The International Knockout Mouse Consortium (IKMC) introduces its targeted constructs into C57BL/6N embryonic stem cells. However, breeding with a Cre-recombinase and/or Flp-recombinase mouse is required for the generation of a null allele with the IKMC cassette. Many recombinase strains are in the C57BL/6J background, resulting in knockout animals on a mixed strain background. This can lead to variability in metabolic data and the use of improper control groups. While C57BL/6N and C57BL/6J are derived from the same parental C57BL/6 strain, there are key genotypic and phenotypic differences between these substrains. Many researchers may not even be aware of these differences, as the shorthand C57BL/6 is often used to describe both substrains. We found that 58% of articles involving genetically modified mouse models did not completely address background strain. This review will describe these two substrains and highlight the importance of separate consideration in mouse model development. Our aim is to increase awareness of this issue in the diabetes research community and to provide practical strategies to enable researchers to avoid mixed strain animals when using IKMC knockout mice.
Prostaglandin E2 (PGE2) is derived from arachidonic acid, whereas PGE3 is derived from eicosapentaenoic acid (EPA) using the same downstream metabolic enzymes. Little is known about the impact of EPA and PGE3 on β-cell function, particularly in the diabetic state. In this work, we determined that PGE3 elicits a 10-fold weaker reduction in glucose-stimulated insulin secretion through the EP3 receptor as compared with PGE2. We tested the hypothesis that enriching pancreatic islet cell membranes with EPA, thereby reducing arachidonic acid abundance, would positively impact β-cell function in the diabetic state. EPA-enriched islets isolated from diabetic BTBR Leptinob/ob mice produced significantly less PGE2 and more PGE3 than controls, correlating with improved glucose-stimulated insulin secretion. NAD(P)H fluorescence lifetime imaging showed that EPA acts downstream and independently of mitochondrial function. EPA treatment also reduced islet interleukin-1β expression, a proinflammatory cytokine known to stimulate prostaglandin production and EP3 expression. Finally, EPA feeding improved glucose tolerance and β-cell function in a mouse model of diabetes that incorporates a strong immune phenotype: the NOD mouse. In sum, increasing pancreatic islet EPA abundance improves diabetic β-cell function through both direct and indirect mechanisms that converge on reduced EP3 signaling.
Recently, a novel type 1 diabetes association locus was identified at human chromosome 6p31.3, and transcription factor 19 (TCF19) is a likely causal gene. Little is known about Tcf19, and we now show that it plays a role in both proliferation and apoptosis in insulinoma cells. Tcf19 is expressed in mouse and human islets, with increasing mRNA expression in nondiabetic obesity. The expression of Tcf19 is correlated with β-cell mass expansion, suggesting that it may be a transcriptional regulator of β-cell mass. Increasing proliferation and decreasing apoptotic cell death are two strategies to increase pancreatic β-cell mass and prevent or delay diabetes. siRNA-mediated knockdown of Tcf19 in the INS-1 insulinoma cell line, a β-cell model, results in a decrease in proliferation and an increase in apoptosis. There was a significant reduction in the expression of numerous cell cycle genes from the late G1 phase through the M phase, and cells were arrested at the G1/S checkpoint. We also observed increased apoptosis and susceptibility to endoplasmic reticulum (ER) stress after Tcf19 knockdown. There was a reduction in expression of genes important for the maintenance of ER homeostasis (Bip, p58IPK, Edem1, and calreticulin) and an increase in proapoptotic genes (Bim, Bid, Nix, Gadd34, and Pdia2). Therefore, Tcf19 is necessary for both proliferation and survival and is a novel regulator of these pathways.
Background Signal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts innate immunity through engagement of its cell surface ligand CD47. Blockade of the CD47–SIRPα interaction synergizes with tumor-specific antibodies and T-cell checkpoint inhibitors by promoting myeloid-mediated antitumor functions leading to the induction of adaptive immunity. Inhibition of the CD47–SIRPα interaction has focused predominantly on targeting CD47, which is expressed ubiquitously and contributes to the accelerated blood clearance of anti-CD47 therapeutics. Targeting SIRPα, which is myeloid-restricted, may provide a differential pharmacokinetic, safety, and efficacy profile; however, SIRPα polymorphisms and lack of pan-allelic and species cross-reactive agents have limited the clinical translation of antibodies against SIRPα. Here, we report the development of humanized AB21 (hAB21), a pan-allelic anti-SIRPα antibody that binds human, cynomolgus monkey, and mouse SIRPα alleles with high affinity and blocks the interaction with CD47. Methods Human macrophages derived from donors with various SIRPα v1 and v2 allelic status were used to assess the ability of hAB21 to enhance phagocytosis. HAB21_IgG subclasses were evaluated for targeted depletion of peripheral blood mononuclear cells, phagocytosis and in vivo efficacy in xenograft models. Combination therapy with anti-PD1/anti-PD-L1 in several syngeneic models was performed. Immunophenotyping of tissues from MC38 tumor-bearing mice treated with AB21 and anti-PD-1 was evaluated. PK, PD and tolerability of hAB21 were evaluated in cynomolgus monkeys. Results SIRPα blockade with hAB21 promoted macrophage-mediated antibody-dependent phagocytosis of tumor cells in vitro and improved responses to rituximab in the Raji human tumor xenograft mouse model. Combined with PD-1/PD-L1 blockade, AB21 improved response rates by facilitating monocyte activation, dendritic cell activation, and T cell effector functions resulting in long term, durable antitumor immunity. In cynomolgus monkeys, hAB21 has a half-life of 5.3 days at 10 mg/kg and complete target occupancy with no hematological toxicity or adverse findings at doses up to 30 mg/kg. Conclusions The in vitro and in vivo antitumor activity of hAB21 broadly recapitulates that of CD47 targeted therapies despite differences in ligand expression, binding partners, and function, validating the CD47–SIRPα axis as a fundamental myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of human malignancies.
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