RESUMO
Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.
Assuntos
Histona-Lisina N-Metiltransferase , Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Fusão GênicaRESUMO
Mitochondrial membranes are pointed out as the site of cardiotoxic action of local anaesthetics. Its three main phospholipids components are phosphatidylcholine, phosphatidylethanolamine and cardiolipin. Cardiolipins, in eukaryotes, are only found in mitochondria and are essential for the maintenance of its integrity and dynamics. Fluorescence and nuclear magnetic resonance spectroscopy were used to study the interactions of a local anaesthetics, Dibucaine (DBC), with different mitochondrial membrane models constituted by combinations of its three main lipid components in which cardiolipin was a natural extract (CLmix). Both CLmix presence/absence and its percentage in the model membranes were evaluated. Fluorescence spectroscopy showed that DBC lowered the transition temperature of all membrane models understudy. DBC partition showed to be dependent of CLmix presence and phosphatidylethanolamine:CL ratio. Furthermore, the maximum emission wavelength (λmax) exhibited a notorious decreased with increasing phospholipid to DBC ratio, in all the membrane models containing CLmix. Nevertheless, it remained approximately the same in the membrane without CLmix. This indicates a differential membrane localization of the anaesthetics, dependent on the membrane models used. NMR results showed that DBC interaction and location in the membrane models is mainly influenced by CLmix presence, and DBC can significant alter lipid systems properties e.g. percentage and type of lipid phase present. Taken all together it was shown that DBC interaction and location are largely dependent on the membrane model system. Furthermore, DBC is able to produce significant changes in the lipidic systems which might help to explain its high toxicity.
Assuntos
Anestésicos Locais/metabolismo , Cardiolipinas/metabolismo , Dibucaína/metabolismo , Membranas Mitocondriais/metabolismo , Fosfatidiletanolaminas/metabolismo , Sítios de Ligação , Modelos Biológicos , TemperaturaRESUMO
Cardiolipins are essential for the integrity and the dynamics of the mitochondria membrane, where they exclusively exist in eukaryotes. Changes in cardiolipins membrane levels have been related to several cardiac health disorders. To evaluate cardiolipins impact on membrane properties a physico-chemical study was conducted using steady-state fluorescence anisotropy, dynamic light scattering and Nuclear Magnetic Resonance (1H and 31P NMR). Different binary and ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and a natural extract of bovine heart cardiolipin were used as models of mitochondrial membrane. The main transition temperatures, obtained by the first two techniques, revealed to be cardiolipins dependent. Cardiolipins also showed to act as a bidirectional regulator of membrane fluidity. 1H and 31P NMR results revealed that cardiolipins affects the conformation, mobility and structural order of the phospholipid molecules. According to 1H NMR results, cardiolipins disturbs the overall structure and packing order of membrane demonstrated with the decrease of the line broadening and shift of all resonances. The 31P NMR line shape analysis confirmed that, at distinct temperatures, different lipid phases coexist in the systems, and their type and quantitative distribution are cardiolipins dependent. In summary, cardiolipins presence/absence dramatically changes the membrane properties and has a major impact in the construction of a mitochondrial membrane model.
Assuntos
Cardiolipinas/metabolismo , Bicamadas Lipídicas/metabolismo , Animais , Cardiolipinas/química , Bovinos , Difusão Dinâmica da Luz , Polarização de Fluorescência , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Temperatura de TransiçãoRESUMO
Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.
Assuntos
Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Adulto , Criança , Aberrações Cromossômicas , Quebra Cromossômica , Feminino , Rearranjo Gênico/genética , Humanos , Lactente , Masculino , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genéticaRESUMO
We formerly hypothesized a mechanism whereby the antimycobacterial efficiency of a set of rhodamine labelled iron chelators is improved via the rhodamine fluorophore which enhances the chelators' permeation properties through membranes. To validate our hypothesis in a cellular context and to understand the influence of the structure of the fluorophore on the chelator's uptake and distribution within macrophages we now report comparative confocal microscopy studies performed with a set of rhodamine labelled chelators. We identify the functional groups of the chelator's framework that favor uptake by macrophages and conclude that the antimycobacterial effect is strongly related with the capacity of the chelator to distribute within the host cell and its compartments, a property that is closely related with the chelators' ability to interact with membranes. The quantification of the chelators' interaction with membranes was assessed through measurement of the corresponding partition constants in liposomes. The overall results support that the compounds which are preferentially taken up are the most efficient antimycobacterial chelators and for that reason we infer that the biological activity is modulated by the structural features of the fluorophore.
Assuntos
Antibacterianos , Quelantes , Macrófagos/microbiologia , Mycobacterium avium/metabolismo , Rodaminas , Tuberculose/tratamento farmacológico , Animais , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Quelantes/química , Quelantes/farmacocinética , Quelantes/farmacologia , Feminino , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Rodaminas/química , Rodaminas/farmacocinética , Rodaminas/farmacologia , Tuberculose/metabolismo , Tuberculose/veterináriaRESUMO
A major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that ensure a high incorporation degree and protein directionality and topology that mimics its in vivo conditions. For this kind of studies, protein reconstitution in membrane systems via a detergent-mediated pathway is usually successfully adopted because detergents are generally used in the initial isolation and purification of membrane proteins. This study reports OmpF reconstitution in preformed Escherichia coli liposomes followed by detection of its insertion by analyzing modifications on membrane structure by two different techniques: steady-state fluorescence anisotropy and dynamic light scattering. Another important issue is protein directionality. For OmpF, it is known that interaction with polyamines promotes channel blockage. In this work, the spermine-OmpF interaction was evaluated using surface plasmon resonance, and protein directionality was confirmed.
Assuntos
Escherichia coli/química , Lipídeos/química , Lipossomos/química , Porinas/química , Escherichia coli/metabolismo , Polarização de Fluorescência , Metabolismo dos Lipídeos , Lipossomos/metabolismo , Porinas/metabolismo , Espermina/metabolismoRESUMO
PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.
Assuntos
Imunoglobulinas/genética , Transtornos Linfoproliferativos/diagnóstico , Receptores de Antígenos de Linfócitos T/genética , DNA/análise , Rearranjo Gênico , Guias como Assunto , Humanos , Transtornos Linfoproliferativos/genética , Reação em Cadeia da Polimerase MultiplexRESUMO
Infiltration galleries are among the oldest known means used for small public water fountains. Owing to its ancestral origin they are usually associated with high quality water. Thirty-one compounds, including pesticides and estrogens from different chemical families, were analysed in waters from infiltration galleries collected in Alto Douro Demarcated Wine region (North of Portugal). A total of twelve compounds were detected in the water samples. Nine of these compounds are described as presenting evidence or potential evidence of interfering with the hormone system of humans and wildlife. Although concentrations of the target analytes were relatively low, many of them below their limit of quantification, four compounds were above quantification limit and two of them even above the legal limit of 0.1 µg/L: dimethoate (30.38 ng/L), folpet (64.35 ng/L), terbuthylazine-desethyl (22.28 to 292.36 ng/L) and terbuthylazine (22.49 to 369.33 ng/L).
Assuntos
Drenagem Sanitária , Disruptores Endócrinos/análise , Monitoramento Ambiental , Praguicidas/análise , Poluentes Químicos da Água/análise , Abastecimento de Água/análise , Animais , Animais Selvagens , Dimetoato/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Ftalimidas/análise , Portugal , Medição de Risco , Triazinas/análise , Abastecimento de Água/normasRESUMO
Probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. Previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. These differences in uptake mechanism can be paramount in counteracting bacterial resistance. In view of lipids role in bacterial drug uptake, the interaction of these compounds with different Escherichia coli model membranes were studied by fluorescence spectroscopy. Partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. These results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological pH, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association.
Assuntos
Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Complexos de Coordenação/farmacologia , Escherichia coli/química , Fluoroquinolonas/farmacologia , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Membrana Celular/química , Complexos de Coordenação/metabolismo , Cobre/metabolismo , Farmacorresistência Bacteriana , Enrofloxacina , Fluoroquinolonas/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Lipossomos/química , Fenantrolinas/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Espectrometria de Fluorescência , Eletricidade EstáticaRESUMO
The phase transition temperatures of several lipidic systems were determined using two different techniques: dynamic light scattering (DLS) and steady-state fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA-DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize different lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for Escherichia coli membranes. Given the important role of cardiolipin (CL) in natural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared with those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture.
Assuntos
Cardiolipinas/metabolismo , Escherichia coli/metabolismo , Polarização de Fluorescência , Membrana Celular/metabolismo , Corantes Fluorescentes , Luz , Lipossomos , Lipídeos de Membrana/metabolismo , Microscopia de Força Atômica , Espalhamento de RadiaçãoRESUMO
Primary cutaneous follicle center lymphoma (PCFCL) is characterized by a proliferation of follicle center cells in the skin. A definitive diagnosis is frequently delayed because of difficulties in interpretation of the histopathologic findings. It has an excellent prognosis with a 5-year survival over 95% and its risk of transformation has not been established. We describe a case report of man with a gastric diffuse large B-cell lymphoma (DLBCL) referred to our clinic because of nodules in the back that had gradually developed over a period of 10 years. A biopsy performed 3 years before was interpreted as reactive follicular hyperplasia. A new skin biopsy revealed a diffuse large B-cell lymphoma and immunoglobulin heavy chain gene rearrangements from the initial skin biopsy (PCBCL) and the DLBCL gastric biopsy were studied by polymerase chain reaction and an identical clonal rearrangement was detected which was highly suggestive of a transformation lymphoma.
RESUMO
A multi-residue methodology based on a solid phase extraction followed by gas chromatography-tandem mass spectrometry was developed for trace analysis of 32 compounds in water matrices, including estrogens and several pesticides from different chemical families, some of them with endocrine disrupting properties. Matrix standard calibration solutions were prepared by adding known amounts of the analytes to a residue-free sample to compensate matrix-induced chromatographic response enhancement observed for certain pesticides. Validation was done mainly according to the International Conference on Harmonisation recommendations, as well as some European and American validation guidelines with specifications for pesticides analysis and/or GC-MS methodology. As the assumption of homoscedasticity was not met for analytical data, weighted least squares linear regression procedure was applied as a simple and effective way to counteract the greater influence of the greater concentrations on the fitted regression line, improving accuracy at the lower end of the calibration curve. The method was considered validated for 31 compounds after consistent evaluation of the key analytical parameters: specificity, linearity, limit of detection and quantification, range, precision, accuracy, extraction efficiency, stability and robustness.
Assuntos
Disruptores Endócrinos/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Análise dos Mínimos Quadrados , Praguicidas/análise , Poluentes Químicos da Água/análise , Estabilidade de Medicamentos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.
Assuntos
Genes de Imunoglobulinas , Leucemia de Células T/genética , Linfoma de Células T/genética , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T/genética , Amplificação de Genes , Rearranjo Gênico , Genótipo , Humanos , Imuno-Histoquímica , Leucemia Prolinfocítica/genética , Leucemia Prolinfocítica/imunologia , Leucemia Prolinfocítica/patologia , Leucemia de Células T/imunologia , Leucemia de Células T/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Linfócitos T/imunologiaRESUMO
Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.
Assuntos
Genes de Imunoglobulinas , Leucemia de Células B/genética , Linfoma de Células B/genética , Reação em Cadeia da Polimerase/métodos , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Rearranjo Gênico , Genótipo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia de Células B/diagnóstico , Leucemia de Células B/imunologia , Linfoma de Células B/diagnóstico , Linfoma de Células B/imunologia , Receptores de Antígenos de Linfócitos T/genética , Translocação GenéticaRESUMO
The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.
Assuntos
Linfoma/genética , Linfoma/patologia , Reação em Cadeia da Polimerase/métodos , Reações Falso-Negativas , Rearranjo Gênico , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Linfoma de Células T/genética , Linfoma de Células T/patologia , Receptores de Antígenos de Linfócitos T/genética , Reprodutibilidade dos TestesRESUMO
A 44-month old girl with congenital amegakaryocytic thrombocytopenia, already with pancytopenia, underwent an unrelated allogeneic cord blood transplantation with recovery of normal blood cell counts. The patient was a compound heterozygote for two c-mpl missense mutations inherited from both parents, one of them, a G578A exon 4 mutation leading to a cysteine to tyrosine replacement of codon 193, previously unreported.
Assuntos
Megacariócitos , Mutação de Sentido Incorreto , Receptores de Trombopoetina/genética , Trombocitopenia/congênito , Trombocitopenia/genética , Pré-Escolar , Feminino , Impressão Genômica , Heterozigoto , Humanos , Megacariócitos/patologia , Transplante de Células-Tronco , Trombocitopenia/patologia , Trombocitopenia/cirurgia , Resultado do TratamentoRESUMO
The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes.
Assuntos
Clordiazepóxido/química , Dibucaína/química , Isoniazida/química , Rifampina/química , Espectrofotometria/métodos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas , Fosfatidilgliceróis , Eletricidade EstáticaRESUMO
Notch signaling is known to differentially affect the development of lymphoid B and T cell lineages, but it remains unclear whether such effects are specifically dependent on distinct Notch ligands. Using a cell coculture assay we observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B cell lineage while promoting the emergence of cells with a phenotype of T cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B or T cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population coexpressing CD4 and CD8. Our results thus indicate that distinct Notch ligands can mediate differential effects of Notch signaling and provide a useful system to further address cell-fate decision processes in lymphopoiesis.
Assuntos
Linfócitos/citologia , Tecido Linfoide/citologia , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Antígenos CD34 , Subpopulações de Linfócitos B/citologia , Linfócitos B/citologia , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Linhagem da Célula , Técnicas de Cocultura , Sangue Fetal/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-15/farmacologia , Interleucina-7/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Jagged-1 , Células Matadoras Naturais , Ligantes , Proteínas Proto-Oncogênicas/farmacologia , Receptores Proteína Tirosina Quinases/farmacologia , Receptores Notch , Proteínas Serrate-Jagged , Transdução de Sinais , Células Estromais/citologia , Linfócitos T/citologia , Tirosina Quinase 3 Semelhante a fmsRESUMO
Oligoclonal B cell proliferation, as defined by the presence of more than one leukemic clone, has been detected in approximately 20% to 30% of patients with acute lymphoblastic leukemia (ALL) using PCR or Southern blotting. An accurate assessment of these populations is required to avoid false negative measurements of minimal residual disease (MRD) in follow-up bone marrow (BM) samples of ALL patients. In this study, we analysed 29 ALL patients with two or more immunoglobulin heavy (IGH) chain gene rearrangements in the presentation samples using IGH fingerprinting PCR and sequence analysis. Thirty-nine (51%) of 76 sequences (from 15 patients), shared no VNDNJ homology (ie different CDR3 regions). In the remaining 14 patients, at least two related VH sequences were identified in each patient (identical DNJ sequences). Numerical abnormalities of chromosome 14 was detected in 10 patients. Eight patients were analysed at presentation and relapse. In four of them, expansion of a minor presentation-clone was detected at relapse while the major presentation clone disappeared, confirming 'subclonal evolution'. Finally, in our cohort of patients, the presence of related or unrelated IGH clones did not influence overall survival.
Assuntos
Linfoma de Burkitt/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Adolescente , Adulto , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Criança , Pré-Escolar , Aberrações Cromossômicas/diagnóstico , Cromossomos Humanos Par 14/genética , Células Clonais/metabolismo , Células Clonais/patologia , Estudos de Coortes , Análise Citogenética , Feminino , Heterogeneidade Genética , Humanos , Cadeias J de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Lactente , Masculino , Reação em Cadeia da Polimerase , Recidiva , Análise de Sequência , Resultado do Tratamento , Trissomia/genéticaRESUMO
BACKGROUND AND OBJECTIVES: Detection of PML-RAR alpha transcripts by RT-PCR is now established as a rapid and sensitive method for diagnosis of acute promyelocytic leukemia (APL). Although the majority of patients in long-term clinical remission are negative by consecutive reverse transcription polymerase chain reaction (RT-PCR) assays, negative tests are still observed in patients who ultimately relapse. Conversion from negative to positive PCR has been observed after consolidation and found to be a much stronger predictor of relapse. This study reports on 47 APL patients to determine the correlation between minimal residual disease (MRD) status and clinical outcome in our cohort of patients. DESIGN AND METHODS: The presence of PML-RAR alpha t transcripts was investigated in 47 APL patients (37 adults and 10 children) using a semi-nested reverse transcriptase-polymerase chain reaction to evaluate the prognostic value of RT-PCR tests. RESULTS: All patients achieved complete clinical remission (CCR) following induction treatment with all-trans retinoic acid (ATRA) and chemotherapy (CHT) or ATRA alone. Patients were followed up between 2 and 117.6 months (median: 37 months). Relapses occurred in 11 patients (9 adults and 2 children) between 11.4 and 19 months after diagnosis (median: 15.1 months) while 36 patients (28 adults and 8 children) remained in CCR. Seventy-five percent of patients carried the PML-RAR alpha long isoform (bcr 1/2) which also predominated among the relapsed cases (9 of 11) but did not associate with any adverse outcome (p= 0.37). For the purpose of this analysis, minimal residual disease tests were clustered into four time-intervals: 0-2 months, 3-5 months, 6-9 months and 10-24 months. INTERPRETATION AND CONCLUSIONS: Children showed persisting disease for longer than adults during the first 2 months of treatment. At 2 months, 10 (50%) of 20 patients who remained in CCR and 4 (80%) of 5 patients who subsequently relapsed were positive. Patients who remained in CCR had repeatedly negative results beyond 5.5 months from diagnosis. A positive MRD test preceded relapse in 3 of 4 tested patients. The ability of a negative test to predict CCR (predictive negative value, PNV) was greater after 6 months (>83%), while the ability of a positive test to predict relapse (predictive positive value, PPV) was most valuable only beyond 10 months (100%). This study (i) highlights the prognostic value of RT-PCR monitoring after treatment of APL patients but only from the end of treatment, (ii) shows an association between conversion to a positive test and relapse and (iii) suggests that PCR assessments should be carried out at 3-month intervals to provide a more accurate prediction of hematologic relapses but only after the end of treatment.