Abstract Growth hormone deficiency results in growth retardation in children and the GH deficiency syndrome in adults and they need to receive recombinant-GH in order to rectify the GH deficiency symptoms. Mammalian cells have become the favorite system for production of recombinant proteins for clinical application compared to prokaryotic systems because of their capability for appropriate protein folding, assembly, post-translational modification and proper signal. However, production level in mammalian cells is generally low compared to prokaryotic hosts. Taguchi has established orthogonal arrays to describe a large number of experimental situations mainly to reduce experimental errors and to enhance the efficiency and reproducibility of laboratory experiments. In the present study, rhGH was produced in CHO cells and production of rhGH was assessed using Dot blotting, western blotting and Elisa assay. For optimization of rhGH production in CHO cells using Taguchi method An M16 orthogonal experimental design was used to investigate four different culture components. The biological activity of rhGH was assessed using LHRE-TK-Luciferase reporter gene system in HEK-293 and compared to the biological activity of prokaryotic rhGH. A maximal productivity of rhGH was reached in the conditions of 1%DMSO, 1%glycerol, 25 µM ZnSO4 and 0 mM NaBu. Our findings indicate that control of culture conditions such as the addition of chemical components helps to develop an efficient large-scale and industrial process for the production of rhGH in CHO cells. Results of bioassay indicated that rhGH produced by CHO cells is able to induce GH-mediated intracellular cell signaling and showed higher bioactivity when compared to prokaryotic GH at the same concentrations.
Erythrocytes are impressive tools for drug delivery, especially to macrophages. Therefore, berberine was loaded into erythrocytes using both hypotonic pre-swelling and endocytosis methods to target macrophages. Physicochemical and kinetic parameters of the resulting carrier cells, such as drug loading/release kinetics, osmotic fragility, and hematological indices, were determined. Drug loading was optimized for the study using Taguchi experimental design and lab experiments. Loaded erythrocytes were targeted to macrophages using ZnCl
A novel infectious disease, caused by 2019 Novel Coronavirus (2019-nCoV) is responsible for the recent outbreak of severe respiratory disease. The 2019-nCoV spread rapidly and reaching epidemic proportions in many countries of the world. ACE2 was identified as a key receptor for 2019-nCoV infections. Excessive form of soluble ACE2 rescues cellular ACE2 activity which has a protective role in acute lung failure and neutralizes the virus. The short half-life of ACE2 is a major limitation to its practical application. Nanoparticle-based drug delivery systems are one of the most widely investigated approaches for developing novel therapies for a variety of diseases. Nevertheless, nanoparticles suffer from the rapid removal from the bloodstream by the reticuloendothelial system (RES). A noncovalent attachment of nanoparticles to RBCs increases their half-life in blood and allows transient accumulation in the lungs, while decreases their uptake by the liver and spleen. Connecting the recombinant ACE2 into the surface of nanoparticles that were attached to RBCs can be a potential therapeutic approach for 2019-nCoV infection through increasing their lung targeting to naturalize the virus and also acting as a bioreactor in the blood circulation to decrease serum level of Angiotensin II and protects lungs from injury/ARDS.
Abstract Development of new growth hormone (GH) agonists and antagonists (GHA) requires animal models for pre-clinical testing. Ideally, effects of treatment can be monitored using the same pharmacodynamic marker later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I - the most sensitive pharmacodynamic marker for GH-action in humans - shows no response to recombinant human GH (rhGH) treatment and there is little evidence for effects of GHA except when administered at very high doses or overexpressed. As an alternative, more suitable model we explored pharmacodynamic markers of GH action in intact rabbits. We performed the first validation of an IGF-I assay for rabbit serum and tested precision, sensitivity, linearity, and recovery using an automated human IGF-I assay (IDS-iSYS). Furthermore, IGF-I was measured in rabbits of different strains, age groups and sexes, and we monitored IGF-I response to treatment with rhGH or GHA. In a subset of samples we used LC-MS/MS to measure IGF-I and quantitative Western-ligand blot to analyze IGF-binding proteins. Results: Although recovery of recombinant rabbit IGF-I was only 50% in the human IGF-I assay, sensitivity, precision (1.7-3.3%CV) and linearity (90.4-105.6%) were excellent in rabbit samples. As expected, sex, age and genetic background were major determinants of IGF-I in rabbits. IGF-I and IGFBP-2 levels increased after single and multiple rhGH injections (IGF-I: 286±22 vs. 434±26ng/ml; p<0.01) and were highly correlated (p<0.0001). GHA treatment lowered IGF-I from the fourth injection onwards (p<0.01). In summary, we demonstrated that the IDS-iSYS IGF-I immunoassay can be used in rabbits. Similar to rodents, rabbits display variations in IGF-I depending on sex, age and genetic background. Unlike in rodents, the IGF-I response to rhGH or GHA treatment closely mimics the pharmacodynamics seen in humans suggesting rabbits as a suitable new model to test human GH agonists and antagonists.
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