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1.
Curr Protoc ; 1(9): e222, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34492732

RESUMO

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large flow cytometry panels reaching up to 43 colors at the single-cell level. However, as panel size and complexity increase, so too does the detail involved in designing and optimizing successful high-quality panels fit for downstream high-dimensional data analysis. In contrast to conventional flow cytometers, full-spectrum flow cytometers measure the entire emission spectrum of each fluorophore across all lasers. This allows for fluorophores with very similar emission maxima but unique overall spectral fingerprints to be used in conjunction, enabling relatively straightforward design of larger panels. Although a protocol for best practices in full-spectrum flow cytometry panel design has been published, there is still a knowledge gap in going from the theoretically designed panel to the necessary steps required for panel optimization. Here, we aim to guide users through the theory of optimizing a high-dimensional full-spectrum flow cytometry panel for immunophenotyping using comprehensive step-by-step protocols. These protocols can also be used to troubleshoot panels when issues arise. A practical application of this approach is exemplified with a 24-color panel designed for identification of conventional T-cell subsets in human peripheral blood. © 2021 Malaghan Institute of Medical Research, Cytek Biosciences. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and evaluation of optimal spectral reference controls Support Protocol 1: Antibody titration Support Protocol 2: Changing instrument settings Basic Protocol 2: Unmixing evaluation of fully stained sample Basic Protocol 3: Evaluation of marker resolution Support Protocol 3: Managing heterogeneous autofluorescence Basic Protocol 4: Assessment of data quality using expert gating and dimensionality reduction algorithms.


Assuntos
Corantes Fluorescentes , Lasers , Citometria de Fluxo , Humanos , Imunofenotipagem , Subpopulações de Linfócitos T
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1093-1100, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362487

RESUMO

OBJECTIVE: To investigate the clinicopathologic characteristics, diagnosis and therapy of aggressive natural killer cell leukemia (ANKL) patients. METHODS: Clinical manifestations, cellular morphology, immunophenotypic analysis by flow cytometry (FCM), TCR gene rearrangement, pathology and Immunohistochemical analysis of bone marrow (BM) were combined to diagnose the six patients with ANKL. RESULTS: The median age of the patients were 35.5 years old. All the patients with fever, cytopenia and liver dysfunction. Imageological examination presented hepatosplenomegaly (6/6), and PET/CT presented diffusely increased metabolism in liver, spleen and BM (3/3). BM cytologic examination presented increased hematophagocyte at the early stage and 1%-42% leukemic cell were detected in BM with the progression of diseases. FCM showed the leukemic cells were positive for CD2(6/6), CD56(5/6), CD16(2/6), CD94(3/6), CD38(3/6), cCD3(1/5), CD8(1/6), CD7(2/6), CD57(1/6) and negative for CD3, CD4, TdT, cMPO, TCR α/ß, TCR γ/δ. The neoplastic cells were negative for TCR gene rearrangement. Five cases showed increased quantitation of whole blood Epstein-Barr virus (EBV) DNA. CONCLUSION: ANKL is a highly aggressive disease. Prompt and repeating BM examination is important to patient with fever, cytopenia and liver dysfunction. The diagnosis of ANKL relies mainly on the integration of clinical, morphologic, immunophenotypic finding and EBV-DNA increasement.


Assuntos
Infecções por Vírus Epstein-Barr , Leucemia Linfocítica Granular Grande , Adulto , Herpesvirus Humano 4 , Humanos , Imunofenotipagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1195-1202, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362502

RESUMO

OBJECTIVE: To investigate the expression of CD319 and CD269 in plasma cells of patients with multiple myeloma (MM) and the feasibility of using CD319 instead of CD38 as a gating antigen in immunophenotyping and minimal residual disease (MRD) monitoring. METHODS: The expression levels of CD319 and CD269 antigens in clonal bone marrow plasma cells of 387 patients were detected by CD38/CD138 gating strategy with 8-color flow cytometry, and the stability of antigens was also analyzed, and the sensitivity and correlation of two different gating strategies employing CD319/CD138 and CD38/CD138 were compared as well. The control group consisted of 53 cases with non-malignant blood disease matched by age and sex. RESULTS: Monoclonal plasma cells were detected in 303 of 387 MM patients, among which 277 cases (91.42%) were positive for CD269, and all cases were positive for CD319 (100%). In newly diagnosed MM (NDMM) and recurrent refractory MM (RRMM) patients, the expression levels of CD269 were 97.53% (0-99.92%) and 94.96% (0.22%-99.99%), respectively, while levels of CD319 were 99.90% (87.77%-100%) and 99.78% (63.12%-100%), respectively. The expression levels of CD269 and CD319 in the control group were 97.00% (77.00%-100%) and 100% (89.00%-100%), respectively. There were no significant differences in the expression levels of CD269 and CD319 among NDMM, RRMM and the control group. Patients acquiring therapeutic effects were divided into complete remission (CR) group, very good partial response (VGPR) group and partial response (PR) group. Gating with CD38/CD138, median MRD values were 0.76% (0-1.88%), 0.77% (0-4.96%) and 1.75% (0.09%-10.90%) in the three groups, respectively, while gating with CD319/CD138, median MRD values were 0.57% (0.18%-1.96%), 1.07% (0.12%-4.85%) and 1.77% (0.08%-8.22%), respectively. There was no significant difference in MRD level by the two gating strategies, but a good correlation between the two (r=0.808, P<0.05). In addition, in 4 patients treated by CD38 monoclonal antibody (DARA), the expression level of CD38 was observed to be down-regulated or even negative after treatment. When the MRD level was very low, CD38/CD138 gating resulted in false MRD-, while CD319/CD138 gating resulted in MRD+. CONCLUSION: CD319 and CD269 express stably and continuously in plasma cells of MM patients at different disease stages. CD319 can be used as an alternative of CD38 for immunophenotyping and MRD detection, especially for MRD detection after DARA treatment, while CD269 is suitable for detection before BCMA-CAR-T treatment.


Assuntos
Mieloma Múltiplo , Citometria de Fluxo , Humanos , Imunofenotipagem , Recidiva Local de Neoplasia , Neoplasia Residual , Plasmócitos
4.
Sci Immunol ; 6(62)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376481

RESUMO

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.


Assuntos
COVID-19/genética , COVID-19/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunofenotipagem , SARS-CoV-2/imunologia , Transcriptoma , Adulto , Idoso , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , COVID-19/virologia , Plasticidade Celular/genética , Plasticidade Celular/imunologia , Evolução Clonal/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Isotipos de Imunoglobulinas/imunologia , Memória Imunológica , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
5.
Zhonghua Xue Ye Xue Za Zhi ; 42(6): 480-486, 2021 Jun 14.
Artigo em Chinês | MEDLINE | ID: mdl-34384154

RESUMO

Objective: To analyze the genetic landscape of multiple fusion genes in patients with de novo acute myeloid leukemia (AML) and investigate the characteristics of immunophenotypes and mutations. Methods: The results of multiple fusion genes from 4192 patients with de novo AML were retrospectively analyzed from 2016 to 2020. In addition, the immunophenotypical data and the mutational results from high-through put method were statistically investigated and correlated as well. Results: ①Among the 52 targets, 29 different types of fusion genes were detected in 1948 patients (46.47%) with AML, which demonstrated an "exponential distribution" . ② As the age increased, the number of patients with fusion gene increased first and then decreased gradually. The total incidence rate of fusion genes and MLL rearrangment in children were significantly higher than those in adults (69.18% vs 44.76%, 15.35% vs 8.36%) . ③The mutations involving FLT3 and RAS signaling pathway contributed most in patients with MLL rearrangment. ④No specific immunophenotypic characteristics were found in AML patients with MLL or NUP98 rearrangements. Conclusion: Nearly half of AML patients were accompanied by specific fusion gene expression, the proportions of different fusion genes in pediatric and adults patients were different by multiple PCR. The gene mutations and immunophenotype of these AML patients have certain rules.


Assuntos
Leucemia Mieloide Aguda , Adulto , Criança , Expressão Gênica , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Estudos Retrospectivos
6.
Sci Immunol ; 6(62)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376480

RESUMO

The IMmunoPhenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective longitudinal study designed to enroll 1000 hospitalized patients with COVID-19 (NCT04378777). IMPACC collects detailed clinical, laboratory and radiographic data along with longitudinal biologic sampling of blood and respiratory secretions for in depth testing. Clinical and lab data are integrated to identify immunologic, virologic, proteomic, metabolomic and genomic features of COVID-19-related susceptibility, severity and disease progression. The goals of IMPACC are to better understand the contributions of pathogen dynamics and host immune responses to the severity and course of COVID-19 and to generate hypotheses for identification of biomarkers and effective therapeutics, including optimal timing of such interventions. In this report we summarize the IMPACC study design and protocols including clinical criteria and recruitment, multi-site standardized sample collection and processing, virologic and immunologic assays, harmonization of assay protocols, high-level analyses and the data sharing plans.


Assuntos
Biomarcadores , COVID-19/imunologia , COVID-19/virologia , Imunofenotipagem , SARS-CoV-2/imunologia , Biomarcadores/sangue , COVID-19/diagnóstico , COVID-19/epidemiologia , Biologia Computacional/métodos , Análise de Dados , Perfilação da Expressão Gênica , Hospitalização , Humanos , Estudos Longitudinais , Técnicas de Diagnóstico Molecular/métodos , Estudos Prospectivos , Proteômica/métodos , Estados Unidos/epidemiologia
7.
Medicine (Baltimore) ; 100(31): e26745, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34397816

RESUMO

BACKGROUND: To assess the prognostic capability of the maximum standardized uptake values (SUVmax) measured in the primary tumor and axillary lymph nodes (ALNs) by pretreatment fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography and analyze outcomes according to the molecular breast cancer subtypes. METHODS: The databases were systematically searched using keywords for breast cancer, positron emission tomography/computed tomography, and SUVmax; the extracted studies reported at least 1 form of survival data, event-free survival (EFS) and overall survival. Comparative analyses of the pooled hazard ratios (HRs) for EFS and overall survival were performed to assess their correlations with SUVmax. The pooled HR was estimated using random-effects model according to the results of heterogeneity. RESULTS: Thirteen eligible studies comprising 3040 patients with breast cancer were included. The pooled HRs of high SUVmax in the primary tumor and ALN were 3.01 (95% CI 1.83-4.97, P < .00001; I2 = 82%) and 3.72 (95% CI 1.15-12.01; I2 = 92%; P = .03), respectively. Patients with higher SUVmax demonstrated a poorer survival prognosis. Furthermore, comparative analyses according to the molecular subtypes demonstrated that the SUVmax in the primary tumor or ALN can be a predictive parameter in patients with the luminal subtype disease. Subtype analysis results indicated a significant association of the luminal group, with a HR of 2.65 (95% CI 1.31-5.37; I2 = 27%; P = .007). CONCLUSIONS: SUVmax from pretreatment is a significant prognostic factor for EFS in patients with breast cancer. Despite several limitations, correlation with molecular subtype (luminal type) was demonstrated. Further large-scale studies are required to investigate the precise prognostic capability of SUVmax.


Assuntos
Neoplasias da Mama/complicações , Neoplasias da Mama/genética , Valor Preditivo dos Testes , Neoplasias da Mama/mortalidade , Feminino , Humanos , Imunofenotipagem
8.
Clin Immunol ; 230: 108827, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34428741

RESUMO

BAL cellularity and lymphocyte immunophenotyping offer insights into lung inflammatory status. Natural killer (NK) cells are efficient effector cells, producing pro-inflammatory cytokines. A better understanding of the biology of NK cells in BAL in the lungs is necessary to improve the pathogenesis of fibrotic ILD and develop prospective targeted treatments. Our aim was to analyse NK and NKT-like cell percentages in BAL from 159 patients with different ILD: f-HP, f-NSIP, IPF and CTD-ILD, to evaluate their potential diagnostic/prognostic role. BAL NK cell percentages showed significantly higher values in IPF than in f-HP and f-NSIP, while BAL NKT-like cells showed significantly lower values in the f-NSIP than the f-HP and IPF. A cut-off of 4%NK cells in BAL of IPF showed a significant difference in survival rate. It suggests a possible new marker of survival and raises the possibility of new targeted approach in treatment and management of IPF.


Assuntos
Células Matadoras Naturais/imunologia , Doenças Pulmonares Intersticiais/imunologia , Idoso , Idoso de 80 Anos ou mais , Alveolite Alérgica Extrínseca/imunologia , Alveolite Alérgica Extrínseca/patologia , Alveolite Alérgica Extrínseca/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Doenças do Tecido Conjuntivo/imunologia , Doenças do Tecido Conjuntivo/patologia , Doenças do Tecido Conjuntivo/fisiopatologia , Feminino , Fibrose , Humanos , Fibrose Pulmonar Idiopática/imunologia , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/fisiopatologia , Imunofenotipagem , Estimativa de Kaplan-Meier , Células Matadoras Naturais/classificação , Células Matadoras Naturais/patologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/fisiopatologia , Doenças Pulmonares Intersticiais/patologia , Doenças Pulmonares Intersticiais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Células T Matadoras Naturais/classificação , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/patologia , Prognóstico , Testes de Função Respiratória
9.
Zhonghua Xue Ye Xue Za Zhi ; 42(7): 577-582, 2021 Jul 14.
Artigo em Chinês | MEDLINE | ID: mdl-34455745

RESUMO

Objective: The study aimed to analyze the clinical features and prognosis of chronic lymphocytic leukemia (CLL) with t (14;18) (q32;q21) and conduct a literature review. Methods: The clinical data of 8 patients with CLL carrying t (14;18) (q32;q21) seen in Jiangsu Province Hospital from November 2009 to November 2019 were collected and analyzed. Results: Among the 8 cases, 7 were male and 1 was female. The median age at diagnosis was 70 years old. The immunophenotype score was 5 in 3 patients. 4 patients were scored 4 and the remaining one scored 3. The bone marrow histopathology showed the typical manifestation of CLL. Karyotype analysis showed that all the cases carried t (14;18) (q32;q21) in the stemline. The t (14;18) (q32;q21) showed as the sole abnormality in 3 cases, with +12 in 4, and with 13q- in 1 case. 13q- was found in another 3 patients by FISH. Immunoglobulin heavy chain gene (IGHV) mutation status was detected in 6 cases and all of them were mutated. None of them used IGHV3-21. Only 1 case harbored TP53 mutation and no TP53, SF3B1, NOTCH1, or MYD88 mutations were found in the remaining cases who underwent the relevant tests. At a median follow-up of 30.9 months, 1 case died. The remaining 7 cases survived and 3 of them have not reached the treatment indication. 4 patients who received chemotherapy or immunotherapy were stable. Conclusions: The t (14;18) (q32;q21) is rare in CLL and often accompanied by +12 and mutated IGHV. CLL with t (14; 18) (q32; q21) tends to have a good prognosis.


Assuntos
Leucemia Linfocítica Crônica de Células B , Idoso , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Mutação , Prognóstico
10.
Science ; 373(6552)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34437125

RESUMO

Germinal centers (GCs) are the site of immunoglobulin somatic hypermutation and affinity maturation, processes essential to an effective antibody response. The formation of GCs has been studied in detail, but less is known about what leads to their regression and eventual termination, factors that ultimately limit the extent to which antibodies mature within a single reaction. We show that contraction of immunization-induced GCs is immediately preceded by an acute surge in GC-resident Foxp3+ T cells, attributed at least partly to up-regulation of the transcription factor Foxp3 by T follicular helper (TFH) cells. Ectopic expression of Foxp3 in TFH cells is sufficient to decrease GC size, implicating the natural up-regulation of Foxp3 by TFH cells as a potential regulator of GC lifetimes.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/genética , Centro Germinativo/imunologia , Células T Auxiliares Foliculares/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Genes Codificadores dos Receptores de Linfócitos T , Centro Germinativo/citologia , Imunização , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Célula Única , Células T Auxiliares Foliculares/imunologia , Linfócitos T Reguladores/fisiologia , Regulação para Cima
11.
Rinsho Ketsueki ; 62(7): 717-720, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-34349053

RESUMO

This study reports a case of a 49-year-old woman having B-cell acute lymphoblastic leukemia with glycophorin A, a representative erythroid marker, expression. According to the WHO criteria for mixed phenotype acute leukemia (MPAL), erythroid lineage is not defined, and to the best of our knowledge, only one other case with erythroid/B-cell biphenotypic acute leukemia has been reported previously. To establish the disease entity and clarify the pathophysiology of erythroid/lymphoid MPAL, additional cases need to be analyzed.


Assuntos
Leucemia Aguda Bifenotípica , Leucemia-Linfoma Linfoblástico de Células Precursoras , Doença Aguda , Linfócitos B , Feminino , Glicoforinas , Humanos , Imunofenotipagem , Pessoa de Meia-Idade
12.
Int J Lab Hematol ; 43 Suppl 1: 78-81, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34288445

RESUMO

Philadelphia (BCR-ABL)-negative myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). MPN can transform into an accelerated or a blast phase, which is associated with poor response to standard therapy and low overall median survival. We present an interesting case of a patient with a history of PMF and progression and summarize the current studies on genetic features of myeloproliferative neoplasms in blast phase (MPN-BP) with an emphasis on PMF. Although MPN-BP show ≥20% blasts in peripheral blood or bone marrow, it is not considered as acute myeloid leukemia (AML) according to the WHO classification. While MPNs-BP typically lack genetic mutations seen in de novo AML, they commonly harbor IDH1/2, SRSF2, ASXL1, and TP53 mutations, similar to the genetic profiles of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC).


Assuntos
Suscetibilidade a Doenças , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/etiologia , Biomarcadores , Medula Óssea/patologia , Análise Citogenética , Progressão da Doença , Índices de Eritrócitos , Predisposição Genética para Doença , Humanos , Imunofenotipagem , Pessoa de Meia-Idade , Mielofibrose Primária/metabolismo , Mielofibrose Primária/terapia
13.
Int J Lab Hematol ; 43 Suppl 1: 43-53, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34288449

RESUMO

Multiple myeloma (MM) is a heterogeneous group of mature B-cell diseases that are typically characterized by the presence and accumulation of abnormal plasma cells (PCs), which results in the excess production of monoclonal immunoglobulin and/or light chain found in the serum and/or urine. Multiparametric flow cytometry (MFC) is an indispensable tool to supplement the diagnosis, classification and monitoring of the disease due to its high patient applicability, excellent sensitivity and encouraging results from various clinical trials. In this regard, minimal or, more appropriately, measurable residual disease (MRD) negativity by MFC has been recognized as a powerful predictor of favourable long-term outcomes. Before flow cytometry can be effectively implemented in the clinical setting for MM MRD testing, sample preparation, panel configuration, analysis and gating strategies must be optimized to ensure accurate results. This manuscript will discuss the current consensus guidelines for flow cytometric processing of samples and reporting of results for MM MRD testing. We also discuss alternative approaches to detect plasma cells in the presence of daratumumab treatment. Finally, there is a lack of information describing the subclonal distribution of myeloma cells based on their protein expression. The advent of high-dimensional analysis may assist in following the evolution of antigen expression patterns on abnormal plasma cells in patients with relapsed/refractory disease. This in turn can help identify clonal subtypes that are more aggressive for potential informed decision. An analysis using t-SNE to identify the emergence of PCs subclones by MFC, along with the analysis of their immunophenotypic profiles are presented as a future perspective.


Assuntos
Citometria de Fluxo , Imunofenotipagem , Mieloma Múltiplo/diagnóstico , Neoplasia Residual/diagnóstico , Biomarcadores Tumorais , Análise de Dados , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Imunofenotipagem/métodos , Imunofenotipagem/normas , Guias de Prática Clínica como Assunto , Reprodutibilidade dos Testes , Projetos de Pesquisa , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Manejo de Espécimes/normas
14.
Int J Lab Hematol ; 43 Suppl 1: 82-85, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34288451

RESUMO

Unexplained blood cytopenias, in particular anemia, are often found in older individuals. The relationship between these cytopenias and myeloid neoplasms like myelodysplastic syndromes is currently poorly defined. Terminology used to describe patients with unexplained cytopenias and with clonally restricted hematopoiesis can be confusing and is evolving. This review uses a complex clinical case with borderline morphology and somatic mutations with high variant allele frequencies to illustrate a diagnostic approach to clonal cytopenias, and differentiation from myeloid neoplasms with a focus on appropriate ancillary testing. Testing for somatic mutations and variant allele frequency is helpful in assessing risk for progression to myeloid malignancy. The interpretation of mutation profiles in patients with cytopenia has been challenging, as some of these genes are commonly detected in elderly adults showing a normal blood count as well as in individuals with nonmalignant bone marrow failure syndromes. For patients with unexplained cytopenias, longitudinal follow-up including monitoring of blood counts may also be appropriate.


Assuntos
Hematopoiese Clonal , Pancitopenia/diagnóstico , Pancitopenia/etiologia , Idoso , Biomarcadores , Medula Óssea/patologia , Hematopoiese Clonal/genética , Diagnóstico Diferencial , Gerenciamento Clínico , Suscetibilidade a Doenças , Testes Hematológicos , Humanos , Imunofenotipagem , Masculino
15.
Blood Adv ; 5(14): 2890-2900, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34297047

RESUMO

Early T-cell precursor phenotype acute lymphoblastic leukemia (ETP-ALL) is a subtype of T-ALL with a unique immunophenotype and genetic abnormalities distinct from conventional T-ALL. A subset of T lymphoblastic lymphoma (T-LLy) also demonstrates the early T-cell precursor immunophenotype and may be a counterpart of ETP-ALL. Unlike ETP-ALL, the incidence, clinical features, and genomic features of ETP-LLy are unknown. We reviewed the immunophenotyping data of 218 T-LLy patients who enrolled in the Children's Oncology Group AALL0434 clinical trial and identified 9 cases (4%) exhibiting a definitive ETP immunophenotype. We performed single-nucleotide polymorphism array profiling on 9 ETP-LLy and 15 non-ETP T-LLy cases. Compared with non-ETP T-LLy, ETP-LLy showed less frequent deletion of 9p (CKDN2A/B), more frequent deletion of 12p (ETV6) and 1p (RPL22), and more frequent absence of biallelic T-cell receptor γ deletions. Recurrent abnormalities previously described in ETP-ALL such as deletions of 5q and 13q and gain of 6q were not observed in ETP-LLy cases. There were no failures of therapy among the ETP-LLy subtype with a 4-year event-free survival of 100%. Overall, ETP-LLy does not exhibit unifying genetic alterations but shows some distinct genomic features from non-ETP T-LLy suggesting that ETP-LLy may be a distinct entity from non-ETP T-LLy.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Células Precursoras de Linfócitos T , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Criança , Genômica , Humanos , Imunofenotipagem , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética
16.
Int J Hematol ; 114(3): 363-372, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34213732

RESUMO

Autologous stem cell transplantation (ASCT) is a standard of care in newly-diagnosed multiple myeloma (MM) patients. Several studies before the introduction of novel therapies in MM, demonstrated a pegylated G-CSF to be successful in mobilizing peripheral blood stem cells (PBSCs). Lipegfilgrastim is a novel long-acting G-CSF that is produced by the conjugation of a single 20-kDa polyethelene glycol to the natural O-glycosylation site of G-CSF. Twenty-four MM patients were included for PBSCs mobilization with a single SC injection of 6 mg lipegfilgrastim. PBSC collection was started when the CD34+ count was > 10 × 106 cells/L. The target progenitor cells were 6 × 106 cells/kg. The median day of apheresis was + 3 (range 2-5) following lipegfilgrastim. Median peripheral blood CD34+ count pre-mobilization was of 22.65 (range 3.36-105) × 106 cells/L. The median number of leukaphaeresis procedures was 2 (range 1-4). The median mobilized CD34+ cells/kg were 8.26 (range 0.77-12.42). One patient failed to mobilize and two patients mobilized < 6 × 106 cells/kg. Toxicity was mild and transient. Twenty-three patients underwent ASCT following high dose melphalan. All patients engrafted. As lipegfilgrastim is administered only once, it is conceivable that it improves both compliance and quality-of-life (NCT02488382).


Assuntos
Filgrastim/administração & dosagem , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo/terapia , Polietilenoglicóis/administração & dosagem , Adulto , Idoso , Remoção de Componentes Sanguíneos , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transplante Autólogo , Resultado do Tratamento
17.
Methods Mol Biol ; 2352: 73-96, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324181

RESUMO

Progressive aging is a physiological process that represents a central risk factor for the development of several human age-associated chronic diseases, including neurodegenerative diseases. A major focus in biomedical research is the pursuit for appropriate model systems to better model the biology of human aging and the interface between aging and disease mechanisms. Direct conversion of human fibroblasts into induced neurons (iNs) has emerged as a novel technology for the in vitro modeling of age-dependent neurological diseases. Similar to other cellular reprogramming techniques, e.g., iPSC-based cellular reprograming, direct conversion relies on the ectopic overexpression of transcription factors, typically including well-known pioneer factors. However, in contrast to alternative technologies to generate neurons, the entire process of direct conversion bypasses any proliferative or stem cell-like stage, which in fact renders it the unique aptitude of preserving age-associated hallmarks from the initial fibroblast source. In this chapter, we introduce direct conversion as a practical and easy-to-approach disease model for aging and neurodegenerative disease research. A focus here is to provide a stepwise protocol for the efficient and highly reproducible generation of iNs from adult dermal fibroblasts from human donors.


Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Biomarcadores , Reprogramação Celular/genética , Derme/citologia , Citometria de Fluxo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Lentivirus/genética , Transdução Genética
18.
Front Immunol ; 12: 557994, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220791

RESUMO

The immunosuppressive mechanisms of the surrounding microenvironment and distinct immunogenomic features in glioblastoma (GBM) have not been elucidated to date. To fill this gap, useful data were extracted from The Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA), GSE16011, GSE43378, GSE23806, and GSE12907. With the ssGSEA method and the ESTIMATE and CIBERSORT algorithms, four microenvironmental signatures were used to identify glioma microenvironment genes, and the samples were reasonably classified into three immune phenotypes. The molecular and clinical features of these phenotypes were characterized via key gene set expression, tumor mutation burden, fraction of immune cell infiltration, and functional enrichment. Exhausted CD8+ T cell (GET) signature construction with the predictive response to commonly used antitumor drugs and peritumoral edema assisted in further characterizing the immune phenotype features. A total of 2,466 glioma samples with gene expression profiles were enrolled. Tumor purity, ESTIMATE, and immune and stromal scores served as the 4 microenvironment signatures used to classify gliomas into immune-high, immune-middle and immune-low groups, which had distinct immune heterogeneity and clinicopathological characteristics. The immune-H phenotype had higher expression of four immune signatures; however, most checkpoint molecules exhibited poor survival. Enriched pathways among the subtypes were related to immunity. The GET score was similar among the three phenotypes, while immune-L was more sensitive to bortezomib, cisplatin, docetaxel, lapatinib, and rapamycin prescriptions and displayed mild peritumor edema. The three novel immune phenotypes with distinct immunogenetic features could have utility for understanding glioma microenvironment regulation and determining prognosis. These results contribute to classifying glioma subtypes, remodeling the immunosuppressive microenvironment and informing novel cancer immunotherapy in the era of precision immuno-oncology.


Assuntos
Neoplasias Encefálicas/genética , Linfócitos T CD8-Positivos/imunologia , Glioblastoma/genética , Glioma/genética , Linfócitos do Interstício Tumoral/imunologia , Algoritmos , Antineoplásicos , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/mortalidade , Edema , Regulação Neoplásica da Expressão Gênica , Genoma , Glioblastoma/diagnóstico , Glioblastoma/mortalidade , Glioma/diagnóstico , Glioma/mortalidade , Humanos , Imunogenética/métodos , Imunofenotipagem , Prognóstico , Análise de Sobrevida , Transcriptoma , Microambiente Tumoral/genética
19.
Clin Immunol ; 229: 108797, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34273585

RESUMO

The global obesity epidemic is contributing to increased prevalence of diseases fuelled by chronic inflammation, including cancer. Oesophageal adenocarcinoma (OAC) is an obesity-associated malignancy with increasing prevalence, dismal prognosis, and severely dysregulated immune processes. We previously reported that αß T cells migrate to omentum and liver in OAC and contribute to inflammation in these tissues. Here, we assessed the tissue distribution and phenotype of gamma/delta (γδ) T cells in the blood, omentum, liver and tumour of OAC patients. Our data show that the Vδ1 and Vδ3 subsets of γδ T cells are most prevalent in omentum and liver of OAC patients. Furthermore, γδ T cells are predominantly pro-inflammatory in these tissues, and co-express IFN-γ and IL-17. Moreover, γδ T cells exhibit cytotoxic capabilities in OAC omentum and liver. This study provides the first indication that γδ T cells contribute to obesity-associated inflammation in OAC and might be exploited therapeutically.


Assuntos
Adenocarcinoma/imunologia , Neoplasias Esofágicas/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Adenocarcinoma/etiologia , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Degranulação Celular , Neoplasias Esofágicas/etiologia , Neoplasias Esofágicas/patologia , Feminino , Humanos , Imunofenotipagem , Inflamação/complicações , Interferon gama/metabolismo , Interleucina-17/metabolismo , Fígado/imunologia , Fígado/patologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Omento/imunologia , Omento/patologia , Receptores CCR6/metabolismo , Subpopulações de Linfócitos T/patologia , Subpopulações de Linfócitos T/fisiologia , Distribuição Tecidual
20.
EBioMedicine ; 69: 103464, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34233260

RESUMO

BACKGROUND: The current desirable endpoint of treatment against chronic hepatitis B virus infection (cHBV) is to achieve a functional cure, which is defined as HBsAg loss (sAg-L) with or without anti-HBs seroconversion. However, the immunological features that are associated with functional cure have not been studied in detail. METHODS: 172 cHBV patients (67 HBeAg+ and 105 HBeAg-), including 141 HBsAg retained (sAg-R) patients (115 chronic hepatitis and 26 asymptomatic carriers), 31 sAg-L patients, and 24 healthy individuals (vaccinated but not infected with HBV) were examined for their T cell phenotypic profile and HBV-specific T cell responses by flow cytometry. 18 cHBV patients with low serum HBsAg levels were also longitudinally followed for their T cell phenotypic profile and HBV-specific T cell responses up to 60 weeks. FINDINGS: sAg-L patients showed distinct CD4+ and CD8+ T cell phenotype fingerprints compared to those of sAg-R patients, as mainly indicated by the upregulation of HLA-DR on both CD4+ and CD8+ T cells, and a potent HBcAg-specific CD8+ T cell response. The changes in the T cell phenotype in cHBV patients were even more profound during rapid HBsAg decrease and was associated with interferon α treatment. The expression of HLA-DR (r = 0·3269, p = 0·0037), CD95 (r = 0·2796, p = 0·0151), CD40L (r = 0·2747, p = 0·0156), CTLA-4 (r = 0·2786, p = 0·0148), TIM-3 (r = 0·3082, p = 0·0068), CD107a (r = 0·3597, p = 0·0013) on CD4+ T cells, and HLA-DR (r = 0·3542, p = 0·0016), CD69 (r = 0·2507, p = 0·0279), CD107a (r = 0·2875, p = 0·0112) on CD8+ T cells were positively correlated with the rate of HBsAg decrease. The expression of HLA-DR (r = 0·2846, p = 0·0009) and CD95 (r = 0·2442, p = 0·0049) on CD8+ T cells were positively correlated with the magnitude of the HBcAg-specific T cell responses in cHBV patients. Importantly, CTLA-4, CD95 and CD107a expression on CD4+ T cells, as well as HLA-DR and TIM-3 expression on CD8+ T cells in combination with HBsAg quantification were identified as potential predictive factors for sAg-L within 48 weeks in cHBV patients. INTERPRETATION: The onset of HBsAg decrease and subsequent loss in cHBV patients on treatment is associated with significant alterations of both CD4+ and CD8+ T cell phenotypes. Characterization of the T cell phenotype in cHBV patients may present predicative value for sAg-L. FUNDING: National Natural Science Foundation of China, National Scientific and Technological Major Project of China, Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, "Double-First Class" Project for the International Cooperation Center on Infection and Immunity, HUST.


Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Hepatite B Crônica/sangue , Linfócitos T/imunologia , Adulto , Biomarcadores/sangue , Feminino , Citometria de Fluxo/métodos , Antígenos HLA-DR/imunologia , Hepatite B Crônica/imunologia , Humanos , Imunofenotipagem/métodos , Masculino
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