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- Resistance to recombinant human erythropoietin therapy in haemodialysis patientsPublication . Costa, Elísio; Lima, Margarida; Rocha, Susana; Pereira, Petronila Rocha; Reis, Flávio; Castro, Elisabeth; Teixeira, Frederico; Miranda, Vasco; Faria, Maria do Sarmento; Loureiro, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, AliceTo better clarify the mechanism of resistance to recombinan thuman erythropoietin (rhEPO) therapy in haemodialysis patients, we studied systemic changes associated with resistance to rhEPO therapy in haemodialysis patients under rhEPO therapies, with particular interest on inflammation, leukocyte activation, iron status, oxidative stress and erythrocyte damage. We studied 63 chronic kidney disease (CKD) patients under haemodialysis and rhEPO therapies (32 responders and 31 non-responders to rhEPO therapy) and 26 healthy volunteers. In 20 of the CKD patients (10 responders and 10 non-responders to rhEPO therapy), blood samples were also collected immediately after dialysis to study the effect of the haemodialysis procedure. When compared to controls, haemodialysis patients presented lymphopenia, which results, at least in part, from a decrease in total circulating CD3+ T-lymphocytes and affect both the CD4+ and the CD8+ T-cell subsets. These lymphocytes presented markers of enhanced continuous activation state and enhanced ability to produce Th1 related cytokines. Furthermore, haemodialysis patients presented raised markers of an inflammatory process, and of an enhanced neutrophil activation, as showed by the high serum levels of elastase. Concerning to iron status, patients showed increased ferritin and prohepcidin serum levels, and a decrease in transferrin. Furthermore, some changes were observed in erythrocyte membrane protein composition and in band 3 profile, being the decrease in spectrin the most significant change. Higher plasma levels of total antioxidant status (TAS), lipidic peroxidation (TBA) and TBA/TAS ratio were also found. When comparing the two groups of patients, we found that non-responders presented a significant decrease in total lymphocyte and CD4+ T-cell counts, a more accentuated inflammatory process and indicators of enhanced neutrophil activation. No significant differences were found in serum iron status markers between the two groups of patients, except for the soluble transferrin receptor, which was higher among nonresponders. Prohepcidin serum levels were significantly lower in non-responders, but were higher than those in the control group. An accentuated decrease in erythrocyte membrane spectrin, alterations in band 3 profile [decrease in band 3 proteolytic fragments (Pfrag) and in Pfrag/band 3 monomer ratio], and a trend to higher values of membrane bound haemoglobin were also found in non-responders patients. In conclusion, although the etiology of resistance to rhEPO therapy is still unknown, our work confirms that inflammationseems to have an important role in its pathophysiology. We also showed that resistance to rhEPO therapy is associated with “functional” iron deficiency, lymphopenia and CD4+ lymphopenia, higher elastase plasma levels, increased interleukin-7 serum levels, and alterations in erythrocyte membrane protein structure and in band 3 profile. Further studies are needed to understand the rise in inflammation with the associated need in higher doses of rhEPO and the reduced iron availability.
- Recombinant human erythropoietin treatment protects the cardio-renal axis in a model of moderate chronic renal failurePublication . Teixeira, Ana Margarida; Garrido, Patrícia; Santos, Paulo; Alves, Rui; Parada, Belmiro; Costa, Elísio; Almeida, Anabela; Teixeira-Lemos, Edite; Sereno, José; Pinto, Rui; Belo, Luís; Santos-Silva, Alice; Teixeira, Frederico; Reis, FlávioChronic kidney disease (CKD) patients develop anemia because of the low kidney erythropoietin (EPO) production, thus promoting cardiovascular complications. The degree of renal insufficiency might determine the moment to start recombinant human erythropoietin (rhEPO) therapy, but the molecular basis for these options deserves better elucidation. This study aimed to clarify the cardio-renal effects of earlier rhEPO ther- 20 apy in rats with moderate chronic renal failure (CRF). Four groups of rats were evaluated for 15 weeks (control; rhEPO - 50 Ill/kg/week; CRF - 3/4 nephrectomy; CRF + rhEPO) to assess renal and hematology data, EPO levels, blood pressure, heart rate, peripheral catecholamines contents, serum-transforming growth factor-31 (TGF-f11), kidney gene expression of EPO, Caspase 9 (Casp9), and vascular endothelial growth factor (Vegf). This model of moderate CRF showed moderate and corrected anemia, hypertension, tachycardia, sympa- 25 thetic overactivity, and increased serum TGF-31 content. The remnant kidney showed a proliferative profile, with hypertrophy, downregulated gene expression of EPO, and upregulated gene expression of Vegf and Casp9. rhEPO treatment promoted erythrocytosis and prevented tachycardia and catecholamines increment, with a rise of serum TGF-$1. Furthermore, the decreased kidney gene expression of EPO and the overexpression of Casp9 were prevented, demonstrating a renoprotective action on the remnant kidney. In conclusion, 30 rhEPO therapy promotes a protective effect on the cardio-renal axis, which might be mainly attributed to its pro-proliferative and anti-apoptotic properties. These findings might recommend its use in earlier stages of CRF, acting as an erythropoiesis stimulating agent, to efficiently correct not only the anemia, one of the major complications in these patients, but also the succeeding adverse cardio-renal effects.
- Plant aqueous extracts: antioxidant capacity via haemolysis and bacteriophage P22 protectionPublication . Gião, Maria S.; Leitão, Isabel; Pereira, Ana; Borges, André B.; Guedes, Catarina J.; Fernandes, João C.; Belo, Luís; Santos-Silva, Alice; Hogg, Tim A.; Pintado, Manuela E.; Malcata, F. XavierThe bacteriophage P22/Salmonella Typhimurium system, as well as human erythrocytes have been used to assay for protection, against forced oxidation caused by hydrogen peroxide, brought about by several aqueous extracts of selected adventitious plants grown in Portugal. This study proved, for the first time, that the aforementioned bacteriophage-based system is a suitable method to assess the antioxidant activity of plant extracts; among the 12 plants tested, raspberry (Rubus idaeus), sage (Salvia sp.), savory (Satureja montana) and yarrow (Achillea millefolium) were found to effectively protect against oxidative damage caused by H2O2. Haemolysis was inhibited via pre-treatment with every plant extract tested, except heath at 0.1% (w/v). The two analytical methods produced different results – and for some plants, there was a dependence (either direct or inverse) of the quantitative protection effect on extract concentration, whereas for others no significant dependence was found at all. Savory yielded the most promising results, using either method. Therefore, the P22/Salmonella system can be used as a suitable in vivo assay, and human erythrocytes as a suitable in vitro assay to confirm (or not) the antioxidant capacity of plant extracts in biological matrices.
- Cardiovascular risk factors in Portuguese obese children and adolescents: impact of small reductions in body mass index imposed by lifestyle modificationsPublication . Nascimento, Henrique; Costa, Elísio; Rocha-Pereira, Petronila; Rego, Carla; Mansilha, Helena Ferreira; Quintanilha, Alexandre; Santos-Silva, Alice; Belo, LuísObjectives: Evaluate cardiovascular risk factors in Portuguese obese children and adolescents and the long-term effects of lifestyle modifications on such risk factors. Design: Transversal cohort study and longitudinal study. Setting: University Hospital S. João and Children's Hospital Maria Pia, Porto. Patients/Participants: 148 obese children and adolescents [81 females (54.7%); mean age of 11.0 years]and 33 controls (sex and age matched) participated in a cross-sectional study. Sixty obese patients agreed to participate in an one year longitudinal study after medical and nutritionist appointments to improve lifestyle modification; a substantial body mass index (BMI) reduction was defined by a decrease in BMI z-score (BMI z-sc) of 0.3 or more over the studied period. Main Outcome measures: Lipid profile (triglycerides, cholesterol, HDLc, LDLc, lipoprotein (a), apolipoproteins A and B) and circulating levels of C-reactive protein (CRP), adiponectin, glucose, and insulin. Results: Compared with the lean children, obese patients demonstrated statistically significantly higher insulin resistance index [Homeostasis model assessment (HOMA)], and triglycerides, LDLc, apolipoprotein (apo) B, insulin and CRP concentrations, whereas their HDLc and apo A levels were significantly lower (cross-sectional study). In the longitudinal study (n=60), a substantial BMI reduction occurred in 17 (28.3%) obese patients which led to a significant reduction in triglycerides, cholesterol, LDLc, apo B, glucose and insulin levels and in HOMA. The ΔBMI values over the studied period correlated inversely and significantly with BMI (P<0.001) and HOMA (P=0.026) values observed at baseline. In multiple linear regression analysis, BMI at baseline remained associated to changes in BMI over the studied period (standardised Beta: -0.271, P=0.05). Conclusion: Our data demonstrates that small reductions in BMI-zc, imposed by lifestyle modifications in obese children and adolescents, improve the cardiovascular risk profile of such patients. Furthermore, patients with higher BMI and/or insulin resistance seem to experience a greater relative reduction in their BMI after lifestyle improvements.
- 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.
- 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
- Antioxidant activity of chitooligosaccharides upon two biological systems: erythrocytes and bacteriophagesPublication . Fernandes, João C.; Eaton, Peter; Nascimento, Henrique; Gião, Maria S.; Ramos, Oscar. L.; Belo, Luís; Santos-Silva, Alice; Pintado, Manuela E.; Malcata, F. XavierMost of the reports to date on the antioxidant capacity of chitosans and chitooligosaccharides (COS) are based on strictly chemical methods. When studying antioxidants with potential in vivo applications, the method used to evaluate the antioxidant activity should be representative of the conditions in which the antioxidant might have a protective effect. In this work we evaluate the antioxidant activity of two COS mixtures and a low MW chitosan (LMWC) upon two biological oxidizable substrates – erythrocytes and phages, subjected to accelerated oxidation conditions. Our results suggest that COS/LMWC can be used as antioxidants in biological systems. All the tested compounds reduced either the hemolytic and DNA damage, by inhibiting H2O2- and AAPH-radicals. However, the results obtained for these biological assays did not reveal a dose dependence, contrary to the chemical assay, suggesting that the protective concentrations should be established, in order to prevent enhancement of the oxidative damage – i.e. a prooxidant effect.
- Neutrophil and monocyte activation in chronic kidney disease patients under hemodialysis and its relationship with resistance to recombinant human erythropoietin and to the hemodialysis procedurePublication . Pereira, Rui; Costa, Elísio; Gonçalves, Marta; Miranda, Vasco; Faria, Maria do Sameiro; Quintanilha, Alexandre; Belo, Luís; Lima, Margarida; Santos-Silva, AliceThe aim of the present work was to further clarify leukocyte activation due to hemodialysis (HD) procedures and to investigate its relationship with recombinant human erythropoietin resistance. Therefore, we studied the expression of CXCR1 and CD11b on neutrophils, as well as the monocyte expression of CD11b, HLA-DR, and CD14. We studied 34 chronic kidney disease (CKD) patients under HD and recombinant human erythropoietin treatment (26 responders and 8 nonresponders to recombinant human erythropoietin therapy). All CKD patients' blood samples were collected before and immediately after the HD procedure. Eighteen healthy individuals (blood donors) were also studied as a control group. Hematological data, neutrophil (CD11b and CXCR1), and monocyte (CD11b, HLA-DR, and CD14) cell surface markers were measured in all patients (before and after the HD procedure) and controls. When compared with the controls, CKD patients presented a significant decrease in CXCR1 neutrophil expression, and in CD14 monocyte expression, accompanied by a significant increase in HLA-DR monocyte expression. When comparing the 2 groups of patients, we found that nonresponders showed an additional decrease in CXCR1 neutrophil expression. After the HD procedure, a statistically significant increase in CD14 and CD11b monocyte surface markers and a decrease in CXCR1 neutrophil expression and in HLA-DR monocyte expression was found. These data further strengthen our previous studies, showing that neutrophils and monocytes are activated in CKD patients, particularly in nonresponder patients. Moreover, this activation is due, at least in part, to the HD procedure, although we should not exclude that it can also be due to the enhanced inflammatory process observed in nonresponder patients.