Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization

A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable X P^ promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product

Postoperative ileus concealing intra-abdominal complications in enhanced recovery programs—a retrospective analysis of the GRACE database

Purpose Postoperative ileus (POI) occurrence within enhanced recovery programs (ERPs) has decreased. Also, intra-abdominal complications (IAC) such as anastomotic leakage (AL) generally present late. The aim was to characterize the link between POI and the other complications occurring after surgery. Methods This retrospective analysis of a prospective database was conducted by the Francophone Group for Enhanced Recovery after Surgery. POI was considered to be present if gastrointestinal functions had not been recovered within 3 days following surgery or if a nasogastric tube replacement was required. Results Of the 2773 patients who took part in the study, 2335 underwent colorectal resections (83.8%) for cancer, benign tumors, inflammatory bowel disease, and diverticulosis. Among the 2335 patients, 309 (13.2%) experienced POI, including 185 (59.9%) cases of secondary POI. Adjusted for well-known risk factors (male gender, need for stoma, right hemicolectomy, surgery duration, laparotomy, and conversion to open surgery), POI was associated with abdominal complications (OR = 4.55; 95% confidence interval (CI): 3.30–6.28), urinary retention (OR = 1.75; 95% CI: 1.05–2.92), pulmonary complications (OR = 4.55; 95% CI: 2.04–9.97), and cardiological complications (OR = 3.01; 95% CI: 1.15–8.02). Among the abdominal complications, AL and IAC were most strongly associated with POI (respectively, OR = 5.97; 95% CI: 3.74–8.88 and OR = 5.76; 95% CI: 3.56–10.62). Conclusion Within ERPs, POI should not be considered as usual. There is a significant link between POI and IAC. Since POI is an early-onset clinical sign, its occurrence should alert the physician and prompt them to consider performing CT scans in order to investigate other potential morbidities

PPARβ/δ directs the therapeutic potential of mesenchymal stem cells in arthritis

Objectives To define how peroxisome proliferator-activated receptor (PPAR) β/δ expression level in mesenchymal stem cells (MSCs) could predict and direct both their immunosuppressive and therapeutic properties. PPARβ/δ interacts with factors such as nuclear factor-kappa B (NF-κB) and regulates the expression of molecules including vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1. Since these molecules are critical for MSC function, we investigated the role of PPARβ/δ on MSC immunosuppressive properties. Methods We either treated human MSCs (hMSCs) with the irreversible PPARβ/δ antagonist (GSK3787) or derived MSCs from mice deficient for PPARβ/δ (PPARβ/δ−/− MSCs). We used the collagen-induced arthritis (CIA) as model of immune-mediated disorder and the MSC-immune cell coculture assays. Results Modulation of PPARβ/δ expression in hMSCs either using GSK3787 or hMSCs from different origin reveals that MSC immunosuppressive potential is inversely correlated with Ppard expression. This was consistent with the higher capacity of PPARβ/δ−/− MSCs to inhibit both the proliferation of T lymphocytes, in vitro, and arthritic development and progression in CIA compared with PPARβ/δ+/+ MSCs. When primed with proinflammatory cytokines to exhibit an immunoregulatory phenotype, PPARβ/δ−/− MSCs expressed a higher level of mediators of MSC immunosuppression including VCAM-1, ICAM-1 and nitric oxide (NO) than PPARβ/δ+/+ MSCs. The enhanced NO2 production by PPARβ/δ−/− MSCs was due to the increased retention of NF-κB p65 subunit on the κB elements of the inducible nitric oxide synthase promoter resulting from PPARβ/δ silencing. Conclusions Our study is the first to show that the inhibition or knockdown of PPARβ/δ in MSCs primes their immunoregulatory functions. Thus, the regulation of PPARβ/δ expression provides a new strategy to generate therapeutic MSCs with a stable regulatory phenotype