Pure red cell aplasia and anti-erythropoietin antibodies in patients on hemodialysis: a report of two cases and a literature review / Aplasia pura de células vermelhas e anticorpo antieritropoietina em pacientes de hemodiálise: relato de dois casos e revisão da literatura

ABSTRACT Introduction: Anemia is a frequent multifactorial complication of CKD seen in patients on dialysis derived mainly from impaired erythropoietin (EPO) production. A less common cause of anemia in individuals with CKD is pure red cell aplasia (PRCA) secondary to the production of anti-EPO antibodies. Objective: This paper aimed two describe two cases of PRCA secondary to the production of anti-EPO antibodies including choice of treatment, patient progression, and a literature review. Materials: This study included the cases of two patients with CKD on hemodialysis with severe anemia in need of specific investigation and management. Results: Patient 1 with CKD secondary to hypertension treated with EPO for 7 months showed persistent decreases in hemoglobin (Hb) levels despite the subcutaneous administration of increasing doses of EPO; the patient required recurring blood transfusions. Workup and imaging tests were negative for the main causes of anemia in individuals with CKD on dialysis. Patient 2 with CKD secondary to adult polycystic kidney disease had been taking EPO for 2 years. The patient developed severe abrupt anemia the month he was started on HD, and required recurring transfusions to treat the symptoms of anemia. Workup and imaging findings were inconclusive. Specific laboratory tests confirmed the patients had anti-EPO antibodies. After six months of immunosuppressant therapy (corticosteroids + cyclosporine) the patients were stable with Hb > 9.0 g/dl. Conclusion: PRCA is a rare condition among patients on dialysis treated with rhEPO and should be considered as a possible cause of refractory anemia. Treating patients with PRCA may be challenging, since the specific management and diagnostic procedures needed in this condition are not always readily available.

RESUMO Introdução: Anemia é complicação frequente da Doença Renal Crônica (DRC) em pacientes dialíticos. Apresenta caráter multifatorial principalmente pela insuficiente produção de eritropoietina (EPO). Situação rara causadora de anemia na DRC é Aplasia Pura de Células Vermelhas (APCV), em decorrência da produção de anticorpos anti-EPO. Objetivo: Descrever 2 casos de APCV com formação de anticorpos anti-EPO, sua abordagem clínica, evolução e revisão de literatura. Métodos: Dois pacientes em hemodiálise que desenvolveram anemia grave, necessitando investigação e manejo específico. Resultados: Paciente nº 1: feminina, 75 anos, DRC secundária à hipertensão arterial. Após 7 meses com EPO desenvolveu queda persistente em valores de hemoglobina (Hb) mesmo com incremento em doses EPO SC, necessitando transfusões de sangue recorrentes. Extensa investigação laboratorial e de imagem resultou negativa para principais causas de anemia. Paciente nº 2: masculino, 66 anos, DRC secundária à DRPA, há 2 anos em uso de EPO. No mês de entrada em HD desenvolveu anemia severa, também exigindo transfusões recorrentes para tratamento da anemia sintomática. Extensa investigação laboratorial e por imagem, sem chegar a uma conclusão definitiva. Em ambos os casos a presença de anticorpos anti-EPO foi confirmada por exames laboratoriais específicos. Terapia imunossupressora resultou em estabilização do quadro e Hb > 9,0 g/dl em ambos os pacientes, 6 meses após início do tratamento. Conclusão: APCV é condição rara entre pacientes dialíticos que recebem EPOHuR e deve ser lembrada como causa de anemia refratária. Seu manejo específico e diagnóstico laboratorial nem sempre acessível, tornando desafiadora a condução dos casos para o nefrologista.

Pure red cell aplasia and anti-erythropoietin antibodies in patients on hemodialysis: a report of two cases and a literature review.

INTRODUCTION: Anemia is a frequent multifactorial complication of CKD seen in patients on dialysis derived mainly from impaired erythropoietin (EPO) production. A less common cause of anemia in individuals with CKD is pure red cell aplasia (PRCA) secondary to the production of anti-EPO antibodies. OBJECTIVE: This paper aimed two describe two cases of PRCA secondary to the production of anti-EPO antibodies including choice of treatment, patient progression, and a literature review. MATERIALS: This study included the cases of two patients with CKD on hemodialysis with severe anemia in need of specific investigation and management. RESULTS: Patient 1 with CKD secondary to hypertension treated with EPO for 7 months showed persistent decreases in hemoglobin (Hb) levels despite the subcutaneous administration of increasing doses of EPO; the patient required recurring blood transfusions. Workup and imaging tests were negative for the main causes of anemia in individuals with CKD on dialysis. Patient 2 with CKD secondary to adult polycystic kidney disease had been taking EPO for 2 years. The patient developed severe abrupt anemia the month he was started on HD, and required recurring transfusions to treat the symptoms of anemia. Workup and imaging findings were inconclusive. Specific laboratory tests confirmed the patients had anti-EPO antibodies. After six months of immunosuppressant therapy (corticosteroids + cyclosporine) the patients were stable with Hb > 9.0 g/dl. CONCLUSION: PRCA is a rare condition among patients on dialysis treated with rhEPO and should be considered as a possible cause of refractory anemia. Treating patients with PRCA may be challenging, since the specific management and diagnostic procedures needed in this condition are not always readily available.

On-line prediction of the feeding phase in high-cell density cultivation of rE. coli using constructive neural networks.

Streptococcus pneumoniae (pneumococcus) is a bacterium responsible for a wide spectrum of illnesses. The surface of the bacterium consists of three distinctive membranes: plasmatic, cellular and the polysaccharide (PS) capsule. PS capsules may mediate several biological processes, particularly invasive infections of human beings. Prevention against pneumococcal related illnesses can be provided by vaccines. There is a sound investment worldwide in the investigation of a proteic antigen as a possible alternative to pneumococcal vaccines based exclusively on PS. A few proteins which are part of the membrane of the pneumococcus seem to have antigen potential to be part of a vaccine, particularly the PspA. A vital aspect in the production of the intended conjugate pneumococcal vaccine is the efficient production (in industrial scale) of both, the chosen PS serotypes as well as the PspA protein. Growing recombinant Escherichia coli (rE. coli) in high-cell density cultures (HCDC) under a fed-batch regime requires a refined continuous control over various process variables where the on-line prediction of the feeding phase is of particular relevance and one of the focuses of this paper. The viability of an on-line monitoring software system, based on constructive neural networks (CoNN), for automatically detecting the time to start the fed-phase of a HCDC of rE. coli that contains a plasmid used for PspA expression is investigated. The paper describes the data and methodology used for training five different types of CoNNs, four of them suitable for classification tasks and one suitable for regression tasks, aiming at comparatively investigate both approaches. Results of software simulations implementing five CoNN algorithms as well as conventional neural networks (FFNN), decision trees (DT) and support vector machines (SVM) are also presented and discussed. A modified CasCor algorithm, implementing a data softening process, has shown to be an efficient candidate to be part of an on-line HCDC monitoring system for detecting the feeding phase of the HCDC process.

A single monoclonal antibody as probe to detect the entire set of native and partially unfolded rhEPO glycoforms.

Human erythropoietin (hEPO) is a highly heterogeneous glycosylated protein that requires well-characterised immunochemical reagents to evaluate the glycoform profile along its biotechnological production as a recombinant hormone. These reagents should be suitable for several assay conditions (like those used for immunoblotting analysis, liquid or solid-phase quantitative assays, immunoaffinity purification) with no glycoform selectivity. Five anti-recombinant hEPO monoclonal antibodies (mAbs) were characterised with the aim of selecting the appropriate reagent. These antibodies mapped two spatially distinct epitopes and neutralised the in vitro biological activity of the cytokine. All of them were able to bind to both, the partially denatured and the native form of the protein. Isoelectric focusing analysis followed by immunoblotting confirmed that all the mAbs, herein described, were able to bind to each glycoform, recognising amino acid sequences of the hEPO. Nevertheless, only mAb 2B2 preserved the ability to bind to soluble recombinant human erythropoietin (rhEPO) when it was coated to polystyrene plates or immobilised on CNBr-activated Sepharose matrix. Besides, mAb 2B2 was able to bind to the complete set of soluble rhEPO glycoforms, showing the same affinity for the glycosylated and deglycosylated cytokine. Thus, mAb 2B2 was useful as a capture antibody to develop a sandwich enzyme-linked immunosorbent assay (ELISA), performing a simple, specific and fast assay to quantify rhEPO with a detection limit of 7 ng ml(-1). mAb 2B2 was also satisfactorily employed as affinity ligand to purify rhEPO. Our work led us to find a suitable and single reagent to perform a variety of immunochemical approaches, where the binding of each glycoform in the native or partially unfolded form of rhEPO is required.

Erythropoietin improves cardiac contractility in post-hypoxic mice.

Mice myocardia, in which plasma erythropoietin (EPO) concentrations were modified in response to different experimental conditions, were studied to evaluate contractility (dF/dt). CF1 mice were randomly separated into four main groups: group I, normocythaemic normoxic; group II-a, normocythaemic intermittently exposed to hypobaria for 72 h; group II-b, normocythaemic intermittently exposed to hypobaria for 3 weeks; group III, hypertransfused polycythaemic exposed to 72 h hypobaria; and group IV, hypertransfused polycythaemic maintained in normobaric air. Plasma EPO, contractile studies and binding assays were performed. The dF/dt was significantly higher in group II-a than in group I and group II-b; but in groups III and IV, the dF/dt was reduced. The toxic action of ouabain was reduced and delayed in its onset, accompanied by increased numbers of 3H-ouabain binding sites in group II-a. Contractility was positively correlated with plasma EPO (pEPO) in the different groups. Treating group I with recombinant human (rHu)-EPO enhanced contractility while treating group II-a with a monoclonal anti-EPO decreased the dF/dt. The inhibition of enzymatic pathway(s) known to participate in the cytokines signal transduction, decreased the basal dF/dt values on atria from group II-a and on group I atria treated with rHu-EPO. The results demonstrated: (1) a cardiac non-haematopoietic effect of EPO; (2) that mice in which the pEPO concentration increased showed improvement in contractility and in the therapeutic action of ouabain; and (3) it is possible that EPO may act as a cardioprotective agent by modulating the cardiac Na+-K+ pump.

Serum erithropoietin levels in normal population of Mexico City

La ciudad de México está localizada a una gran altitud sobre el nivel del mar (2240 m), es una de las metrópolis más contaminadas del mundo, y dado que la presión barométrica afecta la concentración de oxígeno en la sangre, asumimos que esto se podría ver reflejado en los niveles séricos de eritropoyetina. Para establecer circulante de determinó en 100 sujetos sanos, 52 hombres y 48 mujeres, utilizando un ensayo inmunoenzimático. El valor promedio obtenido fue de 18.67 ñ 7.6 mU/mL, que resulta similar al reportado previamente en ciudades al nivel del mar (18.0 ñ 5.8 mU/mL). El hábito de fumar no modifica en forma significativa las concentraciones séricas de eritropoyetina (21.03 ñ 10 mU/mL). No encontramos correlación entre los valores de hematocrito y las concentraciones séricas de eritropoyetina. Estos hallazgos sugieren la existencia de otro u otros factores que pudieran tener un papel en la regulación de la cantidad de eritrocitos circulantes

Lack of erythropoietic inhibitory effect of serum from patients withe congenital pure red cell aplasia.

The serum from five children with congenital pure red cell aplasia demonstrated no erythropoietic inhibitory effect either in vivo in animals or in vitro. The significance of immunosuppressive therapy in this disease is discussed.