Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies

The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state‐of‐the‐art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow

Peripheral immune status in the GFAP-V12 HA-ras B8 spontaneous astrocytoma model

In questo lavoro sperimentale, è stata in particolare studiata l’interazione tra tumore e sistema immunitario

Hypomethylating agents and glioma: from sensitisation to cell death by cytotoxic immune mediators

Malignant gliomas are heterogeneous, aggressive tumours. A small fraction of glioma cells is undifferentiated and defined as glioma initiating cells (GICs), whereas the majority of glioma cells are differentiated (GDCs). Malignant glioma patients have a dismal prognosis, even with standard-of-care treatment. The use of T-cell immunotherapy is a promising approach, but needs to be potentiated. The hypomethylating agent Decitabine (DAC) up-regulated expression of glioma surface molecules involved in cell-cell interactions and enhanced cytotoxic T-cell (CTL) killing of both GDCs and GICs. Thus, DAC sensitises glioma cells (with diverse differentiation levels) to T-cell effector responses. However, in vivo results showed no benefit of systemic DAC administration before CTL adoptive transfer, with limited hypomethylating effects on brain tumour. Overall, the promising in vitro immunosensitisation of glioma cells using DAC should encourage development of efficient drug delivery to the tumour site in vivo to explore the therapeutic potential of hypomethylating drugs and immunotherapy

Glioma Stemlike Cells Enhance the Killing of Glioma Differentiated Cells by Cytotoxic Lymphocytes

Glioblastoma multiforme, the most aggressive primary brain tumor, is maintained by a subpopulation of glioma cells with self-renewal properties that are able to recapitulate the entire tumor even after surgical resection or chemo-radiotherapy. This typifies the vast heterogeneity of this tumor with the two extremes represented on one end by the glioma stemlike cells (GSC) and on the other by the glioma differentiated cells (GDC). Interestingly, GSC are more sensitive to immune effector cells than the GDC counterpart. However, how GSC impact on the killing on the GDC and vice versa is not clear. Using a newly developed cytotoxicity assay allowing to simultaneously monitor cytotoxic lymphocytes-mediated killing of GSC and GDC, we found that although GSC were always better killed and that their presence enhanced the killing of GDC. In contrast, an excess of GDC had a mild protective effect on the killing of GSC, depending on the CTL type. Overall, our results suggest that during combination therapy, immunotherapy would be the most effective after prior treatment with conventional therapies

Glioma Stemlike Cells Enhance the Killing of Glioma Differentiated Cells by Cytotoxic Lymphocytes

<div><p>Glioblastoma multiforme, the most aggressive primary brain tumor, is maintained by a subpopulation of glioma cells with self-renewal properties that are able to recapitulate the entire tumor even after surgical resection or chemo-radiotherapy. This typifies the vast heterogeneity of this tumor with the two extremes represented on one end by the glioma stemlike cells (GSC) and on the other by the glioma differentiated cells (GDC). Interestingly, GSC are more sensitive to immune effector cells than the GDC counterpart. However, how GSC impact on the killing on the GDC and <i>vice versa</i> is not clear. Using a newly developed cytotoxicity assay allowing to simultaneously monitor cytotoxic lymphocytes-mediated killing of GSC and GDC, we found that although GSC were always better killed and that their presence enhanced the killing of GDC. In contrast, an excess of GDC had a mild protective effect on the killing of GSC, depending on the CTL type. Overall, our results suggest that during combination therapy, immunotherapy would be the most effective after prior treatment with conventional therapies.</p></div

Synergy between CD8 T cells and Th1 or Th2 polarised CD4 T cells for adoptive immunotherapy of brain tumours

The feasibility of cancer immunotherapy mediated by T lymphocytes is now a clinical reality. Indeed, many tumour associated antigens have been identified for cytotoxic CD8 T cells, which are believed to be key mediators of tumour rejection. However, for aggressive malignancies in specialised anatomic sites such as the brain, a limiting factor is suboptimal tumour infiltration by CD8 T cells. Here we take advantage of recent advances in T cell biology to differentially polarise CD4 T cells in order to explore their capacity to enhance immunotherapy. We used an adoptive cell therapy approach to work with clonal T cell populations of defined specificity. Th1 CD4 T cells preferentially homed to and accumulated within intracranial tumours compared with Th2 CD4 T cells. Moreover, tumour-antigen specific Th1 CD4 T cells enhanced CD8 T cell recruitment and function within the brain tumour bed. Survival of mice bearing intracranial tumours was significantly prolonged when CD4 and CD8 T cells were co-transferred. These results should encourage further definition of tumour antigens recognised by CD4 T cells, and exploitation of both CD4 and CD8 T cell subsets to optimise T cell therapy of cancer

Glioma stemlike cells are better killed by CTL.

<p>(A) Messenger RNA expression of the stem cell markers CD90, MSl1, CD133, CD44, PTCH, MELK, CXCR4 and differentiation marker GFAP for GL261 and mNS cells assessed by qRT-PCR. (B) Phenotyping of GL261 and mNS cells for the surface expression of MHC class I, CD54, CD80, CD86. Filled dark and light grey histograms are for mNS and GL261 respectively, open dark and light grey histograms are the corresponding controls. (C) GL261 and mNS were treated with a sublytic dose of perforin (P) and granzyme B (GB) for one hour and cell death monitored by annexin V-PI staining. (D) Same as in C mean +/-SD of three independent experiment. (E) GL261 and mNS were pulsed or not with peptide and used as target cells for day 6 PMEL CTL in a 4 hour calcein AM release assay. All data are representative of 3 independent experiments. Bar graphs represent mean +/- SD of three independent experiments. P value * p ≤ 0.05, ** p ≤ 0.01, one-sided t-test.</p

GSC enhance the killing of GDC by day 9 PMEL CTL.

<p>(A) GL261 and mNS were loaded with calcein AM and calcein Red Orange respectively, then pulsed with peptide and mixed in 1:1 ratio before simultaneous incubation with day 9 PMEL CTL in a dual color cytotoxicity assay. The cytotoxicity toward each target is followed by measuring the release of the respective calcein in the supernatant. (B) Peptide-pulsed mNS cells alone were used as target for day 9 PMEL CTL in a classical calcein AM release assay (mNS alone), while on the other side peptide-pulsed mNS were mixed in 9:1 ratio with peptide-pulsed GL261 (mNS:GL 9:1) and incubated simultaneously with day 9 PMEL CTL in a dual color cytotoxicity assay. (C) The killing of GL261 facing CTL alone compared with the killing of GL261 in mNS:GL ratio of 9:1 obtained from B. (D) Same as in B for an mNS:GL ratio of 1:9. (E) Same as in C for mNS:GL ratio of 1:9. All bar graphs are mean +/- SD of three independent experiments. P value * p ≤ 0.05, ** p ≤ 0.01, one-sided t-test.</p