Aglair B. Garcia

Determination of P‐glycoprotein, MDR‐related protein 1, breast cancer resistance protein, and lung‐resistance protein expression in leukemic stem cells of acute myeloid leukemia

The most primitive leukemic precursor in acute myeloid leukemia (AML) is thought to be the leukemic stem cell (LSC), which retains the properties of self‐renewal and high proliferative capacity and quiescence of the hematopoietic stem cell. LSC seems to be immunophenotypically distinct and more resistant to chemotherapy than the more committed blasts. Considering that the multidrug resistance (MDR) constitutive expression may be a barrier to therapy in AML, we have investigated whether various MDR transporters were differentially expressed at the protein level by different leukemic subsets.

Age-related changes of lymphocyte subsets in normal bone marrow biopsies

There is a paucity of information in the literature concerning the age-related changes of the lymphocyte subsets in bone marrow (BM), and the available reports disagree about the characteristics of the population studied and the methods for obtaining, handling, and analyzing the samples. The purpose of the present study was to determine the distribution of lymphoid subsets in the BM from infants, children, and adults by analyzing fragments of sternum obtained during cardiovascular surgery. The samples were studied by flow cytometry employing the whole blood lysis method and excluding from the analysis the contamination of the lymphoid window by erythroid precursors. We observed that in the first 4 years of life the B subset represented more than 65% of all cells in the lymphoid window, most of them (80%) exhibiting the immature phenotype CD19+CD100+. Conversely, the T subset was composed of mature CD4+ or CD8+ cells, with the CD4/CD8 ratio being less than 1 in all age groups. With age there was a progressive decrease in the percentage of B cells and an increase of T cells, reaching similar proportions in the BM from adults (33.6% and 34.8%, respectively). Furthermore, the percentage of CD10+ cells in the B subset decreased independently, whereas the CD20 expression increased. The percentage of NK cells did not change with age.

Adhesion molecules and differentiation syndrome: phenotypic and functional analysis of the effect of ATRA, As2O3, phenylbutyrate, and G-CSF in acute promyelocytic leukemia

Background and Objectives Differentiation Syndrome (DS) is a treatment complication which can occur in patients treated with acute promyelocytic leukemia (APL) with all transretinoic acid (ATRA) or As2O3, and is characterized by enhanced leukocyte transmigration. As2O3, Phenylbutyrate (PB) and G-CSF are known to potentiate ATRA effects. Our aim was to analyze the changes in expression and function of adhesion molecules induced by ATRA, As2O3, G-CSF and PB, and their association. Design and Methods APL blasts and NB4 cells were treated with ATRA, As2O3, PB, G-CSF or their association and the expression of adhesion molecules was determined by flow cytometry. Cell adhesion was evaluated in vitro using Matrigel and for the in vivo analysis, Balb-c mice were injected with NB4 cells pre-treated with ATRA, As2O3, ATRA+G-CSF or ATRA+As2O3. In addition, CD54 and CD18 knock-out mice were injected with NB4 cells and concomitantly treated with ATRA. In both models, the MPO activity in the lungs was determined 6 hours after the injection of the cells. Results In NB4 and APL blasts, ATRA and As2O3 increased CD54 expression, but no synergism was detected. CD11b and CD18 were also up-regulated by ATRA in primary cells. PB and G-CSF had no effect, but the latter potentiated ATRA-induced CD18 up-regulation. These changes were accompanied by increased adhesion to Matrigel and to lung microvasculature, and reversed by anti-CD54, anti-CD18 antibodies. In CD54 and CD18 knock-out mice the ATRA effect was canceled. Interpretation and Conclusions The use of As2O3, PB and G-CSF in association with ATRA should not aggravate DS in APL.

Blastic CD4 NK cell leukemia/lymphoma: a distinct clinical entity.

We report the findings of three new cases of a distinct clinicopathologic natural killer (NK) cell malignancy characterized by cutaneous, nodal and bone marrow infiltration by CD3-CD4+CD56+ NK blastic cells. Tumor cells were detected in bone marrow and in peripheral blood smears and showed finely distributed nuclear chromatin with nucleoli and a moderate amount of cytoplasm. Epstein-Barr virus (EBV) DNA was negative in the two tested cases. The immunophenotypes determined by flow cytometry were identical concerning mCD3-cytCD3-CD4+weakCD56+ HLA-DR+. The TCR was in germline configuration in the two cases tested. NK cell activity was demonstrated only in one out of the two cases tested. The negative reactions with alpha-naphthyl-acetate-esterase (ANAE), CD11b and CD14 strongly suggested that the tumor cells were not of the monocytic lineage.

Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes

The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years) were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

Increased lymphocyte death by neglect-apoptosis is associated with lymphopenia and autoantibodies in lupus patients presenting with neuropsychiatric manifestations

Abstract To evaluate lymphocyte death by neglect-apoptosis features in systemic lupus erythematosus (SLE) patients presenting with neuropsychiatric (NPSLE) involvement we studied 40 SLE patients with active disease, 20 with and 20 without neuropsychiatric manifestations, and 20 control individuals. Lymphocyte apoptosis was evaluated by means of DNA staining using flow cytometry, immediately after cell isolation and after incubation with culture medium or autologous serum. Compared with controls, NPSLE and non-NPSLE patients exhibited increased rates of neglect-apoptosis immediately after cell isolation. Only NPSLE patients exhibited an increased neglect-apoptosis rate after incubation with culture medium; however, the neglect-apoptosis rate was associated with lymphopenia in both series of patients. After lymphocyte incubation with autologous serum, only NPSLE patients exhibited a significant negative correlation between the neglect-apoptosis rate and the number of peripheral lymphocytes. The incubation of lymphocytes with autologous serum containing antiphospholipid or anti-SSA/Ro antibodies significantly increased the neglect-apoptosis in NPSLE when compared with non-NPSLE patients with a similar autoantibody profile. In conclusion, NPSLE and non-NPSLE patients shared several abnormalities in terms of lymphocyte neglect-apoptosis. Peculiar findings were observed in NPSLE patients particularly after incubation with autologous serum, such as the fact that the increased lymphocyte death by neglect-apoptosis was associated with lymphopenia and with the presence of antiphospholipid and anti-SSA/Ro antibodies.

Increased dexamethasone-induced apoptosis of thymocytes from mice exposed to long-term extremely low frequency magnetic fields.

To address the effect of extremely low frequency electromagnetic fields on programmed cell death we assessed both the spontaneous and dexamethasone (Dex)-induced apoptosis of thymocytes and spleen cells from mice submitted to a long-term continuous exposure of a 0.4-1.0 microT 60 Hz magnetic field or an 8-20 microT direct current (DC) magnetic field. Dex-induced apoptosis but not spontaneous apoptosis was substantially increased in thymocytes from 0.4 to 1.0 microT 60 Hz field-exposed animals. Spontaneous apoptosis and Dex-induced apoptosis of spleen cells were not affected by the 0.4-1.0 microT 60 Hz field exposure. In addition, spontaneous apoptosis and Dex-induced apoptosis of thymocytes and spleen cells from mice exposed to an 8-20 microT DC field were similar to the controls. These findings represent the first demonstration that thymocytes from mice exposed to a long-term 0.4-1.0 microT 60 Hz field may show abnormal response to Dex apoptotic stimuli.

Characterization of acute lymphoblastic leukemia subtypes in Brazilian patients

The distribution of the acute lymphoblastic leukemia (ALL) subsets in 225 consecutive Brazilian patients was determined by an immunophenotypic study with an extensive panel of monoclonal antibodies. All subsets were detected and their relative frequencies were similar to those described in developed countries, except for the B-mature subset which had a higher frequency, especially in adults. Associated myeloid markers were expressed by 11% of the ALL and CD10 by 15.9% of T-ALL cases. Besides, the incidence rates determined for the region of Ribeirão Preto showed that the overall incidence of ALL was 12.5 cases/10(6) people years (PY) (5 cases/10(6) PY in non-Whites versus 14 cases/10(6) PY in Whites); the incidence of childhood ALL was 25.5 cases/10(6) PY (8.1 versus 29.8 cases/10(6) PY in non-Whites and Whites, respectively) and the incidence of ALL in adults was 6.2 cases/10(6) PY (5.5 versus 6.1 cases/10(6) Py in non-Whites and Whites, respectively). The significantly lower incidence rate of ALL in non-White children was associated with a selective deficit of the common subtype and a lack of the typical age peak of incidence in this group. The ALL features demonstrated here in Brazilian non-White children resemble those described in the American non-Whites before the seventies and those in British and American Whites at the beginning of the century.

Halofuginone has anti-proliferative effects in acute promyelocytic leukemia by modulating the transforming growth factor beta signaling pathway

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.

Expression of CD117 and CD11b in Bone Marrow Can Differentiate Acute Promyelocytic Leukemia From Recovering Benign Myeloid Proliferation

The morphologic characteristics of bone marrow aspirates from patients recovering from acute agranulocytosis may be closely similar to the pattern observed in cases of acute promyelocytic leukemia (APL). The clinical manifestation also can be ambiguous in a substantial number of cases. The immunophenotypic features of bone marrow from 5 patients recovering from acute agranulocytosis, showing an increase in the percentage of promyelocytes (26%-66%), were compared with the immunophenotype of 31 consecutive patients with APL whose diagnosis was confirmed by PML-RAR alpha gene rearrangement. All markers were similarly expressed, except for CD117 and CD11b. CD117 was positive in 24 (77%) of the APL cases and in none of the acute agranulocytosis cases. On the other hand, CD11b was positive in 5 (100%) of the acute agranulocytosis cases and in only 2 (6%) of the APL cases. Thus, the CD117-CD11b+ phenotype was detected in all patients recovering from agranulocytosis and in only 1 (3%) of 31 APL cases. Therefore, we suggest that the combination of both markers is helpful in the differentiation of APL from recovering benign myeloid proliferation.