RESUMEN
We report a case of catecholamine-induced acute myocardial stunning that occurred in a six-year-old girl. This was triggered by an accidental noradrenaline injection during general anaesthesia for dental surgery. The clinical course was favourable, although cardiac enzymes and echocardiography were significantly altered. The child was discharged home on the second postoperative day, after complete clinical resolution. We emphasise the need to consider shortening the surgical procedure, and to closely monitor patients following a medication error involving vasopressors even in the absence of symptoms. We highlight the importance of a controlled process for storing, identifying, preparing, and handling medications. The identification of weaknesses in the overall process of drug prescription and administration is of utmost importance.
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BACKGROUND: Tonsillectomy is one of the most common major surgical procedures performed in children. In 2013, the use of codeine in children was severely restricted. French guidelines for treating tonsillectomy's postoperative pain at home have been reconsidered OBJECTIVE: The aim of our study was to measure effectiveness and safety of two schedules: acetaminophen + ibuprofen (A + I) and acetaminophen + tramadol (A + T) in children who underwent tonsillectomy. SETTING AND PATIENTS: We undertook a 1 year prospective and observational single-center study. All children who underwent tonsillectomy were eligible. The choice of the regimen, A + I group or A + T group, was left for the anesthesiologist in charge, done during the pre-anesthetic assessment. After hospital discharge, parents had to give systematically A + I or A + T, 4 times a day during 5 days and then acetaminophen alone for the next 5 days The primary endpoint was the home pain assessed using Parents' Postoperative Pain Measurement Short Form (PPPM-SF) scale. Secondary endpoints were the rate of further hospitalization and/or surgery due to tonsillectomy-related adverse events. RESULTS: Over the study period, 342 tonsillectomies were performed. The return rate of PPPM-SF scales was 58%. Two hundred patients were analyzed. The median age was 4 [3; 5.2] years and was lower in group A + I (4 [3; 5]; 5 [4; 7]; p < 0.0001). PPPM-SF scores were greater than or equal to 3 in both groups during the first 6 postoperative days. The mean decrease of PPPM-SF score over time was higher in group A + I than in group A + T (p = 0.007). Readmission rate was significantly higher in group A + T (A + I: 0; A + T: 7; p = 0.002) as the rate of reoperation for bleeding (A + I: 0; A + T: 3; p = 0.049). CONCLUSION: Home pain management after tonsillectomy should be improved. In clinical practice, A + I seems at least as effective as the combination A + T, without increasing readmission and/or additional surgery for bleeding.
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Acetaminofén/uso terapéutico , Ibuprofeno/uso terapéutico , Dolor Postoperatorio/prevención & control , Tonsilectomía , Tramadol/uso terapéutico , Analgésicos no Narcóticos/uso terapéutico , Analgésicos Opioides/uso terapéutico , Niño , Preescolar , Quimioterapia Combinada , Femenino , Humanos , Masculino , Dimensión del Dolor , Readmisión del Paciente/estadística & datos numéricos , Estudios Prospectivos , Reoperación/estadística & datos numéricosRESUMEN
BACKGROUND: In adults, the Post-Anesthetic Discharge Scoring System (PADSS) was built to secure the discharge after outpatient surgery. We evaluate a pediatric adaptation: the Pediatric-PADSS (Ped-PADSS). STUDY DESIGN: Prospective cohort. METHODS: This was a prospective, observational, monocentric study for ambulatory patients. Ped-PADSS is built on 5 items each quoted 0, 1, or 2: hemodynamics, state of awakening, nausea/vomiting, pain and bleeding. A result ≥9/10 validated discharge if the anesthetist did not wish to review the patient, if the parents did not wish to revisit the anesthetist or if there was no hoarseness or dyspnea. The discharge was validated by the anesthetist and the surgeon. Ped-PADSS was made without the knowledge of the nursing team, one hour after return in service and repeated hourly. Addition of patient demographic data, the collection included the hours of leave by the anesthetist, surgeon and Ped-PADSS, the duration of hospital stay post procedure. RESULTS: On 150 patients, 148 patients were allowed to go out with the Ped-PADSS, one patient was released despite a Ped-PADSS<9. One patient was hospitalized for a surgical bleeding in agreement with the anesthetist, surgeon and the Ped-PADSS. Ninety-five percent of patients had a Ped-PADSS ≥9 after 2hours monitoring in the ambulatory unit. CONCLUSION: The majority of the children have met the criteria for discharge at the end of 2hours postoperative monitoring. The use of this score could reduce the hospitalization time in ambulatory unit.
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Procedimientos Quirúrgicos Ambulatorios/normas , Alta del Paciente/normas , Pediatría/normas , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Lactante , Masculino , Hemorragia Posoperatoria/diagnóstico , Náusea y Vómito Posoperatorios/diagnóstico , Estudios ProspectivosRESUMEN
In France, ambulatory anaesthesia and surgery seem to be well codified. Many recommendations have been published by the Health Authority and the professional associations: they are summarized in this review. However, numerous practical problems persist: for example, two situations specific to paediatric practice are problematic parental comprehension and application of the information provided and poor access to strong analgesics outside the hospital. Despite this, the paediatric population is an ideal target for ambulatory care because of its usual good health and quicker recovery after minor injury as proven by the small percentage of failure and readmission.
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Procedimientos Quirúrgicos Ambulatorios/legislación & jurisprudencia , Procedimientos Quirúrgicos Ambulatorios/tendencias , Francia , Humanos , Legislación Médica , Cuidados Posoperatorios , Cuidados Preoperatorios , Sociedades MédicasRESUMEN
Currently, day-case surgery has a significant development. In pediatrics, a big part of interventions can be performed as a day-case surgery. However, postoperative pain, often wrongly regarded as minor, should not be underestimated or undertreated. The aim of this paper is to review the available systemic analgesics and to propose a way to use them in order to improve children's comfort and experiences in their own families.
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Procedimientos Quirúrgicos Ambulatorios/métodos , Analgesia/métodos , Servicios de Atención de Salud a Domicilio , Dolor Postoperatorio/tratamiento farmacológico , Analgésicos Opioides/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Niño , Humanos , Dolor Postoperatorio/diagnósticoRESUMEN
BACKGROUND/AIMS: The simultaneous use of alcohol and cannabis is common among adolescents, but has been little studied. In this study, we examine predictors and consequences of this behavior in a population-based sample of high school students. METHOD: Self-reports were obtained from students in Quebec (Canada) followed throughout high school (N=6589). Logistic regressions were used to test the association between individual, family, and peer-related predictors in grades 7-8 and simultaneous alcohol and cannabis use in grade 10, as well as between simultaneous alcohol and cannabis use in grade 10 and experiencing 3 or more substance-related problems of various types (legal, physical, etc.) in grade 11. RESULTS: Most predictors in grades 7-8 were associated with simultaneous alcohol and cannabis use in grade 10. Only variables reflecting early-onset substance use involvement - alcohol intoxication, cannabis use, and drug use by close friend(s) - remained predictive in a multivariate model. Simultaneous alcohol and cannabis use was associated with increased substance-related problems in grade 11, above and beyond baseline problems and the concurrent use of the two substances in separate episodes in grade 10. CONCLUSIONS: Simultaneous alcohol and cannabis use 1) is anticipated by multiple psychosocial risk factors which come together with individual and peer substance use in early high school and 2) is independently predictive of subsequent substance-related problems. Providing adolescents with adequate information regarding the potential harm of simultaneous use may be a useful prevention strategy.
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Consumo de Bebidas Alcohólicas/epidemiología , Trastornos Relacionados con Alcohol/epidemiología , Abuso de Marihuana/epidemiología , Adolescente , Conducta del Adolescente/psicología , Consumo de Bebidas Alcohólicas/psicología , Trastornos Relacionados con Alcohol/psicología , Niño , Familia , Femenino , Humanos , Modelos Logísticos , Masculino , Abuso de Marihuana/psicología , Grupo Paritario , Estudios Prospectivos , Quebec/epidemiología , Factores de Riesgo , Instituciones Académicas , Autoinforme , Estudiantes/psicologíaRESUMEN
Childhood disruptiveness is one of the most important antecedents of heavy substance use in adolescence, especially among boys. The first aim of the present study is to verify whether parental monitoring and friend conventionality protect disruptive boys from engaging in heavy substance-use in adolescence. The second purpose is to examine whether these protective effects are strengthened by attachment to parents or friends respectively. Finally, the third objective is to verify whether the expected protective effect of parental monitoring could be mediated through exposure to conventional friends. A sample of 1037 boys from low socioeconomic neighbourhoods was followed from childhood (age 6) to adolescence (age 15). Parent, teacher, and self-reported measures were used to measure disruptiveness, parental monitoring, family attachment, friend conventionality, and attachment to friends. Results suggest that parental monitoring and friends' conventionality mitigated the relationship between childhood disruptiveness and adolescence heavy substance use. Exposure to conventional friends further mediated the protective effect of parent monitoring. The postulated enhancement of attachment quality on the protective effect of parents or peer behaviors was not confirmed, but low attachment was related to heavier substance use in highly monitored disruptive boys. Parental monitoring, family attachment, and peer conventionality are factors amenable to intervention, and thus represent promising targets for future prevention strategies aimed at-risk boys. Our results underscore the importance of simultaneously addressing the behavioral and the affective dimensions in interventions with parents.
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Trastornos de la Conducta Infantil/psicología , Relaciones Padres-Hijo , Grupo Paritario , Trastornos Relacionados con Sustancias/prevención & control , Adolescente , Niño , Amigos , Humanos , Relaciones Interpersonales , Estudios Longitudinales , Masculino , QuebecRESUMEN
Plasmacytoid dendritic cell (PDC) leukemia/lymphoma is a rare neoplasm presenting cutaneous lesions at the time of diagnosis, followed by dissemination to bone marrow, lymph nodes, and other lymphoid and nonlymphoid organs. Since these leukemic counterparts of human PDC are similar to normal PDC, we studied their chemokine receptor equipment and their migratory capacities. We found both in skin lesions and in invaded lymph nodes an expression by tumor cells of CXCR3, CXCR4, and CCR7, and the concomitant expression by cells in the microenvironment of their respective ligands CXCL9, CXCL12, and CCL19. Moreover, flow cytometry phenotype of leukemic PDC (LPDC) revealed an unexpected expression of CCR6. We show that fresh tumor cells are able to migrate in response to CXCR4, CCR2, CCR5, CCR6, and CCR7 ligands, and the ability of CXCR3 ligands to increase the responsiveness to CXCL12. IL-3- or virus-induced activation of LPDC leads to downregulation of CXCR3 and CXCR4, and upregulation of CCR7, associated with the loss of response to CXCL12, and the acquisition of sensitivity to CCL19. Altogether, these results suggest that the preferential accumulation of LPDC in the skin or lymph nodes could be orchestrated by CXCR3, CXCR4, CCR6, and CCR7 ligands, found in nontumoral structures of invaded organs.
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Movimiento Celular , Células Dendríticas/metabolismo , Leucemia/metabolismo , Ganglios Linfáticos/metabolismo , Receptores CXCR4/metabolismo , Receptores de Quimiocina/metabolismo , Enfermedades de la Piel/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Quimiocina CCL19 , Quimiocina CXCL12 , Quimiocina CXCL9 , Quimiocinas CC/metabolismo , Quimiocinas CXC/metabolismo , Quimiotaxis , Niño , Células Dendríticas/inmunología , Células Dendríticas/patología , Femenino , Citometría de Flujo , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leucemia/inmunología , Leucemia/patología , Ligandos , Ganglios Linfáticos/patología , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Receptores CCR7 , Receptores CXCR3 , Enfermedades de la Piel/patologíaRESUMEN
Dendritic cells (DC) can facilitate immune responses that might help in the induction of effective antitumor T cell responses. We reported previously that leukemic blasts from selected patients with acute myeloid leukemia (AML) were able to differentiate in vitro into cells with mature DC features. However, despite the use of a wide variety of cytokine combinations, leukemic DC could not be obtained from all AML patients. In this study, we investigated in a wide range of AML patients (n = 30), the nature and functional characteristics of the blast compartment that can be induced to acquire DC features in vitro. Our results demonstrate that leukemic DC generated in the presence of GM-CSF, IL-4 and matured with CD40L, are composed of two major subsets: DC derived from CD14(+) leukemic cells and leukemic DC derived from in vivo expanded circulating blood myeloid DC (MDC). Leukemic DC of both subsets exhibited DC morphology, had a phenotype of mature DC, and could induce a potent proliferative response of naive CD4(+) T cells. Moreover, both subsets produced large amounts of IL-12p70 and leukemic CD14(+)-derived DC could induce a potent Th1 response. These results can be considered as a prerequisite before the design of vaccine immunotherapy protocols for the adjuvant treatment of AML patients.
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Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Leucemia Mieloide/inmunología , Receptores de Lipopolisacáridos/análisis , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Ligando de CD40 , Citocinas/metabolismo , Células Dendríticas/clasificación , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Técnicas In Vitro , Leucemia Mieloide/patología , Masculino , Microscopía Confocal , Persona de Mediana Edad , Receptores CCR7 , Receptores de Quimiocina/metabolismo , Células TH1/metabolismo , Células Tumorales CultivadasRESUMEN
Dendritic cells (DCs) are responsible for the initiation of immune responses. Two distinct subsets of blood DCs have been characterized thus far. Myeloid DCs (MDCs) and plasmacytoid monocytes (PDCs) were shown to be able to promote polarization of naive T cells. This study shows a dramatic quantitative imbalance in both circulating blood DC subsets in 37 patients with acute myeloid leukemias. Eleven patients (30%) displayed a normal quantitative profile (MDC mean, 0.37% +/- 0.21%; range, 0.01% to 0.78%; PDC mean, 0.21% +/- 0.24%; range, 0.04% to 0.62%), whereas 22 (59%) showed a tremendous expansion of MDCs (9 patients: mean, 16.76% +/- 14.03%; range, 1.36% to 41%), PDCs (4 patients: mean, 7.28% +/- 6.84%; range, 1% to 14%), or both subsets (9 patients: MDC mean, 10.86% +/- 12.36%; range, 1.02% to 37.1%; PDC mean, 4.25% +/- 3.78%; range, 1.14% to 13.04%). Finally, in 4 patients (11%), no DC subsets were detectable. Both MDC and PDC subsets exhibited the original leukemic chromosomal abnormality. Ex vivo, leukemic PDCs, but not leukemic MDCs, had impaired capacity for maturation and decreased allostimulatory activity. Also, leukemic PDCs were altered in their ability to secrete interferon-alpha. These data provide evidence that DC subsets in vivo may be affected by leukemogenesis and may contribute to leukemia escape from immune control.
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Aberraciones Cromosómicas , Células Dendríticas/inmunología , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/genética , Animales , Ligando de CD40/farmacología , Células Cultivadas , Células Dendríticas/ultraestructura , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Antígenos HLA-DR/análisis , Humanos , Inmunofenotipificación , Interferón-alfa/biosíntesis , Interferón-alfa/metabolismo , Interleucina-3/farmacología , Interleucina-4/farmacología , Leucemia Mieloide Aguda/inmunología , Ratones , Microscopía Confocal , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
We show here that mouse interferon-alpha (IFN-alpha)-producing cells (mIPCs) are a unique subset of immature antigen-presenting cells (APCs) that secrete IFN-alpha upon stimulation with viruses. mIPCs have a plasmacytoid morphology, can be stained with an antibody to Ly6G and Ly6C (anti-Ly6G/C) and are Ly6C+B220+CD11cloCD4+; unlike other dendritic cell subsets, however, they do not express CD8alpha or CD11b. Although mIPCs undergo apoptosis in vitro, stimulation with viruses, IFN-alpha or CpG oligonucleotides enhanced their survival and T cell stimulatory activity. In vivo, mIPCs were the main producers of IFN-alpha in cytomegalovirus-infected mice, as depletion of Ly6G+/C+ cells abrogated IFN-alpha production. mIPCs produced interleukin 12 (IL-12) in response to viruses and CpG oligodeoxynucleotides, but not bacterial products. Although different pathogens can selectively engage various APC subsets for IL-12 production, IFN-alpha production is restricted to mIPCs' response to viral infection.
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Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/ultraestructura , Interferón-alfa/biosíntesis , Animales , Células Presentadoras de Antígenos/clasificación , Células de la Médula Ósea/inmunología , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Femenino , Infecciones por Herpesviridae/inmunología , Inmunofenotipificación , Interferón-alfa/farmacología , Interleucina-12/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Muromegalovirus/fisiología , Oligodesoxirribonucleótidos/farmacología , Orthomyxoviridae/fisiología , Bazo/inmunologíaRESUMEN
This work aims to demonstrate that CD4(+)CD56(+) malignancies arise from transformed cells of the lymphoid-related plasmacytoid dendritic cell (pDC) subset. The analysis of malignant cells from 7 patients shows that in all cases, like pDCs, leukemic cells are negative for lineage markers CD3, CD19, CD13, CD33, and CD11c but express high levels of interleukin-3 receptor alpha chain (IL-3Ralpha), HLA-DR, and CD45RA. Tumor cells produce interferon-alpha in response to influenza virus, while upon maturation with IL-3 they become a powerful inducer of naive CD4(+) T-cell proliferation and promote their T-helper 2 polarization. As pDCs, leukemic cells also express pre-Talpha and lambda-like 14.1 transcripts, arguing in favor of a lymphoid origin. In addition, malignant cells express significant levels of CD56 and granzyme B. Overall, those observations suggest that CD4(+)CD56(+) leukemic cells could represent the malignant counterpart of pDCs, both of which are closely related to B, T, and NK cells.
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Células Dendríticas/patología , Leucemia/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD4/análisis , Antígenos CD40/genética , Antígenos CD40/fisiología , Antígeno CD56/análisis , Diferenciación Celular , Niño , Células Dendríticas/inmunología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Granzimas , Antígenos HLA-DR/análisis , Humanos , Interferón-alfa/biosíntesis , Interleucina-3/farmacología , Leucemia/inmunología , Antígenos Comunes de Leucocito/análisis , Masculino , Persona de Mediana Edad , Receptores de Interleucina-3/análisis , Serina Endopeptidasas/análisis , Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Transfección , Células Tumorales CultivadasRESUMEN
Three distinct dendritic cell (DC) subsets capable of stimulating allogeneic naive T cells were isolated from human thymus. The most abundant subset was represented by plasmacytoid DCs (pDCs), which secreted high amounts of IFN-alpha upon stimulation with inactivated influenza virus and thus likely correspond to the recently identified peripheral blood natural IFN-alpha/beta-producing cells (IPCs). Like those latter cells, thymic pDCs had distinctive phenotypic features (i.e., Lin(-), HLA-DR(int), IL-3R alpha(hi), CD45RA(hi), CD11c(-), CD13(-), and CD33(lo)) and developed into mature DCs upon culture in IL-3 and CD40L. Of the two other DC subsets, one displayed a phenotype of immature myeloid DCs (imDCs) (HLA-DR(int), CD11c(+), CD13(+), CD33(+)), and the other represented HLA-DR(hi) CD11c(+) mature DCs (mDCs). Since they also expressed DC-LAMP, these mDCs appear to correspond to interdigitating dendritic cells (IDCs). Thymic pDCs, but not myeloid imDCs, strongly expressed lymphoid-specific transcripts such as pre-T alpha, lambda-like, and Spi-B, thereby suggesting a possible lymphoid origin. The detection of Spi-B mRNA, not only upon in vitro maturation of pDCs, but also in freshly purified IDCs, suggests that in vivo pDCs may differentiate into IDCs.
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Células Dendríticas/clasificación , Integrina alfaXbeta2 , Timo/citología , Adolescente , Ligando de CD40/farmacología , Separación Celular , Niño , Preescolar , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Lactante , Interferón-alfa/farmacología , Interleucina-3/farmacología , Orthomyxoviridae/inmunología , ARN Mensajero , Receptores de Interleucina-3/genéticaRESUMEN
Dendritic cells (DCs) are antigen-presenting cells with a unique ability to induce primary immune responses. DCs capture and transfer information from the outside world to the cells of the adaptive immune system. DCs are not only critical for the induction of primary immune responses, but may also be important for the induction of immunological tolerance, as well as for the regulation of the type of T cell-mediated immune response. Although our understanding of DC biology is still in its infancy, we are now beginning to use DC-based immunotherapy protocols to elicit immunity against cancer and infectious diseases.
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Células Dendríticas/inmunología , Animales , Presentación de Antígeno/inmunología , Antígenos/inmunología , Linfocitos B/inmunología , Células Dendríticas/clasificación , Humanos , Activación de Linfocitos/inmunología , Linfocitos T/inmunologíaRESUMEN
Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA. RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb RoBPI mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs. RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs. Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs.
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Proteínas Portadoras/genética , Ribonucleoproteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas Portadoras/metabolismo , Extractos Celulares , Núcleo Celular/metabolismo , Clonación Molecular , Proteínas de Unión al ADN , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa , Humanos , Microscopía Confocal , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Unión Proteica , Factores de Empalme de ARN , Proteínas de Unión al ARN , Conejos , Proteínas Recombinantes/metabolismo , Proteínas Represoras , Ribonucleoproteínas/metabolismo , LevadurasRESUMEN
We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs). To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA. The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones. RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA.
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Técnicas de Sonda Molecular , Ribonucleoproteínas , Saccharomyces cerevisiae/genética , Sitios de Unión , ADN Complementario/análisis , Escherichia coli/genética , Biblioteca de Genes , Células HeLa , Humanos , Plásmidos/genética , ARN/metabolismo , Proteínas Recombinantes , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
CD40 is a 50-kDa protein expressed on B cells, dendritic cells, monocytes and epithelial cells, but the distribution of CD40 expression in humans is not completely known. It binds to a ligand (CD40L) which is expressed essentially on activated T cells. The interaction between CD40 and CD40L plays important roles in immune responses. CD40 expression was investigated on bronchial tissues and human bronchial cell lines using immunohistochemistry, immunofluorescence staining and analysis with a cytometer, respectively. Constitutive CD40 expression, but not that of CD40L, was slightly detectable on normal human bronchial epithelial cells (HBEC) in situ and on an adult lung adenocarcinoma (SKLU1) cell line, while another cell line, a bronchial transformed SV40 cell line (WI26VA4), was negative for CD40. Among the various cytokines tested, only interferon (IFN)-gamma was found to induce CD40 expression on WI26VA4. Tumour necrosis factor (TNF)-alpha was the best cytokine able to up-regulate CD40 in SKLU1 cells. A combination of IFN-gamma and TNF-alpha was slightly more effective than the cytokine alone at up-regulating CD40 expression on both cell lines. We further investigated the functional consequences of CD40 ligation on both cell lines. These bronchial cells expressed CD40, HLADR and CD54 under basal conditions or when stimulated by cytokines. Stimulation through CD40 did not affect cell-surface-antigen expression on either cell line. The production of cytokines such as interleukin (IL)-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) by HBEC has been described. SKLU1 and WI26VA4 cells released IL-6 and GM-CSF spontaneously. Whatever the case, CD40 engagement did not modulate spontaneous or TNF-alpha-induced production of these two cytokines. These data indicate for the first time that normal HBEC express CD40 in situ. Further investigations are required in order to determine the role of CD40 on normal HBEC.
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Bronquios/inmunología , Bronquios/metabolismo , Antígenos CD40/biosíntesis , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Bronquios/citología , Antígenos CD40/fisiología , Línea Celular , Citocinas/farmacología , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
We have reported previously that in vitro generated dendritic cells (DC) can directly regulate B cell responses. Recently, germinal center DC (GCDC) were identified within B cell follicles. Due to their particular localization, we have tested in the present study whether GCDC could contribute to key events characteristic of the GC reaction. Our present results demonstrate that 1) ex vivo GCDC induce a dramatic GC B cell expansion upon CD40 and IL-2 activation and drive plasma cell differentiation, 2) this property is shared by GCDC and blood DC, but not by Langerhans cells, 3) IL-12 production by GCDC is critical in GC B cell expansion and differentiation, and 4) importantly, GCDC also induce IL-10-independent isotype switching toward IgG1. These observations support the novel concept that GCDC directly contribute to the germinal center reaction.
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Linfocitos B/citología , Células Dendríticas/fisiología , Centro Germinal/fisiología , Cambio de Clase de Inmunoglobulina/inmunología , Animales , Linfocitos B/inmunología , Antígenos CD40/fisiología , Diferenciación Celular/inmunología , División Celular/inmunología , Niño , Células Dendríticas/inmunología , Centro Germinal/citología , Centro Germinal/inmunología , Humanos , Interleucina-2/farmacología , Interfase/inmunología , Células L , Activación de Linfocitos , Ratones , Tonsila Palatina , Células Plasmáticas/citología , SolubilidadRESUMEN
It is not known whether subsets of dendritic cells provide different cytokine microenvironments that determine the differentiation of either type-1 T helper (TH1) or TH2 cells. Human monocyte (pDC1)-derived dendritic cells (DC1) were found to induce TH1 differentiation, whereas dendritic cells (DC2) derived from CD4+CD3-CD11c- plasmacytoid cells (pDC2) induced TH2 differentiation by use of a mechanism unaffected by interleukin-4 (IL-4) or IL-12. The TH2 cytokine IL-4 enhanced DC1 maturation and killed pDC2, an effect potentiated by IL-10 but blocked by CD40 ligand and interferon-gamma. Thus, a negative feedback loop from the mature T helper cells may selectively inhibit prolonged TH1 or TH2 responses by regulating survival of the appropriate dendritic cell subset.