M+D: conceptual guidelines for compiling a materials library

This article proposes to present a study conducted by the Raw Materials research group, the results of which comprise the conceptual guidelines for compiling an M+D material library. The study includes the topic, materials and design taking the impact of the changes that came into being in the post industrial era on project methodologies and the search for information regarding materials. Taking into account the importance and complexity that these relationships have taken on currently, we have studied the issue of materials based on Manzini (1983) and Ashby and Johnson (2002). Afterward different databases and materials libraries located in the Brazil, the United States, France and Italy geared toward design professionals and students were analyzed to understand what information and means of access to them were available. The project methodologies were approached based on Löbach (1991), Bürdeck (1994), Schulmann (1994), Baxter (1998), Dantas (1998 and 2005) and Papanek (1995 and 2000). This study sought to identify the key elements of the role of materials in the project process today, to serve as a parameter for the analysis of the models studied. A comparative analysis of the models investigated enabled identification of positive and negative aspects to adapt to the needs previously mentioned and identify conceptual guidelines for compiling a collection of materials for use in design projects. Keywords: Design, Materials, Project Methodology, Library</p

Stattic and metformin inhibit brain tumor initiating cells by reducing STAT3-phosphorylation

Glioblastoma (GBM) is the most common and malignant type of primary brain tumor and associated with a devastating prognosis. Signal transducer and activator of transcription number 3 (STAT3) is an important pathogenic factor in GBM and can be specifically inhibited with Stattic. Metformin inhibits GBM cell proliferation and migration. Evidence from other tumor models suggests that metformin inhibits STAT3, but there is no specific data on brain tumor initiating cells (BTICs). We explored proliferation and migration of 7 BTICs and their differentiated counterparts (TCs) after treatment with Stattic, metformin or the combination thereof. Invasion was measured in situ on organotypic brain slice cultures. Protein expression of phosphorylated and total STAT3, as well as AMPK and mTOR signaling were explored using Western blot. To determine functional relevance of STAT3 inhibition by Stattic and metformin, we performed a stable knock-in of STAT3 in selected BTICs. Inhibition of STAT3 with Stattic reduced proliferation in all BTICs, but only in 4 out of 7 TCs. Migration and invasion were equally inhibited in BTICs and TCs. Treatment with metformin reduced STAT3-phosphorylation in all investigated BTICs and TCs. Combined treatment with Stattic and metformin led to significant additive effects on BTIC proliferation, but not migration or invasion. No additive effects on TCs could be detected. Stable STAT3 knock-in partly attenuated the effects of Stattic and metformin on BTICs. In conclusion, metformin was found to inhibit STAT3-phosphorylation in BTICs and TCs. Combined specific and unspecific inhibition of STAT3 might represent a promising new strategy in the treatment of glioblastoma

Metabolic Heterogeneity of Brain Tumor Cells of Proneural and Mesenchymal Origin

Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. BTICs of different subtypes were challenged with oxidative phosphorylation (OXPHOS) inhibition with metformin to assess the differential effects of metabolic intervention on key resistance features. Whereas mesenchymal BTICs varied according to their invasiveness, they were in general more glycolytic and less responsive to metformin. Proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathway, and responded better to metformin. Targeting glycolysis may be a promising approach to inhibit tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors

NSG mice engrafted with cryoconserved or non-cryoconserved BM-HSPCs by intra-femoral injections.

<p>Adult NSG mice were sublethally irradiated and transplanted with cryoconserved (n = 14, 11 donors) or non-cryoconserved BM-HSPCs (n = 14, 13 donors). At >20 weeks after transplantation mice were analyzed by flow cytometry. (<b>A</b>) The number of injected CD34⁺ HSPCs for each group is given. As the yield of CD34⁺ cells is higher from non-cryoconserved BM samples, the respective group received significantly more HSPCs (p = 0.0045, student‘s t-test). (<b>B</b>) Engraftment in BM and spleen was determined by human CD45 expression. The absolute number of hCD45⁺ cells in BM and spleen of the reconstituted mice is shown, each symbol represents an individual animal. Indicated are values for significant differences between the groups (BM: p = 0.03, Spleen p = 0.0003, Mann-Whitney test) (<b>C</b>) Representative flow cytometric analysis of BM and spleen for CD19⁺ B cells, CD33⁺ myeloid cells and CD3⁺ or CD3⁺CD4⁺/CD8⁺ T cells. B cell developmental stages in BM and spleen are shown for CD24⁺CD38^high/low cells. In BM pro/pre-B cells were defined as CD10⁺ IgM^-, immature B cells as CD10⁺IgM⁺. In spleen mainly transitional (IgD⁺IgM⁺) B cells were detected. (<b>D</b>) Representative flow cytometric analysis of the spleen for plasmacytoid and myeloid dendritic cellls (pDC: Lin^-CD123⁺CD11c^-; mDC: Lin^-CD123⁻CD11c⁺) and their expression of costimulatory molecules (CD40, CD86) (<b>E</b>) Representative flow cytometric analysis of CD3⁺ T cells in the thymus and their expression of CD4 and CD8 at >20 weeks after transplantation. The percentage of mice with CD3⁺ T cells and their absolute number in spleen at >20 weeks after transplantation is given (CB vs. non-cryo BM: p<0.01, Mann-Whitney test).</p

Patient data.

<p>For human bone marrow aspirates of non-metastasized carcinoma patients and cord-blood samples, the median sample size, age and obtained number of BM-MNCs and HSPCs are given. The distribution of the carcinoma type was as follows: lung 29/77, mammary 28/77, prostate 14/77 and oesophageal 6/77.</p

HSPC engraftment in NSG-mice by intra-femoral or intra-hepatic injections.

<p>Adult or neonatal NSG mice were sublethally irradiated and transplanted either intra-hepatically (neonatals: CB-HSPCs n = 8, 3 donors; BM-HSPCs n = 11 donors) or intra-femorally (adult: BM-HSPCs n = 14, 11 donors) with CD34⁺ HSPCs. Mice were analyzed at >20 weeks after transplantation by flow cytometry. (<b>A</b>) The number of injected CD34⁺ HSPCs for each group is given. (<b>B</b>) Engraftment was determined by human CD45 expression. The absolute number of hCD45⁺ cells in BM and spleen of reconstituted mice is shown. Indicated are values for significant differences between the groups. Bone marrow: CB i.h. vs. BM i.h., p = 0.003; CB i.h. vs. BM i.f., p = 0.004; Spleen: CB i.h. vs. BM i.h., p = 0.04; CB i.h. vs. BM i.f., p<0.0001; ANOVA, Kruskal-Wallis). Each symbol represents an individual animal. (<b>C</b>) The proportion of B cells and myeloid cells in BM and spleen is shown as percentage of human CD45⁺ cells. Each symbol represents an individual animal. (<b>D</b>) left: The percentage of mice with T cells in spleen at >20 weeks after transplantation is given. right: The absolute number of CD3⁺ T cells in the spleen of mice with T cells is shown. Due to the limited number of samples in two of the three groups no statistically analysis was performed.</p

Yield of CD34⁺ HSPCs isolated from cryoconserved and non-cryoconserved BM- MNCs.

<p>(<b>A</b>) BM mononuclear cells from BM-aspirates were isolated by density centrifugation and frozen in liquid nitrogen. After maximally 6 months of storage cells were thawed and counted. (<b>B</b>) CD34⁺ HSPCs were isolated from BM-MNCs at the day of arrival (immediate), the next day after overnight storage of BM-MNCs at 4°C or from cryoconserved samples. The number of CD34⁺ cells per 10 million BM-MNCs is shown (p = 0.001 immediate vs. cryo and p = 0.0168 next day vs. cryo; ANOVA Kruskal-Wallis).</p

Determination of the minimal number of BM-HSPCs, BM-MNCs and volume of BM-aspirate required for the reconstitution of NSG mice.

<p>Six-to eight week-old sublethally irradiated NSG mice were transplanted intra-femorally with non-cryoconserved CD34⁺ HSPCs (n = 14, 11 donors). (<b>A</b>) At >20 weeks after transplantation mice were analyzed by flow cytometry and engraftment in BM and spleen was determined by human CD45 expression and plotted vs. the number of injected HSPCs. The number of BM-HSPCs is calculated, which is required to achieve reconstitution in BM and spleen comparable to the lowest number of transplanted CB-HSPCs (4.8×10⁴ injected CD34⁺ HSPCs) that gives a reconstitution level matching with published data (BM: 6×10⁶ hCD45⁺; spleen 1.7×10⁶ hCD45⁺). Linear regression analysis revealed that at least 1.7–2.1×10⁵ BM-HSPCs are needed. (<b>B</b>) The number of BM-MNCs at sample receipt is plotted vs. the number of isolated HSPCs. The number of BM-MNCs (10⁸) is calculated that is required to isolate HSPCs sufficient for the reconstitution of six mice from one BM sample, given that each mouse receives 2.1×10⁵ HSPCs. (<b>C</b>) The number of CD34⁺ HSPCs per 10 million BM-MNCs is plotted vs. the age of carcinoma (n = 77) or control-patients (n = 7). No correlation is observed. (<b>D</b>) Percentage of samples and the volume of samples at receipt, which contained ≥10⁸ BM-MNCs allowing the isolation of HSPCs sufficient for the transplantation of six mice.</p

Reconstitution of a functional, HLA-restricted and self-tolerant T cell repertoire.

<p>Adult sublethally irradiated NSG-HLA-A2/HHD mice were transplanted intra-venously with cryoconserved CD34⁺ HSPCs (n = 23 mice, 13 donors). At different time points after transplantation mice were analyzed by flow cytometry and final analysis of organs was performed at 18–20 weeks after reconstitution. (<b>A</b>) Flow cytometric analysis of peripheral blood of reconstituted mice (n = 12 mice) at 7–19 weeks after BM-HSPC transplantation. Shown is the % of hCD45⁺, hCD45⁺CD19⁺ (B cells), hCD45⁺CD33⁺ (myeloid cells) or hCD45⁺CD3^{+ (}T cells) cells (median with interquartile range). (<b>B</b>) Engraftment in BM and spleen was determined by human CD45 expression. The absolute number of hCD45⁺ cells in BM and spleen of the reconstituted mice is shown and compared to NSG-mice reconstituted with non-cryoconserved BM-HSPCs (see Fig. 3B). Each symbol represents an individual animal. Indicated are values for significant differences between the groups (BM: p = 0.64; Spleen: p = 0.19, Mann-Whitney test). (<b>C</b>) In comparison to CB- (n = 8 mice) and BM-HSPC (n = 10 mice) transplanted NSG mice (i.h. and i.f. respectively), the absolute number of splenic CD3⁺ T cells is given for NSG-HLA-A2/HHD mice (n = 19 mice). Each symbol represents an individual animal. Indicated are values for significant differences between the groups (CB vs BM-NSG: p = 0.009; CB vs BM-HHD: p = 0.0027, Kruskal-Wallis test). (<b>D</b>) Representative flow cytometric analysis of splenic CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells and their respective expression of CD27 and CD45RA. In addition, CD27 and CD45RA expression of peripheral blood T cells of a healthy volunteer is shown. (<b>E</b>) Mixed lymphocyte reaction of human T cells isolated from NSG-HLA-A2/HHD mice ( = chimeric T cells, cT) transplanted with CB- (n = 1 donor, 2 mice) or BM-HSPCs (n = 4 donors, each 2–3 mice). CFSE-labeled cT cells were stimulated with human T cell-depleted and irradiated peripheral blood mononuclear cells (hAPC, d5∶2×10⁵ cells/well; d6∶2×10⁵ cells/well). As control, human T cells from peripheral blood of the same (auto, n = 2) or different (alllo, n = 2) HLA-A2 negative donor were isolated. Self-tolerance was tested incubating cT cells or hT cells on autologous T cell depleted, irradiated cAPC or hAPC from the same humanized mouse or human volunteer. Proliferation of T cells was measured after 5 or 6 days based on the gradual loss of the CFSE-label.</p