RESUMO
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.
Assuntos
Células da Medula Óssea/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Modelos Biológicos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Medula Óssea/fisiologia , Medula Óssea/ultraestrutura , Células da Medula Óssea/fisiologia , Células da Medula Óssea/ultraestrutura , Comunicação Celular/fisiologia , Feminino , Citometria de Fluxo , Voluntários Saudáveis , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Masculino , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Pessoa de Meia-Idade , Transplante Heterólogo , Adulto JovemRESUMO
An α1 -Dawson polyanion bearing a lateral side chain with a 4-aminopyridine end group was synthesized. This organopolyoxometalate catalyzes the addition of indenyl allyl silanes to cinnamoyl fluorides. The polyanionic framework influences the organocatalyst activity and selectivity. A moderate but nonzero chirality transfer from the chiral inorganic framework to the organic substrate was observed.
RESUMO
An asymmetric Torgov cyclization, catalyzed by a novel, highly Brønsted acidic dinitro-substituted disulfonimide, is described. The reaction delivers the Torgov diene and various analogues with excellent yields and enantioselectivity. This method was applied in a very short synthesis of (+)-estrone.
Assuntos
Estrona/síntese química , Ácidos/química , Catálise , Ciclização , Estrona/química , Imidas/química , Estereoisomerismo , Sulfonas/químicaRESUMO
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.
Assuntos
Preservação de Sangue , Criopreservação , Hematopoese/fisiologia , Imunidade/fisiologia , Transplante de Células-Tronco de Sangue Periférico , Adolescente , Adulto , Idoso , Preservação de Sangue/efeitos adversos , Preservação de Sangue/instrumentação , Preservação de Sangue/métodos , Criança , Pré-Escolar , Estudos de Coortes , Criopreservação/métodos , Feminino , Congelamento/efeitos adversos , Sobrevivência de Enxerto , Células-Tronco Hematopoéticas/fisiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco de Sangue Periférico/métodos , Fatores de Tempo , Transplante Autólogo , Resultado do Tratamento , Adulto JovemRESUMO
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.
Assuntos
Citometria de Fluxo/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Piperazinas/farmacocinética , Pirimidinas/farmacocinética , Antineoplásicos/farmacocinética , Benzamidas , Forma Celular , Meios de Cultura/metabolismo , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Fluorescência , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Piperazinas/sangue , Pirimidinas/sangue , Sensibilidade e Especificidade , Raios UltravioletaRESUMO
Grafting of a gold complex to an organo-polyoxometalate delivers catalytically active bitopic hybrids. The gold end activates allenes, while the metal-oxide surface can capture protons (see scheme). The scope of the gold-catalyzed oxacyclization of allenols is expanded to highly sensitive tertiary benzylic alcohols.
RESUMO
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.
Assuntos
Grupo Borrelia Burgdorferi , Borrelia , Doença de Lyme , Linfoma não Hodgkin , Humanos , Hospedeiro Imunocomprometido , Doença de Lyme/diagnósticoRESUMO
The [ε-PMo(V)(8)Mo(VI)(4)O(36)(OH)(4){Ln(III)(H(2)O)}(4)](5+) (Ln=La, Ce, Nd, Sm) polyoxocations, called εLn(4), have been synthesized at room temperature as chloride salts soluble in water, MeOH, EtOH, and DMF. Rare-earth metals can be exchanged, and (31)P NMR spectroscopic studies have allowed a comparison of the affinity of the reduced {ε-PMo(12)} core, thus showing that the La(III) ions have the highest affinity and that rare earths heavier than Eu(III) do not react with the ε-Keggin polyoxometalate. DFT calculations provide a deeper insight into the geometries of the systems studied, thereby giving more accurate information on those compounds that suffer from disorder in crystalline form. It has also been confirmed by the hypothetical LaâGd substitution reaction energy that Ln ions beyond Eu cannot compete with La in coordinating the surface of the ε-Keggin molybdate. Two of these clusters (Ln=La, Ce) have been tested to evidence that such systems are representative of a new efficient Lewis acid catalyst family. This is the first time that the catalytic activity of polyoxocations has been evaluated.
RESUMO
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.
Assuntos
Doença de Gaucher/enzimologia , Glucosilceramidase/deficiência , Hematopoese/fisiologia , Células da Medula Óssea/enzimologia , Proliferação de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo/métodos , Doença de Gaucher/fisiopatologia , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/sangue , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Inositol/análogos & derivados , Inositol/farmacologia , Modelos Biológicos , Técnicas de Cultura de TecidosRESUMO
The preparation of new organosoluble Lewis acidic polyoxometalates (POMs) is reported. These complexes were prepared by the incorporation of Zr, Sc, and Y atoms into the corresponding monolacunary Dawson [P(2)W(17)O(61)](10-) and Keggin [PW(11)O(39)](7-) polyoxotungstates. The catalytic activity of these compounds was evaluated for C-C bond formation in the Diels-Alder, Mannich, and Mukaiyama-type reactions. Comparisons with previously described Lewis acidic POMs are reported. Competitive reactions between imines and aldehydes or between various imines demonstrated that fine tuning of the reactivity could be reached by varying the metal atom incorporated into the polyanionic framework. A series of experiments that employed pyridine derivatives allowed us to distinguish between the Lewis and induced Brønsted acidity of the POMs. These catalysts activate imines in a Lewis acidic way, whereas aldehydes are activated by indirect Brønsted catalysis.
RESUMO
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.
Assuntos
Eritropoetina/sangue , Hematócrito , Janus Quinase 2/genética , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Mutação Puntual , Policitemia Vera/diagnóstico , Substituição de Aminoácidos , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Frequência do Gene , Humanos , Janus Quinase 2/sangue , Masculino , Proteínas Mutantes/sangue , Policitemia/sangue , Policitemia/diagnóstico , Policitemia Vera/sangue , Policitemia Vera/enzimologia , Policitemia Vera/genética , Valor Preditivo dos TestesRESUMO
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.
Assuntos
Crise Blástica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Tirosina Quinases/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores Tumorais , Criança , Doença Crônica , Dasatinibe , Progressão da Doença , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Oxazinas/farmacologia , Fosforilação , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/genética , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Quinase Syk , Tiazóis/farmacologia , Células Tumorais Cultivadas , Adulto JovemAssuntos
Avaliação Geriátrica/métodos , Enfermagem Geriátrica/organização & administração , Avaliação em Enfermagem/organização & administração , Assistentes de Enfermagem/organização & administração , Recursos Humanos de Enfermagem Hospitalar/organização & administração , Equipe de Enfermagem/organização & administração , Idoso , França , Unidades Hospitalares/organização & administração , Humanos , Programas de Rastreamento/organização & administração , Planejamento de Assistência ao Paciente/organização & administração , Assistência Centrada no Paciente/organização & administraçãoRESUMO
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.
Assuntos
Linfócitos B/citologia , Leucemia Linfocítica Crônica de Células B/patologia , Células-Tronco Mesenquimais/citologia , Células Progenitoras Mieloides/citologia , Nicho de Células-Tronco , Idoso , Células Cultivadas , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Exocitose , Fibroblastos/citologia , Fibroblastos/metabolismo , Hematopoese , Humanos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Células Progenitoras Mieloides/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
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.
Assuntos
Terapia de Reposição de Enzimas , Fíbula/lesões , Consolidação da Fratura , Doença de Gaucher/terapia , Glucosilceramidase/uso terapêutico , Doenças Hematológicas/terapia , Pseudoartrose/terapia , Fraturas da Tíbia/terapia , Acidentes por Quedas , Células da Medula Óssea/patologia , Feminino , Fíbula/diagnóstico por imagem , Fixação Interna de Fraturas , Doença de Gaucher/sangue , Doença de Gaucher/complicações , Doença de Gaucher/enzimologia , Doenças Hematológicas/sangue , Doenças Hematológicas/enzimologia , Doenças Hematológicas/etiologia , Humanos , Pessoa de Meia-Idade , Pseudoartrose/diagnóstico por imagem , Pseudoartrose/enzimologia , Pseudoartrose/etiologia , Radiografia , Fraturas da Tíbia/complicações , Fraturas da Tíbia/diagnóstico por imagem , Fraturas da Tíbia/enzimologia , Fatores de Tempo , Resultado do TratamentoRESUMO
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.
Assuntos
Células-Tronco Mesenquimais/citologia , 5'-Nucleotidase/análise , Células da Medula Óssea/imunologia , Proliferação de Células , Separação Celular/métodos , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Humanos , Ílio , Antígenos Comuns de Leucócito , Receptores de Lipopolissacarídeos , Células-Tronco Mesenquimais/imunologia , Controle de Qualidade , Transplante de Células-Tronco/normasRESUMO
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.
Assuntos
Células-Tronco Adultas/citologia , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Adulto , Animais , Pesquisa Biomédica , Células Cultivadas , HumanosRESUMO
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.