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.

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

Unfortunately, the original version of this article contained a mistake in the arrangement of representative cell images in Fig. 2. In this figure, the same representative image for Aß group was mistakenly placed for Aß + LY group. The corrected form of this figure is provided in this correction.

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.

CEPO-Fc (An EPO Derivative) Protects Hippocampus Against Aß-induced Memory Deterioration: A Behavioral and Molecular Study in a Rat Model of Aß Toxicity.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease, characterized by extracellular deposition of senile plaques, mostly amyloid ß-protein (Aß) and neuronal loss. The neuroprotective effects of erythropoietin (EPO) have been reported in some models of neurodegenerative disease, but because of its hematopoietic side effects, its derivatives lacking hematopoietic bioactivity is recommended. In this study, the neuroprotective effects of carbamylated erythropoietin-Fc (CEPO-Fc) against beta amyloid-induced memory deficit were evaluated. Adult male Wistar rats weighing 250-300 g were bilaterally cannulated into CA1. Aß25-35 was administered intrahippocampally for 4 consecutive days (5 µg/2.5 µL/each side/day). CEPO-Fc (500 or 5000 IU) was injected intraperitoneally during days 4-9. Learning and memory performance of rats was assessed on days 10-13 using Morris Water Maze, then hippocampi were isolated and the amount of activated forms of hippocampal MAPKs' subfamily, Akt/GSK-3ß and MMP-2 were analyzed using Western blot. From the behavioral results, it was revealed that CEPO-Fc treatment in both 500 and 5000 IU significantly reversed Aß-induced learning and memory deterioration. From the molecular analysis, an increment of MAPKs and MMP-2 activity and an imbalance in Akt/GSK-3ß signaling after Aß25-35 administration was observed. CEPO-Fc treatment prevented the elevation of hippocampal of P38, ERK, MMP-2 activity and also Akt/GSK-3ß signaling impairment induced by Aß25-35 but it had no effect on JNK. It seems that CEPO-Fc prevents Aß-induced learning and memory deterioration, and also modulates hippocampal MAPKs, Akt/GSK-3ß and MMP-2 activity. This study suggests that CEPO-Fc can be considered as a potential therapeutic strategy for memory deficits like AD.

Novel human renal proximal tubular cell line for the production of complex proteins.

Human host cell lines for the production of biopharmaceutical proteins are of interest due to differences in the glycosylation patterns of human and animal cell lines. Specifically, sialylation, which has a major impact on half-life and immunogenicity of recombinant biopharmaceuticals, differs markedly. Here, we established and characterized an immortalized well documented and serum-free host cell line, RS, from primary human renal proximal tubular epithelial cells (RPTEC). In order to test its capacity to produce complex glycosylated proteins, stable recombinant human erythropoietin (rhEpo) producing clones were generated. The clone with highest productivity, RS-1C9 was further characterized and showed stable productivity. Biological activity was observed in in vitro assays and 28% of rhEpo glyco-isoforms produced by RS-1C9 were in range and distribution of the biological reference standard (BRP) isoform, as compared to 11.5% of a CHO based rhEpo. Additionally, cellular α-2,6 sialylation, Galactose-alpha-1,3-galactose (alpha-Gal) and N-glycolylneuraminic acid (NeuGc) patterns compare favourably to CHO cells. While productivity of RS still needs optimization, its amenability to upscaling in bioreactors, its production of glyco-isoforms that will increase yields after down-stream processing of about 2.5 fold, presence of sialylation and lack of Neu5Gc recommend RS as alternative human host cell line for production of biopharmaceuticals.

IgM characterization directly performed in crude culture supernatants by a new simple electrophoretic method.

A new electrophoretic technique for the qualitative and quantitative analyses of IgM isoforms and fragments has been developed. IgMs which are more complex than many other recombinantly expressed immunoglobulins are characterized by their high molecular weighted active forms and many additional isoforms and fragments in the molecular range between 25 and 1200kDa. To analyze the multimers, isoforms and fragments simultaneously a high-resolution method, which enables sufficient migration and separation is required. Furthermore, this method should be appropriate to analyze IgMs in crude culture supernatants as well as purified samples. Simple sample preparation avoiding unspecific protein loss has been established. Currently no standard method to analyze all of them accordingly is available. The IgM-SDS-PAGE investigated for this purpose includes all these aspects. The combination of simple sample preparation and the application of precast gels make this electrophoretic method suitable for research but also quality control. The selective quantification of the multimers and the relative isoform distribution were performed by sensitive Sypro Ruby staining obtaining reliable and reproducible data in clone screening and process development which has been demonstrated by recombinantly expressed IgMs with significantly different isoform pattern.

Confocal microscopy of giant vesicles supports the absence of HIV-1 neutralizing 2F5 antibody reactivity to plasma membrane phospholipids.

The broadly neutralizing anti-HIV-1 2F5 monoclonal antibody recognizes a gp41 epitope proximal to the viral membrane. Potential phospholipid autoreactivity at cell surfaces has raised concerns about the use of this antibody for development of vaccines or immunotherapy. In this study, confocal microscopy of giant unilamellar vesicles (GUVs) was used to assess 2F5 reactivity with phospholipids assembled into bilayers with surface charge and curvature stress approximating those of the eukaryotic plasma membranes. Antibody partitioning into lipid bilayers required the specific recognition of membrane-inserted epitope, indicating that 2F5 was unable to directly react with GUV phospholipids, even under fluid phase segregation conditions. Our results thus support the feasibility of raising 2F5-like neutralizing responses through vaccination, and the medical safety of mAb infusions.

Proline is not uniquely capable of providing the pivot point for domain swapping in 2G12, a broadly neutralizing antibody against HIV-1.

The human monoclonal antibody 2G12 is a member of a small group of broadly neutralizing antibodies against human immunodeficiency virus type 1. 2G12 adopts a unique variable heavy domain-exchanged dimeric configuration that results in an extensive multivalent binding surface and the ability to bind with high affinity to densely clustered high mannose oligosaccharides on the "silent" face of the gp120 envelope glycoprotein. Here, we further define the amino acids responsible for this extraordinary domain-swapping event in 2G12.

Topological transformation of liposomes by a membrane-affecting domain of recombinant human erythropoietin.

Recombinant human erythropoietin (rh-Epo) is well accepted as a hematopoietic drug, but many other pleiotropic properties are currently under investigation. Rh-Epo-induced receptor-mediated signal transductions are accompanied with membrane dynamic processes, which facilitate the activation of individual pathways. However, its direct effect on membrane dynamics is still unknown. In the present study, we have proven the capability of rh-Epo to associate to and transform artificial lipid membranes. Association studies using neutral, negatively, and positively charged liposomes with the native as well as modified rh-Epo were performed and analyzed by transmission electron microscopy and differential scanning calorimetry. By these studies, we demonstrated that rh-Epo has the capability to transform negatively charged unilamellar vesicles into so-called disc-like micelles. Rh-Epo association to the negatively charged head groups via lysine and arginine initiates this transformation. At physiological temperatures, conformational changes within the rh-Epo structure expose a defined amino-acid sequence, which is able to induce the formation of discoid membrane structures. Enzymatic digestion, analysis, and isolation of related peptides by rp-HPLC and characterization by MS/MS enabled the identification of the membrane-affecting domain of rh-Epo (MAD-E) that represents the exposed helix B of rh-Epo. Finally, association studies performed with these peptides confirmed that the MAD-E is responsible for the formation of disc-like micelles. Since this helix B of rh-Epo has recently been supposed to be involved in the activation of neuroprotective pathways, we believe that the membrane-transforming capacity of rh-Epo participates in the proliferative activity of rh-Epo.

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection.

The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.

Blom7alpha is a novel heterogeneous nuclear ribonucleoprotein K homology domain protein involved in pre-mRNA splicing that interacts with SNEVPrp19-Pso4.

The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEV(Prp19-Pso4) complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEV(Prp19-Pso4), is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7alpha. Blom7alpha is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7alpha, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7alpha co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7alpha to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7alpha influences splice site selection using two different minigene constructs. Indeed, both 5'- as well as 3'-site selection was altered upon Blom7alpha overexpression. Thus we suggest that Blom7alpha is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEV(Prp19-Pso4) complex at the splice sites.

Characterization of a trimeric MPER containing HIV-1 gp41 antigen.

The membrane-proximal external region (MPER) of gp41 is considered as a prime target for the induction of neutralizing antibodies, since it contains the epitopes for three broadly neutralizing antibodies (2F5, 4E10 and Z13). Here we present a novel gp41 construct (HA-gp41) comprising gp41 HR2 and MPER fused to two triple-stranded coiled-coil domains at both ends. HA-gp41 is trimeric, has a high helical content in solution and forms rod-like structures as revealed by negative staining electron microscopy. Immunization of rabbits with HA-gp41 induced antibodies directed against MPER, which failed to exert significant neutralization capacity against envelopes from primary isolates. Thus trimerisation of MPER regions does not suffice to induce a potent neutralizing antibody response specific for conserved regions within gp41.

CHO-recombinant human growth hormone as a protease sensitive reporter protein.

Protein-free media are gaining more and more interest in mammalian cell culture technology. However, the range of commercially available protein-free media is wide, but lack of serum causes the lack of various substances (Keenan et al. in Cytotechnology, 50(1-3):49-56, 2006) which must be substituted case by case. Details on the composition of protein-free media are often unavailable or inaccessible in some cases, and as a consequence, there is an obvious need for testing procedures in order to evaluate the various commercialised products for their performance. Additionally, negative effects of tryptic meat digests on product quality have been reported in the literature (Gu et al. in Biotech Bioeng 56 (4):353-341, 1997). In the present studies of comparing various protein-free media for their suitability in propagation of recombinant CHO cells expressing human growth hormone (hGH), we have found somatotropin to be an excellent candidate for detection of protease activity. Somatotropin contains protease recognition sites for numerous proteases located around amino-acid residues 134-150. In this study, we demonstrate highly specific cleavage of recombinant hGH during batch cultivation. Analysis of the digested molecule was then performed by convergent methods like SDS-PAGE, HPLC and mass spectroscopy, and the results indicate hGH to be an ideal candidate for media and component screening in mammalian cell culture.

Broad neutralization of human immunodeficiency virus type 1 (HIV-1) elicited from human rhinoviruses that display the HIV-1 gp41 ELDKWA epitope.

In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.

Large-scale production and characterization of novel CD4+ cytotoxic T cells with broad tumor specificity for immunotherapy.

Immune-cell-based approaches using cytotoxic and dendritic cells are under constant scrutiny to design novel therapies for the treatment of tumors. These strategies are hampered by the lack of efficient and economical large-scale production methods for effector cells. Here we describe the propagation of large amounts of a unique population of CD4(+) cytotoxic T cells, which we termed tumor killer T cells (TKTC), because of their potent and broad antitumor cell activity. With this cultivation strategy, TKTCs from peripheral blood mononuclear cells are generated within a short period of time using a pulse with a stimulating cell line followed by continuous growth in serum-free medium supplemented with a mixture of interleukin-2 and cyclosporin A. Expression and functional profiling did not allow a classification of TKTCs to any thus far defined subtype of T cells. Cytotoxic assays showed that TKTCs kill a panel of tumor targets of diverse tissue origin while leaving normal cells unaffected. Blocking experiments revealed that TKTC killing was, to a significant extent, mediated by tumor necrosis factor-related apoptosis-inducing ligand and was independent of MHC restriction. These results suggest that TKTCs have a high potential as a novel tool in the adoptive immunotherapy of cancer.

Telomerase immortalized human amnion- and adipose-derived mesenchymal stem cells: maintenance of differentiation and immunomodulatory characteristics.

Cell banking of mesenchymal stem cells (SCs) from various human tissues has significantly increased the feasibility of SC-based therapies. Sources such as adipose tissue and amnion offer outstanding possibilities for allogeneic transplantation due to their high differentiation potential and their ability to modulate immune reaction. Limitations, however, concern the reduced replicative potential as a result of progressive telomere erosion, which hampers scaleable production and long-term analysis of these cells. Here we report the establishment and characterization of two human amnion-derived and two human adipose-derived SC lines immortalized by ectopic expression of the catalytic subunit of human telomerase (hTERT). hTERT overexpression resulted in continuously growing SC lines that were largely unaltered concerning surface marker profile, morphology, karyotype, and immunosuppressive capacity with similar or enhanced differentiation potential for up to 87 population doublings. While all generated lines showed equal immunomodulation compared to the parental cells, one of the amnion-derived immortalized lines resulted in significantly increased immunogenicity. Although telomerase proves as important tool for immortalizing cells, our data emphasize the need for careful and standardized characterization of each individual cell population for cell banks.

The absence of effect of gene copy number and mRNA level on the amount of mAb secretion from mammalian cells.

Recombinant human antibody production represents a major growing class of biopharmaceuticals based on the technological progress within the last decades especially in CHO cells. The HIV neutralizing human monoclonal antibody 2F5 was developed as hybridoma from human lymphocyte preparations. In order to estimate the potential of recombinant 2F5-expressing CHO cells, we generated different recombinant CHO cell lines by varying regulatory sequences, the codon usage, the signal peptides, and the transfection technique. These 2F5-expressing cell lines were developed by selection of the best producer, clone homogeneity, and clone stability. The gene copy number of the clones differed significantly due to methotrexate amplification. In one cell line, we identified only one copy of heavy chain and two copies of light chain. Neither the gene copy number nor the promoter was found to influence the amount of transcript exclusively emphasizing the positioning effect of the transgene. Messenger RNA levels were highest in 2F5/CO and may have resulted from a combination of the promoter and codon-optimized sequences, but unexpectedly, the amount of secreted product was not elevated in this configuration. In our example, translational and post-translational limitations are responsible for decreased antibody secretion.

hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics.

Telomere-dependent replicative senescence is one of the mechanisms that limit the number of population doublings of normal human cells. By overexpression of telomerase, cells of various origins have been successfully immortalized without changing the phenotype. While a limited number of telomerase-immortalized cells of epithelial origin are available, none of renal origin has been reported so far. Here we have established simple and safe conditions that allow serial passaging of renal proximal tubule epithelial cells (RPTECs) until entry into telomere-dependent replicative senescence. As reported for other cells, senescence of RPTECs is characterized by arrest in G1 phase, shortened telomeres, staining for senescence-associated beta-galactosidase, and accumulation of gamma-H2AX foci. Furthermore, ectopic expression of the catalytic subunit of telomerase (TERT) was sufficient to immortalize these cells. Characterization of immortalized RPTEC/TERT1 cells shows characteristic morphological and functional properties like formation of tight junctions and domes, expression of aminopeptidase N, cAMP induction by parathyroid hormone, sodium-dependent phosphate uptake, and the megalin/cubilin transport system. No genomic instability within up to 90 population doublings has been observed. Therefore, these cells are proposed as a valuable model system not only for cell biology but also for toxicology, drug screening, biogerontology, as well as tissue engineering approaches.