Cartilage is an avascular, aneural, and alymphatic connective tissue with a limited capacity caused by low mitotic activity of its resident cells, chondrocytes. Natural repair of full thickness cartilage defects usually leads to the formation of fibrocartilage with lower function and mechanical force compared with the original hyaline cartilage and further deterioration can occur. Tissue engineering and regenerative medicine is a promising strategy to repair bone and articular cartilage defects and rehabilitate joint functions by focusing on the optimal combination of cells, material scaffolds, and signaling molecules. The unique physical and topographical properties of nanofibrous structures allow them to mimic the extracellular matrix of native cartilage, making an appropriate resemblance to induce cartilage tissue regeneration and reconstruction. To improve simulation of native cartilage, the incorporation of nanofibrous scaffolds with suitable corresponsive cells could be effective. In this review article, an attempt was made to present the current state of cartilage tissue engineering using nanofibrous scaffolds and stem cells as high proliferative immune privilege cells with chondrogenic differentiation ability. The comprehensive information was retrieved by search of relevant subject headings in Medline/Pubmed and Elsevier databases. Because of limited capacity for spontaneous repair, cartilage tissue cannot be restored to its normal function and structure after damages caused by trauma, osteoarthritis disease, accidents and so forth. Surgical strategies to repair cartilage chondral or osteochondral defects have been used to restore joint function and eliminate associated pain, including stimulation of the marrow by microfracture, mosaicplasty and cell-based therapies. Although surgical strategies reduce patient pain and increase joint mobility, the regenerated tissue is morphologically, biochemically and biomechanically inferior to the native cartilage. Additional surgery is often required to regain complete function, resulting in the progression to partial or total knee replacement. Therefore, there is a tremendous need for new regenerative medicine approaches to augment the repair process and to facilitate adequate tissue regeneration and longevity.1. Biomaterial as a proper microenvironment for the cells provides mechanical support for engineered tissues. Recently, commercially available synthetic and natural matrix has been tested in animal models or clinical trials for repair of cartilage and the overall short-term clinical outcome is favorable 2. Therefore, tissue engineering is a promising option for the treatment of cartilage defects. Using different materials and production methods, many forms of biomaterial scaffolds with different properties have been developed for cartilage tissue engineering.The novel strategy for regeneration of cartilage defects involves cells seeded biomaterials with appropriate growth factors 3.In the past decade, nanofibrous structures have attracted much interest as tissue engineered scaffolds because of their unique physical and topographical properties. The nanosized structure of a scaffold plays an important role to mimic the Extracellular Matrix (ECM) Structure Nanofiber scaffolds composed of ultra-fine biodegradable polymeric fibers morphologically similar to natural ECM have been widely emerged as potential scaffolds for cartilage tissue engineering.4. Due to some characteristics of stem cells such as self-renewal, high proliferation and trans-differentiation capacity that reduce the challenges propounded about chondrocytes 5, these non-specialized cells are the focus of interest in tissue engineering and regenerative medicine field. This article reviewed and presented actual status of in vitro and in vivo studies on the application of nanofibrous structures and stem cells for cartilage tissue reconstruction. For extraction of related publications, keywords of cartilage tissue engineering, nanofibers and stem cells as MeSH terms in PubMed were used. All data belong to the publications and efforts in the field of cartilage tissue engineering and nanofibers that was achieved to date.It is worth mentioning that while nanofibrous structures could mimic similar fiber diameters, composition, and alignment of the ECM of articular cartilage, the synchronization of these scaffolds with suitable corresponsive cells could help us to achieve the best tissue engineering results for articular cartilage 7. The biomemetic properties and good physiochemical features of nano-materials play a key role in stimulation of chondrocyte growth and cartilage tissue regeneration 9. Their physical characteristics promote advantageous biological responses of seeded cells in vitro, including increased cell proliferation and attachment while maintaining chondrocytic phenotype 10. In addition, application of nanofibrous scaffolds enables incorporation of nanospheres containing different growth factors. Exogenous transforming growth factor (TGF-β) family has been proved to stimulate cell proliferation and chondrogenesis both in vivo and in vitro. The factor TGF-β1 is naturally found in human platelets, bone, and other tissues and has been shown as an inducer of chondrogenesis 11. The controlled release of Bone Morphogenetic Protein (BMP-7) from nanospheres-containing scaffolds has induced significant ectopic bone formation in vivo12. Based on these findings, nanofibrous scaffold and nanospheres, combined with chondrogenic and osteogenic factors, have been introduced as potential candidates to reconstruct the osteochondral defect for the regeneration of bone, cartilage, and their interface simultaneously 13. To provide ECM-like nanofibrous scaffolds, a variety of techniques have been developed, including electrospinning, self-assembly, phase separation and drawing 14.Different synthetic nanomaterials have been fabricated to create the microenvironment that seeded cells can be encouraged to expand and differentiate into desired lineages, including chondrocytes 10. Electrospun nanofibers have a high specific surface area and can be functionalized with bioactive macromolecules 16. Electrospinning outcome is influenced by several parameters, including molecular weight of polymer, polymer solution properties, electric potential, distance between capillary and metal collector, etc.The most conventional method for processing of polymeric biomaterials into nanofibrous scaffolds is electrospinning with promising results for tissue engineering applications. This process is a simple economical technique to produce nanofibers from a wide range of synthetic and natural polymers in randomly-oriented or aligned manner 17, and the production of two-dimensional (2D) matrices with small pores, which inhibits cell penetration and vascular ingrowth 18. In order to elicit the maximum benefit from this method, there are some advancements or modifications to the processing conditions 19. Coaxial electrospinning technique 20 enables the controlled release of active bio-molecules by producing core-shell nanofibers trapping drugs or bioactive molecules. Several attempts have been made to fabricate three-dimensional macroporous nanofibrous electrospun scaffolds by modifying the electrospinning conditions or using post-treatments. Process modifications include low-temperature electrospinning 21, needleless electrospinning using disc as spinneret 22, application of different collector plates, such as parallel plate 23 and screws 24, and introducing micrometer-sized fibers 27 or inert particle spacers, such as salts 29, poly(ethylene oxide) (PEO) 30, gas 31, etc. Using solutions with polyelectrolyte nature  leads to the extension of fibers outwards from the collector under conditions which induce repulsion between neighboring fibers 32. In brief, post-treatments include photo-masking 33 or stacking layered mats 34.In spite of the benefits electrospinning has to offer, it suffers from limitations including jet instability, toxic solvent, packaging, handling 35. Two significant approaches have been proposed to proximate peptide nanofiber scaffolds to ECM: (1) modification with functional motifs  and (2) controlled release of molecular signals such as Fibroblast Growth Factor (FGF-2) and Vascular Endothelial Growth Factor (VEGF). In self-assembly, intermolecular forces determine the properties and shape of nanofibers. Nanofibers can be assembled with various polymeric configurations such as diblock copolymers, triblock copolymers, triblock polymers (of peptide amphiphile and dendrimers), and bolaform (of glucosamide and its deacetylated derivatives) 36. In vitro assessment of many peptide nanofiber scaffolds have revealed the ability to induce cell proliferation, differentiation, migration and ECM production 39. Poor mechanical property of peptide nanofiber scaffolds might limit its application to non-load-bearing sites 40.Novel nanofibrous scaffolds have been fabricated by self-assembling peptides through molecular self-assembly by mimicking regulatory mechanisms of natural ECM. Self-assembly is a manufacturing process in which small molecules-as basic building blocks- will be added-up to form nanofibres. These structures have gained much progress in repairing different injured tissues such as cartilage, bone, nerve, heart and blood vessel nm) 41. This technique is based on the physical incompatibility of polymers and their tendency to separate into two phases for nanofiber production 17. Phase-separation provides the possibilities of scaffold fabrication for a desired anatomical shape and presenting the nano and macro architecture simultaneously 42. Although the fabrication process is convenient and requires simple instrumentation, it is limited to only certain specific polymer-solvent combinations. Also, fiber dimensions cannot be controlled and the mechanical properties of the fiber are not suitable for load-bearing applications due to the highly porous structure. The controlling parameters include polymer type, polymer concentration, solvent type and thermal treatment 41.Phase-separation is a method for fabrication of 3D nanofibrous structures with nanofibers that closely mimic dimension of collagen fibrils of ECM  and induced Pluripotent Stem Cells (iPSCs) are cell sources with high chondrogenic potentials; however, there are concerns on their immunogenicity, potential for malignancy, ethical issues (for ESCs), and heterogeneous differentiation. Therefore, these cell sources cannot be the best candidate for cartilage tissue engineering 50. Compared with adult chondrocytes, they can easily be obtained and manipulated as they are able to undergo several passages before losing their differentiation potential.As shown in in vitro or in vivo52. They can provide a suitable cell source for osteochondral tissue reconstruction 13 and have also been previously explored for the engineering of fibro-cartilaginous tissues such as the annulus fibrous of the inter-vertebral disc and the knee meniscus 54. For example, Li et al stated that adult BMMSCs seeded on electrospun polycaprolactone (PCL) combined with TGF-β1 differentiated into a chondrocytic phenotype at levels comparable to traditional pellet cultures. The designed constructs showed a zonal morphology with a layer of cartilaginous matrix composed of collagen type II, cartilage proteoglycan link protein, and aggrecan 55.Bone Marrow Mesenchymal Stem Cells (BMMSCs), for example, are multi-potential stem cells with the capacity to differentiate into a variety of tissue types including bone, cartilage, fat, muscle, tendon and other tissues when induced by the appropriate cues both et al have studied the in vitro characteristics and chondrogenic capacity of four available human adult stem/progenitor cell sources using aligned electrospun polycaprolactone/poly (L-lactic acid) (PCL/PLLA) nanofibers. The studied cells include BMMSCs, adipose tissue-derived MSC (AD-MSC), Articular Chondrocyte Progenitors (ACP), and nasal septum-derived progenitors (NSPs). Accordingly, NSPs exhibited the highest proliferation potential and chondrogenic capacity 47.Shafiee 57. Our group presented the evidence introducing menstrual blood stem cells (MenSCs) as a suitable stem cell population candidate for cartilage tissue engineering. Indeed, the chondrogenic capacity of MenSCs is a major issue which may support future application of MenSCs as a reliable source for cell therapy of cartilage defects.More recently, menstrual blood has been identified as an easily accessible and renewable stem cell source with the higher proliferative rate compared with umbilical cord and bone marrow derived mesenchymal stem cells 59 and poly  (PLGA) 61. Wise et al found that cell orientation is minimally influenced by soluble factors and is mainly controlled by physical cues (oriented micro- and nano-fibers in this study); however, cell shape was affected by chondrogenic factors 58. Cells cultured in chondrogenic media on nanofibers showed a significant increase in the sGAG content and expression of collagen type II in comparison with culturing in normal growth media and on microfiber scaffolds 58. Alves da Silva et al cultured BMMSCs on electrospun PCL nanofiber mesh in a multi-chamber flow perfusion bioreactor to produce cartilagineous extracellular matrix 59. Statically cultured cells had a fibroblast-like morphology, while dynamic condition induced round-shaped morphology with increased amount of sGAG and collagen type I and II. However, there was no significant difference between gene expression of chondrogenic markers in two culture conditions. Another study has shown that PLGA electrospun nanofibers assisted the growth and differentiation of human BMMSCs as well as their osteogenic and chondrogenic potential 60.Electro-spun nanofibers with different compositions have been widely studied for osteochondral differentiation . Chondro57. In addition, culturing on PCL nanofibers improved level of sGAG and proteoglycan production compared to PCL film  for chondrogenic differentiation on PLGA and PCL electrospun nanofibers 48. Cell culturing on nanofibers resulted in the production of higher levels of sGAG and sulfated proteoglycans. The ratio of collagen type II to type I expression was considered as the differentiation index (DI) in cartilage tissue engineering. There was a significant increase in the DI between PLGA and pellet control while no differences between PCL and PLGA cultures or between the PCL and pellet cultures were detected. While the expression level of elastin was not different between pellet controls and the two nanofiber conditions, significant increase in collagen type X on PLGA nanofiber scaffolds was found when compared to pellet controls.Dahl Randomly-oriented nanofibers: Shafieeet al have proved the potential of hybrid PVA/PCL nanofiber mesh seeded with rabbit BMMSCs in terms of cartilage tissue engineering in vitro and in vivo3. Electrospinning PVA concurrently with PCL improved the capacity of nanofibrous scaffold for cell attachment and interactions and consequently improved cell proliferation rate.et al suggested that soft scaffolds composed of the highly biodegradable PLGA and collagen, in two ratios (40:60 and 60:40) were optimal for chondrogenesis 62. Most recently, Ching et al suggested that P(3HB)/P(3HO) nanofiber scaffolds fabricated by electrospinning reduce the risk of developing secondary osteoarthritis and may be suitable for clinical use 63. Moreover, it has been newly indicated that the PLLA/silk fibroin (PLLA/SF) composite scaffold supports adhesion, proliferation, and growth of chondrocyte more than PLLA scaffold without SF, introducing this scaffold a suitable material with potential application in cartilage tissue engineering 64.In another study, Ahmed Aligned nanofibers: A study has been conducted on seeding BMMSCs and fibrochondrocytes on PCL-PEO aligned nanofibrous meshes and demonstrated that aligned nanofibrous topography could influence human BMMSCs fibrochondrogenesis by mimicking the naturally-occurring ECM more closely than micro-patterned features such as ridges or grooves 65.et al evaluated cell proliferation and chondrogenesis on aligned (A) and randomly (R) oriented electrospun PLLA/PCL hybrid scaffolds. They demonstrated that NSPs exhibit different behavior in two scaffolds. NSPs seeded on R fibers were expanded in all directions and exhibited a polygonal morphology and displayed multipolar shape. Conversely, NSPs were oriented only in the longitudinal direction of A fibers and showed bipolar extension along the fiber course alignment. Also, NSPs cultured on A fibers showed significantly higher expression of markers related to chondrogenesis process compared to cells cultivated on R fibers. The authors emphasized the role of the physical and topographical characteristics of scaffolds in the development of efficient stem cell-scaffold complexes and concluded that the aligned nanofibrous scaffolds can significantly enhance chondrogenic differentiation of nasal septum derived progenitors 66.In this way, Shafiee 68. Therefore, fabrication of multi-scale scaffolds combining microfibers with nanofibers has been considered with the aim of providing larger pore sizes and improving cell differentiation and ECM production 69. For this purpose, Levorson et al fabricated electrospun scaffolds consisting of two differently scaled fibers interspersed evenly throughout an entire construct as well as scaffolds containing fibers of fibrin and PCL. The prepared samples were scaffolds containing PCL microfibers (Pμ), PCL microfibers with PCL nanofibers (PμPn), and PCL microfibers and fibrin nanofibers (PμFn) being electrospun by a dual extrusion process. Both PμFn and PμPn scaffolds displayed similar porosities higher than microfibers alone. However, the Pμ scaffolds bore significantly larger pore sizes than the scaffolds containing nanofibers. Additionally, the PμPn scaffolds had the highest density of nanofibers and the smallest average pore size of all samples. The seeded human UCMSCs on PμPn scaffolds appeared to exhibit a flattened, broad polygonal morphology and spread along microfibers while cells on the Pμ and PμFn scaffolds showed more elongated and spindle-like morphologies and extended between the microfibers. Furthermore, analysis of cellular infiltration by Fast Green staining showed more scattered distribution of cells within the PμPn scaffolds while cells were primarily located on the surface of the other two scaffold types. Histological examination also exhibited more deposition of sGAG in PμPn and PμFn in contrast to the scaffolds composed of microfibers alone suggesting that the inclusion of nanofibers within a microfiber is useful towards the production and distribution of sGAG and may be beneficial for cartilage regeneration. The authors emphasized on tuning the density of nanofibers with respect to microfibers in an effort to control the positive influence of nanofibers on cell attachment and ECM production while minimizing any negative effects such as limited infiltration 70.While nanoscale features are desired due to mimicking the ECM components such as collagen fibers, it is believed that high concentrations of nanoscale fibers could increase cell spreading and limit cellular infiltration et al used a phase separation method to fabricate a desirable scaffold made of PLLA. They showed that fabricated nanofiber scaffolds could efficiently support chondrogenesis of human BMMSCs in the presence of TGF-β1. The expression of chondrogenic markers in human BMMSCs grown on nanofiber matrix was significantly higher compared with cells raised on smooth film culture 13.To achieve 3D highly porous nanofibrous structure for cartilage tissue engineering, Hu et al examined the differentiation of adult BMMSCs to chondrocytic phenotype on a nanofibrous PCL scaffold. They found that in Nanofibrous Scaffold (NFS) chondrocyte-like cells produced higher level of cartilaginous ECM compared with high-density Cell Pellet (CP) culture. In addition, specifically, collagen type IX was expressed to an upper level in nanofibrous system compared to CP culture. Furthermore, the level of sulfated Glycosaminoglycan (sGAG) synthesis in NFS culture was over two-fold higher than CP culture over a 21-day culture period. Their experimental results suggested that, while a 3D environment and TGF-β1 were both necessary to induce chondrogenesis, the PCL-based NFS significantly enhanced the chondrogenic differentiation of BMMSCs compared to the CP culture and could be considered as a candidate scaffold for cell-based tissue engineering approaches to cartilage repair compared to the CP culture 55.Li et al. They found that the augmentation of nanofibrous texture with or without TGF-β1 and/or hyaluronan was helpful in terms of directing the implanted BMMSCs toward a chondrogenic end. In addition, their results demonstrated that nanofibrous scaffold groups have different trends with microfibrous scaffolds via release level of TGF-β1 and chondrogenic development. The microfibrous scaffolds release TGF-β1 more than nanofibrous scaffolds; however, expression of cartilage marker in nanofibrous scaffold groups was higher than that in microfibrous scaffolds 48.The potential of electrospun nanofibrous and micro-fibrous PCL scaffolds to release TGF-β1 and stimulate chondrogenic differentiation of BMMSCs has also been investigated by Schagemann et al, Transforming Growth Factor-β1 mimicking peptide cytomodulin (CM), was conjugated onto the functional nanofibrous hollow microspheres (FNF-HMS) to induce distinct differentiation pathways of rabbit BMMSCs. Their finding indicated that novel FNF-HMS effectively presents CM to BMMSCs and successfully induces their chondrogenesis for cartilage formation in both in vitro and in vivo studies 71.Recently, injectable microspheres were suggested as an attractive stem cell and growth factors carriers for tissue regeneration. In a study by Zhang 72.Implantation of nanofibers-based tissue engineered cartilage eliminates the need for an extra covering material to secure and protect the implant, such as periosteum which is used in the current autologous chondrocyte transplantation procedure. Harvesting periosteum comes with morbidity and complications, thus it is clinically preferable to avoid the use of periosteum et al demonstrated the potential of BMMSCs-seeded PCL-based nanofibrous scaffolds to repair full-thickness cartilage defects in a swine model. This cell-scaffold construct renewed hyaline cartilage-like tissue and restored a smooth cartilage surface as compared with other groups, including acellular constructs and untreated group. Furthermore, the studied group, which was chondrocyte-seeded scaffold, produced fibrocartilage-like tissue with an irregular superficial cartilage contour 72.In the recent decade, effectiveness of implanted electrospun PCL nanofibrous scaffold with/without cells has been evaluated for repair of cartilage defects in animal models . Li et ain vitro culture. In this study, PCL nanofiber scaffolds 73 (with/without chitosan coating) were implanted under periosteum in six months old rabbits with injection of GF-β1 into the implant site. Cell infiltration was observed in all groups while sGAG production and cartilage formation was more typical in the uncoated scaffolds compared to chitosan-coated scaffolds. In addition, TGF-β1-injection and application of uncoated scaffolds resulted in significantly more mineral deposition.An autologous cell-based cartilage repair approach has been developed to eliminate harvesting of healthy cartilage and in vivo74.The iPSCs can be produced by reprogramming of terminally differentiated cells to primary stem cells with pluripotency. To benefit from the breakthrough of iPSCs, the effect of electrospun PCL/gelatin nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration was investigated. It was indicated that iPSCs expressed higher levels of chondrogenic markers on the scaffolds than the culture plate. Additionally, in an animal model, cartilage defects implanted with the scaffold-iPSCs composite exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, it was shown that scaffolds enhanced the chondrogenesis of iPSCs and that iPSCs-containing scaffolds improved the re-establishment of cartilage defects to a greater degree than did scaffolds alone 3.In another study, efficiency of the fabricated hybrid PVA/PCL nanofibers seeded with autologous BMMSCs was evaluated in the knees defect of rabbits. The authors indicated improved regeneration of cartilage in full-thickness defects that treated with BMMSCs-loaded PVA/PCL electrospun scaffolds compared to scaffold alone or untreated defects. After 12 weeks of implantation, almost all defects that were treated with cell-scaffold constructs were completely enclosed with smooth tissue and edges of the grafted areas were hardly detectable. In addition, unlike the group who received only PVA/PCL scaffolds, a high similarity in ECM patterns between regenerated and normal tissues was observed and collagen type II staining was positive et al, the cell seeded electrospun nanofibers containing collagen-poly (L-lactic acid-co-ε-caprolactone) (collagen-PLCL) and chondrocytes were implanted subcutaneously into nude mice followed by evaluation of the quality of neocartilage. Their results revealed that collagen-PLCL membranes facilitate the formation of cartilage-like tissue in animals and thus could mimic the natural ECM with good cell affinity 75.In another study by He 74. Combination of collagen and electrospun nanofibers as bi-layer scaffold has been demonstrated to help cartilage and bone regeneration. In 2013, Zhang et al reported efficiency of a fabricated bi-layer microporous scaffold with collagen and electrospun PLLA nanofibers (collagen-PLLA) for repair of osteochondral defects. Compared with collagen scaffold, implantation of collagen-PLLA scaffold seeded with BMMSCs induced more rapid subchondral bone appearance and better cartilage development, which led to better functional repair of deep osteochondral interfacial tissue structure in rabbits 2.Recently, bi-layer scaffolds have gained considerable attention for the restoration of osteochondral defects affecting both the articular cartilage and the underlying subchondral bone et al have also studied the efficiency of a biphasic complex to repair the osteochondral defects in a rabbit model, which was composed of oriented electrospun poly (L-lactic acid)-co poly(e-caprolactone) P(LLA-CL) Yarn-collagen type I/hyaluronate hybrid scaffold (Yarn-CH) as a chondral phase and porous beta tricalcium phosphate (TCP) as a osseous phase. The regenerated defects treated by differentiated BMMSCs/Yarn-CH/TCP and BMMSCs/CH/TCP (control) biphasic scaffold groups were completely repaired by hyaline-like tissue that appeared to be integrated with the surrounding tissues. In undifferentiated BMMSCs/Yarn-CH/TCP and BMMSCs/CH/TCP biphasic scaffold groups, defects were covered by rough tissue with irregular surfaces which were clearly distinguishable from the normal cartilage. In addition, the cell distribution and cell morphology in the regenerated cartilage were almost identical to the native host cartilage including the superficial zone in differentiated groups compared to undifferentiated groups. Furthermore, immuno-histochemical staining showed higher level of collagen type II in the BMMSCs/Yarn-CH/TCP biphasic scaffold groups than in the other groups. Greater improvement of the compressive modulus was also shown in differentiated-BMMSCs/Yarn-CH/TCP biphasic scaffold group compared to other groups 76.Liu 77.In 2010, a three-dimensional PLGA/nano-hydroxyapatite (PLGA/NHA) scaffold was fabricated by a thermally induced phase separation method and its efficacy to repair articular osteochondral defects in murine model was investigated. The defects in the PLGA/NHA-MSCs treated group were filled with smooth and hyaline-like cartilage with profuse glycosaminoglycan and collagen type II deposition12 weeks after operation 43. In 2012, 3D electrospun nanofiber network composed of PVA-methacrylate (PVA-MA) and a composite of PVA-MA/chondroitin sulfate-methacrylate (CS-MA) (PVA-MA/CS-MA) were used for articular cartilage repair. After evaluation of the scaffolds for cartilage-like tissue formation in vitro, the constructs were implanted into rat osteochondral defects. Their results showed that nanoscaffolds with chondroitin sulfate (PVA-MA/CS-MA) supported chondrogenesis more than PVA-MA alone, judged by collagen type II and proteoglycan production in defects 43.For interacting cells with the surrounding ECM, which gives rise to a dynamic transfer of information between the extracellular and intracellular space, researchers have introduced several biological signals, including chondroitin sulfate, hyaluronic acid, and collagen into tissue-engineered scaffolds to encourage tissue specificity et al assessed efficacy of a bi-layer porous hydroxyapatite/collagen (HAp/collagen) nano-composite prepared by coprecipitation method and impregnated with FGF-2 for repairing the osteochondral defects in a rabbit model. Their results showed that HAp/collagen scaffolds impregnated with FGF-2 not only regenerate bone tissue but also resulted in development of satisfactory cartilage regeneration with a hyaline-like appearance. Their finding suggested that porous HAp/collagen with FGF-2 augmented the cartilage repair 78.Some investigators evaluated impregnation of nanofibrous scaffolds with various growth factors to promote repair of articular cartilage defects. For example, Maehara et al developed a novel drug delivery system using nano-composite for repair of osteochondral defects in miniature pigs. The fabricated cell-free composite scaffolds that contained PVA nanofibers enriched with liposomes, FGF-2, and insulin were subsequently embedded in a fibrin gel including hyaluronate/collagen type I. Interestingly, the cell-free composite scaffold improved migration of the BMMSCs into the defect and their differentiation into chondrocytes as compared with untreated group. As a result, composite scaffold containing nanofibers with liposomes functionalized with growth factors was able to enhance the regeneration of osteochondral defects towards hyaline cartilage and/or fibrocartilage compared with untreated defects that were filled predominantly with fibrous tissue 1.Filova 14.One key challenge in tissue engineering especially cartilage reconstruction is mimicking the architecture of ECM. At present, nanofibrous scaffolds irrespective of their method of synthesis are the most promising matrix to generate artificial ECM. These scaffolds are characterized by high surface area and enhanced porosity, which are highly desired for tissue engineering and drug delivery applications. There are four dominant methods to fabricate nanofibers for cartilage tissue engineering: electrospinning, molecular self-assembly, phase separation, and drawing 80. Due to difficulties in controlling porosity and pore size and architecture, three other techniques have been utilized less than electrospinning for cartilage tissue engineering purposes.Of these methodologies, electrospinning is the most common approach for cartilage tissue engineering since this technique offers great flexibility in terms of the choice of scaffold material and fiber diameter from the micrometer down to nanometer range. Electrospun polymeric fibrous meshes also present a higher surface area for cell attachment. Indeed, fabrication of electrospun nanofibers is easy, inexpensive and relatively reproducible 81. The most common electrospun nanofibers designed for cartilage tissue engineering are made of poly (α-hydroxyesters) 82. Although the synthetic nanofibers prepared form these materials are capable to support chondrocyte proliferation and differentiation, some strategies have been applied to improve cell tendency of these materials that help us to achieve better results in future repair of cartilage defects. One applied strategy is hybridization of these synthetic materials by natural polymers like collagen 62. The combination of synthetic materials with natural polymers in nanofibers has resulted in better cell attachment, proliferation and chondrogenic development compared to synthetic polymers alone 64. Moreover, some in vivo studies implied better repair of cartilage defects by hybrid nanofibers compared to simple nanofiber composed of only synthetic polymers. Another strategy is modifying the surface of scaffolds through physical and chemical methods to improve the bioactivity of materials for cell adhesion and distribution.The availability of a wide range of natural and synthetic biomaterials has broadened the scope for development of nanofibrous scaffolds. Synthetic polymer-based systems offer additional advantages with their adjustable mechanical properties, as well as ease of surface modification via protein coatings, or conjugation of specific signaling molecules 83. Some others have improved the cell affinity of nanofibers by attaching Arg-Gly-Asp (RGD) peptides to the nanofibers surfaces. Indeed, these strategies play an important role in governing cellular responses and helping the scaffold to play a more efficient role as bioactive systems rather than just passive cell carriers. To prove this assumption, different in vitro and in vivo studies narrated that integration of fabrication techniques with surface modification methods has resulted in closer properties of nanofibrous scaffolds to native ECM, encouraged cell attachment and development into chondrocyte lineage. To repair the osteochondral defects that have two different structures, incorporation of stem cells with biphasic scaffolds containing hybrid nanofibers for chondral phase and porous sponge scaffolds for osseous phase seems to be a good strategy (One approach to improve cell affinity is surface modification of the nanofibers by plasma treatment strategy .In spite of the great achievements behind the design of nanofibrous scaffolds, there is still plenty of room for improvement. Integration of nanofibers into micro-fabricated 3D scaffolds has resulted in obtaining more desirable scaffolds with providing larger pore sizes and improving cell differentiation and ECM production.The future research on nanofibrous architecture may be focused on the new nanofabrication techniques. In combination with new nanofabrication technologies, nanofibrous scaffold could be decorated with nano-topographic patterns, such as ridges and grooves to better match the nanostructure of ECM achieving a better control of ECM-mimicry.via secretion of growth factors and cytokines help sGAG production and mediate better situation to mimic ECM environment 8.Based on this review, the efficiency of cell-seeded nanofibers in repair of cartilage defects is significantly more than the scaffold alone. It sounds that the seeded cells 84.Two primarily considered criteria to determine the optimal source of cells for cartilage repair are the performance of the cells and their accessibility. Regarding performance, primary or low passage articular chondrocytes provide several advantages due to their high level of matrix synthesis and lack of hypertrophy. However, for larger defects, which require a larger number of cells, it is generally accepted that the dedifferentiation which occurs during monolayer expansion is a significant hurdle 85.On the other hand, due to requirements of two-step intra-articular procedures for clinical use of autologous chondrocytes, one to harvest the cartilage and one to re-implant, many groups are attempting to develop allogenic sources of cells to be used in articular cartilage repair. Although cartilage is considered an immune privileged site, newer data indicate that chondrocytes have immunological properties that limit host immune reaction Following the search for immune privilege cell source that can readily provide large numbers of undifferentiated progenitors with chondrogenic potential, adult stem cells were introduced as interesting cells for tissue engineering and regenerative medicine purposes.86. However, due to some problems such as invasive techniques for sample collection and low availability, BMMSCs are introduced as not an ideal source and still some challenges for tissue engineering application exist. With introducing more available and accessible stem cell sources with similar immunological properties and great proliferation and trans-differentiation ability such as menstrual blood and adipose tissue stem cells, it is expected that these newer stem cells would be synchronized with nanofibers for future studies on cartilage tissue engineering.The most commonly used stem cells for cartilage tissue engineering especially in nanofibrous structures are the stem cells derived from bone marrow. It is due to the high chondrogenic differentiation ability and the availability of great knowledge about immunological characteristics and nature of this source of stem cells compared to other adult mesenchymal stem cells 87 are future research priorities of cartilage reconstruction. In this manner, designing and applying suitable bioreactors that ultimately help in more ECM production and achievement of artificial constructs simulating native cartilage tissues, should not be ignored. In conclusion, although many experiments have been carried out to simulate native cartilage using nanofibers and stem cells with some promising reports about efficiency of these constructs for repair of cartilage defects in animal models, much joint effort by scientists from multiple disciplines is still required for transition of the data from in vitro to in vivo phase. To facilitate the future applicability of constructs composed of stem cells and nanofibers, a time frame is required for development of bench to bedside strategies.Notably, besides improvement of nanofiber fabrication technique, utilization of other stem cell sources instead of BMMSCs and incorporation of nanofibers with differentiation promoting growth factors such as BMP-6


Adaptive servo-ventilation (ASV) therapy is a recent non-invasive positive pressure ventilation therapy that was developed for patients with heart failure (HF) refractory to optimal medical therapy. However, it is likely that ASV therapy at relatively higher pressure setting worsens some of the patients’ prognosis compared with optimal medical therapy. Therefore, identification of optimal pressure settings of ASV therapy is warranted.We present the case of a 42-year-old male with HF, which was caused by dilated cardiomyopathy, who was admitted to our institution for evaluating his eligibility for heart transplantation. To identify the optimal pressure setting [peak end-expiratory pressure (PEEP) ramp test], we performed an ASV support test, during which the PEEP settings were set at levels ranging from 4 to 8 mmHg, and a heart rate variability (HRV) analysis using the MemCalc power spectral density method. Clinical parameters varied dramatically during the PEEP ramp test. Over incremental PEEP levels, pulmonary capillary wedge pressure, cardiac index and high-frequency level (reflecting parasympathetic activity) decreased; however, the low-frequency level increased along with increase in plasma noradrenaline concentrations.An inappropriately high PEEP setting may stimulate sympathetic nerve activity accompanied by decreased cardiac output. This was the first report on the PEEP ramp test during ASV therapy. Further research is warranted to determine whether use of optimal pressure settings using HRV analyses may improve the long-term prognosis of such patients. As a non-invasive positive pressure ventilation therapy, adaptive servo-ventilation (ASV) therapy was recently developed for treating patients with heart failure that was refractory to optimal medical therapy . This thRecently, Cowie et al published a prospective randomized study and indicated that ASV therapy unexpectedly decreased the survival rate compared with optimal medical therapy for patients with heart failure and SDB . In thisA 42-year-old man with normal sinus rhythm was admitted to our institution for the evaluation of his eligibility for heart transplantation owing to advanced heart failure , which was classified as New York Heart Association functional class IV due to dilated cardiomyopathy diagnosed by previous endomyocardial biopsy. He had been receiving 10 mg/d of carvedilol, 2.5 mg/d of enalapril and 25 mg/d of spironolactone. At admission, he had slight edema in bilateral leg, and his plasma B-type natriuretic peptide level was 423 pg/mL. He had no history of hypertension or diabetes at admission. Transthoracic echocardiography showed that the left ventricular diastolic dimeter was 67 mm, and as calculated by the modified Simpson method, the left ventricular ejection fraction was 23%; mild mitral and tricuspid regurgitations were present as well. He had no SDB or active respiratory infection. For further improvement of his heart failure symptoms, he was subjected to ASV support testing accompanied by haemodynamic assessment using right-heart catheterization as well as HRV and noradrenaline plasma level analyses at 10 days after the admission ; each pressure setting was maintained for 3 min.An advanced bi-level positive airway pressure unit  together with a best-fitting full-face mask (ResMed) was used for the ASV support testing, as previously described . Before 2O , i.e., PEEP of 5 cmHortality . Therefo2O would be the optimal pressure setting at least in this case.Positive pressure ventilation generally relieves elevated intracardiac pressure due to the decrease in preload and increase in cardiac output . However2O may also be the optimal pressure setting at least in this case.We recently showed the usefulness of real-time HRV assessment during ASV support . In the The validity of this PEEP ramp test should be confirmed in many patients. Considering our previous study results , we atteWe performed here for the first time a PEEP ramp test during ASV therapy. Whether optimization of PEEP setting using HRV analyses may improve the long-term prognosis should be confirmed in the prospective study.


Neglecting tree size and stand structure dynamics might bias the interpretation of the diversity-productivity relationship in forests. Here we show evidence that complementarity is contingent on tree size across large-scale climatic gradients in Europe. We compiled growth data of the 14 most dominant tree species in 32,628 permanent plots covering boreal, temperate and Mediterranean forest biomes. Niche complementarity is expected to result in significant growth increments of trees surrounded by a larger proportion of functionally dissimilar neighbours. Functional dissimilarity at the tree level was assessed using four functional types: i.e. broad-leaved deciduous, broad-leaved evergreen, needle-leaved deciduous and needle-leaved evergreen. Using Linear Mixed Models we show that, complementarity effects depend on tree size along an energy availability gradient across Europe. Specifically: (i) complementarity effects at low and intermediate positions of the gradient (coldest-temperate areas) were stronger for small than for large trees; (ii) in contrast, at the upper end of the gradient (warmer regions), complementarity is more widespread in larger than smaller trees, which in turn showed negative growth responses to increased functional dissimilarity. Our findings suggest that the outcome of species mixing on stand productivity might critically depend on individual size distribution structure along gradients of environmental variation. Growing evidence supporting a causal relationship between increased tree functional diversity and above-ground wood production123567Classical experiments, mostly conducted on short-lived communities , point to niche complementarity, and the greater potential for species packing, as a chief mechanism by which the productivity of species mixtures is enhanced compared to the respective monocultures27To understand the functioning of long-lived communities an appreciation of their size structure dynamics is essential2328313234Here, we investigated whether complementarity effects are contingent on tree size across the main climatic gradient of Europe from southern Spain to northern Scandinavian forests. Niche complementarity at the tree level would imply improved individual tree growth rates when growing in neighbourhoods surrounded by a larger proportion of functionally dissimilar neighbours. Although tree growth does not necessarily correlate in a one-to-one relationship with stand productivity, forest ecosystem production can, as a first approximation, be evaluated as the collective sum of individual tree productivity as individual tree growth plays a pivotal role in ecosystem functioning13372 pointed to a high goodness of fit, with more than 70% of variance explained by the final model (see The backward selection of predictor variables in the Linear Mixed Model suggested that one out of the four possible three-variable interactions (i.e. SIZE × POFT × PET) and two pairwise interactions (e.g. SIZE × SBA and SBA × PET) should be included in the fixed effects model , our results indicate complementarity effects on tree growth of small and large trees . SpecifiWe found tree size to be a critical factor driving complementarity effects in forests across Europe. This agrees with our expectations associated limiting resources along the large-scale PET gradient. Hence, complementarity effects can emerge from niche separation along spatial/temporal gradients of resource availability and thus tree size plays a pivotal role in determining resource use partitioning. In space, the combination of different light intercepting strategies and size stratification have been reported to significantly enhance resource yielding and biomass packing in mixed forest stands4145464950In boreo-alpine forests (400–500 mm) our results indicated that strong complementarity effects on small individuals notably weakens with size development. Complementarity in nitrogen uptakeIn temperate forests, complementarity effects depict a similar pattern to the one observed in boreal forests: e.g. strong complementarity effects decrease with tree development. Young broad-leaved deciduous trees are known to benefit from higher light availability when part of the forest overstorey is occupied by early successional conifersIn Mediterranean forests, our results point to strong complementarity effects in large trees, and contrary, negative effects of functional dissimilarity in small trees. Assuming that larger trees have a comparatively higher contribution to total stand biomass and stand basal area change, this finding is consistent with the existing literature posing significant positive diversity-productivity relationships in Mediterranean forests3Neglecting tree size and stand structure and assessing stand growth as an aggregated tree growth measurement might bias the interpretation of the diversity-productivity relationship in forests142954Our broad scale approach, however, lacks information on potential drivers of productivity such as soil nutrients, herbivory, disturbances and slight deviations from long-term climatic conditions at each sampling period might affect our findings since all these uncertainties might alter the diversity-productivity relationship. Given the impossibility of long-term experiments for exploring biodiversity-ecosystem functioning relationships in forests (at least for some years to come), we rely on improved large-scale data analyses and modelling. Therefore, further efforts are needed to improve forest inventory protocols and large-scale data harmonization to account for potential confounding drivers of productivity such as soil information, management, secondary succession and herbivory in forest ecosystems.Abies alba , Acer pseudoplatanus , Betula pendula , Betula pubescens , Carpinus betulus , Castanea sativa , Fagus sylvativa , Juniperus thurifera , Picea abies , Pinus halepensis , Pinus sylvestris , Quercus pyrenaica , Quercus robur , and Quercus ilex . These species represent more than one third of the total stand basal area measured in the whole dataset and are the dominant tree species in at least one of the three principal forest biomes of Europe . The final number of plots was 32,628 and the number of surviving trees 275,558 after removing dead trees or surviving trees with negative growth rates . We used data compiled from the National Forest Inventories (NFIs) of five European countries  relative to the initial tree basal area and the number of years between consecutive surveys . Tree basal area in each inventory was calculated using dbh measurements as:where dbh is the diameter at breast height (i.e. 1.3 meters above ground surface) in metres.2 ha−1) occupied by functional types other than the focal tree (POFT) as follows:Complementarity effects (CE) at the tree level imply that any given tree will grow faster when, keeping biotic and abiotic sources of variability constant, its neighborhood is composed of a larger proportion of functionally dissimilar trees. With this in mind we assessed the proportion of the stand basal area . Specific leaf area has been identified as a critical trait linked to species competitive ability (i.e. broad-leaved species tend to be more tolerant to competition and exhibit stronger competitive effects relative to needle-leaved species1441where SBA2 ha−1) in which the tree resided. Mean climate , annual precipitation  and mean potential evapotranspiration ) for each plot were extracted from the WorldClim databaseFor each individual tree we noted the species identity and calculated the basal area of the tree  and the plot basal area  and between PET and WAI (r = −0.71), we selected PET as an integrative variable that summarizes the two main climatic stress gradients in continental Europe, i.e. water deficit towards southern latitudes and low temperature towards northern latitudes and alpine areas .We fitted Linear Mixed Models (LMM) to model tree growth as function of SIZE, SBA, PET and POFT. Plot nested in country and species were included as two independent random terms in the model. Species was considered to affect the intercept parameter and the slopes associated with main effects of both POFT and SIZE under the assumption that (i) growth responses to an increasing proportion of other functional types might vary among the species considered in this study, and (ii) that growth patterns throughout the ontogeny might differ among species. Plot nested in country was considered to affect the intercept parameter and the effects of POFT on tree growth assuming that differences in plot size and sampling methodologies might affect the POFT assessment and thus the sensitivity of tree growth to this index. The model equation takes the form:TG is tree growth, p parameters associated with the main and interactive effects of ∈ran is the variance component associated with the random terms, and ∈res is the residual normally-distributed error. We considered a fixed-effect term in the maximal model that included all the potential pair-wise interactions as well as the three variables interactions . SIZE was included in a log-transformed form and PET as a second order polynomial. Tree growth was log-transformed to meet normality and homogeneity of variance.where 2 was assessed following Nakagawa and Schielzeth2 the goodness of fit of the fixed effect term only and the conditional R2 the goodness of fit of the whole model including the random term.Model selection was conducted using a backward procedure of predictor variables starting with the maximal modelHow to cite this article: Madrigal-González, J. et al. Complementarity effects on tree growth are contingent on tree size and climatic conditions across Europe. Sci. Rep.6, 32233; doi: 10.1038/srep32233 (2016).


This article aims to review both the pathological and biological evidence implicating a key role for the breast stromal compartment in MD, how this may be modified and the clinical significance of these findings. The epidemiological context will be briefly discussed but will not be covered in detail.There is strong epidemiological data indicating a role for increased mammographic density (MD) in predisposing to breast cancer, however, the biological mechanisms underlying this phenomenon are less well understood. Recently, studies of human breast tissues have started to characterise the features of mammographically dense breasts, and a number of  Mammographic density (MD) refers to the proportion of a mammogram occupied by radiologically dense fibroglandular tissue and is a major independent risk factor for breast cancer. A comprehensive meta-analysis of 14,000 cases observed that women with > 75% breast density had 4-6 times the risk of developing breast cancer compared to women with breasts of the lowest 25% density . High MDMD appears to be highly heritable; studies of twins have attributed up to 65% of the variation in MD to genetic factors  with theThe majority of studies investigating the genetic contribution to MD have focused on investigating the single nucleotide polymorphisms (SNPs) associated with increased breast cancer risk and their relationship with MD. So far SNPs in the region of ESR1, CCDC170, EBF1, LSP1, MIR1972-2:FTO, RAD51L1, ZNF365, MKL1, TNRC9/TOX 3, NTN4, NEK10, TAB2 and loci at 2p24.1 and 12q24 –14 have A key feature of MD compared to other established risk factors for breast cancer is that it is dynamic and modifiable. This modifiability offers significant therapeutic potential in the form of cancer prevention strategies targeted to reduce breast density. MD typically decreases with age, whereas breast cancer incidence conversely increases with advancing age. The Pike model attempts to resolve this apparent paradox by suggesting that cumulative lifetime exposure of the breast to dense tissue and associated growth factors and hormones, also referred to as ‘breast tissue ageing’, confers the age-related risk of developing breast cancer .This is reflected in the apparent hormonally responsive nature of MD; women with known risk factors for breast cancer related to prolonged oestrogen exposure, such as late first pregnancy and early menarche, show a higher degree of MD. In addition, the use of combined hormone replacement therapy also increases MD , 19. ConGiven that MD appears to be hormonally responsive, one might expect high MD to predispose to the development of oestrogen receptor (ER) positive tumours, however to date studies have reported inconsistent findings. McCormack and dos Santos Silva have recently performed a systematic meta-analysis incorporating over 24,000 cases, finding a similar magnitude of association with ER+ and ER- tumours and no difference in the association with human epidermal growth factor receptor 2 (HER2) status .Tamoxifen is a selective oestrogen receptor modulator, demonstrated in the IBIS-1 study to reduce MD and breast cancer risk . A furthPresently the mechanisms conferring susceptibility to the protective effect of tamoxifen treatment remain unknown, however, an association of cytochrome P450 2D6 (CYP2D6) metabolizer status with MD change in response to tamoxifen has recently been reported .In an animal model of breast cancer, tamoxifen treatment initiated remodelling of the mammary stroma to a tumour inhibitory phenotype with lower levels of fibronectin and reduced extra-cellular matrix (ECM) turnover . FurtherMD reflects variations in the tissue composition of the breast and is positively associated with collagen, epithelial and stromal cells and negatively associated with fat . Stroma A comprehensive study by Li and colleagues examined histological features of breast tissue obtained at forensic autopsy . They foIt has been hypothesised that MD may represent the influence of local oestrogen production on the breast . HoweverDespite the association of stromal content and high MD breast tissue, there is no standardised approach to measuring MD in histological sections. This, combined with the fact that MD is often heterogeneous in nature throughout the breast, poses a significant challenge to the planning of translational studies using human tissue.Whilst there is a strong clinical correlation, MD has not yet been causally linked to tumour formation and there have been a limited number of studies investigating the biological pathways mediating MD. Given that the stroma forms the major constituent component of dense breast tissue, the pathways contributing to the density-associated breast cancer risk are likely to involve the stromal cells, ECM proteins, and their interaction with the epithelial component.via multiple diverse mechanisms including the influence of growth factors secreted by cancer associated fibroblasts and remodelling of the ECM [The stromal microenvironment is known to have an important role in the progression of established invasive breast cancer , acting  the ECM , 49. In  the ECM , respons the ECM  and endo the ECM . Similarin-vitro work from another group who have observed that cell density, shape and ECM proteins are capable of inducing stromal aromatase expression [It has been hypothesised that in areas of high MD, abundant stromal fibroblasts may aberrantly secrete growth factors and hormones/cytokines that stimulate epithelial cell proliferation , 53. Onepression , thus prIt has been suggested that increased collagen production by stromal fibroblasts contributes to a stiffer ECM. The inability of normal breast epithelium to contract stiff matrix causes a tensional imbalance, reflected by altered signalling pathways that have the potential to induce epithelial cell proliferation .tmJae transgene which reduces collagen proteolysis, and examined the propensity for tumour formation with increased mammary collagen. They observed that higher stromal collagen density in mouse mammary tissue resulted in a threefold increase in tumour number and that the tumours which developed displayed a more invasive phenotype with greater local invasion and metastasis. The authors propose a functional link between increased stromal collagen density and breast cancer initiation and progression [Patricia Keely and colleagues developed a bi-transgenic mouse to model high breast density, utilising the Col1a1gression .Two possible mechanisms are suggested by which increased collagen density might promote tumour development. The first is a direct effect of increased matrix stiffness, resulting in a higher mechanical force and resistance to contractility on the epithelial cells. These forces might alter focal adhesion and Rho GTPase (Rho) signalling, resulting in increased proliferation . The secFurther work by this group examined whether collagen density alone, in the absence of fibroblasts, could alter mammary epithelial cell behaviour in 3D culture models. Supporting this hypothesis, greater collagen density alone increased matrix stiffness and promoted epithelial cell proliferation and invasion . They shThe authors propose that continued mechanical stimulation from a dense stromal matrix results in sustained activation of FAK-Rho signalling which, in turn, up-regulates other pathways such as Ras- Mitogen activated protein kinase (MAPK). ERK acts as a crucial bottleneck, regulating the response and inducing transcription of proliferation associated genes.via inhibition of notch homolog 1 (NOTCH1) [Soon et al examined the effect of high density collagen matrices on mammary epithelial cell behaviour. They found that high density matrices up-regulated the expression and activity of Rho-associated coiled-coil-containing protein kinase 1 (ROCK 1) (NOTCH1) . ROCK- 1As well as collagen density, the significance of collagen fibre alignment within the ECM in facilitating mammary tumour cell invasion has also been investigated by the Keely group . Collagein-vitro and tumourigenesis in-vivo [Weaver and colleagues have investigated how collagen cross-linking mediated by lysyl oxidase (LOX), a family of ECM cross-linking enzymes, can influence mammary epithelial cell behaviour. They used the MMTV-Neu mouse model of breast cancer to show that fibroblasts overexpressing LOX form a stiffer mammary fat pad and promote the growth and invasion of premalignant mammary epithelial cells. Similarly, inhibition of LOX resulted in a less stiff matrix and reduction in tumourigenesis . LOX-med in-vivo . Thus it in-vivo , has bee in-vivo  and is p in-vivo .More recent work by the same group, focusing on the molecular determinants of mammary epithelial cell mechanotransduction, has highlighted a crucial role for the focal adhesion component vinculin in translating the mechanical cues from a stiff ECM into tumour promoting intracellular signalling pathways . It is svia integrin-dependent activation of β-catenin and MYC. miR-18a subsequently interacts and inhibits expression of the tumour suppressor gene PTEN, which itself is a negative regulator of PI3-K activity - previously demonstrated as a key pathway promoting mammary epithelial cell malignant progression [via decreasing levels of homeobox A9 (HOXA9)  signalling. The activated JNK-1 stress signalling mediates the onset of a ‘myofibroblast’ phenotype characterised by ongoing inflammation and fibrosis, resulting in pro-tumourigenic, high density stroma [In addition, when the high density fibroblast gene signature was compared to the profile of tumour stroma, c-Jun N-terminal kinase 1 (JNK-1) stress signalling emerged as the most significant biological process shared between the two data sets . The auty stroma . The autWhether such markers of an activated stroma could be used to measure patient risk and as indicators of response to treatment strategies is an intriguing possibility.Martin and Boyd have proposed that the combined effects of cell proliferation (mitogenesis) and genetic damage to proliferating cells by mutagens (mutagenesis) may account for the increased risk of breast cancer with high MD ; a hypotThese data further emphasize the importance of stromal biology in mediating MD. The revised Martin and Boyd model incorporates and highlights the role of the stromal microenvironment in breast tumourigenesis.Reduced TGF-β signalling in dense breast tissue has also been observed by Yang and colleagues who compared gene expression in high and low density non-tumour breast tissue taken 5cm away from invasive carcinoma . A numbeA summary of our current understanding of the complex stromal biological pathways conferring MD-associated breast cancer risk is displayed in Figure Despite the progress made in these preclinical studies towards identifying potential stromal markers and signalling pathways contributing to breast density, this has not readily translated into clinically relevant markers or targets. Further clinically applicable approaches are needed to identify the key stromal molecules driving density-associated risk in humans.The influence of MD on breast cancer risk has a number of important clinical implications, particularly with regard to identification of high risk individuals, screening and prevention. Four studies have investigated whether including a measure of MD improves risk estimation compared to standard risk models –80. All Screening at more frequent intervals with more than one imaging modality may be appropriate for women with dense breasts due to their increased risk of developing cancer and their increased risk of tumour ‘masking’. A recent analysis of personalised mammographic screening according to MD, age, family history and history of breast biopsy was found to be cost effective .It has been postulated that change in MD could be used as a marker in clinical trials evaluating breast cancer prevention strategies . This coMD is more strongly associated with breast cancer risk than other variables such as family history and reproductive factors. Therefore, knowledge of an individual's mammographic density and in particular, their expression levels of genes coding for ECM proteins and other stromal biomarkers, may allow individual risk prediction for breast cancer. MD also offers the advantage over other breast cancer risk factors of being modifiable, which provides exciting potential for therapeutic intervention to reduce both MD and breast cancer risk.Targeting components of the microenvironment is already an established strategy in the adjuvant treatment setting for a number of different tumour types , 84. Thevia integrin inhibition is also the subject of clinical trials with agents showing limited anti-tumour efficacy at present [Components of tumour-associated inflammation are also a focus of new agents targeted against the microenvironment. An interleukin 6 (IL-6) neutralising antibody is currently undergoing evaluation in clinical trials , and neu present , 91. Spe present , utilisi present , and vac present .Harnessing similar approaches in the preventive setting to target the stromal molecules and pathways mediating MD could provide an effective cancer prevention strategy. Thus, further work to dissect the complex biological pathways mediating MD is urgently needed to identify novel, clinically relevant, biomarkers driving the density-associated risk.


Aedes aegypti and malaria mosquito Anopheles gambiae. Inhibition of these channels with small molecules inhibits transepithelial K+ and fluid secretion in Malpighian tubules, leading to a disruption of hemolymph K+ and fluid homeostasis in adult female mosquitoes. In addition, we have used next-generation sequencing to characterize the transcriptome of Malpighian tubules in the Asian tiger mosquito Aedes albopictus, before and after blood meals, to reveal new molecular targets for potentially disrupting Malpighian tubule function. Within 24 h after a blood meal, the Malpighian tubules enhance the mRNA expression of genes encoding mechanisms involved with the detoxification of metabolic wastes produced during blood digestion . The development of chemical tools targeting these molecular mechanisms in Malpighian tubules may offer a promising avenue for the development of mosquitocides that are highly-selective against hematophagous females, which are the only life stage that transmits pathogens.The Malpighian tubules and hindgut are the renal excretory tissues of mosquitoes; they are essential to maintaining hemolymph water and solute homeostasis. Moreover, they make important contributions to detoxifying metabolic wastes and xenobiotics in the hemolymph. We have focused on elucidating the molecular mechanisms of Malpighian tubule function in adult female mosquitoes and developing chemical tools as prototypes for next-generation mosquitocides that would act via a novel mechanism of action . To date, we have targeted inward rectifier potassium (Kir) channels expressed in the Malpighian tubules of the yellow fever mosquito  Adult female mosquitoes are vectors of numerous pathogens that cause diseases of significance to global health. Notably, hundreds of millions of people are infected with mosquito-borne malaria and dengue fever each year, leading to over 500,000 deaths ,2. MoreoChemical control of mosquitoes with insecticides remains a cornerstone to integrated vector management, especially in regions of the world where biological and genetic approaches are cost prohibitive or not logistically feasible. Most insecticides used for mosquito control target the nervous system, such as pyrethroids, carbamates, organophosphates, and organochlorides. Although these neurotoxins are highly effective at killing mosquitoes, the overuse of a limited number of active compounds has exerted a strong, selective pressure for traits that make mosquitoes resistant to conventional insecticides  , analogoA physiological system in mosquitoes (and other insect pests/vectors) that has not been exploited to date for insecticide development is the renal excretory system. In the adult female mosquito, the only life stage that feeds on the blood of—and transmits pathogens to—vertebrate hosts, the Malpighian tubules play especially important roles in excreting (1) the excess water and ions that are absorbed into the extracellular fluid (hemolymph) after engorging on blood, and (2) the metabolic wastes that enter the hemolymph during the digestion and metabolism of blood . Thus, the Malpighian tubules offer a potential physiological target to exploit for developing insecticides that not only have novel mechanisms of action, but may also be highly selective to hematophagous adult females. The following pages highlight recent efforts by our group to understand the basic molecular mechanisms of Malpighian tubule function in adult female mosquitoes with the ultimate aim of developing novel mosquitocides.+, K+ and Cl−) are actively transported by the tubule epithelial cells from the hemolymph to the tubule lumen, thereby generating an osmotic gradient for water to follow. The resulting urine is isosmotic to the hemolymph. The hindgut receives and modifies the urine , before excreting it from the mosquito via muscular contractions.The morphology and physiology of mosquito Malpighian tubules have been the focus of recent comprehensive reviews ,9,10. InThe cellular architecture of the Malpighian tubule epithelium is rather simple, consisting of only two different cell types: principal and stellate B,C. The +-ATPase (V-ATPase) in the brush border pumps protons from the cytosol into the tubule lumen, thereby establishing an inside-negative voltage across both the apical and basolateral membranes of the epithelium. A Na+-K+-ATPase (NKA) on the basolateral membrane of stellate cells may play an accessory role to the V-ATPase. The negative membrane voltages drive ion uptake from the hemolymph across the basolateral membrane through (1) ion channels , and (2) potentially electrogenic transporters in the apical membranes . In addition to the conductive pathways, several electroneutral mechanisms for ion transport driven by chemical gradients are present in the basolateral and apical membranes, such as cation chloride cotransporters , K+, Cl− cotransporter (KCC1)), Na+/H+ exchangers , and anion exchangers , Na+-driven anion exchanger (NDAE)). The combined actions of the conductive and electroneutral ion transport mechanisms contribute to the movements of NaCl and KCl from the hemolymph into the tubule lumen, which provides the osmotic gradient for water to follow. This most likely occurs through stellate cells via aquaporin water channels  and/or the paracellular pathway via septate junctions (SJ)  .+, ~145% of the K+, and ~60% of the Cl− from the ingested blood plasma within 2 h [+ channels expressed in the Malpighian tubules of adult female mosquitoes and develop small-molecule inhibitors of these channels to disrupt the capacity of Malpighian tubules to produce urine, with the ultimate aim of disrupting K+ and fluid homeostasis in mosquitoes.In theory, any of the above molecular mechanisms represent potential targets to exploit for disrupting urine production and thereby osmotic/ionic homeostasis in mosquitoes. For adult female mosquitoes, this disruption could be especially debilitating after engorging on blood, when they ingest over the equivalent of their body mass. In response to this engorgement, the Malpighian tubules mediate a pronounced diuresis that eliminates ~40% of the water and Nathin 2 h . Moreove+ (Kir) channels are major routes of K+ uptake across the basolateral membrane of mosquito Malpighian tubules, making them potential molecular targets for disrupting urine production. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti, the Beyenbach laboratory has shown that barium, a canonical blocker of Kir channels, inhibits (1) over 50% of the transepithelial secretion of K+ and fluid, and (2) ~60% of the basolateral membrane conductance [+ >> K+ > Rb+ >> NH4+) consistent with the presence of Kir channels [Several lines of evidence strongly support the hypothesis that inward rectifier Kductance ,15. Morechannels .Ae. aegypti are characterized by the enriched expression of three distinct mRNAs encoding Kir channel subunits: AeKir1, AeKir2B, and AeKir3 [Xenopus oocytes, AeKir1 and AeKir2B each form functional channels mediating barium-inhibitable, inward-rectifying K+ currents [AeKir1 and AeKir2B to the basolateral membranes of stellate and principal cells, respectively [AeKir3 does not form functional K+ channels in the plasma membrane when heterologously-expressed in Xenopus oocytes [AeKir1 and AeKir2B appear to be the best candidates for mediating the barium-sensitive transepithelial transport of K+ and fluid in mosquito Malpighian tubules.Notably, we have shown that the Malpighian tubules of adult female d AeKir3   flux assay for AeKir1 (expressed heterologously in HEK-293 cells) to allow for the high-throughput discovery of novel small-molecule inhibitors of mosquito Kir channels [+-sensitive fluorescent dye and follows the principle that Kir channels mediate the uptake of Tl+ into cells. Thus, upon the addition of Tl+ to the extracellular bath of cells expressing functional Kir channels, intracellular fluorescence increases as Tl+ moves through the Kir channel pore . That is, the K+ currents mediated by AeKir2B are (1) stimulated by VU573, (2) not affected by VU590, and (3) are inhibited by VU625 [To further explore this hypothesis, the Denton laboratory developed a thallium  with a high calculated permeability coefficient (cLogP), which makes a molecule more likely to penetrate the cuticle of insects [Anopheles gambiae (AnKir1) [AeKir1 [Ae. aegypti and An. gambiae within 24 h of topical application to the cuticle [Ae. aegypti, consistent with an effect on the Malpighian tubules [An. gambiae, topical treatment with VU041 leads to apparent fluid retention in the abdomen 24 h after a blood meal [Despite the promising activities of VU573, VU590, and VU625, they each share one common limitation for mosquitocide development: they are unable to elicit toxic effects when applied topically to the cuticle. Thus, our most recent effort has focused on a small molecule inhibitor of  insects . VU041 w(AnKir1) ; it is e [AeKir1 . Notably cuticle . Moreove cuticle . We have tubules  , have not been widely investigated. Thus, we initiated transcriptomic studies on the Malpighian tubules of the Asian tiger mosquito, Aedes albopictus, before and after a blood meal to generate insights.As described above, the Malpighian tubules of mosquitoes play a key role in mediating the post-prandial diuresis, which alleviates the acute challenges to hemolymph salt and water balance after engorging on blood. However, once the post-prandial diuresis subsides , the potential roles of the Malpighian tubules in detoxifying and excreting metabolites generated during blood meal digestion, such as heme and nitrogenous wastes . Within 24 h post-blood meal, over 80% of the ingested protein is digested [Ae. albopictus are in the midst of a functional transition after a blood meal.In collaboration with Dr. Bryan Cassone (Brandon University), we used RNA-Seq to characterize and quantify global transcript expression in Malpighian tubules isolated from digested . Moreovedigested . FurtherS-transferases, glutathione peroxidases, glutaredoxins, xanthine dehydrogenases) associated with redox homeostasis and metabolite detoxification [S-transferase (GST) activity at 3–24 h post-blood meal and (2) uric acid at 12–24 h post-blood meal, relative to the Malpighian tubules of non-blood fed controls [Ae. albopictus undergo a functional transition after a blood meal, wherein molecular resources appear to be diverted away from mechanisms of diuresis and are reinvested into mechanisms associated with redox homeostasis and detoxification of blood-meal metabolites.To further explore this notion, we conducted a Database for Annotation, Visualization and Integrated Discovery (DAVID) functional cluster analysis ,27 on thfication ,25. ConsAn. gambiae, a transcript for kynurenine 3-monoxygenase was also highly enriched at 3 h post-blood meal [Although additional efforts are required to validate that the up-regulated metabolic pathways and transcripts in the Malpighian tubules are vital for mosquito survival and/or fecundity after a blood meal, they may offer unexploited molecular targets for developing insecticides with novel modes of action. For example, several transcripts that encode putative antioxidant enzymes are up-regulated in Malpighian tubules within 24 h post-blood meal . Notablyood meal . These fHigh-throughput assays for discovering small-molecule inhibitors of mammalian thioredoxin reductase, tryptophan 2,3-dioxygenase, and kynurenine 3-monoxygenase, which are human drug targets, have already been developed ,31,32. M+ and fluid homeostasis, leading to toxicity via a completely novel mode of action from conventional insecticides. Transcriptomic studies on Malpighian tubules of blood fed mosquitoes reveal an up-regulation of numerous molecular mechanisms associated with the detoxification and excretion of harmful metabolites, such as heme. Targeting one or more of these renal mechanisms with small molecules could lead to the development of insecticides with novel modes of action that shorten the mosquito life span and/or decrease fecundity by disrupting blood meal processing. Future studies validating the importance of specific up-regulated mechanisms in the survival and fecundity of blood-fed mosquitoes will facilitate the prioritization of molecular targets for insecticide development.Resistance of mosquitoes to conventional insecticides targeting the nervous system limits our capacity to control the spread of emerging and re-emerging mosquito-borne diseases. Thus, development of insecticides with novel modes of action is critical to enhance vector control capabilities. The work highlighted in the present review demonstrates that the Malpighian tubules offer a new physiological target to exploit for insecticide development. In particular, inhibiting Kir channels of adult female mosquitoes with small molecules disrupts the renal regulation of hemolymph K


Background: The spatiotemporal patterns of correlated neural activity during the transition from wakefulness to general anesthesia have not been fully characterized. Correlation analysis of blood-oxygen-level dependent (BOLD) functional magnetic resonance imaging (fMRI) allows segmentation of the brain into resting-state networks (RSNs), with functional connectivity referring to the covarying activity that suggests shared functional specialization. We quantified the persistence of these correlations following the induction of general anesthesia in healthy volunteers and assessed for a dynamic nature over time.Methods: We analyzed human fMRI data acquired at 0 and 1.2% vol sevoflurane. The covariance in the correlated activity among different brain regions was calculated over time using bounded Kalman filtering. These time series were then clustered into eight orthogonal motifs using a K-means algorithm, where the structure of correlated activity throughout the brain at any time is the weighted sum of all motifs.Results: Across time scales and under anesthesia, the reorganization of interactions between RSNs is related to the strength of dynamic connections between member pairs. The covariance of correlated activity between RSNs persists compared to that linking individual member pairs of different RSNs.Conclusions: Accounting for the spatiotemporal structure of correlated BOLD signals, anesthetic-induced loss of consciousness is mainly associated with the disruption of motifs with intermediate strength within and between members of different RSNs. In contrast, motifs with higher strength of connections, predominantly with regions-pairs from within-RSN interactions, are conserved among states of wakefulness and sevoflurane general anesthesia. The induction of general anesthesia incurs a dramatic change in phenotype compared to wakefulness. Substantial progress has been made at identifying the molecular targets Franks,  and subcThe disruption of resting-state networks (RSNs) is a candidate mechanism whereby anesthetic agents induce sedation and unconsciousness. Each RSN encompasses brain regions of presumably shared functional specialization that demonstrate marked zero time lag correlation among surrogates of neural activity. This phenomenon of functional connectivity recapitulates the patterns of brain activation associated with particular stimulus or task. These findings evoke a complementary line of inquiring regarding which components of the functional neuroarchitecture underlying wakefulness remain preserved during the anesthetized state. General anesthesia appear to perturb RSNs in both animals  method of obtaining a single correlation metric over an entire recording. Such an approach obscures changes that might occur in the temporal dynamics of the FC, i.e., dynamic functional connectivity. We hypothesized that analyzing these temporal dynamics  would reveal additional anesthesia-induced disruptions within and between RSNs. While different approaches  of resting-state echoplanar BOLD images were acquired. Structural images were also acquired, including a T1-weighed  and T2-weighted scans .Secondary analysis of data was carried out as previously described  to promote sparsity  to generate covariance matrices 1), correlation trajectories for each region pair were calculated using a bounded Kalman filtering approach explained in Supplementary Material, Section where the regularization parameter, λ, allows us to balance the tradeoff between log-likelihood and the number of non-zero coefficients in the inverse covariance matrix, which was set to 0.1 in our analyses. After estimating the covariance matrix from  Akaike, , Supple. More deOur spatiotemporal motif analysis can be summarized as follow: (1) Extracting correlation trajectory for all region-pairs from each subject and at a specific arousal state, and filtering the correlation trajectories utilizing our developed bounded Kalman filter Figure . (2) AppSimulations on synthesized data demonstrate efficacy and utility of our approach for differentiating different patterns of dynamic fluctuations among spatially distributed clusters of brain regions with identical mean average correlation, .We quantified the similarity between motifs during wakefulness and 1.2% sevoflurane, compared the number of edges that were in each motif among the two conditions, and found the distributions to have no statistical difference .As the correlations of signals within a resting-state network show the greatest correlation Figure , one pos−2; 1.2%: mean 0.21, SD 7.7 × 10−2, F-test, dF1 = 2059, dF2 = 1539, p ≪ 0.001; Figures In contrast, motif 3 has varying membership of intra-RSN and inter-RSN pairs Figure . As withFigure Figure It can be seen in motif 4 that connections/interactions between DAN and VIS networks represent similar dynamic covariation during 0% sevoflurane, which groups them in the same motif, while such a similarity is broken in 1.2% sevoflurane. This reorganization of interactions between RSNs can be seen between VIS and SMN networks in motif 4 with intermediate strength of average correlation as well as DMN and SMN in motif 6 with intermediate average correlation.We also assessed the consistency of motifs as a function across individual subjects Figures . Figure We quantified the extent of conservation of functional connectivity, defined by the correlation of intrinsic activity, within the cerebral cortex between wakefulness and sevoflurane general anesthesia. This was addressed in multiple ways. First, we assessed the similarity between the functional connectivity by computing correlations of the entire BOLD time-series  for each regime. Second, we estimated how the correlation between region-pairs evolves as a function of time, thus enabling us to disambiguate region-pairs with similar average correlations into groups with different correlation dynamics. In particular, we decomposed the correlation at each moment in time into a finite set of spatiotemporal motifs whose superposition recovers the average functional connectivity. Our results indicate that the transition from 0 to 1.2% sevoflurane is associated with altered interaction between resting state networks.The correlation structure of intrinsic neural activity can be decomposed into spatiotemporal motifs whose members show covariation in their temporal correlation patterns. Our clustering approach generates similar motifs for wakefulness compared to sevoflurane general anesthesia for region pairs within RSNs . Other motifs  with intermediate average correlation strength exhibit dissimilar membership between the baseline and anesthetic conditions. Finally, the large bulk of interactions between regions of different RSNs differ between motifs and have low mean correlation strength .Our results complement our prior findings of susceptibility within the DMN and VAN . Although there is a rich body of literature on dynamic functional connectivity which suggests meaningful correlation fluctuations in resting state networks, such fluctuations may nonetheless be a simple reflection of physiological noise, head motion, sampling variability intrinsic to small quantities of data, or bona-fide signal changes related to sleep state , the Washington University Institute of Clinical and Translational Sciences grant UL1 TR000448, subgrant KL2TR000450 from the National Center for Advancing Translational Sciences, and grant 1R21AG052821-01 from the National Institute of Aging. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The mixed chimerism approach is an exceptionally potent strategy for the induction of donor-specific tolerance in organ transplantation and so far the only one that was demonstrated to work in the clinical setting. Regulatory T cells (Tregs) have been shown to improve chimerism induction in experimental animal models. This review summarizes the development of innovative BMT protocols using therapeutic Treg transfer for tolerance induction.Treg cell therapy promotes BM engraftment in reduced conditioning protocols in both, mice and non-human primates. In mice, transfer of polyclonal recipient Tregs was sufficient to substitute cytotoxic recipient conditioning. Treg therapy prevented chronic rejection of skin and heart allografts related to tissue-specific antigen disparities, in part by promoting intragraft Treg accumulation.Adoptive Treg transfer is remarkably effective in facilitating BM engraftment in reduced-intensity protocols in mice and non-human primates. Furthermore, it promotes regulatory mechanisms that prevent chronic rejection. The age-long history of organ transplantation is a story of miracles, myths, and science fiction until six decades ago, when the first successful kidney transplant was performed between identical twins . Some yeRegulatory T cells (Tregs) are not only the key mediators of self-tolerance, preventing fatal autoimmunity , but areTolerance per se is defined by as immunological non-reactivity to a specific antigen while maintaining reactivity to others, in the field of transplantation this means “the absence of graft rejection without the (chronic) use of immunosuppressive drugs” . ExtensiAlthough mouse models have been an invaluable tool for uncovering the basic principles of allorecognition and defined strain combinations as well as genetically modified mice allowed dissection of individual pathways in transplantation immunology, there are major limitations when it comes to translation from mice to men . A majorClinicalTrials.gov Identifier: NCT01780844). Currently, next-generation anti-CD40L mAbs devoid of the Fc-mediated toxicity are under development and offer promise for clinical application [Another barrier for translation of tolerance approaches from mice to humans is the limited availability of pharmaceuticals. Blockade of the CD40:CD40L pathway using an anti-CD40L mAb (MR1) was the most successful and promising approach in murine models. It is the backbone of many non-myeloablative mixed chimerism protocols, obviating the need for global T cell depletion and thymic irradiation . A clinilication .Preclinical animal studies using knockout, immunodeficient, or TCR transgenic strains are definitely important for mechanistic understanding of allorecognition and signaling pathways. However, for the development of tolerance strategies, more realistic approaches using stringent strain combinations including minor antigen disparities, housing in non-SPF environment and clinically available pharmaceuticals are warranted.Mixed chimerism denotes a state of co-existence of donor and recipient hematopoietic cells after allogeneic BMT in a pre-conditioned recipient . InductiAs current clinical protocols for HLA-mismatched BMT are not ready for widespread clinical application for non-life-threatening diseases due to the toxicity related to recipient pre-conditioning and the risk of GVHD, a lot of effort has been made to develop “safer” BMT protocols. Considering the importance of regulatory mechanisms and the potency of Tregs to suppress allo-reactivity, adoptive Treg transfer has emerged as attractive possibility that might be the key to translating murine chimerism-based tolerance protocols to the clinic.+CD25+ Tregs, even in the presence of hematopoietic donor cells. However, neither regulation through Tregs nor classical anergy appear crucial several months after BMT for the maintenance of tolerance in established chimeras [Physiological self-tolerance not only involves central deletion but also critically requires non-deletional mechanisms  in order to maintain peripheral tolerance as autoreactive clones can escape negative selection. Deficits in central or peripheral tolerance cause autoimmune disease or in terms of transplantation tolerance result in graft rejection. In protocols using myeloablative and T cell depleting conditioning regimens, non-deletional tolerance mechanisms play a minor or even dispensable role likely due to the fact that potentially allo-reactive T cells are destroyed at the time of BMT and high levels of intrathymic chimerism retain central deletion of newly developing allo-reactive T cells. Experiments employing reduced intensity BMT protocols have revealed the importance for regulatory mechanisms in the induction phase of tolerance when deletion of allo-reactive T cell clones is still incomplete . Toleranchimeras . In sharchimeras ••, 31. Achimeras , 33, modchimeras , or adopchimeras . Chimerichimeras ••, 36 anchimeras •. This rchimeras –41. The chimeras  and it wchimeras .The original observation that the chimerism approach is successful in the clinical setting derives from numerous case reports in which conventional BMT recipients  subsequently received an organ from the same donor for unrelated treatment of organ failure . In suchThe MGH approach is based on decade-long studies in mice and NHPs and initially tested in patient who suffered from coincidental multiple myeloma and end-stage kidney disease, legitimating BMT and cytotoxic recipient conditioning –59. The In contrast, in murine models, stable macrochimerism and sustained presence of discrete populations of donor APCs in the thymus is a pre-requisite for durable tolerance as it ensures intrathymic deletion of alloreactive T cells. Nevertheless, high levels of chimerism do not necessarily predict long-term allograft survival and donor T cell engraftment was shown to be critical for long-term tolerance . Dissoci+CD25+FoxP3+ Tregs for tolerance induction have been envisioned since the (re)discovery of these powerful suppressor cells more than two decades ago [onestudy.org).Cell therapy approaches using CD4ades ago  and is cThe therapeutic potential of Tregs was underlined by numerous reports, showing potent effects in pre-clinical autoimmune and allo-transplantation models; however, in these reports, the use of lymphopenic hosts , 70 or TAlthough the hype has stagnated a little bit during the last years due to the realization of several hurdles associated with adoptive Treg therapy , a recenGiven the potency of both approaches, combination of the mixed chimerism approach and adoptive Treg transfer seems to be the next logical step on the search for the holy grail of tolerance . The pro6 which corresponds to ≈ 1 × 109 cells/kg). Recipient preconditioning was non-myeloablative and consisted mostly of mild doses of irradiation and/or costimulation blockade targeting the CD40:CD40L or CD28:B7 pathway. Notably, Treg transfer was capable of obviating the need for recipient irradiation (or cytotoxic drug treatment), allowing for the first time engraftment of fully mismatched BM without myelosuppression [6 cells. The most interesting and critical question is the source of Tregs (donor- vs recipient- vs third party-derived) and the need for specificity . Several groups reported that antigen-specific Tregs are more potent compared to a polyclonal Treg population in models with [Most of the protocols with combined Treg and BMT are employing clinically achievable doses of allogeneic BM immune responses is recognized as a complex network of multiple regulatory cell types, CD4