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Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve in vitro platelet production have been hampered by the low amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbβ3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.

CD4+ T regulatory cells (Tregs) comprise a heterogeneous population of cells the regulate immune responses and prevent autoimmunity. Most reports on human Tregs are derived from studies of peripheral blood, although Tregs mainly exert their functions in the periphery. Here we performed a detailed analysis of Tregs in the human upper airway mucosa under non-inflammatory conditions, and found that 10% of all CD4+ T cells expressed the transcription factor FOXP3 and the memory marker CD45RO, as well as high levels of CTLA-4. The majority of FOXP3+CD4+ T cells co-expressed the transcription factor Helios and produced very little cytokines, compatible with being thymus-derived Tregs. FOXP3+Helios-CD4+ T cells were more heterogeneous. A mean of 24% produced the immunomodulatory cytokine IL-10, whereas a large fraction also produced IL-2, IFN-μ or IL-17. A significant population (6%) of FOXP3-negative T cells also produced IL-10, usually in combination with IFN-μ. Together, we found that CD4+ T cells in the upper airways differed functionally from their counterparts in peripheral blood, including higher expression of IL-10. Moreover, our findings suggest that several subsets of CD4+ T cells with functionally distinct regulatory properties reside in the upper airway mucosa which should be taken into account when targeting Tregs for therapy.

Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis.

Background: Inhibitors of the mammalian target of rapamycin (mTOR) might become a novel tool to treat advanced prostate cancer. However, chronic drug exposure may trigger resistance, limiting the utility of mTOR inhibitors. Methods: Metastatic potential of PC3 prostate cancer cells, susceptible (PC3 par ) or resistant (PC3 res ) to the mTOR-inhibitor RAD001 was investigated. Adhesion to vascular endothelium or immobilised collagen, fibronectin and laminin was quantified. Motility, migration and invasion were explored by modified Boyden chamber assay. Integrin α and β subtypes were analysed by flow cytometry, western blotting and real-time PCR. Integrin-related signalling, EGFr, Akt, p70S6kinase and ERK1/2 activation were determined. Results: Adhesion was reduced, whereas motility, migration and invasion were enhanced in PC3 res . The α 2 and β 1 integrin subtypes were dramatically elevated, integrins α 1 and α 6 were lowered, whereas α 5 was nearly lost in PC3 res . Activation of the Akt signalling pathway was strongly upregulated in these cells. Treating PC3 par cells with RAD001 reduced motility, migration and invasion and deactivated Akt signalling. Blocking studies revealed that α 2 and β 1 integrins significantly trigger the motile behaviour of the tumour cells. Conclusion: Chronic RAD001 treatment caused resistance development characterised by distinct modification of the integrin-expression profile, driving prostate cancer cells towards high motility.

Curcumin is a potent anti-cancer drug in several types of human cancers. Despite of several preclinical and clinical studies of curcumin, the precise mechanism of curcumin in cancer prevention has remained unclear. In our study, we for the first time investigated whole transcriptome alteration in A549 non–small cell lung cancer (NSCLC) cell lines after treatment with curcumin using RNA sequencing. We found that lots of genes and signaling pathways were significantly altered after curcumin treatment in A549 cells. With bioinformatics approaches (gene ontology, Kyoto Encyclopedia of Genes and Genomes, and STRING), we found that those curcumin altered genes were not only the genes that induce cell death but also those extracellular matrix receptors and mitogen-activated protein kinase signaling pathway genes which regulate cell migration and proliferation. Among those significantly altered genes, eight genes (COL1A1, COL4A1, COL5A1, LAMA5, ITGA3, ITGA2B, DDIT3, and DUSP1) were further examined by quantitative reverse transcription polymerase chain reaction and western blot analysis in four non–small cell lung cancer cell lines. Both in cell lines and in mouse model, the extracellular matrix receptors including the integrin (ITGA3 and ITGA2B), collagen (COL5A1), and laminin (LAMA5) were significantly inhibited by curcumin at messenger RNA and protein levels. Functional studies confirmed that curcumin not only induced A549 cell death but also repressed cell proliferation and migration by regulating extracellular matrix receptors. Collectively, our study suggests that curcumin may be used as a promising drug candidate for intervening lung cancer in future studies.

Background There is increasing evidence to suggest that pericytes play a crucial role in regulating the remodeling state of blood vessels. As cerebral pericytes are embedded within the extracellular matrix (ECM) of the vascular basal lamina, it is important to understand how individual ECM components influence pericyte remodeling behavior, and how cytokines regulate these events. Methods The influence of different vascular ECM substrates on cerebral pericyte behavior was examined in assays of cell adhesion, migration, and proliferation. Pericyte expression of integrin receptors was examined by flow cytometry. The influence of cytokines on pericyte functions and integrin expression was also examined, and the role of specific integrins in mediating these effects was defined by function-blocking antibodies. Expression of pericyte integrins within remodeling cerebral blood vessels was analyzed using dual immunofluorescence (IF) of brain sections derived from the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Results Fibronectin and collagen I promoted pericyte proliferation and migration, but heparan sulfate proteoglycan (HSPG) had an inhibitory influence on pericyte behavior. Flow cytometry showed that cerebral pericytes express high levels of α5 integrin, and lower levels of α1, α2, and α6 integrins. The pro-inflammatory cytokine tumor necrosis factor (TNF)-α strongly promoted pericyte proliferation and migration, and concomitantly induced a switch in pericyte integrins, from α1 to α2 integrin, the opposite to the switch seen when pericytes differentiated. Inhibition studies showed that α2 integrin mediates pericyte adhesion to collagens, and significantly, function blockade of α2 integrin abrogated the pro-modeling influence of TNF-α. Dual-IF on brain tissue with the pericyte marker NG2 showed that while α1 integrin was expressed by pericytes in both stable and remodeling vessels, pericyte expression of α2 integrin was strongly induced in remodeling vessels in EAE brain. Conclusions Our results suggest a model in which ECM constituents exert an important influence on pericyte remodeling status. In this model, HSPG restricts pericyte remodeling in stable vessels, but during inflammation, TNF-α triggers a switch in pericyte integrins from α1 to α2, thereby stimulating pericyte proliferation and migration on collagen. These results thus define a fundamental molecular mechanism in which TNF-α stimulates pericyte remodeling in an α2 integrin-dependent manner.

The tumor stem cell theory could explain how patients with metastatic disease show clinical relapse several months after starting treatment due to the survival of a small group of cells with unique characteristics. We examined the distribution and expression of a panel of stem cell markers in human breast cancer primary tumors. Human breast tissues were processed for immunohistochemistry, and RNA was extracted for analysis by quantitative-PCR. Immunohistochemical assay revealed that CD44 was strongly expressed in background endothelia and epithelia. CD133 expression was lost in tumor-associated endothelial cells. Conversely, CD49b was strongly stained in the tumors, associated vessels and ducts but was weakly stained in the background epithelia. q-PCR analysis revealed that CD44 and PSCA were reduced in patients with poor outcome (metastatic disease and death from breast cancer), with a marked reduction in ductal carcinoma, particularly with metastasis to bone although these did not reach significant difference. CD133 was significantly reduced in patients with metastatic disease and was also significantly reduced in patients with ductal carcinoma/bone metastasis. Conversely, CD49F was increased in patients with a poor outcome and those with ductal cancer and bone metastases. This is the first study to determine the distribution and expression pattern of these stem cell markers in human breast cancer. There was a significant association between loss of expression and metastatic disease in patients with breast cancer. Such differential expression may play a part in breast cancer disease progression, and suggests that the current stem cell theory may not hold true for all cancer types.

Background Viable embryos secrete preimplantation factor (PIF), a peptide that has autocrine effects where levels correlate with cultured embryos development. sPIF (PIF synthetic analog) promotes implantation by regulating decidual-cells immunity, adhesion, apoptosis and enhances trophoblastic cell invasion. Herein sPIF priming effects on non-decidualized endometrium and decidualized-stroma are investigated, assessing elements critical for effective embryo-maternal cross-talk, prior to and at implantation. Methods We tested sPIF effect on human non-pregnant endometrial epithelial and non-decidualized stroma α2β3 integrin expression (IHC and flow cytometry), comparing with scrambled PIF (PIFscr-control). We examined sPIF effect on decidualized non-pregnant human endometrial stromal cells (HESC) determining pro-inflammatory mediators expression and secretion (ELISA) and growth factors (GFs) expression (Affymetrix global gene array). We tested sPIF effect on HESC Phospho-kinases (BioPlex) and isolated kinases activity (FastKinase). Results sPIF up-regulates α2β3 integrin expression in epithelial cells, (P < 0.05) while PIFscr had no effect. In contrast, in stromal cell cultures sPIF had no effect on the same. In HESC, sPIF up-regulates pro-inflammatory cytokines; IL8, IL1β and IL6 expression. The major increase in GRO-α, ICAM-1 and MCP-3 expression is coupled with same ligands secretion (P < 0.05). sPIF modulates in HESC GFs expression: up-regulates amphiregulin and epiregulin- critical for implantation and enhances several fibroblast growth factors (FGF) relevant for decidual function. In contrast, sPIF down-regulates major pro-proliferative ligands, betacellulin and IGF1 expression. sPIF modulatory effect on GFs is exerted by down-regulating pro-proliferative phospho-activated MAPkinases, p-MEK1 and p-ERK (P < 0.01, P < 0.04, respectively). Stress-induced p-38-MAPK (P = 0.04) and c-Jun kinase signaling involved MAPK8IP2 (−2.1 fold) expression decreased which protects against reactive oxygen species. Although pro-inflammatory p-NFkB (P = 0.06) decrease was mild, its promoter TNFRS11 expression markedly (−25-fold) decreased. In contrast, anti-proliferative phosphatases PTPRZ1 and PPP2R2C expression increased. Conclusions sPIF post-fertilization primes endometrial-epithelium, while during implantation creates a beneficial pro-inflammatory milieu. PIF acts by balancing decidual pro-implantation properties while controlling excessive pro-proliferative and inflammatory signals expression. Overall, PIF influences critical peri-implantation events in a sequential coordinated fashion which facilitates embryo implantation.

Natural killer (NK) cells are part of the first line defense against tumors, parasites and virus‐infected cells. Therefore, factors that control NK‐cell numbers and their function are important. CD27 is constitutively expressed on NK cells and its expression correlates with sequential phases in NK‐cell development, discriminating phenotypically and functionally different subsets within the NK‐cell population. Although CD27 has been described to have an important regulatory role in effector and memory T and B lymphocytes, its role in NK‐cell biology remains to be addressed. In this study, we used CD27−/− mice to investigate the role of CD27 in NK‐cell development and function, both during the resting state and upon stimulation. The results show that NK‐cell numbers are not impaired in CD27−/− mice. Moreover, CD27−/− NK cells reach full phenotypic maturity, evidenced by normal expression of CD49b, CD43 and CD11b. Expression of activating receptors is unaltered, whereas expression of several inhibitory receptors is increased. Cytotoxicity and interferon‐γ production by NK cells from CD27−/− mice in the resting state are normal. However, upon in vivo anti‐CD40‐ or poly‐I:C‐mediated activation, or in vitro interleukin‐15 priming plus anti‐NKp46 stimulation, the absence of CD27 results in decreased cytolytic activity and cytokine production by spleen and liver NK cells. In conclusion, this study demonstrates that CD27 is dispensable for the development of functional NK cells. However, upon stimulation of NK cells, CD27 displays an important role in their activation and functionality.

Background Cancer cells are typically surrounded by stromal cells and embedded in extracellular matrix (ECM). The stromal compartment interacts with cancer cells to promote growth and metastasis. For decades, autologous fasciocutaneous flaps have been safely applied for breast reconstruction after mastectomy. In contrast, the safety of fat grafting (lipofilling) procedure has been under debate regarding the risk of cancer recurrence. Methods Harvested fat tissue (lipoaspirates) and dissected abdominal fat (DAF) were co-cultured with MCF-7 breast cancer cells. The vitality of MCF-7 cells was measured using AlamarBlue ® consecutively for 5 days. ECM degradation was determined by detection of matrix metalloproteinase-1 (MMP-1) expression in MCF-7 cells. Integrin α2 was measured by Western blot to assess the degree of adhesion and motility of MFC-7 cells. Results The MCF-7 proliferation increased substantially when co-cultured with fat tissue. However, there was no significant difference between the proliferation stimulating effects of lipoaspirates and DAF. Similarly, MMP-1 protein expression was equally elevated in MCF-7 cells by both lipoaspirates and DAF. Importantly, MCF-7 cells showed an increased level of integrin α2 once co-cultured with either lipoaspirates or DAF. Conclusion Fat tissue increases the proliferation of MCF-7 cells in vitro. Our data suggest that lipoaspirates as well as DAF might possess a considerable potency to promote tumorigenic growth of breast cancer cells. Thus, clinical trials are needed to address the safety of lipofilling by breast reconstruction surgery after mastectomy.