CD28 inhibits T cell adhesion by recruiting CAPRI to the plasma membrane.

CD28 is a coreceptor expressed on T lymphocytes. Signaling downstream of CD28 promotes multiple T cell functions such as proliferation, survival, and cytokine secretion. Adhesion to APCs is another function of T cells; however, little is known with regard to the role of CD28 in this process. Our previous studies have shown that CD28 inhibits T cell adhesion, but the underlying mechanism that mediates this process remains unknown. In the present study we discovered that signaling downstream of CD28 resulted in inhibition of Rap1 activity and decreased LFA-1-mediated adhesion. We showed that this was regulated by the recruitment of calcium-promoted Ras inactivator (CAPRI), a GTPase-activating protein, to the plasma membrane downstream of CD28 signaling. CAPRI trafficking to the plasma membrane was secondary to calcium influx and was mediated by its C2A and C2B domains. We conclude that CD28 inhibits Rap1-mediated adhesion by recruiting the protein CAPRI to the plasma membrane.

Noninvasive PET Imaging of CDK4/6 Activation in Breast Cancer.

The cell cycle is a progression of 4 distinct phases (G1, S, G2, and M), with various cycle proteins being essential in regulating this process. We aimed to develop a radiolabeled cyclin-dependent kinase 4/6 (CDK4/6) inhibitor for breast cancer imaging. Our transfluorinated analog (18F-CDKi) was evaluated and validated as a novel PET imaging agent to quantify CDK4/6 expression in estrogen receptor (ER)-positive human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Methods:18F-CDKi was synthesized and assayed against CDK4/6 kinases. 18F-CDKi was prepared with a 2-step automated synthetic strategy that yielded the final product with remarkable purity and molar activity. In vitro and in vivo biologic specificity was assessed in a MCF-7 cell line and in mice bearing MCF-7 breast tumors. Nonradioactive palbociclib was used as a blocking agent to investigate the binding specificity and selectivity of 18F-CDKi. Results:18F-CDKi was obtained with an overall radiochemical uncorrected yield of 15% and radiochemical purity higher than 98%. The total time from the start of synthesis to the final injectable formulated tracer is 70 min. The retention time reported for 18F-CDKi and 19F-CDKi is 27.4 min as demonstrated by coinjection with 19F-CDKi in a high-pressure liquid chromatograph. In vivo blood half-life (weighted, 7.03 min) and octanol/water phase partition coefficient (1.91 ± 0.24) showed a mainly lipophilic behavior. 18F-CDKi is stable in vitro and in vivo (>98% at 4 h after injection) and maintained its potent targeting affinity to CDK4/6. Cellular uptake experiments performed on the MCF-7 breast cancer cell line (ER-positive and HER2-negative) demonstrated specific uptake with a maximum intracellular concentration of about 65% as early as 10 min after incubation. The tracer uptake was reduced to less than 5% when cells were coincubated with a molar excess of palbociclib. In vivo imaging and ex vivo biodistribution of ER-positive, HER2-negative MCF-7 breast cancer models showed a specific uptake of approximately 4% injected dose/g of tumor (reduced to ∼0.3% with a 50-fold excess of cold palbociclib). A comprehensive biodistribution analysis also revealed a significantly lower activation of CDK4/6 in nontargeting organs. Conclusion:18F-CDKi represents the first 18F PET CDK4/6 imaging agent and a promising imaging agent for ER-positive, HER2-negative breast cancer.

APOE Genotype Differentially Modulates Effects of Anti-Aß, Passive Immunization in APP Transgenic Mice.

BACKGROUND: APOE genotype is the foremost genetic factor modulating ß-amyloid (Aß) deposition and risk of sporadic Alzheimer's disease (AD). Here we investigated how APOE genotype influences response to anti-Aß immunotherapy. METHODS: APPSW/PS1dE9 (APP) transgenic mice with targeted replacement of the murine Apoe gene for human APOE alleles received 10D5 anti-Aß or TY11-15 isotype control antibodies between the ages of 12 and 15 months. RESULTS: Anti-Aß immunization decreased both the load of fibrillar plaques and the load of Aß immunopositive plaques in mice of all APOE backgrounds. Although the relative reduction in parenchymal Aß plaque load was comparable across all APOE genotypes, APP/ε4 mice showed the greatest reduction in the absolute Aß plaque load values, given their highest baseline. The immunization stimulated phagocytic activation of microglia, which magnitude adjusted for the post-treatment plaque load was the greatest in APP/ε4 mice implying association between the ε4 allele and impaired Aß phagocytosis. Perivascular hemosiderin deposits reflecting ensued microhemorrhages were associated with vascular Aß (VAß) and ubiquitously present in control mice of all APOE genotypes, although in APP/ε3 mice their incidence was the lowest. Anti-Aß immunization significantly reduced VAß burden but increased the number of hemosiderin deposits across all APOE genotypes with the strongest and the weakest effect in APP/ε2 and APP/ε3 mice, respectively. CONCLUSIONS: Our studies indicate that APOE genotype differentially modulates microglia activation and Aß plaque load reduction during anti-Aß immunotherapy. The APOE ε3 allele shows strong protective effect against immunotherapy associated microhemorrhages; while, conversely, the APOE ε2 allele increases risk thereof.