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Lymphocytes within the intestinal epithelial layer (IEL) in mammals have unique composition compared with their counterparts in the lamina propria. Little is known about the role of some of the key colonic IEL subsets, such as TCRαβ+CD8+ T cells, in inflammation. We have recently described liver-enriched innate-like TCRαβ+CD8αα regulatory T cells, partly controlled by the non-classical MHC molecule, Qa-1b, that upon adoptive transfer protect from T cell–induced colitis. In this study, we found that TCRαβ+CD8αα T cells are reduced among the colonic IEL during inflammation, and that their activation with an agonistic peptide leads to significant Qa-1b–dependent protection in an acute model of colitis. Cellular expression of Qa-1b during inflammation and corresponding dependency in peptide-mediated protection suggest that Batf3-dependent CD103+CD11b− type 1 conventional dendritic cells control the protective function of TCRαβ+CD8αα T cells in the colonic epithelium. In the colitis model, expression of the potential barrier-protective gene, Muc2, is enhanced upon administration of a Qa-1b agonistic peptide. Notably, in steady state, the mucin metabolizing Akkermansia muciniphila was found in significantly lower abundance amid a dramatic change in overall microbiome and metabolome, increased IL-6 in explant culture, and enhanced sensitivity to dextran sulfate sodium in Qa-1b deficiency. Finally, in patients with inflammatory bowel disease, we found upregulation of HLA-E, a Qa-1b analog with inflammation and biologic non-response, in silico, suggesting the importance of this regulatory mechanism across species.

Recombinant human lecithin-cholesterol acyltransferase Fc fusion (huLCAT-Fc) is a chimeric protein produced by fusing human Fc to the C-terminus of the human enzyme via a linker sequence. The huLCAT-Fc homodimer contains five N-linked glycosylation sites per monomer. The heterogeneity and site-specific distribution of the various glycans were examined using enzymatic digestion and LC-MS/MS, followed by automatic processing. Almost all of the N-linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N-linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N-linked site exclusively contain typical asialobiantennary structures. HuLCAT-Fc was also confirmed to have mucin-type glycans attached at T407 and S409. When LCAT-Fc fusions were constructed using a G-S-G-G-G-G linker, an unexpected +632 Da xylose-based glycosaminoglycan (GAG) tetrasaccharide core of Xyl-Gal-Gal-GlcA was attached to S418. Several minor intermediate species including Xyl, Xyl-Gal, Xyl-Gal-Gal, and a phosphorylated GAG core were also present. The mucin-type O-linked glycans can be effectively released by sialidase and O-glycanase; however, the GAG could only be removed and localized using chemical alkaline β-elimination and targeted LC-MS/MS. E416 (the C-terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O-xylosyltransferase. HuLCAT-Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPPE416GS418GGGGDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.

The effect of VM-26, a topoisomerase II targeting drug, on IW32 murine erythroleukemia cells was investigated. The VM-26 induced IW32 cells to differentiate at a non-toxic but cytostatic concentration (0.01 μg/ml). More than 40% of the cells were induced to synthesize hemoglobin, and cells were arrested in G 2 /M phase of the cell cycle. Levels of β-globin mRNA also increased significantly. Cells became committed to erythroid maturation after 16 h of continuous drug exposure. Replacement with fresh VM-26 after 48 h of drug treatment further increased the hemoglobin containing cells to greater than 80%. Unlike other drug induced erythroleukemia cell differentiation, c- myc mRNA expression was not affected by VM-26. Inhibition of topoisomerase II activity was observed during the first 12 h of VM-26 treatment; however, elevated enzyme activity was found thereafter. Northern blot analysis showed significant increase in the expression of topoisomerase IIα mRNA at 12 and 24 h after VM-26 addition. These findings indicate that VM-26 inhibited the activity of topoisomerase II and promoted the committed differentiation of IW32 cells along the erythroid pathway. In addition, a parallel increase in mRNA and activity levels of topoisomerase II in differentiated cells suggests that regulation of the enzyme expression occurred in the VM-26 induced erythroid maturation.

Adiponectin is an adipocyte-derived hormone that has been shown to play important roles in the regulation of glucose and energy homeostasis. It exists as homotrimers or complexes containing multiple homotrimer units in plasma. The recombinant adiponectin proteins have been difficult to produce, making it challenging for both research as well as potential therapeutic development. Here, we show a novel approach for the generation of globular adiponectin that involves linking three monomer sequences together in tandem to generate one continuous polypeptide, which we have termed single-chain globular adiponectin (sc-gAd). To improve the pharmacokinetic properties of sc-gAd further, we fused it to an Fc fragment. The combined effects of single-chain and Fc fusion improved the plasma half-life from less than 2 h to close to 2 weeks. Using adeno-associated virus as a delivery method, we demonstrate that Fc-sc-gAd improved both fasting glucose levels and the tolerance to a glucose challenge in ob/ob mice without changes in body weight. Therefore, our results demonstrated the feasibility of generating globular adiponectin trimers from a single polypeptide and a long-acting globular adiponectin that could serve as a starting point for adiponectin-based therapeutics. This novel approach could also be applied to other complement factor C1q family members; in particular, this opens the possibility to study the biological functions of precisely defined heterotrimers of various family members that had not been previously possible.