The indoleamine 2,3-dioxygenase (IDO) enzyme can act as an immunoregulator by inhibiting T cell function via the degradation of the essential amino acid tryptophan (trp) into kynurenine (kyn) and its derivates. The kyn/trp ratio in serum is a prognostic factor for cervical cancer patients; however, information about the relationship between serum levels and IDO expression in the tumor is lacking. IDO expression was studied in 71 primary and 14 paired metastatic cervical cancer samples by various immunohistochemical (IHC) techniques, including 7-color fluorescent multiparameter IHC, and the link between the concentration of IDO metabolites in serum, clinicopathological characteristics, and the presence of (proliferating) T cells (CD8, Ki67, and FoxP3) was examined. In addition, we compared the relationships between IDO1 and IFNG gene expression and clinical parameters using RNAseq data from 144 cervical tumor samples published by The Cancer Genome Atlas (TCGA). Here, we demonstrate that patchy tumor IDO expression is associated with an increased systemic kyn/trp ratio in cervical cancer (P = 0.009), whereas marginal tumor expression at the interface with the stroma is linked to improved disease-free (DFS) (P = 0.017) and disease-specific survival (P = 0.043). The latter may be related to T cell infiltration and localized IFNγ release inducing IDO expression. Indeed, TCGA analysis of 144 cervical tumor samples revealed a strong and positive correlation between IDO1 and IFNG mRNA expression levels (P < 0.001) and a significant association with improved DFS for high IDO1 and IFNG transcript levels (P = 0.031). Unexpectedly, IDO+ tumors had higher CD8+Ki67+ T cell rates (P = 0.004). Our data thus indicate that the serum kyn/trp ratio and IDO expression in primary tumor samples are not clear-cut biomarkers for prognosis and stratification of patients with early stage cervical cancer for clinical trials implementing IDO inhibitors. Rather, a marginal IDO expression pattern in the tumor dominantly predicts favorable outcome, which might be related to IFNγ release in the cervical tumor microenvironment.

Astroglial cells from mouse cerebral hemispheres, cerebellum, olfactory bulbs, and medulla oblongata were grown in the presence of either hormones (hydrocortisone, insulin) or cell second messengers (dBcAMP, dBcGMP). Glutamine synthetase (GS) specific activity, GS protein level, and GS translation were investigated under the effect of these factors. Hydrocortisone produced a simultaneous increase in GS translation, GS level, and activity. This increase was observed in the astrocytes cultured from the four brain areas but at a variable magnitude depending on the area. The hydrocortisone effect appeared at the transcriptional level. Inversely, insulin decreased both the GS activity and the in vitro translated GS. This effect was seen only in the olfactory bulbs and the medulla. DBcAMP increased the GS biological activity only in the cerebral hemisphere cultures. It raised, however, the level of translated GS and GS protein in astrocytes from all the areas, suggesting a post-translational effect for intracellular cAMP. DBcGMP only affected GS in the astrocytes from cerebral hemispheres and the medulla modulating either the GS transcription or the messenger RNA stability. These results suggest specific regulation for GS expression, depending on the brain area from which the cells were dissociated or on the astroglial cell population present in these cultures affecting either the transcription, the mRNA stability, or the biological activity of the protein.

Human papillomavirus (HPV) drives high-grade intraepithelial neoplasia and cancer; for unknown reasons, this occurs most often in the cervical transformation zone. Either mutation or HPV E6–driven inhibition of Notch1 can drive neoplastic development in stratified squamous epithelia. However, the contribution of Notch1 and its Delta-like ligands (DLL) to site susceptibility remains poorly understood. Here, we map DLL1/DLL4 expression in cell populations present in normal cervical biopsies by immunofluorescence. In vitro keratinocyte 2D monolayer models, growth assays, and organotypic raft cultures were used to assess the functional role of DLL–Notch signaling in uninfected cells and its modulation by HPV16 in neoplasia. An RNA sequencing–based gene signature was used to suggest the cell of origin of 279 HPV-positive cervical carcinomas from The Cancer Genome Atlas and to relate this to disease prognosis. Finally, the prognostic impact of DLL4 expression was investigated in three independent cervical cancer patient cohorts. Three molecular cervical carcinoma subtypes were identified, with reserve cell tumors the most common and linked to relatively good prognosis. Reserve cells were characterized as DLL1−/DLL4+, a proliferative phenotype that is temporarily observed during squamous metaplasia and wound healing but appears to be sustained by HPV16 E6 in raft models of low-grade and, more prominently, high-grade neoplasia. High expression of DLL4 was associated with an increased likelihood of cervical cancer–associated death and recurrence. Taken together, DLL4–Notch1 signaling reflects a proliferative cellular state transiently present during physiologic processes but inherent to cervical reserve cells, making them strongly resemble neoplastic tissue even before HPV infection has occurred. Significance: This study investigates cervical cancer cell-of-origin populations and describes a DLL–Notch1 phenotype that is associated with disease prognosis and that might help identify cells that are susceptible to HPV-induced carcinogenesis.

Primary cultures from various areas of newborn mouse brain were developed and characterized. Enriched astroglial cultures of the cerebral hemispheres, cerebellum, medulla oblongata and olfactory bulbs contained about 80–90% glial fibrillary acidic protein (GFA) immunolabelled cells. These cultures were composed of a majority of flat, ''protoplasmic-like'' cells. The aim of this culture model was used: (1) to study glutamine synthetase (GS) activity during in vitro astroglial development; (2) to consider the hydrocortisone effect on GS activity during both growth and maturation periods, and (3) to determine the development of GS immunoreactivity in the cells and eventual GFA and GS expression in these cells. We observed that GS increased during brain maturation in vivo and in vitro, and that addition of hydrocortisone (1 µM, 48 h) to the culture medium induced varying GS activity depending on the developmental stage and the area. In the four areas studied, the number and intensity of GS-immunolabelled cells reached an optimum between 18 and 30 days in vitro. Only about 50–70% of the cell population was GS positive. Double-labelling experiments showed that three groups of cells coexist whatever the considered area. Two expressed both GFA and GS proteins, the last marker at either a low or a high level, and the third was devoid of GS immunoreactivity. Regional differences in GS-specific activity, GS inducibility and GS immunoreactivity exist in the astroglial population, but the factors responsible for these variations are not yet known.

Astroglial cultures from newborn mouse cerebral cortex contain [125I]Insulin binding sites. Binding was specific, reversible, time dependent and reached equilibrium after 45 min. Insulin analogues compete for this [125I]Insulin binding. Incubation of cerebral cortex astroglial cultures with insulin induced a time- and dose-dependent inhibition of the [3H]GABA high affinity uptake. A decrease in the Vmax rather than an effect on the Km was observed. This effect was dose-dependent and effective at 10(-10) M. Autoradiographic observations on the cell monolayer showed the presence of two groups of cells: one which strongly takes up [3H]GABA and consist in smaller GFAP positive process-bearing cells and another group of much flatter and larger GFAP positive cells which uptake was lower. The smaller stellate cells were apparently the most sensitive to insulin effect. These results: 1) confirm the presence of insulin binding sites on astroglial primary cultures, 2) show an effect of insulin on [3H]GABA high affinity uptake of these cells; this effect being optimal on a stellate-like population of astrocytes, and 3) indicate that insulin may interfere in neuromodulation through astroglial signals.