ABSTRACT
Obesity related glomerulopathy (ORG), as a disease with an increasing incidence of metabolic kidney injury, has become a new hot spot in today's society. A variety of factors are involved in the occurrence and development of ORG. Among them, the ectopic deposition of kidney lipids is not only a significant feature of ORG, but also a key link to promote the progress of ORG. This article reviews the related mechanisms of lipid deposition in ORG and the treatment of ORG with lipid deposition as the target.
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An enzyme linked immunosorbent assay ( ELISA ) and a flow cytometry assay ( FCA ) based on Wil2-S cells were developed and systematically compared for quantification of recombinant anti-CD20 humanized monoclonal antibody ( rh-anti-CD20zumab) in biological matrix. The specificity, precision and accuracy of each method at correspondingly different linear range showed good results. For ELISA, the precisions of intra-day and inter-day were both <19 . 5%, the relative error was from-18 . 2% to 17 . 6%;For FCA, the precisions of intra-day and inter-day were both <19. 0%, the relative error was from -18. 9% to 18. 4%. The sensitivity of ELISA was significantly higher than that of FCA. The quantitative ranges of ELISA and FCA methods were 0. 04-5. 0 mg/L and 3. 1-200 mg/L, respectively. The concentrations in serum samples and pharmacokinetics analysis were determined by both of two methods after vein drip administration of rh-anti-CD20zumab in rhesus monkeys. Pharmacokinetics data showed that there was excellent consistency between results obtained by two methods at the given dose. We believe that the novel FCA with high speed and high sensitivity can be used to perform PK and PD study of cell surface antigen-targeted antibody derivatives.
ABSTRACT
As targeted drugs to B-cell malignancies, anti-CD20 monoclonal antibodies have been proved to be important in therapeutic antibody field. With three generations in more than ten years' development, the structures of these drugs have been improved, and many new indications have been found. Nowadays, these kinds of antibodies are not only used in the treatment of lymphoid malignancies, but also been proved to be useful in some autoimmune diseases treatment, and their new indications are still being expanded. With the optimization of their clinical dosage regimens, drug reaction has been increased, thus, therapeutic and side effects of anti-CD20 monoclonal antibody have been further improved as well. However, the exact mechanism of action of their combination therapy with other chemical drugs is still unclear, which remains to be further studied. This article reviewed new development of anti-CD20 therapeutic monoclonal antibodies research in recent years.
ABSTRACT
As targeted drugs to B-cell malignancies, anti-CD20 monoclonal antibodies have been proved to be important in therapeutic antibody field. With three generations in more than ten years' development, the structures of these drugs have been improved, and many new indications have been found. Nowadays, these kinds of antibodies are not only used in the treatment of lymphoid malignancies, but also been proved to be useful in some autoimmune diseases treatment, and their new indications are still being expanded. With the optimization of their clinical dosage regimens, drug reaction has been increased, thus, therapeutic and side effects of anti-CD20 monoclonal antibody have been further improved as well. However, the exact mechanism of action of their combination therapy with other chemical drugs is still unclear, which remains to be further studied. This article reviewed new development of anti-CD20 therapeutic monoclonal antibodies research in recent years.
Subject(s)
Humans , Antibodies, Monoclonal , Therapeutic Uses , Antibodies, Monoclonal, Humanized , Therapeutic Uses , Antigens, CD20 , Allergy and Immunology , Antineoplastic Agents , Therapeutic Uses , Arthritis, Rheumatoid , Drug Therapy , Leukemia, Lymphocytic, Chronic, B-Cell , Drug Therapy , Lymphoma, Non-Hodgkin , Drug Therapy , Rituximab , Therapeutic UsesABSTRACT
Objective To obtain the disulfide stabilized Fv fragment (dsFv) against N-terminal fragment of human lip polysaccharide binding protein (NH-LBP) and to identify its biological vitality. Methods The disulfide stabilized Fv fragment antibody (dsFv) was obtained after the inclusion bodies of dsFvVH and dsFvVL had been refolded and purified. Then the characteristics of dsFv were determined in vitro by ELISA and by detecting the secretion of tumor necrosis factor alpha (TNF-?) in rats. Results There was 2.1 mg protein of dsFv obtained. dsFv had good combination with NH-LBP and could restrain inflammatory reaction caused by lip polysaccharide (LPS) in vivo. Conclusion It is feasible to get dsFv against NH-LBP by respective expression of VL and VH. The partial inhibition of the biological function of LBP by dsFv is a new way to restrain the over-inflammatory reaction in vivo.
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Objective To introduce the mutated gene coding cysteine into the gene of dsFv VL of human antibody to N terminal fragment of lipopolysaccharide binding protein(LBP)and to express,purify the mutated dsFv VL in bacterium.Methods We reconstructed and sequenced the mutated gene of VL of human mAb Fab to LBP by Mega-primer PCR based on point mutagenesis method.Some codes of FWR1 of VL had been replaced by TGT in order to code cysteine.The DNA sequence of reconstructed VL was inserted into vector pET-28a(+),then VL was expressed by E.coli.BL21 star(DE3)and was purified by chromatography.Finally the activity of VL to bind NH-LBP was determined by ELISA.Results The results showed that the cysteine was introduced into the position 21 amino acid of VL to replace the threonine.The gene of VL was about 650 bp and relative molecular weight of VL was 28?103.VL could bind NH-LBP directly.Conclusion These have laid a foundation for producing the dsFv against NH-LBP.
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Objective To employ 2 approaches to construct and express reconstructed LL-37 (rLL-37) in procaryotic system, and to explore a better preparation method. Methods The first method: the rLL-37 was inserted into vector pET-28a (+), then was induced to express in E.coli. BL21 (DE3) and purified by chromatography; the second method: the rare codons in the rLL-37 gene sequence were substituted by the preferred codons of procaryotic cell, and a fragment of carrier protein molecule (CPM) was added to the N termination of the objective sequence to construct expression plasmid pET-30a(+)-CPM-rLL-37, then the rLL-37 was expressed in E.coli. BL21 Star(DE3) and purified by chromatography. The productive rates of the 2 methods were compared and the antimicrobial effects of obtained rLL-37 was studied. Results The first method: the DNA sequence of rLL-37 was obtained successively by Touch-Down PCR. The expression plasmid pET-30a(+)-CPM-rLL-37 was expressed with fusion protein in E.coli BL21 (DE3). The expression rate accounted for 20% of total bacterio-protein, then the expressed product was purified by using high positive ion exchange column Macro-Prep High S; The second method: a fragment of carrier protein molecule was designed that contained 28 amino-acid residue and its pHi was 2.7, net charge was-6.0 at pH 7.4. After the expression plasmid pET-30a(+)-CPM-rLL-37 was constructed successively, it was expressed in E.coli BL21 Star (DE3). The expressed fusion protein accounted for 35% of total bacterio-protein, then the expressed product was purified by using affinity binding chromatography with TALON resins successfully. The obtained 2 kinds of rLL-37 were able to kill both Gram-negative and-positive bacteria by the means of inhibitory zone. Conclusion It’s feasible to prepare efficiently rLL-37 in procaryotic system, which founds the basis for the further research on bactericidal activity of rLL-37.
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Objective To obtain and identify monoclonal antibody against human lipopolysaccharide binding protein (LBP). Methods Three clones of antibody with better activity against human LBP were isolated after human antibody library screening by human LBP and 5 rounds of enrichment. After plasmid extracting, geneⅢ cutting, self-recircling and electric transforming, soluble Fab was expressed in E.coli. XL1-blue by the induction of IPTG and its characteristics were determined. Results The positive antibody was concentrated to 8.16?104 folds and the highest activity of the three clones was 106 . Conclusion Phage antibody against human LBP has been obtained successfully, which lays a solid foundation for researching its function.
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Objective To reconstruct the proteinic sequence of human cathelicidin LL-37 to increase the bactericidal activity of LL-37 and to express the reconstructed LL-37 (rLL-37) in bacterium. Methods The two dimensional structure, three dimensional structure and chemical characteristic of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL. Without the three dimensional changes of LL-37, some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased. According to the proteinic sequence changes of rLL-37, the DNA sequence of rLL-37 was reconstructed by Touch-Down PCR and recombined with vector pET-28a (+), thus rLL-37 was expressed in E.coli. BL21 (DE3) by the induction of IPTG and was purified by chromatography. Results Glu~ 16 , Asp~ 26 , Glu~ 36 of LL-37 were replaced by Gln~ 16 , Asn~ 26 , Gln~ 36 and the static charge of LL-37 was increased from +5.8 to +9.0 at pH 7.4. The DNA sequence of rLL-37 was reconstructed and inserted into vector pET-28a (+), the rLL-37 was expressed in E.coli. BL21 (DE3) and purified by strong cation exchange supports Macro-Prep High S successfully. The rLL-37 was proved by the means of inhibitory zone to be able to kill Gram-negative bacteria and Gram-positive bacteria. Conclusion It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion, which make it possible to produce more rLL-37 and study its biological function deeply.
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Objective To study the effects of NEP1-40 on axon regeneration, motor function recovery of affected limbs and RhoA signal pathway in cerebral ischemia-reperfusion rats for exploring the possible mechanisms. Methods A total of 60 adult male Sprague-Dawley (SD) rats were equally divided into sham operation group (sham group), cerebral ischemia-reperfusion control group (control group), intra-lateral ventricle injection of PBS group (PBS group), and intra-lateral ventricle injection of NEP1-40 group (NEP1-40 group). The middle cerebral artery ischemia-reperfusion model (MCAI/R) was established by nylon monofilament occlusion method in rats. The changes in growth associated protein-43 (GAP-43) and RhoA expressions were determined by Western blotting technique. The motor function of affected limbs was tested by the "staircase test" of Montoya’s design. Results ① The expression of GAP-43 in NEP1-40 group was higher than that in the control and the PBS groups at 7 d and 14 d after MCAI/R, peaking at 7 d; ② The expression of RhoA in NEP1-40 group was significantly lower than that in the control and the PBS groups at each time point; ③ The results of "staircase test" in NEP1-40 group were much higher than those in the control group and the PBS groups at each time point after MCAI/R. Conclusion NEP1-40 can improve axon regeneration in cerebral ischemia-reperfusion rats and promote the recovery of motor function. The mechanism may be associated with the inhibition of RhoA signal pathway.