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Recombinant human erythropoietin (rhEPO) has been abused as a performance enhancer in sports for several years, but with advancements in detection methods, even micro‐doses can be detected in dried blood spot (DBS) samples. Here, we present the results from an Eporatio® (epoetin theta) micro‐dose administration study to detect rhEPO in DBS samples. Five healthy male volunteers received a 15 IU/kg subcutaneous dose of Eporatio®. Urine and DBS samples (Mitra® VAMS and Capitainer® B50) were collected 1, 10, 24, 36, 48 and 72 h after drug administration. After 1 h, all urine samples were negative for rhEPO, whereas 40% of DBS samples were considered suspicious. All samples between 10 and 48 h were suspicious for the presence of Eporatio®, except one urine sample that was negative at 48 h. After 72 h, 40% of urine samples and 60% of DBS samples were suspicious and would have proceeded to a confirmation analysis. DBS is an efficient complementary matrix to urine for detection of rhEPO micro‐doses. [ABSTRACT FROM AUTHOR]

The abuse of recombinant erythropoietin (rEPO) and other erythropoietin (EPO) receptor agonists (ERAs) in sports prompted the need for sensitive detection methods of these substances. Dried blood spot (DBS) samples offer an easy solution for simultaneous collection of blood and urine during a doping control, but sensitivity issues are often presented as a challenge for routine EPO analysis from DBS. Its potential use for detecting rEPO micro‐doses and the EPO gene c.577del variant thus needed further demonstration. Here, capillary blood collected from the arm skin of 111 athletes with Tasso‐M20 (17.5 μL/spot), collected during professional triathlon competitions, were analysed. Also, venous blood samples from healthy volunteers were used to prepare several spots of 20 μL on Mitra VAMS (from an rEPO micro‐dose study) and Whatman filter paper (from an EPO gene variant study). Immunopurification of 2 spots with MAIIA EPO Purification Gel Kit and analysis with sodium N‐lauroylsarcosinate polyacrylamide gel electrophoresis (SAR‐PAGE)/Western blot resulted in sensitive detection of (1) micro‐doses of rEPO from Mitra VAMS, (2) endogenous EPO from Tasso‐M20 in all in‐competition subjects, and (3) the EPO c.577del variant from Whatman filter paper. Additionally, in‐competition endogenous EPO was detected in DBS even when matching urine samples had undetectable EPO. In conclusion, this work demonstrated that DBS can be a useful complementary matrix to urine samples for EPO detection. [ABSTRACT FROM AUTHOR]

Several studies have demonstrated interesting results considering the implication of three growth factors (GFs), namely nerve growth factor (NGF), erythropoietin (EPO), and the insulin-like growth factor-I (IGF-1) in the physiology of male reproductive functions. This review provides insights into the effects of NGF, EPO, and IGF-1 on the male reproductive system, emphasizing mainly their effects on sperm motility and vitality. In the male reproductive system, the expression pattern of the NGF system varies according to the species and testicular development, playing a crucial role in morphogenesis and spermatogenesis. In humans, it seems that NGF positively affects sperm motility parameters and NGF supplementation in cryopreservation media improves post-thaw sperm motility. In animals, EPO is found in various male reproductive tissues, and in humans, the protein is present in seminal plasma and testicular germ cells. EPO receptors have been discovered in the plasma membrane of human spermatozoa, suggesting potential roles in sperm motility and vitality. In humans, IGF-1 is expressed mainly in Sertoli cells and is present in seminal plasma, contributing to cell development and the maturation of spermatozoa. IGF-1 seems to modulate sperm motility, and treatment with IGF-1 has a positive effect on sperm motility and vitality. Furthermore, lower levels of NGF or IGF-1 in seminal plasma are associated with infertility. Understanding the mechanisms of actions of these GFs in the male reproductive system may improve the outcome of sperm processing techniques. [ABSTRACT FROM AUTHOR]

Abstract Chinese Hamster Ovary (CHO) cells are widely employed as host cells for biopharmaceutical production. The manufacturing of biopharmaceuticals poses several challenges, including restricted growth potential and inadequate productivity of the host cells. MicroRNAs play a crucial role in regulating gene expression and are considered highly promising tools for cell engineering to enhance protein production. Our study aimed to evaluate the effects of miR-107, which is recognized as an onco-miR, on erythropoietin-producing CHO cells (CHO-hEPO). To assess the impact of miR-107 on CHO cells, a DNA plasmid containing miR-107 was introduced to CHO-hEPO cells through transfection. Cell proliferation and viability were assessed using the trypan blue dye exclusion method. Cell cycle analysis was conducted by utilizing propidium iodide (PI) staining. The quantification of EPO was determined using an immunoassay test. Moreover, the impact of miR-107 on the expression of downstream target genes was evaluated using qRT-PCR. Our findings highlight and underscore the substantial impact of transient miR-107 overexpression, which led to a remarkable 2.7-fold increase in EPO titers and a significant 1.6-fold increase in the specific productivity of CHO cells (p

Summary: Anemia is a common finding in patients with solid or hematological malignancies. The underlying causes of cancer-related anemia can be multifactorial, including toxicity of cancer therapy, raised inflammatory conditions by the cancer, chronic bleeding and malnutrition. Therapeutic approaches for the treatment of chemotherapy induced anemia encompass red blood cell (RBC) transfusions and erythropoietin-stimulating agents (ESAs). The latter are approved for the treatment of patients with symptomatic anemia caused by palliative chemotherapy to reduce the number of RBC transfusions and gradually improve anemia-related symptoms. Before the treatment with ESA, a baseline Hb level < 10 g/dl is mandatory and iron deficiency must be ruled out. ESAs are linked to an increase in thromboembolic events and potentially raised mortality. Therefore, the risk-benefit ratio should be carefully assessed. [ABSTRACT FROM AUTHOR]

Organ transplantation is the most effective treatment for all categories of end-stage organ diseases. To resolve the shortage of donors in organ transplantation, widespread attention has been diverted to xenotransplantation. At present, clinicians mainly highlight the problems related to xenotransplantation rejection and viral infection. The physiology of xenotransplantation has been rarely studied. Kidney performs endocrine function by producing erythropoietin (EPO), renin and activating vitamin D. Although these pathways are usually well preserved in allogeneic transplantation, species-specific differences, especially those between pigs and non-human primates, may still affect the physiological function of transplant organs. In this article, the changes of EPO, renin-angiotensin-aldosterone system (RAAS) and active vitaminD3 of pig and human after xenotransplantation were illustrated, aiming to provide reference for subclinical research of xenotransplantation. [ABSTRACT FROM AUTHOR]

Hematological parameters and erythropoiesis are known to be influenced by anabolic androgenic steroid (AAS) use. However, little is known in relation to supra‐physiological doses of AAS. Therefore, the aim of this study was to evaluate the effect of supra‐physiological doses of AAS on serum and urinary erythropoietin (EPO), and blood parameters, from self‐reported AAS users. Serum EPO levels were higher in testosterone positive AAS users (11.83 mIU/mL ± 4.19) than nonpositive (6.60 mIU/mL ± 2.70, p = 0.03), while no differences in urinary EPO levels were noted. There were positive correlations between serum EPO and testosterone levels (rs = 0.46, p = 0.01) and reticulocyte percentage (rs = 0.43, p = 0.02). Individuals with AAS‐induced hypogonadism (ASIH; luteinizing hormone levels <1.4 IU/L) had approximately 75% higher serum EPO (p < 0.05) and 140% higher high fluorescence reticulocyte fractions (p < 0.001), as well as other affected hematological parameters, compared with non‐ASIH individuals. The results extend the knowledge of how endocrine and hematological biomarkers are affected by AAS doping. [ABSTRACT FROM AUTHOR]

[Display omitted] Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We discovered DS44470011 in our previous study, which showed potent in vitro activity and in vivo efficacy based on HIF-PHD inhibition. However, DS44470011 was also found to exert genotoxic effects. By converting the biphenyl structure, which is suspected to be the cause of this genotoxicity, to a 1-phenylpiperidine structure, we were able to avoid genotoxicity and further improve the in vitro activity and in vivo efficacy. Furthermore, through the optimization of pyrimidine derivatives, we discovered DS-1093a , which has a wide safety margin with potent in vitro activity and an optimal pharmacokinetic profile. DS-1093a achieved an increase in hemoglobin levels in an adenine-induced rat model of chronic kidney disease after its continuous administration for 4 days. [ABSTRACT FROM AUTHOR]

Abstract Background Treatment with erythropoietin is well established for anemia in chronic kidney disease patients but not well studied in acute kidney injury. Methods This is a multicenter, randomized, pragmatic controlled clinical trial. It included 134 hospitalized patients with anemia defined as hemoglobin

Erythropoietin (EPO) is a pleiotropic cytokine that classically drives erythropoiesis but can also induce bone loss by decreasing bone formation and increasing resorption. Deletion of the EPO receptor (EPOR) on osteoblasts or B cells partially mitigates the skeletal effects of EPO, thereby implicating a contribution by EPOR on other cell lineages. This study was designed to define the role of monocyte EPOR in EPO-mediated bone loss, by using two mouse lines with conditional deletion of EPOR in the monocytic lineage. Low-dose EPO attenuated the reduction in bone volume (BV/TV) in Cx3cr1Cre EPORf/f female mice (27.05%) compared to controls (39.26%), but the difference was not statistically significant. To validate these findings, we increased the EPO dose in LysMCre model mice, a model more commonly used to target preosteoclasts. There was a significant reduction in both the increase in the proportion of bone marrow preosteoclasts (CD115+) observed following high-dose EPO administration and the resulting bone loss in LysMCre EPORf/f female mice (44.46% reduction in BV/TV) as compared to controls (77.28%), without interference with the erythropoietic activity. Our data suggest that EPOR in the monocytic lineage is at least partially responsible for driving the effect of EPO on bone mass. [ABSTRACT FROM AUTHOR]

Objective(s): Myocardial infarction caused by ischemia of heart tissue is the main reason for death worldwide; therefore, early detection can reduce mortality and treatment costs. Erythropoietin (EPO) has protection effects on ischemic tissue due to nonhematopoietic peptide (pHBSP; ARA-290) which is derived from the B-subunit of EPO. Materials and Methods: We designed and synthesized a modified DOTA-(Lys-Dabcyl6, Phe7)-ARA-290 using Fmoc solid-phase peptide synthesis strategies. To improve serum stability, Fmoc-Lys-(Dabcyl)-OH as lipophilic amino acid was synthesized along with Fmoc-Phe-OH which then were substituted with Arg6 and Ala7, respectively; they were then investigated for the ability to detect ischemic cardiac imaging. DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 was labeled with technetium 99m, and its radiochemical purity (RCP), stability in the presence of human serum and, specific bind to hypoxic H9c2 cells were evaluated. In vivo studies for biodistribution and SPECT scintigraphy were checked in a normal and cardiac ischemia rat model. Results: Radiolabeling purity was obtained more than 96% by ITLC, and in vitro stability of the radiopeptide up to 6 hr was 85%. The binding of 99mTc-ARA-290 to hypoxic cells was remarkably higher than normoxic cells (3 times higher than normoxic cells at 1 hr). Biodistribution and SPECT imaging on the cardiac ischemic model showed that radiopeptide considerably accumulated in the ischemic region (cardiac ischemic-to-lung rate = 3.65 ID/g % at 0.5 hr).Conclusion: The results of studies, in vitro and in vivo, indicated that 99mTc-DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 could be an appropriate candidate for early diagnosis of cardiac ischemia.

Neurogenic erectile dysfunction (nED) is one of the most common and intractable postoperative complications of rectal and prostate cancer surgery and sometimes accompanies patients lifelong. The transplantation of stem cells has been proved a promising way for treatment. However, the therapeutic efficacy is severely impaired by excessive cell loss and death and poor accumulation in the injury site along with the traditional implantation strategy. Herein, an EPO‐loaded multifunctional hydrogel was designed. The hydrogels' adhesive property and mechanical strength were enhanced by adding catechol‐catechol adducts, thus significantly improving adipose‐derived stem cells (ADSC) retention and rescuing cell loss in the injury site. Meanwhile, the sustained release of EPO effectively ameliorated the viability and paracrine activity of ADSC, leading to enhanced migration of Schwann cells and differentiation of PC12 cells in vivo. On a bilateral cavernous nerve injury rat model, the present stem cell‐EPO‐hydrogel implanted strategy could significantly alleviate erectile dysfunction. The higher expression of Tuj1 and lower expression of GFAP in the major pelvic ganglia (MPG) indicated the acceleration of neural differentiation while the suppressing development of astrocytes. Also, the combined therapy restored the expression levels of eNOs, nNOs, and α‐SMA in penile tissues, suggesting the rehabilitation of the penis. Further analysis of Masson trichrome staining and apoptosis evaluation of the corpus cavernosum showed the preservation of vascular endothelium content and the prevention of penile fibrosis after denervation. Overall, we believe that this combined strategy presents a promising way not only for restoring neurogenic erectile function but also for the clinical translation of stem cell therapy. [ABSTRACT FROM AUTHOR]

Cardiac dysfunction is an important prognostic predictor of cardiovascular mortality in patients on hemodialysis (HD). Erythropoietin (EPO) has been reported to improve cardiac function by binding to the EPO receptor (EPOR) on cardiomyocytes. This study investigated whether anti-EPOR antibodies were associated with left ventricular cardiac function in patients undergoing HD. This multicenter, cross-sectional observational study included 377 patients (median age, 70 years; 267 (70.8%) males) with chronic kidney disease (CKD) undergoing stable maintenance HD. Serum levels of anti-EPOR antibodies were measured, and echocardiography was used to assess the left ventricular mass index (LVMI) and left ventricular ejection fraction (LVEF). Anti-EPOR antibodies were found in 17 patients (4.5%). LVMI was greater (median of 135 g/m2 vs. 115 g/m2, p = 0.042), and the prevalence of LVEF < 50% was higher (35.3% vs. 15.6%, p = 0.032) in patients with anti-EPOR antibodies than in those without. Multivariable linear regression and logistic regression analysis (after adjusting for known risk factors of heart failure) revealed that anti-EPOR antibodies were independently associated with LVMI (coefficient 16.2%; 95% confidence interval (CI) 1.0–35.0%, p = 0.043) and LVEF <50% (odds ratio 3.20; 95% CI 1.05–9.73, p = 0.041). Thus, anti-EPOR antibody positivity was associated with left ventricular dysfunction in patients undergoing HD. [ABSTRACT FROM AUTHOR]

Burns are leading causes of mortality and morbidity, Including prolonged hospitalization, disfigurement, and disability. Erythropoietin (EPO) is a well-known hormone causing erythropoiesis. However, EPO may play a role in healing acute and chronic wounds due to its anti-inflammatory and pro-regenerative effects. Therefore, the large, prospective, placebo-controlled, randomized, double-blind, multi-center clinical trial "EPO in Burns" was initiated to investigate the effects of EPO versus placebo treatment in severely burned patients. The primary endpoint of "EPO in Burns" was defined as the time elapsed until complete re-epithelialization of a defined split skin graft donor site. Additional analyses of post hoc defined subgroups were performed in view of the primary endpoint. The verum (n 45) and control (n 39) groups were compared with regard to the time it took for study wounds (a predefined split skin graft donor site) to reach the three stages of wound healing (re-epithelialization levels). In addition, the effects of gender (females n 18) and concomitant medications insulin (n 36), non-steroidal anti-inflammatory drugs (NSAIDs) (n 41), and vasopressor agents (n 43) were tested. Life tables were used to compare study groups (EPO vs. placebo) within subgroups. The Cox regression model was applied to evaluate interactions between the study drug (EPO) and concomitant medications for each re-epithelialization level. Using our post hoc defined subgroups, we observed a lower chance of wound healing for women compared to men (in terms of hazard ratio: hr100%: 5.984 [95%-CI: (0.805-44.490), p = 0.080]) in our study population, regardless of the study medication. In addition, results indicated an earlier onset of re-epithelialization in the first days of EPO treatment (EPO: 10% vs. Placebo: 3%). Moreover, the interpretation of the hazard ratio suggested EPO might have a positive, synergistic effect on early stages of re-epithelialization when combined with insulin [hr50%: 1.307 (p = 0.568); hr75%: 1,199 (p = 0.715)], as well as a stabilizing effect on critically ill patients [reduced need for vasopressors in the EPO group (EPO: 44% vs. Placebo 59%)]. However, additional high-quality data from clinical trials designed to address these endpoints are required to gain further insight into these effects. [ABSTRACT FROM AUTHOR]

Abstract Neurogenic erectile dysfunction (nED) is one of the most common and intractable postoperative complications of rectal and prostate cancer surgery and sometimes accompanies patients lifelong. The transplantation of stem cells has been proved a promising way for treatment. However, the therapeutic efficacy is severely impaired by excessive cell loss and death and poor accumulation in the injury site along with the traditional implantation strategy. Herein, an EPO‐loaded multifunctional hydrogel was designed. The hydrogels' adhesive property and mechanical strength were enhanced by adding catechol‐catechol adducts, thus significantly improving adipose‐derived stem cells (ADSC) retention and rescuing cell loss in the injury site. Meanwhile, the sustained release of EPO effectively ameliorated the viability and paracrine activity of ADSC, leading to enhanced migration of Schwann cells and differentiation of PC12 cells in vivo. On a bilateral cavernous nerve injury rat model, the present stem cell‐EPO‐hydrogel implanted strategy could significantly alleviate erectile dysfunction. The higher expression of Tuj1 and lower expression of GFAP in the major pelvic ganglia (MPG) indicated the acceleration of neural differentiation while the suppressing development of astrocytes. Also, the combined therapy restored the expression levels of eNOs, nNOs, and α‐SMA in penile tissues, suggesting the rehabilitation of the penis. Further analysis of Masson trichrome staining and apoptosis evaluation of the corpus cavernosum showed the preservation of vascular endothelium content and the prevention of penile fibrosis after denervation. Overall, we believe that this combined strategy presents a promising way not only for restoring neurogenic erectile function but also for the clinical translation of stem cell therapy.

Burns are leading causes of mortality and morbidity, including prolonged hospitalization, disfigurement, and disability. Erythropoietin (EPO) is a well-known hormone causing erythropoiesis. However, EPO may play a role in healing acute and chronic wounds due to its anti-inflammatory and pro-regenerative effects. Therefore, the large, prospective, placebo-controlled, randomized, double-blind, multi-center clinical trial “EPO in Burns” was initiated to investigate the effects of EPO versus placebo treatment in severely burned patients. The primary endpoint of “EPO in Burns” was defined as the time elapsed until complete re-epithelialization of a defined split skin graft donor site. Additional analyses of post hoc defined subgroups were performed in view of the primary endpoint. The verum (n 45) and control (n 39) groups were compared with regard to the time it took for study wounds (a predefined split skin graft donor site) to reach the three stages of wound healing (re-epithelialization levels). In addition, the effects of gender (females n 18) and concomitant medications insulin (n 36), non-steroidal anti-inflammatory drugs (NSAIDs) (n 41), and vasopressor agents (n 43) were tested. Life tables were used to compare study groups (EPO vs. placebo) within subgroups. The Cox regression model was applied to evaluate interactions between the study drug (EPO) and concomitant medications for each re-epithelialization level. Using our post hoc defined subgroups, we observed a lower chance of wound healing for women compared to men (in terms of hazard ratio: hr100%: 5.984 [95%-CI: (0.805–44.490), p = 0.080]) in our study population, regardless of the study medication. In addition, results indicated an earlier onset of re-epithelialization in the first days of EPO treatment (EPO: 10% vs. Placebo: 3%). Moreover, the interpretation of the hazard ratio suggested EPO might have a positive, synergistic effect on early stages of re-epithelialization when combined with insulin [hr50%: 1.307 (p = 0.568); hr75%: 1,199 (p = 0.715)], as well as a stabilizing effect on critically ill patients [reduced need for vasopressors in the EPO group (EPO: 44% vs. Placebo 59%)]. However, additional high-quality data from clinical trials designed to address these endpoints are required to gain further insight into these effects.

Abstract Background The purpose of periodontal treatments is to reduce inflammation, restore gingival health and clinical attachment level gain by controlling microbial plaque formation and other etiological factors. One of the drugs that has been tested in many areas and shown good anti-inflammatory properties is erythropoietin (EPO). We evaluated the effect of this drug on the improvement of periodontitis after the phase I treatment. Methods This study was conducted on 30 patients with stage III periodontitis who had at least two bilateral teeth with CAL of ≥ 5 mm and PPD ≥ 6 mm at ≥ 2 non‐adjacent teeth and bleeding on probing. After oral hygiene instruction and scaling and root planning (SRP), EPO gel containing a solution of 4000 units was applied deeply in the test group and placebo gel was deeply administered in the control pockets (5 times, every other day). The clinical parameters of the plaque index (PI), gingival index (GI), clinical attachment level (CAL), probing depth (PD) and bleeding index (BI) were measured at baseline and after three months of follow up. The P-value was set at 0.05. Results All clinical variables improved after treatment in both groups. The BI and GI scores (which reflects the degree of gingival inflammation) showed statistically more reduction in test group. The CAL decreased from 5.1 ± 4.1 to 3.40 ± 2.71 mm; and 5.67 ± 4.32 to 4.33 ± 3.19 mm in test and control group, respectively (P

• This review clearly describes and defines the peripheral nerve injury, EPO, ARA290 and CHBP. • This review condenses for the first time the mechanism of action of EPO in repairing peripheral nerve damage. • This review summarizes for the first time the role of ARA290 and CHBP, derivatives of EPO in peripheral nerve injury. Peripheral nerve injury seriously endangers human life and health, but there is no clinical drug for the treatment of peripheral nerve injury, so it is imperative to develop drugs to promote the repair of peripheral nerve injury. Erythropoietin (EPO) not only has the traditional role of promoting erythropoiesis, but also has a tissue-protective effect. Over the past few decades, researchers have confirmed that EPO has neuroprotective effects. However, side effects caused by long-term use of EPO limited its clinical application. Therefore, EPO derivatives with low side effects have been explored. Among them, ARA290 has shown significant protective effects on the nervous system, but the biggest disadvantage of ARA290, its short half-life, limits its application. To address the short half-life issue, the researchers modified ARA290 with thioether cyclization to generate a thioether cyclized helical B peptide (CHBP). ARA290 and CHBP have promising applications as peptide drugs. The neuroprotective effects they exhibit have attracted continuous exploration of their mechanisms of action. This article will review the research on the role of EPO, ARA290 and CHBP in the nervous system around this developmental process, and provide a certain reference for the subsequent research. [ABSTRACT FROM AUTHOR]

[Display omitted] Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We identified a pyrimidine core with HIF-PHD inhibitory activity based on scaffold hopping of FG-2216 using crystal structures of HIF-PHD2 in complex with compound. By optimizing the substituents at the 2- and 6- positions of the pyrimidine core, we discovered DS44470011, which improves the effectiveness of erythropoietin (EPO) release in cells. Oral administration of DS44470011 to cynomolgus monkeys increased plasma EPO levels. [ABSTRACT FROM AUTHOR]

Background: Emerging data suggest that erythropoietin (EPO) promotes neural plasticity and that iron homeostasis is needed to maintain normal physiological brain function. Cognitive functioning could therefore be influenced by endogenous EPO levels and disturbances in iron status. Objective: To determine whether endogenous EPO levels and disturbances in iron status are associated with alterations in cognitive functioning in the general population. Materials and Methods: Community-dwelling individuals from the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study, a general population-based cohort in Groningen, Netherlands, were surveyed between 2003 and 2006. Additionally, endogenous EPO levels and iron status, consisting of serum iron, transferrin, ferritin, and transferrin saturation were analyzed. Cognitive function was assessed by scores on the Ruff Figural Fluency Test (RFFT), as a reflection of executive function, and the Visual Association Test (VAT), as a reflection of associative memory. Results: Among 851 participants (57% males; mean age 60 ± 13 years), higher endogenous EPO levels were independently associated with an improved cognitive function, reflected by RFFT scores (ß = 0.09, P = 0.008). In multivariable backward linear regression analysis, EPO levels were among the most important modifiable determinants of RFFT scores (ß = 0.09, P = 0.002), but not of VAT scores. Of the iron status parameters, only serum ferritin levels were inversely associated with cognitive function, reflected by VAT scores, in multivariable logistic regression analysis (odds ratio, 0.77; 95% confidence interval 0.63–0.95; P = 0.02 for high performance on VAT, i.e., ≥11 points). No association between iron status parameters and RFFT scores was identified. Conclusion: The findings suggest that endogenous EPO levels and serum ferritin levels are associated with specific cognitive functioning tests in the general population. Higher EPO levels are associated with better RFFT scores, implying better executive function. Serum ferritin levels, but not other iron status parameters, were inversely associated with high performance on the VAT score, implying a reduced ability to create new memories and recall recent past. Further research is warranted to unravel underlying mechanisms and possible benefits of therapeutic interventions. [ABSTRACT FROM AUTHOR]

Erythropoietin (ЕPО) is a glycoprotein hormone, mainly known for its haemopoietic function. For orthopaedics, its pleiotropic effects – osteogenic and angiogenic potential, are of primary interest. The exact mechanism of EPO action is still unclear. The effects of EPO on bone healing were investigated through experiments with rats, mice, rabbits and pigs. Each of used models for experimental bone defects (calvarial models, long bone segmental defects, posterolateral spinal fusion and corticosteroid-induced femoral head osteonecrosis) has specific advantages and flaws. Obtaining specific and correct results is largely dependent on the used model. The brief evaluation of models could serve for standardisation of preclinical studies on bone regeneration.

BackgroundEmerging data suggest that erythropoietin (EPO) promotes neural plasticity and that iron homeostasis is needed to maintain normal physiological brain function. Cognitive functioning could therefore be influenced by endogenous EPO levels and disturbances in iron status.ObjectiveTo determine whether endogenous EPO levels and disturbances in iron status are associated with alterations in cognitive functioning in the general population.Materials and MethodsCommunity-dwelling individuals from the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study, a general population-based cohort in Groningen, Netherlands, were surveyed between 2003 and 2006. Additionally, endogenous EPO levels and iron status, consisting of serum iron, transferrin, ferritin, and transferrin saturation were analyzed. Cognitive function was assessed by scores on the Ruff Figural Fluency Test (RFFT), as a reflection of executive function, and the Visual Association Test (VAT), as a reflection of associative memory.ResultsAmong 851 participants (57% males; mean age 60 ± 13 years), higher endogenous EPO levels were independently associated with an improved cognitive function, reflected by RFFT scores (ß = 0.09, P = 0.008). In multivariable backward linear regression analysis, EPO levels were among the most important modifiable determinants of RFFT scores (ß = 0.09, P = 0.002), but not of VAT scores. Of the iron status parameters, only serum ferritin levels were inversely associated with cognitive function, reflected by VAT scores, in multivariable logistic regression analysis (odds ratio, 0.77; 95% confidence interval 0.63–0.95; P = 0.02 for high performance on VAT, i.e., ≥11 points). No association between iron status parameters and RFFT scores was identified.ConclusionThe findings suggest that endogenous EPO levels and serum ferritin levels are associated with specific cognitive functioning tests in the general population. Higher EPO levels are associated with better RFFT scores, implying better executive function. Serum ferritin levels, but not other iron status parameters, were inversely associated with high performance on the VAT score, implying a reduced ability to create new memories and recall recent past. Further research is warranted to unravel underlying mechanisms and possible benefits of therapeutic interventions.

Long-term use of immunosuppressant in kidney transplant recipients leads to poor immune function and infection with various pathogens. In recent years, along with the advancement of detection technique of human parvovirus B19 (HPV-B19) infection and the increasing quantity of kidney transplantation, the infection rate of HPV-B19 after kidney transplantation has been elevated year by year, becoming one of the major causes of pure red cell aplasia (PRCA), affecting the recovery of renal allograft function, and even leading to the injury or poor prognosis of renal allograft. To further standardize the diagnosis and treatment of HPV-B19 infection in kidney transplant recipients, Branch of Organ Transplantation of Chinese Medical Association and National Kidney Transplantation Quality Control Center jointly organized experts to formulate the clinical diagnosis and treatment specification for HPV-B19 infection after kidney transplantation from the perspectives of etiology, epidemiological characteristics, clinical manifestations, diagnosis, prevention, treatment, existing problems and prospects of HPV-B19, aiming to provide guidance for standardized prevention and treatment of HPV-B19 infection post-kidney transplantation in China. [ABSTRACT FROM AUTHOR]

Background: Treatment with erythropoietin is well established for anemia in chronic kidney disease patients but not well studied in acute kidney injury. Methods: This is a multicenter, randomized, pragmatic controlled clinical trial. It included 134 hospitalized patients with anemia defined as hemoglobin < 11 g/dL and acute kidney injury defined as an increase of serum creatinine of ≥ 0.3 mg/dL within 48 h or 1.5 times baseline. One arm received recombinant human erythropoietin 4000 UI subcutaneously every other day (intervention; n = 67) and the second received standard of care (control; n = 67) during the hospitalization until discharge or death. The primary outcome was the need for transfusion; secondary outcomes were death, renal recovery, need for dialysis. Results: There was no statistically significant difference in transfusion need (RR = 1.05, 95%CI 0.65,1.68; p = 0.855), in renal recovery full or partial (RR = 0.96, 95%CI 0.81,1.15; p = 0.671), in need for dialysis (RR = 11.00, 95%CI 0.62, 195.08; p = 0.102) or in death (RR = 1.43, 95%CI 0.58,3.53; p = 0.440) between the erythropoietin and the control group. Conclusions: Erythropoietin treatment had no impact on transfusions, renal recovery or mortality in acute kidney injury patients with anemia. The trial was registered on ClinicalTrials.gov (NCT03401710, 17/01/2018). [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) is a glycoprotein hormone that has been used to treat anemia in patients with chronic kidney disease and in cancer patients who are receiving chemotherapy. Here, we investigated the accessibility of the glutamine (Gln, Q) residues of recombinant human erythropoietin (rHuEPO) towards a thermoresistant variant microbial transglutaminase (mTGase), TG16 with the aim of developing novel rHuEPO conjugates that may potentially enhance its biological efficacy. As a model bioconjugation, we studied the reactivity of rHuEPO towards TG16 with a low molar mass amine group containing substrate, monodansyl cadaverine (MDC). The reactions were carried out at a T m of 54.3 °C, the transition temperature of rHuEPO. Characterization by SDS-PAGE and mass spectrometry confirmed the conjugates formation. Then, we examined the conjugation of rHuEPO with a biodegradable and biocompatible polyester, poly(D -sorbitol adipate) (PDSA). To achieve this, PDSA was enzymatically synthesized using lipase B from Candida antartica (CAL-B), chemically modified with side chains having free primary amine (NH 2) groups that can be acyl acceptor substrate of TG16, thoroughly characterized by 1H NMR spectroscopy, and then applied for the TG16-mediated conjugation reaction with rHuEPO. rHuEPO conjugates generated by this approach were identified by SDS-PAGE proving that the amine-grafted PDSA is accepted as a substrate for TG16. The successful conjugation was further verified by the detection of high molar mass fluorescent bands after labelling of amine-grafted PDSA with rhodamine B-isothiocyanate. Overall, this enzymatic procedure is considered as an effective approach to prepare biodegradable rHuEPO-polymer conjugates even in the presence of N- and O-glycans. [Display omitted] • A novel microbial transglutaminase (mTGase)-recognizable substrate was designed based on poly(D-sorbitol adipate) (PDSA). • Recombinant human erythropoietin (rHuEPO) was successfully conjugated with monodansyl cadaverine using variant mTGase-TG16. • rHuEPO was successfully conjugated with amine-modified PDSA in the presence of N- and O-glycans using variant mTGase-TG16. • This study provides a helpful approach to develop novel rHuEPO conjugates with potential enhanced in vivo potency. [ABSTRACT FROM AUTHOR]

Despite advances in neonatal intensive care in the recent decade, a large number of very preterm infants (VPIs) remain at risk for significant neurodevelopmental impairment (NDI). Given that there are many interventions need to be implemented during the critical perinatal period so that complications of these vulnerable VPIs could be minimized, it is urgent to develop multi-discipline strategies based on evidence to be carried out. The objective of this new term evidence-based perinatal critical strategies (EBPCS), is to provide beneficial intervention towards better neurodevelopmental outcomes, specifically for preterm infants below 28 weeks gestational age. EBPCS is defined as the management of the VPIs during the perinatal period which would include antenatal counseling with team briefing and share decision making, treat the chorioamnionitis, antenatal MgS04, antenatal steroid, delayed cord clamping/milking, neonatal resuscitation team preparation, prevention of hypothermia, immediate respiratory support with continuous positive airway pressure at delivery room, less invasive surfactant administration, early surfactant with budesonide therapy, support of cardiovascular system, early initiate of probiotics administration, early caffeine, early parenteral and enteral nutrition, promptly initiating antibiotics. These critical strategies will be discussed detail in the text; nonetheless, standardized protocols, technical skills and repeated training are the cornerstones of successful of EBPCS. Further experience from different NICU is needed to prove whether these very complicate and comprehensive perinatal critical strategies could translate into daily practice to mitigate the incidence of NDI in high-risk VPIs.

Erythropoietin (EPO) is a therapeutic protein that is widely used to increase red blood cell production in chronic kidney failure. EPO protein can be produced quickly with a transient gene expression system (TGE). However, the titer produced using TGE is usually lower than the stable gene expression system (SGE). It has been known that TGE can be improved by histone deacetylase inhibitors (iHDACs) such as valproic acid (VPA). This study was conducted to examine the VPA effect on EPO protein expression in CHO‐K1 suspension adapted cells and to find the optimum concentration of VPA on transient EPO protein production. EPO proteins was quantified using the enzyme‐linked immunosorbent assay (ELISA) method. The optimization of VPA concentrations showed that VPA increased the EPO protein yield by up to 2‐fold in transient EPO production, and the optimum concentration of VPA was 4 mM. VPA optimization was very helpful to obtain the maximum increase in the transiently expressed protein. Furthermore, this study can be used as a model to produce EPO proteins or other recombinant proteins rapidly with TGE of CHO‐K1 suspension adapted cells.

The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins. [ABSTRACT FROM AUTHOR]

Objective(s): Myocardial infarction caused by ischemia of heart tissue is the main reason for death worldwide; therefore, early detection can reduce mortality and treatment costs. Erythropoietin (EPO) has protection effects on ischemic tissue due to nonhematopoietic peptide (pHBSP; ARA-290) which is derived from the B-subunit of EPO. Materials and Methods: We designed and synthesized a modified DOTA-(Lys-Dabcyl6, Phe7)-ARA-290 using Fmoc solid-phase peptide synthesis strategies. To improve serum stability, Fmoc-Lys-(Dabcyl)- OH as lipophilic amino acid was synthesized along with Fmoc-Phe-OH which then were substituted with Arg6 and Ala7, respectively; they were then investigated for the ability to detect ischemic cardiac imaging. DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 was labeled with technetium 99m, and its radiochemical purity (RCP), stability in the presence of human serum and, specific bind to hypoxic H9c2 cells were evaluated. In vivo studies for biodistribution and SPECT scintigraphy were checked in a normal and cardiac ischemia rat model. Results: Radiolabeling purity was obtained more than 96% by ITLC, and in vitro stability of the radiopeptide up to 6 hr was 85%. The binding of 99mTc-ARA-290 to hypoxic cells was remarkably higher than normoxic cells (3 times higher than normoxic cells at 1 hr). Biodistribution and SPECT imaging on the cardiac ischemic model showed that radiopeptide considerably accumulated in the ischemic region (cardiac ischemic-to-lung rate = 3.65 ID/g % at 0.5 hr). Conclusion: The results of studies, in vitro and in vivo, indicated that 99mTc-DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 could be an appropriate candidate for early diagnosis of cardiac ischemia. [ABSTRACT FROM AUTHOR]

Abstract Currently, most patients with higher‐risk MDS are treated with 5‐azacitidine or decitabine. These agents are toxic. The treatment described here is safe, devoid of toxicity, fosters improved quality of life, and helps reduce transfusion requirements.

Currently, most patients with higher‐risk MDS are treated with 5‐azacitidine or decitabine. These agents are toxic. The treatment described here is safe, devoid of toxicity, fosters improved quality of life, and helps reduce transfusion requirements. [ABSTRACT FROM AUTHOR]

Cardiac dysfunction is an important prognostic predictor of cardiovascular mortality in patients on hemodialysis (HD). Erythropoietin (EPO) has been reported to improve cardiac function by binding to the EPO receptor (EPOR) on cardiomyocytes. This study investigated whether anti-EPOR antibodies were associated with left ventricular cardiac function in patients undergoing HD. This multicenter, cross-sectional observational study included 377 patients (median age, 70 years; 267 (70.8%) males) with chronic kidney disease (CKD) undergoing stable maintenance HD. Serum levels of anti-EPOR antibodies were measured, and echocardiography was used to assess the left ventricular mass index (LVMI) and left ventricular ejection fraction (LVEF). Anti-EPOR antibodies were found in 17 patients (4.5%). LVMI was greater (median of 135 g/m2 vs. 115 g/m2, p = 0.042), and the prevalence of LVEF < 50% was higher (35.3% vs. 15.6%, p = 0.032) in patients with anti-EPOR antibodies than in those without. Multivariable linear regression and logistic regression analysis (after adjusting for known risk factors of heart failure) revealed that anti-EPOR antibodies were independently associated with LVMI (coefficient 16.2%; 95% confidence interval (CI) 1.0–35.0%, p = 0.043) and LVEF p = 0.041). Thus, anti-EPOR antibody positivity was associated with left ventricular dysfunction in patients undergoing HD.

Background: The purpose of periodontal treatments is to reduce inflammation, restore gingival health and clinical attachment level gain by controlling microbial plaque formation and other etiological factors. One of the drugs that has been tested in many areas and shown good anti-inflammatory properties is erythropoietin (EPO). We evaluated the effect of this drug on the improvement of periodontitis after the phase I treatment. Methods: This study was conducted on 30 patients with stage III periodontitis who had at least two bilateral teeth with CAL of ≥ 5 mm and PPD ≥ 6 mm at ≥ 2 non‐adjacent teeth and bleeding on probing. After oral hygiene instruction and scaling and root planning (SRP), EPO gel containing a solution of 4000 units was applied deeply in the test group and placebo gel was deeply administered in the control pockets (5 times, every other day). The clinical parameters of the plaque index (PI), gingival index (GI), clinical attachment level (CAL), probing depth (PD) and bleeding index (BI) were measured at baseline and after three months of follow up. The P-value was set at 0.05. Results: All clinical variables improved after treatment in both groups. The BI and GI scores (which reflects the degree of gingival inflammation) showed statistically more reduction in test group. The CAL decreased from 5.1 ± 4.1 to 3.40 ± 2.71 mm; and 5.67 ± 4.32 to 4.33 ± 3.19 mm in test and control group, respectively (P < 0.00). After the treatment, there was a significant greater reduction in CAL and also PD values in test group (P < 0.01). Conclusion: Local application of EPO gel in adjunct to SRP can improve clinical inflammation and CAL gain in periodontitis. Trial registration: This study was registered at 2017-11-06 in IRCT. All procedures performed in this study were approved with ID number of IR.TUMS.DENTISTRY.REC.1396.3139 in Tehran University of medical science. [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) is a glycoprotein hormone, mainly known for its haemopoietic function. For orthopaedics, its pleiotropic effects - osteogenic and angiogenic potential, are of primary interest. The exact mechanism of EPO action is still unclear. The effects of EPO on bone healing were investigated through experiments with rats, mice, rabbits and pigs. Each of used models for experimental bone defects (calvarial models, long bone segmental defects, posterolateral spinal fusion and corticosteroid-induced femoral head osteonecrosis) has specific advantages and flaws. Obtaining specific and correct results is largely dependent on the used model. The brief evaluation of models could serve for standardisation of preclinical studies on bone regeneration. [ABSTRACT FROM AUTHOR]

Potent beneficial immunomodulatory and anti-inflammatory effects of whole-molecule erythropoietin have been demonstrated in a variety of animal disease models including experimental autoimmune encephalomyelitis (EAE); however, excessive hematopoiesis limits its use in clinical applications. Our group previously generated an Epo-derived small peptide JM4 that is side-effect free and has strong neuroprotective activity without hematologic effects. Here, we investigated the long-term clinical effects of brief treatment with JM4 in chronic relapsing EAE using bioluminescence imaging (BLI) in transgenic mice containing the luciferase gene driven by the murine GFAP promoter. EAE mice treated with JM4 exhibited marked improvement in clinical scores and showed fewer disease flareups than control animals. JM4 therapy concomitantly led to markedly decreased GFAP bioluminescence in the brain and spinal cord in both acute and chronic relapsing EAE mouse models. We found a marker for toxic A1 astrocytes, complement component C3, that is upregulated in the brain and cord of EAE mice and sharply reduced in JM4-treated animals. In addition, an abnormally leaky neurovascular unit permeability was rapidly normalized within 5 days by JM4 therapy. The prolonged therapeutic benefit seen following brief JM4 treatment in EAE mice closely resemble that recently described in humans receiving pulsed immune reconstitution therapy with the disease-modifying compounds, alemtuzumab and cladribine. Our study suggests that JM4 therapy may have widespread clinical applicability for long-term treatment of inflammatory demyelinating diseases and that BLI is a useful noninvasive means of monitoring murine disease activity of the central nervous system. [ABSTRACT FROM AUTHOR]

Background and objective While hypertension (HTN) is a major health-related threat globally, it is often an under-reported clinical condition as most of the stage I hypertensive patients do not present with any symptoms. The relationship between endogenous oxygen-sensing protein [erythropoietin (EPO) and vascular endothelial growth factor (VEGF)] levels and vascular stress in hypertensive patients is not fully understood as the mechanistic pathway by which these oxygen-sensing proteins alter the vascular physiology and cause hypertension is still a matter of debate. In light of this, we explored the role of these two proteins in the development of vascular stress including increased pulse wave velocity (PWV). We aimed to examine the correlation between oxygen-sensing proteins and vascular stress markers including PWV in hypertensive patients. Materials and methods We conducted a cross-sectional study involving age-matched participants classified into three groups (group 1: normotensive persons, n=36; group 2: stage I hypertensive patients, n=36; and group 3, stage II hypertensive patients, n=36). Adiposity-related parameters such as waist circumference (WC), hip circumference (HC), BMI, and waist-hip ratio (WHR) were measured. BP was recorded manually in resting posture by using a sphygmomanometer. PWV, which predicts the progression of BP and the development of HTN, was recorded using a periscope, which works based on the oscillometric method. Vascular stress-induced oxidative stress parameters [serum malondialdehyde (MDA) and serum nitric oxide (NO)] were also estimated by using a UV spectrophotometer. Quantitative estimations of oxygen-sensing proteins (serum EPO and serum VEGF) were done by using the ELISA kit method. The results were expressed as mean ± standard deviation (SD). The correlation between the variables was done using Spearman's correlation. A p-value <0.05 was considered statistically significant. Results Adiposity indices and vascular stiffness parameters were found to be significantly (p <0.05) increased in group 2 and group 3 compared to group 1. The levels of serum MDA were found to be significantly (p<0.05) increased in group 2 and group 3 than group 1, whereas the levels of serum NO were significantly (p<0.05) decreased in group 3 and group 2 than group 1. A significant (p<0.05) positive correlation was observed between the PWV and EPO (r=0.492) while a significant (p<0.05) negative correlation was observed between PWV and VEGF (r=-0.406) among the study population. Conclusion The results are indicative of the influence of vascular stress in stage I and II hypertensive patients. Furthermore, the relationship between oxygen-sensing proteins and vascular stress in hypertensive patients has also been established., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Institutional Ethics Committee of BLDE (Deemed to be University) issued approval IEC/No-09/2021, dated 22/01/2021. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Patil et al.)

Background: Both EPO levels and anemia have shown prognostic value in several cardiac disorders. An observational study with a prospective follow-up was performed to investigate their independent prognostic roles in severe aortic stenosis.Methods: An up to 36-month follow-up of consecutive patients with severe aortic stenosis undergoing TAVR in a high-volume center was performed. Patients with eGRF

The objective of this study was to explore the neuroprotective molecular mechanisms of erythropoietin (EPO) in rats following spinal cord injury (SCI). First, a standard SCI model was established. After drug or saline treatment was administered, locomotor function was evaluated in rats using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. H&E, Nissl, and TUNEL staining were performed to assess the ratio of cavities, number of motor neurons, and apoptotic cells in the damaged area. The relative protein and mRNA expressions were examined using western blot and qRT-PCR analyses, and the inflammatory markers, axon special protein, and neuromuscular junctions (NMJs) were detected by immunofluorescence. Both doses of EPO notably improved locomotor function, but high-dose EPO was more effective than low-dose EPO. Moreover, EPO reduced the cavity ratio, cell apoptosis, and motor neuron loss in the damaged area, but enhanced the autophagy level and extracellular-regulated protein kinase (ERK) activity. Treatment with an ERK inhibitor significantly prevented the effect of EPO on SCI, and an activator mimicked the benefits of EPO. Further investigation revealed that EPO promoted SCI-induced autophagy via the ERK signaling pathway. EPO activates autophagy to promote locomotor function recovery in rats with SCI via the ERK signaling pathway. [ABSTRACT FROM AUTHOR]

Despite advances in neonatal intensive care in the recent decade, a large number of very preterm infants (VPIs) remain at risk for significant neurodevelopmental impairment (NDI). Given that there are many interventions need to be implemented during the critical perinatal period so that complications of these vulnerable VPIs could be minimized, it is urgent to develop multi-discipline strategies based on evidence to be carried out. The objective of this new term evidence-based perinatal critical strategies (EBPCS), is to provide beneficial intervention towards better neurodevelopmental outcomes, specifically for preterm infants below 28 weeks gestational age. EBPCS is defined as the management of the VPIs during the perinatal period which would include antenatal counseling with team briefing and share decision making, treat the chorioamnionitis, antenatal MgS04, antenatal steroid, delayed cord clamping/milking, neonatal resuscitation team preparation, prevention of hypothermia, immediate respiratory support with continuous positive airway pressure at delivery room, less invasive surfactant administration, early surfactant with budesonide therapy, support of cardiovascular system, early initiate of probiotics administration, early caffeine, early parenteral and enteral nutrition, promptly initiating antibiotics. These critical strategies will be discussed detail in the text; nonetheless, standardized protocols, technical skills and repeated training are the cornerstones of successful of EBPCS. Further experience from different NICU is needed to prove whether these very complicate and comprehensive perinatal critical strategies could translate into daily practice to mitigate the incidence of NDI in high-risk VPIs. [ABSTRACT FROM AUTHOR]

The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.

The Nobel Prize in Physiology or Medicine, 2019 was awarded jointly to William G. Kaelin Jr. of Dana-Farber Cancer Institute, Sir Peter J. Ratcliffe of Francis Crick Institute and University of Oxford and Gregg L. Semenza of Johns Hopkins University for their discovery of how cells sense and respond to varying oxygen levels. [ABSTRACT FROM AUTHOR]

In order to study the impact of zinc and copper on the titer levels of mAb and recombinant protein in CHO cells, the IgG-expressing (DP12) and EPO-expressing (SK15) cell lines were cultured in chemically defined media with increasing concentrations of either metal. Supplementation with 25 mg/l in CDM media resulted in a significant increase in EPO (1.7-fold) and IgG (2.6-fold) titers compared to control (no added zinc). Titers at this Zn concentration in CDM containing the insulin replacing agent aurintricarboxylic acid (ATA) (CDM + A) showed a 1.8-fold (EPO) and 1.2-fold (IgG) titers increase compared to control. ATA appeared to also reduce the specific productivity (Qp) enhancement induced by Zn-25, with up to 4.9-fold (DP12) and 1.9-fold (SK15) Qp increase in CDM compared to the 1.6-fold (DP12) and 1.5-fold (SK15) Qp increase observed in CDM + A. A 31% reduced Viable Cell Density (VCD) in DP12 was observed in both Zn-supplemented media (3 × 106 cells/ml vs 4.2 × 106 cells/ml, day 5), whereas SK15 Zn-25 cultures displayed a 24% lower peak only in CDM + A (2.2 × 106 cells/ml vs 3.2 × 106 cells/ml, day 5). Supplementation with copper at 13.7–20 mg/l resulted in less significant cell line/product-type dependent effects on titer, VCD and Viability. Analysis of the energetic phenotype of both cell lines in 25 mg/l Zn-supplemented CDM media revealed a twofold increase in the oxygen consumption rate (OCR) compared to non-supplemented cells. Together, these data suggest that high zinc supplementation may induce an increase in oxidative respiration metabolism that results in increased Qp and titers in suspension CHO cultures. [ABSTRACT FROM AUTHOR]

The hydroxylation of prolyl-residues in eukaryotes is important in collagen biosynthesis and in hypoxic signalling. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are drug targets for the treatment of anaemia, while the procollagen prolyl hydroxylases and other 2-oxoglutarate dependent oxygenases are potential therapeutic targets for treatment of cancer, fibrotic disease, and infection. We describe assay development and inhibition studies for a procollagen prolyl hydroxylase from Paramecium bursaria chlorella virus 1 (vCPH). The results reveal HIF PHD inhibitors in clinical trials also inhibit vCPH. Implications for the targeting of the human PHDs and microbial prolyl hydroxylases are discussed. [ABSTRACT FROM AUTHOR]

Existing therapies for Parkinson’s disease (PD) are only symptomatic. As erythropoietin (EPO) is emerging for its benefits in neurodegenerative diseases, here, we test the protective effect driven by EPO in in vitro (SH-SY5Y cells challenged by MPP+) and in vivo (C57BL/6J mice administered with MPTP) PD models. EPO restores cell viability in both protective and restorative layouts, enhancing the dopaminergic recovery. Specifically, EPO rescues the PD-induced damage to mitochondria, as shown by transmission electron microscopy, Mitotracker assay and PINK1 expression. Moreover, EPO promotes a rescue of mitochondrial respiration while markedly enhancing the glycolytic rate, as shown by the augmented extracellular acidification rate, contributing to elevated ATP levels in MPP+-challenged cells. In PD mice, EPO intrastriatal infusion markedly improves the outcome of behavioral tests. This is associated with the rescue of dopaminergic markers and decreased neuroinflammation. This study demonstrates cellular and functional recovery following EPO treatment, likely mediated by the 37 Kda isoform of the EPO-receptor. We report for the first time, that EPO-neuroprotection is exerted through restoring ATP levels by accelerating the glycolytic rate. In conclusion, the redox imbalance and neuroinflammation associated with PD may be successfully treated by EPO.

The aim of this study was to optimize the formulation of erythropoietin (EPO) using amino acids instead of human serum albumin (HSA) and to evaluate its in vivo stability in order to avoid the risk of viral contamination and antigenicity. Different EPO formulations were developed in such a way as to allow studying the effects of amino acids and surfactants on the EPO stability profile. The main techniques applied for EPO analysis were ELISA, Bradford method, and SDS gel electrophoresis. The in vivo stability was evaluated in a Balb-c mouse animal model. The results showed that the presence of surfactant was very useful in preventing the initial adsorption of EPO on the walls of vials and in minimizing protein aggregation. Amino acid combinations, glycine with glutamic acid, provided maximum stability. Formulation F4 (containing glycine, glutamic acid and Tween 20) showed minimum aggregation and degradation and in vivo activity equivalent to commercially available HSA-stabilized EPO (Eprex®).

OBJECTIVES: Exogenously administered recombinant human erythropoietin (rhEPO) has been reported to exhibit neuroprotective effects in animal models. However, there are still have some controversies that combination of EPO and tissue plasminogen activator (tPA) in acute ischemic stroke. In the present study, we investigated the effects of local intra-arterial infusion of low-dose EPO in combination with tPA on focal cerebral ischemic stroke. MATERIALS AND METHODS: Sixty adult male Sprague-Dawley rats were randomly divided into five groups, including sham, vehicle, EPO, tPA, and EPO+tPA groups. Rats were subjected to middle cerebral artery occlusion (MCAO) and administrated with EPO (800 U/kg, middle cerebral artery injection), tPA (10 mg/kg, tail vein injection), EPO+tPA, or saline (vehicle) onset of reperfusion. Neurobehavioral deficits, infarct volume, brain edema, the expression of tight junction proteins (Claudin-5, Occludin), and AQP4 were assessed following 2 h ischemia and 24 h reperfusion. The number of apoptotic cells in the periinfarct region was detected by the terminal deoxyribonucleotide transferase dUTP nick end labeling (TUNEL) staining. RESULTS: The neurobehavioral deficits, brain infarct volume, edema volume, TUNEL-positive cells and downregulation of Claudin-5 and Occludin were alleviated by EPO or EPO plus tPA, following the ischemia/reperfusion (I/R) in rats. The EPO and EPO plus tPA both reduced the upregulation of AQP4 in the ischemic brain tissue. CONCLUSION: Our data demonstrate local intra-arterial infusion of low-dose EPO in combination with tPA protected against focal cerebral ischemia in rats manifested by a decrease in brain edema and blood-brain barrier (BBB) disruption after 2 h ischemia and 24 h reperfusion.

In adult’s burn injuries belong to the top 15 causes of injury. Annually more than a million patients receive specialized treatment. Improving burned patients’ outcomes is still a challenge. Effects of erythropoietin (EPO) are reported to be pro-angiogenic, pro-regenerative, anti-inflammatory, immunomodulatory and hypoxia/ischemia protective. Study objectives were to demonstrate cytoprotective and regenerative effects of EPO in burned patients in terms of improved wound healing, reduced morbidity and mortality. This was a prospective, placebo-controlled, randomized, double-blind trial. The trial was conducted in 13 specialized burn care centers in Germany. Adult Patients with 2b° or 3° burn injuries were included. Patients received state of the art burn care including obligatory split skin graft transplantation. Study medication was EPO or placebo every other day for 21 days. Between 12/08 and 06/14, 116 patients were randomized, 84 received study medication (EPO 45, Placebo 39). Primary endpoint analysis revealed inconclusive results, as only a minority of patients reached the primary endpoint [100% re-epithelialization: EPO: 23% (9/40); Placebo 30% (11/37)]. Several secondary endpoints such as SOFA score (morbidity), EPO level in blood and wound healing onset revealed clinical, and statistically significant results in favor of the EPO group. Adverse Events (AEs) and Severe Adverse Events (SAEs) were in expected ranges; AEs EPO: 80%, (36/45), Placebo: 77%, (30/39); SAEs EPO: 24%, (11/45), Placebo: 24%, (8/39). Out of 84 patients two died, one per group, thus mortality was lower than expected. Results (SOFA score) indicate a lower morbidity of the EPO group, suggesting pro-regenerative effects of EPO in burned patients. Higher EPO levels might influence the faster onset of re-epithelialization in the first 10 days of the treatment. Both effects could reveal new therapeutic options.Clinical Trial Registration: ISRCT Number: ISRCTN95777824 and EudraCT Number: 2006-002886-38, Protocol Number: 0506.