Browsing by Author "Rocha, Susana"
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- Band 3 profile as a marker of erythrocyte changes in chronic kidney disease patientsPublication . Costa, Elísio; Rocha, Susana; Rocha-Pereira, Petronila; Castro, Elisabeth; Miranda, Vasco; Faria, Maria do Sameiro; Loureio, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, AliceOur aim was to study changes in red blood cell (RBC) membrane band 3 profile, as a cumulative marker of RBC changes, in chronic kidney disease (CKD) patients under haemodialysis and recombinant human erythropoietin (rhEPO) therapy and its linkage with resistance to this therapy. We studied 63 CKD patients, 32 responders and 31 non-responders to rhEPO therapy, and 26 healthy individuals. We evaluated the band 3 profile [% of band 3 monomer, high molecular weight aggregates (HMWAg), and proteolytic fragments (Pfrag)], membrane-bound haemoglobin (MBH), haematological data, total serum bilirubin, glutathione peroxidase (GPx) and superoxide dismutase activities, total antioxidant status (TAS) and plasma lipid peroxidation (TBA). Compared to controls, band 3 profile presented by CKD patients showed statistically significant lower HMWAg and Pfrag values and a significant higher value in band 3 monomer. GPx, TBA and TAS activities, and TBA/TAS ratio were also significantly higher in CKD patients. Comparing responders to non-responders CKD patients, significantly lower value in Pfrag and a trend for a higher value in MBH were found in non-responders. Our data suggest that CKD patients present younger RBC population, which could be related to the rhEPO therapy. The adverse plasma environment associated to CKD patients under hemodialysis imposes changes in band 3 profile, particularly in non-responders, suggesting that resistance to rhEPO therapy in CKD patients seems to be associated to an increase in RBC damage.
- Changes in red blood cells membrane protein composition during hemodialysis procedurePublication . Costa, Elísio; Rocha, Susana; Rocha-Pereira, Petronila; Castro, Elisabeth; Miranda, Vasco; Faria, Maria do Sameiro; Loureio, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, AliceOur aim was to evaluate the influence of the hemodialysis (HD) procedure in red blood cells (RBC) membrane protein composition. We evaluated hematological data (RBC count, hemoglobin concentration, and hematimetric indices) and RBC membrane protein composition (linear and exponential gradient polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate [SDS-PAGE] followed by densitometry analysis of RBC membrane proteins) before and immediately after the HD procedure in 20 patients (10 responders and 10 non-responders to recombinant human erythropoietin therapy [rhEPO]) and 26 healthy controls. Before HD, patients presented anaemia and significant changes in membrane protein composition, namely, a statistically significant reduction in spectrin associated with a significant increase in bands 6, as well as an altered membrane protein interaction (protein 4.1/spectrin, protein 4.1/band 3, protein 4.2/band 3 and spectrin/band 3). After HD, we found that patients showed a statistically significant increase in RBC count and hemoglobin, a further and statistically significant decrease in spectrin, an increase in band 3, and an altered spectrin/band 3 ratio. When comparing responders and non-responders patients after HD, we found that the non-responders presented a trend to a higher reduction in spectrin. Our data suggest that HD procedure seems to contribute to a reduction in spectrin, which is normally associated with a reduction in RBC deformability, being that reduction in spectrin is higher in non-responder patients.
- Cross-talk between inflammation, coagulation/fibrinolysis and vascular access in hemodialysis patientsPublication . Costa, Elísio; Rocha, Susana; Rocha-Pereira, Petronila; Castro, E.; Reis, Flávio; Teixeira, Frederico; Miranda, Vasco; Faria, Maria do Sameiro; Loureiro, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, AliceThis work aimed to study the association between fibrinolytic/endothelial cell function and inflammatory markers in chronic kidney disease (CKD) patients undergoing hemodialysis (HD) and recombinant human erythropoietin (rhEPO) therapies, and its relationship with the type of vascular access (VA) used for the HD procedure. As fibrinolytic/endothelial cell function markers we evaluated plasminogen activator inhibitor type-1 (PAI-1), tissue plasminogen activator (tPA) and D-dimers, and as inflammatory markers; C-reactive protein (CRP), soluble interleukin (IL)-2 receptor (s-IL2R), IL-6 and serum albumin levels. The study was performed in 50 CKD patients undergoing regular HD, 11 with a central venous dialysis catheter (CVC) and 39 with an arteriovenous fistula (AVF), and in 25 healthy controls. Compared to controls, CKD patients presented with significantly higher levels of CRP, s-IL2R, IL-6 and D-dimers, and significantly lower levels of PAI-1. The tPA/PAI-1 ratio was significantly higher in CKD patients. We also found statistical significant correlations in CKD patients between D-dimers levels and inflammatory markers: CRP, albumin, s-IL2R and IL-6. When comparing the two groups of CKD patients, we found that those with a CVC presented statistically significant lower levels of hemoglobin concentration and albumin, and higher levels of CRP, IL-6, D-dimers and tPA. Our results showed an association between fibrinolytic/endothelial cell function and increased inflammatory markers in CKD patients. The increased levels of Ddimer, tPA and inflammatory markers in CKD patients using a CVC, led us to propose a relationship between the type of VA chosen for HD, and the risk of thrombogenesis.
- DMT1 (NRAMP2/DCT1) genetic variability and resistance to recombinant human erythropoietin therapy in chronic kidney disease patients under haemodialysisPublication . Costa, Elísio; Rocha, Susana; Rocha-Pereira, Petronila; Reis, Flávio; Castro, Elisabeth; Teixeira, Frederico; Miranda, Vasco; Faria, Maria do Sameiro; Loureio, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, Alice
- Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structurePublication . Fernandes, João C.; Eaton, Peter; Nascimento, Henrique; Belo, Luís; Rocha, Susana; Vitorino, Rui; Amado, Francisco; Gomes, Joana; Santos-Silva, Alice; Pintado, Manuela E.; Malcata, F. XavierRecent studies of chitosan have increased the interest in its conversion to chitooligosaccharides (COSs) because these compounds are water-soluble and have potential use in several biomedical applications. Furthermore, such oligomers may be more advantageous than chitosans because of their much higher absorption profiles at the intestinal level, which permit their facilitated access to systemic circulation and potential distribution throughout the entire human body. In that perspective, it is important to clarify their effect on blood further, namely, on human red blood cells (RBCs). The aim of this work was thus to study the effect of two COS mixtures with different molecular weight (MW) ranges, <3 and <5 kDa, at various concentrations (5.0-0.005 mg/mL) on human RBCs. The interactions of these two mixtures with RBC membrane proteins and with hemoglobin were assessed, and the RBC morphology and surface structure were analyzed by optical microscopy (OM) and atomic force microscopy (AFM). In the presence of either COS mixture, no significant hemolysis was observed; however, at COS concentrations >0.1 mg/mL, changes in membrane binding hemoglobin were observed. Membrane protein changes were also observed with increasing COS concentration, including a reduction in both α- and β-spectrin and in band 3 protein, and the development of three new protein bands: peroxiredoxin 2, calmodulin, and hemoglobin chains. Morphologic evaluation by OM showed that at high concentrations COSs interact with RBCs, leading to RBC adhesion, aggregation, or both. An increase in the roughness of the RBC surface with increasing COS concentration was observed by AFM. Overall, these findings suggest that COS damage to RBCs was dependent on the COS MW and concentration, and significant damage resulted from either a higher MW or a greater concentration (>0.1 mg/mL).
- Erythrocyte membrane protein destabilization versus clinical outcome in 160 portuguese hereditary spherocytosis patientsPublication . Rocha, Susana; Costa, Elísio; Rocha-Pereira, Petronila; Ferreira, Fátima; Cleto, Esmeralda; Barbot, José; Quintanilha, Alexandre; Belo, Luis; Santos-Silva, AliceHereditary Spherocytosis (HS) is a haemolytic anaemia caused by erythrocyte protein membrane defects - spectrin, ankyrin, band 3 or protein 4.2 - that lead to membrane destabilization. This study aimed to evaluate the prevalence of protein deficiencies and the role of membrane proteins or membrane-linked proteins in membrane disturbance and in HS clinical outcome. A total of 215 Portuguese individuals were studied - 203 from 71 families plus 12 individual unrelated subjects; 160 of them were diagnosed with HS. They were classified as presenting mild, moderate or severe forms of HS according to the degree of haemolytic anaemia. Standardized electrophoretic erythrocyte membrane protein analysis was used to identify and quantify protein deficiencies. Band 3 and ankyrin were found to account for the majority of the erythrocyte protein defects underlying HS. Increasing isolated protein deficiency or increasing imbalance between combined protein deficiencies seemed to underlie HS severity, by increasing membrane destabilization. There was an increased membrane linkage of the cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and peroxiredoxin 2, and of denatured haemoglobin, suggesting that this linkage could interfere with membrane structure. Our data suggest that the quantification and the analysis of RBC membrane proteins may be helpful in predicting the clinical outcome of HS.
- Hepcidin serum levels and resistance to recombinant human erythropoietin therapy in haemodialysis patientsPublication . Costa, Elísio; Swinkels, Dorine W.; Laarakkers, Coby M.; Rocha-Pereira, Petronila; Rocha, Susana; Reis, Flávio; Teixeira, Frederico; Miranda, Vasco; Faria, Maria do Sameiro; Loureiro, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, Alice
- Hereditary spherocytosis and the (TA)nTAA polymorphism of UGT1A1 gene promoter region - a comparison of the bilirubin plasmatic levels in the different clinical formsPublication . Rocha, Susana; Costa, Elísio; Belo, Luís; Santos-Silva, Alice
- Linkage of cytosolic peroxiredoxin 2 to erythrocyte membrane imposed by hydrogen peroxide-induced oxidative stressPublication . Rocha, Susana; Costa, Elísio; Coimbra, Susana; Nascimento, Henrique; Catarino, Cristina; Rocha-Pereira, Petronila; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, AliceHuman erythrocyte peroxiredoxin 2 (Prx2) is a typical 2-cys cytosolic peroxiredoxin with thiol-dependent hydrogen peroxide scavenger activity. In a previous work, we reported Prx2 erythrocyte membrane linkage in some Hereditary Spherocytosis patients and that it seemed to be related to oxidative stress. The aim of the present work was to determine if Prx2 linkage to erythrocyte membrane could be induced by oxidative stress mediated by H2O2 and to further understand how and why this process occurs. We performed in vitro assays in which catalase or both Hb autoxidation and catalase were inhibited, under H2O2-induced oxidative stress conditions. Erythrocyte membrane linked Prx2 was detected by immunoblotting and quantified by densitometry. As oxidative stress markers, we determined membrane bound hemoglobin and lipid peroxidation, and we found that their values increased with H2O2 concentration. Prx2 linkage to the membrane also rose with increasing H2O2 concentration, and was only observed when the oxidized form of the enzyme was present in the cytosol. Oxidized Hb and Prx2 membrane linkages appear to be independent processes, although, both result from oxidative stress and may be useful as oxidative stress and/or erythrocyte damage/senescence markers.
- Neutrophil activation and resistance to recombinant human erythropoietin therapy in Hemodialysis PatientsPublication . Costa, Elísio; Rocha, Susana; Rocha-Pereira, Petronila; Nascimento, Henrique; Castro, Elisabeth; Miranda, Vasco; Faria, Maria do Sameiro; Loureiro, Alfredo; Belo, Luís; Santos-Silva, AliceThe aim of this work was to evaluate the neutrophil activation state in chronic kidney disease (CKD) patients under hemodialysis, and its linkage with resistance to recombinant human erythropoietin (rhEPO) therapy. Methods: We studied 63 CKD patients under hemodialysis and rhEPO treatment (32 responders and 31 non-responders to rhEPO therapy). In 20 of the CKD patients (10 responders and 10 non-responders to rhEPO therapy), blood samples were also collected immediately after dialysis. Twenty-six healthy volunteers were included in a control group. Hemoglobin levels, total and differential leukocyte counts, and circulating levels of C-reactive protein (CRP), elastase and lactoferrin were measured in all patients and controls. Results: Compared with controls, CKD patients presented with significantly higher CRP, neutrophil and elastase levels. When we compared the 2 groups of patients, we found that non-responders presented statistically significantly higher elastase plasma levels. A positive significant correlation was found between elastase levels and weekly rhEPO dose and CRP serum levels. After the hemodialysis procedure, a statistically significant rise in elastase, lactoferrin and, elastase/neutrophil and lactoferrin/neutrophil ratios were found. Conclusions: Our data show that CKD patients under hemodialysis present higher elastase levels (particularly in non-responding patients), which could be related to the rise in neutrophils, and to be part of the enhanced inflammatory process found in these patients