RESUMO
Reciprocal communication between hematopoietic cells and their surrounding bone marrow stroma is crucial for normal progression of hematopoiesis. This complex network of cell-to-cell signals in the microenvironment involves both cell contact-mediated and paracrine cues. In hematological malignancies the intricate balance is, however, disrupted to support cancer progression. In order to detect altered microenvironmental reactivity of a hematopoietic cell sample, cellular functional assays can be designed to measure the cells' capacity to modulate stromal stress reactions, such as inflammation.Recently, we showed that human leukemic cell lines of monocytic origin can actively participate in modulation of stromal inflammation. In order to further functionally evaluate the hematopoietic cells' capacity to modulate stromal inflammation, we utilized an in vitro model of nemosis-induced inflammation of fibroblasts in a three-dimensional culture setting. This process of stromal inflammation in fibroblast aggregates is consistent, requires both cell-contact and paracrine signals, and can be produced on a large scale to support dose-dependent analyses. To extend our previous observations, we evaluated the effect of a wide panel of leukemia cell lines on cyclooxygenase- 2 induction in fibroblast aggregates in co-culture. We also assessed the feasibility of the model to support clinical functional testing by utilizing the hematopoietic fraction of leukemia patients' bone marrow aspirates after immunophenotyping. Our results suggest that the stromal inflammation-modulating activity of these samples is differently modulated in cancer and in normal bone marrow. Moreover, differences in the samples' anti-inflammatory activity may reflect disease state.
Assuntos
Anti-Inflamatórios/metabolismo , Medula Óssea/patologia , Ciclo-Oxigenase 2/metabolismo , Fibroblastos/patologia , Células-Tronco Hematopoéticas/patologia , Leucemia/patologia , Microambiente Tumoral , Adolescente , Adulto , Medula Óssea/enzimologia , Medula Óssea/imunologia , Comunicação Celular , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Derme/enzimologia , Derme/imunologia , Derme/patologia , Feminino , Fibroblastos/enzimologia , Fibroblastos/imunologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Immunoblotting , Imunofenotipagem , Lactente , Leucemia/enzimologia , Leucemia/imunologia , Masculino , Pessoa de Meia-Idade , Células Estromais/enzimologia , Células Estromais/imunologia , Células Estromais/patologia , Adulto JovemRESUMO
Oxygen tension levels may modulate immune responses. Evidence shows that hyperoxia influences the risk of infection, autoimmunity and alloreactivity and hence is a possible therapeutic option in a number of disorders. Regulatory T cells (Tregs) play a central role in tolerance maintenance, but their behaviour under hyperoxia is largely unknown. We investigated in vitro the impact of normobaric hyperoxia on human Tregs and their cellular network. Peripheral blood mononuclear cells isolated from six healthy men were cultured under normoxia and escalating duration of normobaric hyperoxia (10 min, 1, 16, 88 h) under resting conditions and at the presence of anti-CD3/CD28 beads. Foxp3+ Tregs' and other T cell subsets' survival, proliferation, activation, maturation and Th1/Th2 markers were assessed by flow cytometry. We observed decreasing CD4+ cell survival with increasing duration of hyperoxia irrespectively of the presence of stimulators. The prevalence of CD4+ CD45RA+ cells increased under stimulation (P=0.001). In stimulated samples, the proliferation and induced Foxp3 expression decreased after 88 h of hyperoxia (both P=0.001). In conclusion, normobaric hyperoxia up to 16 h does not induce significant changes in basic human T cell subsets, including the prevalence naturally occurring Tregs. Prolonged exposure to hyperoxia likely affects all unstimulated T cell subsets in a similar way. In stimulated T lymphocytes, the proliferation is hampered and cell death increases more evidently after prolonged hyperoxia (several days). Inducible Foxp3 expression is likely closely related to these processes. Naive CD4+ T cells are maintained by stimulation during exposure to hyperoxia.
Assuntos
Hiperóxia/imunologia , Linfócitos T Reguladores/imunologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Fatores de Transcrição Forkhead/imunologia , Humanos , Hiperóxia/fisiopatologia , Imunofenotipagem , Ativação Linfocitária/imunologia , Masculino , Subpopulações de Linfócitos T/imunologiaRESUMO
Acute lymphoblastic leukemia is the most common form of cancer in children. The 10-year event-free survival ranged from 77 to 85% after having achieved complete remission rates of 93% or higher. The main cause of treatment failure is relapse arising from outgrowth of residual leukemic cells that are refractory to therapy. An intense effort has been made to develop methods to determine the degree of minimal residual leukemia cells present in patients considered to be in morphological remission. Because of the strong correlation between minimal residual disease (MRD) levels and risk of relapse, monitoring of MRD provides unique information regarding treatment response. The MRD monitoring based on real-time quantitative PCR detection of patient-specific immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements is currently considered to be the most reliable tool for MRD-based diagnosis in ALL. Because the significance of MRD monitoring has been strongly supported by several studies and because it has been implemented in the latest protocols, there has been a significant effort to develop MRD monitoring in the Slovak Republic since 2005. Between October 2006 and December 2009, 50 children with ALL who were treated at three Slovak centers were included in the RQ PCR MRD pilot project. A total of 40 patients with BCP-ALL ( B cell precursor ALL) and 4 patients with T ALL were analyzed for Ig/TCR rearrangement. We identified 106 different rearrangements in the 44 ALL patients analyzed. Based on MRD stratification, we identified 26 patients who were stratified into the HRG ( high risk group) (n = 3; 11.5%), IRG ( intermediate risk group) (n = 14; 54%) and SRG ) standard risk group) (n = 9; 34.5%). Morphology-based risk stratification allows the identification of most HRG patients identified also by MRD-based stratification, but fails to discriminate the IRG assigned to therapy reduction. Patients in the SRG and the IRG could profit from MRD-based risk assignment