos de Grupos Sanguíneos/inmunología , Transfusión Sanguínea , Hemólisis , Isoanticuerpos/sangre , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Prueba de Coombs , Transfusión de Eritrocitos , Eritrocitos/inmunología , Femenino , Humanos , Isoanticuerpos/inmunología , Japón , Masculino , Embarazo , Sensibilidad y Especificidad , Reacción a la TransfusiónRESUMEN
BACKGROUND AND OBJECTIVES: Obtaining informed consent (IC) for a blood transfusion is an absolute requirement. In this study, we compared the depth of understanding of blood transfusion among patients with or without an explanation by the transfusion unit staff and evaluated the usefulness of this intervention in obtaining IC. MATERIALS AND METHODS: Expert staff from the transfusion unit started to provide patients with a basic explanation of blood transfusion (intervention group, n = 129). The efficacy of this strategy was assessed by comparison with explanation given by the primary doctors only (conventional group, n = 31). We performed a questionnaire survey to analyze the length of time spent providing information of blood transfusion and the depth of understanding of blood transfusion in the two groups. RESULTS: The median time in providing information in the conventional and intervention groups was 6 and 20 minutes, respectively (P < 0.0001). Patients in the intervention group had a better understanding of several key points on blood transfusion than those in the conventional group. CONCLUSION: Our results show that expert staff from the transfusion unit should be involved in obtaining IC for a blood transfusion. Patients who were provided information by transfusion unit staff were more likely to have a better understanding of the risks and benefits of transfusion.
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Transfusión Sanguínea , Personal de Salud , Consentimiento Informado , Anciano , Enfermedades Transmisibles/etiología , Comprensión , Demografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Riesgo , Encuestas y Cuestionarios , Factores de Tiempo , Reacción a la TransfusiónRESUMEN
BACKGROUND: Hemovigilance is an important aspect of transfusion medicine. However, the frequency of the adverse reactions often varies using different reporters. Recently, we have employed a new information technology (IT)-based in-hospital hemovigilance system. Here, we evaluated changes in practice after implementation of an IT-based reporting system. STUDY DESIGN AND METHODS: We compared the rate of frequency and details of blood transfusion-related adverse reactions 3 years before and after introduction of the IT-based reporting system. Contents and severity of the adverse reactions were reported in a paper-based reporting system, but input by selecting items in an IT-based reporting system. The details of adverse reactions are immediately sent to the blood transfusion unit online. RESULTS: After we introduced the IT-based reporting system, the reported rate of transfusion-related adverse reactions increased approximately 10-fold from 0.20% to 2.18% (p < 0.001), and frequencies of urticaria, pruritus, rash, fever (p < 0.001), hypertension (p = 0.001), tachycardia (p = 0.003), and nausea and vomiting (p = 0.010) increased significantly. Although there was no error report in the paper-based reporting, incorrect reports were observed in 90 cases (0.52%) in the IT-based reporting (p < 0.001). CONCLUSION: The advantages of IT-based reporting were: 1) a significant increase in the frequency of adverse reaction reporting and 2) a significant decrease in underreporting, although the true frequency has yet to be clarified. The disadvantage of the IT-based reporting was an increased incidence of incorrect inputs, all of which was unnoticed by the reporters. Our results showed several important points in need of monitoring after introduction of an IT-based reporting system.
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Seguridad de la Sangre , Informática Médica/métodos , Reacción a la Transfusión , Humanos , Medicina TransfusionalRESUMEN
MYH7 (also referred to as cardiac myosin heavy chain ß) gene expression is known to be repressed by thyroid hormone (T3). However, the molecular mechanism by which T3 inhibits the transcription of its target genes (negative regulation) remains to be clarified, whereas those of transcriptional activation by T3 (positive regulation) have been elucidated in detail. Two MCAT (muscle C, A, and T) sites and an A/T-rich region in the MYH7 gene have been shown to play a critical role in the expression of this gene and are known to be recognized by the TEAD/TEF family of transcription factors (TEADs). Using a reconstitution system with CV-1 cells, which has been utilized in the analysis of positive as well as negative regulation, we demonstrate that both T3 receptor (TR) ß1 and α1 inhibit TEAD-dependent activation of the MYH7 promoter in a T3 dose-dependent manner. TRß1 bound with GC-1, a TRß-selective T3 analog, also repressed TEAD-induced activity. Although T3-dependent inhibition required the DNA-binding domain (DBD) of TRß1, it remained after the putative negative T3-responsive elements were mutated. A co-immunoprecipitation study demonstrated the in vivo association of TRß1 with TEAD-1, and the interaction surfaces were mapped to the DBD of the TRß1 and TEA domains of TEAD-1, both of which are highly conserved among TRs and TEADs, respectively. The importance of TEADs in MYH7 expression was also validated with RNA interference using rat embryonic cardiomyocyte H9c2 cells. These results indicate that T3-bound TRs interfere with transactivation by TEADs via protein-protein interactions, resulting in the negative regulation of MYH7 promoter activity.
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Regulación de la Expresión Génica/fisiología , Cadenas Pesadas de Miosina/genética , Receptores de Hormona Tiroidea/fisiología , Factores de Transcripción/fisiología , Triyodotironina/fisiología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Regiones Promotoras Genéticas , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
The initial step of blood transfusion therapy is blood type grouping. ABO-mismatch blood transfusion results in serious adverse effects. Several incidents in the process of blood sampling had been experienced in our hospital since 2006 to 2008. Therefore, we have introduced the computed identification system, and the transfusion unit has taken a part of blood sampling. Just after we introduced it in July 2010, only 7% of the doctors and the nurses used the system in blood sampling. Repeated training programs for doctors and nurses on blood sampling procedure improved the utilization to 95%. We realized the importance of our management in face of its introduction. We have to make continuous efforts on the safety of transfusion therapy, because new type of incidents can appear.
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Recolección de Muestras de Sangre , Sistemas de Identificación de Pacientes , Flebotomía , Tipificación y Pruebas Cruzadas Sanguíneas/instrumentación , Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Transfusión Sanguínea , Sistemas de Computación , Humanos , Sistemas de Identificación de Pacientes/métodosRESUMEN
Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin ß gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity, which is inhibited by T3/TR.
Asunto(s)
ADN Complementario/genética , Elementos de Facilitación Genéticos/genética , Luciferasas de Luciérnaga/genética , Receptores de Hormona Tiroidea/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Animales , Secuencia de Bases , Línea Celular , Genes Reporteros/genética , Humanos , Ligandos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Eliminación de Secuencia/genética , Acetato de Tetradecanoilforbol/farmacología , Timidina Quinasa/metabolismo , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Triyodotironina/farmacologíaRESUMEN
Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHß and α-glycoprotein (αGSU) subunit genes. TSHß expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHß gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHß gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHß promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHß gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHß promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHß expression.
Asunto(s)
Factor de Transcripción GATA2/fisiología , Tirotropina de Subunidad beta/genética , Hormona Liberadora de Tirotropina/fisiología , Activación Transcripcional/fisiología , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Mutación , Regiones Promotoras Genéticas , Transducción de Señal , Hormona Liberadora de Tirotropina/metabolismoRESUMEN
Peroxisome proliferator-activated receptor gamma-2 (PPARG2) is a ligand-dependent transcriptional factor involved in the pathogenesis of insulin resistance. In the presence of a ligand, PPARG2 associates with co-activators, while it recruits co-repressors (CoRs) in the absence of a ligand. It has been reported that the interaction of liganded PPARG2 with co-activators is regulated by the amino-terminal A/B domain (NTD) via inter-domain communication. However, the role of the NTD is unknown in the case of the interaction between unliganded PPARG2 and CoRs. To elucidate this, total elimination of the influence of ligands is required, but the endogenous ligands of PPARG2 have not been fully defined. PPARG1-P467L, a naturally occurring mutant of PPARG1, was identified in a patient with severe insulin resistance. Reflecting its very low affinity for various ligands, this mutant does not have transcriptional activity in the PPAR response element, but exhibits dominant negative effects (DNEs) on liganded wild-type PPARG2-mediated transactivation. Using the corresponding PPARG2 mutant, PPARG2-P495L, we evaluated the role of the NTD in the interaction between unliganded PPARG2 and CoRs. Interestingly, the DNE of PPARG2-P495L was increased by the truncation of its NTD. NTD deletion also enhanced the DNE of a chimeric receptor, PT, in which the ligand-binding domain of PPARG2 was replaced with that of thyroid hormone receptor beta-1. Moreover, NTD deletion facilitated the in vitro binding of nuclear receptor CoR with wild-type PPARG2, mutant P495L, and the PT chimera (PPARG2-THRB). Inter-domain communication in PPARG2 regulates not only ligand-dependent transactivation but also ligand-independent silencing.
Asunto(s)
Proteínas Co-Represoras/metabolismo , PPAR gamma/química , PPAR gamma/metabolismo , Animales , Línea Celular , Genes Dominantes , Humanos , Ligandos , Ratones , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia/genética , Relación Estructura-Actividad , Receptores beta de Hormona Tiroidea/genética , Receptores beta de Hormona Tiroidea/metabolismoRESUMEN
Thyrotropin (TSH) is a heterodimer consisting of alpha and beta chains, and the beta chain (TSHbeta) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHbeta expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHbeta promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHbeta promoter without PIT1. The deleted region (nt -82/-52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHbeta promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.
Asunto(s)
Factor de Transcripción GATA2/metabolismo , Regiones Promotoras Genéticas/genética , Tirotropina de Subunidad beta/genética , Factor de Transcripción Pit-1/metabolismo , Factor de Transcripción Pit-1/fisiología , Activación Transcripcional/fisiología , Animales , Sitios de Unión , Línea Celular , Chlorocebus aethiops , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Factor de Transcripción GATA2/genética , Modelos Biológicos , Unión Proteica , Factor de Transcripción Pit-1/genética , Activación Transcripcional/genéticaRESUMEN
Transcriptional repression of the TSH-specific beta subunit (TSHbeta) gene has been regarded to be specific to thyroid hormone (tri-iodothyronine, T(3)) and its receptors (TRs) in physiological conditions. However, TSHbeta mRNA levels in the pituitary were reported to decrease in the administration of pharmacologic doses of estrogen (17-beta-estradiol, E(2)) and increase in E(2) receptor (ER)-alpha null mice. Here, we investigated the molecular mechanism of inhibition of the TSHbeta gene expression by E(2)-bound E(2)-estrogen receptor 1 (E(2)-ERalpha). In kidney-derived CV1 cells, transcriptional activity of the TSHbeta promoter was stimulated by GATA2 and suppressed by THRBs and ERalpha in a ligand-dependent fashion. Overexpression of PIT1 diminished the E(2)-ERalpha-induced inhibition, suggesting that PIT1 may protect GATA2 from E(2)-ERalpha targeting by forming a stable complex with GATA2. Interacting surfaces between ERalpha and GATA2 were mapped to the DNA-binding domain (DBD) of ERalpha and the Zn finger domain of GATA2. E(2)-dependent inhibition requires the ERalpha amino-terminal domain but not the tertiary structure of the second Zn finger motif in E(2)-ERalpha-DBD. In the thyrotroph cell line, TalphaT1, E(2) treatment reduced TSHbeta mRNA levels measured by the reverse transcription PCR. In the human study, despite similar free thyroxine levels, the serum TSH level was small but significantly higher in post- than premenopausal women who possessed no anti-thyroid antibodies (1.90 microU/ml+/-0.13 S.E.M. vs 1.47 microU/ml+/-0.12 S.E.M., P<0.05). Our findings indicate redundancy between T(3)-TR and E(2)-ERalpha signaling exists in negative regulation of the TSHbeta gene.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Factor de Transcripción GATA2/metabolismo , Factor de Transcripción GATA2/farmacología , Tirotropina de Subunidad beta/genética , Activación Transcripcional/efectos de los fármacos , Adulto , Animales , Línea Celular , Chlorocebus aethiops , Ensayo de Cambio de Movilidad Electroforética , Estradiol/sangre , Estradiol/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/química , Factor de Transcripción GATA2/genética , Humanos , Posmenopausia/sangre , Premenopausia/sangre , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiroxina/sangre , Factor de Transcripción Pit-1/genética , Factor de Transcripción Pit-1/metabolismo , Triyodotironina/sangre , Dedos de Zinc/fisiologíaRESUMEN
Previously we reported that the negative regulation of the TSHbeta gene by T(3) and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSHbeta inhibition. The negative regulatory element (NRE), which had been reported to mediate T(3)-bound TR (T(3)-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHbeta-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T(3)-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T(3)-independent manner. The suppression by T(3)-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TalphaT1, revealed that T(3) treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15-30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSHbeta gene by T(3)-TR we report that 1) GATA2 is the major transcriptional activator of the TSHbeta gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important.