Antonio López

The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes

Mesenchymal stem cells are multipotent non-hematopoietic progenitor cells capable of differentiating into various lineages including osteoblasts, chondrocytes and adipocytes. The findings of this study indicate that mesenchymal stem cells promote survival and inhibit proliferation and maturation of B cells, and support a role of these cells in the immune response. Background Mesenchymal stem cells are multilineage non-hematopoietic progenitor cells that play a key role in supporting the lymphohematopoietic system. Their distribution in bone marrow and secondary lymphoid organs allows an intimate interaction with T- and B-lymphocytes. While their effect on T-lymphocytes has been extensively analyzed, data on the effect of mesenchymal stem cells on B cells are more limited. We analyzed the effects of mesenchymal stem cells on B-lymphocytes and the pathways involved in these effects. Design and Methods The effect of MSC on the proliferation and viability of B cells was evaluated using MTT assays, annexin/7-amino-actinomycin D and propidium iodide staining. The B-cell maturation pattern was established using flow cytometry based on the expression of different markers related to the differentiation of B cells, such as CD38, CD138, CD19 and CCR7, and to the expression of surface and intracellular immunoglobulins. Finally, western blot assays were used to identify the pathways involved in the effects of mesenchymal stem cells on B-lymphocytes. Results Mesenchymal stem cells increased viability and blocked the cell cycle of B-lymphocytes in the G0/G1 phase. In vitro exposure of B cells to plasmacytoid dendritic cells induced B-cell differentiation as shown by an increased number of CD38++/CD138++ cells, which also displayed higher levels of cytoplasmic immunoglobulin and lower levels of CD19, CCR7 and surface immunoglobulin. Interestingly, this maturation pattern was inhibited by adding mesenchymal stem cells to the culture. Finally, mesenchymal stem cells modified the phosphorylation pattern of the extracellular response kinase 1/2 and p38 pathways which are both involved in B-cell viability, proliferation and activation. Conclusions Mesenchymal stem cells increase B-cell viability while inhibiting proliferation, arresting B-lymphocytes in the G0/G1 phase of the cell cycle. The presence of mesenchymal stem cells blocked B-cell differentiation as assessed by flow cytometry. Finally, mesenchymal stem cells modified the activation pattern of the extracellular response kinase and the p38 mitogen-activated protein kinase pathways in B-lymphocytes.

Identification of Leptomeningeal Disease in Aggressive B-Cell Non-Hodgkin's Lymphoma: Improved Sensitivity of Flow Cytometry

PURPOSE Here, we evaluate the sensitivity and specificity of a new 11-parameter flow cytometry (FCM) approach versus conventional cytology (CC) for detecting neoplastic cells in stabilized CSF samples from newly diagnosed aggressive B-cell non-Hodgkins lymphoma (B-NHL) at high risk of CNS relapse, using a prospective, multicentric study design. PATIENTS AND METHODS Moreover, we compared the distribution of different subpopulations of CSF leukocytes and the clinico-biologic characteristics of CSF+ versus CSF-, patients, in an attempt to define new algorithms useful for predicting CNS disease. RESULTS Overall, 27 (22%) of 123 patients showed infiltration by FCM, while CC was positive in only seven patients (6%), with three other cases being suspicious (2%). CC+/FCM+ samples typically had more than 20% neoplastic B cells and/or >or= one neoplastic B cell/microL, while FCM+/CC- samples showed lower levels (P < .0001) of infiltration. Interestingly, in Burkitt lymphoma, presence of CNS disease by FCM could be predicted with a high specificity when increased serum beta2-microglobulin and neurological symptoms coexisted, while peripheral blood involvement was the only independent parameter associated with CNS disease in diffuse large B-cell lymphoma, with low predictive value. CONCLUSION FCM significantly improves the sensitivity of CC for the identification of leptomeningeal disease in aggressive B-NHL at higher risk of CNS disease, particularly in paucicellular samples.

Utility of Flow Cytometry Immunophenotyping in Multiple Myeloma and Other Clonal Plasma Cell-Related Disorders

In recent years, multiparameter flow cytometry (MFC) immunophenotyping has become mandatory in the clinical management of hematological malignancies, both for diagnostic and monitoring purposes. Multiple myeloma (MM) and other clonal plasma cell‐related (PC) disorders should be no exception to this paradigm, but incorporation of immunophenotypic studies in the management of patients with PC disorders is still far from being routinely established in many diagnostic flow cytometry laboratories. For clonal PC disorders, MFC is of clear and established clinical relevance in: (1) the differential diagnosis between MM and other PC‐related disorders; (2) the identification of high‐risk MGUS and smoldering MM; (3) minimal residual disease investigation after therapy; additionally it may also be useful for (4) the definition of prognosis‐associated antigenic profiles; and (5) the identification of new therapeutic targets. In this article, we review the clinical value of MFC in the study of PC disorders, with specific emphasis in those areas where consensus exists on the need to incorporate MFC into routine evaluation of MM and other clonal PC‐related disorders.

Immunophenotypic analysis of mast cells in mastocytosis: When and how to do it. Proposals of the Spanish Network on Mastocytosis (REMA)

Mastocytosis is a term used for a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow, liver, spleen, and lymph nodes, among others.

Aberrant expression of tetraspanin molecules in B-cell chronic lymphoproliferative disorders and its correlation with normal B-cell maturation

Tetraspanin proteins form signaling complexes between them and with other membrane proteins and modulate cell adhesion and migration properties. The surface expression of several tetraspanin antigens (CD9, CD37, CD53, CD63, and CD81), and their interacting proteins (CD19, CD21, and HLA-DR) were analyzed during normal B-cell maturation and compared to a group of 67 B-cell neoplasias. Three patterns of tetraspanin expression were identified in normal B cells. The first corresponded to bone marrow CD10+ B-cell precursors (BCP) which showed high expression of CD81 and CD9, low reactivity for CD53 and negativity for CD37. CD10− B-lymphocytes showed downregulation of CD9/CD81 and upregulation of CD53/CD37. Plasma cells showed re-expressed CD9 and downregulated CD37. Hierarchical clustering analysis of flow cytometry immunophenotypic data showed a good correlation between the tumor differentiation stage and the pattern of tetraspanin expression, with all analyzed individual samples classified into three major groups, independently of their normal or neoplastic origin. Despite this, neoplastic B-cells frequently showed aberrantly high/low expression of the different markers analyzed. Interestingly, in B-cell chronic lymphocytic leukemia, abnormal expression of CD53 and CD9 were associated with different patterns of disease infiltration, which would support the role of these molecules on modulating adhesion and migration of neoplastic B cells.

Immunophenotyping of acute leukemias and myelodysplastic syndromes

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain

Bone marrow cells from myelodysplastic syndromes show altered immunophenotypic profiles that may contribute to the diagnosis and prognostic stratification of the disease: A pilot study on a series of 56 patients

A heterogeneous spectrum of immunophenotypic abnormalities have been reported in myelodysplastic syndromes (MDS). However, most studies are restricted to the analysis of CD34+ cells and/or other major subsets of CD34− cells, frequently not exploring the diagnostic and prognostic impact of immunophenotyping.

Impact of trisomy 12, del(13q), del(17p), and del(11q) on the immunophenotype, DNA ploidy status, and proliferative rate of leukemic B‐cells in chronic lymphocytic leukemia

B‐cell chronic lymphocytic leukemia (B‐CLL) is a well‐defined clinical entity with heterogeneous molecular and cytogenetic features. Here, we analyze the impact of trisomy 12, del(13q), del(17p), and del(11q) as determined by interphase fluorescence in situ hybridization analysis of purified neoplastic B‐CLL cells on their immunophenotype, DNA ploidy status and proliferative rate.

Overexpression of complement receptors and related antigens on the surface of bone marrow mast cells in patients with systemic mastocytosis

Summary. Depending on their stage of maturation and other factors, mast cell (MC) subsets differ from each other in terms of the expression of complement‐associated antigens. This study analysed the expression of various complement‐related cell surface antigens (CD11b/CR3, CD11c/CR4, CD35/CR1, CD55/DAF, CD59/MIRL, CD88/C5aR) on bone marrow mast cells (BMMC) in patients suffering from systemic mastocytosis (SM), other haematological diseases and non‐haematological disorders (control groups). Expression of complement‐associated cell surface antigens was analysed by flow cytometry. There were clear immunophenotypic differences between BMMC obtained from patients with SM and those from the control subjects: the percentage of patients expressing surface CD11c, CD35 and CD88 was significantly higher in patients with SM (76%, 100%, 54%) than in the control subjects (58%, 11%, 18%) (P < 0·05). In addition, the levels of CD11c, CD35 and CD88 expressed per MC (sites per cell) were significantly higher (P < 0·05) in SM than in the control group. Expression of the complement regulatory molecules CD55 and CD59 was detected in BMMC in all patients analysed. However, the levels of CD59 per BMMC were higher in patients with SM as compared with the control subjects, which could help to explain the formation of BMMC aggregates in the former group of individuals. Together, our results showed that BMMC in systemic mastocytosis overexpressed the cell surface membrane receptors involved in binding of complement components and complement‐mediated cell activation. Whether this pathological expression of complement receptors is of pathophysiological significance remains to be determined.

Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas

Barrena S, Almeida J, García‐Macias M D C, López A, Rasillo A, Sayagués J M, Rivas R A, Gutiérrez M L, Ciudad J, Flores T, Balanzategui A, Caballero M D & Orfao A
(2011) Histopathology 58, 906–918
Flow cytometry immunophenotyping of fine‐needle aspiration specimens: utility in the diagnosis and classification of non‐Hodgkin lymphomas