Gil Cunha De Santis

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.

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

BACKGROUND AIMS Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues. METHODS MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays. RESULTS Post-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure. CONCLUSIONS A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.

Hematological abnormalities in HIV-infected patients.

BACKGROUND Anemia, neutropenia, and thrombocytopenia are commonly observed in HIV-infected patients. This study was undertaken to evaluate the prevalence of cytopenias and their association with CD4 count. Furthermore, the association of hemoglobin concentration with mortality was also investigated. METHODS We reviewed the data of 701 HIV-infected patients followed at our institution. Blood cell counts, hemoglobin concentration, CD4 count, and viral load were recorded. We also recorded the mortality rate after 1 year in the groups with CD4 <200/μl and ≥ 200/μl according to hemoglobin concentration. RESULTS Of the total patients, 37.5% had anemia; 61.1% (110/180) were in the low CD4 group and 29.4% (153/521) were in the high CD4 group (p<0.01). Mean neutrophil counts were 2.610 × 10(9)/l and 3.204 × 10(9)/l in the low CD4 and high CD4 groups, respectively (p<0.01); mean platelet counts were 218.639 × 10(9)/l and 234.807 × 10(9)/l for the low CD4 and the high CD4 groups, respectively (p=0.03). Patients whose hemoglobin concentration was below the median value had a higher death rate in both the low CD4 (14 vs. 4 deaths, p=0.013) and high CD4 (8 vs. 1 death, p=0.0158) groups. CONCLUSIONS We found an association between CD4 count and hemoglobin level, neutrophil count, and platelet count, and that anemia was independently associated with a higher mortality.

Therapeutic Leukapheresis in Patients with Leukostasis Secondary to Acute Myelogenous Leukemia

Leukostasis is a relatively uncommon but potentially catastrophic complication of acute myelogenous leukemia (AML). Prompt leukoreduction is considered imperative to reduce the high mortality rate in this condition. Leukapheresis, usually associated with chemotherapy, is an established approach to diminish blast cell counts. We report a single center experience in managing leukostasis with leukapheresis. Fifteen patients with leukostasis of 187 patients with AML (8.02%) followed at our institution were treated with leukapheresis associated with chemotherapy. The procedures were scheduled to be performed on a daily basis until clinical improvement was achieved and WBC counts were significantly reduced. Overall and early mortalities, defined as that occurred in the first 7 days from diagnosis, were reported. A high proportion of our patients with leukostasis (46.66%) had a monocytic subtype AML (M4/M5, according to French‐American‐British classification). The median overall survival was 10 days, despite a significant WBC reduction after the first apheresis procedure (from 200.7 × 109/L to 150.3 × 109/L). Almost half of patients (7/15) had an early death. Therapeutic leukapheresis, associated or not to chemotherapy, is an effective approach to reduce WBC counts in patients with AML and leukostasis; however, this therapeutic procedure does not appear to change significantly the sombre prognosis observed in the majority of patients with this complication. Other forms of treatment must be found to reduce the high mortality rate related to leukostasis. J. Clin. Apheresis, 2011.

Effects of high-dose chemotherapy on bone marrow multipotent mesenchymal stromal cells isolated from lymphoma patients

OBJECTIVE High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation is a widely applied treatment for hematological and autoimmune diseases. Little is known about the effects of this therapy on multipotent mesenchymal stromal cells (MSCs). We aimed to characterize, morphologically and functionally, MSCs isolated from bone marrow aspirates of patients after HDCT. MATERIALS AND METHODS We studied 12 consecutive lymphoma patients submitted to BEAM conditioning regimen followed by autologous stem cell transplantation 28 to 1836 days before the sample collection. Thirteen normal donors were used as control. MSCs were isolated by adherence to plastic and expanded ex vivo by culture in flasks containing alpha-minimum essential medium plus 15% fetal bovine serum. RESULTS The cell population isolated showed a typical MSC morphology, immunophenotype, and differentiation capacity into adipogenic, osteogenic, and chondrogenic lineages. The MSCs obtained from patients with Hodgkins disease and non-Hodgkins lymphoma showed decreased fibroblastoid colony-forming unit count (p = 0.023) and increased doubling time (p = 0.031) related to the control group. The total cell expansion of MSCs from normal subjects was marginally superior to the patient group (p = 0.064). There were no differences in gene expression profile, MSCs plasticity, or hematopoiesis support capability between control and patient group. CONCLUSIONS Results suggest that HDCT applied to lymphoma patients damaged MSCs, which was demonstrated by their reduced clonogenic potential, doubling time, and cell expansion rates when compared to controls.

Clinical and hematological effects of hydroxyurea therapy in sickle cell patients: a single-center experience in Brazil

CONTEXT AND OBJECTIVES Sickle cell disease (SCD) is the most common genetic disorder among people of African descent, affecting approximately 3,500 newborns each year in Brazil. Hydroxyurea (HU) is the only effective drug to treating patients with SCD, thereby reducing morbidity and mortality. The objective was to analyze the effects of HU on SCD patients at our institution. DESIGN AND SETTING Retrospective study conducted at a sickle cell centre in Ribeirão Preto, São Paulo, Brazil. METHODS We analyzed clinical and laboratory data on 37 patients. The hematological parameters and clinical events that occurred during the year before and the first year of treatment with HU were analyzed. The mean dose of HU was 24.5 ± 5.5 mg/kg/day. RESULTS There were rises in three parameters: hemoglobin (8.3 g/dl to 9.0 g/dl, P = 0.0003), fetal hemoglobin (HbF) (2.6% to 19.8%, P < 0.0001) and mean cell volume MCV (89 to 105 fl, P = 0.001); and reductions in the numbers of leukocytes (10,050/µl to 5,700/µl, P < 0.0001), neutrophils (6,200/µl to 3,400/µl, P = 0.001), platelets (459,000/µl to 373,000/µl, P = 0.0002), painful crises (1.86 to 0.81, P = 0.0014), acute chest syndromes (0.35 to 0.08, P = 0.0045), infections (1.03 to 0.5, P = 0.047), hospitalizations (1.63 to 0.53, P = 0.0013) and transfusions (1.23 to 0.1, P = 0.0051). CONCLUSION The patients presented clinical and hematological improvements, with an increase in HbF and a reduction in the infection rate, which had not been addressed in most previous studies.

Cardiac stunning as a manifestation of ATRA differentiation syndrome in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a specific type of acute myelogenous leukemia characterized by the reciprocal translocations between the long arms of chromosomes 15 and 17 [t(15;17)] leading to the fusion of the retinoic acid receptor and the promyelocytic leukemia (PML) genes, called PML/RARalpha. The PML/RARalpha gene product acts as a transcription repressor, resulting in the blocking of the differentiation of APL blasts at the stage of promyelocytes. Pharmacological doses of all-trans retinoic acid (ATRA) reverse this blockage and induce disease remission. However, although ATRA is well tolerated in the majority of the APL patients treated with ATRA and idarubicin regimen (AIDA), approximately one quarter of them develop a complication denominated ATRA differentiation syndrome (DS), formerly known as retinoic acid syndrome [1–3]. The onset of DS ranges from 2 to 21 days (median of 10 days) after initiating ATRA therapy. The full development of DS is characterized by unexplained fever, weight gain, edema, interstitial pulmonary infiltrates with dyspnea, pleural and pericardial effusions, episodic hypotension, and acute renal failure. The most frequent manifestations are respiratory distress and fever, affecting more than 80% of DS patients. Acute myocardial ischemia is not commonly observed in DS. As far as we know, there is only one case report describing this complication [4]. Through early diagnosis, DS responds well to the administration of dexamethasone, with decrease in mortality from 30% to less than 10% in the patients suffering from it. The pathogenesis of this complication is not completely clarified, but increased expression of adhesion molecules on blasts and on endothelial cells and the release of inflammatory cytokines may be the main explanation [5].

Intrahepatic Cholestasis in Sickle Cell Disease: A Case Report

Intrahepatic cholestasis (SCIC) is an uncommon but potentially fatal complication of sickle cell disease (SCD), with a high death rate, observed mainly in patients with homozygous sickle cell anemia. Herein, we describe a case of severe SCIC treated successfully with aggressive manual exchange transfusion (ET). The patient was admitted with enlarged liver and signs of hepatic failure, such as hyperbilirubinemia and coagulopathy. There was no evidence of viral hepatitis or biliary obstruction. We performed several sessions of ET in order to reduce the percentage of HbS to levels inferior to 30%, which was successfully accomplished. The patient had a complete recovery of hepatic function. This case has shown that ET is an effective treatment of SCIC and should be introduced early on the onset of this severe complication.

Hydroxyurea increases plasma concentrations of microparticles and reduces coagulation activation and fibrinolysis in patients with sickle cell anemia.

Microparticles (MPs) are present in healthy subjects and their concentration increases in patients at high risk of thrombosis. We evaluated 10 patients with sickle cell anemia (SCA) treated with hydroxyurea (HU) and 13 SCA patients without this treatment. MP concentrations were determined by flow cytometry. Coagulation was evaluated using the thrombin-antithrombin complex (TAT) and D-dimers. Total MP concentrations were increased in the HU-treated group (265 × 106/ml vs. 67.45 × 106/ml; p = 0.0026), as well as MPs derived from RBC (67.83 × 106/ml vs. 26.31 × 106/ml; p = 0.05), monocytes (51.31 × 106/ml vs. 9.03 × 106/ml; p = 0.0084), monocytes with tissue factor (TF) expression (2.27 × 106/ml vs. 0.27 × 106/ml; p = 0.0058), endothelium (49.42 × 106/ml vs. 7.23 × 106/ml; p = 0.007) and endothelium with TF (1.42 × 106/ml vs. 0.26 × 106/ml; p = 0.0043). Furthermore, the concentrations of TAT (7.56 vs. 10.98 µg/l; p = 0.014) and D-dimers (0.65 vs. 1.29 µg/ml; p = 0.007) were reduced with HU. The MP elevation may suggest a direct cytotoxic effect of HU. Another explanation is a cell surface increase secondary to a megaloblastic process, resulting in increased vesicle release. In our opinion, the known benefits of HU on SCA patients, along with the reduction in coagulation activation, surpass its potential detrimental effect on MPs. Future studies should elucidate the role of MPs and demonstrate their significance in different contexts.

Preoperative variables associated with transfusion requirements in orthotopic liver transplantation

BACKGROUND Patients with end-stage chronic liver disease (CLD) and submitted to orthotopic liver transplantation (OLT) usually require blood transfusion during the procedure or in the post-operative period due to hemorrhage. Risk factors for transfusion need are not fully known. This study aimed to identify the factors associated with blood components requirements. METHODS In this retrospective study a total of 166 consecutive patients submitted to OLT with the piggyback technique, between 2001 and 2011, were evaluated for number of blood components transfused during surgical procedure and the four subsequent days (total of 5 days). We evaluated the association between the number of units transfused and clinical variables, such as: Child-Turcotte-Pugh (CTP) and MELD scores, hemoglobin concentration (Hb), INR, serum creatinine, bilirubin and albumin concentrations, and total, hypothermic and normothermic time of graft ischemia. RESULTS 152 (91.6%) Patients were transfused (median of 24 units of blood components). Risk factors for higher blood transfusion requirements were CTP, INR, Hb and total time of graft ischemia. The group with CTP-A score received less blood components than CTP-B/C (11.5 vs 27; P=0.002). The group with Hb<10 required a higher number of blood units (34.5 vs 23; P=0.003). The group with INR<1.5 received less blood units (20.5 vs 31; P=0.012). The group transplanted with a graft exposed to less than the median of 555 min of ischemia received less transfusion (21 vs 27; P=0.03). MELD score and the other factors were not associated with blood requirements. CONCLUSION These results demonstrate that CTP, but not MELD score, hemoglobin concentration, INR, and total time of graft ischemia are preoperative variables associated with blood requirements during OLT and in the subsequent days.