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1.
Blood ; 143(12): 1139-1156, 2024 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-38064663

RESUMEN

ABSTRACT: The World Health Organization (WHO) classification of hematolymphoid tumors and the International Consensus Classification (ICC) of 2022 introduced major changes to the definition of chronic myelomonocytic leukemia (CMML). To assess its qualitative and quantitative implications for patient care, we started with 3311 established CMML cases (according to WHO 2017 criteria) and included 2130 oligomonocytosis cases fulfilling the new CMML diagnostic criteria. Applying both 2022 classification systems, 356 and 241 of oligomonocytosis cases were newly classified as myelodysplastic (MD)-CMML (WHO and ICC 2022, respectively), most of which were diagnosed as myelodysplastic syndrome (MDS) according to the WHO 2017 classification. Importantly, 1.5 times more oligomonocytosis cases were classified as CMML according to WHO 2022 than based on ICC, because of different diagnostic criteria. Genetic analyses of the newly classified CMML cases showed a distinct mutational profile with strong enrichment of MDS-typical alterations, resulting in a transcriptional subgroup separated from established MD and myeloproliferative CMML. Despite a different cytogenetic, molecular, immunophenotypic, and transcriptional landscape, no differences in overall survival were found between newly classified and established MD-CMML cases. To the best of our knowledge, this study represents the most comprehensive analysis of routine CMML cases to date, both in terms of clinical characterization and transcriptomic analysis, placing newly classified CMML cases on a disease continuum between MDS and previously established CMML.


Asunto(s)
Leucemia Mielomonocítica Crónica , Síndromes Mielodisplásicos , Humanos , Consenso , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/genética , Leucemia Mielomonocítica Crónica/diagnóstico , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/patología , Leucocitosis , Organización Mundial de la Salud , Pronóstico , Compuestos Orgánicos
2.
Platelets ; 32(5): 601-607, 2021 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-32726163

RESUMEN

Investigation of the bone marrow as the main compartment of hematopoiesis is critical in many research fields. Here, we adapted a centrifugation-based method for the isolation of murine bone marrow and compared it to the traditional flushing method. Analysis of primary hematopoietic stem cells, immune cells, and megakaryocytes revealed a comparable distribution of cellular (sub)populations. Furthermore, in vitro differentiated megakaryocytes displayed unaltered proplatelet formation. Strikingly, bone marrow isolation by centrifugation was considerably faster than the flushing method and significantly increased the cell yield. Thus, the centrifugation-based isolation method is highly suitable for the study of murine bone marrow cells.


Asunto(s)
Médula Ósea/metabolismo , Separación Celular/métodos , Centrifugación/métodos , Células Madre Hematopoyéticas/metabolismo , Animales , Humanos , Masculino , Ratones
3.
J Cell Mol Med ; 24(5): 2942-2955, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31957290

RESUMEN

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR-ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR-ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1-mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell-mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Resistencia a Antineoplásicos , Técnicas de Inactivación de Genes , Proteínas con Dominio LIM/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Inhibidores de Proteínas Quinasas/farmacología , Receptores CXCR4/metabolismo , Adenosina Trifosfato/metabolismo , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Biosíntesis de Proteínas/efectos de los fármacos , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Transcripción Genética/efectos de los fármacos , Resultado del Tratamiento
4.
J Immunol ; 198(5): 1944-1951, 2017 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-28100681

RESUMEN

The integrin LFA-1 is essential for efficient activation and for cytotoxicity of NK cells because it initiates the assembly of the immunological synapse and mediates firm adhesion to the target. LFA-1 is also needed to polarize the cytotoxic machinery of the NK cell toward the target cell. The binding affinity and avidity of integrins can be regulated via inside-out signals from other receptors. In this article, we investigate the signals necessary to activate LFA-1 in human NK cells. Our data show that LFA-1 has a low ligand-binding activity in resting human NK cells, but it can be stimulated by triggering activating receptors, such as 2B4 or CD16, or by coactivation of different receptor combinations. Short-term stimulation of freshly isolated NK cells with cytokines, such as IL-15, IL-12, or IL-18, does not activate LFA-1 but increases the responsiveness of the cells to subsequent receptor stimulation. Different NK cell subsets vary in their ability to induce LFA-1 binding activity after activating receptor stimulation. Interestingly, the NK cell subsets that are more mature and possess higher cytotoxic potential also show the highest activation of LFA-1, which correlated with the expression of the small calcium-binding protein S100A4. Our data suggest that regulation of LFA-1 is one reason for the different activity of NK cells during differentiation.


Asunto(s)
Diferenciación Celular , Citocinas/inmunología , Células Asesinas Naturales/fisiología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Transducción de Señal , Proteínas Portadoras/inmunología , Citocinas/genética , Citotoxicidad Inmunológica , Proteínas Ligadas a GPI/inmunología , Regulación de la Expresión Génica , Humanos , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-15/genética , Interleucina-15/inmunología , Interleucina-18/genética , Interleucina-18/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/genética , Receptores de IgG/inmunología
5.
Exp Cell Res ; 325(1): 10-7, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24680986

RESUMEN

In experimental settings, lymphocyte cytotoxicity has been recognized as a central mechanism for immune defense against infected and neoplastic cells. More recently, molecular determinants of lymphocyte cytotoxicity have been identified through studies of rare, inherited hyperinflammatory and lymphoproliferative syndromes that include hemophagocytic lymphohistiocytosis (HLH). These studies have unraveled a set of genes pivotal for the biogenesis and directed release of perforin-containing lysosomes that mediate target cell killing, in addition to other pathways including Fas that also contribute to induction of cell death. Furthermore, studies of such human primary immunodeficiencies have highlighted non-redundant roles of perforin for maintenance of immune homeostasis. Besides providing mechanistic insights to lymphocyte cytotoxicity, studies of individuals with rare hyperinflammatory diseases are highlighting the relevance of lymphocyte cytotoxicity to more common human diseases. It is increasingly recognized that mutations abrogating lymphocyte cytotoxicity not only cause HLH, but also are associated with susceptibility to cancer and autoimmune syndromes. In addition, patients may initially be present with neurological symptoms or severe infectious disease masquerading as variable immunodeficiency syndrome. Here, we highlight new knowledge regarding the molecular mechanisms regulating lymphocyte cytotoxicity and review how mutations in genes associated with HLH cause disease. We also discuss the wider implications of impairments in lymphocyte cytotoxicity for human disease predisposition.


Asunto(s)
Citotoxicidad Inmunológica , Linfohistiocitosis Hemofagocítica/inmunología , Animales , Autoinmunidad , Degranulación de la Célula , Predisposición Genética a la Enfermedad , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/inmunología , Humanos , Células Asesinas Naturales/inmunología , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/fisiopatología , Linfocitos T Citotóxicos/inmunología
7.
Haematologica ; 98(5): 760-4, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23100279

RESUMEN

Experimental model systems have delineated an important role for cytotoxic lymphocytes in the immunosurveillance of cancer. In humans, perforin-deficiency has been associated with occurrence of hematologic malignancies. Here, we describe an Epstein-Barr virus-positive classical Hodgkin's lymphoma in a patient harboring biallelic mutations in STXBP2, a gene required for exocytosis of perforin-containing lytic granules and associated with familial hemophagocytic lymphohistocytosis. Cytotoxic T lymphocytes were found infiltrating the tumor, and a high frequency of Epstein-Barr virus-specific cytotoxic T lymphocytes were detected in peripheral blood. However, lytic granule exocytosis and cytotoxicity by cytotoxic T lymphocytes, as well as natural killer cells, were severely impaired in the patient. Thus, the data suggest a link between defective lymphocyte exocytosis and development of lymphoma in STXBP2-deficient patients. Therefore, with regards to treatment of familial hemophagocytic lymphohistocytosis patients with mutations in genes required for lymphocyte exocytosis, it is important to consider both the risks of hemophagocytic lymphohistocytosis and malignancy.


Asunto(s)
Alelos , Transformación Celular Neoplásica/genética , Enfermedad de Hodgkin/genética , Proteínas Munc18/genética , Mutación , Adolescente , Transformación Celular Viral , Femenino , Expresión Génica , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/virología , Humanos , Linfocitos Infiltrantes de Tumor/patología , Imagen Multimodal , Proteínas Munc18/metabolismo , Linfocitos T Citotóxicos/patología
11.
J Cell Biol ; 210(1): 135-51, 2015 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-26124288

RESUMEN

Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.


Asunto(s)
Membrana Celular/metabolismo , Endosomas/metabolismo , Proteínas R-SNARE/fisiología , Linfocitos T Citotóxicos/inmunología , Degranulación de la Célula , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Sinapsis Inmunológicas/metabolismo , Fusión de Membrana , Transporte de Proteínas , Proteínas Qa-SNARE/metabolismo , Transducción de Señal
12.
Front Immunol ; 4: 515, 2014 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-24459464

RESUMEN

Familial hemophagocytic lymphohistiocytosis (FHL) is an often-fatal hyperinflammatory disorder caused by autosomal recessive mutations in PRF1, UNC13D, STX11, and STXBP2. We identified a homozygous STX11 mutation, c.173T > C (p.L58P), in three patients presenting clinically with hemophagocytic lymphohistiocytosis from unrelated Pakistani families. The mutation yields an amino acid substitution in the N-terminal Habc domain of syntaxin-11 and resulted in defective natural killer cell degranulation. Notably, syntaxin-11 expression was decreased in patient cells. However, in an ectopic expression system, syntaxin-11 L58P was expressed at levels comparable to wild-type syntaxin-11, but did not bind Munc18-2. Moreover, another N-terminal syntaxin-11 mutant, R4A, also did not bind Munc18-2. Thus, we have identified a novel missense STX11 mutation causative of FHL type 4. The syntaxin-11 R4A and L58P mutations reveal that both the N-terminus and Habc domain of syntaxin-11 are required for binding to Munc18-2, implying similarity to the dynamic binary binding of neuronal syntaxin-1 to Munc18-1.

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