Loss of Zbtb32 in NOD mice does not significantly alter T cell responses. [version 2; referees: 2 approved]

Background: We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2+ dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2+ DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods: To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results: Although NOD.Zbtb32-/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32-/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions: The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype

Loss of Zbtb32 in NOD mice does not significantly alter T cell responses. [version 1; referees: 2 approved]

Background: We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2+ dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2+ DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods: To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results: Although NOD.Zbtb32-/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32-/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions: The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype

Structural requirements for roxatidine in the stimulant effect of rat gastric mucin synthesis and the participation of nitric oxide in this mechanism

1. The structural requirements of the histamine H(2)-receptor antagonist, roxatidine (2-acetoxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide hydrochloride), for the stimulant effect on mucin biosynthesis and their relation to histamine H(2)-receptor antagonism were identified by considering the structural analogues of this drug using an organ culture system of the rat stomach and competition studies with [(125)I]iodoaminopotentidine ([(125)I]-APT) binding to membranes of the guinea pig striatum. 2. [(3)H]Glucosamine incorporation into mucin during 5 h incubation period was stimulated by roxatidine and its structural analogues A (2-hydroxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide) and B (N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide). This effect was seen in mucosal cultures of the corpus, but not antrum, region. 3. Structural analogues, in which the length of the flexible chain between the benzene ring and the amide structure differs from that of roxatidine, failed to activate mucin synthesis. No significant change in mucus synthesis occurred with the addition of analogues in which the piperidine ring attached to the benzene ring via a methylene bridge was changed. 4. Specific [(125)I]-APT binding to the histamine H(2) receptor of guinea pig brain membranes was inhibited by roxatidine and all structural analogues used in this study, except F (N-(3-[m-(N, N-dimethyl-aminomethyl)phenoxy]-propyl)acetamide). 5. Ranitidine at 10(−4) M did not suppress the roxatidine-induced increase in [(3)H]glucosamine incorporation into mucin. 6. Roxatidine-induced stimulation of [(3)H]glucosamine incorporation into mucin was completely blocked by the addition of either N(G)-nitro-L-arginine (10(−5) M) or 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide sodium salt (10(−5) M). The inhibitory action of N(G)-nitro-L-arginine was totally reversed by L-arginine (5×10(−3) M). 7. These results suggest that the cardinal chemical features of roxatidine for the activation of mucin biosynthesis in the corpus region of the rat stomach are the appropriate length of the flexible chain between the amide structure and the aromatic ring system bearing the methylpiperidinyl group at the meta position. The activity of roxatidine and its analogues to stimulate mucin synthesis is not related to their histamine H(2) receptor antagonistic activity. Roxatidine-induced activation of mucin biosynthesis in the corpus tissue is mediated by nitric oxide