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Dendritic cell (DC) vaccines have demonstrated good efficacy in preventing relapse and in increasing survival of patients affected by a variety of both solid and hematological tumors. Most protocols used to generate these cells involve the automated separation of peripheral blood monocytes from patients. This approach requires specialized equipment, which elevates the cost of this type of therapy, potentially limiting the widespread access to patients.In this study, we compare the yield and quality of dendritic cells generated from monocytes and isolated by an automated method or by manual methods using gradient centrifugation.The results demonstrate the equivalence of the 3 methods in relation to the yield and final quality of the product, however with considerable differences between the costs of these procedures. In addition, this study also demonstrates the feasibility of the antigenic pulse with autologous tumor cell lysates, constituting a source of antigens, not only easily obtained and manipulated, but also specific to the patient's tumor.These findings may have important implications for emerging centers interested in using this medical approach and potentially increase the access of a greater number of patients to this therapeutic option.

Background Patients with SCD are among one of transfused populations more often alloimmunized to red blood cell (RBC) antigens. Although transfusion services establish protocols of serologic matching and genotypic matching to reduce alloimmunization, there is still no consensus to the best practical approach although the obvious goal is to provide blood that will survive maximally. There are no studies comparing prophylactic genotypic matching to serologic matching. Based on this, our aim was to evaluate the effect prophylactic genotypic matching and serologic matching have on alloimmunization and hemolytic transfusion reactions in patients with SCD receiving regular transfusions. Methods A prospective study was conducted in 29 patients with SCD undergoing red cell transfusions in a period of 5 years. Fifteen patients were receiving prophylactic extended (ABO, Rh, K, Fya/Fyb, S/s) serologic matching and 14 patients were transfused with RBC units genotypic matched for Rh, K, Fya/Fyb, S/s, Dia, Doa/Dob including Rh variants when possible. Serologic typing was performed by gel test (Biorad) and genotyping was performed by HEA, RHD and RHCE BeadChips (Immucor). Results The patients received a range of 15-407 RBC units and the median age was 36. Of the 15 patients receiving prophylactic serologic matching, 6 developed alloantibodies (1 anti-D, 2 anti-e, 1 anti-S, 1 anti-Fya, -Doa and 1 anti-C, -Dia). Three of those patients were not having a good in vivo RBC survival at the time of antibody detection as assessed by a decrease in their hemoglobin levels and an increase in the frequency of transfusions. Molecular analyses showed that the 4 patients who developed Rh antibodies had RH variant alleles predicting partial antigens and the patients who developed anti-S and anti-Fya had discrepancies between the previous phenotype and genotype-derived phenotype. Of the 14 patients transfused with prophylactic genotypic matching RBC units, 2 were alloimmunized to Rh antigens (1 anti-D, 1 anti-e). Unexpected Rh antibodies found in these patients were not linked to expression of partial Rh phenotypes according to serological and molecular analyses but a result of altered Rh epitopes on transfused red cells. Discussion and conclusion Our results showed that prophylactic genotypic matching is superior to phenotypic matching in patients with SCD especially if RH molecular matching is included, with the potential to decrease risk of transfusion reactions and to prevent alloimmunization.

Deferasirox is an oral iron chelator with established dose-dependent efficacy for the treatment of iron overload secondary to transfusion. However, there is few data reporting the use of Desferasirox in adult patients with sickle cell disease (SCD) and transfusional iron overload.We conducted a prospective, single center, nonrandomized study from January 2014 to March 2015 in Campinas, Brazil. Seven patients (five women, median age 50 y.o.) who were followed up on regular transfusion program were treated with a single daily dose of deferasirox (median dose 20 mg/kg). They were monitored for clinical symptoms, renal function and hepatotoxicity.One patient discontinued the study due to lack of compliance. Two patients reported mild to moderate adverse events (gastrointestinal disturbances). Five patients had the drug discontinued due to worsening of renal function. One patient had the drug discontinued due to severe hepatotoxicity that evolved to death; no patient finished the study.

Key Clinical Message This manuscript describes the case of a patient with sickle cell anemia who died of fulminant hepatitis after therapy with the iron chelator Deferasirox. The patient was homozygous for the −1774delG polymorphism in the Abcc2 gene, which raises the concern about the use of hepatotoxic drugs in this specific context.

Introduction: Heart disease represents an important cause of mortality and morbidity in sickle cell disease and beta-thalassemia, however the impairment of cardiac function in compound heterozygotes, namely sickle cell/β-thalassemia (HbS/β-thal) remains less known. Studies with patients of Greek origin demonstrated abnormal diastolic function in both ventricules, with unchanged systolic function, and biventricular dilatation along with pulmonary hypertension (Aessopos, A., et al., Ann Hematol, 88(6), 2009, Moyssakis, I., et al., Postgrad Med J, 81(961), 2005). The aim of this study was to evaluate echocardiographic findings and clinical and laboratory parameters in a cohort of Brazilian HbS/β-thal patients to better understand the cardiac involvement in this particular ethnic background. Methods: A retrospective chart review was performed on thirty-six HbS/β-thal patients followed from 1998 to 2016. Medical records were reviewed for laboratory and clinical data, and hospital admissions were recorded for sickle related complications: acute chest syndrome, retinopathy, avascular bone necrosis, stroke, priapism, leg ulcers and venous thromboembolism. Echocardiographic evaluation was performed in all patients in steady-state disease, with pulsed, continuous, two-dimensional and color Doppler. Results: Among the studied population, 21 (58%) were women and 15 (42%) were men. The median age was 38 (18-70) years; 22 (61%) patients were diagnosed as Sβ0, and 14 (39%) as Sβ+. As expected, when the Sβ-thalassemia phenotype was considered, the two groups of patients differed significantly: hemoglobin levels were lower in Sβ0 patients, who also presented a higher proportion of HbS and HbF and higher transferrin saturation indexes. Sβ0 patients also demonstrated significantly lower body mass index and higher number of platelets highlighting disease severity in this subgroup of patients. Left atrial (LA) and left ventricular (LV) dilation were found in 19.5 and 11% of patients, respectively. These prevalences are considerably lower than observed in patients with SS phenotype: 78 and 35%, respectively (Damy et al, Eur Heart J, 37(14), 2016). Systolic LV disfunction (defined as LV ejection fraction Discussion: In this cohort of Brazilian patients, we observed significant differences in hematological and clinical parameters between Sβ+ and Sβ0 patients, highlighting the difference in disease severity between the two groups. However, the profile of cardiac involvement analyzed by echocardiography was similar in both groups: higher prevalence of diastolic dysfunction with little systolic function impairment, which follows the patterns of the patients of greek origin. Nevertheless, parameters of myocardial hypertrophy were related to multiorgan impairment, and rendered a higher propensity for stroke in older patients. Thus, cardiac involvement in this disease seems to not depend on the thalassemia phenotype. This may also reflect the older age of subjects evaluated, demonstrating that the exposure to the disease features (anemia/hemolysis/inflammation) renders homogeneity in cardiac damage over time, and represents an alert for greater vigilance and control of associated factors, as hypertension and diabetes. Disclosures No relevant conflicts of interest to declare.

O envolvimento renal na doença falciforme compreende uma gama de desordens glomerulares e tubulares sendo sua incidência diretamente proporcional ao aumento da sobrevida dessa população sendo importante causador de morbidade e mortalidade desses pacientes. Os distúrbios renais nas doenças falciformes podem apresentar-se de forma assintomática, ou através de sinais como hematúria. Envolvidos na fisiopatologia desses eventos estão diversos mecanismos, como alterações na concentração e acidificação urinárias, microtromboses e infarto renal. A isquemia medular pode levar a inabilidade em manter o gradiente de íon hidrogênio, levando a uma forma incompleta de acidose tubular renal distal (tipo IV), que pode estar associada a hipercalemia independente da aldosterona. Há escassez de dados na literatura avaliando hipercalemia e acidose tubular renal especificamente em pacientes portadores de síndromes falciformes. Pouco se sabe também sobre a melhor maneira de avaliar a função renal nesse grupo de pacientes, cuja doença é caracterizada, nos estágios iniciais, por hiperfluxo glomerular, levando a falsas estimativas do real acometimento da função dos rins. Há escassez de dados na literatura avaliando hipercalemia e acidose tubular renal especificamente em pacientes portadores de síndromes falciformes. O objetivo deste estudo é fazer esse diagnóstico, e analisar a eficiência do N-GAL(Neutrophil Gelatinase Associated Lipocalin) para este diagnóstico.

BACKGROUND The reason for the difference in susceptibility to red blood cell (RBC) alloimmunization among patients with sickle cell disease (SCD) is not clearly understood and is probably the result of multiple factors. Our hypothesis is that genetic polymorphisms are associated with RBC alloimmunization. STUDY DESIGN AND METHODS We investigated the possible association of susceptibility to RBC alloimmunization with polymorphisms of HLA and cytokines genes in 161 SCD patients prior exposed to RBC transfusion. Cytokine gene polymorphisms were analyzed by polymerase chain reaction (PCR) and TaqMan assays. HLA Class I genotyping was performed using PCR-specific sequence of oligonucleotides. Polymorphism frequencies were compared using the Fisher's exact test. RESULTS Our results revealed increased percentage of the A allele and the GA genotype of the TNFA −308G/A cytokine among alloimmunized patients when compared to nonalloimmunized patients (A allele, 16.4% vs. 6.8%, p = 0.004; GA genotype, 32.8% vs. 11.7%, p = 0.0021). In addition, the IL1B −511T allele and the IL1B −511TT and CT genotype frequencies were overrepresented among alloimmunized patients (T allele, 53.0% vs. 37.5%, p = 0.0085; CT + TT genotypes, 81.82% vs. 60.87%, p = 0.0071). In relation to HLA Class I, we found a higher frequency of HLA-DRB1*15 among patients alloimmunized to Rh antigens when compared to nonalloimmunized patients (15.63% vs. 6.98%, p = 0.044). CONCLUSION Brazilian SCD patients with the TNFA, IL1B, and HLA-DRB1 gene polymorphisms were at increased risk of becoming alloimmunized by RBC transfusions. These findings may contribute to the development of future therapeutic strategies for patients with SCD with higher susceptibility of alloimmunization.

Objectives: In sickle cell/β-thalassemia, mutations in the corresponding β-globin genes are responsible for complex pathological events resulting in diverse clinical complications. The objective of this study was to provide an overview of the clinical and laboratory characteristics of patients with the syndrome, and of the degree of severity of clinical manifestations resulting from the β-thalassemia mutation.Methods: A retrospective chart review was performed on 46 patients with sickle cell/β-thalassemia (31 Sβ° and 15 Sβ+), evaluating hematological parameters and end organ damage. Statistical analyzes were carried out in order to highlight differences between the two groups according to the nature of the thalassemia mutation.Results: As expected, patients with the Sβ0 phenotype had a higher degree of hematological involvement in comparison to Sβ+ patients; with lower hemoglobin levels, and signs of more intense chronic hemolysis. However, Sβ+ patients were more prone to the occurrence of acute chest syn...

Disturbances in the physiological regulation of erythropoietin (EPO) in patients with sickle cell disease (SCD) may contribute to worsening anaemia and increased transfusion requirements, but the use of recombinant EPO in this group of patients is controversial. The objective of this study was to evaluate the use of this drug in adult patients with SCD and its effects on haemoglobin levels and transfusion requirements. We conducted a retrospective analysis at the University of Campinas, with nineteen adults with a diagnosis of SCD (HbSS and HbS/β+ thalassaemia), who had received at least 1 year of EPO therapy between 2007 and 2014. Haemoglobin concentrations and trends of variation in transfused RBC volumes were compared before and after EPO administration. We observed that seven patients had a good response to treatment (Hb increment higher than 1·5 g/dl) and nine had a partial response (0·5-1·5 g/dl increment) and there was a significant decrease in the need for transfusion amongst those who usually required regular transfusions. There were no increases in the rates of vaso-occlusive crisis or venous thromboembolism in comparison to the year before the onset of the therapy. Erythropoietin therapy led to a marked increase in haemoglobin concentration with a concomitant decrease in the demand for transfusion. Considering all complications related to allogeneic transfusion, we believe that EPO therapy represents an important therapeutic tool in sickle cell anaemia.

BACKGROUND Pregnancy represents a challenge for women with sickle cell disease (SCD), with higher rates of both maternal and fetal complications. The aim of this study was to evaluate the impact of prophylactic transfusion support administered specifically to pregnant women with sickle hemoglobin C disease. MATERIALS AND METHODS Patients were divided into two groups according to the type of transfusion support received: 10 women received prophylactic erythrocytapheresis or manual exchange transfusion at 28 weeks of gestation, and 14 received transfusions only on demand, due to acute complications, or received no transfusions at all. RESULTS Our results indicated higher frequencies of SCD-related complications in the group that did not receive prophylactic transfusion support (35.7% vs. only 10% in the erythrocytapheresis group). Furthermore, these complications were more severe in this group and included all cases of acute chest syndrome. A significant difference was observed concerning gestational age at birth (38.7 weeks in the transfusion group vs. 34.4 weeks, p = 0.037), with a higher frequency of preterm births in the nontransfused group (69.23% vs. 30% in the transfusion group). CONCLUSION We demonstrated a clear reduction of unfavorable outcomes in patients receiving prophylactic transfusions, probably reflecting better maternal and fetal conditions, which corroborated to the more satisfactory indices of vitality, observed in newborns. Considering that erythrocytapheresis or manual exchange transfusions both represent feasible and safe procedures, they could represent important tools for the optimal management of these patients.

Bone marrow is organized in specialized microenvironments known as ‘marrow niches’. These are important for the maintenance of stem cells and their hematopoietic progenitors whose homeostasis also depends on other cell types present in the tissue. Extrinsic factors, such as infection and inflammatory states, may affect this system by causing cytokine dysregulation (imbalance in cytokine production) and changes in cell proliferation and self-renewal rates, and may also induce changes in the metabolism and cell cycle. Known to relate to chronic inflammation, obesity is responsible for systemic changes that are best studied in the cardiovascular system. Little is known regarding the changes in the hematopoietic system induced by the inflammatory state carried by obesity or the cell and molecular mechanisms involved. The understanding of the biological behavior of hematopoietic stem cells under obesity-induced chronic inflammation could help elucidate the pathophysiological mechanisms involved in other inflammatory processes, such as neoplastic diseases and bone marrow failure syndromes.

Dendritic cells (DCs) recently revealed as a potent tumor vaccine component, are commonly differentiated from monocytes by cultivation with IL-4 and GM-CSF. Despite the different opinions, the use of IFNalpha can promote DCs differentiation and activation. The aim of this study was to compare the functionality and phenotypic characterization of monocyte-derived DC generated by IL-4 (IL4DC) and IFNalpha (IFNalphaDC) modified protocols. To this aim, we investigated the expression of maturation markers, co-stimulatory molecules, relevant miRNA, cytokine and migratory profiles and the functional ability of these cells to stimulate autologous T cells in vitro. We herein investigated the molecular mechanism underlying the parameters previously described, as the relative expression of NF-kB p65, c-fos and c-jun, transcription factors. Our results demonstrated that IL4DC presented a stable phenotype, an increase in migratory capacity and NF-KB activation, in addition to lower levels of miR-146 a and miR-221. We believe that the IL4DC migratory potential and increase in NFkBp65 expression may be involved in higher IL12 expression and migration, suggesting a preferential activation of TH1 immune responses by IL4DC.

In sickle cell/β-thalassemia, mutations in the corresponding β-globin genes are responsible for complex pathological events resulting in diverse clinical complications. The objective of this study was to provide an overview of the clinical and laboratory characteristics of patients with the syndrome, and of the degree of severity of clinical manifestations resulting from the β-thalassemia mutation.A retrospective chart review was performed on 46 patients with sickle cell/β-thalassemia (31 Sβ° and 15 SβAs expected, patients with the SβThis study identified significant differences among sickle cell/β-thalassemia patients according to the beta mutation involvement, pointing to an important predictor of disease severity.

Purpose Despite not yet explored, the serum lactate dehydrogenase (LDH) level in hemoglobinopathy SC (HbSC) patients could be a marker of disease severity as this association is strong in sickle cell patients. We hypothesized that the degree of hemolysis in HbSC patients is a key determinant influencing a spectrum of complications that reflect the severity of HbSC vasculopathy. The aim of this study was to analyze the associations between hemolytic parameters and chronic complications in adult SC patients. Findings We demonstrated that LDH reflects the overall rate of hemolysis and presents a correlation with the complications related to the hemolytic subphenotype: retinopathy, venous thromboembolism and leg ulcers in HbSC patients. Remarkably, this simple biomarker was associated with a clinical subphenotype of complications in patients with HbSC disease. Conclusions We propose that LDH elevation identifies HbSC patients with hemolysis, which could be a marker of endothelial dysfunction and end-organ vasculopathy. The use of this test as a marker of disease severity could complement the decisions taken during HbSC patient management.

Sickle cell disorders are inherited hemolytic anemias, associated with the presence of Hemoglobin S. This group of disorders comprises homozygotes (HbSS), compound heterozygotes for hemoglobin C (HbSC) or β-thalassemia (Sβ thalassemia) (the most frequent associations) and, uncommonly, hemoglobin D (HbSD) and hemoglobin E (HbSE). This abnormal phenotype is caused by mutations in the Beta globin genes of both chromosomes 11. Thus, these disorders are recessively inherited and abnormalities in both alleles lead to structural defects in the beta-globin chain (HbS, HbC, HbD, HbE), or a reduction in its expression (thalassemia and HbE). Consequently, normal HbA, which is an α2β2 tetramer, is absent and substituted by the mutated hemoglobins, containing an α2β2Mutated tetramer. Ultimately, the clinical phenotype is caused by the relatively high amounts of the α2β2sickle tetramer, which allows hemoglobin polymerization and, in turn, leads to vasoocclusion, the hallmark of all sickle cell disorders. In this chapter, we will discuss clinical and laboratorial aspects of the compound sickle cell disorders SC, SD and Sβ thalassemia. In this book, sickle cell anemia is a synonymous for the homozygote state and it is approached elsewhere.

Thrombocytopenia is common in patients with myelodysplastic syndromes (MDS) and immune destruction of platelets could be an important factor for its occurrence. We prospectively analyzed platelet-associated IgG (PAIgG) through platelet immunofluorescence test (PIFT), mean platelet volume (MPV), platelet size deviation width (PDW) and glycocalicin index (GCI) of 54 patients with MDS, classified according to the International Prognostic Scoring System (IPSS). Thrombocytopenia (platelet count

Sickle cell disease is a chronic inflammatory condition characterized by elevated levels of inflammatory cytokines, which may be regulated by genetic polymorphisms and could be associated with diverse disease presentations and alloimmunization. The aim of this study was to evaluate Treg and Th17 cell frequencies, cytokine gene polymorphisms, and their association with cytokine expression profile in patients with sickle cell disease. For that purpose, we evaluated the IL intron 3 variable number tandem repeat (VNTR, genotypes 1.1, 1.2, 2.2, and 2.3), IL4-T590C>T, IL6-174G>C, TNFα-308G>A, IL10-819T>C, IL10-592A>C, and IL10-1082A>G polymorphisms and their correlation with TGFβ, IL4, IL6, and IL10 gene expression in sickle cell patients. We observed a significant decrease in Treg frequency together with a substantial increase in Th17 response in patients with sickle cell disease compared with healthy controls (p

Pregnancy represents a challenge for women with sickle cell disease (SCD), with higher rates of both maternal and fetal complications. The aim of this study was to evaluate the impact of prophylactic transfusion support administered specifically to pregnant women with sickle hemoglobin C disease.Patients were divided into two groups according to the type of transfusion support received: 10 women received prophylactic erythrocytapheresis or manual exchange transfusion at 28 weeks of gestation, and 14 received transfusions only on demand, due to acute complications, or received no transfusions at all.Our results indicated higher frequencies of SCD-related complications in the group that did not receive prophylactic transfusion support (35.7% vs. only 10% in the erythrocytapheresis group). Furthermore, these complications were more severe in this group and included all cases of acute chest syndrome. A significant difference was observed concerning gestational age at birth (38.7 weeks in the transfusion group vs. 34.4 weeks, p = 0.037), with a higher frequency of preterm births in the nontransfused group (69.23% vs. 30% in the transfusion group).We demonstrated a clear reduction of unfavorable outcomes in patients receiving prophylactic transfusions, probably reflecting better maternal and fetal conditions, which corroborated to the more satisfactory indices of vitality, observed in newborns. Considering that erythrocytapheresis or manual exchange transfusions both represent feasible and safe procedures, they could represent important tools for the optimal management of these patients.

Background : Transfusion-transmitted infections (TTI) continue to be a problem in many parts of the world, and multi-transfused patients (MTP) are at a particularly increased risk of TTI. Objectives : to estimate the prevalence of TTI among multi-transfused patientsin Brazil, and to understand the epidemiological characteristics of TTI among these patients. Study design : cross-sectional study of 353 MTP, who were interviewed using a structured questionnaire and tested for serological markers of hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infection. Results : the overall prevalence of HCV, HIV, HBV and co-infection among MTP were 16.7%, 1.7%, 0.8% and 1.7% respectively. A dose-effect relationship could be detected between the number of units transfused and HCV infection. Other non-transfusion related (NTR) risk factors for HCV did not confer any excess risk of HCV infection to MTP. Conclusions : HCV infection was the most prevalent TTI among MTP, and remains a major health problem for these patients.A dose-effect relationship could be detected between HCV and the number of units transfused. The implementation of measures such as donor education programs, standards for donor selection criteria, and of improved serological screening protocols, paralleled the decline in the prevalence of TTI, specially of HCV, observed in MTP, underscoring the importance of such measures for the reduction of the residual risk of TTI.

Leg ulcers represent a particularly disabling complication in patients with sickle cell disease (SCD). Platelet gel (PG) is a novel therapeutic strategy used for accelerating wound healing of a wide range of tissues through the continuous release of platelet growth factors. Here, we describe the use of PG preparation according to Anitua’s PRGF (preparations rich in growth factors) protocol for treating chronic nonhealing ulcers in patients with SCD. A positive response occurred in 3 patients with an area reduction of 85.7% to 100%, which occurred within 7 to 10 weeks, and a 35.2% and 20.5% of area reduction in 2 other patients, who however, had large ulcers. After calcium chloride addition, the platelet-rich plasmas demonstrated enhanced platelet-derived growth factors–BB ( P < .001), transforming growth factor-β1 ( P = .015), vascular endothelial growth factors ( P = .03), and hepatocyte growth factors (nonsignificant) secretion. Furthermore, calcium chloride addition induced a significant decrease in platelet number ( P = .0134) and there was no leukocyte detection in the PG product. These results demonstrate that PG treatment might impact the healing of leg ulcers in sickle cell disease, especially in patients with small ulcers.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on "good manufacture practices" (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

A polymorphism at codon 129 of the prion protein gene has been shown to confer genetic susceptibility to prion diseases, and to influence the epidemic course of variant Creutzfeldt-Jakob disease. We employed a PCR-endonuclease digestion-based assay to investigate this genetic trait in Brazil, and then compared our results to previously published data from several European and Asian countries.

OBJECTIVE: To evaluate the usefulness of DNA methods to provide a means to precisely genotypically match donor blood units for the antigen-negative type of 35 sickle cell disease patients

Introduction: Regular blood transfusions may result in hemochromatosis, leading to damage of various organs. Deferasirox exhibits good efficacy as iron chelation therapy, however changes in liver parameters may occur, as well as, despite being rare, more serious complications such as liver failure. Multidrug resistance protein 2 (MRP2) is a member of the ATP-binding cassette (ABC) transporter family of membrane proteins encoded by the ABCC2 gene and is expressed in the canalicular part of the hepatocyte. MRP2 acts in the biliary transport of several endogenous compounds and many drugs, including Deferasirox. Thus, genetic polymorphisms related to the ABCC2 gene, which encodes the MRP2 protein, may influence individual susceptibility to hepatotoxicity related to Deferasirox. Methods: Two functional genetic variants, rs369192412 (-1774delG) and rs717620 (-24C> T), of the ABCC2 gene, were studied using the DNA sequencing technique in 31 genomic DNA samples from patients with iron overload who were currently using or had previously used Deferasirox. The sequencing results were compared to clinical data related to the presence or absence of hepatotoxicity during the use of this drug. Results: Of all patients studied, 9 (29%) of them developed hepatotoxicity based on elevation of AST and/or ALT >5 X ULN or elevation of bilirubins >3 X ULN, which occurred in an average of 239 (14-983) days after initiation of treatment. From the 9 (29%) patients with hepatotoxicity, only 2 (22.2%) of them had the polymorphism -24C> T in only one allele, and the remaining 7 (77.8%) patients did not (OR = 0.77, 95% CI = 0.06 to 5.91, p = 1). Despite no association was detected between the -17774delG polymorphism and hepatotoxicity (OR = lower, 95% CI = 0.06 to bottom, p = 0.29), this polymorphism was present in homozygosis in 1 patient with sickle cell anemia, who, coincidently, had fulminant hepatitis leading to death, with no other causes for liver toxicity and despite all introduced therapies. Conclusions: Our study showed no correlation between ABCC2 polymorphisms (-24C> T, -1774delG) and hepatotoxicity to Deferasirox, however, the rs369192412 (-1774delG) polymorphism was present in homozygosis in the only patient who died due to fulminant hepatitis secondary to the drug. This data may indicate a possible influence of the presence of this polymorphism in the severity of liver damage related to Deferasirox. Disclosures No relevant conflicts of interest to declare.

The role of the immune system in myelodysplastic syndrome (MDS) progression has been widely accepted, although mechanisms underlying this immune dysfunction are not clear. CD4+ and CD8+ lymphocyte profiles in the peripheral blood of MDS patients were evaluated and correlated with clinical characteristics, the expression of FOXP3 and the anti-inflammatory cytokines IL10, TGFβ1 and CTLA4. IL10 expression inversely correlated with the percentage of CD8+ cells and was higher in high-risk MDS. Our findings provide further evidence for the role of T cell-mediated IL10 production in MDS and strengthen the idea of distinct cytokine profiles in low and high-risk MDS.

Background. Patients presenting sickle cell disease may develop different types of hepatic complications. Intrahepatic cholestasis is a potentially fatal complication of the disease, and sometimes the only possible solution is transplantation. Postoperative transfusion management has not yet been well established. In this report, we describe the transfusional program of a patient presenting sickle cell disease and intrahepatic cholestasis who underwent liver transplantation 2 years ago. Methods. Data were obtained from the chart and the blood bank records. Results. The liver transplantation was performed successfully. Despite mild allograft dysfunction 3 months after surgery, secondary to intrahepatic sickling, the patient has been doing well with the transfusional management adopted (sickle-cell hemoglobin

GATA3 is a specific T-cell transcription factor involved in the expression of T-cell receptor (TCR). In order to characterize the relationship between HTLV-1 infection, which has been reported to be associated with down-regulation of genes belonging to the TCR/CD3 complex, and the transcription factor GATA3, we evaluated, by semi-quantitative RT-PCR, the expression of GATA3 gene in HTLV-1 carriers and individuals with related diseases. The study included 4 asymptomatic carriers, 2 patients with adult T-cell leukaemia/lymphoma (ATLL), 1 patient with HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis (TSP) and 7 healthy blood donors. A considerable decrease in the expression of the GATA3 mRNA was observed in all subjects infected by HTLV-1 and no expression of GATA3 mRNA was observed in 1 subject with ATLL and in 1 with HAM/TSP.

Background Blood transfusion has always been an important route for Chagas Disease (CD) transmission. The high prevalence of CD in Latin America and its lifelong asymptomatic clinical picture pose a threat for the safety of the blood supply. The outcome of measures designed to improve transfusion safety can be assessed by evaluating the prevalence of CD among multitransfused patients Methods In order to assess the impact of CD control measures on the safety of the blood supply, an observational cross-sectional study was designed to determine the prevalence of CD in 351 highly transfused patients, in which vectorial transmission was excluded. This study compared patients that received transfusion products before (n = 230) and after (n = 121) 1997, when measures to control transfusion-transmitted CD were fully implemented in Brazil. Results The study group consisted of 351 patients exposed to high numbers of blood products during their lifetime (median number of units transfused = 51, range 10–2086). A higher prevalence of transfusion-transmitted CD (1.30%) was observed among multitransfused patients that received their first transfusion before 1997, compared with no cases of transfusion-transmitted CD among multitransfused patients transfused after that year. The magnitude of the exposure to blood products was similar among both groups (mean number of units transfused per year of exposure = 25.00 ± 26.46 and 23.99 ± 30.58 respectively; P = 0.75, Mann-Whitney test). Conclusion Multiple initiatives aimed to control vector and parental transmission of CD can significantly decrease transfusion-transmitted CD in Brazil. Our data suggest that mandatory donor screening for CD represents the most important measure to interrupt transmission of CD by blood transfusions.

The HTLV-I nonstructural protein Tax plays a crucial role in cellular transformation. It activates the transcription factors of various cellular genes and interacts with cellular proteins. There is limited data available on the interaction between specific T cell transcription factor GATA3 and Tax. Implications for the significance of GATA3 in T-cell development and function, (Th2) differentiation, and a role of GATA3 during the immune response have been reported. We had previously demonstrated decreased GATA3 mRNA expression in HTLVI infected individuals and in patients with ATLL and HAM/TSP [Gilli et al., 2000]. To determine the effect of the Tax protein on GATA3 gene expression, we investigated the interaction between this protein and its promoter and repressor regions by DNA-protein interaction and “in vitro” transcription assays. Results demonstrated an interaction between Tax and the GATA3 promoter via the transcription factor Sp1 and a role for Tax in the negative regulation of GATA3 expression, through its interaction with the repressor ZEB. This interaction may be involved in the pathophysiology of adult T cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-I-associated myelopathy.

Hydroxyurea is one of the most successfully used therapies for sickle cell disease. Results of many clinical trials point to hydroxyurea administration for patients with frequent painful crises and acute chest syndrome. Priapism is one of the complications that could be prevented by hydroxyurea, but there are few reports demonstrating the results. Since November 1993, hydroxyurea has been used in our clinic for preventing priapism in patients with stuttering or major attacks who are still capable of achieving intercourse on demand. Five patients were enrolled in the study, and 4 cases benefited by this treatment. After the initial treatment for the acute attack, all five patients developed stuttering priapism. Hydroxyurea was then introduced at the initial dose of 10 mg/kg, and as the hydroxyurea dosage increased, the number or length of priapism episodes decreased. One to two months after the maximal dose (20-35 mg/kg) was introduced, the episodes disappeared. In two patients, we were forced to administer over 30 mg hydroxyurea/kg to abort the episodes, and, in another patient, 25 mg/kg was necessary. All patients present normal sexual activity. Hydroxyurea was discontinued in two patients, but stuttering priapism reappeared. Hydroxyurea was then re-introduced, and priapism disappeared. One patient, using 20 mg hydroxyurea/kg, had a 6-year remission of priapism after hydroxyurea administration; however, he experienced stuttering priapism, 1 month before a major attack, that progressed to impotence. During that month, he did not seek medical attention. In conclusion, the data here presented suggests that hydroxyurea may prevent priapism attacks in sickle cell disease, probably at higher doses than usually prescribed for painful crisis prevention.

Orientador: Sara T. O. Saad Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas Resumo: A infecção pelo vírus linfotrópico de células T tipo I (HTLV I) tem sido associada à leucemia/linfoma T do adulto (LLTA), à paraparesia espástica tropical! mielopatia associada ao HTLV I (PET/MAH), à uveíte e, recentemente, à Síndrome de Sjõgren e outras doenças do sistema conjuntivo. Os fatores que detenninam a evolução para essas doenças relacionadas à infecção são desconhecidos, mas podem estar ligados à predisposição genética e à resposta imune do hospedeiro. Camundongos com ausência do gene GATA3 demonstram várias e graves anormalidades morfológicas e fisiológicas no sistema nervoso central e periférico, além de comprometimento da hematopoese durante o desenvolvimento embrionário. Há, portanto, semelhanças entre os sistemas comprometidos na ausência do gene GATA3 e aqueles alterados secundariamente à infecção pelo HTLV I. Vários estudos sugerem que uma fosfoproteína viral presente no HTLVI, denominada Tax, ative a transcrição de vários genes envolvidos na produção de citocinas ou na resposta e na proliferação celular, como c-fos, c-myc, erg-I, IL-I, IL-2, GM-CSF. Entretanto, a crelação entre a infecção pelo vírus HTLV I e o fator de transcrição GATA3 ainda não havia sido determinada. Os objetivos do presente trabalho foram caracterizar a relação entre o fator de transcrição GATA3 e o vírus HTLV I, utilizando-se, para tanto, a técnica de RT-PCR semiquantitativo; analisar a relação entre o fator de transcrição GATA3 e o vírus HTLV I, por meio de estudos de interação DNA/proteína; e demonstrar, por estudos funcionais em modelos celulares in vitro, a resposta das regiões de controle transcricional do gene GATA3 à proteína Tax. Demonstramos, através do RT-PCR semi-quantitativo que ocorre uma evidente redução na expressão do gene GATA3 em portadores saudávies da infecção pelo HTLVI, e também de forma mais acentuada nos portadores de Leucemia Linfoma T do Adulto e Paraparesia Espástica Tropical/Mielopatia associada ao HTLVI. Estudos in vitro, que utilizaram construções com o gene reporter CAT direcionado pelo promotor e silenciador do gene GATA3 co-transfectados com vetores de expressão da proteína Tax e seu mutante, revelaram que Tax exerce atividade discreta no promotor de gene GATA3, mas reprime de modo marcante a atividade do promotor na presença de seu silenciador. Essa repressão provavelmente ocorre através da interação de tax com o fator de trans.crição ZEB, o silenciador do promotor do gene GATA3, uma vez que interação deste com a proteína Tax foi demonstrada no estudo de retardamento em gel. O estudo demonstrou, pela primeira vez, a regulação do gene GATA3 pelo vírus HTLVI. Essa regulação pode estar envolvida na fisiopatologia das doenças relacionadas à infecção pelo HTLVI Abstract: The HTLV-I nonstructural protein Tax plays a crucial role in cellular transformation. It activates the transcription factors of various cellular genes and interacts with cellular proteins. Limited data are available on the interaction between specific T cell transcription factor GATA3 and Tax. hnplication for the significance ofGATA3 on T-cell development and function, (Th2) differentiation, and a role ofGATA3 during immune response has been reported. To determine the effect of the Tax protein on GATA3 gene expression, we investigated the interaction between this protein and the GATA3 promoter and repressor regions. The semi quantitative RT-PCR demonstrated a considerable decrease in the expression of the GATA 3 cDNA all subjects infected by HTLV I and no expression of GATA 3 mRNA was observed in one subject with ATLL and another with HAM/TSP. Results demonstrated an interaction between Tax and GATA3 gene and a role ofTax in the negative regulation of GATA3 expression, through its interaction with the repressor, ZEB. This interaction may be involved in the pathophysiology of adult T cell leukemiallymphoma and tropical spastic paraparesis/HTLV-I-associated myelophathy Doutorado Clínica Médica Doutor em Clínica Médica

Abstract 1050 Background: Tregs and proinflammatory Th17 cells have been described as two distinct subsets from Th1 and Th2 cells and have opposite effects in autoimmunity and inflammation. Th17/Treg balance controls inflammation and is important in the pathogenesis of autoimmune, infectious and chronic diseases. In view of growing evidence linking sickle cell disease (SS) and chronic inflammation and the potential anti-inflammatory role of Tregs cells, we hypothesized that SS is associated with decreased Treg subpopulation and consequent T cell dysfunction. Furthermore, the effects of hydroxyurea (HU) in this balance are not understood. To assess whether the Th17/Treg balance is impaired in patients with sickle cell disease, we investigated the peripheral blood Th17 and Treg frequencies and the expression of TGFβ1 and RORγT, a master regulator of Th17 differentiation, in sickle cell patients under HU therapy (SS-HU) and without HU (SSw/oHU). Methods. Thirty-eight patients with steady-state sickle cell disease (mean age 43 ± 10.5 yo; range 19–57 yo; 21 female/17 male), 19 SS-HU, 19 SSw/oHU and 20 healthy blood donors were evaluated. Tregs and Th17 quantification was performed by flow cytometry analysis and the expression of TGFβ1 and RORγT by q-PCR. Results. We found significant decrease in the Treg frequency (Foxp3+CD25+CD4+ population) comparing total SS patients and healthy controls (p=0.001) and this difference was more expressive between SS-HU and healthy controls (P=0.0006). Moreover, we found statistically increase of Th17 in SS-HU (p=0.02) and in SSw/oHU (p=0.01), comparing both with healthy controls. The TGFβ1 expression was increased in SS-HU (p=0.04) and SSw/oHU patients (p=0.01) when compared to control group. Interestingly, RORγT expression was significantly elevated only in SSw/oHU vs controls (p=0.03). Conclusion: Our results demonstrated a numerical imbalance of Th17/Treg in the peripheral blood of patients with SS disease. HU has no role mediating Th17/Treg plasticity and even SS patients under HU therapy remains Treg depleted. However, HU is able to modulated RORγT, corroborating the importance of other regulating factors during Th17 differentiation. Moreover, the higher expression of TGFβ1 may be an important positive instructive signal for Th17 differentiation. Th17/Treg imbalance might impact the disease presentation and phenotype and HU therapy may be involved in this issue. Disclosures: No relevant conflicts of interest to declare.

Abstract 2943 Background. Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by ineffective and dysplastic hematopoiesis, progressive bone marrow failure, cytopenias and a high risk of transformation into acute leukemia. Thrombocytopenia is detected in up to two thirds of patients with MDS and severe thrombocytopenia is present in approximately 10%. Besides ineffective thrombopoiesis, immune destruction of platelets could be an additional factor in the genesis of thrombocytopenia, since immunological abnormalities are also frequent in patients with MDS. The detection of platelet associated IgG (PAIgG) by immunofluorescence (platelet immunofluorescence test or PIFT) is a highly sensitive assay. In addition, some morphological platelet indices (PDW and MPV) are correlated with the occurrence of immune thrombocytopenia. We prospectively analysed platelet-bound IgG and platelet indices (PDW and MPV) in 35 patients with MDS. Methods: Thirty-five patients with MDS (mean age ± SD: 63 ± 19 yo; range 21–89 yo; 15female/20male) were evaluated. According to FAB, 27 patients were classified as RA, 5 as RARS and 3 as RAEB. Clinical manifestations of immunological disorder were not present in this population. Blood samples were analyzed by PIFT, in order to detect platelet associated IgG and results were expressed as a ratio of patient fluorescence/negative control fluorescence (R). Cell-dyn Sapphire blood cell analyzer (Abbott, Illinois, USA) was used to measure platelet count, mean platelet volume (MPV) and platelet size deviation width (PDW). Thrombocytopenia was defined as a platelet count Results. Platelet counts of the entire population ranged from 6.7 to 708 ×109/L, with median of 95.4 × 109/L. Eighteen patients (51.43%) had platelet count Conclusion: Thus, we propose that a combination of a simple index as MPV and a highly sensitive and easy to perform screening test for PAIgG as PIFT could be applied to select a subset of MDS patients in which we would be able to prevent the overuse of unnecessary platelet transfusions and who could be candidates for an immunosuppressive therapeutic approach. Supported by INCTS, FAPESP, CNPq. Disclosures: No relevant conflicts of interest to declare.

Background Overall therapeutic outcomes of advanced non-small-cell lung cancer (NSCLC) are poor. The dendritic cell (DC) immunotherapy has been developed as a new strategy for the treatment of lung cancer. The purpose of this study was to evaluate the feasibility, safety and immunologic responses in use in mature, antigen-pulsed autologous DC vaccine in NSCLC patients. Methods Five HLA-A2 patients with inoperable stage III or IV NSCLC were selected to receive two doses of 5 × 107 DC cells administered subcutaneous and intravenously two times at two week intervals. The immunologic response, safety and tolerability to the vaccine were evaluated by the lymphoproliferation assay and clinical and laboratorial evolution, respectively. Results The dose of the vaccine has shown to be safe and well tolerated. The lymphoproliferation assay showed an improvement in the specific immune response after the immunization, with a significant response after the second dose (p = 0.005). This response was not long lasting and a tendency to reduction two weeks after the second dose of the vaccine was observed. Two patients had a survival almost twice greater than the expected average and were the only ones that expressed HER-2 and CEA together. Conclusion Despite the small sample size, the results on the immune response, safety and tolerability, combined with the results of other studies, are encouraging to the conduction of a large clinical trial with multiples doses in patients with early lung cancer who underwent surgical treatment. Trial Registration Current Controlled Trials: ISRCTN45563569