scholarly journals PTD-mediated delivery of α-globin chain into Κ-562 erythroleukemia cells and α-thalassemic (HBH) patients’ RBCs ex vivo in the frame of Protein Replacement Therapy

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Androulla N. Miliotou ◽  
Dionysia Papagiannopoulou ◽  
Efthymia Vlachaki ◽  
Martina Samiotaki ◽  
Dimitra Laspa ◽  
...  
Keyword(s):  
Replacement Therapy ◽  
Inclusion Bodies ◽  
Ex Vivo ◽  
Research Work ◽  
Size Exclusion ◽  
Globin Chain ◽  
Therapy Approach ◽  
Globin Chains ◽  
Exclusion Chromatography

Abstract Background α-Thalassemia, a congenital hemoglobinopathy, is characterized by deficiency and/or reduced levels of α-globin chains in serious forms of α-thalassemia (HbH disease/Hb Bart’s). This research work deals with a Protein Replacement Therapy approach in order to manage α-thalassemia manifestations, caused by the excess of β-globin chain into HbH RBCs. The main goal was to produce the recombinant human α-globin chain in fusion with TAT, a Protein Transduction Domain, to ex vivo deliver it into HbH patients RBCs, to replace the endogenous missing α-globin chain. Results Cloning of the α-globin coding sequence, fused to the nucleotide sequence of TAT peptide was conducted and the human recombinant fusion proteins, 10xHis-XaSITE-α-globin-HA and 10xHis-XaSITE-TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-XaSITE-α-globin-HA to interact in vitro with the previously produced 10xHis-XaSITE-TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and confirmed by size exclusion chromatography. The recombinant 10xHis-XaSITE-TAT-α-globin-HA was successfully delivered into human proerythroid K-562 cells, during the preliminary transduction evaluation experiments. Finally, the recombinant, TAT-fused α-globin was successfully transduced into RBCs, derived from HbH patients and reduced the formation of HbH-Inclusion Bodies, known to contain harmful β4-globin chain tetramers. Conclusions Our data confirm the successful ex vivo transduction of recombinant α-globin chains in HbH RBCs to replace the missing a-globin chain and reduce the HbH-inclusion bodies, seen in α-thalassemias. These findings broaden the possibility of applying a Protein Replacement Therapy approach to module sever forms of α-thalassemia, using recombinant α-globin chains, through PTD technology.

scholarly journals Restricted immune activation and internalisation of anti-idiotype complexes between drug and antidrug antibodies

2018 ◽  
Vol 77 (10) ◽  
pp. 1471-1479 ◽  
Author(s):  
Karin A van Schie ◽  
Simone Kruithof ◽  
Pleuni Ooijevaar-de Heer ◽  
Ninotska I L Derksen ◽  
Fleur S van de Bovenkamp ◽  
...  
Keyword(s):  
Adverse Events ◽  
Immune Activation ◽  
Immune Complexes ◽  
Ex Vivo ◽  
Biological Activities ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Soluble Immune Complexes ◽  
High Concentrations

ObjectivesTherapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these ‘anti-idiotype’ complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies.MethodsSize distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA.ResultsSize and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions.ConclusionsAnti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

Design and Evaluation of Long Acting Biodegradable PLGA Microspheres for Ocular Drug Delivery

2019 ◽  
Vol 10 ◽  
Author(s):  
Anjali Pandya ◽  
Rajani Athawale ◽  
Durga Puro ◽  
Geeta Bhagwat
Keyword(s):  
Particle Size ◽  
Drug Release ◽  
Degradation Products ◽  
Ex Vivo ◽  
Research Work ◽  
Entrapment Efficiency ◽  
Rational Approach ◽  
Plga Microspheres ◽  
Long Acting

Background: The research work involves development of PLGA biodegradable microspheres loaded with dexamethasome for intraocular delivery. Objective: To design and evaluate long acting PLGA microspheres for ocular delivery of dexamethasone. Method: Present formulation involves the development of long acting dexamethasone loaded microspheres composed of a biodegradable controlled release polymer, Poly(D, L- lactide-co-glycolide) (PLGA), for the treatment of posterior segment eye disorders intravitreally. PLGA with monomer ratio of 50:50 of lactic acid to glycolic acid was used to achieve a drug release up to 45 days. Quality by Design approach was utilized for designing the experiments. Single emulsion solvent evaporation technique along with high pressure homogenization was used to facilitate formation of microspheres. Results: Particle size evaluation, drug content and drug entrapment efficiency were determined for the microspheres. Particle size and morphology was observed using Field Emission Gun-Scanning Electron Microscopy (FEG-SEM) and microspheres were in the size range of 1-5 μm. Assessment of drug release was done using in vitro studies and transretinal permeation was observed by ex vivo studies using goat retinal tissues. Conclusion: Considering the dire need for prolonged therapeutic effect in diseases of the posterior eye, an intravitreal long acting formulation was designed. Use of biodegradable polymer with biocompatible degradation products was a rational approach to achieve this aim. Outcome from present research shows that developed microspheres would provide a long acting drug profile and reduce the frequency of administration thereby improving patient compliance.

scholarly journals A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 157
Author(s):  
Kinga Böszörményi ◽  
Janet Hirsch ◽  
Gwendoline Kiemenyi Kayere ◽  
Zahra Fagrouch ◽  
Nicole Heijmans ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Neutralizing Antibodies ◽  
Size Exclusion ◽  
Vitro Assay ◽  
Usutu Virus ◽  
Transmission Electron ◽  
Exclusion Chromatography ◽  
Efficacious Vaccine ◽  
Human Infections

Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

scholarly journals Erythroblastic Inclusions in Dominantly Inherited β Thalassemias

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 322-328 ◽  
Author(s):  
P. Joy Ho ◽  
Sunitha N. Wickramasinghe ◽  
David C. Rees ◽  
Matthew J. Lee ◽  
Ayed Eden ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Inclusion Bodies ◽  
Serum Transferrin ◽  
Mrna Transcript ◽  
Globin Chain ◽  
Prominent Feature ◽  
Ineffective Erythropoiesis ◽  
Serum Transferrin Receptor ◽  
Globin Chains ◽  
Cellular Pathology

While the precipitation of unstable variant β-globin chains has been implicated as a major pathogenic mechanism in dominantly inherited β thalassemia, their instability and presence in intra-erythroblastic inclusions have not been conclusively shown. We report the investigation of two cases of dominantly inherited β thalassemia due to heterozygosity for the β-codon 121 G-T mutation. In one case, we were able to demonstrate the presence of an abnormal β-globin chain in both peripheral blood reticulocytes and bone marrow erythroblasts, and to assess its stability in relation to the substantial amounts of mutant β mRNA transcript. The serum transferrin receptor (TfR) level was markedly increased, an indication of increased erythropoietic activity. In both cases, we could show by immunoelectron microscopy that the intra-erythroblastic inclusion bodies, a prominent feature of diseases in this category, contained not only precipitated α-globin chains, but also β chains. The data confirm previous suggestions that the cellular pathology underlying this group of β thalassemias is related to the synthesis of highly unstable β-globin chain variants, which fail to form functional tetramers and precipitate intracellularly with the concomitant excess α chains, leading to increased ineffective erythropoiesis.

scholarly journals Asynchronous globin chain synthesis in rabbit reticulocyte lystates induced by hemin-controlled repressor

Blood ◽  
1978 ◽  
Vol 51 (4) ◽  
pp. 653-658 ◽  
Author(s):  
RS Franco ◽  
JW Hogg ◽  
OJ Martelo
Keyword(s):  
Globin Chain ◽  
Free System ◽  
Globin Chains ◽  
Reticulocyte Lysate ◽  
Chain Imbalance ◽  
Globin Synthesis ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

Abstract To define further the role of hemin-controlled repressor (HCR) in globin synthesis, we studied its effect on the synthesis of individual globin chains in a rabbit reticulocyte lysate cell-free system. In the presence of HCR there was a marked globin chain imbalance, resulting in a lowered alpha/beta ratio. These findings in vitro may have relevance to certain clinical heme deficiency states in which a similar globin chain imbalance has been observed.

scholarly journals Globin-chain specificity of oxidation-induced changes in red blood cell membrane properties

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1586-1592 ◽  
Author(s):  
SL Schrier ◽  
N Mohandas
Keyword(s):  
Membrane Damage ◽  
Membrane Skeleton ◽  
Material Behavior ◽  
Membrane Properties ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Globin Chain ◽  
Globin Chains ◽  
Alpha Globin

Abstract We have previously shown that excess unpaired alpha- and beta-globin chains in severe alpha- and beta-thalassemia interacting with the membrane skeleton induce different changes in membrane properties of red blood cells (RBCs) in these two phenotypes. We suggest that these differences in membrane material behavior may reflect the specificity of the membrane damage induced by alpha- and beta-globin chains. To further explore this hypothesis, we sought in vitro models that induce similar membrane alterations in normal RBCs. We found that treatment of normal RBCs with phenylhydrazine produced rigid and mechanically unstable membranes in conjunction with selective association of oxidized alpha-globin chains with the membrane skeleton, features characteristic of RBCs in severe beta-thalassemia. Methylhydrazine, in contrast, induced selective association of oxidized beta-globin chains with the membrane skeleton and produced rigid but hyperstable membranes, features that mimicked those of RBCs in severe alpha- thalassemia. These findings suggest that consequences of oxidation induced by globin chains are quite specific in that those agents that cause alpha-globin chain accumulation at the membrane produce rigid but mechanically unstable membranes, whereas membrane accumulation of beta- globin chains results in rigid but mechanically stable membranes. These in vitro experiments lend further support to the hypothesis that membrane-associated alpha- and beta-chains induce oxidative damage to highly specific different skeletal components and that the specificity of this skeletal damage accounts for the differences in material membrane properties of these oxidatively attacked RBCs and perhaps of alpha- and beta-thalassemic RBCs as well.

scholarly journals Isolation, characterization and in vitro anti-salmonellal activity of compounds from stem bark extract of Canarium schweinfurthii

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Jean Baptiste SOKOUDJOU ◽  
Olubunmi ATOLANI ◽  
Guy Sedar Singor NJATENG ◽  
Afsar KHAN ◽  
Cyrille Ngoufack TAGOUSOP ◽  
...  
Keyword(s):  
Gallic Acid ◽  
Food Poisoning ◽  
Ethanolic Extract ◽  
Stem Bark ◽  
Size Exclusion ◽  
Bark Extract ◽  
Main Stream ◽  
Exclusion Chromatography ◽  
Stream Control

Abstract Background Bacteria belonging to the Salmonella genus are major concern for health, as they are widely reported in many cases of food poisoning. The use of antibiotics remains a main stream control strategy for avian salmonellosis as well as typhoid and paratyphoid fevers in humans. Due to the growing awareness about drug resistance and toxicities, the use of antibiotics is being discouraged in many countries whilst advocating potent benign alternatives such as phyto-based medicine. The objective of this work was to isolate, characterise the bioactive compounds of Canarium schweinfurthii; and evaluate their anti-salmonellal activity. Methods The hydro-ethanolic extract of Canarium schweinfurthii was fractionated and tested for their anti-salmonellal activity. The most active fractions (i.e. chloroform and ethyl acetate partition fractions) were then explored for their phytochemical constituents. Fractionation on normal phase silica gel column chromatography and size exclusion chromatography on Sephadex LH-20 led to the isolation of four compounds (maniladiol, scopoletin, ethyl gallate and gallic acid) reported for the first time in Canarium schweinfurthii. Results Result indicated that scopoletin and gallic acid had greater activity than the crude extracts and partition fractions. Among the isolated compounds, scopoletin showed the highest inhibitory activity with a MIC of 16 μg/ml against Salmonella Typhimurium and Salmonella Enteritidis. Conclusions The overall results of this study indicates that the hydro-ethanolic extract as well as some of isolated compounds have interesting anti-salmonellal activities that could be further explored for the development of potent therapy for salmonellosis. Furthermore, the study adds credence to the folkloric applications of the plant.

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