The phosphoinositide 3-kinase (PI3-kinase) signaling pathway is frequently aberrantly activated in glioblastoma multiforme (GM) by mutation or loss of the 3' phospholipid phosphatase PTEN. PTEN abnormalities result in inappropriate signaling to downstream molecules including protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR). PI3-kinase activation increases resistance to radiation-induced cell death; conversely, PI3-kinase inhibition enhances the sensitivity of tumors to radiation. The effects of LY294002, a biochemical inhibitor of PI3-kinase, on the response to radiation were examined in the PTEN mutant glioma cell line U251 MG. Low doses of LY294002 sensitized U251 MG to clinically relevant doses of radiation. In contrast to LY294002, rapamycin, an inhibitor of mTOR, did not result in radiosensitization. We demonstrate that among multiple known targets of LY294002, PI3-kinase is the most likely molecule responsible for LY294002-induced radiosensitization. Furthermore, using a myristoylated PKB/Akt construct, we identified PKB/Akt as the downstream molecule that mediates the synergistic cytotoxicity between LY294002 and radiation. Thus PI3-kinase dysregulation may contribute to the notable radioresistance of GM tumors and inhibition of PKB/Akt offers an excellent target to enhance radiosensitivity.

Summary Normal dynamics between microbiota and dendritic cells (DCs) support modest numbers of T cells, yet these do not cause inflammation. The DCs that induce inflammatory T cells and the signals that drive this process remains unclear. Here we demonstrate that small intestine DCs lacking the signaling attenuator A20 induce inflammatory T cells and that the signals perceived and antigen presenting cell (APC) functions are unique for different DC subsets. Thus, while CD103+CD11b− DCs exclusively instruct IFNγ+ T cells, CD103+CD11b+ DCs exclusively instruct IL-17+ T cells. Surprisingly, APC functions of both DC subsets are upregulated in a MyD88-independent fashion. In contrast, CD103−CD11b+ DCs instruct both IFNγ+ and IL-17+ T cells and only the IL-17-inducing APC functions require MyD88. In disease pathogenesis both CD103− CD11b+ and CD103+CD11b+ DCs expand pathologic Th17 cells. Thus, in disease pathogenesis specific DCs instruct specific inflammatory T cells.

The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.

Malignant gliomas are almost uniformly fatal and display exquisite radiation resistance. Glioma cells lacking wild-type (WT) p53 function are more susceptible to radiation-induced apoptosis than their isogenic counterparts expressing WT p53. We explored the mechanisms of such apoptosis and found that, in the absence of WT p53, radiation increases caspase-8 expression and activity. Inhibition of caspase-8 expression using caspase-8 antisense or small interfering RNA (siRNA) oligonucleotides partially blocks radiation-induced apoptosis. In contrast, inhibition of the mitochondrial death pathway by expression of Bcl-2 has no effect on radiation-induced caspase-8 activity or apoptosis. Our data indicate that, in contrast to commonly accepted models of p53-dependent radiation-induced apoptosis, in our cell system, radiation relies on caspase-8 activity to help mediate p53-independent cell death. In a system of inducible E2F1 activity, E2F1 activated caspase-8 and, accordingly, decreased cellular viability, effects that were abolished by caspase-8 siRNA. In this model, in the absence of WT p53, p21Cip1 is not induced, and E2F1 activity is sustained and allows transcription and activation of caspase-8. This model may explain why p53 mutations in adult gliomas paradoxically correlate with improved survival and enhanced response to radiation.

The remarkable success of SARS CoV-2 mRNA-based vaccines and the ensuing interest in mRNA vaccines and therapeutics have highlighted the need for a scalable clinical-enabling manufacturing process to produce such products, and robust analytical methods to demonstrate safety, potency, and purity. To date, production processes have either not been disclosed or are bench-scale in nature and cannot be readily adapted to clinical and commercial scale production. To address these needs, we have advanced an aqueous-based scalable process that is readily adaptable to GMP-compliant manufacturing, and developed the required analytical methods for product characterization, quality control release and stability testing. We also have demonstrated the products produced at manufacturing scale under such approaches display good potency and protection in relevant animal models with mRNA products encoding both vaccine immunogens and antibodies. Finally, we discuss continued challenges in raw material identification, sourcing and supply, and the cold chain requirements for mRNA therapeutic and vaccine products. While ultimate solutions have yet to be elucidated, we discuss approaches that can be taken that are aligned with regulatory guidance.