Biphasic Calcium Phosphate and Activated Carbon Microparticles in a Plasma Clot for Bone Reconstruction and In Situ Drug Delivery: A Feasibility Study.

The development of bone-filling biomaterials capable of delivering in situ bone growth promoters or therapeutic agents is a key area of research. We previously developed a biomaterial constituting biphasic calcium phosphate (BCP) microparticles embedded in an autologous blood or plasma clot, which induced bone-like tissue formation in ectopic sites and mature bone formation in orthotopic sites, in small and large animals. More recently, we showed that activated carbon (AC) fiber cloth is a biocompatible material that can be used, due to its multiscale porosity, as therapeutic drug delivery system. The present work aimed first to assess the feasibility of preparing calibrated AC microparticles, and second to investigate the properties of a BCP/AC microparticle combination embedded in a plasma clot. We show here, for the first time, after subcutaneous (SC) implantation in mice, that the addition of AC microparticles to a BCP/plasma clot does not impair bone-like tissue formation and has a beneficial effect on the vascularization of the newly formed tissue. Our results also confirm, in this SC model, the ability of AC in particle form to adsorb and deliver large molecules at an implantation site. Altogether, these results demonstrate the feasibility of using this BCP/AC/plasma clot composite for bone reconstruction and drug delivery.

Long-Term Fate and Efficacy of a Biomimetic (Sr)-Apatite-Coated Carbon Patch Used for Bone Reconstruction.

Critical bone defect repair remains a major medical challenge. Developing biocompatible materials with bone-healing ability is a key field of research, and calcium-deficient apatites (CDA) are appealing bioactive candidates. We previously described a method to cover activated carbon cloths (ACC) with CDA or strontium-doped CDA coatings to generate bone patches. Our previous study in rats revealed that apposition of ACC or ACC/CDA patches on cortical bone defects accelerated bone repair in the short term. This study aimed to analyze in the medium term the reconstruction of cortical bone in the presence of ACC/CDA or ACC/10Sr-CDA patches corresponding to 6 at.% of strontium substitution. It also aimed to examine the behavior of these cloths in the medium and long term, in situ and at distance. Our results at day 26 confirm the particular efficacy of strontium-doped patches on bone reconstruction, leading to new thick bone with high bone quality as quantified by Raman microspectroscopy. At 6 months the biocompatibility and complete osteointegration of these carbon cloths and the absence of micrometric carbon debris, either out of the implantation site or within peripheral organs, was confirmed. These results demonstrate that these composite carbon patches are promising biomaterials to accelerate bone reconstruction.

Impact of thermogenesis induced by chronic ß3-adrenergic receptor agonist treatment on inflammatory and infectious response during bacteremia in mice.

White adipocytes store energy differently than brown and brite adipocytes which dissipate energy under the form of heat. Studies have shown that adipocytes are able to respond to bacteria thanks to the presence of Toll-like receptors at their surface. Despite this, little is known about the involvement of each class of adipocytes in the infectious response. We treated mice for one week with a ß3-adrenergic receptor agonist to induce activation of brown adipose tissue and brite adipocytes within white adipose tissue. Mice were then injected intraperitoneally with E. coli to generate acute infection. The metabolic, infectious and inflammatory parameters of the mice were analysed during 48 hours after infection. Our results shown that in response to bacteria, thermogenic activity promoted a discrete and local anti-inflammatory environment in white adipose tissue characterized by the increase of the IL-1RA secretion. More generally, activation of brown and brite adipocytes did not modify the host response to infection including no additive effect with fever and an equivalent bacteria clearance and inflammatory response. In conclusion, these results suggest an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes in response to acute bacterial infection and open a way to characterize their effect along more chronic infection as septicaemia.

Modulation of the inflammatory response to LPS by the recruitment and activation of brown and brite adipocytes in mice.

Numerous studies have shown that the recruitment and activation of thermogenic adipocytes, which are brown and beige/brite, reduce the mass of adipose tissue and normalize abnormal glycemia and lipidemia. However, the impact of these adipocytes on the inflammatory state of adipose tissue is still not well understood, especially in response to endotoxemia, which is a major aspect of obesity and metabolic diseases. First, we analyzed the phenotype and metabolic function of white and brite primary adipocytes in response to lipopolysaccharide (LPS) treatment in vitro. Then, 8-wk-old male BALB/c mice were treated for 1 wk with a ß3-adrenergic receptor agonist (CL316,243, 1 mg/kg/day) to induce recruitment and activation of brown and brite adipocytes and were subsequently injected with LPS (Escherichia coli lipopolysaccharide, 100 µg/mouse ip) to generate acute endotoxemia. The metabolic and inflammatory parameters of the mice were analyzed 6 h later. Our results showed that in response to LPS, thermogenic activity promoted a local anti-inflammatory environment with high secretion of IL-1 receptor antagonist (IL-1RA) without affecting other anti- or proinflammatory cytokines. Interestingly, activation of brite adipocytes reduced the LPS-induced secretion of leptin. However, thermogenic activity and adipocyte function were not altered by LPS treatment in vitro or by acute endotoxemia in vivo. In conclusion, these results suggest an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes specifically in response to endotoxemia. This encourages potential therapy involving brown and brite adipocytes for the treatment of obesity and associated metabolic diseases.NEW & NOTEWORTHY Recruitment and activation of brown and brite adipocytes in the adipose tissue of mice lead to a local low-grade anti-inflammatory phenotype in response to acute endotoxemia without alteration of adipocyte phenotype and function.

Breast cancer mammospheres secrete Adrenomedullin to induce lipolysis and browning of adjacent adipocytes.

BACKGROUND: Cancer cells cooperate with cells that compose their environment to promote tumor growth and invasion. Among them, adipocytes provide lipids used as a source of energy by cancer cells and adipokines that contribute to tumor expansion. Mechanisms supporting the dynamic interactions between cancer cells and stromal adipocytes, however, remain unclear. METHODS: We set-up a co-culture model with breast cancer cells grown in 3D as mammospheres and human adipocytes to accurately recapitulate intrinsic features of tumors, such as hypoxia and cancer cell-adipocytes interactions. RESULTS: Herein, we observed that the lipid droplets' size was reduced in adipocytes adjacent to the mammospheres, mimicking adipocyte morphology on histological sections. We showed that the uncoupling protein UCP1 was expressed in adipocytes close to tumor cells on breast cancer histological sections as well as in adipocytes in contact with the mammospheres. Mammospheres produced adrenomedullin (ADM), a multifactorial hypoxia-inducible peptide while ADM receptors were detected in adipocytes. Stimulation of adipocytes with ADM promoted UCP1 expression and increased HSL phosphorylation, which activated lipolysis. Invalidation of ADM in breast cancer cells dramatically reduced UCP1 expression in adipocytes. CONCLUSIONS: Breast tumor cells secreted ADM that modified cancer-associated adipocytes through paracrine signaling, leading to metabolic changes and delipidation. Hence, ADM appears to be crucial in controlling the interactions between cancer cells and adipocytes and represents an excellent target to hinder them.

Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma.

Inherent immune suppression represents a major challenge in the treatment of human cancer. The extracellular matrix molecule tenascin-C promotes cancer by multiple mechanisms, yet the roles of tenascin-C in tumor immunity are incompletely understood. Using a 4NQO-induced oral squamous cell carcinoma (OSCC) model with abundant and absent tenascin-C, we demonstrated that tenascin-C enforced an immune-suppressive lymphoid stroma via CCL21/CCR7 signaling, leading to increased metastatic tumors. Through TLR4, tenascin-C increased expression of CCR7 in CD11c+ myeloid cells. By inducing CCL21 in lymphatic endothelial cells via integrin α9ß1 and binding to CCL21, tenascin-C immobilized CD11c+ cells in the stroma. Inversion of the lymph node-to-tumor CCL21 gradient, recruitment of T regulatory cells, high expression of anti-inflammatory cytokines, and matrisomal components were hallmarks of the tenascin-C-instructed lymphoid stroma. Ablation of tenascin-C or CCR7 blockade inhibited the lymphoid immune-suppressive stromal properties, reducing tumor growth, progression, and metastasis. Thus, targeting CCR7 could be relevant in human head and neck tumors, as high tenascin-C expression and an immune-suppressive stroma correlate to poor patient survival.

R-spondin signalling is essential for the maintenance and differentiation of mouse nephron progenitors.

During kidney development, WNT/ß-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of nephron progenitor cells. Here, we show in mice that the signalling molecules RSPO1 and RSPO3 act in a functionally redundant manner to permit WNT/ß-catenin signalling and their genetic deletion leads to a rapid decline of nephron progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss of Bmp7 expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activate Lef1, Fgf8 and Wnt4, thus explaining the observed phenotype on a molecular level. Surprisingly, the full knockout of LGR4/5/6, the cognate receptors of R-spondins, only mildly affects progenitor numbers, but does not interfere with MET. Taken together our data demonstrate key roles for R-spondins in permitting stem cell maintenance and differentiation and reveal Lgr-dependent and independent functions for these ligands during kidney formation.

Kidneys filter waste out of the bloodstream to produce urine. Each kidney contains many structures called nephrons which separate the waste from the blood. The number of nephrons in a kidney varies between people, and those with low numbers have a higher risk of chronic kidney disease. Nephrons are formed before birth from a specific group of so-called progenitor cells. Each of these cells can either divide to make others like itself, or it can specialize to make nephron cells. At the end of embryonic kidney development, all the progenitor cells become nephron cells. Cells that specialize to become part of a nephron first go through a change called a mesenchyme-to-epithelial transition. Epithelial cells move less than mesenchymal cells, and also develop a clear structure where the two ends of the cell adapt to different roles. Evidence suggests that a cell communication process called WNT/ß-catenin signaling controls this transition. Yet the details of how this transition is controlled are not fully understood. One way to activate WNT/ß-catenin signaling is with R-spondin proteins, which have been found in developing kidneys. Vidal et al. studied R-spondins during the embryonic development of kidneys in mice. Removing R-spondins stopped the progenitor cells from producing more of themselves and increased the number that died. The R-spondins were also needed for the progenitor cells to specialize as nephron cells through the mesenchyme-to-epithelial transition. Further results revealed that R-spondins activate WNT/ß-catenin signaling in these cells, even though the proteins that usually act as R-spondin receptors (called LGR4/5/6) could be removed without affecting the results. This suggests that R-spondins interact with different receptor proteins during kidney development. These findings highlight the role of R-spondins and WNT/ß-catenin signaling in kidney development. Future studies will seek the receptor proteins that R-spondins interact with in kidneys. They may also look to understand how R-spondins balance their different roles in progenitor cells and during cell specialization. These results in mice could also be extended to determine their relevance in human health and disease, including chronic kidney disease, which is responsible for more deaths than breast or prostate cancer.

A role for early oral exposure to house dust mite allergens through breast milk in IgE-mediated food allergy susceptibility.

BACKGROUND: Successful prevention of food allergy requires the identification of the factors adversely affecting the capacity to develop oral tolerance to food antigen in early life. OBJECTIVES: This study sought to determine whether oral exposure to Dermatophagoides pteronyssinus through breast milk affects gut mucosal immunity with long-term effects on IgE-mediated food allergy susceptibility. METHODS: Gut immunity was explored in 2-week-old mice breast-fed by mothers exposed to D pteronyssinus, protease-inactivated D pteronyssinus, or to PBS during lactation. We further analyzed oral tolerance to a bystander food allergen, ovalbumin (OVA). In a proof-of-concept study, Der p 1 and OVA levels were determined in 100 human breast milk samples and the association with prevalence of IgE-mediated egg allergy at 1 year was assessed. RESULTS: Increased permeability, IL-33 levels, type 2 innate lymphoid cell activation, and Th2 cell differentiation were found in gut mucosa of mice nursed by mothers exposed to D pteronyssinus compared with PBS. This pro-Th2 gut mucosal environment inhibited the induction of antigen-specific FoxP3 regulatory T cells and the prevention of food allergy by OVA exposure through breast milk. In contrast, protease-inactivated D pteronyssinus had no effect on offspring gut mucosal immunity. Based on the presence of Der p 1 and/or OVA in human breast milk, we identified groups of lactating mothers, which mirror the ones found in mice to be responsible for different egg allergy risk. CONCLUSIONS: This study highlights an unpredicted potential risk factor for the development of food allergy, that is, D pteronyssinus allergens in breast milk, which disrupt gut immune homeostasis and prevents oral tolerance induction to bystander food antigen through their protease activity.

Diet Supplementation in ω3 Polyunsaturated Fatty Acid Favors an Anti-Inflammatory Basal Environment in Mouse Adipose Tissue.

Oxylipins are metabolized from dietary ω3 and ω6 polyunsaturated fatty acids and are involved in an inflammatory response. Adipose tissue inflammatory background is a key factor of metabolic disorders and it is accepted that dietary fatty acids, in terms of quality and quantity, modulate oxylipin synthesis in this tissue. Moreover, it has been reported that diet supplementation in ω3 polyunsaturated fatty acids resolves some inflammatory situations. Thus, it is crucial to assess the influence of dietary polyunsaturated fatty acids on oxylipin synthesis and their impact on adipose tissue inflammation. To this end, mice fed an ω6- or ω3-enriched standard diet (ω6/ω3 ratio of 30 and 3.75, respectively) were analyzed for inflammatory phenotype and adipose tissue oxylipin content. Diet enrichment with an ω3 polyunsaturated fatty acid induced an increase in the oxylipins derived from ω6 linoleic acid, ω3 eicosapentaenoic, and ω3 docosahexaenoic acids in brown and white adipose tissues. Among these, the level of pro-resolving mediator intermediates, as well as anti-inflammatory metabolites, were augmented. Concomitantly, expressions of M2 macrophage markers were increased without affecting inflammatory cytokine contents. In vitro, these metabolites did not activate macrophages but participated in macrophage polarization by inflammatory stimuli. In conclusion, we demonstrated that an ω3-enriched diet, in non-obesogenic non-inflammatory conditions, induced synthesis of oxylipins which were involved in an anti-inflammatory response as well as enhancement of the M2 macrophage molecular signature, without affecting inflammatory cytokine secretion.

Impact of dietary ω3 polyunsaturated fatty acid supplementation on brown and brite adipocyte function.

The recent characterization of functional brown adipose tissue in adult humans has opened new perspectives for regulation of energy expenditure with respect to obesity and diabetes. Furthermore, dietary recommendations have taken into account the insufficient dietary intake of ω3 PUFAs and the concomitant excessive intake of ω6 PUFA associated with the occurrence of overweight/obesity. We aimed to study whether ω3 PUFAs could play a role in the recruitment and function of energy-dissipating brown/brite adipocytes. We show that ω3 PUFA supplementation has a beneficial effect on the thermogenic function of adipocytes. In vivo, a low dietary ω6:ω3 ratio improved the thermogenic response of brown and white adipose tissues to ß3-adrenergic stimulation. This effect was recapitulated in vitro by PUFA treatment of hMADS adipocytes. We pinpointed the ω6-derived eicosanoid prostaglandin (PG)F2α as the molecular origin because the effects were mimicked with a specific PGF2α receptor agonist. PGF2α level in hMADS adipocytes was reduced in response to ω3 PUFA supplementation. The recruitment of thermogenic adipocytes is influenced by the local quantity of individual oxylipins, which is controlled by the ω6:ω3 ratio of available lipids. In human nutrition, energy homeostasis may thus benefit from the implementation of a more balanced dietary ω6:ω3 ratio.

Ectopic expression of Pax4 in pancreatic δ cells results in ß-like cell neogenesis.

The recent demonstration that pancreatic α cells can be continuously regenerated and converted into ß-like cells upon ectopic expression of Pax4 opened new avenues of research in the endocrine cell differentiation and diabetes fields. To determine whether such plasticity was also shared by δ cells, we generated and characterized transgenic animals that express Pax4 specifically in somatostatin-expressing cells. We demonstrate that the ectopic expression of Pax4 in δ cells is sufficient to induce their conversion into functional ß-like cells. Importantly, this conversion induces compensatory mechanisms involving the reactivation of endocrine developmental processes that result in dramatic ß-like cell hyperplasia. Importantly, these ß-like cells are functional and can partly reverse the consequences of chemically induced diabetes.

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

Fibronectin-guided migration of carcinoma collectives.

Functional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvß6 and α9ß1 engagement, rather than α5ß1. Moreover, αvß6-driven migration occurs independently of latent TGF-ß activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour-stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC.

A Positive Feed-forward Loop Associating EGR1 and PDGFA Promotes Proliferation and Self-renewal in Glioblastoma Stem Cells.

Glioblastomas are the most common primary brain tumors, highly vascularized, infiltrating, and resistant to current therapies. This cancer leads to a fatal outcome in less than 18 months. The aggressive behavior of glioblastomas, including resistance to current treatments and tumor recurrence, has been attributed to glioma stemlike/progenitor cells. The transcription factor EGR1 (early growth response 1), a member of a zinc finger transcription factor family, has been described as tumor suppressor in gliomas when ectopically overexpressed. Although EGR1 expression in human glioblastomas has been associated with patient survival, its precise location in tumor territories as well as its contribution to glioblastoma progression remain elusive. In the present study, we show that EGR1-expressing cells are more frequent in high grade gliomas where the nuclear expression of EGR1 is restricted to proliferating/progenitor cells. We show in primary cultures of glioma stemlike cells that EGR1 contributes to stemness marker expression and proliferation by orchestrating a PDGFA-dependent growth-stimulatory loop. In addition, we demonstrate that EGR1 acts as a positive regulator of several important genes, including SHH, GLI1, GLI2, and PDGFA, previously linked to the maintenance and proliferation of glioma stemlike cells.

Evolution of neutral and flowering genes along pearl millet (Pennisetum glaucum) domestication.

BACKGROUND: Pearl millet landraces display an important variation in their cycle duration. This diversity contributes to the stability of crop production in the Sahel despite inter-annual rainfall fluctuation. Conservation of phenological diversity is important for the future of pearl millet improvement and sustainable use. Identification of genes contributing to flowering time variation is therefore relevant. In this study we focused on three flowering candidate genes, PgHd3a, PgDwarf8 and PgPHYC. We tested for signatures of past selective events within polymorphism patterns of these three genes that could have been associated with pearl millet domestication and/or landraces differentiation. In order to implement ad hoc neutrality tests, a plausible demographic history of pearl millet domestication was inferred through Approximate Bayesian Computation by using eight neutral STS loci. RESULTS: Domesticated pearl millet exhibited 84% of the nucleotide diversity level found in the wild population. No specific polymorphisms were found either in the wild or in the domestic populations. The bayesian approach and previous studies suggest that gene flow between wild relatives and domesticated pearl millets is a main factor explaining these results. Early and late landraces did not show significant genetic differentiation at both the neutral and the candidate loci. A positive selection was evidenced in PgHd3a and PgDwarf8 genes of domestic forms but not in the wild population. CONCLUSION: Our results strongly suggest that PgHd3a and PgDwarf8 were likely targeted by selection during domestication. However, a potential role of any of the three candidate genes in the phenological differentiation between early and late landraces was not supported by our data. Reasons why these results contrast with previous results that have shown a slight but significant association between PgPHYC polymorphisms and variation in flowering time in pearl millet are discussed.

Cereal domestication and evolution of branching: evidence for soft selection in the Tb1 orthologue of pearl millet (Pennisetum glaucum [L.] R. Br.).

BACKGROUND: During the Neolithic revolution, early farmers altered plant development to domesticate crops. Similar traits were often selected independently in different wild species; yet the genetic basis of this parallel phenotypic evolution remains elusive. Plant architecture ranks among these target traits composing the domestication syndrome. We focused on the reduction of branching which occurred in several cereals, an adaptation known to rely on the major gene Teosinte-branched1 (Tb1) in maize. We investigate the role of the Tb1 orthologue (Pgtb1) in the domestication of pearl millet (Pennisetum glaucum), an African outcrossing cereal. METHODOLOGY/PRINCIPAL FINDINGS: Gene cloning, expression profiling, QTL mapping and molecular evolution analysis were combined in a comparative approach between pearl millet and maize. Our results in pearl millet support a role for PgTb1 in domestication despite important differences in the genetic basis of branching adaptation in that species compared to maize (e.g. weaker effects of PgTb1). Genetic maps suggest this pattern to be consistent in other cereals with reduced branching (e.g. sorghum, foxtail millet). Moreover, although the adaptive sites underlying domestication were not formerly identified, signatures of selection pointed to putative regulatory regions upstream of both Tb1 orthologues in maize and pearl millet. However, the signature of human selection in the pearl millet Tb1 is much weaker in pearl millet than in maize. CONCLUSIONS/SIGNIFICANCE: Our results suggest that some level of parallel evolution involved at least regions directly upstream of Tb1 for the domestication of pearl millet and maize. This was unanticipated given the multigenic basis of domestication traits and the divergence of wild progenitor species for over 30 million years prior to human selection. We also hypothesized that regular introgression of domestic pearl millet phenotypes by genes from the wild gene pool could explain why the selective sweep in pearl millet is softer than in maize.