Isolation of human mesenchymal stem cells from amnion, chorion, placental decidua and umbilical cord: comparison of four enzymatic protocols.

OBJECTIVE: To compare four enzymatic protocols for mesenchymal stem cells (MSCs) isolation from amniotic (A-MSC) and chorionic (C-MSC) membranes, umbilical cord (UC-MSC) and placental decidua (D-MSC) in order to define a robust, practical and low-cost protocol for each tissue. RESULTS: A-MSCs and UC-MSCs could be isolated from all samples using trypsin/collagenase-based protocols; C-MSCs could be isolated from all samples with collagenase- and trypsin/collagenase-based protocols; D-MSCs were isolated from all samples exclusively with a collagenase-based protocol. CONCLUSIONS: The trypsin-only protocol was least efficient; the collagenase-only protocol was best for C-MSCs and D-MSCs; the combination of trypsin and collagenase was best for UC-MSCs and none of tested protocols was adequate for A-MSCs isolation.

Importance of early absolute lymphocyte count after allogeneic stem cell transplantation: a retrospective study.

INTRODUCTION: Early lymphocyte recovery after allogeneic hematopoietic stem cell transplantation (HSCT) is related to the prevention of serious infections and the clearing of residual tumor cells. METHODS: We analyzed the absolute lymphocyte count at 20 (D+20) and 30 (D+30) days after HSCT in 100 patients with malignant hematologic diseases and correlated with the risk of transplant-related mortality, overall survival (OS), disease-free survival (DFS), nonrelapsed mortality (NRM), and risk of infection. RESULTS: Patients presenting with lymphocyte counts of <300 × 103/µL on D+30 have a 3.76 times greater risk of death in <100 days. Over a medium follow-up of 20 months OS, DFS, and NRM were similar between the groups. CONCLUSION: In our group of patients delayed lymphocyte recovery after HSCT was a predictor of early death post-HSCT.

Assessment of immature platelet fraction and immature reticulocyte fraction as predictors of engraftment after hematopoietic stem cell transplantation.

INTRODUCTION: Engraftment is a critical milestone of the hematopoietic stem cell transplantation (HSCT) process. The immature platelet fraction (IPF) and immature reticulocyte fraction (IRF) are considered early indicators of bone marrow recovery. The objective of this study was to assess these parameters as predictors of HSCT engraftment. METHODS: Neutrophil and platelet engraftment were defined as the first of three consecutive days with an absolute neutrophil count >0.5 × 10(9) /L or platelet count >20 × 10(9) /L, respectively. The IRF cutoff was 12%. Two IPF cutoffs were used: >6.2% and >10%. RESULTS: The study sample comprised 44 patients, of whom 24 had undergone autologous HSCT and 20 had undergone allogeneic HSCT. Absolute neutrophil counts >0.5 × 10(9) /L were preceded by IRF >12% in 86% of patients (38 of 44). Platelet counts >20 × 10(9) /L were preceded by an IPF >6.2% in 90% of patients (37 of 41) and by an IPF >10% in 63% of patients (26 of 41). CONCLUSION: The results show that IRF and IPF are engraftment predictors. Peak in IPF was observed before rise in platelet count, while IRF rises before absolute neutrophil count (ANC) and persists increased. This indicates that IRF and IPF can be considered as new tools for hematopoietic assessment after HSCT.

Fungal spore concentrations in two haematopoietic stem cell transplantation (HSCT) units containing distinct air control systems.

Invasive fungal diseases have emerged as important causes of morbidity and mortality in haematological patients. In this study air samples were collected in two haematopoietic stem cell transplantation (HSCT) units, in which distinct air-control systems were in place. In hospital 1 no high-efficiency particulate air (HEPA) filter was available whereas in hospital 2 HSCT rooms were equipped with HEPA filters, with positive air pressure in relation to the corridor. A total of 117 samples from rooms, toilets and corridors were obtained during December 2009 to January 2011, using a six-stage Andersen sampler. In both hospitals, the concentration of potentially pathogenic fungi in the air was reduced in patients' rooms compared to corridors (P < 0·0001). Despite the presence of a HEPA filter in hospital 2, rooms in both hospitals showed similar concentrations of potentially pathogenic fungi (P = 0·714). These findings may be explained by the implementation of additional protective measures in hospital 1, emphasizing the importance of such measures in protected environments.

Invasive fungal diseases in haematopoietic cell transplant recipients and in patients with acute myeloid leukaemia or myelodysplasia in Brazil.

Invasive fungal disease (IFD) shows distinct regional incidence patterns and epidemiological features depending on the geographic region. We conducted a prospective survey in eight centres in Brazil from May 2007 to July 2009. All haematopoietic cell transplant (HCT) recipients and patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS) were followed from admission until 1 year (HCT) or end of consolidation therapy (AML/MDS). The 12-month cumulative incidence (CI) of proven or probable IFD was calculated, and curves were compared using the Grey test. Among 237 AML/MDS patients and 700 HCT recipients (378 allogeneic, 322 autologous), the 1-year CI of IFD in AML/MDS, allogeneic HCT and autologous HCT was 18.7%, 11.3% and 1.9% (p <0.001), respectively. Fusariosis (23 episodes), aspergillosis (20 episodes) and candidiasis (11 episodes) were the most frequent IFD. The 1-year CI of aspergillosis and fusariosis in AML/MDS, allogeneic HCT and autologous HCT were 13.4%, 2.3% and 0% (p <0.001), and 5.2%, 3.8% and 0.6% (p 0.01), respectively. The 6-week probability of survival was 53%, and was lower in cases of fusariosis (41%). We observed a high burden of IFD and a high incidence and mortality for fusariosis in this first multicentre epidemiological study of IFD in haematological patients in Brazil.

Expression of CD55 and CD59 on peripheral blood cells from systemic lupus erythematosus (SLE) patients.

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. CD55 inhibits the formation of C3 convertases, and CD59 prevents the terminal polymerisation of the membrane attack complex. It has been reported that SLE patients seems to have an acquired deficiency of these proteins associated with secondary autoimmune haemolytic anaemia and lymphopenia. The aim of this study was to evaluate the presence of altered CD55 and CD59 expression on peripheral blood cells from SLE patients. Flow cytometric analyses were performed on red and white blood cells from 23 SLE patients and 23 healthy controls. We observed more CD55- and CD59-lymphocytes (p=0.005 and p=0.019, respectively), and CD59-granulocytes (p=0.045) in SLE patients than in controls. These results suggest there is an altered pattern of CD55 and CD59 expression on the peripheral blood cells of SLE patients, and it may play a role in the cytopenias in these patients.

HLA-G polymorphism influences the susceptibility to HCV infection in sickle cell disease patients.

Despite its well known monogenic etiopathogenesis, sickle cell disease (SCD) is characterized by a striking variability of clinical presentation. There is growing evidence that genetic factors may be involved in this variability. Human leukocyte antigen (HLA)-G is a non-classical HLA molecule which was shown to be expressed at sites of inflammation and in inflammatory diseases. Besides its large and highly polymorphic promoter region, the 3' UTR region seems also to play an important role on regulating HLA-G expression. We investigated the influence of the 14 pb (rs1704) and the +3142 (rs1063320) HLA-G polymorphisms in 93 SCD patients in order to evaluate its potential role on clinical parameters. Twenty-one patients presented an HCV infection. Among all SCD patients 16 (22.2%) were homozygous for the +3142C genotype, none of them hepatitis C (HCV) positive. Controlling for blood transfusions in the last year, the C allele represented a dose dependent protection effect for HCV infection (PR = 0.41; 95% CI: 0.24-0.71). The +3142C allele was also underrepresented among patients with history of respiratory-tract infections. Our results support a role of the +3142 polymorphism in the susceptibility to infections, in particular to HCV infection, and suggest a possible interference of the HLA-G molecule in the response to infections, among SCD patients.

Low bone mineral density is associated with insulin resistance in bone marrow transplant subjects.

Post-BMT subjects have an increased bone fracture risk. Additionally, several factors were associated with osteopenia and osteoporosis in these individuals. We aimed to identify other factors associated with osteopenia and osteoporosis in allogeneic post-BMT subjects. We conducted a cross-sectional study with 47 allogeneic post- BMT subjects. Serum 25-hydroxyvitamin D (25(OH)D), parathyroid hormone, ferritin, vitamin B(12), insulin, glucose, cholesterol and triglyceride levels were measured. Insulin resistance and secretion were estimated through the homeostatic model assessment for insulin resistance (HOMA-IR) and homeostatic model assessment for beta-cell function (HOMA-B), respectively. A bone densitometry (BMD) was also obtained. The median time after BMT was 47.7 (12-115) months. Osteoporosis was identified in 17.0% of the subjects and osteopenia in 19.7%. The mean serum ferritin (P=0.002), insulin (P<0.0001), glucose (P=0.003) and triglyceride (P=0.018) levels were higher in individuals with osteopenia/osteoporosis. HOMA-IR (P<0.0001) and HOMA-B (P<0.0001) were increased in post-BMT subjects with osteopenia/osteoporosis. There was no other factor associated with the outcome. After adjustments ferritin, serum 25(OH)D and HOMA-IR remained independently associated with osteopenia/osteoporosis; however triglycerides no longer were. In conclusion, in the present study, low serum 25(OH)D levels, high serum ferritin levels and insulin resistance were associated with osteopenia/osteoporosis in post-BMT subjects.

Patient socioeconomic status as a prognostic factor for allo-SCT.

The aim of the present study was to assess the influence of socioeconomic status (SeS) on the outcome of allo-SCT at a Brazilian SCT center. In total, 201 patients receiving HLA-identical related allo-SCTs were studied. The median age was 30 years. Overall, 163 patients had malignancies (CML 68, ALL/AML 63, myelodysplastic syndrome 12 and others 20). SeS was defined according to the Brazilian Association of Market Research Agencies classification, where people are clustered in groups A-E (richest to poorest). In total, 146 patients (72%) were classified as richest (A+B+C) and 55 (28%) as poorest (D+E). The D+E SeS group was associated with a higher incidence of chronic GVHD and acute GVHD (hazard ratio (HR)=2.61; P=0.001 and HR=2.62; P=0.001, respectively), better platelet and neutrophil engraftment (HR=1.94; P=<0.001 and HR=2.12; P=0.001) and with a higher TRM in multivariate analysis (HR=1.92; P=0.039). Estimated overall survival at 5 years was 55.2%. A D+E SeS (HR=2.13; P=0.001) was associated with a worse survival on multivariate analysis. In conclusion, a lower SeS is a strong prognostic factor in patients undergoing allo-SCT in Brazil, influencing engraftment, TRM and overall survival.

Imatinib mesylate versus allogeneic BMT for patients with chronic myeloid leukemia in first chronic phase.

Imatinib mesylate (IM) is now first-line treatment for CML. To study the results of treatment with IM after IFN failure/intolerance versus allogeneic BMT (allo-BMT), we retrospectively analyzed 264 patients treated for CML in first chronic phase in three different institutions. Over a 6-year period (2001-2006), 174 patients received IM after failure of or intolerance to IFN. During the same period of time, 90 patients received an allo-BMT from an HLA-matched sibling (n=83) or an unrelated donor (n=7). The IM group was older (41 versus 33 years, P<0.001). Five-year EFS was 62% among patients receiving IM and 52% among patients undergoing allo-BMT (P=0.0002). OS at 5 years was 93% for IM-treated patients and 59% for patients undergoing allo-BMT (P<0.0001). Allo-BMT cannot be considered as first-line treatment for CML patients in first chronic phase.

Epidemiology of bacteremia and factors associated with multi-drug-resistant gram-negative bacteremia in hematopoietic stem cell transplant recipients.

The incidence of Gram-negative bacteremia has increased in hematopoietic stem cell transplant (HSCT) recipients. We prospectively collected data from 13 Brazilian HSCT centers to characterize the epidemiology of bacteremia occurring early post transplant, and to identify factors associated with infection due to multi-drug-resistant (MDR) Gram-negative isolates. MDR was defined as an isolate with resistance to at least two of the following: third- or fourth-generation cephalosporins, carbapenems or piperacillin-tazobactam. Among 411 HSCT, fever occurred in 333, and 91 developed bacteremia (118 isolates): 47% owing to Gram-positive, 37% owing to Gram-negative, and 16% caused by Gram-positive and Gram-negative bacteria. Pseudomonas aeruginosa (22%), Klebsiella pneumoniae (19%) and Escherichia coli (17%) accounted for the majority of Gram-negative isolates, and 37% were MDR. These isolates were recovered from 20 patients, representing 5% of all 411 HSCT and 22% of the episodes with bacteremia. By multivariate analysis, treatment with third-generation cephalosporins (odds ratio (OR) 10.65, 95% confidence interval (CI) 3.75-30.27) and being at one of the hospitals (OR 9.47, 95% CI 2.60-34.40) were associated with infection due to MDR Gram-negative isolates. These findings may have important clinical implications in the decision of giving prophylaxis and selecting the empiric antibiotic regimen.

Polymorphisms of chemokine receptors and eNOS in Brazilian patients with sickle cell disease.

Sickle cell disease (SCD) is an inherited disorder that presents extremely variable clinical manifestations. For the past few decades, it has been approached as an inflammatory disorder, and several researchers have tried to determine the factors involved in such characteristic. In order to contribute to the identification of the genetic differences underlying this phenotypic diversity in SCD, we proposed to study the distribution of polymorphic variants of the genes encoding the chemokine receptors CCR2 and CCR5, as well as three polymorphisms in the NOS3 gene, in Brazilian SCD patients. These genes are involved in the development of inflammatory immune reactions, a feature believed to be of extreme importance in SCD pathology. Our results indicate that the polymorphisms studied here are not directly associated with severe clinical manifestations in SCD patients. Nevertheless, we observed a tendency for the development of a severe clinical course in carriers of the variant alleles CCR2-64I and CCR5delta32 and in homozygotes for the -786C variant of the NOS3 gene. Further studies should be carried out in order to assess the role of such variants in the clinical picture of SCD.

Immunosuppressive therapy for kidney transplant prevents vaso-occlusive crisis in a haemoglobin SC disease patient.

Although the molecular basis of sickle cell disease (SCD) is well established, the wide variability in clinical manifestations still puzzles haematologists and clinicians. Recently, SCD started to be considered by different groups as a chronic inflammatory condition, where the inflammatory tendency of each individual could drive more or less severe clinical features. Here we describe a haemoglobin SC disease patient (heterozygous to both HbS and HbC variants) that experienced several vaso-occlusive crises before underwent a successful kidney transplantation. Since then (16 years ago), she is on uninterruped immunosuppressive therapy, and do not experienced any severe vaso-occlusive crisis. Considering SCD associated morbidity as a result of exacerbated immune responses, we suggest that the immunosuppressive therapy directed to the kidney graft maintenance is actually also helping in the control of the chronic inflammatory responses associated to SCD.

Prevalence of TEL/AML1 fusion gene in Brazilian pediatric patients with acute lymphoblastic leukemia.

We studied 58 childhood B-lineage acute lymphoblastic leukemia (B-ALL) in Brazilian sample patients at the time of diagnosis to investigate the prevalence of the cryptic t(12;21)(p13;q22). All bone marrow specimens were G-band karyotyped, and commercial dual-color DNA probes were used to search for fusion signals in nuclei. The karyotype analysis showed hyperdiploidy as the most frequent abnormality. The frequency of patients with TEL/AML1 gene fusion was 19% (11 out of 58 cases). Six of the positive samples had normal karyotypes. Deletion of the wild-type TEL allele was observed in 27.3% of TEL/AML1 fusion-positive cases, but it was also identified in 4.2% of the negative cases. Three cases presented two fusion signals, indicating possible duplication of the der(21). The mean age of the patients with TEL/AML1 fusion was 4.8 years and the mean amount of peripheral leukocytes was 44,270 x 10(6)/L. The higher frequency of females with B-ALL (33/58 cases) observed in our sample was probably due to the selection mode of the study cases. The prevalence of TEL/AML1 fusion in Brazilian children in our study is similar to that found in other populations.

Outcome of treatment in adult acute lymphoblastic leukemia in southern Brazil using a modified german multicenter acute lymphoblastic leukemia protocol.

Reports on treatment outcomes in adults with acute lymphoblastic leukemia (ALL) in Brazil are sparse. To evaluate the outcome of patients with ALL managed by the public healthcare system, we studied 42 adults treated from 1990 to 1997 in the Division of Hematology at Hospital de Clínicas, Porto Alegre, Brazil. Of these patients, 14/42 were females and their median age at diagnosis was 26 (17-64) years. The diagnosis of ALL was based on cytological examination of marrow smears, and immunophenotypic and cytogenetic studies, when available. Fifty percent of the patients expressed CD10, 30% were CD10 negative and CD19 positive and 20% expressed T markers. Philadelphia chromosome was found in 4 (7.14%). The chemotherapy protocol was adapted from the German Multicenter ALL (GMALL) 02-84 protocol. The complete remission rate was 93% and the overall survival at 5 years was 41%. No particular risk factor was identified in our series. These results are comparable to the findings of other international studies.

Generation of activated natural killer (A-NK) cells in patients with chronic myelogenous leukaemia and their role in the in vitro disappearance of BCR/abl-positive targets.

Activated natural killer (A-NK) cells, a subset of CD56(dim)CD3- lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph+ chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP; n=7) contained a mean (+/-SD) of83 +/- 7% of CD3- cells, and those from patients with advanced chronic phase (ACP; n=7) contained 27+/-33% CD56+CD3- cells. In three patients with leukaemia in a blastic phase (BP) it was only possible to obtain one culture enriched in CD56+CD3- cells (81%). Cellular aggregates of myeloid cells and large granular lymphocytes were observed in early A-NK cell cultures. Paired freshly-adherent and cultured A-NK cells were tested for the presence of BCR/abl mRNA by RT-PCR. The BCR/abl+ cells were detected in all 12 preparations of the freshly adherent A-NK cells tested. In 6/12 the BCR/abl+ cells were no longer detectable by RT-PCR on day 14 of culture. Both proliferation and antileukaemic cytotoxicity were significantly higher (P=0.002 and P=0.029, respectively) in the BCR/abl- cultures than those in the six BCR/abl+ cultures. 5/6 BCR/abl- cultures were highly enriched in A-NK cells on day 14, and 1/6 contained predominantly CD56+CD3+ cells. Only 2/6 BCR/abl + cultures were enriched in A-NK cells on day 14, but they had poor cytotoxicity and a low proliferative index. Myeloid cells (CD33+) were more frequently detected in the BCR/abl+ than BCR/abl- A-NK cell cultures (P=0.028). These observations suggest that: (1) populations of benign A-NK cells can be generated from the peripheral blood of CML patients; (2) the ability to generate A-NK cells is impaired in patients with advanced CML; and (3) the ability to generate A-NK cells with antileukaemic activity correlates with the disappearance of BCR/abl+ cells from these cultures.

The role of natural killer cells in the treatment of chronic myeloid leukemia.

The success of chemotherapy in patients with leukemia whose marrow appears to be replaced by leukemia cells must be due to the persistence of normal stem cells. In this normal population are the progenitors of the cells of the immune system. Natural killer (NK) cells originate in the bone marrow. On maturation and activation with interleukin 2 (IL-2) or other cytokines, NK cells develop cytotoxic activity against a variety of leukemic blasts, including those from patients with chronic myeloid leukemia (CML). In the past few years, bone marrow transplantation (BMT) and alpha-interferon (IFN-alpha) have proved to be the most promising therapies for the treatment of CML. In both these therapies, NK cells may play a prominent role. In this article, we discuss the antitumor/antileukemia activity of human NK cells, the presence of benign NK cell precursors in the different stages of CML, the role of NK cells in BMT and IFN-alpha treatment, and the potential therapeutic applications of NK cells in patients with hematologic malignancies.

Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (Fc gamma RIII) expressed by human natural killer (NK) cells.

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.