RESUMO
Interprofessional team collaboration (ITC) in the Swedish Occupational Health Service is an important part of the service given to the customer. The Occupational Health Service (OHS) could be more competitive if they were able to show how successful is their ITC. The Assessment of Interprofessional Team Collaboration Scale (AITCS) is an instrument that measures ITC in teams working with the client as part of the team. The aim of this study was to adapt the Swedish version of the instrument for use in OHS and to evaluate the psychometric properties of the adapted version and the adapted short version. The study included 472 participants from different OHSs, all members of the trade association of occupational health care in Sweden. Face and content validity of the instrument were assessed, and floor and ceiling effects were measured. Internal consistency was measured with Cronbach's alpha and an exploratory factor analysis was conducted on the 42-item adapted instrument and the short, 24-item version. The exploratory factor analysis gave a three-factor solution with an eigenvalue >1 and explaining a total variance of 57.1% and 62.3% for the short version. The study concludes that AITCS-S-(OHS) as well as the short version, is a reliable and valid questionnaire. Further development of the AITCS-S-(OHS) needs to be undertaken and assessed by confirmatory factor analysis.
Assuntos
Relações Interprofissionais , Serviços de Saúde do Trabalhador , Humanos , Psicometria , Suécia , Reprodutibilidade dos Testes , Inquéritos e QuestionáriosRESUMO
Nitrous oxide emissions can contribute significantly to the carbon footprint of municipal wastewater treatment plants even though emissions from conventional nitrogen removal processes are assumed to be moderate. An increased risk for high emissions can occur in connection with process disturbances and nitrite (NO2-) accumulation. This work describes the findings at a large municipal wastewater treatment plant where the levels of NO2- in the activated sludge process effluent were spontaneously and strongly increased on several activated sludge lines which was suspected to be due to shortcut nitrogen removal that stabilized for several months. The high NO2- levels were linked to a dramatic increase in nitrous oxide (N2O) emissions. As much as over 20% of the daily influent nitrogen load was emitted as N2O. These observations indicate that highly increased NO2- levels can occur in conventional activated sludge processes and result in high nitrous oxide emissions. They also raise questions concerning the risk of increased greenhouse gas (GHG) emissions of the nitritation-denitritation processes - although the uncontrolled nature of the event described here must be taken into consideration - and underline the importance of continuous monitoring and control of N2O emissions.
Assuntos
Esgotos , Águas Residuárias , Reatores Biológicos , Nitrogênio/análise , Óxido Nitroso/análiseRESUMO
In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.
Assuntos
Antineoplásicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Adenina/análogos & derivados , Idoso , Antineoplásicos/uso terapêutico , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Linfonodos/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Piperidinas , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , RNA Neoplásico/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle.
Assuntos
Distrofina/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Terapia Genética , Masculino , Camundongos , Camundongos Endogâmicos mdx , MicroRNAs/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Distrofia Muscular de Duchenne/terapia , Mutação , Proteômica , RNA Mensageiro/metabolismoRESUMO
X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.
Assuntos
Agamaglobulinemia/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Tirosina Quinase da Agamaglobulinemia , Processamento Alternativo , Animais , Humanos , Mutação , Proteínas Tirosina Quinases/genética , RNA Mensageiro/genética , Transdução de SinaisRESUMO
Transcriptional pathways controlling the development of CD44(hi) memory phenotype (MP) T cells with "innate-like" functions are not well understood. Here we show that the BTB (bric-a-brac, tramtrack, broad complex) domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44(hi), but not in CD44(lo), CD4(+) T cells. Transgenic expression of PLZF during T cell development and in CD4(+) and CD8(+) T cells induced a T cell intrinsic program leading to an increase in peripheral CD44(hi) MP CD4(+) and CD8(+) T cells and a corresponding decrease of naïve CD44(lo) T cells. The MP CD4(+) and CD8(+) T cells produced IFNgamma upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in single-positive thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted natural killer T cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4(+) transgenic T cells showed reduced IL-2 and IFNgamma production but increased IL-4 secretion as a result of enhanced IL-4 production of the CD44(hi)CD62L(+) subset. Our data indicate that PLZF is a novel regulator of the development of CD44(hi) MP T cells with a characteristic partial innate-like phenotype.
Assuntos
Receptores de Hialuronatos/imunologia , Memória Imunológica/imunologia , Fatores de Transcrição Kruppel-Like/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Selectina L/metabolismo , Camundongos , Camundongos Transgênicos , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Fenótipo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/imunologiaRESUMO
Bruton tyrosine kinase (Btk) is critical for B-cell development. Btk regulates a plethora of signaling proteins, among them nuclear factor-[kappa]B (NF-kappaB). Activation of NF-kappaB is a hallmark of B cells, and NF-kappaB signaling is severely compromised in Btk deficiency. We here present strong evidence indicating that NF-kappaB is required for efficient transcription of the Btk gene. First, we found that proteasome blockers and inhibitors of NF-kappaB signaling suppress Btk transcription and intracellular expression. Similar to Btk, proteasome inhibitors also reduced the expression of other members of this family of kinases, Itk, Bmx, and Tec. Second, 2 functional NF-kappaB-binding sites were found in the Btk promoter. Moreover, in live mice, by hydrodynamic transfection, we show that bortezomib (a blocker of proteasomes and NF-kappaB signaling), as well as NF-kappaB binding sequence-oligonucleotide decoys block Btk transcription. We also demonstrate that Btk induces NF-kappaB activity in mice. Collectively, we show that Btk uses a positive autoregulatory feedback mechanism to stimulate transcription from its own promoter via NF-kappaB.
Assuntos
Homeostase , NF-kappa B/fisiologia , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Tirosina Quinases/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Sítios de Ligação , Retroalimentação Fisiológica , Camundongos , Transcrição GênicaRESUMO
BACKGROUND: The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. RESULTS: The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. CONCLUSION: Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Perfilação da Expressão Gênica , Proteínas Tirosina Quinases/genética , Animais , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Ciclosporina/farmacologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Subpopulações de Linfócitos T/metabolismo , Transcrição GênicaRESUMO
Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, plays an important role in the differentiation and activation of B cells. Mutations affecting Btk cause immunodeficiency in both humans and mice. In this study we set out to investigate the potential role of Btk in Toll-like receptor 9 (TLR9) activation and the production of pro-inflammatory cytokines such as interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and IL-12p40. Our data show that Btk-deficient B cells respond more efficiently to CpG-DNA stimulation, producing significantly higher levels of pro-inflammatory cytokines but lower levels of the inhibitory cytokine IL-10. The quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis presented in this work shows that mRNA production of one of the important new members of the IL-12 family, IL-27, was significantly increased in Btk-deficient B cells after CpG-DNA stimulation. In this study, we demonstrate significant differences in CpG responsiveness between transitional 1 (T1) and T2 B cells for survival and maturation. Furthermore, TLR9 expression, measured both as protein and as mRNA, was increased in Btk-defective cells, especially after TLR9 stimulation. Collectively, these data provide evidence in support of the theory that Btk regulates both TLR9 activation and expression in mouse splenic B cells.
Assuntos
Linfócitos B/imunologia , Citocinas/biossíntese , Proteínas Tirosina Quinases/imunologia , Receptor Toll-Like 9/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Mediadores da Inflamação/metabolismo , Interleucina-10/biossíntese , Interleucinas/biossíntese , Interleucinas/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Oligodesoxirribonucleotídeos/imunologia , Proteínas Tirosina Quinases/deficiência , Baço/imunologia , Regulação para Cima/imunologiaRESUMO
The DT40 cell-line has been extensively used to create deletion mutants, one of which lacks Bruton's tyrosine kinase (Btk). Btk is a cytoplasmic tyrosine kinase important for B-lymphocyte maturation. It was previously shown that there are differences in gene expression between wild-type and Btk-deficient animals. Global gene expression profiling of the avian B-lymphoma DT40 cell-line was used as a model to differentiate among Btk knockout (KO) and Btk KO cells reconstituted with human Btk. Differences in the gene expression pattern showed statistically significant changes between parental DT40 and all the Btk KO cell populations irrespective of whether they are reconstituted or not. These results imply that in the process of generating a knockout cell-line, sub-clones are selected, which have multiple changes in their gene expression pattern (p<0.01). Although other parameters could also influence the expression profile, this potentially has important implications when interpreting microarray data from gene-deleted cell-lines.
Assuntos
Perfilação da Expressão Gênica , Linfoma de Células B/genética , Proteínas Tirosina Quinases/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Linhagem Celular Tumoral , Galinhas , Células Clonais , Técnicas de Inativação de Genes , Humanos , MutaçãoRESUMO
Nursing today faces numerous challenges. Societal changes lead to reorganization of health care, changing workloads with sicker patients in hospital and home care, and limited economic resources. The increasing and changing nature of knowledge needed for expert care provision challenges nurses to continually update their competencies. These are issues demanding proactive and dynamic changes in the way nurses conceive their mandates and practice. The aim of the action-research project presented here was to foster improved quality of care for patients with advanced cancer through collaborative endeavours integrating cancer nursing clinical practice, research and education in a knowledge exchange programme. The programme was based on input about caregiving needs from multi-professional staff caring for patients with advanced cancer in a variety of healthcare settings. Undergraduate baccalaureate nursing students were then engaged in literature studies to help address these needs. Results of the studies were communicated back to the involved clinicians in a variety of ways. In this paper, we discuss what we have experienced as opportunities and obstacles in conducting the project, based on our reflections and external evaluations. This is linked to a broader discussion of ways of integrating cancer nursing research, education and practice.
Assuntos
Neoplasias/enfermagem , Pesquisa Metodológica em Enfermagem , Cuidados Paliativos/normas , Estudantes de Enfermagem , Humanos , Enfermagem Oncológica/normas , Desenvolvimento de Programas , Qualidade da Assistência à Saúde/normasRESUMO
Extensive research on molecular genetics in recent decades has provided a wealth of information regarding the underlying mechanisms of primary immunodeficiency diseases. The microarray technology has made its entry into the molecular biology research area and hereby enabled signature expression profiling of whole species genomes. Perhaps no other methodological approach has transformed molecular biology more in recent years than the use of microarrays. Microarray technology has led the way from studies of the individual biological functions of a few related genes, proteins or, at best, pathways towards more global investigations of cellular activity. The development of this technology immediately yielded new and interesting information, and has produced more data than can be currently dealt with. It has also helped to realize that even a 'horizontally exhaustive' molecular analysis is insufficient. Applications of this tool in primary immunodeficiency studies have generated new information, which has led to a better understanding of the underlying basic biology of the diseases. Also, the technology has been used as an exploratory tool to disease genes in immunodeficiency diseases of unknown cause as in the case of the CD3Delta-chain and the MAPBPIP deficiency. For X-linked agammaglobulinemia, the technique has provided better understanding of the genes influenced by Btk. There is considerable hope that the microarray technology will lead to a better understanding of disease processes and the molecular phenotypes obtained from microarray experiments may represent a new tool for diagnosis of the disease.
Assuntos
Modelos Animais de Doenças , Perfilação da Expressão Gênica , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Modelos Biológicos , Animais , Humanos , CamundongosRESUMO
Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance.
Assuntos
Substituição de Aminoácidos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Tirosina Quinases/genética , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Biocatálise , Linhagem Celular , Cisteína , Humanos , Piperidinas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Treonina , TransfecçãoRESUMO
Duchenne muscular dystrophy (DMD) is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA) microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1) the determination of gene expression changes associated with dystrophic pathology, (2) identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3) investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis), and (4) prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE64420.
RESUMO
Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells. In conclusion, we demonstrate that cells release distinct exosome subpopulations with unique compositions that elicit differential effects on recipient cells. Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics.
Assuntos
Exossomos/metabolismo , Melanoma Experimental/patologia , Animais , Linhagem Celular Tumoral , CamundongosRESUMO
A time-resolved fluorometric assay for the simultaneous measurement of natural killer cell activity against three different lanthanide diethylenetriaminopentaacetate (LaDTPA) labelled target cell lines is described. The target cell line K-562 was labelled with SmDTPA, the cell line Molt with TbDTPA and the cell line Raji with EuDTPA. After co-incubation of the three target cell lines with effector cells the fluorescence of the lanthanides released from the lysed target cells was measured in an enhancer solution in which they formed highly fluorescent complexes. It was possible to differentiate the specific release from the three target cell lines because the emission lines of the europium, samarium and terbium complexes formed in the enhancer solution are well separated from each other. The autofluorescence from culture media supplemented with serum was avoided by the use of time-resolved fluorometry. The results show that applying fluorometry based on the combination of spectral and temporal resolution to natural killer cell assays, makes possible the simultaneous determination of lysis in up to three target cell lines in complex culture medium.
Assuntos
Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Meios de Cultura , Testes Imunológicos de Citotoxicidade/métodos , Európio , Fluorimunoensaio/métodos , Humanos , Cinética , Ácido Pentético , Samário , Térbio , Células Tumorais CultivadasRESUMO
We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay.
Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Células Matadoras Naturais/imunologia , Linhagem Celular , Quelantes , Testes Imunológicos de Citotoxicidade/estatística & dados numéricos , Európio , Estudos de Avaliação como Assunto , Corantes Fluorescentes , Fluorometria/métodos , Humanos , Técnicas In Vitro , Metais Terras Raras , Sensibilidade e Especificidade , Temperatura , Térbio , Células Tumorais Cultivadas/imunologiaRESUMO
A time-resolved fluorometric assay for the detection of natural killer cell activity against target cells labelled with the fluorescent chelate europium-6,6"-bis[N,N-bis(carboxymethyl)-aminomethyl]-4'-phenyl-2,2',6', 2"-terpyridine (EuCAPT) has been developed. In the assay released EuCAPT from lysed K-562 cells is measured in the supernatant after co-incubation of the target cells with effector cells. Thus, the performance of the assay is essentially similar to the previously described EuDTPA assay and the widely used 51Cr assay. EuCAPT is released from target cells lysed by effector cells faster than 51CrO4(2-) but somewhat slower than EuDTPA. In contrast to methods based on prompt fluorometry the autofluorescence from culture medium supplemented with serum can be avoided by the use of time-resolved fluorometry. The result shows that fluorescent europium chelates provide an alternative to radioactive markers currently used for the assessment of in vitro cellular cytotoxicity.
Assuntos
Quelantes , Citotoxicidade Imunológica/imunologia , Európio , Fluorimunoensaio/métodos , Células Matadoras Naturais/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Células Tumorais CultivadasRESUMO
Time-resolved fluorometry was used to detect the cytolysis of concanavalin A-stimulated blast cells by employing cells stimulated in a mixed lymphocyte culture as effectors. The target cells were labelled with europium diethylenetriaminopentaacetate (EuDTPA) chelates. The results obtained showed that this method, which has been successfully applied to the measurement of natural killer cell activity, was also applicable to the more specific cytotoxic T lymphocyte reaction. The specific release was higher with EuDTPA-labelled target cells than with 51chromium-labelled target cells. As with 51chromium, EuDTPA can be used to distinguish between the cytolysis of specific and non-specific target cells.
Assuntos
Concanavalina A , Testes Imunológicos de Citotoxicidade , Európio , Fluorometria , Ativação Linfocitária , Linfócitos T Citotóxicos/imunologia , Radioisótopos de Cromo/metabolismo , Testes Imunológicos de Citotoxicidade/métodos , Európio/metabolismo , Fluorometria/métodos , Humanos , Teste de Cultura Mista de Linfócitos/métodos , Ácido Pentético/metabolismo , Linfócitos T Citotóxicos/metabolismo , Fatores de TempoRESUMO
The use of a new marker for labelling cells used as targets for natural killer cells is described. The human erythroleukaemic cell line K-562 was used as target. The cells were labelled with europium diethylenetriaminopentaacetate (EuDTPA) chelates. The detection of the released marker is based on time-resolved fluorometry. The results obtained show that the method is sensitive, specific and rapid. The high specific activity of the marker and the sensitivity of the detection apparatus result in numeric values (counts per second) which are 10-20 times higher than the values (counts per minute) obtained with 51chromium. In comparison with 51chromium release assay the labelling of target cells is less time consuming, the marker release more rapid and the detection time of released marker only 1 second per tube. The use of this non-radioactive marker is an alternative way of measuring natural killer cell mediated cytolysis of target cells.