Transcriptional Profile of Cytokines, Regulatory Mediators and TLR in Mesenchymal Stromal Cells after Inflammatory Signaling and Cell-Passaging.

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1ß, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFß. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches.

The importance of exosomal PDL1 in tumour immune evasion.

The interaction of programmed cell death 1 ligand 1 (PDL1) with its receptor programmed cell death 1 (PD1) inhibits T cell responses, and blockade of this interaction has proven to be an effective immunotherapy for several different cancers. PDL1 can be expressed on the surface of tumour cells, immune cells and other cells in the tumour microenvironment but is also found in extracellular forms. Recent studies have explored the importance of different forms of extracellular PDL1, such as on exosomes or as a freely soluble protein, and have shown that PDL1-expressing exosomes can inhibit antitumour immune responses. In patients with melanoma, exosomal PDL1 is also a marker of immune activation early after initiation of therapy with PD1-blocking antibodies and predicts a clinical response to PD1 blockade. In this Progress article, we highlight recent insights into the role of exosomal PDL1 in immune oncology and how it may be useful as a biomarker for the management of cancer or to define a subset of patients who would benefit from therapeutics that block exosome production.

Differential expression patterns of MafB and c-Maf in macrophages in vivo and in vitro.

The large Maf transcription factors c-Maf and MafB are expressed in macrophage-lineage hematopoietic cells, but the expression patterns of MafB and c-Maf in macrophage subtypes and tissue-resident macrophages have not been fully analyzed. First, we analyzed MafB and c-Maf protein expression in tissue-resident macrophages. Mouse lymph nodes, spleens, lungs, and kidneys were subjected to immunohistochemistry using anti-MafB and anti-c-Maf. Both MafB and c-Maf signals were observed in lymph node macrophages. In the splenic macrophages the MafB signal was detected by anti-MafB, but the c-Maf signal was not detected. No expression of c-Maf or MafB was detected in macrophages in the lung and kidney. Flow cytometry analysis revealed a similar pattern of GFP expression in Mafb/GFP knock-in heterozygous mice. To analyze these different expression patterns in greater detail, we examined the expression of MafB and c-Maf by quantitative RT-PCR in different cytokine- or LPS-induced macrophages in vitro. MafB expression was induced by IL-10 or IL-4 with IL-13 and was reduced by LPS or GM-CSF. By contrast, c-Maf expression was induced by IL-10 and reduced by IL-4 with IL-13 or GM-CSF. These results indicate that MafB and c-Maf have different expression patterns in macrophages, suggesting differences in function.

Optimisation of the critical medium components for better growth of Picochlorum sp. and the role of stressful environments for higher lipid production.

BACKGROUND: Coastal countries that suffer from a scarcity of water, such as Tunisia, have to cultivate marine microalgae on non-arable land in order to produce feedstock and overcome their demands of nutrition and energy. In this framework, a green microalga, CTM 20019, was isolated, identified as Picochlorum sp. and tested for its lipid production. RESULTS: The dry weight of Picochlorum sp. is composed of 163 g kg(-1) lipids, 225 g kg(-1) total sugars, 440 g kg(-1) proteins and 112 g kg(-1) ash rich in potassium, calcium, iron, magnesium and zinc. Gas chromatography-mass spectrometry analysis showed that the main fatty acids were palmitic acid (29%), linolenic acid (26.5%), linoleic acid (23.5%), hexadecatrienoic acid (11%) and hexadecadienoic acid (8.5%). As it is known that culture conditions greatly influence the composition of microalgae, the experiments were designed to optimise the composition of the medium in order to increase Picochlorum sp. growth from OD680nm = 0.53 to OD680nm = 2.2 and lipid accumulation from 163 g kg(-1) to 190 g kg(-1) . The highest lipid contents of 570 and 585 g kg(-1) were achieved under phosphate starvation and sodium carbonate supplementation, respectively. Under these conditions, the fatty acid profile is dominated by mono-unsaturated and polyunsaturated acids, and is therefore suitable for aqua-culture feeding. However, under high salinity, growth and lipid synthesis are inhibited, and the fatty acids are saturate, and the product is therefore suitable for biodiesel. CONCLUSION: This high lipid content rich in essential fatty acids, omega-6 and omega-3, endorses this wild strain of Picochlorum sp. as a promising feedstock for aqua-culture and human nutrition or for the production of biodiesel. © 2013 Society of Chemical Industry.

Bioluminescence imaging of ß cells and intrahepatic insulin gene activity under normal and pathological conditions.

In diabetes research, bioluminescence imaging (BLI) has been applied in studies of ß-cell impairment, development, and islet transplantation. To develop a mouse model that enables noninvasive imaging of ß cells, we generated a bacterial artificial chromosome (BAC) transgenic mouse in which a mouse 200-kbp genomic fragment comprising the insulin I gene drives luciferase expression (Ins1-luc BAC transgenic mouse). BLI of mice was performed using the IVIS Spectrum system after intraperitoneal injection of luciferin, and the bioluminescence signal from the pancreatic region analyzed. When compared with MIP-Luc-VU mice [FVB/N-Tg(Ins1-luc)VUPwrs/J] expressing luciferase under the control of the 9.2-kbp mouse insulin I promoter (MIP), the bioluminescence emission from Ins1-luc BAC transgenic mice was enhanced approximately 4-fold. Streptozotocin-treated Ins1-luc BAC transgenic mice developed severe diabetes concomitant with a sharp decline in the BLI signal intensity in the pancreas. Conversely, mice fed a high-fat diet for 8 weeks showed an increase in the signal, reflecting a decrease or increase in the ß-cell mass. Although the bioluminescence intensity of the islets correlated well with the number of isolated islets in vitro, the intensity obtained from a living mouse in vivo did not necessarily reflect an absolute quantification of the ß-cell mass under pathological conditions. On the other hand, adenovirus-mediated gene transduction of ß-cell-related transcription factors in Ins1-luc BAC transgenic mice generated luminescence from the hepatic region for more than 1 week. These results demonstrate that BLI in Ins1-luc BAC transgenic mice provides a noninvasive method of imaging islet ß cells and extrapancreatic activity of the insulin gene in the liver under normal and pathological conditions.