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Abstract(s)
Com o objectivo de clarificar o mecanismo de resistĂȘncia Ă terapĂȘutica com eritropoietina humana recombinante (EPOhr) em doentes hemodializados, estudamos alteraçÔes a ela associada, com particular interesse na inflamação, activação leucocitĂĄria, ciclo do ferro, stress oxidativo e lesĂŁo eritrocitĂĄria. Foram estudados 63 doentes renais crĂłnicos (DRC) em hemodiĂĄlise e terapĂȘutica com EPOhr (32 respondedores e 31 nĂŁo respondedores Ă terapĂȘutica com EPOhr) e 26 indivĂduos controlo. Em 20 dos DRC (10 respondedores e 10 nĂŁo respondedores Ă terapĂȘutica com EPOhr), foram tambĂ©m colhidas amostras de sangue imediatamente apĂłs a hemodiĂĄlise para estudar os efeitos deste procedimento. Quando comparados com os controlos, os DRC em hemodiĂĄlise apresentaram linfocitopenia, resultante de uma diminuição da contagem dos linfĂłcitos CD3+ e em que ambos os subtipos de linfĂłcitos T CD4+ e CD8+ se encontravam diminuĂdos. Estes linfĂłcitos apresentavam marcadores celulares de estimulação continuada aumentados e capacidade aumentada de produzir citoquinas associadas com a resposta imune do tipo Th1. Adicionalmente, estes doentes apresentavam marcadores inflamatĂłrios, e aumento na activação dos neutrĂłfilos. No que se refere ao estudo do ciclo do ferro, os DRC apresentavam aumento dos nĂveis sĂ©ricos de ferritina e prohepcidina, e uma diminuição na transferrina. Adicionalmente, foram tambĂ©m encontradas alteraçÔes na composição proteica da membrana dos eritrĂłcitos e no perfil da banda 3, sendo a diminuição da espectrina a alteração mais significativa. Aumento na capacidade antioxidante total (TAS), na peroxidação lipĂdica (TBA) e da razĂŁo TBA/TAS foram tambĂ©m demonstrados. Quando comparamos os dois grupos de DRC, verificamos que os nĂŁo respondedores Ă terapĂȘutica com EPOhr apresentavam diminuição no nĂșmero total de linfĂłcitos e nos linfĂłcitos T CD4+, e aumento nos marcadores inflamatĂłrios e na activação dos neutrĂłfilos. NĂŁo encontramos diferenças significativas nos parĂąmetros relacionados com o ciclo do ferro, com excepção do receptor solĂșvel da transferrina, que se encontrava aumentado nos nĂŁo respondedores. Os nĂveis sĂ©ricos de prohepcidina encontravam-se diminuĂdos nos nĂŁo respondedores; no entanto, encontravam-se mais elevados que no grupo controlo. Diminuição acentuada no conteĂșdo em espectrina, alteraçÔes no perfil de banda 3 [diminuição fragmentos proteolĂticos da banda 3 (Pfrag) e na razĂŁo Pfrag/monĂłmero de banda 3], e uma tendĂȘncia para valores aumentados de hemoglobina ligada Ă membrana foram tambĂ©m encontrados nos DRC nĂŁo respondedores Ă terapĂȘutica com EPOhr. Em conclusĂŁo, apesar da etiologia Ă resistĂȘncia Ă terapĂȘutica com EPOhr nĂŁo estar ainda completamente esclarecidaos nossos resultados confirmam que a inflamação parece ter um papel muito importante. Encontramos tambĂ©m relação entre resistĂȘncia Ăš terapĂȘutica com EPOhr com dĂ©fice funcional em ferro, linfocitopenia e linfocitopenia T CD4+, nĂveis plasmĂĄticos aumentados de elastase, nĂveis sĂ©ricos aumentados de interleucina-7, e alteraçÔes na estrutura das proteĂnas de membrana do eritrĂłcito e no perfil de banda 3. Mais estudos serĂŁo necessĂĄrios para se entender a associação entre a inflamação, e resistĂȘncia Ă terapĂȘutica com EPOhr e diminuição na disponibilidade em ferro.
To better clarify the mechanism of resistance to recombinant human erythropoietin (rhEPO) therapy in haemodialysis patients, we studied systemic changes associated with this resistence in haemodialysis patients under rhEPO therapies, with particular interest on inflammation, leukocyte activation, ironstatus, 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 non-responders. 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 inflammation seems to have an important role in its pathophysiology. We also showed that resistance to rhEPO therapy is associated with âfunctionalâ 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 tounderstand the rise in inflammation with the associated need inhigher doses of rhEPO and the reduced iron availability.
To better clarify the mechanism of resistance to recombinant human erythropoietin (rhEPO) therapy in haemodialysis patients, we studied systemic changes associated with this resistence in haemodialysis patients under rhEPO therapies, with particular interest on inflammation, leukocyte activation, ironstatus, 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 non-responders. 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 inflammation seems to have an important role in its pathophysiology. We also showed that resistance to rhEPO therapy is associated with âfunctionalâ 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 tounderstand the rise in inflammation with the associated need inhigher doses of rhEPO and the reduced iron availability.
Description
Keywords
Eritropoietina humana recombinante Hemodiålise Inflamação Ciclo do ferro Activação leucocitåria Resistance to recombinant human erythropoietin Haemodialysis Erythropoietin Inflammation Iron status Leukocyte activation