Preliminary study on the safety and efficacy of a new polymyxin B-immoblized resin column in treatment of LPS-induced sepsis beagles.

BACKGROUND: This study aims to assess the safety and efficacy of direct hemoperfusion using a new polymyxin B-immobilized resin column (disposable endotoxin adsorber, KCEA) in an endotoxin/ lipopolysaccharide (LPS)-induced sepsis model. METHODS: Eighteen beagles were randomized into 1 intervention group (KCEA group, n = 6) and 2 control groups (sham group and model group, n = 6 each). Sepsis was induced by continuous intravenous application of 0.5 mg/kg body weight of endotoxin for 60 min. An extracorporeal hemoperfusion device made with KCEA for endotoxin adsorption was used. Model group beagles received standard treatment with fluids and vasoactive drugs, KCEA group beagles received standard treatment and direct hemoperfusion of KCEA for 2 h, and sham group beagles were treated with standard treatment and direct hemoperfusion of a sham column for 2 h. RESULTS: Good blood compatibility of KCEA was confirmed by assessing clinical parameters. Blood endotoxin peak levels in the KCEA group were significantly lower, resulting in a significant suppression of IL-6, TNF-α and procalcitonin, which improved mean arterial pressure and significantly lowered vasopressor demand, thereby protecting organ function and improving survival time and rate. In the KCEA group, MAP was significantly higher over 6 h than those recorded both in the sham group and model group. The 7-day survival rates of the KCEA, sham and model groups were 50%, 0% and 0%, respectively. CONCLUSION: KCEA hemoadsorption was effective at detoxifying circulatory endotoxin and inflammatory mediators and contributed to the decreased mortality rate in the sepsis beagles.

Bioinformatics analysis of differentially expressed proteins in alcoholic fatty liver disease treated with recombinant human cytoglobin.

Cytoglobin (Cygb) is a globin molecule that is ubiquitously expressed in all tissues and has a protective role under oxidative stress. It has also been demonstrated to be effective in the treatment of alcoholic fatty liver disease (AFLD). In order to study the molecular mechanisms underlying its beneficial effects for the treatment of alcoholic liver, two­dimensional electrophoresis and mass spectrometric analysis were performed on serum and liver tissues from an in vivo rat model of AFLD. A total of 26 differentially expressed proteins were identified in the serum and 20 differentially expressed proteins were identified in liver specimens. Using online bioinformatics tools, it was indicated that these differentially expressed proteins were primarily associated with pathways including binding and uptake of ligands by scavenger receptors, response to corticosteroid, plasma lipoprotein remodeling, regulation of complement cascade, hydrogen peroxide catabolic process, as well as response to nutrient and monosaccharide. The present results suggested that recombinant human Cygb exerts its role in the treatment of AFLD primarily through affecting nutrient metabolism, monocarboxylic acid biosynthesis, regulation of glutathione expression, plasma lipoprotein remodeling and removal of metabolic waste from the blood.

[Preparation and identification of mouse monoclonal antibodies against human IgE].

Objective To prepare the high-affinity and high-specificity mouse anti-human IgE monoclonal antibody (mAb) as a candidate immunosorbent. Methods BALB/c mice were immunized using recombinant antigen IgECepsilon2-4. Coated ELISA plate with IgECepsilon2-4 was used to screen positive cell lines by indirect ELISA, then coated ELISA plate with natural IgE, IgG, IgM, IgA, IgD to detect the affinity and specificity of serum-free culture supernatant of positive hybridoma cells with natural IgE. The cell line stably secreting specific anti-IgE monoclonal antibodies was expanded and inoculated into the abdominal cavity of BALB/c mice to prepare mAbs. The secreted mAbs were identified by ELISA kit followed by the identification of mAb subtypes. Results The 29 positive hybridoma cells were obtained after five cell fusions, of which 11 strains had strong affinity with natural IgE and 2 strains did not cross-react with other immunoglobulins 3E9 and 7B4. Conclusion The study successfully prepared mAbs against human IgE with high titer, affinity and specificity.

[Eukaryotic expression, purification of human IgECepsilon2-4 protein and its target fishing].

Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region (IgECepsilon2-4) and purify the recombinant protein, and then capture its interacted proteins by surface plasmon resonance (SPR). Methods Three recombinant eukaryotic expression vectors of IgECepsilon2-4 containing different signal peptides were constructed and transiently transfected into HEK293FT suspension cells separately. The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount, and then the recombinant protein was purified by Ni-NTA affinity chromatography. The interaction between high-affinity IgE receptor (FcepsilonR I) of KU812 cell surface and IgECepsilon2-4 was identified by immunofluorescence cytochemistry. The unknown proteins that specifically interacted with IgECepsilon2-4 were captured from human serum by SPR technique. Results The recombinant plasmid containing the signal peptide III showed the highest expression (6.2 mg/L). Highly purified recombinant protein IgECepsilon2-4 was obtained by affinity purification. Immunofluorescence cytochemistry showed that the recombinant protein IgECepsilon2-4 could be combined with the surface receptor of KU812 cells. Thirty-nine kinds of proteins which were likely to interact with IgECepsilon2-4 were captured from human serum by SPR. Conclusion We obtained the purified recombinant protein IgECepsilon2-4 that could be combined with KU812 cell surface receptor. Target fishing experiment revealed that the recombinant protein IgECepsilon2-4 was likely to interact with 39 kinds of proteins in human serum.