Spinal Stroke: Outcome Attenuation by Erythropoietin and Carbamylated Erythropoietin and Its Prediction by Sphingosine-1-Phosphate Serum Levels in Mice.

Spinal strokes may be associated with tremendous spinal cord injury. Erythropoietin (EPO) improves the neurological outcome of animals after spinal cord ischemia (SCI) and its effects on ischemia-induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are considered possible molecular mechanisms. Furthermore, sphingosin-1-phosphate (S1P) is suggested to correlate with SCI. In this study, the effect of recombinant human EPO (rhEPO) and carbamylated EPO (cEPO-Fc) on the outcome of mice after SCI and a prognostic value of S1P were investigated. SCI was induced in 12-month-old male mice by thoracic aortal cross-clamping after administration of rhEPO, cEPO-Fc, or a control. The locomotory behavior of mice was evaluated by the Basso mouse scale and S1P serum levels were measured by liquid chromatography-tandem mass spectrometry. The spinal cord was examined histologically and the expressions of key UPR proteins (ATF6, PERK, and IRE1a, caspase-12) were analyzed utilizing immunohistochemistry and real-time quantitative polymerase chain reaction. RhEPO and cEPO-Fc significantly improved outcomes after SCI. The expression of caspase-12 significantly increased in the control group within the first 24 h of reperfusion. Animals with better locomotory behavior had significantly higher serum levels of S1P. Our data indicate that rhEPO and cEPO-Fc have protective effects on the clinical outcome and neuronal tissue of mice after SCI and that the ER is involved in the molecular mechanisms. Moreover, serum S1P may predict the severity of impairment after SCI.

Carbamylated erythropoietin improves recognition memory by modulating microglia in a rat model of pain.

Patients with chronic pain often complain about memory impairments. Experimental studies have shown neuroprotective effects of Carbamylated erythropoietin (Cepo-Fc) in the treatment of cognitive dysfunctions. However, little is currently known about its precise molecular mechanisms in a model of inflammatory pain. Therefore, this study aimed to investigate neuroprotective effects of Cepo-Fc against cognitive impairment induced by the inflammatory model of Complete Freund's Adjuvant (CFA). Carbamylated erythropoietin was administrated Intraperitoneally (i.p) on the day CFA injection, continued for a 21-days period. After conducting the behavioral tests (thermal hyperalgesia and novel object recognition test), western blot and ELISA were further preformed on days 0, 7, and 21. The results of this study indicate that Cepo-Fc can effectively reverse the CFA induced thermal hyperalgesia and recognition memory impairment. Additionally, Cepo-Fc noticeably decreased the hippocampal microglial expression, production of hippocampal IL-1ß, and hippocampal apoptosis and necroptosis induced by the inflammatory pain. Therefore, our findings suggest that neuroprotective effects of Cepo-Fc in the treatment of pain related recognition memory impairment may be mediated through reducing hippocampal microglial expression as well as IL-1ß production.

Addressing the 'hypoxia paradox' in severe COVID-19: literature review and report of four cases treated with erythropoietin analogues.

BACKGROUND: Since fall 2019, SARS-CoV-2 spread world-wide, causing a major pandemic with estimated ~ 220 million subjects affected as of September 2021. Severe COVID-19 is associated with multiple organ failure, particularly of lung and kidney, but also grave neuropsychiatric manifestations. Overall mortality reaches > 2%. Vaccine development has thrived in thus far unreached dimensions and will be one prerequisite to terminate the pandemic. Despite intensive research, however, few treatment options for modifying COVID-19 course/outcome have emerged since the pandemic outbreak. Additionally, the substantial threat of serious downstream sequelae, called 'long COVID' and 'neuroCOVID', becomes increasingly evident. Among candidates that were suggested but did not yet receive appropriate funding for clinical trials is recombinant human erythropoietin. Based on accumulating experimental and clinical evidence, erythropoietin is expected to (1) improve respiration/organ function, (2) counteract overshooting inflammation, (3) act sustainably neuroprotective/neuroregenerative. Recent counterintuitive findings of decreased serum erythropoietin levels in severe COVID-19 not only support a relative deficiency of erythropoietin in this condition, which can be therapeutically addressed, but also made us coin the term 'hypoxia paradox'. As we review here, this paradox is likely due to uncoupling of physiological hypoxia signaling circuits, mediated by detrimental gene products of SARS-CoV-2 or unfavorable host responses, including microRNAs or dysfunctional mitochondria. Substitution of erythropoietin might overcome this 'hypoxia paradox' caused by deranged signaling and improve survival/functional status of COVID-19 patients and their long-term outcome. As supporting hints, embedded in this review, we present 4 male patients with severe COVID-19 and unfavorable prognosis, including predicted high lethality, who all profoundly improved upon treatment which included erythropoietin analogues. SHORT CONCLUSION: Substitution of EPO may-among other beneficial EPO effects in severe COVID-19-circumvent downstream consequences of the 'hypoxia paradox'. A double-blind, placebo-controlled, randomized clinical trial for proof-of-concept is warranted.

Carbamylated Erythropoietin Alleviates Kidney Damage in Diabetic Rats by Suppressing Oxidative Stress.

The oxidative stress response plays an important role in the occurrence and development of diabetic kidney disease (DKD). It has become a new treatment target for DKD. In the current study, the effects of carbamylated erythropoietin (CEPO) on renal oxidative stress and damage in diabetic rats were examined. Thirty Sprague Dawley rats were intraperitoneally administered with 60 mg/kg streptozotocin to establish the diabetes model. The diabetic rats were randomly allocated into 4 groups (n=6 each): diabetes model group (DM group), DM + CEPO treatment group (DC group), DM + CEPO + EPO receptor (EPOR) blocking peptide treatment group (DCEB group), and DM + CEPO + CD131 blocking peptide treatment group (DCCB group). Meanwhile, a normal control group (NC group, n=6) was set up. Kidney tissues and blood samples were obtained for evaluation of oxidative stress and renal function. The results showed that diabetic rats exhibited increased oxidative stress in the kidney and early pathological changes associated with DKD. Treatment with CEPO reduced oxidative stress and attenuated renal dysfunction. However, diabetic rats treated with the combination of CEPO and EPOR blocking peptide or CD131 blocking peptide showed increased oxidative stress and reduced renal function when compared with CEPO treatment alone group. These results suggested that CEPO can protect against kidney damage in DKD by inhibiting oxidative stress injury via EPOR-CD131 heterodimers.

Improving oncology biosimilar launches in the EU, the USA, and Japan: an updated Policy Review from the Southern Network on Adverse Reactions.

The EU, the USA, and Japan account for the majority of biological pharmacotherapy use worldwide. Biosimilar regulatory approval pathways were authorised in the EU (2006), in Japan (2009), and in the USA (2015), to facilitate approval of biological drugs that are highly similar to reference products and to encourage market competition. Between 2007 and 2020, 33 biosimilars for oncology were approved by the European Medicines Agency (EMA), 16 by the US Food and Drug Administration (FDA), and ten by the Japan Pharmaceuticals and Medical Devices Agency (PMDA). Some of these approved applications were initially rejected because of manufacturing concerns (four of 36 [11%] with the EMA, seven of 16 [44%] with the FDA, none of ten for the PMDA). Median times from initial regulatory submission before approval of oncology biosimilars were 1·5 years (EMA), 1·3 years (FDA), and 0·9 years (PMDA). Pharmacists can substitute biosimilars for reference biologics in some EU countries, but not in the USA or Japan. US regulation prohibits substitution, unless the biosimilar has been approved as interchangeable, a designation not yet achieved for any biosimilar in the USA. Japan does not permit biosimilar substitution, as prescribers must include the product name on each prescription and that specific product must be given to the patient. Policy Reviews published in 2014 and 2016 in The Lancet Oncology focused on premarket and postmarket policies for oncology biosimilars before most of these drugs received regulatory approval. In this Policy Review from the Southern Network on Adverse Reactions, we identify factors preventing the effective launch of oncology biosimilars. Introduction to the market has been more challenging with therapeutic than for supportive care oncology biosimilars. Addressing region-specific competition barriers and educational needs would improve the regulatory approval process and market launches for these biologics, therefore expanding patient access to these products in the EU, the USA, and Japan.

The analytical approach for detection of carbamylated erythropoietin for doping control purposes.

Erythropoietin (EPO) has protective effects in several tissues and could be used for therapeutic purposes, but the doses of EPO that can be beneficial in case of hypoxic-ischemic conditions due to overinduced erythropoiesis could be detrimental in treated patients. Carbamylation of erythropoietin maintains the tissue-protective effects of EPO but without erythropoietic effects. Carbamylated EPO (CEPO) is listed in WADA Prohibited List in class S2 as "Innate repair receptor agonists." The CEPO was synthesized using the method described previously. Digestion with endoproteinase Lys-C was used to distinguish rhEPO from CEPO. The digested samples containing recombinant EPO, urinary EPO (uEPO), or CEPO were analyzed by the SAR-PAGE method (sarcosyl polyacrylamide gel electrophoresis-PAGE). Endoproteinase Lys-C breaks the peptide chains of lysine. Lysine residues, converted to homocitrulline by carbamylation, cannot be cleaved by endoproteinase Lys-C. Therefore, the CEPO protein chain remained unchanged in contrast to rhEPO and uEPO, which allows for easily differentiation of them.

Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling.

Modulation of alloimmune responses is critical to improving transplant outcome and promoting long-term graft survival. To determine mechanisms by which a nonhematopoietic erythropoietin (EPO) derivative, carbamylated EPO (CEPO), regulates innate and adaptive immune cells and affects renal allograft survival, we utilized a rat model of fully MHC-mismatched kidney transplantation. CEPO administration markedly extended the survival time of kidney allografts compared with the transplant alone control group. This therapeutic effect was inhibited when the recipients were given LY294002, a selective inhibitor of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway or anti-EPO receptor (EPOR) antibody, in addition to CEPO. In vitro, CEPO inhibited the differentiation and function of dendritic cells and modulated their production of pro-inflammatory and anti-inflammatory cytokines, along with activating the PI3K/AKT signaling pathway and increasing EPOR mRNA and protein expression by these innate immune cells. Moreover, after CD4+ T cells were exposed to CEPO the Th1/Th2 ratio decreased and the regulatory T cell (Treg)/Th17 ratio increased. These effects were abolished by LY294002 or anti-EPOR antibody, suggesting that CEPO regulates immune responses and promotes kidney allograft survival by activating the PI3K/AKT signaling pathway in an EPOR-dependent manner. The immunomodulatory and specific signaling pathway effects of CEPO identified in this study suggest a potential therapeutic approach to promoting kidney transplant survival.

Single-and repeat-dose toxicity of HM10760A, a long-acting erythropoietin, in rats and monkeys.

Anemia is a frequent complication of chronic kidney disease (CKD) that causes an increase in morbidity and mortality and accelerates the rate of disease progression. Treatment with recombinant human erythropoietin (rhEPO) is a major breakthrough in the therapy of renal anemia. HM10760A, a long-acting EPO, has been developed as a treatment for anemia in CKD patients. A series of preclinical toxicology studies, such as acute, 4 week repeat-dose, and 13 week repeat-dose, was completed to support the safety of human exposure to HM10760A for up to 13 weeks. The rodent and non-rodent species used in the pivotal preclinical general toxicity studies were rats and monkeys, respectively. A once-a-week or once-every-two-week i.v dosing regimen was applied for 4 week and 13 week repeat-dose toxicity studies, respectively, in consideration of the expected administration frequency in humans. Based on the 13 week repeat-dose toxicity studies, 2.61 µg/kg and 22.03 µg/kg can be considered as the NOAELs (no observed adverse effect levels) in rats and monkeys, respectively. Almost all observations recorded at the low- and mid-dose levels are typical pharmacological effects of EPO and not uniquely attributed HM10760A toxicity. To account for the differences between human being and animal physiologies, the safety of HM10760A needs to be further confirmed in future clinical studies.

CEPO (carbamylated erythropoietin)-Fc protects hippocampal cells in culture against beta amyloid-induced apoptosis: considering Akt/GSK-3ß and ERK signaling pathways.

The tissue-protective properties of erythropoietin (EPO) have been described in several neurodegenerative diseases models, but erythrocytosis following EPO treatment may lead to deleterious effects. Carbamylated erythropoietin, an EPO derivative lacking hematopoietic side effects, has shown protective properties in some studies. However, it is not known if CEPO protects primary hippocampal cells against Aß25-35 toxicity. The present study aimed to investigate the effect of CEPO-Fc on biochemical alterations in Akt, GSK-3ß, and ERK signaling and cell death induced by Aß25-35 in isolated hippocampal cell culture. The embryonic hippocampal cells were obtained from 18-19 day rat embryos. The cells were exposed with Aß25-35 (20 µM) in the absence or presence of CEPO-Fc (1 or 5 IU) and PI3k and ERK inhibitors. CEPO-Fc at the dose of 5 IU significantly prevented the cell loss and caspase-3 cleavage caused by Aß25-35. Additionally, CEPO-Fc noticeably reversed Aß mediated decrement of Akt and GSK-3ß phosphorylation. With exposure to LY294002, PI3 kinase inhibitor, Akt phosphorylation diminished and CEPO-Fc protective effects disappeared. Furthermore, while CEPO-Fc nullified Aß-induced increment of phospho-ERK, inhibition of ERK activity by PD98059, had no effect on Aß25-35-mediated caspase-3 cleavage and cell toxicity. These results imply that protective effects of CEPO-Fc seem to be mainly exerted through the PI3K/Akt pathway rather than ERK signaling. This study suggested that CEPO-Fc prevents Aß-induced cell toxicity as well as Akt/GSK-3ß and ERK alterations in isolated hippocampal cells. These findings might provide a new perspective on CEPO-Fc protective properties as a prospective remedial factor for neurodegenerative diseases like AD.

Effect of carbamylated erythropoietin Fc fusion protein (CEPO-Fc) on learning and memory impairment and hippocampal apoptosis induced by intracerebroventricular administration of streptozotocin in rats.

Intracerebroventricular (icv) administration of streptozotocin (STZ) has been used as a metabolic model of sporadic Alzheimer's disease (AD). Erythropoietin (EPO) possesses neuroprotective and memory-improving effects, which might be advantageous in treating different characteristics of AD. Nevertheless, the hematopoietic effect of EPO has hindered its application as a neuroprotective agent. Previous studies have shown that a new Epo derivative called carbamylated Erythropoietin-Fc (CEPO-Fc), yield noticeable neuroprotective effects without affecting hematopoiesis. In this study, the neuroprotective effects of CEPO-Fc on icv-STZ induced memory impairment and hippocampal apoptosis were examined. Adult male Wistar rats weighing 250-300 g were used. STZ was administered on days 1 and 3 (3 mg/kg in divided doses/icv), and CEPO-Fc was administered at the dose of 5000 IU/ip/daily during days 4-14. The animals were trained in Morris water maze during days 15-17, and the memory retention test was performed on the 18th day. Following behavioral studies, the animals were sacrificed and their hippocampi isolated to determine the amounts of cleaved caspase-3 (the landmark of apoptosis). The results showed that CEPO-Fc treatment at the dose of 5000 IU/kg/ip was able to prevent the learning and memory deficit induced by icv-STZ. Western blot analysis revealed that STZ prompted the cleavage of caspase-3 in the hippocampus while pretreatment with CEPO-Fc significantly reduced the cleavage of this protein. Collectively, our findings suggest that CEPO-Fc could restore STZ-induced learning and memory impairment as well as apoptosis in the hippocampal region in a rat model of sporadic AD induced by icv-STZ.

Erythropoietin and its derivatives: from tissue protection to immune regulation.

Erythropoietin (EPO) is an evolutionarily conserved hormone well documented for its erythropoietic role via binding the homodimeric EPO receptor (EPOR)2. In past decades, evidence has proved that EPO acts far beyond erythropoiesis. By binding the tissue-protective receptor (TPR), EPO suppresses proinflammatory cytokines, protects cells from apoptosis and promotes wound healing. Very recently, new data revealed that TPR is widely expressed on a variety of immune cells, and EPO could directly modulate their activation, differentiation and function. Notably, nonerythropoietic EPO derivatives, which mimic the structure of helix B within EPO, specifically bind TPR and show great potency in tissue protection and immune regulation. These small peptides prevent the cardiovascular side effects of EPO and are promising as clinical drugs. This review briefly introduces the receptors and tissue-protective effects of EPO and its derivatives and highlights their immunomodulatory functions and application prospects.

Helix B surface peptide reduces sepsis-induced kidney injury via PI3K/Akt pathway.

AIM: Helix B-Surface peptide (HBSP) is the latest discovered erythropoietin (EPO) analogue that can retain the activity of EPO. EPO, which is widely used for treating renal anemia, has recently been proved to have protective effects on ischemia-reperfusion injury of brain, heart and kidney. The protective effects of EPO and HBSP on cardiac function were found in rats with myocardial ischemia. However, the effect of HBSP on sepsis-induced renal injury is still unclear. METHODS: Establishment of rat kidney injury model and treated with HBSP and lipoposaccharide. Renal injury in rats was observed by hematoxylin-eosin staining and injury index score. Levels of serum creatinine (SCr), blood urea nitrogen (BUN) and Cystatin C (Cys C) were detected using fully automatic biochemical analyzer, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß were detected by enzyme-linked immunosorbent assay. Western blot analysis was performed to determine the role of HBSP in phosphatidylinositol 3-kinase (PI3K)/Akt pathway. RESULTS: Acute kidney injury (AKI) appeared after modeling, however, HBSP alleviated the pathological conditions of the kidney injury. In addition, HBSP lowered kidney injury index score in the rats, and decreased the levels of SCr, BUN, Cys C, TNF-α, IL-6 and IL-1ß, moreover, HBSP also showed the effect of activating PI3K/Akt pathway. CONCLUSION: HBSP alleviated lipoposaccharide-induced AKI and improved kidney function of the rats with sepsis. More importantly, the effects of HBSP on lipoposaccharid-induced AKI were realized via activating PI3K/Akt pathway. The findings in the current study provide new insights into the therapeutic mechanism for treating the disease.

Evaluación de la indicación de los agentes estimulantes de la eritropoyesis en el tratamiento de la anemia asociada a enfermedad renal crónica / Evaluation of the indication of erythropoiesis stimulating agents in the treatment of anaemia associated with chronic kidney disease

OBJETIVOS: Describir las características de los pacientes con anemia asociada a enfermerdad renal crónica (ERC) que inician tratamiento con agentes estimulantes de la eritropoyesis (AEE) y evaluar su indicación. MÉTODOS: Estudio descriptivo transversal. Se incluyeron pacientes ≥ 18 años que iniciaron tratamiento con epoetina beta o darbepoetina alfa entre el 1 de enero 2014 y el 31 de diciembre 2015. Se excluyeron pacientes en tratamiento renal sustitutivo (TRS) y portadores de trasplante renal (TxR) funcionante. Variables estudiadas: sociodemográficas, analíticas, comorbilidades, tratamientos famacológicos concomitantes y relacionadas con la terapia de estudio. Indicación correcta de los AEE: valores de Hb <10,0 g/dL una vez corregida la ferropenia (ferritina < 100 ng/mL e ISAT < 25%). Fuente de datos: Farmatools®; sistema informatizado de historias clínicas. El estudio obtuvo dictamen favorable del Comité Ético de Investigación Clínica de Aragón (CEICA). RESULTADOS: 269 pacientes (59,9% varones, edad media: 74,7±13,2 años), ERC estadio 4 (60,0%). El 64,7% inició tratamiento con darbepoetina α (dosis mediana: 18,7 (4,7-120,0) mig/semanal); el 35,3% con epoetina β (dosis mediana: 6.000,0 (466,7-24.000,0) UI/semanal). Los pacientes tratados con darbepoetina α presentaban estadios de ERC más avanzados (p < 0,001). Aquellos que iniciaron terapia con epoetina β más comorbilidad cardiovascular: insuficiencia cardiaca (IC) (p = 0,002) y cardiopatía isquémica (p = 0,028). El 65,7% presentaba ferropenia y un 37,5% tomaba suplementos con hierro. Hb media basal: 10,2±1,3 g/dL; el 75,8% presentaba niveles medios de Hb < 11,0 g/dL y el 40,8% valores de Hb<10,0 g/dL. CONCLUSIONES: El inicio del tratamiento se ajustó a los parámetros definidos por consensos internacionales

OBJECTIVES: Describe the characteristics of patients with anemia associated with chronic kidney disease (CKD) who start treatment with erythropoiesis-stimulating agents (ESAs) and evaluate their indication. METHODS: Cross-sectional descriptive study. Patients ≥18 years of age who started treatment with epoetin β or darbepoetin α between January 1, 2014 and December 31, 2015 were included. Patients on renal replacement therapy (TRS) and carriers of functioning kidney transplant (TxR) were excluded. Variables studied: sociodemographic, analytical, comorbidities, concomitant drug treatment and related to study therapy. Correct indication of EEE: Hb values <10.0 g / dL after correction of iron deficiency (ferritin <100 ng / mL and ISAT <25%). Data source: Farmatools®; computerized medical record system. The study obtained a favorable opinion from the Ethical Committee for Clinical Research of Aragon (CEICA). RESULTS: 269 patients (59.9% male, mean age: 74.7 ± 13.2 years), stage 4 CKD (60.0%). 64.7% started treatment with darbepoetin α (median dose: 18.7 (4.7-120.0) μg / weekly); 35.3% with epoetin β (median dose: 6,000.0 (466.7-24,000.0) IU / weekly). Patients treated with darbepoetin α had more advanced stages of CKD (p <0.001). Those who started therapy with epoetin β plus cardiovascular comorbidity: heart failure (HF) (p = 0.002) and ischemic heart disease (p = 0.028). 65.7% had iron deficiency and 37.5% took iron supplements. Basal mean Hb: 10.2 ± 1.3 g / dL; 75.8% had mean Hb levels <11.0 g / dL and 40.8% Hb values <10.0 g / dL. CONCLUSIONS: The start of the treatment was adjusted to the parameters defined by international consensus

Effect of carbamylated erythropoietin on neuronal apoptosis in fetal rats during intrauterine hypoxic-ischemic encephalopathy.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a common disease that occurs during the perinatal period. The primary cause of neonatal HIE is related to fetal intrauterine anoxia. Carbamylated erythropoietin (CEPO), a derivative of erythropoietin (EPO), does not exert any erythropoietic effect; however, the neuroprotective effects resemble those of EPO. Previous studies have shown the potential benefits of CEPO on the central nervous system. The present study aimed to investigate the role of CEPO in neuronal apoptosis during intrauterine HIE and the underlying mechanisms. RESULTS: To validate our hypothesis, we established an intrauterine HIE model by occluding the bilateral utero-ovarian arteries of pregnant Sprague-Dawley rats. Compared to the I/R group, neuronal apoptosis in the CEPO group was significantly lower at 4, 12, 24, and 48 h (P < 0.05). CEPO significantly inhibited CC3 expression (P < 0.05) during the early-stages after ischemia-reperfusion (0.5, 4, 8, 12 and 24 h), upregulated Bcl-2 expression, and downregulated Bax expression at 4, 8, 12, and 24 h (P < 0.05). CONCLUSIONS: Carbamylated erythropoietin pretreatment inhibited the expression of proapoptotic protein CC3 in brain and regulated the Bcl-2/Bax ratio, resulting in reduced neuronal apoptosis and thus resulting in a protective effect on intrauterine HIE.

Effect of carbamylated erythropoietin on neuronal apoptosis in fetal rats during intrauterine hypoxic-ischemic encephalopathy

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a common disease that occurs during the perinatal period. The primary cause of neonatal HIE is related to fetal intrauterine anoxia. Carbamylated erythropoietin (CEPO), a derivative of erythropoietin (EPO), does not exert any erythropoietic effect; however, the neuroprotective effects resemble those of EPO. Previous studies have shown the potential benefits of CEPO on the central nervous system. The present study aimed to investigate the role of CEPO in neuronal apoptosis during intrauterine HIE and the underlying mechanisms. RESULTS: To validate our hypothesis, we established an intrauterine HIE model by occluding the bilateral uteroovarian arteries of pregnant Sprague-Dawley rats. Compared to the I/R group, neuronal apoptosis in the CEPO group was significantly lower at 4, 12, 24, and 48 h (P < 0.05). CEPO significantly inhibited CC3 expression (P < 0.05) during the early-stages after ischemia-reperfusion (0.5, 4, 8, 12 and 24 h), upregulated Bcl-2 expression, and downregulated Bax expression at 4, 8, 12, and 24 h (P < 0.05). CONCLUSIONS: Carbamylated erythropoietin pretreatment inhibited the expression of proapoptotic protein CC3 in brain and regulated the Bcl-2/Bax ratio, resulting in reduced neuronal apoptosis and thus resulting in a protective effect on intrauterine HIE.

Patterns of Erythropoiesis-Stimulating Agent Use in European Hemodialysis Patients: The Dialysis Outcomes and Practice Patterns Study.

BACKGROUND: Clinicians providing dialysis care have numerous erythropoiesis-stimulating agents (ESAs) available for treating anemia. We sought to provide a contemporary description of ESA types used in hemodialysis (HD) settings in nine European countries. METHODS: Our study uses Dialysis Outcomes and Practice Patterns Study phase 5 (2012-2015) data from nine European countries (Belgium, France, -Germany, Italy, Russia, Spain, Sweden, Turkey, and the United Kingdom). A total of 164 facilities and 3,281 patients contributed cross-sectional data. ESA types captured included short-acting epoetins (e.g., epoetin alfa, beta, etc., including biosimilars), darbepoetin alfa, and continuous erythropoietin receptor agonist (CERA; methoxy polyethylene glycol-epoetin beta). RESULTS: We observed broad variability across countries in prescription of ESA types: prescription of epoetin alfa or epoetin beta ranged from 22% (France) to 78% (Russia), darbepoetin alfa prescription ranged from 13% (Russia) to 53% (UK), and CERA prescription ranged from <3% (Sweden) to 26% (France). Prescription of different ESA types varied substantially within some European countries from 2012-2015 but not across all countries in aggregate. Number of ESA types prescribed by a facility varied from 1, 2, 3, or 4 different ESA types in 32, 40, 21, and 8% of facilities, respectively. No differences were seen in the unadjusted distributions of achieved hemoglobin values by ESA type. CONCLUSION: A variety of short- and long-acting ESAs are commonly used in European HD facilities to maintain hemoglobin at remarkably similar levels with each ESA type. The availability of numerous ESA options for managing anemia has allowed European providers to optimize anemia management according to the particular circumstances of each patient.

Home Therapy to Reduce Office Visits for Patients with Chronic Kidney Disease and Anemia.

Anemia is a common complication of chronic kidney disease (CKD) and a predictor of increased mortality. This project integrated erythropoietin-stimulating agent (ESA) with CKD care under one practice setting, co-managing anemia with CKD while reducing frequency of office visits in a rural setting. Patients self-administered their weekly dosage of erythropoietin with monthly follow-ups. As a result, office visits decreased by 56% for patients with CKD Stage 4 and by 54% for patients with CKD Stage 5.

Distribution of follicles in canine ovarian tissues and xenotransplantation of cryopreserved ovarian tissues with even distribution of follicles.

Ovarian follicles are not homogeneously distributed within the ovarian cortex in several species of mammals. Yet to maximize the reproducibility of experimental results of ovarian transplantation, it is essential to assess the degree of density and distribution of follicles in ovarian tissues before their transplantation. In this study, the ovarian cortex from 13 immature bitches (ten purebred and three mongrels) was sectioned into 1.0- to 1.5-mm3 cubes, those were fixed, sectioned and stained with haematoxylin and eosin. To evaluate the density and distribution of follicles, the mean number of all stages of follicles per square millimetre was calculated after observation under a microscope. The distribution of follicles was considered even when the variance value was lower than 10 or between 10 and 16, with an absolute value of distortion inferior to 1. The mean number of follicles ranged from 3.24 to 28.34/mm2 in 25 ovaries from 13 bitches examined. The variance and distortion ranged from 0.35 to 119.64 and -1.87 to 4.40, respectively. The distribution of follicles within the ovarian cortex was judged uneven in 12 of 25 ovaries. These results indicated that follicles were not homogeneously distributed within the ovarian cortex in a large proportion of ovaries. In addition, cryopreserved ovarian fragments with even distribution of follicles were transplanted to NSG mice with or without 400 U/kg of disialylated erythropoietin (asialo EPO). After removing both sides of ovary, a piece of ovarian fragment was placed under the kidney capsule in both sides of kidney. At 4 weeks after transplantation, the fragments were recovered from the mice and the number of primordial, primary, secondary and antral follicles was counted. Total number of follicles and survival rates of follicles in transplanted fragments with asialo EPO were higher than without asialo EPO in four bitches examined. These findings suggest that asialo EPO might be effective on the follicular survival of canine ovarian tissues after xenotransplantation. Knowing the degree of density and distribution of follicles in ovarian tissues before transplantation is expected to contribute to the precise interpretation of results after transplantation of the ovarian tissues.

Neuroprotection and CD131/GDNF/AKT Pathway of Carbamylated Erythropoietin in Hypoxic Neurons.

Carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to neuroprotective effects without erythropoiesis. However, little is known about molecular mechanisms behind CEPO-mediated neuroprotection. In primary neurons with oxygen-glucose deprivation (OGD) and mice with hypoxia-reoxygenation, the neuroprotection and possible molecular mechanism of CEPO were performed by immunohistochemistry and immunocytochemistry, Western blot, RT-PCR, and ELISA. The comparisons were analyzed by ANOVA followed by unpaired two-tailed Student's t test. Both CEPO and EPO showed the neuroprotective effects in OGD model and hypoxic brain. CEPO did not trigger JAK-2 but activated AKT through glial cell line-derived neurotrophic factor (GDNF). It has been shown that CEPO acts upon a heteroreceptor complex comprising both the EPO receptor and the common ß receptor subunit (ßcR, also known as CD131). The blockage of CD131 reduced CEPO-mediated GDNF production, while GFR receptor blockage and GDNF neutralization inhibited CEPO-induced neurogenesis. Addition of GDNF to cultured neurons increased phosphorylation of AKT. CEPO protects neurons possible through the CD131/GDNF/AKT pathway.