Relationship between Injury Time and Expressions of EPO and Its Receptors in Rats Brain after Cerebral Injury / 法医学杂志

OBJECTIVE@#To explore the relationship between injury age and expressions of erythropoietin (EPO) and its receptor EPOR in the brain tissue of rats after cerebral injury.@*METHODS@#Seventy-two rats were randomly divided into control group (36 rats) and cerebral injury group (36 rats). The rats were sacrificed at 1, 2, 4, 8, 12, 24 h after cerebral injury (6 rats at each time point) and the brain tissues were extracted. The expressions of mRNA and protein of EPO and EPOR at different time points were detected by real-time fluorescent quantitative PCR and Western bloting.@*RESULTS@#The expressions of EPO and EPOR increased within 24 h after injury. The expressions of mRNA and protein of EPO were related to the injury age, and the correlations were 0.875, 0.911, respectively (P < 0.05). The expressions of mRNA and protein of EPOR were related to the injury age, and the correlation coefficients were 0.936, 0.905, respectively (P < 0.05).@*CONCLUSION@#The expressions of EPO and EPOR increase gradually in the early stage of the rat's cerebral injury, which are associated with the injury age and could be a useful value for estimating injury age.

Expression of GPI anchored human recombinant erythropoietin in CHO cells is devoid of glycosylation heterogeneity.

Erythropoietin is a glycohormone involved in the regulation of the blood cell levels. It is a 166 amino acid protein having 3 N-glycosylation and one O-linked glycosylation sites, and is used to treat anaemia related illness. Though human recombinant erythropoietin (rEPO) is produced in CHO cells, the loss in quality control is 80% due to incomplete glycosylation of the rEPO with low levels of fully glycosylated active rEPO. Here, we describe the expression from CHO cells of fully glycosylated human rEPO when expressed as a GPI anchored molecule (rEPO-g). The results demonstrated the production of a homogenous completely glycosylated human rEPO-g as a 42 kD band without any low molecular weight glycoform variants as shown by affinity chromatography followed by SDS-PAGE and anti-human EPO specific western blot. The western blot using specific monoclonal antibody is the available biochemical technique to prove the presence of homogeneity in the expressed recombinant protein. The GPI anchor can be removed during the purification process to yield a therapeutically relevant recombinant erythropoietin molecule cells with a higher in vivo biological activity due to its high molecular weight of 40 kD. This is possibly the first report on the production of a homogenous and completely glycosylated human rEPO from CHO cells for efficient therapy.

Cyclooxygenase-2 and hypoxia-regulated proteins are modulated by basic fibroblast growth factor in acute renal failure

Acute renal failure (ARF) can be caused by injuries that induce tissue hypoxia, which in turn can trigger adaptive or inflammatory responses. We previously showed the participation of basic fibroblast growth factor (FGF-2) in renal repair. Based on this, the aim of this study was to analyze the effect of FGF-2 signaling pathway manipulation at hypoxia-induced protein levels, as well as in key proteins from the vasoactive systems of the kidney. We injected rat kidneys with FGF-2 recombinant protein (r-FGF) or FGF-2 receptor antisense oligonucleotide (FGFR2-ASO) after bilateral ischemia, and evaluated the presence of iNOS, EPO and HO-1, in representation of hypoxia-induced proteins, as well as COX-2, renin, kallikrein, and B2KR, in representation of the vasoactive systems of the kidney. A reduction in iNOS, HO-1, EPO, renin, kallikrein, B2KR, and in renal damage was observed in animals treated with r-FGF. The opposite effect was found with FGF-2 receptor down-regulation. In contrast, COX-2 protein levels were higher in kidneys treated with r-FGF and lower in those that received FGFR2-ASO, as compared to saline treated kidneys. These results suggest that the protective role of FGF-2 in the pathogenesis of ARF induced by I/R is a complex process, through which a differential regulation of metabolic pathways takes place.

Recombinant human erythropoietin (rhEPO) preconditioning on nuclear factor-kappa B (NF-kB) activation & proinflammatory cytokines induced by myocardial ischaemia-reperfusion.

BACKGROUND & OBJECTIVES: Erythropoietin (EPO), originally identified for its critical hormonal role in promoting erythrocyte survival and differentiation, has shown to a protective effect in myocardial ischaemia-reperfusion (I-R) injury in animal model. However, the precise mechanisms remain unclear. The objective of this study was to determine the roles of nuclear factor-kappa B (NF-kB) and associated cytokines induced by I-R in the cardioprotection by recombinant human erythropoietin (rhEPO). Morphopathological observations were also made on the ultrastructure of myocardial tissue. METHODS: Myocardial I-R rat model was established by 30 min ligation of left descending coronary and 3 h reperfusion. RhEPO or saline solution was intraperitoneally injected 24 h before I-R insult. The infarct sizes were measured by triphenyltetrazolium chloride (TTC)-Evans blue technique and ultrastructural organizations were observed by a transmission electron microscope. Tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 concentrations were analyzed by enzyme-linked immunosorbance assays and NF-kB by electrophoretic mobility shift assay. TNF-alpha and IL-6 mRNA expression were studied by the reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: A single bolus injection of 5,000 units/kg of rhEPO 24 h before insult remarkably reduced infarct size and improved ultrastructural organization of I-R myocardium. It greatly suppressed TNF-alpha and IL-6 expression, but enhanced IL-10 production. It modestly activated NF-kB before I-R insult and markedly attenuated subsequent NF-kB activation during sustained I-R. INTERPRETATION & CONCLUSION: The suppression of proinflammatory cytokines expression may act by inhibiting NF-kB activation during I-R, but not by induction of IL-10. This might be one of the molecular mechanisms of rhEPO in cardioprotection. In addition, NF-kB was suggested to play a dual role in cardioprotective effects of rhEPO.

Myomatous erythrocytosis syndrome.

Uterine myomas are common but erythrocytosis caused by these is rarely seen. We report a case that illustrates the conjunction of various aetiological factors required for this clinical entity to evolve. A voluminous, retroperitoneally located and focally degenerated myoma was associated with severe secondary erythrocytosis (haematocrit: 65.5%) which resolved after hysterectomy. It has been demonstrated previously that myomatous tissue is the source of excessive production of erythropoietin. Local tissue hypoxia, which is more prone to develop in a pedunculated myoma, stimulates the process. Other prerequisites are a very large size of the myoma and the absence of menometrorrhagia of a severity such as to cause a depletion in iron reserves.

Tratamiento con eritropoyetina humana recombinante en período neonatal: indicaciones y controversias / Treatment with recombinant human erythropoietin in the neonate: progress and controversies

La eritropoyetina recombinante (rHuEPO) se utiliza en dos patologías del neonato que se caracterizan por tener niveles inadecuadamente bajos de eritropoyetina circulante: la anemia del prematuro (AP) de muy bajo peso de nacimiento y la anemia hiporregenerativa tardía de la enfermedad hemolítica por incompatibilidad Rh (EHIRh). La experiencia recogida luego de 5 años de utilización en la AP ha demostrado que : a) parece no traer complicaciones; b) para ser efectiva, debe usarse a dosis no inferiores a 500 Ul/kg/semana; c) induce una marcada estimulación de la eritropoyesis, evidenciable por la importante respuesta reticulocitaria observada; d) disminuye significativamente los requerimientos transfuncionales cuando se administra a partir de la 2a. semana de vida; e) no está aún establecida la utilidad de su empleo desde las primeras horas de vida; f) el tratamiento debe durar de 6 a 8 semanas; g) el principal factor limitante de su eficacia terapéutica es la depleción de hierro, por lo que el mismo debe administrarse a dosis de 6 mg/kg/día o superiores mientras dure el tratamiento con rHuEPO. Por lo tanto, su utilización rutinaria parece estar justificada para disminuir la probabilidad de ocurrencia de complicaciones transfuncionales, aunque simultáneamente se deberán realizar los máximos esfuerzos para establecer criterios transfusionales estrictos y disminuir los volúmenes de sangre extraídos para análisis. En niños con EHIRh que han recibido transfusiones intrauterinas se produce una marcada inhibición de la eritropoyesis que demora 2 a 4 meses en recuperarse espontáneamente. El tratamiento con rHuEPO, en los casos comunicados, logró evitar la indicación de transfusiones adicionales. Se encuentran en marcha algunos estudios prospectivos

A simple method for concentrating erythropoietin activity in plasma and in other body fluids

El método aquí descripto se basa en la propiedad de la eritropoyetina de permanecer en solución acuosa en presencia de hasta 60% de etanol. En este proceso las proteínas inertes precipitan y pueden ser eliminadas sin pérdidas significativas de la actividad. El agregado de éter sulfúrico produce la separación de la fase acuosa y de sus solutos, los que se recogen con el material sólido obtenido por liofilización de aquélla. Con esta metodología se puede detectar una marcada actividad en los extractos obtenidos del plasma de ratones después de una moderada reducción del hematocrito