Variation in recombination rate may bias human genetic disease mapping studies.

PubMed

Boyle, A Susannah; Noor, Mohamed A F

2004-11-01

The availability of the human genome sequence and variability information (as from the International HapMap project) will enhance our ability to map genetic disorders and choose targets for therapeutic intervention. However, several factors, such as regional variation in recombination rate, can bias conclusions from genetic mapping studies. Here, we examine the impact of regional variation in recombination rate across the human genome. Through computer simulations and literature surveys, we conclude that genetic disorders have been mapped to regions of low recombination more often than expected if such diseases were randomly distributed across the genome. This concentration in low recombination regions may be an artifact, and disorders appearing to be caused by a few genes of large effect may be polygenic. Future genetic mapping studies should be conscious of this potential complication by noting the regional recombination rate of regions implicated in diseases.

Prevalence of IgG antibodies to human parvovirus B19 in haemophilia children treated with recombinant factor (F)VIII only or with at least one plasma-derived FVIII or FIX concentrate: results from the French haemophilia cohort.

PubMed

Gaboulaud, Valérie; Parquet, Armelle; Tahiri, Cedric; Claeyssens, Ségolène; Potard, Valérie; Faradji, Albert; Peynet, Jocelyne; Costagliola, Dominique

2002-02-01

Human parvovirus B19 (B19) has been transmitted by some brands of virally attenuated plasma-derived factor VIII (FVIII) or IX (FIX) concentrates. To quantify the differences of human parvovirus B19 risk transmission between albumin-stabilized recombinant factor and plasma-derived factor, we studied the prevalence of IgG antibodies to B19 (anti-B19) in 193 haemophiliac children between 1 and 6-years of age who had previously been treated with albumin-stabilized recombinant FVIII only (n = 104), and in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates (n = 89). Association between the prevalence of anti-B19 and the treatment group was analysed using multivariate logistic regression. Age, severity and type of haemophilia, number of cumulative days of exposure to factor VIII or IX, previous history of red blood cells or plasma transfusion were considered as potential confounding variables. A higher prevalence of anti-B19 was found in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates than in children treated with albumin- stabilized recombinant FVIII only (OR: 22.3; CI: 7.9-62.8), independently of the other factors studied.

Tissue engineering skeletal muscle for orthopaedic applications

NASA Technical Reports Server (NTRS)

Payumo, Francis C.; Kim, Hyun D.; Sherling, Michael A.; Smith, Lee P.; Powell, Courtney; Wang, Xiao; Keeping, Hugh S.; Valentini, Robert F.; Vandenburgh, Herman H.

2002-01-01

With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

A review of three stand-alone topical thrombins for surgical hemostasis.

PubMed

Cheng, Christine M; Meyer-Massetti, Carla; Kayser, Steven R

2009-01-01

Topical thrombins are active hemostatic agents that can be used to minimize blood loss during surgery. Before 2007, the only topical thrombins available were derived from bovine plasma. Antibody formation to bovine thrombin and/or factor V, with subsequent risk of cross-reactivity with human factor V, and hemorrhagic complications associated with human factor-V deficiencies have been described in case reports of surgeries in which bovine thrombins were used. This risk is now included in the boxed warning section of the bovine thrombin prescribing information. In 2007 and 2008, 2 new topical thrombins from nonbovine sources received approval for use from the US Food and Drug Administration. The 3 active topical thrombins that are currently marketed are bovine plasma-derived thrombin, human plasma-derived thrombin, and human recombinant thrombin. The purpose of this review was to evaluate the literature on the efficacy and safety of topical thrombins and discuss the pharmacoeconomic considerations associated with their use. PubMed, EMBASE, and International Pharmaceutical Abstracts were searched for relevant papers published in English through October 10,2008, using the terms thrombin, human recombinant thrombin, bovine thrombin, plasma derived thrombin, and topical thrombin. Manufacturer-provided materials were also reviewed. Abstracts and unpublished data, as well as evaluations of sealants, adhesives, glues, and other hemostats that contain thrombin mixed with fibrinogen and other clotting factors, were excluded. Four randomized, double-blind studies involving the active, stand-alone topical thrombins were found. The bovine thrombin involved in these studies was the predecessor to the currently marketed, highly purified bovine formulation. No studies comparing the human products, studies involving the highly purified bovine preparation, or placebo-controlled studies involving bovine thrombin were found. In a Phase III comparison of human recombinant thrombin and bovine thrombin, the percentages of patients who achieved hemostasis within 10 minutes of topical thrombin application were 95.4% and 95.1%, respectively (95% CI, -3.7 to 5.0). The incidence of hemostasis within 10 minutes was also similar in a Phase III comparison of human plasma-derived thrombin and bovine thrombin (both, 97.4% [95% CI, 0.96 to 1.05]). In the study that compared human recombinant and bovine thrombin, the incidence of antiproduct antibody formation was 21.5% (43/200) in the bovine thrombin group and 1.5% (3/198) in the human recombinant thrombin group (P < 0.001); patients with antibodies to bovine thrombin had numerically higher incidences of bleeding or thromboembolic events than did patients without these antibodies (19% vs 13%; P value not reported). Human plasma-derived thrombin is available as a frozen sterile solution that must be thawed before application, whereas the human recombinant and bovine plasma-derived products are supplied as unrefrigerated sterile powders that must be reconstituted before use. The human thrombins are more costly than bovine thrombin on a per-vial basis. The average wholesale prices (US $, 2008) for 5000-IU vials of bovine thrombin and human recombinant thrombin were $87.85 and $103.20, respectively; the average wholesale price for a 4000- to 6000-IU vial of human plasma-derived thrombin was $96.00. Topical thrombins vary in the ways in which they are manufactured and their safety profiles, storage requirements, and costs. Human recombinant thrombin and human plasma-derived thrombin have each been shown to have hemostatic efficacy comparable to that of bovine thrombin. Bovine thrombin carries the risk of formation of cross-reactive antibodies to bovine thrombin, factor V, and other impurities that may be present in these formulations. Immunogenicity data for the currently marketed, highly purified bovine thrombin relative to older formulations of bovine thrombin could not be found. Whether the potential safety advantage justifies the added cost of the human products remains to be established.

A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis

PubMed Central

Noor, Nudrat; Bitoun, Emmanuelle; Tumian, Afidalina; Imbeault, Michael; Chapman, J Ross; Aricescu, A Radu

2017-01-01

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers. PMID:29072575

Monitoring of treatment with vitamin K antagonists: recombinant thromboplastins are more sensitive to factor VII than tissue-extract thromboplastins.

PubMed

Biedermann, J S; van den Besselaar, A M H P; de Maat, M P M; Leebeek, F W G; Kruip, M J H A

2017-03-01

Essentials Differences in sensitivity to factor VII (FVII) have been suggested between thromboplastins. FVII-induced International Normalized Ratio (INR) changes differ between commercial reagents. Recombinant human thromboplastins are more sensitive to FVII than tissue-extract thromboplastins. Thromboplastin choice may affect FVII-mediated INR stability. Background Differences regarding sensitivity to factor VII have been suggested for recombinant human and tissue-extract thromboplastins used for International Normalized Ratio (INR) measurement, but the evidence is scarce. Differences in FVII sensitivity are clinically relevant, as they can affect INR stability during treatment with vitamin K antagonists (VKAs). Objectives To determine whether commercial thromboplastins react differently to changes in FVII. Methods We studied the effect of addition of FVII on the INR in plasma by using three tissue-extract (Neoplastin C1+, Hepato Quick, and Thromborel S) and three recombinant human (Recombiplastin 2G, Innovin, and CoaguChek XS) thromboplastins. Three different concentrations of purified human FVII (0.006, 0.012 and 0.062 μg mL -1 plasma), or buffer, were added to five certified pooled plasmas of patients using VKAs (INR of 1.5-3.5). Changes in FVII activity were measured with two bioassays (Neoplastin and Recombiplastin), and relative INR changes were compared between reagents. Results After addition of 0.062 μg mL -1 FVII, FVII activity in the pooled plasmas increased by approximately 20% (Neoplastin) or 32% (Recombiplastin) relative to the activity in pooled normal plasma. All thromboplastins showed dose-dependent INR decreases. The relative INR change in the pooled plasmas significantly differed between the six thromboplastins. No differences were observed among recombinant or tissue-extract thromboplastins. Pooled results indicated that the FVII-induced INR change was greater for recombinant than for tissue-extract thromboplastins. Conclusions Differences regarding FVII sensitivity exist between various thromboplastins used for VKA monitoring. Recombinant human thromboplastins are more sensitive to FVII than tissue-extract thromboplastins. Therefore, thromboplastin choice may affect FVII-mediated INR stability. © 2017 International Society on Thrombosis and Haemostasis.

Recombinant Factor VIIa Reduces Rebleed Hemorrhage Volume in a Swine Aortotomy Model: A Randomized Double-Blinded Study

DTIC Science & Technology

2008-06-01

Bpops,[ a dose of 90 Hg kgj1 will produce serum concentrations of factor VII high enough to reduce hemorrhage by accelerating thrombin production and...interaction is species-specific, and human FVII seems to have only between 5% and 50% activity when exposed to porcine tissue factor (15). The results...Copyright @ 200 by the Shock Society. Unauthorized reproduction of this article is prohibited.8 RECOMBINANT FACTOR VIIA REDUCES REBLEED HEMORRHAGE

Purification and Characterization of Tagless Recombinant Human Elongation Factor 2 Kinase (eEF-2K) Expressed in Escherichia coli

PubMed Central

Abramczyk, Olga; Tavares, Clint D. J.; Devkota, Ashwini K.; Ryazanov, Alexey G.; Turk, Benjamin E.; Riggs, Austen F.; Ozpolat, Bulent; Dalby, Kevin N.

2012-01-01

The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His6-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ~ 85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme. PMID:21605678

Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

PubMed Central

Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

2008-01-01

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. PMID:18456721

The future of recombinant growth factors in wound healing.

PubMed

Robson, M C; Mustoe, T A; Hunt, T K

1998-08-01

For more than a decade, clinical trials have been conducted of the application of topical exogenous recombinant growth factors in attempts to accelerate the healing of chronic wounds. Although the results of some of these trials have been encouraging, overall the results have been somewhat discouraging. Much of the difficulty lies in the paucity of carefully controlled clinical trials of wound healing. Since wound healing is a complex process that can be influenced, both positively and negatively, by many factors, designing these trials has proved difficult. To date, only a single recombinant growth factor-recombinant human platelet-derived growth factor-BB (rhPDGF-BB)- has been approved by the US Food and Drug Administration; and that only for use in diabetic foot ulcers. It is unlikely, however, that a single growth factor will be able to resolve all issues of repair or strengthen all vulnerabilities of chronic wounds. Our expectation, therefore, is that growth factors, cytokines, and other biologic agents will be used more specifically in the future, for example, by targeting growth factor therapy at those specific components or processes that a given wound uses to heal.

A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

PubMed

Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

2014-03-03

Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

Recombinant factor VIIa (eptacog alfa): a review of its use in congenital hemophilia with inhibitors, acquired hemophilia, and other congenital bleeding disorders.

PubMed

Croom, Katherine F; McCormack, Paul L

2008-01-01

Recombinant factor VIIa (NovoSeven; also known as recombinant activated factor VII or eptacog alfa) is structurally similar to human plasma-derived coagulation factor VIIa, but is manufactured using DNA biotechnology. Recombinant factor VIIa interacts with thrombin-activated platelets to produce a thrombin burst leading to accelerated fibrin clot formation localized to the site of vascular injury. It is approved in many countries for use as an intravenous hemostatic agent in patients with congenital hemophilia with inhibitors, and also for acquired hemophilia, factor VII deficiency, and Glanzmann thrombasthenia in some countries. Studies have shown it to be effective and generally well tolerated when used intravenously to treat bleeding episodes or provide hemostatic cover during surgery in patients with congenital hemophilia with inhibitors, acquired hemophilia, factor VII deficiency or Glanzmann thrombasthenia. Based on available data, its efficacy in terms of patient-assessed response may be similar to that of activated prothrombin complex concentrate (aPCC), but treatment with a single 270 microg/kg dose of recombinant factor VIIa might reduce the need for rescue therapy compared with aPCC. Recombinant factor VIIa is not immunogenic in patients with hemophilia, does not produce an anamnestic response in hemophilia patients with inhibitors, and has very low thrombogenicity. It is recommended in guidelines as the treatment of choice for bleeds in patients with hemophilia B with high-responding inhibitors and for patients with factor VII deficiency, and is also a first-line therapeutic option for high-responder hemophilia A patients with inhibitors and those with acquired hemophilia. Cost data from pharmacoeconomic analyses support its use in hemophilia patients with inhibitors. Thus, recombinant factor VIIa is a valuable treatment option for patients with these rare, but potentially serious, bleeding disorders.

Four-Week Studies of Oral Hypoxia-Inducible Factor–Prolyl Hydroxylase Inhibitor GSK1278863 for Treatment of Anemia

PubMed Central

Meadowcroft, Amy M.; Maier, Rayma; Johnson, Brendan M.; Jones, Delyth; Rastogi, Anjay; Zeig, Steven; Lepore, John J.; Cobitz, Alexander R.

2016-01-01

Hypoxia-inducible factor prolyl hydroxylase inhibitors stabilize levels of hypoxia-inducible factor that upregulate transcription of multiple genes associated with the response to hypoxia, including production of erythropoietin. We conducted two phase 2a studies to explore the relationship between the dose of the hypoxia-inducible factor–prolyl hydroxylase inhibitor GSK1278863 and hemoglobin response in patients with anemia of CKD (baseline hemoglobin 8.5–11.0 g/dl) not undergoing dialysis and not receiving recombinant human erythropoietin (nondialysis study) and in patients with anemia of CKD (baseline hemoglobin 9.5–12.0 g/dl) on hemodialysis and being treated with stable doses of recombinant human erythropoietin (hemodialysis study). Participants were randomized 1:1:1:1 to a once-daily oral dose of GSK1278863 (0.5 mg, 2 mg, or 5 mg) or control (placebo for the nondialysis study; continuing on recombinant human erythropoietin for the hemodialysis study) for 4 weeks, with a 2-week follow-up. In the nondialysis study, GSK1278863 produced dose-dependent effects on hemoglobin, with the highest dose resulting in a mean increase of 1 g/dl at week 4. In the hemodialysis study, treatment with GSK1278863 in the 5-mg arm maintained mean hemoglobin concentrations after the switch from recombinant human erythropoietin, whereas mean hemoglobin decreased in the lower-dose arms. In both studies, the effects on hemoglobin occurred with elevations in endogenous erythropoietin within the range usually observed in the respective populations and markedly lower than those in the recombinant human erythropoietin control arm in the hemodialysis study, and without clinically significant elevations in plasma vascular endothelial growth factor concentrations. GSK1278863 was generally safe and well tolerated at the doses and duration studied. GSK1278863 may prove an effective alternative for managing anemia of CKD. PMID:26494831

Intravenous rFVIIa Administered for Hemorrhage Control in Hypothermic Coagulopathic Swine with Grade V Liver Injuries

DTIC Science & Technology

2001-04-01

Hendriks H, Meijer K, Hagenaars A, et al. Recombinant factor VIIa (rFVIIa, NovoSeven) decreases blood loss and blood product requirements during...and John R. Hess, MD, MPH Background: Intravenous adminis- tration of recombinant activated human clotting factor VII (rFVIIa) has been used...onds, fibrinogen was 91 6 20 mg/dL, and platelets were 221 6 57 3 105/mL, with no differences between groups (p > 0.05). Clotting factor levels

Treatment of Ebola Virus Infection With a Recombinant Inhibitor of Factor Vlla/Tissue Factor: A Study in Rhesus Monkeys

DTIC Science & Technology

2003-12-13

ameliorate the effects of Ebola haemorrhagic fever . Here, we tested the notion that blockade of fVIIa/tissue factor is beneficial after infection with...Ebola virus. Methods We used a rhesus macaque model of Ebola haemorrhagic fever , which produces near 100% mortality. We administered recombinant...severe haemorrhagic fever in primates.1,2 Acute mortality caused by the Zaire species of Ebola virus has been about 80% in outbreaks in human beings1

Epigenetic functions enriched in transcription factors binding to mouse recombination hotspots.

PubMed

Wu, Min; Kwoh, Chee-Keong; Przytycka, Teresa M; Li, Jing; Zheng, Jie

2012-06-21

The regulatory mechanism of recombination is a fundamental problem in genomics, with wide applications in genome-wide association studies, birth-defect diseases, molecular evolution, cancer research, etc. In mammalian genomes, recombination events cluster into short genomic regions called "recombination hotspots". Recently, a 13-mer motif enriched in hotspots is identified as a candidate cis-regulatory element of human recombination hotspots; moreover, a zinc finger protein, PRDM9, binds to this motif and is associated with variation of recombination phenotype in human and mouse genomes, thus is a trans-acting regulator of recombination hotspots. However, this pair of cis and trans-regulators covers only a fraction of hotspots, thus other regulators of recombination hotspots remain to be discovered. In this paper, we propose an approach to predicting additional trans-regulators from DNA-binding proteins by comparing their enrichment of binding sites in hotspots. Applying this approach on newly mapped mouse hotspots genome-wide, we confirmed that PRDM9 is a major trans-regulator of hotspots. In addition, a list of top candidate trans-regulators of mouse hotspots is reported. Using GO analysis we observed that the top genes are enriched with function of histone modification, highlighting the epigenetic regulatory mechanisms of recombination hotspots.

Epigenetic functions enriched in transcription factors binding to mouse recombination hotspots

PubMed Central

2012-01-01

The regulatory mechanism of recombination is a fundamental problem in genomics, with wide applications in genome-wide association studies, birth-defect diseases, molecular evolution, cancer research, etc. In mammalian genomes, recombination events cluster into short genomic regions called "recombination hotspots". Recently, a 13-mer motif enriched in hotspots is identified as a candidate cis-regulatory element of human recombination hotspots; moreover, a zinc finger protein, PRDM9, binds to this motif and is associated with variation of recombination phenotype in human and mouse genomes, thus is a trans-acting regulator of recombination hotspots. However, this pair of cis and trans-regulators covers only a fraction of hotspots, thus other regulators of recombination hotspots remain to be discovered. In this paper, we propose an approach to predicting additional trans-regulators from DNA-binding proteins by comparing their enrichment of binding sites in hotspots. Applying this approach on newly mapped mouse hotspots genome-wide, we confirmed that PRDM9 is a major trans-regulator of hotspots. In addition, a list of top candidate trans-regulators of mouse hotspots is reported. Using GO analysis we observed that the top genes are enriched with function of histone modification, highlighting the epigenetic regulatory mechanisms of recombination hotspots. PMID:22759569

Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System.

PubMed

An, Mahru C; O'Brien, Robert N; Zhang, Ningzhe; Patra, Biranchi N; De La Cruz, Michael; Ray, Animesh; Ellerby, Lisa M

2014-04-15

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR.

A randomized case-controlled study of recombinant human granulocyte colony stimulating factor for the treatment of sepsis in preterm neutropenic infants.

PubMed

Aktaş, Doğukan; Demirel, Bilge; Gürsoy, Tuğba; Ovalı, Fahri

2015-06-01

To investigate the efficacy and safety of recombinant human granulocyte colony-stimulating factor, recombinant human granulocyte-macrophage colony-stimulating factor (rhG-CSF) to treat sepsis in neutropenic preterm infants. Fifty-six neutropenic preterm infants with suspected or culture-proven sepsis hospitalized in Zeynep Kamil Maternity and Children's Educational and Training Hospital, Kozyatağı/Istanbul, Turkey between January 2008 and January 2010 were enrolled. Patients were randomized either to receive rhG-CSF plus empirical antibiotics (Group I) or empirical antibiotics alone (Group II). Clinical features were recorded. Daily complete blood count was performed until neutropenia subsided. Data were analyzed using SPSS version 11.5. Thirty-three infants received rhG-CSF plus antibiotic treatment and 23 infants received antibiotic treatment. No drug-related adverse event was recorded. Absolute neutrophil count values were significantly higher on the 2(nd) study day and 3(rd) study day in Group I. Short-term mortality did not differ between the groups. Treatment with rhG-CSF resulted in a more rapid recovery of ANC in neutropenic preterm infants. However, no reduction in short-term mortality was documented. Copyright © 2014. Published by Elsevier B.V.

Hemophilia and von Willebrand's disease: 2. Management. Association of Hemophilia Clinic Directors of Canada.

PubMed Central

1995-01-01

OBJECTIVE: To present current strategies for the treatment of hemophilia and von Willebrand's disease. OPTIONS: Prophylactic and corrective therapy with hemostatic and adjunctive agents: DDAVP (1-desamino-8-D-arginine vasopressin [desmopressin acetate]), recombinant coagulation products (human Factor VIII and human Factor VIIa) or virally inactivated plasma-derived products (high- or ultra-high-purity human Factor VIII or human Factor VIII concentrate containing von Willebrand factor activity, porcine Factor VIII, high-purity human Factor IX, human prothrombin-complex concentrate, human activated prothrombin-complex concentrate), adjunctive antifibrinolytic agents, topical thrombin and fibrin sealant. The induction of immune tolerance in patients in whom inhibitors develop should also be considered. OUTCOMES: Morbidity and quality of life associated with bleeding and treatment. EVIDENCE: Relevant clinical studies and reports published from 1974 to 1994 were examined. A search was conducted of our reprint files, MEDLINE, citations in the articles reviewed and references provided by colleagues. In the MEDLINE search the following terms were used singly or in combination: "hemophilia," "von Willebrand's disease," "Factor VIII," "Factor IX," "von Willebrand factor," "diagnosis," "management," "home care," "comprehensive care," "inhibitor," "AIDS," "hepatitis," "life expectancy," "complications," "practice guidelines," "consensus statement" and "controlled trial." The in-depth review included only articles written in English from North America and Europe that were relevant to human disease and pertinent to a predetermined outline. The availability of treatment products in Canada was also considered. VALUES: Minimizing morbidity and maximizing functional status and quality of life were given a high value. BENEFITS, HARMS AND COSTS: Proper prophylactic or early treatment with appropriate hemostatic agents minimizes morbidity and functional disability and improves quality of life. Economic gains are realized through the reduction of mortality and morbidity and their associated costs. The patient has a better opportunity to contribute to society through gainful employment and the fulfillment of social roles. Potential harms include HIV infection, hepatitis B, hepatitis C and the development of inhibitor antibodies to clotting-factor concentrates. The risk of viral transmission has been minimized through the development of procedures for the viral inactivation of plasma-derived clotting-factor concentrates and through the use of recombinant coagulation-factor concentrates and other non-plasma-derived hemostatic agents. RECOMMENDATIONS: DDAVP is the drug of choice for patients with mild hemophilia or type 1 or 2 (except 2B) von Willebrand's disease whose response to DDAVP in previous testing has been found to be adequate. Therapeutic blood components of choice include recombinant products and virally inactivated plasma-derived products. In Canada the recommended products are recombinant Factor VIII for hemophilia A, high-purity plasma-derived Factor IX for hemophilia B and plasma-derived Factor VIII concentrates containing adequate von Willebrand factor (e.g., Haemate P) for von Willebrand's disease. Dosages vary according to specific indications. Adjunctive antifibrinolytic agents, topical thrombin and fibrin sealant are useful for the treatment of oral or dental bleeds and localized bleeds in accessible sites. In patients with inhibitor antibodies, high-dose human or porcine Factor VIII is usually effective when the inhibitor titre is less than 5 Bethesda units/mL. In nonresponsive patients, or in those whose inhibitor titre is higher, "bypassing" agents (e.g., activated prothrombin-complex concentrate and recombinant Factor VIIa) are useful. Long-term management may include immune-tolerance induction.VALIDATION: These recommendations were reviewed and approved by the Association of Hemophilia Clinic Directors of Canada (AHCDC) and the Medical and Scientific Advisory Committee of the Canadian Hemophilia Society. No similar consensus statements or practice guidelines are available for comparison. SPONSORS: These recommendations were developed at the request of the Canadian Blood Agency, which funds the provision of all coagulation-factor concentrates for people with congenital bleeding disorders, and were developed and endorsed by the AHCDC and the Medical and Scientific Advisory Committee of the Canadian Hemophilia Society. PMID:7600466

Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle.

PubMed

Wajih, Nadeem; Hutson, Susan M; Owen, John; Wallin, Reidar

2005-09-09

Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent gamma-carboxylation system that converts the proteins to gamma-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully gamma-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the gamma-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of gamma-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent gamma-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.

Recombinant epidermal growth factor-like domain-1 from coagulation factor VII functionalized iron oxide nanoparticles for targeted glioma magnetic resonance imaging.

PubMed

Liu, Heng; Chen, Xiao; Xue, Wei; Chu, Chengchao; Liu, Yu; Tong, Haipeng; Du, Xuesong; Xie, Tian; Liu, Gang; Zhang, Weiguo

The highly infiltrative and invasive nature of glioma cells often leads to blurred tumor margins, resulting in incomplete tumor resection and tumor recurrence. Accurate detection and precise delineation of glioma help in preoperative delineation, surgical planning and survival prediction. In this study, recombinant epidermal growth factor-like domain-1, derived from human coagulation factor VII, was conjugated to iron oxide nanoparticles (IONPs) for targeted glioma magnetic resonance (MR) imaging. The synthesized EGF1-EGFP-IONPs exhibited excellent targeting ability toward tissue factor (TF)-positive U87MG cells and human umbilical vein endothelial cells in vitro, and demonstrated persistent and efficient MR contrast enhancement up to 12 h for preclinical glioma models with high targeting specificity in vivo. They hold great potential for clinical translation and developing targeted theranostics against brain glioma.

Evidence of reduced recombination rate in human regulatory domains.

PubMed

Liu, Yaping; Sarkar, Abhishek; Kheradpour, Pouya; Ernst, Jason; Kellis, Manolis

2017-10-20

Recombination rate is non-uniformly distributed across the human genome. The variation of recombination rate at both fine and large scales cannot be fully explained by DNA sequences alone. Epigenetic factors, particularly DNA methylation, have recently been proposed to influence the variation in recombination rate. We study the relationship between recombination rate and gene regulatory domains, defined by a gene and its linked control elements. We define these links using expression quantitative trait loci (eQTLs), methylation quantitative trait loci (meQTLs), chromatin conformation from publicly available datasets (Hi-C and ChIA-PET), and correlated activity links that we infer across cell types. Each link type shows a "recombination rate valley" of significantly reduced recombination rate compared to matched control regions. This recombination rate valley is most pronounced for gene regulatory domains of early embryonic development genes, housekeeping genes, and constitutive regulatory elements, which are known to show increased evolutionary constraint across species. Recombination rate valleys show increased DNA methylation, reduced doublestranded break initiation, and increased repair efficiency, specifically in the lineage leading to the germ line. Moreover, by using only the overlap of functional links and DNA methylation in germ cells, we are able to predict the recombination rate with high accuracy. Our results suggest the existence of a recombination rate valley at regulatory domains and provide a potential molecular mechanism to interpret the interplay between genetic and epigenetic variations.

Immunogenicity of HILDA/LIF either in a soluble or in a membrane anchored form expressed in vivo by recombinant vaccinia viruses.

PubMed

Taupin, J L; Acres, B; Dott, K; Schmitt, D; Kieny, M P; Gualde, N; Moreau, J F

1993-09-01

Insertion of various cDNAs in the genome of the vaccinia virus (VV) enables the in vivo and in vitro study of the functional role and/or the immunogenicity of the virally encoded recombinant proteins. We have prepared a recombinant VV expressing the cDNA of the human cytokine HILDA/LIF (human interleukin for DA cells/leukaemia inhibitory factor), and used this virus to immunize mice against this protein, which is very homologous to its murine counterpart (approximately 80% homology). We also constructed and expressed by the same system a chimeric gene encoding the HILDA/LIF protein fused to the 37 COOH-terminal amino-acids of the human decay accelerating factor (DAF). This sequence proved to be sufficient for the targeting of the fusion protein to the cell membrane, where it is linked to the phosphatidylinositols. Both recombinant VVs induced cytokine-specific antibodies in mice as analysed with an ELISA where the recombinant HILDA/LIF was plastic-coated and a cytofluorometric assay where the LIF-DAF molecule was present at the cell surface of stably transfected P815. In the latter case HILDA/LIF remained biologically active suggesting that it was expressed in its native form. The LIF-DAF fusion protein was found to exhibit a better capacity to elicit an antibody response against the native form of the cytokine as detected in cytofluorometric assays. Whatever the recombinant virus used to immunize the mice, the MoAbs obtained were positive either in the ELISA or in the cytofluorometric assays but one, which suggested that the plastic coating induced a conformational change of HILDA/LIF.

Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a recombinant brachyury-yeast vaccine

PubMed Central

Jales, Alessandra; Huang, Bruce; Fernando, Romaine I.; Hodge, James W.; Ardiani, Andressa; Apelian, David

2013-01-01

The embryonic T-box transcription factor brachyury is aberrantly expressed in a range of human tumors. Previous studies have demonstrated that brachyury is a driver of the epithelial-mesenchymal transition (EMT), a process associated with cancer progression. Brachyury expression in human tumor cells enhances tumor invasiveness in vitro and metastasis in vivo, and induces resistance to various conventional therapeutics including chemotherapy and radiation. These characteristics, and the selective expression of brachyury for a range of human tumor types vs. normal adult tissues, make brachyury an attractive tumor target. Due to its intracellular localization and the “undruggable” character of transcription factors, available options to target brachyury are currently limited. Here we report on the development and characterization of an immunological platform for the efficient targeting of brachyury-positive tumors consisting of a heat-killed, recombinant Saccharomyces cerevisiae (yeast)–brachyury vector-based vaccine (designated as GI-6301) that expresses the full-length human brachyury protein. We demonstrate that human dendritic cells treated with recombinant yeast-brachyury can activate and expand brachyury-specific CD4+ and CD8+ T cells in vitro that, in turn, can effectively lyse human tumor cells expressing the brachyury protein. Vaccination of mice with recombinant yeast-brachyury is also shown here to elicit brachyury-specific CD4+ and CD8+ T-cell responses, and to induce anti-tumor immunity in the absence of toxicity. Based on these results, a Phase I clinical trial of GI-6301 is currently ongoing in patients with advanced tumors; to our knowledge, this is the first vaccine platform aimed at targeting a driver of tumor EMT that has successfully reached the clinical stage. PMID:24125763

B-domain deleted recombinant factor VIII formulation and stability.

PubMed

Osterberg, T; Fatouros, A; Neidhardt, E; Warne, N; Mikaelsson, M

2001-04-01

B-domain deleted recombinant factor VIII (BDDrFVIII) is a deletion form of human coagulation factor VIII. A lyophilized formulation of highly purified BDDrFVIII has been developed that does not require the use of blood-derived products such as human serum albumin (HSA). By avoiding the use of blood-derived products, the BDDrFVIII formulation minimizes the risk of transmitting blood-borne pathogens that may be present in plasma-derived factor VIII or in other recombinant factor VIII products that contain HSA in their formulation. Upon reconstitution with saline (4 mL), the composition of the reconstituted product (62.5 to 250 IU/mL BDDrFVIII) is 18 mg/mL sodium chloride, 3.0 mg/mL sucrose, 1.5 mg/mL L-histidine, 0.25 mg/mL calcium chloride dihydrate, and 0.1 mg/mL polysorbate 80. The optimal combination of these excipients in the lyophilized BDDrFVIII formulation provides long-term stability, as measured by a variety of analytical methods. The formulation preserves factor VIII activity of lyophilized BDDrFVIII during storage for at least 24 months at 8 degrees C, and for up to 6 months at room temperature (25 degrees C). The reconstituted product retains its factor VIII potency for at least 100 hours at 25 degrees C, which would allow it to be continuously administered via an infusion pump, assuming the product is handled under aseptic conditions.

The new albumin-free recombinant factor VIII concentrates for treatment of hemophilia: do they represent an actual incremental improvement?

PubMed

Josephson, Cassandra D; Abshire, Thomas

2004-07-01

The goal of eliminating the low levels of infectious disease risk from hemophilia treatment has resulted in the development of multiple generations of recombinant factor VIII (rFVIII) products. The ideal product should be devoid of human and animal proteins, which may transmit infectious agents. These products should also maintain molecular integrity, hemostatic efficacy, similar immunogenicity, and acceptable side effect profiles as compared to plasma-derived factor VIII. Currently available first-, second-, and third-generation rFVIII products include Recombinate; Kogenate FS/Helixate FS and ReFacto; and Advate, respectively. During the evolution of rFVIII products, either full-length or B-domain-deleted factor VIII were transfected into immortalized cell lines. The B-domain-deleted product, ReFacto, has resulted in an additional method to monitor factor VIII levels. The third-generation products offer the theoretical advantage of being produced without human and/or animal proteins. Upon initial introduction into the marketplace, the newer products have a higher cost. However, when analyzing historical trends, the prices of these products are almost equivalent to first-generation products within 3 years of licensure. Thus, the initial cost of the product may be a minimal issue in the medical decision process when selecting rFVIII replacement therapy.

Differentiation of PDX1 gene-modified human umbilical cord mesenchymal stem cells into insulin-producing cells in vitro.

PubMed

He, Dongmei; Wang, Juan; Gao, Yangjun; Zhang, Yuan

2011-12-01

Mesenchymal stem cells (MSCs) have significant advantages over other stem cell types, and greater potential for immediate clinical application. MSCs would be an interesting cellular source for treatment of type 1 diabetes. In this study, MSCs from human umbilical cord were differentiated into functional insulin-producing cells in vitro by introduction of the pancreatic and duodenal homeobox factor 1 (PDX1) and in the presence of induction factors. The expressions of cell surface antigens were detected by flow cytometry. After induction in an adipogenic medium or an osteogenic medium, the cells were observed by Oil Red O staining and alkaline phosphatase staining. Recombinant adenovirus carrying the PDX1 gene was constructed and MSCs were infected by the recombinant adenovirus, then treated with several inducing factors for differentiation into islet β-like cells. The expression of the genes and protein related to islet β-cells was detected by immunocytochemistry, RT-PCR and Western blot analysis. Insulin and C-peptide secretion were assayed. Our results show that the morphology and immunophenotype of MSCs from human umbilical cord were similar to those present in human bone marrow. The MSCs could be induced to differentiate into osteocytes and adipocytes. After induction by recombined adenovirus vector with induction factors, MSCs were aggregated and presented islet-like bodies. Dithizone staining of these cells was positive. The genes' expression related to islet β-cells was found. After induction, insulin and C-peptide secretion in the supernatant were significantly increased. In conclusion, our results demonstrated that PDX1 gene-modified human umbilical cord mesenchymal stem cells could be differentiated into insulin-producing cells in vitro.

High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

PubMed

Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

2016-07-30

Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

[Clinical study of recombinant human granulocyte colony stimulating factor (rhG-CSF) on leukopenia induced by chemotherapy in cancer patients].

PubMed

Shi, Y K; Zhou, J C; Feng, F Y

1994-05-01

The clinical usefulness of Recombinant Human Granulocyte Colony Stimulating Factor (rhG-CSF, Filgrastim, GRAN) was evaluated in patients with leukopenia and neutropenia following chemotherapy for non-Hodgkin's lymphoma, lung cancer and breast cancer. During chemotherapy when patients' leukocyte count (WBC) fell below 4.0 x 10(9)/L.rhG-CSF(GRAN) at a dose of 75 micrograms/body.day was given subcutaneously 48 hours after the termination of chemotherapy. The results indicated that rhG-CSF(GRAN) could elevate nadirs of WBC and significantly shortened leukopenic period with WBC below 4.0 x 10(9)/L and expedited the recovery of WBC. rhG-CSF (GRAN)'s side effects were mild.

The Use of Recombinant Human Platelet-Derived Growth Factor for Maxillary Sinus Augmentation.

PubMed

Kubota, Atsushi; Sarmiento, Hector; Alqahtani, Mohammed Saad; Llobell, Arturo; Fiorellini, Joseph P

The maxillary sinus augmentation procedure has become a predictable treatment to regenerate bone for implant placement. The purpose of this study was to evaluate the effect of recombinant human platelet-derived growth factor BB (rhPDGF-BB) combined with a deproteinized cancellous bovine bone graft for sinus augmentation. The lateral window approach was used for maxillary sinuses with minimal residual bone. After a healing period of 4 months, dental implants were placed and then restored following a 2-month osseointegration period. The result demonstrated increased bone height and ISQ values and a 100% survival rate. This study indicates that the addition of rhPDGF-BB to deproteinized cancellous bovine bone accelerated the healing period in maxillary sinuses with minimal native bone.

Vertical ridge augmentation using an equine bone and collagen block infused with recombinant human platelet-derived growth factor-BB: a randomized single-masked histologic study in non-human primates.

PubMed

Nevins, Myron; Al Hezaimi, Khalid; Schupbach, Peter; Karimbux, Nadeem; Kim, David M

2012-07-01

This study tests the effectiveness of hydroxyapatite and collagen bone blocks of equine origin (eHAC), infused with recombinant human platelet-derived growth factor-BB (rhPDGF-BB), to augment localized posterior mandibular defects in non-human primates (Papio hamadryas). Bilateral critical-sized defects simulating severe atrophy were created at the time of the posterior teeth extraction. Test and control blocks (without growth factor) were randomly grafted into the respective sites in each non-human primate. All sites exhibited vertical ridge augmentation, with physiologic hard- and soft-tissue integration of the blocks when clinical and histologic examinations were done at 4 months after the vertical ridge augmentation procedure. There was a clear, although non-significant, tendency to increased regeneration in the test sites. As in the first two preclinical studies in this series using canines, experimental eHAC blocks infused with rhPDGF-BB proved to be a predictable and technically viable method to predictably regenerate bone and soft tissue in critical-sized defects. This investigation supplies additional evidence that eHAC blocks infused with rhPDGF-BB growth factor is a predictable and technically feasible option for vertical augmentation of severely resorbed ridges.

Production of biologically active recombinant human factor H in Physcomitrella.

PubMed

Büttner-Mainik, Annette; Parsons, Juliana; Jérôme, Hanna; Hartmann, Andrea; Lamer, Stephanie; Schaaf, Andreas; Schlosser, Andreas; Zipfel, Peter F; Reski, Ralf; Decker, Eva L

2011-04-01

The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bayne, M.L.; Cascieri, M.A.; Kelder, B.

1987-05-01

A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fibroblasts, directs synthesis of the fusion protein, subsequent proteolytic removal of the bovine growth hormone signal peptide, and secretion of hIGF-I into the culture medium. Conditioned medium frommore » transfected cells inhibits binding of /sup 125/I-labeled IGF-I to type I IGF receptors on human placental membranes and to acid-stable human serum carrier proteins. The recombinant hIGF-I produced is biologically active, as monitored by the stimulation of DNA synthesis in vascular smooth muscle cells.« less

[The induction of neovascularization in chorioallantoic membrane of chicken embryos transfected by a recombinant plasmid containing human angiogenin gene].

PubMed

Avdeeva, S V; Khaĭdarova, N V; Logunov, D Iu; Neugodova, G L; Sevast'ianova, G A; Tarantul, V Z; Naroditskiĭ, B S

2003-01-01

A method was elaborated to evaluate the biological activity of expression products of gene in the plasmid vectors, which are crucial for the synthesis of growth factor of blood vessels. It was proven as possible that the chrioallantonic membrane (CAM) of chicken's embryos could be transfected by recombinant plasmids containing both the reporter and target genes. The efficiency of CAM transfection was assessed by a plasmid carrying the reporter gene of green fluorescent protein (GFP). Finally, it was demonstrated that, at an infiltration of the recombinant plasmid containing the human angiogenine gene, its expression products induce the neovascularization in the CAM cells of chicken's embryos and stimulate an accretion in vessels of the 1st, 2nd and 3d orders.

Bounds on the minimum number of recombination events in a sample history.

PubMed Central

Myers, Simon R; Griffiths, Robert C

2003-01-01

Recombination is an important evolutionary factor in many organisms, including humans, and understanding its effects is an important task facing geneticists. Detecting past recombination events is thus important; this article introduces statistics that give a lower bound on the number of recombination events in the history of a sample, on the basis of the patterns of variation in the sample DNA. Such lower bounds are appropriate, since many recombination events in the history are typically undetectable, so the true number of historical recombinations is unobtainable. The statistics can be calculated quickly by computer and improve upon the earlier bound of Hudson and Kaplan 1985. A method is developed to combine bounds on local regions in the data to produce more powerful improved bounds. The method is flexible to different models of recombination occurrence. The approach gives recombination event bounds between all pairs of sites, to help identify regions with more detectable recombinations, and these bounds can be viewed graphically. Under coalescent simulations, there is a substantial improvement over the earlier method (of up to a factor of 2) in the expected number of recombination events detected by one of the new minima, across a wide range of parameter values. The method is applied to data from a region within the lipoprotein lipase gene and the amount of detected recombination is substantially increased. Further, there is strong clustering of detected recombination events in an area near the center of the region. A program implementing these statistics, which was used for this article, is available from http://www.stats.ox.ac.uk/mathgen/programs.html. PMID:12586723

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.

PubMed

Cunha, Nicolau B; Murad, André M; Ramos, Gustavo L; Maranhão, Andréia Q; Brígido, Marcelo M; Araújo, Ana Cláudia G; Lacorte, Cristiano; Aragão, Francisco J L; Covas, Dimas T; Fontes, Aparecida M; Souza, Gustavo H M F; Vianna, Giovanni R; Rech, Elíbio L

2011-08-01

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

Chitosan-plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies.

PubMed

Jean, M; Smaoui, F; Lavertu, M; Méthot, S; Bouhdoud, L; Buschmann, M D; Merzouki, A

2009-09-01

Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

Neutrophil kinetics of recombinant human granulocyte colony-stimulating factor-induced neutropenia in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okada, Yuji; Kawagishi, Mayumi; Kusaka, Masaru

Single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) immediately induced a decrease in the number of circulating neutrophils in rats. This neutropenia occurred 10 minutes after the injection but disappeared 40 minutes after injection. This transient neutropenia was dose-dependently induced by rhG-CSF and also induced by repeated injections. We studied the kinetics of circulating neutrophils in transient neutropenia. rhG-CSF markedly decreased the number of {sup 3}H-diisopropylfluorophosphate ({sup 3}H-DFP) labeled neutrophils in the circulation 10 minutes after injection but the labeled neutrophils recovered to near the control level 40 minutes after the injection. These results indicate that the neutrophil marginationmore » accounts for the neutrophenia and the marginated neutrophils return to the circulation.« less

[Recombinant granulocyte-colony stimulating factor (filgrastim): optimization of conditions of isolation and purification from inclusion body].

PubMed

Kononova, N V; Iakovlev, A V; Zhuravko, A M; Pankeev, N N; Minaev, S V; Bobruskin, A I; Mart'ianov, V A

2014-01-01

We developed a unified process platform for two recombinant human GCSF medicines--one with the non-prolonged and the other with prolonged action. This unified technology led to a simpler and cheaper production while introduction of the additional pegylation stage to the technological line eased obtaining of the medicines with different action and allowed to standardize technological process documenting according to GMP requirements.

High level expression, purification and characterization of active fusion human C1q and tumor necrosis factor related protein 2 (hCTRP2) in Escherichia coli.

PubMed

Li, Hongbo; Gao, Xuefei; Zhou, Yi; Li, Na; Ge, Caozuo; Hui, Xiaoyan; Wang, Yu; Xu, Aimin; Jin, Shouguang; Wu, Donghai

2011-09-01

C1q and tumor necrosis factor related proteins (CTRPs) are a family of adiponectin paralogues. Among them, CTRP2 is the only CTRP protein that has been shown to possess similar biological activities as adiponectin. To further explore the physiological roles of human CTRP2 and its mechanisms of action, hCTRP2 gene was expressed in Escherichia coli and Pichia pastoris, respectively. In the P. pastoris expression system, recombinant hCTRP2 could be secreted into the culture medium under induction condition, however, the resultant recombinant protein was highly unstable, resulting two main degradation products with molecular masses of approximately 20 and 26 kDa, respectively. In the E. coli expression system, a large amount of soluble thioredoxin (Trx)-hCTRP2 fusion protein could be produced, which accounts about 42% of the total soluble bacterial proteins. The recombinant Trx-hCTRP2 fusion protein was purified to an approximately 95% purity using Ni-NTA affinity chromatography and Superdex G-75 column with a yield of about 15 mg/l protein from 1l bacterial culture. The purified recombinant Trx-hCTRP2 was shown to be active under in vitro assay conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells.

PubMed

Lee, Ji-Hye; Lee, Ji-Eun; Kang, Kyung-Jung; Jang, Young-Joo

2017-07-01

Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H 6 ) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN 6 ) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H 6 -FGF2, FGF2-H 6 , HN 6 -FGF2, and FGF2-HN 6 ) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H 6 -FGF2, FGF2-H 6 , HN 6 -FGF2, and FGF2-HN 6 were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility. Copyright © 2017 Elsevier Inc. All rights reserved.

Tumour necrosis factors modulate the affinity state of the leukotriene B4 receptor on human neutrophils.

PubMed Central

Brom, J; Knöller, J; Köller, M; König, W

1988-01-01

Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity. PMID:2851543

Large-scale purification and characterization of recombinant human stem cell factor in Escherichia coli.

PubMed

Chen, Liang-Hua; Cai, Feng; Zhang, Dan-Ju; Zhang, Li; Zhu, Peng; Gao, Shun

2017-07-01

The pharmacological importance of recombinant human stem cell factor (rhSCF) has increased the demand to establish effective and large-scale production and purification processes. A good source of bioactive recombinant protein with capability of being scaled-up without losing activity has always been a challenge. The objectives of the study were the rapid and efficient pilot-scale expression and purification of rhSCF. The gene encoding stem cell factor (SCF) was cloned into pBV220 and transformed into Escherichia coli. The recombinant SCF was expressed and isolated using a procedure consisting of isolation of inclusion bodies (IBs), denaturation, and refolding followed by chromatographic steps toward purification. The yield of rhSCF reached 835.6 g/20 L, and the expression levels of rhSCF were about 33.9% of the total E. coli protein content. rhSCF was purified by isolation of IBs, denaturation, and refolding, followed by SP-Sepharose chromatography, Source 30 reversed-phase chromatography, and Q-Sepharose chromatography. This procedure was developed to isolate 5.5 g of rhSCF (99.5% purity) with specific activity at 0.96 × 10 6  IU/mg, endotoxin levels of pyrogen at 1.0 EU/mg, and bacterial DNA at 10 ng/mg. Pilot-scale fermentations and purifications were set up for the production of rhSCF that can be upscaled for industry. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Doping control analysis of filgrastim in equine plasma and its application to a co-administration study of filgrastim and recombinant human erythropoietin in the horse.

PubMed

Ho, Emmie N M; Kwok, W H; Lau, M Y; Wong, April S Y; Lam, Kenneth K H; Stewart, Brian D; Wan, Terence S M

2014-04-18

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor regulating granulopoiesis. The recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used for the treatment of granulopenia in humans. Filgrastim is a rhG-CSF analogue and is marketed under various brand names, including Neupogen(®) (Amgen), Imumax(®) (Abbott Laboratories), Neukine(®) (Intas Biopharmaceuticals) and others. It is banned in both human and equine sports owing to its potential for misuse. In order to control the abuse of filgrastim in equine sports, a method to identify unequivocally its prior use in horses is required. This study describes an effective screening method for filgrastim in equine plasma by enzyme-linked immunosorbant assays (ELISA), and a follow-up confirmatory method for the unequivocal identification of filgrastim by analysing its highly specific tryptic peptide (1)MTPLGPASSLPQSFLLK(17). Filgrastim was isolated from equine plasma by immunoaffinity purification. After trypsin digestion, the mixture was analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Filgrastim could be detected and confirmed at 0.2ng/mL in equine plasma. The applicability of the ELISA screening method and the LC/MS/MS confirmation method was demonstrated by analysing post-administration plasma samples collected from horses having been co-administered with epoetin alfa as recombinant human erythropoietin (rhEPO) and filgrastim as rhG-CSF. rhEPO and filgrastim could be detected in plasma samples collected from horses for at least 57 and 101h respectively. To our knowledge, this is the first identification of filgrastim in post-administration samples from horses. Copyright © 2014 Elsevier B.V. All rights reserved.

Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50).

PubMed

Li De La Sierra, I M; Vincent, M; Padron, G; Gallay, J

1992-01-01

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).

The remarkable frequency of human immunodeficiency virus type 1 genetic recombination.

PubMed

Onafuwa-Nuga, Adewunmi; Telesnitsky, Alice

2009-09-01

The genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from a combination of point mutations and genetic recombination, and rates of both processes are unusually high. This review focuses on the mechanisms and outcomes of HIV-1 genetic recombination and on the parameters that make recombination so remarkably frequent. Experimental work has demonstrated that the process that leads to recombination--a copy choice mechanism involving the migration of reverse transcriptase between viral RNA templates--occurs several times on average during every round of HIV-1 DNA synthesis. Key biological factors that lead to high recombination rates for all retroviruses are the recombination-prone nature of their reverse transcription machinery and their pseudodiploid RNA genomes. However, HIV-1 genes recombine even more frequently than do those of many other retroviruses. This reflects the way in which HIV-1 selects genomic RNAs for coencapsidation as well as cell-to-cell transmission properties that lead to unusually frequent associations between distinct viral genotypes. HIV-1 faces strong and changeable selective conditions during replication within patients. The mode of HIV-1 persistence as integrated proviruses and strong selection for defective proviruses in vivo provide conditions for archiving alleles, which can be resuscitated years after initial provirus establishment. Recombination can facilitate drug resistance and may allow superinfecting HIV-1 strains to evade preexisting immune responses, thus adding to challenges in vaccine development. These properties converge to provide HIV-1 with the means, motive, and opportunity to recombine its genetic material at an unprecedented high rate and to allow genetic recombination to serve as one of the highest barriers to HIV-1 eradication.

Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice.

PubMed

Zeng, Qingchun; Song, Rui; Fullerton, David A; Ao, Lihua; Zhai, Yufeng; Li, Suzhao; Ballak, Dov B; Cleveland, Joseph C; Reece, T Brett; McKinsey, Timothy A; Xu, Dingli; Dinarello, Charles A; Meng, Xianzhong

2017-02-14

Calcific aortic valve disease is a chronic inflammatory process, and aortic valve interstitial cells (AVICs) from diseased aortic valves express greater levels of osteogenic factors in response to proinflammatory stimulation. Here, we report that lower cellular levels of IL-37 in AVICs of diseased human aortic valves likely account for augmented expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) following stimulation of Toll-like receptor (TLR) 2 or 4. Treatment of diseased AVICs with recombinant human IL-37 suppresses the levels of BMP-2 and ALP as well as calcium deposit formation. In mice, aortic valve thickening is observed when exposed to a TLR4 agonist or a high fat diet for a prolonged period; however, mice expressing human IL-37 exhibit significantly lower BMP-2 levels and less aortic valve thickening when subjected to the same regimens. A high fat diet in mice results in oxidized low-density lipoprotein (oxLDL) deposition in aortic valve leaflets. Moreover, the osteogenic responses in human AVICs induced by oxLDL are suppressed by recombinant IL-37. Mechanistically, reduced osteogenic responses to oxLDL in human AVICs are associated with the ability of IL-37 to inhibit NF-κB and ERK1/2. These findings suggest that augmented expression of osteogenic factors in AVICs of diseased aortic valves from humans is at least partly due to a relative IL-37 deficiency. Because recombinant IL-37 suppresses the osteogenic responses in human AVICs and alleviates aortic valve lesions in mice exposed to high fat diet or a proinflammatory stimulus, IL-37 has therapeutic potential for progressive calcific aortic valve disease.

Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice

PubMed Central

Zeng, Qingchun; Song, Rui; Fullerton, David A.; Ao, Lihua; Zhai, Yufeng; Li, Suzhao; Ballak, Dov B.; Cleveland, Joseph C.; Reece, T. Brett; McKinsey, Timothy A.; Xu, Dingli; Dinarello, Charles A.; Meng, Xianzhong

2017-01-01

Calcific aortic valve disease is a chronic inflammatory process, and aortic valve interstitial cells (AVICs) from diseased aortic valves express greater levels of osteogenic factors in response to proinflammatory stimulation. Here, we report that lower cellular levels of IL-37 in AVICs of diseased human aortic valves likely account for augmented expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) following stimulation of Toll-like receptor (TLR) 2 or 4. Treatment of diseased AVICs with recombinant human IL-37 suppresses the levels of BMP-2 and ALP as well as calcium deposit formation. In mice, aortic valve thickening is observed when exposed to a TLR4 agonist or a high fat diet for a prolonged period; however, mice expressing human IL-37 exhibit significantly lower BMP-2 levels and less aortic valve thickening when subjected to the same regimens. A high fat diet in mice results in oxidized low-density lipoprotein (oxLDL) deposition in aortic valve leaflets. Moreover, the osteogenic responses in human AVICs induced by oxLDL are suppressed by recombinant IL-37. Mechanistically, reduced osteogenic responses to oxLDL in human AVICs are associated with the ability of IL-37 to inhibit NF-κB and ERK1/2. These findings suggest that augmented expression of osteogenic factors in AVICs of diseased aortic valves from humans is at least partly due to a relative IL-37 deficiency. Because recombinant IL-37 suppresses the osteogenic responses in human AVICs and alleviates aortic valve lesions in mice exposed to high fat diet or a proinflammatory stimulus, IL-37 has therapeutic potential for progressive calcific aortic valve disease. PMID:28137840

Recombinant human leptin in women with hypothalamic amenorrhea.

PubMed

Welt, Corrine K; Chan, Jean L; Bullen, John; Murphy, Robyn; Smith, Patricia; DePaoli, Alex M; Karalis, Aspasia; Mantzoros, Christos S

2004-09-02

Disruptions in hypothalamic-gonadal and other endocrine axes due to energy deficits are associated with low levels of the adipocyte-secreted hormone leptin and may result in hypothalamic amenorrhea. We hypothesized that exogenous recombinant leptin replacement would improve reproductive and neuroendocrine function in women with hypothalamic amenorrhea. Eight women with hypothalamic amenorrhea due to strenuous exercise or low weight were studied for one month before receiving recombinant human leptin and then while receiving treatment for up to three months. Six control subjects with hypothalamic amenorrhea received no treatment and were studied for a mean (+/-SD) of 8.5+/-8.1 months. Luteinizing hormone (LH) pulsatility, body weight, ovarian variables, and hormone levels did not change significantly over time in the controls and during a one-month control period before recombinant leptin therapy in the treated subjects. In contrast, recombinant leptin treatment increased mean LH levels and LH pulse frequency after two weeks and increased maximal follicular diameter, the number of dominant follicles, ovarian volume, and estradiol levels over a period of three months. Three patients had an ovulatory menstrual cycle (P<0.05 for the comparison with an expected rate of spontaneous ovulation of 10 percent); two others had preovulatory follicular development and withdrawal bleeding during treatment (P<0.05). Recombinant leptin significantly increased levels of free triiodothyronine, free thyroxine, insulin-like growth factor 1, insulin-like growth factor-binding protein 3, bone alkaline phosphatase, and osteocalcin but not cortisol, corticotropin, or urinary N-telopeptide. Leptin administration for the relative leptin deficiency in women with hypothalamic amenorrhea appears to improve reproductive, thyroid, and growth hormone axes and markers of bone formation, suggesting that leptin, a peripheral signal reflecting the adequacy of energy stores, is required for normal reproductive and neuroendocrine function. Copyright 2004 Massachusetts Medical Society

Molecular farming of human cytokines and blood products from plants: challenges in biosynthesis and detection of plant-produced recombinant proteins.

PubMed

da Cunha, Nicolau B; Vianna, Giovanni R; da Almeida Lima, Thaina; Rech, Elíbio

2014-01-01

Plants have emerged as an attractive alternative to the traditional mammalian cell cultures or microbial cell-based systems system for the production of valuable recombinant proteins. Through recombinant DNA technology, plants can be engineered to produce large quantities of pharmaceuticals and industrial proteins of high quality at low costs. The recombinant production, by transgenic plants, of therapeutic proteins normally present in human plasma, such as cytokines, coagulation factors, anticoagulants, and immunoglobulins, represents a response to the ongoing challenges in meeting the demand for therapeutic proteins to treat serious inherited or acquired bleeding and immunological diseases. As the clinical utilization of fractionated plasma molecules is limited by high production costs, using recombinant biopharmaceuticals derived from plants represents a feasible alternative to provide efficient treatment. Plant-derived pharmaceuticals also reduce the potential risks to patients of infection with pathogens or unwanted immune responses due to immunogenic antigens. In this review, we summarize the recent advances in molecular farming of cytokines. We also examine the technological basis, upcoming challenges, and perspectives for the biosynthesis and detection of these molecules in different plant production platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Doping in the recombinant era: strategies and counterstrategies.

PubMed

Azzazy, Hassan M E; Mansour, Mai M H; Christenson, Robert H

2005-11-01

Advances in recombinant DNA technology have created one of the most powerful weapons in the current doping arsenal: recombinant proteins [Sweeney HL. Gene doping. Sci Am 2004;291:62-9; Unal M, Ozer Unal D. Gene doping in sports. Sports Med 2004;34:357-62]. Recombinant erythropoietin (EPO) and human growth hormone (hGH) are currently being abused but are fortunately detectable either directly by employing isoelectric focusing and immunoassays or indirectly by assessing changes in selected hematopoietic parameters. The detection is technically demanding due to the extent of similarity between the recombinant proteins and their endogenous counterparts. Another issue facing detection efforts is the speed and conditions at which blood samples are collected and analyzed in a sports setting. Recently, gene doping, which stemmed out of legitimate gene therapy trials, has emerged as the next level of doping. Erythropoietin (EPO), human growth hormone (hGH), insulin-like growth factor-1 (IGF-1), peroxisome proliferator-activated receptor-delta (PPAR delta), and myostatin inhibitor genes have been identified as primary targets for doping. Sports clinical scientists today are racing against the clock because assuring the continued integrity of sports competition depends on their ability to outpace the efforts of dopers by developing new detection strategies.

Does granulocyte colony-stimulating factor ameliorate the proinflammatory response in human meningococcal septic shock?

PubMed

Rojahn, Astrid; Brusletto, Berit; Øvstebø, Reidun; Haug, Kari B F; Kierulf, Peter; Brandtzaeg, Petter

2008-09-01

To test the hypothesis that granulocyte colony-stimulating factor acts cooperatively with interleukin-10 in down-regulating monocyte function in severe meningococcal septic shock. 1) We quantified the plasma levels of granulocyte colony-stimulating factor, interleukin-10, Neisseria meningitidis lipopolysaccharide and the number of N. meningitidis DNA copies in 28 patients with systemic meningococcal disease. 2) We studied the inhibitory effect of recombinant human granulocyte colony-stimulating factor on normal human monocytes stimulated with purified meningococcal lipopolysaccaride. 3) We monitored the inhibitory effects of endogenously produced granulocyte colony-stimulating factor and interleukin-10 in meningococcal shock plasmas on monocytes. Comparative, experimental study. University Hospital and laboratory. Twenty-eight patients with systemic meningococcal disease, 13 with persistent shock, 7 died, and 15 without shock. The median levels of granulocyte colony-stimulating factor in shock and nonshock patients were 1.7 x 10(6) and 8.1 x 10(2) pg/mL; interleukin-10, 2.1 x 10(4) and 4 x 10(1) pg/mL; number of N. meningitidis DNA copies, 2.9 x 10(7) and <10(3)/mL; and lipopolysaccharide, 105 and <0.04 endotoxin units/mL, respectively. The plasma levels of granulocyte colony-stimulating factor were reduced by 50% within 4 to 6 hrs after initiation of antibiotic treatment. In model experiments with lipopolysaccharide-stimulated human monocytes, recombinant human granulocyte colony-stimulating factor and interleukin-10 reduced the release of tumor necrosis factor-alpha by mean 30% and 92%, respectively. When plasmas from three shock patients were depleted of native granulocyte colony-stimulating factor or interleukin-10 by immunoprecipitation, no increase in tumor necrosis factor-alpha release occurred after removal of granulocyte colony-stimulating factor, whereas removal of interleukin-10 increased the tumor necrosis factor-alpha release eight-fold. Although granulocyte colony-stimulating factor in plasma increases by five orders of magnitude in patients with meningococcal shock, the anti-inflammatory effect on patients' monocytes is uncertain.

Age-Dependent Recombination Rates in Human Pedigrees

PubMed Central

Hussin, Julie; Roy-Gagnon, Marie-Hélène; Gendron, Roxanne; Andelfinger, Gregor; Awadalla, Philip

2011-01-01

In humans, chromosome-number abnormalities have been associated with altered recombination and increased maternal age. Therefore, age-related effects on recombination are of major importance, especially in relation to the mechanisms involved in human trisomies. Here, we examine the relationship between maternal age and recombination rate in humans. We localized crossovers at high resolution by using over 600,000 markers genotyped in a panel of 69 French-Canadian pedigrees, revealing recombination events in 195 maternal meioses. Overall, we observed the general patterns of variation in fine-scale recombination rates previously reported in humans. However, we make the first observation of a significant decrease in recombination rates with advancing maternal age in humans, likely driven by chromosome-specific effects. The effect appears to be localized in the middle section of chromosomal arms and near subtelomeric regions. We postulate that, for some chromosomes, protection against non-disjunction provided by recombination becomes less efficient with advancing maternal age, which can be partly responsible for the higher rates of aneuploidy in older women. We propose a model that reconciles our findings with reported associations between maternal age and recombination in cases of trisomies. PMID:21912527

Efficient production of human acidic fibroblast growth factor in pea (Pisum sativum L.) plants by agroinfection of germinated seeds

PubMed Central

2011-01-01

Background For efficient and large scale production of recombinant proteins in plants transient expression by agroinfection has a number of advantages over stable transformation. Simple manipulation, rapid analysis and high expression efficiency are possible. In pea, Pisum sativum, a Virus Induced Gene Silencing System using the pea early browning virus has been converted into an efficient agroinfection system by converting the two RNA genomes of the virus into binary expression vectors for Agrobacterium transformation. Results By vacuum infiltration (0.08 Mpa, 1 min) of germinating pea seeds with 2-3 cm roots with Agrobacteria carrying the binary vectors, expression of the gene for Green Fluorescent Protein as marker and the gene for the human acidic fibroblast growth factor (aFGF) was obtained in 80% of the infiltrated developing seedlings. Maximal production of the recombinant proteins was achieved 12-15 days after infiltration. Conclusions Compared to the leaf injection method vacuum infiltration of germinated seeds is highly efficient allowing large scale production of plants transiently expressing recombinant proteins. The production cycle of plants for harvesting the recombinant protein was shortened from 30 days for leaf injection to 15 days by applying vacuum infiltration. The synthesized aFGF was purified by heparin-affinity chromatography and its mitogenic activity on NIH 3T3 cells confirmed to be similar to a commercial product. PMID:21548923

Can indirect tests detect a known recombination event in human mtDNA?

PubMed

White, Daniel James; Gemmell, Neil John

2009-07-01

Whether human mitochondrial DNA (mtDNA) recombines sufficiently to influence its evolution, evolutionary analysis, and disease etiology, remains equivocal. Overall, evidence from indirect studies of population genetic data suggests that recombination is not occurring at detectable levels. This may be explained by no, or low, recombination or, alternatively, current indirect tests may be incapable of detecting recombination in human mtDNA. To investigate the latter, we have tested whether six well-established indirect tests of recombination could detect recombination in a human mtDNA data set, in which its occurrence had been empirically confirmed. Three showed statistical evidence for recombination (r(2) vs. distance, the Homoplasy test, Neighborhood Similarity Score), and three did not (D' vs. distance, Max Chi Squared, Pairwise Homoplasy Index). Possible reasons for detection failure are discussed. Further, evidence from earlier studies suggesting a lack of recombination in mtDNA in humans is reconsidered, taking into account the appropriateness of the tests used, based on our new findings.

Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

PubMed

Tran, Anh-Minh; Nguyen, Thanh-Thao; Nguyen, Cong-Thuan; Huynh-Thi, Xuan-Mai; Nguyen, Cao-Tri; Trinh, Minh-Thuong; Tran, Linh-Thuoc; Cartwright, Stephanie P; Bill, Roslyn M; Tran-Van, Hieu

2017-04-04

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.

[Clinical study on a concomitant therapy with fluconazole and human recombinant granulocyte colony stimulating factor in the treatment of systemic fungal infections with hematological disorders].

PubMed

Kitamura, K; Miyagawa, K; Urabe, A; Sato, H; Obayashi, Y; Aoki, I; Takaku, F; Togawa, A; Shindou, E; Wakabayashi, Y; Ohshima, T; Horikoshi, A; Nomura, T; Ohki, I; Suzuki, K; Kamakura, M; Oguchi, A; Toyama, K; Yaguchi, M; Aoki, N; Kato, A; Mizoguchi, H; Masuda, M; Irie, S; Fujioka, S

1996-12-01

The clinical efficacy and the safety of concomitant therapy with fluconazole and recombinant human granulocyte colony stimulating factor (rhG-CSF) was compared with fluconazole monotherapy in neutropenic patients with hematological disorders. The clinical efficacy rate was 73.5% (25/34) in the combination therapy and 48.1% (37/77) in monotherapy. The difference between the two is statistically significant. Side effects were not observed in the combination group, but laboratory abnormalities were found in 6 patients with an incident rate of 11%. The combination therapy with fluconazole and rhG-CSF may be selected as empiric therapy for systemic fungal infection associated with hematological disorders, since this combination therapy showed high efficacy and low incident of side effects. Some patients, however, did not show increased neutrophil counts in spite of rhG-CSF administration.

Immunogenicity of a meningococcal native outer membrane vesicle vaccine with attenuated endotoxin and over-expressed factor H binding protein in infant rhesus monkeys

PubMed Central

Koeberling, Oliver; Seubert, Anja; Santos, George; Colaprico, Annalisa; Ugozzoli, Mildred; Donnelly, John; Granoff, Dan M.

2011-01-01

We previously investigated immunogenicity of meningococcal native outer membrane vesicle (NOMV) vaccines prepared from recombinant strains with attenuated endotoxin (ΔLpxL1) and over-expressed factor H binding protein (fHbp) in a mouse model. The vaccines elicited broad serum bactericidal antibody responses. While human toll-like receptor 4 (TLR-4) is mainly stimulated by wildtype meningococcal endotoxin, mouse TLR-4 is stimulated by both the wildtype and mutant endotoxin. An adjuvant effect in mice of the mutant endotoxin would be expected to be much less in humans, and may have contributed to the broad mouse bactericidal responses. Here we show that as previously reported for humans, rhesus primate peripheral blood mononuclear cells incubated with a NOMV vaccine from ΔLpxL1 recombinant strains had lower proinflammatory cytokine responses than with a control wildtype NOMV vaccine. The cytokine responses to the mutant vaccine were similar to those elicited by a detergent-treated, wildtype outer membrane vesicle vaccine that had been safely administered to humans. Monkeys (N=4) were immunized beginning at ages 2 to 3 months with three doses of a NOMV vaccine prepared from ΔLpxL1 recombinant strains with over-expressed fHbp in the variant 1 and 2 groups. The mutant NOMV vaccine elicited serum bactericidal titers ≥1:4 against all 10 genetically diverse strains tested, including 9 with heterologous PorA to those in the vaccine. Negative-control animals had serum bactericidal titers <1:4. Thus, the mutant NOMV vaccine elicited broadly protective serum antibodies in a non-human infant primate model that is more relevant for predicting human antibody responses than mice. PMID:21571025

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21*

PubMed Central

Dunshee, Diana Ronai; Bainbridge, Travis W.; Kljavin, Noelyn M.; Zavala-Solorio, Jose; Schroeder, Amy C.; Chan, Ruby; Corpuz, Racquel; Wong, Manda; Zhou, Wei; Deshmukh, Gauri; Ly, Justin; Sutherlin, Daniel P.; Ernst, James A.; Sonoda, Junichiro

2016-01-01

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders. PMID:26797127

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India.

PubMed

Bondre, Vijay P; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N

2016-11-01

Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections.

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India

PubMed Central

Bondre, Vijay P.; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N.

2016-01-01

Background & objectives: Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Methods: Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Results: Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Interpretation & conclusions: Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections. PMID:28361829

Batch and fed-batch cultivation for excretive production of human epidermal growth factor (hEGF) with recombinant E. Coli K12 system.

PubMed

Wang, J; Chen, J; Xu, R; Xu, Z

2008-01-01

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32 degrees C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

PubMed

Shirvan, Ali Nazari; Aitken, Robert

2016-01-01

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

Human granulocyte colony-stimulating factor (hG-CSF) expression in plastids of Lactuca sativa.

PubMed

Sharifi Tabar, Mehdi; Habashi, Ali Akbar; Rajabi Memari, Hamid

2013-01-01

Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.

Drug eruption caused by recombinant human G-CSF.

PubMed

Sasaki, O; Yokoyama, A; Uemura, S; Fujino, S; Inoue, Y; Kohno, N; Hiwada, K

1994-10-01

Two types of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are available, and equally used for mitigation of neutropenia. One is a glycosylated natural product from mammalian cells, and the other a non-glycosylated form from Escherichia coli. Though only minimal adverse effects have been reported for both, we treated two patients with rhG-CSF-induced systemic eruption. Based on these patients, the following should be noted: 1) drug eruption may occur in both types of rhG-CSF without detectable antibodies, 2) intradermal test is useful for determination of the causal drug, and 3) if one rhG-CSF product causes eruption, the alternative one may possibly be safe and effective.

Bone marrow hematopoietic stem cells behavior with or without growth factors in trauma hemorrhagic shock

PubMed Central

Kumar, Manoj; Bhoi, Sanjeev; Mohanty, Sujata; Kamal, Vineet Kumar; Rao, D. N.; Mishra, Pravas; Galwankar, Sagar

2016-01-01

Background: Hemorrhagic shock (HS) is the major leading cause of death after trauma. Up to 50% of early deaths are due to massive hemorrhage. Excessive release of pro-inflammatory cytokine and hypercatecholamine induces hematopoietic progenitor cells (HPCs) apoptosis, leading to multiorgan failure and death. However, still, result remains elusive for hematopoietic stem cells (HSCs) behavior in trauma HS (T/HS). Objectives: Therefore, our aim was to evaluate the in vitro HSCs behavior with or without recombinant human erythropoietin (rhEPO), recombinant human granulocyte macrophage-colony-stimulating factor (rhGM-CSF), recombinant human interleukin-3 (rhIL-3) alone, and combination with rhEPO + rhGM-CSF + rhIL-3 (EG3) in T/HS patients. Methodology: Bone marrow (BM) aspirates (n = 14) were collected from T/HS patients, those survived on day 3. BM cells were cultured for HPCs: Colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, monocyte/macrophage colonies growth. HPCs were counted with or without rhEPO, rhGM-CSF, rhIL-3 alone, and combination with EG3 in T/HS patients. Results: BM HSCs growth significantly suppressed in T/HS when compared with control group (P < 0.05). In addition, CFU-E and BFU-E colony growth were increased with additional growth factor (AGF) (rhEPO, rhGM-CSF, and rhIL-3) as compared to baseline (without AGF) (P < 0.05). Conclusion: Suppressed HPCs may be reactivated by addition of erythropoietin, GM-CSF, IL-3 alone and with combination in T/HS. PMID:27722113

Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones.

PubMed

Kaplan, Ondřej; Zárubová, Jana; Mikulová, Barbora; Filová, Elena; Bártová, Jiřina; Bačáková, Lucie; Brynda, Eduard

2016-01-01

We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-α2-PI1-8-VEGF121 fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7. The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF121. This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.

Effect of recombinant human insulin-like growth factor-I on progression of ALS. A placebo-controlled study. The North America ALS/IGF-I Study Group.

PubMed

Lai, E C; Felice, K J; Festoff, B W; Gawel, M J; Gelinas, D F; Kratz, R; Murphy, M F; Natter, H M; Norris, F H; Rudnicki, S A

1997-12-01

The objective of this study was to investigate the safety and efficacy of recombinant human insulinlike growth factor-I (rhIGF-I) in the treatment of sporadic ALS. A double-blind, placebo-controlled, randomized study of 266 patients was conducted at eight centers in North America. Placebo or rhIGF-I (0.05 mg/kg/day or 0.10 mg/kg/day) was administered for 9 months. The primary outcome measure was disease symptom progression, assessed by the rate of change (per patient slope) in the Appel ALS rating scale total score. The Sickness Impact Profile (SIP), a patient-perceived, health-related quality of life assessment, was a secondary outcome variable. Progression of functional impairment in patients receiving high-dose (0.10 mg/kg/day) rhIGF-I was 26% slower than in patients receiving placebo (p = 0.01). The high-dose treatment group was less likely to terminate the study due to protocol-defined markers of disease symptom progression, and members in this group exhibited a slower decline in quality of life, as assessed by the SIP. Patients receiving 0.05 mg/kg/day of rhIGF-I exhibited trends similar to those associated with high-dose treatment, suggesting a dose-dependent response. The incidence of clinically significant adverse experiences was comparable among the three treatment groups. Recombinant human insulin-like growth factor-I slowed the progression of functional impairment and the decline in health-related quality of life in patients with ALS with no medically important adverse effects.

Attenuating homologous recombination stimulates an AID-induced antileukemic effect

PubMed Central

Lamont, Kristin R.; Hasham, Muneer G.; Donghia, Nina M.; Branca, Jane; Chavaree, Margaret; Chase, Betsy; Breggia, Anne; Hedlund, Jacquelyn; Emery, Ivette; Cavallo, Francesca; Jasin, Maria; Rüter, Jens

2013-01-01

Activation-induced cytidine deaminase (AID) is critical in normal B cells to initiate somatic hypermutation and immunoglobulin class switch recombination. Accumulating evidence suggests that AID is also prooncogenic, inducing cancer-promoting mutations or chromosome rearrangements. In this context, we find that AID is expressed in >40% of primary human chronic lymphocytic leukemia (CLL) cases, consistent with other reports. Using a combination of human B lymphoid leukemia cells and mouse models, we now show that AID expression can be harnessed for antileukemic effect, after inhibition of the RAD51 homologous recombination (HR) factor with 4,4′-diisothiocyanatostilbene-2-2′-disulfonic acid (DIDS). As a proof of principle, we show that DIDS treatment inhibits repair of AID-initiated DNA breaks, induces apoptosis, and promotes cytotoxicity preferentially in AID-expressing human CLL. This reveals a novel antineoplastic role of AID that can be triggered by inhibition of HR, suggesting a potential new paradigm to treat AID-expressing tumors. Given the growing list of tumor types with aberrant AID expression, this novel therapeutic approach has potential to impact a significant patient population. PMID:23589568

Production of transgenic-cloned pigs expressing large quantities of recombinant human lysozyme in milk.

PubMed

Lu, Dan; Liu, Shen; Shang, Shengzhe; Wu, Fangfang; Wen, Xiao; Li, Zhiyuan; Li, Yan; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Qiuyan; Li, Ning

2015-01-01

Human lysozyme is a natural non-specific immune factor in human milk that plays an important role in the defense of breastfed infants against pathogen infection. Although lysozyme is abundant in human milk, there is only trace quantities in pig milk. Here, we successfully generated transgenic cloned pigs with the expression vector pBAC-hLF-hLZ-Neo and their first generation hybrids (F1). The highest concentration of recombinant human lysozyme (rhLZ) with in vitro bioactivity was 2759.6 ± 265.0 mg/L in the milk of F0 sows. Compared with wild-type milk, rhLZ milk inhibited growth of Escherichia coli K88 during the exponential growth phase. Moreover, rhLZ in milk from transgenic sows was directly absorbed by the intestine of piglets with no observable anaphylactic reaction. Our strategy may provide a powerful tool for large-scale production of this important human protein in pigs to improve resistance to pathogen infection.

Administration of Interleukin-6 Stimulates Multilineage Hematopoiesis and Accelerates Recovery from Radiation-Induced Hematopoietic Depression

DTIC Science & Technology

1991-02-01

Htirano T, Kishimoto T, Nakahata T. Asano S: In vitro hernatopoietic growth factors. J1 Natl Cancer Inst 81: t370. 1989 expansion of the murine...a.i phas S. %’ink A. Billiau A. VanSnick 1: Identification of the nutohlbyrcmiatntrekn.Cllmuolt12. human Zh-kd protein, interferon beta ,, as a B... beta . B-cell %timulatory factor type 2 shares identity T. Takaku F. Akivama Y: In vivo effects of recombinant human with rnsanc~tc-derived

α-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes

PubMed Central

Potapovich, AI; Pastore, S; Kostyuk, VA; Lulli, D; Mariani, V; De Luca, C; Dudich, EI; Korkina, LG

2009-01-01

Background and purpose: The immunomodulatory effects of α-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. Experimental approach: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFκB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFκB DNA binding activity was measured by specific assays. Nitric oxide and H2O2 production and redox status were assessed by fluorescent probe and biochemical methods. Key results: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H2O2 and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFκB was activated by AFP alone or by its combination with UVA. Conclusions and implications: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin. PMID:19785658

How good are indirect tests at detecting recombination in human mtDNA?

PubMed

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-07-08

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D' and r(2), Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ(2)) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7-70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed.

How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

PubMed Central

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-01-01

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

Efficient mobilization of haematopoietic progenitors after a single injection of pegylated recombinant human granulocyte colony-stimulating factor in mouse strains with distinct marrow-cell pool sizes.

PubMed

de Haan, G; Ausema, A; Wilkens, M; Molineux, G; Dontje, B

2000-09-01

We have compared the efficacy of a single injection of SD/01, a newly engineered, pegylated form of recombinant human granulocyte colony stimulating factor (rhG-CSF), with a single injection of glycosylated rhG-CSF (Filgrastim). SD/01 was administered to regular and recombinant inbred strains of mice (AKR, C57L/J, DBA/2, C57BL/6, AKXL) known to have widely distinct marrow-cell pool sizes and proliferation kinetics. A single injection of G-CSF was unable to mobilize granulocyte-macrophage colony-forming units (CFU-GM). In sharp contrast, a single dose of SD/01 resulted in massive mobilization of progenitors and stem cells. Although all mice strains showed qualitatively similar mobilization responses, large interstrain differences remained. C57L and C57BL/6 mice mobilized relatively poorly, whereas AKR and DBA/2 mice showed threefold to tenfold superior responses. In order to explain these different phenotypes, we studied the effects of SD/01 in nine AKXL recombinant inbred strains, derived from well-responding AKR and poorly responding C57L parental strains. The best predictor for SD/01 responsiveness in these strains was marrow cellularity prior to mobilization. Comparison of the AKXL strain distribution pattern for marrow cellularity with loci previously mapped in these strains showed complete concordance with Aat, a serine protease inhibitor mapping to chromosome 12.

Prevalence of nucleic acid sequences specific for human parvoviruses, hepatitis A and hepatitis E viruses in coagulation factor concentrates.

PubMed

Modrow, S; Wenzel, J J; Schimanski, S; Schwarzbeck, J; Rothe, U; Oldenburg, J; Jilg, W; Eis-Hübinger, A M

2011-05-01

Due to their high resistance to inactivation procedures, nonenveloped viruses such as parvovirus B19, human bocavirus (HBoV), human parvovirus 4 (PARV4), hepatitis A (HAV) and hepatitis E virus (HEV) pose a particular threat to blood products. Virus transmission to patients treated with blood products presents an additional burden to disease. We determined the frequency and the amount of nucleic acid specific for nonenveloped viruses in recently manufactured preparations of commercial coagulation factor concentrates. At least three different batches of each of 13 different plasma-derived and recombinant coagulation factor products were tested for the presence and the amount of nucleic acid for parvovirus B19, HBoV, human parvovirus 4, hepatitis A virus and HEV by using quantitative polymerase chain reaction. Whereas none of the recombinant products tested positive for any of these viruses, parvovirus B19 DNA with amounts ranging between 2×10(1) and 1.3×10(3) genome equivalents/ml was detected in five plasma-derived products. In addition to parvovirus B19 genotype 1, genotypes 2 and 3 were observed in two batches of a factor VIII/von-Willebrand factor product. In two products (one factor VIII concentrate and one activated prothrombin complex concentrate), a combination of both genotypes 1 and 2 of parvovirus B19 was detected. The data show that nucleic acids from several relevant nonenveloped viruses are not found at detectable levels in coagulation factor concentrates. In some cases, parvovirus B19 DNA was detectable at low levels. Testing of the plasma pools for the full range of parvovirus genotypes is advocated for ensuring product safety. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

A novel recombinant anti-epidermal growth factor receptor peptide vaccine capable of active immunization and reduction of tumor volume in a mouse model.

PubMed

Asadi-Ghalehni, Majid; Rasaee, Mohamad Javad; RajabiBazl, Masoumeh; Khosravani, Masood; Motaghinejad, Majid; Javanmardi, Masoud; Khalili, Saeed; Modjtahedi, Helmout; Sadroddiny, Esmaeil

2017-12-01

Over-expression of epidermal growth factor receptor (EGFR) has been reported in a number of human malignancies. Strong expression of this receptor has been associated with poor survival in many such patients. Active immunizations that elicit antibodies of the desired type could be an appealing alternative to conventional passive immunization. In this regard, a novel recombinant peptide vaccine capable of prophylactic and therapeutic effects was constructed. A novel fusion recombinant peptide base vaccine consisting of L2 domain of murine extra-cellular domain-EGFR and EGFR mimotope (EM-L2) was constructed and its prophylactic and therapeutic effects in a Lewis lung carcinoma mouse (C57/BL6) model evaluated. Constructed recombinant peptide vaccine is capable of reacting with anti-EGFR antibodies. Immunization of mice with EM-L2 peptide resulted in antibody production against EM-L2. The constructed recombinant peptide vaccine reduced tumor growth and increased the survival rate. Designing effective peptide vaccines could be an encouraging strategy in contemporary cancer immunotherapy. Investigating the efficacy of such cancer immunotherapy approaches may open exciting possibilities concerning hyperimmunization, leading to more promising effects on tumor regression and proliferation. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Mortality Benefit of Recombinant Human Interleukin-1 Receptor Antagonist for Sepsis Varies by Initial Interleukin-1 Receptor Antagonist Plasma Concentration.

PubMed

Meyer, Nuala J; Reilly, John P; Anderson, Brian J; Palakshappa, Jessica A; Jones, Tiffanie K; Dunn, Thomas G; Shashaty, Michael G S; Feng, Rui; Christie, Jason D; Opal, Steven M

2018-01-01

Plasma interleukin-1 beta may influence sepsis mortality, yet recombinant human interleukin-1 receptor antagonist did not reduce mortality in randomized trials. We tested for heterogeneity in the treatment effect of recombinant human interleukin-1 receptor antagonist by baseline plasma interleukin-1 beta or interleukin-1 receptor antagonist concentration. Retrospective subgroup analysis of randomized controlled trial. Multicenter North American and European clinical trial. Five hundred twenty-nine subjects with sepsis and hypotension or hypoperfusion, representing 59% of the original trial population. Random assignment of placebo or recombinant human interleukin-1 receptor antagonist × 72 hours. We measured prerandomization plasma interleukin-1 beta and interleukin-1 receptor antagonist and tested for statistical interaction between recombinant human interleukin-1 receptor antagonist treatment and baseline plasma interleukin-1 receptor antagonist or interleukin-1 beta concentration on 28-day mortality. There was significant heterogeneity in the effect of recombinant human interleukin-1 receptor antagonist treatment by plasma interleukin-1 receptor antagonist concentration whether plasma interleukin-1 receptor antagonist was divided into deciles (interaction p = 0.046) or dichotomized (interaction p = 0.028). Interaction remained present across different predicted mortality levels. Among subjects with baseline plasma interleukin-1 receptor antagonist above 2,071 pg/mL (n = 283), recombinant human interleukin-1 receptor antagonist therapy reduced adjusted mortality from 45.4% to 34.3% (adjusted risk difference, -0.12; 95% CI, -0.23 to -0.01), p = 0.044. Mortality in subjects with plasma interleukin-1 receptor antagonist below 2,071 pg/mL was not reduced by recombinant human interleukin-1 receptor antagonist (adjusted risk difference, +0.07; 95% CI, -0.04 to +0.17), p = 0.230. Interaction between plasma interleukin-1 beta concentration and recombinant human interleukin-1 receptor antagonist treatment was not statistically significant. We report a heterogeneous effect of recombinant human interleukin-1 receptor antagonist on 28-day sepsis mortality that is potentially predictable by plasma interleukin-1 receptor antagonist in one trial. A precision clinical trial of recombinant human interleukin-1 receptor antagonist targeted to septic patients with high plasma interleukin-1 receptor antagonist may be worthy of consideration.

PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans.

PubMed

Berg, Ingrid L; Neumann, Rita; Lam, Kwan-Wood G; Sarbajna, Shriparna; Odenthal-Hesse, Linda; May, Celia A; Jeffreys, Alec J

2010-10-01

PRDM9 has recently been identified as a likely trans regulator of meiotic recombination hot spots in humans and mice. PRDM9 contains a zinc finger array that, in humans, can recognize a short sequence motif associated with hot spots, with binding to this motif possibly triggering hot-spot activity via chromatin remodeling. We now report that human genetic variation at the PRDM9 locus has a strong effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Subtle changes within the zinc finger array can create hot-spot nonactivating or enhancing variants and can even trigger the appearance of a new hot spot, suggesting that PRDM9 is a major global regulator of hot spots in humans. Variation at the PRDM9 locus also influences aspects of genome instability-specifically, a megabase-scale rearrangement underlying two genomic disorders as well as minisatellite instability-implicating PRDM9 as a risk factor for some pathological genome rearrangements.

PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans

PubMed Central

Berg, Ingrid L.; Neumann, Rita; Lam, Kwan-Wood G.; Sarbajna, Shriparna; Odenthal-Hesse, Linda; May, Celia A.; Jeffreys, Alec J.

2011-01-01

PRDM9 has recently been identified as a likely trans-regulator of meiotic recombination hot spots in humans and mice1-3. The protein contains a zinc finger array that in humans can recognise a short sequence motif associated with hot spots4, with binding to this motif possibly triggering hot-spot activity via chromatin remodelling5. We now show that variation in the zinc finger array in humans has a profound effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Very subtle changes within the array can create hot-spot non-activating and enhancing alleles, and even trigger the appearance of a new hot spot. PRDM9 thus appears to be the preeminent global regulator of hot spots in humans. Variation at this locus also influences aspects of genome instability, specifically a megabase-scale rearrangement underlying two genomic disorders6 as well as minisatellite instability7, implicating PRDM9 as a risk factor for some pathological genome rearrangements. PMID:20818382

Systemic delivery of factor IX messenger RNA for protein replacement therapy

PubMed Central

Ramaswamy, Suvasini; Tonnu, Nina; Tachikawa, Kiyoshi; Limphong, Pattraranee; Vega, Jerel B.; Karmali, Priya P.; Chivukula, Pad; Verma, Inder M.

2017-01-01

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable. PMID:28202722

The kinetochore prevents centromere-proximal crossover recombination during meiosis

PubMed Central

Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel; Oke, Ashwini; Kerr, Alastair RW; Hochwagen, Andreas; Fung, Jennifer; Keeney, Scott; Vader, Gerben; Marston, Adèle L

2015-01-01

During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. DOI: http://dx.doi.org/10.7554/eLife.10850.001 PMID:26653857

Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng Zhiming; Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia; Wang Ping

2013-02-01

Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials:more » Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.« less

Combining heavy ion radiation and artificial microRNAs to target the homologous recombination repair gene efficiently kills human tumor cells.

PubMed

Zheng, Zhiming; Wang, Ping; Wang, Hongyan; Zhang, Xiangming; Wang, Minli; Cucinotta, Francis A; Wang, Ya

2013-02-01

Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of pegylated recombinant human megakaryocyte growth and development factor on thrombocytopenia induced by a new myelosuppressive chemotherapy regimen in mice.

PubMed

Akahori, H; Shibuya, K; Ozai, M; Ida, M; Kabaya, K; Kato, T; Miyazaki, H

1996-11-01

Thrombopoietin, the endogenous c-Mpl ligand, is a novel lineage-specific hematopoietic factor that plays a pivotal role in the regulation of megakaryocytopoiesis and thrombopoiesis. In this study, we examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human c-Mpl ligand derivatized with polyethylene glycol, on myelosuppressive chemotherapy-induced thrombocytopenia in mice. We developed a new murine model of thrombocytopenia induced by i.v. injections of mitomycin C (MMC) for two consecutive days. In control mice, platelet counts began to decrease on day 6, reached a nadir of less than 5% of basal level on day 14, and could not recover to basal level by day 26. Administration of PEG-rHuMGDF greatly enhanced recovery of the number of megakaryocyte progenitor cells and the megakaryocytes in bone marrow, and markedly reduced the severity of thrombocytopenia; it also accelerated platelet recovery in a dose-dependent manner in myelosuppressed mice. Mice receiving consecutive administration of higher doses of PEG-rHuMGDF showed no thrombocytopenia but rather had platelet counts being increased over basal level. Although absolute neutrophil counts and red cell counts also were decreased following MMC treatment, administration of PEG-rHuMGDF also improved neutropenia and anemia. Administration of PEG-rHuMGDF on alternate days or once a week after chemotherapy was almost as effective as consecutive administration in improving thrombocytopenia. Combined administration of PEG-rHuMGDF and rHuG-CSF had an additive effect on improvement of thrombocytopenia and neutropenia. These results suggest that PEG-rHuMGDF is a therapeutically effective agent in the treatment of thrombocytopenia associated with chemotherapy.

Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons

NASA Astrophysics Data System (ADS)

Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

2015-11-01

With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

One-step refolding and purification of recombinant human tumor necrosis factor-α (rhTNF-α) using ion-exchange chromatography.

PubMed

Wang, Yan; Ren, Wenxuan; Gao, Dong; Wang, Lili; Yang, Ying; Bai, Quan

2015-02-01

Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic-based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor-α (rhTNF-α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF-α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF-α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 10(8) IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF-α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF-α refolding in terms of specific bioactivity and mass recovery. Copyright © 2014 John Wiley & Sons, Ltd.

Treatment of dwarfism with recombinant human insulin-like growth factor-1.

PubMed

Ranke, Michael B; Wölfle, Joachim; Schnabel, Dirk; Bettendorf, Markus

2009-10-01

The growth hormone-IGF (insulin-like growth factor) system plays a central role in hormonal growth regulation. Recombinant human (rh) growth hormone (GH) has been available since the late 1980s for replacement therapy in GH-deficient patients and for the stimulation of growth in patients with short stature of various causes. Growth promotion by GH occurs in part indirectly through the induction of IGF-1 synthesis. In primary disturbances of IGF-1 production, short stature can only be treated with recombinant human IGF-1 (rhIGF-1). rhIGF-1 was recently approved for this indication but can also be used to treat other conditions. Selective review of the literature on IGF-1 therapy, based on a PubMed search. In children with severe primary IGF-1 deficiency (a rare condition whose prevalence is less than 1:10,000), the prognosis for final height is very poor (ca. 130 cm), and IGF-1 therapy is the appropriate form of pathophysiologically based treatment. There is no alternative treatment at present. The subcutaneous administration of IGF-1 twice daily in doses of 80 to 120 microg/kg accelerates growth and increases final height by 12 to 15 cm, according to current data. There is, however, a risk of hypoglycemia, as IGF-1 has an insulin-like effect. As treatment with IGF-1 is complex, this new medication should only be prescribed, for the time being, by experienced pediatric endocrinologists and diabetologists.

Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells.

PubMed

Asano, Ryutaro; Kawaguchi, Hiroko; Watanabe, Yasuhiro; Nakanishi, Takeshi; Umetsu, Mitsuo; Hayashi, Hiroki; Katayose, Yu; Unno, Michiaki; Kudo, Toshio; Kumagai, Izumi

2008-10-01

Recently, recombinant antibodies have been dissected into antigen-binding regions and rebuilt into multivalent high-avidity formats. These new structural designs are expected to improve in vivo pharmacokinetics and efficacy in clinical use. Here, we designed effective recombinant bispecific antibody (BsAb) formats based on hEx3, a humanized bispecific diabody with epidermal growth factor receptor and CD3 retargeting. The bispecific and bivalent IgG-like antibodies engineered from hEx3 (or its single-chain form, hEx3-scDb) and the human Fc region showed stronger binding to each target cell than did monovalent diabody formats, and their affinity was identical to that of the corresponding parent IgG. The bivalent effect of the constructed IgG-like BsAbs resulted in cell cytotoxicity 10 times that of monovalent diabodies, and further, the fusion of Fc portion contributed intense cytotoxicity in peripheral blood mononuclear cells by the induction of the antibody-dependent cellular cytotoxicity. The growth-inhibition effects of IgG-like BsAbs were superior to those of the approved therapeutic antibody cetuximab, which recognizes the same epidermal growth factor receptor antigen, even when peripheral blood mononuclear cells were used as effector cells. We thus demonstrated a critical improvement in the effect of hEx3 by the bottom-up construction of IgG-like BsAbs; in adoptive immunotherapy, monotherapy without supplemental molecules may be able to induce antibody-dependent cellular cytotoxicity.

Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect.

PubMed

Mathijssen, Natascha C J; Masereeuw, Rosalinde; Holme, Pal Andre; van Kraaij, Marian G J; Laros-van Gorkom, Britta A P; Peyvandi, Flora; van Heerde, Waander L

2013-08-01

Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. Ten factor VII deficient patients were treated with either recombinant activated (20 μg/kg) or plasma-derived (25 IU/kg) factor VII in a cross-over design. Pharmacokinetic parameters were analyzed through activated factor VII activity, factor VII clotting activity, and factor VII antigen levels on depicted time points. Factor VII activity half-lifes, determined by non-compartmental and one-compartmental analysis (results in brackets), were shorter for recombinant activated (1.4h; 0.7h) than for plasma-derived factor VII (6.8h; 3.2h); both recombinant activated (5.1h; 2.1h and plasma-derived factor VII (5.8h; 3.2h) resulted in longer half-lives of factor VII antigen. Activated factor VII half-lives (based on activated factor VII activity levels) were significantly higher compared to factor VII clotting activity (1.6h; 0.9h). Volumes of distribution were significantly higher for activated factor VII (236 ml/kg; 175 ml/kg, measured by activated factor VII) as compared to plasma-derived factor VII (206 ml/kg; 64 ml/kg, measured by factor FVII activity), suggesting a plasma- and extracellular fluid distribution for recombinant activated factor VII. Recombinant activated factor VII showed significantly shorter half-lifes than plasma-derived factor VII. Volumes of distribution were significantly higher for treatment with recombinant activated factor VII. The longer half-life for plasma-derived factor VII, compared to recombinant activated factor VII, and the increased volume of distribution for recombinant activated factor VII, compared to plasma-derived factor VII may further elucidate the beneficial effect of prophylactic treatment of both products. Copyright © 2013 Elsevier Ltd. All rights reserved.

A review of luteinising hormone and human chorionic gonadotropin when used in assisted reproductive technology.

PubMed

Ezcurra, Diego; Humaidan, Peter

2014-10-03

Gonadotropins extracted from the urine of post-menopausal women have traditionally been used to stimulate folliculogenesis in the treatment of infertility and in assisted reproductive technology (ART). Products, such as human menopausal gonadotropin (hMG), consist not only of a mixture of the hormones, follicle-stimulating hormone (FSH), luteinising hormone (LH) and human chorionic gonadotropin (hCG), but also other biologically active contaminants, such as growth factors, binding proteins and prion proteins. The actual amount of molecular LH in hMG preparations varies considerably due to the purification process, thus hCG, mimicking LH action, is added to standardise the product. However, unlike LH, hCG plays a different role during the natural human menstrual cycle. It is secreted by the embryo and placenta, and its main role is to support implantation and pregnancy. More recently, recombinant gonadotropins (r-hFSH and r-hLH) have become available for ART therapies. Recombinant LH contains only LH molecules. In the field of reproduction there has been controversy in recent years over whether r-hLH or hCG should be used for ART. This review examines the existing evidence for molecular and functional differences between LH and hCG and assesses the clinical implications of hCG-supplemented urinary therapy compared with recombinant therapies used for ART.

Human thioredoxin-1 attenuates the rate of lipopolysaccharide-induced preterm delivery in mice in association with its anti-inflammatory effect.

PubMed

Namba, Fumihiko; Kobayashi-Miura, Mikiko; Goda, Taro; Nakura, Yukiko; Nishiumi, Fumiko; Son, Aoi; Kubota, Akio; Yodoi, Junji; Yanagihara, Itaru

2016-09-01

Maternal intrauterine infection/inflammation represents the major etiology of preterm delivery and the leading cause of neonatal mortality and morbidity. The aim of this study was to investigate the anti-inflammatory properties of thioredoxin-1 in vivo and its potential ability to attenuate the rate of inflammation-induced preterm delivery. Two intraperitoneal injections of lipopolysaccharide from Escherichia coli were administered in pregnant mice on gestational day 15, with a 3-h interval between the injections. From either 1 h before or 1 h after the first lipopolysaccharide injection, mice received three intravenous injections of either recombinant human thioredoxin-1, ovalbumin, or vehicle, with a 3-h interval between injections. Intraperitoneal injection of lipopolysaccharide induced a rise of tumor necrosis factor-α, interferon-γ, monocyte chemotactic protein 1, and interleukin-6 in maternal serum levels and provoked preterm delivery. Recombinant human thoredoxin-1 prevented the rise in these proinflammatory cytokine levels. After the inflammatory challenge, placentas exhibited severe maternal vascular dilatation and congestion and a marked decidual neutrophil activation. These placental pathological findings were ameliorated by recombinant human thioredoxin-1, and the rate of inflammation-induced preterm delivery was attenuated. Thioredoxin-1 may thus represent a novel effective treatment to delay inflammation-induced preterm delivery.

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.

PubMed

Watanabe, M; Fukamachi, H; Uzumaki, H; Kabaya, K; Tsumura, H; Ishikawa, M; Matsuki, S; Kusaka, M

1991-05-15

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Mediation of mouse natural cytotoxic activity by tumour necrosis factor

NASA Astrophysics Data System (ADS)

Ortaldo, John R.; Mason, Llewellyn H.; Mathieson, Bonnie J.; Liang, Shu-Mei; Flick, David A.; Herberman, Ronald B.

1986-06-01

Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors1. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.

Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Besemer, J.; Hujber, A.; Kuhn, B.

1989-10-15

The interaction of {sup 125}I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4{degree}C to pH 3 resistance at 37{degree}C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37{degree}C led to nonlinearmore » curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.« less

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

PubMed

Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

2016-12-01

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Streptococcal 5′-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion*

PubMed Central

Zheng, Lisa; Khemlani, Adrina; Lorenz, Natalie; Loh, Jacelyn M. S.; Langley, Ries J.; Proft, Thomas

2015-01-01

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5′-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 μm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5′-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg2+, Ca2+, or Mn2+. However, Zn2+ inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5′-nucleotidase activity and immune evasion properties. PMID:26527680

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

PubMed

Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

2004-03-07

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

Recombinant activated factor VII in cardiac surgery: single-center experience.

PubMed

Singh, Sarvesh Pal; Chauhan, Sandeep; Choudhury, Minati; Malik, Vishwas; Choudhary, Shiv Kumar

2014-02-01

The widespread off-label use of recombinant activated factor VII for the control of refractory postoperative hemorrhage continues despite a warning from the Food and Drug Administration. Although effective in reducing the need for transfusion of blood and blood products, safety concerns still prevail. To compare the dosing and efficacy of recombinant activated factor VII between pediatric and adult patients, and in the operating room and intensive care unit. The records of 69 patients (33 children and 36 adults) who underwent cardiovascular surgery and received recombinant activated factor VII were reviewed retrospectively. The dose of recombinant activated factor VII, mediastinal drainage, use of blood and blood products, incidence of thrombosis, and 28-day mortality were studied. the efficacy of recombinant activated factor VII was comparable in adults and children, despite the lower dose in adults. Prophylactic use of recombinant activated factor VII decreased the incidence of mediastinal exploration and the duration of intensive care unit stay. A 4.3% incidence of thrombotic complications was observed in this study. The efficacious dose of recombinant activated factor VII is much less in adults compared to children. Prophylactic use of recombinant activated factor VII decreases the dose required, the incidence of mediastinal exploration, and intensive care unit stay, with no survival benefit.

Establishment of human hair follicle mesenchymal stem cells with overexpressed human hepatocyte growth factor.

PubMed

Zhou, Dan; Cheng, Hongjing; Liu, Jinyu; Zhang, Lei

2017-06-01

Chronic liver disease has become a major health problem that causes serious damage to human health. Since the existing treatment effect was not ideal, we need to seek new treatment methods. We utilized the gene recombination technology to obtain the human hair mesenchymal stem cells which overexpression of human hepatocyte growth factor (hHGF). Furthermore, we verified the property of transfected cells through detecting surface marker by flow cytometry. We show here establishment of the hHGF-overexpressing lentivirus vector, and successfully transfection to human hair follicle mesenchymal stem cells. The verified experiments could demonstrate the human hair follicle mesenchymal stem cells which have been transfected still have the properties of stem cells. We successfully constructed human hair follicle mesenchymal stem cells which overexpression hHGF, and maintain the same properties compared with pro-transfected cells.

Heterologous mitochondrial DNA recombination in human cells.

PubMed

D'Aurelio, Marilena; Gajewski, Carl D; Lin, Michael T; Mauck, William M; Shao, Leon Z; Lenaz, Giorgio; Moraes, Carlos T; Manfredi, Giovanni

2004-12-15

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

High-Resolution Patterns of Meiotic Recombination across the Human Major Histocompatibility Complex

PubMed Central

Cullen, Michael; Perfetto, Stephen P.; Klitz, William; Nelson, George; Carrington, Mary

2002-01-01

Definitive characteristics of meiotic recombination events over large (i.e., >1 Mb) segments of the human genome remain obscure, yet they are essential for establishing the haplotypic structure of the genome and for efficient mapping of complex traits. We present a high-resolution map of recombination at the kilobase level across a 3.3-Mb interval encompassing the major histocompatibility complex (MHC). Genotyping of 20,031 single sperm from 12 individuals resulted in the identification and fine mapping of 325 recombinant chromosomes within genomic intervals as small as 7 kb. Several principal characteristics of recombination in this region were observed: (1) rates of recombination can differ significantly between individuals; (2) intense hot spots of recombination occur at least every 0.8 Mb but are not necessarily evenly spaced; (3) distribution in the location of recombination events can differ significantly among individuals; (4) between hot spots, low levels of recombination occur fairly evenly across 100-kb segments, suggesting the presence of warm spots of recombination; and (5) specific sequence motifs associate significantly with recombination distribution. These data provide a plausible model for recombination patterns of the human genome overall. PMID:12297984

Decreased prothrombotic effects of pegylated recombinant human megakaryocyte growth and development factor in thrombocytopenic state in a rat thrombosis model.

PubMed

Nishiyama, U; Kuwaki, T; Akahori, H; Kato, T; Ikeda, Y; Miyazaki, H

2005-02-01

Previous in vitro studies demonstrated that thrombopoietin (TPO) acts on platelets to activate a variety of intracellular signaling pathways and to enhance platelet sensitivity to multiple agonists. Little is known, however, about whether TPO exerts prothrombotic effects in vivo. The aim of this study was to examine the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a pegylated N-terminal domain of human TPO, in a rat model of venous thrombosis. A microthrombus was photochemically induced on the vessel wall of a mesenteric venule, but the vessel was not occluded by it. A single intravenous injection of PEG-rHuMGDF (3 microg kg(-1)) after the thrombus generation into normal rats enhanced the thrombus size, resulting in transient thrombotic occlusion in the majority of rats. Stimulatory effects on thrombus growth were also observed following administration of glycosylated recombinant human full-length TPO (6 microg kg(-1)). In rats rendered thrombocytopenic by total body irradiation, however, PEG-rHuMGDF, even at 300 microg kg(-1), did not induce a significant increase in thrombus size or thrombotic occlusion. Platelets from thrombocytopenic rats had decreased surface levels of c-Mpl and decreased sensitivity to PEG-rHuMGDF in an in vitro aggregation response. Thus, decreased prothrombotic effects of PEG-rHuMGDF in thrombocytopenic rats might be the result not only of low platelet counts but also of decreased platelet reactivity to PEG-rHuMGDF. These results indicate that PEG-rHuMGDF has little effect on venous thrombus formation in thrombocytopenic states associated with high endogenous TPO levels.

RANKL/RANK interaction promotes the growth of cervical cancer cells by strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion.

PubMed

Shang, Wen-Qing; Li, Hui; Liu, Li-Bing; Chang, Kai-Kai; Yu, Jia-Jun; Xie, Feng; Li, Ming-Qing; Yu, Jin-Jin

2015-12-01

Receptor activator for nuclear factor κB ligand (RANKL) is a member of the tumor necrosis factor (TNF) family. The interaction between RANKL and its receptor RANK plays an important role in the development and function of diverse tissues. However, the expression and role of RANKL in cervical cancer are still unknown. In the present study, we found that RANKL and RANK were highly co-expressed in cervical cancer. HeLa and SiHa cells secreted soluble RANKL (sRANKL), expressed member RANKL (mRANKL) and RANK. Recombinant human RANKL protein had no effect on the viability of HeLa and SiHa cells. Yet, blocking RANKL with an anti-human RANKL neutralizing antibody (α-RANKL) or recombinant human osteoprotegrin (OPG) protein resulted in the downregulation of Ki-67 and B-cell lymphoma 2 (Bcl-2) expression and an increase in Fas and Fas ligand (FasL) expression, as well as a high level of viability and a low level of apoptosis in the HeLa and SiHa cells. In addition, α-RANKL led to a decrease in IL-8 secretion. Recombinant human IL-8 protein reversed the effect of α-RANKL on the expression of proliferation- and apoptosis‑related molecules, and proliferation and apoptosis in the HeLa and SiHa cells. The present study suggests that a high level of mRANKL/RANK expression in cervical cancer lesions plays an important role in the rapid growth of cervical cancer cells possibly through strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion, which may be a possible target for cervical cancer therapy.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization.

PubMed

Vanz, Ana Ls; Renard, Gaby; Palma, Mario S; Chies, Jocelei M; Dalmora, Sérgio L; Basso, Luiz A; Santos, Diógenes S

2008-04-04

Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

PubMed Central

Vanz, Ana LS; Renard, Gaby; Palma, Mario S; Chies, Jocelei M; Dalmora, Sérgio L; Basso, Luiz A; Santos, Diógenes S

2008-01-01

Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community. PMID:18394164

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

PubMed

Zsurka, Gábor; Kraytsberg, Yevgenia; Kudina, Tatiana; Kornblum, Cornelia; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2005-08-01

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

Expression and characterization of bioactive recombinant human alpha-lactalbumin in the milk of transgenic cloned cows.

PubMed

Wang, J; Yang, P; Tang, B; Sun, X; Zhang, R; Guo, C; Gong, G; Liu, Y; Li, R; Zhang, L; Dai, Y; Li, N

2008-12-01

Improvement of the nutritional value of cow milk with transgenic expression of recombinant human alpha-lactalbumin (alpha-LA) has been previously attempted. However, the detailed characterization of the recombinant protein and analysis of the transgenic milk components are not explored yet. Here, we first report production of healthy transgenic cows by somatic cell nuclear transfer, in which expression of up to 1.55 g/L of recombinant human alpha-LA was achieved. The recombinant human alpha-LA was purified from transgenic milk and displayed physicochemical properties similar to its natural counterpart with respect to molecular weight, structure, and regulatory activity for beta-1,4-galactosyltransferase. Additionally, no N-glycosylation was found in the recombinant human alpha-LA, whereas the endogenous bovine alpha-LA was glycosylated at the unusual site (71)Asn-Ile-(73)Cys. Compared with milk from nontransgenic cows, expression of the transgene did not materially alter milk composition, such as fat and protein content. Our research thus provides scientific evidence supporting the feasibility of humanizing cow milk.

Safety of recombinant human factor XIII in a cynomolgus monkey model of extracorporeal blood circulation.

PubMed

Ponce, R; Armstrong, K; Andrews, K; Hensler, J; Waggie, K; Heffernan, J; Reynolds, T; Rogge, M

2005-01-01

Factor XIII (FXIII) is a thrombin-activated plasma coagulation factor critical for blood clot stabilization and longevity. Administration of exogenous FXIII to replenish depleted stores after major surgery, including cardiopulmonary bypass, may reduce bleeding complications and transfusion requirements. Thus, a model of extracorporeal circulation (ECC) was developed in adult male cynomolgus monkeys (Macaca fascicularis) to evaluate the nonclinical safety of recombinant human FXIII (rFXIII). The hematological and coagulation profile in study animals during and after 2 h of ECC was similar to that reported for humans during and after cardiopulmonary bypass, including observations of anemia, thrombocytopenia, and activation of coagulation and platelets. Intravenous slow bolus injection of 300 U/kg (2.1 mg/kg) or 1000 U/kg (7 mg/kg) rFXIII after 2 h of ECC was well tolerated in study animals, and was associated with a dose-dependent increase in FXIII activity. No clinically significant effects in respiration, ECG, heart rate, blood pressure, body temperature, clinical chemistry, hematology (including platelet counts), or indicators of thrombosis (thrombin:anti-thrombin complex and D-Dimer) or platelet activation (platelet factor 4 and beta-thromboglobulin) were related to rFXIII administration. Specific examination of brain, heart, lung, liver, and kidney from rFXIII-treated animals provided no evidence of histopathological alterations suggestive of subclinical hemorrhage or thrombosis. Taken as a whole, the results demonstrate the ECC model suitably replicated the clinical presentation reported for humans during and after cardiopulmonary bypass surgery, and do not suggest significant concerns regarding use of rFXIII in replacement therapy after extracorporeal circulation.

Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

PubMed

Winge, Stefan; Yderland, Louise; Kannicht, Christoph; Hermans, Pim; Adema, Simon; Schmidt, Torben; Gilljam, Gustav; Linhult, Martin; Tiemeyer, Maya; Belyanskaya, Larisa; Walter, Olaf

2015-11-01

Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Reduced bleeding events with subcutaneous administration of recombinant human factor IX in immune-tolerant hemophilia B dogs.

PubMed

Russell, Karen E; Olsen, Eva H N; Raymer, Robin A; Merricks, Elizabeth P; Bellinger, Dwight A; Read, Marjorie S; Rup, Bonita J; Keith, James C; McCarthy, Kyle P; Schaub, Robert G; Nichols, Timothy C

2003-12-15

Intravenous administration of recombinant human factor IX (rhFIX) acutely corrects the coagulopathy in hemophilia B dogs. To date, 20 of 20 dogs developed inhibitory antibodies to the xenoprotein, making it impossible to determine if new human FIX products, formulations, or methods of chronic administration can reduce bleeding frequency. Our goal was to determine whether hemophilia B dogs rendered tolerant to rhFIX would have reduced bleeding episodes while on sustained prophylactic rhFIX administered subcutaneously. Reproducible methods were developed for inducing tolerance to rhFIX in this strain of hemophilia B dogs, resulting in a significant reduction in the development of inhibitors relative to historical controls (5 of 12 versus 20 or 20, P <.001). The 7 of 12 tolerized hemophilia B dogs exhibited shortened whole blood clotting times (WBCTs), sustained detectable FIX antigen, undetectable Bethesda inhibitors, transient or no detectable antihuman FIX antibody titers by enzyme-linked immunosorbent assay (ELISA), and normal clearance of infused rhFIX. Tolerized hemophilia B dogs had 69% reduction in bleeding frequency in year 1 compared with nontolerized hemophilia B dogs (P =.0007). If proven safe in human clinical trials, subcutaneous rhFIX may provide an alternate approach to prophylactic therapy in selected patients with hemophilia B.

Oximes: Inhibitors of Human Recombinant Acetylcholinesterase. A Structure-Activity Relationship (SAR) Study

PubMed Central

Sepsova, Vendula; Karasova, Jana Zdarova; Korabecny, Jan; Dolezal, Rafael; Zemek, Filip; Bennion, Brian J.; Kuca, Kamil

2013-01-01

Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring. PMID:23959117

Producing recombinant human milk proteins in the milk of livestock species.

PubMed

Bösze, Zsuzsanna; Baranyi, Mária; Whitelaw, C Bruce A

2008-01-01

Recombinant human proteins produced by the mammary glands of genetically modified transgenic livestock mammals represent a special aspect of milk bioactive components. For therapeutic applications, the often complex posttranslational modifications of human proteins should be recapitulated in the recombinant products. Compared to alternative production methods, mammary gland production is a viable option, underlined by a number of transgenic livestock animal models producing abundant biologically active foreign proteins in their milk. Recombinant proteins isolated from milk have reached different phases of clinical trials, with the first marketing approval for human therapeutic applications from the EMEA achieved in 2006.

A monoclonal antibody based elisa for quantitation of human leukaemia inhibitory factor.

PubMed

Taupin, J L; Gualde, N; Moreau, J F

1997-02-01

The authors report on the development of a new sandwich enzyme-linked immunoabsorbent assay (ELISA) for the quantitation of the human cytokine leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) with high accuracy and sensitivity (23 pg/ml), in less than 5 h and in various biological fluids. The antibodies used in this assay were raised against recombinant glycosylated LIF expressed in vivo following inoculation of recombinant vaccinia viruses, and screened with the biologically active cytokine in a flow cytometry assay using cells expressing a membrane-bound form of LIF. Furthermore, this home-made assay was compared with two commercially available ELISA kits. The results led to the conclusion that these three assays are far from being equivalent between each other, in terms of sensitivity towards non-glycosylated vs glycosylated LIF. Two major parameters must be incriminated: the glycosylation status of the LIF molecule used as the calibrator, and the binding characteristics of the monoclonal antibodies used to set up these assays toward LIF derived from Escherichia coli or from eukaryotic cells. This points out the importance of these parameters for the design of ELISAs meant for the quantitation of glycosylated cytokines in biological fluids.

Evaluation of two recombinant Leishmania proteins identified by an immunoproteomic approach as tools for the serodiagnosis of canine visceral and human tegumentary leishmaniasis.

PubMed

Coelho, Eduardo Antonio Ferraz; Costa, Lourena Emanuele; Lage, Daniela Pagliara; Martins, Vívian Tamietti; Garde, Esther; de Jesus Pereira, Nathália Cristina; Lopes, Eliane Gonçalves Paiva; Borges, Luiz Felipe Nunes Menezes; Duarte, Mariana Costa; Menezes-Souza, Daniel; de Magalhães-Soares, Danielle Ferreira; Chávez-Fumagalli, Miguel Angel; Soto, Manuel; Tavares, Carlos Alberto Pereira

2016-01-15

Serological diagnostic tests for canine and human leishmaniasis present problems related with their sensitivity and/or specificity. Recently, an immunoproteomic approach performed with Leishmania infantum proteins identified new parasite antigens. In the present study, the diagnostic properties of two of these proteins, cytochrome c oxidase and IgE-dependent histamine-releasing factor, were evaluated for the serodiagnosis of canine visceral (CVL) and human tegumentary (HTL) leishmaniasis. For the CVL diagnosis, sera samples from non-infected dogs living in an endemic or non-endemic area of leishmaniasis, sera from asymptomatic or symptomatic visceral leishmaniasis (VL) dogs, from Leish-Tec(®)-vaccinated dogs, and sera from animals experimentally infected by Trypanosoma cruzi or Ehrlichia canis were used. For the HTL diagnosis, sera from non-infected subjects living in an endemic area of leishmaniasis, sera from active cutaneous or mucosal leishmaniasis patients, as well as those from T. cruzi-infected patients were employed. ELISA assays using the recombinant proteins showed both sensitivity and specificity values of 100% for the serodiagnosis of both forms of disease, with high positive and negative predictive values, showing better diagnostic properties than the parasite recombinant A2 protein or a soluble Leishmania antigen extract. In this context, the two new recombinant proteins could be considered to be used in the serodiagnosis of CVL and HTL. Copyright © 2015 Elsevier B.V. All rights reserved.

Bystander effects in radiation-induced genomic instability

NASA Technical Reports Server (NTRS)

Morgan, William F.; Hartmann, Andreas; Limoli, Charles L.; Nagar, Shruti; Ponnaiya, Brian

2002-01-01

Exposure of GM10115 hamster-human hybrid cells to X-rays can result in the induction of chromosomal instability in the progeny of surviving cells. This instability manifests as the dynamic production of novel sub-populations of cells with unique cytogenetic rearrangements involving the "marker" human chromosome. We have used the comet assay to investigate whether there was an elevated level of endogenous DNA breaks in chromosomally unstable clones that could provide a source for the chromosomal rearrangements and thus account for the persistent instability observed. Our results indicate no significant difference in comet tail measurement between non-irradiated and radiation-induced chromosomally unstable clones. Using two-color fluorescence in situ hybridization we also investigated whether recombinational events involving the interstitial telomere repeat-like sequences in GM10115 cells were involved at frequencies higher than random processes would otherwise predict. Nine of 11 clones demonstrated a significantly higher than expected involvement of these interstitial telomere repeat-like sequences at the recombination junction between the human and hamster chromosomes. Since elevated levels of endogenous breaks were not detected in unstable clones we propose that epigenetic or bystander effects (BSEs) lead to the activation of recombinational pathways that perpetuate the unstable phenotype. Specifically, we expand upon the hypothesis that radiation induces conditions and/or factors that stimulate the production of reactive oxygen species (ROS). These reactive intermediates then contribute to a chronic pro-oxidant environment that cycles over multiple generations, promoting chromosomal recombination and other phenotypes associated with genomic instability.

Effectiveness of Recombinant Human Growth Hormone for Pharyngocutaneous Fistula Closure.

PubMed

Kucuk, Nurten; Sari, Murat; Midi, Ahmet; Yumusakhuylu, Ali Cemal; Findik, Ozan; Binnetoglu, Adem

2015-12-01

In laryngeal cancer, which comprises 25% of head and neck cancer, chemotherapy has come into prominence with the increase in organ-protective treatments. With such treatment, salvage surgery has increased following recurrence; the incidence of pharyngocutaneous fistula has also increased in both respiratory and digestive system surgery. We investigated the effects of recombinant human growth hormone on pharyngocutaneous fistula closure in Sprague-Dawley rats, based on an increase in amino acid uptake and protein synthesis for wound healing, an increase in mitogenesis, and enhancement of collagen formation by recombinant human growth hormone. This study was experimental animal study. Forty Sprague-Dawley rats were separated into two groups, and pharyngoesophagotomy was performed. The pharyngoesophagotomy was sutured with vicryl in both groups. Rats in group 1 (control group) received no treatment, while those in group 2 were administered a subcutaneous injection of recombinant human growth hormone daily. On day 14, the pharynx, larynx, and upper oesophagus were excised and examined microscopically. Pharyngocutaneous fistula exhibited better closure macroscopically in the recombinant human growth hormone group. There was a significant difference in collagen formation and epithelisation in the recombinant human growth hormone group compared to the control group. This study is believed to be the first in which the effect of recombinant human growth hormone on pharyngocutaneous fistula closure was evaluated, and the findings suggest the potential of use of growth hormone for treatment of pharyngocutaneous fistula.

Recombinant Newcastle disease virus expressing human TRAIL as a potential candidate for hepatoma therapy

USDA-ARS?s Scientific Manuscript database

Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently proved for clinical trials. We have previously reported, for the first time, NDV Anhinga strain has an efficient cancer therapeutic efficacy in hepatoma. Tumor necrosis factor-related apo...

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

PubMed

Varghese, Anju; Raina, Opinder K; Chandra, Dinesh; Mirdha, Bijay R; Kelawala, Naresh H; Solanki, Jayesh B; Kumar, Niranjan; Ravindran, Reghu; Arun, Anandanarayanan; Rialch, Ajayta; Lalrinkima, Hniang; Kelawala, Rohan N; Samanta, Subhamoy

2017-12-20

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

PubMed

Tiefenbacher, S; Bohra, R; Amiral, J; Bowyer, A; Kitchen, S; Lochu, A; Rosén, S; Ezban, M

2017-10-01

Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA ® -Cephascreen ® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective assay qualifications. Conclusion The one-stage clotting assay with the STA-Cephascreen APTT reagent, the ROX Factor IX chromogenic assay and the BIOPHEN Factor IX chromogenic assay are considered to be qualified for the measurement of N9-GP in 3.2% (0.109 m) citrated human plasma. © 2017 International Society on Thrombosis and Haemostasis.

Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur

2009-07-31

Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increasedmore » levels of immunoreactive phosphoAkt in treated NB41A3-hGFR{alpha}-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.« less

Recombinant granulocyte colony-stimulating factor administered enterally to neonates is not absorbed.

PubMed

Calhoun, Darlene A; Maheshwari, Akhil; Christensen, Robert D

2003-08-01

Granulocyte colony-stimulating factor (G-CSF) is present in liquids swallowed by the fetus and neonate; specifically, amniotic fluid, colostrum, and human milk. The swallowed G-CSF has local effects on enteric cells, which express the G-CSF receptor. However, some portion of the G-CSF ingested by the fetus and neonate might be absorbed into the circulation and have systemic actions, such as stimulating neutrophil production. To assess this possibility we sought to determine if circulating G-CSF concentrations of neonates increase after enteral administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF). This was a single-center, prospective, blinded, randomized, 2 x 2 crossover study, with each infant receiving 1 dose of rhG-CSF (100 microg/kg) and 1 dose of placebo. Plasma G-CSF concentrations were measured at 2 and 4 hours after administration of the test solution. No significant change in plasma G-CSF concentration was observed after the enteral administration of rhG-CSF. On this basis, we conclude that orally administered rhG-CSF is not absorbed in significant quantities, and we speculate that the G-CSF swallowed by the fetus and neonate has local but not systemic effects.

Genetic recombination as a major cause of mutagenesis in the human globin gene clusters.

PubMed

Borg, Joseph; Georgitsi, Marianthi; Aleporou-Marinou, Vassiliki; Kollia, Panagoula; Patrinos, George P

2009-12-01

Homologous recombination is a frequent phenomenon in multigene families and as such it occurs several times in both the alpha- and beta-like globin gene families. In numerous occasions, genetic recombination has been previously implicated as a major mechanism that drives mutagenesis in the human globin gene clusters, either in the form of unequal crossover or gene conversion. Unequal crossover results in the increase or decrease of the human globin gene copies, accompanied in the majority of cases with minor phenotypic consequences, while gene conversion contributes either to maintaining sequence homogeneity or generating sequence diversity. The role of genetic recombination, particularly gene conversion in the evolution of the human globin gene families has been discussed elsewhere. Here, we summarize our current knowledge and review existing experimental evidence outlining the role of genetic recombination in the mutagenic process in the human globin gene families.

Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

PubMed

Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

2012-09-01

Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

Granulocyte colony stimulating factor treatment for neonatal neutropenia.

PubMed Central

Russell, A. R.; Davies, E. G.; Ball, S. E.; Gordon-Smith, E.

1995-01-01

In a pilot study recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered to 12 neutropenic preterm infants to determine if neonatal neutropenia is secondary to decreased endogenous G-CSF production. Respiratory variables were monitored because of the possible link between inflammatory cells and hyaline membrane disease. All infants showed increased neutrophil counts. The only possible side effect observed was an exacerbation of thrombocytopenia. PMID:7538031

The Development of Novel Recombinant Human Gelatins as Replacements for Animal-Derived Gelatin in Pharmaceutical Applications

NASA Astrophysics Data System (ADS)

Olsen, David; Chang, Robert; Williams, Kim E.; Polarek, James W.

We have developed a recombinant expression system to produce a series of novel recombinant human gelatins that can substitute for animal sourced gelatin preparations currently used in pharmaceutical and nutraceutical applications. This system allows the production of human sequence gelatins, or, if desired, gelatins from any other species depending on the availability of the cloned gene. The gelatins produced with this recombinant system are of defined molecular weight, unlike the animal-sourced gelatins, which consist of numerous polypeptides of varying size. The fermentation and purification process used to prepare these recombinant gelatins does not use any human- or animal-derived components and thus this recombinant material should be free from viruses and agents that cause transmissible spongiform encephalopathies. The recombinant gelatins exhibit lot-to-lot reproducibility and we have performed extensive analytical testing on them. We have demonstrated the utility of these novel gelatins as biological stabilizers and plasma expanders, and we have shown they possess qualities that are important in applications where gel formation is critical. Finally, we provide examples of how our system allows the engineering of these recombinant gelatins to optimize the production process.

High-Mobility Group Box 1 From Hypoxic Trophoblasts Promotes Endothelial Microparticle Production and Thrombophilia in Preeclampsia.

PubMed

Hu, Yae; Yan, Ruhong; Zhang, Ce; Zhou, Zhichao; Liu, Meng; Wang, Can; Zhang, Hong; Dong, Liang; Zhou, Tiantian; Wu, Yi; Dong, Ningzheng; Wu, Qingyu

2018-04-12

Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1-stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia. © 2018 American Heart Association, Inc.

Therapeutic inhibition of the complement system. Y2K update.

PubMed

Asghar, S S; Pasch, M C

2000-09-01

Activation of complement is an essential part of the mechanism of pathogenesis of a large number of human diseases; its inhibition by pharmacological means is likely to suppress disease processes in complement mediated diseases. From this point of view low molecular weight synthetic inhibitors of complement are being developed and high molecular weight natural inhibitors of human origin present in plasma or embedded in cell membrane are being purified or produced in their recombinant forms. This review is concerned with high molecular weight inhibitors, some of which are already in clinical use but may be efficacious in many other diseases in which they have not yet been tried. C1-esterase inhibitor (C1-INH) concentrate prepared from human plasma is being successfully used for the treatment of hereditary angioneurotic edema. Recently, C1-INH has been found to be consumed in severe inflammation and has been shown to exert beneficial effects in several inflammatory conditions such as human sepsis, post-operative myocardial dysfunction due to reperfusion injury, severe capillary leakage syndrome after bone marrow transplantation, reperfusion injury after lung transplantation, burn, and cytotoxicity caused by IL-2 therapy in cancer. Factor I has been used for the treatment of factor I deficiency. Recombinant soluble forms of membrane cofactor protein (MCP), and decay accelerating factor (DAF) have not yet been tried in humans but have been shown to be effective in immune complex mediate inflammation in animals. Organs of pigs transgenic for one or more of human membrane regulators of complement namely membrane cofactor protein (MCP), decay accelerating factor (DAF) or CD59, are being produced for transplantation into humans. They have been shown to be resistant to hyperacute rejection in non-human primates; acute vascular rejection is still a problem in their clinical use. It is hoped that these observations together with future developments will make xeno-transplantation in clinical practice a reality. Several recombinant variants of complement receptor 1 (CR1) have been produced. The most effective of these appears to be sCR1-SLe x, sCR1 part of which inhibits complement and carbohydrate Sle x moiety inhibits selectin mediated interactions of neutrophils and lymphocytes with endothelium. Although clinical trials of sCR1 in humans is eagerly awaited, several of the recombinant versions of sCR1 have been shown to suppress ischemia/reperfusion injury, thermal trauma, and immune complex mediated inflammation. They have also been shown to be effective in experimental models of systemic sclerosis, arthritis, myasthenia gravis, Guillain Barré syndrome and glomerulonephritis. Intravenous immunoglobulin, three of the most prominent properties of which are neutralization of autoantibody activity, suppression of autoantibody production and inhibition of complement activity, is being used in several diseases. These include autoimmune thrombocyopenic purpura, Kawasaki disease and several neurological diseases such as myasthenia gravis and Guillain Barre syndrome. In many uncontrolled small scale studies intravenous immunoglobulin has been shown to be effective in many immunological including dermatological diseases; controlled clinical trials in a large number of patients with these diseases is needed to establish the efficacy. It is hoped that in future therapeutic inhibition of complement will be one of the major approaches to combat many human diseases.

LDSplitDB: a database for studies of meiotic recombination hotspots in MHC using human genomic data.

PubMed

Guo, Jing; Chen, Hao; Yang, Peng; Lee, Yew Ti; Wu, Min; Przytycka, Teresa M; Kwoh, Chee Keong; Zheng, Jie

2018-04-20

Meiotic recombination happens during the process of meiosis when chromosomes inherited from two parents exchange genetic materials to generate chromosomes in the gamete cells. The recombination events tend to occur in narrow genomic regions called recombination hotspots. Its dysregulation could lead to serious human diseases such as birth defects. Although the regulatory mechanism of recombination events is still unclear, DNA sequence polymorphisms have been found to play crucial roles in the regulation of recombination hotspots. To facilitate the studies of the underlying mechanism, we developed a database named LDSplitDB which provides an integrative and interactive data mining and visualization platform for the genome-wide association studies of recombination hotspots. It contains the pre-computed association maps of the major histocompatibility complex (MHC) region in the 1000 Genomes Project and the HapMap Phase III datasets, and a genome-scale study of the European population from the HapMap Phase II dataset. Besides the recombination profiles, related data of genes, SNPs and different types of epigenetic modifications, which could be associated with meiotic recombination, are provided for comprehensive analysis. To meet the computational requirement of the rapidly increasing population genomics data, we prepared a lookup table of 400 haplotypes for recombination rate estimation using the well-known LDhat algorithm which includes all possible two-locus haplotype configurations. To the best of our knowledge, LDSplitDB is the first large-scale database for the association analysis of human recombination hotspots with DNA sequence polymorphisms. It provides valuable resources for the discovery of the mechanism of meiotic recombination hotspots. The information about MHC in this database could help understand the roles of recombination in human immune system. DATABASE URL: http://histone.scse.ntu.edu.sg/LDSplitDB.

Expression of human factor IX gene in murine plasma through lentiviral vector-infected haematopoietic stem cells.

PubMed

Chen, Haoming; Yao, Hengmei; Huang, Lu; Shen, Qi; Jia, William; Xue, Jinglun

2006-12-01

1. Haematopoietic stem cells (HSC) are an attractive target for gene therapy. Gene transfer to HSC can provide a potential cure for many inherited diseases. Moreover, recombinant lentiviral vectors can transfer genes efficiently to HSC. In the present study, we used the recombinant lentiviruses FUGW (Flip, ubiquitin promoter, GFP and WRE vector) and FUXW (Flip, ubiquitin promoter, F IX and WRE vector), which carry the enhanced green fluorescent protein (EGFP) and human factor IX (hFIX) gene, respectively, to infect HSC. 2. High titres of recombinant lentivirus were prepared from 293T cells by calcium phosphate-mediated transient cotransfection. Murine mononuclear cells (MNC) separated from murine bone marrow and HSC separated by magnetic cell sorting were cultured in vitro. Cells they were infected by the recombinant lentiviruses FUGW and FUXW. The expression of EGFP was observed under a fluorescent microscope and was analysed by fluorescence-activated cell sorting, whereas the expression of hFIX was detected by ELISA. 3. The results show that the lentiviral vectors can efficiently infect murine HSC in vitro and that transduction was more efficient following cytokine treatment with interleukin (IL)-3, IL-6 and stem cell factor. 4. Haematopoietic stem cells infected with lentivirus FUXW were transplanted into [(60)Co]-irradiated non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice. The expression of hFIX in the blood plasma of the transplanted mice reached a peak of 44.9 +/- 7.6 ng/mL on Day 7. An assay of transaminase levels and a histological study of the liver showed that there was no significant damage following HSC transplantation to mice. 5. The results of the present study suggest that transplantation of HSC results in the persistant expression of hFIX in mice, which may be useful in haemophilia B gene therapy.

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein.

PubMed

McCue, Justin; Kshirsagar, Rashmi; Selvitelli, Keith; Lu, Qi; Zhang, Mingxuan; Mei, Baisong; Peters, Robert; Pierce, Glenn F; Dumont, Jennifer; Raso, Stephen; Reichert, Heidi

2015-07-01

Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc. Copyright © 2015 Biogen. Published by Elsevier Ltd.. All rights reserved.

Population Demographic History Can Cause the Appearance of Recombination Hotspots

PubMed Central

Johnston, Henry R.; Cutler, David J.

2012-01-01

Although the prevailing view among geneticists suggests that recombination hotspots exist ubiquitously across the human genome, there is only limited experimental evidence from a few genomic regions to support the generality of this claim. A small number of true recombination hotspots are well supported experimentally, but the vast majority of hotspots have been identified on the basis of population genetic inferences from the patterns of linkage disequilibrium (LD) seen in the human population. These inferences are made assuming a particular model of human history, and one of the assumptions of that model is that the effective population size of humans has remained constant throughout our history. Our results show that relaxation of the constant population size assumption can create LD and variation patterns that are qualitatively and quantitatively similar to human populations without any need to invoke localized hotspots of recombination. In other words, apparent recombination hotspots could be an artifact of variable population size over time. Several lines of evidence suggest that the vast majority of hotspots identified on the basis of LD information are unlikely to have elevated recombination rates. PMID:22560089

Streptococcal 5'-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion.

PubMed

Zheng, Lisa; Khemlani, Adrina; Lorenz, Natalie; Loh, Jacelyn M S; Langley, Ries J; Proft, Thomas

2015-12-25

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5'-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 μm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of In Vitro Hemodilution, Hypothermia and rFVIIa Addition on Coagulation in Human Blood

DTIC Science & Technology

2012-03-30

primary fluids used by many trauma units and the US Army for pre-hospital resuscitation [17]. HX, a hetastarch-based product in a balanced electro...and has been associated with dilution of coagulation factors and hypothermia. Recombinant activated Factor VII (rFVIIa) has been used, often as a...of rFVIIa results in an enhancement of thrombin generation on the platelet surface at the site of injury independent of the presence of Factor VIII

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma.

PubMed

Coppen, S R; Newsam, R; Bull, A T; Baines, A J

1995-04-20

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.

Quantification of GC-biased gene conversion in the human genome

PubMed Central

Glémin, Sylvain; Arndt, Peter F.; Messer, Philipp W.; Petrov, Dmitri; Galtier, Nicolas; Duret, Laurent

2015-01-01

Much evidence indicates that GC-biased gene conversion (gBGC) has a major impact on the evolution of mammalian genomes. However, a detailed quantification of the process is still lacking. The strength of gBGC can be measured from the analysis of derived allele frequency spectra (DAF), but this approach is sensitive to a number of confounding factors. In particular, we show by simulations that the inference is pervasively affected by polymorphism polarization errors and by spatial heterogeneity in gBGC strength. We propose a new general method to quantify gBGC from DAF spectra, incorporating polarization errors, taking spatial heterogeneity into account, and jointly estimating mutation bias. Applying it to human polymorphism data from the 1000 Genomes Project, we show that the strength of gBGC does not differ between hypermutable CpG sites and non-CpG sites, suggesting that in humans gBGC is not caused by the base-excision repair machinery. Genome-wide, the intensity of gBGC is in the nearly neutral area. However, given that recombination occurs primarily within recombination hotspots, 1%–2% of the human genome is subject to strong gBGC. On average, gBGC is stronger in African than in non-African populations, reflecting differences in effective population sizes. However, due to more heterogeneous recombination landscapes, the fraction of the genome affected by strong gBGC is larger in non-African than in African populations. Given that the location of recombination hotspots evolves very rapidly, our analysis predicts that, in the long term, a large fraction of the genome is affected by short episodes of strong gBGC. PMID:25995268

Atelocollagen sponge and recombinant basic fibroblast growth factor combination therapy for resistant wounds with deep cavities.

PubMed

Nakanishi, Asako; Hakamada, Arata; Isoda, Ken-ichi; Mizutani, Hitoshi

2005-05-01

Recent advances in bioengineering have introduced materials that enhance wound healing. Even with such new tools, some deep ulcers surrounded by avascular tissues, including bone, tendon, and fascia, are resistant to various therapies and easily form deep cavities with loss of subcutaneous tissue. Atelocollagen sponges have been used as an artificial dermis to cover full-thickness skin defects. Topical recombinant human basic fibroblast growth factor has been introduced as a growth factor to induce fibroblast proliferation in skin ulcers. We applied these materials in combination in two patients with deep resistant wounds: one with a cavity reaching the mediastinum through a divided sternum and one with deep necrotic wounds caused by electric burns. These wounds did not respond to the topical basic fibroblast growth factor alone. In contrast, the combination therapy closed the wounds rapidly without further surgical treatment. This combination therapy is a potent treatment for resistant wounds with deep cavities.

Rat Stem-Cell Factor Induces Splenocytes Capable Of Regenerating The Thymus

PubMed Central

Migita, Russell T.; Trebasky, Lisa D.; Housman, Jerry M.; Elliott, Gary S.; Hendren, R. Wayne; Deprince, Randolph B.; Greiner, Dale L.

1992-01-01

Cytokine regulation of prethymic T-lymphoid progenitor-cell proliferation and/or differentiation has not been well-defined, although much is known of cytokine regulation of hemopoietic stem- and progenitor-cell development. Here we use a recently identified hemopoietic growth factor, stem-cell factor (SCF) (a form of the c-kit ligand), and a transplant model of thymocyte regeneration to assess the effect of SCF on the in vivo generation of prethymic, thymocyte progenitor-cell activity. We show that recombinant rat SCF (rrSCF164 administered to weanling rats selectively induces an increase in thymocyte progenitor activity in the spleens of treated rats as compared to rats treated with vehicle, polyethylene glycol (PEG)-conjugated rat albumin, or recombinant human granulocyte colony-stimulating factor (rhG-CSF). These data demonstrate that administration of SCF in vivo affects extrathymic-origin thymocyte regenerating cells and may influence, directly or indirectly, early prethymic stages of T-cell lymphopoiesis in addition to its known effect on early stages of myelopoiesis and erythropoiesis. PMID:1285280

Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

PubMed

Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained*

PubMed Central

Zheng, Hongying; Nagaraja, Ganachari M.; Kaur, Punit; Asea, Edwina E.; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72bv can now be used to unlock the important role Hsp72 plays in modulating immune function. PMID:19861412

Effectiveness of Recombinant Human Growth Hormone for Pharyngocutaneous Fistula Closure

PubMed Central

Sari, Murat; Midi, Ahmet; Yumusakhuylu, Ali Cemal; Findik, Ozan; Binnetoglu, Adem

2015-01-01

Objectives In laryngeal cancer, which comprises 25% of head and neck cancer, chemotherapy has come into prominence with the increase in organ-protective treatments. With such treatment, salvage surgery has increased following recurrence; the incidence of pharyngocutaneous fistula has also increased in both respiratory and digestive system surgery. We investigated the effects of recombinant human growth hormone on pharyngocutaneous fistula closure in Sprague-Dawley rats, based on an increase in amino acid uptake and protein synthesis for wound healing, an increase in mitogenesis, and enhancement of collagen formation by recombinant human growth hormone. Methods This study was experimental animal study. Forty Sprague-Dawley rats were separated into two groups, and pharyngoesophagotomy was performed. The pharyngoesophagotomy was sutured with vicryl in both groups. Rats in group 1 (control group) received no treatment, while those in group 2 were administered a subcutaneous injection of recombinant human growth hormone daily. On day 14, the pharynx, larynx, and upper oesophagus were excised and examined microscopically. Results Pharyngocutaneous fistula exhibited better closure macroscopically in the recombinant human growth hormone group. There was a significant difference in collagen formation and epithelisation in the recombinant human growth hormone group compared to the control group. Conclusion This study is believed to be the first in which the effect of recombinant human growth hormone on pharyngocutaneous fistula closure was evaluated, and the findings suggest the potential of use of growth hormone for treatment of pharyngocutaneous fistula. PMID:26622960

Evaluation of Recombinant Human FGF-2 and PDGF-BB in Periodontal Regeneration: A Systematic Review and Meta-Analysis.

PubMed

Li, Feifei; Yu, Fanyuan; Xu, Xin; Li, Chunjie; Huang, Dingming; Zhou, Xuedong; Ye, Ling; Zheng, Liwei

2017-03-06

The prognosis for successful treatment of periodontal diseases is generally poor. Current therapeutic strategies often fail to regenerate infected periodontium. Recently an alternative strategy has been developed that combines conventional treatment with the application of recombinant human growth factors (rhGFs). But ambiguities in existed studies on the clinical efficacy of rhGFs do not permit either the identification of the specific growth factors effective for therapeutic interventions or the optimal concentration of them. Neither is it known whether the same rhGF can stimulate regeneration of both soft tissue and bone, or whether different patient populations call for differential use of the growth factors. In order to explore these issues, a meta-analysis was carried out. Particular attention was given to the therapeutic impact of fibroblast growth factor 2(FGF-2) and platelet derived growth factor BB (PDGF-BB). Our findings indicate that 0.3% rhFGF-2 and 0.3 mg/ml rhPDGF-BB show a greater capacity for periodontal regeneration than other concentrations and superiority to control groups with statistical significance. In the case of patients suffering only from gingival recession, however, the application of rhPDGF-BB produces no significant regenerative advantage. The findings of this study can potentially endow clinicians with guidelines for the appropriate application of these two rhGFs.

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4).

PubMed

Bertling, Anne; Brodde, Martin F; Visser, Mayken; Treffon, Janina; Fennen, Michelle; Fender, Anke C; Kelsch, Reinhard; Kehrel, Beate E

2017-09-01

Hemarthrosis, or bleeding into the joints, is a hallmark of hemophilia. Heme triggers oxidative stress, inflammation, and destruction of cartilage and bone. The haptoglobin-CD163-heme oxygenase-1 (HO-1) pathway circumvents heme toxicity through enzymatic degradation of heme and transcription of antioxidant genes. Plasma-derived factor concentrates contain many proteins that might impact on cellular pathways in joints, blood, and vessels. Activation of platelets from healthy volunteers was assessed by flow cytometry analysis of fibrinogen binding and CD62P expression. Platelet CXCL4 release was measured by ELISA. Human peripheral blood mononuclear cells were exposed to CXCL4 or platelet supernatants (untreated or pre-stimulated with factor VIII (FVIII) products) during their differentiation to macrophages and analyzed for CD163 expression. Some macrophage cultures were additionally incubated with autologous hemoglobin for 18 h for analysis of HO-1 expression. Platelet CXCL4 release was increased by all 8 tested plasma-derived FVIII products but not the 3 recombinant products. Macrophages exposed to supernatant from platelets treated with some plasma-derived FVIII products downregulated CD163 surface expression and failed to upregulate the athero- and joint protective enzyme HO-1 in response to hemoglobin. Plasma-derived FVIII products might promote bleeding-induced joint injury via generation of macrophages that are unable to counteract redox stress.

A Recombinant Probiotic, Lactobacillus casei, Expressing the Clostridium perfringens α-toxoid, as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis.

PubMed

Alimolaei, Mojtaba; Golchin, Mehdi; Abshenas, Jalil; Ezatkhah, Majid; Bafti, Mehrdad Shamsaddini

2018-06-01

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa 247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.

TOPBP1Dpb11 plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1Rad9

PubMed Central

Sims, Jennie Rae; Freire, Raimundo

2017-01-01

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1–TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair. PMID:28228534

The substitution of cysteine 17 of recombinant human G-CSF with alanine greatly enhanced its stability.

PubMed

Ishikawa, M; Iijima, H; Satake-Ishikawa, R; Tsumura, H; Iwamatsu, A; Kadoya, T; Shimada, Y; Fukamachi, H; Kobayashi, K; Matsuki, S

1992-02-01

Human recombinant granulocyte-colony stimulating factor (rhG-CSF) has one free cysteine at position 17 and has two disulfide bridges (Cys36-Cys42 and Cys64-Cys74). The Cys17 of rhG-CSF was substituted with Gly, Ala, Ser, Ile, Tyr, Arg, and Pro, or deleted using site-directed mutagenesis in order to improve its thermostability. With the exception of Pro17-rhG-CSF, all mutant proteins retained biological activity which promotes the growth of mouse bone marrow cells in vitro. Among these mutant proteins, Ala17-rhG-CSF had more than 5 times higher stability than rhG-CSF. But Ser17-rhG-CSF had almost same stability as rhG-CSF and other mutant proteins had only lower stability.

Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

PubMed

Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

2010-10-01

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens. 2010 Elsevier Inc. All rights reserved.

The serum concentration of tumor necrosis factor alpha is not an index of growth-hormone- or obesity-induced insulin resistance.

PubMed

Pincelli, A I; Brunani, A; Scacchi, M; Dubini, A; Borsotti, R; Tibaldi, A; Pasqualinotto, L; Maestri, E; Cavagnini, F

2001-01-01

The tumor necrosis factor alpha (TNF-alpha) might play a central role in insulin resistance, a frequent correlate of obesity likely contributing to some obesity-associated complications. Adult growth hormone (GH) deficiency syndrome (GHDA) shares with obesity excessive fat mass, hyperlipidemia, increased cardiovascular risk, and insulin resistance. On the other hand, GH has been shown to induce transient deterioration of glucose metabolism and insulin resistance when administered in normal humans and in GHDA patients. No information is presently available on the relationship between serum TNF-alpha levels and insulin sensitivity in GHDA. We compared the serum TNF-alpha levels found in 10 GHDA patients before and after a 6-month recombinant human GH therapy (Genotropin), in an insulin resistance prone population of 16 obese (OB) patients and in 38 normal-weight healthy blood donors (controls). The insulin sensitivity was assessed by a euglycemic-hyperinsulinemic glucose clamp in all the GHDA patients and in 10 OB and in 6 control subjects. The serum TNF-alpha levels were not significantly different in OB patients (42.2 +/- 12.81 pg/ml), in GHDA patients at baseline (71.3 +/- 23.97 pg/ml), and in controls (55.3 +/- 14.28 pg/ml). A slight decrease of TNF-alpha values was noted in GHDA patients after 6 months of recombinant human GH treatment (44.5 +/- 20.19 pg/ml; NS vs. baseline). The insulin sensitivity (M) was significantly reduced in OB patients (2.4 +/- 0.30 mg/kg/min) as compared with control subjects (7.5 +/- 0.39 mg/kg/min) and in GHDA patients both at baseline (6.6 +/- 0.6 mg/kg/min) and after recombinant human GH therapy (5.6 +/- 0.7 mg/kg/min). The insulin sensitivity in the GHDA patients, similar to that of controls at baseline, worsened after recombinant human GH treatment (p < 0.05 vs. baseline; p = 0.05 vs. controls). Linear regression analysis showed no correlation between TNF-alpha and M values (see text) in all patient groups. These data indicate that circulating concentrations of TNF-alpha do not reflect the degree of insulin resistance in obesity and GHDA. They, however, do not exclude that TNF-alpha may induce insulin resistance at tissue level. Copyright 2001 S. Karger AG, Basel

The ESA scenario gets complex: from biosimilar epoetins to activin traps.

PubMed

Jelkmann, Wolfgang

2015-04-01

Recombinant human erythropoietin (rhEpo, epoetin) has proved beneficial in preventing transfusion-dependent anaemia in patients with chronic kidney disease. Apart from copied epoetins distributed in less regulated markets, 'biosimilar' epoetins have gained currency in many regions, where they compete with the originals and with rhEpo analogues with prolonged survival in circulation ('biobetter'). Recombinant erythropoiesis stimulating agents are potent and well tolerated. However, their production is costly, and they must be administered by the parenteral route. Hence, other anti-anaemia treatments are being evaluated. Clinical trials are being performed with stabilizers of the hypoxia-inducible transcription factors (HIFs), which increase endogenous Epo production. HIF stabilizers are chemical drugs and they are active on oral administration. However, there is fear that they may promote tumour growth. Epo mimetic peptides have also raised expectations. Yet the prototype peginesatide was recalled after just 1 year of its widespread use in the USA because of serious side-effects including cases of death. Most recently, clinical trials have been initiated with sotatercept, a recombinant soluble activin receptor type 2A IgG-Fc fusion protein. Sotatercept binds distinct members of the transforming growth factor-β family, thereby preventing the inhibitory action of these factors in erythropoiesis. Taken together, rhEpo and its long-acting recombinant analogues will likely remain mainstay of anti-anaemia therapies in the near future. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

PubMed

Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

2016-06-21

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

Perioperative recombinant human granulocyte colony-stimulating factor (Filgrastim) treatment prevents immunoinflammatory dysfunction associated with major surgery.

PubMed

Schneider, Christian; von Aulock, Sonja; Zedler, Siegfried; Schinkel, Christian; Hartung, Thomas; Faist, Eugen

2004-01-01

To examine the effects of perioperative rhG-CSF administration on immune function in patients subjected to major surgery. Severe trauma, such as major surgery, initiates acute immunodysfunction which predisposes the patient towards infectious complications. Sixty patients undergoing elective surgery received either recombinant human granulocyte colony-stimulating factor/rh G-CSF (Filgrastim) or a placebo perioperatively. At several time points before and after the surgical intervention immunofunctional parameters were assessed. RESULTS Leukocyte counts and serum levels of anti-inflammatory mediators (IL-1ra and TNF-R) were increased in Filgrastim-treated patients, while the post-operative acute phase response was attenuated. Monocyte deactivation (reduced TNF-alpha release and HLA-DR expression) and lymphocyte anergy (impaired mitogenic proliferation and reduced TH1 lymphokine release) were blunted and the incidence and severity of infectious complications were reduced. These results suggest that Filgrastim treatment reinforces innate immunity, enabling better prevention of infection. Thus, this unique combination of hematopoietic, anti-inflammatory and anti-infectious effects on the innate immune system warrants further study of clinical efficacy and sepsis prophylaxis.

Perioperative Recombinant Human Granulocyte Colony-Stimulating Factor (Filgrastim) Treatment Prevents Immunoinflammatory Dysfunction Associated With Major Surgery

PubMed Central

Schneider, Christian; von Aulock, Sonja; Zedler, Siegfried; Schinkel, Christian; Hartung, Thomas; Faist, Eugen

2004-01-01

Objective: To examine the effects of perioperative rhG-CSF administration on immune function in patients subjected to major surgery. Summary Background Data: Severe trauma, such as major surgery, initiates acute immunodysfunction which predisposes the patient towards infectious complications. Methods: Sixty patients undergoing elective surgery received either recombinant human granulocyte colony-stimulating factor/rh G-CSF (Filgrastim) or a placebo perioperatively. At several time points before and after the surgical intervention immunofunctional parameters were assessed. Results: Leukocyte counts and serum levels of anti-inflammatory mediators (IL-1ra and TNF-R) were increased in Filgrastim-treated patients, while the post-operative acute phase response was attenuated. Monocyte deactivation (reduced TNF-α release and HLA-DR expression) and lymphocyte anergy (impaired mitogenic proliferation and reduced TH1 lymphokine release) were blunted and the incidence and severity of infectious complications were reduced. Conclusions: These results suggest that Filgrastim treatment reinforces innate immunity, enabling better prevention of infection. Thus, this unique combination of hematopoietic, anti-inflammatory and anti-infectious effects on the innate immune system warrants further study of clinical efficacy and sepsis prophylaxis. PMID:14685103

Expression of bioactive recombinant human fibroblast growth factor 10 in Carthamus tinctorius L. seeds.

PubMed

Huang, Jian; Yang, Jing; Guan, Lili; Yi, Shanyong; Du, Linna; Tian, Haishan; Guo, Yongxin; Zhai, Feng; Lu, Zhen; Li, Haiyan; Li, Xiaokun; Jiang, Chao

2017-10-01

Fibroblast growth factor 10 (FGF10) is a member of the FGF superfamily. It exhibits diverse biological functions, and is extensively used for fundamental research and clinical applications involving hair growth, tissue repair, and burn wounds. Oil bodies, obtained from oil seeds, have been exploited for a variety of biotechnology applications. The use of oil bodies reduces purification steps and costs associated with the production of heterogonous proteins. Here, recombinant human FGF10 (rhFGF10) was expressed in safflower (Carthamus tinctorius L.) seeds using oilbody-oleosin technology. A plant expression vector, pOTBar-oleosin-rhFGF10, was constructed and introduced into safflower using Agrobacterium tumefaciens transformation, and mature safflower plants were obtained by grafting. Oleosin-rhFGF10 was successfully transformed and expressed in safflower seeds and inherited to the T 3 generation. Moreover, MTT assays demonstrated that oil bodies expressed oleosin-FGF10 had a dose-dependent effect on cellular proliferation. In conclusion, this may provide a method of producing oleosin-rhFGF10, and help us meet the increasing pharmacological demands for the protein. Copyright © 2016. Published by Elsevier Inc.

Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

PubMed

Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

2015-01-30

Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

Clec11a/osteolectin is an osteogenic growth factor that promotes the maintenance of the adult skeleton

PubMed Central

Yue, Rui; Shen, Bo; Morrison, Sean J

2016-01-01

Bone marrow stromal cells maintain the adult skeleton by forming osteoblasts throughout life that regenerate bone and repair fractures. We discovered that subsets of these stromal cells, osteoblasts, osteocytes, and hypertrophic chondrocytes secrete a C-type lectin domain protein, Clec11a, which promotes osteogenesis. Clec11a-deficient mice appeared developmentally normal and had normal hematopoiesis but reduced limb and vertebral bone. Clec11a-deficient mice exhibited accelerated bone loss during aging, reduced bone strength, and delayed fracture healing. Bone marrow stromal cells from Clec11a-deficient mice showed impaired osteogenic differentiation, but normal adipogenic and chondrogenic differentiation. Recombinant Clec11a promoted osteogenesis by stromal cells in culture and increased bone mass in osteoporotic mice in vivo. Recombinant human Clec11a promoted osteogenesis by human bone marrow stromal cells in culture and in vivo. Clec11a thus maintains the adult skeleton by promoting the differentiation of mesenchymal progenitors into mature osteoblasts. In light of this, we propose to call this factor Osteolectin. DOI: http://dx.doi.org/10.7554/eLife.18782.001 PMID:27976999

Age-related increase in Wnt inhibitor causes a senescence-like phenotype in human cardiac stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Tamami; Hosoyama, Tohru; Regenerative Medicine Institute, Yamaguchi University Graduate School of Medicine

Aging of cardiac stem/progenitor cells (CSCs) impairs heart regeneration and leads to unsatisfactory outcomes of cell-based therapies. As the precise mechanisms underlying CSC aging remain unclear, the use of therapeutic strategies for elderly patients with heart failure is severely delayed. In this study, we used human cardiosphere-derived cells (CDCs), a subtype of CSC found in the postnatal heart, to identify secreted factor(s) associated with CSC aging. Human CDCs were isolated from heart failure patients of various ages (2–83 years old). Gene expression of key soluble factors was compared between CDCs derived from young and elderly patients. Among these factors, SFRP1,more » a gene encoding a Wnt antagonist, was significantly up-regulated in CDCs from elderly patients (≥65 years old). sFRP1 levels was increased significantly also in CDCs, whose senescent phenotype was induced by anti-cancer drug treatment. These results suggest the participation of sFRP1 in CSC aging. We show that the administration of recombinant sFRP1 induced cellular senescence in CDCs derived from young patients, as indicated by increased levels of markers such as p16, and a senescence-associated secretory phenotype. In addition, co-administration of recombinant sFRP1 could abrogate the accelerated CDC proliferation induced by Wnt3A. Taken together, our results suggest that canonical Wnt signaling and its antagonist, sFRP1, regulate proliferation of human CSCs. Furthermore, excess sFRP1 in elderly patients causes CSC aging. - Highlights: • Wnt signaling regulates proliferation of human cardiac stem cells. • Expression of sFRP1, which is a Wnt antagonist, is up-regulated in elderly patients with heart failure. • Expression of sFRP1 is increased in anti-cancer drug-induced senescent human cardiac stem cells. • sFRP1 causes cellular senescence of young patients-derived cardiac stem cells.« less

Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses.

PubMed

Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line; Delpeyroux, Francis

2014-08-05

Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3' end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. Importance: The multiplication of circulating vaccine-derived polioviruses (cVDPVs) in poorly immunized human populations can render these viruses pathogenic, causing poliomyelitis outbreaks. Most cVDPVs are intertypic recombinants between a poliovirus (PV) strain and another human enterovirus, such as type 17 coxsackie A viruses (CA17). For further studies of the genetic exchanges between PV and CA17, we have developed a model of recombination, making it possible to rescue defective PV RNA genomes with a short deletion by cotransfecting cells with the defective PV genome and CA17 genomic RNA. Numerous recombinants were found, including homologous PV/CA17 recombinants, but mostly nonhomologous recombinants presenting duplications of parental sequences preferentially located in particular regions. Long duplications were excised by passages in cultured cells or in mice, generating diverse homologous recombinants. Recombination leading to nonhomologous recombinants, which evolve into homologous recombinants, may therefore be seen as a model of genetic plasticity in enteroviruses and, possibly, in other RNA viruses. Copyright © 2014 Holmblat et al.

An amorphous silicon photodiode microfluidic chip to detect nanomolar quantities of HIV-1 virion infectivity factor.

PubMed

Vistas, Cláudia R; Soares, Sandra S; Rodrigues, Rogério M M; Chu, Virginia; Conde, João P; Ferreira, Guilherme N M

2014-08-07

A hydrogenated amorphous silicon (a-Si:H) photosensor was explored for the quantitative detection of a HIV-1 virion infectivity factor (Vif) at a detection limit in the single nanomolar range. The a-Si:H photosensor was coupled with a microfluidic channel that was functionalized with a recombinant single chain variable fragment antibody. The biosensor selectively recognizes HIV-1 Vif from human cell extracts.

Hematopoietic growth factors and human acute leukemia.

PubMed

Löwenberg, B; Touw, I

1988-10-22

The study of myelopoietic maturation arrest in acute myeloblastic leukemia (AML) has been eased by availability of the human recombinant hemopoietic growth factors, macrophage colony stimulating factor (M-CSF), granulocyte-(G-CSF), granulocyte-macrophage-(GM-CSF) and multilineage stimulating factor (IL-3). Nonphysiological expansion of the leukemic population is not due to escape from control by these factors. Proliferation in vitro of AML cells is dependent on the presence of one or several factors in most cases. The pattern of factor-dependency does not correlate with morphological criteria in individual cases, and may thus offer a new tool for classification of AML. Overproduction of undifferentiated cells is not due to abnormal expression of receptors for the stimulating factors acting at an immature level. Rather, autocrine secretion of early acting lymphokines maintains proliferation of the leukemic clone. When looking at causes of leukemic dysregulation, yet undefined inhibitors of differentiation probably are of equal importance as dysequilibrated stimulation by lymphokines.

Recombinant factor VIIa (eptacog alfa): a pharmacoeconomic review of its use in haemophilia in patients with inhibitors to clotting factors VIII or IX.

PubMed

Lyseng-Williamson, Katherine A; Plosker, Greg L

2007-01-01

Recombinant factor VIIa (NovoSeven; also known as recombinant activated factor VII or eptacog alfa) is indicated as an intravenous haemostatic agent in haemophilia patients with inhibitors to clotting factors VIII or IX. In noncomparative trials in haemophilia patients with inhibitors, on-demand home treatment with recombinant factor VIIa was effective in controlling episodes of mild to moderate bleeding and well tolerated, with early treatment being associated with a greater rate of success and the need for fewer doses than delayed treatment. Prophylactic treatment with recombinant factor VIIa was also effective in maintaining haemostasis in patients with this indication undergoing surgery. Relative to prior treatment with plasma-derived agents, treatment with recombinant factor VIIa was associated with improvements in health-related quality of life in a cost-utility study in haemophilia patients with inhibitors in Australia. In well designed decision-model cost analyses conducted from a healthcare payer perspective in several countries, on-demand treatment with recombinant factor VIIa to control mild to moderate bleeding episodes in this patient population was predicted to be cost saving or cost neutral relative to on-demand treatment with intravenous activated prothrombin complex concentrate (aPCC). Although the acquisition cost of recombinant factor VIIa was greater than that of aPCC in some studies, the greater initial efficacy of recombinant factor VIIa than aPCC resulted in lower predicted total medical costs. Results were generally robust to plausible changes in key parameters. Orthopaedic surgery with recombinant factor VIIa to maintain haemostasis in haemophilia patients with inhibitors was generally predicted to be cost saving, relative to not having surgery, over the medium to long term in modelled cost analyses from a healthcare payer perspective in the UK and US. The initial cost of surgery was high, but the difference in costs between patients undergoing or not undergoing surgery was predicted to decline over time, as savings were realised from the decrease in the number of bleeding episodes requiring treatment in patients who underwent surgery. In haemophilia patients with inhibitors, recombinant factor VIIa is clinically effective in controlling mild to moderate bleeds, and in maintaining haemostasis in patients undergoing orthopaedic surgery. Available pharmacoeconomic data from several countries, despite inherent limitations, support the use of recombinant factor VIIa as a treatment option that is at least cost neutral relative to aPCC in treating mild to moderate bleeds in this patient population. In addition, orthopaedic surgery with recombinant factor VIIa to maintain haemostasis in haemophilia patients with inhibitors is generally cost saving relative to not having surgery over the medium to long term, as the acquisition costs of recombinant factor VIIa are offset by cost savings resulting from the decrease in the number of joint-related bleeds.

Recombinant platelet factor 4: a therapeutic, anti-neoplastic chimera?

PubMed

Lippi, Giuseppe; Favaloro, Emmanuel J

2010-07-01

Angiogenesis plays a pivotal role in many serious and life-threatening disorders (e.g., cancer, atherosclerosis, diabetes, arthritis, psoriasis, nephropathy, and retinopathy) and is regulated by a delicate equilibrium between a variety of pro- and anti-angiogenic factors. Although recombinant platelet factor 4 (PF4) was originally developed and evaluated as a clinical alternative to protamine for heparin neutralization, the current scientific evidence supports a role for this protein and derivative peptides in inhibiting tumor growth and spread, by suppression of tumor-induced neovascularization in many different types of solid tumors. As a heparin-binding tetramer, recombinant PF4 interferes with several steps of endothelial cell proliferation, migration, and angiogenesis, regulates apoptotic death through activation of distinct signal transduction pathways, inhibits growth factor receptor binding, amplifies the inflammatory response of natural killer cells through regulation of cytokines production, and induces and maintains a nonspecific immune response to cancer cells. These biological evidences paved the way for the development and marketing of novel PF4-based angiostatic agents characterized by reduced toxicity and improved bioavailability, thus raising the possibility of an alternative approach for preventing and treating growth and metastasis of tumors. Some PF4-derived molecules such as carboxyl-terminal fragments of recombinant human PF4 and modified and chimeric peptides have already been developed that exhibit stronger anti-angiogenic properties than the parent molecule and may serve as leads for further therapeutic developments. Newer means of delivering of this anti-angiogenic agent are also being attempted, including PF4-bearing polymeric microspheres, vector-mediated PF4 transduction, transgene transfection into oncolytic viruses, and molecular targeting therapy against PF4 and rHuPF4 conjugates. These delivery systems aim to produce high concentrations of the therapeutic agent in a local area for a sustained period, thereby avoiding the typical problems encountered with long-term administration of recombinant proteins.

Human cells: new platform for recombinant therapeutic protein production.

PubMed

Swiech, Kamilla; Picanço-Castro, Virgínia; Covas, Dimas Tadeu

2012-07-01

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production. Copyright © 2012 Elsevier Inc. All rights reserved.

Genetic battle between Helicobacter pylori and humans. The mechanism underlying homologous recombination in bacteria, which can infect human cells.

PubMed

Hanada, Katsuhiro; Yamaoka, Yoshio

2014-10-01

Helicobacter pylori is a gram-negative pathogenic bacterium that colonises the human stomach. The chronic infection it causes results in peptic ulcers and gastric cancers. H. pylori can easily establish a chronic infection even if the immune system attacks this pathogen with oxidative stress agents and immunoglobulins. This is attributed to bacterial defence mechanisms against these stresses. As a defence mechanism against oxidative stresses, in bacterial genomes, homologous recombination can act as a repair pathway of DNA's double-strand breaks (DSBs). Moreover, homologous recombination is also involved in the antigenic variation in H. pylori. Gene conversion alters genomic structures of babA and babB (encoding outer membrane proteins), resulting in escape from immunoglobulin attacks. Thus, homologous recombination in bacteria plays an important role in the maintenance of a chronic infection. In addition, H. pylori infection causes DSBs in human cells. Homologous recombination is also involved in the repair of DSBs in human cells. In this review, we describe the roles of homologous recombination with an emphasis on the maintenance of a chronic infection. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Cloning, expression and functional characterization of Schizosaccharomyces pombe TFIIB.

PubMed

Tamayo, Evelyn; Maldonado, Edio

2002-09-27

The transcription factor TFIIB has been identified and cloned from the yeast Schizosaccharomyces pombe. The cloned polypeptide is highly homologous to human TFIIB and to Saccharomyces cerevisiae TFIIB. S. pombe TFIIB is a 340-amino-acid-long protein and it possesses a repeated motif of 75 amino acids near the carboxy-terminal region. The purified recombinant protein is able to bind to the TBP-DNA promoter complex in gel retardation experiments. Recombinant S. pombe TFIIB is active in in vitro transcription assays, since it can complement the transcription activity of a S. pombe cell extract in which TFIIB was depleted by using antibodies.

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

PubMed Central

Vergis, James M.; Wiener, Michael C.

2011-01-01

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (Enterokinase, Factor Xa, Human Rhinovirus 3C Protease, SUMOstar, Tobacco Etch Virus Protease, and Thrombin) by use of a panel of ninety-four individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and Factor Xa were only affected by a small number of detergents, making them good choices as well. PMID:21539919

New developments in the treatment of chemotherapy-induced neutropenia: focus on balugrastim.

PubMed

Ghidini, Michele; Hahne, Jens Claus; Trevisani, Francesco; Panni, Stefano; Ratti, Margherita; Toppo, Laura; Tomasello, Gianluca

2016-01-01

Neutropenia and febrile neutropenia are two major complications of chemotherapy. Dose reductions, delays in treatment administration, and the use of granulocyte colony-stimulating factors are equally recommended options to preserve absolute neutrophil count in case of chemotherapy regimens bringing a risk of febrile neutropenia of 20% or higher. Recombinant granulocyte colony-stimulating factors, such as filgrastim and lenograstim, have a short elimination half-life (t1/2) and need to be used daily, while others, like pegfilgrastim and lipegfilgrastim, are characterized by a long t1/2 requiring only a single administration per cycle. Balugrastim is a novel long-acting recombinant granulocyte colony-stimulating factor obtained by means of a genetic fusion between recombinant human serum albumin and granulocyte colony-stimulating factor. Albumin binding increases the molecular weight and determines a high plasmatic stability leading to a t1/2 of ~19 days. Balugrastim's efficacy, safety, and tolerability have been assessed in four different clinical trials involving breast cancer patients treated with doxorubicin and docetaxel. Pegfilgrastim was chosen as a comparator. Balugrastim was noninferior to pegfilgrastim with regard to the reduction of mean duration of severe neutropenia during cycle 1. Moreover, both treatments were comparable in terms of efficacy and safety profile. Balugrastim was well tolerated, with the only related adverse event being mild to moderate bone pain. The aim of this review is to summarize the currently available literature data on balugrastim.

Suppression of genetic recombination in the pseudoautosomal region and at subtelomeres in mice with a hypomorphic Spo11 allele.

PubMed

Smagulova, Fatima; Brick, Kevin; Pu, Yongmei; Sengupta, Uttara; Camerini-Otero, R Daniel; Petukhova, Galina V

2013-07-22

Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes - normally the hottest region in the genome. Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions.

Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

PubMed

Leitner, Gerda C; Faschingbauer, Martin; Wenda, Sabine; Weigel, Günter; Fischer, Gottfried

2014-12-01

The short-term safety profile of recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) in the allogeneic stem cell setting seems acceptable; only few data on long-term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year. In this study, 20 stem cell donors (14 male/six female; median age, 40 years; range, 22-54 years) and 20 sex- and age-matched blood component donors (controls) were included. Sampling was performed before rHuG-CSF administration; at the time of donation; and on Days (+1), 7, 30, 100, 180, and 360 in both groups. Analysis of DNMT activity in nuclear extracts was performed using a modified radionuclide assay. We performed methylation-specific polymerase chain reaction to detect the methylation status of promoter CpG islands of the genes of the retinoic acid receptor beta (RAR-B) and the Ras association domain family 1A (RASSF1A). DNMT activity increased significantly on the day of donation and 1 day after (p < 0.05). By Day +7 baseline values were reached. No further significant alterations of DNMT activity in the treated group compared to the controls were observed. We could not detect any differences in the gene methylation of RAR-B and RASSF1A between both groups. In our prospective study no evidence of long-lasting increased DNMT activity or enhanced DNA methylation in a limited panel of target genes after recombinant human G-CSF administration was observed in healthy stem cell donors. © 2014 AABB.

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

PubMed

Böhm, Ernst; Seyfried, Birgit K; Dockal, Michael; Graninger, Michael; Hasslacher, Meinhard; Neurath, Marianne; Konetschny, Christian; Matthiessen, Peter; Mitterer, Artur; Scheiflinger, Friedrich

2015-09-18

BACKGROUND & Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

Chemical heterogeneity as a result of hydroxylamine cleavage of a fusion protein of human insulin-like growth factor I.

PubMed Central

Canova-Davis, E; Eng, M; Mukku, V; Reifsnyder, D H; Olson, C V; Ling, V T

1992-01-01

Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent. Images Fig. 2. PMID:1637301

N-/O-glycosylation analysis of human FVIIa produced in the milk of transgenic rabbits

PubMed Central

Chevreux, Guillaume; Faid, Valegh; Scohyers, Jean-Marc; Bihoreau, Nicolas

2013-01-01

Human coagulation factor VIIa is a glycoprotein that promotes haemostasis through activation of the coagulation cascade extrinsic pathway. Most haemophilia A/B patients with inhibitors are treated by injection of plasma-derived or recombinant FVIIa. The use of recombinant products raises questions about the ability of the host cell to produce efficiently post-translationally modified proteins. Glycosylation is especially critical considering that it can modulate protein safety and efficacy. The present paper reports the N-/O-glycosylation pattern of a new recombinant human factor VIIa expressed in the mammary glands of transgenic rabbits. Glycosylation was investigated by chromatography and advanced mass spectrometry techniques for glycan identification and quantitation. Mass spectrometry (MS)/MS analyses were performed to confirm the glycan structures as well as the position and branching of specific monosaccharides or substituents. The two N-glycosylation sites were found to be fully occupied mostly by mono- and bi-sialylated biantennary complex-type structures, the major form being A2G2S1. Some oligomannose/hybrid structures were retrieved in lower abundance, the major ones being GlcNAcα1,O-phosphorylated at the C6-position of a Man residue (Man-6-(GlcNAcα1,O-)phosphate motif) as commonly observed on lysosomal proteins. No immunogenic glycotopes such as Galili (Galα1,3Gal) and HD antigens (N-glycolylneuraminic acid (NeuGc)) were detected. Concerning O-glycosylation, the product exhibited O-fucose and O-glucose-(xylose)0, 1, 2 motifs as expected. The N-glycosylation consistency was also investigated by varying production parameters such as the period of lactation, the number of consecutive lactations and rabbit generations. Results show that the transgenesis technology is suitable for the long-term production of rhFVIIa with a reproducible glycosylation pattern. PMID:24092837

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

PubMed Central

Chappell, William H.; Gautam, Dipendra; Ok, Suzan T.; Johnson, Bryan A.; Anacker, Daniel C.

2015-01-01

ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. PMID:26699641

Human buccal plate extraction socket regeneration with recombinant human platelet-derived growth factor BB or enamel matrix derivative.

PubMed

Nevins, Marc L; Camelo, Marcelo; Schupbach, Peter; Nevins, Myron; Kim, Soo-Woo; Kim, David M

2011-01-01

The objective of this study was to assess the osseous healing of buccal plate extraction socket defects. There were four cohorts: group A (mineral collagen bone substitute [MCBS] scaffold alone), group B (MCBS with recombinant human platelet-derived growth factor BB [rhPDGF-BB; 0.3 mg/mL]), group C (MCBS with enamel matrix derivative [EMD]), and group D (combination of EMD with bone ceramic). The primary outcome of bone quality was evaluated using light microscopy, backscatter scanning electron microscopy, and histomorphometrics. Reentry surgery provided an opportunity for clinical observation of the healed ridge morphology. Sixteen patients with buccal wall extraction socket defects were randomized into four treatment groups of equal size. Grafting was provided at the time of extraction with advancement of the buccal flap for primary closure. A trephine core biopsy of the implant site preparation was performed after 5 months for implant placement. Histologic examination identified new bone healing around the biomaterial scaffolds. Statistically significant differences in new bone formation were not observed among the treatment groups. There was a histomorphometric trend toward more new bone for the rhPDGF-BB-treated group (group B). This group had the most favorable ridge morphology for optimal implant placement.

Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions.

PubMed

Du, Jinping; Rehm, Bernd H A

2017-11-02

Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction. By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b). Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

Characterization of the post-translational modification of recombinant human BMP-15 mature protein

PubMed Central

Saito, Seiji; Yano, Keiichi; Sharma, Shweta; McMahon, Heather E.; Shimasaki, Shunichi

2008-01-01

Bone morphogenetic protein-15 (BMP-15) is an oocyte-secreted factor critical for the regulation of ovarian physiology. When recombinant human BMP-15 (rhBMP-15) produced in human embryonic kidney 293 cells was subjected to SDS-PAGE analysis, two mature protein forms corresponding to 16 kDa (P16) and 17 kDa (P17) were observed. Despite the physiological relevance and critical function of BMP-15 in female reproduction, little is known about the structure of rhBMP-15. Here, we have analyzed the structure of the rhBMP-15 mature proteins (P16 and P17) using state-of-the-art proteomics technology. Our findings are as follows: (1) the N-terminal amino acid of P16 and P17 is pyroglutamic acid; (2) the Ser residue at the sixth position of P16 is phosphorylated; (3) P17 is O-glycosylated at Thr10; and (4) the C-terminal amino acid of P16 and P17 is truncated. These findings are the first knowledge of the structure of rhBMP-15 mature protein toward understanding the molecular basis of BMP-15 function and could provide an important contribution to the rapidly progressing research area involving oocyte-specific growth factors in modulation of female fertility. PMID:18227435

Epidemiology of a Novel Recombinant Middle East Respiratory Syndrome Coronavirus in Humans in Saudi Arabia

PubMed Central

Assiri, Abdullah M.; Midgley, Claire M.; Abedi, Glen R.; Saeed, Abdulaziz Bin; Almasri, Malak M.; Lu, Xiaoyan; Al-Abdely, Hail M.; Abdalla, Osman; Mohammed, Mutaz; Algarni, Homoud S.; Alhakeem, Raafat F.; Sakthivel, Senthilkumar K.; Nooh, Randa; Alshayab, Zainab; Alessa, Mohammad; Srinivasamoorthy, Ganesh; AlQahtani, Saeed Yahya; Kheyami, Ali; HajOmar, Waleed Husein; Banaser, Talib M.; Esmaeel, Ahmad; Hall, Aron J.; Curns, Aaron T.; Tamin, Azaibi; Alsharef, Ali Abraheem; Erdman, Dean; Watson, John T.; Gerber, Susan I.

2017-01-01

Background Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans. Fundamental questions about circulating viruses and transmission routes remain. Methods We assessed routinely collected epidemiologic data for MERS-CoV cases reported in Saudi Arabia during 1 January– 30 June 2015 and conducted a more detailed investigation of cases reported during February 2015. Available respiratory specimens were obtained for sequencing. Results During the study period, 216 MERS-CoV cases were reported. Full genome (n = 17) or spike gene sequences (n = 82) were obtained from 99 individuals. Most sequences (72 of 99 [73%]) formed a discrete, novel recombinant subclade (NRC-2015), which was detected in 6 regions and became predominant by June 2015. No clinical differences were noted between clades. Among 87 cases reported during February 2015, 13 had no recognized risks for secondary acquisition; 12 of these 13 also denied camel contact. Most viruses (8 of 9) from these 13 individuals belonged to NRC-2015. Discussions Our findings document the spread and eventual predominance of NRC-2015 in humans in Saudi Arabia during the first half of 2015. Our identification of cases without recognized risk factors but with similar virus sequences indicates the need for better understanding of risk factors for MERS-CoV transmission. PMID:27302191

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

PubMed

Jin, Qingwen; Chen, Hong; Wang, Xingxia; Zhao, Liandong; Xu, Qingchen; Wang, Huijuan; Li, Guanyu; Yang, Xiaofan; Ma, Hongming; Wu, Haoquan; Ji, Xiaohui

2015-01-01

Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

Genetic Recombination between Human and Animal Parasites Creates Novel Strains of Human Pathogen

PubMed Central

Gibson, Wendy; Peacock, Lori; Ferris, Vanessa; Fischer, Katrin; Livingstone, Jennifer; Thomas, James; Bailey, Mick

2015-01-01

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT. PMID:25816228

Genetic recombination between human and animal parasites creates novel strains of human pathogen.

PubMed

Gibson, Wendy; Peacock, Lori; Ferris, Vanessa; Fischer, Katrin; Livingstone, Jennifer; Thomas, James; Bailey, Mick

2015-03-01

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

High rates of recombination in otitis media isolates of non-typeable Haemophilus influenzae✩

PubMed Central

Cody, Alison J.; Field, Dawn; Feil, Edward J.; Stringer, Suzanna; Deadman, Mary E.; Tsolaki, Anthony G.; Gratz, Brett; Bouchet, Valérie; Goldstein, Richard; Hood, Derek W.; Moxon, E. Richard

2008-01-01

Non-typeable (NT) or capsule-deficient, Haemophilus influenzae (Hi) is a common commensal of the upper respiratory tract of humans and can be pathogenic resulting in diseases such as otitis media, sinusitis and pneumonia. The lipopolysaccharide (LPS) of NTHi is a major virulence factor that displays substantial intra-strain and inter-strain variation of its oligosaccharide structures. To investigate the genetic basis of LPS variation we sequenced internal regions of each of seven genes required for the biosynthesis of either the inner or the outer core oligosaccharide structures. These sequences were obtained from 25 representative NTHi isolates from episodes of otitis media. We found abundant evidence of recombination among LPS genes of NTHi, a finding in marked contrast to previous analyses of biosynthetic genes for capsular polysaccharide, a well-documented virulence factor of Hi. We found mosaic sequences, linkage equilibrium between loci and a lack of congruence between gene trees. These high rates were not confined to LPS genes since evidence for similar amounts of recombination was also found in eight housekeeping genes in a subset of the same 25 isolates. These findings provide a population based foundation for a better understanding of the role of NTHi LPS as a virulence factor and its potential as a candidate vaccine. PMID:12797973

Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: a prospective clinical trial.

PubMed

Mannucci, Pier Mannuccio; Kempton, Christine; Millar, Carolyn; Romond, Edward; Shapiro, Amy; Birschmann, Ingvild; Ragni, Margaret V; Gill, Joan Cox; Yee, Thynn Thynn; Klamroth, Robert; Wong, Wing-Yen; Chapman, Miranda; Engl, Werner; Turecek, Peter L; Suiter, Tobias M; Ewenstein, Bruce M

2013-08-01

Safety and pharmacokinetics (PK) of recombinant von Willebrand factor (rVWF) combined at a fixed ratio with recombinant factor VIII (rFVIII) were investigated in 32 subjects with type 3 or severe type 1 von Willebrand disease (VWD) in a prospective phase 1, multicenter, randomized clinical trial. rVWF was well tolerated and no thrombotic events, inhibitors, or serious adverse events were observed. The PK of rVWF ristocetin cofactor activity, VWF antigen, and collagen-binding activity were similar to those of the comparator plasma-derived (pd) VWF-pdFVIII. In vivo cleavage of ultra-large molecular-weight rVWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; the endogenous VWF protease) and generation of characteristic satellite bands were demonstrated. In 2 subjects with specific nonneutralizing anti-VWF-binding antibodies already detectable before rVWF infusion, a reduction in VWF multimers and VWF activity was observed. Stabilization of endogenous FVIII was enhanced following post-rVWF-rFVIII infusion as shown by the difference in area under the plasma concentration curve compared with pdVWF-pdFVIII (AUC0-∞) (P < .01). These data support the concept of administering rVWF alone once a therapeutic level of endogenous FVIII is achieved.

Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

PubMed

Mamat, Uwe; Wilke, Kathleen; Bramhill, David; Schromm, Andra Beate; Lindner, Buko; Kohl, Thomas Andreas; Corchero, José Luis; Villaverde, Antonio; Schaffer, Lana; Head, Steven Robert; Souvignier, Chad; Meredith, Timothy Charles; Woodard, Ronald Wesley

2015-04-16

Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product. As an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels. This paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

Facilitated recycling protects human RNA polymerase III from repression by Maf1 in vitro.

PubMed

Cabart, Pavel; Lee, JaeHoon; Willis, Ian M

2008-12-26

Yeast cells synthesize approximately 3-6 million molecules of tRNA every cell cycle at a rate of approximately 2-4 transcripts/gene/s. This high rate of transcription is achieved through many rounds of reinitiation by RNA polymerase (pol) III on stable DNA-bound complexes of the initiation factor TFIIIB. Studies in yeast have shown that the rate of reinitiation is increased by facilitated recycling, a process that involves the repeated reloading of the polymerase on the same transcription unit. However, when nutrients become limiting or stress conditions are encountered, RNA pol III transcription is rapidly repressed through the action of the conserved Maf1 protein. Here we examine the relationship between Maf1-mediated repression and facilitated recycling in a human RNA pol III in vitro system. Using an immobilized template transcription assay, we demonstrate that facilitated recycling is conserved from yeast to humans. We assessed the ability of recombinant human Maf1 to inhibit different steps in transcription before and after preinitiation complex assembly. We show that recombinant Maf1 can inhibit the recruitment of TFIIIB and RNA pol III to immobilized templates. However, RNA pol III bound to preinitiation complexes or in elongation complexes is protected from repression by Maf1 and can undergo several rounds of initiation. This indicates that recombinant Maf1 is unable to inhibit facilitated recycling. The data suggest that additional biochemical steps may be necessary for rapid Maf1-dependent repression of RNA pol III transcription.

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

PubMed Central

Evers, R; Grummt, I

1995-01-01

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597036

Validation of biological activity testing procedure of recombinant human interleukin-7.

PubMed

Lutsenko, T N; Kovalenko, M V; Galkin, O Yu

2017-01-01

Validation procedure for method of monitoring the biological activity of reсombinant human interleukin-7 has been developed and conducted according to the requirements of national and international recommendations. This method is based on the ability of recombinant human interleukin-7 to induce proliferation of T lymphocytes. It has been shown that to control the biological activity of recombinant human interleukin-7 peripheral blood mononuclear cells (PBMCs) derived from blood or cell lines can be used. Validation charac­teristics that should be determined depend on the method, type of product or object test/measurement and biological test systems used in research. The validation procedure for the method of control of biological activity of recombinant human interleukin-7 in peripheral blood mononuclear cells showed satisfactory results on all parameters tested such as specificity, accuracy, precision and linearity.

Potential Resuscitation Strategies for Treatment of Hemorrhagic Shock

DTIC Science & Technology

2004-09-01

thrombus (the “pop-clot” pressure); 2) an injectable clot stabilizer (“fix-a-leak”) that is a naturally occurring factor in the clotting cascade (human...recombinant Factor VIIa); and 3) the maximum time up to 24 hours for hypotensive resuscitation below the “pop-the-clot” pressure (“how low for how long...To prevent this blood products are given as soon as possible in the emergency department. Only crystalloids and colloids are currently available on

High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

PubMed

Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

2016-01-01

Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H 10 . The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination.

PubMed

Marin-Vicente, Consuelo; Domingo-Prim, Judit; Eberle, Andrea B; Visa, Neus

2015-03-15

The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs. © 2015. Published by The Company of Biologists Ltd.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.

Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression hostmore » offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.« less

Efficient expression and purification of recombinant therapeutic protein candidates, human midkine and pleiotrophin.

PubMed

Murasugi, Akira

2013-01-01

Midkine is a heparin-binding growth factor that promotes cell growth, survival, and migration. Externally added midkine prevents ventricular remodeling and improves long-term survival after myocardial infarction in the mouse. Preclinical testing of this protein is in progress. Externally added pleiotrophin, a member of the midkine protein family, promotes functional recovery after neural transplantation in rats. Thus, pleiotrophin is also a candidate therapeutic protein. Large amounts of these proteins were obtained by using the heterologous protein expression system of Pichia pastoris, and the recombinant P. pastoris clones were cultured in a controlled fermentor. Intracellular expression yielded about 300 mg/L recombinant human (rh)-midkine, which was extracted, renatured, and purified. From 1 L of the culture, 64 mg of rh-midkine was purified. Secretory expression induced by the midkine secretion signal resulted in about 100 mg of rhmidkine in 1 L of the culture supernatant, but over 70% of the rh-midkine had yeast-specific glycosylation. Three threonyl residues that are targets for glycosylation were substituted with alanyl residues, and nonglycosylated, active rh-midkine was obtained. In secretory expression using α-mating factor prepro-sequence, about 640 mg/L rh-midkine was obtained, but it was partially truncated. Therefore, a protease-deficient host was used, and about 360 mg/L intact rh-midkine was then obtained. The rh-midkine was recovered and purified, with 70% final yield. All purified rh-midkine, regardless of expression method, was able to promote mammalian cell proliferation. In secretory expression of rh-pleiotrophin using α- mating factor prepro-sequence, 260 mg/L rh-pleiotrophin could be secreted. The rh-pleiotrophin was recovered and efficiently purified with 72% final yield.

Properties of a recombinant bovine tissue factor expressed by Silkworm pupae and its performance as an Owren-type prothrombin time reagent for warfarin monitoring.

PubMed

Okuda, Masahiro; Taniguchi, Tomokuni; Takamiya, Osamu

2012-09-01

Tissue factor (TF), or thromboplastin, is a glycoprotein that triggers the extrinsic coagulation pathway. In blood coagulation testing, TF has been used as a natural source for determining Quick prothrombin time (PT) or the Owren PT (OBT). Currently, natural sources are being replaced with recombinant proteins because of their uniform characteristics and the possibility of stable mass production of PT reagents. Because bovine spongiform encephalopathy (BSE)-infected cows are widespread in Japan, we prepared a recombinant bovine TF (rbTF) with a baculovirus expression system using silkworms. To overcome the limitations of natural TF, especially in bovine brain, we expressed a full-length rbTF protein in Silkworm pupae with a baculovirus expression system. Baculovirus inactivation and the presence of DNA fragments in the rbTF fraction were confirmed using Reed-Muench and polymerase chain reaction methods after inactivation with a detergent. The rbTF fraction prepared by an immobilized anti-Silkworm pupae fluid protein Sepharose 4B column was identified as a visible band on western blots with a polyclonal antibody against human TF with cross-reactivity with TFs. The inhibition of the polyclonal antibody against human TF by the clotting assay for PT was identified, and amidolytic biological activity through activated factor VII on S-2288 substrate was observed. In conclusion, the rbTF expressed by the baculovirus system using Silkworm pupae was uniformly specific for bovine TF. The OBT reagent incorporated by this rbTF was similar to those of commercial reagents. It also showed a suitable International Sensitivity Index and reproducibility precision, thereby allowing for diagnostic use. Copyright © 2012 Elsevier Ltd. All rights reserved.

Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

PubMed

Karyagina, A S; Boksha, I S; Grunina, T M; Demidenko, A V; Poponova, M S; Sergienko, O V; Lyashchuk, A M; Galushkina, Z M; Soboleva, L A; Osidak, E O; Bartov, M S; Gromov, A V; Lunin, V G

2017-05-01

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.

New thrombopoietin receptor agonists for platelet disorders.

PubMed

Homeida, S; Ebdon, C; Batty, P; Jackson, B; Kolade, S; Bateman, C; Peng, Y Y; Stasi, R

2012-04-01

Since thrombopoietin (TPO) was cloned in 1994, TPO receptor (TPO-R) agonists have been developed which have shown significant clinical activity in various conditions characterized by thrombocytopenia. First-generation TPO-R agonists were recombinant forms of human TPO. The clinical development of these molecules was discontinued after one of them, pegylated recombinant human megakaryocyte growth and development factor, was associated with the development of neutralizing autoantibodies cross-reacting with endogenous TPO. Second-generation TPO-R agonists are now available, which present no sequence homology to endogenous TPO. Two of these new agents, romiplostim and eltrombopag, have been granted marketing authorization for use in patients with primary immune thrombocytopenia unresponsive to conventional treatments. Clinical trials with TPO-R agonists are also ongoing in other thrombocytopenias, such as hepatitis C virus-related thrombocytopenia and the myelodysplastic syndromes. Copyright 2012 Prous Science, S.A.U. or its licensors. All rights reserved.

Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

PubMed

Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

2013-06-01

Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

Plasmodium circumsporozoite protein suppresses the growth of A549 cells via inhibiting nuclear transcription factor κB.

PubMed

Deng, Xu-Feng; Zhou, Dong; Liu, Quan-Xing; Zheng, Hong; Ding, Yan; Xu, Wen-Yue; Min, Jia-Xin; Dai, Ji-Gang

2018-05-01

Blocking the activation of nuclear factor κB (NF-κB) is a promising strategy for the treatment of non-small cell lung cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was previously reported to block NF-κB activation in hepatocytes. Therefore, in the present study, the effect of CSP on the growth of the human lung cancer cell line, A549, was investigated. It was demonstrated that transfection with a recombinant plasmid expressing CSP was able to inhibit the proliferation of A549 cells in a dose-dependent manner and induce the apoptosis of A549 cells. A NF-κB gene reporter assay indicated that CSP and its nuclear localization signal (NLS) motif were able to equally suppress the activation of NF-κB following stimulation with human recombinant tumor necrosis factor (TNF)-α in A549 cells. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but was able to markedly inhibit the nuclear translocation of NF-κB in TNF-α stimulated A549 cells. Therefore, the data suggest that CSP may be investigated as a potential novel NF-κB inhibitor for the treatment of lung cancer.

In Vitro Effect of Activated Recombinant Factor VII (rFVIIa) on Coagulation Properties of Human Blood at Hypothermic Temperatures

DTIC Science & Technology

2007-11-01

purified soy phosphatides in ellagic acid (Actin FS Activated PTT Reagent, Dade Behring, Marburg, Germany) was added to plasma samples in a 1:1 volume...acetylsali- cylic acid or any other nonsteroidal anti-inflammatory drugs for the 7 days before blood sampling. A smooth cubital venipuncture was

Evaluation of human recession defects treated with coronally advanced flaps and either purified recombinant human platelet-derived growth factor-BB with beta tricalcium phosphate or connective tissue: a histologic and microcomputed tomographic examination.

PubMed

McGuire, Michael K; Scheyer, Todd; Nevins, Myron; Schupbach, Peter

2009-02-01

The current study examined the histologic and microcomputed tomographic (micro CT) outcomes of the treatment of gingival recession defects with either a subepithelial connective tissue graft (CTG) or 0.3 mg/mL recombinant human platelet-derived growth factor (rhPDGF-BB) on a beta tricalcium phosphate (beta-TCP) matrix. Gingival recession defects were surgically created in six premolar teeth with no more than 3 mm of keratinized marginal tissue, an osseous crest 2 to 3 mm apical to the newly created gingival margin, and recession depth of at least 3 mm. The defects were left untouched for 2 months; then, four defects were grafted with rhPDGF-BB + beta-TCP + a wound healing dressing, and two defects received CTGs. A coronally advanced flap covered each grafted site. Nine months later, sections were obtained for examination. All four sites treated with rhPDGF-BB + beta-TCP showed connective tissue fibers (Sharpey fibers) perpendicularly inserting into newly formed cementum and alveolar bone. In the two sites treated with CTGs, a long junctional epithelium was seen coronal to the osseous crest and connective tissue fibers ran parallel to the adjacent root surfaces, with no evidence of insertion into cementum or bone. There was no evidence of regeneration of cementum, inserting connective tissue fibers, or supporting alveolar bone. Regeneration of the periodontium in gingival recession defects is possible through growth factor-mediated therapy.

A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells.

PubMed

Xue, Haipeng; Wu, Sen; Papadeas, Sophia T; Spusta, Steve; Swistowska, Anna Maria; MacArthur, Chad C; Mattson, Mark P; Maragakis, Nicholas J; Capecchi, Mario R; Rao, Mahendra S; Zeng, Xianmin; Liu, Ying

2009-08-01

In this study, we targeted Olig2, a basic helix-loop-helix transcription factor that plays an important role in motoneuron and oligodendrocyte development, in human embryonic stem cell (hESC) line BG01 by homologous recombination. One allele of Olig2 locus was replaced by a green fluorescent protein (GFP) cassette with a targeting efficiency of 5.7%. Targeted clone R-Olig2 (like the other clones) retained pluripotency, typical hESC morphology, and a normal parental karyotype 46,XY. Most importantly, GFP expression recapitulated endogenous Olig2 expression when R-Olig2 was induced by sonic hedgehog and retinoic acid, and GFP-positive cells could be purified by fluorescence-activated cell sorting. Consistent with previous reports on rodents, early GFP-expressing cells appeared biased to a neuronal fate, whereas late GFP-expressing cells appeared biased to an oligodendrocytic fate. This was corroborated by myoblast coculture, transplantation into the rat spinal cords, and whole genome expression profiling. The present work reports an hESC reporter line generated by homologous recombination targeting a neural lineage-specific gene, which can be differentiated and sorted to obtain pure neural progenitor populations.

Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits.

PubMed

van Veen, Harrie A; Koiter, Jaco; Vogelezang, Carla J M; van Wessel, Noucha; van Dam, Tijtje; Velterop, Ingeborg; van Houdt, Kristina; Kupers, Luc; Horbach, Danielle; Salaheddine, Mourad; Nuijens, Jan H; Mannesse, Maurice L M

2012-12-31

C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M(r)) of 67,200. Differences in M(r) on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest(®)) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE. Copyright © 2012 Elsevier B.V. All rights reserved.

Partial white and grey matter protection with prolonged infusion of recombinant human erythropoietin after asphyxia in preterm fetal sheep.

PubMed

Wassink, Guido; Davidson, Joanne O; Dhillon, Simerdeep K; Fraser, Mhoyra; Galinsky, Robert; Bennet, Laura; Gunn, Alistair J

2017-03-01

Perinatal asphyxia in preterm infants remains a significant contributor to abnormal long-term neurodevelopmental outcomes. Recombinant human erythropoietin has potent non-haematopoietic neuroprotective properties, but there is limited evidence for protection in the preterm brain. Preterm (0.7 gestation) fetal sheep received sham asphyxia (sham occlusion) or asphyxia induced by umbilical cord occlusion for 25 min, followed by an intravenous infusion of vehicle (occlusion-vehicle) or recombinant human erythropoietin (occlusion-Epo, 5000 international units by slow push, then 832.5 IU/h), starting 30 min after asphyxia and continued until 72 h. Recombinant human erythropoietin reduced neuronal loss and numbers of caspase-3-positive cells in the striatal caudate nucleus, CA3 and dentate gyrus of the hippocampus, and thalamic medial nucleus ( P < 0.05 vs. occlusion-vehicle). In the white matter tracts, recombinant human erythropoietin increased total, but not immature/mature oligodendrocytes ( P < 0.05 vs. occlusion-vehicle), with increased cell proliferation and reduced induction of activated caspase-3, microglia and astrocytes ( P < 0.05). Finally, occlusion-Epo reduced seizure burden, with more rapid recovery of electroencephalogram power, spectral edge frequency, and carotid blood flow. In summary, prolonged infusion of recombinant human erythropoietin after severe asphyxia in preterm fetal sheep was partially neuroprotective and improved electrophysiological and cerebrovascular recovery, in association with reduced apoptosis and inflammation.

Partial white and grey matter protection with prolonged infusion of recombinant human erythropoietin after asphyxia in preterm fetal sheep

PubMed Central

Wassink, Guido; Davidson, Joanne O; Dhillon, Simerdeep K; Fraser, Mhoyra; Galinsky, Robert; Bennet, Laura

2016-01-01

Perinatal asphyxia in preterm infants remains a significant contributor to abnormal long-term neurodevelopmental outcomes. Recombinant human erythropoietin has potent non-haematopoietic neuroprotective properties, but there is limited evidence for protection in the preterm brain. Preterm (0.7 gestation) fetal sheep received sham asphyxia (sham occlusion) or asphyxia induced by umbilical cord occlusion for 25 min, followed by an intravenous infusion of vehicle (occlusion-vehicle) or recombinant human erythropoietin (occlusion-Epo, 5000 international units by slow push, then 832.5 IU/h), starting 30 min after asphyxia and continued until 72 h. Recombinant human erythropoietin reduced neuronal loss and numbers of caspase-3-positive cells in the striatal caudate nucleus, CA3 and dentate gyrus of the hippocampus, and thalamic medial nucleus (P < 0.05 vs. occlusion-vehicle). In the white matter tracts, recombinant human erythropoietin increased total, but not immature/mature oligodendrocytes (P < 0.05 vs. occlusion-vehicle), with increased cell proliferation and reduced induction of activated caspase-3, microglia and astrocytes (P < 0.05). Finally, occlusion-Epo reduced seizure burden, with more rapid recovery of electroencephalogram power, spectral edge frequency, and carotid blood flow. In summary, prolonged infusion of recombinant human erythropoietin after severe asphyxia in preterm fetal sheep was partially neuroprotective and improved electrophysiological and cerebrovascular recovery, in association with reduced apoptosis and inflammation. PMID:27207167

Effects of recombinant human growth hormone and nandrolone phenylpropionate on the healing of ischemic colon anastomosis in rats.

PubMed

Yarimkaya, Ali; Apaydin, Berat; Unal, Ethem; Karabicak, Ilhan; Aydogan, Fatih; Uslu, Ezel; Erginoz, Ethem; Artis, Tarik; Eyuboglu, Erhun

2003-12-01

Recombinant human growth hormone and nandrolone phenylpropionate are two different anabolic agents. This study was designed to investigate the effects of these anabolic agents on the healing of ischemic colon anastomosis in rats. Seventy adult male Wistar rats were divided into five groups (n = 14). Group I was the sham laparotomy group. In the other groups, surgical procedures consisting of transsection and anastomosis were made at a distance 3 cm from the peritoneal reflection. Group II was the nonischemic control group. Ischemic colon model was produced in the remaining groups. Group III was the untreated control group. Groups IV and V received recombinant human growth hormone and nandrolone phenylpropionate, respectively. Bursting pressure and hydroxyproline levels were measured on the third and seventh postoperative days to evaluate anastomotic healing. Recombinant human growth hormone increased both collagen deposition and bursting pressure significantly at postoperative Days 3 and 7 compared with the sham and untreated control groups (P < 0.005). When compared with the untreated control, nandrolone phenylpropionate significantly increased collagen deposition at postoperative Days 3 and 7 (P < 0.005) and bursting pressure only at postoperative Day 3 (P < 0.005). Recombinant human growth hormone has more favorable therapeutic effects on the healing of ischemic colonic anastomoses than nandrolone phenylpropionate. Recombinant human growth hormone also improves healing of nonischemic colonic anastomosis.

Population-specific recombination sites within the human MHC region.

PubMed

Lam, T H; Shen, M; Chia, J-M; Chan, S H; Ren, E C

2013-08-01

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.

ELF5 in epithelial ovarian carcinoma tissues and biological behavior in ovarian carcinoma cells.

PubMed

Yan, Hongchao; Qiu, Linglin; Xie, Xiaolei; Yang, He; Liu, Yongli; Lin, Xiaoman; Huang, Hongxiang

2017-03-01

The expression of E74-like factor 5 (ELF5) in epithelial ovarian carcinoma tissues and its effects on biological behavior in ovarian carcinoma cells were assessed in search for a new approach for gene treatment of epithelial ovarian carcinoma. RT-PCR technology was applied to detect the expression of ELF5 mRNA in epithelial ovarian carcinoma (n=49), borderline ovarian epithelial tumor (n=19), benign ovarian epithelial tumor (n=31) and normal ovarian tissues (n=40). Then, we transfected recombinant plasmid pcDNA3.1‑ELF5+EGFP into human ovarian carcinoma SKOV3 cells (recombinant plasmid group) in vitro and screened out stably transfected cells to conduct multiplication culture. Western blot analysis was performed to detect the expression of ELF5 protein in the different groups. Flow cytometry was employed to detect cell apoptosis and cycles. ELF5 mRNA in epithelial ovarian carcinoma and borderline ovarian epithelial tumor tissues were significantly lower (P<0.05) than those in benign ovarian epithelial tumor and normal ovarian tissues. ELF5 protein expression in the cells of recombinant plasmid group was significantly higher compared with empty plasmid and blank control groups. The capacity of cell reproductive recombinant plasmid group at each time point decreased (P<0.05). Flow cytometry detection showed that 67.03% of cells in recombinant plasmid group was blocked in G0/G1 phase (P<0.05), compared with empty plasmid group (37.17%) and blank control group (38.24%). Apoptotic rate of recombinant plasmid group was significantly lower (31.4±1.9%; P<0.05), compared with that of empty plasmid group (9.1±2.2%) and blank control group (8.7±1.5%), and the differences were statistically significant. In conclusion, ELF5 interfered with cell cycle of human ovarian carcinoma SKOV3 cells and promoted apoptosis of human ovarian carcinoma SKOV3 cells inhibiting their growth and invasive capacity; and thus providing a new approach to gene treatment of ovarian carcinoma.

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4)

PubMed Central

Bertling, Anne; Brodde, Martin F.; Visser, Mayken; Treffon, Janina; Fennen, Michelle; Fender, Anke C.; Kelsch, Reinhard; Kehrel, Beate E.

2017-01-01

Background Hemarthrosis, or bleeding into the joints, is a hallmark of hemophilia. Heme triggers oxidative stress, inflammation, and destruction of cartilage and bone. The haptoglobin-CD163-heme oxygenase-1 (HO-1) pathway circumvents heme toxicity through enzymatic degradation of heme and transcription of antioxidant genes. Plasma-derived factor concentrates contain many proteins that might impact on cellular pathways in joints, blood, and vessels. Methods Activation of platelets from healthy volunteers was assessed by flow cytometry analysis of fibrinogen binding and CD62P expression. Platelet CXCL4 release was measured by ELISA. Human peripheral blood mononuclear cells were exposed to CXCL4 or platelet supernatants (untreated or pre-stimulated with factor VIII (FVIII) products) during their differentiation to macrophages and analyzed for CD163 expression. Some macrophage cultures were additionally incubated with autologous hemoglobin for 18 h for analysis of HO-1 expression. Results Platelet CXCL4 release was increased by all 8 tested plasma-derived FVIII products but not the 3 recombinant products. Macrophages exposed to supernatant from platelets treated with some plasma-derived FVIII products downregulated CD163 surface expression and failed to upregulate the athero- and joint protective enzyme HO-1 in response to hemoglobin. Conclusion Plasma-derived FVIII products might promote bleeding-induced joint injury via generation of macrophages that are unable to counteract redox stress. PMID:29070980

Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli.

PubMed

Geng, Zhirong; Song, Xiaoli; Xing, Zhi; Geng, Jinlong; Zhang, Sichun; Zhang, Xinrong; Wang, Zhilin

2009-05-01

The effects of Se(IV) on the structure and function of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT) purified from the cytoplasm of Escherichia coli were studied. The coding region of human AS3MT complementary DNA was amplified from total RNA extracted from HepG2 cell by reverse transcription PCR. Soluble and active human AS3MT was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25 degrees C. Spectra (UV-vis, circular dichroism, and fluorescence) were first used to probe the interaction of Se(IV) and recombinant human AS3MT and the structure-function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% alpha-helix, 23.9% beta-pleated sheet, 17.9% beta-turn, and 29.2% random coil. When Se(IV) was added, the content of the alpha-helix did not change, but that of the beta-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. Se(IV) inhibited the enzymatic methylation of inorganic As(III) in a concentration-dependent manner. The IC(50) value for Se(IV) was 2.38 muM. Double-reciprocal (1/V vs. 1/[inorganic As(III)]) plots showed Se(IV) to be a noncompetitive inhibitor of the methylation of inorganic As(III) by recombinant human AS3MT with a K (i) value of 2.61 muM. We hypothesized that Se(IV) interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When Se(IV) was combined with cysteine(s) in the structural residues, the conformation of recombinant human AS3MT changed and the enzymatic activity decreased. Considering the quenching of tryptophan fluorescence, Cys72 and/or Cys226 are deduced to be primary targets for Se(IV).

Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

PubMed Central

Dälken, Benjamin; Jabulowsky, Robert A.; Oberoi, Pranav; Benhar, Itai; Wels, Winfried S.

2010-01-01

Background The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins. PMID:21203542

[Production of recombinant human interleukin-38 and its inhibitory effect on the expression of proinflammatory cytokines in THP-1 cells].

PubMed

Yuan, X L; Li, Y; Pan, X H; Zhou, M; Gao, Q Y; Li, M C

2016-01-01

Interleukin (IL)-38 is the latest member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-38, its precise functions are poorly understood. In the present study, the cloning, expression, purification, and activity analysis of recombinant human IL-38 was described. Human IL-38 cDNA was cloned into the prokaryotic expression vector pET-44. The recombinant IL-38 containing a C-hexahistidine tag was expressed in Escherichia coli BL21 (DE3) which induced by isopropyl-β-D-thiogalactoside. The expressed fusion protein was purified by Ni-NTA affinity chromatography. IL-38 protein was largely found in the soluble fraction. The purified IL-38 appeared a single band on SDS-PAGE, the yield of IL-38 was 4 mg from 1 L of bacterial culture, and the purity was more than 98% with low endotoxin level (<0.1 EU/μg). Western blotting confirmed the identity of the purified protein. Activity analysis showed that IL-38 can inhibit effectively the expression of proinflammatory cytokines, such as tumor necrosis factor-α, IL-1β, IL-17, and monocyte chemoattractant protein-1 in lipopolysaccharide-activated THP-1 cells. The production and characterization of biologically active IL-38 will be beneficial for its potential role in clinical applications.

International Validation of Two Human Recombinant Estrogen Receptor (ERa) Binding Assays

EPA Science Inventory

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay...

Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

PubMed

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-03-16

There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

PubMed Central

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-01-01

Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-Self CD4 Antibody Responses with Anti-Human Immunodeficiency Virus Activity

NASA Astrophysics Data System (ADS)

Watanabe, Mamoru; Boyson, Jonathan E.; Lord, Carol I.; Letvin, Norman L.

1992-06-01

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

PubMed Central

Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

2018-01-01

ABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota. PMID:29925671

Recombinant cathepsin E has no proteolytic activity at neutral pH.

PubMed

Zaidi, Nousheen; Herrmann, Timo; Voelter, Wolfgang; Kalbacher, Hubert

2007-08-17

Cathepsin E (CatE) is a major intracellular aspartic protease reported to be involved in cellular protein degradation and several pathological processes. Distinct cleavage specificities of CatE at neutral and acidic pH have been reported previously in studies using CatE purified from human gastric mucosa. Here, in contrast, we have analyzed the proteolytic activity of recombinant CatE at acidic and neutral pH using two separate approaches, RP-HPLC and FRET-based proteinase assays. Our data clearly indicate that recombinant CatE does not possess any proteolytic activity at all at neutral pH and was unable to cleave the peptides glucagon, neurotensin, and dynorphin A that were previously reported to be cleaved by CatE at neutral pH. Even in the presence of ATP, which is known to stabilize CatE, no proteolytic activity was observed. These discrepant results might be due to some contaminating factor present in the enzyme preparations used in previous studies or may reflect differences between recombinant CatE and the native enzyme.

Evidence for human meiotic recombination interference obtained through construction of a short tandem repeat-polymorphism linkage map of chromosome 19

PubMed Central

Weber, James L.; Wang, Zhenyuan; Hansen, Kevin; Stephenson, Matt; Kappel, Clarisse; Salzman, Sherry; Wilkie, Patricia J.; Keats, Bronya; Dracopoli, Nicholas C.; Brandriff, Brigitte F.; Olsen, Anne S.

1993-01-01

An improved linkage map for human chromosome 19 containing 35 short tandem repeat polymorphisms (STRPs) and one VNTR (D19S20) was constructed. The map included 12 new (GATA)n tetranucleotide STRPs. Although total lengths of the male (114 cM) and female (128 cM) maps were similar, at both ends of the chromosome male recombination exceeded female recombination, while in the interior portion of the map female recombination was in excess. Cosmid clones containing the STRP sequences were identified and were positioned along the chromosome by fluorescent in situ hybridization. Four rounds of careful checking and removal of genotyping errors allowed biologically relevant conclusions to be made concerning the numbers and distributions of recombination events on chromosome 19. The average numbers of recombinations per chromosome matched closely the lengths of the genetic maps computed by using the program CRIMAP. Significant numbers of chromosomes with zero, one, two, or three recombinations were detected as products of both female and male meioses. On the basis of the total number of observed pairs of recombination events in which only a single informative marker was situated between the two recombinations, a maximal estimate for the rate of meiotic STRP “gene” conversion without recombination was calculated as 3 × 10−4/meiosis. For distances up to 30 cM between recombinations, many fewer chromosomes which had undergone exactly two recombinations were observed than were expected on the basis of the assumption of independent recombination locations. This strong new evidence for human meiotic interference will help to improve the accuracy of interpretation of clinical DNA test results involving polymorphisms flanking a genetic abnormality. PMID:8213834

Evolution of collagen arthritis in mice is arrested by treatment with anti-tumour necrosis factor (TNF) antibody or a recombinant soluble TNF receptor.

PubMed Central

Piguet, P F; Grau, G E; Vesin, C; Loetscher, H; Gentz, R; Lesslauer, W

1992-01-01

Immunization of DBA/1 mice with type II collagen within complete Freund's adjuvant leads to arthritis, lasting more than 3 months. Injection of anti-tumour necrosis factor (TNF) IgG, 2 and 3 weeks after immunization prevented the development of arthritis in the following months. This treatment had no effect when started 2 months after induction of the disease. A soluble form of the human recombinant TNF receptor type-beta (rsTNFR-beta), continuously infused at a rate of 20 micrograms/day during the second and third week after immunization, also had a long-term protective effect. Anti-TNF antibody had no effect upon the production of anti-type II collagen antibodies. These results indicate that TNF is critically involved in an early phase of this arthritis. Images Figure 1 Figure 2 PMID:1337334

Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Protective effects of granulocyte colony-stimulating factor on endotoxin shock in mice with retrovirus-induced immunodeficiency syndrome.

PubMed

Toki, S; Hiromatsu, K; Aoki, Y; Makino, M; Yoshikai, Y

1997-10-01

Mice with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) were hypersensitive to lipopolysaccharide (LPS)-induced lethal shock accompanied by marked elevations of systematic interleukin 1beta (IL-beta) and interferon gamma (IFN-gamma) after LPS challenge. Pretreatment with 10 microg of recombinant human granulocyte colony-stimulating factor (rhG-CSF) protected MAIDS mice from hypersensitivity to LPS-induced lethal shock and this protection was concomitant with suppression of IFN-gamma production. Copyright 1997 Academic Press Limited.

Design of Recombinant Stem Cell Factor macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

NASA Astrophysics Data System (ADS)

Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan

2005-05-01

Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

Expression of a functional recombinant human basic fibroblast growth factor from transgenic rice seeds.

PubMed

An, Na; Ou, Jiquan; Jiang, Daiming; Zhang, Liping; Liu, Jingru; Fu, Kai; Dai, Ying; Yang, Daichang

2013-02-07

Basic fibroblast growth factor (FGF-2) is an important member of the FGF gene family. It is widely used in clinical applications for scald and wound healing in order to stimulate cell proliferation. Further it is applied for inhibiting stem cell differentiation in cultures. Due to a shortage of plasma and low expression levels of recombinant rbFGF in conventional gene expression systems, we explored the production of recombinant rbFGF in rice grains (Oryza sativa bFGF, OsrbFGF). An expression level of up to 185.66 mg/kg in brown rice was obtained. A simple purification protocol was established with final recovery of 4.49% and resulting in a yield of OsrbFGF reaching up to 8.33 mg/kg OsrbFGF. The functional assay of OsrbFGF indicated that the stimulating cell proliferation activity on NIH/3T3 was the same as with commercialized rbFGF. Wound healing in vivo of OsrbFGF is equivalent to commercialized rbFGF. Our results indicate that rice endosperm is capable of expressing small molecular mass proteins, such as bFGF. This again demonstrates that rice endosperm is a promising system to express various biopharmaceutical proteins.

The use of recombinant factor VIIa in a patient with Noonan syndrome and life-threatening bleeding.

PubMed

Tofil, Nancy M; Winkler, Margaret K; Watts, Raymond G; Noonan, Jacqueline

2005-05-01

To present a case report of a patient with Noonan syndrome who developed life-threatening gastrointestinal bleeding shortly after cardiac surgery that was successfully treated with recombinant factor VIIa. Case report. Pediatric intensive care unit of a children's hospital. Ten-month-old with Noonan syndrome and massive gastrointestinal bleeding resulting in severe hypovolemic shock. Recombinant factor VIIa was used in this patient's severe bleeding associated with Noonan syndrome after no other supportive measures were successful. Recombinant Factor VIIa significantly decreased the patient's bleeding and allowed his hypovolemic shock to improve. Ultimately, the patient made a complete recovery. Noonan syndrome has a constellation of both cardiac and noncardiac malformations including an increased risk of bleeding, and recombinant factor VIIa is an important agent in the treatment of significant bleeding.

Effect of a structurally modified human granulocyte colony stimulating factor, G-CSFa, on leukopenia in mice and monkeys

PubMed Central

2011-01-01

Background Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, and differentiation of neutrophilic granulocyte precursors, Recombinant G-CSF has been used for the treatment of congenital and therapy-induced neutropenia and stem cell mobilization. Due to its intrinsic instability, recombinant G-CSF needs to be excessively and/or frequently administered to patients in order to maintain a plasma concentration high enough to achieve therapeutic effects. Therefore, there is a need for the development of G-CSF derivatives that are more stable and active in vivo. Methods Using site-direct mutagenesis and recombinant DNA technology, a structurally modified derivative of human G-CSF termed G-CSFa was obtained. G-CSFa contains alanine 17 (instead of cysteine 17 as in wild-type G-CSF) as well as four additional amino acids including methionine, arginine, glycine, and serine at the amino-terminus. Purified recombinant G-CSFa was tested for its in vitro activity using cell-based assays and in vivo activity using both murine and primate animal models. Results In vitro studies demonstrated that G-CSFa, expressed in and purified from E. coli, induced a much higher proliferation rate than that of wild-type G-CSF at the same concentrations. In vivo studies showed that G-CSFa significantly increased the number of peripheral blood leukocytes in cesium-137 irradiated mice or monkeys with neutropenia after administration of clyclophosphamide. In addition, G-CSFa increased neutrophil counts to a higher level in monkeys with a concomitant slower declining rate than that of G-CSF, indicating a longer half-life of G-CSFa. Bone marrow smear analysis also confirmed that G-CSFa was more potent than G-CSF in the induction of granulopoiesis in bone marrows of myelo-suppressed monkeys. Conclusion G-CSFa, a structurally modified form of G-CSF, is more potent in stimulating proliferation and differentiation of myeloid cells of the granulocytic lineage than the wild-type counterpart both in vitro and in vivo. G-CSFa can be explored for the development of a new generation of recombinant therapeutic drug for leukopenia. PMID:21668998

Effect of a structurally modified human granulocyte colony stimulating factor, G-CSFa, on leukopenia in mice and monkeys.

PubMed

Jiang, Yongping; Jiang, Wenhong; Qiu, Yuchang; Dai, Wei

2011-06-13

Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, and differentiation of neutrophilic granulocyte precursors, Recombinant G-CSF has been used for the treatment of congenital and therapy-induced neutropenia and stem cell mobilization. Due to its intrinsic instability, recombinant G-CSF needs to be excessively and/or frequently administered to patients in order to maintain a plasma concentration high enough to achieve therapeutic effects. Therefore, there is a need for the development of G-CSF derivatives that are more stable and active in vivo. Using site-direct mutagenesis and recombinant DNA technology, a structurally modified derivative of human G-CSF termed G-CSFa was obtained. G-CSFa contains alanine 17 (instead of cysteine 17 as in wild-type G-CSF) as well as four additional amino acids including methionine, arginine, glycine, and serine at the amino-terminus. Purified recombinant G-CSFa was tested for its in vitro activity using cell-based assays and in vivo activity using both murine and primate animal models. In vitro studies demonstrated that G-CSFa, expressed in and purified from E. coli, induced a much higher proliferation rate than that of wild-type G-CSF at the same concentrations. In vivo studies showed that G-CSFa significantly increased the number of peripheral blood leukocytes in cesium-137 irradiated mice or monkeys with neutropenia after administration of cyclophosphamide. In addition, G-CSFa increased neutrophil counts to a higher level in monkeys with a concomitant slower declining rate than that of G-CSF, indicating a longer half-life of G-CSFa. Bone marrow smear analysis also confirmed that G-CSFa was more potent than G-CSF in the induction of granulopoiesis in bone marrows of myelo-suppressed monkeys. G-CSFa, a structurally modified form of G-CSF, is more potent in stimulating proliferation and differentiation of myeloid cells of the granulocytic lineage than the wild-type counterpart both in vitro and in vivo. G-CSFa can be explored for the development of a new generation of recombinant therapeutic drug for leukopenia.

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

PubMed

Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

2015-01-01

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins. © 2015 S. Karger AG, Basel.

Recombinant IκBα-loaded curcumin nanoparticles for improved cancer therapeutics

NASA Astrophysics Data System (ADS)

Banerjee, Subhamoy; Sahoo, Amaresh Kumar; Chattopadhyay, Arun; Sankar Ghosh, Siddhartha

2014-08-01

The field of recombinant protein therapeutics has been evolving rapidly, making significant impact on clinical applications for several diseases, including cancer. However, the functional aspects of proteins rely exclusively on their structural integrity, in which nanoparticle mediated delivery offers unique advantages over free proteins. In the present work, a novel strategy has been developed where the nanoparticles (NPs) used for the delivery of the recombinant protein could contribute to enhancing the therapeutic efficacy of the recombinant protein. The transcription factor, NFκB, involved in cell growth and its inhibitor, IκBα, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NFκB, is curcumin. Hence, we have developed a ‘green synthesis’ method for preparing water-soluble curcumin nanoparticles to stabilize recombinant IκBα protein. The NPs were characterized by UV-vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering before administration into human cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Experimental results demonstrated that this combined module had enhanced therapeutic efficacy, causing apoptotic cell death, which was confirmed by cytotoxicity assay and flowcytometry analyses. The expression of apoptotic genes studied by semi-quantitative reverse transcription PCR delineated the molecular pathways involved in cell death. Thus, our study revealed that the functional delivery of recombinant IκBα-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells.

Recombination in Eukaryotic Single Stranded DNA Viruses

PubMed Central

Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

2011-01-01

Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

Glycoprotein from street rabies virus BD06 induces early and robust immune responses when expressed from a non-replicative adenovirus recombinant.

PubMed

Wang, Shuchao; Sun, Chenglong; Zhang, Shoufeng; Zhang, Xiaozhuo; Liu, Ye; Wang, Ying; Zhang, Fei; Wu, Xianfu; Hu, Rongliang

2015-09-01

The rabies virus (RABV) glycoprotein (G) is responsible for inducing neutralizing antibodies against rabies virus. Development of recombinant vaccines using the G genes from attenuated strains rather than street viruses is a regular practice. In contrast to this scenario, we generated three human adenovirus type 5 recombinants using the G genes from the vaccine strains SRV9 and Flury-LEP, and the street RABV strain BD06 (nrAd5-SRV9-G, nrAd5-Flury-LEP-G, and nrAd5-BD06-G). These recombinants were non-replicative, but could grow up to ~10(8) TCID50/ml in helper HEK293AD cells. Expression of the G protein was verified by immunostaining, quantitative PCR and cytometry. Animal experiments revealed that immunization with nrAd5-BD06-G can induce a higher seroconversion rate, a higher neutralizing antibody level, and a longer survival time after rabies virus challenge in mice when compared with the other two recombinants. Moreover, the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly higher in mice immunized with nrAd5-BD06-G, which might also contribute to the increased protection. These results show that the use of street RABV G for non-replicative systems may be an alternative for developing effective recombinant rabies vaccines.

Recombinant IκBα-loaded curcumin nanoparticles for improved cancer therapeutics.

PubMed

Banerjee, Subhamoy; Sahoo, Amaresh Kumar; Chattopadhyay, Arun; Ghosh, Siddhartha Sankar

2014-08-29

The field of recombinant protein therapeutics has been evolving rapidly, making significant impact on clinical applications for several diseases, including cancer. However, the functional aspects of proteins rely exclusively on their structural integrity, in which nanoparticle mediated delivery offers unique advantages over free proteins. In the present work, a novel strategy has been developed where the nanoparticles (NPs) used for the delivery of the recombinant protein could contribute to enhancing the therapeutic efficacy of the recombinant protein. The transcription factor, NFκB, involved in cell growth and its inhibitor, IκBα, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NFκB, is curcumin. Hence, we have developed a 'green synthesis' method for preparing water-soluble curcumin nanoparticles to stabilize recombinant IκBα protein. The NPs were characterized by UV-vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering before administration into human cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Experimental results demonstrated that this combined module had enhanced therapeutic efficacy, causing apoptotic cell death, which was confirmed by cytotoxicity assay and flowcytometry analyses. The expression of apoptotic genes studied by semi-quantitative reverse transcription PCR delineated the molecular pathways involved in cell death. Thus, our study revealed that the functional delivery of recombinant IκBα-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells.

Genetic engineering approach to develop next-generation reagents for endotoxin quantification.

PubMed

Mizumura, Hikaru; Ogura, Norihiko; Aketagawa, Jun; Aizawa, Maki; Kobayashi, Yuki; Kawabata, Shun-Ichiro; Oda, Toshio

2017-02-01

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.

Genetic engineering approach to develop next-generation reagents for endotoxin quantification

PubMed Central

Ogura, Norihiko; Aketagawa, Jun; Aizawa, Maki; Kobayashi, Yuki; Kawabata, Shun-ichiro; Oda, Toshio

2016-01-01

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources. PMID:27913792

Effect of recombinant human granulocyte colony-stimulating factor on variations of morphologically identifiable bone marrow cells in myelosuppressed mice.

PubMed

Kabaya, K; Kusaka, M; Seki, M

1994-01-01

To examine the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on neutrophilic recovery after cytotoxic agents, the variations of marrow colony-forming units of granulocytes and macrophages (CFU-GM) and morphologically identifiable bone marrow cells were investigated in cyclophosphamide (CPA)-treated mice. In mice treated with CPA at 200mg/kg intraperitoneally (day 0), marked decreases in peripheral neutrophils and nucleated cells in the femur were observed. In the femur of mice treated with CPA, the greatest depression in number occurred firstly with CFU-GM and the most immature granulocytes, such as myeloblasts and promyelocytes, followed in turn by myelocytes, metamyelocytes and mature neutrophils. Administration of rhG-CSF for four successive days (days 1-4) after CPA treatment completely prevented the neutropenia. In the femur, rhG-CSF enhanced the recovery of progenitors and immature granulocytes from their depression in the order of their differentiation, and recovery of marrow neutrophils was also promoted. From these studies, we confirmed that rhG-CSF effects an increase in peripheral neutrophils by enhancing the proliferation and differentiation of CFU-GM and immature marrow granulocytes.

Recombinant AAV8-mediated intrastriatal gene delivery of CDNF protects rats against methamphetamine neurotoxicity

PubMed Central

Wang, Lizheng; Wang, Zixuan; Xu, Xiaoyu; Zhu, Rui; Bi, Jinpeng; Liu, Wenmo; Feng, Xinyao; Wu, Hui; Zhang, Haihong; Wu, Jiaxin; Kong, Wei; Yu, Bin; Yu, Xianghui

2017-01-01

Methamphetamine (METH) exerts significant neurotoxicity in experimental animals and humans when taken at high doses or abused chronically. Long-term abusers have decreased dopamine levels, and they are more likely to develop Parkinson's disease (PD). To date, few medications are available to treat the METH-induced damage of neurons. Glial cell line-derived neurotrophic factor (GDNF) has been previously shown to reduce the dopamine-depleting effects of neurotoxic doses of METH. However, the effect of cerebral dopamine neurotrophic factor (CDNF), which has been reported to be more specific and efficient than GDNF in protecting dopaminergic neurons against 6-OHDA toxicity, in attenuating METH neurotoxicity has not been determined. Thus, the present study aimed to evaluate the neuroprotective effect of CDNF against METH-induced damage to the dopaminergic system in vitro and in vivo. In vitro, CDNF protein increased the survival rate and reduced the tyrosine hydroxylase (TH) loss of METH-treated PC12 cells. In vivo, METH was administered to rats following human CDNF overexpression mediated by the recombinant adeno-associated virus. Results demonstrated that CDNF overexpression in the brain could attenuate the METH-induced dopamine and TH loss in the striatum but could not lower METH-induced hyperthermia. PMID:28553166

Delayed treatment with recombinant human tissue factor pathway inhibitor improves survival in rabbits with gram-negative peritonitis.

PubMed

Camerota, A J; Creasey, A A; Patla, V; Larkin, V A; Fink, M P

1998-03-01

To determine whether treatment with recombinant human tissue factor pathway inhibitor (TFPI), an inhibitor of the extrinsic coagulation pathway, can improve survival in a clinically relevant model of gram-negative sepsis, rabbits were given an intraperitoneal inoculation of a suspension containing hemoglobin (40 microg/mL), porcine mucin (150 microg/mL), and viable Escherichia coli O18:K1 (1.0 +/- 0.5 x 10(5) cfu/kg). Treatment with gentamicin (5 mg/kg every 12 h for five doses) was instituted 4 h after induction of peritonitis. At the same time point, rabbits were randomized to receive a 24-h infusion of vehicle or one of three different doses of TFPI. Treatment groups, 7-day survival rates, and significance versus control were as follows: control, 1 of 20; TFPI(LOW DOSE) (0.1 mg/kg, then 1 microg/kg/min), 3 of 12 (P = .14); TFPI(MID DOSE), (0.5 mg/kg, then 5 microg/kg/min), 7 of 12 (P = .002); TFPI(HIGH DOSE) (10 mg/kg, then 10 microg/kg/min), 4 of 13 (P = .04). Thus, delayed treatment with TFPI improves survival in septic rabbits.

Recombinant Human Factor IX Produced from Transgenic Porcine Milk

PubMed Central

Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

2014-01-01

Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk. PMID:24955355

Spotlight on the human factor: building a foundation for the future of haemophilia A management: report from a symposium on human recombinant FVIII at the World Federation of Hemophilia World Congress, Melbourne, Australia on 12 May 2014.

PubMed

Kessler, C; Oldenburg, J; Ettingshausen, C Escuriola; Tiede, A; Khair, K; Négrier, C; Klamroth, R

2015-01-01

Inhibitor development is the most serious and challenging complication in the treatment of severe haemophilia A. Up to 38% of such patients develop inhibitors with current recombinant factor VIII (rFVIII) products produced in hamster cell lines. Human-cl rhFVIII is a new generation fully sulfated B-domain-deleted FVIII coagulant glycoprotein, which is generated from a human cell line. Thus, there are no non-human epitopes which would be potentially immunogenic. This molecule has significantly higher VWF-binding affinity compared with existing full-length rFVIII produced in hamster cell lines. The development aim of Human-cl rhFVIII is to address the challenges of FVIII inhibitors and frequent infusions during prophylaxis. Human-cl rhFVIII's mean half-life is very comparable to some of the newer products which involve modification of the FVIII molecule to extend the circulating half-life. There are promising data concerning the use of a personalized prophylaxis regimen with Human-cl rhFVIII. Preliminary data indicate a median dosing interval of 3.5 days with 66.7% of the patients on a twice per week or fewer infusions schedule combined with a low bleeding rate and no increased FVIII consumption when compared to standard prophylaxis. No product-specific laboratory assay is required to monitor the coagulation activity for Human-cl rhFVIII. The results of registration clinical trials with Human-cl rhFVIII as well as the ongoing studies in previously untreated patients (NuProtect) and personalized prophylaxis study in previously treated patients (NuPreviq), will be discussed. The manufacturer has received marketing authorization for Human-cl rhFVIII in Europe and Canada under the name Nuwiq(®) and plans to launch it in the USA and globally in 2015. © 2014 John Wiley & Sons Ltd.

Effect of the combination of fibrin glue and growth hormone on intestinal anastomoses in a pig model of traumatic shock associated with peritonitis.

PubMed

Wang, Pengfei; Wang, Jian; Zhang, Wenbo; Li, Yousheng; Li, Jieshou

2009-03-01

Intra-abdominal sepsis and hemorrhagic shock have been found to impair the healing of intestinal anastomoses. The present study examined whether fibrin glue (FG) and recombinant human growth hormone (GH) can improve intestinal primary anastomotic healing in a pig model of traumatic shock associated with peritonitis. Further, the study was designed to investigate the probable mechanism of these agents. Female anesthetized pigs were divided into five groups. Group sham (n = 7), pigs without traumatic shock had small bowel resection anastomoses; group control (n = 14), pigs had bowel resection anastomoses 24 h after abdominal gunshot plus exsanguination/resuscitation; group FG (n = 14); group GH (n = 14); group FG/GH (n = 14), pigs received FG, recombinant GH, or both, respectively. Recombinant GH was given daily for 7 days. Blood samples were collected daily for measurement of interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha levels. Investigations also included adhesion formation, anastomotic bursting pressure, tensile strength, hydroxyproline (HP) content, myeloperoxidase (MPO), tumor necrosis factor (NF)-kappaB activity, and histology analysis 10 days later. A second experiment (n = 20 subjects assigned to each of the five groups) was designed to study survival during the first 20 postoperative days. Traumatic shock associated with peritonitis led to significant decreases in intestinal anastomotic bursting pressures, tensile strengths, and tissue hydroxyproline content, along with severe adhesion formation, increases in MPO activity and NF-kappaB activity, and plasma levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Both FG and recombinant GH treatment led to early significant increases in plasma levels of TNF-alpha and IL-6. At the same time, FG alone, unlike recombinant GH alone, led to significant increases in anastomotic bursting pressures, tensile strength, and tissue HP content, along with decreases in anastomotic MPO and NF-kappaB activity and later plasma levels of TNF-a and IL-6. The FG group also developed more marked neoangiogenesis and collagen deposition on histology analysis. However, FG and recombinant GH synergistically effected improved anastomotic healing, abolishing the infaust effects promoted by recombinant GH. Adhesion formation after intestinal anastomosis could not be lowered by FG alone or by the combination of FG and recombinant GH. Both FG alone and FG/GH, in contrast to GH alone and control treatment, significantly prolonged the survival time of experimental animals. We found that FG, but not recombinant GH, could lower the risk of anastomotic leakage, improve intestinal anastomotic healing, and prolong survival in a pig model of traumatic shock associated with peritonitis. Both FG and recombinant GH synergistically effected improved intestinal anastomotic healing. It was suggested that GH could be used locally to promote intestinal anastomotic healing in intra-abdominal peritonitis.

Essential components for ex vivo proliferation of mesenchymal stromal cells.

PubMed

Fekete, Natalie; Rojewski, Markus Thomas; Lotfi, Ramin; Schrezenmeier, Hubert

2014-02-01

Mesenchymal stromal cells (MSCs) are highly interesting candidates for clinical applications in regenerative medicine. Due to their low occurrence in human tissues, extensive in vitro expansion is necessary to obtain sufficient cell numbers applicable as a clinical dose in the context of cellular therapy. Current cell culture media formulations for the isolation and expansion of MSCs include fetal calf serum (FCS), human AB serum (ABS), or human platelet lysate (PL) as a supplement. However, these established supplements are inherently ill-defined formulations that contain a variety of bioactive molecules in varying batch-to-batch compositions and the risk of transmitting pathogens that escape routine screening procedures. In this study, we have comparatively characterized the capacity of commonly used basal media, such as the Minimum Essential Medium alpha (αMEM), Dulbecco's modified Eagle's medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), and RPMI 1640 as well as human- and animal-derived supplements, that is, PL, ABS, and FCS to stimulate cell proliferation. MSC proliferation was observed to be optimal in the PL-supplemented αMEM. Using a combinatorial approach, we then assessed a library of soluble factors, including mitogens (TGF-β1, Activin A, bFGF, EGF, IGF-I, PDGF-BB, and VEGF), chemokines (CCL21, CCL25, CXCL12, and RANTES), proteins (human serum albumin), lipids (e.g., oleic acid, linoleic acid, and arachidonic acid), and hormones (dexamethasone, insulin, and TSH), to create a defined medium as well as coating of cell culture surfaces to promote robust MSC proliferation in vitro. A combination of recombinant human factors partially met the nutritional requirements of bone marrow-derived MSCs, and was able to promote cell proliferation comparable to about 5% PL if supplemented with auxiliary 0.6%-1.2% PL. Maximal MSC proliferation was achieved by combining 5% PL with a cocktail of recombinant factors and did not depend on coating of cell culture surfaces.

Using Recombinant Lactococci as an Approach to Dissect the Immunomodulating Capacity of Surface Piliation in Probiotic Lactobacillus rhamnosus GG

PubMed Central

Nummenmaa, Elina; Mäkinen, Veli-Matti; Reunanen, Justus; Satokari, Reetta; de Vos, Willem M.; Palva, Ilkka; Palva, Airi

2013-01-01

Primarily arising from their well understood beneficial health effects, many lactobacilli strains are considered good candidates for use as probiotics in humans and animals. Lactobacillar probiosis can itself be best typified by the Lactobacillus rhamnosus GG strain, which, with its well-documented clinical benefits, has emerged as one of the most widely used probiotics in the food and health-supplement industries. Even so, many facets of its molecular mechanisms and limitations as a beneficial commensal bacterium still remain to be thoroughly explored and dissected. Because L. rhamnosus GG is one of only a few such strains exhibiting surface piliation (called SpaCBA), we sought to examine whether this particular type of cell-surface appendage has a discernible immunomodulating capacity and is able to trigger targeted responses in human immune-related cells. Thus, presented herein for this study, we recombinantly engineered Lactococcus lactis to produce native (and pilin-deleted) SpaCBA pili that were assembled in a structurally authentic form and anchored to the cell surface, and which had retained mucus-binding functionality. By using these recombinant lactococcal constructs, we were able to demonstrate that the SpaCBA pilus can be a contributory factor in the activation of Toll-like receptor 2-dependent signaling in HEK cells as well as in the modulation of pro- and anti-inflammatory cytokine (TNF-α, IL-6, IL-10, and IL-12) production in human monocyte-derived dendritic cells. From these data, we suggest that the recombinant-expressed and surface-anchored SpaCBA pilus, given its projected functioning in the gut environment, might be viewed as a new microbe-associated molecular pattern (MAMP)-like modulator of innate immunity. Accordingly, our study has brought some new insight to the molecular immunogenicity of the SpaCBA pilus, thus opening the way to a better understanding of its possible role in the multifaceted nature of L. rhamnosus GG probiosis within the human gut. PMID:23691212

78 FR 78838 - Grant of Interim Extension of the Term of U.S. Patent No. 5,496,801; Recombinant Human...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-27

... Hormone AGENCY: United States Patent and Trademark Office, Commerce. ACTION: Notice of interim patent term... No. 5,496,801. The patent claims the human biological product recombinant human parathyroid hormone... human parathyroid hormone, was filed on October 24, 2013, and is currently undergoing regulatory review...

Trap-assisted and Langevin-type recombination in organic light-emitting diodes

NASA Astrophysics Data System (ADS)

Wetzelaer, G. A. H.; Kuik, M.; Nicolai, H. T.; Blom, P. W. M.

2011-04-01

Trapping of charges is known to play an important role in the charge transport of organic semiconductors, but the role of traps in the recombination process has not been addressed. Here we show that the ideality factor of the current of organic light-emitting diodes (OLEDs) in the diffusion-dominated regime has a temperature-independent value of 2, which reveals that nonradiative trap-assisted recombination dominates the current. In contrast, the ideality factor of the light output approaches unity, demonstrating that luminance is governed by recombination of the bimolecular Langevin type. This apparent contradiction can be resolved by measuring the current and luminance ideality factor for a white-emitting polymer, where both free and trapped charge carriers recombine radiatively. With increasing bias voltage, Langevin recombination becomes dominant over trap-assisted recombination due to its stronger dependence on carrier density, leading to an enhancement in OLED efficiency.

IFN-beta1b augments glucocorticoid-induced suppression of tumor necrosis factor-alpha production by increasing the number of glucocorticoid receptors on a human monocytic cell line.

PubMed

Uitdehaag, B M; Hoekstra, K; Koper, J W; Polman, C H; Dijkstra, C D

2001-03-01

We studied the effect of recombinant interferon-beta1b (IFN-beta1b) on the sensitivity to glucocorticoids (GC) and on the number of GC receptors (GCR) in the human monocytic cell line THP-1. We found that IFN-beta1b augments the suppressive effect that dexamethasone has on the stimulated production of tumor necrosis factor-alpha (TNF-alpha), most likely related to the increased number of GCR observed after exposure to IFN-beta1b. This provides a possible clue to the mechanism of action of IFN-beta in multiple sclerosis.

A reanalysis of the indirect evidence for recombination in human mitochondrial DNA.

PubMed

Piganeau, G; Eyre-Walker, A

2004-04-01

In an attempt to resolve the controversy about whether recombination occurs in human mtDNA, we have analysed three recently published data sets of complete mtDNA sequences along with 10 RFLP data sets. We have analysed the relationship between linkage disequilibrium (LD) and distance between sites under a variety of conditions using two measures of LD, r2 and /D'/. We find that there is a negative correlation between r2 and distance in the majority of data sets, but no overall trend for /D'/. Five out of six mtDNA sequence data sets show an excess of homoplasy, but this could be due to either recombination or hypervariable sites. Two additional recombination detection methods used, Geneconv and Maximum Chi-Square, showed nonsignificant results. The overall significance of these findings is hard to quantify because of nonindependence, but our results suggest a lack of evidence for recombination in human mtDNA.

Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

PubMed

Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

1996-07-15

Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

Activated protein C attenuates systemic lupus erythematosus and lupus nephritis in MRL-Fas(lpr) mice.

PubMed

Lichtnekert, Julia; Rupanagudi, Khader Valli; Kulkarni, Onkar P; Darisipudi, Murthy Narayana; Allam, Ramanjaneyulu; Anders, Hans-Joachim

2011-09-15

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease leading to inflammatory tissue damage in multiple organs (e.g., lupus nephritis). Current treatments including steroids, antimalarials, and immunosuppressive drugs have significant side effects. Activated protein C is a natural protein with anticoagulant and immunomodulatory effects, and its recombinant version has been approved by the U.S. Food and Drug Administration to treat severe sepsis. Given the similarities between overshooting immune activation in sepsis and autoimmunity, we hypothesized that recombinant activated protein C would also suppress SLE and lupus nephritis. To test this concept, autoimmune female MRL-Fas(lpr) mice were injected with either vehicle or recombinant human activated protein C from week 14-18 of age. Activated protein C treatment significantly suppressed lupus nephritis as evidenced by decrease in activity index, glomerular IgG and complement C3 deposits, macrophage counts, as well as intrarenal IL-12 expression. Further, activated protein C attenuated cutaneous lupus and lung disease as compared with vehicle-treated MRL-Fas(lpr) mice. In addition, parameters of systemic autoimmunity, such as plasma cytokine levels of IL-12p40, IL-6, and CCL2/MCP-1, and numbers of B cells and plasma cells in spleen were suppressed by activated protein C. The latter was associated with lower total plasma IgM and IgG levels as well as lower titers of anti-dsDNA IgG and rheumatoid factor. Together, recombinant activated protein C suppresses the abnormal systemic immune activation in SLE of MRL-Fas(lpr) mice, which prevents subsequent kidney, lung, and skin disease. These results implicate that recombinant activated protein C might be useful for the treatment of human SLE.

Detection of biomarkers using recombinant antibodies coupled to nanostructured platforms

PubMed Central

Kierny, Michael R.; Cunningham, Thomas D.; Kay, Brian K.

2012-01-01

The utility of biomarker detection in tomorrow's personalized health care field will mean early and accurate diagnosis of many types of human physiological conditions and diseases. In the search for biomarkers, recombinant affinity reagents can be generated to candidate proteins or post-translational modifications that differ qualitatively or quantitatively between normal and diseased tissues. The use of display technologies, such as phage-display, allows for manageable selection and optimization of affinity reagents for use in biomarker detection. Here we review the use of recombinant antibody fragments, such as scFvs and Fabs, which can be affinity-selected from phage-display libraries, to bind with both high specificity and affinity to biomarkers of cancer, such as Human Epidermal growth factor Receptor 2 (HER2) and Carcinoembryonic antigen (CEA). We discuss how these recombinant antibodies can be fabricated into nanostructures, such as carbon nanotubes, nanowires, and quantum dots, for the purpose of enhancing detection of biomarkers at low concentrations (pg/mL) within complex mixtures such as serum or tissue extracts. Other sensing technologies, which take advantage of ‘Surface Enhanced Raman Scattering’ (gold nanoshells), frequency changes in piezoelectric crystals (quartz crystal microbalance), or electrical current generation and sensing during electrochemical reactions (electrochemical detection), can effectively provide multiplexed platforms for detection of cancer and injury biomarkers. Such devices may soon replace the traditional time consuming ELISAs and Western blots, and deliver rapid, point-of-care diagnostics to market. PMID:22833780

Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes.

PubMed

Pan, XiaoPing; Wang, Yini; Yu, XiaoPeng; Li, JianZhou; Zhou, Ning; Du, WeiBo; Zhang, YanHong; Cao, HongCui; Zhu, DanHua; Chen, Yu; Li, LanJuan

2015-01-01

The liver-specific functions of hepatocytes are improved by co-culturing hepatocytes with primary hepatic stellate cells (HSC). However, primary HSC have a short lifespan in vitro, which is considered a major limitation for their use in various applications. This study aimed to establish immortalized human HSC using the simian virus 40 large T antigen (SV40LT) for applications in co-culturing with hepatocytes and HSC in vitro. Primary human HSC were transfected with a recombinant retrovirus containing SV40LT. The immortalized human HSC were characterized by analyzing their gene expression and functional characteristics. The liver-specific functions of hepatocytes were evaluated in a co-culture system incorporating immortalized human hepatocytes with HSC-Li cells. The immortalized HSC line, HSC-Li, was obtained after infection with a recombinant retrovirus containing SV40LT. The HSC-Li cells were longitudinally spindle-like and had numerous fat droplets in their cytoplasm as shown using electron microscopy. Hepatocyte growth factor (HGF), VEGF Receptor 1(Flt-1), collagen type Iα1 and Iα2 mRNA expression levels were observed in the HSC-Li cells by RT-PCR. Immunofluorescence staining showed that the HSC-Li cells were positive for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-beta (PDGFR-β), vimentin, and SV40LT protein expression. The HSC-Li cells produced both HGF and transforming growth factor-beta1 (TGF-β1) in a time-dependent manner. Real-time PCR showed that albumin, CYP3A5, CYP2E1, and UGT2B7 mRNA expression generally increased in the co-culture system. The enzymatic activity of CYP1A2 under the co-culture conditions also generally increased as compared to the monoculture of immortalized human hepatocytes. We successfully established the immortalized human HSC cell line HSC-Li. It has the specific phenotypic and functional characteristics of primary human HSC, which would be a useful tool to develop anti-fibrotic therapies. Co-culturing with the HSC-Li cells improved the liver-specific functions of hepatocytes, which may be valuable and applicable for bioartificial liver systems.

Short-term effects of recombinant human growth hormone and feeding on gluconeogenesis in humans

USDA-ARS?s Scientific Manuscript database

After a short-term fast, lactating women have increased rates of glucose production but not gluconeogenesis (GNG) despite relative hypoinsulinemia. We explored the effects of non-insulin-dependent increase in glucose utilization and recombinant human growth hormone (rhGH) on glucose production, glyc...

Site-specific genetic recombination: hops, flips, and flops.

PubMed

Sadowski, P D

1993-06-01

Genetic recombination plays a key role in the life of organisms as diverse as bacteriophages and humans. Contrary to our idea that chromosomes are stable structures, studies of recombination over the past few decades have shown that in fact DNA replicons are remarkably plastic, undergoing frequent recombination-induced rearrangements. This review summarizes our recent knowledge of the biochemistry of the two major classes of site-specific recombination: 1) transpositional recombination, and 2) conservative site-specific recombination.

Enhanced In Vivo Tumor Detection by Active Tumor Cell Targeting Using Multiple Tumor Receptor-Binding Peptides Presented on Genetically Engineered Human Ferritin Nanoparticles.

PubMed

Kwon, Koo Chul; Ko, Ho Kyung; Lee, Jiyun; Lee, Eun Jung; Kim, Kwangmeyung; Lee, Jeewon

2016-08-01

Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

PubMed Central

Gründel, Anne; Pfeiffer, Melanie; Jacobs, Enno

2015-01-01

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract. PMID:26667841

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells.

PubMed

Wan, Aini; Xu, Dongsheng; Liu, Kedong; Peng, Lin; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

2017-08-09

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500 µg/L) and the half-life of IGF-1 in blood circulation is only 4.5 min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA-IGF-1 reached 100 mg/L. The fusion protein HSA-IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA-IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA-IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA-IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.

Production of coagulation factor VII in human cell lines Sk-Hep-1 and HKB-11.

PubMed

Corrêa de Freitas, Marcela Cristina; Bomfim, Aline de Sousa; Mizukami, Amanda; Picanço-Castro, Virgínia; Swiech, Kamilla; Covas, Dimas Tadeu

2017-09-01

Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 μg and 202.6 μg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

High pH solubilization and chromatography-based renaturation and purification of recombinant human granulocyte colony-stimulating factor from inclusion bodies.

PubMed

Li, Ming; Fan, Hua; Liu, Jiahua; Wang, Minhong; Wang, Lili; Wang, Chaozhan

2012-03-01

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin

PubMed Central

Zhao, Shuqiang; Zhang, Yu; Tian, Hong; Chen, Xiaofei; Cai, Di; Yao, Wenbing; Gao, Xiangdong

2013-01-01

Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned into pPICZαA along with the open reading frame of the α-factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed into Pichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF. PMID:24151579

Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

PubMed

Shinohara, Mayuka; Sumino, Yasuhiro; Sato, Fuminori; Kiyono, Tohru; Hashimoto, Naohiro; Mimata, Hiromitsu

2017-06-01

To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence. © 2017 The Japanese Urological Association.

Potentiation by Tumor Necrosis Factor of Mitoxantrone Cytotoxicity to Human Ovarian Cancer Cell Lines

PubMed Central

Parodi, Silvio; Billi, Giovanna; Oliva, Cristina; Venturing, Marco; Noviello, Elvira; Conte, PierFranco

1992-01-01

The cytotoxic activity of human recombinant tumor necrosis factor (rHuTNF) (from 0.01 to 10000 U/ml) was assayed on six human ovarian cancer cell lines and one human cervical carcinoma cell line using a crystal violet assay. rHuTNF was cytotoxic to four cell lines (A2780, A2774, SW626, PAD, while 3 cell lines (IGROV1, SKOV3, Mel80) were marginally sensitive to its activity. However, under the same experimental conditions rHuTNF markedly enhanced the cytotoxicity of mitoxantrone, a chemotherapeutic drug targeted at DNA topoisomerase II, in six cell lines. The potentiation of mitoxantrone cytotoxicity was not caused by increased drug accumulation after rHuTNF treatment. No significant increase in cytotoxicity to Me180 cell line was seen when rHuTNF was added to mitoxantrone. PMID:1517145

Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

PubMed

Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

2014-09-11

The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with recombination hotspots. The trans-regulators predicted by our pipeline are enriched with epigenetic functions (e.g., histone modifications), demonstrating the epigenetic regulatory mechanisms of recombination hotspots. The identified protein complexes also provide us with candidates to further investigate the molecular machineries for recombination hotspots. Moreover, the experimental data and results are available on our web site http://www.ntu.edu.sg/home/zhengjie/data/RecombinationHotspot/NetPipe/.

Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant Escherichia coli.

PubMed

Khalilzadeh, Rasoul; Mohammadian-Mosaabadi, Jafar; Bahrami, Ali; Nazak-Tabbar, Ahmad; Nasiri-Khalili, Mohammad Ali; Amouheidari, Alireza

2008-12-01

The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF at 48 g dry cell wt l(-1) during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as approximately 64 g dry cell wt l(-1), 223 mg hG-CSF g(-1) dry cell wt and 775 mg hG-CSF l(-1) h(-1), respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose. Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show valuable decrease in activity in purified form.

Neuregulin-1β induces proliferation, survival and paracrine signaling in normal human cardiac ventricular fibroblasts.

PubMed

Kirabo, Annet; Ryzhov, Sergey; Gupte, Manisha; Sengsayadeth, Seng; Gumina, Richard J; Sawyer, Douglas B; Galindo, Cristi L

2017-04-01

Neuregulin-1β (NRG-1β) is critical for cardiac development and repair, and recombinant forms are currently being assessed as possible therapeutics for systolic heart failure. We previously demonstrated that recombinant NRG-1β reduces cardiac fibrosis in an animal model of cardiac remodeling and heart failure, suggesting that there may be direct effects on cardiac fibroblasts. Here we show that NRG-1β receptors (ErbB2, ErbB3, and ErbB4) are expressed in normal human cardiac ventricular (NHCV) fibroblast cell lines. Treatment of NHCV fibroblasts with recombinant NRG-1β induced activation of the AKT pathway, which was phosphoinositide 3-kinase (PI3K)-dependent. Moreover, the NRG-1β-induced PI3K/AKT signaling in these cells required phosphorylation of both ErbB2 and ErbB3 receptors at tyrosine (Tyr)1248 and Tyr1289 respectively. RNASeq analysis of NRG-1β-treated cardiac fibroblasts obtained from three different individuals revealed a global gene expression signature consistent with cell growth and survival. We confirmed enhanced cellular proliferation and viability in NHCV fibroblasts in response to NRG-1β, which was abrogated by PI3K, ErbB2, and ErbB3 inhibitors. NRG-1β also induced production and secretion of cytokines (interleukin-1α and interferon-γ) and pro-reparative factors (angiopoietin-2, brain-derived neurotrophic factor, and crypto-1), suggesting a role in cardiac repair through the activation of paracrine signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cholesteryl group- and acryloyl group-bearing pullulan nanogel to deliver BMP2 and FGF18 for bone tissue engineering.

PubMed

Fujioka-Kobayashi, Masako; Ota, Masato S; Shimoda, Asako; Nakahama, Ken-ichi; Akiyoshi, Kazunari; Miyamoto, Youji; Iseki, Sachiko

2012-10-01

To create a drug delivery system that allows the controlled release of proteins, such as growth factors, over a long-term period, cholesteryl group- and acryloyl group-bearing pullulan (CHPOA) nanogels were aggregated to form fast-degradable hydrogels (CHPOA/hydrogels) by cross-linking with thiol-bearing polyethylene glycol. The gold standard of clinical bone reconstruction therapy with a physiologically active material is treatment with recombinant human bone morphogenetic protein 2 (BMP2); however, this approach has limitations, such as inflammation, poor cost-efficiency, and varying interindividual susceptibility. In this study, two distinct growth factors, BMP2 and recombinant human fibroblast growth factor 18 (FGF18), were applied to a critical-size skull bone defect for bone repair by the CHPOA/hydrogel system. The CHPOA-FGF18/hydrogel displayed identical results to the control CHPOA-PBS/hydrogel, and the CHPOA-BMP2/hydrogel treatment imperfectly induced bone repair. By contrast, the CHPOA-FGF18 + BMP2/hydrogel treatment strongly enhanced and stabilized the BMP2-dependent bone repair, inducing osteoprogenitor cell infiltration inside and around the hydrogel. This report indicates that the CHPOA/hydrogel system can successfully deliver two different proteins to the bone defect to induce effective bone repair. The combination of the CHPOA/hydrogel system with the growth factors FGF18 and BMP2 might be a step towards efficient bone tissue engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

PubMed

Chappell, William H; Gautam, Dipendra; Ok, Suzan T; Johnson, Bryan A; Anacker, Daniel C; Moody, Cary A

2015-12-23

High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

PubMed

Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

The Potential Role of Recombinant Activated Factor VIIa (rFVIIa) in Military Pre-Hospital Setting

DTIC Science & Technology

2004-09-01

coagulation factors and platelets by crystalloids, colloids, or blood products The severity of dilutional coagulopathy is determined by both volume and...RTO-MP-HFM-109 3 - 1 The Potential Role of Recombinant Activated Factor VIIa (rFVIIa) in Military Pre-Hospital Setting LTC (ret.) Uri...decrease mortality from exsanguinations. Recombinant factor VIIa (rFVIIa) has been shown to overcome a variety of coagulation and platelet disorders

Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein

PubMed Central

McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

2014-01-01

Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc. PMID:24811361

Immunologic Intervention in HIV Infection: Anti-Polymerase Responses and Hormonal Regulation

DTIC Science & Technology

1993-09-01

chronic human immunodeficiency virus infection is blocked in vitro by a methylphosphonate oligodeoxynucleoside targeted to a U3/enhancer element. J...Grimison B, Gonenne A. 1992. Effect of recombinant human growth hormone on acute and chronic human immunodeficiency virus infection in vitro. Blood 79...Kong X-B, Chou T-C. Interactions of recombinant human growth hormone with dideoxynucleoside inhibitors of human immunodeficiency virus. Blood, in

Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein.

PubMed

Peters, R T; Toby, G; Lu, Q; Liu, T; Kulman, J D; Low, S C; Bitonti, A J; Pierce, G F

2013-01-01

Hemophilia A results from a deficiency in factor VIII activity. Current treatment regimens require frequent dosing, owing to the short half-life of FVIII. A recombinant FVIII-Fc fusion protein (rFVIIIFc) was molecularly engineered to increase the half-life of FVIII, by 1.5-2-fold, in several preclinical animal models and humans. To perform a biochemical and functional in vitro characterization of rFVIIIFc, with existing FVIII products as comparators.  rFVIIIFc was examined by utilizing a series of structural and analytic assays, including mass spectrometry following lysyl endopeptidase or thrombin digestion. rFVIIIFc activity was determined in both one-stage clotting (activated partial thromboplastin time) and chromogenic activity assays, in the context of the FXase complex with purified components, and in both in vitro and ex vivo rotational thromboelastometry (ROTEM) assays performed in whole blood.  rFVIIIFc contained the predicted primary structure and post-translational modifications, with an FVIII moiety that was similar to other recombinant FVIII products. The von Willebrand factor-binding and specific activity of rFVIIIFc were also found to be similar to those of other recombinant FVIII molecules. Both chromogenic and one-stage assays of rFVIIIFc gave similar results. Ex vivo ROTEM studies demonstrated that circulating rFVIIIFc activity was prolonged in mice with hemophilia A in comparison with B-domain-deleted or full-length FVIII. Clot parameters at early time points were similar to those for FVIII, whereas rFVIIIFc showed prolonged improvement of clot formation.  rFVIIIFc maintains normal FVIII interactions with other proteins necessary for its activity, with prolonged in vivo activity, owing to fusion with the Fc region of IgG(1) . © 2012 International Society on Thrombosis and Haemostasis.

Mechanisms and Factors that Influence High Frequency Retroviral Recombination

PubMed Central

Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga; Mens, Helene; Pathak, Vinay K.; Hu, Wei-Shau

2011-01-01

With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development. PMID:21994801

The contribution of alu elements to mutagenic DNA double-strand break repair.

PubMed

Morales, Maria E; White, Travis B; Streva, Vincent A; DeFreece, Cecily B; Hedges, Dale J; Deininger, Prescott L

2015-03-01

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

NASA Astrophysics Data System (ADS)

Zuo, Ping; Manley, James L.

1994-04-01

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

Effects of CASP5 gene overexpression on angiogenesis of HMEC-1 cells.

PubMed

Li, Haiyan; Li, Yuzhen; Cai, Limin; Bai, Bingxue; Wang, Yanhua

2015-01-01

The efficacy of gene overexpression of CASP5, a caspase family member, in angiogenesis in vitro and its mechanisms were clarified. Human full-length CASP5 gene was delivered into human microvascular endothelial HMEC-1 cells by recombinant lentivirus. The infection was estimated by green fluorescent protein. MTT method was used to analyze the efficacy of gene overexpression in cell proliferation ability, and Matrigel was used to estimate its effects in angiogenesis ability of cells. Meanwhile, Western blot was used to analyze the effects of CASP5 gene overexpression on the expression levels of angpt-1, angpt-2, Tie2 and VEGF-1 in the cells, which were signaling pathway factors related to angiogenesis. Recombinant lentivirus containing human full-length CASP5 gene was packed and purified successfully, with virus titer of 1×10(8) TU/ml. The recombinant lentivirus was used to infect HMEC-1 cells with MOI of 1, leading to a cell infection rate of 100%. There were no significant effects of CASP5 gene overexpression on both cell proliferation ability and the expression level of angpt-1. Meanwhile, expressions of angpt-2 and VEGF-1 were both enhanced, while Tie2 expression was inhibited. Results indicated that CASP5 gene overexpression promoted angiogenesis of HMEC-1 cells. CASP5 gene overexpression significantly promoted angiogenesis ability of HMEC-1 cells, which was probably achieved by inhibiting angpt-1/Tie2 and promoting VEGF-1 signal pathway.

Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.

PubMed

Xue, Haipeng; Wu, Jianbo; Li, Shenglan; Rao, Mahendra S; Liu, Ying

2016-01-01

Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability.

Modulating Cellular Recombination Potential through Alterations in RecA Structure and Regulation

PubMed Central

Bakhlanova, Irina V.; Dudkina, Alexandra V.; Baitin, Dima M.; Knight, Kendall L.; Cox, Michael M.; Lanzov, Vladislav A.

2010-01-01

The wild type E. coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to 6 fold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to 4 fold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50 fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of Escherichia coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSB-coated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function – filament formation and the inherent DNA pairing activity of the formed filaments. PMID:21143322

Current Ebola vaccines

PubMed Central

Hoenen, Thomas; Groseth, Allison; Feldmann, Heinz

2012-01-01

Introduction Ebolaviruses cause severe viral hemorrhagic fever in humans and non-human primates, with case fatality rates of up to 90%. Currently, neither a specific treatment nor a vaccine licensed for use in humans is available. However, a number of vaccine candidates have been developed in the last decade that are highly protective in non-human primates, the gold standard animal model for Ebola hemorrhagic fever. Areas covered This review analyzes a number of scenarios for the use of ebolavirus vaccines, discusses the requirements for ebolavirus vaccines in these scenarios, and describes current ebolavirus vaccines. Among these vaccines are recombinant Adenoviruses, recombinant Vesicular Stomatitis viruses, recombinant Human Parainfluenza viruses and virus-like particles. Interestingly, one of these vaccine platforms, based on recombinant Vesicular Stomatitis viruses, has also demonstrated post-exposure protection in non-human primates. Expert opinion The most pressing remaining challenge is now to move these vaccine candidates forward into human trials and towards licensure. In order to achieve this, it will be necessary to establish the mechanisms and correlates of protection for these vaccines, and to continue to demonstrate their safety, particularly in potentially immunocompromised populations. However, already now there is sufficient evidence that, from a scientific perspective, a vaccine protective against ebolaviruses is possible. PMID:22559078

Accelerators of Osteogenesis by Recombinant Human Bone Morphogenetic Protein-2

PubMed Central

Okubo, Yasunori; Kusumoto, Kenji; Bessho, Kazuhisa

2007-01-01

Bone morphogenetic protein (BMP) appears to be one of the most promising cytokine and for clinical use in reconstructive surgery for bony defects and augmentation. To evaluate the effect of basic fibroblast growth factor (bFGF), FK506, elcatonin, and hyperbaric oxygenation (HBO) on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), 2 or 5 μg of rhBMP-2 was implanted into intramuscular sites of rats. At 21 days after implantation, the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and histologically. The amount of new bone in the treatment group was significantly greater than that in the control group. The alkaline phosphatase activity and calcium content in the treatment group were significantly higher than those in the control group. These results suggest that bFGF, FK506, elcatonin, and HBO accelerated the activity and rate of osteoinduction by rhBMP2. These results may be useful when BMP is applied clinically in near future. PMID:21901062

Induction of chemokine receptor CXCR4 expression by transforming growth factor-β1 in human basal cell carcinoma cells.

PubMed

Chu, Chia-Yu; Sheen, Yi-Shuan; Cha, Shih-Ting; Hu, Yeh-Fang; Tan, Ching-Ting; Chiu, Hsien-Ching; Chang, Cheng-Chi; Chen, Min-Wei; Kuo, Min-Liang; Jee, Shiou-Hwa

2013-11-01

Higher CXCR4 expression enhances basal cell carcinoma (BCC) invasion and angiogenesis. The underlying mechanism of increased CXCR4 expression in invasive BCC is still not well understood. To investigate the mechanisms involved in the regulation of CXCR4 expression in invasive BCC. We used qRT-PCR, RT-PCR, Western blot, and flow cytometric analyses to examine different CXCR4 levels among the clinical samples, co-cultured BCC cells and BCC cells treated with recombinant transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF). Immunohistochemical studies were used to demonstrate the correlation between TGF-β1 and CXCR4 expressions. The signal transduction pathway and transcriptional regulation were confirmed by treatments with chemical inhibitors, neutralizing antibodies, or short interfering RNAs, as well as luciferase reporter activity. Invasive BCC has higher TGF-β1 and CTGF levels compared to non-invasive BCC. Non-contact dermal fibroblasts co-culture with human BCC cells also increases the expression of CXCR4 in BCC cells. Treatment with recombinant human TGF-β1, but not CTGF, enhanced the CXCR4 levels in time- and dose-dependent manners. The protein level and surface expression of CXCR4 in human BCC cells was increased by TGF-β1 treatment. TGF-β1 was intensely expressed in the surrounding fibroblasts of invasive BCC and was positively correlated with the CXCR4 expression of BCC cells. The transcriptional regulation of CXCR4 by TGF-β1 is mediated by its binding to the TGF-β receptor II and phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2)-ETS-1 pathway. TGF-β1 induces upregulation of CXCR4 in human BCC cells by phosphorylation of ERK1/2-ETS-1 pathway. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The Murine Factor H-Related Protein FHR-B Promotes Complement Activation.

PubMed

Cserhalmi, Marcell; Csincsi, Ádám I; Mezei, Zoltán; Kopp, Anne; Hebecker, Mario; Uzonyi, Barbara; Józsi, Mihály

2017-01-01

Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

Protein Replacement Therapy and Gene Transfer in Canine Models of Hemophilia A, Hemophilia B, von Willebrand Disease, and Factor VII Deficiency

PubMed Central

Nichols, Timothy C.; Dillow, Aaron M.; Franck, Helen W.G.; Merricks, Elizabeth P.; Raymer, Robin A.; Bellinger, Dwight A.; Arruda, Valder R.; High, Katherine A.

2011-01-01

Dogs with hemophilia A, hemophilia B, von Willebrand disease (VWD), and factor VII deficiency faithfully recapitulate the severe bleeding phenotype that occurs in humans with these disorders. The first rational approach to diagnosing these bleeding disorders became possible with the development of reliable assays in the 1940s through research that used these dogs. For the next 60 years, treatment consisted of replacement of the associated missing or dysfunctional protein, first with plasma-derived products and subsequently with recombinant products. Research has consistently shown that replacement products that are safe and efficacious in these dogs prove to be safe and efficacious in humans. But these highly effective products require repeated administration and are limited in supply and expensive; in addition, plasma-derived products have transmitted bloodborne pathogens. Recombinant proteins have all but eliminated inadvertent transmission of bloodborne pathogens, but the other limitations persist. Thus, gene therapy is an attractive alternative strategy in these monogenic disorders and has been actively pursued since the early 1990s. To date, several modalities of gene transfer in canine hemophilia have proven to be safe, produced easily detectable levels of transgene products in plasma that have persisted for years in association with reduced bleeding, and correctly predicted the vector dose required in a human hemophilia B liver-based trial. Very recently, however, researchers have identified an immune response to adeno-associated viral gene transfer vector capsid proteins in a human liver-based trial that was not present in preclinical testing in rodents, dogs, or nonhuman primates. This article provides a review of the strengths and limitations of canine hemophilia, VWD, and factor VII deficiency models and of their historical and current role in the development of improved therapy for humans with these inherited bleeding disorders. PMID:19293459

Distribution of recombination hotspots in the human genome--a comparison of computer simulations with real data.

PubMed

Mackiewicz, Dorota; de Oliveira, Paulo Murilo Castro; Moss de Oliveira, Suzana; Cebrat, Stanisław

2013-01-01

Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar.

Distribution of Recombination Hotspots in the Human Genome – A Comparison of Computer Simulations with Real Data

PubMed Central

Mackiewicz, Dorota; de Oliveira, Paulo Murilo Castro; Moss de Oliveira, Suzana; Cebrat, Stanisław

2013-01-01

Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar. PMID:23776462

Effect of cyclophilin A on gene expression in human pancreatic cancer cells.

PubMed

Li, Min; Wang, Hao; Li, Fei; Fisher, William E; Chen, Changyi; Yao, Qizhi

2005-11-01

We previously found that cyclophilin A (CypA) is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. In this study, we further investigated the effect of CypA on gene expression of several key molecules that are involved in pancreatic cancer cell proliferation. Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3) and human pancreatic ductal epithelial (HPDE) cells were used. The messenger RNA (mRNA) levels of CypA, CypB, CD147, neuropilins (NRPs), vascular endothelial growth factor (VEGF), and VEGF receptors upon the treatment of exogenous recombinant human CypA were determined by real-time reverse-transcription polymerase chain reaction. Exogenous human recombinant CypA reduced the mRNA levels of NRP-1 and VEGF, but not endogenous CypA, CypB, and CD147, in Panc-1, MIA PaCa-2, and BxPC-3 cells. In contrast, HPDE cells showed a decrease of endogenous CypA and CD147 mRNA, but not detectable changes of CypB, NRPs, and VEGF mRNA levels upon exogenous CypA treatment. These data show that exogenous CypA downregulates NRP-1 and VEGF expression in pancreatic cancer cells. This effect is different in normal HPDE cells. Thus, soluble CypA may affect cell growth of pancreatic cancer.

Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L)

NASA Technical Reports Server (NTRS)

Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy; Chatterjee, A. (Principal Investigator)

2002-01-01

Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

PubMed

Portolano, Nicola; Watson, Peter J; Fairall, Louise; Millard, Christopher J; Milano, Charles P; Song, Yun; Cowley, Shaun M; Schwabe, John W R

2014-10-16

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

Multilineage hematopoietic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in myelosuppressed mice.

PubMed

Shibuya, K; Akahori, H; Takahashi, K; Tahara, E; Kato, T; Miyazaki, H

1998-01-01

Previous studies have shown that daily multiple administration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) markedly stimulates thrombopoiesis and effectively ameliorates thrombocytopenia, and in most cases anemia and neutropenia, in myelosuppressed animals. In this study, we evaluated the effects of a single intravenous injection of PEG-rHuMGDF on hematopoietic recovery after sublethal total-body irradiation in mice. A single injection of PEG-rHuMGDF (1 to 640 microg/kg) 1 hour after irradiation accelerated platelet, red blood cell (RBC), and white blood cell (WBC) recovery in a dose-dependent fashion. In the bone marrow of vehicle-treated mice, megakaryocytic, erythroid, and myeloid progenitors, as well as day 12 colony-forming unit-spleen (CFU-S), were dramatically decreased much earlier than the nadirs of peripheral blood cells, whereas megakaryocytes were modestly decreased. Treatment with PEG-rHuMGDF (80 microg/kg, an optimal dose) 1 hour after irradiation resulted in more rapid recovery of these four hematopoietic progenitors and also significantly facilitated megakaryocyte recovery. In addition, the same PEG-rHuMGDF administration schedule expanded bone marrow cells capable of rescuing lethally irradiated recipient mice. As the interval between irradiation and PEG-rHuMGDF treatment was longer, its effects on hematopoietic recovery were attenuated. In contrast to the effects of PEG-rHuMGDF, a single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 1 hour after irradiation exclusively accelerated WBC recovery, but only to a similar extent as PEG-rHuMGDF (80 microg/kg) treatment even when rhG-CSF doses were escalated to 1,000 microg/kg. This appeared related to different pharmacokinetics of these two factors after a single injection in irradiated mice. The concentrations of PEG-rHuMGDF after injection persisted in the plasma for a longer time compared with rhG-CSF. These results indicate that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.

Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme

PubMed Central

Foster, Barbara A.; Gangavarapu, Kalyan J.; Mathew, Grinu; Azabdaftari, Gissou; Morrison, Carl D.; Miller, Austin; Huss, Wendy J.

2013-01-01

Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population. PMID:23383057

Enhanced functional recombinant factor VII production by HEK 293 cells stably transfected with VKORC1 where the gamma-carboxylase inhibitor calumenin is stably suppressed by shRNA transfection.

PubMed

Wajih, Nadeem; Owen, John; Wallin, Reidar

2008-01-01

Recombinant members of the vitamin K-dependent protein family (factors IX and VII and protein C) have become important pharmaceuticals in treatment of bleeding disorders and sepsis. However, because the in vivo gamma-carboxylation system in stable cell lines used for transfection has a limited capacity of post translational gamma-carboxylation, the recovery of fully gamma-carboxylated and functional proteins is low. In this work we have engineered recombinant factor VII producing HEK 293 cells to stably overexpress VKORC1, the reduced vitamin K gamma-carboxylase cofactor and in addition stably silenced the gamma-carboxylase inhibitory protein calumenin. Stable cell lines transfected with only a factor VII cDNA had a 9% production of functional recombinant factor VII. On the other hand, these recombinant factor VII producing cells when engineered to overexpress VKORC1 and having calumenin stably suppressed more than 80% by shRNA expression, produced 68% functional factor VII. The technology presented should be applicable to all vertebrae members of the vitamin K-dependent protein family and should lower the production cost of the clinically used factors VII, IX and protein C.

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

PubMed

Masoomi Dezfooli, Seyedehsara; Tan, Wen Siang; Tey, Beng Ti; Ooi, Chien Wei; Hussain, Siti Aslina

2016-01-01

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers.

Expression of recombinant human flavin monooxygenase and moclobemide-N-oxide synthesis on multi-mg scale.

PubMed

Hanlon, Steven P; Camattari, Andrea; Abad, Sandra; Glieder, Anton; Kittelmann, Matthias; Lütz, Stephan; Wirz, Beat; Winkler, Margit

2012-06-18

A panel of human flavin monooxygenases were heterologously expressed in E. coli to obtain ready-to-use biocatalysts for the in vitro preparation of human drug metabolites. Moclobemide-N-oxide (65 mg) was the first high-priced metabolite prepared with recombinant hFMO3 on the multi-milligram scale.

Nonhomologous Recombination between Defective Poliovirus and Coxsackievirus Genomes Suggests a New Model of Genetic Plasticity for Picornaviruses

PubMed Central

Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line

2014-01-01

ABSTRACT Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3′ end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. PMID:25096874

BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

PubMed

Maas Enriquez, Monika; Thrift, John; Garger, Stephen; Katterle, Yvonne

2016-11-01

BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Recombinant Allergens in Structural Biology, Diagnosis, and Immunotherapy

PubMed Central

Tscheppe, Angelika; Breiteneder, Heimo

2017-01-01

The years 1988–1995 witnessed the beginning of allergen cloning and the generation of recombinant allergens, which opened up new avenues for the diagnosis and research of human allergic diseases. Most crystal and solution structures of allergens have been obtained using recombinant allergens. Structural information on allergens allows insights into their evolutionary biology, illustrates clinically observed cross-reactivities, and makes the design of hypoallergenic derivatives for allergy vaccines possible. Recombinant allergens are widely used in molecule-based allergy diagnosis such as protein microarrays or suspension arrays. Recombinant technologies have been used to produce well-characterized, noncontaminated vaccine components with known biological activities including a variety of allergen derivatives with reduced IgE reactivity. Such recombinant hypoallergens as well as wild-type recombinant allergens have been used successfully in several immunotherapy trials for more than a decade to treat birch and grass pollen allergy. As a more recent application, the development of antibody repertoires directed against conformational epitopes during immunotherapy has been monitored by recombinant allergen chimeras. Although much progress has been made, the number and quality of recombinant allergens will undoubtedly increase and keep improving our knowledge in basic scientific investigations, diagnosis, and therapy of human allergic diseases. PMID:28467993

Kinetic Modeling Sheds Light on the Mode of Action of Recombinant Factor VIIa on Thrombin Generation

DTIC Science & Technology

2011-01-01

Regular Article Kinetic modeling sheds light on the mode of action of recombinant factor VIIa on thrombin generation Alexander Y. Mitrophanov...its effects on the quantitative parameters of thrombin generation. For recombinant activated factor VII (rFVIIa) ― a promising hemostasis-inducing...modulate thrombin production , it is necessary to identify rFVIIa-induced effects that are compatible with the available biochemical knowledge about

Expression, purification, and characterization of recombinant human and murine milk fat globule-epidermal growth factor-factor 8.

PubMed

Castellanos, Erick R; Ciferri, Claudio; Phung, Wilson; Sandoval, Wendy; Matsumoto, Marissa L

2016-08-01

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8. Copyright © 2016 Elsevier Inc. All rights reserved.

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

PubMed Central

Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

2017-01-01

Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

Termini of human chromosomes display elevated rates of mitotic recombination.

PubMed

Cornforth, M N; Eberle, R L

2001-01-01

The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.

The Red Queen Model of Recombination Hotspots Evolution in the Light of Archaic and Modern Human Genomes

PubMed Central

Lesecque, Yann; Glémin, Sylvain; Lartillot, Nicolas; Mouchiroud, Dominique; Duret, Laurent

2014-01-01

Recombination is an essential process in eukaryotes, which increases diversity by disrupting genetic linkage between loci and ensures the proper segregation of chromosomes during meiosis. In the human genome, recombination events are clustered in hotspots, whose location is determined by the PRDM9 protein. There is evidence that the location of hotspots evolves rapidly, as a consequence of changes in PRDM9 DNA-binding domain. However, the reasons for these changes and the rate at which they occur are not known. In this study, we investigated the evolution of human hotspot loci and of PRDM9 target motifs, both in modern and archaic human lineages (Denisovan) to quantify the dynamic of hotspot turnover during the recent period of human evolution. We show that present-day human hotspots are young: they have been active only during the last 10% of the time since the divergence from chimpanzee, starting to be operating shortly before the split between Denisovans and modern humans. Surprisingly, however, our analyses indicate that Denisovan recombination hotspots did not overlap with modern human ones, despite sharing similar PRDM9 target motifs. We further show that high-affinity PRDM9 target motifs are subject to a strong self-destructive drive, known as biased gene conversion (BGC), which should lead to the loss of the majority of them in the next 3 MYR. This depletion of PRDM9 genomic targets is expected to decrease fitness, and thereby to favor new PRDM9 alleles binding different motifs. Our refined estimates of the age and life expectancy of human hotspots provide empirical evidence in support of the Red Queen hypothesis of recombination hotspots evolution. PMID:25393762

The red queen model of recombination hotspots evolution in the light of archaic and modern human genomes.

PubMed

Lesecque, Yann; Glémin, Sylvain; Lartillot, Nicolas; Mouchiroud, Dominique; Duret, Laurent

2014-11-01

Recombination is an essential process in eukaryotes, which increases diversity by disrupting genetic linkage between loci and ensures the proper segregation of chromosomes during meiosis. In the human genome, recombination events are clustered in hotspots, whose location is determined by the PRDM9 protein. There is evidence that the location of hotspots evolves rapidly, as a consequence of changes in PRDM9 DNA-binding domain. However, the reasons for these changes and the rate at which they occur are not known. In this study, we investigated the evolution of human hotspot loci and of PRDM9 target motifs, both in modern and archaic human lineages (Denisovan) to quantify the dynamic of hotspot turnover during the recent period of human evolution. We show that present-day human hotspots are young: they have been active only during the last 10% of the time since the divergence from chimpanzee, starting to be operating shortly before the split between Denisovans and modern humans. Surprisingly, however, our analyses indicate that Denisovan recombination hotspots did not overlap with modern human ones, despite sharing similar PRDM9 target motifs. We further show that high-affinity PRDM9 target motifs are subject to a strong self-destructive drive, known as biased gene conversion (BGC), which should lead to the loss of the majority of them in the next 3 MYR. This depletion of PRDM9 genomic targets is expected to decrease fitness, and thereby to favor new PRDM9 alleles binding different motifs. Our refined estimates of the age and life expectancy of human hotspots provide empirical evidence in support of the Red Queen hypothesis of recombination hotspots evolution.

The complete genome sequence of human adenovirus 84, a highly recombinant new Human mastadenovirus D type with a unique fiber gene.

PubMed

Kaján, Győző L; Kajon, Adriana E; Pinto, Alexis Castillo; Bartha, Dániel; Arnberg, Niklas

2017-10-15

A novel human adenovirus was isolated from a pediatric case of acute respiratory disease in Panama City, Panama in 2011. The clinical isolate was initially identified as an intertypic recombinant based on hexon and fiber gene sequencing. Based on the analysis of its complete genome sequence, the novel complex recombinant Human mastadenovirus D (HAdV-D) strain was classified into a new HAdV type: HAdV-84, and it was designated Adenovirus D human/PAN/P309886/2011/84[P43H17F84]. HAdV-D types possess usually an ocular or gastrointestinal tropism, and respiratory association is scarcely reported. The virus has a novel fiber type, most closely related to, but still clearly distant from that of HAdV-36. The predicted fiber is hypothesised to bind sialic acid with lower affinity compared to HAdV-37. Bioinformatic analysis of the complete genomic sequence of HAdV-84 revealed multiple homologous recombination events and provided deeper insight into HAdV evolution. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioinformatic Analysis of the Human Recombinant Iduronate 2-Sulfate Sulfatase

PubMed Central

Morales-Álvarez, Edwin D.; Rivera-Hoyos, Claudia M.; Landázuri, Patricia; Poutou-Piñales, Raúl A.; Pedroza-Rodríguez, Aura M.

2016-01-01

Mucopolysaccharidosis type II is a human recessive disease linked to the X chromosome caused by deficiency of lysosomal enzyme Iduronate 2-Sulfate Sulfatase (IDS), which leads to accumulation of glycosaminoglycans in tissues and organs. The human enzyme has been expressed in Escherichia coli and Pichia pastoris in attempt to develop more successful expression systems that allow the production of recombinant IDS for Enzyme Replacement Therapy (ERT). However, the preservation of native signal peptide in the sequence has caused conflicts in processing and recognition in the past, which led to problems in expression and enzyme activity. With the main object being the improvement of the expression system, we eliminate the native signal peptide of human recombinant IDS. The resulting sequence showed two modified codons, thus, our study aimed to analyze computationally the nucleotide sequence of the IDSnh without signal peptide in order to determine the 3D structure and other biochemical properties to compare them with the native human IDS (IDSnh). Results showed that there are no significant differences between both molecules in spite of the two-codon modifications detected in the recombinant DNA sequence. PMID:27335624

A novel platform for biologically active recombinant human interleukin-13 production.

PubMed

Wang, David J; Brandsma, Martin; Yin, Ziqin; Wang, Aiming; Jevnikar, Anthony M; Ma, Shengwu

2008-06-01

Interleukin-13 (IL-13) is a pleiotropic regulatory cytokine with the potential for treating several human diseases, including type-1 diabetes. Thus far, conventional expression systems for recombinant IL-13 production have proven difficult and are limited by efficiency. In this study, transgenic plants were used as a novel expression platform for the production of human IL-13 (hIL-13). DNA constructs containing hIL-13 cDNA were introduced into tobacco plants. Transcriptional expression of the hIL-13 gene in transgenic plants was confirmed by reverse transcriptase-polymerase chain reaction and Northern blotting. Western blot analysis showed that the hIL-13 protein was efficiently accumulated in transgenic plants and present in multiple molecular forms, with an expression level as high as 0.15% of total soluble protein in leaves. The multiple forms of plant-derived recombinant hIL-13 (rhIL-13) are a result of differential N-linked glycosylation, as revealed by enzymatic and chemical deglycosylation, but not of disulphide-linked oligomerization. In vitro trypsin digestion indicated that plant rhIL-13 was more resistant than unglycosylated control rhIL-13 to proteolysis. The stability of plant rhIL-13 to digestion was further supported with simulated gastric and intestinal fluid digestion. In vitro bioassays using a factor-dependent human erythroleukaemic cell line (TF-1 cells) showed that plant rhIL-13 retained the biological functions of the authentic hIL-13 protein. These results demonstrate that transgenic plants are superior to conventional cell-based expression systems for the production of rhIL-13. Moreover, transgenic plants synthesizing high levels of rhIL-13 may prove to be an attractive delivery system for direct oral administration of IL-13 in the treatment of clinical diseases such as type-1 diabetes.

Recombinant human type II collagen hydrogel provides a xeno-free 3D micro-environment for chondrogenesis of human bone marrow-derived mesenchymal stromal cells.

PubMed

Muhonen, Virpi; Narcisi, Roberto; Nystedt, Johanna; Korhonen, Matti; van Osch, Gerjo J V M; Kiviranta, Ilkka

2017-03-01

Recombinant human type II collagen (rhCII) hydrogel was tested as a xeno-free micro-environment for the chondrogenesis of human bone marrow-derived mesenchymal stromal cells (BM-MSCs). The rhCII hydrogels were seeded with BM-MSCs and cultured in a xeno-free chondro-inductive medium for 14, 28 and 84 days. High-density pellet cultures served as controls. The samples were subjected to biochemical, histological and gene expression analyses. Although the cells deposited glycosaminoglycans into the extracellular space significantly more slowly in the rhCII hydrogels compared to the high-density pellets, a similar potential of matrix deposition was reached by the end of the 84-day culture. At day 28 of culture, the gene expression level for cartilage marker genes (i.e. genes encoding for Sox9 transcription factor, Collagen type II and Aggrecan) were considerably lower in the rhCII hydrogels than in the high-density pellets, but at the end of the 84-day culture period, all the cartilage marker genes analysed were expressed at a similar level. Interestingly, the expression of the matrix metallopeptidases (MMP)-13, MMP-14 and MMP-8, i.e. extracellular collagen network-degrading enzymes, were transiently upregulated in the rhCII hydrogel, indicating active matrix reorganization. This study demonstrated that the rhCII hydrogel functions as a xeno-free platform for BM-MSC chondrogenesis, although the process is delayed. The reversible catabolic reaction evoked by the rhCII hydrogel might be beneficial in graft integration in vivo and pinpoints the need to further explore the use of hydrogels containing recombinant extracellular matrix (ECM) proteins to induce the chondrogenesis of MSCs. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

PubMed Central

Courtney, Harry S.; Li, Yi

2013-01-01

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG1>IgG4>IgG2>IgG3. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host. PMID:24205299

Fine-scale maps of recombination rates and hotspots in the mouse genome.

PubMed

Brunschwig, Hadassa; Levi, Liat; Ben-David, Eyal; Williams, Robert W; Yakir, Benjamin; Shifman, Sagiv

2012-07-01

Recombination events are not uniformly distributed and often cluster in narrow regions known as recombination hotspots. Several studies using different approaches have dramatically advanced our understanding of recombination hotspot regulation. Population genetic data have been used to map and quantify hotspots in the human genome. Genetic variation in recombination rates and hotspots usage have been explored in human pedigrees, mouse intercrosses, and by sperm typing. These studies pointed to the central role of the PRDM9 gene in hotspot modulation. In this study, we used single nucleotide polymorphisms (SNPs) from whole-genome resequencing and genotyping studies of mouse inbred strains to estimate recombination rates across the mouse genome and identified 47,068 historical hotspots--an average of over 2477 per chromosome. We show by simulation that inbred mouse strains can be used to identify positions of historical hotspots. Recombination hotspots were found to be enriched for the predicted binding sequences for different alleles of the PRDM9 protein. Recombination rates were on average lower near transcription start sites (TSS). Comparing the inferred historical recombination hotspots with the recent genome-wide mapping of double-strand breaks (DSBs) in mouse sperm revealed a significant overlap, especially toward the telomeres. Our results suggest that inbred strains can be used to characterize and study the dynamics of historical recombination hotspots. They also strengthen previous findings on mouse recombination hotspots, and specifically the impact of sequence variants in Prdm9.

Immune responses in macaques to a prototype recombinant adenovirus live oral human papillomavirus 16 vaccine.

PubMed

Berg, Michael G; Adams, Robert J; Gambhira, Ratish; Siracusa, Mark C; Scott, Alan L; Roden, Richard B S; Ketner, Gary

2014-09-01

Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Methamphetamine enhances Hepatitis C virus replication in human hepatocytes

PubMed Central

Ye, L.; Peng, J. S.; Wang, X.; Wang, Y. J.; Luo, G. X.; Ho, W. Z.

2009-01-01

SUMMARY Very little is known about the interactions between hepatitis C virus (HCV) and methamphetamine, which is a highly abused psychostimulant and a known risk factor for human immunodeficiency virus (HIV)/HCV infection. This study examined whether methamphetamine has the ability to inhibit innate immunity in the host cells, facilitating HCV replication in human hepatocytes. Methamphetamine inhibited intracellular interferon alpha expression in human hepatocytes, which was associated with the increase in HCV replication. In addition, methamphetamine also compromised the anti-HCV effect of recombinant interferon alpha. Further investigation of mechanism(s) responsible for the methamphetamine action revealed that methamphetamine was able to inhibit the expression of the signal transducer and activator of transcription 1, a key modulator in interferon-mediated immune and biological responses. Methamphetamine also down-regulated the expression of interferon regulatory factor-5, a crucial transcriptional factor that activates the interferon pathway. These in vitro findings that methamphetamine compromises interferon alpha-mediated innate immunity against HCV infection indicate that methamphetamine may have a cofactor role in the immunopathogenesis of HCV disease. PMID:18307590

High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.

PubMed

Su, Lin; Chen, Song-Sen; Yang, Ke-Gong; Liu, Chang-Zheng; Zhang, Yan-Li; Liang, Zhi-Quan

2006-06-01

Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.

Efficient Isolation and Propagation of Human Immunodeficiency Virus on Recombinant Colony-Stimulating Factor 1-Treated Monocytes

DTIC Science & Technology

1988-04-01

search Library /Reprint Deor.: renc Dept of Iimunology, Wasington, DC 20307-5100 Washington, DC: 20�-5100, Ba NAVE OF FNING, SPONSORING an )::Z C- S VBOL...released~~O into)int cultures \\luids wauheeigy na clox Mooc ~es hr ncaly ifeced 4(1 i) ith11 V- (pate IND20A~. second J~pas- sage)~ ~ ~ ~ ~ ~~~~~(l intill

Proposed Multicenter Studies

DTIC Science & Technology

2009-02-01

One plasma- derived AT product is Thrombate, produced by Bayer. Recombinant AT (rhAT) is made on a large scale in the milk of transgenic goats and is...infusions of rhAT to increase AT levels to 200 and 500% of normal, followed by infusions of endotoxin . AT dose dependently decreased tissue factor...injury. REFERENCES 1. Edmunds T, Van Patten SM, Pollock J, et al. Transgenically produced human antithrombin: structural and functional comparison to

LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during late mitosis.

PubMed

Hong, Ye; Sonneville, Remi; Wang, Bin; Scheidt, Viktor; Meier, Bettina; Woglar, Alexander; Demetriou, Sarah; Labib, Karim; Jantsch, Verena; Gartner, Anton

2018-02-20

Faithful chromosome segregation and genome maintenance requires the removal of all DNA bridges that physically link chromosomes before cells divide. Using C. elegans embryos we show that the LEM-3/Ankle1 nuclease defines a previously undescribed genome integrity mechanism by processing DNA bridges right before cells divide. LEM-3 acts at the midbody, the structure where abscission occurs at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane. LEM-3 locally processes chromatin bridges that arise from incomplete DNA replication, unresolved recombination intermediates, or the perturbance of chromosome structure. Proper LEM-3 midbody localization and function is regulated by AIR-2/Aurora B kinase. Strikingly, LEM-3 acts cooperatively with the BRC-1/BRCA1 homologous recombination factor to promote genome integrity. These findings provide a molecular basis for the suspected role of the LEM-3 orthologue Ankle1 in human breast cancer.

Host cell proteins in biotechnology-derived products: A risk assessment framework.

PubMed

de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

2015-11-01

To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant)

USDA-ARS?s Scientific Manuscript database

Action of human small intestinal brush border carbohydrate digesting enzymes is thought to involve only final hydrolysis reactions of oligosaccharides to monosaccharides. In vitro starch digestibility assays use fungal amyloglucosidase to provide this function. In this study, recombinant N-terminal ...

Recombinant human granulocyte colony-stimulating factor administration in a case of neutropenia due to increased neutrophil sequestration.

PubMed

Carulli, G; Lazzeri, E; Lagomarsini, G; Zucca, A; Cannizzo, E; Riccioni, R; Petrini, M

2007-01-01

A 55-year-old female was admitted with fever which followed an episode of pseudomembranous colitis. Despite an accurate clinical investigation, there was no evidence for specific sites of infection. Remission of fever was not obtained with antibiotic therapy (gentamycin plus carbepenem) and progressive neutropenia was observed. Neutrophils fell to 0.3 x 10(9)/1. The diagnostic approach, including a bone marrow aspirate, excluded mechanisms leading to impaired neutrophil production, and in the suspect of increased neutrophil sequestration/destruction, whole-body scintigraphy with (99m)technetium-hexamethylpropyleneamineoxime ((99m)Tc-HMPAO)-labeled autologous leukocytes was performed. As a result, a site of leukocyte sequestration localized at the medium lobe of the right lung was detected. In an attempt to enhance neutrophil functions and achieve remission of infection, recombinant human granulocyte colony-stimulating factor (Filgrastim, Granulokine 30, Roche) at the dosage of 300 microg/day, subcutaneously, was added. As a results, fever disappeared in three days, but neutrophil recovery was slower, and normalization of the absolute neutrophil count (ANC) was obtained on day +7. The results obtained in this peculiar case of neutropenia, and the kinetics of both fever and ANC, suggest the possible combination of neutrophil function enhancement and an anti-inflammatory effect of rhG-CSF.

Effect of recombinant human granulocyte colony-stimulating factor on combination therapy with aztreonam and clindamycin for infections in neutropenic patients with hematologic diseases.

PubMed

Toyama, K; Yaguchi, M; Mizoguchi, H; Masuda, M; Urabe, A; Ikeda, Y; Aoki, I; Shinbo, T; Togawa, A; Hirashima, K; Miura, Y; Hirose, S; Tsuruoka, N; Omine, M; Kamakura, M; Saito, T; Arimori, S; Aoki, N; Kuraishi, Y; Hirai, H; Asano, S; Mori, M; Shirai, T; Muto, Y; Takaku, F

1996-12-01

The present multicenter study was performed to evaluate the effect of recombinant human granulocyte-colony stimulating factor (rhG-CSF) on combination therapy using aztreonam (AZT) and clindamycin (CLDM) to treat severe infection in neutropenic patients with hematologic diseases. Forty-three neutropenic patients with infections (rhG-CSF group) were treated with AZT (2 g) and CLDM (600 mg) 2-3 times daily as well as rhG-CSF (Lenograstim or Filgrastim: 2-5 mu/kg/day). The clinical efficacy of this regimen was compared to that obtained in 44 febrile neutropenic patients, with hematologic diseases, who received only AZT and CLDM in a previous study (historical control group). The overall efficacy rate was 69.8% (30/43) in the rhG-CSF group and 65.9% (29/44) in the historical control group. Although the neutrophil count was significantly increased and C-reactive protein tended to be lower in the rhG-CSF group, the daily maximum body temperature profiles of the 2 groups were nearly the same. These results suggest that rhG-CSF is of little benefit in the treatment of single infectious episodes in neutropenic patients, and that appropriate antibiotic therapy is more important.

Effects of recombinant human granulocyte colony-stimulating factor on the hematologic recovery and survival of irradiated mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanikawa, S.; Nose, M.; Aoki, Y.

1990-08-01

We studied the effects of intraperitoneal injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) according to various administration schedules on the recovery of spleen colony-forming units (CFU-S) and peripheral blood counts, and on the survival of irradiated mice. The sooner and more frequently the mice were injected with rhG-CSF after irradiation, the more enhanced the recovery of CFU-S in bone marrow was obtained on day 7. Twice-daily injections of rhG-CSF from day 0 to day 2 significantly enhanced the recovery of platelets and hematocrit, but two injections of rhG-CSF on only day 0 did not. Twice-daily injections of rhG-CSF frommore » day 0 to day 6 enhanced the recovery of platelets more effectively than twice-daily injections of rhG-CSF from day 1 to day 7, and increased the survival of irradiated mice more effectively than any other examined administration schedules. Twice-daily injections of rhG-CSF from day 0 to day 6 were significantly effective in enhancing the survival of mice irradiated with 8.5-, 9.0-, and 9.5-Gy x-rays, although not effective after irradiation of 10.5-Gy x-rays.« less

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

PubMed Central

Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

2013-01-01

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80.

PubMed

Clark, Jakson; Montgomery, Jade; Squires, Ryan; McGuire, Joseph

2016-03-01

Detection of enhanced surface tension depression by surfactant in the presence of protein was recently suggested as a basis for determining whether protein stabilization by that surfactant is owing to surfactant forming a steric barrier at interfaces or surfactant association with the protein. In particular, protein interaction with surfactant aggregates may lead to an increased concentration of monomers thus enhancing surfactant adsorption, or to formation of surfactant-protein complexes having little or no effect on adsorption. We compared the initial rates of surface tension depression by poloxamer 188 and polysorbate 80 (PS 80) in the presence and absence of a human recombinant factor VIII (rFVIII). Indirect evidence had suggested poloxamer 188 enters into stable associations with rFVIII in solution but does not form a steric barrier at the interface, while PS 80 behaves in contrary fashion. In this study, we show the presence of rFVIII caused an increase in the rate (reduction in the activation energy) of PS 80 adsorption, while no such change was recorded in the case of poloxamer 188. Thus, we provide substantiation for detection of protein-mediated acceleration of surfactant adsorption as a means to compare different surfactants in relation to their favored mechanism for protein stabilization.

Stability of nonaqueous suspension formulations of plasma derived factor IX and recombinant human alpha interferon at elevated temperatures.

PubMed

Knepp, V M; Muchnik, A; Oldmark, S; Kalashnikova, L

1998-07-01

To identify a suitable nonaqueous, parenterally acceptable suspending vehicle whereby a therapeutic protein is delivered as a stable flowable powder, making it amenable to delivery from sustained delivery systems maintained at body temperature. Formulations of plasma derived Factor IX (pdFIX) and recombinant human alpha interferon (rhalpha-IFN) were formulated as dry powders, suspended in various vehicles (perfluorodecalin, perfluorotributylamine, methoxyflurane, polyethylene glycol 400, soybean oil, tetradecane or octanol) and stored at 37 degrees C. Stability was assessed by size exclusion chromatography, reverse phase chromatography, ion exchange chromatography, and bioassay, and was compared to the stability of dry powder formulations stored at 37 degrees C and -80 degrees C. PdFIX was stable when stored at 37 degrees C as a dry powder, or when the dry powder was suspended in the pharmaceutically acceptable vehicles perfluorodecalin or perfluorotributylamine. Suspensions of the powder in other pharmaceutically/parenterally acceptable vehicles such as soybean oil or PEG 400 resulted in aggregation and loss of bioactivity. A dry powder formulation of rhalpha-IFN suspended in perfluorodecalin was also stable at 37 degrees C. This study shows the potential utility of perfluorinated hydrocarbons as nonaqueous suspending vehicles for long term in-vivo delivery of therapeutic proteins.

Intratracheal Administration of Recombinant Human Keratinocyte Growth Factor Promotes Alveolar Epithelial Cell Proliferation during Compensatory Lung Growth in Rat

PubMed Central

Furukawa, Katsuro; Matsumoto, Keitaro; Nagayasu, Takeshi; Yamamoto-Fukuda, Tomomi; Tobinaga, Shuichi; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Kamohara, Ryotaro; Nanashima, Atsushi; Obatake, Masayuki; Koji, Takehiko

2013-01-01

Keratinocyte growth factor (KGF) is considered to be one of the most important mitogens for lung epithelial cells. The objectives of this study were to confirm the effectiveness of intratracheal injection of recombinant human KGF (rhKGF) during compensatory lung growth and to optimize the instillation protocol. Here, trilobectomy in adult rat was performed, followed by intratracheal rhKGF instillation with low (0.4 mg/kg) and high (4 mg/kg) doses at various time-points. The proliferation of alveolar cells was assessed by the immunostaining for proliferating cell nuclear antigen (PCNA) in the residual lung. We also investigated other immunohistochemical parameters such as KGF, KGF receptor and surfactant protein A as well as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Consequently, intratracheal single injection of rhKGF in high dose group significantly increased PCNA labeling index (LI) of alveolar cells in the remaining lung. Surprisingly, there was no difference in PCNA LI between low and high doses of rhKGF with daily injection, and PCNA LI reached a plateau level with 2 days-consecutive administration (about 60%). Our results indicate that even at low dose, daily intratracheal injection is effective to maintain high proliferative states during the early phase of compensatory lung growth. PMID:24610965

Intratracheal Administration of Recombinant Human Keratinocyte Growth Factor Promotes Alveolar Epithelial Cell Proliferation during Compensatory Lung Growth in Rat.

PubMed

Furukawa, Katsuro; Matsumoto, Keitaro; Nagayasu, Takeshi; Yamamoto-Fukuda, Tomomi; Tobinaga, Shuichi; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Kamohara, Ryotaro; Nanashima, Atsushi; Obatake, Masayuki; Koji, Takehiko

2013-12-28

Keratinocyte growth factor (KGF) is considered to be one of the most important mitogens for lung epithelial cells. The objectives of this study were to confirm the effectiveness of intratracheal injection of recombinant human KGF (rhKGF) during compensatory lung growth and to optimize the instillation protocol. Here, trilobectomy in adult rat was performed, followed by intratracheal rhKGF instillation with low (0.4 mg/kg) and high (4 mg/kg) doses at various time-points. The proliferation of alveolar cells was assessed by the immunostaining for proliferating cell nuclear antigen (PCNA) in the residual lung. We also investigated other immunohistochemical parameters such as KGF, KGF receptor and surfactant protein A as well as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Consequently, intratracheal single injection of rhKGF in high dose group significantly increased PCNA labeling index (LI) of alveolar cells in the remaining lung. Surprisingly, there was no difference in PCNA LI between low and high doses of rhKGF with daily injection, and PCNA LI reached a plateau level with 2 days-consecutive administration (about 60%). Our results indicate that even at low dose, daily intratracheal injection is effective to maintain high proliferative states during the early phase of compensatory lung growth.

Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor

PubMed Central

Chi, Eva Y.; Krishnan, Sampathkumar; Kendrick, Brent S.; Chang, Byeong S.; Carpenter, John F.; Randolph, Theodore W.

2003-01-01

We studied the non-native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (ΔGunf) and osmotic second virial coefficient (B22), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large ΔGunf and negative B22), the first step is rate-limiting, and increasing ΔGunf (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B22 values) the second step is rate-limiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate-limiting. PMID:12717013

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

Productive replication of human papillomavirus 31 requires DNA repair factor Nbs1.

PubMed

Anacker, Daniel C; Gautam, Dipendra; Gillespie, Kenric A; Chappell, William H; Moody, Cary A

2014-08-01

Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

PubMed Central

Anacker, Daniel C.; Gautam, Dipendra; Gillespie, Kenric A.; Chappell, William H.

2014-01-01

ABSTRACT Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication. PMID:24850735

Recombinant human basic fibroblast growth factor (bFGF) stimulates periodontal regeneration in class II furcation defects created in beagle dogs.

PubMed

Murakami, S; Takayama, S; Kitamura, M; Shimabukuro, Y; Yanagi, K; Ikezawa, K; Saho, T; Nozaki, T; Okada, H

2003-02-01

Several growth factors (or cytokines) have been recently investigated for their use as potential therapeutics for periodontal tissue regeneration. The objective of this study was to evaluate periodontal tissue regeneration, including new bone and cementum formation, following topical application of recombinant basic fibroblast growth factor (bFGF, FGF-2) to furcation class II defects. Twelve furcation class II bone defects were surgically created in six beagle dogs, then recombinant bFGF (30 micro g/site) + gelatinous carrier was topically applied to the bony defects. Six weeks after application, periodontal regeneration was analyzed. In all sites where bFGF was applied, periodontal ligament formation with new cementum deposits and new bone formation was observed histomorphometrically, in amounts greater than in the control sites. Basic FGF-applied sites exhibited significant regeneration as represented by the new bone formation rate (NBR) (83.6 +/- 14.3%), new trabecular bone formation rate (NTBR) (44.1 +/- 9.5%), and new cementum formation rate (NCR) (97.0 +/- 7.5%). In contrast, in the carrier-only sites, the NBR, NTBR, and NCR were 35.4 +/- 8.9%, 16.6 +/- 6.2%, and 37.2 +/- 15.1%, respectively. Moreover, no instances of epithelial down growth, ankylosis, or root resorption were observed in the bFGF-applied sites examined. The present results indicate that topical application of bFGF can enhance considerable periodontal regeneration in artificially created furcation class II bone defects of beagle dogs.

Activin receptor ligand traps in chronic kidney disease.

PubMed

Jelkmann, Wolfgang

2018-05-29

Sotatercept and luspatercept are recombinant soluble activin type-II receptor-IgG-Fc fusion proteins that are tested in clinical trials for the treatment of various types of anemias, including renal anemia. The mechanism of the action of the novel drugs is incompletely understood, but it seems to be based on the inactivation of soluble proteins of the transforming growth factor-ß (TGFß) family. This review considers pros and cons of the clinical use of the drugs in reference to the current therapy with recombinant erythropoiesis-stimulating agents (ESAs). One or more activin type-II receptor (ActRII) ligands appear to inhibit erythroid precursors, for example growth and differentiation factor 11. Trapping of these ligands by the recombinant ActRII fusion proteins, sotatercept and luspatercept increases red blood cell numbers and hemoglobin levels in humans. Reportedly, the novel compounds were well tolerated in trials on healthy volunteers and patients suffering from anemia due to chronic kidney disease or malignancies. On approval, the drugs may prove particularly useful in patients suffering from ineffective erythropoiesis, such as in myelodysplastic syndrome, multiple myeloma or ß-thalassemia, where ESAs are of little use. Independent of their effect on erythropoiesis, ActRII ligand traps were found to exert beneficial effects on renal tissue in experimental animals. ESAs are likely to remain standard of care in renal anemia. There is a need for a better understanding of the effects of ActRII ligand traps on TGFß-like proteins. The novel drugs have not been approved for sale as therapeutics so far. Their long-term efficacy and safety still needs to be proven, particularly with respect to immunogenicity. Antifibrotic effects may be worthy to be investigated in humans.

Ovarian response to recombinant human follicle-stimulating hormone in luteinizing hormone-depleted women: examination of the two cell, two gonadotropin theory.

PubMed

Ben-Chetrit, A; Gotlieb, L; Wong, P Y; Casper, R F

1996-04-01

To evaluate the relative contribution of FSH to ovarian estrogen production. Nonrandomized, prospective study. University of Toronto teaching hospital reproductive biology unit. Five women who had been treated with depot GnRH agonist with hormonal add-back for 4 to 48 months and who were confirmed to be gonadotropin depleted by both bioassay and RIA. Subjects received 75 IU SC recombinant human FSH daily for 7 days followed by 150 IU daily for 7 days and 225 IU daily for the third week. Serum steroid determination and vaginal sonography for follicle size and endometrial thickness were performed serially and follicular fluid hormone levels were measured in two subjects. Bioactive LH and FSH activity were less than the detection limit of the assay (0.1 mIU/mL; conversion factor to SI units, 1.00 for LH and FSH) before recombinant FSH treatment in all five women. In all subjects, at least one preovulatory follicle developed by the end of two to three weeks. Endometrial thickness increased to between 7 and 9 mm in four women. Mean serum E2 in the five subjects increased from 17 pg/mL (range: 5 to 33 pg/mL; conversion factor to SI unit, 3.671) at baseline to 230 pg/mL (range: 37 to 489 pg/mL) at the end of the study. Follicular fluid E2 concentrations ranged from 44,296 to 69,367 pg/mL in the four follicles aspirated. Our results indicate that LH is not necessary for ovarian E2 production. We speculate that the granulosa cells, in the absence of detectable LH bioactivity, can use circulating adrenal androgens or constitutive or FSH-stimulated thecal androgens, to produce intrafollicular E2.

ARG-walker: inference of individual specific strengths of meiotic recombination hotspots by population genomics analysis.

PubMed

Chen, Hao; Yang, Peng; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

2015-01-01

Meiotic recombination hotspots play important roles in various aspects of genomics, but the underlying mechanisms for regulating the locations and strengths of recombination hotspots are not yet fully revealed. Most existing algorithms for estimating recombination rates from sequence polymorphism data can only output average recombination rates of a population, although there is evidence for the heterogeneity in recombination rates among individuals. For genome-wide association studies (GWAS) of recombination hotspots, an efficient algorithm that estimates the individualized strengths of recombination hotspots is highly desirable. In this work, we propose a novel graph mining algorithm named ARG-walker, based on random walks on ancestral recombination graphs (ARG), to estimate individual-specific recombination hotspot strengths. Extensive simulations demonstrate that ARG-walker is able to distinguish the hot allele of a recombination hotspot from the cold allele. Integrated with output of ARG-walker, we performed GWAS on the phased haplotype data of the 22 autosome chromosomes of the HapMap Asian population samples of Chinese and Japanese (JPT+CHB). Significant cis-regulatory signals have been detected, which is corroborated by the enrichment of the well-known 13-mer motif CCNCCNTNNCCNC of PRDM9 protein. Moreover, two new DNA motifs have been identified in the flanking regions of the significantly associated SNPs (single nucleotide polymorphisms), which are likely to be new cis-regulatory elements of meiotic recombination hotspots of the human genome. Our results on both simulated and real data suggest that ARG-walker is a promising new method for estimating the individual recombination variations. In the future, it could be used to uncover the mechanisms of recombination regulation and human diseases related with recombination hotspots.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

PubMed

Cappelletti, Pamela; Binda, Elisa; Tunesi, Marta; Albani, Diego; Giordano, Carmen; Molla, Gianluca; Pollegioni, Loredano

2017-10-01

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Control of erythropoietin gene expression and its use in medicine.

PubMed

Jelkmann, Wolfgang

2007-01-01

Erythropoietin (EPO) gene expression is under the control of inhibitory (GATA-2, NF-kappaB) and stimulatory (hypoxia-inducible transcription factor [HIF]-2, hepatocyte nuclear factor [HNF]-4alpha [alpha]) transcription factors. EPO deficiency is the main cause of the anemia in chronic kidney disease (CKD) and a contributing factor in the anemias of inflammation and cancer. Small, orally active compounds capable of stimulating endogenous EPO production are in preclinical or clinical trials for treatment of anemia. These agents include stabilizers of the HIFs that bind to the EPO enhancer and GATA inhibitors which prevent GATA from suppressing the EPO promoter. While HIF stabilizing drugs may prove useful as inexpensive second-line choices, at present, their side effects--particularly tumorigenicity--preclude their use as first-choice therapy. As an alternative, EPO gene therapy has been explored in animal studies and in trials on CKD patients. Here, a major problem is immunogenicity of ex vivo transfected implanted cells and of the recombinant protein produced after ex vivo or in vivo EPO complementary DNA (cDNA) transfer. Recombinant human EPO (rhEPO) engineered in Chinese hamster ovary (CHO) cell cultures (epoetin alpha and epoetin beta [beta]) and its hyperglycosylated analogue darbepoetin alpha are established and safe drugs to avoid allogeneic red blood cell transfusion. Gene-activated EPO (epoetin delta [delta]) from human fibrosarcoma cells (HT-1080) has recently been launched for use in CKD. It is important to know the basics of the technologies, production processes, and structural properties of the novel anti-anemic strategies and drugs.

A role for very low-dose recombinant activated factor VII in refractory bleeding after cardiac surgery: Lessons from an observational study.

PubMed

Hoffmann, Till; Assmann, Alexander; Dierksen, Angelika; Roussel, Elisabeth; Ullrich, Sebastian; Lichtenberg, Artur; Albert, Alexander; Sixt, Stephan

2018-04-18

Although off-label use of recombinant activated factor VII against refractory bleeding is incorporated in current guideline recommendations, safety concerns persist predominantly with respect to thromboembolic complications. We analyzed the safety and efficacy of recombinant activated factor VII at a very low dose in cardiosurgical patients with refractory bleeding. This prospective study includes 1180 cardiosurgical patients at risk of bleeding. Goal-directed substitution was based on real-time laboratory testing and clinical scoring of the bleeding intensity. All patients who fulfilled the criteria for enhanced risk of bleeding (n = 281) were consequently included in the present analysis. Patients in whom refractory bleeding developed despite substitution with specific hemostatic compounds (n = 167) received a single shot of very low-dose recombinant activated factor VII (≤20 μg/kg). Mortality and risk of thromboembolic complications, and freedom from stroke and acute myocardial infarction in particular, were analyzed (vs patients without recombinant activated factor VII) by multivariable logistic and Cox regression analyses, as well as Kaplan-Meier estimates. There was no increase in rates of mortality (30-day mortality 4.2% vs 7.0% with P = .418; follow-up survival 85.6% at 13.0 [interquartile range, 8.4-15.7] months vs 80.7% at 10.2 [interquartile range, 7.2-16.1] months with P = .151), thromboembolic complications (6.6% vs 9.6% with P = .637), renal insufficiency, need for percutaneous coronary intervention, duration of ventilation, duration of hospital stay, or rehospitalization in patients receiving very low-dose recombinant activated factor VII compared with patients not receiving recombinant activated factor VII. Complete hemostasis without any need for further hemostatic treatment was achieved after very low-dose recombinant activated factor VII administration in the majority of patients (up to 88.6% vs 0% with P < .001). The key results were confirmed after adjustment by propensity score-based analyses. When combined with early and specific restoration of hemostatic reserves after cardiac surgery, very low-dose recombinant activated factor VII treatment of refractory bleeding is effective and not associated with any apparent increase in adverse events. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Lessons learned from successful human vaccines: Delineating key epitopes by dissecting the capsid proteins

PubMed Central

Zhang, Xiao; Xin, Lu; Li, Shaowei; Fang, Mujin; Zhang, Jun; Xia, Ningshao; Zhao, Qinjian

2015-01-01

Recombinant VLP-based vaccines have been successfully used against 3 diseases caused by viral infections: Hepatitis B, cervical cancer and hepatitis E. The VLP approach is attracting increasing attention in vaccine design and development for human and veterinary use. This review summarizes the clinically relevant epitopes on the VLP antigens in successful human vaccines. These virion-like epitopes, which can be delineated with molecular biology, cryo-electron microscopy and x-ray crystallographic methods, are the prerequisites for these efficacious vaccines to elicit functional antibodies. The critical epitopes and key factors influencing these epitopes are discussed for the HEV, HPV and HBV vaccines. A pentamer (for HPV) or a dimer (for HEV and HBV), rather than a monomer, is the basic building block harboring critical epitopes for the assembly of VLP antigen. The processing and formulation of VLP-based vaccines need to be developed to promote the formation and stabilization of these epitopes in the recombinant antigens. Delineating the critical epitopes is essential for antigen design in the early phase of vaccine development and for critical quality attribute analysis in the commercial phase of vaccine manufacturing. PMID:25751641

In vivo characterization of fusion protein comprising of A1 subunit of Shiga toxin and human GM-CSF: Assessment of its immunogenicity and toxicity.

PubMed

Oloomi, Mana; Bouzari, Saeid; Shariati, Elaheh

2010-10-01

Most cancer cells become resistant to anti-cancer agents. In the last few years, a new approach for targeted therapy of human cancer has been developed using immunotoxins which comprise both the cell targeting and the cell killing moieties. In the present study, the recombinant Shiga toxin A1 subunit fused to human granulocyte-macrophage colony stimulating factor (A1-GM-CSF), previously produced in E. coli, was further characterized. The recombinant protein could cause 50% cytotoxicity and induced apoptosis in cells bearing GM-CSF receptors. The non-specific toxicity of the fusion protein was assessed in C57BL/6 and BALB/c mice. No mortality was observed in either group of mice, with different concentration of fusion protein. The lymphocyte proliferation assay, induction of specific IgG response and a mixed (Th1/Th2) response were observed only in BALB/c mice. The mixed response in BALB/c mice (Th1/Th2) could be explained on the basis of the two components of the fusion protein i.e. A1 and GM-CSF.

Epimorphin expression in interstitial pneumonia

PubMed Central

Terasaki, Yasuhiro; Fukuda, Yuh; Suga, Moritaka; Ikeguchi, Naoki; Takeya, Motohiro

2005-01-01

Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP), and lungs with usual interstitial pneumonia (UIP); we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells in vitro. Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling. PMID:15651999

Debates, divisions, and decisions: recombinant DNA advisory committee (RAC) authorization of the first human gene transfer experiments.

PubMed Central

Carmen, I H

1992-01-01

Possibly the most far-reaching, controversial research currently being conducted in the international biological science community involves human gene therapy experimentation. In this paper, I report the dynamics of the political process which ultimately found the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health approving for the first time protocols of this genre. A full appreciation of the policy-making dialogue shows that significant participants perceived the process from very different vantage points regarding the way in which the American political system works and the way in which it ought to work. I argue that, if we are to understand how the RAC should proceed in orchestrating a human gene therapy policy agenda, then we must flesh out and critically analyze these competing vantage points. To that end, I postulate seven possible "action models" for characterizing how protocol assessments of the type at issue might be developed given the nature of our politics, reaching the conclusion that one of these models holds out the most promise for synthesizing efficaciously the key factors involved. In conclusion, I discuss how the RAC might profitably employ this preferred strategy in these and other cases. PMID:1734711

Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination.

PubMed

Shao, Renfu; Barker, Stephen C

2011-02-15

The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse. Copyright © 2010 Elsevier B.V. All rights reserved.

Mesenchymal stem cells protect podocytes from apoptosis induced by high glucose via secretion of epithelial growth factor

PubMed Central

2013-01-01

Introduction The apoptosis and subsequent injury of podocytes plays a pathogenic role in diabetic nephropathy (DN). Mesenchymal stem cells (MSCs) are promising therapeutic cells for preventing apoptosis and reducing cellular injury. Our previous study found that MSCs could protect kidneys from diabetes-induced injury without obvious engraftment. So we evaluated the effects of human adipose-derived MSCs (hAd-MSCs) on podocytic apoptosis and injury induced by high glucose (HG) and the underlying mechanisms. Methods We used flow cytometry, Western blot and confocal fluorescence microscopy to study podocytic apoptosis and injury induced by HG at 24 hours, 48 hours, and 72 hours in the presence or absence of MSC-conditioned medium (CM). An antibody-based cytokine array was used to identify the mediating factor, which was verified by adding the neutralizing antibody (NtAb) to block its function or adding the recombinant cytokine to the medium to induce its function. Results hAd-MSC-CM reduced podocytic apoptosis in a dose-dependent manner, decreased the expression of podocytic cleaved caspase-3, and prevented the reduced expression and maintained the normal arrangement of podocytic synaptopodin and nephrin. However, human embryonic lung cell (Wi38)-CM failed to ameliorate podocytic apoptosis or injury. Twelve cytokines with concentration ratios (MSC-CM/Wi38-CM) >10-fold were identified. Epithelial growth factor (EGF) was singled out for its known ability to prevent apoptosis. Recombinant human EGF (rhEGF) prevented podocytic apoptosis and injury similarly to hAd-MSC-CM but, upon blockade of EGF, the beneficial effect of hAd-MSC-CM decreased dramatically. Conclusions hAd-MSCs prevent podocytic apoptosis and injury induced by HG, mainly through secreting soluble EG. PMID:24004644

Mesenchymal stem cells protect podocytes from apoptosis induced by high glucose via secretion of epithelial growth factor.

PubMed

Li, Diangeng; Wang, Nan; Zhang, Li; Hanyu, Zhu; Xueyuan, Bai; Fu, Bo; Shaoyuan, Cui; Zhang, Weiguang; Xuefeng, Sun; Li, Rongshan; Chen, Xiangmei

2013-01-01

The apoptosis and subsequent injury of podocytes plays a pathogenic role in diabetic nephropathy (DN). Mesenchymal stem cells (MSCs) are promising therapeutic cells for preventing apoptosis and reducing cellular injury. Our previous study found that MSCs could protect kidneys from diabetes-induced injury without obvious engraftment. So we evaluated the effects of human adipose-derived MSCs (hAd-MSCs) on podocytic apoptosis and injury induced by high glucose (HG) and the underlying mechanisms. We used flow cytometry, Western blot and confocal fluorescence microscopy to study podocytic apoptosis and injury induced by HG at 24 hours, 48 hours, and 72 hours in the presence or absence of MSC-conditioned medium (CM). An antibody-based cytokine array was used to identify the mediating factor, which was verified by adding the neutralizing antibody (NtAb) to block its function or adding the recombinant cytokine to the medium to induce its function. hAd-MSC-CM reduced podocytic apoptosis in a dose-dependent manner, decreased the expression of podocytic cleaved caspase-3, and prevented the reduced expression and maintained the normal arrangement of podocytic synaptopodin and nephrin. However, human embryonic lung cell (Wi38)-CM failed to ameliorate podocytic apoptosis or injury. Twelve cytokines with concentration ratios (MSC-CM/Wi38-CM) >10-fold were identified. Epithelial growth factor (EGF) was singled out for its known ability to prevent apoptosis. Recombinant human EGF (rhEGF) prevented podocytic apoptosis and injury similarly to hAd-MSC-CM but, upon blockade of EGF, the beneficial effect of hAd-MSC-CM decreased dramatically. hAd-MSCs prevent podocytic apoptosis and injury induced by HG, mainly through secreting soluble EG.

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

PubMed Central

Dubreuil, Patrice; Letard, Sébastien; Ciufolini, Marco; Gros, Laurent; Humbert, Martine; Castéran, Nathalie; Borge, Laurence; Hajem, Bérengère; Lermet, Anne; Sippl, Wolfgang; Voisset, Edwige; Arock, Michel; Auclair, Christian; Leventhal, Phillip S.; Mansfield, Colin D.; Moussy, Alain; Hermine, Olivier

2009-01-01

Background The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity. PMID:19789626

Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

PubMed

McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

2014-07-01

Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc. © 2014 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

Cysteine proteinase from Streptococcus pyogenes enables evasion of innate immunity via degradation of complement factors.

PubMed

Honda-Ogawa, Mariko; Ogawa, Taiji; Terao, Yutaka; Sumitomo, Tomoko; Nakata, Masanobu; Ikebe, Kazunori; Maeda, Yoshinobu; Kawabata, Shigetada

2013-05-31

Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors and aimed to elucidate the mechanism involved in occurrence of streptococcal toxic shock syndrome from the aspect of the complement system. First, we revealed that culture supernatant of a given S. pyogenes strain and recombinant SpeB degraded the C1-INH. Then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity than normal as compared with heat-inactivated serum, whereas it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of C9, one of the components of membrane the attack complex, in greater amounts on the surface of the speB mutant, whereas lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.

Enhanced Functional Recombinant Factor VII Production by HEK 293 Cells Stably Transfected with VKORC1 where the Gamma-Carboxylase Inhibitor Calumenin is Stably Suppressed by shRNA Transfection

PubMed Central

Wajih, Nadeem; Owen, John; Wallin, Reidar

2008-01-01

Introduction Recombinant members of the vitamin K-dependent protein family (factors IX and VII and protein C) have become important pharmaceuticals in treatment of bleeding disorders and sepsis. However, because the in vivo γ-carboxylation system in stable cell lines used for transfection has a limited capacity of post translational γ carboxylation, the recovery of fully γ-carboxylated and functional proteins is low. Materials and Methods In this work we have engineered recombinant factor VII producing HEK 293 cells to stably overexpress VKORC1, the reduced vitamin K γ-carboxylase cofactor and in addition stably silenced the γ-carboxylase inhibitory protein calumenin. Results and Conclusions Stable cell lines transfected with only a factor VII cDNA had a 9% production of functional recombinant factor VII. On the other hand, these recombinant factor VII producing cells when engineered to overexpress VKORC1 and having calumenin stably suppressed more than 80% by shRNA expression, produced 68% functional factor VII. The technology presented should be applicable to all vertebrae members of the vitamin K-dependent protein family and should lower the production cost of the clinically used factors VII, IX and protein C. PMID:18177690

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

PubMed Central

Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

2016-01-01

Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel.

PubMed

Mazzeffi, Michael; Szlam, Fania; Jakubowski, Joseph A; Tanaka, Kenichi A; Sugidachi, Atsuhiro; Levy, Jerrold H

2013-07-01

Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings. Copyright © 2013 Elsevier Ltd. All rights reserved.

Preparation and characterization of human recombinant protein 1/Clara cell M(r) 10,000 protein.

PubMed

Okutani, R; Itoh, Y; Yamada, T; Yamaguchi, T; Singh, G; Yagisawa, H; Kawai, T

1996-09-01

Protein 1, which is identical to human Clara cell M(r) 10(4) protein, is a homodimeric, low molecular mass protein (M(r) 14,000) and an effective inhibitor of phospholipase A2 activity. We have expressed this protein in E. coli and characterized its physiochemical and biological properties. Using a pET expression system, about 1.7 mg of purified recombinant protein 1 was obtained from 250 ml of E. coli culture. The amino-terminal sequence of recombinant protein 1 up to the 20th residue was identical to that of native protein 1 except for an extra methionine at the amino-terminus. On reversed-phase HPLC, recombinant protein 1 eluted at the same retention time as native protein 1. The dose-response curves of recombinant protein 1 and native protein 1 in an enzyme-linked immunosorbent assay for protein 1 were identical. Recombinant protein 1 inhibited both porcine pancreas and cobra venom phospholipase A2 activities. These results indicated that recombinant protein 1 is structurally and biologically identical to native protein 1. We found that recombinant protein 1 also inhibits phosphatidylinositol-specific phospholipase C activity.

Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History

PubMed Central

Tao, Ying; Shi, Mang; Chommanard, Christina; Queen, Krista; Zhang, Jing; Markotter, Wanda; Kuzmin, Ivan V.; Holmes, Edward C.

2017-01-01

ABSTRACT Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5′ and 3′ ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination “hot spot” in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances. IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential. PMID:28077633

Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

PubMed

Dagar, Vikas Kumar; Adivitiya; Devi, Nirmala; Khasa, Yogender Pal

2016-10-01

Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni-NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % β-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.

[The effect of retrovirus-mediated hTRT transfection into cultured oral keratinocytes].

PubMed

Huang, Ji-yan; Liu, Wei; Zhou, Zeng-tong; Zhou, Hai-wen

2014-06-01

Human telomerase reverse transcriptase (hTRT) was transfected into cultured oral keratinocytes (OKC) mediated by pBABE-tert recombined retrovirus to investigate the effect on OKC lifespan. pBABE-tert recombined retrovirus loaded with hTRT gene was amplified by transfected PT67 cells, and then transfected into cultured OKC in vitro. The positive clones of OKC were separated by puromycin and subcultured. Telomerase activity was analyzed by telomerase PCR-ELISA and PCR-PAGE. The hTRT positive clones of OKC showed telomerase expression, with extending lifespan to 8-9 passages. The hTRT transfected OKC can prolong doubly lifespan but not be immortalized, which indicates that cellular immortality mechanism is complicated and multi-controled. Telomerase activity is the key for cell immortalization but not the only impact factor.

Role of Insulin-Like Growth Factor-1 Signaling Pathway in Cisplatin-Resistant Lung Cancer Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Yunguang; Zheng Siyuan; Torossian, Artour

2012-03-01

Purpose: The development of drug-resistant phenotypes has been a major obstacle to cisplatin use in non-small-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatin resistance using microarray expression analysis. Methods and Materials: H460 cells were treated with cisplatin. The differences between cisplatin-resistant lung cancer cells and parental H460 cells were studied using Western blot, MTS, and clonogenic assays, in vivo tumor implantation, and microarray analysis. The cisplatin-R cells were treated with human recombinant insulin-like growth factor (IGF) binding protein-3 and siRNA targeting IGF-1 receptor. Results: Cisplatin-R cells illustrated greater expression of the markers CD133more » and aldehyde dehydrogenase, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposide-induced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, cisplatin-R demonstrated decreased expression of insulin-like growth factor binding protein-3 and increased activation of IGF-1 receptor signaling compared with parental H460 cells in the presence of IGF-1. Human recombinant IGF binding protein-3 reversed cisplatin resistance in cisplatin-R cells and targeting of IGF-1 receptor using siRNA resulted in sensitization of cisplatin-R-cells to cisplatin and radiation. Conclusions: The IGF-1 signaling pathway contributes to cisplatin-R to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment.« less

Isolated growth hormone deficiency (GHD) in childhood and adolescence: recent advances.

PubMed

Alatzoglou, Kyriaki S; Webb, Emma Alice; Le Tissier, Paul; Dattani, Mehul T

2014-06-01

The diagnosis of GH deficiency (GHD) in childhood is a multistep process involving clinical history, examination with detailed auxology, biochemical testing, and pituitary imaging, with an increasing contribution from genetics in patients with congenital GHD. Our increasing understanding of the factors involved in the development of somatotropes and the dynamic function of the somatotrope network may explain, at least in part, the development and progression of childhood GHD in different age groups. With respect to the genetic etiology of isolated GHD (IGHD), mutations in known genes such as those encoding GH (GH1), GHRH receptor (GHRHR), or transcription factors involved in pituitary development, are identified in a relatively small percentage of patients suggesting the involvement of other, yet unidentified, factors. Genome-wide association studies point toward an increasing number of genes involved in the control of growth, but their role in the etiology of IGHD remains unknown. Despite the many years of research in the area of GHD, there are still controversies on the etiology, diagnosis, and management of IGHD in children. Recent data suggest that childhood IGHD may have a wider impact on the health and neurodevelopment of children, but it is yet unknown to what extent treatment with recombinant human GH can reverse this effect. Finally, the safety of recombinant human GH is currently the subject of much debate and research, and it is clear that long-term controlled studies are needed to clarify the consequences of childhood IGHD and the long-term safety of its treatment.

[Promotive effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on recovery from neutropenia induced by fractionated irradiation in mice].

PubMed

Kabaya, K; Watanabe, M; Kusaka, M; Seki, M; Fushiki, M

1994-08-25

The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the recovery from neutropenia induced by fractionated whole-body irradiation was investigated in mice. Male 7-week old C3H/HeN mice received a total of ten exposures of 0.25 Gy/day from day 1 to 5 and from day 8 to 12. Peripheral neutropenia with a nadir on day 17 was caused by the fractionated irradiation. Daily subcutaneous injections of rhG-CSF at 0.25 and 2.5 micrograms/body/day from day 1 to 21 promoted the recovery of neutrophils in a dose-dependent manner. The kinetics of morphologically identifiable bone marrow cells were studied to clarify the mechanism behind the promotive effect of this factor. A slight decrease in mitotic immature granulocytes, such as myeloblasts, promyelocytes and myelocytes on day 5, and a drastic decrease in metamyelocytes and marrow neutrophils on days 5, 9, and 17 were seen in the femur of irradiated mice. Treatment using rhG-CSF caused an increase in immature granulocytes of all differential stages in the femur. Microscopic findings of the femurs and spleens also revealed an increase in immature granulocytes in these organs in mice injected with rhG-CSF. These results indicate that rhG-CSF accelerates granulopoiesis in the femur and spleen, thereby promoting recovery from neutropenia induced by fractionated irradiation.

Integrated Summary Report: Validation of Two Binding Assays Using Human Recombinant Estrogen Receptor Alpha (hrERa)

EPA Science Inventory

This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (h...

QUANTITATIVE EVALUATION OF BROMODICHLOROMETHANE METABOLISM BY RECOMBINANT RAT AND HUMAN CYTOCHROME P450S

EPA Science Inventory

ABSTRACT
We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. BDCM is a drinking water disinfectant byproduct that has been implicated in liver, kidn...

Establishment and Characterization of an Immortalized Human Hepatic Stellate Cell Line for Applications in Co-Culturing with Immortalized Human Hepatocytes

PubMed Central

Pan, XiaoPing; Wang, Yini; Yu, XiaoPeng; Li, JianZhou; Zhou, Ning; Du, WeiBo; Zhang, YanHong; Cao, HongCui; Zhu, DanHua; Chen, Yu; Li, LanJuan

2015-01-01

Background and objective. The liver-specific functions of hepatocytes are improved by co-culturing hepatocytes with primary hepatic stellate cells (HSC). However, primary HSC have a short lifespan in vitro, which is considered a major limitation for their use in various applications. This study aimed to establish immortalized human HSC using the simian virus 40 large T antigen (SV40LT) for applications in co-culturing with hepatocytes and HSC in vitro. Methods. Primary human HSC were transfected with a recombinant retrovirus containing SV40LT. The immortalized human HSC were characterized by analyzing their gene expression and functional characteristics. The liver-specific functions of hepatocytes were evaluated in a co-culture system incorporating immortalized human hepatocytes with HSC-Li cells. Results. The immortalized HSC line, HSC-Li, was obtained after infection with a recombinant retrovirus containing SV40LT. The HSC-Li cells were longitudinally spindle-like and had numerous fat droplets in their cytoplasm as shown using electron microscopy. Hepatocyte growth factor (HGF), VEGF Receptor 1(Flt-1), collagen type Iα1 and Iα2 mRNA expression levels were observed in the HSC-Li cells by RT-PCR. Immunofluorescence staining showed that the HSC-Li cells were positive for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-beta (PDGFR-β), vimentin, and SV40LT protein expression. The HSC-Li cells produced both HGF and transforming growth factor-beta1 (TGF-β1) in a time-dependent manner. Real-time PCR showed that albumin, CYP3A5, CYP2E1, and UGT2B7 mRNA expression generally increased in the co-culture system. The enzymatic activity of CYP1A2 under the co-culture conditions also generally increased as compared to the monoculture of immortalized human hepatocytes. Conclusions. We successfully established the immortalized human HSC cell line HSC-Li. It has the specific phenotypic and functional characteristics of primary human HSC, which would be a useful tool to develop anti-fibrotic therapies. Co-culturing with the HSC-Li cells improved the liver-specific functions of hepatocytes, which may be valuable and applicable for bioartificial liver systems. PMID:25678842

Effect of summer daylight exposure and genetic background on growth in growth hormone-deficient children.

PubMed

De Leonibus, C; Chatelain, P; Knight, C; Clayton, P; Stevens, A

2016-11-01

The response to growth hormone in humans is dependent on phenotypic, genetic and environmental factors. The present study in children with growth hormone deficiency (GHD) collected worldwide characterised gene-environment interactions on growth response to recombinant human growth hormone (r-hGH). Growth responses in children are linked to latitude, and we found that a correlate of latitude, summer daylight exposure (SDE), was a key environmental factor related to growth response to r-hGH. In turn growth response was determined by an interaction between both SDE and genes known to affect growth response to r-hGH. In addition, analysis of associated networks of gene expression implicated a role for circadian clock pathways and specifically the developmental transcription factor NANOG. This work provides the first observation of gene-environment interactions in children treated with r-hGH.

Effect of summer daylight exposure and genetic background on growth in growth hormone-deficient children

PubMed Central

De Leonibus, C; Chatelain, P; Knight, C; Clayton, P; Stevens, A

2016-01-01

The response to growth hormone in humans is dependent on phenotypic, genetic and environmental factors. The present study in children with growth hormone deficiency (GHD) collected worldwide characterised gene–environment interactions on growth response to recombinant human growth hormone (r-hGH). Growth responses in children are linked to latitude, and we found that a correlate of latitude, summer daylight exposure (SDE), was a key environmental factor related to growth response to r-hGH. In turn growth response was determined by an interaction between both SDE and genes known to affect growth response to r-hGH. In addition, analysis of associated networks of gene expression implicated a role for circadian clock pathways and specifically the developmental transcription factor NANOG. This work provides the first observation of gene–environment interactions in children treated with r-hGH. PMID:26503811

Oral recombinant human or mouse lactoferrin reduces Mycobacterium tuberculosis TDM induced granulomatous lung pathology.

PubMed

Hwang, Shen-An; Kruzel, Marian L; Actor, Jeffrey K

2017-02-01

Trehalose 6'6-dimycolate (TDM) is the most abundant glycolipid on the cell wall of Mycobacterium tuberculosis (MTB). TDM is capable of inducing granulomatous pathology in mouse models that resembles those induced by MTB infection. Using the acute TDM model, this work investigates the effect of recombinant human and mouse lactoferrin to reduce granulomatous pathology. C57BL/6 mice were injected intravenously with TDM at a dose of 25 μg·mouse -1 . At day 4 and 6, recombinant human or mouse lactoferrin (1 mg·(100 μL) -1 ·mouse -1 ) were delivered by gavage. At day 7 after TDM injection, mice were evaluated for lung pathology, cytokine production, and leukocyte populations. Mice given human or mouse lactoferrin had reduced production of IL-12p40 in their lungs. Mouse lactoferrin increased IL-6 and KC (CXCL1) in lung tissue. Increased numbers of macrophages were observed in TDM-injected mice given human or mouse lactoferrin. Granulomatous pathology, composed of mainly migrated leukocytes, was visually reduced in mice that received human or mouse lactoferrin. Quantitation of granulomatous pathology demonstrated a significant decrease in mice given human or mouse lactoferrin compared with TDM control mice. This report is the first to directly compare the immune modulatory effects of both heterologous recombinant human and homologous mouse lactoferrin on the development of TDM-induced granulomas.

Homologous Recombination between Genetically Divergent Campylobacter fetus Lineages Supports Host-Associated Speciation

PubMed Central

Duim, Birgitta; van der Graaf-van Bloois, Linda; Wagenaar, Jaap A; Zomer, Aldert L

2018-01-01

Abstract Homologous recombination is a major driver of bacterial speciation. Genetic divergence and host association are important factors influencing homologous recombination. Here, we study these factors for Campylobacter fetus, which shows a distinct intraspecific host dichotomy. Campylobacter fetus subspecies fetus (Cff) and venerealis are associated with mammals, whereas C. fetus subsp. testudinum (Cft) is associated with reptiles. Recombination between these genetically divergent C. fetus lineages is extremely rare. Previously it was impossible to show whether this barrier to recombination was determined by the differential host preferences, by the genetic divergence between both lineages or by other factors influencing recombination, such as restriction-modification, CRISPR/Cas, and transformation systems. Fortuitously, a distinct C. fetus lineage (ST69) was found, which was highly related to mammal-associated C. fetus, yet isolated from a chelonian. The whole genome sequences of two C. fetus ST69 isolates were compared with those of mammal- and reptile-associated C. fetus strains for phylogenetic and recombination analysis. In total, 5.1–5.5% of the core genome of both ST69 isolates showed signs of recombination. Of the predicted recombination regions, 80.4% were most closely related to Cft, 14.3% to Cff, and 5.6% to C. iguaniorum. Recombination from C. fetus ST69 to Cft was also detected, but to a lesser extent and only in chelonian-associated Cft strains. This study shows that despite substantial genetic divergence no absolute barrier to homologous recombination exists between two distinct C. fetus lineages when occurring in the same host type, which provides valuable insights in bacterial speciation and evolution. PMID:29608720

Recombination gives a new insight in the effective population size and the history of the old world human populations.

PubMed

Melé, Marta; Javed, Asif; Pybus, Marc; Zalloua, Pierre; Haber, Marc; Comas, David; Netea, Mihai G; Balanovsky, Oleg; Balanovska, Elena; Jin, Li; Yang, Yajun; Pitchappan, R M; Arunkumar, G; Parida, Laxmi; Calafell, Francesc; Bertranpetit, Jaume

2012-01-01

The information left by recombination in our genomes can be used to make inferences on our recent evolutionary history. Specifically, the number of past recombination events in a population sample is a function of its effective population size (Ne). We have applied a method, Identifying Recombination in Sequences (IRiS), to detect specific past recombination events in 30 Old World populations to infer their Ne. We have found that sub-Saharan African populations have an Ne that is approximately four times greater than those of non-African populations and that outside of Africa, South Asian populations had the largest Ne. We also observe that the patterns of recombinational diversity of these populations correlate with distance out of Africa if that distance is measured along a path crossing South Arabia. No such correlation is found through a Sinai route, suggesting that anatomically modern humans first left Africa through the Bab-el-Mandeb strait rather than through present Egypt.

[Studies on fermentation conditions and purification of mutant human interleukin-2 expressed in Pichia pastoris].

PubMed

Liu, Yan; Su, Chang; Hu, Ying-He; Ouyang, Ke-Qing; Cai, Shao-Xi

2005-05-01

Interleukin-2 (IL-2) was initially isolated as a T cell growth factor and had been shown to direct the expansion and differentiation of several hematopoietic cell types. Clinical studies using IL-2 in the treatment of AIDS have been encouraging, due to its critical role as a proliferative signal for activated T-lymphocytes. IL-2 has also undergone trials in the treatment of several types of cancer, based on its stimulation of cytotoxic, antitumor cells. Today, human IL-2 is produced completely by genetically engineered method, and it has been proved that genetically engineered recombinant human IL-2 has almost the same function and clinical effect as wild IL-2. In the former study, recombinant human IL-2 usually comes from E. coli, in this paper the mutant IL-2 was successfully expressed and purified in Pichia pastoris for the first time. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression level. Expression conditions of human mutant interleukin-2(the codon for cysteine-125 of human IL-2 with alanine; the codon for leucine-18 with methionine; the codon for leucine-19 with serine) in the recombinant Pichia pastoris strain were optimized via test of some factors such as the rate of aeration, the inductive duration, the initial pH and the concentration of methanol. The results from tests showed that the most important parameter for efficient expression of interleukin-2 in recombinant Pichia pastoris strain is adequate aeration during methanol induction, and the optimum inductive condition for interleukin-2 expression was: more than 80% aeration, 2 days for induction, the initial pH of 6.0, the final methanol concentration of 1.0%. With this condition, the expressed IL-2 was secreted into fermentation broth and reached a yield of 30%, approximately 200 mg/L. Expressed interleutin-2 (MvIL-2) was isolated and purified by centrifugation, millipore filtration to concentration, Econo-PacS strongly acidic cation exchanger cartridge and molecular sieve chromatography and the yield of MvIL-2 was 27%. MvIL-2 was purified to electrophoretic purity by SDS-PAGE and only one peak being loaded on HPLC. Purified MvIL-2 protein had stimulating activity similar to the wild type of IL-2 as assayed by IL-2-dependent CTLL-2 cells. However, the stability of MvIL-2 was superior than that of IL-2 at different temperatures. The activity of obtained MvIL-2 was 4 - 5 times of the wild type of IL-2, So MvIL-2 had an advantage over wild type of rhIL-2 in storage stability and activity.

Recombinant spider silk genetically functionalized with affinity domains.

PubMed

Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

2014-05-12

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

The Effect of Recombinant Activated Factor VII on Mortality in Combat-Related Casualties With Severe Trauma and Massive Transfusion

DTIC Science & Technology

2008-02-01

The Effect of Recombinant Activated Factor VII on Mortality in Combat-Related Casualties With Severe Trauma and Massive Transfusion Philip C...REPORT TYPE 3. DATES COVERED 00-00-2007 to 00-00-2007 4. TITLE AND SUBTITLE The Effect of Recombinant Activated Factor VII on Mortality in Combat...Boffard study,17 the effect of rFVIIa on 24-hour blood product ad- ministration was only determined for patients who lived for at least 24 hours

Leukemia inhibitory factor: a novel bone-active cytokine.

PubMed

Reid, L R; Lowe, C; Cornish, J; Skinner, S J; Hilton, D J; Willson, T A; Gearing, D P; Martin, T J

1990-03-01

A number of cytokines have been found to be potent regulators of bone resorption and to share the properties originally attributed to osteoclast-activating factor. One such activity, differentiation-inducing factor (DIF, D-factor) from mouse spleen cells, shares a number of biological and biochemical properties with the recently characterized and cloned leukemia inhibitory factor (LIF). We have assessed the effects of recombinant LIF on bone resorption and other parameters in neonatal mouse calvaria. Both recombinant murine and human (h) LIFs stimulated 45Ca release from prelabeled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in the number of osteoclasts per mm2 bone. The osteolytic effect of hLIF were blocked by 10(-7) M indomethacin. hLIF also stimulated incorporation of [3H] thymidine into calvaria, but the dose-response relationship was distinct from that for bone resorption, and this effect was not blocked by indomethacin. Similarly, hLIF increased [3H]phenylalanine incorporation into calvaria, and this was also not inhibited by indomethacin. It is concluded that LIF stimulates bone resorption by a mechanism involving prostaglandin production, but that a distinct mechanism is responsible for its stimulation of DNA and protein synthesis. The primary structure of LIF differs from that of other fully characterized, bone-active cytokines, and it, thus, represents a novel factor which may be involved in the normal regulation of bone cell function.

Generation of Recombinant Human AChE OP-Scavengers with Extended Circulatory Longevity

DTIC Science & Technology

2006-11-01

PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Avigdor Shafferman Ph.D. 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail: avigdor...glycans of rHuAChE, and in particular the distal termini of these glycan 8 projections , constitute a major factor in determining the circulatory...experiments funded by sources other than the USAMRMC contract, since they may have a major impact on the present project . Section III focuses on the

Incomplete lineage sorting patterns among human, chimpanzee, and orangutan suggest recent orangutan speciation and widespread selection

PubMed Central

Hobolth, Asger; Dutheil, Julien Y.; Hawks, John; Schierup, Mikkel H.; Mailund, Thomas

2011-01-01

We search the complete orangutan genome for regions where humans are more closely related to orangutans than to chimpanzees due to incomplete lineage sorting (ILS) in the ancestor of human and chimpanzees. The search uses our recently developed coalescent hidden Markov model (HMM) framework. We find ILS present in ∼1% of the genome, and that the ancestral species of human and chimpanzees never experienced a severe population bottleneck. The existence of ILS is validated with simulations, site pattern analysis, and analysis of rare genomic events. The existence of ILS allows us to disentangle the time of isolation of humans and orangutans (the speciation time) from the genetic divergence time, and we find speciation to be as recent as 9–13 million years ago (Mya; contingent on the calibration point). The analyses provide further support for a recent speciation of human and chimpanzee at ∼4 Mya and a diverse ancestor of human and chimpanzee with an effective population size of about 50,000 individuals. Posterior decoding infers ILS for each nucleotide in the genome, and we use this to deduce patterns of selection in the ancestral species. We demonstrate the effect of background selection in the common ancestor of humans and chimpanzees. In agreement with predictions from population genetics, ILS was found to be reduced in exons and gene-dense regions when we control for confounding factors such as GC content and recombination rate. Finally, we find the broad-scale recombination rate to be conserved through the complete ape phylogeny. PMID:21270173

[Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

PubMed

Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

2015-10-01

To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

Early onset of disc degeneration in SM/J mice is associated with changes in ion transport systems and fibrotic events.

PubMed

Zhang, Ying; Xiong, Chi; Kudelko, Mateusz; Li, Yan; Wang, Cheng; Wong, Yuk Lun; Tam, Vivian; Rai, Muhammad Farooq; Cheverud, James; Lawson, Heather A; Sandell, Linda; Chan, Wilson C W; Cheah, Kathryn S E; Sham, Pak C; Chan, Danny

2018-04-09

Intervertebral disc degeneration (IDD) causes back pain and sciatica, affecting quality of life and resulting in high economic/social burden. The etiology of IDD is not well understood. Along with aging and environmental factors, genetic factors also influence the onset, progression and severity of IDD. Genetic studies of risk factors for IDD using human cohorts are limited by small sample size and low statistical power. Animal models amenable to genetic and functional studies of IDD provide desirable alternatives. Despite differences in size and cellular content as compared to human intervertebral discs (IVDs), the mouse is a powerful model for genetics and assessment of cellular changes relevant to human biology. Here, we provide evidence for early onset disc degeneration in SM/J relative to LG/J mice with poor and good tissue healing capacity respectively. In the first few months of life, LG/J mice maintain a relatively constant pool of notochordal-like cells in the nucleus pulposus (NP) of the IVD. In contrast, chondrogenic events are observed in SM/J mice beginning as early as one-week-old, with progressive fibrotic changes. Further, the extracellular matrix changes in the NP are consistent with IVD degeneration. Leveraging on the genomic data of two parental and two recombinant inbred lines, we assessed the genetic contribution to the NP changes and identified processes linked to the regulation of ion transport systems. Significantly, "transport" system is also in the top three gene ontology (GO) terms from a comparative proteomic analysis of the mouse NP. These findings support the potential of the SM/J, LG/J and their recombinant inbred lines for future genetic and biological analysis in mice and validation of candidate genes and biological relevance in human cohort studies. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD008784. Copyright © 2017. Published by Elsevier B.V.

Current ebola vaccines.

PubMed

Hoenen, Thomas; Groseth, Allison; Feldmann, Heinz

2012-07-01

Ebolaviruses cause severe viral hemorrhagic fever in humans and non-human primates (NHPs), with case fatality rates of up to 90%. Currently, neither a specific treatment nor a vaccine licensed for use in humans is available. However, a number of vaccine candidates have been developed in the last decade that are highly protective in NHPs, the gold standard animal model for ebola hemorrhagic fever. This review analyzes a number of scenarios for the use of ebolavirus vaccines, discusses the requirements for ebolavirus vaccines in these scenarios and describes current ebolavirus vaccines. Among these vaccines are recombinant adenoviruses, recombinant vesicular stomatitis viruses (VSVs), recombinant human parainfluenza viruses and virus-like particles. Interestingly, one of these vaccine platforms, based on recombinant VSVs, has also demonstrated post-exposure protection in NHPs. The most pressing remaining challenge is now to move these vaccine candidates forward into human trials and toward licensure. In order to achieve this, it will be necessary to establish the mechanisms and correlates of protection for these vaccines, and to continue to demonstrate their safety, particularly in potentially immunocompromised populations. However, already now there is sufficient evidence that, from a scientific perspective, a vaccine protective against ebolaviruses is possible.

Posterior reversible encephalopathy syndrome (PRES) after granulocyte-colony stimulating factor (G-CSF) therapy: a report of 2 cases.

PubMed

Stübgen, Joerg-Patrick

2012-10-15

Two patients with recurrent lymphoma developed an acute, transient encephalopathy following administration of recombinant human granulocyte-colony stimulating factor (rhG-CSF), filgrastim, in anticipation of leukapheresis for hematopoietic stem cell transplantation. Head magnetic resonance imaging showed evidence of blood-brain barrier (BBB) breakdown, compatible with posterior reversible encephalopathy syndrome (PRES). The proposed pathogenesis of PRES was rhG-CSF-induced neutrophil mobilization and activation with the release of inflammatory mediators, resulting in transient alteration of barrier permeability and capillary leakage. Copyright © 2012 Elsevier B.V. All rights reserved.

Dramatic Differences in Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes

DTIC Science & Technology

2009-01-01

Literature 3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE Dramatic Differences in Organophosphorus Hydrolase Activity between Human and 5a... activity , V-agents, VX, bioscavenger, medical countermeasures 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES...Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes† Tamara C. Otto,‡ Christina K. Harsch,§ David T

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

PubMed

Lennon, V A; Lambert, E H; Leiby, K R; Okarma, T B; Talib, S

1991-04-01

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

Designing ternary blend bulk heterojunction solar cells with reduced carrier recombination and a fill factor of 77%

NASA Astrophysics Data System (ADS)

Gasparini, Nicola; Jiao, Xuechen; Heumueller, Thomas; Baran, Derya; Matt, Gebhard J.; Fladischer, Stefanie; Spiecker, Erdmann; Ade, Harald; Brabec, Christoph J.; Ameri, Tayebeh

2016-09-01

In recent years the concept of ternary blend bulk heterojunction (BHJ) solar cells based on organic semiconductors has been widely used to achieve a better match to the solar irradiance spectrum, and power conversion efficiencies beyond 10% have been reported. However, the fill factor of organic solar cells is still limited by the competition between recombination and extraction of free charges. Here, we design advanced material composites leading to a high fill factor of 77% in ternary blends, thus demonstrating how the recombination thresholds can be overcome. Extending beyond the typical sensitization concept, we add a highly ordered polymer that, in addition to enhanced absorption, overcomes limits predicted by classical recombination models. An effective charge transfer from the disordered host system onto the highly ordered sensitizer effectively avoids traps of the host matrix and features an almost ideal recombination behaviour.

Competition between recombination and extraction of free charges determines the fill factor of organic solar cells

PubMed Central

Bartesaghi, Davide; Pérez, Irene del Carmen; Kniepert, Juliane; Roland, Steffen; Turbiez, Mathieu; Neher, Dieter; Koster, L. Jan Anton

2015-01-01

Among the parameters that characterize a solar cell and define its power-conversion efficiency, the fill factor is the least well understood, making targeted improvements difficult. Here we quantify the competition between charge extraction and recombination by using a single parameter θ, and we demonstrate that this parameter is directly related to the fill factor of many different bulk-heterojunction solar cells. Our finding is supported by experimental measurements on 15 different donor:acceptor combinations, as well as by drift-diffusion simulations of organic solar cells in which charge-carrier mobilities, recombination rate, light intensity, energy levels and active-layer thickness are all varied over wide ranges to reproduce typical experimental conditions. The results unify the fill factors of several very different donor:acceptor combinations and give insight into why fill factors change so much with thickness, light intensity and materials properties. To achieve fill factors larger than 0.8 requires further improvements in charge transport while reducing recombination. PMID:25947637

Elevated plasma factor VIII enhances venous thrombus formation in rabbits: contribution of factor XI, von Willebrand factor and tissue factor.

PubMed

Sugita, Chihiro; Yamashita, Atsushi; Matsuura, Yunosuke; Iwakiri, Takashi; Okuyama, Nozomi; Matsuda, Shuntaro; Matsumoto, Tomoko; Inoue, Osamu; Harada, Aya; Kitazawa, Takehisa; Hattori, Kunihiro; Shima, Midori; Asada, Yujiro

2013-07-01

Elevated plasma levels of factor VIII (FVIII) are associated with increased risk of deep venous thrombosis. The aim of this study is to elucidate how elevated FVIII levels affect venous thrombus formation and propagation in vivo. We examined rabbit plasma FVIII activity, plasma thrombin generation, whole blood coagulation, platelet aggregation and venous wall thrombogenicity before and one hour after an intravenous infusion of recombinant human FVIII (rFVIII). Venous thrombus induced by the endothelial denudation of rabbit jugular veins was histologically assessed. Thrombus propagation was evaluated as indocyanine green fluorescence intensity. Argatroban, a thrombin inhibitor, and neutralised antibodies for tissue factor (TF), factor XI (FXI), and von Willebrand factor (VWF) were infused before or after thrombus induction to investigate their effects on venous thrombus formation or propagation. Recombinant FVIII (100 IU/kg) increased rabbit plasma FVIII activity two-fold and significantly enhanced whole blood coagulation and total plasma thrombin generation, but did not affect initial thrombin generation time, platelet aggregation and venous wall thrombogenicity. The rFVIII infusion also increased the size of venous thrombus 1 hour after thrombus induction. Argatroban and the antibodies for TF, FXI or VWF inhibited such enhanced thrombus formation and all except TF suppressed thrombus propagation. In conclusion, elevated plasma FVIII levels enhance venous thrombus formation and propagation. Excess thrombin generation by FXI and VWF-mediated FVIII recruitment appear to contribute to the growth of FVIII-driven venous thrombus.

Efficacy and safety of BAY 81-8973, a full-length recombinant factor VIII: results from the LEOPOLD I trial.

PubMed

Saxena, K; Lalezari, S; Oldenburg, J; Tseneklidou-Stoeter, D; Beckmann, H; Yoon, M; Maas Enriquez, M

2016-09-01

BAY 81-8973 (Kovaltry(®) ) is a full-length, unmodified recombinant human factor VIII (FVIII) with the same amino acid sequence as sucrose-formulated recombinant FVIII and is produced using additional advanced manufacturing technologies. To demonstrate efficacy and safety of BAY 81-8973 for treatment of bleeds and as prophylaxis based on two different potency assignments. In LEOPOLD I (ClinicalTrials.gov identifier, NCT01029340), males aged 12-65 years with severe haemophilia A and ≥150 exposure days received BAY 81-8973 20-50 IU kg(-1) two or three times per week for 12 months. Potency was based on chromogenic substrate assay per European Pharmacopoeia and label adjusted to mimic one-stage assay potency. Patients were randomized for potency sequence and crossed over potency groups after 6 months, followed by an optional 12-month extension. Primary efficacy endpoint was annualized bleeding rate (ABR). Patients also received BAY 81-8973 during major surgeries. Sixty-two patients received BAY 81-8973 prophylaxis and were included in the analysis. Median ABR was 1.0 (quartile 1, 0; quartile 3, 5.1) without clinically relevant differences between potency periods. Median ABR was similar for twice-weekly vs. three times-weekly dosing (1.0 vs. 2.0). Haemostasis was maintained during 12 major surgeries. Treatment-related adverse event (AE) incidence was ≤7% overall; no patient developed inhibitors. One patient with risk factors for cardiovascular disease developed a myocardial infarction. BAY 81-8973 was efficacious in preventing and treating bleeding episodes, irrespective of the potency assignment method, with few treatment-related AEs. Caution should be used when treating older patients with cardiovascular risk factors. © 2016 Bayer. Haemophilia Published by John Wiley & Sons Ltd.

A glycosylated recombinant human granulocyte colony stimulating factor produced in a novel protein production system (AVI-014) in healthy subjects: a first-in human, single dose, controlled study

PubMed Central

Varki, Roslyn; Pequignot, Ed; Leavitt, Mark C; Ferber, Andres; Kraft, Walter K

2009-01-01

Background AVI-014 is an egg white-derived, recombinant, human granulocyte colony-stimulating factor (G-CSF). This healthy volunteer study is the first human investigation of AVI-014. Methods 24 male and female subjects received a single subcutaneous injection of AVI-014 at 4 or 8 mcg/kg. 16 control subjects received 4 or 8 mcg/kg of filgrastim (Neupogen, Amgen) in a partially blinded, parallel fashion. Results The Geometric Mean Ratio (GMR) (90% CI) of 4 mcg/kg AVI-014/filgrastim AUC(0–72 hr) was 1.00 (0.76, 1.31) and Cmax was 0.86 (0.66, 1.13). At the 8 mcg/kg dose, the AUC(0–72) GMR was 0.89 (0.69, 1.14) and Cmax was 0.76 (0.58, 0.98). A priori pharmacokinetic bioequivalence was defined as the 90% CI of the GMR bounded by 0.8–1.25. Both the white blood cell and absolute neutrophil count area under the % increase curve AUC(0–9 days) and Cmax (maximal % increase from baseline)GMR at 4 and 8 mcg/kg fell within the 0.5–2.0 a priori bound set for pharmacodynamic bioequivalence. The CD 34+ % increase curve AUC(0–9 days) and Cmax GMR for both doses was ~1, but 90% confidence intervals were large due to inherent variance, and this measure did not meet pharmacodynamic bioequivalence. AVI-014 demonstrated a side effect profile similar to that of filgrastim. Conclusion AVI-014 has safety, pharmacokinetic, and pharmacodynamic properties comparable to filgrastim at an equal dose in healthy volunteers. These findings support further investigation in AVI-014. PMID:19175929

Inductive potential of recombinant human granulocyte colony-stimulating factor to mature neutrophils from x-irradiated human peripheral blood hematopoietic progenitor cells.

PubMed

Katsumori, Takeo; Yoshino, Hironori; Hayashi, Masako; Takahashi, Kenji; Kashiwakura, Ikuo

2009-11-01

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been used for treatment of neutropenia. Filgrastim, Nartograstim, and Lenograstim are clinically available in Japan. However, the differences in potential benefit for radiation-induced disorder between these types of rhG-CSFs remain unknown. Therefore, the effects of three different types of rhG-CSFs on granulocyte progenitor cells and expansion of neutrophils from nonirradiated or 2 Gy X-irradiated human CD34+ hematopoietic progenitor cells were examined. For analysis of granulocyte colony-forming units (CFU-G) and a surviving fraction of CFU-G, nonirradiated or X-irradiated CD34+ cells were cultured in methylcellulose containing rhG-CSF. These cells were cultured in serum-free medium supplemented with rhG-CSF, and the expansion and characteristics of neutrophils were analyzed. All three types of rhG-CSFs increased the number of CFU-G in a dose-dependent manner; however, Lenograstim is superior to others because of CFU-G-derived colony formation at relatively low doses. The surviving fraction of CFU-G was independent of the types of rhG-CSFs. Expansion of neutrophils by rhG-CSF was largely attenuated by X-irradiation, though no significant difference in neutrophil number was observed between the three types of rhG-CSFs under both nonirradiation and X-irradiation conditions. In terms of functional characteristics of neutrophils, Lenograstim-induced neutrophils produced high levels of reactive oxygen species compared to Filgrastim, when rhG-CSF was applied to nonirradiated CD34(+) cells. In conclusion, different types of rhG-CSFs lead to different effects when rhG-CSF is applied to nonirradiated CD34+ cells, though Filgrastim, Nartograstim, and Lenograstim show equal effects on X-irradiated CD34+ cells.

Application of the relative activity factor approach in scaling from heterologously expressed cytochromes p450 to human liver microsomes: studies on amitriptyline as a model substrate.

PubMed

Venkatakrishnan, K; von Moltke, L L; Greenblatt, D J

2001-04-01

The relative activity factor (RAF) approach is being increasingly used in the quantitative phenotyping of multienzyme drug biotransformations. Using lymphoblast-expressed cytochromes P450 (CYPs) and the tricyclic antidepressant amitriptyline as a model substrate, we have tested the hypothesis that the human liver microsomal rates of a biotransformation mediated by multiple CYP isoforms can be mathematically reconstructed from the rates of the biotransformation catalyzed by individual recombinant CYPs using the RAF approach, and that the RAF approach can be used for the in vitro-in vivo scaling of pharmacokinetic clearance from in vitro intrinsic clearance measurements in heterologous expression systems. In addition, we have compared the results of two widely used methods of quantitative reaction phenotyping, namely, chemical inhibition studies and the prediction of relative contributions of individual CYP isoforms using the RAF approach. For the pathways of N-demethylation (mediated by CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and E-10 hydroxylation (mediated by CYPs 2B6, 2D6, and 3A4), the model-predicted biotransformation rates in microsomes from a panel of 12 human livers determined from enzyme kinetic parameters of the recombinant CYPs were similar to, and correlated with the observed rates. The model-predicted clearance via N-demethylation was 53% lower than the previously reported in vivo pharmacokinetic estimates. Model-predicted relative contributions of individual CYP isoforms to the net biotransformation rate were similar to, and correlated with the fractional decrement in human liver microsomal reaction rates by chemical inhibitors of the respective CYPs, provided the chemical inhibitors used were specific to their target CYP isoforms.

Inhibition of Human MCF-7 Breast Cancer Cells and HT-29 Colon Cancer Cells by Rice-Produced Recombinant Human Insulin-Like Growth Binding Protein-3 (rhIGFBP-3)

PubMed Central

Liu, Lizhong; Liu, Qiaoquan; Lan, Linlin; Tong, Peter C. Y.; Sun, Samuel S. M.

2013-01-01

Background Insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I) to form a complex (IGF-I/IGFBP-3) that can treat growth hormone insensitivity syndrome (GHIS) and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells. Methodology/Principal Findings We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3) in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL) were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 ± 1.72% vs 45.00 ± 0.86%, p < 0.05; 50.84 ± 1.97% vs 45.00 ± 0.86%, p < 0.01 respectively) and HT-29 colon cancer cells (65.14 ±3.84% vs 18.01 ± 13.81%, p < 0.05 and 54.7 ± 9.44% vs 18.01 ± 13.81%, p < 0.05 respectively) when compared with wild type rice. Conclusion/Significance These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future. PMID:24143239

Characterization of diverse subvariants of the meningococcal factor H (fH) binding protein for their ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies.

PubMed

Seib, Kate L; Brunelli, Brunella; Brogioni, Barbara; Palumbo, Emmanuelle; Bambini, Stefania; Muzzi, Alessandro; DiMarcello, Federica; Marchi, Sara; van der Ende, Arie; Aricó, Beatrice; Savino, Silvana; Scarselli, Maria; Comanducci, Maurizio; Rappuoli, Rino; Giuliani, Marzia M; Pizza, Mariagrazia

2011-02-01

Neisseria meningitidis is a commensal of the human nasopharynx but is also a major cause of septicemia and meningitis. The meningococcal factor H binding protein (fHbp) binds human factor H (fH), enabling downregulation of complement activation on the bacterial surface. fHbp is a component of two serogroup B meningococcal vaccines currently in clinical development. Here we characterize 12 fHbp subvariants for their level of surface exposure and ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies. Flow cytometry and Western analysis revealed that all strains examined expressed fHbp on their surface to different extents and bound fH in an fHbp-dependent manner. However, differences in fH binding did not always correlate with the level of fHbp expression, indicating that this is not the only factor affecting the amount of fH bound. To overcome the issue of strain variability in fHbp expression, the MC58ΔfHbp strain was genetically engineered to express different subvariants from a constitutive heterologous promoter. These recombinant strains were characterized for fH binding, and the data confirmed that each subvariant binds different levels of fH. Surface plasmon resonance revealed differences in the stability of the fHbp-fH complexes that ranged over 2 orders of magnitude, indicating that differences in residues between and within variant groups can influence fH binding. Interestingly, the level of survival in human sera of recombinant MC58 strains expressing diverse subvariants did not correlate with the level of fH binding, suggesting that the interaction of fHbp with fH is not the only function of fHbp that influences serum resistance. Furthermore, cross-reactive bactericidal activity was seen within each variant group, although the degree of activity varied, suggesting that amino acid differences within each variant group influence the bactericidal antibody response.

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

PubMed

Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

2017-11-26

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

The role of recombination in the origin and evolution of Alu subfamilies.

PubMed

Teixeira-Silva, Ana; Silva, Raquel M; Carneiro, João; Amorim, António; Azevedo, Luísa

2013-01-01

Alus are the most abundant and successful short interspersed nuclear elements found in primate genomes. In humans, they represent about 10% of the genome, although few are retrotransposition-competent and are clustered into subfamilies according to the source gene from which they evolved. Recombination between them can lead to genomic rearrangements of clinical and evolutionary significance. In this study, we have addressed the role of recombination in the origin of chimeric Alu source genes by the analysis of all known consensus sequences of human Alus. From the allelic diversity of Alu consensus sequences, validated in extant elements resulting from whole genome searches, distinct events of recombination were detected in the origin of particular subfamilies of AluS and AluY source genes. These results demonstrate that at least two subfamilies are likely to have emerged from ectopic Alu-Alu recombination, which stimulates further research regarding the potential of chimeric active Alus to punctuate the genome.

Oocyte-somatic cell interactions in the human ovary-novel role of bone morphogenetic proteins and growth differentiation factors.

PubMed

Chang, Hsun-Ming; Qiao, Jie; Leung, Peter C K

2016-12-01

Initially identified for their capability to induce heterotopic bone formation, bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor β superfamily. Using cellular and molecular genetic approaches, recent studies have implicated intra-ovarian BMPs as potent regulators of ovarian follicular function. The bi-directional communication of oocytes and the surrounding somatic cells is mandatory for normal follicle development and oocyte maturation. This review summarizes the current knowledge on the physiological role and molecular determinants of these ovarian regulatory factors within the human germline-somatic regulatory loop. The regulation of ovarian function remains poorly characterized in humans because, while the fundamental process of follicular development and oocyte maturation is highly similar across species, most information on the regulation of ovarian function is obtained from studies using rodent models. Thus, this review focuses on the studies that used human biological materials to gain knowledge about human ovarian biology and disorders and to develop strategies for preventing, diagnosing and treating these abnormalities. Relevant English-language publications describing the roles of BMPs or growth differentiation factors (GDFs) in human ovarian biology and phenotypes were comprehensively searched using PubMed and the Google Scholar database. The publications included those published since the initial identification of BMPs in the mammalian ovary in 1999 through July 2016. Studies using human biological materials have revealed the expression of BMPs, GDFs and their putative receptors as well as their molecular signaling in the fundamental cells (oocyte, cumulus/granulosa cells (GCs) and theca/stroma cells) of the ovarian follicles throughout follicle development. With the availability of recombinant human BMPs/GDFs and the development of immortalized human cell lines, functional studies have demonstrated the physiological role of intra-ovarian BMPs/GDFs in all aspects of ovarian functions, from follicle development to steroidogenesis, cell-cell communication, oocyte maturation, ovulation and luteal function. Furthermore, there is crosstalk between these potent ovarian regulators and the endocrine signaling system. Dysregulation or naturally occurring mutations within the BMP system may lead to several female reproductive diseases. The latest development of recombinant BMPs, synthetic BMP inhibitors, gene therapy and tools for BMP-ligand sequestration has made the BMP pathway a potential therapeutic target in certain human fertility disorders; however, further clinical trials are needed. Recent studies have indicated that GDF8 is an intra-ovarian factor that may play a novel role in regulating ovarian functions in the human ovary. Intra-ovarian BMPs/GDFs are critical regulators of folliculogenesis and human ovarian functions. Any dysregulation or variations in these ligands or their receptors may affect the related intracellular signaling and influence ovarian functions, which accounts for several reproductive pathologies and infertility. Understanding the normal and pathological roles of intra-ovarian BMPs/GDFs, especially as related to GC functions and follicular fluid levels, will inform innovative approaches to fertility regulation and improve the diagnosis and treatment of ovarian disorders. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

PubMed

Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

The yeast Fun30 and human SMARCAD1 chromatin remodelers promote DNA end resection

PubMed Central

Costelloe, Thomas; Louge, Raphaël; Tomimatsu, Nozomi; Mukherjee, Bipasha; Martini, Emmanuelle; Khadaroo, Basheer; Dubois, Kenny; Wiegant, Wouter W.; Thierry, Agnès; Burma, Sandeep; van Attikum, Haico; Llorente, Bertrand

2012-01-01

Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5′-3′ degradation of DSB ends that yields 3′ single-stranded DNA (ssDNA) required for loading of checkpoint and recombination proteins. The Mre11-Rad50-Xrs2/NBS1 complex and Sae2/CtIP initiate end resection while long-range resection depends on the exonuclease Exo1 or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 with the endonuclease Dna21-6. DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood7. Here, we show that the yeast S. cerevisiae Fun30 protein and its human counterpart SMARCAD18, two poorly characterized ATP-dependent chromatin remodelers of the Snf2 ATPase family, are novel factors that are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates repair of camptothecin (CPT)-induced DNA lesions, and it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs and the kinetics of recruitment is similar to that of Exo1. Loss of SMARCAD1 impairs end resection, recombinational DNA repair and renders cells hypersensitive to DNA damage resulting from CPT or PARP inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodelers in controlling end resection, homologous recombination and genome stability in the context of chromatin. PMID:22960744

Cell-based delivery of glucagon-like peptide-1 using encapsulated mesenchymal stem cells.

PubMed

Wallrapp, Christine; Thoenes, Eric; Thürmer, Frank; Jork, Anette; Kassem, Moustapha; Geigle, Peter

2013-01-01

Glucagon-like peptide-1 (GLP-1) CellBeads are cell-based implants for the sustained local delivery of bioactive factors. They consist of GLP-1 secreting mesenchymal stem cells encapsulated in a spherically shaped immuno-isolating alginate matrix. A highly standardized and reproducible encapsulation method is described for the manufacturing of homogeneous CellBeads. Viability and sustained secretion was shown for the recombinant GLP-1 and the cell endogenous bioactive factors like vascular endothelial growth factor, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor. Manufacturing and quality control is performed in compliance with good manufacturing practice and fulfils all regulatory requirements for human clinical use. GLP-1 CellBeads combine the neuro- and cardioprotective properties of both GLP-1 and mesenchymal stem cells. First promising results were obtained from preclinical studies and an ongoing safety trial in humans but further studies have to prove the overall potential of CellBead technology in cell-based regenerative medicine.

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

PubMed

Shcherbakov, Victor P; Kudryashova, Elena

2014-09-01

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i×j) and three-factor (i k×j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1×i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1-i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1×i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1×i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII(+) recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3-8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants. Copyright © 2014 Elsevier B.V. All rights reserved.

Development, Validation, and Application of a Novel Ligand-Binding Assay to Selectively Measure PEGylated Recombinant Human Coagulation Factor VIII (BAX 855).

PubMed

Weber, Alfred; Engelmaier, Andrea; Hainzelmayer, Sandra; Minibeck, Eva; Anderle, Heinz; Schwarz, Hans Peter; Turecek, Peter L

2015-10-21

BAX 855 is a PEGylated recombinant factor VIII preparation that showed prolonged circulatory half-life in nonclinical and clinical studies. This paper describes the development, validation, and application of a novel ligand-binding assay (LBA) to selectively measure BAX 855 in plasma. The LBA is based on PEG-specific capture of BAX 855, followed by immunological factor VIII (FVIII)-specific detection of the antibody-bound BAX 855. This assay principle enabled sensitive measurement of BAX 855 down to the low nanomolar range without interference from non-PEGylated FVIII as demonstrated by validation data for plasma from animals typically used for nonclinical characterization of FVIII. The selectivity of an in-house-developed anti-PEG and a commercially available preparation, shown by competition studies to primarily target the terminating methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, this new LBA adds to the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. The feasibility and convenience of using this method was demonstrated during extensive nonclinical characterization of BAX 855.

Recombinant factor VIIa treatment for asymptomatic factor VII deficient patients going through major surgery.

PubMed

Livnat, Tami; Shenkman, Boris; Spectre, Galia; Tamarin, Ilia; Dardik, Rima; Israeli, Amnon; Rivkind, Avraham; Shabtai, Moshe; Marinowitz, Uri; Salomon, Ophira

2012-07-01

Factor VII deficiency is the most common among the rare autosomal recessive coagulation disorders worldwide. In factor VII deficient patients, the severity and clinical manifestations cannot be reliably determined by factor VII levels. Severe bleeding tends to occur in individuals with factor VII activity levels of 2% or less of normal. Patients with 2-10% factor VII vary between asymptomatic to severe life threatening haemorrhages behaviour. Recombinant factor VIIa (rFVIIa) is the most common replacement therapy for congenital factor VII deficiency. However, unlike haemophilia patients for whom treatment protocols are straight forward, in asymptomatic factor VII deficiency patients it is still debatable. In this study, we demonstrate that a single and very low dose of recombinant factor VIIa enabled asymptomatic patients with factor VII deficiency to go through major surgery safely. This suggestion was also supported by thrombin generation, as well as by thromboelastometry.

Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives

PubMed Central

Bujarski, Jozef J.

2013-01-01

RNA recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA–RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings) along with non-replicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (1) How various factors modulate the ability of viral replicase to switch templates, (2) What is the intracellular location of RNA–RNA template switchings, (3) Mechanisms and factors responsible for non-replicative RNA recombination, (4) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (5) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented. PMID:23533000

Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives.

PubMed

Bujarski, Jozef J

2013-01-01

RNA recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings) along with non-replicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (1) How various factors modulate the ability of viral replicase to switch templates, (2) What is the intracellular location of RNA-RNA template switchings, (3) Mechanisms and factors responsible for non-replicative RNA recombination, (4) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (5) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

Organization and roles of nucleosomes at mouse meiotic recombination hotspots

PubMed Central

Getun, Irina V.; Wu, Zhen K.; Bois, Philippe R.J.

2012-01-01

Meiotic double strand breaks (DSBs) occur at discrete regions in the genome coined hotspots. Precisely what directs site selection of these DSBs is hotly debated and in particular it is unclear which chromatin features, and regulatory factors are necessary for a genomic region to initiate and resolve DSBs as a crossover (CO) event. In human and mouse, one layer of hotspot selection control is a recognition sequence element present at these sites that is bound by the Prdm9 zinc-finger protein. Furthermore, an overall open chromatin structure is thought to be required to allow access of the recombination machinery, and this is often dictated by the packaging of DNA around nucleosomes. We recently defined the nucleosome occupancy maps of four mouse recombination hotspots throughout meiosis. These analyses revealed no obvious dynamic changes in nucleosome occupancy, suggesting an intrinsic nature of recombinogenic sites, yet they also revealed that nucleosomes define zones of exclusion for CO resolution. Here, we discuss new evidence implicating nucleosome occupancy in recombinogenic repair and its potential roles in controlling chromatin structure at mouse meiotic hotspots. PMID:22572955

Analysis of Pressure Variations in a Low-Pressure Nickel-Hydrogen Battery – Part 1

PubMed Central

Purushothaman, B. K.; Wainright, J. S.

2012-01-01

A low pressure nickel-hydrogen battery using either a metal hydride or gaseous hydrogen for H2 storage has been developed for use in implantable neuroprosthetic devices. In this paper, pressure variations inside the cell for the gaseous hydrogen version are analyzed and correlated with oxygen evolution side reaction at the end of charging, the recombination of oxygen with hydrogen during charging and a subsequent rest period, and the self-discharge of the nickel electrode. About 70% of the recombination occurred simultaneously with oxygen evolution during charging and the remaining oxygen recombined with hydrogen during the 1st hour after charging. Self-discharge of the cell varies linearly with hydrogen pressure at a given state of charge and increased with increasing battery charge levels. The coulometric efficiency calculated based on analysis of the pressure-time data agreed well with the efficiency calculated based on the current-time data. Pressure variations in the battery are simulated accurately to predict coulometric efficiency and the state of charge of the cell, factors of extreme importance for a battery intended for implantation within the human body. PMID:22423175

Analysis of Pressure Variations in a Low-Pressure Nickel-Hydrogen Battery - Part 1.

PubMed

Purushothaman, B K; Wainright, J S

2012-05-15

A low pressure nickel-hydrogen battery using either a metal hydride or gaseous hydrogen for H(2) storage has been developed for use in implantable neuroprosthetic devices. In this paper, pressure variations inside the cell for the gaseous hydrogen version are analyzed and correlated with oxygen evolution side reaction at the end of charging, the recombination of oxygen with hydrogen during charging and a subsequent rest period, and the self-discharge of the nickel electrode. About 70% of the recombination occurred simultaneously with oxygen evolution during charging and the remaining oxygen recombined with hydrogen during the 1(st) hour after charging. Self-discharge of the cell varies linearly with hydrogen pressure at a given state of charge and increased with increasing battery charge levels. The coulometric efficiency calculated based on analysis of the pressure-time data agreed well with the efficiency calculated based on the current-time data. Pressure variations in the battery are simulated accurately to predict coulometric efficiency and the state of charge of the cell, factors of extreme importance for a battery intended for implantation within the human body.

Hematologic improvement in dogs with parvovirus infection treated with recombinant canine granulocyte-colony stimulating factor.

PubMed

Duffy, A; Dow, S; Ogilvie, G; Rao, S; Hackett, T

2010-08-01

Previously, dogs with canine parvovirus-induced neutropenia have not responded to treatment with recombinant human granulocyte-colony stimulating factor (rhG-CSF). However, recombinant canine G-CSF (rcG-CSF) has not been previously evaluated for treatment of parvovirus-induced neutropenia in dogs. We assessed the effectiveness of rcG-CSF in dogs with parvovirus-induced neutropenia with a prospective, open-label, nonrandomized clinical trial. Endpoints of our study were time to recovery of WBC and neutrophil counts, and duration of hospitalization. 28 dogs with parvovirus and neutropenia were treated with rcG-CSF and outcomes were compared to those of 34 dogs with parvovirus and neutropenia not treated with rcG-CSF. We found that mean WBC and neutrophil counts were significantly higher (P < 0.05) in the 28 dogs treated with rcG-CSF compared to disease-matched dogs not treated with rcG-CSF. In addition, the mean duration of hospitalization was reduced (P = 0.01) in rcG-CSF treated dogs compared to untreated dogs. However, survival times were decreased in dogs treated with rcG-CSF compared to untreated dogs. These results suggest that treatment with rcG-CSF was effective in stimulating neutrophil recovery and shortening the duration of hospitalization in dogs with parvovirus infection, but indicate the need for additional studies to evaluate overall safety of the treatment.

Internalization and Recycling of the HER2 Receptor on Human Breast Adenocarcinoma Cells Treated with Targeted Phototoxic Protein DARPinminiSOG

PubMed Central

Shilova, O. N.; Proshkina, G. M.; Lebedenko, E. N.; Deyev, S. M.

2015-01-01

Design and evaluation of new high-affinity protein compounds that can selectively and efficiently destroy human cancer cells are a priority research area in biomedicine. In this study we report on the ability of the recombinant phototoxic protein DARPin-miniSOG to interact with breast adenacarcinoma human cells overexpressing the extracellular domain of human epidermal growth factor receptor 2 (HER2). It was found that the targeted phototoxin DARPin-miniSOG specifically binds to the HER2 with following internalization and slow recycling back to the cell membrane. An insight into the role of DARPin-miniSOG in HER2 internalization could contribute to the treatment of HER2-positive cancer using this phototoxic protein. PMID:26483969

Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant E. coli.

PubMed

Fakruddin, Md; Mohammad Mazumdar, Reaz; Bin Mannan, Khanjada Shahnewaj; Chowdhury, Abhijit; Hossain, Md Nur

2013-01-01

E. coli is the most frequently used host for production of enzymes and other proteins by recombinant DNA technology. E. coli is preferable for its relative simplicity, inexpensive and fast high-density cultivation, well-known genetics, and large number of compatible molecular tools available. Despite all these advantages, expression and production of recombinant enzymes are not always successful and often result in insoluble and nonfunctional proteins. There are many factors that affect the success of cloning, expression, and mass production of enzymes by recombinant E. coli. In this paper, these critical factors and approaches to overcome these obstacles are summarized focusing controlled expression of target protein/enzyme in an unmodified form at industrial level.

Role of tumor necrosis factor in macrophage leishmanicidal activity in vitro and resistance to cutaneous leishmaniasis in vivo.

PubMed Central

Theodos, C M; Povinelli, L; Molina, R; Sherry, B; Titus, R G

1991-01-01

Recombinant human tumor necrosis factor (TNF) and purified murine TNF were both able to activate macrophages to destroy intracellular Leishmania major in vitro. In addition, parasitizing macrophages with L. major markedly increased the ability of the cells to produce TNF. Finally, when mice were vaccinated with an avirulent form of L. major, the animals produced large amounts of TNF but no gamma interferon in response to infection with virulent L. major. Treating these mice with a neutralizing anti-TNF antibody led to partial but not complete inhibition of the resistant state, which suggests that factors other than TNF and gamma interferon contribute to resistance to L. major. PMID:1906844

Repression of the DCL2 and DCL4 genes in Nicotiana benthamiana plants for the transient expression of recombinant proteins.

PubMed

Matsuo, Kouki; Matsumura, Takeshi

2017-08-01

The production of recombinant proteins in plants has many advantages, including safety and reduced costs. However, this technology still faces several issues, including low levels of production. The repression of RNA silencing seems to be particularly important for improving recombinant protein production because RNA silencing effectively degrades transgene-derived mRNAs in plant cells. Therefore, to overcome this, we used RNA interference technology to develop DCL2- and DCL4-repressed transgenic Nicotiana benthamiana plants (ΔD2, ΔD4, and ΔD2ΔD4 plants), which had much lower levels of NbDCL2 and/or NbDCL4 mRNAs than wild-type plants. A transient gene expression assay showed that the ΔD2ΔD4 plants accumulated larger amounts of green fluorescent protein (GFP) and human acidic fibroblast growth factor (aFGF) than ΔD2, ΔD4, and wild-type plants. Furthermore, the levels of GFP and aFGF mRNAs were also higher in ΔD2ΔD4 plants than in ΔD2, ΔD4, and wild-type plants. These findings demonstrate that ΔD2ΔD4 plants express larger amounts of recombinant proteins than wild-type plants, and so would be useful for recombinant protein production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

PubMed

Knoll, Alexander; Higgins, James D; Seeliger, Katharina; Reha, Sarah J; Dangel, Natalie J; Bauknecht, Markus; Schröpfer, Susan; Franklin, F Christopher H; Puchta, Holger

2012-04-01

The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

Molecular Cloning and Characterization of Growth Factor Receptor Bound-Protein in Clonorchis sinensis

PubMed Central

Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong

2014-01-01

Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892

Diminished survival of human cytotrophoblast cells exposed to hypoxia/reoxygenation injury and associated reduction of heparin-binding epidermal growth factor-like growth factor.

PubMed

Leach, Richard E; Kilburn, Brian A; Petkova, Anelia; Romero, Roberto; Armant, D Randall

2008-04-01

The antiapoptotic action of heparin-binding epidermal growth factor (HBEGF)-like growth factor and its regulation by O(2) constitutes a key factor for trophoblast survival. The hypothesis that cytotrophoblast survival is compromised by exposure to hypoxia-reoxygenation (H/R) injury, which may contribute to preeclampsia and some missed abortions, prompted us to investigate HBEGF regulation and its role as a survival factor during H/R in cytotrophoblast cells. A transformed human first-trimester cytotrophoblast cell line HTR-8/SVneo was exposed to H/R (2% O(2) followed by 20% O(2)) and assessed for HBEGF expression and cell death. Cellular HBEGF declined significantly within 30 minutes of reoxygenation after culture at 2% O(2). H/R significantly reduced proliferation and increased cell death when compared with trophoblast cells cultured continuously at 2% or 20% O(2). Restoration of cell survival also was achieved by adding recombinant HBEGF during reoxygenation. HBEGF inhibited apoptosis through its binding to either human epidermal receptor (HER)-1 or HER4, its cognate receptors. These results provide evidence that cytotrophoblast exposure to H/R induces apoptosis and decreased cell proliferation. HBEGF accumulation is diminished under these conditions, whereas restoration of HBEGF signaling improves trophoblast survival.

Severe Toxoplasma gondii I/III Recombinant-Genotype Encephalitis in a Human Immunodeficiency Virus Patient▿

PubMed Central

Genot, Séverine; Franck, Jacqueline; Forel, Jean-Marie; Rebaudet, Stanislas; Ajzenberg, Daniel; de Paula, Andre Maues; Dardé, Marie-Laure; Stein, Andreas; Ranque, Stéphane

2007-01-01

The reactivation of an uncommon type I/III recombinant-genotype Toxoplasma gondii strain resulted in unusually severe encephalitis and chorioretinitis associated with a cerebral salt wasting syndrome in an African human immunodeficiency virus patient. This observation suggests an influence of the parasite genotype on disease expression in immunocompromised patients. PMID:17634310

Comparison of Chemical Binding to Recombinant Fathead minnow and Human Estrogen Receptor alpha (ERα) in Whole Cell and Cell-Free Assay Systems.

EPA Science Inventory

Our objectives were to assess whether binding of chemicals differs significantly between recombinant estrogen receptors from fathead minnow (fhERα) and human (hERα) and to evaluate the performance of these receptors using two different in vitro assay systems: a COS whole cell bin...

Recombinant human bone morphogenetic protein-2 use in the off-label treatment of nonunions and acute fractures: a retrospective review.

PubMed

Starman, James S; Bosse, Michael J; Cates, Casey A; Norton, H James

2012-03-01

Recombinant human bone morphogenetic protein-2 (BMP-2) is Food and Drug Administration-approved for use in acute open tibial shaft fractures. Some surgeons, however, also use BMP-2 in an "off-label" application for other acute fractures and for nonunion care. This retrospective study was performed to assess radiographic outcomes of off-label uses of BMP-2 for acute fractures and nonunions at our institution. All eligible off-label BMP-2 applications between 2004 and 2008 for acute fractures or nonunions were reviewed. Univariate and multivariate analyses were completed to identify patient and clinical factors that could predict radiographic success or failure of the procedure. One hundred sixteen of 145 BMP-2 applications in 104 of 128 patients met inclusion and exclusion criteria. The overall radiographic union rate was 66% (76 of 116). In the univariate analysis, five factors correlated with significantly higher union rate: volume of bone defect <4 cm3, >2 cortices in contact at the index procedure, male gender, body mass index <30, and history of closed fracture pattern. Within the multivariate analysis, factors independently predictive of radiographic union included open versus closed fracture, gender, and volume of bone defect. Off-label use of BMP-2 in acute fractures and nonunions resulted in a 66% success rate. It remains uncertain whether there is any clinical advantage to this approach, but it appears that female gender, open injury, and higher volumes of bone defect may be important negative prognostic factors for obtaining radiographic union. Appropriately powered prospective randomized trials are needed for further clarification, especially in light of the high cost of this treatment.

Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biver, Emmanuel, E-mail: ebiver@yahoo.fr; Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex; Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14

2012-11-02

Highlights: Black-Right-Pointing-Pointer FGF modulates BMPs pathway in HMSCs by down-regulating BMP/BMPR expression. Black-Right-Pointing-Pointer This effect is mediated by ERK and JNK MAPKs pathways. Black-Right-Pointing-Pointer Crosstalk between FGF and BMPs must be taken into account in skeletal bioengineering. Black-Right-Pointing-Pointer It must also be considered in the use of recombinant BMPs in orthopedic and spine surgeries. -- Abstract: Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exertmore » their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.« less

Analysis of European mtDNAs for recombination.

PubMed

Elson, J L; Andrews, R M; Chinnery, P F; Lightowlers, R N; Turnbull, D M; Howell, N

2001-01-01

The standard paradigm postulates that the human mitochondrial genome (mtDNA) is strictly maternally inherited and that, consequently, mtDNA lineages are clonal. As a result of mtDNA clonality, phylogenetic and population genetic analyses should therefore be free of the complexities imposed by biparental recombination. The use of mtDNA in analyses of human molecular evolution is contingent, in fact, on clonality, which is also a condition that is critical both for forensic studies and for understanding the transmission of pathogenic mtDNA mutations within families. This paradigm, however, has been challenged recently by Eyre-Walker and colleagues. Using two different tests, they have concluded that recombination has contributed to the distribution of mtDNA polymorphisms within the human population. We have assembled a database that comprises the complete sequences of 64 European and 2 African mtDNAs. When this set of sequences was analyzed using any of three measures of linkage disequilibrium, one of the tests of Eyre-Walker and colleagues, there was no evidence for mtDNA recombination. When their test for excess homoplasies was applied to our set of sequences, only a slight excess of homoplasies was observed. We discuss possible reasons that our results differ from those of Eyre-Walker and colleagues. When we take the various results together, our conclusion is that mtDNA recombination has not been sufficiently frequent during human evolution to overturn the standard paradigm.

Recombinant neutral endopeptidase attenuates substance P-induced plasma extravasation in the guinea pig skin.

PubMed

Rubinstein, I; Iwamoto, I; Ueki, I F; Borson, D B; Nadel, J A

1990-01-01

To determine whether exogenously administered neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) inhibits the substance P-induced increase in vascular permeability in the skin, we examined the effects of recombinant human NEP on plasma extravasation induced by intradermal injection of substance P in guinea pig skin. Injection of substance P (2.5 X 10(-8) M) induced significant plasma extravasation in the skin (53 +/- 4 mm2 of Evans blue extravasation; mean +/- 1 SEM). In vitro incubation of substance P with recombinant human NEP prior to injection prevented the substance P-induced plasma extravasation in the skin in a dose-dependent fashion. Intradermal preinjection of recombinant human NEP partially inhibited plasma extravasation induced by subsequent injection of substance P (52 +/- 9% of the control without NEP). The H1 and H2 histamine antagonists pyrilamine and cimetidine, and a muscarinic antagonist, atropine, had no effects on substance P-induced responses. Two products of substance P degradation by NEP containing the carboxy-terminal portion, substance P7-11 and substance P8-11, were also without effect. These findings suggest that recombinant human NEP can attenuate substance P-induced increases in vascular permeability in guinea pig skin and, therefore, may be useful in treating dermatologic disorders in which abnormal responses to substance P or other neuropeptides cleaved by NEP may occur.

[Experimental study on human periodontal ligament cells transfected with human amelogenin gene].

PubMed

Yu, Guang; Shu, Rong; Sun, Ying; Cheng, Lan; Song, Zhong-Chen; Zhang, Xiu-Li

2008-02-01

To construct the recombinant lentiviral vector of human amelogenin gene, infect human periodontal ligament cells with the recombinant lentivirus, and evaluate the feasibility of applying modified PDLCs as seeds for a further periodontal reconstruction. The mature peptide of hAm cDNA was cloned and linked into the vector plasmid, the recombinant plasmid FUAmW was confirmed by double enzyme digestion and sequence analysis. Recombinant lentivirus was prepared from 293T cells by polytheylenimine (PEI)-mediated transient cotransfection. The hPDLCs and 293T cells were infected with the generated lentivirus. The infection efficiency was analysed by detection of green fluorescence protein (GFP) with fluorescent microscope and flow cytometer 72 hours later. The expression of hAm gene was detected by reverse transcription polymerase chain reaction (RT-PCR). The sequence of inserted fragment in recombinant plasmid was identical to the hAm sequence reported in Genebank. Green fluorescence was visible under fluorescent microscope, FCM assay showed that positive percentage was 69.46% and 33.99% in 293T and hPDLCs, respectively. The targeted gene was obtained in the experimental groups by RT-PCR. The recombinan lentiviral vector of hAm gene is constructed successfully and it could be transfected into cultured hPDLCs. hAm gene and seed cells may be used for further study in the fields periodontal tissue engineering. Supported by National Natural Science Foundation of China (Grant No. 30672315).

Sequential promotion of normal and leukemic hemopoiesis by recombinant human granulocyte colony-stimulating factor during the course of myelodysplastic syndrome.

PubMed

Ueda, T; Kawai, Y; Sugiyama, T; Takeuchi, N; Yoshida, A; Iwasaki, H; Wano, Y; Tsutani, H; Kamada, N; Nakamura, T

1993-12-01

A 48-year-old man developed refractory anemia with excess of blasts in transformation. Complete response was achieved by low-dose ara-C therapy, but he relapsed 15 months later, with pancytopenia and 13.0% myeloblasts in normocellular marrow. He was treated unsuccessfully with prednisolone, metenolone, and 1-alpha-hydroxyvitamin D3 for 8 weeks. He then developed life-threatening pneumonia and was treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF Filgrastim; 125 micrograms/day s.c.). The pneumonia resolved and, interestingly, he achieved a partial response, with normal blood cell counts and only a few dysmyelopoietic cells in the marrow. However, thrombocytopenia progressed when rhG-CSF administration was tapered. When the dose was increased again, leukemic blasts were found to proliferate. When rhG-CSF was discontinued, blasts rapidly decreased in the peripheral blood. Chromosomal analysis revealed a complex abnormality during the first relapse, a normal 46,XY karyotype during the partial response, and recurrence of the same complex abnormality during leukemic transformation. The stimulation index of marrow mononuclear cells cultured with rhG-CSF increased with disease progression. These findings suggest that rhG-CSF initially stimulated the selective proliferation of normal hemopoietic cells, but the evolution or selection of a leukemic clone responsive to rhG-CSF appears to have occurred subsequently.

A Randomized Controlled Phase III Trial of Recombinant Human Granulocyte Colony-Stimulating Factor (Filgrastim) for Treatment of Severe Chronic Neutropenia

PubMed Central

Dale, David C.; Bonilla, Mary Ann; Davis, Mark W.; Nakanishi, Arline M.; Hammond, William P.; Kurtzberg, Joanne; Wang, Winfred; Jakubowski, Ann; Winton, Elliott; Lalezari, Parviz; Robinson, William; Glaspy, John A.; Emerson, Steve; Gabrilove, Janice; Vincent, Martha; Boxer, Laurence A.

2014-01-01

Patients with idiopathic, cyclic, and congenital neutropenia have recurrent severe bacterial infections. One hundred twenty-three patients with recurrent infections and severe chronic neutropenia (absolute neutrophil count < 0.5 × 109/L) due to these diseases were enrolled in this multi-center phase III trial. They were randomized to either immediately beginning recombinant human granulocyte colony-stimulating factor (filgrastim) (3.45 to 11.50 μg/kg/d, subcutaneously) or entering a 4-month observation period followed by filgrastim administration. Blood neutrophil counts, bone marrow (BM) cell histology, and incidence and duration of infection-related events were monitored. Of the 123 patients enrolled, 120 received filgrastim. On therapy, 108 patients had a median absolute neutrophil count of ≥ 1.5 × 109/L. Examination of BM aspirates showed increased proportions of maturing neutrophils. Infection-related events were significantly decreased (P < .05) with approximately 50% reduction in the incidence and duration of infection-related events and almost 70% reduction in duration of antibiotic use. Asymptomatic splenic enlargement occurred frequently: adverse events frequently reported were bone pain, headache, and rash, which were generally mild and easily manageable. These data indicate that treatment of patients with severe chronic neutropenia with filgrastim results in a stimulation of BM production and maturation of neutrophils, an increase in circulating neutrophils, and a reduction in infection-related events. PMID:8490166

Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

PubMed

Nair, Nisha R; Chidambareswaren, M; Manjula, S

2014-09-01

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

RECOMBINANT FACTOR XIII DIMINISHES MULTIPLE ORGAN DYSFUNCTION IN RATS CAUSED BY GUT ISCHEMIA-REPERFUSION INJURY

PubMed Central

Zaets, Sergey B.; Xu, Da-Zhong; Lu, Qi; Feketova, Eleonora; Berezina, Tamara L.; Gruda, Maryann; Malinina, Inga V.; Deitch, Edwin A.; Olsen, Eva H. N.

2010-01-01

Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. Because FXIII has also been shown to modulate inflammation and endothelial permeability, we hypothesized that FXIII diminishes multiple organ dysfunction caused by gut I/R injury. A model of superior mesenteric artery occlusion (SMAO) was used to induce gut I/R injury. Rats were subjected to 45-min SMAO or sham SMAO and treated with recombinant human FXIII A2 subunit (rFXIII) or placebo at the beginning of the reperfusion period. Lung permeability, lung and gut myeloperoxidase activity, gut histology, neutrophil respiratory burst, and microvascular blood flow in the liver and muscles were measured after a 3-h reperfusion period. The effect of activated rFXIII on transendothelial resistance of human umbilical vein endothelial cells was tested in vitro. Superior mesenteric artery occlusion–induced lung permeability as well as lung and gut myeloperoxidase activity was significantly lower in rFXIII-treated versus untreated animals. Similarly, rFXIII-treated rats had lower neutrophil respiratory burst activity and ileal mucosal injury. Rats treated with rFXIII also had higher liver microvascular blood flow compared with the placebo group. Superior mesenteric artery occlusion did not cause FXIII consumption during the study period. In vitro, activated rFXIII caused a dose-dependent increase in human umbilical vein endothelial cell monolayer resistance to thrombin-induced injury. Thus, administration of rFXIII diminishes SMAO-induced multiple organ dysfunction in rats, presumably by preservation of endothelial barrier function and the limitation of polymorphonuclear leukocyte activation. PMID:18948851

Effects of a human recombinant alkaline phosphatase during impaired mitochondrial function in human renal proximal tubule epithelial cells.

PubMed

Peters, Esther; Schirris, Tom; van Asbeck, Alexander H; Gerretsen, Jelle; Eymael, Jennifer; Ashikov, Angel; Adjobo-Hermans, Merel J W; Russel, Frans; Pickkers, Peter; Masereeuw, Rosalinde

2017-02-05

Sepsis-associated acute kidney injury is a multifactorial syndrome in which inflammation and renal microcirculatory dysfunction play a profound role. Subsequently, renal tubule mitochondria reprioritize cellular functions to prevent further damage. Here, we investigated the putative protective effects of human recombinant alkaline phosphatase (recAP) during inhibition of mitochondrial respiration in conditionally immortalized human proximal tubule epithelial cells (ciPTEC). Full inhibition of mitochondrial oxygen consumption was obtained after 24h antimycin A treatment, which did not affect cell viability. While recAP did not affect the antimycin A-induced decreased oxygen consumption and increased hypoxia-inducible factor-1α or adrenomedullin gene expression levels, the antimycin A-induced increase of pro-inflammatory cytokines IL-6 and IL-8 was attenuated. Antimycin A tended to induce the release of detrimental purines ATP and ADP, which reached statistical significance when antimycin A was co-incubated with lipopolysaccharide, and were completely converted into cytoprotective adenosine by recAP. As the adenosine A 2A receptor was up-regulated after antimycin A exposure, an adenosine A 2A receptor knockout ciPTEC cell line was generated in which recAP still provided protection. Together, recAP did not affect oxygen consumption but attenuated the inflammatory response during impaired mitochondrial function, an effect suggested to be mediated by dephosphorylating ATP and ADP into adenosine. Copyright © 2016 Elsevier B.V. All rights reserved.

Progranulin expression in advanced human atherosclerotic plaque.

PubMed

Kojima, Yoji; Ono, Koh; Inoue, Katsumi; Takagi, Yasushi; Kikuta, Ken-ichiro; Nishimura, Masaki; Yoshida, Yoshinori; Nakashima, Yasuhiro; Matsumae, Hironobu; Furukawa, Yutaka; Mikuni, Nobuhiro; Nobuyoshi, Masakiyo; Kimura, Takeshi; Kita, Toru; Tanaka, Makoto

2009-09-01

Progranulin (PGRN) is a unique growth factor that plays an important role in cutaneous wound healing. It has an anti-inflammatory effect and promotes cell proliferation. However, when it is degraded to granulin peptides (GRNs) by neutrophil proteases, a pro-inflammatory reaction occurs. Since injury, inflammation and repair are common features in the progression of atherosclerosis, it is conceivable that PGRN plays a role in atherogenesis. Immunohistochemical analysis of human carotid endoatherectomy specimens indicated that vascular smooth muscle cells (vSMCs) in the intima expressed PGRN. Some macrophages in the plaque also expressed PGRN. We assessed the effect of PGRN on a human monocytic leukemia cell line (THP-1) and human aortic smooth muscle cells (HASMCs). PGRN alone had no effect on HASMC or THP-1 proliferation or migration. However, when THP-1 cells were stimulated with MCP-1, the number of migrated cells decreased in a PGRN-dose-dependent manner. TNF-alpha-induced HASMC migration was enhanced only at 10nM of PGRN. Interleukin-8 (IL-8) secretion from HASMCs was reduced by forced expression of PGRN and increased by RNAi-mediated knockdown of PGRN. While exogenous treatment with recombinant PGRN decreased IL-8 secretion, degraded recombinant GRNs increased IL-8 secretion from HASMCs. The expression of PGRN mainly reduces inflammation and its degradation into GRNs enhances inflammation in atherosclerotic plaque and may contribute to the progression of atherosclerosis.

Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs.

PubMed

Farahmand, Mahin; Nahrevanian, Hossein

2016-07-01

Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

Evaluation of off-label recombinant activated factor VII for multiple indications in children.

PubMed

Reiter, Pamela D; Valuck, Robert J; Taylor, Ruston S

2007-07-01

Despite a paucity of safety and efficacy data, the use of recombinant activated factor VII in children for off-label indications has now surpassed its use in hemophilia. A retrospective chart review was conducted of 46 subjects (age, 6.7 +/- 6 years; weight, 26 +/- 20 kg) who received recombinant activated factor VII for nonhemophiliac indications between January 1, 2004, and September 1, 2005. Indications for use included prevention (n = 6) or treatment (n = 40) of bleeding due to general surgery, hepatic failure, gastrointestinal bleeding, severe traumatic brain injury, bone marrow transplant, cardiac, acetaminophen overdose, and multiorgan system failure. Decreases in prothrombin time, partial thromboplastin time, and international normalized ratio were observed. No inappropriate thrombotic events were noted. Administration of recombinant activated factor VII was associated with a reduction in coagulation markers without obvious adverse thrombotic events at cost of $4189 per dose. These findings should be confirmed in a prospective trial.

Vaccine development against the Taenia solium parasite: the role of recombinant protein expression in Escherichia coli.

PubMed

Gauci, Charles; Jayashi, César; Lightowlers, Marshall W

2013-01-01

Taenia solium is a zoonotic parasite that causes cysticercosis. The parasite is a major cause of human disease in impoverished communities where it is transmitted to humans from pigs which act as intermediate hosts. Vaccination of pigs to prevent transmission of T. solium to humans is an approach that has been investigated to control the disease. A recombinant vaccine antigen, TSOL18, has been remarkably successful at reducing infection of pigs with T. solium in several experimental challenge trials. The vaccine has been shown to eliminate transmission of naturally acquired T. solium in a field trial conducted in Africa. We recently reported that the vaccine was also effective in a field trial conducted in Peru. The TSOL18 recombinant antigen for each of these trials has been produced by expression in Escherichia coli. Here we discuss research that has been undertaken on the TSOL18 antigen and related antigens with a focus on improved methods of preparation of recombinant TSOL18 and optimized expression in Escherichia coli.

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

PubMed Central

Miller, A M; Savinelli, E A; Couture, S M; Hannigan, G M; Han, Z; Selden, R F; Treco, D A

1993-01-01

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8367472

Human basic fibroblast growth factor fused with Kringle4 peptide binds to a fibrin scaffold and enhances angiogenesis.

PubMed

Zhao, Wenxue; Han, Qianqian; Lin, Hang; Sun, Wenjie; Gao, Yuan; Zhao, Yannan; Wang, Bin; Wang, Xia; Chen, Bing; Xiao, Zhifeng; Dai, Jianwu

2009-05-01

Appropriate three-dimensional (3D) scaffolds and signal molecules could accelerate tissue regeneration and wound repair. In this work, we targeted human basic fibroblast growth factor (bFGF), a potent angiogenic factor, to a fibrin scaffold to improve therapeutic angiogenesis. We fused bFGF to the Kringle4 domain (K4), a fibrin-binding peptide from human plasminogen, to endow bFGF with specific fibrin-binding ability. The recombinant K4bFGF bound specifically to the fibrin scaffold so that K4bFGF was delivered in a site-specific manner, and the fibrin scaffold provided 3D support for cell migration and proliferation. Subcutaneous implantation of the fibrin scaffolds bound with K4bFGF but not with bFGF induced neovascularization. Immunohistochemical analysis showed significantly more proliferation cells in the fibrin scaffolds incorporated with K4bFGF than in those with bFGF. Moreover, the regenerative tissues were integrated well with the fibrin scaffolds, suggesting its good biocompatibility. In summary, targeted delivery of K4bFGF could potentially improve therapeutic angiogenesis.

Expression of exogenous human hepatic nuclear factor-1α by a lentiviral vector and its interactions with Plasmodium falciparum subtilisin-like protease 2.

PubMed

Liao, Shunyao; Liu, Yunqiang; Zheng, Bing; Cho, Pyo Yun; Song, Hyun Ok; Lee, Yun-Seok; Jung, Suk-Yul; Park, Hyun

2011-12-01

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors as well as by individual host responses to these determinants. In both humans and mice, liver injury follows parasite entry, persisting to the erythrocytic stage in the case of infection with the fatal strain of Plasmodium falciparum. Hepatic nuclear factor (HNF)-1α is a master regulator of not only the liver damage and adaptive responses but also diverse metabolic functions. In this study, we analyzed the expression of host HNF-1α in relation to malaria infection and evaluated its interaction with the 5'-untranslated region of subtilisin-like protease 2 (subtilase, Sub2). Recombinant human HNF-1α expressed by a lentiviral vector (LV HNF-1α) was introduced into mice. Interestingly, differences in the activity of the 5'-untranslated region of the Pf-Sub2 promoter were detected in 293T cells, and LV HNF-1α was observed to influence promoter activity, suggesting that host HNF-1α interacts with the Sub2 gene.

atlant: Advanced Three Level Approximation for Numerical Treatment of Cosmological Recombination

NASA Astrophysics Data System (ADS)

Kholupenko, E. E.; Ivanchik, A. V.; Balashev, S. A.; Varshalovich, D. A.

2011-10-01

atlant is a public numerical code for fast calculations of cosmological recombination of primordial hydrogen-helium plasma is presented. This code is based on the three-level approximation (TLA) model of recombination and allows us to take into account some "fine" physical effects of cosmological recombination simultaneously with using fudge factors.

Large-scale analysis of pedigree and sperm-typing data reveals PRDM9 allele-specific recombination maps in cattle

USDA-ARS?s Scientific Manuscript database

Meiotic recombination is a major driving force in promoting genetic and phenotypic variations in sexually reproducing organisms. Although PRDM9 is known to modulate the binding-specificity and location of recombination hotspots in humans and mice, its role, especially in domesticated animals like ca...

Recombination or mutational hot spots in human mtDNA?

PubMed

Innan, Hideki; Nordborg, Magnus

2002-07-01

Awadalla, Eyre-Walker, and Maynard Smith (1999) recently argued that there might be recombination in human mitochondrial DNA (mtDNA). Their claim was based on their observation of decaying linkage disequilibrium (LD) as a function of physical distance. Their study was much criticized, and follow-up studies have failed to find any evidence for recombination. We argue that the criticisms levied, even if correct, could not possibly explain the findings of Awadalla, Eyre-Walker, and Maynard Smith (1999). Nonetheless, the test proposed by Awadalla, Eyre-Walker, and Maynard Smith (1999 ) is not robust because recombination is not the only explanation for decay of LD. We show that such a pattern can be caused by mutational hot spots as well. However, a closer look at the data suggests that the pattern observed was not caused by mutational hot spots but rather by chance. Thus, there appears to be no evidence for recombination in the mtDNA polymorphism data. In conclusion, we discuss the possibility of detecting recombination in mtDNA and the implications of its existence.

Reactions of chicken sera to recombinant Campylobacter jejuni flagellar proteins.

PubMed

Yeh, Hung-Yueh; Hiett, Kelli L; Line, John E

2015-03-01

Campylobacter jejuni is a Gram-negative spiral rod bacterium and is the leading but underreported bacterial food-borne pathogen that causes human campylobacteriosis worldwide. Raw or undercooked poultry products are regarded as a major source for human infection. C. jejuni flagella have been implicated in colonization and adhesion to the mucosal surface of chicken gastrointestinal tracts. Therefore, flagellar proteins would be the excellent targets for further investigation. In this report, we used the recombinant technology to generate a battery of C. jejuni flagellar proteins, which were purified by His tag affinity chromatography and determined antigenic profiles of these recombinant flagellar proteins using sera from chickens older than 6 weeks of age. The immunoblot results demonstrate that each chicken serum reacted to various numbers of recombinant flagellar proteins. Among these recombinant proteins, chicken sera reacted predominantly to the FlgE1, FlgK, FlhF, FliG and FliY proteins. These antibody screening results provide a rationale for further evaluation of these recombinant flagellar proteins as potential vaccines for chickens to improve food safety as well as investigation of host immune response to C. jejuni.

Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2, DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death

PubMed Central

Malecki, Marek; LaVanne, Christine; Alhambra, Dominique; Dodivenaka, Chaitanya; Nagel, Sarah; Malecki, Raf

2014-01-01

Introduction The worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors. Specific aim The specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases). Methods The DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers’ bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies’ guided DNA vectors delivered the transgenes for the human recombinant DNases’ into proliferating stem cells. Results Differentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases resulted in complete collapse of the chromatin architecture and degradation of the proliferating cells’ genomic DNA. The proliferating stem cells, but not the differentiating ones, were effectively induced to die. Conclusion Herein, we describe attaining the proof-of-concept for the strategy, whereby transgenic expression of the genetically engineered human recombinant DNases in proliferating and directed differentiation resisting stem cells leads to their death. This novel strategy reduces the risk of iatrogenic neoplasms in stem cell therapy. PMID:25045589

Recombinant Human Insulin in Global Diabetes Management – Focus on Clinical Efficacy

PubMed Central

Mbanya, Jean Claude; Sandow, Juergen; Landgraf, Wolfgang

2017-01-01

Abstract Biosynthetic human insulin and insulin analogues are the mainstay of insulin therapy for both type 1 and type 2 diabetes although access to human insulin at affordable prices remains a global issue. The world is experiencing an exponential rise in the prevalence of diabetes presenting an urgent need to establish effective diabetes therapy in countries burdened by inadequate health care budgets, malnutrition and infectious diseases. Recombinant human insulin has replaced animal insulins and animal-based semisynthetic human insulin thereby available in sufficient quantities and at affordable prices able to provide global access to insulin therapy. In many patients, analog insulins can offer additional clinical benefit, although at a considerably higher price thus severely restricting availability in low income countries. The approval process for recombinant human insulins (i.e. biosimilars) and analogue insulins is highly variable in the developing countries in contrast to Europe and in North America, where it is well established within a strict regulatory framework. This review aims to discuss the future access to human insulin therapy in a global context with an ever increasing burden of diabetes and significant economic implications. PMID:29632602

Identification of Recombinant Human Rhinovirus A and C in Circulating Strains from Upper and Lower Respiratory Infections

PubMed Central

Kim, Hak; Kim, Kisoon; Kim, Dae-Won; Jung, Hee-Dong; Min Cheong, Hyang; Kim, Ki Hwan; Soo Kim, Dong; Kim, You-Jin

2013-01-01

Human rhinoviruses (HRVs), in the Enterovirus genus within the family Picornaviridae, are a highly prevalent cause of acute respiratory infection (ARI). Enteroviruses are genetically highly variable, and recombination between serotypes is known to be a major contribution to their diversity. Recently it was reported that recombination events in HRVs cause the diversity of HRV-C. This study analyzed parts of the viral genes spanning the 5′ non- coding region (NCR) through to the viral protein (VP) encoding sequences of 105 HRV field isolates from 51 outpatient cases of Acute Respiratory Infectious Network (ARINET) and 54 inpatient cases of severe lower respiratory infection (SLRI) surveillance, in order to identify recombination in field samples. When analyzing parts of the 5′NCR and VP4/VP2 encoding sequences, we found intra- and interspecies recombinants in field strains of HRV-A and -C. Nineteen cases of recombination events (18.1%) were found among 105 field strains. For HRV-A, there were five cases (4.8%) of intraspecies recombination events and three cases (2.8%) of interspecies recombination events. For HRV-C, there were four cases (3.8%) of intraspecies recombination events and seven cases (6.7%) of interspecies recombination events. Recombination events were significantly more frequently observed in the ARINET samples (18 cases) than in the SLRI samples (1 case; P< 0.0001). The recombination breakpoints were located in nucleotides (nt) 472–554, which comprise stem-loop 5 in the internal ribosomal entry site (IRES), based on the HRV-B 35 sequence (accession no. FJ445187). Our findings regarding genomic recombination in circulating HRV-A and -C strains suggest that recombination might play a role in HRV fitness and could be a possible determinant of disease severity caused by various HRV infections in patients with ARI. PMID:23826363

Cloning and expression of porcine Colony Stimulating Factor-1 (CSF-1) and Colony Stimulating Factor-1 Receptor (CSF-1R) and analysis of the species specificity of stimulation by CSF-1 and Interleukin 34

PubMed Central

Gow, Deborah J.; Garceau, Valerie; Kapetanovic, Ronan; Sester, David P.; Fici, Greg J.; Shelly, John A.; Wilson, Thomas L.; Hume, David A.

2012-01-01

Macrophage Colony Stimulating Factor (CSF-1) controls the survival, differentiation and proliferation of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, Interleukin 34 (IL-34), has been described, but its physiological role is not yet known. The domestic pig provides an alternative to traditional rodent models for evaluating potential therapeutic applications of CSF-1R agonists and antagonists. To enable such studies, we cloned and expressed active pig CSF-1. To provide a bioassay, pig CSF-1R was expressed in the factor-dependent Ba/F3 cell line. On this transfected cell line, recombinant porcine CSF-1 and human CSF-1 had identical activity. Mouse CSF-1 does not interact with the human CSF-1 receptor but was active on pig. By contrast, porcine CSF-1 was active on mouse, human, cat and dog cells. IL-34 was previously shown to be species-specific, with mouse and human proteins demonstrating limited cross-species activity. The pig CSF-1R was equally responsive to both mouse and human IL-34. Based upon the published crystal structures of CSF-1/CSF-1R and IL34/CSF-1R complexes, we discuss the molecular basis for the species specificity. PMID:22974529

Biobusiness in the pharmaceutical industry.

PubMed

Werner, R G

1987-09-01

Although conventional biotechnology used for the synthesis of antibiotics, vitamins, amino acids, nucleotides, enzyme inhibitors and immunomodulating compounds has still a major impact in the production of pharmaceutical compounds, the importance of the new biotechnology is increasing. Whereas in conventional biotechnology naturally occurring strains are screened for production of pharmacologically active compounds, in new biotechnology known organisms are programmed by genetic engineering to produce a distinct protein or glycoprotein of human origin for substitution therapy. Such complex compounds from new biotechnology can be divided into products which might replace compounds which are already on the market by safer recombinant products such as human insulin, human growth hormone, urokinase, factor VIII and products which are new on the market such as interferons, lymphokines, tissue plasminogen activator, oligonucleotide probes, monoclonal antibodies and subunit vaccines. However, only a few of these recombinant products have reached the market such as human insulin, interferon alpha, interferon beta, human growth hormone and recombivax HB. In most cases, depending on the difficulties in demonstrating clinical efficacy, the investigated drugs have reached the marketing phase much faster than conventional chemical drugs. Return on investment of biotechnical produced pharmaceutics mainly depends on the issues of whether the product has to compete with chemically synthesized drugs, whether it is totally new but competes with other bioproducts, whether it is exceptional but the proof of clinical efficacy is difficult, or whether it is totally new and clinical studies are promising.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know.

PubMed

Hayat, Seyed Mohammad Gheibi; Farahani, Najmeh; Golichenari, Behrouz; Sahebkar, Amir Hosein

2018-01-31

Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

PubMed

Mell, Joshua Chang; Viadas, Cristina; Moleres, Javier; Sinha, Sunita; Fernández-Calvet, Ariadna; Porsch, Eric A; St Geme, Joseph W; Nislow, Corey; Redfield, Rosemary J; Garmendia, Junkal

2016-04-01

Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe "transformed recombinant enrichment profiling" (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation.

Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

PubMed

Freyberger, Alexius; Weimer, Marc; Tran, Hoai-Son; Ahr, Hans-Jürgen

2010-08-01

Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation. Copyright 2009 Elsevier Inc. All rights reserved.

Pharmacokinetics, pharmacodynamics and safety of recombinant canine FVIIa in a study dosing one haemophilia A and one haemostatically normal dog.

PubMed

Knudsen, T; Kristensen, A T; Nichols, T C; Agersø, H; Jensen, A L; Kjalke, M; Ezban, M; Tranholm, M

2011-11-01

Recombinant human FVIIa (rhFVIIa) corrects the coagulopathy in hemophilia A and B as well as FVII deficiency. This is also the case in dogs until canine anti-human FVIIa antibodies develop (~2 weeks). Recombinant canine factor VIIa (rcFVIIa), successfully over-expressed by gene transfer in haemophilia dogs, has provided long-term haemostasis (>2 years). However, pharmacokinetics (PK), pharmacodynamics (PD) and safety of rcFVIIa after pharmacological administration have not been reported. We therefore wanted to explore the safety, PK and PD of rcFVIIa in dogs. A pilot study was set up to evaluate the safety as well as PK and PD of rcFVIIa after a single intravenous dose of 270 μg kg(-1) to one HA and one haemostatically normal dog and to directly compare rcFVIIa with rhFVIIa in these two dogs. Single doses of rcFVIIa and rhFVIIa were well tolerated. No adverse events were observed. Pharmacokinetic characteristics including half-life (FVIIa activity: 1.2-1.8 h; FVIIa antigen 2.8-3.7 h) and clearance were comparable for rcFVIIa and rhFVIIa. Kaolin-activated thromboelastography approached normal in the HA dog with the improvement being most pronounced after rcFVIIa. This study provided the first evidence that administering rcFVIIa intravenously is feasible, safe, well tolerated and efficacious in correcting the haemophilic coagulopathy in canine HA and that rcFVIIa exhibits pharmacokinetic characteristics comparable to rhFVIIa in haemophilic and haemostatically competent dogs. This strengthens the hypothesis that rcFVIIa can be administered to dogs to mimic the administration of rhFVIIa to humans. © 2011 Blackwell Publishing Ltd.

Healthy human T-Cell Responses to Aspergillus fumigatus antigens.

PubMed

Chaudhary, Neelkamal; Staab, Janet F; Marr, Kieren A

2010-02-17

Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T(H)1-T(H)17) and destructive allergic (T(H)2) immunity. How Aspergillus allergens (Asp f proteins) participate in the development of allergic sensitization is unknown. To determine whether Asp f proteins are strictly associated with T(H)2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-gamma, IL-4 or IL-17) to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-gamma more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T(H)1-responses to Asp f3 (a putative peroxismal membrane protein), Asp f9/16 (cell wall glucanase), Asp f11 (cyclophilin type peptidyl-prolyl isomerase) and Asp f22 (enolase). Strong IFN-gamma responses were reproduced in most subjects tested over 6 month intervals. Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.

Quality-of-Life Outcomes following Thoracolumbar and Lumbar Fusion with and without the Use of Recombinant Human Bone Morphogenetic Protein-2: Does Recombinant Human Bone Morphogenetic Protein-2 Make a Difference?

PubMed Central

Lubelski, Daniel; Alvin, Matthew D.; Torre-Healy, Andrew; Abdullah, Kalil G.; Nowacki, Amy S.; Whitmore, Robert G.; Steinmetz, Michael P.; Benzel, Edward C.; Mroz, Thomas E.

2014-01-01

Design Retrospective study. Objectives (1) To investigate the quality-of-life (QOL) outcomes in the population undergoing lumbar spine surgery with versus without recombinant human bone morphogenetic protein-2 (rhBMP-2); (2) to determine QOL outcomes for those patients who experience postoperative complications; and (3) to identify the effect of patient characteristics on postoperative QOL outcomes. Methods A retrospective review of QOL questionnaires, including the Patient Health Questionnaire-9, Patient Disability Questionnaire (PDQ), EuroQol-5D (EQ-5D), and quality of life-year (QALY), was performed for all patients who underwent thoracolumbar and lumbar fusion surgery with versus without rhBMP-2 between March 2008 and September 2010. Individual preoperative and postoperative QOL data were compared for each patient. Demographic factors and complications were reviewed. Results We identified 266 patients, including 60 with and 206 without rhBMP-2. Questionnaires were completed an average of 10.3 ± 5 months after surgery. For all measures, average scores improved postoperatively compared with preoperatively. No differences in postoperative QOL outcomes were identified between the rhBMP-2 and the control cohorts. Median annual household income was positively associated with EQ-5D and QALY. Compared with those without, patients with postoperative complications had fewer QOL improvements. Conclusions There was no difference in QOL outcomes in the rhBMP-2 compared with the control group. Socioeconomic status and postoperative complications affected QOL outcomes following surgery. The QOL questionnaires provide the clinician with information regarding the patients' self-perceived well-being and can be helpful in the selection of surgical candidates and for understanding the effectiveness of a given surgical procedure. PMID:25396105

Effect of Recombinant Human Growth Hormone and Rosiglitazone for HIV-Associated Abdominal Fat Accumulation on Adiponectin and other Markers of Inflammation

PubMed Central

Leung, Vivien; Chiu, Ya-Lin; Kotler, Donald P.; Albu, Jeanine; Zhu, Yuan-Shan; Ham, Kirsis; Engelson, Ellen S.; Hammad, Hoda; Christos, Paul; Donovan, Daniel S.; Ginsberg, Henry N.; Glesby, Marshall J.

2016-01-01

Background/Objective In a previous report of HIV-infected patients with fat redistribution, we found that recombinant human growth hormone (rhGH) therapy reduced visceral adipose tissue (VAT) but increased insulin resistance, and that the addition of rosiglitazone reversed the negative effects of rhGH on insulin sensitivity. In this study, we sought to determine the effects of recombinant human growth hormone (rhGH) and rosiglitazone therapy on an array of inflammatory and fibrinolytic markers. Methods 72 patients with HIV-associated abdominal obesity and insulin resistance were randomized to treatment with rhGH, rosiglitazone, the combination of rhGH and rosiglitazone, or placebo for 12 weeks. Subjects with plasma and serum samples available at weeks 0 (n = 63) and 12 (n = 46-48) were assessed for adiponectin, C-reactive protein (CRP), homocysteine, interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), fibrinogen, plasminogen activator inhibitor-1 (PAI-1) antigen, and tissue plasminogen activator (tPA) antigen. Results Treatment with both rosiglitazone alone and the combination of rosiglitazone and rhGH for 12 weeks resulted in significant increases in adiponectin levels from baseline. Adiponectin levels did not change significantly in the rhGH alone arm. There were no significant changes in the other biomarkers amongst the different treatment groups. Discussion In this study of HIV-infected patients with altered fat distribution, treatment with rosiglitazone had beneficial effects on adiponectin concentrations, an effect that was also seen with combination rosiglitazone and rhGH. RhGH administration alone, however, did not demonstrate any significant impact on adiponectin levels despite reductions in VAT. PMID:27077672

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding

NASA Astrophysics Data System (ADS)

Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben S.; Wengel, Jesper; Howard, Kenneth A.

2017-05-01

Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding.

PubMed

Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben S; Wengel, Jesper; Howard, Kenneth A

2017-05-19

Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

Comparative Biochemical and Functional Studies on a Branded Human Recombinant Factor VIIa and a Biosimilar Equivalent Product.

PubMed

Sadeghi, Nasiredin; Kahn, Daniel; Syed, Daneyal; Iqbal, Omer; Abro, Schuharazad; Eshraghi, Reza; Hoppensteadt, Debra; Fareed, Jawed

2014-09-01

Recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Copenhagen, Denmark) is used to control bleeding in patients with hemophilia. A generic version of FVIIa was developed by AryoGen (Tehran, Iran). This study compared the composition and functional activities of AryoSeven and NovoSeven. Each product was compared at equigravimetric (1 mg/mL) stock solution for protein content. The proteomic profile was obtained using surface-enhanced laser desorption ionization mass spectrometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was carried out to determine the protein profile and Western blotting was performed using a polyclonal rabbit antihuman FVIIa antibody. The FVIIa-related antigen was also measured using a commercially available enzyme-linked immunosorbent assay method. Functional assay included the prothrombin time correction in FVII-deficient plasma. The protein content was comparable in 2 products and the mass spectra analysis showed a single peak at 50 kDa in all products. The SDS-PAGE and immunoblotting studies were comparable. Both products exhibited similar coagulant properties in different assays. © The Author(s) 2014.

Successful ovulation induction, conception, and normal delivery after chronic therapy with etanercept: a recombinant fusion anti-cytokine treatment for rheumatoid arthritis.

PubMed

Sills, E S; Perloe, M; Tucker, M J; Kaplan, C R; Palermo, G D

2001-11-01

Etanercept (Enbrel; Wyeth-Ayerst/Immunex Inc, Seattle, WA, USA) is a subcutaneously administered novel fusion protein consisting of the extracellular ligand-binding domain of the 75 kD receptor for tumor necrosis factor-alpha (anti-TNFalpha) and the Fc portion of human IgG1. The agent is synthesized by plasmid transfection of a Chinese hamster ovary cell line, utilizing recombinant DNA technology. Etanercept was approved by the US FDA for treatment of multi-drug resistant rheumatoid arthritis in 1998, but no human data exist regarding the impact of anti-TNFalpha therapy on human reproductive function or its use before ovulation induction. As TNFalpha potentiates collagenolysis via matrix metalloproteinase gene expression (thereby facilitating ovulation), there exists a theoretical risk that TNFalpha-inhibition could exert an undesirable effect on ovulation and pregnancy. In this report, we describe the first case of ovulation induction, intrauterine insemination, normal pregnancy and singleton delivery of a healthy infant following chronic ( > 1 year) pre-ovulatory TNFalpha-inhibitor therapy for rheumatoid arthritis. Reproductive endocrinologists and obstetrician-gynecologists should be familiar with etanercept therapy in the context of severe rheumatic disease, and offer appropriate reassurance regarding its safe use for infertility patients planning ovulation induction.

Sites of instability in the human TCF3 (E2A) gene adopt G-quadruplex DNA structures in vitro

PubMed Central

Williams, Jonathan D.; Fleetwood, Sara; Berroyer, Alexandra; Kim, Nayun; Larson, Erik D.

2015-01-01

The formation of highly stable four-stranded DNA, called G-quadruplex (G4), promotes site-specific genome instability. G4 DNA structures fold from repetitive guanine sequences, and increasing experimental evidence connects G4 sequence motifs with specific gene rearrangements. The human transcription factor 3 (TCF3) gene (also termed E2A) is subject to genetic instability associated with severe disease, most notably a common translocation event t(1;19) associated with acute lymphoblastic leukemia. The sites of instability in TCF3 are not randomly distributed, but focused to certain sequences. We asked if G4 DNA formation could explain why TCF3 is prone to recombination and mutagenesis. Here we demonstrate that sequences surrounding the major t(1;19) break site and a region associated with copy number variations both contain G4 sequence motifs. The motifs identified readily adopt G4 DNA structures that are stable enough to interfere with DNA synthesis in physiological salt conditions in vitro. When introduced into the yeast genome, TCF3 G4 motifs promoted gross chromosomal rearrangements in a transcription-dependent manner. Our results provide a molecular rationale for the site-specific instability of human TCF3, suggesting that G4 DNA structures contribute to oncogenic DNA breaks and recombination. PMID:26029241

Treatment With Recombinant Human Insulin-Like Growth Factor-1 Improves Growth in Patients With PAPP-A2 Deficiency

PubMed Central

Muñoz-Calvo, María T.; Barrios, Vicente; Pozo, Jesús; Chowen, Julie A.; Martos-Moreno, Gabriel Á.; Hawkins, Federico; Dauber, Andrew; Domené, Horacio M.; Yakar, Shoshana; Rosenfeld, Ron G.; Pérez-Jurado, Luis A.; Oxvig, Claus; Frystyk, Jan

2016-01-01

Context: Pregnancy-associated plasma protein-A2 (PAPP-A2) is a metalloproteinase that specifically cleaves IGFBP-3 and IGFBP-5. Mutations in the PAPP-A2 gene have recently been shown to cause postnatal growth failure in humans, with specific skeletal features, due to the resulting decrease in IGF-1 bioavailability. However, a pharmacological treatment of this entity is yet to be established. Case Description: A 10.5-year-old girl and a 6-year-old boy, siblings from a Spanish family, with short stature due to a homozygous loss-of-function mutation in the PAPP-A2 gene (p.D643fs25*) and undetectable PAPP-A2 activity, were treated with progressive doses (40, 80, 100, and 120 μg/kg) of recombinant human IGF-1 (rhIGF-1) twice daily for 1 year. There was a clear increase in growth velocity and height in both siblings. Bioactive IGF-1 was increased, and spontaneous GH secretion was diminished after acute administration of rhIGF-1, whereas serum total IGF-1 and IGFBP-3 levels remained elevated. No episodes of hypoglycemia or any other secondary effects were observed during treatment. Conclusion: Short-term treatment with rhIGF-1 improves growth in patients with PAPP-A2 deficiency. PMID:27648969

Chemical modifications of therapeutic proteins induced by residual ethylene oxide.

PubMed

Chen, Louise; Sloey, Christopher; Zhang, Zhongqi; Bondarenko, Pavel V; Kim, Hyojin; Ren, Da; Kanapuram, Sekhar

2015-02-01

Ethylene oxide (EtO) is widely used in sterilization of drug product primary containers and medical devices. The impact of residual EtO on protein therapeutics is of significant interest in the biopharmaceutical industry. The potential for EtO to modify individual amino acids in proteins has been previously reported. However, specific identification of EtO adducts in proteins and the effect of residual EtO on the stability of therapeutic proteins has not been reported to date. This paper describes studies of residual EtO with two therapeutic proteins, a PEGylated form of the recombinant human granulocyte colony-stimulating factor (Peg-GCSF) and recombinant human erythropoietin (EPO) formulated with human serum albumin (HSA). Peg-GCSF was filled in an EtO sterilized delivery device and incubated at accelerated stress conditions. Glu-C peptide mapping and LC-MS analyses revealed residual EtO reacted with Peg-GCSF and resulted in EtO modifications at two methionine residues (Met-127 and Met-138). In addition, tryptic peptide mapping and LC-MS analyses revealed residual EtO in plastic vials reacted with HSA in EPO formulation at Met-328 and Cys-34. This paper details the work conducted to understand the effects of residual EtO on the chemical stability of protein therapeutics. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Losac, the First Hemolin that Exhibits Procogulant Activity through Selective Factor X Proteolytic Activation*

PubMed Central

Alvarez-Flores, Miryam Paola; Furlin, Daniel; Ramos, Oscar H. P.; Balan, Andrea; Konno, Katsuhiro; Chudzinski-Tavassi, Ana Marisa

2011-01-01

Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X. PMID:21177860

Growth factor delivery: How surface interactions modulate release in vitro and in vivo

PubMed Central

King, William J.; Krebsbach, Paul H.

2013-01-01

Biomaterial scaffolds have been extensively used to deliver growth factors to induce new bone formation. The pharmacokinetics of growth factor delivery has been a critical regulator of their clinical success. This review will focus on the surface interactions that control the non-covalent incorporation of growth factors into scaffolds and the mechanisms that control growth factor release from clinically relevant biomaterials. We will focus on the delivery of recombinant human bone morphogenetic protein-2 from materials currently used in the clinical practice, but also suggest how general mechanisms that control growth factor incorporation and release delineated with this growth factor could extend to other systems. A better understanding of the changing mechanisms that control growth factor release during the different stages of preclinical development could instruct the development of future scaffolds for currently untreatable injuries and diseases. PMID:22433783

Effects of recombinant human prothrombin on thrombin generation in plasma from patients with hemophilia A and B.

PubMed

Hansson, K M; Gustafsson, D; Skärby, T; Frison, L; Berntorp, E

2015-07-01

The present study was carried out to investigate the impact of FII levels, and their increase, on the hemostatic potential in plasma from hemophilia A and B patients with and without inhibitors. Recombinant human factor (F) II (rhFII) was added ex vivo to plasma from 68 patients with hemophilia A and B, with or without inhibitors. The hemostatic potential as measured by thrombin generation (calibrated automated thrombogram [CAT]) was focused on the endogenous thrombin potential (ETP) as it has been shown to correlate with the clinical phenotype of bleeding in hemophilia patients and has also been used to guide bypassing therapy in hemophilia patients with inhibitors before elective surgery. The factor eight inhibitor bypassing agent (FEIBA(®) ) was used as a reference to the clinical situation. The study shows that rhFII concentration-dependently increased ETP by a similar magnitude in hemophilia A and B, both with and without inhibitors. Compared with FEIBA, rhFII showed a shallower concentration-response curve. In both types of hemophilia 100 mg L(-1) of rhFII roughly doubled the ETP. A corresponding response was obtained by 0.5 U mL(-1) of FEIBA. These data support the theory that FII is one of the major components responsible for the efficacy of FEIBA. The data also indicate that rhFII may be useful, alone or in combination with other coagulation factors, in some of the conditions for which FEIBA is used today, although more data are needed to substantiate this. © 2015 International Society on Thrombosis and Haemostasis.

Glucose lowering effect of transgenic human insulin-like growth factor-I from rice: in vitro and in vivo studies

PubMed Central

2011-01-01

Background Human insulin-like growth factor-I (hIGF-I) is a growth factor which is highly resemble to insulin. It is essential for cell proliferation and has been proposed for treatment of various endocrine-associated diseases including growth hormone insensitivity syndrome and diabetes mellitus. In the present study, an efficient plant expression system was developed to produce biologically active recombinant hIGF-I (rhIGF-I) in transgenic rice grains. Results The plant-codon-optimized hIGF-I was introduced into rice via Agrobacterium-mediated transformation. To enhance the stability and yield of rhIGF-I, the endoplasmic reticulum-retention signal and glutelin signal peptide were used to deliver rhIGF-I to endoplasmic reticulum for stable accumulation. We found that only glutelin signal peptide could lead to successful expression of hIGF-I and one gram of hIGF-I rice grain possessed the maximum activity level equivalent to 3.2 micro molar of commercial rhIGF-I. In vitro functional analysis showed that the rice-derived rhIGF-I was effective in inducing membrane ruffling and glucose uptake on rat skeletal muscle cells. Oral meal test with rice-containing rhIGF-I acutely reduced blood glucose levels in streptozotocin-induced and Zucker diabetic rats, whereas it had no effect in normal rats. Conclusion Our findings provided an alternative expression system to produce large quantities of biologically active rhIGF-I. The provision of large quantity of recombinant proteins will promote further research on the therapeutic potential of rhIGF-I. PMID:21486461

Heterotopic ossification after the use of recombinant human bone morphogenetic protein-7

PubMed Central

Papanagiotou, Marianthi; Dailiana, Zoe H; Karachalios, Theophilos; Varitimidis, Sokratis; Hantes, Michael; Dimakopoulos, Georgios; Vlychou, Marianna; Malizos, Konstantinos N

2017-01-01

AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7 (rhBMP-7) for the treatment of nonunions. METHODS Bone morphogenetic proteins (BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients (80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent joints were also carried out. Factors related to the patient (age, gender), the nonunion (location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure (graft and fixation type, amount of rhBMP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ2 test was performed. RESULTS Eighty point nine percent of the nonunions treated with rhBMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients (17.8%) and it was apparent in the routine radiological evaluation of the nonunion site, in a mean time of 5.5 mo after the rhBMP-7 application (range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhBMP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient’s gender was the only important factor for the development of heterotopic ossification (P = 0.007). CONCLUSION Heterotopic ossification after the use of rhBMP-7 in nonunions was common but it did not compromise the final clinical outcome in most cases, and affected only male patients. PMID:28144577

Doxorubicin-loaded glycyrrhetinic acid modified recombinant human serum albumin nanoparticles for targeting liver tumor chemotherapy.

PubMed

Qi, Wen-Wen; Yu, Hai-Yan; Guo, Hui; Lou, Jun; Wang, Zhi-Ming; Liu, Peng; Sapin-Minet, Anne; Maincent, Philippe; Hong, Xue-Chuan; Hu, Xian-Ming; Xiao, Yu-Ling

2015-03-02

Due to overexpression of glycyrrhetinic acid (GA) receptor in liver cancer cells, glycyrrhetinic acid modified recombinant human serum albumin (rHSA) nanoparticles for targeting liver tumor cells may result in increased therapeutic efficacy and decreased adverse effects of cancer therapy. In this study, doxorubicin (DOX) loaded and glycyrrhetinic acid modified recombinant human serum albumin nanoparticles (DOX/GA-rHSA NPs) were prepared for targeting therapy for liver cancer. GA was covalently coupled to recombinant human serum albumin nanoparticles, which could efficiently deliver DOX into liver cancer cells. The resultant GA-rHSA NPs exhibited uniform spherical shape and high stability in plasma with fixed negative charge (∼-25 mV) and a size about 170 nm. DOX was loaded into GA-rHSA NPs with a maximal encapsulation efficiency of 75.8%. Moreover, the targeted NPs (DOX/GA-rHSA NPs) showed increased cytotoxic activity in liver tumor cells compared to the nontargeted NPs (DOX/rHSA NPs, DOX loaded recombinant human serum albumin nanoparticles without GA conjugating). The targeted NPs exhibited higher cellular uptake in a GA receptor-positive liver cancer cell line than nontargeted NPs as measured by both flow cytometry and confocal laser scanning microscopy. Biodistribution experiments showed that DOX/GA-rHSA NPs exhibited a much higher level of tumor accumulation than nontargeted NPs at 1 h after injection in hepatoma-bearing Balb/c mice. Therefore, the DOX/GA-rHSA NPs could be considered as an efficient nanoplatform for targeting drug delivery system for liver cancer.

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

PubMed

Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

2006-04-28

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

Discovery of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi in Latin America.

PubMed

Teixeira, Clarissa; Gomes, Regis; Collin, Nicolas; Reynoso, David; Jochim, Ryan; Oliveira, Fabiano; Seitz, Amy; Elnaiem, Dia-Eldin; Caldas, Arlene; de Souza, Ana Paula; Brodskyn, Cláudia I; de Oliveira, Camila Indiani; Mendonca, Ivete; Costa, Carlos H N; Volf, Petr; Barral, Aldina; Kamhawi, Shaden; Valenzuela, Jesus G

2010-03-23

Sand flies deliver Leishmania parasites to a host alongside salivary molecules that affect infection outcomes. Though some proteins are immunogenic and have potential as markers of vector exposure, their identity and vector specificity remain elusive. We screened human, dog, and fox sera from endemic areas of visceral leishmaniasis to identify potential markers of specific exposure to saliva of Lutzomyia longipalpis. Human and dog sera were further tested against additional sand fly species. Recombinant proteins of nine transcripts encoding secreted salivary molecules of Lu. longipalpis were produced, purified, and tested for antigenicity and specificity. Use of recombinant proteins corresponding to immunogenic molecules in Lu. longipalpis saliva identified LJM17 and LJM11 as potential markers of exposure. LJM17 was recognized by human, dog, and fox sera; LJM11 by humans and dogs. Notably, LJM17 and LJM11 were specifically recognized by humans exposed to Lu. longipalpis but not by individuals exposed to Lu. intermedia. Salivary recombinant proteins are of value as markers of vector exposure. In humans, LJM17 and LJM11 emerged as potential markers of specific exposure to Lu. longipalpis, the vector of Leishmania infantum chagasi in Latin America. In dogs, LJM17, LJM11, LJL13, LJL23, and LJL143 emerged as potential markers of sand fly exposure. Testing these recombinant proteins in large scale studies will validate their usefulness as specific markers of Lu. longipalpis exposure in humans and of sand fly exposure in dogs.

[Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

PubMed

Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

2016-01-01

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

Genome-Wide Fine-Scale Recombination Rate Variation in Drosophila melanogaster

PubMed Central

Song, Yun S.

2012-01-01

Estimating fine-scale recombination maps of Drosophila from population genomic data is a challenging problem, in particular because of the high background recombination rate. In this paper, a new computational method is developed to address this challenge. Through an extensive simulation study, it is demonstrated that the method allows more accurate inference, and exhibits greater robustness to the effects of natural selection and noise, compared to a well-used previous method developed for studying fine-scale recombination rate variation in the human genome. As an application, a genome-wide analysis of genetic variation data is performed for two Drosophila melanogaster populations, one from North America (Raleigh, USA) and the other from Africa (Gikongoro, Rwanda). It is shown that fine-scale recombination rate variation is widespread throughout the D. melanogaster genome, across all chromosomes and in both populations. At the fine-scale, a conservative, systematic search for evidence of recombination hotspots suggests the existence of a handful of putative hotspots each with at least a tenfold increase in intensity over the background rate. A wavelet analysis is carried out to compare the estimated recombination maps in the two populations and to quantify the extent to which recombination rates are conserved. In general, similarity is observed at very broad scales, but substantial differences are seen at fine scales. The average recombination rate of the X chromosome appears to be higher than that of the autosomes in both populations, and this pattern is much more pronounced in the African population than the North American population. The correlation between various genomic features—including recombination rates, diversity, divergence, GC content, gene content, and sequence quality—is examined using the wavelet analysis, and it is shown that the most notable difference between D. melanogaster and humans is in the correlation between recombination and diversity. PMID:23284288

Recombinant methionyl human leptin administration activates signal transducer and activator of transcription 3 signaling in peripheral blood mononuclear cells in vivo and regulates soluble tumor necrosis factor-alpha receptor levels in humans with relative leptin deficiency.

PubMed

Chan, Jean L; Moschos, Stergios J; Bullen, John; Heist, Kathleen; Li, Xian; Kim, Young-Bum; Kahn, Barbara B; Mantzoros, Christos S

2005-03-01

Studies of congenital complete leptin deficiency in animals and humans support a role for leptin in regulating immune function. Whether acquired relative leptin deficiency affects immunological parameters in healthy humans remains unknown. We thus used experimental models of relative leptin deficiency and recombinant methionyl human leptin (r-metHuLeptin) administration in humans to investigate whether r-metHuLeptin would activate signaling pathways in peripheral blood mononuclear cells (PBMCs) and whether acquired relative leptin deficiency and/or increasing circulating leptin levels into the physiologic range would change PBMC subpopulations and cytokines important in the T-helper cell and systemic immune responses. We found that r-metHuLeptin administration to healthy humans activates signal transducer and activator of transcription-3 signaling in PBMCs in vivo. Neither short-term leptin deficiency, induced by 3-d complete fasting, nor physiologic r-metHuLeptin replacement for the same period of time had a major effect on PBMC subpopulations or serum cytokines in healthy men. In contrast, normalizing serum leptin levels over 8 wk in lean women with relative leptin deficiency for 5.1 +/- 1.4 yr (mean +/- se) due to chronic energy deficit increased soluble TNFalpha receptor levels, indicating activation of the TNFalpha system. These findings suggest that relative leptin deficiency due to more long-term energy deprivation is associated with defects in immunological parameters that may be corrected with exogenous r-metHuLeptin administration. Further studies are warranted to assess the implications of acquired relative hypoleptinemia and/or r-metHuLeptin administration on the immunosuppression associated with energy- and leptin-deficient states in humans.

GH safety workshop position paper: A critical appraisal of recombinant human GH therapy in children and adults

USDA-ARS?s Scientific Manuscript database

Recombinant human Growth Hormone (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however, t...

Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP).

USDA-ARS?s Scientific Manuscript database

The ability to rescue an infectious, recombinant, RNA virus from a cDNA clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting enhanced green fluorescent protein (EGFP) gene into the human parainfluenza vi...

Brucella melitensis VirB12 recombinant protein is a potential marker for serodiagnosis of human brucellosis.

PubMed

Mirkalantari, Shiva; Zarnani, Amir-Hassan; Nazari, Mahboobeh; Irajian, Gholam Reza; Amirmozafari, Nour

2017-03-03

The numerous drawbacks of current serological tests for diagnosis of brucellosis which mainly results from cross reactivity with LPS from other gram-negative bacteria have generated an increasing interest to find more specific non-LPS antigens. Previous studies had indicated that Brucella VirB12 protein, a cell surface protein and component of type IV secretion system, induces antibody response during animal infection. However, this protein has not yet been tested as a serological diagnostic marker in human brucellosis. Recombinant VirB12 protein was prepared and evaluated the efficacy of it in an indirect enzyme-linked immunosorbent assay (ELISA) for brucellosis with sera collected from different region of Iran and the results were compared with a commercial ELISA kit. Sera from human brucellosis patients strongly reacted to the purified recombinant VirB12. The sensitivity, specificity, accuracy, negative predictive value and positive predictive value of recombinant VirB12-based ELISA related to the commercial-ELISA method were 87.8, 94, 90, 80 and 96.6% respectively. We concluded that antigenic VirB12 have a property value that can be considered as a candidate for using in serodiagnostic tests for human brucellosis.

Local immunotherapy of spontaneous feline fibrosarcomas using recombinant poxviruses expressing interleukin 2 (IL2).

PubMed

Jourdier, T-M; Moste, C; Bonnet, M-C; Delisle, F; Tafani, J-P; Devauchelle, P; Tartaglia, J; Moingeon, P

2003-12-01

We tested the canarypox virus vector ALVAC and the genetically attenuated vaccinia virus vector NYVAC as vehicles for achieving local immunomodulation in domestic animals bearing spontaneous tumours. Following intratumoral administration of ALVAC-, or NYVAC-luciferase in dogs with melanoma, it was demonstrated that viral recombinants remained localized along the needle track, with no virus detectable in the periphery of the tumour. Given these distribution characteristics and their well-documented safety profile, ALVAC- or NYVAC-based recombinants expressing feline or human IL2, respectively, were administered to domestic cats, in order to prevent the recurrence of spontaneous fibrosarcomas. In the absence of immunotherapy, tumour recurrence was observed in 61% of animals within a 12-month follow-up period after treatment with surgery and iridium-based radiotherapy. In contrast, only 39 and 28% of cats receiving either NYVAC-human IL2 or ALVAC-feline IL2, respectively, exhibited tumour recurrences. Based on such results, and in the context of ongoing clinical studies conducted in humans, we discuss the utilization of ALVAC- or NYVAC-based recombinants as viable therapeutic modalities for local immunotherapy or therapeutic vaccination against cancer, both in humans and companion animals.

Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism.

PubMed

Ruvolo, Giovanni; Roccheri, Maria Carmela; Brucculeri, Anna Maria; Longobardi, Salvatore; Cittadini, Ettore; Bosco, Liana

2013-04-01

An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p<0.05), but we found a significant reduction in patients with high basal DFI values (>15 %), while no significant variation occurred in the patients with DFI values ≤ 15 %. Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 % .

[Experimental study of human umbilical cord blood derived stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1 and GFP].

PubMed

Zhang, Xi; Si, Ying-Jian; Chen, Xing-Hua; Liu, Yao; Gao, Li; Gao, Lei; Peng, Xian-Gui; Wang, Qing-Yu

2008-06-01

This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.

[Administration of activated recombinant factor VII (novo seven) for the control of massive bleeding in gynecology].

PubMed

Tanchev, S; Pandurski, F; Georgiev, A; Gesheva, Iu; Platikanov, V; Dinov, P

2004-01-01

We report our clinical opinion for recombinant activated factor VII (NovoSeven, Novo Nordisk, Copenhagen, Denmark) administration in gynecology patients with massive haemorrhage. 3 women with gynecology deseases and severe bleeding in recieved NovoSeven in bolus IV. The blood loss and laboratory changes in hematology and haemostasis parameters are monitored. The bleeding was ceased in all cases. Decrease in values of Hb, Er and PTT was noted. The use of recombinant factor VIIA in gynecology patients with severe bleeding is effective and safe enough and could be an alternative to the extreme surgical procedures.

To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

PubMed Central

Katz, Samantha S.; Gimble, Frederick S.; Storici, Francesca

2014-01-01

Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy. PMID:24558436

The Role of Recombination in the Origin and Evolution of Alu Subfamilies

PubMed Central

Teixeira-Silva, Ana; Silva, Raquel M.; Carneiro, João; Amorim, António; Azevedo, Luísa

2013-01-01

Alus are the most abundant and successful short interspersed nuclear elements found in primate genomes. In humans, they represent about 10% of the genome, although few are retrotransposition-competent and are clustered into subfamilies according to the source gene from which they evolved. Recombination between them can lead to genomic rearrangements of clinical and evolutionary significance. In this study, we have addressed the role of recombination in the origin of chimeric Alu source genes by the analysis of all known consensus sequences of human Alus. From the allelic diversity of Alu consensus sequences, validated in extant elements resulting from whole genome searches, distinct events of recombination were detected in the origin of particular subfamilies of AluS and AluY source genes. These results demonstrate that at least two subfamilies are likely to have emerged from ectopic Alu-Alu recombination, which stimulates further research regarding the potential of chimeric active Alus to punctuate the genome. PMID:23750218

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

PubMed

Warmerdam, Daniël O; van den Berg, Jeroen; Medema, René H

2016-03-22

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Refined genetic maps reveal sexual dimorphism in human meiotic recombination at multiple scales

NASA Astrophysics Data System (ADS)

Bhérer, Claude; Campbell, Christopher L.; Auton, Adam

2017-04-01

In humans, males have lower recombination rates than females over the majority of the genome, but the opposite is usually true near the telomeres. These broad-scale differences have been known for decades, yet little is known about differences at the fine scale. By combining data sets, we have collected recombination events from over 100,000 meioses and have constructed sex-specific genetic maps at a previously unachievable resolution. Here we show that, although a substantial fraction of the genome shows some degree of sexually dimorphic recombination, the vast majority of hotspots are shared between the sexes, with only a small number of putative sex-specific hotspots. Wavelet analysis indicates that most of the differences can be attributed to the fine scale, and that variation in rate between the sexes can mostly be explained by differences in hotspot magnitude, rather than location. Nonetheless, known recombination-associated genomic features, such as THE1B repeat elements, show systematic differences between the sexes.

Characterization of a New HIV-1 CRF01_AE/ CRF07_BC recombinant virus in Tianjin, China.

PubMed

Zhou, Zhehua; Ma, Ping; Feng, Yi; Ou, Weidong; Qian, Jing; Gao, Liying; Zhang, Defa; Shao, Yiming; Wei, Min

2018-05-04

Human immunodeficiency virus (HIV) is notorious for its rapid evolving since its transmissions from money to human. Currently, HIV contains multiple subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Here, from an HIV-positive mother and her child in Tianjin, China, we identified a novel HIV-1 second-generation recombinant virus (TJ20170316 and TJ20170317) between CRF01_AE and CRF07_BC. Near full-length genomes were obtained from both samples, and they shared very close sequences, except some point mutations. Phylogenetic analyses of the near full-length genomes showed that they consist of CRF01_AE backbone and part CRF07_BC sequences. Recombinant Identification Program (RIP) and Simplot software identified four breakpoints in gag, pol, vif, tat genes in TJ20170316, totally different from other reported CRFs and URFs. The emergence of such URF in Tianjin, China, highlights the complexity of HIV-1 epidemic and more measures should be taken to prevent HIV transmissions.

SPAR1/RTEL1 maintains genomic stability by suppressing homologous recombination

PubMed Central

Barber, Louise J.; Youds, Jillian L.; Ward, Jordan D.; McIlwraith, Michael J.; O’Neil, Nigel J.; Petalcorin, Mark I.R.; Martin, Julie S.; Collis, Spencer J.; Cantor, Sharon B.; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C.; Rose, Ann M.; Boulton, Simon J.

2013-01-01

SUMMARY Inappropriate homologous recombination (HR) can cause gross chromosomal rearrangements that in mammalian cells may lead to tumorigenesis. In yeast, the Srs2 protein is an anti-recombinase that eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has proven to be elusive. In this work, we identify C. elegans SPAR-1 as a functional analogue of Srs2 and describe its vertebrate counterpart, SPAR1/RTEL1, which is required for genome stability and tumour avoidance. We find that spar-1 mutant worms and SPAR1 knockdown human cells share characteristic phenotypes with yeast srs2 mutants, including inviability upon deletion of the sgs1/BLM homologue, hyper-recombination, and DNA damage sensitivity. In vitro, purified human SPAR1 antagonises HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control following deregulation of SPAR1/RTEL1 may be a critical event that drives genome instability and cancer. PMID:18957201

Analysis of HIV-1 intersubtype recombination breakpoints suggests region with high pairing probability may be a more fundamental factor than sequence similarity affecting HIV-1 recombination.

PubMed

Jia, Lei; Li, Lin; Gui, Tao; Liu, Siyang; Li, Hanping; Han, Jingwan; Guo, Wei; Liu, Yongjian; Li, Jingyun

2016-09-21

With increasing data on HIV-1, a more relevant molecular model describing mechanism details of HIV-1 genetic recombination usually requires upgrades. Currently an incomplete structural understanding of the copy choice mechanism along with several other issues in the field that lack elucidation led us to perform an analysis of the correlation between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarity to further explore structural mechanisms. Near full length sequences of URFs from Asia, Europe, and Africa (one sequence/patient), and representative sequences of worldwide CRFs were retrieved from the Los Alamos HIV database. Their recombination patterns were analyzed by jpHMM in detail. Then the relationships between breakpoint distributions and (1) the probability of base pairing, and (2) intersubtype genetic similarities were investigated. Pearson correlation test showed that all URF groups and the CRF group exhibit the same breakpoint distribution pattern. Additionally, the Wilcoxon two-sample test indicated a significant and inexplicable limitation of recombination in regions with high pairing probability. These regions have been found to be strongly conserved across distinct biological states (i.e., strong intersubtype similarity), and genetic similarity has been determined to be a very important factor promoting recombination. Thus, the results revealed an unexpected disagreement between intersubtype similarity and breakpoint distribution, which were further confirmed by genetic similarity analysis. Our analysis reveals a critical conflict between results from natural HIV-1 isolates and those from HIV-1-based assay vectors in which genetic similarity has been shown to be a very critical factor promoting recombination. These results indicate the region with high-pairing probabilities may be a more fundamental factor affecting HIV-1 recombination than sequence similarity in natural HIV-1 infections. Our findings will be relevant in furthering the understanding of HIV-1 recombination mechanisms.

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

PubMed

Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2013-03-01

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy. Georg Thieme Verlag KG Stuttgart · New York.

Effects of Recombinant Activated Factor VII in Traumatic Nonsurgical Intracranial Hemorrhage

DTIC Science & Technology

2006-09-01

with inhibitors to factors VIII and IX, and it is ap- proved in Europe for the treatment of patients with acquired hemophilia, congenital FVII deficiency...GARY P. WRATTEN SURGICAL SYMPOSIUM Effects of Recombinant Activated Factor VII in Traumatic Nonsurgical Intracranial Hemorrhage Christopher E. White...OBJECTIVE: To determine whether treatment with recombi- nant activated factor VII (rFVIIa) will prevent progression of bleeding in nonsurgical

Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behçet's disease.

PubMed

Lee, Kwang Hoon; Chung, Hae-Shin; Kim, Hyoung Sup; Oh, Sang-Ho; Ha, Moon-Kyung; Baik, Ja-Hyun; Lee, Sungnack; Bang, Dongsik

2003-07-01

To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD. The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis). Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be alpha-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant alpha-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human alpha-enolase. BD patient sera positive for anti-alpha-enolase did not react with human gamma-enolase. On dot-blotting, reactivity to human alpha-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant alpha-enolase IgM antibody by ELISA. The alpha-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to alpha-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, alpha-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

PubMed

Mead, Emma J; Masterton, Rosalyn J; Feary, Marc; Obrezanova, Olga; Zhang, Lin; Young, Robert; Smales, C Mark

2015-12-15

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity. © 2015 Authors; published by Portland Press Limited.

Limited infection upon human exposure to a recombinant raccoon pox vaccine vector

USGS Publications Warehouse

Rocke, T.E.; Dein, F.J.; Fuchsberger, M.; Fox, B.C.; Stinchcomb, D.T.; Osorio, J.G.

2004-01-01

A laboratory accident resulted in human exposure to a recombinant raccoon poxvirus (RCN) developed as a vaccine vector for antigens of Yersinia pestis for protection of wild rodents (and other animals) against plague. Within 9 days, the patient developed a small blister that healed within 4 weeks. Raccoon poxvirus was cultured from the lesion, and the patient developed antibody to plague antigen (F1) and RCN. This is the first documented case of human exposure to RCN.

Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.

PubMed Central

Gauldie, J; Richards, C; Harnish, D; Lansdorp, P; Baumann, H

1987-01-01

One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Images PMID:2444978

Production of recombinant adenovirus containing human interlukin-4 gene.

PubMed

Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-11-01

Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics.

Effect of transforming growth factor-beta and growth differentiation factor-5 on proliferation and matrix production by human bone marrow stromal cells cultured on braided poly lactic-co-glycolic acid scaffolds for ligament tissue engineering.

PubMed

Jenner, J M G Th; van Eijk, F; Saris, D B F; Willems, W J; Dhert, W J A; Creemers, Laura B

2007-07-01

Tissue engineering of ligaments based on biomechanically suitable biomaterials combined with autologous cells may provide a solution for the drawbacks associated with conventional graft material. The aim of the present study was to investigate the contribution of recombinant human transforming growth factor beta 1 (rhTGF-beta1) and growth differentiation factor (GDF)-5, known for their role in connective tissue regeneration, to proliferation and matrix production by human bone marrow stromal cells (BMSCs) cultured onto woven, bioabsorbable, 3-dimensional (3D) poly(lactic-co-glycolic acid) scaffolds. Cells were cultured for 12 days in the presence or absence of these growth factors at different concentrations. Human BMSCs attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I and collagen III. No differentiation was demonstrated toward cartilage or bone tissue. The addition of rhTGF-beta1 (1-10 ng/mL) and GDF-5 (10-100 ng/mL) increased cell content (p < 0.05), but only TGF-beta1 also increased total collagen production (p < 0.05) and collagen production per cell, which is a parameter indicating differentiation. In conclusion, stimulation with rhTGF-beta1, and to a lesser extent with GDF-5, can modulate human BMSCs toward collagenous soft tissue when applied to a 3D hybrid construct. The use of growth factors could play an important role in the improvement of ligament tissue engineering.