Sequential Stem Cell-Kidney Transplantation in Schimke Immuno-osseous Dysplasia.

Lifelong immunosuppression is required for allograft survival after kidney transplantation but may not ultimately prevent allograft loss resulting from chronic rejection. We developed an approach that attempts to abrogate immune rejection and the need for post-transplantation immunosuppression in three patients with Schimke immuno-osseous dysplasia who had both T-cell immunodeficiency and renal failure. Each patient received sequential transplants of αß T-cell-depleted and CD19 B-cell-depleted haploidentical hematopoietic stem cells and a kidney from the same donor. Full donor hematopoietic chimerism and functional ex vivo T-cell tolerance was achieved, and the patients continued to have normal renal function without immunosuppression at 22 to 34 months after kidney transplantation. (Funded by the Kruzn for a Kure Foundation.).

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

Parathyroid hormone signaling in mature osteoblasts/osteocytes protects mice from age-related bone loss.

Aging is accompanied by osteopenia, characterized by reduced bone formation and increased bone resorption. Osteocytes, the terminally differentiated osteoblasts, are regulators of bone homeostasis, and parathyroid hormone (PTH) receptor (PPR) signaling in mature osteoblasts/osteocytes is essential for PTH-driven anabolic and catabolic skeletal responses. However, the role of PPR signaling in those cells during aging has not been investigated. The aim of this study was to analyze the role of PTH signaling in mature osteoblasts/osteocytes during aging. Mice lacking PPR in osteocyte (Dmp1-PPRKO) display an age-dependent osteopenia characterized by a significant decrease in osteoblast activity and increase in osteoclast number and activity. At the molecular level, the absence of PPR signaling in mature osteoblasts/osteocytes is associated with an increase in serum sclerostin and a significant increase in osteocytes expressing 4-hydroxy-2-nonenals, a marker of oxidative stress. In Dmp1-PPRKO mice there was an age-dependent increase in p16Ink4a/Cdkn2a expression, whereas it was unchanged in controls. In vitro studies demonstrated that PTH protects osteocytes from oxidative stress-induced cell death. In summary, we reported that PPR signaling in osteocytes is important for protecting the skeleton from age-induced bone loss by restraining osteoclast's activity and protecting osteocytes from oxidative stresses.

RNAseq and RNA molecular barcoding reveal differential gene expression in cortical bone following hindlimb unloading in female mice.

Disuse-induced bone loss is seen following spinal cord injury, prolonged bed rest, and exposure to microgravity. We performed whole transcriptomic profiling of cortical bone using RNA sequencing (RNAseq) and RNA molecular barcoding (NanoString) on a hindlimb unloading (HLU) mouse model to identify genes whose mRNA transcript abundances change in response to disuse. Eleven-week old female C57BL/6 mice were exposed to ambulatory loading or HLU for 7 days (n = 8/group). Total RNA from marrow-flushed femoral cortical bone was analyzed on HiSeq and NanoString platforms. The expression of several previously reported genes associated with Wnt signaling and metabolism was altered by HLU. Furthermore, the increased abundance of transcripts, such as Pfkfb3 and Mss51, after HLU imply these genes also have roles in the cortical bone's response to altered mechanical loading. Our study demonstrates that an unbiased approach to assess the whole transcriptomic profile of cortical bone can reveal previously unidentified mechanosensitive genes and may eventually lead to novel targets to prevent disuse-induced osteoporosis.

Role of c-Met/ß1 integrin complex in the metastatic cascade in breast cancer.

Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. Although individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a complex between receptor tyrosine kinase c-Met and ß1 integrin in metastases. Using cell culture and in vivo assays, we found that c-Met/ß1 complex induction promoted intravasation and vessel wall adhesion in triple-negative breast cancer cells, but did not increase extravasation. These effects may have been driven by the ability of the c-Met/ß1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/ß1 complex induction. A ß1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/ß1 binding, decreased breast cancer cell invasion and mesenchymal gene expression. Bone-seeking breast cancer cells exhibited higher levels of c-Met/ß1 complex than parental controls and preferentially adhered to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/ß1 complex than brain metastases. Thus, the c-Met/ß1 complex drove intravasation of triple-negative breast cancer cells and preferential affinity for bone-specific matrix. Pharmacological targeting of the complex may have prevented metastases, particularly osseous metastases.

Global transcriptomic analysis of a murine osteocytic cell line subjected to spaceflight.

Bone loss is a major health concern for astronauts during long-term spaceflight and for patients during prolonged bed rest or paralysis. Growing evidence suggests that osteocytes, the most abundant cells in the mineralized bone matrix, play a key role in sensing mechanical forces applied to the skeleton and integrating the orchestrated response into subcellular biochemical signals to modulate bone homeostasis. However, the precise molecular mechanisms underlying both mechanosensation and mechanotransduction in late-osteoblast-to-osteocyte cells under microgravity (µG) have yet to be elucidated. To unravel the mechanisms by which late osteoblasts and osteocytes sense and respond to mechanical unloading, we exposed the osteocytic cell line, Ocy454, to 2, 4, or 6 days of µG on the SpaceX Dragon-6 resupply mission to the International Space Station. Our results showed that µG impairs the differentiation of osteocytes, consistent with prior osteoblast spaceflight experiments, which resulted in the downregulation of key osteocytic genes. Importantly, we demonstrate the modulation of critical glycolysis pathways in osteocytes subjected to microgravity and discovered a set of mechanical sensitive genes that are consistently regulated in multiple cell types exposed to microgravity suggesting a common, yet to be fully elucidated, genome-wide response to microgravity. Ground-based simulated microgravity experiments utilizing the NASA rotating-wall-vessel were unable to adequately replicate the changes in microgravity exposure highlighting the importance of spaceflight missions to understand the unique environmental stress that microgravity presents to diverse cell types. In summary, our findings demonstrate that osteocytes respond to µG with an increase in glucose metabolism and oxygen consumption.

Intraoperative Stereotactic Frame Registration Using a Three-Dimensional Imaging System with and without Preoperative Computed Tomography for Image Fusion.

BACKGROUND: The O-arm O2 imaging system (OAO2) is an intraoperative cone beam 3D tomogram imaging tool with a wide enough field of view to perform intraoperative fiducial registration with standard stereotactic frames. However, the OAO2 3D images (cone beam CT) provide limited tissue contrast, which may reduce the accuracy of fusion to a preoperative targeting MRI for planning awake deep brain stimulation (DBS) surgeries. Therefore, most users obtain a preoperative CT scan to use as the reference exam for computational fusion with the preoperative targeting MRI and the intraoperative OAO2 cone beam CT. OBJECTIVE: In this study, we retrospectively analyzed the discrepancy between stereotactic coordinates of deep brain targets on MRI derived from intraoperative OAO2 fiducial registration with and without the use of preoperative CT as the reference for image fusion. METHODS: Preoperative stereotactic CT/MRI and intraoperative OAO2 cone beam CT were retrospectively evaluated for 27 consecutive DBS patients, using two commercial surgical planning software packages (BrainLab Elements and Medtronic Stealth 8). The anterior commissure, posterior commissure, and left subthalamic nucleus were identified on preoperative MRI. Each patient had intraoperative fiducial registration using the OAO2 with a Leksell headframe. For each subject, the reference scan for image fusion was set as either the preoperative CT or the preoperative MRI (volumetric T1 with contrast). Computed stereotactic coordinates for each target were then compared. RESULTS: For 8 of 27 subjects, a discrepancy greater than 1.0 mm for at least one designated target was observed utilizing the Medtronic Stealth S8 planning station when a preoperative CT scan was not used. An additional 5 (5/27) had a discrepancy greater than 2 mm. The most common discrepancy was in the z axis. No coordinate discrepancies greater than 1 mm were observed utilizing BrainLab Elements. CONCLUSIONS: Caution is advised in fusing intraoperative OAO2 images directly to preoperative MRI without a preoperative CT as the reference exam for image fusion, as the specific fusion algorithm employed may unpredictably affect targeting accuracy.

A G protein-coupled, IP3/protein kinase C pathway controlling the synthesis of phosphaturic hormone FGF23.

Dysregulated actions of bone-derived phosphaturic hormone fibroblast growth factor 23 (FGF23) result in several inherited diseases, such as X-linked hypophosphatemia (XLH), and contribute substantially to the mortality in kidney failure. Mechanisms governing FGF23 production are poorly defined. We herein found that ablation of the Gq/11α-like, extralarge Gα subunit (XLαs), a product of GNAS, exhibits FGF23 deficiency and hyperphosphatemia in early postnatal mice (XLKO). FGF23 elevation in response to parathyroid hormone, a stimulator of FGF23 production via cAMP, was intact in XLKO mice, while skeletal levels of protein kinase C isoforms α and δ (PKCα and PKCδ) were diminished. XLαs ablation in osteocyte-like Ocy454 cells suppressed the levels of FGF23 mRNA, inositol 1,4,5-trisphosphate (IP3), and PKCα/PKCδ proteins. PKC activation in vivo via injecting phorbol myristate acetate (PMA) or by constitutively active Gqα-Q209L in osteocytes and osteoblasts promoted FGF23 production. Molecular studies showed that the PKC activation-induced FGF23 elevation was dependent on MAPK signaling. The baseline PKC activity was elevated in bones of Hyp mice, a model of XLH. XLαs ablation significantly, but modestly, reduced serum FGF23 and elevated serum phosphate in Hyp mice. These findings reveal a potentially hitherto-unknown mechanism of FGF23 synthesis involving a G protein-coupled IP3/PKC pathway, which may be targeted to fine-tune FGF23 levels.

Large G protein α-subunit XLαs limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo.

Alterations in the activity/levels of the extralarge G protein α-subunit (XLαs) are implicated in various human disorders, such as perinatal growth retardation. Encoded by GNAS, XLαs is partly identical to the α-subunit of the stimulatory G protein (Gsα), but the cellular actions of XLαs remain poorly defined. Following an initial proteomic screen, we identified sorting nexin-9 (SNX9) and dynamins, key components of clathrin-mediated endocytosis, as binding partners of XLαs. Overexpression of XLαs in HEK293 cells inhibited internalization of transferrin, a process that depends on clathrin-mediated endocytosis, while its ablation by CRISPR/Cas9 in an osteocyte-like cell line (Ocy454) enhanced it. Similarly, primary cardiomyocytes derived from XLαs knockout (XLKO) pups showed enhanced transferrin internalization. Early postnatal XLKO mice showed a significantly higher degree of cardiac iron uptake than wild-type littermates following iron dextran injection. In XLKO neonates, iron and ferritin levels were elevated in heart and skeletal muscle, where XLαs is normally expressed abundantly. XLKO heart and skeletal muscle, as well as XLKO Ocy454 cells, showed elevated SNX9 protein levels, and siRNA-mediated knockdown of SNX9 in XLKO Ocy454 cells prevented enhanced transferrin internalization. In transfected cells, XLαs also inhibited internalization of the parathyroid hormone and type 2 vasopressin receptors. Internalization of transferrin and these G protein-coupled receptors was also inhibited in cells expressing an XLαs mutant missing the Gα portion, but not Gsα or an N-terminally truncated XLαs mutant unable to interact with SNX9 or dynamin. Thus, XLαs restricts clathrin-mediated endocytosis and plays a critical role in iron/transferrin uptake in vivo.

Treatment With a Soluble Bone Morphogenetic Protein Type 1A Receptor (BMPR1A) Fusion Protein Increases Bone Mass and Bone Formation in Mice Subjected to Hindlimb Unloading.

Previous work has shown that the soluble murine BMPR1A-fusion protein (mBMPR1A-mFc) binds to BMP2 and BMP4 with high affinity, preventing downstream signaling. Further, treatment of intact and ovariectomized mice with mBMPR1A-mFc leads to increased bone mass, and improved bone microarchitecture and strength, via increased bone formation and reduced resorption. In this study, we tested the effects of mBMPR1A-mFc on disuse-induced bone loss caused by 21 days of hindlimb unloading (HLU) via tail suspension versus cage controls (CONs). Adult female C57BL/6J mice (12 weeks old) were assigned to one of four groups (n = 10 each): CON-VEH; CON-mBMPR1A-mFc; HLU-VEH; and HLU-mBMPR1A-mFc. Mice were injected subcutaneously with VEH or mBMPR1A-mFc (4.5 mg/kg, 2×/week). Leg BMD declined in the HLU-VEH group (-5.3% ± 1.3%), whereas it was unchanged in HLU-mBMPR1A-mFc (-0.3% ± 0.9%, p < 0.05 versus HLU-VEH). Leg BMD increased significantly more in CON-mBMPR1A-mFc than CON-VEH (10.2% ± 0.6% versus 4.4% ± 0.8%). In the femur, trabecular, and cortical bone microarchitecture was worse in the HLU-VEH compared to CON-VEH mice, whereas mBMPR1A-mFc treatment for 3 weeks led to greater Tb.BV/TV, Tb.Th, and midshaft Ct.Th in both the HLU and CON groups compared to comparable VEH-treated counterparts (p < 0.05). HLU-mBMPR1A-mFc mice also had 21% greater failure load (p < 0.05) compared to their VEH-treated counterparts. Dynamic histomorphometry indicated that treatment with mBMPR1A-mFc led to significantly greater mineralizing surface and mineral apposition rate, resulting in a 3.5-fold and fivefold higher bone formation rate in the mBMPR1A-mFc-treated CON and HLU animals versus VEH groups, respectively. mBMPR1A-mFc-treated mice had a similar osteoblast surface but significantly lower osteoclast surface than VEH-treated animals in both the CON and HLU groups. Altogether, these findings suggest that treatment with the soluble BMPR1A fusion protein may be useful for maintenance of skeletal integrity in the setting of disuse-induced bone loss.

Osteocyte-Secreted Wnt Signaling Inhibitor Sclerostin Contributes to Beige Adipogenesis in Peripheral Fat Depots.

Cells of the osteoblast lineage are increasingly identified as participants in whole-body metabolism by primarily targeting pancreatic insulin secretion or consuming energy. Osteocytes, the most abundant bone cells, secrete a Wnt-signaling inhibitor called sclerostin. Here we examined three mouse models expressing high sclerostin levels, achieved through constitutive or inducible loss of the stimulatory subunit of G-proteins (Gsα in mature osteoblasts and/or osteocytes). These mice showed progressive loss of white adipose tissue (WAT) with tendency toward increased energy expenditure but no changes in glucose or insulin metabolism. Interestingly beige adipocytes were increased extensively in both gonadal and inguinal WAT and had reduced canonical ß-catenin signaling. To determine if sclerostin directly contributes to the increased beige adipogenesis, we engineered an osteocytic cell line lacking Gsα which has high sclerostin secretion. Conditioned media from these cells significantly increased expression of UCP1 in primary adipocytes, and this effect was partially reduced after depletion of sclerostin from the conditioned media. Similarly, treatment of Gsα-deficient animals with sclerostin-neutralizing antibody partially reduced the increased UCP1 expression in WAT. Moreover, direct treatment of sclerostin to wild-type mice significantly increased UCP1 expression in WAT. These results show that osteocytes and/or osteoblasts secrete factors regulating beige adipogenesis, at least in part, through the Wnt-signaling inhibitor sclerostin. Further studies are needed to assess metabolic effects of sclerostin on adipocytes and other metabolic tissues. © 2016 American Society for Bone and Mineral Research.

The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.

Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone.

Combined effects of botulinum toxin injection and hind limb unloading on bone and muscle.

Bone receives mechanical stimulation from two primary sources, muscle contractions and external gravitational loading; but the relative contribution of each source to skeletal health is not fully understood. Understanding the most effective loading for maintaining bone health has important clinical implications for prescribing physical activity for the treatment or prevention of osteoporosis. Therefore, we investigated the relative effects of muscle paralysis and reduced gravitational loading on changes in muscle mass, bone mineral density, and microarchitecture. Adult female C57Bl/6J mice (n = 10/group) underwent one of the following: unilateral botulinum toxin (BTX) injection of the hind limb, hind limb unloading (HLU), both unilateral BTX injection and HLU, or no intervention. BTX and HLU each led to significant muscle and bone loss. The effect of BTX was diminished when combined with HLU, though generally the leg that received the combined intervention (HLU+BTX) had the most detrimental changes in bone and muscle. We found an indirect effect of BTX affecting the uninjected (contralateral) leg that led to significant decreases in bone mineral density and deficits in muscle mass and bone architecture relative to the untreated controls; the magnitude of this indirect BTX effect was comparable to the direct effect of BTX treatment and HLU. Thus, while it was difficult to definitively conclude whether muscle force or external gravitational loading contributes more to bone maintenance, it appears that BTX-induced muscle paralysis is more detrimental to muscle and bone than HLU.

Osteocyte biology and space flight.

The last decade has seen an impressive expansion of our understanding of the role of osteocytes in skeletal homeostasis. These amazing cells, deeply embedded into the mineralized matrix, are the key regulators of bone homeostasis and skeletal mechano sensation and transduction. They are the cells that can sense the mechanical forces applied to the bone and then translate these forces into biological responses. They are also ideally positioned to detect and respond to hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts through the production and secretion of molecules such as Sclerostin and RANKL. How osteocytes perceive mechanical forces and translate them into biological responses in still an open question. Novel "in vitro" models as well the opportunity to study these cells under microgravity condition, will allow a closer look at the molecular and cellular mechanisms of mechano transduction. This article highlights novel investigations on osteocytes and discusses their significance in our understanding of skeletal mechano transduction.

Sclerostin antibody inhibits skeletal deterioration due to reduced mechanical loading.

Sclerostin, a product of the SOST gene produced mainly by osteocytes, is a potent negative regulator of bone formation that appears to be responsive to mechanical loading, with SOST expression increasing following mechanical unloading. We tested the ability of a murine sclerostin antibody (SclAbII) to prevent bone loss in adult mice subjected to hindlimb unloading (HLU) via tail suspension for 21 days. Mice (n = 11-17/group) were assigned to control (CON, normal weight bearing) or HLU and injected with either SclAbII (subcutaneously, 25 mg/kg) or vehicle (VEH) twice weekly. SclAbII completely inhibited the bone deterioration due to disuse, and induced bone formation such that bone properties in HLU-SclAbII were at or above values of CON-VEH mice. For example, hindlimb bone mineral density (BMD) decreased -9.2% ± 1.0% in HLU-VEH, whereas it increased 4.2% ± 0.7%, 13.1% ± 1.0%, and 30.6% ± 3.0% in CON-VEH, HLU-SclAbII, and CON-SclAbII, respectively (p < 0.0001). Trabecular bone volume, assessed by micro-computed tomography (µCT) imaging of the distal femur, was lower in HLU-VEH versus CON-VEH (p < 0.05), and was 2- to 3-fold higher in SclAbII groups versus VEH (p < 0.001). Midshaft femoral strength, assessed by three-point bending, and distal femoral strength, assessed by micro-finite element analysis (µFEA), were significantly higher in SclAbII versus VEH-groups in both loading conditions. Serum sclerostin was higher in HLU-VEH (134 ± 5 pg/mL) compared to CON-VEH (116 ± 6 pg/mL, p < 0.05). Serum osteocalcin was decreased by hindlimb suspension and increased by SclAbII treatment. Interestingly, the anabolic effects of sclerostin inhibition on some bone outcomes appeared to be enhanced by normal mechanical loading. Altogether, these results confirm the ability of SclAbII to abrogate disuse-induced bone loss and demonstrate that sclerostin antibody treatment increases bone mass by increasing bone formation in both normally loaded and underloaded environments.

Amphiregulin-EGFR signaling mediates the migration of bone marrow mesenchymal progenitors toward PTH-stimulated osteoblasts and osteocytes.

Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH's anabolic actions on bone.