Sickle cell anemia (SCA) is a common, severe monogenetic disorder characterized by chronic hemolysis, frequent infections, a chronic inflammatory state and recurrent occlusions of the microcirculation, resulting in painful crises, organ damage and premature death. This study evaluated associations between serum levels of IL-18, uric acid, hemolytic markers, and inflammatory molecules in SCA patients. A cross-sectional study was performed including 45 SCA patients (median age of 20.5 years) without general symptoms and who had not undergone blood transfusions. Inclusion criteria for the steady-state SCA patients were the absence of hospitalization and the absence of infections. Interleukin-18 and uric acid levels were correlated closely with markers of hemolysis, endothelial dysfunction and others cytokines levels. These findings suggest probable influences of IL-18 and uric acid in the pathophysiology of vascular occlusion in SCA. Additional studies should be performed to characterize similar prognosis markers and possible therapeutic targets.

PURPOSE: To investigate the association between polymorphisms in genes that encode enzymes involved in folate-and vitamin B12-dependent homocysteine metabolism and recurrent spontaneous abortion (RSA). METHODS: We investigated the C677T and A1298C polymorphisms of the methylenetetrahydrofalate reductase gene (MTHFR), the A2756G polymorphism of the methionine synthase gene (MS) and the 844ins68 insertion of the cystathionine beta synthetase gene (CBS). The PCR technique followed by RFLP was used to assess the polymorphisms; the serum levels of homocysteine, vitamin B 12 and folate were investigated by chemiluminescence. The EPI Info Software version 6.04 was used for statistical analysis. Parametric variables were compared by Student's t-test and nonparametric variables by the Wilcoxon rank sum test. RESULTS: The frequencies of gene polymorphisms in 89 women with a history of idiopathic recurrent miscarriage and 150 controls were 19.1 and 19.6% for the C677T, insertion, 20.8 and 26% for the A1298C insertion, 14.2 and 21.9% for the A2756G insertion, and 16.4 and 18% for the 844ins68 insertion, respectively. There were no significant differences between case and control groups in any of the gene polymorphisms investigated. However, the frequency of the 844ins68 insertion in the CBS gene was higher among women with a history of loss during the third trimester of pregnancy (p=0.003). Serum homocysteine, vitamin B 12 and folate levels id not differ between the polymorphisms studied in the case and control groups. However, linear regression analysis showed a dependence of serum folate levels on the maintenance of tHcy levels. CONCLUSION: The investigated gene polymorphisms and serum homocysteine, vitamin B 12 and folate levels were not associated with idiopathic recurrent miscarriage in the present study. Further investigations are needed in order to confirm the role of the CBS 844ins68 insertion in recurrent miscarriage. ResumoOBJETIVO: Investigar a associação entre polimorfismos nos genes que codificam enzimas envolvidas no metabolismo da homocisteína dependente de folato e vitamina B12 e aborto espontâneo recorrente. MÉTODOS: Investigamos os polimorfismos C677T e A1298C no gene methilenotetrahidrofalato redutase (MTHFR); o polimorfismo A2756G no gene metionina sintase (MS) e a inserção 844ins68 no gene da cistationina beta-sintetase (CBS). A técnica de PCR seguido por RFLP foi utilizada para investigar os polimorfismos. Os níveis séricos de homocisteína, vitamina B12 e de folato foram investigados pela técnica de quimioluminescência. O Software Epi Info versão 6.04 foi utilizado para realizar a análise estatística. As variáveis paramétricas foram comparadas pelo teste t de Student e as variáveis não paramétricas pelo teste de Wilcoxon rank sum. RESULTADOS: As frequências dos polimorfismos gênicos em 89 mulheres com história de aborto recorrente idiopático e 150 controles foram de 19,1 e 19,6% para o C677T; 20,8 e 26% para o A1298C; 14,2 e 21,9% para o A2756G e 16,4 e 18% para a inserção 844ins68, respectiva...

Introduction Sickle cell disease (SCD) has a heterogeneous clinical picture, characterized by hemolysis, chronic inflammation and vaso-occlusive events and painful crisis. Aims Investigate the levels of Alpha 1 antitrypsin (A1AT), C reactive protein (CRP), hemolysis markers and its association with cytokines profile in SCD patients in steady-state and crisis-state. Methods It was developed a cross sectional study in a total of 72 SCD patients in steady-state (SP) and 23 SCD patients in crisis (CP) age-and-sex-matched. Hematological analysis was performed by automatic cell counter, hemoglobin profile by HPLC, and biochemistry analyses of inflammation and infection markers and lipid, hepatic, and kidney metabolism markers were investigated by immunochemistry assays. Plasma levels of TNFα, IFNγ, IL-10, IL-1β, IL-6 and IL-8 were measured using Cytometric Bead Array - CBA (BD Biosciences Pharmingen, USA) according to the manufacturer’s protocol. Results Statistical analysis showed that SCD steady-state patients group had the highest IL-12 concentration (p<0.0001) and there were a decrease in TNFα, IL-10, IL-1β, and IL-6 levels (p< 0.0001) when compared to CP group (figure 1). However, significant differences in IL-8 levels were not finding in the comparison between SCD in steady-state and crisis patients groups. Biomarkers analyses of SCD steady-state patients showed significant negative correlation between IL-8 with A1AT (r= -0.25; p= 0.03) and CRP (r= -0.24; p= 0.04) and significant positive correlation with lactate dehydrogenase (LDH) (r= 0.25; p= 0.03). The IL-6 showed significant positive correlation with white blood cell count (WBC) (r= 0.52; p< 0.0001), ferritin (r= 0.30; p= 0.001) and CRP (r= 0.42; p= 0.0002). TNFα showed a significant positive correlation with leukocyte count (r= 0.28; p= 0.02) and A1AT (r= 0.29; p= 0.01) and IL-10 showed a significant positive correlation with platelets count (r= 0.30; p= 0.01) and A1AT (r= 0.29; p= 0.01). Conclusions Immunological aspects of SCD patients have been increasingly studied. The high levels of Th2 cytokines (IL-6 and IL-10) and proinflammatory cytokines (TNFα and IL-1β) in SCD patients in crisis-state, possibly is related to severe clinical manifestations, despite the fundamental role of these cytokines in the pathophysiology is not yet completely clear. The correlation between these cytokines and biomarkers associated with important clinical manifestations in SCD, such as WBC, platelet count, A1AT and CRP, highlights the important role of inflammatory mediators in SCD and its possible association with an expression or activation of adhesion molecules in neutrophils and vascular endothelium. Disclosures: No relevant conflicts of interest to declare.

Sickle cell anemia is a severe monogenic disorder characterized by the homozygous state of a single beta globin gene mutation, with heterogeneous clinic characteristics, associated with pro-inflammatory profile, oxidant state and hypercoagulable state. Vessels occlusion is likely initiated by intimal proliferation and amplified by inflammation, excessive adhesion of cells to activated endothelium and vascular tone dysregulation, normally modulated by NFkB pathway both endothelium cells as leukocytes. Herein, we investigated the gene expression of tissue factor (Factor III), oxide nitric synthase (NOS) and endothelial protein C receptor (EPCR) associating with biomarkers of prognosis like hemolysis markers, pro-inflammatory and anti-inflammatory profile. Forty two steady-state sickle cell disease (SCD) patients (16.5 ± 13.5 years, 20 female) from Northeast Brazil were enrolled in this study and were diagnosed in attendance of the outpatients’ clinic of the Sickle Cell Disease Center of Itabuna (CERDOFI). The control group was compound by 20 healthy Brazilian individuals with hemoglobin AA pattern matched by age, years and ethnic origin. The study was approved by the UESC ethical committee and informed consents were signed by patients or official responsible. Using real time quantitative PCR, we analyzed tissue factor, NOS and EPCR gene expression. We also measured hematological and hemoglobin parameters by electronic cell counter and HPLC respectively, biochemical profile was evaluated by colorimetric methodology and cytokine by flow cytometry. The statistical analysis was performed using the Kolmogorov–Smirnov test to access distribution of quantitative variables. Mean values of quantitative variables between groups were compared using an unpaired t-test for data distributed normally and a Mann–Whitney test for non-normal data. Oxide nitric synthase gene expression was increased in SCD patients 1.58-fold compared with healthy controls and higher tissue factor and EPCR gene expression were detected in patients than healthy controls, 4.82-fold and 5.46-fold respectively. The SCD cohort comprised pediatric and adult patients, and the medical history data was search from patient’s records where 95% of patients presented painful crisis at least once. Tissue factor gene expression was positive correlated with expression of NOS (p=0.005) and EPCR (p=0.0001). The increase tissue factor gene expression was detected in patients with high serum levels of bilirubin (p=0.026). Tissue factor gene expression above 75th percentile was associated high concentration of serum creatine kinase and serum calcium (p<0.005) in SCD patients. Our study reveals that genes associate to hemostasis and vascular integrity are upregulated in SCD patients, probably associated to chronic oxidative stress and pro-inflammatory state. Enhanced tissue factor, NOS and EPCR gene expression may influences in the pathophysiology of SCD. Studies tissue factor, NOS and EPCR should be carried out in order to explore mechanism, clarify participation and contributing to search of therapeutic strategies on prevention of vascular events among SCD patients. Disclosures: No relevant conflicts of interest to declare.

Vascular occlusions (VOC) underlie most of the acute and chronic sickle cell anemia (SCA) clinical complications and have been correlated to soluble adhesion molecules up regulation and leukocyte activation. A sequential process involving adhesion through selectins and integrins governs leukocyte recruitment to activated endothelium and to sickle red blood cells (RBC), resulting in heterotypic aggregation and VOC. The chronic hemolysis is the major cause of oxidative stress and it can induce transcriptions factors involved in the recruitment of adherent leukocytes in venules. In this work, we assessed the inflammatory potential of leukocytes in venous blood samples by examining cell surface antigens expression by flow cytometry, activation state and its association with hemolytic state in SCA patients group. The study was approved by the FIOCRUZ ethical committee and informed consents were signed by patients or official responsible. Leukocytes were obtained from 22 SCA patients and 22 healthy controls after RBC lyses and labeled with monoclonal anti-CD11b, anti-CD18, anti-CD32, anti-CD62L (L-Selectin) and anti-CD62P (P-Selectin). Leukocyte activation was stimulated by LPS. Statistical analyses were performed using SPSS version 9.0. The basal expression levels on leukocytes cell surface antigens from patients were not different from the control group. However, the CD62L expression levels were associated to C-reactive protein (CRP) higher levels and decrease of fetal hemoglobin in SCA patients (p=0.012). The SCA patients had higher CRP levels when compared to reference levels. Moreover, the data showed a co-expression of CD11b with CD18 (p&lt;0.0001) and CD62P (p=0.011).The platelet count was positively correlated to CD11b expression (p=0.016) and the alanine transaminase high levels with the CD62P expression (p=0.012). Our results demonstrate a leukocyte chronic activation state by expression of CD62L related to CRP higher levels. Interestingly, the platelets could be activated by the indirect activation of CD62P by CD11b, participating of the inflammation state presents at vaso-occlusive events. It seems that the stress oxidative generated by the hemolytic state can contribute to endothelial dysfunction and vaso-occlusive events by CD62L activation in SCA. Fetal hemoglobin is a prognostic factor for several sickle cell complications and we showed that it can ameliorate the CD62L expression on leukocytes, decreasing the chronic inflammatory state of this disease. CD62L serves to as a homing receptor for naïve T lymphocytes and dendritic cells to lymph nodes, mediating the biding of T cells to high endothelial venules, in this view, this can be important marker to inflammatory state and vascular complications in SCA.

1546 Poster Board I-569 Introduction There is emerging consensus that a pro-inflammatory and anti-inflammatory condition contributes to modulate the vaso-occlusive pain crisis in sickle cell anemia (SCA), that have been correlated to red blood cell (RBC), leukocytes, platelets and endothelial cell activation and with the participation of adhesion molecules. The result is a severe clinical outcome related to a chronic pro-inflammatory state with cytokine disruption equilibrium. In this study, we assessed the inflammatory potential of monocytes in venous blood samples by examining cell surface expression of ICAM-1 (CD54) and tissue factor (CD142), measurement of serum levels of cytokine, soluble adhesion molecule (ICAM-1s and VCAM-1s), biochemical markers, searching the potential value of inflammatory mediators as early markers of severity of vaso-occlusive crisis in SCA. Patients and Methods The study was approved by the FIOCRUZ human ethical research board and informed consents were signed by patients or official responsible. A prospective study was developed among 61 steady-state SCA patients aging 7.8±4.3 years, with 42.7% female. Monocytes were obtained after RBC lyses and labeled with monoclonal anti-CD54, anti-CD142 and analyzed by flow cytometry. Biochemical analyses for serum uric acid were measured by colorimetric method, hematologic analysis were performed using an electronic cell counter and VCAM-1 and ICAM-1 soluble molecules, IL-4, IL-8, TNF-alpha, IL-13, IL-17, IL-18, IL-23 and TGF-B were measured by ELISA. Statistical analyses were performed using STATA version 10.0. Results Our results show a positive significant association between CD142 and CD54 expression on monocytes surface (r=0.296, p=0.014). The VCAM-1s and IL-18 were significantly positive associated to ICAM-1s (r=0.813, p=0.0001; r=0.315, p=0.048 respectively). The stratification of TGF-B in low and high concentration groups was statistically associated with uric acid, IL-17 and IL-23 serum levels (p=0.038; p=0.022; p=0.008 respectively). The IL-17 and IL-23 were significantly positive associated (r=0.552, p=0.0001). The serum levels of uric acid were significantly negative associated with RBC number and hematocrit (r=-0.366, p=0.0006; r=-0.283, p=0.035 respectively) and significantly positive associated with mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) (r=0.383, p=0.004; r=0.403, p=0.002 respectively) Conclusion Inflammation, cell adhesion to vascular endothelium, and endothelial injury contribute to sickle cell anemia (SCA) vaso-occlusive crisis. Although alterations in inflammatory cytokines and biomarkers have been related, reports have been conflicting, and a conclusive role for these molecules in the disease remains to be established. Our results showed an association of tissue factor and ICAM-1 on monocytes expression with the participation of IL-18, VCAM-1s and ICAM-1s and TGF-B that had an interaction with uric acid, IL-17 and IL-23 levels, contributing to the establishment of inflammasome multiprotein complex and also with an probably alternative mechanism that include the Th17 cells response. Furthermore, additional studies will elucidate the mechanism involved with the interaction of this complex network of molecule and the endothelial activation and dysfunction described in the pathophysiology of vaso-occlusives episodes of sickle cell anemia. Disclosures No relevant conflicts of interest to declare.

Introduction The vascular alterations play an important role in the SCA chronic inflammatory state with the participation of different cell types, including macrophages and dendritic cells (DCs). Aim Here, we evaluated the mRNA expression of TLRs in DCs cells pre and post LPS stimulus and also measured IL-1β, TNF-α and IL-10 cytokines in plasma and supernatant of DCs pre and post LPS challenge, in order to indentify a possible role of this cell and receptor in the inflammatory state described in sickle cell anemia. Methods In vitro generation of immature DCs (imDC) monocyte-derived was obtained of monocytes from peripheral blood mononuclear cells (PBMC) of seven hospitalized HbSS patients. Relative mRNA abundance of each transcript was investigated by quantitative real time PCR and cytokines levels were measured by ELISA. Results We found that different HbSS patients showed specific DCs TLR expression and a differential levels of IL-1β among HbSS patients with and without pneumonia history (p=0.0253) and with hemoglobin concentration (p=0.0288) (Fig. 1). There was difference of TLR4 mRNA expression between HbSS patients groups without and with leg ulcers history (0.320+0.302 and 1.60+0.014) respectively (p=0.0016); with platelets count between HbSS patients groups with TLR2 expression < 50th (569.7+3.512) and > 50th (304.5+111.7) (p=0.0102) and reticulocytes count between HbSS patients groups with TLR2 expression < 50th (15.97+2.450) and > 50th (2.025+2.417) (p=0.0007) and of TNF-alpha concentration in DCs supernatant culture among HbSS patients with TLR5 expression < 50th (30.57+11.92) and > 50th (7.053+6.918) (p=0.0496) and of IL-10 concentration in DCs supernatant culture among HbSS patients with TLR5 expression < 50th (3.525+0.921) and > 50th (5.790+1.226) (p=0.0374) (Fig.2). It was also found statistic difference of IL-1 beta concentration in DCs supernatant culture post LPS stimulus between HbSS patients without and with pneumonia history (P=0.0253); analysis of TLR4 mRNA expression between HbSS patients without and with leg ulcer history (P=0.0016); analysis of hemoglobin concentration between HbSS patients with different plasma IL-1 beta concentration (P=0.0288) Conclusion Ours results show that these molecules and cell type possibly have an important role in maintain the inflammatory state described in this disease. Disclosures: No relevant conflicts of interest to declare.

Introduction Sickle cell anemia (SCA) is a genetical hemolytic disorder defined as chronic inflammatory disease. Nitrite (NO-2) in SCA (HbSS) patients may be associated with the hemolytic process while fetal hemoglobin (Hb F) presents protective effect in these patients. Serum NO-2 interferes with the role of Hb F in reducing the hemolytic process contributing to vaso-occlusive crises. Aims The aim of this study was to evaluate the correlation of the serum levels of nitrite with fetal hemoglobin, low density lipoprotein cholesterol (LDL-C) and triglycerides levels in HbSS patients in steady-state of the Centro de Referência em Doença Falciforme de Itabuna (CERDOFI), Bahia, Brazil. Methods Forty-two patients diagnosed with SCA were included at baseline. Diagnosis was confirmed by high-performance liquid chromatography (HPLC). Serum levels of nitrite were performed using Griess reaction, and fetal hemoglobin, LDL cholesterol and triglycerides levels were measured by biochemical methods. The experimental protocol was approved by the Researcher Board Committee on Human Research, Gonçalo Moniz Research Center and informed consents were signed by patients. Results Our results showed a significant increase in NO-2 (p<0.0001) in adult patients with SCA compared to the control group (healthy volunteers). Data demonstrated a negative correlation between nitrite and fetal hemoglobin (p=0.004, r=-0.627). No correlation was found between fetal hemoglobin or nitrite with LDL-Cl and triglycerides. Conclusion The results reinforce that the serum nitrite can predict the clinical disease course. Disclosures: No relevant conflicts of interest to declare.