The macrophage is critical to the innate immune response and contributes to human diseases, including inflammatory arthritis and plaque formation in atherosclerosis. Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that is produced by macrophages. To study the regulation of VEGF production in macrophages we show that stimulation of monocyte‐macrophage‐like RAW‐264.7 cells by lipopolysaccharide (LPS) increases expression of VEGF mRNA and protein. Three alternative splicing VEGF mRNA isoforms are produced, and the stability of VEGF mRNA increases following cellular activation. To study post‐transcriptional regulation of the VEGF gene the 3′‐untranslated region (3′ UTR) was introduced into the 3′ UTR of the luciferase gene in a reporter construct. In both RAW‐264.7 cells and thioglycollate‐elicited macrophages, the 3′ UTR sequence dramatically reduces reporter expression. Treatment with activators of macrophages, including LPS, lipoteichoic acid, and VEGF protein, stimulates expression of 3′ UTR reporters. Finally, mapping studies of the 3′ UTR of VEGF mRNA show that deletion of the heterogeneous nuclear ribonucleoprotein l binding site affects basal reporter expression in RAW‐264.7 cells, but does not affect reporter activation with LPS. Together these results demonstrate that a post‐transcriptional mechanism contributes to VEGF gene expression in activated macrophage cells.

Vascular endothelial growth factor (VEGF) expression is regulated by sequence elements in the 3′ UTR of VEGF mRNA. AUF1/hnRNP D suppresses VEGF 3′ UTR–dependent expression. Peptides with arginine–glycine–glycine motifs derived from AUF1 also suppress VEGF expression.

Background: To evaluate whether metformin use assuredly alters overall all-cause death in patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD). Methods: Pubmed, Web of Science, Embase, and Cochrane Central Register of Controlled Trials were systematically searched from inception to Feb. 29, 2020 with no language restriction. All related articles comparing all-cause death of T2DM and CKD patients after metformin use (monotherapy or combination) versus non-metformin treatment were identified. Pooled risk ratios (RR) and 95% confidence intervals (CI) were computed using random-effects models regardless of the heterogeneity quantified by Cochrane c 2 and I 2 statistics. Results: Totally 13 studies (9 cohort studies [CSs], 3 subanalyses or post-hoc analyses of randomized controlled trials [RCTs], and 1 nested case-control article) involving 303,540 patients were included. Metformin-based treatments relative to any other measure displayed significantly lower risks of all-cause mortality (Pooled RRs 0.71, 95%CI 0.61 to 0.84; I 2 = 79.0%) and cardiovascular events (Pooled RRs 0.76, 95%CI 0.60 to 0.97; I 2 = 87.0%) in CKD patients at stage G1-3, with substantial heterogeneity. Metformin use was not significantly related with these end points in advanced CKD patients. Conclusions: Metformin use is connected with significantly less risks of all-cause mortality and cardiovascular events in patients with T2DM and mild/moderate CKD. However, RCTs with large sample sizes are warranted in the future to assess whether these key benefits extend to later stages of CKD by dose adjustment.

Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs.

Monovalent bispecific antibodies (BsAbs) are projected to have broad clinical applications due to their ability to bind two different targets simultaneously. Although they can be produced using recombinant technologies, the correct pairing of heavy and light chains is a significant manufacturing problem. Various approaches exploit mutations or linkers to favor the formation of the desired BsAb, but a format using a single common light chain has the advantage that no other modification to the antibody is required. This strategy reduces the number of formed molecules to three (the BsAb and the two parent mAbs), but the separation of the BsAb from the two monovalent parent molecules still poses a potentially difficult purification challenge. Current methods employ ion exchange chromatography and linear salt gradients, but are only successful if the difference in the observed isoelectric points (pIs) of two parent molecules is relatively large. Here, we describe the use of highly linear pH gradients for the facile purification of common light chain BsAbs. The method is effective at separating molecules with differences in pI as little as 0.10, and differing in their sequence by only a single charged amino acid. We also demonstrate that purification resins validated for manufacturing are compatible with this approach.

As the only effective energy substrate in the absence of O2, glucose (Glc) is central to all cellular strategies for hypoxic adaptation. Obligatory reliance on Glc also explains associated induction of glycolytic enzymes such as hexokinases which catalyze the first committed step of Glc metabolism. HIF1α contributes to HK2 expression in cancer cells, but the specific cis‐acting sequences are undefined and corresponding effects in nontransformed cells are unexplored. We therefore used chimeric reporter genes to delineate hypoxia‐responsive cis‐acting HK2 promoter regions in nontransformed NRK‐52E cells.Major hypoxic control of HK2 expression mapped to a phylogenetically conserved tandem pair of canonical HIF‐binding motifs just 5' to the 157 bp basal promoter region. Targeted mutagenesis of either motif abrogated hypoxia‐responsiveness, as did their spatial separation by a half (+5 bp) ‐ but not a full (+10 bp) ‐ helical turn. Similar results with hypoxia‐mimetics (e.g. CoCl2) and stabilized mutants of HIF1α ‐ but not HIF2α ‐ suggest HIF1α selectivity. Independent hypoxic enhancer activity responsive to both HIF1α and HIF2α ‐ but not other known HK2 transcriptional regulators ‐ was also observed within 90 bp of the transcription start site. These results suggest complex hypoxic gene regulation involving both combinatorial interactions and cis‐acting elements with differing HIFα isoform specificities.

No abstract

No abstract