Luis Escribano

Expression of the c-kit (CD117) Molecule in Normal and Malignant Hematopoiesis

The c-kit proto-oncogen (CD 117) has been shown to be present in several cell types including normal and neoplastic hemopoietic cells. Among normal BM cells, CD117 expression has been found in about half of the CD34+ precursors including progenitors committed to the erythroid, granulo-monocytic, and megakaryocytic cell lineages. In addition, strong CD117 expression is detected in bone marrow mast cells as well as in a small subset of NK cells displaying strong reactivity for CD56, and in a relatively important proportion of CD3 /CD4 /CD8 prothymocytes. These results suggest that CD117 expression can be detected in both myeloid and lymphoid lineages although for the lymphoid lineage it would be restricted to a small NK-cell subset and early T-cell precursors. In acute leukemias CD117 expression was initially associated with AML. Nevertheless, at present it is well established that CD 117 expression may also be found in a relatively important proportion of T-ALL while it is usually absent in B-lineage ALL. Moreover, recent studies have shown that in about one-third of multiple myeloma cases and patients with monoclonal gammopathy of undetermined significance plasma cells display reactivity for CD1117. The prognostic influence of CD117 expression has not yet been clearly established. The analysis of this marker may also be of value for the investigation of minimal residual disease (MRD). It has been suggested that CD117 in combination with other antigens may be of great help for the identification of leukemia-associated phenotypes that could be used to monitor MRD in both acute myeloid leukemias and multiple myeloma patients achieving morphological complete remission.

Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage- cells

Dendritic cells (DC) represent the most powerful professional antigen‐presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single‐cell basis by multiparametric flow cytometry with simultaneous four‐colour staining and a two‐step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA‐II+/lineage− were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16u2003±u20030.06% of the PB nucleated cells and 55.9u2003±u200311.9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12u2003±u20030.04% of PB nucleated cells and 44.53u2003±u200311.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid‐associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA‐DR and HLA‐DQ expression and a higher reactivity for the IL‐6 receptor α‐chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL‐1β (97u2003±u20035% of positive cells), IL‐6 (96u2003±u20031.1% of positive cells), IL‐12 (81.5u2003±u200315.5% of positive cells) and tumour necrosis factor‐alpha (TNF‐α) (84u2003±u200322.1% of positive cells) as well as chemokines such as IL‐8 (99u2003±u20031% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.

Sequential Immunophenotypic Analysis of Mast Cells in a Case of Systemic Mast Cell Disease Evolving to a Mast Cell Leukemia

The immunophenotypic characteristics of both bone marrow (BM) and peripheral blood (PB) mast cells (MC), from a patient suffering from an aggressive systemic mast cell disease (SMCD), were sequentially analyzed by flow cytometry using direct immunofluorescence. Analysis was carried out at diagnosis, during clinical response induced by interferon alfa-2h/prednisone therapy, and later at relapse. Our results show that together with the CD117 and IgE characteristic markers, at diagnosis BM MC showed strong expression of CD11c, CD13, CD29, CD33, CD44, CD45, CD63, and CD71, and they were also positive for CD2, CD22, CD25, and CD54 although at a lower level. PB MC displayed similar immunophenotypic characteristics although they had a lower expression of CD11c, CD25, CD33, CD63, CD69, and CD71 with a higher reactivity for CD117. Unlike BM MC, PB MC were weakly positive for CD41a and CD61. Sequential studies showed decreased numbers of both BM and PB MC during clinical response associated with a higher expression of the CD29 and CD54 adhesion molecules. In turn, clinical relapse was related to increased numbers of PB and BM MC together with lower CD2, CD11c, CD45, and and CD54 expression and a higher reactivity for the CD117 and CD25 antigens. CD2 had become negative at the last follow-up study. In addition, an increased proportion of S-phase MC was observed at relapse. These findings suggest that the assessment of the quantitative expression of cell-adhesion molecules and growth-factor receptors together with cell cycle studies of mast cells could be of value for monitoring therapy and predicting clinical outcome in aggressive SMCD.

Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: Comparative analysis of different stimuli, secretion-blocking agents and incubation periods

In this paper, we comparatively analyze the effects of the following different stimuli on the production and intracellular accumulation of the interleukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) dendritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Staphylococcus aureus cowan I (SAC) in the presence or absence of interferon-(IFN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus brefeldin A plus monensin), and incubation periods (6, 12, and 24 h). For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n = 19). Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CD33(-/+lo) lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAC, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAC. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin. This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone.

The CD69 early activation molecule is overexpressed in human bone marrow mast cells from adults with indolent systemic mast cell disease.

We have analysed the quantitative expression of surface CD69 antigen on human mast cells (MC), from both normal and pathological bone marrow (BM) samples, using flow cytometry. Our major aim was to analyse whether CD69 is constitutively expressed by normal BMMC and to explore the possible differences between CD69 expression by BMMC from normal controls and patients suffering from different pathological conditions.

HUMAN BONE MARROW MAST CELLS FROM INDOLENT SYSTEMIC MAST CELL DISEASE CONSTITUTIVELY EXPRESS INCREASED AMOUNTS OF THE CD63 PROTEIN ON THEIR SURFACE

The quantitative measurement of the expression of both cytoplasmic and surface CD63 antigen by human mast cells from both normal and pathological bone marrow samples was studied by use of flow cytometry. Our major goal was to analyze whether in vivo CD63 expression by human bone marrow mast cells could be useful to discriminate bone marrow mast cells from patients with mastocytosis from other conditions. For that purpose, a total of 65 subjects corresponding to 12 healthy volunteers, 25 B-cell chronic lymphoproliferative disorders, 5 reactive bone marrow samples, 4 myelodysplastic syndromes, and 19 mastocytosis were analyzed. The expression of both surface and cytoplasmic CD63 by human bone marrow mast cells is clearly demonstrated. Our results show that high amounts of CD63 are present in human bone marrow mast cells most of it corresponding to an intracellular localization. No significant differences in CD63 expression were observed as regards both total and cytoplasmic CD63, except for higher CD63 levels in adult patients with mastocytosis (P = 0.05). By contrast, the mean level of surface CD63 significantly varied between the different groups of individuals. Accordingly, patients with monoclonal gammopathies displayed a slight decrease (P = 0.1) in surface CD63 expression, whereas bone marrow mast cells from adults with indolent systemic mast cell disease showed significantly (P = 0.0005) higher levels of surface CD63 as compared to healthy controls.

Expression of high amounts of the CD117 molecule in a case of B‐cell non‐Hodgkin's lymphoma carrying the t(14:18) translocation

The c‐kit proto‐oncogen (CD117) has been described to be present in normal and neoplastic hemopoietic cells including both myeloid and lymphoid lineages. Among the normal lymphoid cells CD117 expression would be restricted to a small subset of NK‐cells, and to early T‐cell precursors and it is not expressed by normal B‐cells. Regarding chronic lymphoproliferative disorders the only data provided up to now suggests that CD117 expression is restricted to cases of Hodgkins disease and anaplastic large‐cell lymphoma. In the present paper we describe a case of a B‐cell chronic lymphoproliferative disorder carrying the t(14:18) translocation as demonstrated by molecular studies, in which the flow cytometric immunophenotypic analysis of both peripheral blood and bone marrow samples revealed the expression of high amounts of the CD117 antigen in the surface of the clonal B‐cell population. Further studies are necessary to explore both the functional role of c‐kit expression in the neoplastic B‐cells from this patient and its potential utility for the diagnosis and follow‐up of patients with B‐cell non‐Hodgkins lymphoma. Am. J. Hematol. 63:226–229, 2000.

Ultrastructural Study of Blood Cells in Toxic Oil Syndrome

The toxic oil syndrome is a new multisystemic disease, linked to the consumption of denatured rapeseed oil, which occurred in Spain in 1981. The hematological symptoms, present in about all the patients since the beginning of this disease have been dominated by leukocytosis and eosinophilia. The ultrastructural study of circulating blood cells has shown the presence of multiple abnormal lipid vacuoles in the eosinophil series and less frequently in neutrophils, monocytes and lymphocytes, associated to a high phagocytic activity. Bone marrow cells contrasted by the absence of lipid vacuoles in any hemopoietic series, while phagocytic activity and cytoplasmic processes were abundant. Even though patients entered the chronic phase of the disease, the hematological symptoms have spontaneously corrected themselves.

Amelanotic bone marrow infiltration secondary to pigmented malignant melanoma.

Amelanotic melanomas account for aproximately 2 to 3% of malignant melanomas. Some authors suggest an increased aggressiveness and a higher rate of metastases for these clinical variants (l). In autopsy series, bone marrow involvement is observed in 45% of patients with malignant melanoma (MM) (2). However, in vivo staging procedures disclose neoplastic infiltration only in 7% of the cases (2). As far as we can determine, bone marrow infiltration by amelanotic cells of malignant melanoma has not been previously described. We report the case of a woman with a personal history of inelanotic MM who developed involvement of bone marrow by amelanotic cells presenting as primary fibrinolysis.