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The prognosis for patients with glioblastoma is grim. Ex vivo expanded tumor-associated antigen (TAA)-reactive T-cells from patients with glioma may represent a viable source for anticancer-directed cellular therapies. Immunohistochemistry was used to test the survivin (n = 40 samples) and NY-ESO-1 (n = 38 samples) protein expression in tumor specimens. T-cells from peripheral blood were stimulated with TAAs (synthetic peptides) in IL-2 and IL-7, or using a combination of IL-2, IL-15 and IL-21. CD4+ and CD8+ T-cells were tested for antigen-specific proliferation by flow cytometry, and IFN-γ production was tested by ELISA. Twenty-eight out of 38 cancer specimens exhibited NY-ESO-1 protein expression, 2/38 showed a strong universal (4+) NY-ESO-1 staining, and 9/40 cancer lesions exhibited a strong (4+) staining for survivin. We could detect antigen-specific IFN-γ responses in 25% blood samples for NY-ESO-1 and 30% for survivin. NY-ESO-1-expanded T-cells recognized naturally processed and presented epitopes. NY-ESO-1 or survivin expression in glioma represents viable targets for anticancer-directed T-cells for the biological therapy of patients with glioma. [ABSTRACT FROM AUTHOR]

Little is known about the molecular mechanisms underlying age-dependent deterioration in [beta]-cell function. We now demonstrate that age-dependent impairment in insulin release, and thereby glucose homeostasis, is associated with subtle changes in Ca(2+) dynamics in mouse [beta]-cells. We show that these changes are likely to be accounted for by impaired mitochondrial function and to involve phospholipase C/inositol 1,4,5-trisphosphate-mediated Ca(2+) mobilization from intracellular stores as well as decreased [beta]-cell Ca(2+) influx over the plasma membrane. We use three mouse models, namely, a premature aging phenotype, a mature aging phenotype, and an aging-resistant phenotype. Premature aging is studied in a genetically modified mouse model with an age-dependent accumulation of mitochondrial DNA mutations. Mature aging is studied in the C57BL/6 mouse, whereas the 129 mouse represents a model that is more resistant to age-induced deterioration. Our data suggest that aging is associated with a progressive decline in [beta]-cell mitochondrial function that negatively impacts on the fine tuning of Ca(2+) dynamics. This is conceptually important since it emphasizes that even relatively modest changes in [beta]-cell signal transduction over time lead to compromised insulin release and a diabetic phenotype. [ABSTRACT FROM AUTHOR]

Little is known about the molecular mechanisms underlying age-dependent deterioration in β-cell function. We now demonstrate that age-dependent impairment in insulin release, and thereby glucose homeostasis, is associated with subtle changes in Ca2+ dynamics in mouse β-cells. We show that these changes are likely to be accounted for by impaired mitochondrial function and to involve phospholipase C/inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization from intracellular stores as well as decreased β-cell Ca2+ influx over the plasma membrane. We use three mouse models, namely, a premature aging phenotype, a mature aging phenotype, and an aging-resistant phenotype. Premature aging is studied in a genetically modified mouse model with an age-dependent accumulation of mitochondrial DNA mutations. Mature aging is studied in the C57BL/6 mouse, whereas the 129 mouse represents a model that is more resistant to age-induced deterioration. Our data suggest that aging is associated with a progressive decline in β-cell mitochondrial function that negatively impacts on the fine tuning of Ca2+ dynamics. This is conceptually important since it emphasizes that even relatively modest changes in β-cell signal transduction over time lead to compromised insulin release and a diabetic phenotype. [ABSTRACT FROM AUTHOR]

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP 7 , is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP 7 regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP 7 . Although the inhibitors have diverse targets, they all perturbed IP 7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP 7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K m for ATP makes IP 7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP 7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP 7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP 7 's role in cellular signal transduction is likely to have been underestimated., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor-infiltrating lymphocytes (TILs) may represent a viable source of T cells for the biological treatment of patients with gliomas. Glioma tissue was obtained from 16 patients, tumor cell lines were established, and TILs were expanded in 16/16 cases using a combination of IL-2/IL-15/IL-21. Intracellular cytokine staining (ICS, IL-2, IL-17, TNFα and IFNγ production) as well as a cytotoxicity assay was used to detect TIL reactivity against autologous tumor cells or shared tumor-associated antigens (TAAs; i.e., NY-ESO-1, Survivin or EGFRvIII). TILs were analyzed by flow cytometry, including T-cell receptor (TCR) Vβ family composition, exhaustion/activation and T-cell differentiation markers (CD45RA/CCR7). IL-2/IL-15/IL-21 expanded TILs exhibited a mixture of CD4 + , CD8 + , as well as CD3 + CD4 - CD8 - T cells with a predominant central memory CD45RA - CCR7 + phenotype. TIL showed low frequencies of T cells testing positive for PD-1, TIM-3 and CTLA-4. LAG3 tested positive in up to 30% of CD8 + TIL, with low (1.25%) frequencies in CD4 + T cells. TIL cultures exhibited preferential usage of Vβ families and recognition of autologous tumor cells defined by cytokine production and cytotoxicity. IL-2/IL-15/IL-21 expanded TILs represent a viable source for the cellular therapy of patients with gliomas.

High performance liquid chromatography (HPLC) is an essential analytical tool in the study of the large number of inositol phosphate isomers. This chapter focuses on the separation of inositol polyphosphates from [(3)H]myo-inositol labeled tissues and cells. We review the different HPLC columns that have been used to separate inositol phosphates and their advantages and disadvantages. We describe important elements of sample preparation for effective separations and give examples of how changing factors, such as pH, can considerably improve the resolving ability of the HPLC chromatogram.

Several inositol compounds undergo rapid cycles of phosphorylation and dephosphorylation. These cycles are dependent on ATP and energy metabolism. Therefore, interfering with the cellular energy metabolism can change the concentration of rapidly turning over inositols. Many pharmacological inhibitors, apart from their intended action, also affect the energy metabolism of the cells and lower ATP. This can unspecifically influence rapidly turning over inositol phosphates. Thus, the ATP concentration should be checked when reduced inositol phosphates are observed after application of pharmacological inhibitors. A luminescence-based assay for the measurement of ATP and ADP is described. ATP is measured luminometrically using firefly luciferase. Detection of ADP is performed in a two-step enzymatic procedure: (1) The sample ATP is degraded to AMP and (2) ADP is phosphorylated to ATP, which can then be measured luminometrically. This method gives a better signal-to-noise ratio than other methods that do not degrade the sample ATP, but convert ADP directly to ATP and then measure the sum of ATP plus ADP.