The RANKL-RANK Axis: A Bone to Thymus Round Trip

The identification of Receptor activator of nuclear factor kappa B ligand (RANKL) and its cognate receptor Receptor activator of nuclear factor kappa B (RANK) during a search for novel tumor necrosis factor receptor (TNFR) superfamily members has dramatically changed the scenario of bone biology by providing the functional and biochemical proof that RANKL signaling via RANK is the master factor for osteoclastogenesis. In parallel, two independent studies reported the identification of mouse RANKL on activated T cells and of a ligand for osteoprotegerin on a murine bone marrow-derived stromal cell line. After these seminal findings, accumulating data indicated RANKL and RANK not only as essential players for the development and activation of osteoclasts, but also for the correct differentiation of medullary thymic epithelial cells (mTECs) that act as mediators of the central tolerance process by which self-reactive T cells are eliminated while regulatory T cells are generated. In light of the RANKL-RANK multi-task function, an antibody targeting this pathway, denosumab, is now commonly used in the therapy of bone loss diseases including chronic inflammatory bone disorders and osteolytic bone metastases; furthermore, preclinical data support the therapeutic application of denosumab in the framework of a broader spectrum of tumors. Here, we discuss advances in cellular and molecular mechanisms elicited by RANKL-RANK pathway in the bone and thymus, and the extent to which its inhibition or augmentation can be translated in the clinical arena

Soluble Factors on Stage to Direct Mesenchymal Stem Cells Fate

Mesenchymal stem cells (MSCs) are multipotent stromal cells that are identified by in vitro plastic adherence, colony-forming capacity, expression of a panel of surface molecules, and ability to differentiate at least toward osteogenic, adipogenic, and chondrogenic lineages. They also produce trophic factors with immunomodulatory, proangiogenic, and antiapoptotic functions influencing the behavior of neighboring cells. On the other hand, a reciprocal regulation takes place; in fact, MSCs can be isolated from several tissues, and depending on the original microenvironment and the range of stimuli received from there, they can display differences in their essential characteristics. Here, we focus mainly on the bone tissue and how soluble factors, such as growth factors, cytokines, and hormones, present in this microenvironment can orchestrate bone marrow-derived MSCs fate. We also briefly describe the alteration of MSCs behavior in pathological settings such as hematological cancer, bone metastasis, and bone marrow failure syndromes. Overall, the possibility to modulate MSCs plasticity makes them an attractive tool for diverse applications of tissue regeneration in cell therapy. Therefore, the comprehensive understanding of the microenvironment characteristics and components better suited to obtain a specific MSCs response can be extremely useful for clinical use

631 rankl knock out mesenchymal stromal cells have an unexpected osteogenic differentiation defect which is improved by a rankl expressing lentiviral vector

Osteoclast-poor RANKL-dependent Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Hematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Therefore Mesenchymal Stromal Cells (MSC) transplantation together with a gene-therapy strategy to correct RANKL defect in MSC could represent a possible effective therapy. Of note, whether also MSC, besides the osteoclasts, are affected by RANKL deficiency is unknown. To verify this, we established and characterized bone marrow derived MSC (BM-MSC) lines from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from wild type (WT) mice. No differences were found between KO and WT MSC in terms of morphology, immunophenotype and proliferation capacity. However, KO MSC displayed a reduced clonogenic potential with a decrease in stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while showed a significantly impaired osteogenic differentiation capacity compared to WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we developed a third generation lentiviral vector expressing human soluble RANKL (hsRL) for the genetic correction of KO MSC. We first investigated lentiviral transduction in 293T cells to optimize transduction efficiency at different multiplicity of infection (MOI) ranging from 1 to 100. hsRL production increased proportionally to the MOI and was stable over time. However, the higher the MOI the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRL transduction in KO MSC at 20 and 50 MOI, to define the optimal transduction conditions. After transduction 99.5% of MSC were GFP+. While in Rankl−/− control cells the cytokine was not detected, in corrected cells hsRL production and secretion was measurable and comparable to sRL levels in WT mouse. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to untransduced KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. Moreover, the expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Our data suggest that restoration of RANKL production in lentiviral-transduced KO MSC might not only allow osteoclast differentiation in Rankl−/− mice upon transplantation, but also improve the osteogenic differentiation defect of KO MSC

Label-free multimodal nonlinear optical microscopy reveals features of bone composition in pathophysiological conditions

Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections

Adverse effects of bisphenol a exposure on glucose metabolism regulation

Bisphenol A (BPA) is used as basic chemical compound in the production of polycarbonate food containers or epoxy resins coating metallic cans for food and beverages conservation. Its xeno-estrogenic activity alters endocrine-metabolic pathways modulating glucose metabolism and increasing the risk of developing diabetes, insulin resistance, and obesity. Based on in vitro and in vivo experimental research, here we report some of the major BPA adverse effects on tissues that play a key role in the regulation on the whole body’s metabolism. Evidences have shown that BPA is able to exert its endocrine disrupting action altering glucose metabolism and contributing to the onset of metabolic disorders, acting on liver functions and affecting insulin production by the pancreas. Exposure to BPA has been reported also to modulate glucose utilization in muscles, as well as to interfere with adipose tissue endocrine function. In addition, to peripheral tissues, recent studies have shown that BPA by acting in the Central Nervous System affects neuroendocrine regulation of glucose metabolism, promoting glucose metabolism dysfunction such as glucose intolerance and insulin resistance. Thus, exposure to BPA seems to be an important risk factor in the onset of obesity and metabolic syndrome. However, its mechanisms of action need to be further investigated to provide a major evaluation of risk assessment

Ethnobotanical investigation on medicinal plants in the Vesuvio National Park (Campania, Southern Italy)

This paper illustrates the results of an ethnobotanical study carried out in the Vesuvio National Park (VNP) (Campania, Southern Italy). It describes the medicinal uses of the plants in an ancient area rich in ethnobiodiversity investigated for the first time. The main aim of the study was to understand at what extent current knowledge on medicinal plant uses is still alive in VNP. The informations were collected using semi-structured and unstructured interviews performed on 136 persons living in the investigated area from March to November 2014 and from April to October 2015. Local plant uses were listed and analyzed in a table and compared with uses in other localities in Italy and in other regions of the Mediterranean basin. In VNP were recorded a total number of 132 plant species, belonging to 110 genera and 51 families mentioned for medicinal purposes. Among the recorded 132 plant species, 70 are spontaneous or subspontaneous and 62 are cultivated above all in the kitchen gardens or in the apartments, as food or as ornamental. The investigated plants were used to cure 116 different human health diseases and 4 veterinary problems. The majority of plants are used in the treatment of gastrointestinal, skin and respiratory problems. The number of medicinal plants reported in this paper reflects a well-preserved traditional popular knowledge (TPK) of the elderly people living in the rural areas and in the small villages of VNP. The conservation of TPK is owed to the persistence of an oral tradition that safeguard the use of plants as herbal medicine. We realized that while the use of some wild plants is decreasing, people continue to gather some cultivated and invasive plants for preparing remedies. Researches like this are necessary to protect ancient memories, to promote the transfer of information to the younger generations, to preserve ethno-biodiversity and to provide a starting point fur further biochemical investigations on medicinal entities