RESUMEN
We report an atypical Borrelia garinii infection in a patient who was immunocompromised. It was first suspected as a transformation of follicular lymphoma into high-grade lymphoma. Spirochetes were directly observed on a peripheral blood smear and the diagnosis was confirmed using molecular methods. The clinical presentation and the diagnosis are unique and contrast with the cases described in the literature in patients who are immunocompromised.
Asunto(s)
Grupo Borrelia Burgdorferi , Borrelia , Enfermedad de Lyme , Linfoma no Hodgkin , Humanos , Huésped Inmunocomprometido , Enfermedad de Lyme/diagnósticoRESUMEN
To understand the complex interactions between hematopoietic stem cells and the bone marrow niche, a human experimental model is needed. Our hypothesis is that hematons are an appropriate ex vivo model of human bone marrow. We analyzed the hierarchical hematopoietic cell content and the tissue organization of single hematons from healthy donors. Most (>90%) hematons contained precursors of all cell lineages, myeloid progenitors, and LTC-ICs without preferential commitment. Approximately, half of the hematons could generate significant levels of lympho-myeloid hematopoiesis after transplantation in an NSG mouse model, despite the low absolute numbers of transplanted CD34+ cells. Mesenchymal STRO-1+ and/or CD271+ cells formed a critical network that preserved hematon cohesion, and STRO-1+ cells colocalized with most hematopoietic CD34+ cells (68%). We observed an influence of age and gender. These structures represent a particularly attractive model for studying the homeostasis of the bone marrow niche and pathological changes that occur during diseases.
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Células de la Médula Ósea/citología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Modelos Biológicos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Médula Ósea/fisiología , Médula Ósea/ultraestructura , Células de la Médula Ósea/fisiología , Células de la Médula Ósea/ultraestructura , Comunicación Celular/fisiología , Femenino , Citometría de Flujo , Voluntarios Sanos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Células Madre Hematopoyéticas/ultraestructura , Humanos , Masculino , Ratones , Microscopía Confocal , Microscopía Electrónica , Persona de Mediana Edad , Trasplante Heterólogo , Adulto JovenRESUMEN
We investigated Syk as a potential marker of CML progression. We observed a significant over-expression of Syk mRNA and constitutive phosphorylation of Syk Y348 in blast cells from six AP or BP-CML, but not in 15 CML in chronic phase. We could follow in vivo the recurrence of pSyk(348) throughout blast cell escape, despite observing storage of dasatinib in blast cells. A combination of dasatinib and R406 did not improve therapeutic efficacy in vitro. Our results strongly suggest that Syk activation could be a relevant biomarker of disease progression and dasatinib resistance but is probably not a molecular target.
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Crisis Blástica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores de Tumor , Niño , Enfermedad Crónica , Dasatinib , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Estudios de Seguimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Oxazinas/farmacología , Fosforilación , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/genética , Piridinas/farmacología , Pirimidinas/farmacología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Quinasa Syk , Tiazoles/farmacología , Células Tumorales Cultivadas , Adulto JovenRESUMEN
The systematic localization of chronic lymphocytic leukemia (CLL) B-cells in the bone marrow (BM), together with the ex vivo protective effect of stromal cells on their spontaneous apoptosis, both indicate a specific role of the BM microenvironment. In vivo, the impact of CLL cells on mesenchymal stromal cells (MSCs) remains a source of debate. Here, we quantified and expanded colony forming unit-fibroblasts (CFU-Fs) from CLL-BM under standard conditions, analyzed the expression of selected genes, and studied secretion profiles. We observed failing of CLL-BM cultures in standard conditions (45.5% vs. <0.1%), and even after adding basic fibroblast growth factor (bFGF), there were fewer CFU-F than from normal BM (1.3 vs. 40/10(6) cells respectively; P<0.01). Furthermore, their polygonal aspect and low proliferative capacity, together with the expression of 384 selected genes and a secreted set of molecules related to senescence-associated secretory phenotype indicated a state of senescence, further confirmed by the higher proportion of senescence-associated ß-galactosidase (SA-ßGAL)-positive cells and p16INK4a overexpression. In our hands, hypoxic conditions (5% O2) did not rescue CFU-Fs. Given the role of MSC in BM tissue organization, we studied hematons that are generally considered to be elementary BM units. These structures were rare or had even disappeared completely. When hematons were present, we systematically observed nodular B-CLL cell invasion only. These data confirm that the B-CLL clone has a marked impact on MSC and disrupts BM organization in vivo, raising new questions about in vivo pathophysiology.
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Linfocitos B/citología , Leucemia Linfocítica Crónica de Células B/patología , Células Madre Mesenquimatosas/citología , Células Progenitoras Mieloides/citología , Nicho de Células Madre , Anciano , Células Cultivadas , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Exocitosis , Fibroblastos/citología , Fibroblastos/metabolismo , Hematopoyesis , Humanos , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Células Progenitoras Mieloides/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: Controlled-rate freezing and storage in nitrogen is the standard technique for cryopreservation of peripheral hematopoietic progenitor cells (PHPCs) but presents high cost and dimethyl sulfoxide (DMSO) toxicity. Cryopreservation at -80°C, by uncontrolled rate freezing with only 3.5% DMSO, preserves the functional capacities of PHPCs, produces successful engraftment, and reduces toxicity during infusion. STUDY DESIGN AND METHODS: Long-term hematopoietic and immunologic reconstitution for 342 autografts (311 adults, 31 children) after PHPCs were cryopreserved at -80°C was studied at 3, 6, and 12 months. The median (range) storage time of PHPCs cryopreserved was 1.7 (0.1-5.99) months. RESULTS: Hemoglobin (Hb), white blood cells, and platelets (PLTs) reach normal values to trilineage at 12 months for 39% patients. Multivariate analysis shows a significant impact on CD34+ infused and on conditioning regimen for PLTs. Hb was influenced by growth factor administration at 3 months. Long-term recovery is also highly dependent on blood counts (Hb, PLT, and neutrophil) at start of high-dose chemotherapy. Only 43% of patients had reached normal lymphocyte values at 12 months after transplant, and a profound CD4+ T-lymphocyte deficit remained, as others reported. CONCLUSION: Transplantation with PHPCs cryopreserved at -80°C for no more than 6 months is satisfactory for long-term hematopoietic and immunologic reconstitution, even if a profound CD4+ T lymphocyte deficit persists at 1 year. This easier and cheaper cryopreservation method also leads to successful engraftment.
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Conservación de la Sangre , Criopreservación , Hematopoyesis/fisiología , Inmunidad/fisiología , Trasplante de Células Madre de Sangre Periférica , Adolescente , Adulto , Anciano , Conservación de la Sangre/efectos adversos , Conservación de la Sangre/instrumentación , Conservación de la Sangre/métodos , Niño , Preescolar , Estudios de Cohortes , Criopreservación/métodos , Femenino , Congelación/efectos adversos , Supervivencia de Injerto , Células Madre Hematopoyéticas/fisiología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Trasplante de Células Madre de Sangre Periférica/métodos , Factores de Tiempo , Trasplante Autólogo , Resultado del Tratamiento , Adulto JovenRESUMEN
One of the essential parameters of targeted therapy efficiency in cancer treatment is the amount of drug reaching the therapeutic target area. Imatinib (IM) was the first specifically targeted drug to be developed and has revolutionized the treatment of patients with chronic myeloid leukemia (CML). To evaluate cellular uptake of IM, we developed a method based on the chemical structure of the molecule and using the natural UV fluorescence that we quantified by flow cytometry. In two CML cell lines, we obtained a satisfactory relationship between intracellular IM (ICIM) levels and media concentrations, and we found a strong correlation between ICIM at 1 h and IM efficacy at 24 h, demonstrating that ICIM at 1 h might be a relevant predictive parameter of cell sensitivity. Our method was more sensitive than the standard physicochemical method. We applied our method to primary cells and found cell morphology-dependent IM accumulation. Moreover, in CML cells from patients at diagnosis, IM accumulation was heterogeneous. In all cases, ICIM at the single-cell level was much higher than in culture media arguing in favor of a predominantly active uptake process. We developed a simple method directly applicable to primary cells that has shown two major advantages: only a small number of cells are required, and cell subsets can be identified according to morphological criteria and/or the presence of particular antigenic sites. This method provides a new tool to assess CML cell sensitivity to IM, and ICIM levels in native CML cells could be used to monitor therapeutic response.
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Citometría de Flujo/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Piperazinas/farmacocinética , Pirimidinas/farmacocinética , Antineoplásicos/farmacocinética , Benzamidas , Forma de la Célula , Medios de Cultivo/metabolismo , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Fluorescencia , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Piperazinas/sangre , Pirimidinas/sangre , Sensibilidad y Especificidad , Rayos UltravioletaRESUMEN
One of the cardinal symptoms of type 1 Gaucher Disease (GD) is cytopenia, usually explained by bone marrow (BM) infiltration by Gaucher cells and hypersplenism. However, some cases of cytopenia in splenectomized or treated patients suggest possible other mechanisms. To evaluate intra-cellular glucocerebrosidase (GlcC) activity in immature progenitors and to prove the conduritol B epoxide (CBE)-induced inhibition of the enzyme, we used an adapted flow cytometric technique before assessing the direct effect of GlcC deficiency in functional assays. Among haematopoietic cells from healthy donors, monocytes showed the highest GlcC activity but immature CD34(+) and mesenchymal cells also had significant GlcC activity. CBE greatly inhibited the enzyme activity of all cell categories. GlcC-deficient CD34(+) cells showed impaired ability to proliferate and differentiate in the expansion assay and had lower frequency of erythroid burst-forming units, granulocyte colony-forming units (CFU) and macrophage CFU progenitors, but the effect of GlcC deficiency on megakaryocyte CFU lineage was not significant. GlcC deficiency strongly impaired primitive haematopoiesis in long-term culture. Furthermore, GlcC deficiency progressively impaired proliferation of mesenchymal progenitors. These data suggest an intrinsic effect of GlcC deficiency on BM immature cells that supplements the pathophysiology of GD and opens new perspectives of therapeutic approach.
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Enfermedad de Gaucher/enzimología , Glucosilceramidasa/deficiencia , Hematopoyesis/fisiología , Células de la Médula Ósea/enzimología , Proliferación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Inhibidores Enzimáticos/farmacología , Citometría de Flujo/métodos , Enfermedad de Gaucher/fisiopatología , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/sangre , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Inositol/análogos & derivados , Inositol/farmacología , Modelos Biológicos , Técnicas de Cultivo de TejidosRESUMEN
OBJECTIVE: This letter reports on the effect of enzyme replacement therapy with imiglucerase on bone healing and bone and blood abnormalities in a woman with previously untreated type 1 Gaucher disease (GD). METHODS: The 49-year-old patient had been diagnosed with GD at the age of 28 years and had previously undergone splenectomy. She presented with pseudarthrosis 14 months after sustaining a traumatic fracture of the tibia and fibula. Therapy was begun with imiglucerase 60 U/kg q2wk. The effects of treatment on bone healing were monitored radiographically, and effects on blood and bone marrow biology were monitored by hemograms, myelograms, and hematopoietic and mesenchymal clonogenic assays. RESULTS: Objective bone healing was observed starting in the third month of imiglucerase treatment. Blood abnormalities normalized and bone marrow parameters improved over the first 9 months, including a decrease in Gaucher cells, an increase in bone marrow CD34+ cell cloning efficiency, and the appearance of mesenchymal progenitors. CONCLUSION: This research letter reports the results of hematologic and bone evaluations during enzyme replacement therapy with imiglucerase in a woman with previously untreated type 1 GD who presented with a traumatic fracture.
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Terapia de Reemplazo Enzimático , Peroné/lesiones , Curación de Fractura , Enfermedad de Gaucher/terapia , Glucosilceramidasa/uso terapéutico , Enfermedades Hematológicas/terapia , Seudoartrosis/terapia , Fracturas de la Tibia/terapia , Accidentes por Caídas , Células de la Médula Ósea/patología , Femenino , Peroné/diagnóstico por imagen , Fijación Interna de Fracturas , Enfermedad de Gaucher/sangre , Enfermedad de Gaucher/complicaciones , Enfermedad de Gaucher/enzimología , Enfermedades Hematológicas/sangre , Enfermedades Hematológicas/enzimología , Enfermedades Hematológicas/etiología , Humanos , Persona de Mediana Edad , Seudoartrosis/diagnóstico por imagen , Seudoartrosis/enzimología , Seudoartrosis/etiología , Radiografía , Fracturas de la Tibia/complicaciones , Fracturas de la Tibia/diagnóstico por imagen , Fracturas de la Tibia/enzimología , Factores de Tiempo , Resultado del TratamientoRESUMEN
The scarcity of mesenchymal stem cells (MSC) in bone marrow (BM) has justified their ex vivo expansion before therapeutic use, but a method to evaluate the quality of initial mesenchymal content and track the modifications induced by graft processing has not yet been proposed. The aim of this study was to establish such a procedure. Flow cytometric and functional assay methods were modified to count CD45(-) CD14(-)/CD73(+) subsets containing all MSC and used them to study BM from spongy bone (SB) and iliac crest aspirate (ICA). These methods detected the target subsets in all BM suspensions derived from SB (n = 154) and ICA, (n = 44) with a satisfactory correlation between immuno-phenotyping and functional tests by low-density plating. We noted a higher overall MSC frequency in SB cell suspensions but a lower plating efficiency of CD45(-) CD14(-)/CD73(+) SB cells under standard culture conditions. We propose a cell quality control on un-manipulated BM cell suspensions to quantify the mesenchymal compartment with regard to varying donor factors, such as age and sampling site, that influence expansion and define a therapeutic threshold value. Furthermore, we were able to confirm differences in plating efficiency and proliferative capacity between two BM origins.
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Células Madre Mesenquimatosas/citología , 5'-Nucleotidasa/análisis , Células de la Médula Ósea/inmunología , Proliferación Celular , Separación Celular/métodos , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Humanos , Ilion , Antígenos Comunes de Leucocito , Receptores de Lipopolisacáridos , Células Madre Mesenquimatosas/inmunología , Control de Calidad , Trasplante de Células Madre/normasRESUMEN
The predictive values of common biological criteria for the diagnosis of polycythemia vera were studied in a cohort of patients with high hematocrit. We found JAK2V617F and erythropoietin assays were the most relevant first tests. Classification of patients according to their JAK2V617F status and erythropoietin levels facilitated the choice of further diagnostic investigations.
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Eritropoyetina/sangre , Hematócrito , Janus Quinasa 2/genética , Proteínas Mutantes/genética , Mutación Missense , Mutación Puntual , Policitemia Vera/diagnóstico , Sustitución de Aminoácidos , Estudios de Cohortes , Diagnóstico Diferencial , Femenino , Frecuencia de los Genes , Humanos , Janus Quinasa 2/sangre , Masculino , Proteínas Mutantes/sangre , Policitemia/sangre , Policitemia/diagnóstico , Policitemia Vera/sangre , Policitemia Vera/enzimología , Policitemia Vera/genética , Valor Predictivo de las PruebasRESUMEN
For most therapeutic strategies using MSC, the preliminary amplification is carried out in media containing fetal calf serum (FCS). The theoretical health risk of using a xenogenic serum, a recent practice for which we have limited data, cannot be underestimated, while amplification using human serum (HS) remains controversial. At present, the available information on multipotentiality, self-renewal, and transplantability does not permit the selection of FCS rather than HS. Cellular modifications observed during cell passage seem to indicate a gradual impairment of cells in relation to native MSC, suggesting the making of short cell cultures without necessarily trying to reinfuse a high number of MSC in patients. With this approach, the volume of HS required would remain limited. While clinical studies have already started, many problems remain, such as evaluating the quality of the initial mesenchymal compartment and the biological properties of the cell suspension with FCS compared to those with HS, and depending on culture time.
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Células Madre Adultas/citología , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/química , Adulto , Animales , Investigación Biomédica , Células Cultivadas , HumanosRESUMEN
We determined the allelic frequency of the JAK2-V617F mutation in DNA and assessed the expression levels of the mutant and wild-type JAK2 mRNA in granulocytes from 60 patients with essential thrombocythemia (ET) and 62 patients with polycythemia vera (PV) at the time of diagnosis. Using allele-specific quantitative polymerase chain reaction (qPCR), we detected JAK2-V617F in 75% of ET and 97% of PV at diagnosis. The total JAK2 mRNA levels were elevated in ET, PV, and secondary and idiopathic erythrocytosis, suggesting that hyperactive hematopoiesis alters JAK2 expression. The expression levels of JAK2-V617F mRNA were variable but strongly correlated with the allelic ratio of JAK2-V617F determined in DNA. Thus, differences in JAK2-V617F expression, markedly lower in ET than in PV, reflected different percentages of granulocytes carrying the mutation. Moreover, allelic ratios higher than 50% JAK2-V617F, indicating the presence of granulocytes homozygous for JAK2-V617F, were found in 70% of PV at diagnosis but never in ET.