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Sample records for primary mouse bone

  1. Cysteine Dioxygenase Type 1 Inhibits Osteogenesis by Regulating Wnt Signaling in Primary Mouse Bone Marrow Stromal Cells

    PubMed Central

    Zhao, Xuefeng; Deng, Peng; Feng, Jie; Wang, Zheng; Xiang, Zichao; Han, Xianglong; Bai, Ding; Pae, Eung-Kwon

    2016-01-01

    Mesenchymal stem cells (MSCs) are multipotent cells, which can give rise to variety of cell types, including adipocytes and osteoblasts. Previously, we have shown that cysteine dioxygenase type 1 (Cdo1) promoted adipogenesis of primary mouse bone marrow stromal cells (BMSCs) and 3T3-L1 pre-adipocytes via interaction with Pparγ. However, the role of Cdo1 in osteogenesis remains unclear. Here, we demonstrated that expression of Cdo1 was elevated during osteoblastic differentiation of BMSCs in vitro. Interestingly, knockdown of Cdo1 by siRNA led to an increased expression of osteogenic related genes, elevated alkaline phosphatase (ALP) activity, and enhanced mineralization. Overexpression of Cdo1 in BMSCs inversely suppressed the osteogenesis. Furthermore, we found that overexpression of Cdo1 impaired Wnt signaling and restricted the Wnt3a induced expression of osteogenic transcriptional factors, such as Runx2 and Dlx5. Collectively, our findings indicate Cdo1 suppresses osteogenic differentiation of BMSCs, through a potential mechanism which involves in Wnt signaling reduction concomitantly. PMID:26763277

  2. Cysteine Dioxygenase Type 1 Inhibits Osteogenesis by Regulating Wnt Signaling in Primary Mouse Bone Marrow Stromal Cells.

    PubMed

    Zhao, Xuefeng; Deng, Peng; Feng, Jie; Wang, Zheng; Xiang, Zichao; Han, Xianglong; Bai, Ding; Pae, Eung-Kwon

    2016-01-01

    Mesenchymal stem cells (MSCs) are multipotent cells, which can give rise to variety of cell types, including adipocytes and osteoblasts. Previously, we have shown that cysteine dioxygenase type 1 (Cdo1) promoted adipogenesis of primary mouse bone marrow stromal cells (BMSCs) and 3T3-L1 pre-adipocytes via interaction with Pparγ. However, the role of Cdo1 in osteogenesis remains unclear. Here, we demonstrated that expression of Cdo1 was elevated during osteoblastic differentiation of BMSCs in vitro. Interestingly, knockdown of Cdo1 by siRNA led to an increased expression of osteogenic related genes, elevated alkaline phosphatase (ALP) activity, and enhanced mineralization. Overexpression of Cdo1 in BMSCs inversely suppressed the osteogenesis. Furthermore, we found that overexpression of Cdo1 impaired Wnt signaling and restricted the Wnt3a induced expression of osteogenic transcriptional factors, such as Runx2 and Dlx5. Collectively, our findings indicate Cdo1 suppresses osteogenic differentiation of BMSCs, through a potential mechanism which involves in Wnt signaling reduction concomitantly. PMID:26763277

  3. Bone disease in primary hyperparathyroidism.

    PubMed

    Bandeira, Francisco; Cusano, Natalie E; Silva, Barbara C; Cassibba, Sara; Almeida, Clarissa Beatriz; Machado, Vanessa Caroline Costa; Bilezikian, John P

    2014-07-01

    Bone disease in severe primary hyperparathyroidism (PHPT) is described classically as osteitis fibrosa cystica (OFC). Bone pain, skeletal deformities and pathological fractures are features of OFC. Bone mineral density is usually extremely low in OFC, but it is reversible after surgical cure. The signs and symptoms of severe bone disease include bone pain, pathologic fractures, proximal muscle weakness with hyperreflexia. Bone involvement is typically characterized as salt-and-pepper appearance in the skull, bone erosions and bone resorption of the phalanges, brown tumors and cysts. In the radiography, diffuse demineralization is observed, along with pathological fractures, particularly in the long bones of the extremities. In severe, symptomatic PHPT, marked elevation of the serum calcium and PTH concentrations are seen and renal involvement is manifested by nephrolithiasis and nephrocalcinosis. A new technology, recently approved for clinical use in the United States and Europe, is likely to become more widely available because it is an adaptation of the lumbar spine DXA image. Trabecular bone score (TBS) is a gray-level textural analysis that provides an indirect index of trabecular microarchitecture. Newer technologies, such as high-resolution peripheral quantitative computed tomography (HR-pQCT), have provided further understanding of the microstructural skeletal features in PHPT. PMID:25166047

  4. Bone impairment in primary hyperoxaluria: a review.

    PubMed

    Bacchetta, Justine; Boivin, Georges; Cochat, Pierre

    2016-01-01

    Deposition of calcium oxalate crystals in the kidney and bone is a hallmark of primary hyperoxaluria (PH). Since the bone compartment can store massive amounts of oxalate, patients present with recurrent low-trauma fractures, bone deformations, severe bone pains, and specific oxalate osteopathy on X-ray. Bone biopsy from the iliac crest displays specific features such as oxalate crystals surrounded by a granulomatous reaction corresponding to an invasion of bone surface by macrophages. The objective of this manuscript is therefore to provide an overview of bone impairment in PH, by reviewing the current literature on bone and dental symptoms as well as imaging techniques used for assessing bone disease. PMID:25631241

  5. Osteoclast derivation from mouse bone marrow.

    PubMed

    Tevlin, Ruth; McArdle, Adrian; Chan, Charles K F; Pluvinage, John; Walmsley, Graham G; Wearda, Taylor; Marecic, Owen; Hu, Michael S; Paik, Kevin J; Senarath-Yapa, Kshemendra; Atashroo, David A; Zielins, Elizabeth R; Wan, Derrick C; Weissman, Irving L; Longaker, Michael T

    2014-01-01

    Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease. As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation. An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts. PMID:25407120

  6. Ultrasound of Primary Aneurysmal Bone Cyst

    PubMed Central

    Glazebrook, Katrina N.; Keeney, Gary L.; Rock, Michael G.

    2014-01-01

    Aneurysmal bone cysts (ABC) are rare, benign, expansile lesions of bone often found in the metaphyses of long bones in pediatric and young adult population. Multiple fluid levels are typically seen on imaging with magnetic resonance imaging (MRI) or computed tomography (CT). We describe a case of a primary ABC in the fibula of a 34-year-old man diagnosed on ultrasound with a mobile fluid level demonstrated sonographically. PMID:24587935

  7. Primary Pseudomyogenic Hemangioendothelioma of Bone.

    PubMed

    Inyang, Alero; Mertens, Fredrik; Puls, Florian; Sumathi, Vaiyapuri; Inwards, Carrie; Folpe, Andrew; Lee, Cheng-Han; Zhang, Yaxia; Symmans, Pennie; Rubin, Brian; Nielsen, Gunnlaugur P; Nguyen, Van-Hung; Rosenberg, Andrew E

    2016-05-01

    Pseudomyogenic hemangioendothelioma (PMH) is a well-recognized neoplasm that usually arises in the soft tissue; concurrent bone involvement occurs in 24% of cases. PMH of bone without soft tissue involvement is rare. We describe the clinicopathologic findings of 10 such cases, the largest series reported to date. The study included 9 male and 1 female patient; their ages ranged from 12 to 74 years (mean 36.7 y). All patients had multiple tumors with a distinct regional distribution: 45% restricted to the lower extremity; 25% to the spine and pelvis; and 15% to the upper extremity. On imaging studies the tumors were well circumscribed and lytic. The neoplasms were composed of spindled cells arranged in intersecting fascicles with scattered epithelioid cells; epithelioid cells predominated in 3 cases. The neoplastic cells contained abundant densely eosinophilic cytoplasm and vesicular nuclei. There was limited cytologic atypia and necrosis, few mitoses (0 to 2/10 high-power fields), and inconspicuous stroma. Unique findings included abundant intratumoral reactive woven bone and hemorrhage with numerous osteoclast-like giant cells. Immunohistochemically, most tumors were positive for keratin, ERG, and CD31; CD34 was negative. The balanced t(7:19)(q22;13) translocation was documented in 3 cases. Follow-up is limited, but no patient developed documented visceral dissemination, and all have stable or progressive osseous disease. PMH exclusively involving bone is rare. It is multicentric, often involves the lower extremity, and has unusual morphology. The differential diagnosis includes epithelioid vascular neoplasms, giant cell tumor, bone forming neoplasms, and metastatic carcinoma. Because of its rarity, unusual presentation, and morphology, accurate diagnosis can be challenging. PMID:26872012

  8. Regional bone change in intramuscular haemangioma mimicking primary bone tumour.

    PubMed

    Shikhare, Sumer; Chacko, Julio K; Chuah, Khoon L

    2015-04-01

    Intramuscular haemangiomas are benign soft-tissue tumours, commonly located in the extremities. We present a right-leg intramuscular haemangioma with florid periosteal reaction in adjacent tibia, mimicking a primary bone tumour. Plain radiograph and magnetic resonance imaging features are illustrated with the surgical and histopathological findings. Radiologists need to be familiar with reactive bone changes secondary to deep-seated intramuscular haemangiomas to avoid potential misdiagnosis. PMID:25224409

  9. Evaluation of Toxicity in Mouse Bone Marrow Progenitor Cells.

    PubMed

    Ezeh, Peace C; Xu, Huan; Wang, Shu Chun; Medina, Sebastian; Burchiel, Scott W

    2016-01-01

    Development of blood cells through hematopoiesis occurs in the bone marrow (BM), and can be adversely impacted by various substances and/or conditions ranging from known therapeutic, intentionally administered xenobiotics to unintentional food additives and exposure to environmental chemicals. The principles underlying the techniques for evaluating toxicity to BM progenitors (erythroid, myeloid, and lymphoid) exploit changes in the normal hematopoietic process, biochemical cell surface and intracellular markers, as well as components of the BM microenvironment. Toxicological investigations following in vivo exposures of mice or in vitro exposures of mouse primary BM cell cultures allow the assessment of the developmental and functional integrity of BM cells, cell population shifts, and adverse biochemical effects due to toxicity. Colony forming unit (CFU) assays and flow cytometry are indispensable techniques in these toxicity studies. © 2016 by John Wiley & Sons, Inc. PMID:26828331

  10. Cilia/Ift protein and motor-related bone diseases and mouse models

    PubMed Central

    Yuan, Xue; Yang, Shuying

    2015-01-01

    Primary cilia are essential cellular organelles projecting from the cell surface to sense and transduce developmental signaling. They are tiny but have complicated structures containing microtubule (MT)-based internal structures (the axoneme) and mother centriole formed basal body. Intraflagellar transport (Ift) operated by Ift proteins and motors are indispensable for cilia formation and function. Mutations in Ift proteins or Ift motors cause various human diseases, some of which have severe bone defects. Over the last few decades, major advances have occurred in understanding the roles of these proteins and cilia in bone development and remodeling by examining cilia/Ift protein-related human diseases and establishing mouse transgenic models. In this review, we describe current advances in the understanding of the cilia/Ift structure and function. We further summarize cilia/Ift-related human diseases and current mouse models with an emphasis on bone-related phenotypes, cilia morphology, and signaling pathways. PMID:25553465

  11. Glycosphingolipid patterns in primary mouse kidney cultures

    SciTech Connect

    Lyerla, T.A.; Gross, S.K.; McCluer, R.H.

    1986-12-01

    Primary kidney cultures from C57BL/6J mice, 6 weeks of age or older, were produced using D-valine medium to select for epithelial cell growth. After allowing the cells to attach and proliferate for 1 week following plating, medium was changed once per week. Cells formed nearly confluent monolayers during the second week of culture. The cultured cells contained all of the glycosphingolipids seen in the adult kidney, analyzed by high performance liquid chromatography as their perbenzoyl derivatives. Glucosylceramide, however, was highly predominant in the cultured cells, whereas dihexosyl- and trihexosylceramides predominate in the intact kidney. Sex differences in glycolipid contents found in the intact kidney were also apparent in these cultured cells: The concentration of neutral glycolipids, in general, was higher in male cells than in those derived from females, and the male-specific glycolipid nonhydroxy fatty acid digalactosylceramide was high in male cells but very low in female cells. Neutral glycosphingolipids were labeled in 2-week-old cultures using (/sup 3/H)palmitate. The (/sup 3/H)palmitate was incorporated into all of the glycolipids within 2 hr of labeling. Hence, adult mouse kidney cells in D-valine medium retain their differentiated characteristics for a sufficient period of time to allow investigation of glycolipid syntheses in monolayer cultures of epithelial cells derived from this organ.

  12. Genetic mouse models for bone studies—Strengths and limitations

    PubMed Central

    Elefteriou, Florent; Yang, Xiangli

    2012-01-01

    Mice have become a preferred model system for bone research because of their genetic and pathophysiological similarities to humans: a relatively short reproductive period, leading to relatively low cost of maintenance and the availability of the entire mouse genome sequence information. The success in producing the first transgenic mouse line that expressed rabbit β-globin protein in mouse erythrocytes three decades ago marked the beginning of the use of genetically engineered mice as model system to study human diseases. Soon afterward the development of cultured pluripotent embryonic stem cells provided the possibility of gene replacement or gene deletion in mice. These technologies have been critical to identify new genes involved in bone development, growth, remodeling, repair, and diseases, but like many other approaches, they have limitations. This review will introduce the approaches that allow the generation of transgenic mice and global or conditional (tissue-specific and inducible) mutant mice. A list of the various promoters used to achieve bone-specific gene deletion or overexpression is included. The limitations of these approaches are discussed, and general guidelines related to the analysis of genetic mouse models are provided. PMID:21907838

  13. The terminator mouse: salvation for primary cell culture.

    PubMed

    Kabgani, Nazanin; Moeller, Marcus J

    2013-11-01

    The Terminator had to come back from the future already several times in an effort to bring salvation to mankind. In the present issue of Kidney International, Guo et al. brought us a novel transgenic mouse model: the terminator mouse. This highly elegant mouse may facilitate significantly the derivation of primary cultures of a specific cell type from a tissue containing multiple cell populations. PMID:24172731

  14. New mouse primary retinal degeneration (rd-3)

    SciTech Connect

    Chang, B.; Hawes, N.L.; Roderick, T.H. ); Heckenlively, J.R. )

    1993-04-01

    A new mouse retinal degeneration that appears to be an excellent candidate for modeling human retinitis pigmentosa is reported. In this degeneration, called rd-3, differentiation proceeds postnatally through 2 weeks, and photoreceptor degeneration starts by 3 weeks. The rod photoreceptor loss is essentially complete by 5 weeks, whereas remnant cone cells are seen through 7 weeks. This is the only mouse homozygous retinal degeneration reported to date in which photoreceptors are initially normal. Crosses with known mouse retinal degenerations rd, Rds, nr, and pcd are negative for retinal degeneration in offspring, and linkage analysis places rd-3 on mouse chromosome 1 at 10 [+-]2.5 cM distal to Akp-1. Homology mapping suggests that the homologous human locus should be on chromosome 1q. 32 refs., 3 figs., 3 tabs.

  15. Quercetin inhibits inflammatory bone resorption in a mouse periodontitis model.

    PubMed

    Napimoga, Marcelo H; Clemente-Napimoga, Juliana T; Macedo, Cristina G; Freitas, Fabiana F; Stipp, Rafael N; Pinho-Ribeiro, Felipe A; Casagrande, Rubia; Verri, Waldiceu A

    2013-12-27

    Periodontitis is a disease that leads to bone destruction and represents the main cause of tooth loss in adults. The development of aggressive periodontitis has been associated with increased inflammatory response that is induced by the presence of a subgingival biofilm containing Aggregatibacter actinomycetemcomitans. The flavonoid quercetin (1) is widespread in vegetables and fruits and exhibits many biological properties for possible medical and clinical applications such as its anti-inflamatory and antioxidant effects. Thus, in the present study, the properties of 1 have been evaluated in bone loss and inflammation using a mouse periodontitis model induced by A. actinomycetemcomitans infection. Subcutaneous treatment with 1 reduced A. actinomycetemcomitans-induced bone loss and IL-1β, TNF-α, IL-17, RANKL, and ICAM-1 production in the gingival tissue without affecting bacterial counts. These results demonstrated that quercetin exhibits protective effects in A. actinomycetemcomitans-induced periodontitis in mice by modulating cytokine and ICAM-1 production. PMID:24246038

  16. Effects of suspension-induced osteopenia on the mechanical behaviour of mouse long bones

    NASA Technical Reports Server (NTRS)

    Simske, S. J.; Greenberg, A. R.; Luttges, M. W.; Spooner, B. S. (Principal Investigator)

    1991-01-01

    Whereas most studies of tail-suspension induced osteopenia have utilized rat femora, the present study investigated the effects of a 14 day tail-suspension on the mechanical behaviour of mice femora, tibiae and humeri. Force-deflection properties were obtained via three-point bending for long bones from suspended and control mice. Whole bone behaviour was characterized by converting the force-deflection values to stiffness, strength, ductility and energy parameters which were not normalized for specimen geometry. The effects of a systematic variation in the deflection rate over the range 0.1-10 mm min-1 were also evaluated. Statistical analysis indicated that the primary effect of the tail-suspension period was lowered bone mass which was manifested mechanically through lower values of the bone strength parameters. These effects were similar in the bones of both the fore and hind limbs. The results also demonstrated that the stiffness, ductility and energy characteristics were much less influenced by the tail-suspension. Whereas a significant dependence of the bone strength values upon deflection rate was observed for the femora and humeri, the other mechanical parameters were less sensitive. Based upon the nature of the physical and mechanical changes observed in the long bones following tail-suspension, the mouse appears to be a suitable animal model for the study of osteopenia.

  17. A Computational Analysis of Bone Formation in the Cranial Vault in the Mouse

    PubMed Central

    Lee, Chanyoung; Richtsmeier, Joan T.; Kraft, Reuben H.

    2015-01-01

    Bones of the cranial vault are formed by the differentiation of mesenchymal cells into osteoblasts on a surface that surrounds the brain, eventually forming mineralized bone. Signaling pathways causative for cell differentiation include the actions of extracellular proteins driven by information from genes. We assume that the interaction of cells and extracellular molecules, which are associated with cell differentiation, can be modeled using Turing’s reaction–diffusion model, a mathematical model for pattern formation controlled by two interacting molecules (activator and inhibitor). In this study, we hypothesize that regions of high concentration of an activator develop into primary centers of ossification, the earliest sites of cranial vault bone. In addition to the Turing model, we use another diffusion equation to model a morphogen (potentially the same as the morphogen associated with formation of ossification centers) associated with bone growth. These mathematical models were solved using the finite volume method. The computational domain and model parameters are determined using a large collection of experimental data showing skull bone formation in mouse at different embryonic days in mice carrying disease causing mutations and their unaffected littermates. The results show that the relative locations of the five ossification centers that form in our model occur at the same position as those identified in experimental data. As bone grows from these ossification centers, sutures form between the bones. PMID:25853124

  18. Suramin inhibits bone resorption and reduces osteoblast number in a neonatal mouse calvarial bone resorption assay.

    PubMed

    Walther, M M; Kragel, P J; Trahan, E; Venzon, D; Blair, H C; Schlesinger, P H; Jamai-Dow, C; Ewing, M W; Myers, C E; Linehan, W M

    1992-11-01

    The antineoplastic properties of suramin, a polyanionic agent with demonstrated antigrowth factor activity, are under evaluation in vitro, in vivo, and in clinical trials. Suramin has been shown to have antitumor activity in patients with advanced, hormone refractory prostate cancer. During these trials, significant resolution of osseous pain was observed in nearly three quarters of the patients treated with suramin. To evaluate the effect of suramin on bone cells, we studied the effect of suramin on bone resorption in a neonatal mouse calvarial assay. Suramin inhibited bone-resorbing activity in a dose-related fashion and had an additive effect with calcitonin. Calvaria pretreated with suramin had less bone-resorbing activity, fewer attached osteoblasts, and less medium alkaline phosphatase activity than control calvaria. Suramin also inhibited osteoclastic release of tritiated proline from labeled bone in a dose-dependent fashion. The effect of metastatic prostate carcinoma on bone is incompletely understood, but may be moderated by tumor-produced factors and/or cytokines. The effects of several such agents, therefore, were examined in combination with suramin. Bone resorption induced by PTH, epidermal growth factor, tumor necrosis factor, and a tumor-produced factor, PTH related-protein, was blocked by suramin. The ability of suramin to inhibit the bone-resorbing effects of several cytokines suggests that its mechanism may involve direct action on bone metabolism. Autoradiography performed on calvaria treated with labeled suramin demonstrated heavy deposition of suramin on the outer surface of the matrix, adjacent to osteoblasts and osteoclasts lining the outer table, suggesting that bone cells may be subject to high local concentrations of the drug, in keeping with this hypothesis. PMID:1425426

  19. A methodology for the investigation of toughness and crack propagation in mouse bone.

    PubMed

    Carriero, Alessandra; Zimmermann, Elizabeth A; Shefelbine, Sandra J; Ritchie, Robert O

    2014-11-01

    Bone fracture is a health concern for those with aged bone and brittle bone diseases. Mouse bone is widely used as a model of human bone, especially to investigate preclinical treatment strategies. However, little is known about the mechanisms of mouse bone fracture and its similarities and differences from fracture in human bone. In this work we present a methodology to investigate the fracture toughness during crack initiation and crack propagation for mouse bone. Mouse femora were dissected, polished on their periosteal surface, notched on the posterior surface at their mid-diaphysis, and tested in three-point bending under displacement control at a rate of 0.1mm/min using an in situ loading stage within an environmental scanning electron microscope. We obtained high-resolution real-time imaging of the crack initiation and propagation in mouse bone. From the images we can measure the crack extension at each step of the crack growth and calculate the toughness of the bone (in terms of stress intensity factor (K) and work to fracture (Wf)) as a function of stable crack length (Δa), thus generating a resistance curve for the mouse bone. The technique presented here provides insight into the evolution of microdamage and the toughening mechanisms that resist crack propagation, which are essential for preclinical development of treatments to enhance bone quality and combat fracture risk. PMID:25084121

  20. [Pathological proximal femur fracture: consider also primary bone tumour].

    PubMed

    van de Sande, Michiel A J; van Rijswijk, Carla S P; Dijkstra, P D Sander; Taminiau, Antonie M H

    2010-01-01

    Two male and one female patient, aged 64, 70 and 51 respectively, were surgically treated for pathological fracture of the proximal femur without preoperative biopsy. In contrast to their benign radiological diagnosis, all three patients were finally diagnosed as having a malignant primary bone tumour. The proximal femur is the primary location of pathological fractures in the appendicular skeleton. Metastases to bone are the most common cause of a destructive lesion of the skeleton in an adult. Although rare, a primary bone tumour must be included in differential diagnosis of a pathological fracture. A systematic diagnostic strategy is critical to avoid complications that make curative treatment impossible. A solitary bone lesion seen on radiography should never be assumed to be a bone metastasis. Without further diagnostic research, surgical treatment for a pathological fracture should never be commenced before a definitive diagnosis is made. PMID:20619031

  1. Bone and bone-marrow blood flow in chronic granulocytic leukemia and primary myelofibrosis

    SciTech Connect

    Lahtinen, R.; Lahtinen, T.; Romppanen, T.

    1982-03-01

    Blood flow in hematopoietic bone marrow and in nonhematopoietic bone has been measured with a Xe-133 washout method in 20 patients with chronic granulocytic leukemia (CGL) and in seven with primary myelofibrosis. Age-matched healthy persons served as controls. Bone-marrow blood flow in CGL was dependent upon the phase of the disease. In the metamorphosis phase, bone-marrow blood flow was high compared with that in the well-controlled phase. Apart from the initial phase, the mean values for bone blood flow in CGL were increased compared with the values of the healthy controls. In myelofibrosis the bone blood flow was also increased. Bone-marrow blood flow in these diseases was dependent upon the cellularity of bone marrow as measured morphometrically.

  2. Parallel input channels to mouse primary visual cortex

    PubMed Central

    Gao, Enquan; DeAngelis, Gregory C.; Burkhalter, Andreas

    2011-01-01

    It is generally accepted that in mammals visual information is sent to the brain along functionally specialized parallel pathways, but whether the mouse visual system uses similar processing strategies is not known. It is important to resolve this issue because the mouse brain provides a tractable system for developing a cellular and molecular understanding of disorders affecting spatiotemporal visual processing. We have used single unit recordings in mouse primary visual cortex to study whether individual neurons are more sensitive to one set of sensory cues than another. Our quantitative analyses show that neurons with short response latencies have low spatial acuity and high sensitivity to contrast, temporal frequency and speed, whereas neurons with long latencies have high spatial acuity, low sensitivities to contrast, temporal frequency and speed. These correlations suggest that neurons in mouse V1 receive inputs from a weighted combination of parallel afferent pathways with distinct spatiotemporal sensitivities. PMID:20427651

  3. Primary aneurysmal bone cyst of coronoid process

    PubMed Central

    Goyal, Amit; Tyagi, Isha; Syal, Rajan; Agrawal, Tanu; Jain, Manoj

    2006-01-01

    Background Aneurysmal bone cysts are relatively uncommon in the facial skeleton. These usually affect the mandible but origin from the coronoid process is even rarer. To the best of our knowledge, this is the first reported case of a coronoid process aneurysmal bone cyst presenting as temporal fossa swelling. Case presentation A 17 year old boy presented with a progressively increasing swelling in the left temporal region developed over the previous 8 months. An expansile lytic cystic lesion originating from the coronoid process of the left mandible and extending into the infratemporal and temporal fossa regions was found on CT scan. It was removed by a superior approach to the infratemporal fossa. Conclusion Aneurysmal bone cyst of the coronoid process can attain enormous dimensions until the temporal region is also involved. A superior approach to the infratemporal fossa is a reasonable approach for such cases, providing wide exposure and access to all parts of the lesion and ensuring better control and complete excision. PMID:16533409

  4. A practical guide to culturing mouse and human bone marrow stromal cells.

    PubMed

    Nemeth, K; Mayer, B; Sworder, B J; Kuznetsov, S A; Mezey, E

    2013-01-01

    Bone marrow stromal cells (BMSCs, frequently also called MSCs) represent a cell population within the bone marrow, a subset of which contains multipotent stem cells. Their primary role is to produce and maintain both bone tissue and bone marrow microenvironment necessary for hematopoiesis. The latter is achieved by secreting a wide variety of different cytokines and growth factors, many of which also have a regulatory role in immune processes. BMSCs have recently been introduced into the field of immunobiology after their successful clinical use in GVHD was reported in 2004. Since then, numerous studies confirmed and expanded the knowledge on the immunosuppressive potential of BMSCs in various in vitro and in vivo models. Although the immunomodulatory capacity of BMSCs is well established, there are still many unanswered questions regarding the cytokines, chemokines, receptors, and molecular pathways that play a role in this effect. To study these cells and answer many of the questions, researchers must be able to reliably and reproducibly isolate, culture, and use these cells. Below a practical guide on how to culture and characterize mouse and human BMSCs, which can then be applied in various in vitro and in vivo assays, is provided. PMID:24510517

  5. Cytogenetic risk assessment of etoposide from mouse bone marrow.

    PubMed

    Choudhury, Ramesh C; Palo, Anil K; Sahu, Prajyoti

    2004-01-01

    Increased clinical applications of the anticancer drug etoposide (a non-intercalative epipodophyllotoxin derivative) and the frequent induction of a second malignancy, particularly leukaemia, in post-etoposide-treated cancer survivors warrant detailed genotoxicity testing of etoposide. The genotoxicity test results available on etoposide are either primarily in in vitro test systems or in lower organisms after treatment with unusually high doses, or after chronic exposures, having little extrapolative value to humans. Therefore, a cytogenetic risk assessment study on etoposide in mouse in vivo was undertaken after a low dose (in accordance with the human therapeutic dose) single exposure. The cytogenetic toxicity of etoposide was assessed from bone marrow of mouse at three separate endpoints: chromosomal aberration and mitotic index studies at 24 h post-treatment and the micronucleus test (MNT) at 30 h post-treatment. The flame drying technique using colchicine, hypotonic sodium citrate, methanol-glacial acetic acid and Giemsa was followed for the preparation of slides for the metaphase chromosomal aberration and mitotic index studies and a simple technique was followed for the MNT. Although induction of chromosomal aberrations, excluding gaps, per 100 metaphases by 10 and 15 mg kg(-1) etoposide was not significant statistically, 20 mg kg(-1) of etoposide induced a significantly higher number of chromosomal aberrations in female (P < or = 0.01) and male (P < or = 0.05) mice. There was no significant change in the induced percentages of dividing cells by any of the doses of etoposide tested. The micronucleus induction also was not significant statistically with the lowest dose but it was significant in female (P mouse bone marrow after a single treatment with such low doses. However, the drug did not interfere with cell cycle progression. Although it is a DNA-non-intercalating agent, etoposide is known for its interference in the activity of DNA topoisomerase IIalpha enzyme, particularly in the proliferative cells where the concentration and activity of the enzyme are greater. This might be the reason for the induction of leukaemia in post-etoposide-treated cancer survivors. Therefore, it has become absolutely necessary to make etoposide target-specific, i.e. specific to the topoisomerase II enzymes of cancerous cells. PMID:15052606

  6. Mouse models of primary Sjögren’s syndrome

    PubMed Central

    Park, Young-Seok; Gauna, Adrienne E.; Cha, Seunghee

    2015-01-01

    Sjögren’s syndrome (SjS) is a chronic autoimmune disorder characterized by immune cell infiltration and progressive injury to the salivary and lacrimal glands. As a consequence, patients with SjS develop xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). SjS is the third most common rheumatic autoimmune disorder, affecting 4 million Americans with over 90% of patients being female. Current diagnostic criteria for SjS frequently utilize histological examinations of minor salivary glands for immune cell foci, serology for autoantibodies, and dry eye evaluation by corneal or conjunctival staining. SjS can be classified as primary or secondary SjS, depending on whether it occurs alone or in association with other systemic rheumatic conditions, respectively. Clinical manifestations typically become apparent when the disease is relatively advanced in SjS patients, which poses a challenge for early diagnosis and treatment of SjS. Therefore, SjS mouse models, because of their close resemblance to the human SjS, have been extremely valuable to identify early disease markers and to investigate underlying biological and immunological dysregulations. However, it is important to bear in mind that no single mouse model has duplicated all aspects of SjS pathogenesis and clinical features, mainly due to the multifactorial etiology of SjS that includes numerous susceptibility genes and environmental factors. As such, various mouse models have been developed in the field to try to recapitulate SjS. In this review, we focus on recent mouse models of primary SjS and describe them under three categories of spontaneous, genetically engineered, and experimentally induced development of SjS-like disease. In addition, we discuss future perspectives of SjS mouse models highlighting pros and cons of utilizing mouse models and demands for improved models. PMID:25777752

  7. Biomechanical properties of bone in a mouse model of Rett syndrome

    PubMed Central

    Kamal, Bushra; Russell, David; Payne, Anthony; Constante, Diogo; Tanner, K. Elizabeth; Isaksson, Hanna; Mathavan, Neashan; Cobb, Stuart R.

    2015-01-01

    Rett syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene are the primary cause of the disorder. Despite the dominant neurological phenotypes, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as scoliosis, reduced bone mineral density and skeletal fractures are also common and important clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any defects, we have conducted a series of histological, imaging and biomechanical tests of bone in a functional knockout mouse model of RTT. Both hemizygous Mecp2stop/y male mice in which Mecp2 is silenced in all cells and female Mecp2stop/+ mice in which Mecp2 is silenced in ~ 50% of cells as a consequence of random X-chromosome inactivation, revealed significant reductions in cortical bone stiffness, microhardness and tensile modulus. Microstructural analysis also revealed alterations in both cortical and cancellous femoral bone between wild-type and MeCP2-deficient mice. Furthermore, unsilencing of Mecp2 in adult mice cre-mediated stop cassette deletion resulted in a restoration of biomechanical properties (stiffness, microhardness) towards wild-type levels. These results show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies. PMID:25445449

  8. The protective effects of ?-cryptoxanthin on inflammatory bone resorption in a mouse experimental model of periodontitis.

    PubMed

    Matsumoto, Chiho; Ashida, Noriyuki; Yokoyama, Satoshi; Tominari, Tsukasa; Hirata, Michiko; Ogawa, Kazunori; Sugiura, Minoru; Yano, Masamichi; Inada, Masaki; Miyaura, Chisato

    2013-01-01

    We examined the effects of ?-cryptoxanthin, a typical carotenoid, on inflammatory periodontitis. ?-Cryptoxanthin suppressed lipopolysaccharide (LPS)-induced osteoclast formation in co-cultures of bone marrow cells and osteoblasts. In a mouse model of periodontitis, it suppressed bone resorption in the mandibular alveolar bone in vitro and restored alveolar bone loss induced by LPS in vivo. ?-Cryptoxanthin might protect against periodontal disease. PMID:23615426

  9. Primary temporal bone secretory meningioma presenting as chronic otitis media

    PubMed Central

    de Bondt, R. B. J.; Lammens, M.; Manni, J. J.

    2008-01-01

    We report an extremely rare case of a secretory meningioma primarily involving the temporal bone. A 56-year old female patient presented to us with a history of a chronic otitis media and unilateral hearing loss. Diagnostic investigations revealed a tumor arising from the temporal bone without signs of intracranial involvement. Histopathological examination showed a meningioma of the secretory type. The tumor was partially resected and serial imaging at follow-up revealed no extension of the tumor. No new symptoms developed 1 year after surgery. Secretory meningioma is a rare meningioma subtype and extracranial presentation in the temporal bone is very unusual. We present the first case of a primary temporal bone secretory meningioma in the otorhinolaryngological literature. As radical as possible surgical excision with serial imaging at follow-up is recommended. PMID:18172659

  10. Imaging of primary bone tumors in veterinary medicine: which differences?

    PubMed

    Vanel, Maïa; Blond, Laurent; Vanel, Daniel

    2013-12-01

    Veterinary medicine is most often a mysterious world for the human doctors. However, animals are important for human medicine thanks to the numerous biological similarities. Primary bone tumors are not uncommon in veterinary medicine and especially in small domestic animals as dogs and cats. As in human medicine, osteosarcoma is the most common one and especially in the long bones extremities. In the malignant bone tumor family, chondrosarcoma, fibrosarcoma and hemangiosarcoma are following. Benign bone tumors as osteoma, osteochondroma and bone cysts do exist but are rare and of little clinical significance. Diagnostic modalities used depend widely on the owner willing to treat his animal. Radiographs and bone biopsy are the standard to make a diagnosis but CT, nuclear medicine and MRI are more an more used. As amputation is treatment number one in appendicular bone tumor in veterinary medicine, this explains on the one hand why more recent imaging modalities are not always necessary and on the other hand, that prognostic on large animals is so poor that it is not much studied. Chemotherapy is sometimes associated with the surgery procedure, depending on the aggressivity of the tumor. Although, the strakes differs a lot between veterinary and human medicine, biological behavior are almost the same and should led to a beneficial team work between all. PMID:22197093

  11. Reference point indentation is not indicative of whole mouse bone measures of stress intensity fracture toughness

    PubMed Central

    Carriero, Alessandra; Bruse, Jan L.; Oldknow, Karla J.; Millán, José Luis; Farquharson, Colin; Shefelbine, Sandra J.

    2014-01-01

    Bone fragility is a concern for aged and diseased bone. Measuring bone toughness and understanding fracture properties of the bone are critical for predicting fracture risk associated with age and disease and for preclinical testing of therapies. A reference point indentation technique (BioDent) has recently been developed to determine bone's resistance to fracture in a minimally invasive way by measuring the indentation distance increase (IDI) between the first and last indentations over cyclic indentations in the same position. In this study, we investigate the relationship between fracture toughness KC and reference point indentation parameters (i.e. IDI, total indentation distance (TID) and creep indentation distance (CID)) in bones from 38 mice from six types (C57Bl/6, Balb, oim/oim, oim/+, Phospho1−/− and Phospho1 wild type counterpart). These mice bone are models of healthy and diseased bone spanning a range of fracture toughness from very brittle (oim/oim) to ductile (Phospho1−/−). Left femora were dissected, notched and tested in 3-point bending until complete failure. Contralateral femora were dissected and indented in 10 sites of their anterior and posterior shaft surface over 10 indentation cycles. IDI, TID and CID were measured. Results from this study suggest that reference point indentation parameters are not indicative of stress intensity fracture toughness in mouse bone. In particular, the IDI values at the anterior mid-diaphysis across mouse types overlapped, making it difficult to discern differences between mouse types, despite having extreme differences in stress intensity based toughness measures. When more locations of indentation were considered, the normalised IDIs could distinguish between mouse types. Future studies should investigate the relationship of the reference point indentation parameters for mouse bone in other material properties of the bone tissue in order to determine their use for measuring bone quality. PMID:25280470

  12. Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes

    PubMed Central

    Ramey, Guillemette; Deschemin, Jean-Christophe; Vaulont, Sophie

    2009-01-01

    Background The circulating hormone hepcidin plays a central role in iron homeostasis. Our goal was to establish an ex vivo iron-sensing model and to characterize the molecular mechanisms linking iron to hepcidin. Design and Methods Murine hepatocytes were isolated by the collagenase method, either from wild type or HFE knockout mice, and cultured 42 h without serum before treatments. Results After 42 h of serum-free culture, hepcidin gene expression was undetectable in the hepatocytes. Hepcidin gene expression could, however, be re-activated by an additional 24 h of incubation with 10% serum. Interestingly, addition of 30 μM holotransferrin consistently increased serum-dependent hepcidin levels 3- to 5-fold. The effects of serum and serum+holotransferrin were direct, transcriptional, independent of de novo protein synthesis and required the presence of bone morphogenetic protein. Transferrin receptor-2 activation by its ligand holotransferrin led to extracellular signal regulated kinase (ERK)/mitogen activated protein kinase pathway stimulation and the ERK specific inhibitor U0-126 blunted holotransferrin-mediated induction of hepcidin. ERK activation by holotransferrin provoked increased levels of phospho-Smad1/5/8 highlighting cross-talk between the bone morphogenetic protein/hemojuvelin and ERK1/2 pathways. Finally, we demonstrated, using hepatocytes isolated from Hfe−/− mice, that HFE was not critical for the hepcidin response to holotransferrin but important for basal hepcidin expression. Conclusions We demonstrate that hepatocytes are liver iron-sensor cells and that transferrin receptor-2, by signaling through the ERK1/2 pathway, and bone morphogenetic protein/hemojuvelin, by signaling through the Smad pathways, coordinately regulate the iron-sensing machinery linking holotransferrin to hepcidin. PMID:19454495

  13. MRI detection of early bone metastases in B16 mouse melanoma models

    PubMed Central

    Gauvain, Karen M.; Garbow, Joel R.; Song, Sheng-Kwei; Hirbe, Angela C.; Weilbaecher, Katherine

    2009-01-01

    Bone metastasis causes significant morbidity in cancer patients, including bone pain, pathologic fractures, nerve compression syndrome, and hypercalcemia. Animal models are utilized to study the pathogenesis of skeletal metastases and to evaluate potential therapeutic agents. Previously published methods for imaging bone metastasis in rodent models have focused on identifying advanced stage metastasis using simple X-rays. Here we report MRI as a method for detecting early bone metastases in mouse models in vivo. B16 mouse melanoma cells were injected into the left cardiac ventricle of C57BL/6 mice and magnetic resonance (MR) images were obtained of the left leg following the development of metastatic disease, when tumor associated bone destruction was histologically present but not visible by X-ray. T1 and T2 relaxation times of bone marrow were measured in healthy control mice and B16 melanoma tumor-bearing mice. Mean T2 values for normal marrow were 28 ms (SD 5) and for diseased bone marrow were 41 ms (SD 3). T2 relaxation time of diseased bone marrow is significantly longer than that of normal bone marrow (P < 0.0001) and can be used as a marker of early bone metastases. These studies demonstrate that MR imaging can detect bone marrow metastases in small animals prior to development of cortical bone loss identified by X-ray. PMID:16283483

  14. Preoperative embolization of primary bone tumors: A case control study

    PubMed Central

    Jha, Roushan; Sharma, Raju; Rastogi, Shishir; Khan, Shah Alam; Jayaswal, Arvind; Gamanagatti, Shivanand

    2016-01-01

    AIM: To study the safety and effectiveness of preoperative embolization of primary bone tumors in relation to intraoperative blood loss, intraoperative blood transfusion volume and surgical time. METHODS: Thirty-three patients underwent preoperative embolization of primary tumors of extremities, hip or vertebrae before resection and stabilization. The primary osseous tumors included giant cell tumors, aneurysmal bone cyst, osteoblastoma, chondroblastoma and chondrosarcoma. Twenty-six patients were included for the statistical analysis (embolization group) as they were operated within 0-48 h within preoperative embolization. A control group (non-embolization group, n = 28) with bone tumor having similar histological diagnosis and operated without embolization was retrieved from hospital record for statistical comparison. RESULTS: The mean intraoperative blood loss was 1300 mL (250-2900 mL), the mean intraoperative blood transfusion was 700 mL (0-1400 mL) and the mean surgical time was 221 ± 76.7 min for embolization group (group I, n = 26). Non-embolization group (group II, n = 28), the mean intraoperative blood loss was 1800 mL (800-6000 mL), the mean intraoperative blood transfusion was 1400 mL (700-8400 mL) and the mean surgical time was 250 ± 69.7 min. On comparison, statistically significant (P < 0.001) difference was found between embolisation group and non-embolisation group for the amount of blood loss and requirement of blood transfusion. There was no statistical difference between the two groups for the surgical time. No patients developed any angiography or embolization related complications. CONCLUSION: Preoperative embolization of bone tumors is a safe and effective adjunct to the surgical management of primary bone tumors that leads to reduction in intraoperative blood loss and blood transfusion volume. PMID:27158424

  15. Bone Windows for Distinguishing Malignant from Benign Primary Bone Tumors on FDG PET/CT

    PubMed Central

    Costelloe, Colleen M.; Chuang, Hubert H.; Chasen, Beth A.; Pan, Tinsu; Fox, Patricia S.; Bassett, Roland L.; Madewell, John E.

    2013-01-01

    Objective. The default window setting on PET/CT workstations is soft tissue. This study investigates whether bone windowing and hybrid FDG PET/CT can help differentiate between malignant and benign primary bone tumors. Materials and methods. A database review included 98 patients with malignant (n=64) or benign primary bone (n=34) tumors. The reference standard was biopsy for malignancies and biopsy or >1 year imaging follow-up of benign tumors. Three radiologists and/or nuclear medicine physicians blinded to diagnosis and other imaging viewed the lesions on CT with bone windows (CT-BW) without and then with PET (PET/CT-BW), and separate PET-only images for malignancy or benignity. Three weeks later the tumors were viewed on CT with soft tissue windows (CT-STW) without and then with PET (PET/CT-STW). Results. Mean sensitivity and specificity for identifying malignancies included: CT-BW: 96%, 90%; CT-STW: 90%, 90%; PET/CT-BW: 95%, 85%, PET/CT-STW: 95%, 86% and PET-only: 96%, 75%, respectively. CT-BW demonstrated higher specificity than PET-only and PET/CT-BW (p=0.0005 and p=0.0103, respectively) and trended toward higher sensitivity than CT-STW (p=0.0759). Malignant primary bone tumors were more avid than benign lesions overall (p<0.0001) but the avidity of benign aggressive lesions (giant cell tumors and Langerhans Cell Histiocytosis) trended higher than the malignancies (p=0.08). Conclusion. Bone windows provided high specificity for distinguishing between malignant and benign primary bone tumors and are recommended when viewing FDG PET/CT. PMID:23983816

  16. Probiotic L. reuteri treatment prevents bone loss in a menopausal ovariectomized mouse model

    PubMed Central

    Britton, Robert A.; Irwin, Regina; Quach, Darin; Schaefer, Laura; Zhang, Jing; Lee, Taehyung; Parameswaran, Narayanan; McCabe, Laura R.

    2014-01-01

    Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post-menopausal bone loss. Recent studies suggest an important role for gut-bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri treated mice. Consistent with this, L. reuteri suppressed Ovx-induced increases in bone marrow CD4+ T-lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identif ied that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost-effective approach to reduce post-menopausal bone loss. PMID:24677054

  17. Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

    PubMed Central

    Mattinzoli, Deborah; Messa, Piergiorgio; Corbelli, Alessandro; Ikehata, Masami; Mondini, Anna; Zennaro, Cristina; Armelloni, Silvia; Li, Min; Giardino, Laura; Rastaldi, Maria Pia

    2014-01-01

    The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line. The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features. After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules. Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications. PMID:24894124

  18. Application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts.

    PubMed

    Mattinzoli, Deborah; Messa, Piergiorgio; Corbelli, Alessandro; Ikehata, Masami; Mondini, Anna; Zennaro, Cristina; Armelloni, Silvia; Li, Min; Giardino, Laura; Rastaldi, Maria Pia

    2014-01-01

    The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line. The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features. After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules. Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications. PMID:24894124

  19. Endochondral bone formation in embryonic mouse pre-metatarsals

    NASA Technical Reports Server (NTRS)

    Klement, B. J.; Spooner, B. S.

    1992-01-01

    Long term exposure to a reduced gravitational environment has a deleterious effect on bone. The developmental events which occur prior to initial bone deposition will provide insight into the regulation of mature bone physiology. We have characterized a system in which the events preceding bone formation take place in an isolated in vitro organ culture environment. We show that cultured pre-metatarsal tissue parallels development of pre-metatarsal tissue in the embryo. Both undergo mesenchyme differentiation and morphogenesis to form a cartilage rod, which resembles the future bone, followed by terminal chondrocyte differentiation in a definite morphogenetic pattern. These sequential steps occur prior to osteoblast maturation and bone matrix deposition in the developing organism. Alkaline phosphatase (ALP) activity is a distinctive enzymatic marker for mineralizing tissues. We have measured this activity throughout pre-metatarsal development and show (a) where in the tissue it is predominantly found, and (b) that this is indeed the mineralizing isoform of the enzyme.

  20. Genetics of primary and timing effects in the mnd mouse

    SciTech Connect

    Messer, A.; Plummer, J.; MacMillen, M.C.

    1995-06-05

    The mnd mouse shows a spontaneous adult-onset hereditary neurological disease, with motor abnormality by 6 months of age, progressing to severe spastic paralysis and premature death. The disease is autosomal recessive, with heterozygote effects seen under stress. It maps to mouse chromosome (chr) 8. Histopathology with Nissl stains documents substantial abnormalities of upper and lower motor neurons, and there is retinal degeneration beginning in the first month, even without light exposure. Increasing levels of autofluorescent lipopigment are found in both neuronal and non-neuronal tissues as the mnd mice age. Recently, NCL-like inclusions and accumulating subunit c have also been described. When mnd is outcrossed to the AKR/J genetic background, ca. 40% of the mnd/mnd F2 progeny show early onset (onset by 4.5-5 months and death by 7 months). This accelerated timing effect seems to be strain-specific, and unlinked to the mnd gene itself. Our current working hypothesis is that the timing effect is due to 2 or 3 unlinked dominant genes with incomplete penetrance at any single locus. In a combined RFLP/PCR fragment genetic analysis, the strongest deviation from the expected ratio of AKR vs B6 alleles occurs with markers on proximal half of chr 1. Additional loci on chrs 5 and 10 may also be involved. The mechanism of interaction of these modifying genes with the primary mnd gene may offer new therapeutic avenues. 22 refs., 2 tabs.

  1. Cadmium stimulates osteoclast-like multinucleated cell formation in mouse bone marrow cell cultures

    SciTech Connect

    Miyahara, Tatsuro; Takata, Masakazu; Miyata, Masaki; Nagai, Miyuki; Sugure, Akemi; Kozuka, Hiroshi; Kuze, Shougo )

    1991-08-01

    Most of cadmium (Cd)-treated animals have been reported to show osteoporosis-like changes in bones. This suggests that Cd may promote bone loss by a direct action on bone. It was found that Cd stimulated prostaglandin E{sub 2}(PGE{sub 2}) production in the osteoblast-like cell, MC3T3-E1. Therefore, Cd stimulates bone resorption by increasing PGE{sub 2} production. Recently, several bone marrow cell culture systems have been developed for examining the formation of osteoclast-like multinucleated cells in vitro. As osteoblasts produce PGE{sub 2} by Cd-induced cyclooxygenase and may play an important role in osteoclast formation, the present study was undertaken to clarify the possibility that Cd might stimulate osteoclast formation in a mouse bone marrow culture system.

  2. Primary homologies of the circumorbital bones of snakes.

    PubMed

    Palci, Alessandro; Caldwell, Michael W

    2013-09-01

    Some snakes have two circumorbital ossifications that in the current literature are usually referred to as the postorbital and supraorbital. We review the arguments that have been proposed to justify this interpretation and provide counter-arguments that reject those conjectures of primary homology based on the observation of 32 species of lizards and 81 species of snakes (both extant and fossil). We present similarity arguments, both topological and structural, for reinterpretation of the primary homologies of the dorsal and posterior orbital ossifications of snakes. Applying the test of similarity, we conclude that the posterior orbital ossification of snakes is topologically consistent as the homolog of the lacertilian jugal, and that the dorsal orbital ossification present in some snakes (e.g., pythons, Loxocemus, and Calabaria) is the homolog of the lacertilian postfrontal. We therefore propose that the terms postorbital and supraorbital should be abandoned as reference language for the circumorbital bones of snakes, and be replaced with the terms jugal and postfrontal, respectively. The primary homology claim for the snake "postorbital" fails the test of similarity, while the term "supraorbital" is an unnecessary and inaccurate application of the concept of a neomorphic ossification, for an element that passes the test of similarity as a postfrontal. This reinterpretation of the circumorbital bones of snakes is bound to have important repercussions for future phylogenetic analyses and consequently for our understanding of the origin and evolution of snakes. PMID:23630161

  3. Exogenous regucalcin suppresses osteoblastogenesis and stimulates adipogenesis in mouse bone marrow culture.

    PubMed

    Yamaguchi, Masayoshi; Weitzmann, M Neale; Baile, Clifton A; Murata, Tomiyasu

    2012-10-01

    Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss and hyperlipidemia. Consistent with these effects exogenous regucalcin has been shown to promote osteoclastogenesis in mouse bone marrow cultures and to suppress the differentiation and mineralization of MC3T3 osteoblast precursors. Regucalcin may induce hyperlipidemia in vivo by suppressing osteoblast differentiation and stimulating adipogenesis in bone marrow mesenchymal stem cells. The present study demonstrates that exogenous regucalcin suppresses differentiation to osteoblasts and stimulates adipogenesis in mouse bone marrow cell culture ex vivo. Moreover, exogenous regucalcin was found to enhance adipogenesis stimulated by insulin which is involved in the extracellular signal-related kinase pathway in 3T3-L1 adipocytes in vitro. PMID:22868942

  4. Mineral metabolism in isolated mouse long bones: Opposite effects of microgravity on mineralization and resorption

    NASA Technical Reports Server (NTRS)

    Veldhuijzen, Jean Paul; Vanloon, Jack J. W. A.

    1994-01-01

    An experiment using isolated skeletal tissues under microgravity, is reported. Fetal mouse long bones (metatarsals) were cultured for 4 days in the Biorack facility of Spacelab during the IML-1 (International Microgravity Laboratory) mission of the Space Shuttle. Overall growth was not affected, however glucose consumption was significantly reduced under microgravity. Mineralization of the diaphysis was also strongly reduced under microgravity as compared to the on-board 1 g group. In contrast, mineral resorption by osteoclasts was signficantly increased. These results indicate that these fetal mouse long bones are a sensitive and useful model to further study the cellular mechanisms involved in the changed mineral metabolism of skeletal tissues under microgravity.

  5. Primary Osteosarcoma of the Rib Identified on Bone Scintigraphy.

    PubMed

    Xie, Peng; Huang, Jianmin

    2016-05-01

    Osteosarcomas generally arise in appendicular skeletons, but rarely in the ribs. We described Tc-MDP bone scan findings from a 23-year-old man with right back pain. The images demonstrated elevated activity in the region overlapping the posterior 8th to 10th ribs and in the L9-10 vertebral bodies. CT showed an 8.8 × 8.3 cm mildly peripherally calcified mass arising from the right 10th rib involving the 9th and 10th thoracic vertebrae. Pathological examination confirmed primary osteosarcoma of the rib. PMID:26704734

  6. Hydroxyproline metabolism in mouse models of primary hyperoxaluria

    PubMed Central

    Holmes, Ross P.; Cramer, Scott D.; Takayama, Tatsuya; Salido, Eduardo

    2012-01-01

    Primary hyperoxaluria type 1 (PH1) and type 2 (PH2) are rare genetic diseases that result from deficiencies in glyoxylate metabolism. The increased oxalate synthesis that occurs can lead to kidney stone formation, deposition of calcium oxalate in the kidney and other tissues, and renal failure. Hydroxyproline (Hyp) catabolism, which occurs mainly in the liver and kidney, is a prominent source of glyoxylate and could account for a significant portion of the oxalate produced in PH. To determine the sensitivity of mouse models of PH1 and PH2 to Hyp-derived oxalate, animals were fed diets containing 1% Hyp. Urinary excretions of glycolate and oxalate were used to monitor Hyp catabolism and the kidneys were examined to assess pathological changes. Both strains of knockout (KO) mice excreted more oxalate than wild-type (WT) animals with Hyp feeding. After 4 wk of Hyp feeding, all mice deficient in glyoxylate reductase/hydroxypyruvate reductase (GRHPR KO) developed severe nephrocalcinosis in contrast to animals deficient in alanine-glyoxylate aminotransferase (AGXT KO) where nephrocalcinosis was milder and with a lower frequency. Plasma cystatin C measurements over 4-wk Hyp feeding indicated no significant loss of renal function in WT and AGXT KO animals, and significant and severe loss of renal function in GRHPR KO animals after 2 and 4 wk, respectively. These data suggest that GRHPR activity may be vital in the kidney for limiting the conversion of Hyp-derived glyoxylate to oxalate. As Hyp catabolism may make a major contribution to the oxalate produced in PH patients, Hyp feeding in these mouse models should be useful in understanding the mechanisms associated with calcium oxalate deposition in the kidney. PMID:22189945

  7. Polarization in Raman spectroscopy helps explain bone brittleness in genetic mouse models

    PubMed Central

    Makowski, Alexander J.; Pence, Isaac J.; Uppuganti, Sasidhar; Zein-Sabatto, Ahbid; Huszagh, Meredith C.; Mahadevan-Jansen, Anita; Nyman, Jeffry S.

    2014-01-01

    Abstract. Raman spectroscopy (RS) has been extensively used to characterize bone composition. However, the link between bone biomechanics and RS measures is not well established. Here, we leveraged the sensitivity of RS polarization to organization, thereby assessing whether RS can explain differences in bone toughness in genetic mouse models for which traditional RS peak ratios are not informative. In the selected mutant mice—activating transcription factor 4 (ATF4) or matrix metalloproteinase 9 (MMP9) knock-outs—toughness is reduced but differences in bone strength do not exist between knock-out and corresponding wild-type controls. To incorporate differences in the RS of bone occurring at peak shoulders, a multivariate approach was used. Full spectrum principal components analysis of two paired, orthogonal bone orientations (relative to laser polarization) improved genotype classification and correlation to bone toughness when compared to traditional peak ratios. When applied to femurs from wild-type mice at 8 and 20 weeks of age, the principal components of orthogonal bone orientations improved age classification but not the explanation of the maturation-related increase in strength. Overall, increasing polarization information by collecting spectra from two bone orientations improves the ability of multivariate RS to explain variance in bone toughness, likely due to polarization sensitivity to organizational changes in both mineral and collagen. PMID:25402627

  8. Polarization in Raman spectroscopy helps explain bone brittleness in genetic mouse models

    NASA Astrophysics Data System (ADS)

    Makowski, Alexander J.; Pence, Isaac J.; Uppuganti, Sasidhar; Zein-Sabatto, Ahbid; Huszagh, Meredith C.; Mahadevan-Jansen, Anita; Nyman, Jeffry S.

    2014-11-01

    Raman spectroscopy (RS) has been extensively used to characterize bone composition. However, the link between bone biomechanics and RS measures is not well established. Here, we leveraged the sensitivity of RS polarization to organization, thereby assessing whether RS can explain differences in bone toughness in genetic mouse models for which traditional RS peak ratios are not informative. In the selected mutant mice-activating transcription factor 4 (ATF4) or matrix metalloproteinase 9 (MMP9) knock-outs-toughness is reduced but differences in bone strength do not exist between knock-out and corresponding wild-type controls. To incorporate differences in the RS of bone occurring at peak shoulders, a multivariate approach was used. Full spectrum principal components analysis of two paired, orthogonal bone orientations (relative to laser polarization) improved genotype classification and correlation to bone toughness when compared to traditional peak ratios. When applied to femurs from wild-type mice at 8 and 20 weeks of age, the principal components of orthogonal bone orientations improved age classification but not the explanation of the maturation-related increase in strength. Overall, increasing polarization information by collecting spectra from two bone orientations improves the ability of multivariate RS to explain variance in bone toughness, likely due to polarization sensitivity to organizational changes in both mineral and collagen.

  9. How tough is Brittle Bone? Investigating Osteogenesis Imperfecta in Mouse Bone††

    PubMed Central

    Carriero, A.; Zimmermann, E. A.; Paluszny, A.; Tang, S. Y.; Bale, H.; Busse, B.; Alliston, T.; Kazakia, G.

    2015-01-01

    The multiscale hierarchical structure of bone is naturally optimized to resist fractures. In osteogenesis imperfecta, or brittle bone disease, genetic mutations affect the quality and/or quantity of collagen, dramatically increasing bone fracture risk. Here we reveal how the collagen defect results in bone fragility in a mouse model of osteogenesis imperfecta (oim), which has homotrimeric α1(I) collagen. At the molecular level we attribute the loss in toughness to a decrease in the stabilizing enzymatic crosslinks and an increase in non-enzymatic crosslinks, which may break prematurely inhibiting plasticity. At the tissue level, high vascular canal density reduces the stable crack growth, and extensive woven bone limits the crack-deflection toughening during crack growth. This demonstrates how modifications at the bone molecular level have ramifications at larger length scales affecting the overall mechanical integrity of the bone; thus, treatment strategies have to address multiscale properties in order to regain bone toughness. In this regard, findings from the heterozygous oim bone, where defective as well as normal collagen are present, suggest that increasing the quantity of healthy collagen in these bones helps to recover toughness at the multiple length scales. PMID:24420672

  10. Primary sclerosing epithelioid fibrosarcoma of bone: analysis of a series.

    PubMed

    Wojcik, John B; Bellizzi, Andrew M; Dal Cin, Paola; Bredella, Miriam A; Fletcher, Christopher D M; Hornicek, Francis J; Deshpande, Vikram; Hornick, Jason L; Nielsen, G Petur

    2014-11-01

    Sclerosing epithelioid fibrosarcoma (SEF) is a rare, aggressive malignant neoplasm characterized by small nests and linear arrays of epithelioid cells embedded in a dense collagenous matrix. Very few primary SEFs of bone have been reported. Recognition is critical, as the dense extracellular collagenous matrix can be interpreted as osteoid, leading to misdiagnosis as-osteosarcoma. MUC4 and SATB2 are 2 recently characterized immunohistochemical markers for SEF and osteosarcoma, respectively. In reports to date, osteosarcomas are positive for SATB2 and negative for MUC4, whereas soft tissue SEFs have shown the opposite immunohistochemical profile (SATB2-/MUC4+). The purpose of this study was to characterize the clinicopathologic and immunohistochemical features of 8 primary SEFs of bone. The patients presented at a wide range of ages (25 to 73 y; median 52 y). Tumors mostly involved long bones of the extremities, with 3 cases involving the femur, 2 involving the ulna, and 1 involving the humerus. Other sites of involvement included the second rib (1) and the C6 vertebra (1). Follow-up information was available for 7 patients, 3 of whom developed metastases within 2 years of diagnosis. The other 4 patients were free of local recurrence or metastases at 1, 5, 12, and >84 months of follow-up, respectively. Radiographically, the tumors were predominantly lytic and poorly marginated. Histologically, 6 tumors showed pure SEF morphology, and 2 showed hybrid SEF/low-grade fibromyxoid sarcoma morphology. Focal dystrophic mineralization was seen in 1 case but was limited to areas of necrosis. None of the tumors showed the lace-like pattern of mineralization typical of osteosarcoma. The majority (6/8) of the tumors strongly expressed MUC4. SATB2 was negative in all but 1 case, which showed variable weak to moderate staining in ∼50% of nuclei. In general, the combination of morphology, MUC4 expression, and the absence of SATB2 expression was highly useful in arriving at the correct diagnosis. PMID:24921641

  11. Hormone Treatment Restores Bone Density for Young Women with Menopause-Like Condition (Primary Ovarian Insufficiency)

    MedlinePlus

    ... determine the effects of hormone treatment on bone mineral density of women with primary ovarian insufficiency. Researchers ... insufficiency (POI) led to increases in their bone mineral density, restoring levels to normal. The study was ...

  12. Transgenic Mouse Model for Reducing Oxidative Damage in Bone

    NASA Technical Reports Server (NTRS)

    Schreurs, A.-S.; Torres, S.; Truong, T.; Kumar, A.; Alwood, J. S.; Limoli, C. L.; Globus, R. K.

    2014-01-01

    Exposure to musculoskeletal disuse and radiation result in bone loss; we hypothesized that these catabolic treatments cause excess reactive oxygen species (ROS), and thereby alter the tight balance between bone resorption by osteoclasts and bone formation by osteoblasts, culminating in bone loss. To test this, we used transgenic mice which over-express the human gene for catalase, targeted to mitochondria (MCAT). Catalase is an anti-oxidant that converts the ROS hydrogen peroxide into water and oxygen. MCAT mice were shown previously to display reduced mitochondrial oxidative stress and radiosensitivity of the CNS compared to wild type controls (WT). As expected, MCAT mice expressed the transgene in skeletal tissue, and in marrow-derived osteoblasts and osteoclast precursors cultured ex vivo, and also showed greater catalase activity compared to wildtype (WT) mice (3-6 fold). Colony expansion in marrow cells cultured under osteoblastogenic conditions was 2-fold greater in the MCAT mice compared to WT mice, while the extent of mineralization was unaffected. MCAT mice had slightly longer tibiae than WT mice (2%, P less than 0.01), although cortical bone area was slightly lower in MCAT mice than WT mice (10%, p=0.09). To challenge the skeletal system, mice were treated by exposure to combined disuse (2 wk Hindlimb Unloading) and total body irradiation Cs(137) (2 Gy, 0.8 Gy/min), then bone parameters were analyzed by 2-factor ANOVA to detect possible interaction effects. Treatment caused a 2-fold increase (p=0.015) in malondialdehyde levels of bone tissue (ELISA) in WT mice, but had no effect in MCAT mice. These findings indicate that the transgene conferred protection from oxidative damage caused by treatment. Unexpected differences between WT and MCAT mice emerged in skeletal responses to treatment.. In WT mice, treatment did not alter osteoblastogenesis, cortical bone area, moment of inertia, or bone perimeter, whereas in MCAT mice, treatment increased these parameters. Taken together, this typically catabolic treatment (disuse and irradiation) appeared to stimulate cortical expansion in MCAT mice but not WT mice. In conclusion, these results reveal the importance of mitochondrial ROS generation in skeletal remodeling and show that MCAT mice provide a useful animal model for bone studies.

  13. Activity of the human carcinogen MeCCNU in the mouse bone marrow micronucleus assay

    SciTech Connect

    Tinwell, H.; Ashby, J. )

    1991-01-01

    The nitrosourea mustard MeCCNU is the most recent organic chemical to be classified as a human carcinogen by IARC. MeCCNU gave a strong positive response when tested in the mouse bone marrow micronucleus assay. Activity was evident using either ip injection or oral gavage of the test chemical. These results further support the correlation between human carcinogens and their genotoxicity.

  14. Decreased Bone Formation Explains Osteoporosis in a Genetic Mouse Model of Hemochromatosiss.

    PubMed

    Doyard, Mathilde; Chappard, Daniel; Leroyer, Patricia; Roth, Marie-Paule; Loréal, Olivier; Guggenbuhl, Pascal

    2016-01-01

    Osteoporosis may complicate iron overload diseases such as genetic hemochromatosis. However, molecular mechanisms involved in the iron-related osteoporosis remains poorly understood. Recent in vitro studies support a role of osteoblast impairment in iron-related osteoporosis. Our aim was to analyse the impact of excess iron in Hfe-/- mice on osteoblast activity and on bone microarchitecture. We studied the bone formation rate, a dynamic parameter reflecting osteoblast activity, and the bone phenotype of Hfe-/- male mice, a mouse model of human hemochromatosis, by using histomorphometry. Hfe-/- animals were sacrificed at 6 months and compared to controls. We found that bone contains excess iron associated with increased hepatic iron concentration in Hfe-/- mice. We have shown that animals with iron overload have decreased bone formation rate, suggesting a direct impact of iron excess on active osteoblasts number. For bone mass parameters, we showed that iron deposition was associated with bone loss by producing microarchitectural impairment with a decreased tendency in bone trabecular volume and trabecular number. A disorganization of trabecular network was found with marrow spaces increased, which was confirmed by enhanced trabecular separation and star volume of marrow spaces. These microarchitectural changes led to a loss of connectivity and complexity in the trabecular network, which was confirmed by decreased interconnectivity index and increased Minkowski's fractal dimension. Our results suggest for the first time in a genetic hemochromatosis mouse model, that iron overload decreases bone formation and leads to alterations in bone mass and microarchitecture. These observations support a negative effect of iron on osteoblast recruitment and/or function, which may contribute to iron-related osteoporosis. PMID:26829642

  15. Decreased Bone Formation Explains Osteoporosis in a Genetic Mouse Model of Hemochromatosiss

    PubMed Central

    Doyard, Mathilde; Chappard, Daniel; Leroyer, Patricia; Roth, Marie-Paule; Loréal, Olivier; Guggenbuhl, Pascal

    2016-01-01

    Osteoporosis may complicate iron overload diseases such as genetic hemochromatosis. However, molecular mechanisms involved in the iron-related osteoporosis remains poorly understood. Recent in vitro studies support a role of osteoblast impairment in iron-related osteoporosis. Our aim was to analyse the impact of excess iron in Hfe-/- mice on osteoblast activity and on bone microarchitecture. We studied the bone formation rate, a dynamic parameter reflecting osteoblast activity, and the bone phenotype of Hfe−/− male mice, a mouse model of human hemochromatosis, by using histomorphometry. Hfe−/− animals were sacrificed at 6 months and compared to controls. We found that bone contains excess iron associated with increased hepatic iron concentration in Hfe−/− mice. We have shown that animals with iron overload have decreased bone formation rate, suggesting a direct impact of iron excess on active osteoblasts number. For bone mass parameters, we showed that iron deposition was associated with bone loss by producing microarchitectural impairment with a decreased tendency in bone trabecular volume and trabecular number. A disorganization of trabecular network was found with marrow spaces increased, which was confirmed by enhanced trabecular separation and star volume of marrow spaces. These microarchitectural changes led to a loss of connectivity and complexity in the trabecular network, which was confirmed by decreased interconnectivity index and increased Minkowski’s fractal dimension. Our results suggest for the first time in a genetic hemochromatosis mouse model, that iron overload decreases bone formation and leads to alterations in bone mass and microarchitecture. These observations support a negative effect of iron on osteoblast recruitment and/or function, which may contribute to iron-related osteoporosis. PMID:26829642

  16. Impact of bone quality and implant type on the primary stability: an experimental study using bovine bone.

    PubMed

    Anil, Sukumaran; Aldosari, Abdullah Alfarraj

    2015-04-01

    The purpose of this in vitro study was to compare the primary stability and removal torque of bone level and tissue level implants in different bone qualities. Twenty tissue level and bone level implants (3.3 × 10 mm and 4.1 × 10 mm) were used for assessing the stability in type II and type IV bone. Forty bovine rib blocks were used in this study. The primary stability of the implant was measured by the resonance frequency using an Osstel device. The removal torque values (RTV) of the implants was assessed using a digital torque gauge instrument. The implant stability quotient (ISQ) values and the RTV showed a marginally higher stability with bone level implants as compared to tissue level implants. However, these differences were not statistically significant in both type of bone used (P > 0.05). On the other hand, compared to type IV, type II bone showed significant differences in the ISQ (P < 0.01) and RTV (P < 0.001) of bone level and tissue level implants. The study concluded that bone quality is an important factor in establishing primary stability than the implant dimension. Bone level and tissue level implants of same dimensions can be selected based on the esthetic demands since they showed similar mechanical properties. PMID:23713963

  17. Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption.

    PubMed

    Canalis, Ernesto; Schilling, Lauren; Yee, Siu-Pok; Lee, Sun-Kyeong; Zanotti, Stefano

    2016-01-22

    Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C→T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2(Q2319X) heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2(Q2319X) cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor κB ligand in vitro were increased in Notch2(Q2319X) mutants. These effects were suppressed by the γ-secretase inhibitor LY450139. In conclusion, Notch2(Q2319X) mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption. PMID:26627824

  18. Measuring the dynamic mechanical response of hydrated mouse bone by nanoindentation

    PubMed Central

    Pathak, Siddhartha; Swadener, J. Gregory; Kalidindi, Surya R.; Courtland, Hayden-William; Jepsen, Karl J.; Goldman, Haviva M.

    2011-01-01

    This study demonstrates a novel approach to characterizing hydrated bones viscoelastic behavior at the lamellar length scales using dynamic indentation techniques. We studied the submicron-level viscoelastic response of bone tissue from two different inbred mouse strains, A/J and B6, with known differences in whole bone and tissue-level mechanical properties. Our results show that bone having a higher collagen content or a lower mineral-to-matrix ratio demonstrates a trend towards a larger viscoelastic response. When normalized for anatomical location relative to biological growth patterns in the antero-medial (AM) cortex, bone tissue from B6 femora, known to have a lower mineral-to-matrix ratio, is shown to exhibit a significantly higher viscoelastic response compared to A/J tissue. Newer bone regions with a higher collagen content (closer to the endosteal edge of the AM cortex) showed a trend towards a larger viscoelastic response. Our study demonstrates the feasibility of this technique to be used to study local composition-property relationships in bone. Further, this technique of viscoelastic nanoindentation mapping of the bone surface at these submicron length scales is shown to be highly advantageous in studying sub-surface features, such as porosity, of wet hydrated biological specimens, which are difficult to identify using other methods. PMID:21094478

  19. Effects of 810 nm laser on mouse primary cortical neurons

    NASA Astrophysics Data System (ADS)

    Kharkwal, Gitika B.; Sharma, Sulbha K.; Huang, Ying-Ying; De Taboada, Luis; McCarthy, Thomas; Hamblin, Michael R.

    2011-03-01

    In the past four decades numerous studies have reported the efficacy of low level light (laser) therapy (LLLT) as a treatment for diverse diseases and injuries. Recent studies have shown that LLLT can biomodulate processes in the central nervous system and has been extensively studied as a stroke treatment. However there is still a lack of knowledge on the effects of LLLT at the cellular level in neurons. The present study aimed to study the effect of 810 nm laser on several cellular processes in primary cortical neurons cultured from mouse embryonic brains. Neurons were irradiated with light dose of 0.03, 0.3, 3, 10 and 30 J/cm2 and intracellular levels of reactive oxygen species, nitric oxide and calcium were measured. The changes in mitochondrial function in response to light were studied in terms of adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP). Light induced a significant increase in calcium, ATP and MMP at lower fluences and a decrease at higher fluence. ROS was induced significantly by light at all light doses. Nitric oxide levels also showed an increase on treatment with light. The results of the present study suggest that LLLT at lower fluences is capable of inducing mediators of cell signaling process which in turn may be responsible for the biomodulatory effects of the low level laser. At higher fluences beneficial mediators are reduced but potentially harmful mediators are increased thus offering an explanation for the biphasic dose response.

  20. Myricetin Prevents Alveolar Bone Loss in an Experimental Ovariectomized Mouse Model of Periodontitis

    PubMed Central

    Huang, Jialiang; Wu, Chuanlong; Tian, Bo; Zhou, Xiao; Ma, Nian; Qian, Yufen

    2016-01-01

    Periodontitis is a common chronic inflammatory disease, which leads to alveolar bone resorption. Healthy and functional alveolar bone, which can support the teeth and enable their movement, is very important for orthodontic treatment. Myricetin inhibited osteoclastogenesis by suppressing the expression of some genes, signaling pathways, and cytokines. This study aimed to investigate the effects of myricetin on alveolar bone loss in an ovariectomized (OVX) mouse model of periodontitis as well as in vitro osteoclast formation and bone resorption. Twenty-four healthy eight-week-old C57BL/J6 female mice were assigned randomly to four groups: phosphate-buffered saline (PBS) control (sham) OVX + ligature + PBS (vehicle), and OVX + ligature + low or high (2 or 5 mg∙kg−1∙day−1, respectively) doses of myricetin. Myricetin or PBS was injected intraperitoneally (i.p.) every other day for 30 days. The maxillae were collected and subjected to further examination, including micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, and tartrate-resistant acid phosphatase (TRAP) staining; a resorption pit assay was also performed in vitro to evaluate the effects of myricetin on receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. Myricetin, at both high and low doses, prevented alveolar bone resorption and increased alveolar crest height in the mouse model and inhibited osteoclast formation and bone resorption in vitro. However, myricetin was more effective at high dose than at low dose. Our study demonstrated that myricetin had a positive effect on alveolar bone resorption in an OVX mouse model of periodontitis and, therefore, may be a potential agent for the treatment of periodontitis and osteoporosis. PMID:27011174

  1. Myricetin Prevents Alveolar Bone Loss in an Experimental Ovariectomized Mouse Model of Periodontitis.

    PubMed

    Huang, Jialiang; Wu, Chuanlong; Tian, Bo; Zhou, Xiao; Ma, Nian; Qian, Yufen

    2016-01-01

    Periodontitis is a common chronic inflammatory disease, which leads to alveolar bone resorption. Healthy and functional alveolar bone, which can support the teeth and enable their movement, is very important for orthodontic treatment. Myricetin inhibited osteoclastogenesis by suppressing the expression of some genes, signaling pathways, and cytokines. This study aimed to investigate the effects of myricetin on alveolar bone loss in an ovariectomized (OVX) mouse model of periodontitis as well as in vitro osteoclast formation and bone resorption. Twenty-four healthy eight-week-old C57BL/J6 female mice were assigned randomly to four groups: phosphate-buffered saline (PBS) control (sham) OVX + ligature + PBS (vehicle), and OVX + ligature + low or high (2 or 5 mg∙kg(-1)∙day(-1), respectively) doses of myricetin. Myricetin or PBS was injected intraperitoneally (i.p.) every other day for 30 days. The maxillae were collected and subjected to further examination, including micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, and tartrate-resistant acid phosphatase (TRAP) staining; a resorption pit assay was also performed in vitro to evaluate the effects of myricetin on receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. Myricetin, at both high and low doses, prevented alveolar bone resorption and increased alveolar crest height in the mouse model and inhibited osteoclast formation and bone resorption in vitro. However, myricetin was more effective at high dose than at low dose. Our study demonstrated that myricetin had a positive effect on alveolar bone resorption in an OVX mouse model of periodontitis and, therefore, may be a potential agent for the treatment of periodontitis and osteoporosis. PMID:27011174

  2. Inhibiting and stimulating effects of TGF-. beta. 1 on osteoclastic bone resorption in fetal mouse bone organ cultures

    SciTech Connect

    Dieudonne, S.C.; Foo, P.; van Zoelen, E.J.; Burger, E.H. )

    1991-05-01

    The effects of TGF-{beta} 1 on osteoclastic resorption of fetal mouse calvaria and long bones at various stages of development was studied in organ culture. In resorbing calvariae and long bones with an established marrow cavity TGF-beta 1 (4-10 ng/ml) had a stimulating effect on 45Ca release that was partially inhibited by indomethacin. In primitive long bones, however, which were explanted before osteoclast invasion and excavation of a marrow cavity had started, TGF-beta 1 (1-4 ng/ml) inhibited 45Ca release by an indomethacin-insensitive mechanism. Histomorphometry of long bones after staining for tartrate-resistant acid phosphatase (TRAP) revealed that TGF-beta 1 treatment inhibited the migration of TRAP-positive cells from periosteum to developing marrow cavity and inhibited cell fusion. However, the formation of (mononuclear) TRAP-positive cells in the periosteum-perichondrium was strongly enhanced. These data suggest that TGF-beta 1 modulates various steps in the cascade of osteoclast development, recruitment, and activation in different ways, involving both prostaglandin-mediated and prostaglandin-independent pathways. Therefore the net effect of exogenous TGF-beta 1 on osteoclastic resorption in bone organ cultures depends on the relative prevalence of osteoclast progenitors, precursors, and mature osteoclasts in the tissue under study.

  3. Comparative analyses of B cell populations in trout kidney and mouse bone marrow; establishing B cell signatures

    PubMed Central

    Zwollo, Patty; Mott, Katrina; Barr, Maggie

    2010-01-01

    This study aimed to identify the frequency and distribution of developing B cell populations in the kidney of the rainbow trout, using four molecular B cell markers that are highly conserved between species, including two transcription factors, Pax5 and EBF1, recombination activating gene RAG1, and the immunoglobulin heavy chain mu. Three distinct B cell stages were defined: early developing B cells (CLP, pro-B, and early pre-B cells), late developing B cell (late pre-B, immature B, and mature B cells), and IgM-secreting cells. Developmental stage-specific, combinatorial expression of Pax5, EBF1, RAG1 and immunoglobulin mu was determined in trout anterior kidney cells by flow cytometry. Trout staining patterns were compared to a well-defined primary immune tissue, mouse bone marrow, and using mouse surface markers B220 and CD43. A remarkable level of similarity was uncovered between the primary immune tissues of both species. Subsequent analysis of the entire trout kidney, divided into five contiguous segments K1-K5, revealed a complex pattern of early developing, late developing, and IgM-secreting B cells. Patterns in anterior kidney segment K1 were most similar to those of mouse bone marrow, while the most posterior part of the kidney, K5, had many IgM-secreting cells, but lacked early developing B cells. A potential second B lymphopoiesis site was uncovered in segment K4 of the kidney. The B cell patterns, or B cell signatures described here provide information on the relative abundance of distinct developing B cell populations in the trout kidney, and can be used in future studies on B cell development in other vertebrate species. PMID:20705088

  4. Interleukin-10 inhibits the osteogenic activity of mouse bone marrow.

    PubMed

    Van Vlasselaer, P; Borremans, B; Van Den Heuvel, R; Van Gorp, U; de Waal Malefyt, R

    1993-10-15

    Murine bone marrow cells synthesize bone proteins, including alkaline phosphatase (ALP), collagen type I, and osteocalcin, and form a mineralized extracellular matrix when cultured in the presence of beta-glycerophosphate and vitamin C. Interleukin-10 (IL-10) suppressed the synthesis of these bone proteins and mineralization without affecting cell proliferation. In addition, mRNA levels for the latter proteins were reduced in IL-10-treated cultures. This inhibitory effect was most outspoken when IL-10 was added before ALP activity peaked, eg, day 15 of culture. No significant effect was observed when IL-10 was added at later time points. This finding suggests that IL-10 acts at osteogenic differentiation stages that precede ALP expression but is ineffective on cells that progressed beyond this maturation stage. Likewise, IL-10 appeared to be unable to block both ALP activity and collagen synthesis in the preosteosteoblastic cell lines MN7 and MC3T3 that constitutively synthesize these proteins. Whereas IL-10 did not alter the number of fibroblast colony-forming cells of the marrow, it significantly reduced their osteogenic differentiation potential. In contrast to control cultures, IL-10-treated stroma was unable to either synthesize osteocalcin or to mineralize when subcultured over a 25-day period in the absence of IL-10. The inhibitory activity of IL-10 coincided with significant changes in stroma morphology. Whereas control cultures contained mainly flat adherent polygonal cells, significant numbers of rounded semiadherent to nonadherent cells were observed in the presence of IL-10. Scanning and transmission electron microscopy showed that, in contrast to control cultures, IL-10-treated stromas completely lacked a mineralized extracellular matrix. Collectively, these data suggest that IL-10 may have important regulatory effects on bone biology because of its capacity to downregulate early steps of osteogenic differentiation. PMID:8400287

  5. Osteoblast isolation from murine calvaria and long bones.

    PubMed

    Bakker, Astrid D; Klein-Nulend, Jenneke

    2012-01-01

    This chapter describes the isolation of primary mouse osteoblasts from adult mouse calvaria and long bones, as well as the process of isolation of bone cells from neonatal mouse calvaria. Osteoblasts from adult mouse bone are obtained as outgrowth from collagenase-treated bone pieces. Isolation of osteoblasts from neonatal calvaria is achieved by sequential enzymatic digestion of the bone matrix. Because of differences in origin and isolation method, each of the primary bone cell cultures described will have their own characteristics. PMID:22130919

  6. Effect of cyclophosphamide and electromagnetic fields on mouse bone marrow

    SciTech Connect

    Cadossi, R.; Zucchini, P.; Emilia, G.; Torelli, G. )

    1990-02-26

    The authors have previously shown that the exposure to low frequency pulsing electromagnetic fields (PEMF) of mice X-ray irradiated resulted in an increased damage to the bone marrow. The series of experiments here reported were designed to investigate the effect of PEMF exposure after intraperitoneum injection of 200mg/kg of cyclophosphamide (CY). Control mice were CY injected only; experimental mice were CY injected and then exposed to PEMF. Exposure to PEMF (24 hours/day) increased the rate of decline of white blood cells in peripheral blood. Spleen weight was statistically higher among control mice than among mice exposed to PEMF at day 6, 8 and 10 after CY injection. Spleen autoradiography proved to be higher among PEMF exposed mice than among controls at day 8 and 9 after CY injection. The grafting efficiency of the bone marrow obtained from control mice was higher than the grafting efficiency of the bone marrow recovered from mice exposed to PEMF. All these data indicate that the exposure to PEMF increases the cytotoxic effect of CY.

  7. Neovascular Niche for Human Myeloma Cells in Immunodeficient Mouse Bone

    PubMed Central

    Miyakawa, Yoshitaka; Nakamura-Ishizu, Ayako; Miyauchi, Yoshiteru; Fujita, Nobuyuki; Miyamoto, Kana; Miyamoto, Takeshi; Ikeda, Eiji; Kizaki, Masahiro; Nojima, Yoshihisa; Suda, Toshio

    2012-01-01

    The interaction with bone marrow (BM) plays a crucial role in pathophysiological features of multiple myeloma (MM), including cell proliferation, chemoresistance, and bone lesion progression. To characterize the MM-BM interactions, we utilized an in vivo experimental model for human MM in which a GFP-expressing human MM cell line is transplanted into NOG mice (the NOG-hMM model). Transplanted MM cells preferentially engrafted at the metaphyseal region of the BM endosteum and formed a complex with osteoblasts and osteoclasts. A subpopulation of MM cells expressed VE-cadherin after transplantation and formed endothelial-like structures in the BM. CD138+ myeloma cells in the BM were reduced by p53-dependent apoptosis following administration of the nitrogen mustard derivative bendamustine to mice in the NOG-hMM model. Bendamustine maintained the osteoblast lining on the bone surface and protected extracellular matrix structures. Furthermore, bendamustine suppressed the growth of osteoclasts and mesenchymal cells in the NOG-hMM model. Since VE-cadherin+ MM cells were chemoresistant, hypoxic, and HIF-2α-positive compared to the VE-cadherin− population, VE-cadherin induction might depend on the oxygenation status. The NOG-hMM model described here is a useful system to analyze the dynamics of MM pathophysiology, interactions of MM cells with other cellular compartments, and the utility of novel anti-MM therapies. PMID:22347385

  8. Primary cilia act as mechanosensors during bone healing around an implant

    PubMed Central

    Leucht, P.; Monica, S.D.; Temiyasathit, S.; Lenton, K.; Manu, A.; Longaker, M.T.; Jacobs, C.R.; Spilker, R.L.; Guo, H.; Brunski, J.B.; Helms, J.A.

    2012-01-01

    The primary cilium is an organelle that senses cues in a cells local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3afl/fl mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective. PMID:22784673

  9. Neuronal Representation of Ultraviolet Visual Stimuli in Mouse Primary Visual Cortex

    PubMed Central

    Tan, Zhongchao; Sun, Wenzhi; Chen, Tsai-Wen; Kim, Douglas; Ji, Na

    2015-01-01

    The mouse has become an important model for understanding the neural basis of visual perception. Although it has long been known that mouse lens transmits ultraviolet (UV) light and mouse opsins have absorption in the UV band, little is known about how UV visual information is processed in the mouse brain. Using a custom UV stimulation system and in vivo calcium imaging, we characterized the feature selectivity of layer 2/3 neurons in mouse primary visual cortex (V1). In adult mice, a comparable percentage of the neuronal population responds to UV and visible stimuli, with similar pattern selectivity and receptive field properties. In young mice, the orientation selectivity for UV stimuli increased steadily during development, but not direction selectivity. Our results suggest that, by expanding the spectral window through which the mouse can acquire visual information, UV sensitivity provides an important component for mouse vision. PMID:26219604

  10. Ex vivo determination of bone tissue strains for an in vivo mouse tibial loading model.

    PubMed

    Carriero, Alessandra; Abela, Lisa; Pitsillides, Andrew A; Shefelbine, Sandra J

    2014-07-18

    Previous studies introduced the digital image correlation (DIC) as a viable technique for measuring bone strain during loading. In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations on the strain contour maps measured with a DIC system. We loaded tibiae of C57BL/6 mice (12 and 18 weeks old male) up to 12 N at 8 N/min. Images of speckles on the bone surface were recorded at 1N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps. This work provides a better understanding of parameters affecting full field strain measurements from DIC in ex vivo murine tibial loading tests. PMID:24835472

  11. Predicting cortical bone adaptation to axial loading in the mouse tibia

    PubMed Central

    Pereira, A. F.; Javaheri, B.; Pitsillides, A. A.; Shefelbine, S. J.

    2015-01-01

    The development of predictive mathematical models can contribute to a deeper understanding of the specific stages of bone mechanobiology and the process by which bone adapts to mechanical forces. The objective of this work was to predict, with spatial accuracy, cortical bone adaptation to mechanical load, in order to better understand the mechanical cues that might be driving adaptation. The axial tibial loading model was used to trigger cortical bone adaptation in C57BL/6 mice and provide relevant biological and biomechanical information. A method for mapping cortical thickness in the mouse tibia diaphysis was developed, allowing for a thorough spatial description of where bone adaptation occurs. Poroelastic finite-element (FE) models were used to determine the structural response of the tibia upon axial loading and interstitial fluid velocity as the mechanical stimulus. FE models were coupled with mechanobiological governing equations, which accounted for non-static loads and assumed that bone responds instantly to local mechanical cues in an on–off manner. The presented formulation was able to simulate the areas of adaptation and accurately reproduce the distributions of cortical thickening observed in the experimental data with a statistically significant positive correlation (Kendall's τ rank coefficient τ = 0.51, p < 0.001). This work demonstrates that computational models can spatially predict cortical bone mechanoadaptation to a time variant stimulus. Such models could be used in the design of more efficient loading protocols and drug therapies that target the relevant physiological mechanisms. PMID:26311315

  12. Automated Cell Detection and Morphometry on Growth Plate Images of Mouse Bone

    PubMed Central

    Ascenzi, Maria-Grazia; Du, Xia; Harding, James I; Beylerian, Emily N; de Silva, Brian M; Gross, Ben J; Kastein, Hannah K; Wang, Weiguang; Lyons, Karen M; Schaeffer, Hayden

    2014-01-01

    Microscopy imaging of mouse growth plates is extensively used in biology to understand the effect of specific molecules on various stages of normal bone development and on bone disease. Until now, such image analysis has been conducted by manual detection. In fact, when existing automated detection techniques were applied, morphological variations across the growth plate and heterogeneity of image background color, including the faint presence of cells (chondrocytes) located deeper in tissue away from the image’s plane of focus, and lack of cell-specific features, interfered with identification of cell. We propose the first method of automated detection and morphometry applicable to images of cells in the growth plate of long bone. Through ad hoc sequential application of the Retinex method, anisotropic diffusion and thresholding, our new cell detection algorithm (CDA) addresses these challenges on bright-field microscopy images of mouse growth plates. Five parameters, chosen by the user in respect of image characteristics, regulate our CDA. Our results demonstrate effectiveness of the proposed numerical method relative to manual methods. Our CDA confirms previously established results regarding chondrocytes’ number, area, orientation, height and shape of normal growth plates. Our CDA also confirms differences previously found between the genetic mutated mouse Smad1/5CKO and its control mouse on fluorescence images. The CDA aims to aid biomedical research by increasing efficiency and consistency of data collection regarding arrangement and characteristics of chondrocytes. Our results suggest that automated extraction of data from microscopy imaging of growth plates can assist in unlocking information on normal and pathological development, key to the underlying biological mechanisms of bone growth. PMID:25525552

  13. Proinflammatory cytokine response and viral replication in mouse bone marrow derived macrophages infected with influenza H1N1 and H5N1 viruses.

    PubMed

    Chan, Renee W Y; Leung, Connie Y H; Nicholls, John M; Peiris, J S Malik; Chan, Michael C W

    2012-01-01

    The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes. PMID:23226456

  14. Isolation of Dendritic Cell Progenitor and Bone Marrow Progenitor Cells from Mouse.

    PubMed

    Onai, Nobuyuki; Ohteki, Toshiaki

    2016-01-01

    Dendritic cells (DCs) comprise two major subsets, conventional DC (cDC) and plasmacytoid DC (pDC) in the steady-state lymphoid organ. These cells have a short half-life and therefore, require continuous generation from hematopoietic stem cells and progenitor cells. Recently, we identified DC-restricted progenitors called common DC progenitors (CDPs) in the bone marrow of mouse. The CDPs can be isolated from mouse bone marrow based on the hematopoietic cytokine receptors, such as Flt3 (Fms-related tyrosine kinase 3) (CD135), c-kit (CD117), M-CSF (macrophage colony-stimulating factor) receptor (CD115), and IL-7 (interleukin-7) receptor-α (CD127). The CDPs comprise of two progenitors, CD115(+) CDPs and CD115(-) CDPs, and give rise to only DC subsets in both in vitro and in vivo. The former CDPs are the main source of cDC, while the later CDPs are the main source of pDC in vivo. Here, we provide a protocol for the isolation of dendritic cell progenitor and bone marrow progenitor cells from mouse. PMID:27142008

  15. Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow

    PubMed Central

    Dyer, Kimberly D.; Moser, Jennifer M.; Czapiga, Meggan; Siegel, Steven J.; Percopo, Caroline M.; Rosenberg, Helene F.

    2009-01-01

    We have devised an ex vivo culture system which generates large numbers of eosinophils at high purity (>90%) from unselected mouse bone marrow progenitors. In response to four days of culture with recombinant mouse (rm)FLT3-L and rmSCF followed by rmIL-5 alone thereafter, the resulting bone-marrow derived eosinophils (bmEos) express immunoreactive major basic protein, Siglec F, IL-5 receptor alpha chain, and transcripts encoding mouse eosinophil peroxidase, CC chemokine receptor 3, the IL-3/IL-5/GMCSF receptor common beta-chain (βc), and the transcription factor GATA-1. BmEos are functionally competent: they undergo chemotaxis toward mouse eotaxin-1 and produce characteristic cytokines, including interferon-γ, IL-4, MIP-1α and IL-6. The rodent pathogen, pneumonia virus of mice (PVM) replicates in bmEos, and elevated levels of IL-6 are detected in supernatants of bmEos cultures in response to active infection. Finally, differentiating bmEos are readily transfected with lentiviral vectors, suggesting a means for rapid production of genetically manipulated cells. PMID:18768855

  16. Adipocytes regulate the bone marrow microenvironment in a mouse model of obesity.

    PubMed

    Xu, Fei; Du, Yu; Hang, Shilong; Chen, Anmin; Guo, Fengjin; Xu, Tao

    2013-09-01

    Obesity is markedly associated with abnormal bone density indicating the importance of adipocytes in bone metabolism. However, the specific function of adipocytes remains unclear, with marked discrepancies in observations of previous studies. In the present study, the effect of adipocytes on osteoblasts/osteoclasts was analyzed. A mouse model of obesity was established and an in vitro co-culture system was utilized containing adipocyte and MC3T3/RAW 264.7 cells in a Transwell plate. Compared with control mice, obese mice exhibited low body weight and bone mineral density of the tibia and fat cells were observed to accumulate in bone marrow. MC3T3/RAW 264.7 cells were co-cultured with adipocytes and the mRNA and protein expression of alkaline phosphatase and osteocalcin was found to be decreased in MC3T3-E1 cells and mRNA and protein expression of tartrate-resistant acid phosphatase and cathepsin K was significantly increased in RAW 264.7 cells. In addition, the effect of adipocytes on the osteoprotegerin (OPG)/receptor activator of nuclear factor κB ligand (RANKL)/RANK system indicated that the RANKL/OPG ratio secreted by osteoblasts increased and RANK expression by osteoclasts increased, leading to increased osteoclastogenesis. These results indicate that bone metabolism is impaired in obese mice leading to decreased osteoblastogenesis and marked increases in osteoclastogenesis and low bone mass. PMID:23835909

  17. Accretion of Bone Quantity and Quality in the Developing Mouse Skeleton

    SciTech Connect

    Miller,L.; Little, W.; Schirmer, A.; Sheik, F.; Busa, B.; Judex, S.

    2007-01-01

    To meet the mechanical challenges during early development, the skeleton requires the rapid accretion of bone quality and bone quantity. Here, we describe early bone development in the mouse skeleton and test the hypothesis that specific compositional properties determine the stiffness of the tissue. Tibias of female BALB mice were harvested at eight time-points (n = 4 each) distributed between 1 and 40 days of age and subjected to morphometric ({mu}CT), chemical (Fourier transform infrared microscpectroscopy), and mechanical (nanoindentation) analyses. Tibias of 450-day-old mice served as fully mineralized control specimens. In this work, we found that bone mineral formation proceeded very rapidly in mice by 1 day of age, where the degree of mineralization, the tissue mineral density, and the mineral crystallinity reached 36%, 51%, and 87% of the adult values, respectively. However, even though significant mineralization had occurred, the elastic modulus of 1-day-old bone was only 14% of its adult value, indicating that the intrinsic stiffening of the bone lags considerably behind the initial mineral formation.

  18. Primary bone natural killer/T cell lymphoma, nasal type without EBV infection: a case report

    PubMed Central

    Tian, Chen; Wang, Yafei; Zhu, Lei; Yu, Yong; Zhang, Yizhuo

    2015-01-01

    Primary bone NK/T cell lymphoma is very rare. We report a case of 52-year-old man of primary bone NK/T cell lymphoma and then progressed to NK leukemia. The patient had low-grade fever for 4-month, and Ultrasonic B revealed a diffuse hepatosplenomegaly without lymphadenopathy. PET scanning showed increased FDG uptake in many bones of the whole body. The diagnosis was established by bone specimen. These neoplastic cells demonstrated a typical immunophenotype of CD56, CD3, CD2 and MPO positive, and CD5, CD20, CD30, PAX-5, CD4 and CD8 negative. Primary bone ENKTL is very rare; it should be made with the combination of clinical feature, PET-CT image, and pathological characteristics, and should be distinguished from other lymphomas or leukemia involved in bone. PMID:26823813

  19. Magnetic assembly-mediated enhancement of differentiation of mouse bone marrow cells cultured on magnetic colloidal assemblies

    NASA Astrophysics Data System (ADS)

    Sun, Jianfei; Liu, Xuan; Huang, Jiqing; Song, Lina; Chen, Zihao; Liu, Haoyu; Li, Yan; Zhang, Yu; Gu, Ning

    2014-05-01

    Here we reported an interesting phenomenon that the field-induced assemblies of magnetic nanoparticles can promote the differentiation of primary mouse bone marrow cells into osteoblasts. The reason was thought to lie in the remnant magnetic interaction inside the assemblies which resulted from the magnetic field-directed assembly. Influence of the assemblies on the cells was realized by means of interface effect rather than the internalization effect. We fabricated a stripe-like assemblies array on the glass plate and cultured cells on this surface. We characterized the morphology of assemblies and measured the mechanic property as well as the magnetic property. The cellular differentiation was measured by staining and quantitative PCR. Finally, Fe uptake was excluded as the reason to cause the phenomenon.

  20. Spectrum of primary bone grafting in cranio maxillofacial trauma at a tertiary care centre in India

    PubMed Central

    Singh, Arun Kumar; Mohapatra, Devi Prasad; Kumar, Vijay

    2011-01-01

    Background: In past several years, traumas following road traffic accidents and other causes have increased, owing to an increase in mechanization and pace of life. These patients frequently have complicated injuries involving soft tissue and the craniofacial skeleton. Assessment of bony injuries and loss of portions of facial skeleton and their management has proved to be a challenge to the reconstructive surgeon. Aims: Primary bone grafting of craniofacial skeletal injuries provides an opportunity for one stage correction of bony defects. The varied spectrum of primary bone grafts for management of craniomaxillofacial injuries are evaluated in this study. Materials and Methods: Patients with a history of acute trauma resulting in facial skeletal injuries with or without bone loss were included in the study. Primary bone grafting was undertaken in situations requiring contour correction, replacement of skeletal losses and for rigid fixation of fracture segments. Olecranon, Iliac crest, ribs, Vascularized as well as nonvascularized outer table calvarial grafts and nonvascularized inner table calvarial grafts were used in this study. Results: Sixty two patients of craniomaxillofacial injury following trauma requiring primary bone grafting were considered in this study. Fifty seven percent of patients (n=32) required primary bone grafting for replacement of bone loss while bone grafting for contour correction was done in twenty three patients. The parietal calvaria overlying the non-dominant hemisphere was used as a source of bone graft in forty-nine patients. Nearly ninety-two percent of the patients were satisfied with the results of primary bone grafting. Conclusions: Functional and aesthetic assessment of each of these patients, managed with primary bone grafting revealed a low rate of disabilities and high percentage of satisfaction in this study. PMID:21713213

  1. Methods and theory in bone modeling drift: comparing spatial analyses of primary bone distributions in the human humerus.

    PubMed

    Maggiano, Corey M; Maggiano, Isabel S; Tiesler, Vera G; Chi-Keb, Julio R; Stout, Sam D

    2016-01-01

    This study compares two novel methods quantifying bone shaft tissue distributions, and relates observations on human humeral growth patterns for applications in anthropological and anatomical research. Microstructural variation in compact bone occurs due to developmental and mechanically adaptive circumstances that are 'recorded' by forming bone and are important for interpretations of growth, health, physical activity, adaptation, and identity in the past and present. Those interpretations hinge on a detailed understanding of the modeling process by which bones achieve their diametric shape, diaphyseal curvature, and general position relative to other elements. Bone modeling is a complex aspect of growth, potentially causing the shaft to drift transversely through formation and resorption on opposing cortices. Unfortunately, the specifics of modeling drift are largely unknown for most skeletal elements. Moreover, bone modeling has seen little quantitative methodological development compared with secondary bone processes, such as intracortical remodeling. The techniques proposed here, starburst point-count and 45° cross-polarization hand-drawn histomorphometry, permit the statistical and populational analysis of human primary tissue distributions and provide similar results despite being suitable for different applications. This analysis of a pooled archaeological and modern skeletal sample confirms the importance of extreme asymmetry in bone modeling as a major determinant of microstructural variation in diaphyses. Specifically, humeral drift is posteromedial in the human humerus, accompanied by a significant rotational trend. In general, results encourage the usage of endocortical primary bone distributions as an indicator and summary of bone modeling drift, enabling quantitative analysis by direction and proportion in other elements and populations. PMID:26471797

  2. Evaluation of an in vitro muscle contraction model in mouse primary cultured myotubes.

    PubMed

    Manabe, Yasuko; Ogino, Shinya; Ito, Miyuki; Furuichi, Yasuro; Takagi, Mayumi; Yamada, Mio; Goto-Inoue, Naoko; Ono, Yusuke; Fujii, Nobuharu L

    2016-03-15

    To construct an in vitro contraction model with the primary cultured myotubes, we isolated satellite cells from the mouse extensor digitorum longus. Differentiated myotubes possessed a greater number of sarcomere assemblies and higher expression levels of myosin heavy chain, cytochrome c oxidase IV, and myoglobin than in C2C12 myotubes. In agreement with these results regarding the sarcomere assemblies and protein expressions, the primary myotubes showed higher contractile activity stimulated by the electric pulses than that in the C2C12 myotubes. These data suggest that mouse primary myotubes will be a valuable research tool as an in vitro muscle contraction model. PMID:26548957

  3. A temporary decrease in mineral density in perinatal mouse long bones.

    PubMed

    Sharir, A; Milgram, J; Dubnov-Raz, G; Zelzer, E; Shahar, R

    2013-01-01

    Fetal and postnatal bone development in humans is traditionally viewed as a process characterized by progressively increasing mineral density. Yet, a temporary decrease in mineral density has been described in the long bones of infants in the immediate postnatal period. The mechanism that underlies this phenomenon, as well as its causes and consequences, remain unclear. Using daily μCT scans of murine femora and tibiae during perinatal development, we show that a temporary decrease in tissue mineral density (TMD) is evident in mice. By monitoring spatial and temporal structural changes during normal growth and in a mouse strain in which osteoclasts are non-functional (Src-null), we show that endosteal bone resorption is the main cause for the perinatal decrease in TMD. Mechanical testing revealed that this temporary decrease is correlated with reduced stiffness of the bones. We also show, by administration of a progestational agent to pregnant mice, that the decrease in TMD is not the result of parturition itself. This study provides a comprehensive view of perinatal long bone development in mice, and describes the process as well as the consequences of density fluctuation during this period. PMID:23044045

  4. Targeting of Primary Breast Cancers and Metastases in a Transgenic Mouse Model Using Rationally Designed Multifunctional SPIONs

    PubMed Central

    Kievit, Forrest M.; Stephen, Zachary R.; Veiseh, Omid; Arami, Hamed; Wang, Tingzhong; Lai, Vy P.; Park, James O.; Ellenbogen, Richard G.; Disis, Mary L.; Zhang, Miqin

    2012-01-01

    Breast cancer remains one of the most prevalent and lethal malignancies in women. The inability to diagnose small volume metastases early has limited effective treatment of stage 4 breast cancer. Here we report the rational development and use of a multifunctional superparamagnetic iron oxide nanoparticle (SPION) for targeting metastatic breast cancer in a transgenic mouse model and imaging with magnetic resonance (MR). SPIONs coated with a copolymer of chitosan and polyethylene glycol (PEG) were labeled with a fluorescent dye for optical detection and conjugated with a monoclonal antibody against the neu receptor (NP-neu). SPIONs labeled with mouse IgG were used as a non-targeting control (NP-IgG). These SPIONs had desirable physiochemical properties for in vivo applications such as near neutral zeta potential and hydrodynamic size around 40 nm, and were highly stable in serum containing medium. Only NP-neu showed high uptake in neu expressing mouse mammary carcinoma (MMC) cells which was reversed by competing free neu antibody, indicating their specificity to the neu antigen. In vivo, NP-neu was able to tag primary breast tumors and significantly, only NP-neu bound to spontaneous liver, lung, and bone marrow metastases in a transgenic mouse model of metastatic breast cancer, highlighting the necessity of targeting for delivery to metastatic disease. The SPIONs provided significant contrast enhancement in MR images of primary breast tumors; thus, they have the potential for MRI detection of micrometastases, and provide an excellent platform for further development of an efficient metastatic breast cancer therapy. PMID:22324543

  5. Neonatal bone marrow transplantation prevents bone pathology in a mouse model of mucopolysaccharidosis type I.

    PubMed

    Pievani, Alice; Azario, Isabella; Antolini, Laura; Shimada, Tsutomu; Patel, Pravin; Remoli, Cristina; Rambaldi, Benedetta; Valsecchi, Maria Grazia; Riminucci, Mara; Biondi, Andrea; Tomatsu, Shunji; Serafini, Marta

    2015-03-01

    Neonatal bone marrow transplantation (BMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth, thus preventing tissue damage. We tested this concept in mucopolysaccharidosis type I (MPS IH; Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-l-iduronidase. MPS IH is characterized by a broad spectrum of clinical manifestations, including severe progressive skeletal abnormalities. Although BMT increases the life span of patients with MPS IH, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal BM into newborn MPS I mice soon after birth can prevent skeletal dysplasia. We observed that neonatal BMT was effective at restoring α-l-iduronidase activity and clearing elevated glycosaminoglycans in blood and multiple organs. At 37 weeks of age, we observed an almost complete normalization of all bone tissue parameters, using radiographic, microcomputed tomography, biochemical, and histological analyses. Overall, the magnitude of improvements correlated with the extent of hematopoietic engraftment. We conclude that BMT at a very early stage in life markedly reduces signs and symptoms of MPS I before they appear. PMID:25298037

  6. A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells

    PubMed Central

    Lima, Djalma S.; Zamboni, Dario S.

    2010-01-01

    The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells. PMID:21179419

  7. Neonatal bone marrow transplantation prevents bone pathology in a mouse model of mucopolysaccharidosis type I

    PubMed Central

    Pievani, Alice; Azario, Isabella; Antolini, Laura; Shimada, Tsutomu; Patel, Pravin; Remoli, Cristina; Rambaldi, Benedetta; Valsecchi, Maria Grazia; Riminucci, Mara; Biondi, Andrea; Tomatsu, Shunji

    2015-01-01

    Neonatal bone marrow transplantation (BMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth, thus preventing tissue damage. We tested this concept in mucopolysaccharidosis type I (MPS IH; Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-l-iduronidase. MPS IH is characterized by a broad spectrum of clinical manifestations, including severe progressive skeletal abnormalities. Although BMT increases the life span of patients with MPS IH, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal BM into newborn MPS I mice soon after birth can prevent skeletal dysplasia. We observed that neonatal BMT was effective at restoring α-l-iduronidase activity and clearing elevated glycosaminoglycans in blood and multiple organs. At 37 weeks of age, we observed an almost complete normalization of all bone tissue parameters, using radiographic, microcomputed tomography, biochemical, and histological analyses. Overall, the magnitude of improvements correlated with the extent of hematopoietic engraftment. We conclude that BMT at a very early stage in life markedly reduces signs and symptoms of MPS I before they appear. PMID:25298037

  8. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading

    PubMed Central

    Jing, Da; Baik, Andrew D.; Lu, X. Lucas; Zhou, Bin; Lai, Xiaohan; Wang, Liyun; Luo, Erping; Guo, X. Edward

    2014-01-01

    Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca2+) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca2+ responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid-induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca2+ spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca2+ oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca2+ oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.—Jing, D., Baik, A. D., Lu, X. L., Zhou, B., Lai, X., Wang, L., Luo, E., Guo, X. E. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. PMID:24347610

  9. Preoperative Computed Tomography-Derived Bone Densities in Hounsfield Units at Implant Sites Acquired Primary Stability

    PubMed Central

    Hiasa, Kyou; Abe, Yasuhiko; Okazaki, Yohei; Nogami, Keisuke; Mizumachi, Wataru; Akagawa, Yasumasa

    2011-01-01

    The purpose of this study was to evaluate preoperative CT-derived bone densities in Hounsfield units (HU) at implant sites that acquired primary stability, and to compare these values to the optimal bone densities proposed in the literature. Fifty-one patients, 18 males (37 implant sites) and 33 females (67 implant sites) between 2003 and 2010 were assessed. CT data for different jaw sections, regions, and operating procedures were compared using the Kruskal-Wallis test and Scheffe's test for multiple comparisons (P < 0.05). The mean bone density in the maxilla was significantly lower than that in the mandible (P < 0.05); the mean bone densities in the 4 jaw regions decreased in the following order: anterior mandible > anterior maxilla > posterior mandible > posterior maxilla. The bone densities assessed by HU fell into the range of optimal bone densities associated with acquired primary implant stability proposed in the literature. PMID:22203910

  10. miRNA-29b improves bone healing in mouse fracture model.

    PubMed

    Lee, Wayne Y; Li, Nan; Lin, Sien; Wang, Bin; Lan, Hui Y; Li, Gang

    2016-07-15

    A number of miRNAs regulates bone remodeling and their levels in circulation were associated with bone fracture, however no miRNAs have yet been shown to improve fracture healing directly. This study aimed to investigate the effect of miR-29b-3p on mice femoral fracture healing through site-specific delivery with microbubble-ultrasound system. miR-29b-3p promoted osteogenesis of mouse bone marrow-derived mesenchymal stem cells as indicated with quantitative real-time polymerase chain reaction (qPCR) and Alizarin red S staining. Animal study showed that single injection of miR-29b-3p at week 2 post fracture improved healing outcome as indicated by significant decrease of callus width and area with radiographic analysis without causing significant weight loss. Static bone histomorphometry analysis showed that miR-29b-3p increased bone volume fraction (BV/TV), and micro-computed tomography (micro-CT) measurement showed increased BV/TV of high density bone and bone mineral density (BMD) of the callus. 3 point bending mechanical test showed improved relative stiffness. However, repeated injection of miR-29b-3p at weeks 2 and 3 did not result in additive therapeutic outcome, and caused increased total tissue volume and reduced BMD of the callus. This is the first report showing significant therapeutic effect of miR-29b-3p on femoral fracture healing through site-specific delivery with microbubble-ultrasound system. Further studies are warranted to investigate the underlying mechanisms and to refine the treatment protocol. PMID:27113026

  11. Assessment of lamellar level properties in mouse bone utilizing a novel spherical nanoindentation data analysis method

    PubMed Central

    Pathak, Siddhartha; Vachhani, Shraddha J.; Jepsen, Karl J.; Goldman, Haviva M.; Kalidindi, Surya R.

    2016-01-01

    In this work, we demonstrate the viability of using our recently developed data analysis procedures for spherical nanoindentation in conjunction with Raman spectroscopy for studying lamellar-level correlations between the local composition and local mechanical properties in mouse bone. Our methodologies allow us to convert the raw load-displacement datasets to much more meaningful indentation stress–strain curves that accurately capture the loading and unloading elastic moduli, the indentation yield points, as well as the post-yield characteristics in the tested samples. Using samples of two different inbred mouse strains, A/J and C57BL/6J (B6), we successfully demonstrate the correlations between the mechanical information obtained from spherical nanoindentation measurements to the local composition measured using Raman spectroscopy. In particular, we observe that a higher mineral-to-matrix ratio correlated well with a higher local modulus and yield strength in all samples. Thus, new bone regions exhibited lower moduli and yield strengths compared to more mature bone. The B6 mice were also found to exhibit lower modulus and yield strength values compared to the more mineralized A/J strain. PMID:22842281

  12. Establishing biomechanical mechanisms in mouse models: practical guidelines for systematically evaluating phenotypic changes in the diaphyses of long bones.

    PubMed

    Jepsen, Karl J; Silva, Matthew J; Vashishth, Deepak; Guo, X Edward; van der Meulen, Marjolein C H

    2015-06-01

    Mice are widely used in studies of skeletal biology, and assessment of their bones by mechanical testing is a critical step when evaluating the functional effects of an experimental perturbation. For example, a gene knockout may target a pathway important in bone formation and result in a "low bone mass" phenotype. But how well does the skeleton bear functional loads; eg, how much do bones deform during loading and how resistant are bones to fracture? By systematic evaluation of bone morphological, densitometric, and mechanical properties, investigators can establish the "biomechanical mechanisms" whereby an experimental perturbation alters whole-bone mechanical function. The goal of this review is to clarify these biomechanical mechanisms and to make recommendations for systematically evaluating phenotypic changes in mouse bones, with a focus on long-bone diaphyses and cortical bone. Further, minimum reportable standards for testing conditions and outcome variables are suggested that will improve the comparison of data across studies. Basic biomechanical principles are reviewed, followed by a description of the cross-sectional morphological properties that best inform the net cellular effects of a given experimental perturbation and are most relevant to biomechanical function. Although morphology is critical, whole-bone mechanical properties can only be determined accurately by a mechanical test. The functional importance of stiffness, maximum load, postyield displacement, and work-to-fracture are reviewed. Because bone and body size are often strongly related, strategies to adjust whole-bone properties for body mass are detailed. Finally, a comprehensive framework is presented using real data, and several examples from the literature are reviewed to illustrate how to synthesize morphological, tissue-level, and whole-bone mechanical properties of mouse long bones. PMID:25917136

  13. Cardiopulmonary dysfunction in the Osteogenesis imperfecta mouse model Aga2 and human patients are caused by bone-independent mechanisms.

    PubMed

    Thiele, Frank; Cohrs, Christian M; Flor, Armando; Lisse, Thomas S; Przemeck, Gerhard K H; Horsch, Marion; Schrewe, Anja; Gailus-Durner, Valerie; Ivandic, Boris; Katus, Hugo A; Wurst, Wolfgang; Reisenberg, Catherine; Chaney, Hollis; Fuchs, Helmut; Hans, Wolfgang; Beckers, Johannes; Marini, Joan C; Hrabé de Angelis, Martin

    2012-08-15

    Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with skeletal dysplasia of varying severity, predominantly caused by mutations in the collagen I genes (COL1A1/COL1A2). Extraskeletal findings such as cardiac and pulmonary complications are generally considered to be significant secondary features. Aga2, a murine model for human OI, was systemically analyzed in the German Mouse Clinic by means of in vivo and in vitro examinations of the cardiopulmonary system, to identify novel mechanisms accounting for perinatal lethality. Pulmonary and, especially, cardiac fibroblast of perinatal lethal Aga2/+ animals display a strong down-regulation of Col1a1 transcripts in vivo and in vitro, resulting in a loss of extracellular matrix integrity. In addition, dysregulated gene expression of Nppa, different types of collagen and Agt in heart and lung tissue support a bone-independent vicious cycle of heart dysfunction, including hypertrophy, loss of myocardial matrix integrity, pulmonary hypertension, pneumonia and hypoxia leading to death in Aga2. These murine findings are corroborated by a pediatric OI cohort study, displaying significant progressive decline in pulmonary function and restrictive pulmonary disease independent of scoliosis. Most participants show mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. Data obtained from human OI patients and the mouse model Aga2 provide novel evidence for primary effects of type I collagen mutations on the heart and lung. The findings will have potential benefits of anticipatory clinical exams and early intervention in OI patients. PMID:22589248

  14. Cardiopulmonary dysfunction in the Osteogenesis imperfecta mouse model Aga2 and human patients are caused by bone-independent mechanisms

    PubMed Central

    Thiele, Frank; Cohrs, Christian M.; Flor, Armando; Lisse, Thomas S.; Przemeck, Gerhard K. H.; Horsch, Marion; Schrewe, Anja; Gailus-Durner, Valerie; Ivandic, Boris; Katus, Hugo A.; Wurst, Wolfgang; Reisenberg, Catherine; Chaney, Hollis; Fuchs, Helmut; Hans, Wolfgang; Beckers, Johannes; Marini, Joan C.; Hrabé de Angelis, Martin

    2012-01-01

    Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with skeletal dysplasia of varying severity, predominantly caused by mutations in the collagen I genes (COL1A1/COL1A2). Extraskeletal findings such as cardiac and pulmonary complications are generally considered to be significant secondary features. Aga2, a murine model for human OI, was systemically analyzed in the German Mouse Clinic by means of in vivo and in vitro examinations of the cardiopulmonary system, to identify novel mechanisms accounting for perinatal lethality. Pulmonary and, especially, cardiac fibroblast of perinatal lethal Aga2/+ animals display a strong down-regulation of Col1a1 transcripts in vivo and in vitro, resulting in a loss of extracellular matrix integrity. In addition, dysregulated gene expression of Nppa, different types of collagen and Agt in heart and lung tissue support a bone-independent vicious cycle of heart dysfunction, including hypertrophy, loss of myocardial matrix integrity, pulmonary hypertension, pneumonia and hypoxia leading to death in Aga2. These murine findings are corroborated by a pediatric OI cohort study, displaying significant progressive decline in pulmonary function and restrictive pulmonary disease independent of scoliosis. Most participants show mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. Data obtained from human OI patients and the mouse model Aga2 provide novel evidence for primary effects of type I collagen mutations on the heart and lung. The findings will have potential benefits of anticipatory clinical exams and early intervention in OI patients. PMID:22589248

  15. Primary Hemangiopericytoma of the Parietal Bone: A Case Report

    PubMed Central

    Sipal, Sare; Demirci, Elif; Calık, Muhammet; Gundogdu, Betul; Sengul, Goksin; Gundogdu, Cemal

    2009-01-01

    Summary Hemangiopericytomas are rare hypervascular tumors arising from Zimmerman’s pericytes. They usually occur in the soft tissue, and intraosseous lesions are very rare. Surgical excision is the first choice for treatment. Many studies show that patients should be monitored for some time following treatment because of a high rate of recurrence and metastasis after radical resection. This report introduces a 56-year-old patient with a hemangiopericytoma in his parietal bone. Keywords: Parietal bone, Hemangiopericytoma PMID:25610105

  16. Trabecular Bone Score (TBS)—A Novel Method to Evaluate Bone Microarchitectural Texture in Patients With Primary Hyperparathyroidism

    PubMed Central

    Boutroy, Stephanie; Zhang, Chiyuan; McMahon, Donald Jay; Zhou, Bin; Wang, Ji; Udesky, Julia; Cremers, Serge; Sarquis, Marta; Guo, Xiang-Dong Edward; Hans, Didier

    2013-01-01

    Context: In the milder form of primary hyperparathyroidism (PHPT), cancellous bone, represented by areal bone mineral density at the lumbar spine by dual-energy x-ray absorptiometry (DXA), is preserved. This finding is in contrast to high-resolution peripheral quantitative computed tomography (HRpQCT) results of abnormal trabecular microstructure and epidemiological evidence for increased overall fracture risk in PHPT. Because DXA does not directly measure trabecular bone and HRpQCT is not widely available, we used trabecular bone score (TBS), a novel gray-level textural analysis applied to spine DXA images, to estimate indirectly trabecular microarchitecture. Objective: The purpose of this study was to assess TBS from spine DXA images in relation to HRpQCT indices and bone stiffness in radius and tibia in PHPT. Design and Setting: This was a cross-sectional study conducted in a referral center. Patients: Participants were 22 postmenopausal women with PHPT. Main Outcome Measures: Outcomes measured were areal bone mineral density by DXA, TBS indices derived from DXA images, HRpQCT standard measures, and bone stiffness assessed by finite element analysis at distal radius and tibia. Results: TBS in PHPT was low at 1.24, representing abnormal trabecular microstructure (normal ≥1.35). TBS was correlated with whole bone stiffness and all HRpQCT indices, except for trabecular thickness and trabecular stiffness at the radius. At the tibia, correlations were observed between TBS and volumetric densities, cortical thickness, trabecular bone volume, and whole bone stiffness. TBS correlated with all indices of trabecular microarchitecture, except trabecular thickness, after adjustment for body weight. Conclusion: TBS, a measurement technology readily available by DXA, shows promise in the clinical assessment of trabecular microstructure in PHPT. PMID:23526463

  17. Caffeine inhibits adipogenic differentiation of primary adipose-derived stem cells and bone marrow stromal cells.

    PubMed

    Su, Shu-Hui; Shyu, Huey-Wen; Yeh, Yao-Tsung; Chen, Kuan-Ming; Yeh, Hua; Su, Shu-Jem

    2013-09-01

    Caffeine consumption has been related to loss of body weight and modulates lipid metabolism. However, impacts of caffeine on adipogenic differentiation have not been well determined yet. The present study evaluated the effects of caffeine on adipogenesis using primary rat adipose-derived stem cells (ADSCs) and a mouse bone marrow stromal cell line (M2-10B4) in vitro. ADSCs and M2-10B4 were continuously exposed to caffeine (0.1-1mM) during adipogenic differentiation for 7 and 12 days, respectively. Oil red O and Nile red staining showed that caffeine reduced lipid droplet and adipocyte levels in both cell types. In addition, Nile red staining and FACScan flow cytometry showed that caffeine dose-dependently decreased adipocyte differentiation from 20% to 50% of the control ADSCs and M2-10B4 cells. Caffeine decreased the expression of adipogenesis-related genes including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α, adipocyte lipid binding protein, lipoprotein lipase, leptin, and TNFα in a dose-dependent manner. Rather, low concentration of caffeine (0.1mM) significantly increased IL-6 expression, but unexpectedly inhibited that at a concentration more than 0.3mM. Taken together, caffeine was able to effectively inhibit adipogenic differentiation of ADSCs and M2-10B4 cells partly through its inhibition of adipogenesis-related factors. PMID:23727198

  18. Genetically engineered mouse models to evaluate the role of Wnt secretion in bone development and homeostasis.

    PubMed

    Williams, Bart O

    2016-03-01

    Alterations in components of the Wnt signaling pathway are associated with altered bone development and homeostasis in several human diseases. We created genetically engineered mouse models (GEMMs) that mimic the cellular defect associated with the Porcupine mutations in patients with Goltz Syndrome/Focal Dermal Hypoplasia. These GEMMs were established by utilizing mice containing a conditionally inactivatable allele of Wntless/GPR177 (a gene encoding a protein required for the transport of Porcupine-modified ligand to the plasma membrane for secretion). We crossed this strain to another which drives cre-mediated gene deletion in mature osteoblasts (Osteocalcin-cre) resulted in mice lacking the ability to secrete Wnt ligands in this cell type. These mice displayed severely reduced bone mass and provide a model to understand the effects of disrupting the ability to secrete Wnt ligands on the skeletal system. © 2016 Wiley Periodicals, Inc. PMID:26818176

  19. Chronic intermittent hypoxia preserves bone density in a mouse model of sleep apnea.

    PubMed

    Torres, Marta; Montserrat, Josep M; Pavía, Javier; Dalmases, Mireia; Ros, Domenec; Fernandez, Yolanda; Barbé, Ferran; Navajas, Daniel; Farré, Ramon

    2013-12-01

    Very recent clinical research has investigated whether obstructive sleep apnea (OSA) may modulate bone homeostasis but the few data available are conflicting. Here we report novel data obtained in a mouse study specifically designed to determine whether chronic intermittent hypoxia realistically mimicking OSA modifies bone mineral density (BMD). Normal male and female mice and orchidectomized mice (N=10 each group) were subjected to a pattern of high-frequency intermittent hypoxia (20s at 5% and 40s at 21%, 60 cycles/h) for 6h/day. Identical groups breathing room air (normoxia) were the controls. After 32 days of intermittent hypoxia/normoxia the trabecular bone mineral density (BMD) in the peripheral femora were measured by micro-CT scanning. When compared with normoxia (two-way ANOVA), intermittent hypoxia did not significantly modify BMD in the three animal groups tested. Data in this study suggest that the type of intermittent hypoxia characterizing OSA, applied as a single challenge, preserves bone homeostasis. PMID:23994179

  20. Mouse basophils reside in extracellular matrix-enriched bone marrow niches which control their motility.

    PubMed

    Smaniotto, Salete; Schneider, Elke; Goudin, Nicolas; Bricard-Rignault, Rachel; Machavoine, François; Dardenne, Mireille; Dy, Michel; Savino, Wilson

    2013-01-01

    Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2β1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (α-4, α-5 and α-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow. PMID:24086246

  1. Effect of oral calcium and calcium + fluoride treatments on mouse bone properties during suspension

    NASA Technical Reports Server (NTRS)

    Simske, S. J.; Luttges, M. W.; Allen, K. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The bone effects of oral dosages of calcium chloride with or without supplementary sodium fluoride were assessed in antiorthostatically suspended mice. Two calcium dosages were used to replace half (3.1 mM) or all(6.3 mM) of the dietary calcium lost due to reduced food intake by the suspended mice. Two groups of 6.3 mM CaCl2-treated mice were additionally treated with 0.25 or 2.5 mM NaF. The results indicate that supplementation of the mouse drinking water with calcium salts prevents bone changes induced by short-term suspension, while calcium salts in combination with fluoride are less effective as fluoride dosage increases. However, the calcium supplements change the relationship between the femur mechanical properties and the mineral composition of the bone. Because of this, it appears that oral calcium supplements are effective through a mechanism other than simple dietary supplementation and may indicate a dependence of bone consistency on systemic and local fluid conditions.

  2. Micrometastases in bone marrow in patients with primary breast cancer: evaluation as an early predictor of bone metastases.

    PubMed Central

    Mansi, J L; Berger, U; Easton, D; McDonnell, T; Redding, W H; Gazet, J C; McKinna, A; Powles, T J; Coombes, R C

    1987-01-01

    The bone marrow of 307 patients with primary breast cancer was examined for tumour cells by immunocytochemistry using an antiserum to epithelial membrane antigen. Micrometastases were found in 81 cases (26.4%) and their presence was related to various poor prognostic factors: spread to lymph nodes, vascular invasion, T stage, and pathological size. The median duration of follow up was 28 months. Seventy five patients relapsed, 60 at distant sites. Of these 60 patients, 26 had micrometastases detected at presentation and 34 were free of micrometastases initially. The relapse free interval was significantly shorter for patients with micrometastases, and these patients had a shorter survival. Analysis of the sites of relapse showed that the test predicted bone metastases only. Thus 10 out of 19 patients (53%) who developed bone metastases at first relapse had micrometastases at presentation compared with only 41 out of 288 patients (14%) who remained free of bone metastases or relapsed in non-skeletal sites. The presence of micrometastases detected at the time of initial surgery in a patient with primary breast cancer is a useful predictor of early relapse in bone and may help in selecting patients for subsequent systemic treatment. Images FIG 1 PMID:3120893

  3. A NEW APPROACH TO PARTIALKNEE ENDOPROSTHESIS IN PRIMARY BONE SARCOMAS

    PubMed Central

    Penna, Valter; Toller, Eduardo Areas; Pinheiro, Carla; Becker, Ricardo Gehrke

    2015-01-01

    Partial knee endoprosthesis to bone sarcomas resections seems to be a good solution to treat this immature skeletal patients. The purpose of this study is to evaluate the functional score in fourteen patients, advantages and the technique indications. Methods: Retrospective analysis was done to assess in this group of patients the functional evolution and the possible complications of the procedure. 14 patients between 10 and 22 years functionally evaluated in Ennekin/ISOLS (International Society of Limb Salvage) criteria, being all of them operated in the same institution by the same surgeon. Were used distal femur and proximal tibia partial endoprosthesis. Results: General analysis demonstrated that the functional results were over than 67 percent (ISOLS criteria) in 78,6 percent of the patients, being considered excellent. 21,4 percent were considered good results, being between 50 and 66 percent. Bone storage was preserved when avoiding the adjacent segment resection. Surgery time was not prolonged in ligament reconstruction. Conclusion: Knee partial endoprosthesis are less damage to bone storage in young patients. The critics about the bad functional results are being supplied by new surgical techniques, excellent rehabilitation protocols, implants technology and the consequent learning curve. This option of treatment permits the preservation of healthy bone and provides the possibility of a revision replacement less aggressive. PMID:26998452

  4. Visual impairment in an optineurin mouse model of primary open-angle glaucoma.

    PubMed

    Tseng, Henry C; Riday, Thorfinn T; McKee, Celia; Braine, Catherine E; Bomze, Howard; Barak, Ian; Marean-Reardon, Carrie; John, Simon W M; Philpot, Benjamin D; Ehlers, Michael D

    2015-06-01

    Primary open-angle glaucoma (POAG) is characterized by progressive neurodegeneration of retinal ganglion cells (RGCs). Why RGCs degenerate in low-pressure POAG remains poorly understood. To gain mechanistic insights, we developed a novel mouse model based on a mutation in human optineurin associated with hereditary, low-pressure POAG. This mouse improves the design and phenotype of currently available optineurin mice, which showed high global overexpression. Although both 18-month-old optineurin and nontransgenic control mice showed an age-related decrease in healthy axons and RGCs, the expression of mutant optineurin enhanced axonal degeneration and decreased RGC survival. Mouse visual function was determined using visual evoked potentials, which revealed specific visual impairment in contrast sensitivity. The E50K optineurin transgenic mouse described here exhibited clinical features of POAG and may be useful for mechanistic dissection of POAG and therapeutic development. PMID:25818176

  5. High extracellular calcium affects osteoclastogenesis in mouse bone marrow cell culture.

    PubMed

    Takahashi, Etsuko; Mukohyama, Hitoshi; Aoki, Kazuhiro; Duarte, Wagner R; Lerner, Ulf H; Ohya, Keiichi; Omura, Ken; Kasugai, Shohei

    2002-12-01

    The effects of high extracellular calcium (high Ca) in the local microenvironment on osteoclasts, osteoclast progenitors and stromal cells are not fully understood. We examined high Ca effect on osteoclastogenesis in mouse bone marrow cell culture. Mouse bone marrow cells were cultured for up to 6 days in the medium supplemented with 1, 25(OH)2 vitamin D3 (D3). High Ca treatment at the early stage of culture (the initial 24 hours) reduced the number of tartrate resistant acid phosphatase-positive multinuclear cells (TRAP(+)MNCs). This treatment slightly up-regulated the mRNA expressions of receptor activator of NF-(B ligand (RANKL), RANK and osteoprotegerin (OPG). This inhibitory effect on the formation of TRAP(+)MNCs was recovered by RANKL. In contrast, high Ca treatment at the later stage of osteoclastogenesis (the last 2 days of culture) stimulated the formation of TRAP(+)MNCs, increased RANKL and RANK mRNA expressions and decreased OPG mRNA. High Ca at neither the early nor the later stage of culture affected the total number of adherent cells and the mRNA expression of alkaline phosphatase and osteopontin. In conclusion, high Ca affects osteoclastogenesis in a manner depending on the stage of osteoclastogenesis, which is partly mediated via the RANKL-RANK-OPG regulatory system. PMID:12641381

  6. AN INVESTIGATION OF THE MINERAL IN DUCTILE AND BRITTLE CORTICAL MOUSE BONE

    PubMed Central

    Rodriguez-Florez, Naiara; Garcia-Tunon, Esther; Mukadam, Quresh; Saiz, Eduardo; Oldknow, Karla J.; Farquharson, Colin; Millán, José Luis; Boyde, Alan; Shefelbine, Sandra J.

    2015-01-01

    Bone is a strong and tough material composed of apatite mineral, organic matter and water. Changes in composition and organization of these building blocks affect bone’s mechanical integrity. Skeletal disorders often affect bone’s mineral phase, either by variations in the collagen or directly altering mineralization. The aim of the current study was to explore the differences in the mineral of brittle and ductile cortical bone at the mineral (nm) and tissue (µm) levels using two mouse phenotypes. Osteogenesis imperfecta murine (oim−/−) mice were used to model brittle bone; PHOSPHO1 mutants (Phospho1−/−) had ductile bone. They were compared to their respective wild-type controls. Femora were defatted and ground to powder to measure average mineral crystal size using X-ray diffraction (XRD), and to monitor the bulk mineral to matrix ratio via thermogravimetric analysis (TGA). XRD scans were run after TGA for phase identification, to assess the fractions of hydroxyapatite and β-tricalcium phosphate. Tibiae were embedded to measure elastic properties with nanoindentation and the extent of mineralization with backscattered electron microscopy (qbSEM). Interestingly, the mineral of brittle oim−/− and ductile Phospho1−/− bones had many similar characteristics. Both pathology models had smaller apatite crystals, lower mineral to matrix ratio, and showed more thermal conversion to β-tricalcium phosphate than their wild-types, indicating deviations from stoichiometric hydroxyapatite in the original mineral. The degree of mineralization of the bone matrix was different for each strain: oim−/− were hypermineralized, while Phospho1−/− were hypomineralized. However, alterations in the mineral were associated with reduced tissue elastic moduli in both pathologies. Results revealed that despite having extremely different whole bone mechanics, the mineral of oim−/− and Phospho1−/− has several similar trends at smaller length scales. This indicates that alterations from normal crystal size, composition, and structure will reduce the mechanical integrity of bone. PMID:25418329

  7. Primary culture of adult mouse olfactory receptor neurons.

    PubMed

    Liu, N; Shields, C B; Roisen, F J

    1998-06-01

    Olfactory receptor neurons (ORNs) are unique because they can be replaced by stem cells throughout life. Previous studies have demonstrated that adult mouse olfactory epithelium (OE) injured by exposure to ZnSO4 through nasal irrigation can stimulate stem cell mitotic activity in situ, which continues when placed in culture. We report on an improved ZnSO4 delivery method, mist inhalation, which produces more consistent and greater yields of OE cells. Cultures established following this method contained bipolar, nest, fusiform, and giant cells. The bipolar cells usually underwent asymmetric process development. Some bipolar cells reacted positively to neuron-specific antibodies and were immunonegative for keratin and glia-specific proteins, suggesting that they were ORNs. Those that were negative for the neuron-specific proteins may represent either neuron progenitors or olfactory ensheathing cells. The fusiform cells were relatively small and undifferentiated, exposure to brain-derived neurotrophic factor resulted in their decrease and an increase in bipolar cells. Therefore, they might be the stem cells. The nest cells had morphological characteristics of epithelia and bound keratin antibodies. The giant cells had the morphology of epithelial cells but were negative for keratin; they may represent a unique cell population induced by the ZnSO4. These results indicate that the major cell types of intact OE are present in our cultures, and each retains characteristics found in situ. The mist inhalation method provides an in vitro population of adult mitotically active neurons for study. PMID:9628752

  8. Osteopontin antisense deoxyoligonucleotides inhibit bone resorption by mouse osteoclasts in vitro.

    PubMed

    Tani-Ishii, N; Tsunoda, A; Umemoto, T

    1997-08-01

    Osteopontin (OPN) is an acidic phosphoprotein synthesized by osteoblasts and osteoclastic cells that are localized in the mineralized phase of bone matrix. OPN is thought to bind to the vitronection receptor on the osteoclast membrane and regulates bone resorption by the osteoclast. In this study, we investigated whether or not OPN can relate to osteoclast differentiation and bone resorption in a co-culture system. When C57Black/6N mouse bone marrow cells suspended on ivory slices coated with collagen were inoculated onto a MC3T3-G2/PA6 cell layer, colonies containing TRAP(+) mononuclear and multinuclear cells were formed in the presence of 1 alpha, 25-dihydroxyvitamin D3 and dexamethasone. At the end of culture period the number of TRAP(+) osteoclast-like cells were counted and the resorption pits were evaluated by reflected light microscopy. The mRNA of OPN was detected by in situ hybridization. Osteoclast-like cells expressed OPN mRNA. The addition of an OPN antisense oligomer (5' AAT CAC TGC CAA TCT CAT 3') at the start of the co-culture period decreased the number of TRAP(+) cells present after 7 d (30.3 +/- 3.4 vs 56.9 +/- 12.4), and the ratio of mononuclear and multinucleated cells was changed (77.6:23.2 vs 60.8:39.3). The total area of pits per ivory slice was also decreased by adding the OPN antisense oligomer (246813 vs 303139 microns2). These results showed that OPN can be an important mechanism for regulating differentiation and bone resorption. PMID:9379315

  9. Therapeutic effects of mouse bone marrow-derived clonal mesenchymal stem cells in a mouse model of inflammatory bowel disease.

    PubMed

    Park, Jin Seok; Yi, Tac-Ghee; Park, Jong-Min; Han, Young Min; Kim, Jun-Hyung; Shin, Dong-Hee; Tak, Seon Ji; Lee, Kyuheon; Lee, Youn Sook; Jeon, Myung-Shin; Hahm, Ki-Baik; Song, Sun U; Park, Seok Hee

    2015-11-01

    Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-α, IFN-γ, IL-1β, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-κB activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy. PMID:26566304

  10. Therapeutic effects of mouse bone marrow-derived clonal mesenchymal stem cells in a mouse model of inflammatory bowel disease

    PubMed Central

    Park, Jin Seok; Yi, Tac-Ghee; Park, Jong-Min; Han, Young Min; Kim, Jun-Hyung; Shin, Dong-Hee; Tak, Seon Ji; Lee, Kyuheon; Lee, Youn Sook; Jeon, Myung-Shin; Hahm, Ki-Baik; Song, Sun U; Park, Seok Hee

    2015-01-01

    Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-?, IFN-?, IL-1?, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-?B activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy. PMID:26566304

  11. Effects of Tanshinone IIA on osteogenic differentiation of mouse bone marrow mesenchymal stem cells.

    PubMed

    Qian, Kejun; Xu, Huazhong; Dai, Teng; Shi, Keqing

    2015-11-01

    Tanshinone IIA (TSA) is a lipophilic diterpene purified from the Chinese herb Danshen, which exhibits potent antioxidant and anti-inflammatory properties. Effect of TSA remains largely uninvestigated on the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs), which are widely used in cell-based therapy of bone diseases. In the present study, both ALP activity at day 7 and calcium content at day 24 were upregulated during the osteogenesis of mouse BM-MSCs treated with TSA (1 and 5 μM), demonstrating that it promoted the osteogenesis at both early and late stages. We found that TSA promoted osteogenesis and inhibited osteoclastogenesis, evident by RT-PCR analysis of osteogenic marker gene expressions. However, osteogenesis was inhibited by TSA at 20 μM. We further revealed that TSA (1 and 5 μM) upregulated BMP and Wnt signaling. Co-treatment with Wnt inhibitor DKK-1 or BMP inhibitor noggin significantly decreased the TSA-promoted osteogenesis, indicating that upregulation of BMP and Wnt signaling plays a significant role and contributes to the TSA-promoted osteogenesis. Of clinical interest, our study suggests TSA as a promising therapeutic strategy during implantation of BM-MSCs for a more effective treatment of bone diseases. PMID:26231349

  12. Tumor-targeting Salmonella typhimurium A1-R inhibits human prostate cancer experimental bone metastasis in mouse models

    PubMed Central

    Toneri, Makoto; Miwa, Shinji; Zhang, Yong; Hu, Cameron; Yano, Shuya; Matsumoto, Yasunori; Bouvet, Michael; Nakanishi, Hayao; Hoffman, Robert M.; Zhao, Ming

    2015-01-01

    Bone metastasis is a frequent occurrence in prostate cancer patients and often is lethal. Zoledronic acid (ZOL) is often used for bone metastasis with limited efficacy. More effective models and treatment methods are required to improve the outcome of prostate cancer patients. In the present study, the effects of tumor-targeting Salmonella typhimurium A1-R were analyzed in vitro and in vivo on prostate cancer cells and experimental bone metastasis. Both ZOL and S. typhimurium A1-R inhibited the growth of PC-3 cells expressing red fluorescent protien in vitro. To investigate the efficacy of S. typhimurium A1-R on prostate cancer experimental bone metastasis, we established models of both early and advanced stage bone metastasis. The mice were treated with ZOL, S. typhimurium A1-R, and combination therapy of both ZOL and S. typhimurium A1-R. ZOL and S. typhimurium A1-R inhibited the growth of solitary bone metastases. S. typhimurium A1-R treatment significantly decreased bone metastasis and delayed the appearance of PC-3 bone metastases of multiple mouse models. Additionally, S. typhimurium A1-R treatment significantly improved the overall survival of the mice with multiple bone metastases. The results of the present study indicate that S. typhimurium A1-R is useful to prevent and inhibit prostate cancer bone metastasis and has potential for future clinical use in the adjuvant setting. PMID:26431498

  13. Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis.

    PubMed

    Canon, Jude R; Roudier, Martine; Bryant, Rebecca; Morony, Sean; Stolina, Marina; Kostenuik, Paul J; Dougall, William C

    2008-01-01

    Bone metastases cause severe skeletal morbidity including fractures and hypercalcemia. Tumor cells in bone induce activation of osteoclasts, which mediate bone resorption and release of growth factors from bone matrix, resulting in a "vicious cycle" of bone breakdown and tumor proliferation. Receptor activator of NF-kappaB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival, and is blocked by a soluble decoy receptor, osteoprotegerin (OPG). In human malignancies that metastasize to bone, dysregulation of the RANK/RANKL/OPG pathway can increase the RANKL:OPG ratio, a condition which favors excessive osteolysis. In a mouse model of bone metastasis, RANKL protein levels in MDA-MB-231 (MDA-231) tumor-bearing bones were significantly higher than tumor-free bones. The resulting tumor-induced osteoclastogenesis and osteolysis was dose-dependently inhibited by recombinant OPG-Fc treatment, supporting the essential role for RANKL in this process. Using bioluminescence imaging in a mouse model of metastasis, we monitored the anti-tumor efficacy of RANKL inhibition on MDA-231 human breast cancer cells in a temporal manner. Treatment with OPG-Fc in vivo inhibited growth of MDA-231 tumor cells in bony sites when given both as a preventative (dosed day 0) and as a therapeutic agent for established bone metastases (dosed day 7). One mechanism by which RANKL inhibition reduced tumor burden appears to be indirect through inhibition of the "vicious cycle" and involved an increase in tumor cell apoptosis, as measured by active caspase-3. Here, we demonstrate for the first time that OPG-Fc treatment of mice with established bone metastases resulted in an overall improvement in survival. PMID:18064531

  14. Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts.

    PubMed

    Jeong, Ji-Cheon; Kang, Sung-Koo; Youn, Cheol-Ho; Jeong, Chang-Whan; Kim, Hyung-Min; Lee, Young-Choon; Chang, Young-Chae; Kim, Cheorl-Ho

    2003-11-01

    In the traditional Korean medicine, Drynariae Rhizoma (DR) [Drynaria fortunei (kunze) J. Sm] has been reported as a good enhancer for bone healing. In this experiment, we investigate the effects of DR on bone resorption using the bone cells culture. Different concentrations of crude extract of DR were added to mouse bone cells culture. The mitochondria activity of the bone cells after exposure was determined by colorimetric MTT assay. It was demonstrated that DR has potential effects on the bone cells culture without any cytotoxicity. The most effective concentration of DR on bone cells was 100 micro g/ml. On the other hand, cathepsin K (Cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. In this study, Mouse long bone cells including osteoclasts and osteoblast were treated with the PI3-kinase inhibitor, wortmannin (WT), and a specific inhibitor of protein kinase C (PKC), calphostin C. Although WT prevented the osteoclast-mediated intracellular processing of Cat K, calphostin C did not. Similarly, treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extracts prevented the intracellular maturation of Cat K, suggesting that DR may disrupt the intracellular trafficking of pro Cat K. This is similar to that of WT. Since secreted proenzymes have the potential to reenter the cell via mannose-6-phosphate (M6P) receptor, to prevent this possibility, we tested WT and DR in the absence or presence of M6P. Inhibition of Cat K processing by WT or DR was observed in a dose-dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of WT and DR. DR dose-dependently inhibited in vitro bone resorption with a potency similar to that observed for inhibition of Cat K processing. PMID:14555293

  15. Guidelines for histopathological specimen examination and diagnostic reporting of primary bone tumours

    PubMed Central

    2011-01-01

    This review is intended to provide histopathologists with guidelines for clinical assessment, specimen handling and diagnostic reporting of benign and malignant primary bone tumours. Information from radiology, surgical, oncology and other clinical colleagues involved in the diagnosis and treatment of primary bone tumours should be properly assessed before undertaking a structured approach to specimen handling and histological reporting. This ensures that the information needed for planning appropriate treatment of these complex tumours is provided. Consistency in diagnostic evaluation with respect to both terminology and report content facilitates liaison at multidisciplinary bone tumour meetings and collaboration between cancer units and networks, as well as providing a common database for audit of the clinical, radiological and pathological aspects of bone tumours. PMID:22613930

  16. A Mouse Model for Studying Nutritional Programming: Effects of Early Life Exposure to Soy Isoflavones on Bone and Reproductive Health

    PubMed Central

    Ward, Wendy E.; Kaludjerovic, Jovana; Dinsdale, Elsa C.

    2016-01-01

    Over the past decade, our research group has characterized and used a mouse model to demonstrate that “nutritional programming” of bone development occurs when mice receive soy isoflavones (ISO) during the first days of life. Nutritional programming of bone development can be defined as the ability for diet during early life to set a trajectory for better or compromised bone health at adulthood. We have shown that CD-1 mice exposed to soy ISO during early neonatal life have higher bone mineral density (BMD) and greater trabecular inter-connectivity in long bones and lumbar spine at young adulthood. These skeletal sites also withstand greater forces before fracture. Because the chemical structure of ISO resembles that of 17-β-estradiol and can bind to estrogen receptors in reproductive tissues, it was prudent to expand analyses to include measures of reproductive health. This review highlights aspects of our studies in CD-1 mice to understand the early life programming effects of soy ISO on bone and reproductive health. Preclinical mouse models can provide useful data to help develop and guide the design of studies in human cohorts, which may, depending on findings and considerations of safety, lead to dietary interventions that optimize bone health. PMID:27187422

  17. Radiation sensitivity and cycling status of mouse bone marrow prothymocytes and day 8 colony forming units spleen (CFUs)

    SciTech Connect

    Boersma, W.J.

    1983-11-01

    Mouse bone marrow prothymocytes as determined in an in vivo thymus regeneration assay have an in vitro gamma radiation sensitivity which is different from that of spleen colony forming cells (CFUs). Determination of Do according to in vivo irradiation revealed similar but insignificant differences. Prothymocytes in normal bone marrow maintain a low but slightly different proliferative state as compared to CFUs, according to determinations using the /sup 3/H-TdR suicide technique. In regenerating bone marrow prothymocytes were found to be sensitive to an inhibitory effect of in vitro incubation with cold thymidine. CFUs and normal bone marrow prothymocytes were not affected by cold thymidine. Taking into account the cold thymidine effect it can be concluded that prothymocytes and CFUs in regenerating bone marrow are fully in cycle. These results are best explained when prothymocytes and CFUs are considered to be different cells.

  18. An in vivo mouse model of primary dysmenorrhea

    PubMed Central

    YANG, Lu; CAO, Zhengyu; YU, Boyang; CHAI, Chengzhi

    2015-01-01

    Primary dysmenorrhea (PD) is a common gynecological disorder. Hitherto, animal models which recapitulate clinical features of PD have not been fully established. We aimed to examine whether a pain model in mice could mimic the clinic features of PD. After pretreated with estradiol benzoate (1 mg/kg/day) intraperitoneally (i.p.) for 3 consecutive days, non-pregnant female Imprinting Control Region mice (6–8 weeks old) was injected with 0.4 U of oxytocin to induce the stretching or writhing response which was recorded for a time period of 30 min. During the writhing period, the uterine artery blood flow alterations were examined by Doppler ultrasound detection. After writhing test, the uterine morphological changes were observed by hematoxylin and eosin (H&E) staining histopathology. In addition, enzyme-linked immunosorbent assay kit was used to measure the levels of prostaglandins F2α/prostaglandins E2 (PGF2α/PGE2) and TXB2 (a metabolite of TXA2)/6-keto-PGF1α (a metabolite of PGI2) in the uterine tissue homogenates and plasma, respectively. Western blot analyses were performed to determine the expressions of oxytocin receptor (OTR), beta2-adrenergic receptor (beta2-AR), and cyclooxygenase-2 (COX-2) in uterine, which are responsible for the uterine contraction. The writhing response only occurred in the estrogen pretreated female mice. The area of uterine myometrium significantly decreased along with the increased thickness in the oxytocin-induced estrogen pretreated mice model. The uterine artery blood flow velocity dropped, while the pulsatility index and resistance index slightly increased after the injection of oxytocin. The PGF2α/PGE2 level significantly increased and the plasma TXB2/6-keto-PGF1α level significantly enhanced. Compared with the control group, the uterine histopathology demonstrated moderate to severe edema of endometrium lamina propria. In consistent with the uterine morphological changes, a significant reduction of beta2-AR and a significant increase of OTR and COX-2 in the uterine tissue were observed. The writhing response was caused by the abnormal contraction of uterus. The uterine spasm and ischemia changes of oxytocin-induced estrogen pretreated female mice model were similar to the pathology of human PD. We reported an in vivo mice model, which can be used to study PD and for clinical therapeutic evaluations. PMID:25912320

  19. The role of the SIBLING, Bone Sialoprotein in skeletal biology - Contribution of mouse experimental genetics.

    PubMed

    Bouleftour, Wafa; Juignet, Laura; Bouet, Guenaelle; Granito, Renata Neves; Vanden-Bossche, Arnaud; Laroche, Norbert; Aubin, Jane E; Lafage-Proust, Marie-Hélène; Vico, Laurence; Malaval, Luc

    2016-01-01

    Bone Sialoprotein (BSP) is a member of the "Small Integrin-Binding Ligand N-linked Glycoproteins" (SIBLING) extracellular matrix protein family of mineralized tissues. BSP has been less studied than other SIBLING proteins such as Osteopontin (OPN), which is coexpressed with it in several skeletal cell types. Here we review the contribution of genetically engineered mice (BSP gene knockout and overexpression) to the understanding of the role of BSP in the bone organ. The studies made so far highlight the role of BSP in skeletal mineralization, as well as its importance for proper osteoblast and osteoclast differentiation and activity, most prominently in primary/repair bone. The absence of BSP also affects the local environment of the bone tissue, in particular hematopoiesis and vascularization. Interestingly, lack of BSP induces an overexpression of OPN, and the cognate protein could be responsible for some aspects of the BSP gene knockout skeletal phenotype, while replacing BSP for some of its functions. Such interplay between the partly overlapping functions of SIBLING proteins, as well as the network of cross-regulations in which they are involved should now be the focus of further work. PMID:26763578

  20. [Effect of osteogenically and adipogenically differentiated bone mesenchymal stem cells from mouse on osteoclast formation].

    PubMed

    Zhu, Heng; Liu, Yuan-Lin; Chen, Ji-De; Li, Hong; Liu, Yu-Xiao; Xu, Fen-Fen; Jiang, Xiao-Xia; Zhang, Yi; Mao, Ning

    2012-10-01

    This study was purposed to investigate the regulatory effects of differentiating mesenchymal stem cells (MSC) on osteoclast formation. The MSC from mouse compact bones were cultured and induced into osteoblasts and adipocytes for one week. To test their regulatory effect on osteoclastogenesis, osteogenically differentiated and adipogenically differentiated MSC were co-cultured with CD11b(+) monocytes and osteoclasts were identified with in situ tartrate-resistant acid phosphatase (TRAP) staining. The results showed that differentiated MSC supported osteoclastogenesis but the osteoclast supporting capacity of osteogenically differentiated MSC decreased as compared with undifferentiated MSC. More interestingly, the adipogenically differentiated MSC significantly promoted osteoclasts formation when co-cultured with monocytes. It is concluded that the regulatory effect of MSC on osteoclast formation has changed while they have differentiated into different types of cells. The findings indicate that MSC may exert alternative effect on osteoclastogenesis by differentiation to descendant cells. PMID:23114145

  1. Mechanical Unloading of Mouse Bone in Microgravity Significantly Alters Cell Cycle Gene Set Expression

    NASA Astrophysics Data System (ADS)

    Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Kaplan, Warren; Burns, Brnedan

    2012-07-01

    Spaceflight factors, including microgravity and space radiation, have many detrimental short-term effects on human physiology, including muscle and bone degradation, and immune system dysfunction. The long-term progression of these physiological effects is still poorly understood, and a serious concern for long duration spaceflight missions. We hypothesized that some of the degenerative effects of spaceflight may be caused in part by an inability of stem cells to proliferate and differentiate normally resulting in an impairment of tissue regenerative processes. Furthermore, we hypothesized that long-term bone tissue degeneration in space may be mediated by activation of the p53 signaling network resulting in cell cycle arrest and/or apoptosis in osteoprogenitors. In our analyses we found that spaceflight caused significant bone loss in the weight-bearing bones of mice with a 6.3% reduction in bone volume and 11.9% decrease in bone thickness associated with increased osteoclastic activity. Along with this rapid bone loss we also observed alterations in the cell cycle characterized by an increase in the Cdkn1a/p21 cell cycle arrest molecule independent of Trp53. Overexpression of Cdkn1a/p21 was localized to osteoblasts lining the periosteal surface of the femur and chondrocytes in the head of the femur, suggesting an inhibition of proliferation in two key regenerative cell types of the femur in response to spaceflight. Additionally we found overexpression of several matrix degradation molecules including MMP-1a, 3 and 10, of which MMP-10 was localized to osteocytes within the shaft of the femur. This, in conjunction with 40 nm resolution synchrotron nano-Computed Tomography (nano-CT) observations of an increase in osteocyte lacunae cross-sectional area, perimeter and a decrease in circularity indicates a potential role for osteocytic osteolysis in the observed bone degeneration in spaceflight. To further investigate the genetic response of bone to mechanical unloading in spaceflight, we conducted genome wide microarray analysis of total RNA isolated from the mouse pelvis. Specifically, 16 week old mice were subjected to 15 days spaceflight onboard NASA's STS-131 space shuttle mission. The pelvis of the mice was dissected, the bone marrow was flushed and the bones were briefly stored in RNAlater. The pelvii were then homogenized, and RNA was isolated using TRIzol. RNA concentration and quality was measured using a Nanodrop spectrometer, and 0.8% agarose gel electrophoresis. Samples of cDNA were analyzed using an Affymetrix GeneChip\\S Gene 1.0 ST (Sense Target) Array System for Mouse and GenePattern Software. We normalized the ST gene arrays using Robust Multichip Average (RMA) normalization, which summarizes perfectly matched spots on the array through the median polish algorithm, rather than normalizing according to mismatched spots. We also used Limma for statistical analysis, using the BioConductor Limma Library by Gordon Smyth, and differential expression analysis to identify genes with significant changes in expression between the two experimental conditions. Finally we used GSEApreRanked for Gene Set Enrichment Analysis (GSEA), with Kolmogorov-Smirnov style statistics to identify groups of genes that are regulated together using the t-statistics derived from Limma. Preliminary results show that 6,603 genes expressed in pelvic bone had statistically significant alterations in spaceflight compared to ground controls. These prominently included cell cycle arrest molecules p21, and p18, cell survival molecule Crbp1, and cell cycle molecules cyclin D1, and Cdk1. Additionally, GSEA results indicated alterations in molecular targets of cyclin D1 and Cdk4, senescence pathways resulting from abnormal laminin maturation, cell-cell contacts via E-cadherin, and several pathways relating to protein translation and metabolism. In total 111 gene sets out of 2,488, about 4%, showed statistically significant set alterations. These alterations indicate significant impairment of normal cellular function in the mechanically unloaded environment of space and could provide important genetic insight into the observed uncoupling of bone formation and resorption in space.

  2. Primary hyperparathyroidism presenting with acute pancreatitis and asymptomatic bone involvement.

    PubMed

    Saif, Aasem

    2015-01-01

    A 15-year-old female patient presented to the emergency room with vomiting and abdominal pain. She had two similar attacks in the previous three months both of them were diagnosed as pancreatitis in two different hospitals. On admission, her serum calcium and parathyroid hormone levels were very high. CT scan revealed left inferior parathyroid adenoma. Investigations to rule out possible multiple endocrine neoplasia were all negative. The patient was managed by intravenous fluids and furosemide to lower her serum calcium level. Then, left inferior parathyroidectomy was done. Postoperatively, the patient had hungry bone syndrome with severe hypocalcaemia and was managed by intravenous calcium infusion for five days in the intensive care unit. Later, she was kept on oral calcium and vitamin D supplementation. She became symptom-free and her serum calcium improved gradually. PMID:26604950

  3. Primary hyperparathyroidism presenting with acute pancreatitis and asymptomatic bone involvement

    PubMed Central

    Saif, Aasem

    2015-01-01

    Summary A 15-year-old female patient presented to the emergency room with vomiting and abdominal pain. She had two similar attacks in the previous three months both of them were diagnosed as pancreatitis in two different hospitals. On admission, her serum calcium and parathyroid hormone levels were very high. CT scan revealed left inferior parathyroid adenoma. Investigations to rule out possible multiple endocrine neoplasia were all negative. The patient was managed by intravenous fluids and furosemide to lower her serum calcium level. Then, left inferior parathyroidectomy was done. Postoperatively, the patient had hungry bone syndrome with severe hypocalcaemia and was managed by intravenous calcium infusion for five days in the intensive care unit. Later, she was kept on oral calcium and vitamin D supplementation. She became symptom-free and her serum calcium improved gradually. PMID:26604950

  4. Induction of bone formation in biphasic calcium phosphate scaffolds by bone morphogenetic protein-2 and primary osteoblasts.

    PubMed

    Strobel, L A; Rath, S N; Maier, A K; Beier, J P; Arkudas, A; Greil, P; Horch, R E; Kneser, U

    2014-03-01

    Bone tissue engineering strategies mainly depend on porous scaffold materials. In this study, novel biphasic calcium phosphate (BCP) matrices were generated by 3D-printing. High porosity was achieved by starch consolidation. This study aimed to characterise the porous BCP-scaffold properties and interactions of osteogenic cells and growth factors under in vivo conditions. Five differently treated constructs were implanted subcutaneously in syngeneic rats: plain BCP constructs (group A), constructs pre-treated with BMP-2 (group B; 1.6 µg BMP-2 per scaffold), seeded with primary osteoblasts (OB) (group C), seeded with OB and BMP-2 (group D) and constructs seeded with OB and pre-cultivated in a flow bioreactor for 6 weeks (group E). After 2, 4 and 6 weeks, specimens were explanted and subjected to histological and molecular biological analyses. Explanted scaffolds were invaded by fibrovascular tissue without significant foreign body reactions. Morphometric analysis demonstrated significantly increased bone formation in samples from group D (OB + BMP-2) compared to all other groups. Samples from groups B-E displayed significant mRNA expression of bone-specific genes after 6 weeks. Pre-cultivation in the flow bioreactor (group E) induced bone formation comparable with group B. In this study, differences in bone distribution between samples with BMP-2 or osteoblasts could be observed. In conclusion, combination of osteoblasts and BMP-2 synergistically enhanced bone formation in novel ceramic scaffolds. These results provide the basis for further experiments in orthotopic defect models with a focus on future applications in orthopaedic and reconstructive surgery. PMID:22740314

  5. Pulsed focused ultrasound treatment of muscle mitigates paralysis-induced bone loss in the adjacent bone: a study in a mouse model.

    PubMed

    Poliachik, Sandra L; Khokhlova, Tatiana D; Wang, Yak-Nam; Simon, Julianna C; Bailey, Michael R

    2014-09-01

    Bone loss can result from bed rest, space flight, spinal cord injury or age-related hormonal changes. Current bone loss mitigation techniques include pharmaceutical interventions, exercise, pulsed ultrasound targeted to bone and whole body vibration. In this study, we attempted to mitigate paralysis-induced bone loss by applying focused ultrasound to the midbelly of a paralyzed muscle. We employed a mouse model of disuse that uses onabotulinumtoxinA-induced paralysis, which causes rapid bone loss in 5 d. A focused 2 MHz transducer applied pulsed exposures with pulse repetition frequency mimicking that of motor neuron firing during walking (80 Hz), standing (20 Hz), or the standard pulsed ultrasound frequency used in fracture healing (1 kHz). Exposures were applied daily to calf muscle for 4 consecutive d. Trabecular bone changes were characterized using micro-computed tomography. Our results indicated that application of certain focused pulsed ultrasound parameters was able to mitigate some of the paralysis-induced bone loss. PMID:24857416

  6. Primary bone microanatomy records developmental aspects of life history in catarrhine primates.

    PubMed

    McFarlin, Shannon C; Terranova, Carl J; Zihlman, Adrienne L; Bromage, Timothy G

    2016-03-01

    A central challenge in human origins research is to understand how evolution has shaped modern human life history. As fossilized remains of our ancestors provide the only direct evidence for life history evolution, efforts to reconstruct life history in paleontological contexts have focused on hard tissues, particularly on dental development. However, among investigators of other vertebrate groups, there is a long tradition of examining primary bone microstructure to decipher growth rates and maturational timing, based on an empirical relationship between the microanatomy of primary bone and the rate at which it is deposited. We examined ontogenetic variation in primary bone microstructure at the midshaft femur of Chlorocebus aethiops, Hylobates lar, and Pan troglodytes to test whether tissue type proportions vary in accordance with predictions based on body mass growth patterns described previously. In all taxa, younger age classes were characterized by significantly higher percent areas of fibro-lamellar and/or parallel-fibered tissues, while older age classes showed significantly higher proportions of lamellar bone. In prior experimental studies, fibro-lamellar and parallel-fibered tissue types have been associated with faster depositional rates than lamellar bone. Principal components analysis revealed differences among taxa in the timing of this transition, and in the particular tissue types observed among individuals of similar dental emergence status. Among M1 and M2 age classes, higher proportions of parallel-fibered and fibro-lamellar tissues were observed in those taxa characterized by reportedly faster body mass growth rates. Further, persistence of fibro-lamellar tissue throughout DECID, M1 and M2 age classes in chimpanzees contrasts with the pattern reported previously for modern humans. Despite the necessary limitations of our cross-sectional study design and the secondary remodeling of bone in primates, large areas of primary bone remain intact and represent a valuable and independent source of information about the evolution of growth and development in the fossil record. PMID:26989017

  7. Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan.

    PubMed

    Cuong, Dang Van; Kim, Hyoung Kyu; Marquez, Jubert; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin

    2016-03-01

    Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca(2+), which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 µg/ml BG, 100 µg/ml peptidoglycan (PGN), or 10 µM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca(2+) uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca(2+) uniporter has an important regulatory role in BG-induced mast cell degranulation. PMID:26937218

  8. Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan

    PubMed Central

    Cuong, Dang Van; Kim, Hyoung Kyu; Marquez, Jubert; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo

    2016-01-01

    Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 µg/ml BG, 100 µg/ml peptidoglycan (PGN), or 10 µM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation. PMID:26937218

  9. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    SciTech Connect

    Kawahara, Takeshi

    2012-11-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed {alpha}, {beta}, and {gamma} subunits of high-affinity immunoglobulin E (IgE) receptor (Fc{epsilon}RI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-{alpha}, and cyclooxygenase 2, and production of prostaglandin D{sub 2} and leukotriene C{sub 4} in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-{alpha} expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on Fc{epsilon}RI- and TLR-mediated effector functions of mast cells.

  10. Cadmium chloride strongly enhances cyclophosphamide-induced chromosome aberrations in mouse bone marrow cells

    SciTech Connect

    Pandurangarao, V.L.; Blazina, S.; Bherje, R.

    1997-10-01

    Earlier we reported that a single 5 mg cadmium chloride (CdCl{sub 2})/kg ip dose enhanced chromosome aberrations (ca) with 50 mg/kg cyclophosphamide (CP) in mouse bone marrow cells. In this report groups of 4 mice were injected ip with saline, 0.31, 0.62, 1.25, 2.5 or 5.0 mg/kg CdCl{sub 2}, followed by saline injections at 24 h. Other mice similarly uninjected at 0 h were injected with 50 mg/kg CP at 24 h. All the mice were injected ip with 4 mg colchicine/kg at 44 h. At 48 h the bone marrow cells were processed for chromosome spreads. After dissection, visual examination revealed obvious internal hemorrhaging of the testes at 1.25 CdCl{sub 2} mg/kg and higher doses. This effect was not further increased by CP treatment. The lowest ca enhancing dose of CdCl{sub 2} on CP was 0.625 mg/kg. Our hypothesis is that Cd replaces zinc presents in numerous DNA repair enzymes and proteins resulting in diminished repair. Subsequently, the excess of unrepaired DNA damage is seen as chromatid breaks, deletions, fragments and exchanges.

  11. Hypomorphic mutation in mouse Nppc gene causes retarded bone growth due to impaired endochondral ossification

    SciTech Connect

    Tsuji, Takehito Kondo, Eri; Yasoda, Akihiro; Inamoto, Masataka; Kiyosu, Chiyo; Nakao, Kazuwa; Kunieda, Tetsuo

    2008-11-07

    Long bone abnormality (lbab/lbab) is a spontaneous mutant mouse characterized by dwarfism with shorter long bones. A missense mutation was reported in the Nppc gene, which encodes C-type natriuretic peptide (CNP), but it has not been confirmed whether this mutation is responsible for the dwarf phenotype. To verify that the mutation causes the dwarfism of lbab/lbab mice, we first investigated the effect of CNP in lbab/lbab mice. By transgenic rescue with chondrocyte-specific expression of CNP, the dwarf phenotype in lbab/lbab mice was completely compensated. Next, we revealed that CNP derived from the lbab allele retained only slight activity to induce cGMP production through its receptor. Histological analysis showed that both proliferative and hypertrophic zones of chondrocytes in the growth plate of lbab/lbab mice were markedly reduced. Our results demonstrate that lbab/lbab mice have a hypomorphic mutation in the Nppc gene that is responsible for dwarfism caused by impaired endochondral ossification.

  12. Modulation of doxorubicin-induced genotoxicity by Aegle marmelos in mouse bone marrow: a micronucleus study.

    PubMed

    Venkatesh, Ponemone; Shantala, Bellary; Jagetia, Ganesh Chandra; Rao, K Koteshwer; Baliga, Manjeshwar Shrinath

    2007-03-01

    The effect of various concentrations of Aegle marmelos (AME) on the doxorubicin (DOX)-induced genotoxic effects in mice bone marrow was studied. Treatment of mice with different concentrations of DOX resulted in a dose-dependent elevation in the frequency of micronucleated polychromatic (MPCE) as well as normochromatic (MNCE) erythrocytes in mouse bone marrow. The frequencies of MPCE and MNCE increased with scoring time, and the greatest elevation for MPCE was observed at 48 hours post-DOX treatment, whereas a maximum increase in MNCE was observed at 72 hours post-DOX treatment. This increase in MPCE and MNCE was accompanied by a decline in the polychromatic erythrocytes-normochromatic erythrocytes (PCE/NCE) ratio, which showed a DOX-dose-dependent decline. Treatment of mice with 200, 250, 300, 350, and 400 mg/kg body weight of AME, orally once daily for 5 consecutive days before DOX treatment, significantly reduced the frequency of DOX-induced micronuclei accompanied by a significant elevation in the PCE/NCE ratio at all scoring times. The greatest protection against DOX-induced genotoxicity was observed at 350 mg/kg AME. The protection against DOX-induced genotoxicity by AME may be due to inhibition of free radicals and increased antioxidant status. PMID:17351026

  13. [Clinical guidelines for bone disease in primary biliary cirrhosis].

    PubMed

    Ishibashi, Hiromi; Komori, Atsumasa

    2015-11-01

    Primary biliary cirrhosis(PBC)is a chronic cholestatic liver disease and the association of osteoporosis is high. In this paper, the practical guidelines for PBC of Japan as well as those of America(AASLD)and Europe(EASL)are mentioned. Description of each guideline is essentially the same;taking sufficient calcium(1,000~1,200 mg/day)with vitamin D and weight-bearing exercise, and thereafter medication such as alendronate is recommended. PMID:26503873

  14. Raman spectroscopy detects deterioration in biomechanical properties of bone in a glucocorticoid-treated mouse model of rheumatoid arthritis

    NASA Astrophysics Data System (ADS)

    Maher, Jason R.; Takahata, Masahiko; Awad, Hani A.; Berger, Andrew J.

    2011-08-01

    Although glucocorticoids are frequently prescribed for the symptomatic management of inflammatory disorders such as rheumatoid arthritis, extended glucocorticoid exposure is the leading cause of physician-induced osteoporosis and leaves patients at a high risk of fracture. To study the biochemical effects of glucocorticoid exposure and how they might affect biomechanical properties of the bone, Raman spectra were acquired from ex vivo tibiae of glucocorticoid- and placebo-treated wild-type mice and a transgenic mouse model of rheumatoid arthritis. Statistically significant spectral differences were observed due to both treatment regimen and mouse genotype. These differences are attributed to changes in the overall bone mineral composition, as well as the degree of phosphate mineralization in tibial cortical bone. In addition, partial least squares regression was used to generate a Raman-based prediction of each tibia's biomechanical strength as quantified by a torsion test. The Raman-based predictions were as accurate as those produced by microcomputed tomography derived parameters, and more accurate than the clinically-used parameter of bone mineral density. These results suggest that Raman spectroscopy could be a valuable tool for monitoring bone biochemistry in studies of bone diseases such as osteoporosis, including tests of drugs being developed to combat these diseases.

  15. Improved Quantitative Analysis of Primary Bone Marrow Megakaryocytes Utilizing Imaging Flow Cytometry

    PubMed Central

    Niswander, Lisa M.; McGrath, Kathleen E.; Kennedy, John C.; Palis, James

    2014-01-01

    Life-threatening thrombocytopenia can develop following bone marrow injury due to decreased platelet production from megakaryocytes (MKs). However, the study of primary MKs has been complicated by their low frequency in the bone marrow and by technical challenges presented by their unique maturation properties. More accurate and efficient methods for the analysis of in vivo MKs are needed to enhance our understanding of megakaryopoiesis and ultimately develop new therapeutic strategies for thrombocytopenia. Imaging flow cytometry (IFC) combines the morphometric capabilities of microscopy with the high-throughput analyses of flow cytometry (FC). Here, we investigate the application of IFC on the ImageStreamX platform to the analysis of primary MKs isolated from murine bone marrow. Our data highlight and address technical challenges for conventional FC posed by the wide range of cellular size within the MK lineage as well as the shared surface phenotype with abundant platelet progeny. We further demonstrate that IFC can be used to reproducibly and efficiently quantify the frequency of primary murine MKs in the marrow, both at steady-state and in the setting of radiation-induced bone marrow injury, as well as assess their ploidy distribution. The ability to accurately analyze the full spectrum of maturing MKs in the bone marrow now allows for many possible applications of IFC to enhance our understanding of megakaryopoiesis and platelet production. PMID:24616422

  16. Cancellous impaction bone grafting of acetabular defects in complex primary and revision total hip arthroplasty.

    PubMed

    Patil, Nilesh; Hwang, Katherine; Goodman, Stuart B

    2012-03-01

    The reconstruction of major acetabular bone defects during revision, conversion, and primary total hip arthroplasties (THAs) is challenging. We reviewed a consecutive series of 168 THAs (108 revisions, 8 conversions, and 52 primary THAs) performed by 1 surgeon (S.B.G.) between 1997 and 2008 using impaction bone grafting for acetabular reconstruction. Autograft, cancellous allograft croutons, and demineralized bone matrix were used to fill bone defects as needed. The acetabular bone deficiency was classified according to the American Academy of Orthopaedic Surgeons: type I, segmental deficiency with significant rim defect; type II, cavitary defects medially or posteriorly; type III, combined cavitary and segmental deficiency; type IV, pelvic discontinuity; and type V, arthrodesis. According to this method, 56 hips had type I, 31 hips had type II, 48 hips had type III, and 27 hips had type IV deficiencies. Of the 168 patients, 19 subsequently died of causes unrelated to the THA, and 11 were lost to follow-up. All patients had at least 2 years of follow-up. Average Harris Hip Score improved from 45.5±17.9 preoperatively to 81.1±16.5 postoperatively (P<.05) for revision THAs, from 40.0±11.3 preoperatively to 85.0±12.8 postoperatively (P<.05) for conversion THAs, and from 42.3±14.9 preoperatively to 85.0±12.0 postoperatively (P<.05) for primary THAs. All impaction grafted bone (allograft, autograft, or a combination) incorporated radiographically, thus restoring bone stock. Complications included 1 early infection, which was managed successfully with debridement and liner exchange, and 2 late infections that were managed successfully with staged revision. Two revisions required subsequent re-revision for late loosening. Two hip dislocations occurred, 1 of which required surgical treatment to place a constrained liner. PMID:22385438

  17. Targeted Mutation of Nuclear Bone Morphogenetic Protein 2 Impairs Secondary Immune Response in a Mouse Model

    PubMed Central

    Olsen, Daniel S.; Goar, Wesley A.; Nichols, Brandt A.; Bailey, K. Tyson; Christensen, S. Loyd; Merriam, Kayla R.; Reynolds, Paul R.; Wilson, Eric; Weber, K. Scott; Bridgewater, Laura C.

    2015-01-01

    We recently identified a nuclear variant of the BMP2 growth factor, called nBMP2. In an effort to understand the function of this variant protein, we generated a mouse line in which BMP2 is expressed and functions normally, but nBMP2 is excluded from the nucleus. This novel mutation allows the study of nBMP2 without compromising BMP2 function. To determine whether nBMP2 plays a role in immune function, we performed a series of experiments in which we compared mouse survival, organ weights, immune cells numbers, and bacterial load in wild type and nBmp2NLStm mice following primary and secondary challenges with Staphylococcus aureus. Following primary challenge with S. aureus, wild type and nBmp2NLStm mice showed no differences in survival or bacterial load and generated similar numbers and types of leukocytes, although mutant spleens were smaller than wild type. Secondary bacterial challenge with S. aureus, however, produced differences in survival, with increased mortality seen in nBmp2NLStm mice. This increased mortality corresponded to higher levels of bacteremia in nBmp2NLStm mice and to a reduced enlargement of mutant spleens in response to the secondary infection. Together, these results suggest that the recently described nuclear variant of BMP2 is necessary for efficient secondary immune responses. PMID:26491697

  18. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    SciTech Connect

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    2009-10-30

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  19. Relative Contribution of Trabecular and Cortical Bone to Primary Implant Stability: An In Vitro Model Study.

    PubMed

    Wang, Russell; Eppell, Steven J; Nguyen, Christian; Morris, Nathan

    2016-04-01

    The specific aim of this study was to examine the relative contributions to the implant insertion torque value (ITV) by cortical and trabecular components of an in vitro bone model. Simulated bone blocks of polyurethane were used with 2 densities of foam (0.08 g/cm(3) to mimic trabecular bone and 0.64 g/cm(3) to mimic cortical bone). We have developed a new platform technology to collect data that enables quantitative evaluation of ITV at different implant locations. Seven groups were used to model varying thicknesses of cortical bone over a lower-quality trabecular bone that have clinical significance: a solid 0.08 g/cm(3) block; 1 mm, 2 mm, and 3 mm thick 0.64 g/cm(3) sheets with no underlayer; and 1 mm, 2 mm, and 3 mm thick 0.64 g/cm(3) sheets laminated on top of a 4 cm thick 0.08 g/cm(3) block. The ITVs were recorded as a function of insertion displacement distance. Relative contributions of ITV ranged from 3% to 18% from trabecular bone, and 62% to 74% from cortical bone depending on the thickness of the cortical layer. Inserting an implant into 2-mm and 3-mm cortical layers laminated atop trabecular blocks had a synergistic effect on ITVs. Finally, an implant with a reverse bevel design near the abutment showed final average torque values that were 14% to 34% less than their maximum torque values. This work provides basic quantitative information for clinicians to understand the influence of composite layers of bone in relation to mechanical torque resistances during implant insertion in order to obtain desired primary implant stability. PMID:26103153

  20. Effect of prostaglandins E1, E2, and F2 alpha on osteoclast formation in mouse bone marrow cultures

    SciTech Connect

    Collins, D.A.; Chambers, T.J. )

    1991-02-01

    Prostaglandins (PG) act as direct inhibitors of mature osteoclasts, but although resorption-inhibition is also observed initially PG increase bone resorption in organ culture. This suggests that PG influence bone resorption in organ culture through actions on cell types other than mature osteoclasts. We have therefore tested the effects of PG E1, E2, and F2 alpha on the differentiation of osteoclastic phenotype in mouse bone marrow cultures using bone resorption and calcitonin receptors (CTR) as markers of osteoclastic differentiation. We found that PGE2 (10{sup {minus} 6}-10{sup {minus} 9} M) and PGE1 (10{sup {minus} 6} - 10{sup {minus} 7} M) induced a significant increase in CTR-positive cell numbers, to levels five to eight times those seen in controls and similar to the number induced by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Bone resorption was increased (10{sup {minus} 7} M PGE2 and 10{sup {minus} 6} M PGE1) in association with the increased CTR-positive cell numbers, suggesting that the PG also induced resorptive function. 1,25-(OH)2D3 increased both the number of CTR-positive cells and the extent of resorption per cell; the additional presence of PG did not affect the number of CTR-positive cells but did reduce bone resorption compared with 1,25-(OH)2D3 alone. PGF2 alpha had no significant effect on CTR-positive cell induction or bone resorption. The results suggest that PGE1 and E2 induce osteoclastic differentiation in mouse bone marrow cultures and inhibit the function of the osteoclasts thus formed.

  1. The impact of pathological fractures on therapy outcome in patients with primary malignant bone tumours

    PubMed Central

    Moradi, Babak; Zahlten-Hinguranage, Anita; Lehner, Burkhard

    2009-01-01

    The primary objective of this study was to investigate the implications of pathological fractures on therapy outcome in patients with primary malignant bone tumours and to determine whether limb salvage can be safely performed. A retrospective analysis of 447 patients with primary malignant bone tumours, treated between 1985 and 2005, was performed. Multivariate Cox regression analysis was used to investigate the influence of pathological fractures and further independent variables on survival rate. In 52 of the 447 patients, the primary malignant bone tumour was complicated by a pathological fracture. These fractures were more common in malignant fibrous histiocytoma (MFH) of the bone and in the tumour stages IIa/b and III. Ablative surgery was performed in ten patients and limb salvage surgery in 42 patients. The mortality risk for patients with pathological fractures was significantly increased by a factor of 1.82 (p = 0.015), and overall duration of survival was significantly lower in the fracture group, with a median of 6.2 years (p < 0.00001). In univariate and multivariate analysis, fracture, higher tumour stages and resection margins remained a significant predictor of worse survival. Overall survival, rate of local recurrence and distant metastases were not affected by the type of surgical treatment selected; there was no difference between the patients who underwent limb salvage and those who underwent an amputation. Pathological fracture in patients with primary malignant bone tumours is a predictor of worse survival and significantly increases mortality risk. Reconstructive surgery did not influence the survival rate, showing that limb salvage therapy is safe when adequate resection margins are achieved. PMID:20012861

  2. Lamin B1 protein is required for dendrite development in primary mouse cortical neurons

    PubMed Central

    Giacomini, Caterina; Mahajani, Sameehan; Ruffilli, Roberta; Marotta, Roberto; Gasparini, Laura

    2016-01-01

    Lamin B1, a key component of the nuclear lamina, plays an important role in brain development and function. A duplication of the human lamin B1 (LMNB1) gene has been linked to adult-onset autosomal dominant leukodystrophy, and mouse and human loss-of-function mutations in lamin B1 are susceptibility factors for neural tube defects. In the mouse, experimental ablation of endogenous lamin B1 (Lmnb1) severely impairs embryonic corticogenesis. Here we report that in primary mouse cortical neurons, LMNB1 overexpression reduces axonal outgrowth, whereas deficiency of endogenous Lmnb1 results in aberrant dendritic development. In the absence of Lmnb1, both the length and complexity of dendrites are reduced, and their growth is unresponsive to KCl stimulation. This defective dendritic outgrowth stems from impaired ERK signaling. In Lmnb1-null neurons, ERK is correctly phosphorylated, but phospho-ERK fails to translocate to the nucleus, possibly due to delocalization of nuclear pore complexes (NPCs) at the nuclear envelope. Taken together, these data highlight a previously unrecognized role of lamin B1 in dendrite development of mouse cortical neurons through regulation of nuclear shuttling of specific signaling molecules and NPC distribution. PMID:26510501

  3. Fast kinetics, high frequency oscillations and sub-primary firing range in adult mouse spinal motoneurons

    PubMed Central

    Manuel, M; Iglesias, C; Donnet, M; Leroy, F; Heckman, CJ; Zytnicki, D

    2009-01-01

    The fast contraction time of mouse motor units creates a unique situation where motoneurons have to fire at low frequencies to produce small forces but also at very high frequency (much higher than in cat or rat motoneurons) to reach the fusion frequency of their motor units. To understand how this problem is solved, we performed intracellular recordings of adult mouse spinal motoneurons and investigated systematically their sub-threshold properties and their discharge pattern. We show that mouse motoneurons have a much wider range of firing frequencies than cat and rat motoneurons because of three salient features. First, they have a short membrane time constant. This results in a higher cut off frequency and a higher resonance frequency, which allow mouse motoneurons to integrate inputs at higher frequencies. Second, their AHP is faster allowing the motoneurons to discharge at a higher rate. Third, motoneurons display high frequency (100–150 Hz) sub-threshold oscillations during the interspike intervals. The fast membrane kinetics greatly favors the appearance of these oscillations, creating a "sub-primary range" of firing. In this range, which has never been reported in cat and in rat spinal motoneurons, the oscillations follow the AHP and trigger spiking after a variable delay, allowing a discharge at low frequency but at the expense of an irregular rate. PMID:19741131

  4. Automatic detection and quantitative analysis of cells in the mouse primary motor cortex

    NASA Astrophysics Data System (ADS)

    Meng, Yunlong; He, Yong; Wu, Jingpeng; Chen, Shangbin; Li, Anan; Gong, Hui

    2014-09-01

    Neuronal cells play very important role on metabolism regulation and mechanism control, so cell number is a fundamental determinant of brain function. Combined suitable cell-labeling approaches with recently proposed three-dimensional optical imaging techniques, whole mouse brain coronal sections can be acquired with 1-μm voxel resolution. We have developed a completely automatic pipeline to perform cell centroids detection, and provided three-dimensional quantitative information of cells in the primary motor cortex of C57BL/6 mouse. It involves four principal steps: i) preprocessing; ii) image binarization; iii) cell centroids extraction and contour segmentation; iv) laminar density estimation. Investigations on the presented method reveal promising detection accuracy in terms of recall and precision, with average recall rate 92.1% and average precision rate 86.2%. We also analyze laminar density distribution of cells from pial surface to corpus callosum from the output vectorizations of detected cell centroids in mouse primary motor cortex, and find significant cellular density distribution variations in different layers. This automatic cell centroids detection approach will be beneficial for fast cell-counting and accurate density estimation, as time-consuming and error-prone manual identification is avoided.

  5. Multiple soft tissue aneurysmal cysts: An occurrence after resection of primary aneurysmal bone cyst of fibula

    PubMed Central

    Karkuzhali, P; Bhattacharyya, Mahuya; Sumitha, P

    2007-01-01

    We report a case of multiple extraosseous aneurysmal cysts occurring in the muscle and subcutaneous plane of postero-lateral aspects of the upper right leg. They appeared about 15 months after resection of aneurysmal bone cyst of the upper end of the fibula. They varied in size from 2 cm to 5 cm. Radiologically they were well-defined lesions with central septate areas surrounded by a rim of calcification. Histologically they showed central cystic spaces separated by septa consisting of fibroblasts, osteoclast type of giant cells and reactive woven bone. Thus they showed histological similarity with aneurysmal bone cysts, but did not show any connection with the bone. Only very few examples of aneurysmal cysts of soft tissue had been described in the past one decade and they were reported in various locations including rare sites such as arterial wall and larynx. Recent cytogenetic analyses have shown abnormalities involving 17p11-13 and/or 16q22 in both osseous and extraosseous aneurysmal cysts indicating its probable neoplastic nature. Our case had unique features like multiplicity and occurrence after resection of primary aneurysmal bone cyst of the underlying bone. PMID:21139755

  6. Bone Marrow Mesenchymal Stem Cells Attenuate Mitochondria Damage Induced by Hypoxia in Mouse Trophoblasts

    PubMed Central

    Wang, Lingjuan; Xu, Xiaoyan; Kang, Lina

    2016-01-01

    Objective We aimed to observe the change of mitochondrial function and structure as well as the cell function induced by hypoxia in mouse trophoblasts, and moreover, to validate the restoration of these changes after co-culture with bone marrow mesenchymal stem cells (hereinafter referred to as “MSCs”). Further, we explored the mechanism of MSCs attenuating the functional damage of trophoblasts caused by hypoxia. Methods Cells were divided into two groups, trophoblasts and MSCs+trophoblasts respectively, and the two groups of cells were incubated with normoxia or hypoxia. Chemiluminescence was used to assay the β-HCG and progesterone in cell culture supernatants quantitatively. Western blotting and PCR were applied to detect the expression of Mfn2, MMP-2, MMP-9 and integrin β1 in the two groups. The mitochondrial membrane potential of each group of cells was detected with JC-1 dye and the ATP content was measured by the phosphomolybdic acid colorimetric method. We utilized transmission electron microscopy for observing the ultrastructure of mitochondria in trophoblasts. Finally, we assessed the cell apoptosis with flow cytometry (FCM) and analyzed the expression of the apoptosis related genes—Bcl-2, Bax, Caspase3 and Caspase9 by western blotting. Results The results showed that the Mfn2 expression was reduced after 4 h in hypoxia compared with that in normoxia, but increased in the co-culture group when compared with that in the separated-culture group (p<0.05). In addition, compared with the separated-culture group, theβ-HCG and progesterone levels in the co-culture group were significantly enhanced (p<0.05), and so were the expressions of MMP-2, MMP-9 and integrin β1 (p<0.05). Moreover, it exhibited significantly higher in ATP levels and intensified about the mitochondrial membrane potential in the co-culture group. TEM revealed disorders of the mitochondrial cristae and presented short rod-like structure and spheroids in hypoxia, however, in the co-culture group, the mitochondrial cristae had a relatively regular arrangement and the mitochondrial ultrastructure showed hyperfusion. The expression of Bax, Caspase3 and Caspase9 was decreased in the co-culture group when compared with that in trophoblast cells cultured alone (p<0.05), while the Bcl-2 levels and the Apoptosis Index (AI) were markedly increased in the co-culture group (p<0.05). Conclusion Bone marrow mesenchymal stem cells can attenuate mitochondria damage and cell apoptosis induced by hypoxia; the mechanism could be upregulating the expression of Mfn2 in mouse trophoblasts and changing mitochondrial structure. PMID:27100996

  7. Properties of the primary somatosensory cortex projection to the primary motor cortex in the mouse

    PubMed Central

    Petrof, Iraklis; Viaene, Angela N.

    2015-01-01

    The primary somatosensory (S1) and primary motor (M1) cortices are reciprocally connected, and their interaction has long been hypothesized to contribute to coordinated motor output. Very little is known, however, about the nature and synaptic properties of the S1 input to M1. Here we wanted to take advantage of a previously developed sensorimotor slice preparation that preserves much of the S1-to-M1 connectivity (Rocco MM, Brumberg JC. J Neurosci Methods 162: 139–147, 2007), as well as available optogenetic methodologies, in order to investigate the synaptic profile of this projection. Our data show that S1 input to pyramidal cells of M1 is highly homogeneous, possesses many features of a “driver” pathway, such as paired-pulse depression and lack of metabotropic glutamate receptor activation, and is mediated through axons that terminate in both small and large synaptic boutons. Our data suggest that S1 provides M1 with afferents that possess synaptic and anatomical characteristics ideal for the delivery of strong inputs that can “drive” postsynaptic M1 cells, thereby potentially affecting their output. PMID:25632081

  8. Variable Bone Fragility Associated With an Amish COL1A2 Variant and a Knock-in Mouse Model

    PubMed Central

    Daley, Ethan; Streeten, Elizabeth A; Sorkin, John D; Kuznetsova, Natalia; Shapses, Sue A; Carleton, Stephanie M; Shuldiner, Alan R; Marini, Joan C; Phillips, Charlotte L; Goldstein, Steven A; Leikin, Sergey; McBride, Daniel J

    2010-01-01

    Osteogenesis imperfecta (OI) is a heritable form of bone fragility typically associated with a dominant COL1A1 or COL1A2 mutation. Variable phenotype for OI patients with identical collagen mutations is well established, but phenotype variability is described using the qualitative Sillence classification. Patterning a new OI mouse model on a specific collagen mutation therefore has been hindered by the absence of an appropriate kindred with extensive quantitative phenotype data. We benefited from the large sibships of the Old Order Amish (OOA) to define a wide range of OI phenotypes in 64 individuals with the identical COL1A2 mutation. Stratification of carrier spine (L1–4) areal bone mineral density (aBMD) Z-scores demonstrated that 73% had moderate to severe disease (less than −2), 23% had mild disease (−1 to −2), and 4% were in the unaffected range (greater than −1). A line of knock-in mice was patterned on the OOA mutation. Bone phenotype was evaluated in four F1 lines of knock-in mice that each shared approximately 50% of their genetic background. Consistent with the human pedigree, these mice had reduced body mass, aBMD, and bone strength. Whole-bone fracture susceptibility was influenced by individual genomic factors that were reflected in size, shape, and possibly bone metabolic regulation. The results indicate that the G610C OI (Amish) knock-in mouse is a novel translational model to identify modifying genes that influence phenotype and for testing potential therapies for OI. © 2010 American Society for Bone and Mineral Research PMID:19594296

  9. Fever and arthralgia as the initial symptoms of primary bone marrow diffuse large B-cell lymphoma: A case report

    PubMed Central

    REN, SAISAI; TAO, YANLING; JIA, LU; CHENG, PANPAN; ZHANG, JILEI; ZHANG, HAO

    2016-01-01

    Primary bone marrow diffuse large B-cell lymphoma (DLBCL) is rare, and only a few cases have been reported. Fever and arthralgia as the initial symptom are extremely rare; however, awareness must be made of this presentation. The current study describes the clinical and pathological findings of a 41-year-old man affected by fever and arthralgia. Blood tests revealed leukopenia and anemia. Multiple bone marrow biopsies were conducted and confirmed the diagnosis of primary bone marrow DLBCL. Primary bone marrow DLBCL is a rare and frequently misdiagnosed subset of non-Hodgkin's lymphoma. The current case demonstrates that utility of bone marrow biopsy for diagnosis should not be ignored, and that repeated bone marrow punctures in multiple locations may be necessary. PMID:27123129

  10. A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

    PubMed Central

    Parikh, Mukti R.; Belch, Andrew R.; Pilarski, Linda M; Kirshner, Julia

    2014-01-01

    Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions. PMID:24637629

  11. Targeted radiotherapy with Sm-153-EDTMP in nine cases of canine primary bone tumours.

    PubMed

    Milner, R J; Dormehl, I; Louw, W K; Croft, S

    1998-03-01

    Nine dogs with primary bone tumours were treated with Samarium-153-EDTMP (Sm-153-EDTMP). Conventional treatment protocols were precluded by the size of the dogs and the owners' refusal of limb amputation. All the tumours were of the appendicular skeleton; 4 were confirmed osteosarcomas. The other 5 tumours were radiologically suspect for osteosarcoma. Bone scans were performed on all dogs using Technetium-99m-methylene diphosphonate (Tc-99m-MDP) before administration of Sm-153-EDTMP. Regions of interest were identified over the contralateral limb at the same site as the tumour and counts per pixel were recorded for the tumour and contralateral limb and expressed as a ratio. The dogs were given 1 injection of 37 MBq/kg (1 mCi/kg) of Sm-153-EDTMP intravenously. Thoracic and primary tumour site radiographs were taken at monthly or 2-monthly intervals to monitor progression of the primary tumour and search for evidence of metastasis. Two dogs showed no response to treatment, with an increase in bone pain, and were euthanased within 1 month. In 1 dog, a tumour of the scapula underwent complete involution and the dog is considered free of disease at 20 months post Sm-153-EDTMP treatment. The overall tumourcidal effect of a single dose of Sm-153-EDTMP on primary bone tumours was difficult to evaluate in this group of dogs, as, with one exception, all the primary tumours progressed over time and the dogs were euthanased. Pain control, for which Sm-155-EDTMP is used in man, was not evident, except in the dog that responded completely to treatment. PMID:9646255

  12. Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development

    NASA Technical Reports Server (NTRS)

    Simske, Steven J.; Bateman, Ted A.; Smith, Erin E.; Ferguson, Virginia L.; Chapes, Stephen K.

    2002-01-01

    We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls.

  13. Transplantation of bone marrow-derived mesenchymal stem cells into the developing mouse eye.

    PubMed

    Lee, Eun-Shil; Yu, Song-Hee; Jang, Yu-Jin; Hwang, Dong-Youn; Jeon, Chang-Jin

    2011-10-26

    Mesenchymal stem cells (MSCs) have been studied widely for their potential to differentiate into various lineage cells including neural cells in vitro and in vivo. To investigate the influence of the developing host environment on the integration and morphological and molecular differentiation of MSCs, human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the developing mouse retina. Enhanced green fluorescent protein (GFP)-expressing BM-MSCs were transplanted by intraocular injections into mice, ranging in ages from 1 day postnatal (PN) to 10 days PN. The survival dates ranged from 7 days post-transplantation (DPT) to 28DPT, at which time an immunohistochemical analysis was performed on the eyes. The transplanted BM-MSCs survived and showed morphological differentiation into neural cells and some processes within the host retina. Some transplanted cells expressed microtubule associated protein 2 (MAP2ab, marker for mature neural cells) or glial fibrillary acid protein (GFAP, marker for glial cells) at 5PN 7DPT. In addition, some transplanted cells integrated into the developing retina. The morphological and molecular differentiation and integration within the 5PN 7DPT eye was greater than those of other-aged host eye. The present findings suggest that the age of the host environment can strongly influence the differentiation and integration of BM-MSCs. PMID:22096261

  14. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    SciTech Connect

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. )

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  15. High MN1 expression increases the in vitro clonogenic activity of primary mouse B-cells.

    PubMed

    Numata, Masashi; Yener, Mehmet Deniz; Ekmekçi, Sema Sırma; Aydın, Müge; Grosveld, Gerard; Cardone, Monica; Terranova, Sabrina; Geltink, Ramon Klein; Özbek, Uğur; Özçelik, Emrah; Güleç, Çağrı; Anak, Sema; Karaman, Serap; Öztürk, Gülyüz; Akbıyık, Meral; Kandilci, Ayten

    2015-08-01

    The MN1 (Meningioma 1) gene is overexpressed in certain subtypes of acute myeloid leukemia (AML) and high levels of MN1 expression in mouse bone marrow cells results in myeloid leukemia. We showed that compared with control bone marrow (BM) MN1 expression was increased (2-fold or more) in 29 out of 73 (40%) pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient BM. Additional analysis of MN1 expression in sub-groups within our cohort carrying different chromosome translocations showed that carriers of the good prognostic marker t(12;21)(TEL-AML1) (n=27) expressed significantly more MN1 than both healthy controls (n=9) (P=0.02) and the group carrying the t(9;22)(BCR-ABL) (n=9) (P=0.001). In addition, AML1 expression was also upregulated in 31 out of 45 (68%) B-ALL patient BM compared with control and there was a significant correlation between MN1 and AML1 expression (r=0.3552, P=0.0167). Retroviral MN1 overexpression increased the colony forming activity of mouse Pro-B/Pre-B cells in vitro. Our results suggest that deregulated MN1 expression contributes to the pathogenesis of pediatric B-ALL. Further investigation into the clinical and biological significance of elevated MN1 expression in TEL-AML1(positive) leukemia might provide insight into additional molecular mechanisms contributing to B-ALL and may lead to improved treatment options for patients. PMID:26111797

  16. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    SciTech Connect

    Cuneo, Kyle C.; Mito, Jeffrey K.; Javid, Melodi P.; Ferrer, Jorge M.; Kim, Yongbaek; Lee, W. David; Bawendi, Moungi G.; Brigman, Brian E.; Kirsch, David G.

    2013-05-01

    Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

  17. Cytokine response in mouse bone marrow derived macrophages after infection with pathogenic and non-pathogenic Rift Valley fever virus.

    PubMed

    Roberts, Kimberly K; Hill, Terence E; Davis, Melissa N; Holbrook, Michael R; Freiberg, Alexander N

    2015-07-01

    Rift Valley fever virus (RVFV) is the most pathogenic member of the genus Phlebovirus within the family Bunyaviridae, and can cause severe disease in humans and livestock. Until recently, limited information has been published on the cellular host response elicited by RVFV, particularly in macrophages and dendritic cells, which play critical roles in stimulating adaptive and innate immune responses to viral infection. In an effort to define the initial response of host immunomodulatory cells to infection, primary mouse bone marrow derived macrophages (BMDM) were infected with the pathogenic RVFV strain ZH501, or attenuated strains MP-12 or MP-12 based Clone13 type (rMP12-C13 type), and cytokine secretion profiles examined. The secretion of T helper (Th)1-associated antiviral cytokines, chemokines and various interleukins increased rapidly after infection with the attenuated rMP12-C13 type RVFV, which lacks a functional NSs virulence gene. In comparison, infection with live-attenuated MP-12 encoding a functional NSs gene appeared to cause a delayed immune response, while pathogenic ZH501 ablates the immune response almost entirely. These data demonstrate that NSs can inhibit components of the BMDM antiviral response and supports previous work indicating that NSs can specifically regulate the type I interferon response in macrophages. Furthermore, our data demonstrate that genetic differences between ZH501 and MP-12 reduce the ability of MP-12 to inhibit antiviral signalling and subsequently reduce virulence in BMDM, demonstrating that viral components other than NSs play a critical role in regulating the host response to RVFV infection. PMID:25759029

  18. Cytokine response in mouse bone marrow derived macrophages after infection with pathogenic and non-pathogenic Rift Valley fever virus

    PubMed Central

    Roberts, Kimberly K.; Hill, Terence E.; Davis, Melissa N.; Holbrook, Michael R.

    2015-01-01

    Rift Valley fever virus (RVFV) is the most pathogenic member of the genus Phlebovirus within the family Bunyaviridae, and can cause severe disease in humans and livestock. Until recently, limited information has been published on the cellular host response elicited by RVFV, particularly in macrophages and dendritic cells, which play critical roles in stimulating adaptive and innate immune responses to viral infection. In an effort to define the initial response of host immunomodulatory cells to infection, primary mouse bone marrow derived macrophages (BMDM) were infected with the pathogenic RVFV strain ZH501, or attenuated strains MP-12 or MP-12 based Clone13 type (rMP12-C13 type), and cytokine secretion profiles examined. The secretion of T helper (Th)1-associated antiviral cytokines, chemokines and various interleukins increased rapidly after infection with the attenuated rMP12-C13 type RVFV, which lacks a functional NSs virulence gene. In comparison, infection with live-attenuated MP-12 encoding a functional NSs gene appeared to cause a delayed immune response, while pathogenic ZH501 ablates the immune response almost entirely. These data demonstrate that NSs can inhibit components of the BMDM antiviral response and supports previous work indicating that NSs can specifically regulate the type I interferon response in macrophages. Furthermore, our data demonstrate that genetic differences between ZH501 and MP-12 reduce the ability of MP-12 to inhibit antiviral signalling and subsequently reduce virulence in BMDM, demonstrating that viral components other than NSs play a critical role in regulating the host response to RVFV infection. PMID:25759029

  19. Benzene-induced micronuclei formation in mouse fetal liver blood, peripheral blood, and maternal bone marrow cells

    SciTech Connect

    Ning, Hansun Ministry of Railways, Beijing ); Kado, N.Y. California Air Resources Board, Sacramento ); Kuzmicky, P.A.; Hsieh, D.P.H. )

    1991-01-01

    The transplacental cytogenetic effects of benzene were studied by using the micronucleus test of polychromatic erythrocytes (PCE) found in both fetal liver and fetal peripheral blood, and were compared with PCE from maternal bone marrow. Timed-pregnant mice received single intraperitoneal doses of benzene on the 14th day of gestation and were sacrificed 21 hours after injection. Benzene elicited a significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in fetal liver blood cells at doses of 219 to 874 mg/kg, and in fetal peripheral blood cells and maternal bone marrow cells at doses of 437 and 874 mg/kg. The data demonstrate that benzene is a moderate transplacental clastogenic agent, and that the mouse transplacental micronucleus test using fetal liver blood cells is a potentially more sensitive indicator of the genotoxicity of benzene than either fetal peripheral blood or maternal bone marrow cells.

  20. Primary Hyperoxaluria Diagnosed Based on Bone Marrow Biopsy in Pancytopenic Adult with End Stage Renal Disease.

    PubMed

    Nematollahi, Pardis; Mohammadizadeh, Fereshteh

    2015-01-01

    Inborn errors of metabolism cause increase of metabolites in serum and their deposition in various organs including bone marrow. Primary hyperoxaluria (PH) is a rare inborn error in the pathway of glyoxylate metabolism which causes excessive oxalate production. The disease is characterized by widespread deposition of calcium oxalate (oxalosis) in multiple organs. Urinary tract including renal parenchyma is the initial site of deposition followed by extrarenal organs such as bone marrow. This case report introduces a 54-year-old woman with end stage renal disease presenting with debilitating fatigue and pancytopenia. The remarkable point in her past medical history was recurrent episodes of nephrolithiasis, urolithiasis, and urinary tract infection since the age of 5 years and resultant end stage renal disease in adulthood in the absence of appropriate medical evaluation and treatment. She had an unsuccessful renal transplantation with transplant failure. The patient underwent bone marrow biopsy for evaluation of pancytopenia. Microscopic study of bone marrow biopsy led to the diagnosis of primary hyperoxaluria. PMID:26634160

  1. Primary amines protect against retinal degeneration in mouse models of retinopathies

    PubMed Central

    Maeda, Akiko; Golczak, Marcin; Chen, Yu; Okano, Kiichiro; Kohno, Hideo; Shiose, Satomi; Ishikawa, Kaede; Harte, William; Palczewska, Grazyna; Maeda, Tadao; Palczewski, Krzysztof

    2011-01-01

    Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore, 11-cis-retinal, and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomered product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing FDA-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by mass spectrometry. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that displays features of Stargardts and age-related retinal degeneration. PMID:22198730

  2. Extensive primary Ewings' sarcoma in the greater wing of the sphenoid bone.

    PubMed

    Apostolopoulos, Kostas; Ferekidis, Eleftherios

    2003-01-01

    We describe a rare case of an extensive primary cranial Ewing's sarcoma located in the greater wing of the sphenoid bone with extension to the orbit, the endocranium, the parapharyngeal and infratemporal space. The patient presented with diplopia, anosmia and prolapse of the left eye. He was given chemo- and radiotherapy and was free of symptoms on re-examination 1.5 years later. The prognosis of Ewing's sarcoma in the absence of surgery is uncertain, but prompt treatment appears to have a satisfactory therapeutic outcome. In the future, more cases should be studied in order to investigate the biological behaviour of a primary cranial Ewing's sarcoma. PMID:14564101

  3. Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain.

    PubMed

    Alterman, Julia F; Hall, Lauren M; Coles, Andrew H; Hassler, Matthew R; Didiot, Marie-Cecile; Chase, Kathryn; Abraham, Jasmin; Sottosanti, Emily; Johnson, Emily; Sapp, Ellen; Osborn, Maire F; Difiglia, Marian; Aronin, Neil; Khvorova, Anastasia

    2015-01-01

    Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context. PMID:26623938

  4. Live-Cell Imaging of Phagosome Motility in Primary Mouse RPE Cells.

    PubMed

    Hazim, Roni; Jiang, Mei; Esteve-Rudd, Julian; Diemer, Tanja; Lopes, Vanda S; Williams, David S

    2016-01-01

    The retinal pigment epithelium (RPE) is a post-mitotic epithelial monolayer situated between the light-sensitive photoreceptors and the choriocapillaris. Given its vital functions for healthy vision, the RPE is a primary target for insults that result in blinding diseases, including age-related macular degeneration (AMD). One such function is the phagocytosis and digestion of shed photoreceptor outer segments. In the present study, we examined the process of trafficking of outer segment disk membranes in live cultures of primary mouse RPE, using high speed spinning disk confocal microscopy. This approach has enabled us to track phagosomes, and determine parameters of their motility, which are important for their efficient degradation. PMID:26427485

  5. Homeobox genes d11–d13 and a13 control mouse autopod cortical bone and joint formation

    PubMed Central

    Villavicencio-Lorini, Pablo; Kuss, Pia; Friedrich, Julia; Haupt, Julia; Farooq, Muhammed; Türkmen, Seval; Duboule, Denis; Hecht, Jochen; Mundlos, Stefan

    2010-01-01

    The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11–/–Hoxd12–/–Hoxd13–/– triple-knockout mice and Hoxd13–/–Hoxa13+/– mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2. PMID:20458143

  6. Endogenous retrovirus induces leukemia in a xenograft mouse model for primary myelofibrosis

    PubMed Central

    Triviai, Ioanna; Ziegler, Marion; Bergholz, Ulla; Oler, Andrew J.; Stübig, Thomas; Prassolov, Vladimir; Fehse, Boris; Kozak, Christine A.; Kröger, Nicolaus; Stocking, Carol

    2014-01-01

    The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdcscid and Il2rgnull alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation. PMID:24912157

  7. Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

    PubMed Central

    Hwang, Geun Hye; Jeon, Yu Jin; Han, Ho Jae; Park, Soo Hyun; Baek, Kyoung Min; Chang, Woochul; Kim, Joong Sun; Kim, Lark Kyun; Lee, You-Mie; Lee, Sangkyu; Bae, Jong-Sup; Jee, Jun-Goo

    2015-01-01

    Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation. PMID:25798044

  8. Establishment of primary cultures for mouse ameloblasts as a model of their lifetime

    SciTech Connect

    Suzawa, Tetsuo . E-mail: suzawa@dent.showa-u.ac.jp; Itoh, Nao; Takahashi, Naoyuki; Katagiri, Takenobu; Morimura, Naoko; Kobayashi, Yasuna; Yamamoto, Toshinori; Kamijo, Ryutaro

    2006-07-07

    To understand how the properties of ameloblasts are spatiotemporally regulated during amelogenesis, two primary cultures of ameloblasts in different stages of differentiation were established from mouse enamel epithelium. Mouse primary ameloblasts (MPAs) prepared from immature enamel epithelium (MPA-I) could proliferate, whereas those from mature enamel epithelium (MPA-M) could not. MPA-M but not MPA-I caused apoptosis during culture. The mRNA expression of amelogenin, a marker of immature ameloblasts, was down-regulated, and that of enamel matrix serine proteiase-1, a marker of mature ameloblasts, was induced in MPA-I during culture. Using green fluorescence protein as a reporter, a visualized reporter system was established to analyze the promoter activity of the amelogenin gene. The region between -1102 bp and -261 bp was required for the reporter expression in MPA-I. These results suggest that MPAs are valuable in vitro models for investigation of ameloblast biology, and that the visualized system is useful for promoter analysis in MPAs.

  9. Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes.

    PubMed

    Akie, Thomas E; Cooper, Marcus P

    2015-01-01

    Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates. PMID:26382148

  10. Maintenance and activation of Cyp2e-1 gene expression in mouse hepatocytes in primary culture.

    PubMed

    Sakurai, J; Funae, Y; Nemoto, N

    1996-08-21

    The expression of Cyp2e-1 mRNA and protein was investigated in the C57BL/6NCrj mouse hepatocytes in primary culture, as well as liver and kidney. The mRNA and protein expression in the liver was in the same range in both sexes and was not affected by orchiectomy or ovariectomy. The mRNA expression was enhanced in the kidney of ovariectomized mice, in which the protein contents were not influenced. Orchiectomy decreased the expression of both mRNA and protein. When the hepatocytes were transferred to primary culture, the amounts of the mRNA were not changed within 24 h and about half remained by day 3. However, the expression was low thereafter. The expression of the protein gradually decreased after the start of culture. Dexamethasone showed a potential as an inducer at more than 10(-8) M. Sex hormones increased the expression of this P-450 species a little in culture, but growth hormone did not. These observations indicated that glucocorticoid hormone plays a role in modifying expression of Cyp2e-1 and that the mouse hepatocyte culture is useful for examining its regulation mechanism. PMID:8781547

  11. Chemokine-Targeted Mouse Models of Human Primary and Metastatic Colorectal Cancer

    PubMed Central

    Chen, Huanhuan Joyce; Sun, Jian; Huang, Zhiliang; Hou, Harry; Arcilla, Myra; Rakhilin, Nikolai; Joe, Daniel J.; Choi, Jiahn; Gadamsetty, Poornima; Milsom, Jeff; Nandakumar, Govind; Longman, Randy; Zhou, Xi Kathy; Edwards, Robert; Chen, Jonlin; Chen, Kai Yuan; Bu, Pengcheng; Wang, Lihua; Xu, Yitian; Munroe, Robert; Abratte, Christian; Miller, Andrew D.; Gm?, Zeynep H.; Shuler, Michael; Nishimura, Nozomi; Edelmann, Winfried; Shen, Xiling; Lipkin, Steven M.

    2015-01-01

    Current orthotopic xenograft models of human colorectal cancer (CRC) require surgery and do not robustly form metastases in the liver, the most common site clinically. CCR9 traffics lymphocytes to intestine and colorectum. We engineered use of the chemokine receptor CCR9 in CRC cell lines and patient-derived cells to create primary gastrointestinal (GI) tumors in immunodeficient mice by tail-vein injection rather than surgery. The tumors metastasize inducibly and robustly to the liver. Metastases have higher DKK4 and NOTCH signaling levels and are more chemoresistant than paired sub-cutaneous xenografts. Using this approach, we generated 17 chemokine-targeted mouse models (CTMMs) that recapitulate the majority of common human somatic CRC mutations. We also show that primary tumors can be modeled in immunocompetent mice by microinjecting CCR9-expressing cancer cell lines into early-stage mouse blastocysts, which induces central immune tolerance. We expect that CTMMs will facilitate investigation of the biology of CRC metastasis and drug screening. PMID:26006007

  12. The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

    SciTech Connect

    Taguchi, Kazuhiro . E-mail: s3061@nms.ac.jp; Ogawa, Rei; Migita, Makoto; Hanawa, Hideki; Ito, Hiromoto; Orimo, Hideo

    2005-05-27

    We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses.

  13. Analysis of differences in bone removal during femoral box osteotomy for primary total knee arthroplasty

    PubMed Central

    GRACEFFA, ANGELO; INDELLI, PIER FRANCESCO; BASNETT, KAITLYN; MARCUCCI, MASSIMILIANO

    2014-01-01

    Purpose this study was conducted to compare the quantity of intercondylar bone removed during femoral box osteotomy for implantation of three contemporary posterior stabilized (PS) total knee arthroplasty designs: Sigma PS (DePuy), Vanguard (Biomet) and Persona (Zimmer). Methods we compared the maximum volumetric bone resection required for the housing of the PS mechanism of these three designs. Bone removal by each PS box cutting jig was three-dimensionally measured. The differences between the three designs were analyzed by the Kruskal-Wallis test. The Mann-Whitney U-test was used for pairwise comparisons. The level of significance was set at p<0.05. Results for small-size implants, the average box osteotomy volume of Persona was significantly smaller than the Vanguard and Sigma PS volumes (p=0.003). The mean difference between Vanguard and Sigma PS (p=0.01) was also significant. For medium size implants, the mean difference between Persona and Sigma PS (p=0.008) and the mean difference between Vanguard and Sigma PS (p=0.01) were statistically significant. For large size implants, the mean difference between Vanguard and Sigma PS (p=0.01) and the mean difference between Sigma PS and Persona (p=0.008) were statistically significant. Conclusions irrespective of implant size, the Persona cutting jig always resected significantly less bone than did Vanguard and Sigma PS. Clinical Relevance although this study does not establish any clinical relevance of removing more or less bone at primary TKA, its results suggest that if a PS design is indicated, it is preferable to select a model which resects less distal femoral bone. PMID:25606547

  14. [Clinical perspectives of the study of RANK/RANKL/OPG system components in primary and metastatic bone tumor].

    PubMed

    Kushlinskiĭ, N E; Timofeev, Iu S; Gershteĭn, E S; Solov'ev, Iu N

    2014-01-01

    Disbalance of bone homeostasis, associated with malfunctioning of RANK/RANKL/OPG system underlies the oncological processes such as the destruction of bone, metastasis development, tumor progression. Pathological activity of system was described in such conditions, as breast cancer, prostate cancer, multiple myeloma, squamous cell carcinoma, Hodgkin's disease, and also metastasis in bones from lung cancer and other malignant diseases. In the literature, there is evidence of involvement of RANK/RANKL/OPG system in the pathogenesis of bone tumors (osteosarcoma, giant cell tumor of bone, chondroblastoma). Experimental data show that RANKL inhibitors can play a role in reducing tumor-induced lesions of bone in multiple myeloma, breast cancer, prostate cancer and lung cancer. Also this review presents data from clinical studies of the drug efficacy targeted on RANK/RANKL/OPG system and results of authors' study of the levels of this system's components and proinflammatory cytokines in blood serum of primary bone sarcoma patients. PMID:25552059

  15. Euphorbia supina inhibits inflammatory mediators in mouse bone marrow-derived mast cells and macrophages.

    PubMed

    Chae, Hee-Sung; Song, Hyuk-Hwan; Kim, Young-Mi; Lee, Hyeong-Kyu; Oh, Sei-Ryang; Chin, Young-Won

    2015-12-01

    Euphorbia supina has been traditionally used for the treatment of furuncle and bloody diarrhea relevant to the inflammatory process. It has been proven to have a variety of pharmacological efficacies including antiarthritic, detoxification, hemostatic, and diuretic activities. RAW 264.7 macrophages and bone marrow-derived mast cells (BMMCs) were used to determine the anti-inflammatory and anti-allergic effects of E. supina (ES). NO production was assayed by measuring the nitrite content of the supernatants of cultured RAW 264.7 cells. β-hexosaminidase, a marker of mast cell degranulation, was quantitated by spectrophotometric analysis. ELISA was used for the analysis of interleukin-6 expression, and Western blotting was used to analyze 5-LOX, iNOS, and MAPK activation. The relevant gene expression upon ES treatment was measured by RT-PCR. ES inhibited inducible nitric oxide synthase (iNOS) in RAW 264.7 cells, and IL-6 and LTC4 production in PMA- and A23187-induced BMMCs along with the downregulation of 5-LOX gene expression. Furthermore, in the present study, a decrease in p-ERK, p-JNK, and p-P38 expression, as well as the suppression of degranulation, were observed by treatment with ES. Further in vivo study revealed that ES treatment also remarkably inhibited xylene-induced mouse ear edema and MPO levels in mice ears. This study demonstrates that ES has a potential regulatory effect on the expression of inflammatory mediators through the inhibition of both the phosphorylation of MAPK signaling and the activation of degranulation. PMID:26386544

  16. Oxidative stress and antioxidant status in primary bone and soft tissue sarcoma

    PubMed Central

    2011-01-01

    Background Oxidative stress is characterised by an increased level of reactive oxygen species (ROS) that disrupts the intracellular reduction-oxidation (redox) balance and has been implicated in various diseases including cancer. Malignant tumors of connective tissue or sarcomas account for approximately 1% of all cancer diagnoses in adults and around 15% of paediatric malignancies per annum. There exists no information on the alterations of oxidant/antioxidant status of sarcoma patients in literature. This study was aimed to determine the levels of oxidative stress and antioxidant defence in patients with primary bone and soft tissue sarcoma and to investigate if there exists any significant differences in these levels between both the sarcomas. Methods The study cohort consisted of 94 subjects; 20 soft tissue sarcoma, 27 primary bone sarcoma and 47 healthy controls. Malondialdehyde (MDA) and protein carbonyls were determined to assess their oxidative stress levels while antioxidant status was evaluated using catalase (CAT), superoxide dismutase (SOD), thiols and trolox equivalent antioxidant capacity (TEAC). Results Sarcoma patients showed significant increase in plasma and urinary MDA and serum protein carbonyl levels (p < 0.05) while significant decreases were noted in TEAC, thiols, CAT and SOD levels (p < 0.05). No significant difference in oxidative damage was noted between both the sarcomas (p > 0.05). Conclusions In conclusion, an increase in oxidative stress and decrease in antioxidant status is observed in both primary bone and soft tissue sarcomas with a similar extent of damage. This study offers the basis for further work on whether the manipulation of redox balance in patients with sarcoma represents a useful approach in the design of future therapies for bone disease. PMID:21871117

  17. Enumeration of the colony-forming units-fibroblast from mouse and human bone marrow in normal and pathological conditions.

    PubMed

    Kuznetsov, Sergei A; Mankani, Mahesh H; Bianco, Paolo; Robey, Pamela G

    2009-01-01

    Bone marrow stromal cell populations, containing a subset of multipotential skeletal stem cells, are increasingly contemplated for use in tissue engineering and stem cell therapy, whereas their involvement in the pathogenetic mechanisms of skeletal disorders is far less recognized. We compared the concentrations of stromal clonogenic cells, colony forming units-fibroblast (CFU-Fs), in norm and pathology. Initially, culture conditions were optimized by demonstrating that fetal bovine serum heat inactivation could significantly repress colony formation. Using non-heat-inactivated fetal bovine serum, the concentration of CFU-Fs (colony-forming efficiency, CFE) ranged from 3.5 +/- 1.0 to 11.5 +/- 4.0 per 1 x 10(5) nucleated cells in five inbred mouse strains. In four transgenic lines with profound bone involvement, CFE was either significantly reduced or increased compared to wild-type littermates. In normal human donors, CFE decreased slightly with age and averaged 52.2 +/- 4.1 for children and 32.3 +/- 3.0 for adults. CFE was significantly altered in patients with several skeletal, metabolic, and hematological disorders: reduced in congenital generalized lipodystrophy, achondroplasia (SADDAN), pseudoachondroplasia, and Paget disease of bone and elevated in alcaptonuria and sickle cell anemia. Our findings indicate that under appropriate culture conditions, CFE values may provide useful insights into bone/bone marrow pathophysiology. PMID:19383412

  18. In vivo visualizing the dynamics of bone marrow stem cells in mouse retina and choroidal-retinal circulation

    NASA Astrophysics Data System (ADS)

    Wang, Heuy-Ching H.; Zwick, Harry; Edsall, Peter R.; Cheramie, Rachel D.; Lund, David J.; Stuck, Bruce

    2007-02-01

    It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. Therefore it is an attractive therapeutic intervention to apply autologous bone marrow-derived stem cells that may offer neuroprotection to laser-induced retinal injuries. The purpose of this study is to develop a method with which to visualize bone marrow stem cells dynamics in mouse retinal circulation. We have used a physiological method, confocal scanning laser ophthalmoscope (SLO), to track the highly enriched stem/progenitor cells circulating in the retina. Stem cells were enriched by immunomagnetic depletion of cells committed to the T- and B lymphocytic, myeloid and erythorid lineages. CellTracker TM Green-labeled stem cells were injected into the tail veins of mice with laser-induced focal retinal injuries. Bone marrow stem cells labeled with CellTracker TM Green were visible in the retinal circulation for as long as 1 hour and 30 minutes. These studies suggest that stem cell-enriched bone marrow cells may have the ability to mobilize into laser-induced retinal injuries and possibly further proliferate, differentiate and functionally integrate into the retina.

  19. Constructing a multi-scan synchrotron X-ray microscope to study the function of osteocyte canaliculi in mouse bone

    SciTech Connect

    Nango, Nobuhito; Kubota, Shogo; Yashiro, Wataru; Momose, Atsushi; Takada, Yasunari; Matsuo, Koichi

    2012-07-31

    Formulating a multi-scan method applied to an X-ray microscope CT with synchrotron radiation, we attempted to analyze the 3D functional structure of osteocyte canaliculi inside the cortical bone of a mouse tibia. We employed a two-method combination to scan the same position of the specimen. To extract the internal bone canalicular structure, we first combined a Talbot interferometer with an X-ray microscope, and applied a differential phase imaging method to measure the absolute value of bone mineral around the canaliculi. Next, we used the X-ray microscope without the Talbot interferometer under a defocus condition, moving the specimen toward the zone plate by 6 mm. This defocus contrast method visualizes the canaliculi by emphasizing the edges of the bone. We performed CT scans by the two configurations and precisely aligned resultant 3D images so that the same position in the specimen is compared. We could extract the osteocyte canaliculi and evaluate the mineral density of their surroundings. The degree of mineralization varied for each osteocyte lacuna and canaliculus. The multi-scan microscopic X-ray CT is a powerful tool for analyzing bone mineralization.

  20. Enumeration of the colony-forming units–fibroblast from mouse and human bone marrow in normal and pathological conditions

    PubMed Central

    Kuznetsov, Sergei A.; Mankani, Mahesh H.; Bianco, Paolo; Robey, Pamela G.

    2009-01-01

    Bone marrow stromal cell populations, containing a subset of multipotential skeletal stem cells, are increasingly contemplated for use in tissue engineering and stem cell therapy, whereas their involvement in the pathogenetic mechanisms of skeletal disorders is far less recognized. We compared the concentrations of stromal clonogenic cells, colony forming units–fibroblast (CFU-Fs), in norm and pathology. Initially, culture conditions were optimized by demonstrating that fetal bovine serum heat inactivation could significantly repress colony formation. Using non-heat-inactivated fetal bovine serum, the concentration of CFU-Fs (colony-forming efficiency, CFE) ranged from 3.5 ± 1.0 to 11.5 ± 4.0 per 1 × 105 nucleated cells in five inbred mouse strains. In four transgenic lines with profound bone involvement, CFE was either significantly reduced or increased compared to wild-type littermates. In normal human donors, CFE decreased slightly with age and averaged 52.2 ± 4.1 for children and 32.3 ± 3.0 for adults. CFE was significantly altered in patients with several skeletal, metabolic, and hematological disorders: reduced in congenital generalized lipodystrophy, achondroplasia (SADDAN), pseudoachondroplasia, and Paget disease of bone and elevated in alcaptonuria and sickle cell anemia. Our findings indicate that under appropriate culture conditions, CFE values may provide useful insights into bone/bone marrow pathophysiology. PMID:19383412

  1. Bone thickening in osteoarthrosis. Observations of an osteoarthrosis-prone strain of mouse.

    PubMed

    Walton, M; Elves, M W

    1979-10-01

    Estimations were made of the amount of bone (histologically) and the rate of bone formation (85Sr incorporation) in the epiphyses of the knees of osteoarthrosis-prone (STR/ORT) and normal (CBA/ORT) mice. Though the bone was significantly thicker in the STR/ORT mice, this was not the cause of the articular degeneration. Bone sclerosis and cartilage breakdown were chronologically very closely related with perhaps the cartilage changes occurring initially. In male STR/ORT mice bone formation was depressed in the cancellous bone of the epiphyses as, unlike the normal mice, it was at the same level as the compact bone of the femoral shaft. As there was no elevation of the osteoblastic activity in knee joints with developing osteoarthrosis, it would appear that bone sclerosis associated with the disease was due to decreased osteoclasis. PMID:525314

  2. Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

    PubMed Central

    Shepherd, Andrew J.; Mohapatra, Durga P.

    2012-01-01

    Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue. PMID:22314687

  3. Cytokine Expression Pattern in Bone Marrow Microenvironment after Allogeneic Stem Cell Transplantation in Primary Myelofibrosis.

    PubMed

    Hussein, Kais; Stucki-Koch, Angelika; Alchalby, Haefaa; Triviai, Ioanna; Kröger, Nicolaus; Kreipe, Hans

    2016-04-01

    The only curative therapy for primary myelofibrosis (PMF) is allogeneic stem cell transplantation (ASCT). However, although we know that patients can benefit from ASCT, we do not know the extent of the changes of the expression profile of cytokines and matrix modulation factors. In this first systematic analysis, we evaluated the expression profile of 103 factors before and after transplantation to identify potential biomarkers. The expression of fibrosis-, inflammation-, and angiogenesis-associated genes was analyzed in a total of 52 bone marrow biopsies: PMF patients (n = 14) before and after ASCT and, for control purposes, post-ASCT multiple myeloma patients (n = 14) and non-neoplastic hematopoiesis (n = 10). In post-ASCT PMF cases, decreased expression of tissue inhibitor of metalloproteinases (TIMP) and platelet-derived growth factor alpha (PDGFA) correlated with bone marrow remodeling and hematological remission. Expression of several other matrix factors remained at high levels and may contribute to post-ASCT remodeling. This is the first systematic analysis of cytokine expression in post-ASCT PMF bone marrow that shows that normalization of bone marrow microenvironment is paralleled by decreased expression of TIMP and PDGFA. PMID:26708839

  4. Receptor-Activator of Nuclear KappaB Ligand Expression as a New Therapeutic Target in Primary Bone Tumors

    PubMed Central

    Yamagishi, Tetsuro; Kawashima, Hiroyuki; Ogose, Akira; Ariizumi, Takashi; Sasaki, Taro; Hatano, Hiroshi; Hotta, Tetsuo; Endo, Naoto

    2016-01-01

    The receptor-activator of nuclear kappaB ligand (RANKL) signaling pathway plays an important role in the regulation of bone growth and mediates the formation and activation of osteoclasts. Osteoclasts are involved in significant bone resorption and destruction. Denosumab is a fully human monoclonal antibody against RANKL that specifically inhibits osteoclast differentiation and bone resorption. It has been approved for use for multiple myeloma and bone metastases, as well as for giant cell tumor of bone. However, there is no previous report quantitatively, comparing RANKL expression in histologically varied bone tumors. Therefore, we analyzed the mRNA level of various bone tumors and investigated the possibility of these tumors as a new therapeutic target for denosumab. We examined RANKL mRNA expression in 135 clinical specimens of primary and metastatic bone tumors using real-time PCR. The relative quantification of mRNA expression levels was performed via normalization with RPMI8226, a human multiple myeloma cell line that is recognized to express RANKL. Of 135 cases, 64 were also evaluated for RANKL expression by using immunohistochemistry. Among all of the tumors investigated, RANKL expression and the RANKL/osteoprotegerin ratio were highest in giant cell tumor of bone. High RANKL mRNA expression was observed in cases of aneurysmal bone cyst, fibrous dysplasia, osteosarcoma, chondrosarcoma, and enchondroma, as compared to cases of multiple myeloma and bone lesions from metastatic carcinoma. RANKL-positive stromal cells were detected in six cases: five cases of GCTB and one case of fibrous dysplasia. The current study findings indicate that some primary bone tumors present new therapeutic targets for denosumab, particularly those tumors expressing RANKL and those involving bone resorption by osteoclasts. PMID:27163152

  5. Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes.

    PubMed

    White, Jennifer; Barro, Marietta V; Makarenkova, Helen P; Sanger, Joseph W; Sanger, Jean M

    2014-09-01

    It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, nonmuscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z-bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. PMID:25125171

  6. Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes

    EPA Science Inventory

    Abstract: Propiconazole is a triazole-containing fungicide that is used agriculturally on grasses, fruits, grains, seeds, hardwoods, and conifers. Propiconazole is a mouse liver hepatotoxicant and a hepatocarcinogen and has adverse reproductive and developmental toxicities in exp...

  7. Culture and Identification of Mouse Bone Marrow-Derived Dendritic Cells and Their Capability to Induce T Lymphocyte Proliferation

    PubMed Central

    Wang, Wenguang; Li, Jia; Wu, Kun; Azhati, Baihetiya; Rexiati, Mulati

    2016-01-01

    Background The aim of this study was to establish a culture method for mouse dendritic cells (DCs) in vitro and observe their morphology at different growth stages and their ability to induce the proliferation of T lymphocytes. Material/Methods Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were used in combination to induce differentiation of mouse bone marrow (BM) mononucleocytes into DCs. The derived DCs were then assessed for morphology, phenotype, and function. Results The mouse BM-derived mononucleocytes had altered cell morphology 3 days after induction by GM-CSF and IL-4 and grew into colonies. Typical dendrites appeared 8 days after induction. Many mature DCs were generated, with typical dendritic morphology observed under scanning electron microscopy. Expression levels of CD11c, a specific marker of BM-derived DCs, and of co-stimulatory molecules such as CD40, CD80, CD86, and MHC-II were elevated in the mature DCs. Furthermore, the mature DCs displayed a strong potency in stimulating the proliferation of syngenic or allogenic T lymphocytes. Conclusions Mouse BM-derived mononucleocytes cultured in vitro can produce a large number of DCs, as well as immature DCs, in high purity. The described in vitro culture method lays a foundation for further investigations of anti-tumor vaccines. PMID:26802068

  8. Efficient nano iron particle-labeling and noninvasive MR imaging of mouse bone marrow-derived endothelial progenitor cells

    PubMed Central

    Chen, Rong; Yu, Hui; Jia, Zhen-Yu; Yao, Qun-Li; Teng, Gao-Jun

    2011-01-01

    In this study, we sought to label mouse bone marrow-derived endothelial progenitor cells (EPCs) with Resovist® in vitro and to image them using 7.0 Tesla (T) magnetic resonance imaging (MRI). Mouse bone marrow-derived EPCs were cultured in endothelial basal medium with endothelial growth supplement. They were then characterized by immunocytochemistry, flow cytometry, and fluorescence quantitative polymerase chain reaction. Their functions were evaluated by measuring their uptake of 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-Ac-LDL), binding of fluorine isothiocyanate (FITC)-labeled Ulex europaeus agglutinin (UEA), and formation of capillary-like networks. EPCs were labeled with superparamagnetic iron oxide (SPIO) and their proliferation was then assessed in a water-soluble tetrazolium (WST-8)-based cell proliferation assay. Spin echo sequence (multislice, multiecho [MSME]) and gradient echo sequence (2D-FLASH) were used to detect differences in the numbers of labeled cells by 7.0 T MRI. The results showed that the cultured cells were of “cobblestone”-like shape and positive for CD133, CD34, CD31, von Willebrand factor, kinase domain receptor, and CD45, but negative for F4/80. They could take up Dil-Ac-LDL, bind FITC-UEA, and form capillary-like networks on Matrigel in vitro. Prussian-blue staining demonstrated that the cells were efficiently labeled with SPIO. The single-cell T2* effect was more obvious in the 2D-FLASH sequence than in the MSME sequence. Further, there were almost no adverse effects on cell vitality and proliferation. In conclusion, mouse bone marrow-derived EPCs can be efficiently labeled with SPIO and imaged with 7.0-T MRI. They may thus be traced by MRI following transplantation for blood vessel disorders and cancer treatment. PMID:21468354

  9. Primary Ewing's sarcoma of the squamous part of temporal bone in a young girl treated with adjuvant volumetric arc therapy.

    PubMed

    Nandi, Moujhuri; Bhattacharya, Jibak; Goswami, Suchanda; Goswami, Chanchal

    2015-01-01

    Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumors usually arise in the long bones of children and young adults. Primary ES of the cranium is unusual. Treatment involves multi-modality therapy incorporating surgery, radiotherapy and chemotherapy; outcomes are similar to those arising from long bones. We report a case of Primary ES of the squamous part of temporal bone with intracranial extension in a 9-year-old girl who was treated with surgery, chemotherapy followed by adjuvant radiotherapy by volumetric arc therapy. Post 1-year of treatment the girl is performing well in her classes. PMID:26881573

  10. Radiotherapy Combined With Androgen Deprivation for Bone Oligometastases After Primary Curative Radiotherapy for Prostate Cancer

    PubMed Central

    Wu, Jun-Xin; Lin, Li-Mei; He, Jun-Yan; Hong, Liang; Li, Jin-Luan

    2016-01-01

    Abstract To evaluate the effects and toxicity of radiotherapy (RT) combined with androgen deprivation (AD) for bone oligometastases after primary curative RT for prostate cancer (PCa). We retrospectively analyzed 30 consecutively treated PCa patients with bone oligometastases from April 2005 to July 2014. All patients underwent RT combined with AD for oligometastatic bones after curative RT for PCa. Measured outcomes included overall survival (OS) rate, local control (LC), progression-free survival (PFS), pain relief, and toxicities. Statistical analysis was performed with SPSS17.0. The median follow-up was 32.5 months (range, 0.6–50.3). The 3-year PFS and OS rates were 22.8% (95% CI, 13.4–37.5%) and 69% (95% CI, 51.7–81.1%), respectively. The number of bone oligometastases and RT schedule were found to be significantly associated with OS on univariate analysis (P < 0.05, respectively). The 3-year OS for patients with 1 and >1 metastases was 78.8% versus 42.2%, respectively (P = 0.037). The long-course RT was associated with better 3-year OS compared with short-course (76.4% vs 44.1%, P = 0.03). A total of 15 (83.3%, 15/18) patients achieved pain relief. No grade 3 toxicity was observed. Long-course RT combined with ADT was effective and well-tolerated in PCa patients with bone oligometastases after curative RT for PCa. Further randomized controlled trials are needed to corroborate the findings. PMID:26871838

  11. Radiogallium Complex-Conjugated Bifunctional Peptides for Detecting Primary Cancer and Bone Metastases Simultaneously.

    PubMed

    Ogawa, Kazuma; Yu, Jing; Ishizaki, Atsushi; Yokokawa, Masaru; Kitamura, Masanori; Kitamura, Yoji; Shiba, Kazuhiro; Odani, Akira

    2015-08-19

    (68)Ga (T(1/2) = 68 min, a generator-produced nuclide) is an interesting radionuclide for clinical positron emission tomography (PET). Recently, it was reported that radiogallium-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated (Asp)n peptide [Ga-DOTA-(Asp)n] has great potential for bone metastases imaging. In the current study, a compound containing an aspartic acid peptide linker (D11) as a carrier to bone metastases, an RGD peptide [c(RGDfK) peptide] as a carrier to the primary cancer, and Ga-DOTA as a stable radiometal complex for imaging in one molecule, Ga-DOTA-D11-c(RGDfK), was designed, prepared, and evaluated to detect both the primary cancer and bone metastases simultaneously using (67)Ga, which is easy to handle. After DOTA-D11-c(RGDfK) was synthesized using Fmoc-based solid-phase methodology, (67)Ga-DOTA-D11-c(RGDfK) was prepared by complexing DOTA-D11-c(RGDfK) with (67)Ga. Hydroxyapatite binding assays, integrin binding assays, biodistribution experiments, and single photon emission tomography (SPECT) imaging using tumor-bearing mice were performed using (67)Ga-DOTA-D11-c(RGDfK). (67)Ga-DOTA-D11-c(RGDfK) was prepared with a radiochemical purity of >97%. In vitro, (67)Ga-DOTA-D11-c(RGDfK) had a high affinity for hydroxyapatite and αvβ3 integrin. In vivo, (67)Ga-DOTA-D11-c(RGDfK) exhibited high uptake in bone and tumor. The accumulation of (67)Ga-DOTA-D11-c(RGDfK) in tumor decreased when it was co-injected with c(RGDfK) peptide. (68)Ga-DOTA-D11-c(RGDfK) has great potential as a PET tracer for the diagnosis of both the primary cancer and bone metastases simultaneously. PMID:26087328

  12. Comparative Study of Radionuclide Uptake Levels between Primary and Metastatic Bone Tumors.

    PubMed

    Huguette, Yigbedeck Yolande Ebele; Kwame, Kyere Augustine; Kojo, Wilson Isaac; Francis, Hasford; Kwabla, Sosu Edem; Otoe, Ankrah Alfred

    2014-01-01

    Study on 95 patients to compare radionuclide uptake levels in patients undergoing bone scintigraphy at a Nuclear Medicine Unit has been performed quantitatively using Image J software. Patients were administered with activity ranging from 0.555 to 1.110 MBq depending on their body weight, and their whole-body bone scans obtained with an installed e.cam single-photon emission computed tomography system. Matrix size of 256 × 1024 was used in acquiring the scintigrams. Quantitative analyses performed with installed Image J software revealed higher radionuclide uptake levels in metastatic tumors compared with primary tumors for all selected skeletal parts. Average normalized count of activity in metastatic tumors was 37.117 ± 27.740 cts/mm(2)/MBq and its corresponding uptake in primary tumors was 23.035 ± 19.542 cts/mm(2)/MBq. The relative higher uptake in metastatic tumors over primary tumors could be attributed to higher osteoblastic activity and blood flow in metastatic tumors. PMID:25191113

  13. Primary Ovarian Insufficiency Induced by Fanconi Anemia E Mutation in a Mouse Model

    PubMed Central

    Fu, Chun; Begum, Khurshida; Overbeek, Paul A.

    2016-01-01

    In most cases of primary ovarian insufficiency (POI), the cause of the depletion of ovarian follicles is unknown. Fanconi anemia (FA) proteins are known to play important roles in follicular development. Using random insertional mutagenesis with a lentiviral transgene, we identified a family with reduced fertility in the homozygous transgenic mice. We identified the integration site and found that the lentivirus had integrated into intron 8 of the Fanconi E gene (Fance). By RT-PCR and in situ hybridization, we found that Fance transcript levels were significantly reduced. The Fance homozygous mutant mice were assayed for changes in ovarian development, follicle numbers and estrous cycle. Ovarian dysplasias and a severe lack of follicles were seen in the mutant mice. In addition, the estrous cycle was disrupted in adult females. Our results suggest that POI has been induced by the Fance mutation in this new mouse model. PMID:26939056

  14. Chitosan-poly(butylene succinate) scaffolds and human bone marrow stromal cells induce bone repair in a mouse calvaria model.

    PubMed

    Costa-Pinto, A R; Correlo, V M; Sol, P C; Bhattacharya, M; Srouji, S; Livne, E; Reis, R L; Neves, N M

    2012-01-01

    Tissue engineering sustains the need of a three-dimensional (3D) scaffold to promote the regeneration of tissues in volume. Usually, scaffolds are seeded with an adequate cell population, allowing their growth and maturation upon implantation in vivo. Previous studies obtained by our group evidenced significant growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded and cultured on melt-based porous chitosan fibre mesh scaffolds (cell constructs). Therefore, it is crucial to test the in vivo performance of these in vitro 3D cell constructs. In this study, chitosan-based scaffolds were seeded and cultured in vitro with hBMSCs for 3 weeks under osteogenic stimulation conditions and analysed for cell adhesion, proliferation and differentiation. Implantation of 2 weeks precultured cell constructs in osteogenic culture conditions was performed into critical cranial size defects in nude mice. The objective of this study was to verify the scaffold integration and new bone formation. At 8 weeks of implantation, scaffolds were harvested and prepared for micro-computed tomography (µCT) analysis. Retrieved implants showed good integration with the surrounding tissue and significant bone formation, more evident for the scaffolds cultured and implanted with human cells. The results of this work demonstrated that chitosan-based scaffolds, besides supporting in vitro proliferation and osteogenic differentiation of hBMSCs, induced bone formation in vivo. Thus, their osteogenic potential in orthotopic location in immunodeficient mice was validated, evidencing good prospects for their use in bone tissue-engineering therapies. PMID:21312336

  15. Positive Selection in Bone Morphogenetic Protein 15 Targets a Natural Mutation Associated with Primary Ovarian Insufficiency in Human

    PubMed Central

    Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Graldine; Persani, Luca; Fabre, Stphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGF?-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGF? family member was performed. A maximum likelihood phylogenetic tree of several TGF?/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGF family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women. PMID:24147118

  16. Positive selection in bone morphogenetic protein 15 targets a natural mutation associated with primary ovarian insufficiency in human.

    PubMed

    Auclair, Sylvain; Rossetti, Raffaella; Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women. PMID:24147118

  17. Effect of osteocalcin deficiency on the nanomechanics and chemistry of mouse bones.

    PubMed

    Kavukcuoglu, N B; Patterson-Buckendahl, P; Mann, A B

    2009-08-01

    In healthy bone there is a balance between bone resorption and formation. When an imbalance occurs there is an overall loss of bone mass leading to an increased risk of fracture. The deterioration is typically accompanied by changes in the non-collagenous proteins in the bone. Osteocalcin (OC) is the most abundant noncollageneous bone matrix protein and it is believed to play a role in bone formation and resorption. Nanoindentation and Raman microspectroscopy have been used to correlate the mechanical and chemical properties of cortical bone from femora of OC -/- (osteocalcin deficient) mice and their wild-type controls (OC +/+). There are significant intra-bone variations in mechanics and crystallinity especially in the mid-cortical section for OC -/- mice compared to OC +/+ mice. Type-B carbonate substitution decreased significantly in the absence of osteocalcin and this appears to affect the hardness more than the elasticity. The results suggest that OC plays a role in the growth of apatite crystals in bone by increasing the degree of carbonate substitutions. The addition of these defects to the apatite's crystal lattice has little effect on elasticity, but does appear to reduce the bone's hardness. PMID:19627841

  18. Altered Bone Development in a Mouse Model of Peripheral Sensory Nerve Inactivation

    PubMed Central

    Heffner, Mollie A.; Anderson, Matthew J.; Yeh, Gregory C.; Genetos, Damian C.; Christiansen, Blaine A.

    2014-01-01

    Objectives The present study sought to determine the effects of decreased peripheral sensory nerve function on skeletal development and bone metabolism in mice. Methods C57BL/6 neonatal mice were treated with capsaicin to induce peripheral sensory nerve degeneration, and compared to vehicle-treated controls at 4, 8 and 12 weeks of age. Changes in bone structure were assessed using micro-computed tomography, mechanical properties and fracture resistance were assessed using three-point bending of radii, and bone turnover was assessed using dynamic histomorphometry and serum biomarkers. Results Capsaicin treatment resulted in small but significant decreases in bone structure, particularly affecting trabecular bone. Capsaicin-treated mice exhibited lower trabecular thickness at the femoral metaphysis and L5 vertebral body compared with vehicle-treated mice. However, capsaicin- and vehicle-treated mice had similar mechanical properties and bone turnover rates. Conclusion Neonatal capsaicin treatment affected trabecular bone during development; however these small changes may not be meaningful with respect to bone strength under normal loading conditions. It is possible that capsaicin-sensitive neurons may be more important for bone under stress conditions such as increased mechanical loading or injury. Future studies will investigate this potential role of peripheral sensory nerves in bone adaptation. PMID:24583535

  19. Bone mass and body composition in children and adolescents with primary hypertension: preliminary data.

    PubMed

    Pludowski, Pawel; Litwin, Mieczyslaw; Sladowska, Joanna; Antoniewicz, Jolanta; Niemirska, Anna; Wierzbicka, Aldona; Lorenc, Roman S

    2008-01-01

    Because primary hypertension (PH) is associated with calcium metabolism, it is hypothesized that PH may be related to osteoporosis risk. The study aimed to evaluate the relationship between body composition and bone strength in hypertensive adolescents. Total body scans using x-ray absorptiometry (DPX-L, GE Healthcare) were performed in 94 PH children aged 6 to 18 years (21 girls and 73 boys). References of healthy control subjects were used for the calculation of Z scores (age and gender matched), SD scores (height and gender matched), and SDs scores (weight and gender matched). Total body bone mineral density, total body bone mineral content (TBBMC), lean body mass (LBM), and fat mass (FM) were investigated. Relative bone strength index was calculated as the TBBMC:LBM ratio. As evidenced by Z scores, PH case subjects had slightly higher total body bone mineral density, TBBMC, and LBM compared with healthy subjects. Reduced LBM/body weight (BW) Z scores of -1.9+/-1.5 and -1.2+/-1.4, increased FM Z scores of +2.5+/-2.5 and +1.7+/-2.0, and increased FM/BW Z scores of +1.6+/-1.3 and +1.1+/-1.4 were noted in girls and boys compared with healthy subjects, respectively (P<0.001). When increased BW was controlled for, PH girls differed in SDs scores for LBM (-1.4+/-1.7; P<0.01), FM (+1.6+/-2.2; P<0.05), FM/BW (+0.9+/-1.0; P<0.05), and FM/LBM (+1.3+/-1.4; P<0.01) but not for total body bone mineral density (+0.2+/-1.0; P value not significant), TBBMC (-1.2+/-1.6; P=0.07), LBM/BW (-0.7+/-1.0; P=0.07), and TBBMC/LBM (-1.0+/-2.1; P value not significant), when compared with respective SDs scores of -0.3+/-1.1, +0.3+/-1.1, +0.3+/-1.0, +0.3+/-1.0, -0.2+/-1.0, -0.6+/-1.9, -0.3+/-1.0, and -0.2+/-1.0 in PH boys. In conclusion, PH adolescents had increased FM and an imbalanced relationship among BW, FM, and LBM. In PH girls, bone strength, although proper for chronological age and body height, was lower than expected for BW. PMID:17984369

  20. Identification and Characterization of Lineage(-)CD45(-)Sca-1(+) VSEL Phenotypic Cells Residing in Adult Mouse Bone Tissue.

    PubMed

    Nakatsuka, Ryusuke; Iwaki, Ryuji; Matsuoka, Yoshikazu; Sumide, Keisuke; Kawamura, Hiroshi; Fujioka, Tatsuya; Sasaki, Yutaka; Uemura, Yasushi; Asano, Hiroaki; Kwon, A-Hon; Sonoda, Yoshiaki

    2016-01-01

    Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs. PMID:26595762

  1. Using digital image correlation to determine bone surface strains during loading and after adaptation of the mouse tibia.

    PubMed

    Sztefek, Pavel; Vanleene, Maximilien; Olsson, Robin; Collinson, Rebecca; Pitsillides, Andrew A; Shefelbine, Sandra

    2010-03-01

    Previous models of cortical bone adaptation, in which loading is imposed on the bone, have estimated the strains in the tissue using strain gauges, analytical beam theory, or finite element analysis. We used digital image correlation (DIC), tracing a speckle pattern on the surface of the bone during loading, to determine surface strains in a murine tibia during compressive loading through the knee joint. We examined whether these surface strains in the mouse tibia are modified following two weeks of load-induced adaptation by comparison with contralateral controls. Results indicated non-uniform strain patterns with isolated areas of high strain (0.5%), particularly on the medial side. Strain measurements were reproducible (standard deviation of the error 0.03%), similar between specimens, and in agreement with strain gauge measurements (between 0.1 and 0.2% strain). After structural adaptation, strains were more uniform across the tibial surface, particularly on the medial side where peak strains were reduced from 0.5% to 0.3%. Because DIC determines local strains over the entire surface, it will provide a better understanding of how strain stimulus influences the bone response during adaptation. PMID:20005517

  2. A primary sclerosing epithelioid fibrosarcoma of the pubic bone, with evidence of divergent epithelial differentiation.

    PubMed

    Wang, Guofeng; Eyden, Brian

    2010-04-01

    Sclerosing epithelioid fibrosarcoma (SEF) is a rare variant of fibrosarcoma, described initially by Meis-Kindblom et al. in 1995 (Meis-Kindblom JM, Kindblom L-G, Enzinger FM. Sclerosing epithelioid fibrosarcoma: a variant of fibrosarcoma simulating carcinoma. Am J Surg Pathol. 1995;19:979-993): more than 80 cases have been documented clinicopathologically since. Bone is a rare primary site for SEF, with only 2 cases so far reported. This paper documents the detailed clinical, histological, immunohistochemical, and ultrastructural features of a case occurring in the pubic bone of a 57-year-old diabetic woman presenting with a history of pain and compromised mobility involving her hip. Radiology revealed a destructive lesion in the right pubic bone. The lesion was resected, and 7 months postoperatively it recurred. The patient died following metastases to multiple bony sites and liver, some 4 years after the onset of symptoms. Histologically, the tumor was consistent with SEF, although it showed some anomalous immunostaining, which, however, is typical of the tumor (for example, for S-100 protein and epithelial membrane antigen). By electron microscopy, some rough endoplasmic reticulum was present, but also tonofibrils and desmosomes. The overall features were of an SEF with the ultrastructural but incomplete immunohistochemical evidence for divergent epithelial differentiation. The differential diagnosis of this tumor is discussed. PMID:20192707

  3. Pattern of primary tumors and tumor-like lesions of bone in children: retrospective survey of biopsy results

    PubMed Central

    Özkan, Esra Akyüz; Göret, Ceren Canbey; Özdemir, Zeynep Tuğba; Yanık, Serdar; Doğan, Meryem; Gönültaş, Aylin; Akkoca, Ayşe Neslin

    2015-01-01

    Background: Although primary bone tumors are relatively uncommon, they constitute the most important tumors in patients less than 20 years. We aimed to determine the frequencies of primary bone tumors and tumor-like lesions of bone and the anatomical sites of their occurrence. Methods: A retrospective review of histopathology reports of all bone specimens received in a private pathology laboratory in Istanbul between 2009 and 2015. Results: A total of 57 patients (aged 5 to 18 years) with a mean of 13.12 years were studied. Thirty five patients (61.4%) were males and 22 (38.6%) were females. Fifty five (94.4%) of the tumors were benign. Osteochondroma was the commonest tumor accounting for 31 cases (54.3%) followed by osteoid osteoma, 9 cases (15.7%). Chondrosarcoma observed in two patients and Ewing sarcoma in one patient as malignant tumors. Of the 57 bone tumors 13 (22.8%) occurred in the upper extremities, while 44 (77.2%) were in the lower extremities. Proximal humerus was the most commonly involved site in upper extremity tumors, with osteochondromas representing the most frequent type of tumor (4 patients; 7%). In the lower extremities again osteochondromas were the most common type of tumor (8 cases, 14%), with the femur being the most common site of involvement (18 patients, 31.5%). Of the patients with tumor-like lesions; four patients had fibrous dysplasia, 4 patients had non-ossified fibromas, 4 patients had simple bone cysts and 3 had aneurismal bone cyst. Conclusion: This study showed that primary bone tumors were mainly benign, settled predominantly in the lower extremities mostly in the femur with a male preponderance. Osteochondroma was the most common benign bone tumor. We didn’t observed osteosarcoma, which is the most frequent malignant bone tumor. PMID:26617888

  4. Benefit of Consolidative Radiation Therapy for Primary Bone Diffuse Large B-Cell Lymphoma

    SciTech Connect

    Tao, Randa; Allen, Pamela K.; Rodriguez, Alma; Shihadeh, Ferial; Pinnix, Chelsea C.; Arzu, Isadora; Reed, Valerie K.; Oki, Yasuhiro; Westin, Jason R.; Fayad, Luis E.; Medeiros, L. Jeffrey; Dabaja, Bouthaina

    2015-05-01

    Purpose: Outcomes for patients with diffuse large B-cell lymphoma (DLBCL) differ according to the site of presentation. With effective chemotherapy, the need for consolidative radiation therapy (RT) is controversial. We investigated the influence of primary bone presentation and receipt of consolidative RT on progression-free survival (PFS) and overall survival (OS) in patients with DLBCL. Methods and Materials: We identified 102 patients with primary bone DLBCL treated consecutively from 1988 through 2013 and extracted clinical, pathologic, and treatment characteristics from the medical records. Survival outcomes were calculated by the Kaplan-Meier method, with factors affecting survival determined by log-rank tests. Univariate and multivariate analyses were done with a Cox regression model. Results: The median age was 55 years (range, 16-87 years). The most common site of presentation was in the long bones. Sixty-five patients (63%) received R-CHOP–based chemotherapy, and 74 (72%) received rituximab. RT was given to 67 patients (66%), 47 with stage I to II and 20 with stage III to IV disease. The median RT dose was 44 Gy (range, 24.5-50 Gy). At a median follow-up time of 82 months, the 5-year PFS and OS rates were 80% and 82%, respectively. Receipt of RT was associated with improved 5-year PFS (88% RT vs 63% no RT, P=.0069) and OS (91% vs 68%, P=.0064). On multivariate analysis, the addition of RT significantly improved PFS (hazard ratio [HR] = 0.14, P=.014) with a trend toward an OS benefit (HR=0.30, P=.053). No significant difference in PFS or OS was found between patients treated with 30 to 35 Gy versus ≥36 Gy (P=.71 PFS and P=.31 OS). Conclusion: Patients with primary bone lymphoma treated with standard chemotherapy followed by RT can have excellent outcomes. The use of consolidative RT was associated with significant benefits in both PFS and OS.

  5. Surgical cytoreduction of the primary tumor reduces metastatic progression in a mouse model of prostate cancer.

    PubMed

    Cifuentes, Federico F; Valenzuela, Rodrigo H; Contreras, Hctor R; Castelln, Enrique A

    2015-12-01

    Metastatic prostate cancer (mPCa) is one of the most prevalent cancers in men worldwide. The main cause of death in these patients is androgen-resistant metastatic disease. Surgery of the primary tumor has been avoided in these patients as there is no strong evidence that supports a beneficial effect. From the biological point of view, it appears rational to hypothesize that the primary tumor may contribute to the establishment and growth of metastases. Considering this, we propose that cytoreductive surgery (CS) in advanced metastatic stage slows the progression of metastatic disease. To test this, we used a mouse model of resectable orthotopic prostate cancer (PCa) and performed CS. After surgery, metastases were smaller and less numerous in the treated mice; an effect that was observable until the end of the experiment. These results suggest that CS alone delays the progression of metastatic disease and that although this effect may be temporary, it may translate to prolonged survival, especially when used with adjuvant therapy. PMID:26503286

  6. Surgical cytoreduction of the primary tumor reduces metastatic progression in a mouse model of prostate cancer

    PubMed Central

    CIFUENTES, FEDERICO F.; VALENZUELA, RODRIGO H.; CONTRERAS, HÉCTOR R.; CASTELLÓN, ENRIQUE A.

    2015-01-01

    Metastatic prostate cancer (mPCa) is one of the most prevalent cancers in men worldwide. The main cause of death in these patients is androgen-resistant metastatic disease. Surgery of the primary tumor has been avoided in these patients as there is no strong evidence that supports a beneficial effect. From the biological point of view, it appears rational to hypothesize that the primary tumor may contribute to the establishment and growth of metastases. Considering this, we propose that cytoreductive surgery (CS) in advanced metastatic stage slows the progression of metastatic disease. To test this, we used a mouse model of resectable orthotopic prostate cancer (PCa) and performed CS. After surgery, metastases were smaller and less numerous in the treated mice; an effect that was observable until the end of the experiment. These results suggest that CS alone delays the progression of metastatic disease and that although this effect may be temporary, it may translate to prolonged survival, especially when used with adjuvant therapy. PMID:26503286

  7. Acute toxicity and cytotoxicity of Bacillus thuringiensis and Bacillus sphaericus strains on fish and mouse bone marrow.

    PubMed

    Grisolia, Cesar Koppe; Oliveira-Filho, Eduardo Cyrino; Ramos, Felipe Rosa; Lopes, Madaí Cruz; Muniz, Daphne Heloisa Freitas; Monnerat, Rose Gomes

    2009-01-01

    The insecticidal properties of delta-endotoxins from Bacillus thuringiensis (Bt) serotypes kurstaki and israelensis and crystal proteins of Bacillus sphaericus (Bs) serotype H5 have been used in insect control for decades. The availability of microbial toxins in biopesticides as well as in plants with incorporated protection has been increasing the concerns about biosafety. Acute toxicity to Danio rerio and cytotoxicity on mouse bone marrow cells and peripheral erythrocytes of Oreochromis niloticus were tested with Bt israelensis, Bt kurstaki and Bs H5 strains. The concentration and dose tested were 10(6) and 10(8) spores/ml, respectively. Neither lethality nor effects on mouse bone marrow were promoted by any strain. In necrosis-apoptosis study on peripheral erythrocytes of O. niloticus an increased frequency of necrotic cells caused by exposure to strains of B. thuringiensis was found. Exposure to B. sphaericus did not show cytotoxic effects in either tested system. None of the strains studied induced apoptosis in contrast with the chemical controls. PMID:18670879

  8. Disrupted bone remodeling leads to cochlear overgrowth and hearing loss in a mouse model of fibrous dysplasia.

    PubMed

    Akil, Omar; Hall-Glenn, Faith; Chang, Jolie; Li, Alfred; Chang, Wenhan; Lustig, Lawrence R; Alliston, Tamara; Hsiao, Edward C

    2014-01-01

    Normal hearing requires exquisite cooperation between bony and sensorineural structures within the cochlea. For example, the inner ear secretes proteins such as osteoprotegrin (OPG) that can prevent cochlear bone remodeling. Accordingly, diseases that affect bone regulation can also result in hearing loss. Patients with fibrous dysplasia develop trabecular bone overgrowth resulting in hearing loss if the lesions affect the temporal bones. Unfortunately, the mechanisms responsible for this hearing loss, which could be sensorineural and/or conductive, remain unclear. In this study, we used a unique transgenic mouse model of increased Gs G-protein coupled receptor (GPCR) signaling induced by expression of an engineered receptor, Rs1, in osteoblastic cells. These ColI(2.3)+/Rs1+ mice showed dramatic bone lesions that histologically and radiologically resembled fibrous dysplasia. We found that ColI(2.3)+/Rs1+ mice showed progressive and severe conductive hearing loss. Ossicular chain impingement increased with the size and number of dysplastic lesions. While sensorineural structures were unaffected, ColI(2.3)+/Rs1+ cochleae had abnormally high osteoclast activity, together with elevated tartrate resistant acid phosphatase (TRAP) activity and receptor activator of nuclear factor kappa-B ligand (Rankl) mRNA expression. ColI(2.3)+/Rs1+ cochleae also showed decreased expression of Sclerostin (Sost), an antagonist of the Wnt signaling pathway that normally increases bone formation. The osteocyte canalicular networks of ColI(2.3)+/Rs1+ cochleae were disrupted and showed abnormal osteocyte morphology. The osteocytes in the ColI(2.3)+/Rs1+ cochleae showed increased expression of matrix metalloproteinase 13 (MMP-13) and TRAP, both of which can support osteocyte-mediated peri-lacunar remodeling. Thus, while the ossicular chain impingement is sufficient to account for the progressive hearing loss in fibrous dysplasia, the deregulation of bone remodeling extends to the cochlea as well. Our findings suggest that factors regulating bone remodeling, including peri-lacunar remodeling by osteocytes, may be useful targets for treating the bony overgrowths and hearing changes of fibrous dysplasia and other bony pathologies. PMID:24788917

  9. In vivo radiometric analysis of glucose uptake and distribution in mouse bone

    PubMed Central

    Zoch, Meredith L; Abou, Diane S; Clemens, Thomas L; Thorek, Daniel L J; Riddle, Ryan C

    2016-01-01

    Bone formation and remodeling occurs throughout life and requires the sustained activity of osteoblasts and osteoclasts, particularly during periods of rapid bone growth. Despite increasing evidence linking bone cell activity to global energy homeostasis, little is known about the relative energy requirements or substrate utilization of bone cells. In these studies, we measured the uptake and distribution of glucose in the skeleton in vivo using positron-emitting 18F-fluorodeoxyglucose ([18F]-FDG) and non-invasive, high-resolution positron emission tomography/computed tomography (PET/CT) imaging and ex vivo autoradiography. Assessment of [18F]-FDG uptake demonstrated that relative to other tissues bone accumulated a significant fraction of the total dose of the glucose analog. Skeletal accumulation was greatest in young mice undergoing the rapid bone formation that characterizes early development. PET/CT imaging revealed that [18F]-FDG uptake was greatest in the epiphyseal and metaphyseal regions of long bones, which accords with the increased osteoblast numbers and activity at this skeletal site. Insulin administration significantly increased skeletal accumulation of [18F]-FDG, while uptake was reduced in mice lacking the insulin receptor specifically in osteoblasts or fed a high-fat diet. Our results indicated that the skeleton is a site of significant glucose uptake and that its consumption by bone cells is subject to regulation by insulin and disturbances in whole-body metabolism. PMID:27088042

  10. Analysis of human primary bone cells by fluorescence activated cell scanning: methodological problems and preliminary results.

    PubMed

    Siggelkow, H; Hilmes, D; Robenstorff, K; Kurre, W; Engel, I; Hüfner, M

    1998-04-27

    We describe the development of flowcytometrical methods to analyse human primary osteoblast-like cultures obtained from trabecular bone explants in comparison to the human osteosarcoma cell line HOS 58. Two antigens typical of osteoblasts were studied: bone alkaline phosphatase and collagen/procollagen I; the non-specific attachment protein fibronectin served as control. The morphology of all different antigens is shown by immunocytochemistry before flowcytometrical analysis. The establishment of flowcytometry is described in detail. While all antigens tested were nearly 100% positive in the HOS 58 cells in immunocytochemistry and flowcytometry, in primary osteoblast-like cells results varied widely between both methods. Cell permeabilisation before flowcytometry improved the homogeneity of results, probably by increasing the accessibility of the specific antibody to intracellular compartments. Though up to 80% of cells were lost during preparation the ratio of positive versus negative cells in specific experiment was not dependent on the cell recovery. Therefore, the cells finally analysed seemed to be representative of the total population. PMID:9641353

  11. TUMOR CONTAMINATION IN THE BIOPSY PATH OF PRIMARY MALIGNANT BONE TUMORS

    PubMed Central

    Oliveira, Marcelo Parente; Lima, Pablo Moura de Andrade; de Mello, Roberto José Vieira

    2015-01-01

    Objective: To study factors possibly associated with tumor contamination in the biopsy path of primary malignant bone tumors. Method: Thirty-five patients who underwent surgical treatment with diagnoses of osteosarcoma, Ewing's tumor and chondrosarcoma were studied retrospectively. The sample was analyzed to characterize the biopsy technique used, histological type of the tumor, neoadjuvant chemotherapy used, local recurrences and tumor contamination in the biopsy path. Results: Among the 35 patients studied, four cases of contamination occurred (11.43%): one from osteosarcoma, two from Ewing's tumor and one from chondrosarcoma. There was no association between the type of tumor and presence of tumor contamination in the biopsy path (p = 0.65). There was also no association between the presence of tumor contamination and the biopsy technique (p = 0.06). On the other hand, there were associations between the presence of tumor contamination and local recurrence (p = 0.01) and between tumor contamination and absence of neoadjuvant chemotherapy (p = 0.02). Conclusion: Tumor contamination in the biopsy path of primary malignant bone tumors was associated with local recurrence. On the other hand, the histological type of the tumor and the type of biopsy did not have an influence on tumor contamination. Neoadjuvant chemotherapy had a protective effect against this complication. Despite these findings, tumor contamination is a complication that should always be taken into consideration, and removal of the biopsy path is recommended in tumor resection surgery.

  12. Array CGH analysis identifies two distinct subgroups of primary angiosarcoma of bone.

    PubMed

    Verbeke, Sofie L J; de Jong, Danielle; Bertoni, Franco; Sciot, Raf; Antonescu, Cristina R; Szuhai, Karoly; Bovée, Judith V M G

    2015-02-01

    Molecular genetic studies on vascular tumors are rare. Recently, possible involvement of MYC and KDR has been documented in a subset of angiosarcomas of soft tissue. We performed a cytogenetic analysis of primary angiosarcomas of bone (n = 13) and soft tissue (n = 5) using high density array-comparative genomic hybridization (array-CGH). Regions of interest were validated by fluorescence in situ hybridization (FISH). Antibodies for candidate genes (SKI, MYC, KDR, and MAPK9) were selected and immunohistochemistry was performed. Six angiosarcomas of bone and four angiosarcomas of soft tissue showed chromosomal losses, gains, and high level amplifications. Cluster analysis identified two groups: a group with a complex genetic profile and a group with only few genetic aberrations. Five regions of interest were selected, which were located at chromosome bands 1p36.23, 2q32-34, 5q35, 8q24, and 17q21.32-24.2. Interphase FISH confirmed the high-level amplifications. Immunohistochemical analysis showed high expression of MYC (16/60), MAPK9 (63/69), and SKI (52/62). There were no differences between the two groups with regards to location, immunohistochemical expression nor survival. In summary, we identified two subgroups of angiosarcoma: those with few or no gross aberrations and those which show numerous genetic aberrations consisting of chromosomal losses, gains and high level amplifications or complex aberrations. The most common finding was amplification of 2q and 17q in both angiosarcoma of bone and soft tissue, suggesting overlap in tumorigenesis irrespective of their location. We show MYC amplification in primary angiosarcoma indicating this is not entirely specific for radiation-induced angiosarcoma. PMID:25231439

  13. Loss of SH3BP2 function suppresses bone destruction in TNF-driven and collagen-induced arthritis mouse models

    PubMed Central

    Mukai, Tomoyuki; Gallant, Richard; Ishida, Shu; Kittaka, Mizuho; Yoshitaka, Teruhito; Fox, David A.; Morita, Yoshitaka; Nishida, Keiichiro; Rottapel, Robert; Ueki, Yasuyoshi

    2014-01-01

    Objective SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. The purpose of this study was to investigate the role of SH3BP2 in arthritis in human TNF-α transgenic (hTNFtg) and collagen-induced arthritis (CIA) models. Methods First, SH3BP2-deficient (Sh3bp2–/–) and wild-type (Sh3bp2+/+) mice were crossed with hTNFtg mice. Inflammation and bone loss were examined by clinical inspection and histological and micro-CT analyses. Osteoclastogenesis was evaluated with primary bone marrow-derived M-CSF-dependent macrophages (BMMs). Second, CIA was induced in Sh3bp2–/– and Sh3bp2+/+ mice, and the incidence and severity of arthritis were evaluated. Anti-mouse type II collagen (CII) antibody levels were measured by ELISA. Lymph node cell responses to CII were also determined. Results SH3BP2-deficiency did not alter the severity of joint swelling but suppressed bone erosion in the hTNFtg model. Bone loss of talus and tibia was prevented in Sh3bp2–/–/hTNFtg mice compared to Sh3bp2+/+/hTNFtg mice. RANKL- and TNF-α-induced osteoclastogenesis was suppressed in Sh3bp2–/– BMM cultures. NFATc1 nuclear localization in response to TNF-α was decreased in Sh3bp2–/– BMMs compared to Sh3bp2+/+ BMMs. In the CIA model, SH3BP2-deficiency suppressed the incidence of arthritis, which was associated with decreased anti-CII antibody production, while the antigen-specific T-cell responses in lymph nodes were not significantly different between Sh3bp2+/+ and Sh3bp2–/– mice. Conclusion SH3BP2-deficiency prevents bone loss via impaired osteoclastogenesis in the hTNFtg model and suppresses the induction of arthritis via decreased autoantibody production in the CIA model. Therefore, SH3BP2 could be a therapeutic target for rheumatoid arthritis. PMID:25470448

  14. Overexpression of spermidine/spermine N1-acetyltransferase impairs osteoblastogenesis and alters mouse bone phenotype.

    PubMed

    Pirnes-Karhu, Sini; Määttä, Jorma; Finnilä, Mikko; Alhonen, Leena; Uimari, Anne

    2015-04-01

    Spermidine/spermine N (1)-acetyltransferase (SSAT) is a catabolic regulator of polyamines, ubiquitous molecules essential for cell proliferation and differentiation. In pathological conditions, the increased polyamine catabolism has been shown to mediate its cellular functions not only by changed polyamine levels but also by the availability of metabolites shared with other metabolic pathways or by production of toxic compounds. Our previous results showed that mice overexpressing SSAT (SSAT mice) developed a myeloproliferative disease and the bone marrow microenvironment partly contributed to its development. In this study, the physiological role of SSAT and polyamines in bone remodeling was characterized. Skeletal development of the SSAT mice appeared outwardly similar to wild-type mice until maturity, after which the SSAT mice developed kyphosis. With aging, the SSAT overexpression elicited increased bone perimeter with strikingly thinned cortical bone, decreased trabecular thickness and increased trabecular number in mice. In vitro studies showed that the maturation of SSAT overexpressing osteoblasts was impaired and the expression of bone formation marker genes was dramatically decreased. The polyamine pattern in osteoblasts of SSAT mice was distorted in comparison with wild-type mice. However, treatment of osteoblasts with a SSAT-inducing functional polyamine analogue suggested that defective osteoblastogenesis resulted rather from other consequences of enhanced SSAT activity than lowered levels of the higher polyamines. In comparison to SSAT overexpressing mice, SSAT deficiency led to opposite changes in osteoblastogenesis and differences in bone phenotype in mice. In conclusion, the level of SSAT enzyme activity affected osteoblastogenesis and hence influenced bone remodeling and the bone phenotype in mice. Furthermore, our results suggest the contribution of the catabolic part of the polyamine cycle, other than polyamine depletion, in pathophysiological processes of bone remodeling. PMID:25231394

  15. Glucocerebrosidase deficiency in zebrafish affects primary bone ossification through increased oxidative stress and reduced Wnt/β-catenin signaling.

    PubMed

    Zancan, Ilaria; Bellesso, Stefania; Costa, Roberto; Salvalaio, Marika; Stroppiano, Marina; Hammond, Chrissy; Argenton, Francesco; Filocamo, Mirella; Moro, Enrico

    2015-03-01

    Loss of lysosomal glucocerebrosidase (GBA1) function is responsible for several organ defects, including skeletal abnormalities in type 1 Gaucher disease (GD). Enhanced bone resorption by infiltrating macrophages has been proposed to lead to major bone defects. However, while more recent evidences support the hypothesis that osteoblastic bone formation is impaired, a clear pathogenetic mechanism has not been depicted yet. Here, by combining different molecular approaches, we show that Gba1 loss of function in zebrafish is associated with defective canonical Wnt signaling, impaired osteoblast differentiation and reduced bone mineralization. We also provide evidence that increased reactive oxygen species production precedes the Wnt signaling impairment, which can be reversed upon human GBA1 overexpression. Type 1 GD patient fibroblasts similarly exhibit reduced Wnt signaling activity, as a consequence of increased β-catenin degradation. Our results support a novel model in which a primary defect in canonical Wnt signaling antecedes bone defects in type 1 GD. PMID:25326392

  16. The impact of bone morphogenetic protein 4 (BMP4) on breast cancer metastasis in a mouse xenograft model.

    PubMed

    Ampuja, M; Alarmo, E L; Owens, P; Havunen, R; Gorska, A E; Moses, H L; Kallioniemi, A

    2016-06-01

    Bone morphogenetic protein 4 (BMP4) is a key regulator of cell proliferation and differentiation. In breast cancer cells, BMP4 has been shown to reduce proliferation in vitro and interestingly, in some cases, also to induce migration and invasion. Here we investigated whether BMP4 influences breast cancer metastasis formation by using a xenograft mouse model. MDA-MB-231 breast cancer cells were injected intracardially into mice and metastasis formation was monitored using bioluminescence imaging. Mice treated with BMP4 developed metastases slightly earlier as compared to control animals but the overall number of metastases was similar in both groups (13 in the BMP4 group vs. 12 in controls). In BMP4-treated mice, bone metastases were more common (10 vs. 7) but adrenal gland metastases were less frequent (1 vs. 5) than in controls. Immunostaining revealed no differences in signaling activation, proliferation rate, blood vessel formation, EMT markers or the number of cancer-associated fibroblasts between the treatment groups. In conclusion, BMP4 caused a trend towards accelerated metastasis formation, especially in bone. More work is needed to uncover the long-term effects of BMP4 and the clinical relevance of these findings. PMID:26970275

  17. Cobalt, titanium and PMMA bone cement debris influence on mouse osteoblast cell elasticity, spring constant and calcium production activity

    PubMed Central

    Preedy, Emily Callard; Perni, Stefano

    2015-01-01

    Periprosthetic osteolysis and implant loosening are the outcomes of wear debris generation in total joint replacements. Wear debris formed from the implanted materials consisting of metals, polymers, ceramic and bone cement initiate the immune system response. Often osteoblasts, the principal cell type in bone tissue adjacent to the prostheses, are directly impacted. In this study, the influence of cobalt, titanium and PMMA bone cement particles of different sizes, charges and compositions on mouse osteoblast adhesion, nanomechanics (elasticity and spring constant) and metabolic activity were investigated. These studies were accompanied by osteoblast mineralisation experiments and cell uptake after exposure to particles at defined time points. Our results demonstrate that alteration of the nanomechanical properties are mainly dependent on the metal type rather than nanoparticles size and concentration. Moreover, despite uptake increasing over exposure time, the cell characteristics exhibit changes predominately after the first 24 hours, highlighting that the cell responses to nanoparticle exposure are not cumulative. Understanding these processes is critical to expanding our knowledge of implant loosening and elucidating the nature of prosthetic joint failure. PMID:27019701

  18. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function

    PubMed Central

    Sato, Kazuya; Feng, Xingmin; Chen, Jichun; Li, Jungang; Muranski, Pawel; Desierto, Marie J.; Keyvanfar, Keyvan; Malide, Daniela; Kajigaya, Sachiko; Young, Neal S.

    2016-01-01

    Acquired aplastic anemia is an immune-mediated disease, in which T cells target hematopoietic cells; at presentation, the bone marrow is replaced by fat. It was reported that bone marrow adipocytes were negative regulators of hematopoietic microenvironment. To examine the role of adipocytes in bone marrow failure, we investigated peroxisomal proliferator-activated receptor gamma, a key transcription factor in adipogenesis, utilizing an antagonist of this factor called bisphenol-A-diglycidyl-ether. While bisphenol-A-diglycidyl-ether inhibited adipogenesis as expected, it also suppressed T cell infiltration of bone marrow, reduced plasma inflammatory cytokines, decreased expression of multiple inflammasome genes, and ameliorated marrow failure. In vitro, bisphenol-A-diglycidyl-ether suppressed activation and proliferation, and reduced phospholipase C gamma 1 and nuclear factor of activated T-cells 1 expression, as well as inhibiting calcium flux in T cells. The in vivo effect of bisphenol-A-diglycidyl-ether on T cells was confirmed in a second immune-mediated bone marrow failure model, using different strains and non-major histocompatibility antigen mismatched: bisphenol-A-diglycidyl-ether ameliorated marrow failure by inhibition of T cell infiltration of bone marrow. Our data indicate that peroxisomal proliferator-activated receptor gamma antagonists may attenuate murine immune-mediated bone marrow failure, at least in part, by suppression of T cell activation, which might hold implications in the application of peroxisomal proliferator-activated receptor gamma antagonists in immune-mediated pathophysiologies, both in the laboratory and in the clinic. Genetically “fatless” mice developed bone marrow failure with accumulation of marrow adipocytes in our model, even in the absence of body fat, suggesting different mechanisms of systematic and marrow adipogenesis and physiologic versus pathophysiologic fat accumulation. PMID:26589913

  19. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function.

    PubMed

    Sato, Kazuya; Feng, Xingmin; Chen, Jichun; Li, Jungang; Muranski, Pawel; Desierto, Marie J; Keyvanfar, Keyvan; Malide, Daniela; Kajigaya, Sachiko; Young, Neal S

    2016-01-01

    Acquired aplastic anemia is an immune-mediated disease, in which T cells target hematopoietic cells; at presentation, the bone marrow is replaced by fat. It was reported that bone marrow adipocytes were negative regulators of hematopoietic microenvironment. To examine the role of adipocytes in bone marrow failure, we investigated peroxisomal proliferator-activated receptor gamma, a key transcription factor in adipogenesis, utilizing an antagonist of this factor called bisphenol-A-diglycidyl-ether. While bisphenol-A-diglycidyl-ether inhibited adipogenesis as expected, it also suppressed T cell infiltration of bone marrow, reduced plasma inflammatory cytokines, decreased expression of multiple inflammasome genes, and ameliorated marrow failure. In vitro, bisphenol-A-diglycidyl-ether suppressed activation and proliferation, and reduced phospholipase C gamma 1 and nuclear factor of activated T-cells 1 expression, as well as inhibiting calcium flux in T cells. The in vivo effect of bisphenol-A-diglycidyl-ether on T cells was confirmed in a second immune-mediated bone marrow failure model, using different strains and non-major histocompatibility antigen mismatched: bisphenol-A-diglycidyl-ether ameliorated marrow failure by inhibition of T cell infiltration of bone marrow. Our data indicate that peroxisomal proliferator-activated receptor gamma antagonists may attenuate murine immune-mediated bone marrow failure, at least in part, by suppression of T cell activation, which might hold implications in the application of peroxisomal proliferator-activated receptor gamma antagonists in immune-mediated pathophysiologies, both in the laboratory and in the clinic. Genetically "fatless" mice developed bone marrow failure with accumulation of marrow adipocytes in our model, even in the absence of body fat, suggesting different mechanisms of systematic and marrow adipogenesis and physiologic versus pathophysiologic fat accumulation. PMID:26589913

  20. The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen.

    PubMed

    Boraschi-Diaz, Iris; Komarova, Svetlana V

    2016-01-01

    Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50 ng/ml) and macrophage colony stimulating factor (50 ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9 days for spleen, as compared to 5 days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm(2) for spleen, as compared to 50,000/cm(2) for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70-80 % of cells were lost during the first week of freezing, during the subsequent 5 weeks the losses were within 2-5 % per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5 weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1 week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics. PMID:25245056

  1. Pasteurella multocida toxin is a mitogen for bone cells in primary culture.

    PubMed Central

    Mullan, P B; Lax, A J

    1996-01-01

    The effect of recombinant Pasteurella multocida toxin (PMT) on primary cultures of embryonic chick bone-derived osteoblastic cells was investigated. It was found that PMT was a potent mitogen for primary derived chicken osteoblasts. The toxin stimulated DNA synthesis and cell proliferation in quiescent osteoblasts at the first passage and accelerated cell growth in subconfluent cultures. Cell viability was not affected by PMT, even at relatively high concentrations. Osteoblast numbers increased in a dose-dependent manner in response to PMT. Intracellular inositol phosphates were elevated in response to PMT, but no elevation in cyclic AMP (cAMP) levels was evident. Indeed, PMT inhibited cAMP elevation in osteoblasts in response to cholera toxin at a stage before other PMT-mediated events take place. In addition to increased cell turnover, PMT down-regulated the expression of several markers of osteoblast differentiation. Both alkaline phosphatase and type I collagen were reduced, but osteonectin was not affected. The in vitro deposition of mineral in cultures of primary osteoblasts and osteoblast-like osteosarcoma cells was also inhibited by the presence of PMT. This suggests that PMT interferes with differentiation at a preosteoblastic stage. PMID:8641807

  2. Model-based analysis of pattern motion processing in mouse primary visual cortex.

    PubMed

    Muir, Dylan R; Roth, Morgane M; Helmchen, Fritjof; Kampa, Björn M

    2015-01-01

    Neurons in sensory areas of neocortex exhibit responses tuned to specific features of the environment. In visual cortex, information about features such as edges or textures with particular orientations must be integrated to recognize a visual scene or object. Connectivity studies in rodent cortex have revealed that neurons make specific connections within sub-networks sharing common input tuning. In principle, this sub-network architecture enables local cortical circuits to integrate sensory information. However, whether feature integration indeed occurs locally in rodent primary sensory areas has not been examined directly. We studied local integration of sensory features in primary visual cortex (V1) of the mouse by presenting drifting grating and plaid stimuli, while recording the activity of neuronal populations with two-photon calcium imaging. Using a Bayesian model-based analysis framework, we classified single-cell responses as being selective for either individual grating components or for moving plaid patterns. Rather than relying on trial-averaged responses, our model-based framework takes into account single-trial responses and can easily be extended to consider any number of arbitrary predictive models. Our analysis method was able to successfully classify significantly more responses than traditional partial correlation (PC) analysis, and provides a rigorous statistical framework to rank any number of models and reject poorly performing models. We also found a large proportion of cells that respond strongly to only one stimulus class. In addition, a quarter of selectively responding neurons had more complex responses that could not be explained by any simple integration model. Our results show that a broad range of pattern integration processes already take place at the level of V1. This diversity of integration is consistent with processing of visual inputs by local sub-networks within V1 that are tuned to combinations of sensory features. PMID:26300738

  3. Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes.

    PubMed

    Zheng, Jinying; Peng, Chuan; Ai, Yanbiao; Wang, Heng; Xiao, Xiaoqiu; Li, Jibin

    2016-01-01

    The increase in fructose consumption is considered to be a risk factor for developing nonalcoholic fatty liver disease (NAFLD). We investigated the effects of docosahexaenoic acid (DHA) on hepatic lipid metabolism in fructose-treated primary mouse hepatocytes, and the changes of Endoplasmic reticulum (ER) stress pathways in response to DHA treatment. The hepatocytes were treated with fructose, DHA, fructose plus DHA, tunicamycin (TM) or fructose plus 4-phenylbutyric acid (PBA) for 24 h. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining. The mRNA expression levels and protein levels related to lipid metabolism and ER stress response were determined by real-time PCR and Western blot. Fructose treatment led to obvious TG accumulation in primary hepatocytes through increasing expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two key enzymes in hepatic de novo lipogenesis. DHA ameliorates fructose-induced TG accumulation by upregulating the expression of carnitine palmitoyltransferase 1A (CPT-1α) and acyl-CoA oxidase 1 (ACOX1). DHA treatment or pretreatment with the ER stress inhibitor PBA significantly decreased TG accumulation and reduced the expression of glucose-regulated protein 78 (GRP78), total inositol-requiring kinase 1 (IRE1α) and p-IRE1α. The present results suggest that DHA protects against high fructose-induced hepatocellular lipid accumulation. The current findings also suggest that alleviating the ER stress response seems to play a role in the prevention of fructose-induced hepatic steatosis by DHA. PMID:26805874

  4. Model-based analysis of pattern motion processing in mouse primary visual cortex

    PubMed Central

    Muir, Dylan R.; Roth, Morgane M.; Helmchen, Fritjof; Kampa, Björn M.

    2015-01-01

    Neurons in sensory areas of neocortex exhibit responses tuned to specific features of the environment. In visual cortex, information about features such as edges or textures with particular orientations must be integrated to recognize a visual scene or object. Connectivity studies in rodent cortex have revealed that neurons make specific connections within sub-networks sharing common input tuning. In principle, this sub-network architecture enables local cortical circuits to integrate sensory information. However, whether feature integration indeed occurs locally in rodent primary sensory areas has not been examined directly. We studied local integration of sensory features in primary visual cortex (V1) of the mouse by presenting drifting grating and plaid stimuli, while recording the activity of neuronal populations with two-photon calcium imaging. Using a Bayesian model-based analysis framework, we classified single-cell responses as being selective for either individual grating components or for moving plaid patterns. Rather than relying on trial-averaged responses, our model-based framework takes into account single-trial responses and can easily be extended to consider any number of arbitrary predictive models. Our analysis method was able to successfully classify significantly more responses than traditional partial correlation (PC) analysis, and provides a rigorous statistical framework to rank any number of models and reject poorly performing models. We also found a large proportion of cells that respond strongly to only one stimulus class. In addition, a quarter of selectively responding neurons had more complex responses that could not be explained by any simple integration model. Our results show that a broad range of pattern integration processes already take place at the level of V1. This diversity of integration is consistent with processing of visual inputs by local sub-networks within V1 that are tuned to combinations of sensory features. PMID:26300738

  5. Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes

    PubMed Central

    Zheng, Jinying; Peng, Chuan; Ai, Yanbiao; Wang, Heng; Xiao, Xiaoqiu; Li, Jibin

    2016-01-01

    The increase in fructose consumption is considered to be a risk factor for developing nonalcoholic fatty liver disease (NAFLD). We investigated the effects of docosahexaenoic acid (DHA) on hepatic lipid metabolism in fructose-treated primary mouse hepatocytes, and the changes of Endoplasmic reticulum (ER) stress pathways in response to DHA treatment. The hepatocytes were treated with fructose, DHA, fructose plus DHA, tunicamycin (TM) or fructose plus 4-phenylbutyric acid (PBA) for 24 h. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining. The mRNA expression levels and protein levels related to lipid metabolism and ER stress response were determined by real-time PCR and Western blot. Fructose treatment led to obvious TG accumulation in primary hepatocytes through increasing expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two key enzymes in hepatic de novo lipogenesis. DHA ameliorates fructose-induced TG accumulation by upregulating the expression of carnitine palmitoyltransferase 1A (CPT-1α) and acyl-CoA oxidase 1 (ACOX1). DHA treatment or pretreatment with the ER stress inhibitor PBA significantly decreased TG accumulation and reduced the expression of glucose-regulated protein 78 (GRP78), total inositol-requiring kinase 1 (IRE1α) and p-IRE1α. The present results suggest that DHA protects against high fructose-induced hepatocellular lipid accumulation. The current findings also suggest that alleviating the ER stress response seems to play a role in the prevention of fructose-induced hepatic steatosis by DHA. PMID:26805874

  6. Pou3f4-Mediated Regulation of Ephrin-B2 Controls Temporal Bone Development in the Mouse

    PubMed Central

    Raft, Steven; Coate, Thomas M.; Kelley, Matthew W.; Crenshaw, E. Bryan; Wu, Doris K.

    2014-01-01

    The temporal bone encases conductive and sensorineural elements of the ear. Mutations of POU3F4 are associated with unique temporal bone abnormalities and X-linked mixed deafness (DFNX2/DFN3). However, the target genes and developmental processes controlled by POU3F4 transcription factor activity have remained largely uncharacterized. Ephrin-B2 (Efnb2) is a signaling molecule with well-documented effects on cell adhesion, proliferation, and migration. Our analyses of targeted mouse mutants revealed that Efnb2 loss-of-function phenocopies temporal bone abnormalities of Pou3f4 hemizygous null neonates: qualitatively identical malformations of the stapes, styloid process, internal auditory canal, and cochlear capsule were present in both mutants. Using failed/insufficient separation of the stapes and styloid process as a quantitative trait, we found that single gene Efnb2 loss-of-function and compound Pou3f4/Efnb2 loss-of-function caused a more severe phenotype than single gene Pou3f4 loss-of-function. Pou3f4 and Efnb2 gene expression domains overlapped at the site of impending stapes-styloid process separation and at subcapsular mesenchyme surrounding the cochlea; at both these sites, Efnb2 expression was attenuated in Pou3f4 hemizygous null mutants relative to control. Results of immunoprecipitation experiments using chromatin isolated from nascent middle ear mesenchyme supported the hypothesis of a physical association between Pou3f4 and specific non-coding sequence of Efnb2. We propose that Efnb2 is a target of Pou3f4 transcription factor activity and an effector of mesenchymal patterning during temporal bone development. PMID:25299585

  7. Basement membrane of mouse bone marrow sinusoids shows distinctive structure and proteoglycan composition: a high resolution ultrastructural study.

    PubMed

    Inoue, S; Osmond, D G

    2001-11-01

    Venous sinusoids in bone marrow are the site of a large-scale traffic of cells between the extravascular hemopoietic compartment and the blood stream. The wall of the sinusoids consists solely of a basement membrane interposed between a layer of endothelial cells and an incomplete covering of adventitial cells. To examine its possible structural specialization, the basement membrane of bone marrow sinusoids has now been examined by high resolution electron microscopy of perfusion-fixed mouse bone marrow. The basement membrane layer was discontinuous, consisting of irregular masses of amorphous material within a uniform 60-nm-wide space between apposing endothelial cells and adventitial cell processes. At maximal magnifications, the material was resolved as a random arrangement of components lacking the "cord network" formation seen in basement membranes elsewhere. Individual components exhibited distinctive ultrastructural features whose molecular identity has previously been established. By these morphological criteria, the basement membrane contained unusually abundant chondroitin sulfate proteoglycan (CSPG) revealed by 3-nm-wide "double tracks," and moderate amounts of both laminin as dense irregular coils and type IV collagen as 1-1.5-nm-wide filaments, together with less conspicuous amounts of amyloid P forming pentagonal frames. In contrast, 4.5-5-nm-wide "double tracks" characteristic of heparan sulfate proteoglycan (HSPG) were absent. The findings demonstrate that, in comparison with "typical" basement membranes in other tissues, the bone marrow sinusoidal basement membrane is uniquely specialized in several respects. Its discontinuous nature, lack of network organization, and absence of HSPG, a molecule that normally helps to maintain membrane integrity, may facilitate disassembly and reassembly of basement membrane material in concert with movements of adventitial cell processes as maturing hemopoietic cells pass through the sinusoidal wall: the exceptionally large quantity of CSPG may represent a reservoir of CD44 receptor for use in hemopoiesis. PMID:11596011

  8. Behavior of Primary Cilia and Tricellular Tight Junction Proteins during Differentiation in Temperature-Sensitive Mouse Cochlear Precursor Hair Cells.

    PubMed

    Kakuki, Takuya; Kaneko, Yakuto; Takano, Kenichi; Ninomiya, Takafumi; Kohno, Takayuki; Kojima, Takashi; Himi, Tetsuo

    2016-01-01

    In the sensory hair cells of the mammalian cochlea, the primary cilia in the planar cell polarity as well as the tight junctions in the epithelial cell polarity and the barrier are important to maintain normal hearing. Temperature-sensitive mouse cochlear precursor hair cells were used to investigate the behavior of primary cilia and tricellular tight junction proteins during the differentiation of sensory hair cells. In undifferentiated cells (incubated at 33°C), many acetylated tubulin-positive primary cilia were observed, and each was accompanied with an x03B3;-tubulin-positive basal body. The primary cilia had a '9 + 0' architecture with nine outer microtubule doublets but lacking a central pair of microtubules. In differentiated cells (incubated at 39°C), acetylated tubulin-positive primary cilia as well as acetylated tubulin-positive cilia-like structures were partially observed on the cell surface. In differentiated cells, the number of primary cilia was markedly reduced compared with undifferentiated cells, and innumerable cilia-like structures with no ciliary pockets were partially observed on the cell surface. In undifferentiated cells, few tricellulin molecules and lipolysis-stimulated lipoprotein receptors (LSRs) were observed in the cytoplasm. In differentiated cells, many tricellulin molecules and LSRs were observed on the membranes and within the cytoplasm. Conditional immortalized mouse cochlear precursor hair cells may be useful to investigate the roles of primary cilia and tricellular tight junctions during cellular differentiation and degeneration such as apoptosis. PMID:27115742

  9. Oxidative damage induced by copper in mouse primary hepatocytes by single-cell analysis.

    PubMed

    Jing, Mingyang; Liu, Yang; Song, Wei; Yan, Yunxing; Yan, Wenbao; Liu, Rutao

    2016-01-01

    Copper can disturb the intracellular redox balance, induce oxidative stress, and subsequently cause irreversible damage, leading to a variety of diseases. In the present study, mouse primary hepatocytes were chosen to elucidate the in vitro oxidative damage of short-term copper exposure (10-200 μM) by single-cell analysis. We evaluated the toxicity of copper by reactive oxygen species (ROS), glutathione (GSH), and oxidative DNA damage at the single-cell level. Oxidative damage induced by copper was verified by the morphological changes, persistent elevations of excessive ROS and malondialdehyde (MDA), a decrease in GSH level, and the oxidative DNA damage. Furthermore, the average ROS generation, GSH consumption, and the indicators in DNA damage did not significantly change at relatively low concentrations (10 or 50 μM), but we can find the alterations of parameters in some single cells clearly. Emphasis on the analysis of single cells is conducive to gain a better understanding on the toxicity of copper. This study will also complement studies on the environmental risk assessment of copper pollution. Graphical abstract ᅟ. PMID:26358217

  10. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

    NASA Astrophysics Data System (ADS)

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Dayem, Ahmed Abdal; Kwon, Deug-Nam; Kim, Jin-Hoi

    2013-09-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a `green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.

  11. Transcriptomics analysis of primary mouse thymocytes exposed to bis(tri-n-butyltin)dioxide (TBTO).

    PubMed

    van Kol, Sandra W M; Hendriksen, Peter J M; van Loveren, Henk; Peijnenburg, Ad

    2012-06-14

    The biocide bis(tri-n-butyltin)oxide (TBTO) causes thymus atrophy in rodents and is toxic to many cell types of which thymocytes are the most sensitive. To obtain insight in the mechanisms of action of TBTO, we exposed primary mouse thymocytes in vitro for 3, 6 and 11 h to 0.1, 0.5, 1 and 2 μM TBTO. Subsequently, the cells were subjected to whole-genome gene expression profiling. Biological interpretation of the gene expression data revealed that TBTO affects a wide range of processes. Cell proliferation related genes were downregulated by all treatments except for 3 and 6 h 0.5 μM TBTO which upregulated these genes. Treatment with TBTO resulted in upregulation of genes involved in endoplasmatic reticulum (ER) stress, NFkB and TNFα pathways, and genes involved in DNA damage, p53 signaling and apoptosis. Remarkably, TBTO also increased the expression of genes that are known to be upregulated during T cell activation or during negative selection of thymocytes. The effect of TBTO on expression of genes involved in ER stress and apoptosis was confirmed by qPCR. Induction of the T cell activation response was corroborated by demonstrating that TBTO exposure resulted in translocation of NFAT to the nucleus, which is an essential event for T cell activation. PMID:22434021

  12. Nonviral direct conversion of primary mouse embryonic fibroblasts to neuronal cells.

    PubMed

    Adler, Andrew F; Grigsby, Christopher L; Kulangara, Karina; Wang, Hong; Yasuda, Ryohei; Leong, Kam W

    2012-01-01

    Transdifferentiation, where differentiated cells are reprogrammed into another lineage without going through an intermediate proliferative stem cell-like stage, is the next frontier of regenerative medicine. Wernig et al. first described the direct conversion of fibroblasts into functional induced neuronal cells (iNs). Subsequent reports of transdifferentiation into clinically relevant neuronal subtypes have further endorsed the prospect of autologous cell therapy for neurodegenerative disorders. So far, all published neuronal transdifferentiation protocols rely on lentiviruses, which likely precludes their clinical translation. Instead, we delivered plasmids encoding neuronal transcription factors (Brn2, Ascl1, Myt1l) to primary mouse embryonic fibroblasts with a bioreducible linear poly(amido amine). The low toxicity and high transfection efficiency of this gene carrier allowed repeated dosing to sustain high transgene expression levels. Serial 0.5g cm(-2) doses of reprogramming factors delivered at 48-hour intervals produced up to 7.6% Tuj1(+) (neuron-specific class III ?-tubulin) cells, a subset of which expressed MAP2 (microtubule-associated protein 2), tau, and synaptophysin. A synapsin-red fluorescent protein (RFP) reporter helped to identify more mature, electrophysiologically active cells, with 24/26 patch-clamped RFP(+) cells firing action potentials. Some non-virally induced neuronal cells (NiNs) were observed firing multiple and spontaneous action potentials. This study demonstrates the feasibility of nonviral neuronal transdifferentiation, and may be amenable to other transdifferentiation processes. PMID:23344148

  13. Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells

    SciTech Connect

    Jeng, A.Y.; Lichti, U.; Strickland, J.E.; Blumberg, P.M.

    1985-11-01

    Interaction of tumor promoting phorbol esters with specific high affinity receptors is probably essential for many of the biological responses elicited by these agents. Since diacylglycerols which can be produced enzymatically from phospholipids by phospholipase C are postulated to be the physiological ligands for the phorbol ester receptor, the authors have examined primary cultures of mouse epidermal basal cells exposed to phospholipase C (Clostridium perfringens) for several biological and biochemical responses characteristic of treatment with 12-O-tetradecanoyl-phorbol-13-acetate, the most potent phorbol ester tumor promoter. Formation of diacylglycerols by treatment with phospholipase C was demonstrated by the dose-dependent release of radioactive diacylglycerols in cells prelabeled with (TH)arachidonic acid. Treatment with phospholipase C led to the morphological changes and to the reduction in epidermal growth factor binding (90%) associated with 12-O-tetradecanoylphorbol-13-acetate treatment. Continuous treatment at the same dose led to the induction of the enzymes ornithine decarboxylase and transglutaminase with a time course and extent similar to the inductions by 12-O-tetradecanoylphorbol-13-acetate. Treatment with phospholipase C yielded substantial suppression of the binding affinity of phorbol-12,13-dibutyrate for its receptors without reduction in total number of binding sites, consistent with the production by phospholipase C of a competitive inhibitor of phorbol ester binding.

  14. ULTRAVIOLET B IRRADIATION AND ACTIVATION OF PROTEIN KINASE D IN PRIMARY MOUSE EPIDERMAL KERATINOCYTES

    PubMed Central

    Arun, Senthil N.; Kaddour-Djebbar, Ismail; Shapiro, Brian A.; Bollag, Wendy B.

    2010-01-01

    Our previous studies demonstrated that protein kinase D (PKD), a serine/threonine kinase implicated in various cell processes, is up-regulated in basal cell carcinoma (BCC), supporting a possible tumorigenic role for PKD in skin. Since the greatest risk factor for BCC is sun exposure, the ability of ultraviolet B (UVB) irradiation to activate PKD in primary mouse keratinocytes was investigated. Using western analysis with two autophosphorylation-specific antibodies, we show for the first time that UVB activated PKD in a time- and dose-dependent manner. UVB-induced PKD activation was verified using an in vitro kinase assay. Furthermore, activation was reduced by antioxidant pretreatment, suggesting a link with oxidative stress. UVB-induced PKD activation was mediated primarily by Src family tyrosine kinases rather than protein kinase C (PKC), and in fact, UVB did not alter PKC-mediated transphosphorylation. UVB induced apoptosis dose-dependently, and this death could be prevented by overexpression of wild-type PKD, but not mutant PKD or the empty adenovirus. Indeed, a mutant that cannot be phosphorylated by Src kinases exacerbated UVB-elicited apoptosis. Thus, our data indicate that UVB irradiation of keratinocytes induces Src-mediated activation of PKD, which protects cells from UVB-stimulated apoptosis, providing a possible explanation for the observed up-regulation of PKD in BCC. PMID:21132013

  15. Spatial organization of excitatory synaptic inputs to layer 4 neurons in mouse primary auditory cortex

    PubMed Central

    Kratz, Megan B.; Manis, Paul B.

    2015-01-01

    Layer 4 (L4) of primary auditory cortex (A1) receives a tonotopically organized projection from the medial geniculate nucleus of the thalamus. However, individual neurons in A1 respond to a wider range of sound frequencies than would be predicted by their thalamic input, which suggests the existence of cross-frequency intracortical networks. We used laser scanning photostimulation and uncaging of glutamate in brain slices of mouse A1 to characterize the spatial organization of intracortical inputs to L4 neurons. Slices were prepared to include the entire tonotopic extent of A1. We find that L4 neurons receive local vertically organized (columnar) excitation from layers 2 through 6 (L6) and horizontally organized excitation primarily from L4 and L6 neurons in regions centered ~300–500 μm caudal and/or rostral to the cell. Excitatory horizontal synaptic connections from layers 2 and 3 were sparse. The origins of horizontal projections from L4 and L6 correspond to regions in the tonotopic map that are approximately an octave away from the target cell location. Such spatially organized lateral connections may contribute to the detection and processing of auditory objects with specific spectral structures. PMID:25972787

  16. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells.

    PubMed

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Dayem, Ahmed Abdal; Kwon, Deug-Nam; Kim, Jin-Hoi

    2013-01-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a 'green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications. PMID:24059222

  17. Preclinical mouse models of osteosarcoma

    PubMed Central

    Uluçkan, Özge; Segaliny, Aude; Botter, Sander; Santiago, Janice M; Mutsaers, Anthony J

    2015-01-01

    Osteosarcoma is the most common form of primary bone tumors with high prevalence in children. Survival rates of osteosarcoma are low, especially in the case of metastases. Mouse models of this disease have been very valuable in investigation of mechanisms of tumorigenesis, metastasis, as well as testing possible therapeutic options. In this chapter, we summarize currently available mouse models for osteosarcoma and provide detailed methodology for the isolation of cell lines from genetically engineered mouse models (GEMMs), gene modification and tumor cell injection methods, as well as imaging techniques. PMID:25987985

  18. Inflammation as a Keystone of Bone Marrow Stroma Alterations in Primary Myelofibrosis

    PubMed Central

    Desterke, Christophe; Martinaud, Christophe; Ruzehaji, Nadira; Le Bousse-Kerdilès, Marie-Caroline

    2015-01-01

    Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm where severity as well as treatment complexity is mainly attributed to a long lasting disease and presence of bone marrow stroma alterations as evidenced by myelofibrosis, neoangiogenesis, and osteosclerosis. While recent understanding of mutations role in hematopoietic cells provides an explanation for pathological myeloproliferation, functional involvement of stromal cells in the disease pathogenesis remains poorly understood. The current dogma is that stromal changes are secondary to the cytokine “storm” produced by the hematopoietic clone cells. However, despite therapies targeting the myeloproliferation-sustaining clones, PMF is still regarded as an incurable disease except for patients, who are successful recipients of allogeneic stem cell transplantation. Although the clinical benefits of these inhibitors have been correlated with a marked reduction in serum proinflammatory cytokines produced by the hematopoietic clones, further demonstrating the importance of inflammation in the pathological process, these treatments do not address the role of the altered bone marrow stroma in the pathological process. In this review, we propose hypotheses suggesting that the stroma is inflammatory-imprinted by clonal hematopoietic cells up to a point where it becomes “independent” of hematopoietic cell stimulation, resulting in an inflammatory vicious circle requiring combined stroma targeted therapies. PMID:26640324

  19. Primary Hyperparathyroidism Misdiagnosed as Giant Cell Bone Tumor of Maxillary Sinus: A Case Report

    PubMed Central

    Aghaghazvini, Leila; Sharifian, Hashem; Rasuli, Bahman

    2016-01-01

    Primary hyperparathyroidism is an endocrine disorder recognized by hyperfunction of parathyroid gland, which can result in persistent bone absorption and brown tumor. Facial involvement of brown tumor is rare and usually involves the mandible. Giant cell tumor ( GCT) is an expansile osteolytic bone tumor which is very similar in clinical, radiological and histological features to brown tumor. Herein, we present a 35-year-old woman with an 11-month history of gradually swelling of the right maxilla and buccal spaces began during pregnancy two years ago. No other clinical or laboratory problems were detected. Postpartum CT scan demonstrated a lytic expansile multi-septated mass lesion containing enhancing areas, which initially described as GCT of the right maxillary sinus following surgery. Four months later, gradual progressive swelling of the bed of tumor was recurred and revised pathological slices were compatible with GCT. Regarding patient recent paresthesia, repeated laboratory tests were performed. Finally, according to laboratory results (elevation of serum calcium and parathyroid hormone), ultrasonographic findings and radioisotope scan (Sestamibi), probable parathyroid mass and brown tumor of maxilla was diagnosed. Pathology confirmed hyperplasia of right inferior parathyroid gland. Our case was thought-provoking due to its interesting clinical presentation and unusual presentation of brown tumor in parathyroid hyperplasia. PMID:27127572

  20. Primary bone marrow lymphoma presenting with cold-type autoimmune hemolytic anemia.

    PubMed

    Kosugi, Shigeki; Watanabe, Mai; Hoshikawa, Masahiro

    2014-09-01

    We report a rare case of primary bone marrow lymphoma with cold-type autoimmune hemolytic anemia (AIHA). A 70-year-old Japanese woman with suspected liver disorder presented to our hospital with palpitation. On physical examination, she had jaundice and signs of anemia. No lymphadenopathy or hepatosplenomegaly was noted. A direct antiglobulin test was positive for complement C3b and C3d. Anti-IgG testing was negative. Cold agglutinin was positive with a titer of 1:≥8,192, and haptoglobin was absent. A diagnosis of cold-type AIHA was made. Bone marrow biopsy revealed involvement with a population of lymphocytes that were positive for CD20 (L-26), CD79a, and Bcl-2. No lymphoma lesion was detected on computerized tomography or on upper and lower endoscopy. The patient was diagnosed with diffuse large B cell lymphoma (DLBCL) presenting with cold-type AIHA. She was treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone, resulting in complete remission after six cycles. As of 22 months after presentation, no signs of cold-type AIHA or lymphoma were present. PMID:25332595

  1. Clinical Outcomes of Surgical Treatments for Primary Malignant Bone Tumors Arising in the Acetabulum

    PubMed Central

    Fujiwara, Tomohiro; Ogura, Koichi; Kobayashi, Eisuke; Tanzawa, Yoshikazu; Nakatani, Fumihiko; Chuman, Hirokazu; Kawai, Akira

    2015-01-01

    The functional and oncologic results of eighteen patients with primary malignant periacetabular tumors were reviewed to determine the impact of surgical treatment. The reconstruction procedures were endoprosthesis (11), hip transposition (4), iliofemoral arthrodesis (2), and frozen bone autograft (1). After a mean follow-up of 62 months, 13 patients were alive and 5 had died of their disease; the 5-year overall survival rate was 67.2%. The corresponding mean MSTS scores of patients with endoprosthesis (11) and other reconstructions (7) were 42% and 55% (49%, 68%, and 50%), respectively. Overall, postoperative complications including deep infection or dislocation markedly worsened the functional outcome. Iliofemoral arthrodesis provided better function than the other procedures, whereas endoprosthetic reconstruction demonstrated poor functional outcome except for patients who were reconstructed with the adequate soft tissue coverage. Avoiding postoperative complications is highly important for achieving better function, suggesting that surgical procedures with adequate soft tissue coverage or without the massive use of nonbiological materials are preferable. Appropriate selection of the reconstructive procedures for individual patients, considering the amount of remaining bone and soft tissues, would lead to better clinical outcomes. PMID:26451129

  2. Mechanical Properties of Calvarial Bones in a Mouse Model for Craniosynostosis

    PubMed Central

    Moazen, Mehran; Peskett, Emma; Babbs, Christian; Pauws, Erwin; Fagan, Michael J.

    2015-01-01

    The mammalian cranial vault largely consists of five flat bones that are joined together along their edges by soft fibrous tissues called sutures. Premature closure of the cranial sutures, craniosynostosis, can lead to serious clinical pathology unless there is surgical intervention. Research into the genetic basis of the disease has led to the development of various animal models that display this condition, e.g. mutant type Fgfr2C342Y/+ mice which display early fusion of the coronal suture (joining the parietal and frontal bones). However, whether the biomechanical properties of the mutant and wild type bones are affected has not been investigated before. Therefore, nanoindentation was used to compare the elastic modulus of cranial bone and sutures in wild type (WT) and Fgfr2C342Y/+mutant type (MT) mice during their postnatal development. Further, the variations in properties with indentation position and plane were assessed. No difference was observed in the elastic modulus of parietal bone between the WT and MT mice at postnatal (P) day 10 and 20. However, the modulus of frontal bone in the MT group was lower than the WT group at both P10 (1.390.30 vs. 5.320.68 GPa; p<0.05) and P20 (5.570.33 vs. 7.140.79 GPa; p<0.05). A wide range of values was measured along the coronal sutures for both the WT and MT samples, with no significant difference between the two groups. Findings of this study suggest that the inherent mechanical properties of the frontal bone in the mutant mice were different to the wild type mice from the same genetic background. These differences may reflect variations in the degree of biomechanical adaptation during skull growth, which could have implications for the surgical management of craniosynostosis patients. PMID:25966306

  3. Transient expression of the cloned mouse c-Ha-ras 5' upstream region in transfected primary SENCAR mouse keratinocytes demonstrates its power as a promoter element.

    PubMed

    Neades, R; Betz, N A; Sheng, X Y; Pelling, J C

    1991-01-01

    The mouse Ha-ras oncogene is activated by point mutation and overexpressed in developing papillomas during two-stage skin carcinogenesis in SENCAR mice. One of our research aims is to characterize the factors regulating Ha-ras gene expression at the transcriptional level in SENCAR mouse epidermis. Towards this goal, we sequenced 1400 bp of the 5' upstream region of the mouse Ha-ras gene so as to characterize various cis-regulatory elements present in the gene. We identified seven sites with the proper consensus sequence for binding the SP1 transcription factor and three potential binding sites for the CTF-1 factor. In addition, we located a 13-base sequence with 92% homology to the consensus sequence for an estrogen response element and two hexamers with consensus sequences identical to the core sequence of the glucocorticoid response element. A series of transient gene expression vectors was constructed in which various regions of the mouse Ha-ras 5' upstream region were fused to the chloramphenicol acetyltransferase (CAT) gene. These expression plasmids were transfected into newborn and adult primary SENCAR epidermal cells, the epidermal cell population that presumably contains the stem cells involved in two-stage skin tumorigenesis. Transient gene expression assays carried out after 48-72 h indicated that a 2.3-kb Ha-ras 5' fragment produced CAT activity comparable to that produced by pSV2CAT and pdolCMVCAT, both of which are plasmids with strong viral promoters and enhancers driving CAT gene expression. Maintenance of transfected keratinocytes under both nondifferentiating (0.05 mM calcium) and differentiating (1.2 mM calcium) culture conditions demonstrated that the mouse Ha-ras upstream region was relatively unresponsive to changes in calcium concentration in transient expression assays carried out in either newborn or adult keratinocytes. Our results demonstrated the power of the cloned mouse Ha-ras promoter and upstream region in driving transient gene expression after transfection into primary keratinocytes. PMID:1910481

  4. Re-evaluation of the need for multiple sampling times in the mouse bone marrow micronucleus assay: results for DMBA

    SciTech Connect

    Ashby, J.; Mirkova, E.

    1987-01-01

    7,12-dimethylbenzanthracene (DMBA) is confirmed as active in the mouse bone marrow micronucleus assay 24 hr after dosing as corn-oil homogenate via either oral gavage or intraperitoneal (ip) injection. These data are consistent with recent observations made by several investigators. However, when dosed via ip injection as a solution in DMSO, peak activity was evident 48 hr after dosing and a dramatic reduction in erythropoiesis was observed. It is suggested that a maximum of two sampling times is adequate and that, as a consequence, the number of animals employed in the conduct of the test could be reduced with no loss of sensitivity. The present data also suggest that the use of a corn-oil homogenate of insoluble test agents may provide an efficient replacement for the use of ground suspensions or solutions in DMSO.

  5. Effects of magnetic resonance imaging (MRI) on the formation of mouse dentin and bone

    SciTech Connect

    Kwong-Hing, A.; Sandhu, H.S.; Prato, F.S.; Frappier, J.R.; Kavaliers, M. )

    1989-10-01

    The effects of magnetic resonance imaging (MRI) on dentin and bone formation in mice were examined using standard autoradiographic and liquid scintillation procedures. It was observed that exposure to a standard 23.2 min clinical multislice MRI (0.15T) procedure caused a significant increase in the synthesis of the collagenous matrix of dentin in the incisors of mice. There were no significant effects on alveolar and tibial bone matrix synthesis. These results suggest that the magnetic fields associated with MRI can affect the activity of cells and/or tissues that are involved in rapid synthetic activity.

  6. Bone Marrow Stromal Cells Modulate Mouse ENT1 Activity and Protect Leukemia Cells from Cytarabine Induced Apoptosis

    PubMed Central

    Macanas-Pirard, Patricia; Leisewitz, Andrea; Broekhuizen, Richard; Cautivo, Kelly; Barriga, Francisco M.; Leisewitz, Francisco; Gidi, Victoria; Riquelme, Erick; Montecinos, Viviana P.; Swett, Pilar; Besa, Pelayo; Ramirez, Pablo; Ocqueteau, Mauricio; Kalergis, Alexis M.; Holt, Matthew; Rettig, Michael; DiPersio, John F.; Nervi, Bruno

    2012-01-01

    Background Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. Methods Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of 3H-adenosine. Results Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. Conclusion The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML. PMID:22629369

  7. Route of administration influences the antitumor effects of bone marrow-derived dendritic cells engineered to produce interleukin-12 in a metastatic mouse prostate cancer model.

    PubMed

    Saika, Takashi; Satoh, Takefumi; Kusaka, Nobuyuki; Ebara, Shin; Mouraviev, Vladimir B; Timme, Terry L; Thompson, Timothy C

    2004-05-01

    Gene-modified dendritic cells (DC) provide unique therapeutic strategies for prostate cancer; however, the comparative evaluation of specific delivery options using appropriate preclinical models has not been described. In this study, bone marrow-derived DC were genetically engineered to express high levels of interleukin-12 (IL-12) with or without the costimulatory molecule B7-1, by ex vivo infection with recombinant adenoviral vectors. We used an orthotopic metastatic mouse prostate cancer preclinical model (178-2 BMA) to compare two therapeutic protocols for DC delivery, in situ and subcutaneous. DC were generated from bone marrow of syngeneic 129/Sv mice by culturing in the presence of GM-CSF and IL-4. In vitro DC/IL-12 or DC/IL-12/B7 produced high levels of biologically active IL-12. In situ delivery of DC/IL-12 or DC/IL-12/B7 induced a significant suppression of primary tumor growth compared to DC/beta gal controls (P=.0328 and P=.0019, respectively), as well as reduced numbers of spontaneous lung metastatic nodules (P=.1404 and P=.0335, respectively). In survival experiments, in situ DC/IL-12 injection demonstrated a small but statistically significant advantage (P=.0041). Subcutaneous, tumor lysate pulsed DC/IL-12 significantly decreased tumor size (P=.0152) and increased survival (P=0.0433) compared to HBSS controls but the decrease in the number of spontaneous lung metastases did not achieve statistical significance. Both in situ and subcutaneous treatments enhanced cytolytic activities of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). In this preclinical model, gene-modified DC-based intratumoral immunotherapy was shown to be an effective therapeutic strategy for locally advanced prostate cancer based on tumor growth suppression, inhibition of metastasis and survival improvement. PMID:15044961

  8. Chimeric Mouse model to track the migration of bone marrow derived cells in glioblastoma following anti-angiogenic treatments.

    PubMed

    Achyut, B R; Shankar, Adarsh; Iskander, A S M; Ara, Roxan; Knight, Robert A; Scicli, Alfonso G; Arbab, Ali S

    2016-03-01

    Bone marrow derived cells (BMDCs) have been shown to contribute in the tumor development. In vivo animal models to investigate the role of BMDCs in tumor development are poorly explored. We established a novel chimeric mouse model using as low as 5 × 10(6) GFP+ BM cells in athymic nude mice, which resulted in >70% engraftment within 14 d. In addition, chimera was established in NOD-SCID mice, which displayed >70% with in 28 d. Since anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in glioblastoma (GBM), which resulted into marked hypoxia and recruited BMDCs to the tumor microenvironment (TME). We exploited chimeric mice in athymic nude background to develop orthotopic U251 tumor and tested receptor tyrosine kinase inhibitors and CXCR4 antagonist against GBM. We were able to track GFP+ BMDCs in the tumor brain using highly sensitive multispectral optical imaging instrument. Increased tumor growth associated with the infiltration of GFP+ BMDCs acquiring suppressive myeloid and endothelial phenotypes was seen in TME following treatments. Immunofluorescence study showed GFP+ cells accumulated at the site of VEGF, SDF1 and PDGF expression, and at the periphery of the tumors following treatments. In conclusion, we developed a preclinical chimeric model of GBM and phenotypes of tumor infiltrated BMDCs were investigated in context of AATs. Chimeric mouse model could be used to study detailed cellular and molecular mechanisms of interaction of BMDCs and TME in cancer. PMID:26797476

  9. Isolation of the stromal-vascular fraction of mouse bone marrow markedly enhances the yield of clonogenic stromal progenitors

    PubMed Central

    Suire, Colby; Brouard, Nathalie; Hirschi, Karen

    2012-01-01

    The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of mouse bone marrow stromal cells (BMSCs), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of stromal cell stem/progenitors, we hypothesized that a potential reason for the low yield of mouse BMSCs is the flushing of the marrow used to remove single-cell suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of BMSCs physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs, which yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1631.8 + 199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32 + 1.9) by at least 2 orders of magnitude (P < .001; N = 8) with an accompanying 113.95-fold enrichment of CFU-F frequency when plated at low oxygen (5%). Purified BMSC populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single-cell suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays. PMID:22262767

  10. Effect of bone marrow derived mesenchymal stem cells on lung pathology and inflammation in ovalbumin-induced asthma in mouse

    PubMed Central

    Mohammadian, Maryam; Boskabady, Mohammad Hosein; Kashani, Iraj Ragerdi; Jahromi, Gila Pirzad; Omidi, Amene; Nejad, Amir Kavian; Khamse, Safoura; Sadeghipour, Hamid Reza

    2016-01-01

    Objective(s): Bone marrow-derived mesenchymal stem cells (BMSCs) have attracted significant interest to treat asthma and its complication. In this study, the effects of BMSCs on lung pathology and inflammation in an ovalbumin-induced asthma model in mouse were examined. Materials and Methods: BALB/c mice were divided into three groups: control group (animals were not sensitized), asthma group (animals were sensitized by ovalbumin), asthma+BMSC group (animals were sensitized by ovalbumin and treated with BMSCs). BMSCs were isolated and characterized and then labeled with Bromodeoxyuridine (BrdU). After that the cells transferred into asthmatic mice. Histopathological changes of the airways, BMSCs migration and total and differential white blood cell (WBC) count in bronchoalveolar lavage (BAL) fluid were evaluated. Results: A large number of BrdU-BMSCs were found in the lungs of mice treated with BMSCs. The histopathological changes, BAL total WBC counts and the percentage of neutrophils and eosinophils were increased in asthma group compared to the control group. Treatment with BMSCs significantly decreased airway pathological indices, inflammatory cell infiltration, and also goblet cell hyperplasia. Conclusion: The results of this study revealed that BMSCs therapy significantly suppressed the lung pathology and inflammation in the ovalbumin induced asthma model in mouse. PMID:27096065

  11. Three-dimensional culture of mouse bone marrow cells on stroma formed within a porous scaffold: influence of scaffold shape and cryopreservation of the stromal layer on expansion of haematopoietic progenitor cells.

    PubMed

    Miyoshi, Hirotoshi; Ohshima, Norio; Sato, Chiaki

    2013-01-01

    This study's primary goal was to develop an effective ex vivo expansion method for haematopoietic cells. 3D culture of mouse bone marrow cells was performed in porous scaffolds using a sheet or cube shape. Bone marrow cells were cultured on bone marrow-derived stromal layers formed within the scaffolds and the effect of scaffold shape on the expansion of haematopoietic cells was examined. In some experiments, stromal layers within cubic scaffolds were frozen and then used to culture bone marrow cells after thawing. Results show that after comparison, total cell density and expansion of haematopoietic cells were greater in cultures using the cubic scaffold, suggesting that it was superior to the sheet-like scaffold for expanding haematopoietic cells. When cryopreserved stroma was used, it effectively supported the expansion of haematopoietic cells, and a greater expansion of haematopoietic cells [(erythroid and haematopoietic progenitor cells (HPCs)] was achieved than in cultures with stromal cells that had not been cryopreserved. Expansion of cells using cryopreserved stroma had several other advantages such as a shorter culture period than the conventional method, a stable supply of stromal cells, and ease of handling and scaling up. As a result, this is an attractive method for ex vivo expansion of haematopoietic stem cells (HSCs) and HPCs for clinical use. PMID:22081538

  12. Is bone marrow biopsy always indicated in patients with primary cutaneous marginal zone B-cell lymphoma?

    PubMed

    Muniesa, C; Hernández-Machín, B

    2013-10-01

    Bone marrow involvement at the time of diagnosis is uncommon in patients with primary cutaneous marginal zone B-cell lymphoma (PCMZL). Moreover, in these patients such involvement is rarely found in isolation on diagnosis. Typically the few patients with PCMZL who have early bone marrow involvement also present secondary nodal or visceral involvement, which is detected by other staging studies (usually computed tomography). In recent years, this has given rise to some debate about whether a bone marrow biopsy should be routinely performed in patients diagnosed with PCMZL in view of the good prognosis and low incidence of bone marrow infiltration and/or extracutaneous involvement in this type of lymphoma. PMID:23954046

  13. GPR18 Controls Reconstitution of Mouse Small Intestine Intraepithelial Lymphocytes following Bone Marrow Transplantation

    PubMed Central

    Becker, Amy M.; Callahan, Derrick J.; Richner, Justin M.; Choi, Jaebok; DiPersio, John F.; Diamond, Michael S.; Bhattacharya, Deepta

    2015-01-01

    Specific G protein coupled receptors (GPRs) regulate the proper positioning, function, and development of immune lineage subsets. Here, we demonstrate that GPR18 regulates the reconstitution of intraepithelial lymphocytes (IELs) of the small intestine following bone marrow transplantation. Through analysis of transcriptional microarray data, we find that GPR18 is highly expressed in IELs, lymphoid progenitors, and mature follicular B cells. To establish the physiological role of this largely uncharacterized GPR, we generated Gpr18-/- mice. Despite high levels of GPR18 expression in specific hematopoietic progenitors, Gpr18-/- mice have no defects in lymphopoiesis or myelopoiesis. Moreover, antibody responses following immunization with hapten-protein conjugates or infection with West Nile virus are normal in Gpr18-/- mice. Steady-state numbers of IELs are also normal in Gpr18-/- mice. However, competitive bone marrow reconstitution experiments demonstrate that GPR18 is cell-intrinsically required for the optimal restoration of small intestine TCRγδ+ and TCRαβ+ CD8αα+ IELs. In contrast, GPR18 is dispensable for the reconstitution of large intestine IELs. Moreover, Gpr18-/- bone marrow reconstitutes small intestine IELs similarly to controls in athymic recipients. Gpr18-/- chimeras show no changes in susceptibility to intestinal insults such as Citrobacter rodentium infections or graft versus host disease. These data reveal highly specific requirements for GPR18 in the development and reconstitution of thymus-derived intestinal IEL subsets in the steady-state and after bone marrow transplantation. PMID:26197390

  14. GPR18 Controls Reconstitution of Mouse Small Intestine Intraepithelial Lymphocytes following Bone Marrow Transplantation.

    PubMed

    Becker, Amy M; Callahan, Derrick J; Richner, Justin M; Choi, Jaebok; DiPersio, John F; Diamond, Michael S; Bhattacharya, Deepta

    2015-01-01

    Specific G protein coupled receptors (GPRs) regulate the proper positioning, function, and development of immune lineage subsets. Here, we demonstrate that GPR18 regulates the reconstitution of intraepithelial lymphocytes (IELs) of the small intestine following bone marrow transplantation. Through analysis of transcriptional microarray data, we find that GPR18 is highly expressed in IELs, lymphoid progenitors, and mature follicular B cells. To establish the physiological role of this largely uncharacterized GPR, we generated Gpr18-/- mice. Despite high levels of GPR18 expression in specific hematopoietic progenitors, Gpr18-/- mice have no defects in lymphopoiesis or myelopoiesis. Moreover, antibody responses following immunization with hapten-protein conjugates or infection with West Nile virus are normal in Gpr18-/- mice. Steady-state numbers of IELs are also normal in Gpr18-/- mice. However, competitive bone marrow reconstitution experiments demonstrate that GPR18 is cell-intrinsically required for the optimal restoration of small intestine TCRγδ+ and TCRαβ+ CD8αα+ IELs. In contrast, GPR18 is dispensable for the reconstitution of large intestine IELs. Moreover, Gpr18-/- bone marrow reconstitutes small intestine IELs similarly to controls in athymic recipients. Gpr18-/- chimeras show no changes in susceptibility to intestinal insults such as Citrobacter rodentium infections or graft versus host disease. These data reveal highly specific requirements for GPR18 in the development and reconstitution of thymus-derived intestinal IEL subsets in the steady-state and after bone marrow transplantation. PMID:26197390

  15. PDGFB-based stem cell gene therapy increases bone strength in the mouse

    PubMed Central

    Chen, Wanqiu; Baylink, David J.; Brier-Jones, Justin; Neises, Amanda; Kiroyan, Jason B.; Rundle, Charles H.; Lau, Kin-Hing William; Zhang, Xiao-Bing

    2015-01-01

    Substantial advances have been made in the past two decades in the management of osteoporosis. However, none of the current medications can eliminate the risk of fracture and rejuvenate the skeleton. To this end, we recently reported that transplantation of hematopoietic stem/progenitor cells (HSCs) or Sca1+ cells engineered to overexpress FGF2 results in a significant increase in lamellar bone matrix formation at the endosteum; but this increase was attended by the development of secondary hyperparathyroidism and severe osteomalacia. Here we switch the therapeutic gene to PDGFB, another potent mitogen for mesenchymal stem cells (MSCs) but potentially safer than FGF2. We found that modest overexpression of PDGFB using a relatively weak phosphoglycerate kinase (PGK) promoter completely avoided osteomalacia and secondary hyperparathyroidism, and simultaneously increased trabecular bone formation and trabecular connectivity, and decreased cortical porosity. These effects led to a 45% increase in the bone strength. Transplantation of PGK-PDGFB–transduced Sca1+ cells increased MSC proliferation, raising the possibility that PDGF-BB enhances expansion of MSC in the vicinity of the hematopoietic niche where the osteogenic milieu propels the differentiation of MSCs toward an osteogenic destination. Our therapy should have potential clinical applications for patients undergoing HSC transplantation, who are at high risk for osteoporosis and bone fractures after total body irradiation preconditioning. It could eventually have wider application once the therapy can be applied without the preconditioning. PMID:26150503

  16. Secondary aneurysmal bone cystic change of the chondroblastoma, mistaken for a primary aneurysmal bone cyst in the patella.

    PubMed

    Chung, Jin Wha; Lee, Hwa Sung

    2014-03-01

    A 29-year-old woman complained of a 3-month history of left knee pain without trauma history. X-ray showed a well-defined osteolytic lesion with a sclerotic margin in the patella and magnetic resonance imaging showed T1-low and T2-high signal intensity with different fluid level. Our impression was an aneurysmal bone cyst. At surgery, the lesion was a blood-filled cystic cavity, surrounded by a gray or brownish tissue. Hemorrhagic soft tissues with recognizable bone fragments were observed. Curettage and autogenous bone graft was done. Microscopically, sheets of tumor cells were intermingled with some areas of eosinophilic chondroid matrix. The tumor cells showed oval-shaped nuclei with moderate eosinophilic cytoplasm. Several multinucleated giant cells and blood filled cystic cavities were observed. The final diagnosis was a chondroblastoma with a secondary aneurysmal bone cyst. At the post-operative 1.5-year follow-up, grafted bones were well incorporated radiographically and there were no recurrent evidence or any other abnormal symptoms. PMID:24639947

  17. A primary phosphorus-deficient skeletal phenotype in juvenile Atlantic salmon Salmo salar: the uncoupling of bone formation and mineralization.

    PubMed

    Witten, P E; Owen, M A G; Fontanillas, R; Soenens, M; McGurk, C; Obach, A

    2016-02-01

    To understand the effect of low dietary phosphorus (P) intake on the vertebral column of Atlantic salmon Salmo salar, a primary P deficiency was induced in post-smolts. The dietary P provision was reduced by 50% for a period of 10 weeks under controlled conditions. The animal's skeleton was subsequently analysed by radiology, histological examination, histochemical detection of minerals in bones and scales and chemical mineral analysis. This is the first account of how a primary P deficiency affects the skeleton in S. salar at the cellular and at the micro-anatomical level. Animals that received the P-deficient diet displayed known signs of P deficiency including reduced growth and soft, pliable opercula. Bone and scale mineral content decreased by c. 50%. On radiographs, vertebral bodies appear small, undersized and with enlarged intervertebral spaces. Contrary to the X-ray-based diagnosis, the histological examination revealed that vertebral bodies had a regular size and regular internal bone structures; intervertebral spaces were not enlarged. Bone matrix formation was continuous and uninterrupted, albeit without traces of mineralization. Likewise, scale growth continues with regular annuli formation, but new scale matrix remains without minerals. The 10 week long experiment generated a homogeneous osteomalacia of vertebral bodies without apparent induction of skeletal malformations. The experiment shows that bone formation and bone mineralization are, to a large degree, independent processes in the fish examined. Therefore, a deficit in mineralization must not be the only cause of the alterations of the vertebral bone structure observed in farmed S. salar. It is discussed how the observed uncoupling of bone formation and mineralization helps to better diagnose, understand and prevent P deficiency-related malformations in farmed S. salar. PMID:26707938

  18. A primary phosphorus‐deficient skeletal phenotype in juvenile Atlantic salmon Salmo salar: the uncoupling of bone formation and mineralization

    PubMed Central

    Owen, M. A. G.; Fontanillas, R.; Soenens, M.; McGurk, C.; Obach, A.

    2015-01-01

    To understand the effect of low dietary phosphorus (P) intake on the vertebral column of Atlantic salmon Salmo salar, a primary P deficiency was induced in post‐smolts. The dietary P provision was reduced by 50% for a period of 10 weeks under controlled conditions. The animal's skeleton was subsequently analysed by radiology, histological examination, histochemical detection of minerals in bones and scales and chemical mineral analysis. This is the first account of how a primary P deficiency affects the skeleton in S. salar at the cellular and at the micro‐anatomical level. Animals that received the P‐deficient diet displayed known signs of P deficiency including reduced growth and soft, pliable opercula. Bone and scale mineral content decreased by c. 50%. On radiographs, vertebral bodies appear small, undersized and with enlarged intervertebral spaces. Contrary to the X‐ray‐based diagnosis, the histological examination revealed that vertebral bodies had a regular size and regular internal bone structures; intervertebral spaces were not enlarged. Bone matrix formation was continuous and uninterrupted, albeit without traces of mineralization. Likewise, scale growth continues with regular annuli formation, but new scale matrix remains without minerals. The 10 week long experiment generated a homogeneous osteomalacia of vertebral bodies without apparent induction of skeletal malformations. The experiment shows that bone formation and bone mineralization are, to a large degree, independent processes in the fish examined. Therefore, a deficit in mineralization must not be the only cause of the alterations of the vertebral bone structure observed in farmed S. salar. It is discussed how the observed uncoupling of bone formation and mineralization helps to better diagnose, understand and prevent P deficiency‐related malformations in farmed S. salar. PMID:26707938

  19. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

    PubMed Central

    2013-01-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet–visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a ‘green’, natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications. PMID:24059222

  20. Bifidobacterium animalis subsp. lactis decreases urinary oxalate excretion in a mouse model of primary hyperoxaluria

    PubMed Central

    Whittamore, Jonathan M.; Hatch, Marguerite

    2015-01-01

    Hyperoxaluria significantly increases the risk of calcium oxalate kidney stone formation. Since several bacteria have been shown to metabolize oxalate in vitro, including probiotic bifidobacteria, we focused on the efficiency and possible mechanisms by which bifidobacteria can infuence oxalate handling in vivo, especially in the intestines, and compared these results with the reported effects of Oxalobacter formigenes. Bifidobacterium animalis subsp. lactis DSM 10140 and B. adolescentis ATCC 15703 were administered to wild-type (WT) mice and to mice defcient in the hepatic enzyme alanine-glyoxylate aminotransferase (Agxt−/−, a mouse model of Primary Hyperoxaluria) that were fed an oxalate-supplemented diet. The administration of B. animalis subsp. lactis led to a significant decrease in urinary oxalate excretion in WT and Agxt−/− mice when compared to treatment with B. adolescent-is. Detection of B. animalis subsp. lactis in feces revealed that 3 weeks after oral gavage with the bacteria 64 % of WT mice, but only 37 % of Agxt−/− mice were colonized. Examining intestinal oxalate fuxes showed there were no significant changes to net oxalate secretion in colonized animals and were therefore not associated with the changes in urinary oxalate excretion. These results indicate that colonization with B. animalis subsp. lactis decreased urinary oxalate excretion by degrading dietary oxalate thus limiting its absorption across the intestine but it did not promote enteric oxalate excretion as reported for O. formigenes. Preventive or therapeutic administration of B. animalis subsp. lactis appears to have some potential to beneficially infuence dietary hyperoxaluria in mice. PMID:25269440

  1. Postnatal Lesion Evidence Against a Primary Role for the Corpus Callosum in Mouse Sociability

    PubMed Central

    Yang, Mu; Clarke, Andrew M.; Crawley, Jacqueline N.

    2009-01-01

    BTBR T+tf/J (BTBR) is an inbred strain of mice that displays prominent social deficits and repetitive behaviors analogous to the defining symptoms of autism, along with a complete congenital agenesis of corpus callosum. BTBR is genetically distant from the widely used C57BL/6J (B6) strain, which exhibits high levels of sociability, low repetitive behaviors, and an intact corpus callosum. Emerging evidence implicates compromised inter-hemispherical connectivity in some cases of autism. We investigated the hypothesis that the disconnection of corpus callosum (CC) fiber tracts contribute to behavioral traits in mice that are relevant to the behavioral symptoms of autism. Surgical lesion of the CC in B6 mice at postnatal day 7 had no effect on juvenile play and adult social approach, and did not elevate repetitive self-grooming. No correlations were detected between rostral-caudal extent of the CC lesion and behavioral scores. In addition, LP/J, the strain that is genetically closest to BTBR but has an intact CC, displayed juvenile play deficits and repetitive self-grooming similar to those seen in BTBR. These corroborative results offer evidence against the hypothesis that the corpus callosum disconnection is a primary cause of low sociability and high repetitive behaviors in inbred mice. Our findings indicate that genes mediating other aspects of neurodevelopment, including those whose mutations underlie more subtle disruptions in white matter pathways and connectivity, are more likely to contribute to the aberrant behavioral phenotypes in the BTBR mouse model of autism. PMID:19419429

  2. Binocular input coincidence mediates critical period plasticity in the mouse primary visual cortex.

    PubMed

    Chen, Xiao-Jing; Rasch, Malte J; Chen, Guang; Ye, Chang-Quan; Wu, Si; Zhang, Xiao-Hui

    2014-02-19

    Classical studies on the development of ocular dominance (OD) organization in primary visual cortex (V1) have revealed a postnatal critical period (CP), during which visual inputs between the two eyes are most effective in shaping cortical circuits through synaptic competition. A brief closure of one eye during CP caused a pronounced shift of response preference of V1 neurons toward the open eye, a form of CP plasticity in the developing V1. However, it remains unclear what particular property of binocular inputs during CP is responsible for mediating this experience-dependent OD plasticity. Using whole-cell recording in mouse V1, we found that visually driven synaptic inputs from the two eyes to binocular cells in layers 2/3 and 4 became highly coincident during CP. Enhancing cortical GABAergic transmission activity by brain infusion with diazepam not only caused a precocious onset of the high coincidence of binocular inputs and OD plasticity in pre-CP mice, but rescued both of them in dark-reared mice, suggesting a tight link between coincident binocular inputs and CP plasticity. In Thy1-ChR2 mice, chronic disruption of this binocular input coincidence during CP by asynchronous optogenetic activation of retinal ganglion cells abolished the OD plasticity. Computational simulation using a feed-forward network model further suggests that the coincident inputs could mediate this CP plasticity through a homeostatic synaptic learning mechanism with synaptic competition. These results suggest that the high-level correlation of binocular inputs is a hallmark of the CP of developing V1 and serves as neural substrate for the induction of OD plasticity. PMID:24553935

  3. Protection of primary neurons and mouse brain from Alzheimer’s pathology by molecular tweezers

    PubMed Central

    Attar, Aida; Ripoli, Cristian; Riccardi, Elisa; Maiti, Panchanan; Li Puma, Domenica D.; Liu, Tingyu; Hayes, Jane; Jones, Mychica R.; Lichti-Kaiser, Kristin; Yang, Fusheng; Gale, Greg D.; Tseng, Chi-hong; Tan, Miao; Xie, Cui-Wei; Straudinger, Jeffrey L.; Klärner, Frank-Gerrit; Schrader, Thomas; Frautschy, Sally A.; Grassi, Claudio

    2012-01-01

    Alzheimer’s disease is a devastating cureless neurodegenerative disorder affecting >35 million people worldwide. The disease is caused by toxic oligomers and aggregates of amyloid β protein and the microtubule-associated protein tau. Recently, the Lys-specific molecular tweezer CLR01 has been shown to inhibit aggregation and toxicity of multiple amyloidogenic proteins, including amyloid β protein and tau, by disrupting key interactions involved in the assembly process. Following up on these encouraging findings, here, we asked whether CLR01 could protect primary neurons from Alzheimer’s disease-associated synaptotoxicity and reduce Alzheimer’s disease–like pathology in vivo. Using cell culture and brain slices, we found that CLR01 effectively inhibited synaptotoxicity induced by the 42-residue isoform of amyloid β protein, including ∼80% inhibition of changes in dendritic spines density and long-term potentiation and complete inhibition of changes in basal synaptic activity. Using a radiolabelled version of the compound, we found that CLR01 crossed the mouse blood–brain barrier at ∼2% of blood levels. Treatment of 15-month-old triple-transgenic mice for 1 month with CLR01 resulted in a decrease in brain amyloid β protein aggregates, hyperphosphorylated tau and microglia load as observed by immunohistochemistry. Importantly, no signs of toxicity were observed in the treated mice, and CLR01 treatment did not affect the amyloidogenic processing of amyloid β protein precursor. Examining induction or inhibition of the cytochrome P450 metabolism system by CLR01 revealed minimal interaction. Together, these data suggest that CLR01 is safe for use at concentrations well above those showing efficacy in mice. The efficacy and toxicity results support a process-specific mechanism of action of molecular tweezers and suggest that these are promising compounds for developing disease-modifying therapy for Alzheimer’s disease and related disorders. PMID:23183235

  4. Delineation of a frequency-organized region isolated from the mouse primary auditory cortex.

    PubMed

    Tsukano, Hiroaki; Horie, Masao; Bo, Takeshi; Uchimura, Arikuni; Hishida, Ryuichi; Kudoh, Masaharu; Takahashi, Kuniyuki; Takebayashi, Hirohide; Shibuki, Katsuei

    2015-04-01

    The primary auditory cortex (AI) is the representative recipient of information from the ears in the mammalian cortex. However, the delineation of the AI is still controversial in a mouse. Recently, it was reported, using optical imaging, that two distinct areas of the AI, located ventrally and dorsally, are activated by high-frequency tones, whereas only one area is activated by low-frequency tones. Here, we show that the dorsal high-frequency area is an independent region that is separated from the rest of the AI. We could visualize the two distinct high-frequency areas using flavoprotein fluorescence imaging, as reported previously. SMI-32 immunolabeling revealed that the dorsal region had a different cytoarchitectural pattern from the rest of the AI. Specifically, the ratio of SMI-32-positive pyramidal neurons to nonpyramidal neurons was larger in the dorsal high-frequency area than the rest of the AI. We named this new region the dorsomedial field (DM). Retrograde tracing showed that neurons projecting to the DM were localized in the rostral part of the ventral division of the medial geniculate body with a distinct frequency organization, where few neurons projected to the AI. Furthermore, the responses of the DM to ultrasonic courtship songs presented by males were significantly greater in females than in males; in contrast, there was no sex difference in response to artificial pure tones. Our findings offer a basic outline on the processing of ultrasonic vocal information on the basis of the precisely subdivided, multiple frequency-organized auditory cortex map in mice. PMID:25695649

  5. Expression of HPV 58 long and short L1 capsid proteins in primary mouse keratinocyte cultures.

    PubMed

    Wang, Xiao; Liu, Juan; Zhao, Wei Ming; Zhao, Kong-Nan

    2009-01-01

    We studied expression of HPV 58 long and short L1 proteins in primary mouse keratinocyte (KC) cultures by transient transfection of the L1 expression constructs. Following transient transfection, long and short L1 open reading frames (ORFs) were transcribed continuously for 9 days; however, no significant difference was detected between the long and short L1 mRNA levels measured by quantitative RT-PCR. Western blot analysis showed that both long and short L1 proteins were continuously detected in L1-transfected KCs for 9 days post-transfection and the significantly increased signals of the L1 proteins over time were associated with KC differentiation. Moreover, L1 protein was more abundant in KCs transfected with the short L1 ORF than the long L1 ORF. In vitro translation of the L1 mRNAs indicated further that the short L1 mRNA had significantly higher translation efficiency than the long L1 mRNA in cell-free lysate system. The L1 proteins expressed from the two L1 mRNAs in KCs were similarly stable. Thus, approximate 40% lower level of expression of the L1 protein in KCs transfected with the long L1 ORF was probably due to a stem-loop structure with high DeltaG value downstream the first AUG codon in its mRNA secondary structure. This stem-loop structure might prevent efficient binding of the ribosome to mRNA and therefore reduced translation. PMID:19149676

  6. Delineation of a frequency-organized region isolated from the mouse primary auditory cortex

    PubMed Central

    Horie, Masao; Bo, Takeshi; Uchimura, Arikuni; Hishida, Ryuichi; Kudoh, Masaharu; Takahashi, Kuniyuki; Takebayashi, Hirohide; Shibuki, Katsuei

    2015-01-01

    The primary auditory cortex (AI) is the representative recipient of information from the ears in the mammalian cortex. However, the delineation of the AI is still controversial in a mouse. Recently, it was reported, using optical imaging, that two distinct areas of the AI, located ventrally and dorsally, are activated by high-frequency tones, whereas only one area is activated by low-frequency tones. Here, we show that the dorsal high-frequency area is an independent region that is separated from the rest of the AI. We could visualize the two distinct high-frequency areas using flavoprotein fluorescence imaging, as reported previously. SMI-32 immunolabeling revealed that the dorsal region had a different cytoarchitectural pattern from the rest of the AI. Specifically, the ratio of SMI-32-positive pyramidal neurons to nonpyramidal neurons was larger in the dorsal high-frequency area than the rest of the AI. We named this new region the dorsomedial field (DM). Retrograde tracing showed that neurons projecting to the DM were localized in the rostral part of the ventral division of the medial geniculate body with a distinct frequency organization, where few neurons projected to the AI. Furthermore, the responses of the DM to ultrasonic courtship songs presented by males were significantly greater in females than in males; in contrast, there was no sex difference in response to artificial pure tones. Our findings offer a basic outline on the processing of ultrasonic vocal information on the basis of the precisely subdivided, multiple frequency-organized auditory cortex map in mice. PMID:25695649

  7. Neurogenic and Neurotrophic Effects of BDNF Peptides in Mouse Hippocampal Primary Neuronal Cell Cultures

    PubMed Central

    Cardenas-Aguayo, Maria del Carmen; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Iqbal, Khalid

    2013-01-01

    The level of brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is down regulated in Alzheimers disease (AD), Parkinsons disease (PD), depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5) corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18) primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706) of the TrkB receptor, which could be blocked by the Trks inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H2O2-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated. PMID:23320097

  8. Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

    PubMed Central

    Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2013-01-01

    Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

  9. Mouse bone marrow-derived mast cells (BMMC) change their phenotype when cultured with fibroblasts

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Stevens, R.L.

    1986-03-05

    The heparin-containing mast cells (HP-MC) that reside in the connective tissues of the mouse, but not the chondroitin sulfate containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse BMMC (the presumptive in vitro counterpart of the in vivo differentiated mucosal mast cell) were cultured for 2-14 days with confluent skin-derived 3T3 fibroblasts in RPMI-1640 containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin-3 in the conditioned medium. The mast cells remained viable throughout the period of co-culture, since they failed to release LDG and because they increased their histamine content per cell approx.15-fold. After 8-9 days of co-culture, >50% of the BMMC changed histochemically becoming safranin positive. At this time, 30-50% of the (/sup 35/S)glycosaminoglycans on the proteoglycans synthesized by these co-cultured mass cells were heparin, whereas the initial BMMC synthesized proteoglycans containing only chondroitin sulfate E. That interleukin 3-dependent mouse BMMC can be induced to undergo a phenotypic change so as to express characteristics of a HP-MC suggests that the tissue microenvironment determines the differentiated characteristics of these cells.

  10. Potential role of proprotein convertase SKI-1 in the mineralization of primary bone.

    PubMed

    Gorski, Jeff P; Huffman, Nichole T; Cui, Chaoying; Henderson, Ellen P; Midura, Ronald J; Seidah, Nabil G

    2009-01-01

    The biochemical mechanism controlling nucleation of mineral crystals in developing bone, along with the growth and propagation of these crystals once formed, remains poorly understood. To define the nucleation mechanism, a proteomics analysis was begun on isolated biomineralization foci (BMF), sites of initial crystal nucleation in osteoblastic cell cultures and in primary bone. Comparative analyses of the protein profile for mineralized BMF with that for total osteoblast cultures revealed the latter were enriched in several proteins including BAG-75 and BSP, as well as fragments of each. When 12 protease inhibitors were added separately to UMR 106-01 osteoblastic cultures, only the serine protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) blocked cleavage of BAG-75 and BSP, and prevented mineral crystal nucleation within BMF. Consideration of the specificities of the inhibitors tested and the fact that AEBSF inhibition was not dependent upon inclusion of FBS in the culture media indicated that mineral nucleation does not require serine protease plasmin, thrombin, kallikrein, urokinase, C1s or furin. In contrast, SKI-1 (S1P or site-1) is a membrane-bound serine protease inhibitable by AEBSF. We show here for the first time that mineralizing UMR 106 cells express a 98-kDa active, soluble form of SKI-1 within BMF. In contrast, nonmineralizing UMR cells appear to differentially process SKI-1 into smaller immunoreactive fragments (<35 kDa). These findings suggest that SKI-1 plays a direct or indirect role in assembly of functional nucleation complexes containing BAG-75 and BSP and their fragments, thus facilitating initial mineral nucleation within these biomineralization foci. PMID:18728345

  11. Analysis of and prognostic information from disseminated tumour cells in bone marrow in primary breast cancer: a prospective observational study

    PubMed Central

    2012-01-01

    Background Disseminated tumour cells (DTCs) in the bone marrow of patients with breast cancer have been identified as an independent predictor of poor prognosis in patients with non-metastatic disease. This prospective study aimed to evaluate the presence and prognostic value of DTCs in the bone marrow of female patients with primary breast cancer. Methods Between 1999 and 2003, bone marrow aspirates were obtained from patients at the time of surgery for primary invasive breast cancer. DTCs in bone marrow were identified using monoclonal antibodies against cytokeratins for detection of epithelial cells. The detection of DTCs was related to clinical follow-up with distant disease-free survival (DDFS) and breast cancer-specific survival as endpoints. Bone marrow aspirates from adult healthy bone marrow donors were analysed separately. Results DTCs were analysed in 401 patients, and cytokeratin-positive cells were found in 152 of these (38%). An immunofluorescence (IF) staining procedure was used in 327 patients, and immunocytochemistry (IC) was performed in 74 patients. The IF-based method resulted in 40% DTC-positive cases, whereas 30% were positive using IC (p?=?0.11). The presence of DTCs in bone marrow was not significantly related to patient or tumour characteristics. The presence of DTCs was not a prognostic factor for DDFS (IF: hazards ratio [HR], 2.2; 95% confidence interval [CI], 0.632.2; p?=?0.60; IC: HR, 0.84; 95% CI, 0.098.1; p?=?0.88). Significant prognostic factors were lymph node metastases, oestrogen receptor positivity, Nottingham histological grade, and tumour size using Cox univariate analysis. The analyses were positive for epithelial cells in bone marrow from adult healthy donors in 19 (25%) samples. Conclusions The detection of DTCs in bone marrow in primary breast cancer was previously shown to be a predictor of poor prognosis. We were not able to confirm these results in a prospective cohort including unselected patients before the standard procedure was established. Future studies with a standardised patient protocol and improved technique for isolating and detecting DTCs may reveal the clinical applications of DTC detection in patients with micrometastases in the bone marrow. PMID:22963449

  12. Bone marrow origin of decidual cell precursors in the pseudopregnant mouse uterus

    SciTech Connect

    Kearns, M.; Lala, P.K.

    1982-05-01

    Decidual cells are considered to be the endproduct of a hormonally induced transformation of endometrial stromal cells of the uterus. However, the source of these precursors remains unknown. This study of evaluated the possibility of their bone marrow origin by an examination of the H-2 phenotype of decidual cells in pseudopregnant bone marrow chimeras. These chimeras were produced by repopulating lethally irradiated CBA/J female (H-2k) mice with bone marrow from (CBA/J x C57BL/6J) F1 female (H-2kb) mice. Pseudopregnancy was produced with a hormonal regimen followed by an oil-induced decidual stimulus. Chimerism was evaluated radioautographically by an identification of the donor-specific Kb phenotype on cells with an immunolabeling technique with monospecific anti-H-2 serum followed by radioiodinated protein A. The extent of chimerism as indicated by the degree of Kb labeling on decidual cells as well as macrophages contained within the decidual nodules was quantitatively compared with that seen on splenic lymphocytes. Fair to good chimerism, as reflected by labeling for the donor-specific marker (Kb), was seen on splenic lymphocytes and macrophages within the decidual nodules in 6 out of 11 animals. A similar level of chimerism was detected on decidual cells in all but one of these six, in which case this was low. One animal showed low chimerism in the spleen but good chimerism on the decidual cells. The remaining four mice were nonchimeric for all three cell types. These results indicate that decidual cells and macrophages appearing within the decidual nodules of pseudopregnant mice are ultimate descendants of bone marrow cells.

  13. ERR{alpha} regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

    SciTech Connect

    Rajalin, Ann-Marie; Pollock, Hanna; Aarnisalo, Piia; Department of Clinical Chemistry, University of Helsinki and Helsinki University Central Hospital

    2010-05-28

    The orphan nuclear receptor estrogen-related receptor-{alpha} (ERR{alpha}) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERR{alpha} in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERR{alpha} deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERR{alpha} deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERR{alpha} in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERR{alpha} deficient MSCs and enhanced upon ERR{alpha} overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERR{alpha}. Under adipogenic conditions, ERR{alpha} deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERR{alpha} in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERR{alpha} may play different roles in bone under different physiological conditions.

  14. Exome sequencing identifies a nonsense mutation in Fam46a associated with bone abnormalities in a new mouse model for skeletal dysplasia.

    PubMed

    Diener, Susanne; Bayer, Sieglinde; Sabrautzki, Sibylle; Wieland, Thomas; Mentrup, Birgit; Przemeck, Gerhard K H; Rathkolb, Birgit; Graf, Elisabeth; Hans, Wolfgang; Fuchs, Helmut; Horsch, Marion; Schwarzmayr, Thomas; Wolf, Eckhard; Klopocki, Eva; Jakob, Franz; Strom, Tim M; Hrabě de Angelis, Martin; Lorenz-Depiereux, Bettina

    2016-04-01

    We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a (E157*Mhda)) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a (E157*Mhda) mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a (E157*Mhda) mice are the first mouse model for a mutation within the Fam46a gene. PMID:26803617

  15. Autologous serum improves bone formation in a primary stable silica-embedded nanohydroxyapatite bone substitute in combination with mesenchymal stem cells and rhBMP-2 in the sheep model

    PubMed Central

    Boos, Anja M; Weigand, Annika; Deschler, Gloria; Gerber, Thomas; Arkudas, Andreas; Kneser, Ulrich; Horch, Raymund E; Beier, Justus P

    2014-01-01

    New therapeutic strategies are required for critical size bone defects, because the gold standard of transplanting autologous bone from an unharmed area of the body often leads to several severe side effects and disadvantages for the patient. For years, tissue engineering approaches have been seeking a stable, axially vascularized transplantable bone replacement suitable for transplantation into the recipient bed with pre-existing insufficient conditions. For this reason, the arteriovenous loop model was developed and various bone substitutes have been vascularized. However, it has not been possible thus far to engineer a primary stable and axially vascularized transplantable bone substitute. For that purpose, a primary stable silica-embedded nanohydroxyapatite (HA) bone substitute in combination with blood, bone marrow, expanded, or directly retransplanted mesenchymal stem cells, recombinant human bone morphogenetic protein 2 (rhBMP-2), and different carrier materials (fibrin, cell culture medium, autologous serum) was tested subcutaneously for 4 or 12 weeks in the sheep model. Autologous serum lead to an early matrix change during degradation of the bone substitute and formation of new bone tissue. The best results were achieved in the group combining mesenchymal stem cells expanded with 60 μg/mL rhBMP-2 in autologous serum. Better ingrowth of fibrovascular tissue could be detected in the autologous serum group compared with the control (fibrin). Osteoclastic activity indicating an active bone remodeling process was observed after 4 weeks, particularly in the group with autologous serum and after 12 weeks in every experimental group. This study clearly demonstrates the positive effects of autologous serum in combination with mesenchymal stem cells and rhBMP-2 on bone formation in a primary stable silica-embedded nano-HA bone grafting material in the sheep model. In further experiments, the results will be transferred to the sheep arteriovenous loop model in order to engineer an axially vascularized primary stable bone replacement in clinically relevant size for free transplantation. PMID:25429218

  16. Incidence of distal bone metastases in patients treated for palliative radiotherapy and associations with primary tumour types

    PubMed Central

    Barnes, Mark; Tiwana, Manpreet S.; Kiraly, Andrew; Hutchison, Mitch; Olson, Robert A.

    2015-01-01

    Purpose This study assesses the incidence of distal bone metastases in palliative radiotherapy (RT) patients. Material and methods All courses of RT for bone metastases from 2007–2011 for patient living in British Columbia (BC) were identified in a provincial RT programme. Treated bone metastases (BoM) were categorized as distal if the BoM was located within or distal to the elbow or knee. Patients were grouped by primary tumour site as breast, lung, prostate gastrointestinal, haematological, melanoma, and other. The incidence of distal bone metastases and associations with primary tumour types were determined. Results From 2007 to 2011, 8008 patients were treated with 16,277 courses of RT, of which 425 (3%) were courses of RT for distal BoM. The incidence of distal BoM in decreasing order by primary tumour type was melanoma (5%), haematological (3%), lung (2%), other (2%), prostate (2%), breast (1%) and gastrointestinal (1%). Distal BoM where more commonly identified in the lower extremity (87%, p<0.001). Single fraction RT was used more commonly for distal vs non-distal BoM (66% vs. 49%; p<0.001). Conclusion The incidence of distal BoM among patients treated with palliative RT was 3% and most commonly identified in patients with melanoma and haematological malignancies. PMID:26730358

  17. Novel Lesions of Bones and Joints Associated with Chikungunya Virus Infection in Two Mouse Models of Disease: New Insights into Disease Pathogenesis

    PubMed Central

    Goupil, Brad A.; McNulty, Margaret A.; Martin, Matthew J.; McCracken, Michael K.; Christofferson, Rebecca C.; Mores, Christopher N.

    2016-01-01

    Chikungunya virus is an arbovirus spread predominantly by Aedes aegypti and Ae. albopictus mosquitoes, and causes debilitating arthralgia and arthritis. While these are common manifestations during acute infection and it has been suggested they can recur in patients chronically, gaps in knowledge regarding the pathogenesis still exist. Two established mouse models were utilized (adult IRF 3/7 -/- -/- and wild-type C57BL/6J mice) to evaluate disease manifestations in bones and joints at various timepoints. Novel lesions in C57BL/6J mice consisted of periostitis (91%) and foci of cartilage of necrosis (50% of mice at 21 DPI). Additionally, at 21 DPI, 50% and 75% of mice exhibited periosteal bone proliferation affecting the metatarsal bones, apparent via histology and μCT, respectively. μCT analysis did not reveal any alterations in trabecular bone volume measurements in C57BL/6J mice. Novel lesions demonstrated in IRF 3/7 -/- -/- mice at 5 DPI included focal regions of cartilage necrosis (20%), periosteal necrosis (66%), and multifocal ischemic bone marrow necrosis (100%). Contralateral feet in 100% of mice of both strains had similar, though milder lesions. Additionally, comparison of control IRF 3/7 -/- -/- and wild-type C57BL/6J mice demonstrated differences in cortical bone. These experiments demonstrate novel manifestations of disease similar to those occurring in humans, adding insight into disease pathogenesis, and representing new potential targets for therapeutic interventions. Additionally, results demonstrate the utility of μCT in studies of bone and joint pathology and illustrate differences in bone dynamics between mouse strains. PMID:27182740

  18. Efficient expansion of mesenchymal stem cells from mouse bone marrow under hypoxic conditions.

    PubMed

    Yew, Tu-Lai; Chang, Ming-Chau; Hsu, Yuan-Tong; He, Fan-Yu; Weng, Wen-Hui; Tsai, Chih-Chien; Chiu, Fang-Yao; Hung, Shih-Chieh

    2013-12-01

    To realize the therapeutic potential of mesenchymal stem cells (MSCs), a large number of high-quality MSCs isolated from different species, such as mouse, were acquired for preclinical animal studies. Surprisingly, isolation and purification of mouse MSCs (mMSCs) is arduous because of the low frequency of MSCs and contamination of haematopoietic cells in culture. We have developed a method based on low density and hypoxic culture to isolate and expand mMSCs from different strains, including BALB/c, C57BL/6J, FVB/N and DBA/2. The cells from all of the strains expanded more rapidly when plated at low density in hypoxic culture compared with normoxic culture. These cells expressed CD44, CD105, CD29 and Sca-1 markers but not CD11b, CD34, CD45 and CD31 markers. Moreover, they were able to differentiate along osteoblastic, adipocytic and chondrocytic lineages. In conclusion, we have developed a robust method for isolation and expansion of mMSCs by combining low-density culture with hypoxic culture. PMID:22623422

  19. A Common Variant in CLDN14 is Associated with Primary Biliary Cirrhosis and Bone Mineral Density.

    PubMed

    Tang, Ruqi; Wei, Yiran; Li, Zhiqiang; Chen, Haoyan; Miao, Qi; Bian, Zhaolian; Zhang, Haiyan; Wang, Qixia; Wang, Zhaoyue; Lian, Min; Yang, Fan; Jiang, Xiang; Yang, Yue; Li, Enling; Seldin, Michael F; Gershwin, M Eric; Liao, Wilson; Shi, Yongyong; Ma, Xiong

    2016-01-01

    Primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, has been associated with increased incidence of osteoporosis. Intriguingly, two PBC susceptibility loci identified through genome-wide association studies are also involved in bone mineral density (BMD). These observations led us to investigate the genetic variants shared between PBC and BMD. We evaluated 72 genome-wide significant BMD SNPs for association with PBC using two European GWAS data sets (n = 8392), with replication of significant findings in a Chinese cohort (685 cases, 1152 controls). Our analysis identified a novel variant in the intron of the CLDN14 gene (rs170183, Pfdr = 0.015) after multiple testing correction. The three associated variants were followed-up in the Chinese cohort; one SNP rs170183 demonstrated consistent evidence of association in diverse ethnic populations (Pcombined = 2.43 × 10(-5)). Notably, expression quantitative trait loci (eQTL) data revealed that rs170183 was correlated with a decline in CLDN14 expression in both lymphoblastoid cell lines and T cells (Padj = 0.003 and 0.016, respectively). In conclusion, our study identified a novel PBC susceptibility variant that has been shown to be strongly associated with BMD, highlighting the potential of pleiotropy to improve gene discovery. PMID:26842849

  20. A Common Variant in CLDN14 is Associated with Primary Biliary Cirrhosis and Bone Mineral Density

    PubMed Central

    Tang, Ruqi; Wei, Yiran; Li, Zhiqiang; Chen, Haoyan; Miao, Qi; Bian, Zhaolian; Zhang, Haiyan; Wang, Qixia; Wang, Zhaoyue; Lian, Min; Yang, Fan; Jiang, Xiang; Yang, Yue; Li, Enling; Seldin, Michael F.; Gershwin, M. Eric; Liao, Wilson; Shi, Yongyong; Ma, Xiong

    2016-01-01

    Primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, has been associated with increased incidence of osteoporosis. Intriguingly, two PBC susceptibility loci identified through genome-wide association studies are also involved in bone mineral density (BMD). These observations led us to investigate the genetic variants shared between PBC and BMD. We evaluated 72 genome-wide significant BMD SNPs for association with PBC using two European GWAS data sets (n = 8392), with replication of significant findings in a Chinese cohort (685 cases, 1152 controls). Our analysis identified a novel variant in the intron of the CLDN14 gene (rs170183, Pfdr = 0.015) after multiple testing correction. The three associated variants were followed-up in the Chinese cohort; one SNP rs170183 demonstrated consistent evidence of association in diverse ethnic populations (Pcombined = 2.43 × 10−5). Notably, expression quantitative trait loci (eQTL) data revealed that rs170183 was correlated with a decline in CLDN14 expression in both lymphoblastoid cell lines and T cells (Padj = 0.003 and 0.016, respectively). In conclusion, our study identified a novel PBC susceptibility variant that has been shown to be strongly associated with BMD, highlighting the potential of pleiotropy to improve gene discovery. PMID:26842849

  1. Quality of Life in Children Following Treatment for a Malignant Primary Bone Tumour Around the Knee

    PubMed Central

    Cool, Paul; Grimer, Robert J.; Carter, Simon R.; Cotter, Imogen M.; Ellis, Ann J.; Kopel, Sheryl

    1997-01-01

    Purpose. We report on the quality of life following treatment for a malignant primary bone tumour around the knee in skeletally immature children. Patients. Patients (n = 41; mean age = 18 years; range 8–28) had all experienced chemotherapy in a neo-adjuvant setting, surgical excision of the tumour and endoprosthetic replacement. Methods. Interviews were conducted separately with the child and mother and focused on mobility, body image and the impact of treatment on schooling, employment and plans for the future. Results. Mobility in the group was variable. Only 12% reported that they could run with any confidence. The proportion who were able to swim (49%) or ride a bike (46%) was higher. All had experienced major disruption in schooling (mean absence following diagnosis = 12 months). Eight had repeated a school year and 41% patients reported that their schoolwork was affected. As a result of their experience, eight (six females and two males) chose health-related employment. Concerns for the future were highest among males and those with manual jobs. Three patients were receiving psychiatric support, in relation to extreme concern about the risk of recurrence. All expressed satisfaction with treatment, and older patients believed that the prosthesis gave a better quality of life than amputation. Discussion. Our data suggest that outcome following limb-salvage surgery is variable. Education is disrupted. Even so, only two left school with no qualifications. Employment is most restricted among males with few qualifications who may benefit from sensitive vocational counselling. PMID:18521199

  2. Allogeneic bone marrow mesenchymal stem cell transplantation in patients with UDCA-resistant primary biliary cirrhosis.

    PubMed

    Wang, Li; Han, Qin; Chen, Hua; Wang, Ke; Shan, Guang-liang; Kong, Fang; Yang, Yun-jiao; Li, Yong-zhe; Zhang, Xuan; Dong, Fen; Wang, Qian; Xu, Dong; Hu, Zhao-jun; Wang, Shi-hua; Keating, Armand; Bi, Ya-lan; Zhang, Feng-chun; Zhao, Robert Chun-hua

    2014-10-15

    The objective of this study was to evaluate the safety and efficacy of allogeneic bone marrow mesenchymal stromal/stem cell transplantation (BM-MSCT) for patients with ursodeoxycholic acid (UDCA)-resistant primary biliary cirrhosis (PBC). Ten patients were enrolled in this trial of BM-MSCT. All patients were permitted to concurrently continue their previous UDCA treatment. The efficacy of BM-MSCT in UDCA-resistant PBC was assessed at various time points throughout the 12-month follow up. No transplantation-related side effects were observed. The life quality of the patients was improved after BM-MSCT as demonstrated by responses to the PBC-40 questionnaire. Serum levels of ALT, AST, γ-GT, and IgM significantly decreased from baseline after BM-MSCT. In addition, the percentage of CD8+ T cells was reduced, while that of CD4+CD25+Foxp3+ T cells was increased in peripheral lymphocytic subsets. Serum levels of IL-10 were also elevated. Notably, the optimal therapeutic outcome was acquired in 3 to 6 months and could be maintained for 12 months after BM-MSCT. In conclusion, allogeneic BM-MSCT in UDCA-resistant PBC is safe and appears to be effective. PMID:24835895

  3. Transplanted bone marrow stem cells relocate to infarct penumbra and co-express endogenous proliferative and immature neuronal markers in a mouse model of ischemic cerebral stroke

    PubMed Central

    2010-01-01

    Background Several studies demonstrate that neurogenesis may be induced or activated following vascular insults, which may be important for neuronal regeneration and functional recovery. Understanding the cellular mechanism underlying stroke-associated neurogenesis is of neurobiological as well as neurological/clinical relevance. The present study attempted to explore potential homing and early development of transplanted bone marrow stem cells in mouse forebrain after focal occlusion of the middle cerebral artery, an experimental model of ischemic stroke. Results Bone marrow stem cells isolated from donor mice were confirmed by analysis of surface antigen profile, and were pre-labeled with a lipophilic fluorescent dye PKH26, and subsequently transfused into recipient mice with middle cerebral artery coagulation. A large number of PKH26-labeled cells were detected surrounding the infarct site, most of which colocalized with immunolabelings for the proliferating cell nuclear antigen (PCNA) and some also colocalized with the immature neuronal marker doublecortin (DCX) during 1-2 weeks after the bone marrow cells transfusion. Conclusions The present study shows that transplanted bone morrow cells largely relocate to the infarct penumbra in ischemic mouse cerebrum. These transplanted bone marrow cells appear to undergo a process of in situ proliferation and develop into putative cortical interneurons during the early phase of experimental vascular injury. PMID:20973978

  4. IL-6 Contributes to the Defective Osteogenesis of Bone Marrow Stromal Cells from the Vertebral Body of the Glucocorticoid-Induced Osteoporotic Mouse

    PubMed Central

    Zhang, Yuan-yuan; Yang, Hui-lin

    2016-01-01

    Osteoporosis is one of the most prevalent skeletal system diseases. It is characterized by a decrease in bone mass and microarchitectural changes in bone tissue that lead to an attenuation of bone resistance and susceptibility to fracture. Vertebral fracture is by far the most prevalent osteoporotic fracture. In the musculoskeletal system, osteoblasts, originated from bone marrow stromal cells (BMSC), are responsible for osteoid synthesis and mineralization. In osteoporosis, BMSC osteogenic differentiation is defective. However, to date, what leads to the defective BMSC osteogenesis in osteoporosis remains an open question. In the current study, we made attempts to answer this question. A mouse model of glucocorticoid-induced osteoporosis (GIO) was established and BMSC were isolated from vertebral body. The impairment of osteogenesis was observed in BMSC of osteoporotic vertebral body. The expression profiles of thirty-six factors, which play important roles in bone metabolisms, were compared through antibody array between normal and osteoporotic BMSC. Significantly higher secretion level of IL-6 was observed in osteoporotic BMSCs compared with normal control. We provided evidences that IL-6 over-secretion impaired osteogenesis of osteoporotic BMSC. Further, it was observed that β-catenin activity was inhibited in response to IL-6 over-secretion. More importantly, in vivo administration of IL-6 neutralizing antibody was found to be helpful to rescue the osteoporotic phenotype of mouse vertebral body. Our study provides a deeper insight into the pathophysiology of osteoporosis and identifies IL-6 as a promising target for osteoporosis therapy. PMID:27128729

  5. Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells.

    PubMed

    Takenaka, T; Murray, G J; Qin, G; Quirk, J M; Ohshima, T; Qasba, P; Clark, K; Kulkarni, A B; Brady, R O; Medin, J A

    2000-06-20

    Fabry disease is a compelling target for gene therapy as a treatment strategy. A deficiency in the lysosomal hydrolase alpha-galactosidase A (alpha-gal A; EC ) leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop vascular occlusions that cause cardiovascular, cerebrovascular, and renal disease. Unlike for some lysosomal storage disorders, there is limited primary nervous system involvement in Fabry disease. The enzyme defect can be corrected by gene transfer. Overexpression of alpha-gal A by transduced cells results in secretion of this enzyme. Secreted enzyme is available for uptake by nontransduced cells presumably by receptor-mediated endocytosis. Correction of bystander cells may occur locally or systemically after circulation of the enzyme in the blood. In this paper we report studies on long-term genetic correction in an alpha-gal A-deficient mouse model of Fabry disease. alpha-gal A-deficient bone marrow mononuclear cells (BMMCs) were transduced with a retrovirus encoding alpha-gal A and transplanted into sublethally and lethally irradiated alpha-gal A-deficient mice. alpha-gal A activity and Gb3 levels were analyzed in plasma, peripheral blood mononuclear cells, BMMCs, liver, spleen, heart, lung, kidney, and brain. Primary recipient animals were followed for up to 26 weeks. BMMCs were then transplanted into secondary recipients. Increased alpha-gal A activity and decreased Gb3 storage were observed in all recipient groups in all organs and tissues except the brain. These effects occurred even with a low percentage of transduced cells. The findings indicate that genetic correction of bone marrow cells derived from patients with Fabry disease may have utility for phenotypic correction of patients with this disorder. PMID:10840053

  6. Primary structure of the mouse protein encoded by the lgh1{sup 3} allele of the n haplotype

    SciTech Connect

    Klebert, S.; Zimmermann, B.; Vaesen, M.; Pardowitz, I.; Kratzin, H.; Hilschmann, N.

    1995-01-11

    The monoclonal mouse IgG2a antibody mAb735 obtained from an autoimmune NZB mouse immunized with live group B memingococci was purified from ascitic fluid. After reduction and carboxymethylation the H chain was digested with trypsin or cyanogen bromide, and peptides isolated by reversed-phase high-performance liquid chromatography were characterized by automated sequence analysis and plasma desorption mass spectrometry either directly or after subfragmentation with endoproteinase Asp-N and {alpha}-chymotrypsin, respectively. Along with previous reports on the L chain and the F(ab`){sub 2} region sequences the H chain sequence reported here completes the primary structure of mAb735. It was determined as the basis for the X-ray structure elucidation which is in progress. 7 refs., 2 figs.

  7. In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow

    PubMed Central

    Lo Celso, Cristina; Lin, Charles P; Scadden, David T

    2011-01-01

    In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ~1 week. PMID:21212779

  8. Functional and transcriptomic recovery of infarcted mouse myocardium treated with bone marrow mononuclear cells.

    PubMed

    Lachtermacher, Stephan; Esporcatte, Bruno L B; Fortes, Fábio da Silva de Azevedo; Rocha, Nazareth Novaes; Montalvão, Fabrício; Costa, Patricia C; Belem, Luciano; Rabischoffisky, Arnaldo; Faria Neto, Hugo C C; Vasconcellos, Rita; Iacobas, Dumitru A; Iacobas, Sanda; Spray, David C; Thomas, Neil M; Goldenberg, Regina C S; de Carvalho, Antonio C Campos

    2012-03-01

    Although bone marrow-derived mononuclear cells (BMNC) have been extensively used in cell therapy for cardiac diseases, little mechanistic information is available to support reports of their efficacy. To address this shortcoming, we compared structural and functional recovery and associated global gene expression profiles in post-ischaemic myocardium treated with BMNC transplantation. BMNC suspensions were injected into cardiac scar tissue 10 days after experimental myocardial infarction. Six weeks later, mice undergoing BMNC therapy were found to have normalized antibody repertoire and improved cardiac performance measured by ECG, treadmill exercise time and echocardiography. After functional testing, gene expression profiles in cardiac tissue were evaluated using high-density oligonucleotide arrays. Expression of more than 18% of the 11981 quantified unigenes was significantly altered in the infarcted hearts. BMNC therapy restored expression of 2099 (96.2%) of the genes that were altered by infarction but led to altered expression of 286 other genes, considered to be a side effect of the treatment. Transcriptional therapeutic efficacy, a metric calculated using a formula that incorporates both recovery and side effect of treatment, was 73%. In conclusion, our results confirm a beneficial role for bone marrow-derived cell therapy and provide new information on molecular mechanisms operating after BMNC transplantation on post ischemic heart failure in mice. PMID:21671060

  9. Thymic repopulation following intrathymic injection of mouse bone marrow cells in MHC matched and mismatched recipients

    SciTech Connect

    Chervenak, R.

    1986-03-01

    T cell precursors (pre-T cells) have traditionally been detected by their ability to repopulate the thymus of heavily irradiated mice following intravenous injection. Recently, Goldschneider et. al. developed an assay system which involves the direct injection of pre-T cells into the thymus. The authors used this technique to evaluate the ability of bone marrow cells to repopulate thymuses in various donor-host strain combinations. Sub-lethally irradiated (600R) mice were injected intrathymically with 2 x 10/sup 6/ bone marrow cells which differed from the recipient with respect to their Thy 1 allotype. The percentage of thymus cells expressing either the donor or recipient type Thy 1 marker was determined 14 to 21 days after injection. These experiments showed that in MHC matched donor-host combinations (AKR/cum ..-->.. AKR/J and CBA/J ..-->.. AKR/J), cells derived from the donor inoculum accounted for 40% to 75% of the total thymus population. MHC mismatched donor-host combinations (C57BL/6J ..-->.. AKR/J and Balb/c ..-->.. AKR/J) resulted in significantly less donor-type repopulation of the thymus. In these cases, donor repopulation typically ranged from 0% to 4%. The ability of the pre-T cells detected by intrathymic injection to proliferate in the thymic environment, therefore, appears to be influenced by the MHC. This may reflect commitment of pre-T cells to MHC haplotype recognition prior to their migration to the thymus.

  10. Development of a novel frontal bone defect mouse model for evaluation of osteogenesis efficiency.

    PubMed

    Kim, Ju-Young; Kwak, Sung Chul; Ahn, Sung-Jun; Baek, Jong Min; Jung, Sung Tae; Yun, Ki Jung; Yoon, Kwon-Ha; Oh, Jaemin; Lee, Myeung Su

    2015-12-01

    The skull defect model is the existing representative osteogenesis model. The skull defect model involves monitoring osteogenesis patterns at the site of a skull defect, which has the advantages that identical defects can be induced across individual experimental animals and the results can be quantitatively evaluated. However, it can damage the cerebrum because it requires a complex surgery performed on the parietal bone. This study aims to develop a new osteogenesis model that compensates for the weak points of the existing model. Male 8-week-old imprinting control region mice were put under inhalational anesthesia, and the surgery area was disinfected with 70% ethanol prior to the creation of a 5-mm incision along the sagittal line between the glabella with a pair of scissors. The incised area was opened and, after we checked the positions of the inferior cerebral vein and the sagittal suture, a 21-gauge needle was used to make two symmetrical holes with respect to the sagittal suture 3 mm below the inferior cerebral vein and 2 mm on either side of the sagittal suture. After images were obtained using micro-computed tomography, the degree of osteogenesis was quantitatively analyzed. In addition, mRNA extracted from the site of the defect confirmed a significant increase in mRNA levels of collagen 1a, alkaline phosphatase, bone sialoprotein, osteocalcin, and Runx2, known markers for osteoblasts. The promotion of osteogenesis could be observed at the site of the defect, by histological analysis. PMID:26053543

  11. Profilin 1 is essential for retention and metabolism of mouse hematopoietic stem cells in bone marrow

    PubMed Central

    Zheng, Junke; Lu, Zhigang; Kocabas, Fatih; Böttcher, Ralph T.; Costell, Mercedes; Kang, Xunlei; Liu, Xiaoye; DeBerardinis, Ralph J.; Wang, Qianming; Chen, Guo-Qiang

    2014-01-01

    How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we demonstrated that the deletion of the cytoskeleton-modulating protein profilin 1 (pfn1) in hematopoietic stem cell (HSCs) led to bone marrow failure, loss of quiescence, and mobilization and apoptosis of HSCs in vivo. A switch from glycolysis to mitochondrial respiration with increased reactive oxygen species (ROS) level was also observed in HSCs on pfn1 deletion. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that the metabolism is mechanistically linked to the cell cycle quiescence of stem cells. The actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Our study provided evidence that pfn1 at least partially acts through the axis of pfn1/Gα13/EGR1 to regulate stem cell retention and metabolism in the bone marrow. PMID:24385538

  12. Functional and Transcriptomic Recovery of Infarcted Mouse Myocardium Treated with Bone Marrow Mononuclear Cells

    PubMed Central

    Lachtermacher, Stephan; Esporcatte, Bruno L. B.; da Silva de Azevedo Fortes, Fábio; Rocha, Nazareth Novaes; Montalvão, Fabrício; Costa, Patricia C.; Belem, Luciano; Rabischoffisky, Arnaldo; Neto, Hugo C. C. Faria; Vasconcellos, Rita; Iacobas, Dumitru A.; Iacobas, Sanda; Spray, David C.; Thomas, Neil M.; Goldenberg, Regina C. S.; de Carvalho, Antonio C. Campos

    2011-01-01

    Although bone marrow-derived mononuclear cells (BMNC) have been extensively used in cell therapy for cardiac diseases, little mechanistic information is available to support reports of their efficacy. To address this shortcoming, we compared structural and functional recovery and associated global gene expression profiles in post-ischaemic myocardium treated with BMNC transplantation. BMNC suspensions were injected into cardiac scar tissue 10 days after experimental myocardial infarction. Six weeks later, mice undergoing BMNC therapy were found to have normalized antibody repertoire and improved cardiac performance measured by ECG, treadmill exercise time and echocardiography. After functional testing, gene expression profiles in cardiac tissue were evaluated using high-density oligonucleotide arrays. Expression of more than 18% of the 11981 quantified unigenes was significantly altered in the infarcted hearts. BMNC therapy restored expression of 2099 (96.2%) of the genes that were altered by infarction but led to altered expression of 286 other genes, considered to be a side effect of the treatment. Transcriptional therapeutic efficacy, a metric calculated using a formula that incorporates both recovery and side effect of treatment, was 73%. In conclusion, our results confirm a beneficial role for bone marrow-derived cell therapy and provide new information on molecular mechanisms operating after BMNC transplantation on post ischemic heart failure in mice. PMID:21671060

  13. Missense Mutations in LRP5 Associated with High Bone Mass Protect the Mouse Skeleton from Disuse- and Ovariectomy-Induced Osteopenia

    PubMed Central

    Niziolek, Paul J.; Bullock, Whitney; Warman, Matthew L.; Robling, Alexander G.

    2015-01-01

    The low density lipoprotein receptor-related protein-5 (LRP5), a co-receptor in the Wnt signaling pathway, modulates bone mass in humans and in mice. Lrp5 knock-out mice have severely impaired responsiveness to mechanical stimulation whereas Lrp5 gain-of-function knock-in and transgenic mice have enhanced responsiveness to mechanical stimulation. Those observations highlight the importance of Lrp5 protein in bone cell mechanotransduction. It is unclear if and how high bone mass-causing (HBM) point mutations in Lrp5 alter the bone-wasting effects of mechanical disuse. To address this issue we explored the skeletal effects of mechanical disuse using two models, tail suspension and Botulinum toxin-induced muscle paralysis, in two different Lrp5 HBM knock-in mouse models. A separate experiment employing estrogen withdrawal-induced bone loss by ovariectomy was also conducted as a control. Both disuse stimuli induced significant bone loss in WT mice, but Lrp5 A214V and G171V were partially or fully protected from the bone loss that normally results from disuse. Trabecular bone parameters among HBM mice were significantly affected by disuse in both models, but these data are consistent with DEXA data showing a failure to continue growing in HBM mice, rather than a loss of pre-existing bone. Ovariectomy in Lrp5 HBM mice resulted in similar protection from catabolism as was observed for the disuse experiments. In conclusion, the Lrp5 HBM alleles offer significant protection from the resorptive effects of disuse and from estrogen withdrawal, and consequently, present a potential mechanism to mimic with pharmaceutical intervention to protect against various bone-wasting stimuli. PMID:26554834

  14. Reciprocal interactions of mouse bone marrow-derived mesenchymal stem cells and BV2 microglia after lipopolysaccharide stimulation

    PubMed Central

    2013-01-01

    Introduction Mesenchymal stem cells (MSCs) are immunosuppressive, but we lack an understanding of how these adult stem cells are in turn affected by immune cells and the surrounding tissue environment. As MSCs have stromal functions and exhibit great plasticity, the influence of an inflamed microenvironment on their responses is important to determine. MSCs downregulate microglial inflammatory responses, and here we describe the mutual effects of coculturing mouse bone marrow MSCs with BV2 microglia in a lipopolysaccharide (LPS) inflammatory paradigm. Methods Mouse MSCs were cultured from femoral and tibial bone marrow aspirates and characterized. MSCs were cocultured with BV2 microglia at four seeding-density ratios (1:0.2, 1:0.1, 1:0.02, and 1:0.01 (BV2/MSC)), and stimulated with 1 μg/ml LPS. In certain assays, MSCs were separated from BV2 cells with a cell-culture insert to determine the influence of soluble factors on downstream responses. Inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) were measured in cocultures, and MSC and BV2 chemotactic ability determined by migration assays. Results We demonstrated MSCs to increase expression of NO and IL-6 and decrease TNF-α in LPS-treated cocultures. These effects are differentially mediated by soluble factors and cell-to-cell contact. In response to an LPS stimulus, MSCs display distinct behaviors, including expressing IL-6 and very high levels of the chemokine CCL2. Microglia increase their migration almost fourfold in the presence of LPS, and interestingly, MSCs provide an equal impetus for microglia locomotion. MSCs do not migrate toward LPS but migrate toward microglia, with their chemotaxis increasing when microglia are activated. Similarly, MSCs do not produce NO when exposed to LPS, but secrete large amounts when exposed to soluble factors from activated microglia. This demonstrates that certain phenotypic changes of MSCs are governed by inflammatory microglia, and not by the inflammatory stimulus. Nonetheless, LPS appears to "prime" the NO-secretory effects of MSCs, as prior treatment with LPS triggers a bigger NO response from MSCs after exposure to microglial soluble factors. Conclusions These effects demonstrate the multifaceted and reciprocal interactions of MSCs and microglia within an inflammatory milieu. PMID:23356521

  15. Zinc finger factor 521 enhances adipogenic differentiation of mouse multipotent cells and human bone marrow mesenchymal stem cells

    PubMed Central

    Tseng, Kuo-Yun; Lin, Shankung

    2015-01-01

    Previously, we found that ZNF521 expression was up-regulated with advancing age in human bone marrow mesenchymal stem cells (bmMSCs). Here, we investigated the regulatory role of ZNF521 in the differentiation of mouse C3H10T1/2 cells and human bmMSCs. Our data show that ZNF521 overexpression repressed osteoblastic differentiation of C3H10T1/2 cells, accompanied by a decrease in Runx2 expression and an increase in PPARγ2 expression. In contrast, ZNF521 overexpression enhanced adipogenic differentiation of C3H10T1/2 cells, concomitant with increased expression of PPARγ2, aP2, adiponectin and C/EBPδ. Chromatin immunoprecipitation followed by quantitative PCR analyses and luciferase reporter assays suggested that ZNF521 overexpression enhances PPARγ2 expression at the transcriptional level. The enhancing effect of ZNF521 overexpression on the adipogenic differentiation of C3H10T1/2 cells was also observed ex vivo. Finally, similar to those noted in C3H10T1/2 cells, ZNF521 overexpression in human bmMSCs was found to promote adipogenic differentiation in vitro and ex vivo, but repressed osteoblastic differentiation in vitro. ZNF521 knockdown significantly repressed adipogenic differentiation in vitro and ex vivo, but promoted osteoblastic differentiation in vitro. We propose that ZNF521 can function as a repressor of osteoblastic differentiation of bmMSCs while promoting adipogenesis, and that elevated ZNF521 expression might play a role in the age-related bone loss. PMID:26008984

  16. Ultra-high performance liquid chromatography-tandem mass spectrometry for the quantification of icaritin in mouse bone.

    PubMed

    Zhang, Shuang-Qing

    2015-01-26

    Icaritin (ICT), a bioactive metabolite of prenylflavonoids from genus Epimedium, has displayed potential benefits for the treatment of osteoporosis, prostate cancer, liver cancer, renal cancer and breast cancer. To investigate the quantity of ICT in bones in vivo, a simple and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed. After a rapid one-step liquid-liquid extraction using ethyl acetate with recovery more than 87.2% at four levels (0.1, 0.2, 8 and 15 ng/mL), ICT and internal standard coumestrol were analyzed on a C18 column using a gradient elution of acetonitrile and water containing ammonium formate and formic acid at a flow rate of 0.3 mL/min. Quantification was performed using selected reaction monitoring mode to monitor precursor-product ion transitions of m/z 367.1→297.1 for ICT and of 267.0→211.1 for coumestrol in the negative ionization mode. A calibration curve with good linearity (r>0.99) within the concentration range of 0.1-20 ng/mL for ICT was obtained with the lower limit of quantification of 0.1 ng/mL. Matrix effect did not interfere with ICT analysis and ICT was stable under three freeze-thaw cycles, short-term temperature, post-preparative and long-term temperature conditions. The method was successfully applied to a dynamic distribution of ICT in mouse bone after a single intraperitoneal administration to ICT to mice. PMID:25531867

  17. Bone tissue ultrastructural defects in a mouse model for osteogenesis imperfecta: a Raman spectroscopy study

    NASA Astrophysics Data System (ADS)

    Chen, Tsoching; Kozloff, Kenneth M.; Goldstein, Steven A.; Morris, Michael D.

    2004-07-01

    Osteogenesis imperfecta (OI) is genetic defect in which the genes that code for the ?1(I) or ?2(I) chains of type I collagen are defective. The defects often result in substitution of a bulky amino acid for glycine, causing formation of collagen that can not form the normal triple helix. Depending on the details of the defects, the outcomes range from controllable to lethal. This study focuses on OI type IV, a more common and moderately severe form of the disease. People with the disease have a substantial increase in the risk and rate of fracture. We examine the spectroscopic consequences of these defects, using a mouse model (BRTL) that mimics OI type IV. We compare Raman images from tibial cortical tissue of wild-type mice and BRTL mice with single copy of mutation and show that both mineral to matrix ratios and collagen inter-fibril cross-links are different in wild-type and mutant mice.

  18. Localization of the mouse gene for secreted phosphoprotein 1 (Spp-1) (2ar, osteopontin, bone sialoprotein 1, 44-kDa bone phosphoprotein, tumor-secreted phosphoprotein) to chromosome 5, closely linked to Ric (Rickettsia resistance).

    PubMed

    Fet, V; Dickinson, M E; Hogan, B L

    1989-08-01

    We have used a cDNA probe for mouse secreted phosphoprotein 1 (Spp-1, also known as 2ar, osteopontin, bone sialoprotein 1, 44-kDa bone phosphotein, tumor-secreted protein) to find a restriction fragment length polymorphism in the gene from C57BL/6J and DBA/2J mice. The strain distribution pattern in 25 BXD recombinant inbred lines is identical with that previously determined for the dominant, autosomal gene, Ricr (Rickettsia resistance). This places Spp-1 on mouse chromosome 5 with a 95% confidence limit of being within 4.32 cM of Ric. Evidence supporting the possibility of allelism between Spp-1 and Ric is briefly discussed. PMID:2571582

  19. Water permeability of primary mouse keratinocyte cultures grown at the air-liquid interface

    SciTech Connect

    Cumpstone, M.B.; Kennedy, A.H.; Harmon, C.S.; Potts, R.O.

    1989-04-01

    In order to study the development of the epidermal permeability barrier in vitro, tritiated water (HTO) flux was measured across murine keratinocytes cultured at the air-liquid interface. Using a micro-diffusion technique, it was shown that air-liquid cultures form areas where the water diffusion is comparable to that of intact neonatal mouse skin. When water permeability is measured over a large area of the culture surface, however, significantly higher flux is obtained. These results show that under the culture conditions used, areas of water barrier comparable to intact neonatal mouse skin coexist with regions of less complete barrier formation.

  20. Combination therapy with BMP-2 and a systemic RANKL inhibitor enhances bone healing in a mouse critical-sized femoral defect.

    PubMed

    Bougioukli, Sofia; Jain, Ashish; Sugiyama, Osamu; Tinsley, Brian A; Tang, Amy H; Tan, Matthew H; Adams, Douglas J; Kostenuik, Paul J; Lieberman, Jay R

    2016-03-01

    Recombinant human BMP-2 (rhBMP-2) is a potent osteoinductive agent, but has been associated not only with bone formation, but also osteoclastogenesis and bone resorption. Osteoprotegerin (OPG) is a RANKL inhibitor that blocks differentiation and function of osteoclasts. We hypothesized that the combination of local BMP-2 (recombinant protein or a product of gene therapy) plus systemic OPG-Fc is more effective than BMP-2 alone in promoting bone repair. To test this hypothesis we used a mouse critical-sized femoral defect model. Col2.3eGFP (osteoblastic marker) male mice were treated with rhBMP-2 (group I), rhBMP-2 and systemic OPG (group II), rhBMP-2 and delayed administration of OPG (group III), mouse BM cells transduced with a lentiviral vector containing the BMP-2 gene (LV-BMP-2; group IV), LV-BMP-2 and systemic OPG (group V), a carrier alone (group VI) and administration of OPG alone (group VII). All bone defects treated with BMP-2 (alone or combined with OPG) healed, whereas minimal bone formation was noted in animals treated with the carrier alone or OPG alone. MicroCT analysis showed that bone volume (BV) in rhBMP-2+OPG and LV-BMP-2+OPG groups was significantly higher compared to rhBMP-2 alone (p<0.01) and LV-BMP-2 alone (p<0.001). Similar results were observed in histomorphometry, with rhBMP-2 alone defects exhibiting significantly lower bone area (B.Ar) compared to rhBMP-2+OPG defects (p<0.005) and LV-BMP-2 defects having a significantly lower B.Ar compared to all BMP-2+OPG treated groups (p≤0.01). TRAP staining demonstrated a major osteoclast response in the groups that did not receive OPG (rhBMP-2, LV-BMP-2 and sponge alone) beginning as early as 7days post-operatively. In conclusion, we demonstrated that locally delivered BMP-2 (recombinant protein or gene therapy) in combination with systemically administered OPG improved bone healing compared to BMP-2 alone in a mouse critical-sized bone defect. These data indicate that osteoclasts can diminish healing responses to BMP-2 and that RANKL inhibition may thus accentuate BMP-2 efficacy. PMID:26723577

  1. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    SciTech Connect

    Jiang Fangxu . E-mail: jiang@wehi.edu.au; Harrison, Leonard C.

    2005-08-01

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its {alpha}{sub 6} integrin and {alpha}-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells.

  2. CXCR4 expression on pathogenic T cells facilitates their bone marrow infiltration in a mouse model of aplastic anemia

    PubMed Central

    Arieta Kuksin, Christina; Gonzalez-Perez, Gabriela

    2015-01-01

    Aplastic anemia (AA) is a disease characterized by T-cellmediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells. Physiologically, T cells migrate to the BM in response to chemokines, such as SDF-1?, the ligand for CXCR4. However, how T cells traffic to the BM in AA is poorly understood. CXCR4 is aberrantly expressed in immune-mediated diseases and its regulation by nuclear factor-?B (NF-?B) in cancer models is well documented. In this study, we show that CXCR4 is highly expressed on BM-infiltrating CD4+ and CD8+ T cells in a mouse model of AA. Inhibiting CXCR4 in AA mice, using CXCR4?/? splenocytes or AMD3100, significantly reduced BM infiltration of T cells. We also report that NF-?B occupancy at the CXCR4 promoter is enhanced in BM-infiltrating CD8+ T cells of AA mice. Moreover, inhibiting NF-?B signaling in AA mice using Bay11 or dehydroxymethylepoxyquinomicin, or transferring p50?/? splenocytes, decreased CXCR4 expression on CD8+ T cells, significantly reduced BM infiltration of T cells, and strongly attenuated disease symptoms. Remarkably, therapeutic administration of Bay11 significantly extended survival of AA mice. Overall, we demonstrate that CXCR4 mediates migration of pathogenic T cells to the BM in AA mice, and inhibiting NF-?B signaling may represent a novel therapeutic approach to treating AA. PMID:25647836

  3. Intravenous transplantation of bone marrow-derived mononuclear cells prevents memory impairment in transgenic mouse models of Alzheimer's disease.

    PubMed

    Kanamaru, Takuya; Kamimura, Naomi; Yokota, Takashi; Nishimaki, Kiyomi; Iuchi, Katsuya; Lee, Hyunjin; Takami, Shinya; Akashiba, Hiroki; Shitaka, Yoshitsugu; Ueda, Masayuki; Katsura, Ken-Ichiro; Kimura, Kazumi; Ohta, Shigeo

    2015-04-24

    Stem cell transplantation therapy is currently in clinical trials for the treatment of ischemic stroke, and several beneficial aspects have been reported. Similarly, in Alzheimer's disease (AD), stem cell therapy is expected to provide an efficient therapeutic approach. Indeed, the intracerebral transplantation of stem cells reduced amyloid-β (Aβ) deposition and rescued memory deficits in AD model mice. Here, we show that intravenous transplantation of bone marrow-derived mononuclear cells (BMMCs) improves cognitive function in two different AD mouse models, DAL and APP mice, and prevents neurodegeneration. GFP-positive BMMCs were isolated from tibiae and femurs of 4-week-old mice and then transplanted intravenously into DAL and APP mice. Transplantation of BMMCs suppressed neuronal loss and restored memory impairment of DAL mice to almost the same level as in wild-type mice. Transplantation of BMMCs to APP mice reduced Aβ deposition in the brain. APP mice treated with BMMCs performed significantly better on behavioral tests than vehicle-injected mice. Moreover, the effects were observed even with transplantation after the onset of cognitive impairment in DAL mice. Together, our results indicate that intravenous transplantation of BMMCs has preventive effects against the cognitive decline in AD model mice and suggest a potential therapeutic effect of BMMC transplantation therapy. PMID:25698614

  4. Blocking LFA-1 Activation with Lovastatin Prevents Graft-versus-Host Disease in Mouse Bone Marrow Transplantation

    PubMed Central

    Wang, Yang; Li, Dan; Jones, Dan; Bassett, Roland; Sale, George E.; Khalili, Jahan; Komanduri, Krishna V.; Couriel, Daniel R.; Champlin, Richard E.; Molldrem, Jeffrey J.; Ma, Qing

    2016-01-01

    Graft-versus-host disease (GVHD) following bone marrow transplantation (BMT) is mediated by alloreactive donor T lymphocytes. Migration and activation of donor-derived T lymphocytes play critical roles in the development of GVHD. Leukocyte function associated antigen-1 (LFA-1) regulates T cell adhesion and activation. We previously demonstrated that the I-domain, the ligand-binding site of LFA-1, changes from the low affinity state to the high affinity state upon LFA-1 activation. Therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by modulating the affinity state of the LFA-1 I-domain. In this study, we demonstrated that lovastatin blocked mouse T cell adhesion, proliferation and cytokine production in vitro. Furthermore, locking LFA-1 in the low affinity state with lovastatin reduced the mortality and morbidity associated with GVHD in a murine BMT model. Specifically, lovastatin prevented T lymphocytes homing to lymph nodes and Peyer’s Patches during the GVHD initiation phase, and following donor lymphocyte infusion after establishment of GVHD. In addition, treatment with lovastatin impaired donor-derived T cell proliferation in vivo. Taken together, these results indicate the important role of lovastatin in the treatment of GVHD. PMID:19896074

  5. STAT3 mutations identified in human hematologic neoplasms induce myeloid malignancies in a mouse bone marrow transplantation model

    PubMed Central

    Couronné, Lucile; Scourzic, Laurianne; Pilati, Camilla; Valle, Véronique Della; Duffourd, Yannis; Solary, Eric; Vainchenker, William; Merlio, Jean-Philippe; Beylot-Barry, Marie; Damm, Frederik; Stern, Marc-Henri; Gaulard, Philippe; Lamant, Laurence; Delabesse, Eric; Merle-Beral, Hélène; Nguyen-Khac, Florence; Fontenay, Michaëla; Tilly, Hervé; Bastard, Christian; Zucman-Rossi, Jessica; Bernard, Olivier A.; Mercher, Thomas

    2013-01-01

    STAT3 protein phosphorylation is a frequent event in various hematologic malignancies and solid tumors. Acquired STAT3 mutations have been recently identified in 40% of patients with T-cell large granular lymphocytic leukemia, a rare T-cell disorder. In this study, we investigated the mutational status of STAT3 in a large series of patients with lymphoid and myeloid diseases. STAT3 mutations were identified in 1.6% (4 of 258) of patients with T-cell neoplasms, in 2.5% (2 of 79) of patients with diffuse large B-cell lymphoma but in no other B-cell lymphoma patients (0 of 104) or patients with myeloid malignancies (0 of 96). Functional in vitro assays indicated that the STAT3Y640F mutation leads to a constitutive phosphorylation of the protein. STA21, a STAT3 small molecule inhibitor, inhibited the proliferation of two distinct STAT3 mutated cell lines. Using a mouse bone marrow transplantation assay, we observed that STAT3Y640F expression leads to the development of myeloproliferative neoplasms with expansion of either myeloid cells or megakaryocytes. Together, these data indicate that the STAT3Y640F mutation leads to constitutive activation of STAT3, induces malignant hematopoiesis in vivo, and may represent a novel therapeutic target in some lymphoid malignancies. PMID:23872306

  6. Resveratrol augments therapeutic efficiency of mouse bone marrow mesenchymal stem cell-based therapy in experimental autoimmune encephalomyelitis.

    PubMed

    Wang, Dong; Li, Shi-Ping; Fu, Jin-Sheng; Bai, Lin; Guo, Li

    2016-04-01

    Experimental autoimmune encephalitis (EAE) is an inflammatory demyelinating disease, which served as a useful model providing considerable insights into the pathogenesis of multiple sclerosis (MS). Mouse bone marrow mesenchymal stem cells (mBM-MSC) were shown to have neuroprotection capabilities in EAE. Resveratrol is a small polyphenolic compound and possess therapeutic activity in various immune-mediated diseases. The sensitivity of mBM-MSCs to resveratrol was determined by an established cell-viability assay. Resveratrol-treated mBM-MSCs were also characterized with flow cytometry using MSC-specific surface markers and analyzed for their multiple differentiation capacities. EAE was induced in C57BL/6 mice by immunization with MOG35-55. Interferon gamma (IFN-γ)/tumor necrosis factor alpha (TNF-α) and interleukin-4 (IL-4)/interleukin-10 (IL-10), the hallmark cytokines that direct T helper type 1 (Th1) and Th2 development, were detected with enzyme-linked immunosorbent assay (ELISA). In vivo efficacy experiments showed that mBM-MSCs or resveratrol alone led to a significant reduction in clinical scores, and combined treatment resulted in even more prominent reduction. The combined treatment with mBM-MSCs and resveratrol enhanced the immunomodulatory effects, showing suppressed proinflammatory cytokines (IFN-γ, TNF-α) and increased anti-inflammatory cytokines (IL-4, IL-10). The combination of mBM-MSCs and resveratrol provides a novel potential experimental protocol for alleviating EAE symptoms. PMID:26827767

  7. Induction of unfolded protein response during neuronal induction of rat bone marrow stromal cells and mouse embryonic stem cells

    PubMed Central

    Cho, Yoon Mi; Jang, Yoon-Seong; Jang, Young-Min; Chung, Sang-Mi; Kim, Ho-Shik; Lee, Jeong-Hwa; Jeong, Seong-Whan; Kim, In-Kyung; Kim, Jung Jin; Kim, Kwang-Soo

    2009-01-01

    When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2α phosphorylation. Transcription of two downstream targets of eIF2α, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation. PMID:19322020

  8. Fourier transform infrared imaging microspectroscopy and tissue-level mechanical testing reveal intraspecies variation in mouse bone mineral and matrix composition.

    PubMed

    Courtland, Hayden-William; Nasser, Philip; Goldstone, Andrew B; Spevak, Lyudmila; Boskey, Adele L; Jepsen, Karl J

    2008-11-01

    Fracture susceptibility is heritable and dependent upon bone morphology and quality. However, studies of bone quality are typically overshadowed by emphasis on bone geometry and bone mineral density. Given that differences in mineral and matrix composition exist in a variety of species, we hypothesized that genetic variation in bone quality and tissue-level mechanical properties would also exist within species. Sixteen-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse femora were analyzed using Fourier transform infrared imaging and tissue-level mechanical testing for variation in mineral composition, mineral maturity, collagen cross-link ratio, and tissue-level mechanical properties. A/J femora had an increased mineral-to-matrix ratio compared to B6. The C3H mineral-to-matrix ratio was intermediate of A/J and B6. C3H femora had reduced acid phosphate and carbonate levels and an increased collagen cross-link ratio compared to A/J and B6. Modulus values paralleled mineral-to-matrix values, with A/J femora being the most stiff, B6 being the least stiff, and C3H having intermediate stiffness. In addition, work-to-failure varied among the strains, with the highly mineralized and brittle A/J femora performing the least amount of work-to-failure. Inbred mice are therefore able to differentially modulate the composition of their bone mineral and the maturity of their bone matrix in conjunction with tissue-level mechanical properties. These results suggest that specific combinations of bone quality and morphological traits are genetically regulated such that mechanically functional bones can be constructed in different ways. PMID:18855037

  9. Fourier Transform Infrared Imaging Microspectroscopy and Tissue-Level Mechanical Testing Reveal Intraspecies Variation in Mouse Bone Mineral and Matrix Composition

    PubMed Central

    Courtland, Hayden-William; Nasser, Philip; Goldstone, Andrew B.; Spevak, Lyudmila; Boskey, Adele L.; Jepsen, Karl J.

    2009-01-01

    Fracture susceptibility is heritable and dependent upon bone morphology and quality. However, studies of bone quality are typically overshadowed by emphasis on bone geometry and bone mineral density. Given that differences in mineral and matrix composition exist in a variety of species, we hypothesized that genetic variation in bone quality and tissue-level mechanical properties would also exist within species. Sixteen-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse femora were analyzed using Fourier transform infrared imaging and tissue-level mechanical testing for variation in mineral composition, mineral maturity, collagen cross-link ratio, and tissue-level mechanical properties. A/J femora had an increased mineral-to-matrix ratio compared to B6. The C3H mineral-to-matrix ratio was intermediate of A/J and B6. C3H femora had reduced acid phosphate and carbonate levels and an increased collagen cross-link ratio compared to A/J and B6. Modulus values paralleled mineral-to-matrix values, with A/J femora being the most stiff, B6 being the least stiff, and C3H having intermediate stiffness. In addition, work-to-failure varied among the strains, with the highly mineralized and brittle A/J femora performing the least amount of work-to-failure. Inbred mice are therefore able to differentially modulate the composition of their bone mineral and the maturity of their bone matrix in conjunction with tissue-level mechanical properties. These results suggest that specific combinations of bone quality and morphological traits are genetically regulated such that mechanically functional bones can be constructed in different ways. PMID:18855037

  10. Early-Stage Primary Bone Lymphoma: A Retrospective, Multicenter Rare Cancer Network (RCN) Study

    SciTech Connect

    Cai Ling; Stauder, Michael C.; Zhang Yujing; Poortmans, Philip; Li Yexiong; Constantinou, Nicolaos; Thariat, Juliette; Kadish, Sidney P.; Nguyen, Tan Dat; Kirova, Youlia M.; Ghadjar, Pirus; Weber, Damien C.; Bertran, Victoria Tuset; Ozsahin, Mahmut; Mirimanoff, Rene-Olivier

    2012-05-01

    Purpose: Primary bone lymphoma (PBL) represents less than 1% of all malignant lymphomas. In this study, we assessed the disease profile, outcome, and prognostic factors in patients with Stages I and II PBL. Patients and Methods: Thirteen Rare Cancer Network (RCN) institutions enrolled 116 consecutive patients with PBL treated between 1987 and 2008 in this study. Eighty-seven patients underwent chemoradiotherapy (CXRT) without (78) or with (9) surgery, 15 radiotherapy (RT) without (13) or with (2) surgery, and 14 chemotherapy (CXT) without (9) or with (5) surgery. Median RT dose was 40 Gy (range, 4-60). The median number of CXT cycles was six (range, 2-8). Median follow-up was 41 months (range, 6-242). Results: The overall response rate at the end of treatment was 91% (complete response [CR] 74%, partial response [PR] 17%). Local recurrence or progression was observed in 12 (10%) patients and systemic recurrence in 17 (15%). The 5-year overall survival (OS), lymphoma-specific survival (LSS), and local control (LC) were 76%, 78%, and 92%, respectively. In univariate analyses (log-rank test), favorable prognostic factors for OS and LSS were International Prognostic Index (IPI) score {<=}1 (p = 0.009), high-grade histology (p = 0.04), CXRT (p = 0.05), CXT (p = 0.0004), CR (p < 0.0001), and RT dose >40 Gy (p = 0.005). For LC, only CR and Stage I were favorable factors. In multivariate analysis, IPI score, RT dose, CR, and CXT were independently influencing the outcome (OS and LSS). CR was the only predicting factor for LC. Conclusion: This large multicenter retrospective study confirms the good prognosis of early-stage PBL treated with combined CXRT. An adequate dose of RT and complete CXT regime were associated with better outcome.

  11. FDG-PET/CT Imaging Predicts Histopathologic Treatment Responses after Neoadjuvant Therapy in Adult Primary Bone Sarcomas

    DOE PAGESBeta

    Benz, Matthias R.; Czernin, Johannes; Tap, William D.; Eckardt, Jeffrey J.; Seeger, Leanne L.; Allen-Auerbach, Martin S.; Dry, Sarah M.; Phelps, Michael E.; Weber, Wolfgang A.; Eilber, Fritz C.

    2010-01-01

    Purpose . Tmore » he aim of this study was to prospectively evaluate whether FDG-PET allows an accurate assessment of histopathologic response to neoadjuvant treatment in adult patients with primary bone sarcomas. Methods . Twelve consecutive patients with resectable, primary high grade bone sarcomas were enrolled prospectively. FDG-PET/CT imaging was performed prior to the initiation and after completion of neoadjuvant treatment. Imaging findings were correlated with histopathologic response. Results . Histopathologic responders showed significantly more pronounced decreases in tumor FDG-SUVmax from baseline to late follow up than non-responders ( 64 ± 19 % versus 29 ± 30 %, resp.; P = .03 ). Using a 60% decrease in tumor FDG-uptake as a threshold for metabolic response correctly classified 3 of 4 histopathologic responders and 7 of 8 histopathologic non-responders as metabolic responders and non-responders, respectively (sensitivity, 75%; specificity, 88%). Conclusion . These results suggest that changes in FDG-SUVmax at the end of neoadjuvant treatment can identify histopathologic responders and non-responders in adult primary bone sarcoma patients.« less

  12. [Construction and identification of primary open angle glaucoma disease MYOC Pro356Leu mutant plasmid in mouse.

    PubMed

    Li, Chunmei; Zhuo, Yehong; Chen, Mengfei; Sun, Xuerong; Qi, Ying; Ge, Jian

    2010-08-01

    PURPOSE:To construct and identify the plasmid of mutant MYOC Pro356Leu in mouse. METHODS:1.Transfering Red recombinantinase- expressing plasmid pKD46 was transferred to RP23-180F15 BAC clone by electroporation.2.Amplifying tThe pStart-K vector was amplified by PCR and electroporating the PCR product was electroporated into the bacteria containing the mMYOC. 3.PCR amplification of the mMyoc AfeI fragment and Subcloningand then subcloned it into pBluescript vector.4.Designing two oligonucleotides was designed and introducingthe Pro356Leu (C to T at codon 356) was introduced into the pBS_mMyoc AfeI clone. 5. the Introducing pBS_mMyoc AfeI mut clone was introduced to pStart-K_mMyoc by digestion and ligation reaction. Identifying correct the final clones were identified by restriction enzyme digestion and sequenceing. RESULTS: pStart-K_mMyoc mut clone,the mouse MYOC Pro356Leu mutant plasmid was constructed and identified well by restriction enzyme digest and sequenceing. CONCLUSIONS: It is a good method to construct a long DNA fragment clone by red recombinase and pStart-K vector. The constructed Mouse MYOC Pro356Leu plasmidwill be very helpful for the studies on the function and biologic effects of MYOC gene in primary open angle glaucoma. PMID:21166044

  13. AAV8-mediated expression of N-acetylglucosamine-1-phosphate transferase attenuates bone loss in a mouse model of mucolipidosis II.

    PubMed

    Ko, Ah-Ra; Jin, Dong-Kyu; Cho, Sung Yoon; Park, Sung Won; Przybylska, Malgorzata; Yew, Nelson S; Cheng, Seng H; Kim, Jung-Sun; Kwak, Min Jung; Kim, Su Jin; Sohn, Young Bae

    2016-04-01

    Mucolipidoses II and III (ML II and ML III) are lysosomal disorders in which the mannose 6-phosphate recognition marker is absent from lysosomal hydrolases and other glycoproteins due to mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Both disorders are caused by the same gene, but ML II represents the more severe phenotype. Bone manifestations of ML II include hip dysplasia, scoliosis, rickets and osteogenesis imperfecta. In this study, we sought to determine whether a recombinant adeno-associated viral vector (AAV2/8-GNPTAB) could confer high and prolonged gene expression of GNPTAB and thereby influence the pathology in the cartilage and bone tissue of a GNPTAB knock out (KO) mouse model. The results demonstrated significant increases in bone mineral density and content in AAV2/8-GNPTAB-treated as compared to non-treated KO mice. We also showed that IL-6 (interleukin-6) expression in articular cartilage was reduced in AAV2/8-GNPTAB treated ML II mice. Together, these data suggest that AAV-mediated expression of GNPTAB in ML II mice can attenuate bone loss via inhibition of IL-6 production. This study emphasizes the value of the MLII KO mouse to recapitulate the clinical manifestations of the disease and highlights its amenability to therapy. PMID:26857995

  14. Mouse host unlicensed NK cells promote donor allogeneic bone marrow engraftment.

    PubMed

    Alvarez, Maite; Sun, Kai; Murphy, William J

    2016-03-01

    Natural killer (NK) cells exist as subsets based on expression of inhibitory receptors that recognize major histocompatibility complex I (MHCI) molecules. NK cell subsets bearing MHCI binding receptors for self-MHCI have been termed as "licensed" and exhibit a higher ability to respond to stimuli. In the context of bone marrow transplantation (BMT), host licensed-NK (L-NK) cells have also been demonstrated to be responsible for the acute rejection of allogeneic and MHCI-deficient BM cells (BMCs) in mice after lethal irradiation. However, the role of recipient unlicensed-NK (U-NK) cells has not been well established with regard to allogeneic BMC resistance. After NK cell stimulation, the prior depletion of host L-NK cells resulted in a marked increase of donor engraftment compared with the untreated group. Surprisingly, this increased donor engraftment was reduced after total host NK cell depletion, indicating that U-NK cells can actually promote donor allogeneic BMC engraftment. Furthermore, direct coculture of U-NK cells with allogeneic but not syngeneic BMCs resulted in increased colony-forming unit cell growth in vitro, which was at least partially mediated by granulocyte macrophage colony-stimulating factor (GM-CSF) production. These data demonstrate that host NK cell subsets exert markedly different roles in allogeneic BMC engraftment where host L- and U-NK cells reject or promote donor allogeneic BMC engraftment, respectively. PMID:26738538

  15. Mouse host unlicensed NK cells promote donor allogeneic bone marrow engraftment

    PubMed Central

    Alvarez, Maite; Sun, Kai

    2016-01-01

    Natural killer (NK) cells exist as subsets based on expression of inhibitory receptors that recognize major histocompatibility complex I (MHCI) molecules. NK cell subsets bearing MHCI binding receptors for self-MHCI have been termed as “licensed” and exhibit a higher ability to respond to stimuli. In the context of bone marrow transplantation (BMT), host licensed-NK (L-NK) cells have also been demonstrated to be responsible for the acute rejection of allogeneic and MHCI-deficient BM cells (BMCs) in mice after lethal irradiation. However, the role of recipient unlicensed-NK (U-NK) cells has not been well established with regard to allogeneic BMC resistance. After NK cell stimulation, the prior depletion of host L-NK cells resulted in a marked increase of donor engraftment compared with the untreated group. Surprisingly, this increased donor engraftment was reduced after total host NK cell depletion, indicating that U-NK cells can actually promote donor allogeneic BMC engraftment. Furthermore, direct coculture of U-NK cells with allogeneic but not syngeneic BMCs resulted in increased colony-forming unit cell growth in vitro, which was at least partially mediated by granulocyte macrophage colony-stimulating factor (GM-CSF) production. These data demonstrate that host NK cell subsets exert markedly different roles in allogeneic BMC engraftment where host L- and U-NK cells reject or promote donor allogeneic BMC engraftment, respectively. PMID:26738538

  16. Mouse Bone Marrow-Derived Endothelial Progenitor Cells Do Not Restore Radiation-Induced Microvascular Damage

    PubMed Central

    Seemann, Ingar; te Poele, Johannes A. M.; Hoving, Saske; Stewart, Fiona A.

    2014-01-01

    Background. Radiotherapy is commonly used to treat breast and thoracic cancers but it also causes delayed microvascular damage and increases the risk of cardiac mortality. Endothelial cell proliferation and revascularization are crucial to restore microvasculature damage and maintain function of the irradiated heart. We have therefore examined the potential of bone marrow-derived endothelial progenitor cells (BM-derived EPCs) for restoration of radiation-induced microvascular damage. Material & Methods. 16?Gy was delivered to the heart of adult C57BL/6 mice. Mice were injected with BM-derived EPCs, obtained from Eng+/+ or Eng+/? mice, 16 weeks and 28 weeks after irradiation. Morphological damage was evaluated at 40 weeks in transplanted mice, relative to radiation only and age-matched controls. Results. Cardiac irradiation decreased microvascular density and increased endothelial damage in surviving capillaries (decrease alkaline phosphatase expression and increased von Willebrand factor). Microvascular damage was not diminished by treatment with BM-derived EPCs. However, BM-derived EPCs from both Eng+/+ and Eng+/? mice diminished radiation-induced collagen deposition. Conclusion. Treatment with BM-derived EPCs did not restore radiation-induced microvascular damage but it did inhibit fibrosis. Endoglin deficiency did not impair this process. PMID:25101181

  17. Bone morphogenetic proteins, eye patterning, and retinocollicular map formation in the mouse

    PubMed Central

    Plas, Daniel T.; Dhande, Onkar; Lopez, Joshua E.; Murali, Deepa; Thaller, Christina; Henkemeyer, Mark; Furuta, Yasuhide; Overbeek, Paul; Crair, Michael C.

    2009-01-01

    Patterning events during early eye formation determine retinal cell fate and can dictate the behavior of retinal ganglion cell (RGC) axons as they navigate toward central brain targets. The temporally and spatially regulated expression of bone morphogenetic proteins (BMPs) and their receptors in the retina are thought to play a key role in this process, initiating gene expression cascades that distinguish different regions of the retina, particularly along the dorsoventral axis. Here, we examine the role of BMP and a potential downstream effector, EphB, in retinotopic map formation in the lateral geniculate nucleus (LGN) and superior colliculus (SC). RGC axon behaviors during retinotopic map formation in wild type mice are compared with those in several strains of mice with engineered defects of BMP and EphB signaling. Normal RGC axon sorting produces axon order in the optic tract that reflects the dorsoventral position of the parent RGCs in the eye. A dramatic consequence of disrupting BMP signaling is a missorting of RGC axons as they exit the optic chiasm. This sorting is not dependent on EphB. When BMP signaling in the developing eye is genetically modified, RGC order in the optic tract and targeting in the LGN and SC are correspondingly disrupted. These experiments show that BMP signaling regulates dorsoventral RGC cell fate, RGC axon behavior in the ascending optic tract and retinotopic map formation in the LGN and SC through mechanisms that are in part distinct from EphB signaling in the LGN and SC. PMID:18614674

  18. Bone morphogenetic proteins, eye patterning, and retinocollicular map formation in the mouse.

    PubMed

    Plas, Daniel T; Dhande, Onkar S; Lopez, Joshua E; Murali, Deepa; Thaller, Christina; Henkemeyer, Mark; Furuta, Yasuhide; Overbeek, Paul; Crair, Michael C

    2008-07-01

    Patterning events during early eye formation determine retinal cell fate and can dictate the behavior of retinal ganglion cell (RGC) axons as they navigate toward central brain targets. The temporally and spatially regulated expression of bone morphogenetic proteins (BMPs) and their receptors in the retina are thought to play a key role in this process, initiating gene expression cascades that distinguish different regions of the retina, particularly along the dorsoventral axis. Here, we examine the role of BMP and a potential downstream effector, EphB, in retinotopic map formation in the lateral geniculate nucleus (LGN) and superior colliculus (SC). RGC axon behaviors during retinotopic map formation in wild-type mice are compared with those in several strains of mice with engineered defects of BMP and EphB signaling. Normal RGC axon sorting produces axon order in the optic tract that reflects the dorsoventral position of the parent RGCs in the eye. A dramatic consequence of disrupting BMP signaling is a missorting of RGC axons as they exit the optic chiasm. This sorting is not dependent on EphB. When BMP signaling in the developing eye is genetically modified, RGC order in the optic tract and targeting in the LGN and SC are correspondingly disrupted. These experiments show that BMP signaling regulates dorsoventral RGC cell fate, RGC axon behavior in the ascending optic tract, and retinotopic map formation in the LGN and SC through mechanisms that are in part distinct from EphB signaling in the LGN and SC. PMID:18614674

  19. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells

    SciTech Connect

    Arora, S.; Jain, J.; Rajwade, J.M.; Paknikar, K.M.

    2009-05-01

    Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 {mu}g/mL and 100 {mu}g/mL SNP, respectively, although with minor decrease in confluence. IC{sub 50} values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 {mu}g/mL and 449 {mu}g/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC{sub 50} SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with {approx} 1/2 IC{sub 50} concentration of SNP (i.e. 30 {mu}g/mL and 225 {mu}g/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH ({approx} 1.2 fold) and depletion of lipid peroxidation ({approx} 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD ({approx} 1.4 fold) and GSH ({approx} 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 {mu}g/mL in primary fibroblasts, 12.5 {mu}g/mL in primary liver cells) than the necrotic concentration (100 {mu}g/mL in primary fibroblasts, 500 {mu}g/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC{sub 50} SNP (resulting in apoptosis) and 2x IC{sub 50}) cells (resulting in necrosis). These results clearly suggest that although silver nanoparticles seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage. This property, in conjunction with the finding that primary cells possess much higher SNP tolerance than the concentration in the gel ({approx} 20 {mu}g/g), indicates preliminary safety of the formulation and warrants further study for possible human application.

  20. TNF-? Regulates the Effects of Irradiation in the Mouse Bone Marrow Microenvironment

    PubMed Central

    Cachao, Ana Sofia; Carvalho, Tnia; Santos, Ana Cristina; Igreja, Ctia; Fragoso, Rita; Osrio, Catarina; Ferreira, Manuela; Serpa, Jacinta; Correia, Sofia; Pinto-do-, Perptua; Dias, Srgio

    2010-01-01

    Background Secondary bone marrow (BM) myelodysplastic syndromes (MDS) are increasingly common, as a result of radio or chemotherapy administered to a majority of cancer patients. Patients with secondary MDS have increased BM cell apoptosis, which results in BM dysfunction (cytopenias), and an increased risk of developing fatal acute leukemias. In the present study we asked whether TNF-?, known to regulate cell apoptosis, could modulate the onset of secondary MDS. Principal Findings We show that TNF-? is induced by irradiation and regulates BM cells apoptosis in vitro and in vivo. In contrast to irradiated wild type (WT) mice, TNF-? deficient (TNF-? KO) mice or WT mice treated with a TNF-?-neutralizing antibody were partially protected from the apoptotic effects of irradiation. Next we established a 3-cycle irradiation protocol, in which mice were sub-lethally irradiated once monthly over a 3 month period. In this model, irradiated WT mice presented loss of microsatellite markers on BM cells, low white blood cell (WBC) counts, reduced megakaryocyte (MK) and platelet levels (thrombocytopenia) and macrocytic anemia, phenoypes that suggest the irradiation protocol resulted in BM dysfunction with clinical features of MDS. In contrast, TNF-? KO mice were protected from the irradiation effects: BM cell apoptosis following irradiation was significantly reduced, concomitant with sustained BM MK numbers and absence of other cytopenias. Moreover, irradiated WT mice with long term (?5 months) BM dysfunction had increased BM angiogenesis, MMPs and VEGF and NFkB p65, suggestive of disease progression. Conclusion Taken together, our data shows that TNF-? induction following irradiation modulates BM cell apoptosis and is a crucial event in BM dysfunction, secondary MDS onset and progression. PMID:20126546

  1. Contribution of Bone Marrow Hematopoietic Stem Cells to Adult Mouse Inner Ear: Mesenchymal Cells and Fibrocytes

    PubMed Central

    Lang, Hainan; Ebihara, Yasuhiro; Schmiedt, Richard A.; Minamiguchi, Hitoshi; Zhou, Daohong; Smythe, Nancy; Liu, Liya; Ogawa, Makio; Schulte, Bradley A.

    2008-01-01

    Bone marrow (BM)-derived stem cells have shown plasticity with a capacity to differentiate into a variety of specialized cells. To test the hypothesis that some cells in the inner ear are derived from BM, we transplanted either isolated whole BM cells or clonally expanded hematopoietic stem cells (HSCs) prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) into irradiated adult mice. Isolated GFP+ BM cells also were transplanted into conditioned newborn mice derived from pregnant mice injected with busulfan (which ablates HSCs in the newborns). Quantification of GFP+ cells was performed 3-20 months after transplant. GFP+ cells were found in the inner ear with all transplant conditions. They were most abundant within the spiral ligament but were also found in other locations normally occupied by fibrocytes and mesenchymal cells. No GFP+ neurons or hair cells were observed in inner ears of transplanted mice. Dual immunofluorescence assays demonstrated that most of the GFP+ cells were negative for CD45, a macrophage and hematopoietic cell marker. A portion of the GFP+ cells in the spiral ligament expressed immunoreactive Na, K-ATPase or the Na-K-Cl transporter (NKCC), proteins used as markers for specialized ion transport fibrocytes. Phenotypic studies indicated that the GFP+ cells did not arise from fusion of donor cells with endogenous cells. This study provides the first evidence for the origin of inner ear cells from BM and more specifically from HSCs. The results suggest that mesenchymal cells, including fibrocytes in the adult inner ear, may be derived continuously from HSCs. PMID:16538683

  2. Suppression of Hepatic Cyp1a2 by Total Ginsenosides in Lipopolysaccharide-Treated Mice and Primary Mouse Hepatocytes.

    PubMed

    Sun, Haiyan; Yan, Yijing; Xu, Chenshu; Wan, Hongxia; Liu, Dong

    2016-03-23

    The roots of Panax ginseng (ginseng) have been extensively used in traditional Chinese medicine. However, herb-drug interactions between ginseng and other co-administered drugs are not fully understood concerning the effect of ginseng on drug metabolism and clearance. The current study aimed to elucidate the effect of total ginsenosides, a typical ginseng extract, on the regulation of Cyp1a2, a key enzyme to regulate drug metabolism under the normal and inflammatory conditions in mice. Female C57BL/6J mice treated with vehicle and lipopolysaccharide (LPS) were intragastrically administered ginseng extract for 7 days before hepatic P450 expression was analyzed. Primary mouse hepatocytes were also employed to further explore the effects of total ginsenosides on Cyp1a2 expression. The results showed that total ginsenosides in P. ginseng extract exhibited a concentration-dependent suppression on Cyp1a2 mRNA and protein level in both mice and primary mouse hepatocytes. Notably, the inhibitory effects of total ginsenosides on Cyp1a2 mRNA and protein expression were further enhanced following LPS treatment. Therefore, future research is warranted to investigate the role of ginsenosides in the regulation of hepatic CYP450s. Moreover, consumption of ginseng as food or supplement should be monitored for patients on combinational therapy, especially those with inflammatory diseases. PMID:26923348

  3. The relationship of excess copper accumulation by fibroblasts from the brindled mouse model of Menkes disease to the primary defect.

    PubMed Central

    Waldrop, G L; Ettinger, M J

    1990-01-01

    Fibroblasts from the brindled mouse model of Menkes disease are known to accumulate excess copper. Most of the copper in the cytosol of these fibroblasts is bound to metallothionein (MT), which is elevated in Menkes or brindled mouse fibroblasts. Copper accumulation by normal fibroblasts containing excess MT was examined to determine if the excess copper accumulation phenotype was secondary to excess MT or associated with the primary defect in fibroblasts from the brindled mice. MT was induced in normal fibroblasts by copper, zinc or dexamethasone to levels comparable with those in brindled mice fibroblasts, as determined by radioimmunoassays. Normal fibroblasts containing excess MT accumulate copper normally, i.e. they do not exhibit the excess copper accumulation phenotype. Consistent with this result, copper efflux from normal fibroblasts containing excess MT was also normal. The data suggest that one function of the protein associated with the primary defect is to help determine how much copper is taken up and retained by fibroblasts and other cell types exhibiting the excess copper phenotype in Menkes disease. The capacity of this protein is apparently exceeded in normal fibroblasts if serum or albumin is not present extracellularly to limit total copper uptake. Consistent with a defect in an intracellular protein, the kinetics of copper transport by brindled mice fibroblasts were found to be normal. PMID:2334401

  4. Photo-Induction and Automated Quantification of Reversible Mitochondrial Permeability Transition Pore Opening in Primary Mouse Myotubes

    PubMed Central

    Smeitink, Jan A. M.; Willems, Peter H. G. M.; Koopman, Werner J. H.

    2014-01-01

    Opening of the mitochondrial permeability transition pore (mPTP) is involved in various cellular processes including apoptosis induction. Two distinct states of mPTP opening have been identified allowing the transfer of molecules with a molecular weight <1500 Da or <300 Da. The latter state is considered to be reversible and suggested to play a role in normal cell physiology. Here we present a strategy combining live-cell imaging and computer-assisted image processing allowing spatial visualization and quantitative analysis of reversible mPTP openings (“ΔΨ flickering”) in primary mouse myotubes. The latter were stained with the photosensitive cation TMRM, which partitions between the cytosol and mitochondrial matrix as a function of mitochondrial membrane potential (ΔΨ). Controlled illumination of TMRM-stained primary mouse myotubes induced ΔΨ flickering in particular parts of the cell (“flickering domains”). A novel quantitative automated analysis was developed and validated to detect and quantify the frequency, size, and location of individual ΔΨ flickering events in myotubes. PMID:25423172

  5. Establishment of Green Fluorescent Protein and Firefly Luciferase Expressing Mouse Primary Macrophages for In Vivo Bioluminescence Imaging

    PubMed Central

    Pajarinen, Jukka; Lin, Tzu-hua; Sato, Taishi; Loi, Florence; Yao, Zhenyu; Konttinen, Yrjö T.; Goodman, Stuart B.

    2015-01-01

    Macrophages play a key role in tissue homeostasis as well as in a range of pathological conditions including atherosclerosis, cancer, and autoimmunity. Many aspects of their in vivo behavior are, however, poorly understood. Bioluminescence imaging (BLI) with green fluorescent protein (GFP) and firefly luciferase (FLUC) labelled autologous reporter macrophages could potentially offer a powerful tool to study macrophage biology, but this approach has been hindered by the relative difficulty of efficient gene transfer into primary macrophages. Here we describe a straightforward method for producing large numbers of GFP/FLUC expressing mouse primary macrophages utilizing lentivirus vector, cyclosporine, and a double infection strategy. Using this method we achieved up to 60% of macrophages to express GFP with correspondingly high FLUC signal. When injected into the circulation using a mouse model of local biomaterial induced inflammation and osteolysis, macrophages were initially detectable within the lungs, followed by systemic homing to the local area of chronic inflammation in the distal femur. In addition, transduced macrophages maintained their ability to assume M1 and M2 phenotypes although the GFP/FLUC expression was altered by the polarizing signals. These reporter macrophages could prove to be valuable tools to study the role of macrophages in health and disease. PMID:26555613

  6. Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D3 Metabolites

    PubMed Central

    Tuohimaa, Pentti; Wang, Jing-Huan; Khan, Sofia; Kuuslahti, Marianne; Qian, Kui; Manninen, Tommi; Auvinen, Petri; Vihinen, Mauno; Lou, Yan-Ru

    2013-01-01

    1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D3 (25(OH)D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) broadened the vitamin D3 endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D3 into 1α,25(OH)2D3 by 1α-hydroxylase (encoded by the gene CYP27B1), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH)2D3, 25(OH)D3, and 24R,25(OH)2D3, respectively. Mouse primary Cyp27b1 knockout fibroblasts (mCyp27b1−/−), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in mCyp27b1−/−. By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH)2D3 and 25(OH)D3 and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D3 hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D3 endocrine system, which may guide the strategic use of vitamin D3 in disease prevention and treatment. PMID:24116037

  7. Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice

    PubMed Central

    Stern, Amber Rath; Stern, Matthew M.; Van Dyke, Mark E.; Jähn, Katharina; Prideaux, Matthew; Bonewald, Lynda F.

    2013-01-01

    The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38.[Q1] The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease. PMID:22668415

  8. Mechanical stimulation and intermittent parathyroid hormone treatment induce disproportional osteogenic, geometric, and biomechanical effects in growing mouse bone

    PubMed Central

    McAteer, Maureen E.; Niziolek, Paul J.; Ellis, Shana N.; Alge, Daniel L.; Robling, Alexander G.

    2011-01-01

    Mechanical loading and intermittent parathyroid (iPTH) treatment are both osteoanabolic stimuli, and are regulated by partially overlapping cellular signaling pathways. iPTH has been shown clinically to be effective in increasing bone mass and reducing fracture risk. Likewise, mechanical stimulation can significantly enhance bone apposition and prevent bone loss, but its clinical effects on fracture susceptibility are less certain. Many of the osteogenic effects of iPTH are localized to biomechanically suboptimal bone surfaces, whereas mechanical loading directs new bone formation to high-stress areas and not to strain-neutral areas. These differences in localization in new tissue, resulting from load-induced vs iPTH-induced bone accumulation, should affect the relation between bone mass and bone strength, or “tissue economy.” We investigated the changes in bone mass and strength induced by 6 wks mechanical loading, and compared them to changes induced by 6 wks iPTH treatment. Loading and iPTH both increased ulnar bone accrual, as measured by bone mineral density and content, and fluorochrome-derived bone formation. iPTH induced a significantly greater increase in bone mass than loading, but ulnar bone strength was increased approximately the same amount by both treatments. Mechanical loading during growth can spatially optimize new bone formation to improve structural integrity with a minimal increase in mass, thereby increasing tissue economy i.e., the amount of strength returned per unit bone mass added. Furthermore, exercise studies in which only small changes in bone mass are detected might be more beneficial to bone health and fracture resistance than has commonly been presumed. PMID:20306026

  9. Primary bone marrow diffuse large B-cell lymphoma accompanying cold agglutinin disease: A case report with review of the literature.

    PubMed

    Yamashita, Tomoko; Ishida, Mitsuaki; Moro, Hiroko; Yumoto, Hirofumi; Uchibayashi, Sachiko; Yoshii, Miyuki; Nakanishi, Ryota; Okuno, Hiroko; Yoshida, Takashi; Okuno, Takafumi; Hodohara, Keiko; Okabe, Hidetoshi

    2014-01-01

    Cold agglutinin disease (CAD) is a well-recognized complication of lymphoproliferative disorders. It has been previously recognized that cases of primary CAD frequently exhibit underlying malignant lymphoma in the bone marrow. Lymphoplasmacytic lymphoma is the most common subtype of malignant lymphoma; however, diffuse large B-cell lymphoma (DLBCL) has also been documented, albeit extremely rare. The current report presents a case of primary bone marrow DLBCL accompanying CAD. A 76-year-old male presented with fever and fatigue. Laboratory tests revealed anemia and elevated bilirubin and cold agglutinins with a titer of 8,192 at 4°C. Bone marrow biopsy demonstrated DLBCL and systemic surveillance failed to detect tumorous lesions or lymphadenopathy. Following R-THP-COP therapy, cold agglutinins titer was markedly decreased (by <4); however, malignant lymphoma relapsed and cold agglutinin levels increased again (4,096). This is the second documented case of primary bone marrow DLBCL accompanying CAD. Previously, malignant lymphoma exclusively involving the bone marrow, namely primary bone marrow lymphoma (PBML), has been recognized as a rare and aggressive subtype. The analyses of the present study revealed that the incidence of hemolytic anemia in primary bone marrow DLBCL may be high compared with conventional DLBCL. Therefore, additional analyses are required to clarify the clinicopathological features of PBML. PMID:24348825

  10. Genotoxic, Cytotoxic, Antigenotoxic, and Anticytotoxic Effects of Sulfonamide Chalcone Using the Ames Test and the Mouse Bone Marrow Micronucleus Test

    PubMed Central

    Borges, Flávio Fernandes Veloso; Bernardes, Aline; Perez, Caridad Noda; Silva, Daniela de Melo e

    2015-01-01

    Chalcones present several biological activities and sulfonamide chalcone derivatives have shown important biological applications, including antitumor activity. In this study, genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activities of the sulfonamide chalcone N-{4-[3-(4-nitrophenyl)prop-2-enoyl]phenyl} benzenesulfonamide (CPN) were assessed using the Salmonella typhimurium reverse mutation test (Ames test) and the mouse bone marrow micronucleus test. The results showed that CPN caused a small increase in the number of histidine revertant colonies in S. typhimurium strains TA98 and TA100, but not statistically significant (p > 0.05). The antimutagenicity test showed that CPN significantly decreased the number of His+ revertants in strain TA98 at all doses tested (p < 0.05), whereas in strain TA100 this occurred only at doses higher than 50 μg/plate (p < 0.05). The results of the micronucleus test indicated that CPN significantly increased the frequency of micronucleated polychromatic erythrocytes (MNPCE) at 24 h and 48 h, revealing a genotoxic effect of this compound. Also, a significant decrease in polychromatic/normochromatic erythrocyte ratio (PCE/NCE) was observed at the higher doses of CPN at 24 h and 48 h (p < 0.05), indicating its cytotoxic action. CPN co-administered with mitomycin C (MMC) significantly decreased the frequency of MNPCE at almost all doses tested at 24 h (p < 0.05), showing its antigenotoxic activity, and also presented a small decrease in MNPCE at 48 h (p > 0.05). Additionally, CPN co-administered with MMC significantly increased PCE/NCE ratio at all doses tested, demonstrating its anticytotoxic effect. In summary, CPN presented genotoxic, cytotoxic, antigenotoxic, and anticytotoxic properties. PMID:26335560

  11. Bacteria-reactive immune response may induce RANKL-expressing T-cells in the mouse periapical bone loss lesion

    PubMed Central

    Silva, Marcelo J.B.; Kajiya, Mikihito; AlShwaimi, Emad; Sasaki, Hajime; Hong, Jennifer; Ok, Peter; Rezende, Taia M.B.; Pagonis, Tom C.; White, Robert R.; Paster, Bruce J; Stashenko, Philip; Kawai, Toshihisa

    2012-01-01

    Introduction The present study investigated if T-cells infiltrating the periapical lesion produce RANKL and whether bacteria infecting the root canal can activate T-cells to produce RANKL. Methods Using a mouse model of periapical lesion induced by artificial dental pulp exposure, the presence of RANKL-positive T-cells and osteoclasts in the periapical lesion was examined by an immuno-histochemical approach. The bacteria colonizing the exposed root canal were identified by 16S ribosomal RNA (rRNA) sequence analysis. The isolated endodontic bacteria were further immunized to normal mice, and sRANKL production by the T-cells isolated from the immunized mice was evaluated by ex vivo culture system. Results RANKL-positive T-cells, along with TARP+ osteoclasts, were identified in periapical bone resorption lesions. The Gram-negative bacterium Pasterurella pnumotropica (P. pnumotropica), which was most frequently detected from root canal of exposed pulp, showed remarkably elevated serum IgG antibody response in pulp-exposed mice compared to control non-treated mice. Immunization of mice with P. pneumotropica induced not only serum IgG antibody but also primed bacteria reactive T-cells that produced sRANKL in response to ex vivo exposure to P. pneumotropica. Conclusion T-cells infiltrating the periapical region express RANKL, and the endodontic bacteria colonizing the root canal appear to induce RANKL expression from bacteria-reactive T-cells, suggesting the possible pathogenic engagement of immune response to endodontic bacteria in the context of developing boneresorptive periapical lesions. PMID:22341072

  12. Prostaglandin E2 Production and T Cell Function in Mouse Adenovirus Type 1 Infection following Allogeneic Bone Marrow Transplantation

    PubMed Central

    McCarthy, Mary K.; Procario, Megan C.; Wilke, Carol A.; Moore, Bethany B.; Weinberg, Jason B.

    2015-01-01

    Adenovirus infections are important complications of bone marrow transplantation (BMT). We demonstrate delayed clearance of mouse adenovirus type 1 (MAV-1) from lungs of mice following allogeneic BMT. Virus-induced prostaglandin E2 (PGE2) production was greater in BMT mice than in untransplanted controls, but BMT using PGE2-deficient donors or recipients failed to improve viral clearance, and treatment of untransplanted mice with the PGE2 analog misoprostol did not affect virus clearance. Lymphocyte recruitment to the lungs was not significantly affected by BMT. Intracellular cytokine staining of lung lymphocytes demonstrated impaired production of INF-? and granzyme B by cells from BMT mice, and production of IFN-?, IL-2, IL-4, and IL-17 following ex vivo stimulation was impaired in lymphocytes obtained from lungs of BMT mice. Viral clearance was not delayed in untransplanted INF-?-deficient mice, suggesting that delayed viral clearance in BMT mice was not a direct consequence of impaired IFN-? production. However, lung viral loads were higher in untransplanted CD8-deficient mice than in controls, suggesting that delayed MAV-1 clearance in BMT mice is due to defective CD8 T cell function. We did not detect significant induction of IFN-? expression in lungs of BMT mice or untransplanted controls, and viral clearance was not delayed in untransplanted type I IFN-unresponsive mice. We conclude that PGE2 overproduction in BMT mice is not directly responsible for delayed viral clearance. PGE2-independent effects on CD8 T cell function likely contribute to the inability of BMT mice to clear MAV-1 from the lungs. PMID:26407316

  13. GATA2 is critical for the maintenance of cellular identity in differentiated mast cells derived from mouse bone marrow.

    PubMed

    Ohmori, Shin'ya; Moriguchi, Takashi; Noguchi, Yuki; Ikeda, Muneharu; Kobayashi, Kota; Tomaru, Nazuki; Ishijima, Yasushi; Ohneda, Osamu; Yamamoto, Masayuki; Ohneda, Kinuko

    2015-05-21

    GATA2 plays a crucial role for the mast cell fate decision. We herein demonstrate that GATA2 is also required for the maintenance of the cellular identity in committed mast cells derived from mouse bone marrow (BMMCs). The deletion of the GATA2 DNA binding domain (GATA2ΔCF) in BMMCs resulted in a loss of the mast cell phenotype and an increase in the number of CD11b- and/or Ly6G/C-positive cells. These cells showed the ability to differentiate into macrophage- and neutrophil-like cells but not into eosinophils. Although the mRNA levels of basophil-specific genes were elevated, CD49b, a representative basophil marker, never appeared on these cells. GATA2 ablation led to a significant upregulation of C/EBPα, and forced expression of C/EBPα in wild-type BMMCs phenocopied the GATA2ΔCF cells. Interestingly, simultaneous deletion of the Gata2 and Cebpa genes in BMMCs restored the aberrant increases of CD11b and Ly6G/C while retaining the reduced c-Kit expression. Chromatin immunoprecipitation assays indicated that GATA2 directly binds to the +37-kb region of the Cebpa gene and thereby inhibits the RUNX1 and PU.1 binding to the neighboring region. Upregulation of C/EBPα following the loss of GATA2 was not observed in cultured mast cells derived from peritoneal fluid, whereas the repression of c-Kit and other mast cell-specific genes were observed in these cells. Collectively, these results indicate that GATA2 maintains cellular identity by preventing Cebpa gene activation in a subpopulation of mast cells, whereas it plays a fundamental role as a positive regulator of mast cell-specific genes throughout development of this cell lineage. PMID:25855601

  14. Aneurysmal bone cyst primary - about eight pediatric cases: radiological aspects and review of the literature

    PubMed Central

    Boubbou, Meryem; Atarraf, Karima; Chater, Lamiae; Afifi, Abderrahmane; Tizniti, Siham

    2013-01-01

    The aneurysmal bone cyst is a pseudotumoral lesion that can take several aspects. This is a rare lesion representing 1% of bone tumors. It appears usually during the first 30 years of life. The pathogenesis is that of a process of dysplasia/hyperplasia, favored by a circulatory deficiency and hemorrhage within the lesion and the phenomena of osteoclasis. The objective of this work is to illustrate with analysis, the specific forms and atypical aneurysmal bone cyst which often pose a diagnostic challenge requiring radiological investigation with histological confirmation. We report eight pediatric cases of aneurysmal cysts collected over a period of 3 years, 3 boys and 5 girls. All patients had standard radiographs. MRI was performed in three patients. The diagnosis was confirmed histologically. The atypia has been in the seat: fibula (1 case), metaphyseal (2 cases), diaphyseal (4 cases) and metatarsal (1 case). Aneurysmal bone cyst is a rare benign tumor with predilection to the metaphysis of long bones. Atypical forms even fewer are dominated by the atypical seat. PMID:24244797

  15. Aneurysmal bone cyst primary--about eight pediatric cases: radiological aspects and review of the literature.

    PubMed

    Boubbou, Meryem; Atarraf, Karima; Chater, Lamiae; Afifi, Abderrahmane; Tizniti, Siham

    2013-01-01

    The aneurysmal bone cyst is a pseudotumoral lesion that can take several aspects. This is a rare lesion representing 1% of bone tumors. It appears usually during the first 30 years of life. The pathogenesis is that of a process of "dysplasia/hyperplasia", favored by a circulatory deficiency and hemorrhage within the lesion and the phenomena of osteoclasis. The objective of this work is to illustrate with analysis, the specific forms and atypical aneurysmal bone cyst which often pose a diagnostic challenge requiring radiological investigation with histological confirmation. We report eight pediatric cases of aneurysmal cysts collected over a period of 3 years, 3 boys and 5 girls. All patients had standard radiographs. MRI was performed in three patients. The diagnosis was confirmed histologically. The atypia has been in the seat: fibula (1 case), metaphyseal (2 cases), diaphyseal (4 cases) and metatarsal (1 case). Aneurysmal bone cyst is a rare benign tumor with predilection to the metaphysis of long bones. Atypical forms even fewer are dominated by the atypical seat. PMID:24244797

  16. Effects of heavy ion to the primary culture of mouse brain cells

    NASA Technical Reports Server (NTRS)

    Nojima, Kumie; Nakadai, Taeko; Kohno, Yukio; Vazquez, Marcelo E.; Yasuda, Nakahiro; Nagaoka, Shunji

    2004-01-01

    To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.

  17. Quantification of Alterations in Cortical Bone Geometry Using Site Specificity Software in Mouse models of Aging and the Responses to Ovariectomy and Altered Loading

    PubMed Central

    Galea, Gabriel L.; Hannuna, Sion; Meakin, Lee B.; Delisser, Peter J.; Lanyon, Lance E.; Price, Joanna S.

    2015-01-01

    Investigations into the effect of (re)modeling stimuli on cortical bone in rodents normally rely on analysis of changes in bone mass and architecture at a narrow cross-sectional site. However, it is well established that the effects of axial loading produce site-specific changes throughout bones’ structure. Non-mechanical influences (e.g., hormones) can be additional to or oppose locally controlled adaptive responses and may have more generalized effects. Tools currently available to study site-specific cortical bone adaptation are limited. Here, we applied novel site specificity software to measure bone mass and architecture at each 1% site along the length of the mouse tibia from standard micro-computed tomography (μCT) images. Resulting measures are directly comparable to those obtained through μCT analysis (R2 > 0.96). Site Specificity analysis was used to compare a number of parameters in tibiae from young adult (19-week-old) versus aged (19-month-old) mice; ovariectomized and entire mice; limbs subjected to short periods of axial loading or disuse induced by sciatic neurectomy. Age was associated with uniformly reduced cortical thickness and site-specific decreases in cortical area most apparent in the proximal tibia. Mechanical loading site-specifically increased cortical area and thickness in the proximal tibia. Disuse uniformly decreased cortical thickness and decreased cortical area in the proximal tibia. Ovariectomy uniformly reduced cortical area without altering cortical thickness. Differences in polar moment of inertia between experimental groups were only observed in the proximal tibia. Aging and ovariectomy also altered eccentricity in the distal tibia. In summary, site specificity analysis provides a valuable tool for measuring changes in cortical bone mass and architecture along the entire length of a bone. Changes in the (re)modeling response determined at a single site may not reflect the response at different locations within the same bone. PMID:25954246

  18. Transplant of Primary Human Hepatocytes Cocultured With Bone Marrow Stromal Cells to SCID Alb-uPA Mice

    PubMed Central

    Mohajerani, S. A.; Nourbakhsh, M.; Cadili, A.; Lakey, J. R.; Kneteman, N. M.

    2010-01-01

    Hepatocytes are vulnerable to loss of function and viability in culture. Modified culture methods have been applied to maintain their functional status. Heterotypic interactions between hepatocytes and nonparenchymal neighbors in liver milieu are thought to modulate cell differentiation. Cocultivation of hepatocyte with various cell types has been applied to mimic the hepatic environment. Bone marrow stromal cells (BMSC) are plastic cell lines capable of transforming to other cell types. In this study hepatocyte coculture with BMSCs achieved long-term function of human hepatocytes in culture for 4 weeks. In vitro functional status of human hepatocytes in BMSC coculture was compared with fibroblast coculture and collagen culture by measuring albumin, human-α-1-antitrypsin (hAAT), urea secretion, CYP450 activity, and staining for intracellular albumin and glycogen. After 2 weeks in culture hepatocytes were retrieved and transplanted to severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID Alb-uPA) mice and engraft-ment capacity was analyzed by human hepatic-specific function measured by hAAT levels in mouse serum, and Alu staining of mouse liver for human hepatocytes. Hepatocytes from BMSC coculture had significantly higher albumin, hAAT secretion, urea production, and cytochrome P450 (CYP450) activity than other culture groups. Staining confirmed the higher functional status in BMSC coculture. Transplantation of hepatocytes detached from BMSC cocultures showed significantly higher engraftment function than hepatocytes from other culture groups measured by hAAT levels in mouse serum. In conclusion, BMSC coculture has excellent potential for hepatocyte function preservation in vitro and in vivo after transplant. It is possible to use BMSC hepatocyte coculture as a supply of cell therapy in liver disease.

  19. Metabolic bone disease as a presenting manifestation of primary Sjögren's syndrome: Three cases and review of literature

    PubMed Central

    Khandelwal, Deepak; Bhattacharya, Saptarshi; Gadodia, Ankur; Khadgawat, Rajesh; Tandon, Nikhil; Ammini, A. C.

    2011-01-01

    Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by a progressive lymphocytic infiltration of the exocrine glands with varying degrees of systemic involvement. Chronic inflammation compromises the glands’ function that leads to dry symptoms in the mouth/eyes. Renal involvement is a well recognized extraglandular manifestation of pSS. Metabolic bone disease (MBD), however, rarely occurs as the primary manifestation of a renal tubule disorder due to pSS. To the best of our knowledge there are only 6 reported cases of metabolic bone disease as the primary manifestation of pSS to date. Four of these had distal renal tubular acidosis (RTA), and 2 had a combined picture of distal and proximal tubular dysfunction. We herein present our experience of 3 cases who presented to us with a clinical picture suggestive of MBD. While investigating these patients, we found evidence of RTA, which was found to be secondary to pSS. PMID:22029009

  20. Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis

    PubMed Central

    Moon, Clara; VanDussen, Kelli L.; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S.

    2013-01-01

    There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining IgA transcytosis across Transwells. IgA transcytosis required induction of polymeric immunoglobulin receptor (pIgR) expression, which could be stimulated by a combination of LPS and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that TNFα, IL-1β, IL-17 and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that amongst these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. IFNγ however did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology. PMID:24220295

  1. Heterogeneity of (TH)phorbol 12,13-dibutyrate binding in primary mouse keratinocytes at different stages of maturation

    SciTech Connect

    Dunn, J.A.; Jeng, A.Y.; Yuspa, S.H.; Blumberg, P.M.

    1985-11-01

    Mouse keratinocytes respond heterogeneously to phorbol esters with distinct subpopulations stimulated to proliferate or induced to differentiate. The maturation state of the epidermal cell at the time of exposure may determine its response. The binding of phorbol esters to primary mouse keratinocytes was studied under culture conditions selecting for proliferating cells or differentiating cells. (20-TH)-12-Deoxyphorbol 13-isobutyrate ((TH)-DPB) bound to both types of cells at one class of binding sites. The dissociation constant (Kd) for (TH)DPB in the proliferative cells was 69 nM and the binding at saturation (Bmax) was 1.3 pmol/mg of protein. The corresponding values in the differentiative cells were 96 nM and 1.5 pmol/mg of protein, respectively. In contrast to the results obtained with (TH)DPB, (20-TH)phorbol 12,13-dibutyrate ((TH)PDBU) bound to both cell types in a heterogeneous fashion. The site for (TH)DPB binding seemed to correspond to the higher affinity (TH)PDBU binding site. The major difference in the cells grown in the medium containing 1.2 mM CaCl2 was an increase in the Bmax of the lower affinity binding site with the other three parameters remaining similar. The state of epidermal differentiation thus appears to modulate the amount of the lower affinity binding sites for phorbol esters.

  2. First Neuromuscular Contact Correlates with Onset of Primary Myogenesis in Rat and Mouse Limb Muscles

    PubMed Central

    Duxson, Marilyn J.; Deries, Marianne

    2015-01-01

    Skeletal muscle development has been the focus of intensive study for many decades. Recent advances in genetic manipulation of the mouse have increased our understanding of the cell signalling involved in the development of muscle progenitors which give rise to adult skeletal muscles and their stem cell populations. However, the influence of a vital tissue type – the peripheral nerve—has largely been ignored since its earliest descriptions. Here we carefully describe the timing in which myogenic progenitors expressing Pax3 and Pax7 (the earliest markers of myogenic cells) enter the limb buds of rat and mouse embryos, as well as the spatiotemporal relationship between these progenitors and the ingrowing peripheral nerve. We show that progenitors expressing Pax3 enter the limb bud one full day ahead of the first neurites and that Pax7-expressing progenitors (associated with secondary myogenesis in the limb) are first seen in the limb bud at the time of nerve entry and in close proximity to the nerve. The initial entry of the nerve also coincides with the first expression of myosin heavy chain showing that the first contact between nerves and myogenic cells correlates with the onset of myogenic differentiation. Furthermore, as the nerve grows into the limb, Pax3 expression is progressively replaced by Pax7 expression in myogenic progenitors. These findings indicate that the ingrowing nerve enters the limb presumptive muscle masses earlier than what was generally described and raises the possibility that nerve may influence the differentiation of muscle progenitors in rodent limbs. PMID:26207754

  3. Severe hypocalcemia complicated by postsurgical hypoparathyroidism and hungry bone syndrome in a patient with primary hyperparathyroidism, Graves' disease, and acromegaly.

    PubMed

    Tachibana, Seigo; Sato, Shinya; Yokoi, Tadao; Nagaishi, Ryoko; Akehi, Yuko; Yanase, Toshihiko; Yamashita, Hiroyuki

    2012-01-01

    We herein report a case of severe postsurgical hypocalcemia associated with primary hyperparathyroidism (pHPT), Graves' disease (GD) and acromegaly (AC). A 54-year-old woman was referred to our clinic for treatment of pHPT and GD. She also had active AC and was clinically diagnosed as multiple endocrine neoplasm type 1 because of pHPT and AC. Two enlarged parathyroid glands were detected by preoperative examinations. We performed total parathyroidectomy and thyroidectomy. After the operation, she showed severe hypocalcemia induced by postsurgical hypoparathyroidism and hungry bone syndrome. This is a rare case of postsurgical severe hypocalcemia associated with pHPT, GD and AC. PMID:22821103

  4. Purkinje cells and Bergmann glia are primary targets of the TR?1 thyroid hormone receptor during mouse cerebellum postnatal development.

    PubMed

    Fauquier, Teddy; Chatonnet, Fabrice; Picou, Frdric; Richard, Sabine; Fossat, Nicolas; Aguilera, Nadine; Lamonerie, Thomas; Flamant, Frdric

    2014-01-01

    Thyroid hormone is necessary for normal development of the central nervous system, as shown by the severe mental retardation syndrome affecting hypothyroid patients with low levels of active thyroid hormone. The postnatal defects observed in hypothyroid mouse cerebellum are recapitulated in mice heterozygous for a dominant-negative mutation of Thra, the gene encoding the ubiquitous TR?1 receptor. Using CRE/loxP-mediated conditional expression approach, we found that this mutation primarily alters the differentiation of Purkinje cells and Bergmann glia, two cerebellum-specific cell types. These primary defects indirectly affect cerebellum development in a global manner. Notably, the inward migration and terminal differentiation of granule cell precursors is impaired. Therefore, despite the broad distribution of its receptors, thyroid hormone targets few cell types that exert a predominant role in the network of cellular interactions that govern normal cerebellum maturation. PMID:24346699

  5. Calcium Intake, Major Dietary Sources and Bone Health Indicators in Iranian Primary School Children

    PubMed Central

    Omidvar, Nasrin; Neyestani, Tirang-Reza; Hajifaraji, Majid; Eshraghian, Mohammad-Reza; Rezazadeh, Arezoo; Armin, Saloumeh; Haidari, Homa; Zowghi, Telma

    2015-01-01

    Background: Adequate calcium intake may have a crucial role with regards to prevention of many chronic diseases, including hypertension, hypercholesterolemia, different types of cancer, obesity and osteoporosis. In children, sufficient calcium intake is especially important to support the accelerated growth spurt during the preteen and teenage years and to increase bone mineral mass to lay the foundation for older age. Objectives: This study aimed to assess daily calcium intake in school-age children to ensure whether they fulfill the FGP dairy serving recommendations, the recommended levels of daily calcium intake and to assess the relationship between dietary calcium intake and major bone health indicators. Patients and Methods: A total of 501 Iranian school-age children were randomly selected. Calcium intake was assessed using a semi-quantitative food frequency questionnaire. Bone health indicators were also assessed. Results: Dairy products contributed to 69.3% of the total calcium intake of the children. Daily adequate intake of calcium was achieved by 17.8% of children. Only 29.8% met the Food guide pyramid recommendations for dairy intake. Dietary calcium intake was not significantly correlated with serum calcium and other selected biochemical indicators of bone health. Conclusions: The need for planning appropriate nutrition strategies for overcoming inadequate calcium intake in school age children in the city of Tehran is inevitable. PMID:26199684

  6. Inhibition of Transforming Growth Factor-β Activation Diminishes Tumor Progression and Osteolytic Bone Disease in Mouse Models of Multiple Myeloma.

    PubMed

    Lu, Ailing; Pallero, Manuel A; Lei, Weiqi; Hong, Huixian; Yang, Yang; Suto, Mark J; Murphy-Ullrich, Joanne E

    2016-03-01

    Transforming growth factor (TGF)-β supports multiple myeloma progression and associated osteolytic bone disease. Conversion of latent TGF-β to its biologically active form is a major regulatory node controlling its activity. Thrombospondin1 (TSP1) binds and activates TGF-β. TSP1 is increased in myeloma, and TSP1-TGF-β activation inhibits osteoblast differentiation. We hypothesized that TSP1 regulates TGF-β activity in myeloma and that antagonism of the TSP1-TGF-β axis inhibits myeloma progression. Antagonists (LSKL peptide, SRI31277) derived from the LSKL sequence of latent TGF-β that block TSP1-TGF-β activation were used to determine the role of the TSP1-TGF-β pathway in mouse models of myeloma. TSP1 binds to human myeloma cells and activates TGF-β produced by cultured human and mouse myeloma cell lines. Antagonists delivered via osmotic pump in an intratibial severe combined immunodeficiency CAG myeloma model or in a systemic severe combined immunodeficiency CAG-heparanase model of aggressive myeloma reduced TGF-β signaling (phospho-Smad 2) in bone sections, tumor burden, mouse IL-6, and osteoclasts, increased osteoblast number, and inhibited bone destruction as measured by microcomputed tomography. SRI31277 reduced tumor burden in the immune competent 5TGM1 myeloma model. SRI31277 was as effective as dexamethasone or bortezomib, and SRI31277 combined with bortezomib showed greater tumor reduction than either agent alone. These studies validate TSP1-regulated TGF-β activation as a therapeutic strategy for targeted inhibition of TGF-β in myeloma. PMID:26801735

  7. MHC-compatible bone marrow stromal/stem cells trigger fibrosis by activating host T cells in a scleroderma mouse model.

    PubMed

    Ogawa, Yoko; Morikawa, Satoru; Okano, Hideyuki; Mabuchi, Yo; Suzuki, Sadafumi; Yaguchi, Tomonori; Sato, Yukio; Mukai, Shin; Yaguchi, Saori; Inaba, Takaaki; Okamoto, Shinichiro; Kawakami, Yutaka; Tsubota, Kazuo; Matsuzaki, Yumi; Shimmura, Shigeto

    2016-01-01

    Fibrosis of organs is observed in systemic autoimmune disease. Using a scleroderma mouse, we show that transplantation of MHC compatible, minor antigen mismatched bone marrow stromal/stem cells (BMSCs) play a role in the pathogenesis of fibrosis. Removal of donor BMSCs rescued mice from disease. Freshly isolated PDGFRα(+) Sca-1(+) BMSCs expressed MHC class II following transplantation and activated host T cells. A decrease in FOXP3(+) CD25(+) Treg population was observed. T cells proliferated and secreted IL-6 when stimulated with mismatched BMSCs in vitro. Donor T cells were not involved in fibrosis because transplanting T cell-deficient RAG2 knock out mice bone marrow still caused disease. Once initially triggered by mismatched BMSCs, the autoimmune phenotype was not donor BMSC dependent as the phenotype was observed after effector T cells were adoptively transferred into naïve syngeneic mice. Our data suggest that minor antigen mismatched BMSCs trigger systemic fibrosis in this autoimmune scleroderma model. PMID:26809474

  8. [Action of two pyrazine-containing chemosignals on cells of bone marrow and testes in male house mouse Mus musculus L].

    PubMed

    Daev, E V; Vyborova, A M; Kazarova, V É; Dukel'skaia, A V

    2012-01-01

    Evolutionary conservative chemosignal 2,5-dimethylpyrazin that is pheromone in female mice has been shown to increase frequency of mitotic aberrations analyzed with aid of metaphasic and ana-telophasic analysis in bone marrow cells. Replacement of one of methyl radicals in the pheromone molecule by the carboxyl radical reveals specificity of action of the used derivative: the frequency of disturbances revealed only by the ana-telophasic analysis increases, whereas by the metaphasic analysis, no induction of disturbance is detected. In the sperm head abnormality test there is shown a rise of the anomalies by both compounds. Possible mechanisms of specific action of the tested substances on stability of genetic apparatus of the bone marrow dividing cells in the house mouse are discussed. PMID:22567971

  9. Calcitonin receptors as markers for osteoclastic differentiation: correlation between generation of bone-resorptive cells and cells that express calcitonin receptors in mouse bone marrow cultures.

    PubMed

    Hattersley, G; Chambers, T J

    1989-09-01

    The osteoclast is the cell that resorbs bone. It is known to derive from hemopoietic precursors, but analysis of lineage and regulation of differentiation has been hampered by lack of a specific marker that enables identification of cells of osteoclastic phenotype. Previously used markers, such as multinuclearity, that are specific for osteoclasts in bone become less specific in culture. Uniquely among bone and bone marrow cells, osteoclasts possess abundant calcitonin (CT) receptors. We therefore tested the correlation between the generation of bone-resorptive function and the formation of CT receptor-positive cells from hemopoietic tissue in vitro. Without 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3], a hormone that induces osteoclastic differentiation in vitro, bone marrow cultures showed very little bone resorption, and only small numbers of CT receptor-positive cells developed. When 1,25-(OH)2D3 was added to the cultures, CT receptor-positive cells developed within 1 day and reached a peak after 7 days. Bone resorption commenced within 2 days of hormone addition. There was a strong parallelism between the cumulative number of CT receptor-positive cells and the extent of bone resorption. The capacity of cultures to generate bone-resorptive activity and CT receptor-positive cells declined progressively when 1,25-(OH)2D3 was added to hemopoietic tissue after a 7- to 21-day hormone-free incubation period. The number of CT receptor-positive cells in these cultures correlated strongly (r = 0.96) with bone resorption. The behavior of these cultures suggests that 1,25-(OH)2D3 acts to induce terminal differentiation of osteoclast precursors present in the cultures, and that precursor cell numbers decreased with increasing time in vitro. All of the CT receptor-positive cells in control cultures and all of those seen shortly after 1,25-(OH)2D3 addition were mononuclear, despite considerable bone resorption; the majority of CT receptor-positive cells remained mononuclear throughout the incubation period. This suggests that mononuclear cells with characteristics of osteoclasts exist that are able to excavate bone. CT receptor-positive cells slightly preceded the development of bone-resorptive function, implying that CT receptors develop before the acquisition of bone-resorptive capacity by osteoclasts. Peritoneal macrophages, blood mononuclear cells, and cells of the J774 macrophage cell line failed to either resorb bone or express CT receptors, even after incubation with 1,25-(OH)2D3 for 14 days. These results show a strong and specific correlation between the generation of bone-resorptive cells and CT receptor-positive cells, and suggest that CT receptor express PMID:2547591

  10. Cured of Primary Bone Cancer, But at What Cost: A Qualitative Study of Functional Impairment and Lost Opportunities

    PubMed Central

    Fauske, Lena; Bruland, Oyvind S.; Grov, Ellen Karine; Bondevik, Hilde

    2015-01-01

    Purpose. Our study aims to explore how former cancer patients experience physical and psychosocial late effects 3–7 years after they underwent treatment for primary bone sarcoma in the hip/pelvic region. A qualitative, phenomenological, and hermeneutic design was applied. Methods. Sarcoma survivors (n = 10) previously treated at Oslo University Hospital, Norwegian Radium Hospital were selected to participate. In-depth and semistructured interviews were conducted. The interviews were analysed using inductive thematic analysis. Results. The participants reported that the late effects had three core spheres of impact: “their current daily life,” “their future opportunities,” and “their identity.” They expressed negative changes in activity, increased dependence on others, and exclusion from participation in different areas. Their daily life, work, sports activities, and social life were all affected. Several of their experiences are similar to those described by people with functional impairment or disability. Conclusion. Patients cured of bone cancer in the hip/pelvic region pay a significant price in terms of functional impairment, practical challenges, exclusion from important aspects of life, and loss of previous identity. It is important to appreciate this in order to help bone cancer survivors who struggle to reorient their life and build a secure new identity. PMID:25949211

  11. In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    PubMed Central

    Abouzaripour, Morteza; Pasbakhsh, Parichehr; Atlasi, Nader; Shahverdi, Abdol Hossein; Mahmoudi, Reza; Kashani, Iraj Ragerdi

    2016-01-01

    Objective Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar- row have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy. Materials and Methods This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza- tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. Results The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem- istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticβ-cell markers). Additionally, our results demonstrate expression of Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. Conclusion Our study clearly demonstrates the potential of SSEA-1 positive cells to differentiate into insulin secreting cells in defined culture conditions for clinical ap- plications. PMID:26862529

  12. In vitro differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in mouse: a proteomic analysis

    PubMed Central

    Cong, Qing; Li, Bin; Wang, Yisheng; Zhang, Wenbi; Cheng, Mingjun; Wu, Zhiyong; Zhang, Xiaoyan; Jiang, Wei; Xu, Congjian

    2014-01-01

    Objective: Mouse bone marrow mesenchymal stem cells (BMSCs) have been demonstrated to differentiate into female endometrial epithelial cells (EECs) in vivo. Our previous studies demonstrated that BMSCs can differentiate in the direction of EECs when co-cultured with endometrial stromal cells in vitro. Here, we obtain and analyse differential proteins and their relevant pathways in the process of BMSCs differentiating into EECs by isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis. Methods: A 0.4-μm pore size indirect co-culture system was established with female mice endometrial stromal cells (EStCs) restricted in the upper Transwell chamber and BMSCs in the lower well plate. After indirect co-culture for several days, the BMSCs were revealed to progressively differentiate towards EECs in vitro. Then, four groups were divided according to different co-culture days with single culture groups of BMSCs as controls. Proteins were detected using iTRAQ based on 2DLC-ESI-MS/MS and data were analysed by bioinformatics. Results: A total number of 311 proteins were detected, of which 210 proteins were identified with relative quantitation. Among them, 107 proteins were differentially expressed with a 1.2-fold change as the benchmark, with 61 up-regulated and 46 down-regulated proteins. Differential proteins CK19 and CK8 were epithelial markers and upregulated. Stromal marker vimentin were downregulated. Top canonical pathways was “remodeling of epithelial adhesions junctions” and “actin cytoskeleton signaling”. Top networks was “cell-to-cell signaling and interaction, tissue development and cellular movement” regulated by ERK/MAPK and α-catenin. Conclusion: To the best of our knowledge, this is the first preliminary study of differential protein expression in the differentiation process of BMSCs into EECs in vitro. We further elucidated BMSCs differentiated in the direction of EECs. In addition, ERK/MAPK and α-catenin played important roles by regulating core differential proteins in the “cell-to-cell signaling and interaction, tissue development and cellular movement” network. PMID:25120742

  13. Mechanisms of benzene-induced hematotoxicity and leukemogenicity: cDNA microarray analyses using mouse bone marrow tissue.

    PubMed Central

    Yoon, Byung-Il; Li, Guang-Xun; Kitada, Kunio; Kawasaki, Yasushi; Igarashi, Katsuhide; Kodama, Yukio; Inoue, Tomoaki; Kobayashi, Kazuko; Kanno, Jun; Kim, Dae-Yong; Inoue, Tohru; Hirabayashi, Yoko

    2003-01-01

    Although the mechanisms underlying benzene-induced toxicity and leukemogenicity are not yet fully understood, they are likely to be complicated by various pathways, including those of metabolism, growth factor regulation, oxidative stress, DNA damage, cell cycle regulation, and programmed cell death. With this as a background, we performed cDNA microarray analyses on mouse bone marrow tissue during and after a 2-week benzene exposure by inhalation. Our goal was to clarify the mechanisms underlying the hematotoxicity and leukemogenicity induced by benzene at the level of altered multigene expression. Because a few researchers have postulated that the cell cycle regulation mediated by p53 is a critical event for benzene-induced hematotoxicity, the present study was carried out using p53-knockout (KO) mice and C57BL/6 mice. On the basis of the results of large-scale gene expression studies, we conclude the following: (a) Benzene induces DNA damage in cells at any phase of the cell cycle through myeloperoxidase and in the redox cycle, resulting in p53 expression through Raf-1 and cyclin D-interacting myb-like protein 1. (b) For G1/S cell cycle arrest, the p53-mediated pathway through p21 is involved, as well as the pRb gene-mediated pathway. (c) Alteration of cyclin G1 and Wee-1 kinase genes may be related to the G2/M arrest induced by benzene exposure. (d) DNA repair genes such as Rad50 and Rad51 are markedly downregulated in p53-KO mice. (e) p53-mediated caspase 11 activation, aside from p53-mediated Bax gene induction, may be an important pathway for cellular apoptosis after benzene exposure. Our results strongly suggest that the dysfunction of the p53 gene, possibly caused by strong and repeated genetic and epigenetic effects of benzene on candidate leukemia cells, may induce fatal problems such as those of cell cycle checkpoint, apoptosis, and the DNA repair system, finally resulting in hemopoietic malignancies. Our cDNA microarray data provide valuable information for future investigations of the mechanisms underlying the toxicity and leukemogenicity of benzene. PMID:12928149

  14. Creation of Primary Cell Lines from Lineage-Labeled Mouse Models of Cancer

    PubMed Central

    Rhim, Andrew D.

    2015-01-01

    Frequently, it is necessary to isolate pure populations of cancer cells for downstream assays, such as transcriptional analysis, signaling studies, and the creation of noncontaminated primary cell lines. Genetic lineage labeling with fluorescent reporter alleles allows for the identification of epithelial-derived cells within tumors. This protocol describes a method to isolate lineage-labeled pancreatic epithelial cells for ex vivo analysis, but it can be adapted for any type of lineage-labeled tumor. PMID:25934932

  15. Combined zoledronic acid and meloxicam reduced bone loss and tumour growth in an orthotopic mouse model of bone-invasive oral squamous cell carcinoma.

    PubMed

    Martin, C K; Dirksen, W P; Carlton, M M; Lanigan, L G; Pillai, S P; Werbeck, J L; Simmons, J K; Hildreth, B E; London, C A; Toribio, R E; Rosol, T J

    2015-09-01

    Oral squamous cell carcinoma (OSCC) is common in cats and humans and invades oral bone. We hypothesized that the cyclooxygenase (COX)-2 inhibitor, meloxicam, with the bisphosphonate, zoledronic acid (ZOL), would inhibit tumour growth, osteolysis and invasion in feline OSCC xenografts in mice. Human and feline OSCC cell lines expressed COX-1 and COX-2 and the SCCF2 cells had increased COX-2 mRNA expression with bone conditioned medium. Luciferase-expressing feline SCCF2Luc cells were injected beneath the perimaxillary gingiva and mice were treated with 0.1 mg kg(-1) ZOL twice weekly, 0.3 mg kg(-1) meloxicam daily, combined ZOL and meloxicam, or vehicle. ZOL inhibited osteoclastic bone resorption at the tumour-bone interface. Meloxicam was more effective than ZOL at reducing xenograft growth but did not affect osteoclastic bone resorption. Although a synergistic effect of combined ZOL and meloxicam was not observed, combination therapy was well-tolerated and may be useful in the clinical management of bone-invasive feline OSCC. PMID:23651067

  16. In vitro assessment of nanosilver-functionalized PMMA bone cement on primary human mesenchymal stem cells and osteoblasts.

    PubMed

    Pauksch, Linda; Hartmann, Sonja; Szalay, Gabor; Alt, Volker; Lips, Katrin S

    2014-01-01

    Peri-prosthetic infections caused by multidrug resistant bacteria have become a serious problem in surgery and orthopedics. The aim is to introduce biomaterials that avoid implant-related infections caused by multiresistant bacteria. The efficacy of silver nanoparticles (AgNP) against a broad spectrum of bacteria and against multiresistant pathogens has been repeatedly described. In the present study polymethylmethacrylate (PMMA) bone cement functionalized with AgNP and/or gentamicin were tested regarding their biocompatibility with bone forming cells. Therefore, influences on viability, cell number and differentiation of primary human mesenchymal stem cells (MSCs) and MSCs cultured in osteogenic differentiation media (MSC-OM) caused by the implant materials were studied. Furthermore, the growth behavior and the morphology of the cells on the testing material were observed. Finally, we examined the induction of cell stress, regarding antioxidative defense and endoplasmatic reticulum stress. We demonstrated similar cytocompatibility of PMMA loaded with AgNP compared to plain PMMA or PMMA loaded with gentamicin. There was no decrease in cell number, viability and osteogenic differentiation and no induction of cell stress for all three PMMA variants after 21 days. Addition of gentamicin to AgNP-loaded PMMA led to a slight decrease in osteogenic differentiation. Also an increase in cell stress was detectable for PMMA loaded with gentamicin and AgNP. In conclusion, supplementation of PMMA bone cement with gentamicin, AgNP, and both results in bone implants with an antibacterial potency and suitable cytocompatibility in MSCs and MSC-OM. PMID:25485700

  17. In Vitro Assessment of Nanosilver-Functionalized PMMA Bone Cement on Primary Human Mesenchymal Stem Cells and Osteoblasts

    PubMed Central

    Pauksch, Linda; Hartmann, Sonja; Szalay, Gabor; Alt, Volker; Lips, Katrin S.

    2014-01-01

    Peri-prosthetic infections caused by multidrug resistant bacteria have become a serious problem in surgery and orthopedics. The aim is to introduce biomaterials that avoid implant-related infections caused by multiresistant bacteria. The efficacy of silver nanoparticles (AgNP) against a broad spectrum of bacteria and against multiresistant pathogens has been repeatedly described. In the present study polymethylmethacrylate (PMMA) bone cement functionalized with AgNP and/or gentamicin were tested regarding their biocompatibility with bone forming cells. Therefore, influences on viability, cell number and differentiation of primary human mesenchymal stem cells (MSCs) and MSCs cultured in osteogenic differentiation media (MSC-OM) caused by the implant materials were studied. Furthermore, the growth behavior and the morphology of the cells on the testing material were observed. Finally, we examined the induction of cell stress, regarding antioxidative defense and endoplasmatic reticulum stress. We demonstrated similar cytocompatibility of PMMA loaded with AgNP compared to plain PMMA or PMMA loaded with gentamicin. There was no decrease in cell number, viability and osteogenic differentiation and no induction of cell stress for all three PMMA variants after 21 days. Addition of gentamicin to AgNP-loaded PMMA led to a slight decrease in osteogenic differentiation. Also an increase in cell stress was detectable for PMMA loaded with gentamicin and AgNP. In conclusion, supplementation of PMMA bone cement with gentamicin, AgNP, and both results in bone implants with an antibacterial potency and suitable cytocompatibility in MSCs and MSC-OM. PMID:25485700

  18. Biorhythms, deciduous enamel thickness, and primary bone growth: a test of the Havers-Halberg Oscillation hypothesis.

    PubMed

    Mahoney, Patrick; Miszkiewicz, Justyna J; Pitfield, Rosie; Schlecht, Stephen H; Deter, Chris; Guatelli-Steinberg, Debbie

    2016-06-01

    Across mammalian species, the periodicity with which enamel layers form (Retzius periodicity) in permanent teeth corresponds with average body mass and the pace of life history. According to the Havers-Halberg Oscillation hypothesis (HHO), Retzius periodicity (RP) is a manifestation of a biorhythm that is also expressed in lamellar bone. Potentially, these links provide a basis for investigating aspects of a species' biology from fossilized teeth. Here, we tested intra-specific predictions of this hypothesis on skeletal samples of human juveniles. We measured daily enamel growth increments to calculate RP in deciduous molars (n = 25). Correlations were sought between RP, molar average and relative enamel thickness (AET, RET), and the average amount of primary bone growth (n = 7) in humeri of age-matched juveniles. Results show a previously undescribed relationship between RP and enamel thickness. Reduced major axis regression reveals RP is significantly and positively correlated with AET and RET, and scales isometrically. The direction of the correlation was opposite to HHO predictions as currently understood for human adults. Juveniles with higher RPs and thicker enamel had increased primary bone formation, which suggests a coordinating biorhythm. However, the direction of the correspondence was, again, opposite to predictions. Next, we compared RP from deciduous molars with new data for permanent molars, and with previously published values. The lowermost RP of 4 and 5 days in deciduous enamel extends below the lowermost RP of 6 days in permanent enamel. A lowered range of RP values in deciduous enamel implies that the underlying biorhythm might change with age. Our results develop the intra-specific HHO hypothesis. PMID:26914945

  19. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

    PubMed Central

    Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

  20. Brg1 Controls the Expression of Pax7 to Promote Viability and Proliferation of Mouse Primary Myoblasts.

    PubMed

    Padilla-Benavides, Teresita; Nasipak, Brian T; Imbalzano, Anthony N

    2015-12-01

    Brg1 (Brahma-related gene 1) is a catalytic component of the evolutionarily conserved mammalian SWI/SNF ATP-dependent chromatin remodeling enzymes that disrupt histone-DNA contacts on the nucleosome. While the requirement for the SWI/SNF enzymes in cell differentiation has been extensively studied, its role in precursor cell proliferation and survival is not as well defined. Muscle satellite cells constitute the stem cell pool that sustains and regenerates myofibers in adult skeletal muscle. Here, we show that deletion of Brg1 in primary mouse myoblasts derived from muscle satellite cells cultured ex vivo leads to a cell proliferation defect and apoptosis. We determined that Brg1 regulates cell proliferation and survival by controlling chromatin remodeling and activating transcription at the Pax7 promoter, which is expressed during somite development and is required for controlling viability of the satellite cell population. Reintroduction of catalytically active Brg1 or of Pax7 into Brg1-deficient satellite cells rescued the apoptotic phenotype and restored proliferation. These data demonstrate that Brg1 functions as a positive regulator for cellular proliferation and survival of primary myoblasts. Therefore, the regulation of gene expression through Brg1-mediated chromatin remodeling is critical not just for skeletal muscle differentiation but for maintaining the myoblast population as well. PMID:26036967

  1. Zonisamide Enhances Neurite Elongation of Primary Motor Neurons and Facilitates Peripheral Nerve Regeneration In Vitro and in a Mouse Model

    PubMed Central

    Yagi, Hideki; Ohkawara, Bisei; Nakashima, Hiroaki; Ito, Kenyu; Tsushima, Mikito; Ishii, Hisao; Noto, Kimitoshi; Ohta, Kyotaro; Masuda, Akio; Imagama, Shiro; Ishiguro, Naoki; Ohno, Kinji

    2015-01-01

    No clinically applicable drug is currently available to enhance neurite elongation after nerve injury. To identify a clinically applicable drug, we screened pre-approved drugs for neurite elongation in the motor neuron-like NSC34 cells. We found that zonisamide, an anti-epileptic and anti-Parkinson’s disease drug, promoted neurite elongation in cultured primary motor neurons and NSC34 cells in a concentration-dependent manner. The neurite-scratch assay revealed that zonisamide enhanced neurite regeneration. Zonisamide was also protective against oxidative stress-induced cell death of primary motor neurons. Zonisamide induced mRNA expression of nerve growth factors (BDNF, NGF, and neurotrophin-4/5), and their receptors (tropomyosin receptor kinase A and B). In a mouse model of sciatic nerve autograft, intragastric administration of zonisamide for 1 week increased the size of axons distal to the transected site 3.9-fold. Zonisamide also improved the sciatic function index, a marker for motor function of hindlimbs after sciatic nerve autograft, from 6 weeks after surgery. At 8 weeks after surgery, zonisamide was protective against denervation-induced muscle degeneration in tibialis anterior, and increased gene expression of Chrne, Colq, and Rapsn, which are specifically expressed at the neuromuscular junction. We propose that zonisamide is a potential therapeutic agent for peripheral nerve injuries as well as for neuropathies due to other etiologies. PMID:26571146

  2. Variation and genetic control of gene expression in primary immunocytes across inbred mouse strains.

    PubMed

    Mostafavi, Sara; Ortiz-Lopez, Adriana; Bogue, Molly A; Hattori, Kimie; Pop, Cristina; Koller, Daphne; Mathis, Diane; Benoist, Christophe

    2014-11-01

    To determine the breadth and underpinning of changes in immunocyte gene expression due to genetic variation in mice, we performed, as part of the Immunological Genome Project, gene expression profiling for CD4(+) T cells and neutrophils purified from 39 inbred strains of the Mouse Phenome Database. Considering both cell types, a large number of transcripts showed significant variation across the inbred strains, with 22% of the transcriptome varying by 2-fold or more. These included 119 loci with apparent complete loss of function, where the corresponding transcript was not expressed in some of the strains, representing a useful resource of "natural knockouts." We identified 1222 cis-expression quantitative trait loci (cis-eQTL) that control some of this variation. Most (60%) cis-eQTLs were shared between T cells and neutrophils, but a significant portion uniquely impacted one of the cell types, suggesting cell type-specific regulatory mechanisms. Using a conditional regression algorithm, we predicted regulatory interactions between transcription factors and potential targets, and we demonstrated that these predictions overlap with regulatory interactions inferred from transcriptional changes during immunocyte differentiation. Finally, comparison of these and parallel data from CD4(+) T cells of healthy humans demonstrated intriguing similarities in variability of a gene's expression: the most variable genes tended to be the same in both species, and there was an overlap in genes subject to strong cis-acting genetic variants. We speculate that this "conservation of variation" reflects a differential constraint on intraspecies variation in expression levels of different genes, either through lower pressure for some genes, or by favoring variability for others. PMID:25267973

  3. Mouse bone marrow micronucleus test results do not predict the germ cell mutagenicity of N-hydroxymethylacrylamide in the mouse dominant lethal assay.

    PubMed

    Witt, Kristine L; Hughes, Lori A; Burka, Leo T; McFee, Alfred F; Mathews, James M; Black, Sherry L; Bishop, Jack B

    2003-01-01

    N-Hydroxymethylacrylamide (NHMA), a mouse carcinogen inactive in the Salmonella assay and mouse micronucleus (MN) assay, was tested for reproductive effects in a mouse continuous breeding study. In that study, increased embryonic deaths were observed after 13 weeks exposure of parental animals to NHMA via drinking water (highest dose, 360 ppm); the results indicated the possible induction of chromosome damage in germ cells of treated males. An additional mouse MN test was conducted using a 31-day treatment period to better match the dosing regimen used in the breeding study; the results were negative. Additional studies were conducted to explore the germ cell activity of NHMA. A male mouse dominant lethal study was conducted using a single intraperitoneal injection of 150 mg/kg NHMA; the results were negative. A follow-up study was conducted using fractionated dosing, 50 mg/kg/day for 5 days; again, no increase in dominant lethal mutations was observed. NHMA (180-720 ppm) was then administered to male mice in drinking water for 13 weeks, during which three sets of matings occurred. Two weeks after mating, females were killed and the uterine contents were analyzed. Large, dose-related increases in dominant lethal mutations were observed with increasing length of exposure. The magnitude of the increases stabilized after 8 weeks of treatment. However, the frequency of micronucleated peripheral blood erythrocytes was not elevated in mice treated for 13 weeks with NHMA in drinking water. Thus, NHMA appears to be unique in inducing genetic damage in germ cells but not somatic cells of male mice. PMID:12605380

  4. Dual energy micro-CT imaging of radiation-induced vascular changes in primary mouse sarcomas

    PubMed Central

    Moding, Everett J.; Clark, Darin P.; Qi, Yi; Li, Yifan; Ma, Yan; Ghaghada, Ketan; Johnson, G. Allan; Kirsch, David G.; Badea, Cristian T.

    2013-01-01

    Purpose To evaluate the effects of radiation therapy on primary tumor vasculature using dual energy (DE) micro-computed tomography (micro-CT). Methods and Materials The Cre-loxP system was used to generate primary sarcomas with mutant Kras and p53. Unirradiated tumors were compared to tumors irradiated with 20 Gy. A long-circulating PEGylated liposomal-iodinated contrast agent was administered one day after treatment, and mice were imaged immediately after injection (day 1) and three days later (day 4) using DE micro-CT. CT-derived tumor sizes were used to assess tumor growth. After DE decomposition, iodine maps were used to assess tumor fractional blood volume (FBV) at day 1 and tumor vascular permeability at day 4. For comparison, tumor vascularity and vascular permeability were also evaluated histologically using CD31 immunofluorescence and fluorescently-labeled dextrans. Results Radiation treatment significantly decreased tumor growth (P<0.05). There was a positive correlation between CT-measurement of tumor FBV and extravasated iodine with microvascular density (MVD) (R2=0.53) and dextran accumulation (R2=0.63), respectively. Despite no change in MVD measured by histology, tumor FBV significantly increased after irradiation as measured by DE micro-CT (0.070 vs. 0.091, P<0.05). Both dextran and liposomal-iodine accumulation in tumors increased significantly after irradiation with dextran fractional area increasing 4.2-fold and liposomal-iodine concentration increasing 3.0-fold. Conclusions DE micro-CT is an effective tool for non-invasive assessment of vascular changes in primary tumors. Tumor blood volume and vascular permeability increased after a single therapeutic dose of radiation treatment. PMID:23122984

  5. Makings of a brittle bone: Unexpected lessons from a low protein diet study of a mouse OI model.

    PubMed

    Mertz, E L; Makareeva, E; Mirigian, L S; Koon, K Y; Perosky, J E; Kozloff, K M; Leikin, S

    2016-01-01

    Glycine substitutions in type I collagen appear to cause osteogenesis imperfecta (OI) by disrupting folding of the triple helix, the structure of which requires Gly in every third position. It is less clear, however, whether the resulting bone malformations and fragility are caused by effects of intracellular accumulation of misfolded collagen on differentiation and function of osteoblasts, effects of secreted misfolded collagen on the function of bone matrix, or both. Here we describe a study originally conceived for testing how reducing intracellular accumulation of misfolded collagen would affect mice with a Gly610 to Cys substitution in the triple helical region of the α2(I) chain. To stimulate degradation of misfolded collagen by autophagy, we utilized a low protein diet. The diet had beneficial effects on osteoblast differentiation and bone matrix mineralization, but also affected bone modeling and suppressed overall animal growth. Our more important observations, however, were not related to the diet. They revealed how altered osteoblast function and deficient bone formation by each cell caused by the G610C mutation combined with increased osteoblastogenesis might make the bone more brittle, all of which are common OI features. In G610C mice, increased bone formation surface compensated for reduced mineral apposition rate, resulting in normal cortical area and thickness at the cost of altering cortical modeling process, retaining woven bone, and reducing the ability of bone to absorb energy through plastic deformation. Reduced collagen and increased mineral density in extracellular matrix of lamellar bone compounded the problem, further reducing bone toughness. The latter observations might have particularly important implications for understanding OI pathophysiology and designing more effective therapeutic interventions. PMID:27039252

  6. Genetic deletion of keratin 8 corrects the altered bone formation and osteopenia in a mouse model of cystic fibrosis.

    PubMed

    Le Henaff, Carole; Faria Da Cunha, Mélanie; Hatton, Aurélie; Tondelier, Danielle; Marty, Caroline; Collet, Corinne; Zarka, Mylène; Geoffroy, Valérie; Zatloukal, Kurt; Laplantine, Emmanuel; Edelman, Aleksander; Sermet-Gaudelus, Isabelle; Marie, Pierre J

    2016-04-01

    Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-β-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-β-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-β-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis. PMID:26769674

  7. Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation

    SciTech Connect

    Li, Ju; Reed, Sarah A.; Johnson, Sally E.

    2009-08-01

    Niche localized HGF plays an integral role in G{sub 0} exit and the return to mitotic activity of adult skeletal muscle satellite cells. HGF actions are regulated by MET initiated intracellular signaling events that include recruitment of SHP2, a protein tyrosine phosphatase. The importance of SHP2 in HGF-mediated signaling was examined in myoblasts and primary cultures of satellite cells. Myoblasts stably expressing SHP2 (23A2-SHP2) demonstrate increased proliferation rates by comparison to controls or myoblasts expressing a phosphatase-deficient SHP2 (23A2-SHP2DN). By comparison to 23A2 myoblasts, treatment of 23A2-SHP2 cells with HGF does not further increase proliferation rates and 23A2-SHP2DN myoblasts are unresponsive to HGF. Importantly, the effects of SHP2 are independent of downstream ERK1/2 activity as inclusion of PD98059 does not blunt the HGF-induced proliferative response. SHP2 function was further evaluated in primary satellite cell cultures. Ectopic expression of SHP2 in satellite cells tends to decrease proliferation rates and siSHP2 causes an increase the percentage of dividing myogenic cells. Interestingly, treatment of satellite cells with high concentrations of HGF (50 ng/ml) inhibits proliferation, which can be overcome by knockdown of SHP2. From these results, we conclude that HGF signals through SHP2 in myoblasts and satellite cells to directly alter proliferation rates.

  8. Murine Stem Cell-Based Retrovirus Production for Marking Primary Mouse Mammary Cells for Metastasis Studies.

    PubMed

    Beverly, Levi J; Podsypanina, Katrina

    2016-01-01

    Since the introduction of retroviral vector technology, permanent genetic marking of cells has considerably contributed to the understanding of different physiological and disease processes in vivo. Recent marking strategies aim to elucidate the contribution of cells on the clonal level, and the advent of fluorescent proteins has opened new avenues for the in vivo analysis of gene-marked cells. Gene-modified cells are easily identifiable (e.g., via the introduced fluorescent protein) within whole organ structures, allowing one to measure the contribution of transduced cells to malignant outgrowth. In our laboratory, we use the tetracycline-inducible system to study oncogene cooperation in metastatic progression. We use bicistronic retroviruses expressing the tetracycline transactivator (tTA) and the candidate gene (MIT-gene) or the tTA alone (MIT-Rx) to infect primary mammary cells from mice harboring tetracycline-inducible transgenes. This allows for constitutive expression of the candidate gene and tTA-dependent expression of the inducible oncogene. We also use MIG-based vectors, which allow for constitutive expression of the candidate gene and a green fluorescent protein. Here we describe how to produce retroviral particles carrying both MIT- and MIG-based vectors. Because of the fragility of the retroviral envelope, we do not attempt to concentrate the virus, and we directly use packaging cell media to infect primary epithelial cells (either normal or tumor). Infected cells can be transplanted into recipient mice to investigate metastatic colonization. PMID:26832680

  9. Comparison of the Treatment Efficiency of Bone Marrow-Derived Mesenchymal Stem Cell Transplantation via Tail and Portal Veins in CCl4-Induced Mouse Liver Fibrosis

    PubMed Central

    Truong, Nhung Hai; Nguyen, Nam Hai; Le, Trinh Van; Vu, Ngoc Bich; Huynh, Nghia; Nguyen, Thanh Van; Le, Huy Minh; Phan, Ngoc Kim

    2016-01-01

    Because of self-renewal, strong proliferation in vitro, abundant sources for isolation, and a high differentiation capacity, mesenchymal stem cells are suggested to be potentially therapeutic for liver fibrosis/cirrhosis. In this study, we evaluated the treatment effects of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) on mouse liver cirrhosis induced by carbon tetrachloride. Portal and tail vein transplantations were examined to evaluate the effects of different injection routes on the liver cirrhosis model at 21 days after transplantation. BM-MSCs transplantation reduced aspartate aminotransferase/alanine aminotransferase levels at 21 days after injection. Furthermore, BM-MSCs induced positive changes in serum bilirubin and albumin and downregulated expression of integrins (600- to 7000-fold), transforming growth factor, and procollagen-α1 compared with the control group. Interestingly, both injection routes ameliorated inflammation and liver cirrhosis scores. All mice in treatment groups had reduced inflammation scores and no cirrhosis. In conclusion, transplantation of BM-MSCs via tail or portal veins ameliorates liver cirrhosis in mice. Notably, there were no differences in treatment effects between tail and portal vein administrations. In consideration of safety, we suggest transfusion of bone marrow-derived mesenchymal stem cells via a peripheral vein as a potential method for liver fibrosis treatment. PMID:26839564

  10. Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse

    PubMed Central

    Coelho, Paula A.; Bury, Leah; Shahbazi, Marta N.; Liakath-Ali, Kifayathullah; Tate, Peri H.; Wormald, Sam; Hindley, Christopher J.; Huch, Meritxell; Archer, Joy; Skarnes, William C.; Zernicka-Goetz, Magdalena; Glover, David M.

    2015-01-01

    To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development. PMID:26701933

  11. Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse.

    PubMed

    Coelho, Paula A; Bury, Leah; Shahbazi, Marta N; Liakath-Ali, Kifayathullah; Tate, Peri H; Wormald, Sam; Hindley, Christopher J; Huch, Meritxell; Archer, Joy; Skarnes, William C; Zernicka-Goetz, Magdalena; Glover, David M

    2015-12-01

    To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development. PMID:26701933

  12. Epidermal growth factor does not act as a primary cue for inducing developmental changes in suckling mouse jejunum.

    PubMed

    Ménard, D; Arsenault, P; Gallo-Payet, N

    1986-01-01

    The direct influence of epidermal growth factor (EGF) on the differentiation and proliferation of small intestine was studied in organ culture. Eight-day-old mouse small intestine was cultured during 2 days in serum-free Leibovitz L-15 medium alone or supplemented with EGF (50, 100, and 500 ng/ml) either at room temperature or at 37 degrees C. Brush border membrane hydrolytic activities, namely, sucrase, lactase, glucoamylase, trehalase, maltase, and alkaline phosphatase, were assayed in the intestinal tissue as well as in the culture medium. None of the brush border enzymic activities was affected by the addition of EGF to the culture medium. This lack of effect is not temperature dependent since it occurred both at room temperature and at 37 degrees C. The addition of hydrocortisone (10(-6) M) to the culture medium induced the appearance of sucrase activity and increased the activity of the other brush border enzymes. The simultaneous addition of EGF with hydrocortisone did not influence the response of the intestinal explants to hydrocortisone. The deoxyribonucleic acid (DNA) content was determined while DNA synthesis was evaluated by the incorporation of (3H)-thymidine. The addition of EGF did not affect DNA content or (3H)-thymidine incorporation into DNA either at room temperature or at 37 degrees C. The EGF binding to epithelial cells did not significantly vary throughout the culture period and a down-regulation process occurred in presence of EGF. These observations strongly suggest that EGF does not act as a primary cue for inducing developmental changes in suckling mouse small intestine. It is proposed that EGF induces a systemic reaction in vivo that then influences the neonatal small intestine. PMID:3491891

  13. Effects of Fatty Acids on CYP2A5 and Nrf2 Expression in Mouse Primary Hepatocytes.

    PubMed

    Cui, Yizhe; Wang, Qiuju; Yi, Xing; Zhang, Xiuying

    2016-02-01

    Abnormal fatty acid metabolism is observed throughout nonalcoholic fatty liver disease (NAFLD) pathogenesis, and fatty acid storage is an important inducing factor in insulin resistance, lipid oxidation, hepatic cell damage, and inflammation. During NAFLD pathogenesis, changes in blood and liver contents of different fatty acid types also vary. Cytochrome P450 2A5 (CYP2A5), an important enzyme in mouse liver, metabolizes many drugs and activates multiple pro-carcinogens with widely varying structures. According to the changes in liver fatty acid profiles observed in NAFLD animal models developed in our laboratory and others, saturated (PA/palmitic, and SA/stearic acids) and unsaturated (OA/oleic, LA/linoleic, ALA/α-linolenic and AA/arachidonic acids) fatty acids were selected to induce mouse primary hepatocytes, at concentrations under 1 mM, as detected by MTT assay. After 24 h treatment with various fatty acid concentrations and types, CYP2A5 mRNA and protein amounts, and enzyme activity were determined by real-time PCR, Western blot, and Coumarin 7-hydroxylation, respectively. Meanwhile, Nrf2 mRNA and protein levels were evaluated by real-time PCR and Western blot. The results indicated that saturated fatty acids are more potent in inducing CYP2A5 than unsaturated ones, except arachidonic acid. In addition, the changes in CYP2A5 expression were consistent with the alterations observed in Nrf2 expression, indicating that Nrf2 might play a regulatory role in CYP2A5 expression. PMID:26423681

  14. Impaired bone remodeling and its correction by combination therapy in a mouse model of mucopolysaccharidosis-I.

    PubMed

    Kuehn, Sonja C; Koehne, Till; Cornils, Kerstin; Markmann, Sandra; Riedel, Christoph; Pestka, Jan M; Schweizer, Michaela; Baldauf, Christina; Yorgan, Timur A; Krause, Matthias; Keller, Johannes; Neven, Mona; Breyer, Sandra; Stuecker, Ralf; Muschol, Nicole; Busse, Bjoern; Braulke, Thomas; Fehse, Boris; Amling, Michael; Schinke, Thorsten

    2015-12-15

    Mucopolysaccharidosis-I (MPS-I) is a lysosomal storage disease (LSD) caused by inactivating mutations of IDUA, encoding the glycosaminoglycan-degrading enzyme α-l-iduronidase. Although MPS-I is associated with skeletal abnormalities, the impact of IDUA deficiency on bone remodeling is poorly defined. Here we report that Idua-deficient mice progressively develop a high bone mass phenotype with pathological lysosomal storage in cells of the osteoblast lineage. Histomorphometric quantification identified shortening of bone-forming units and reduced osteoclast numbers per bone surface. This phenotype was not transferable into wild-type mice by bone marrow transplantation (BMT). In contrast, the high bone mass phenotype of Idua-deficient mice was prevented by BMT from wild-type donors. At the cellular level, BMT did not only normalize defects of Idua-deficient osteoblasts and osteocytes but additionally caused increased osteoclastogenesis. Based on clinical observations in an individual with MPS-I, previously subjected to BMT and enzyme replacement therapy (ERT), we treated Idua-deficient mice accordingly and found that combining both treatments normalized all histomorphometric parameters of bone remodeling. Our results demonstrate that BMT and ERT profoundly affect skeletal remodeling of Idua-deficient mice, thereby suggesting that individuals with MPS-I should be monitored for their bone remodeling status, before and after treatment, to avoid long-term skeletal complications. PMID:26427607

  15. A novel mouse model for the study of the inhibitory effects of chronic ethanol exposure on direct bone formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Excessive alcohol consumption has been reported to interfere with human bone homeostasis and repair in multiple ways. Previous studies have demonstrated that chronic ethanol exposure in the rat via an intragastric dietary delivery system inhibits direct bone formation during distraction osteogenesis...

  16. Synchronous double primary cancer - intrahepatic cholangiocarcinoma with bone metastases and thyroid carcinoma: A case report

    PubMed Central

    WANG, QING-LIANG; LI, XIAO-JIE; ZHAO, KUN; LIU, BO; YE, XIAO-MING

    2015-01-01

    There is a low incidence of multiple primary cancer, particularly when the cancer is synchronous. The present report presents a case of synchronous double primary malignancies. A 58-year-old woman was admitted to Ling Nan Hospital (Guangzhou, China) complaining of pain in the left hip. X-ray revealed an osteolytic lesion and further examination indicated the presence of double primary cancer, consisting of hepatic cholangiocarcinoma and thyroid carcinoma. Biopsy of the osteolytic lesion showed a metastatic adenocarcinoma of unknown origin. Subsequently, final diagnosis was confirmed by I-131 scan and liver lesion biopsy. The patient received positive multidisciplinary treatments and survived for 9 months following diagnosis. The results of the present case suggest that multiplicity of primary malignancy is not necessarily an indicator of poor prognosis, as long as effective diagnosis and adequate disease management are achieved. PMID:26788211

  17. RNA interference-mediated silencing of Atp6i prevents both periapical bone erosion and inflammation in the mouse model of endodontic disease.

    PubMed

    Ma, Junqing; Chen, Wei; Zhang, Lijie; Tucker, Byron; Zhu, Guochun; Sasaki, Hajime; Hao, Liang; Wang, Lin; Ci, Hongliang; Jiang, Hongbing; Stashenko, Philip; Li, Yi-Ping

    2013-04-01

    Dental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7 gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6i inhibition impaired osteoclast function in vitro and in vivo and decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7 knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i(+/-) mice with haploinsufficiency of Atp6i exhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7 knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease. PMID:23166162

  18. P15, MDM2, NF-κB, and Bcl-2 expression in primary bone tumor and correlation with tumor formation and metastasis

    PubMed Central

    Qian, Guibin; Hao, Songnan; Yang, Dawei; Meng, Qinggang

    2015-01-01

    Primary bone tumor is one of the most common malignant tumors in skeletal system. It seriously affected bone movement and development with unclear pathogenesis. In this paper, rabbit VX-2 malignant bone tumor model was applied to explore apoptotic genes P15, MDM2, NF-κB and Bcl-2 correlation with primary bone tumor occurrence and metastasis. 0.3 ml rabbit VX-2 tumor cell suspension (1×106/ml) was injected to the marrow cavity of the right tibia condyle to establish the rabbit malignant bone tumor model, while equal amount of the saline was injected to the left tibia as control. Real-time PCR was applied to determine P15, MDM2, NF-κB and Bcl-2 expression level. Immunohistochemistry was performed to detect the abovementioned genes expression in lung, stomach, kidney and bladder. Compared with control, P15 expression level in the inoculation site surrounding tissues decreased obviously following the inoculate time elongation (P<0.05), while Bcl-2, MDM2 and NF-κB expression significantly increased (P<0.05). Bcl-2 showed significant correlation with MDM2 and NF-κB (P<0.05). At the 2, 4, 6 weeks, Bcl-2, MDM2 and NF-κB in lung, Bcl-2 in kidney, and Bcl-2 and MDM2 in bladder positively expressed (P<0.05), whereas P15 gene exhibited no significant positive expression in these tissues (P>0.05). P15, MDM2, NF-κB, and Bcl-2 genes expression levels can effectively reflect malignant bone tumor growth of rabbit tibia. MDM2, NF-κB and Bcl-2 genes involved in primary bone tumors metastasis directly. It has important clinical significance for early diagnosis and treatment of primary bone tumor. PMID:26823818

  19. siDNMT1 Increases γ-globin Expression in Chemical-Inducer-of-Dimerization (CID)-Dependent Mouse βYAC Bone Marrow Cells and in Baboon Erythroid Progenitor Cell Cultures

    PubMed Central

    Banzon, Virryan; Ibanez, Vinzon; Vaitkus, Kestis; Ruiz, Maria Armila; Peterson, Kenneth; DeSimone, Joseph; Lavelle, Donald

    2014-01-01

    1) Objective These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult stage erythroid cells. 2) Methods DNMT1 levels were reduced by nucleofection of siRNA targeting DNMT1 in chemical-inducer-of-dimerization (CID)-dependent multipotential mouse bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and in primary cultures of erythroid progenitor cells derived from CD34+ baboon BM cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real time PCR and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabelling of globin chains followed by HPLC analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. 3) Results Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin mRNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. 4) Conclusion DNMT1 is required to maintain DNA methylation of the γ-globin gene promoters and repress γ-globin gene expression in adult-stage erythroid cells. PMID:20974210

  20. Effects of Carbon Ions on Primary Cultures of Mouse Brain Cells

    NASA Astrophysics Data System (ADS)

    Nojima, K.; Ando, K.; Fujiwara, H.; Ando, S.

    Primary mixed cultures of astrocytes and microglia were obtained from neonatal mice, and were irradiated with high-LET carbon ions. Immunohistochemical staining showed astrocytes survived more prominently than microglia. Tagged with specific antibodies, astrocytes and microglia surviving after irradiation were counted by flow cytometry. Decreases in the number of microglia and astrocytes were detected at a dose as small as 2 Gy when Day 5 cultures were irradiated with 13 keV/μm carbon ions. When the cultures were irradiated on Day 10, the dose-dependent decrease of microglia was more prominent for 13 keV/μun carbon ions than 70 keV/μm carbon ions. Astrocytes showed a marginal decrease at Day 10 and Day 14. We concluded that microglia are more sensitive than astrocytes to carbon ions and X-rays, and that the radiosensitivity of microglia depends on both differentiation/proliferation status and radiation quality

  1. Functional improvement of damaged adult mouse muscle by implantation of primary myoblasts.

    PubMed Central

    Irintchev, A; Langer, M; Zweyer, M; Theisen, R; Wernig, A

    1997-01-01

    1. Myoblasts from expanded primary cultures were implanted into cryodamaged soleus muscles of adult BALB/c mice. One to four months later isometric tension recordings were performed in vitro, and the male donor cells implanted into female hosts were traced on histological sections using a Y-chromosome-specific probe. The muscles were either mildly or severely cryodamaged, which led to reductions in tetanic muscle force to 33% (n = 9 muscles, 9 animals) and 70% (n = 11) of normal, respectively. Reduced forces resulted from deficits in regeneration of muscle tissue as judged from the reduced desmin-positive cross-sectional areas (34 and 66% of control, respectively). 2. Implantation of 10(6) myogenic cells into severely cryodamaged muscles more than doubled muscle tetanic force (to 70% of normal, n = 14), as well as specific force (to 66% of normal). Absolute and relative amount of desmin-positive muscle cross-sectional areas were significantly increased indicating improved microarchitecture and less fibrosis. Newly formed muscle tissue was fully innervated since the tetanic forces resulting from direct and indirect (nerve-evoked) stimulation were equal. Endplates were found on numerous Y-positive muscle fibres. 3. As judged from their position under basal laminae of muscle fibres and the expression of M-cadherin, donor-derived cells contributed to the pool of satellite cells on small- and large-diameter muscle fibres. 4. Myoblast implantation after mild cryodamage and in undamaged muscles had little or no functional or structural effects; in both preparations only a few Y-positive muscle nuclei were detected. It is concluded that myoblasts from expanded primary cultures-unlike permanent cell lines-significantly contribute to muscle regeneration only when previous muscle damage is extensive and loss of host satellite cells is severe. Images Figure 1 Figure 2 Figure 3 PMID:9161990

  2. Transcapillary fluid balance consequences of missing initial lymphatics studied in a mouse model of primary lymphoedema

    PubMed Central

    Karlsen, Tine V; Karkkainen, Marika J; Alitalo, Kari; Wiig, Helge

    2006-01-01

    To investigate the phenotypic consequences of a deranged lymphangiogenesis in relation to tissue fluid accumulation and the possible role of inflammation in the pathogenesis of lymphoedema, we measured determinants of transcapillary fluid filtration and inflammatory mediators in the interstitial fluid in genetically engineered Chy mice, a model for primary congenital lymphoedema (Milroy's disease). Although initial lymphatics were not present in dermis in any of the areas studied (fore paw, hind paw, thigh and back skin) interstitial fluid pressure (Pif), measured with micropipettes, and tissue fluid volumes were significantly increased only in the areas with visible swelling – the fore and hind paw, whereas interstitial colloid osmotic pressure (COPif) was increased in all the skin areas examined. A volume load of 15% of body weight resulted in a more pronounced increase in Pif as well as a four-fold increase in interstitial fluid volume in Chy relative to wild-type (wt) mice, showing the quantitative importance of lymphatics for fluid homeostasis during acute perturbations. A similar level of proinflammatory markers in interstitial fluid in early established lymphoedema (3–4 months) in Chy and wt suggests that inflammation does not have a major pathogenetic role for the development of lymphoedema, whereas a reduced level of the immunomodulatory cytokine interleukin (IL)-4 may result in a reduced immunological defence ability and thus lead to the increase in inflammatory cytokines IL-2 and IL-6 observed at a later stage (11–13 months). Our data suggest that primary lymphoedema results in a high interstitial fluid protein concentration that does not induce an interstitial inflammatory reaction per se, and furthermore shows the paramount importance of the initial lymphatics in tissue fluid homeostasis, especially during perturbations of transcapillary fluid balance. PMID:16675495

  3. Pathologic fracture after radiation therapy for primary non-Hodgkin's malignant lymphoma of bone

    SciTech Connect

    Stokes, S.H.; Walz, B.J.

    1983-08-01

    Between 1963 and 1981, 32 patients with biopsy proven non-Hodgkin's lymphoma involving bone were treated at the Mallinckrodt Institute of Radiology either with radiation alone or in conjunction with chemotherapy. An unexpectedly high rate of fracture at the site of the tumor was observed. Six patients were excluded because they survived less than six months after the completion of radiotherapy or were lost to follow-up within six months. There were 15 appendicular and 17 axial sites treated. Local control was achieved in 30 of 32. There were 10 patients with appendicular lesions of which seven suffered a fracture. Of the seven patients with lesions in a weight bearing bone, six suffered fractures. Twenty-six sites of involvement received less than 5000 rad. Of the six patients receiving high dose, two presented with pathologic fractures of the femur requiring surgical stabilization and the remaining four patients suffered subsequent fractures 7 to 30 months after completion of therapy. Two of these six had local recurrence of disease. It appears that involvement of the appendicular skeleton by lymphoma frequently results in fracture. Doses of 5000 rad or greater do not increase the probability of local control but may contribute to the risk of fracture following radiotherapy.

  4. Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model

    PubMed Central

    Hu, M; Yang, J-L; Teng, H; Jia, Y-Q; Wang, R; Zhang, X-W; Wu, Y; Luo, Y; Chen, X-C; Zhang, R; Tian, L; Zhao, X; Wei, Y-Q

    2008-01-01

    Background Bone marrow-derived stromal cells (BMSCs) are important for development, tissue cell replenishment, and wound healing in physiological and pathological conditions. BMSCs were found to preferably reach sites undergoing the process of cell proliferation, such as wound and tumor, suggesting that BMSCs may be used as a vehicle for gene therapy of tumor. Methods Mouse BMSCs were loaded with recombinant adenoviruses which express soluble Vascular Endothelial Growth Factor Receptor-1 (sFlt-1). The anti-angiogenesis of sFlt-1 in BMSCs was determined using endothelial cells proliferation inhibition assay and alginate encapsulation assay. The anti-tumor effects of BMSCs expressing sFlt-1 through tail-vein infusion were evaluated in two mouse tumor metastases models. Results BMSCs genetically modified with Adv-GFP-sFlt-1 could effectively express and secret sFlt-1. BMSCs loaded with sFlt-1 gene could preferentially home to tumor loci and decrease lung metastases and prolong lifespan in mouse tumor model through inducing anti-angiogenesis and apoptosis in tumors. Conclusion We demonstrated that BMSCs might be employed as a promising vehicle for tumor gene therapy which can effectively not only improve the concentration of anticancer therapeutics in tumors, but also modify the tumor microenvironment. PMID:18947384

  5. Space Radiation and Bone Loss.

    PubMed

    Willey, Jeffrey S; Lloyd, Shane A J; Nelson, Gregory A; Bateman, Ted A

    2011-01-01

    Exposure to ionizing radiation may negatively impact skeletal integrity during extended spaceflight missions to the moon, Mars, or near-Earth asteroids. However, our understanding of the effects of radiation on bone is limited when compared to the effects of weightlessness. In addition to microgravity, astronauts will be exposed to space radiation from solar and cosmic sources. Historically, radiation exposure has been shown to damage both osteoblast precursors and local vasculature within the irradiated volume. The resulting suppression of bone formation and a general state of low bone-turnover is thought to be the primary contributor to bone loss and eventual fracture. Recent investigations using mouse models have identified a rapid, but transient, increase in osteoclast activity immediately after irradiation with both spaceflight and clinically-relevant radiation qualities and doses. Together with a chronic suppression of bone formation after radiation exposure, this acute skeletal damage may contribute to long-term deterioration of bone quality, potentially increasing fracture risk. Direct evidence for the damaging effects of radiation on human bone are primarily demonstrated by the increased incidence of fractures at sites that absorb high doses of radiation during cancer therapy: exposures are considerably higher than what could be expected during spaceflight. However, both the rapidity of bone damage and the chronic nature of the changes appear similar between exposure scenarios. This review will outline our current knowledge of space and clinical exploration exposure to ionizing radiation on skeletal health. PMID:22826632

  6. Mouse primary visual cortex is used to detect both orientation and contrast changes.

    PubMed

    Glickfeld, Lindsey L; Histed, Mark H; Maunsell, John H R

    2013-12-11

    In mammals, the lateral geniculate nucleus (LGN) and the superior colliculus (SC) are the major targets of visual inputs from the retina. The LGN projects mainly to primary visual cortex (V1) while the SC targets the thalamus and brainstem, providing two potential pathways for processing visual inputs. Indeed, cortical lesion experiments in rodents have yielded mixed results, leading to the hypothesis that performance of simple visual behaviors may involve computations performed entirely by this subcortical pathway through the SC. However, these previous experiments have been limited by both their assays of behavioral performance and their use of lesions to change cortical activity. To determine the contribution of V1 to these tasks, we trained mice to perform threshold detection tasks in which they reported changes in either the contrast or orientation of visual stimuli. We then reversibly inhibited V1 by optogenetically activating parvalbumin-expressing inhibitory neurons with channelrhodopsin-2. We found that suppressing activity in V1 substantially impaired performance in visual detection tasks. The behavioral deficit depended on the retinotopic position of the visual stimulus, confirming that the effect was due to the specific suppression of the visually driven V1 neurons. Behavioral effects were seen with only moderate changes in neuronal activity, as inactivation that raised neuronal contrast thresholds by a median of only 14% was associated with a doubling of behavioral contrast detection threshold. Thus, detection of changes in either orientation or contrast is dependent on, and highly sensitive to, the activity of neurons in V1. PMID:24336708

  7. Astaxanthin prevents and reverses the activation of mouse primary hepatic stellate cells.

    PubMed

    Yang, Yue; Bae, Minkyung; Kim, Bohkyung; Park, Young-Ki; Koo, Sung I; Lee, Ji-Young

    2016-03-01

    Activation of hepatic stellate cells (HSCs) is a critical step that leads to the development of liver fibrosis. We showed that astaxanthin (ASTX), a xanthophyll carotenoid, displays antifibrogenic effects in LX-2 cells, a human HSC cell line. In this study, we further determined the effect of ASTX on HSC activation and inactivation using primary HSCs from C57BL/6J mice. Quiescent and activated HSCs were incubated with ASTX (25μM) at different stages of activation. ASTX prevented the activation of quiescent HSCs, as evidenced by the presence of intracellular lipid droplets and reduction of α-smooth muscle actin, an HSC activation marker. Also, ASTX reverted activated HSCs to a quiescent phenotype with the reappearance of lipid droplets with a concomitant increase in lecithin retinol acyltransferase mRNA. Cellular accumulation of reactive oxygen species was significantly reduced by ASTX, which was attributable to a decrease in NADPH oxidase 2 expression. The antifibrogenic effect of ASTX was independent of nuclear erythroid 2-related factor 2 as it was observed in HSCs from wild-type and Nrf2(-/-) mice. In conclusion, ASTX inhibits HSC activation and reverts activated HSCs to a quiescent state. Further investigation is warranted to determine if ASTX effectively prevents the development of liver fibrosis. PMID:26895661

  8. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

    PubMed Central

    López-Islas, Anayelly; Chagoya-Hazas, Victoria; Pérez-Aguilar, Benjamin; Palestino-Domínguez, Mayrel; Souza, Verónica; Miranda, Roxana U.; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Gutiérrez-Ruiz, María-Concepción

    2016-01-01

    Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol. PMID:26788255

  9. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes.

    PubMed

    Lpez-Islas, Anayelly; Chagoya-Hazas, Victoria; Prez-Aguilar, Benjamin; Palestino-Domnguez, Mayrel; Souza, Vernica; Miranda, Roxana U; Bucio, Leticia; Gmez-Quiroz, Luis Enrique; Gutirrez-Ruiz, Mara-Concepcin

    2016-01-01

    Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol. PMID:26788255

  10. Triptolide inhibits osteoclast formation, bone resorption, RANKL-mediated NF-қB activation and titanium particle-induced osteolysis in a mouse model.

    PubMed

    Huang, Jianbin; Zhou, Lin; Wu, Huafei; Pavlos, Nathan; Chim, Shek Man; Liu, Qian; Zhao, Jinmin; Xue, Wei; Tan, Ren Xiang; Ye, Jiming; Xu, Jun; Ang, Estabelle S; Feng, Haotian; Tickner, Jennifer; Xu, Jiake; Ding, Yue

    2015-01-01

    The RANKL-induced NF-κB signaling pathway is required for osteoclast formation and function. By screening for compounds that inhibit RANKL-induced NF-κB activation using a luciferase reporter gene assay in RAW264.7 cells, we identified triptolide (PG490), as a candidate compound targeting osteoclast differentiation and osteoclast-mediated osteolysis. Triptolide (PG490) is an active compound of the medicinal herb Tripterygium wilfordii Hook F (TWHF) or Lei Gong Teng with known anti-inflammatory properties. We found that triptolide inhibited osteoclastogenesis and bone resorption, as well as RANKL-induced NF-қB activities as monitored by luciferase reporter gene assays and the nuclear translocation of p65. In vivo studies showed that triptolide attenuates titanium-induced osteolysis and osteoclast formation in a mouse calvarial model. Considering that drugs which protect against localized bone loss are critically needed for the effective treatment of particle-induced osteolysis, our data suggest that triptolide might have therapeutic potential for the treatment of bone lytic diseases caused by prosthetic wear particles. PMID:25448849

  11. Global miRNA expression and correlation with mRNA levels in primary human bone cells

    PubMed Central

    Laxman, Navya; Rubin, Carl-Johan; Mallmin, Hans; Nilsson, Olle; Pastinen, Tomi; Grundberg, Elin; Kindmark, Andreas

    2015-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA–mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA–mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, α1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression. PMID:26078267

  12. Bone-grafting for acetabular deficiency during primary and revision total hip arthroplasty. A radiographic and clinical analysis.

    PubMed

    Knight, J L; Fujii, K; Atwater, R; Grothaus, L

    1993-08-01

    The use of bone-graft to augment the deficient acetabulum in primary and revision total hip arthroplasty (THA) is controversial. To identify factors affecting cup loosening in patients who received a bone-graft during THA, two orthopaedic surgeons retrospectively examined sequential radiographs. The surgeons also obtained independent computer measurements of hip center and cup abduction migration from preoperative, initial, and latest postoperative radiographs. Variables studied included host factors, graft factors, and technique factors. All conclusions were based on Kaplan-Meier log-rank analysis to account for differing lengths of follow-up periods among the cases. The authors report a series of 74 consecutive cases with a minimum 24-month follow-up period (mean, 40 months). All grafts appeared to unite. The clinicians found 80% stable cups, 8% possibly loose cups, and 12% (n = 9) definitely loose cups. In retrospect, technical errors were seen in six loose cups. Five revisions for loosening (6.7% of cases) were performed. Computer measurement found cup loosening in a higher percentage of cases than detected by the clinicians and did so an average of 18 months sooner. Acetabular cup loosening was associated with the American Academy of Orthopaedic Surgeons type III defects, use of allograft versus autograft, and initial cup abduction of 50 degrees or more. Kaplan-Meier survivorship analysis found 31% of cups radiographically loose and 15% revised at 5 years or more since surgery. Acetabular bone-grafting is technically demanding and should be employed when alternative reconstructions will not give a durable result. PMID:8409988

  13. Pediatric primary bone lymphoma-diffuse large B-cell lymphoma: morphologic and immunohistochemical characteristics of 10 cases.

    PubMed

    Zhao, X Frank; Young, Ken H; Frank, Dale; Goradia, Ami; Glotzbecker, Michael P; Pan, Wilbur; Kersun, Leslie S; Leahey, Ann; Dormans, John P; Choi, John K

    2007-01-01

    Most primary bone lymphomas (PBLs) are diffuse large B-cell lymphomas (DLBCLs). Pediatric PBL-DLBCL has a favorable prognosis but remains poorly characterized. Herein, 10 such cases are detailed. They involved 11- to 20-year-old males with bone lesions that were often painful. They were diagnosed often after months to years of symptoms, suggesting an indolent disease. All were successfully treated with chemotherapy with or without radiotherapy (0.5- to 24-year followup). Biopsy revealed that the lymphomas were paratrabecular or diffuse and were medium- to large-sized with round to irregular nuclei, dispersed chromatin, indistinct to small nucleoli, and abundant cytoplasm. Other features included varying levels of necrosis, cytoplasmic retraction, and myeloid hyperplasia. All cases marked as mature B cells, and most were CD10+ (7/10). Typical centroblastic morphologic features with nucleoli were rare, multilobated nuclei were uncommon, and CD10 negativity did not predict poor prognosis, unlike in the adult PBL-DLBCL. These findings suggest that pediatric and adult PBL-DLBCLs are distinct entities. PMID:17145622

  14. Noise-induced cell death in the mouse medial geniculate body and primary auditory cortex.

    PubMed

    Basta, Dietmar; Tzschentke, Barbara; Ernst, Arne

    Noise-induced effects within the inner ear have been well investigated for several years. However, this peripheral damage cannot fully explain the audiological symptoms in noise-induced hearing loss (NIHL), e.g. tinnitus, recruitment, reduced speech intelligibility, hyperacusis. There are few reports on central noise effects. Noise can induce an apoptosis of neuronal tissue within the lower auditory pathway. Higher auditory structures (e.g. medial geniculate body, auditory cortex) are characterized by metabolic changes after noise exposure. However, little is known about the microstructural changes of the higher auditory pathway after noise exposure. The present paper was therefore aimed at investigating the cell density in the medial geniculate body (MGB) and the primary auditory cortex (AI) after noise exposure. Normal hearing mice were exposed to noise (10 kHz center frequency at 115 dB SPL for 3 h) at the age of 21 days under anesthesia (Ketamin/Rompun, 10:1). After 1 week, auditory brainstem response recordings (ABR) were performed in noise exposed and normal hearing animals. After fixation, the brain was microdissected and stained (Kluever-Barrera). The cell density in the MGB subdivisions and the AI were determined by counting the cells within a grid. Noise-exposed animals showed a significant ABR threshold shift over the whole frequency range. Cell density was significantly reduced in all subdivisions of the MGB and in layers IV-VI of AI. The present findings demonstrate a significant noise-induced change of the neuronal cytoarchitecture in central key areas of auditory processing. These changes could contribute to the complex psychoacoustic symptoms after NIHL. PMID:15882817

  15. Primary definitive radiotherapy with or without chemotherapy for squamous cell carcinoma of the temporal bone.

    PubMed

    Kitani, Yosuke; Kubota, Akira; Furukawa, Madoka; Sato, Kaname; Nakayama, Yuko; Nonaka, Tetsuo; Mizoguchi, Nobutaka

    2016-05-01

    We aimed to evaluate the impact of concurrent chemoradiotherapy (CCRT) on the survival of patients with squamous cell carcinoma of the temporal bone. We retrospectively analyzed the data of 13 consecutive patients who were treated by definitive radiation therapy (RT) or CCRT as the initial treatment between 1999 and 2012. There were 5 patients with stage II disease, 5 with stage III, and 3 with stage IV, as classified according to the University of Pittsburgh system. Among these, 2, 4, and 3 patients, respectively, were treated by CCRT; whereas the remaining (3 patients with stage II and 1 with stage III) were treated by RT alone. Median follow-up duration was 39 months (12-106 months) in all cases, and 61.5 months (17-70 months) in censored cases. The 5-year overall survival (OS) rates were 51 % in all patients, and 40, 100, and 0 % in patients with stage II, stage III, and stage IV disease, respectively. In patients with stage II and III disease, the 5-year OS rates were 80 % in the CCRT group and 50 % in the RT-alone group. We found better prognosis in patients with stage II and III disease who were treated by CCRT. Only 2 patients treated by CCRT experienced adverse events more than grade 3, which were neutropenia and dermatitis. There was no late adverse event of bony necrosis. Our study results indicate that CCRT is safe and very effective as a first-line treatment for stage II and III squamous cell carcinoma of the temporal bone. PMID:25822291

  16. Primary neuroendocrine carcinoma of breast with liver and bone metastasis detected with fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography

    PubMed Central

    Kamaleshwaran, Koramadai Karuppusamy; Mohanan, Vyshak; Shibu, Deepu; Radhakrishnan, Edathuruthy Kalarikal; Shinto, Ajit Sugunan

    2014-01-01

    Cases of primary neuroendocrine carcinoma (NEC) of the breast have been reported, though rare. We report the case of a 45-year-old woman presented with jaundice and evaluated to have liver metastasis from neuroendocrine origin. She underwent whole body positron emission tomography/computed tomography, which showed left breast lesion and bone metastasis. Fine-needle aspiration (FNA) of breast revealed a NEC. A diagnosis of a primary NEC of the breast was rendered with hepatic and bone metastasis. She was treated with peptide receptor radionuclide therapy and is on follow-up. PMID:24591780

  17. Primary neuroendocrine carcinoma of breast with liver and bone metastasis detected with fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography.

    PubMed

    Kamaleshwaran, Koramadai Karuppusamy; Mohanan, Vyshak; Shibu, Deepu; Radhakrishnan, Edathuruthy Kalarikal; Shinto, Ajit Sugunan

    2014-01-01

    Cases of primary neuroendocrine carcinoma (NEC) of the breast have been reported, though rare. We report the case of a 45-year-old woman presented with jaundice and evaluated to have liver metastasis from neuroendocrine origin. She underwent whole body positron emission tomography/computed tomography, which showed left breast lesion and bone metastasis. Fine-needle aspiration (FNA) of breast revealed a NEC. A diagnosis of a primary NEC of the breast was rendered with hepatic and bone metastasis. She was treated with peptide receptor radionuclide therapy and is on follow-up. PMID:24591780

  18. Strain-Dependent Brain Defects in Mouse Models of Primary Ciliary Dyskinesia with Mutations in Pcdp1 and Spef2

    PubMed Central

    Finn, Rozzy; Evans, Claire C.; Lee, Lance

    2014-01-01

    Hydrocephalus is caused by the accumulation of cerebrospinal fluid in the cerebral ventricular system which results in an enlargement of the cranium due to increased intraventricular pressure. The increase in pressure within the brain typically results in sloughing of ciliated ependymal cells, loss of cortical grey matter, and increased gliosis. Congenital hydrocephalus is associated with several syndromes including primary ciliary dyskinesia (PCD), a rare, genetically heterogeneous, pediatric syndrome that results from defects in motile cilia and flagella. We have examined the morphological and physiological defects in the brains of two mouse models of PCD, nm1054 and bgh, which have mutations in Pcdp1 (also known as Cfap221) and Spef2, respectively. Histopathological and immunohistochemical analyses of mice with these mutations on the C57BL/6J and 129S6/SvEvTac genetic backgrounds demonstrate strain-dependent morphological brain damage. Alterations in astrocytosis, microglial activation, myelination, and the neuronal population were identified and are generally more severe on the C57BL/6J background. Analysis of ependymal ciliary clearance ex vivo and cerebrospinal fluid flow in vivo demonstrate a physiological defect in nm1054 and bgh mice on both genetic backgrounds, indicating that abnormal cilia-driven flow is not the sole determinant of the severity of hydrocephalus in these models. These results suggest that genetic modifiers play an important role in susceptibility to severe PCD-associated hydrocephalus. PMID:25073043

  19. The structure of pairwise correlation in mouse primary visual cortex reveals functional organization in the absence of an orientation map.

    PubMed

    Denman, Daniel J; Contreras, Diego

    2014-10-01

    Neural responses to sensory stimuli are not independent. Pairwise correlation can reduce coding efficiency, occur independent of stimulus representation, or serve as an additional channel of information, depending on the timescale of correlation and the method of decoding. Any role for correlation depends on its magnitude and structure. In sensory areas with maps, like the orientation map in primary visual cortex (V1), correlation is strongly related to the underlying functional architecture, but it is unclear whether this correlation structure is an essential feature of the system or arises from the arrangement of cells in the map. We assessed the relationship between functional architecture and pairwise correlation by measuring both synchrony and correlated spike count variability in mouse V1, which lacks an orientation map. We observed significant pairwise synchrony, which was organized by distance and relative orientation preference between cells. We also observed nonzero correlated variability in both the anesthetized (0.16) and awake states (0.18). Our results indicate that the structure of pairwise correlation is maintained in the absence of an underlying anatomical organization and may be an organizing principle of the mammalian visual system preserved by nonrandom connectivity within local networks. PMID:23689635

  20. Therapeutic Touch Has Significant Effects on Mouse Breast Cancer Metastasis and Immune Responses but Not Primary Tumor Size

    PubMed Central

    Gronowicz, Gloria; Secor, Eric R.; Flynn, John R.; Jellison, Evan R.; Kuhn, Liisa T.

    2015-01-01

    Evidence-based integrative medicine therapies have been introduced to promote wellness and offset side-effects from cancer treatment. Energy medicine is an integrative medicine technique using the human biofield to promote well-being. The biofield therapy chosen for study was Therapeutic Touch (TT). Breast cancer tumors were initiated in mice by injection of metastatic 66cl4 mammary carcinoma cells. The control group received only vehicle. TT or mock treatments were performed twice a week for 10 minutes. Two experienced TT practitioners alternated treatments. At 26 days, metastasis to popliteal lymph nodes was determined by clonogenic assay. Changes in immune function were measured by analysis of serum cytokines and by fluorescent activated cells sorting (FACS) of immune cells from the spleen and lymph nodes. No significant differences were found in body weight gain or tumor size. Metastasis was significantly reduced in the TT-treated mice compared to mock-treated mice. Cancer significantly elevated eleven cytokines. TT significantly reduced IL-1-a, MIG, IL-1b, and MIP-2 to control/vehicle levels. FACS demonstrated that TT significantly reduced specific splenic lymphocyte subsets and macrophages were significantly elevated with cancer. Human biofield therapy had no significant effect on primary tumor but produced significant effects on metastasis and immune responses in a mouse breast cancer model. PMID:26113869

  1. Therapeutic Touch Has Significant Effects on Mouse Breast Cancer Metastasis and Immune Responses but Not Primary Tumor Size.

    PubMed

    Gronowicz, Gloria; Secor, Eric R; Flynn, John R; Jellison, Evan R; Kuhn, Liisa T

    2015-01-01

    Evidence-based integrative medicine therapies have been introduced to promote wellness and offset side-effects from cancer treatment. Energy medicine is an integrative medicine technique using the human biofield to promote well-being. The biofield therapy chosen for study was Therapeutic Touch (TT). Breast cancer tumors were initiated in mice by injection of metastatic 66cl4 mammary carcinoma cells. The control group received only vehicle. TT or mock treatments were performed twice a week for 10 minutes. Two experienced TT practitioners alternated treatments. At 26 days, metastasis to popliteal lymph nodes was determined by clonogenic assay. Changes in immune function were measured by analysis of serum cytokines and by fluorescent activated cells sorting (FACS) of immune cells from the spleen and lymph nodes. No significant differences were found in body weight gain or tumor size. Metastasis was significantly reduced in the TT-treated mice compared to mock-treated mice. Cancer significantly elevated eleven cytokines. TT significantly reduced IL-1-a, MIG, IL-1b, and MIP-2 to control/vehicle levels. FACS demonstrated that TT significantly reduced specific splenic lymphocyte subsets and macrophages were significantly elevated with cancer. Human biofield therapy had no significant effect on primary tumor but produced significant effects on metastasis and immune responses in a mouse breast cancer model. PMID:26113869

  2. Human A53T α-synuclein causes reversible deficits in mitochondrial function and dynamics in primary mouse cortical neurons.

    PubMed

    Li, Li; Nadanaciva, Sashi; Berger, Zdenek; Shen, Wei; Paumier, Katrina; Schwartz, Joel; Mou, Kewa; Loos, Paula; Milici, Anthony J; Dunlop, John; Hirst, Warren D

    2013-01-01

    Parkinson's disease (PD) is the second most common neurodegenerative disease. A key pathological feature of PD is Lewy bodies, of which the major protein component is α-synuclein (α-syn). Human genetic studies have shown that mutations (A53T, A30P, E46K) and multiplication of the α-syn gene are linked to familial PD. Mice overexpressing the human A53T mutant α-syn gene develop severe movement disorders. However, the molecular mechanisms of α-syn toxicity are not well understood. Recently, mitochondrial dysfunction has been linked with multiple neurodegenerative diseases including Parkinson's disease. Here we investigated whether mitochondrial motility, dynamics and respiratory function are affected in primary neurons from a mouse model expressing the human A53T mutation. We found that mitochondrial motility was selectively inhibited in A53T neurons while transport of other organelles was not affected. In addition, A53T expressing neurons showed impairment in mitochondrial membrane potential and mitochondrial respiratory function. Furthermore, we found that rapamycin, an autophagy inducer, rescued the decreased mitochondrial mobility. Taken together, these data demonstrate that A53T α-syn impairs mitochondrial function and dynamics and the deficit of mitochondrial transport is reversible, providing further understanding of the disease pathogenesis and a potential therapeutic strategy for PD. PMID:24392030

  3. Skp2 promotes adipocyte differentiation via a p27{sup Kip1}-independent mechanism in primary mouse embryonic fibroblasts

    SciTech Connect

    Okada, Mitsuru; Sakai, Tamon; Nakamura, Takehiro; Tamamori-Adachi, Mimi; Kitajima, Shigetaka; Matsuki, Yasushi; Watanabe, Eijiro; Hiramatsu, Ryuji; Sakaue, Hiroshi Kasuga, Masato

    2009-02-06

    Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27{sup Kip1}, a principal target of the SCF{sup Skp2} complex. Genetic ablation of p27{sup Kip1} in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27{sup Kip1} by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2{sup -/-} MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), largely restored lipid accumulation and PPAR{gamma} gene expression in Skp2{sup -/-} MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27{sup Kip1} expression.

  4. Insulin-like growth factor I is required for the anabolic actions of parathyroid hormone on mouse bone

    NASA Technical Reports Server (NTRS)

    Bikle, Daniel D.; Sakata, Takeshi; Leary, Colin; Elalieh, Hashem; Ginzinger, David; Rosen, Clifford J.; Beamer, Wesley; Majumdar, Sharmila; Halloran, Bernard P.

    2002-01-01

    Parathyroid hormone (PTH) is a potent anabolic agent for bone, but the mechanism(s) by which it works remains imperfectly understood. Previous studies have indicated that PTH stimulates insulin-like growth factor (IGF) I production, but it remains uncertain whether IGF-I mediates some or all of the skeletal actions of PTH. To address this question, we examined the skeletal response to PTH in IGF-I-deficient (knockout [k/o]) mice. These mice and their normal littermates (NLMs) were given daily injections of PTH (80 microg/kg) or vehicle for 2 weeks after which their tibias were examined for fat-free weight (FFW), bone mineral content, bone structure, and bone formation rate (BFR), and their femurs were assessed for mRNA levels of osteoblast differentiation markers. In wild-type mice, PTH increased FFW, periosteal BFR, and cortical thickness (C.Th) of the proximal tibia while reducing trabecular bone volume (BV); these responses were not seen in the k/o mice. The k/o mice had normal mRNA levels of the PTH receptor and increased mRNA levels of the IGF-I receptor but markedly reduced basal mRNA levels of the osteoblast markers. Surprisingly, these mRNAs in the k/o bones increased several-fold more in response to PTH than the mRNAs in the bones from their wild-type littermates. These results indicate that IGF-I is required for the anabolic actions of PTH on bone formation, but the defect lies distal to the initial response of the osteoblast to PTH.

  5. Rictor is required for optimal bone accrual in response to anti-sclerostin therapy in the mouse.

    PubMed

    Sun, Weiwei; Shi, Yu; Lee, Wen-Chih; Lee, Seung-Yon; Long, Fanxin

    2016-04-01

    Wnt signaling has emerged as a major target pathway for the development of novel bone anabolic therapies. Neutralizing antibodies against the secreted Wnt antagonist sclerostin (Scl-Ab) increase bone mass in both animal models and humans. Because we have previously shown that Rictor-dependent mTORC2 activity contributes to Wnt signaling, we test here whether Rictor is required for Scl-Ab to promote bone anabolism. Mice with Rictor deleted in the early embryonic limb mesenchyme (Prx1-Cre;Rictor(f/f), hereafter RiCKO) were subjected to Scl-Ab treatment for 5weeks starting at 4months of age. In vivo micro-computed tomography (μCT) analyses before the treatment showed that the RiCKO mice displayed normal trabecular, but less cortical bone mass than the littermate controls. After 5weeks of treatment, Scl-Ab dose-dependently increased trabecular and cortical bone mass in both control and RiCKO mice, but the increase was significantly blunted in the latter. Dynamic histomorphometry revealed that the RiCKO mice formed less bone than the control in response to Scl-Ab. In addition, the RiCKO mice possessed fewer osteoclasts than normal under the basal condition and exhibited lesser suppression in osteoclast number by Scl-Ab. Consistent with the fewer osteoclasts in vivo, bone marrow stromal cells (BMSC) from the RiCKO mice expressed less Rankl but normal levels of Opg or M-CSF, and were less effective than the control cells in supporting osteoclastogenesis in vitro. The reliance of Rankl on Rictor appeared to be independent of Wnt-β-catenin or Wnt-mTORC2 signaling as Wnt3a had no effect on Rankl expression by BMSC from either control or RICKO mice. Overall, Rictor in the limb mesenchymal lineage is required for the normal response to the anti-sclerostin therapy in both bone formation and resorption. PMID:26780446

  6. Expression profiling of Dexamethasone-treated primary chondrocytes identifies targets of glucocorticoid signalling in endochondral bone development

    PubMed Central

    James, Claudine G; Ulici, Veronica; Tuckermann, Jan; Underhill, T Michael; Beier, Frank

    2007-01-01

    Background Glucocorticoids (GCs) are widely used anti-inflammatory drugs. While useful in clinical practice, patients taking GCs often suffer from skeletal side effects including growth retardation in children and adolescents, and decreased bone quality in adults. On a physiological level, GCs have been implicated in the regulation of chondrogenesis and osteoblast differentiation, as well as maintaining homeostasis in cartilage and bone. We identified the glucocorticoid receptor (GR) as a potential regulator of chondrocyte hypertrophy in a microarray screen of primary limb bud mesenchyme micromass cultures. Some targets of GC regulation in chondrogenesis are known, but the global effects of pharmacological GC doses on chondrocyte gene expression have not been comprehensively evaluated. Results This study systematically identifies a spectrum of GC target genes in embryonic growth plate chondrocytes treated with a synthetic GR agonist, dexamethasone (DEX), at 6 and 24 hrs. Conventional analysis of this data set and gene set enrichment analysis (GSEA) was performed. Transcripts associated with metabolism were enriched in the DEX condition along with extracellular matrix genes. In contrast, a subset of growth factors and cytokines were negatively correlated with DEX treatment. Comparing DEX-induced gene expression data to developmental changes in gene expression in micromass cultures revealed an additional layer of complexity in which DEX maintains the expression of certain chondrocyte marker genes while inhibiting factors that promote vascularization and ultimately ossification of the cartilaginous template. Conclusion Together, these results provide insight into the mechanisms and major molecular classes functioning downstream of DEX in primary chondrocytes. In addition, comparison of our data with microarray studies of DEX treatment in other cell types demonstrated that the majority of DEX effects are tissue-specific. This study provides novel insights into the effects of pharmacological GC on chondrocyte gene transcription and establishes the foundation for subsequent functional studies. PMID:17603917

  7. Connexin43 gap junctions in normal, regenerating, and cultured mouse bone marrow and in human leukemias: their possible involvement in blood formation.

    PubMed Central

    Krenacs, T.; Rosendaal, M.

    1998-01-01

    Communicating channels called gap junctions are thought to play a ubiquitous part in cell growth and development. Based on earlier work, we have recently found functional evidence of their presence in human and mouse bone marrow. In this study we studied the cell-type association of the gap junction channel-forming protein, connexin, in mouse and human bone marrow under different physiological and pathological conditions and tested the pathway of communication in bone marrow cultures. For high-resolution antigen demonstration we took advantage of semi-thin resin sections, antigen retrieval methods, immunofluorescence, and confocal laser scanning microscopy. Connexin43 (Cx43) and its mRNA were consistently expressed in human and rodent marrow. Cx37 was found only in the arteriolar endothelium, but neither Cx32 nor -26 were expressed. In tissue sections, the immunostained junctions appeared as dots, which were digitally measured and counted. Their average size was 0.40 mm in human and 0.49 mm in mice marrow. There were at least twice as many gap junctions in the femoral midshaft of 6-week-old mice (1.75 x 10(5)/mm3) as in those older than 12 weeks (0.89 x 10(5)/mm3). Most Cx43 was associated with collagen III+ endosteal and adventitial stromal cells and with megakaryocytes. Elsewhere, they were few and randomly distributed between all kinds of hematopoietic cells. In the femoral epiphysis of juvenile mice, stromal cell processes full of Cx43 enmeshed three to six layers of hematopoietic cells near the endosteum. The same pattern was seen in the midshaft of regenerating mouse marrow 3 to 5 days after cytotoxic treatment with 5-fluorouracil. Functional tests in cultures showed the transfer of small fluorescent dyes, Lucifer Yellow and 2',7'-bis-(2-carboxyethyl)-5, 6-carboxyfluorescein, between stromal cells and in rare cases between stromal and hematopoietic cells too. The stromal cells were densely packed with Cx43 and we found aggregates of connexon particles in their membrane replicas. In normocellular human bone marrow, gap junctions were as rare as in adult mouse and similarly distributed, except that they were also on adipocytic membranes. In a few leukemic samples, characterized by an increased stromal/hematopoietic cell ratio, there were two- to fourfold more Cx43 (2.8 x 10(5) to 3.9 x 10(5)/mm3) than in the normal (1.0 x 10(5) to 1.2 x 10(5)/mm3). The cases included a hypoplastic acute lymphoblastic leukemia, an acute myeloid leukemia (French-American-British classification M4-5), a case of myelodysplastic syndrome with elevated number of megakaryocytes, and a CD34+ acute hemoblastosis, probably acute myeloid leukemia (French-American-British classification M7). Taken together, our results indicate that direct cell-cell communication may be involved in hematopoiesis, ie, in developmentally active epiphyseal bone marrow and when there is a demand for progenitors in regeneration. However, gap junctions may not play as important a role in resting adult hematopoiesis and in leukemias. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 10 PMID:9546360

  8. Connexin43 gap junctions in normal, regenerating, and cultured mouse bone marrow and in human leukemias: their possible involvement in blood formation.

    PubMed

    Krenacs, T; Rosendaal, M

    1998-04-01

    Communicating channels called gap junctions are thought to play a ubiquitous part in cell growth and development. Based on earlier work, we have recently found functional evidence of their presence in human and mouse bone marrow. In this study we studied the cell-type association of the gap junction channel-forming protein, connexin, in mouse and human bone marrow under different physiological and pathological conditions and tested the pathway of communication in bone marrow cultures. For high-resolution antigen demonstration we took advantage of semi-thin resin sections, antigen retrieval methods, immunofluorescence, and confocal laser scanning microscopy. Connexin43 (Cx43) and its mRNA were consistently expressed in human and rodent marrow. Cx37 was found only in the arteriolar endothelium, but neither Cx32 nor -26 were expressed. In tissue sections, the immunostained junctions appeared as dots, which were digitally measured and counted. Their average size was 0.40 mm in human and 0.49 mm in mice marrow. There were at least twice as many gap junctions in the femoral midshaft of 6-week-old mice (1.75 x 10(5)/mm3) as in those older than 12 weeks (0.89 x 10(5)/mm3). Most Cx43 was associated with collagen III+ endosteal and adventitial stromal cells and with megakaryocytes. Elsewhere, they were few and randomly distributed between all kinds of hematopoietic cells. In the femoral epiphysis of juvenile mice, stromal cell processes full of Cx43 enmeshed three to six layers of hematopoietic cells near the endosteum. The same pattern was seen in the midshaft of regenerating mouse marrow 3 to 5 days after cytotoxic treatment with 5-fluorouracil. Functional tests in cultures showed the transfer of small fluorescent dyes, Lucifer Yellow and 2',7'-bis-(2-carboxyethyl)-5, 6-carboxyfluorescein, between stromal cells and in rare cases between stromal and hematopoietic cells too. The stromal cells were densely packed with Cx43 and we found aggregates of connexon particles in their membrane replicas. In normocellular human bone marrow, gap junctions were as rare as in adult mouse and similarly distributed, except that they were also on adipocytic membranes. In a few leukemic samples, characterized by an increased stromal/hematopoietic cell ratio, there were two- to fourfold more Cx43 (2.8 x 10(5) to 3.9 x 10(5)/mm3) than in the normal (1.0 x 10(5) to 1.2 x 10(5)/mm3). The cases included a hypoplastic acute lymphoblastic leukemia, an acute myeloid leukemia (French-American-British classification M4-5), a case of myelodysplastic syndrome with elevated number of megakaryocytes, and a CD34+ acute hemoblastosis, probably acute myeloid leukemia (French-American-British classification M7). Taken together, our results indicate that direct cell-cell communication may be involved in hematopoiesis, ie, in developmentally active epiphyseal bone marrow and when there is a demand for progenitors in regeneration. However, gap junctions may not play as important a role in resting adult hematopoiesis and in leukemias. PMID:9546360

  9. Effect of microgravity and mechanical stimulation on the in vitro mineralization and resorption of fetal mouse long bones (7-IML-1)

    NASA Technical Reports Server (NTRS)

    Veldhuijzen, J. Paul

    1992-01-01

    Mechanical forces play an important role in the differentiation, growth, and remodeling of skeletal tissues. An increase in the normal loading pattern of the skeleton leads to an increase in bone mass. An overall decrease in the functional load exerted on the skeleton produces mineral loss and osteoporosis. However, the responses of the skeletal tissue cells to various loading conditions are still largely unresolved, as is the mechanism of the cellular response to changed mechanical environment. Using an in vitro approach, we hope to avoid some problems encountered in the use of in vivo animal and man models, which have been extensively used in the past. In a number of experiments we have demonstrated that 16 and 17 day old fetal mouse long bone rudiments (metatarsalia), cultured in a liquid culture medium, are very suitable to study mineralization and resorption, respectively. We have also demonstrated that under hydrostatic compression, mineralization is increased while resorption is decreased. Culture of long bone rudiments under noncompressed control conditions can be regarded as a situation of partial unloading, showing some phenomena of a disuse situation. Under microgravity conditions, responses of osteoblasts and chondrocytes (involved in mineralization) and osteoclasts (involved in mineral resorption), to culture with and without compression, may be much more outspoken. This will have advantages for the study and the interpretation of the role of cellular events in the process of mineralization and resorption of developing skeletal tissues under various loading conditions. The BONES Experiment is carried out in four type I/O and four type I/E containers. Various aspects of the investigation are discussed.

  10. Cellular Mechanism of Decreased Bone in Brtl Mouse Model of OI: Imbalance of Decreased Osteoblast Function and Increased Osteoclasts and Their Precursors

    PubMed Central

    Uveges, Thomas E; Collin-Osdoby, Patricia; Cabral, Wayne A; Ledgard, Felicia; Goldberg, Leah; Bergwitz, Clemens; Forlino, Antonella; Osdoby, Philip; Gronowicz, Gloria A; Marini, Joan C

    2008-01-01

    The Brtl mouse, a knock-in model for moderately severe osteogenesis imperfecta (OI), has a G349C substitution in half of type I collagen α1(I) chains. We studied the cellular contribution to Brtl bone properties. Brtl cortical and trabecular bone are reduced before and after puberty, with BV/TV decreased 40–45%. Brtl ObS/BS is comparable to wildtype, and Brtl and wildtype marrow generate equivalent number of colony-forming units (CFUs) at both ages. However, OcS/BS is increased in Brtl at both ages (36–45%), as are TRACP+ cell numbers (57–47%). After puberty, Brtl ObS/BS decreases comparably to wildtype mice, but osteoblast matrix production (MAR) decreases to one half of wildtype values. In contrast, Brtl OcS falls only moderately (∼16%), and Brtl TRACP staining remains significantly elevated compared with wildtype. Consequently, Brtl BFR decreases from normal at 2 mo to one half of wildtype values at 6 mo. Immunohistochemistry and real-time RT-PCR show increased RANK, RANKL, and osteoprotegerin (OPG) levels in Brtl, although a normal RANKL/OPG ratio is maintained. TRACP+ precursors are markedly elevated in Brtl marrow cultures and form more osteoclasts, suggesting that osteoclast increases arise from more RANK-expressing precursors. We conclude that osteoblasts and osteoclasts are unsynchronized in Brtl bone. This cellular imbalance results in declining BFR as Brtl ages, consistent with reduced femoral geometry. The disparity in cellular number and function results from poorly functioning osteoblasts in addition to increased RANK-expressing precursors that respond to normal RANKL/OPG ratios to generate more bone-resorbing osteoclasts. Interruption of the stimulus that increases osteoclast precursors may lead to novel OI therapies. PMID:18684089

  11. Age-Related Modulation of the Effects of Obesity on Gene Expression Profiles of Mouse Bone Marrow and Epididymal Adipocytes

    PubMed Central

    Liu, Li-Fen; Shen, Wen-Jun; Ueno, Masami; Patel, Shailja; Azhar, Salman; Kraemer, Fredric B.

    2013-01-01

    This study aimed to characterize and compare the effects of obesity on gene expression profiles in two distinct adipose depots, epididymal and bone marrow, at two different ages in mice. Alterations in gene expression were analyzed in adipocytes isolated from diet-induced obese (DIO) C57BL/6J male mice at 6 and 14 months of age and from leptin deficient mice (ob/ob) at 6 months of age using microarrays. DIO affected gene expression in both depots at 6 and 14 months, but more genes were altered in epididymal than bone marrow adipocytes at each age and younger mice displayed more changes than older animals. In epididymal adipocytes a total of 2789 (9.6%) genes were differentially expressed at 6-months with DIO, whereas 952 (3.3%) were affected at 14-months. In bone marrow adipocytes, 347 (1.2%) genes were differentially expressed at 6-months with DIO, whereas only 189 (0.66%) were changed at 14-months. 133 genes were altered by DIO in both fat depots at 6-months, and 37 genes at 14-months. Only four genes were altered in both depots at both ages with DIO. Bone marrow adipocytes are less responsive to DIO than epididymal adipocytes and the response of both depots to DIO declines with age. This loss of responsiveness with age is likely due to age-associated changes in expression of genes related to adipogenesis, inflammation and mitochondrial function that are similar to and obscure the changes commonly associated with DIO. Patterns of gene expression were generally similar in epididymal adipocytes from ob/ob and DIO mice; however, several genes were differentially expressed in bone marrow adipocytes from ob/ob and DIO mice, perhaps reflecting the importance of leptin signaling for bone metabolism. In conclusion, obesity affects age-associated alterations in gene expression in both epididymal and bone marrow adipocytes regardless of diet or genetic background. PMID:23967297

  12. Quality of Life in Survivors of a Primary Bone Tumour: A Systematic Review

    PubMed Central

    Grimer, Robert J.

    1999-01-01

    Purpose. We conducted a systematic search of published literature, to assess (i) quality of life (QoL) for survivors of a bone tumour compared with the normal population; (ii) QoL implications following amputation, successful or failed limb salvage; (iii) adaptation of young children to amputation compared with older children or adolescents. Methods. Electronic databases were searched including Medline, PsycLIT and Cinahl covering the years 1982– 1998. Results. We identified 11 studies. Regardless of treatment, physical functioning was poor compared with population norms or healthy siblings.There was less consistent evidence regarding emotional functioning. Seven studies compared functioning in amputees and limb salvage patients.Two reported advantages in physical function for the limb salvage group, one for the amputees and the rest no differences. Evidence about social functioning or marriage is inconclusive, but there are suggestions that amputees report more job discrimination. Discussion. The literature is inconclusive, largely because of methodological problems. These include small and non-representative samples, and lack of sensitive and appropriate measures. Specific gaps in the literature include very little work concerned with psychological outcomes for children, or for those experiencing failed limb salvage. More attention needs to be given to gender differences in emotional response to traumatic surgery.The implications of the results for helping families balance the merits of different treatments are discussed. PMID:18521283

  13. Radiation and mechanical unloading effects on mouse vertebral bone: Ground-based models of the spaceflight environment

    NASA Astrophysics Data System (ADS)

    Alwood, Joshua Stewart

    Astronauts on long-duration space missions experience increased ionizing radiation background levels and occasional acute doses of ionizing radiation from solar particle events, in addition to biological challenges introduced by weightlessness. Previous research indicates that cancer radiotherapy damages bone marrow cell populations and reduces mechanical strength of bone. However, the cumulative doses in radiotherapy are an order of magnitude or greater than dose predictions for long-duration space missions. Further detriments to the skeletal system are the disuse and mechanical unloading experienced during weightlessness, which causes osteopenia in weight-bearing cancellous bone (a sponge-like bony network of rods, plates and voids) and cortical bone (dense, compact bone). Studies of radiation exposure utilizing spaceflight-relevant types and doses, and in combination with mechanical unloading, have received little attention. Motivated by the future human exploration of the solar system, the effects of acute and increased background radiation on astronaut skeletal health are important areas of study in order to prevent osteopenic deterioration and, ultimately, skeletal fracture. This dissertation addresses how spaceflight-relevant radiation affects bone microarchitecture and mechanical properties in the cancellous-rich vertebrae and compares results to that of mechanical unloading. In addition, a period of re-ambulation is used to test whether animals recover skeletal tissue after irradiation. Whether radiation exposure displays synergism with mechanical unloading is further investigated. Finite element structural and statistical analyses are used to investigate how changes in architecture affect mechanical stress within the vertebra and to interpret the mechanical testing results. In this dissertation, ground-based models provide evidence that ionizing radiation, both highly energetic gamma-rays and charged iron ions, resulted in a persistent loss of cancellous bone in male mice. Mechanical unloading, by contrast, is shown to cause bone loss in the vertebrae via cancellous and cortical thinning that resulted in decreased whole-bone mechanical properties. The effects of mechanical unloading were altogether reversible in the vertebra after re-ambulation, though some residual alteration of trabecular morphology persisted. The combination of unloading and radiation exposure appeared to worsen the reductions of strength. Under either environmental condition, cancellous bone loss occurred near the vertebral endplates and at the centrum midplane. Finite element analysis suggested that tissue-level stresses increase in the centrum after either unloading or irradiation in agreement with the cellular-solid model of dense, plate-like trabeculae. Force-sharing between cancellous and cortical bone decreased after radiation, with stress concentrating on the cortex. In conclusion, acute exposure to spaceflight-relevant ionizing radiation altered trabecular microarchitecture and stress distribution, without a loss of whole-bone strength at the endpoints investigated, while unloading presented the greater immediate detriment to whole-bone mechanical properties. From a skeletal-health perspective, strategies to mitigate and counteract astronaut exposure to acute doses of radiation and mechanical unloading should be developed in preparation for long-term human spaceflight.

  14. Osteocyte-directed bone demineralization along canaliculi.

    PubMed

    Nango, Nobuhito; Kubota, Shogo; Hasegawa, Tomoka; Yashiro, Wataru; Momose, Atsushi; Matsuo, Koichi

    2016-03-01

    The mammalian skeleton stores calcium and phosphate ions in bone matrix. Osteocytes in osteocyte lacunae extend numerous dendrites into canaliculi less than a micron in diameter and which are distributed throughout bone matrix. Although osteoclasts are the primary bone-resorbing cells, osteocytes also reportedly dissolve hydroxyapatite at peri-lacunar bone matrix. However, robust three-dimensional evidence for peri-canalicular bone mineral dissolution has been lacking. Here we applied a previously reported Talbot-defocus multiscan tomography method for synchrotron X-ray microscopy and analyzed the degree of bone mineralization in mouse cortical bone around the lacuno-canalicular network, which is connected both to blood vessels and the peri- and endosteum. We detected cylindrical low mineral density regions spreading around canaliculi derived from a subset of osteocytes. Transmission electron microscopy revealed both intact and demineralized bone matrix around the canaliculus. Peri-canalicular low mineral density regions were also observed in osteopetrotic mice lacking osteoclasts, indicating that osteoclasts are dispensable for peri-canalicular demineralization. These data suggest demineralization can occur from within bone through the canalicular system, and that peri-canalicular demineralization occurs not uniformly but directed by individual osteocytes. Blockade of peri-canalicular demineralization may be a therapeutic strategy to increase bone mass and quality. PMID:26709236

  15. Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis Can Promote Mouse and Human Primary Mast Cell Angiogenic Potential through Upregulation of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-2

    PubMed Central

    Chumanevich, Alena; Wedman, Piper; Oskeritzian, Carole A.

    2016-01-01

    Mast cells (MC) are present in most vascularized tissues around the vasculature likely exerting immunomodulatory functions. Endowed with diverse mediators, resident MC represent first-line fine-tuners of local microenvironment. Sphingosine-1-phosphate (S1P) functions as a pluripotent signaling sphingolipid metabolite in health and disease. S1P formation occurs at low levels in resting MC and is upregulated upon activation. Its export can result in type 2 S1P receptor- (S1PR2-) mediated stimulation of MC, further fueling inflammation. However, the role of S1PR2 ligation in proangiogenic vascular endothelial growth factor- (VEGF-) A and matrix metalloproteinase- (MMP-) 2 release from MC is unknown. Using a preclinical MC-dependent model of acute allergic responses and in vitro stimulated primary mouse bone marrow-derived MC (BMMC) or human primary skin MC, we report that S1P signaling resulted in substantial amount of VEGF-A release. Similar experiments using S1pr2-deficient mice or BMMC or selective S1P receptor agonists or antagonists demonstrated that S1P/S1PR2 ligation on MC is important for VEGF-A secretion. Further, we show that S1P stimulation triggered transcriptional upregulation of VEGF-A and MMP-2 mRNA in human but not in mouse MC. S1P exposure also triggered MMP-2 secretion from human MC. These studies identify a novel proangiogenic axis encompassing MC/S1P/S1PR2 likely relevant to inflammation. PMID:26884643

  16. Stimulation of minerals by carbon nanotube grafted glucosamine in mouse mesenchymal stem cells for bone tissue engineering.

    PubMed

    Venkatesan, Jayachandran; Kim, Se Kwon

    2012-08-01

    In the recent years, Single Walled Carbon Nanotube (SWCNT) is known to play a vital role in preparation of artificial bone materials. For this, we have prepared certain complexes of SWCNT grafted with different molecular weight polysaccharides, named as SWCNT-Glucosamine, SWCNT-Chitooligosaccharide (< 1 KDa), SWCNT-Chitooligosaccharide (1-3 KDa), SWCNT-Chitosan (310 KDa) and SWCNT-Chitosan (510 KDa) and subjected to bone tissue engineering application in vitro with mesenchymal stem cells. The physicochemical characteristic results revealed the existence of covalent bond between SWCNT and their respective polysaccharides. SWCNT-Glucosamine has been used for the cell culture experiment due to its highly soluble nature. As compared to glucosamine alone, no cytotoxic effect, higher alkaline phosphatase activity and enhanced mineralization have been observed in SWCNT-glucosamine. We propose that SWCNT-Polysaccharide derivatives are promising biomaterials for bone tissue engineering. PMID:22852477

  17. Resistance to infection with Eimeria vermiformis in mouse radiation chimeras is determined by donor bone-marrow cells

    SciTech Connect

    Joysey, H.S.; Wakelin, D.; Rose, M.E.

    1988-05-01

    The course of infection with Eimeria vermiformis was determined in BALB/b, BALB/c, and C57BL/10ScSn (B10) mice and in radiation chimeras prepared from the H-2-compatible BALB/b and B10 mice. The BALB strains, irrespective of H-2 haplotype, were resistant, the B10 mice were susceptible, and in the chimeras infection was characterized by the genotype of the donated bone-marrow cells and not by the phenotype of the recipient. Thus, the genetic control of relative resistance or susceptibility to infection with this parasite is expressed through bone-marrow-derived cells.

  18. Evaluation of the Therapeutic Potential of Bone Marrow-Derived Myeloid Suppressor Cell (MDSC) Adoptive Transfer in Mouse Models of Autoimmunity and Allograft Rejection

    PubMed Central

    Bouchet-Delbos, Laurence; Beriou, Gaelle; Merieau, Emmanuel; Hill, Marcelo; Delneste, Yves; Cuturi, Maria Cristina; Louvet, Cedric

    2014-01-01

    Therapeutic use of immunoregulatory cells represents a promising approach for the treatment of uncontrolled immunity. During the last decade, myeloid-derived suppressor cells (MDSC) have emerged as novel key regulatory players in the context of tumor growth, inflammation, transplantation or autoimmunity. Recently, MDSC have been successfully generated in vitro from naive mouse bone marrow cells or healthy human PBMCs using minimal cytokine combinations. In this study, we aimed to evaluate the potential of adoptive transfer of such cells to control auto- and allo-immunity in the mouse. Culture of bone marrow cells with GM-CSF and IL-6 consistently yielded a majority of CD11b+Gr1hi/lo cells exhibiting strong inhibition of CD8+ T cell proliferation in vitro. However, adoptive transfer of these cells failed to alter antigen-specific CD8+ T cell proliferation and cytotoxicity in vivo. Furthermore, MDSC could not prevent the development of autoimmunity in a stringent model of type 1 diabetes. Rather, loading the cells prior to injection with a pancreatic neo-antigen peptide accelerated the development of the disease. Contrastingly, in a model of skin transplantation, repeated injection of MDSC or single injection of LPS-activated MDSC resulted in a significant prolongation of allograft survival. The beneficial effect of MDSC infusions on skin graft survival was paradoxically not explained by a decrease of donor-specific T cell response but associated with a systemic over-activation of T cells and antigen presenting cells, prominently in the spleen. Taken together, our results indicate that in vitro generated MDSC bear therapeutic potential but will require additional in vitro factors or adjunct immunosuppressive treatments to achieve safe and more robust immunomodulation upon adoptive transfer. PMID:24927018

  19. Bone Histology and Primary Growth Rates in Hatchling Titanosaurs from Madagascar: New Insights from Micro-Computed Tomography

    NASA Astrophysics Data System (ADS)

    Bagley, B. C.; Whitney, M.; Rogers, K. C.

    2012-12-01

    Sauropods are the largest known terrestrial vertebrates and exhibit a greater ontogenetic variation in body size than any other taxon. More than 120 species of sauropods are known from the Jurassic and Cretaceous, and a wealth of specimens documents their enormous adult body sizes. Juvenile sauropods, in contrast, are rare. Though titanosaur eggs containing embryos have been recovered, to date the smallest known post-hatching juveniles are only a little less than half of known adult size, and details of the earliest stages of sauropod ontogeny remain particularly poorly understood. Here we report on two partial skeletons of hatchling Rapetosaurus krausei, a titanosaur from the Upper Cretaceous Maevarano Formation of Madagascar, and provide important new data on primary early stage growth rates in sauropods. The two partial skeletons come from different localities in the Anembalemba Member of the Maevarano Formation. There is no duplication of elements for either specimen. Comparison of greatest length ratios for appendicular elements to those of a complete sub-adult Rapetosaurus confirms that there are only two individuals present, that there is no significant allometry in Rapetosaurus postcranial ontogeny, and that each individual is less than 15% adult size. The smaller specimen includes a sacral neural arch, three caudal centra, three caudal neural arches, left pubis, right femur (maximum length [ml] = 19.3 cm), tibia (ml = 12.7 cm), and metacarpal III, left and right fibulae, humeri, and metatarsal I, and a phalanx. The larger specimen includes a caudal centrum and neural arch, right metacarpal I, right tibia (ml = 17.9 cm), and left metacarpal IV. In order to non-destructively sample these exceptional Rapetosaurus juvenile elements, we employed micro-computed tomography to garner bone histology data. The micro-computed tomography was carried out using an X5000 high-resolution microfocus X-ray CT system located in the Department of Earth Sciences, University of Minnesota. The microfocus head has a minimum focal spot size of < 6 microns and the detector has a pixel pitch of 74.8 μm. Machine parameters (e.g. voltage, current, tube to detector distance) vary based on sample size and desired magnification. For this study 70-100 kV (260-370 μA) was sufficient to penetrate the samples and obtain good contrast. We were able to achieve an effective pixel pitch of 36-48 μm for the larger samples and 14-28 μm for sub-volumes. 2-D radiographs were collected and these data were reconstructed to produce a 3-D volume for visual analysis, and slices of the 3-D volume for quantitative analysis. Our results indicate that primary bone growth in Rapetosaurus is highly vascularized woven and fibrolamellar bone. However, even in these very small juvenile individuals, endosteal remodeling is common at the mid-diaphysis and extends in some areas into the mid-cortex. The presence of a single line of arrested growth is recorded in each individual. These results are surprising given the small size of the elements, and support the hypothesis that intensive remodeling observed in the bones of older juvenile Rapetosaurus may be dictated, at least in part, by resource limitations during periods of drought/ecological stress recorded in the Maevarano Formation of Madagascar.

  20. Melanocortin-4 Receptor Expression in Different Classes of Spinal and Vagal Primary Afferent Neurons in the Mouse

    PubMed Central

    Gautron, Laurent; Lee, Charlotte E.; Lee, Syann; Elmquist, Joel K.

    2013-01-01

    Melanocortin-4 receptor (MC4R) ligands are known to modulate nociception, but the site of action of MC4R signaling on nociception remains to be elucidated. The current study investigated MC4R expression in dorsal root ganglia (DRG) of the MC4R-GFP reporter mouse. Because MC4R is known to be expressed in vagal afferent neurons in the nodose ganglion (NG), we also systematically compared MC4R-expressing vagal and spinal afferent neurons. Abundant green fluorescent protein (GFP) immunoreactivity was found in about 45% of DRG neuronal profiles (at the mid-thoracic level), the majority being small-sized profiles. Immunohistochemistry combined with in situ hybridization confirmed that GFP was genuinely produced in MC4R-expressing neurons in the DRG. While a large number of GFP profiles in the DRG coexpressed Nav1.8 mRNA (84%) and bound isolectin B4 (72%), relatively few GFP profiles were positive for NF200 (16%) or CGRP (13%), suggesting preferential MC4R expression in C-fiber nonpeptidergic neurons. By contrast, GFP in the NG frequently colocalized with Nav1.8 mRNA (64%) and NF200 (29%), but only to a moderate extent with isolectin B4 (16%). Lastly, very few GFP profiles in the NG expressed CGRP (5%) or CART (4%). Together, our findings demonstrate variegated MC4R expression in different classes of vagal and spinal primary afferent neurons, and underscore the role of the melanocortin system in modulating nociceptive and nonnociceptive peripheral sensory modalities. PMID:22592759

  1. Coactivator PGC-1{alpha} regulates the fasting inducible xenobiotic-metabolizing enzyme CYP2A5 in mouse primary hepatocytes

    SciTech Connect

    Arpiainen, Satu; Jaervenpaeae, Sanna-Mari; Manninen, Aki; Viitala, Pirkko; Lang, Matti A.; Pelkonen, Olavi; Hakkola, Jukka

    2008-10-01

    The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1{alpha} and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1{alpha} expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1{alpha} expression vector demonstrated that PGC-1{alpha} is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4{alpha} response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1{alpha} binds, together with HNF-4{alpha}, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1{alpha} mediates the expression of CYP2A5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4{alpha}. This strongly suggests that PGC-1{alpha} is the major factor mediating the fasting response of CYP2A5.

  2. XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer.

    PubMed

    Morton, J J; Bird, G; Keysar, S B; Astling, D P; Lyons, T R; Anderson, R T; Glogowska, M J; Estes, P; Eagles, J R; Le, P N; Gan, G; McGettigan, B; Fernandez, P; Padilla-Just, N; Varella-Garcia, M; Song, J I; Bowles, D W; Schedin, P; Tan, A-C; Roop, D R; Wang, X-J; Refaeli, Y; Jimeno, A

    2016-01-21

    The limitations of cancer cell lines have led to the development of direct patient-derived xenograft models. However, the interplay between the implanted human cancer cells and recruited mouse stromal and immune cells alters the tumor microenvironment and limits the value of these models. To overcome these constraints, we have developed a technique to expand human hematopoietic stem and progenitor cells (HSPCs) and use them to reconstitute the radiation-depleted bone marrow of a NOD/SCID/IL2rg(-/-) (NSG) mouse on which a patient's tumor is then transplanted (XactMice). The human HSPCs produce immune cells that home into the tumor and help replicate its natural microenvironment. Despite previous passage on nude mice, the expression of epithelial, stromal and immune genes in XactMice tumors aligns more closely to that of the patient tumor than to those grown in non-humanized mice-an effect partially facilitated by human cytokines expressed by both the HSPC progeny and the tumor cells. The human immune and stromal cells produced in the XactMice can help recapitulate the microenvironment of an implanted xenograft, reverse the initial genetic drift seen after passage on non-humanized mice and provide a more accurate tumor model to guide patient treatment. PMID:25893296

  3. KINETICS OF IN VIVO SISTER CHROMATID EXCHANGE INDUCTION IN MOUSE BONE MARROW CELLS BY ALKYLATING AGENTS: CYCLOPHOSPHAMIDE

    EPA Science Inventory

    Administration of cyclophosphamide (5, 10, 20 and 25 mg/kg body weight) to male CD-1 mice 2 hours after subcutaneous implantation of a 5-bromo-2'-deoxyuridine (BrdU) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Tre...

  4. Mechanical Stretching of Mouse Calvarial Osteoblasts In Vitro Models Changes in MMP-2 and MMP-9 Expression at the Bone-Implant Interface.

    PubMed

    Nichols, Richard A; Niagro, Frank D; Borke, James L; Cuenin, Michael F

    2016-04-01

    Bone to mechanical loading elicits a biological response that has clinical significance for several areas in dental medicine, including orthodontic tooth movement, tempromandibular joint disease, and endosseous dental implant osseointegration. Human orthopedic studies of failed hip implant sites have identified increased mRNA expression of several collagen-degrading matrix metalloproteinases (MMPs), while in vitro experiments have shown increases in MMP secretion after exposure to inflammatory mediators. This investigation evaluates the effects of mechanical deformation on in vitro osteoblasts by assessing changes in MMP gene expression and enzyme activity. We seeded mouse neonatal calvarial osteoblasts onto flexible 6-well plates and subjected to continuous cyclic mechanical stretching. The expression and activity of mRNA for several MMPs (2, 3, 9, and 10) was assessed. When subjected to mechanical stress in culture, only mRNA specific for MMP-9 was significantly increased compared to nonstretched controls (P < .005). Measurement of MMP activity by gelatin zymography demonstrated that none of the MMPs showed increased activity with stretching; however, MMP-2 activity decreased. Our results suggest that in response to stretch, MMP-2 responds rapidly by inhibiting conversion of a MMP-2 to the active form, while a slower up-regulation of MMP-9 may play a role in the long-term remodeling of extracellular matrix in response to continuous mechanical loading. This study suggests that the regulation of metalloproteinases at both the mRNA and protein level are important in the response of bone to mechanical stress. PMID:25961753

  5. Cocaine- and amphetamine-regulated transcript promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells into neural cells

    PubMed Central

    2011-01-01

    Background Neural tissue has limited potential to self-renew after neurological damage. Cell therapy using BM-MSCs (bone marrow mesenchymal stromal cells) seems like a promising approach for the treatment of neurological diseases. However, the neural differentiation of stem cells influenced by massive factors and interactions is not well studied at present. Results In this work, we isolated and identified MSCs from mouse bone marrow. Co-cultured with CART (0.4 nM) for six days, BM-MSCs were differentiated into neuron-like cells by the observation of optical microscopy. Immunofluorescence demonstrated that the differentiated BM-MSCs expressed neural specific markers including MAP-2, Nestin, NeuN and GFAP. In addition, NeuN positive cells could co-localize with TH or ChAT by double-labled immunofluorescence and Nissl bodies were found in several differentiated cells by Nissl stain. Furthermore, BDNF and NGF were increased by CART using RT-PCR. Conclusion This study demonstrated that CART could promote the differentiation of BM-MSCs into neural cells through increasing neurofactors, including BNDF and NGF. Combined application of CART and BM-MSCs may be a promising cell-based therapy for neurological diseases. PMID:21756347

  6. MHC-compatible bone marrow stromal/stem cells trigger fibrosis by activating host T cells in a scleroderma mouse model

    PubMed Central

    Ogawa, Yoko; Morikawa, Satoru; Okano, Hideyuki; Mabuchi, Yo; Suzuki, Sadafumi; Yaguchi, Tomonori; Sato, Yukio; Mukai, Shin; Yaguchi, Saori; Inaba, Takaaki; Okamoto, Shinichiro; Kawakami, Yutaka; Tsubota, Kazuo; Matsuzaki, Yumi; Shimmura, Shigeto

    2016-01-01

    Fibrosis of organs is observed in systemic autoimmune disease. Using a scleroderma mouse, we show that transplantation of MHC compatible, minor antigen mismatched bone marrow stromal/stem cells (BMSCs) play a role in the pathogenesis of fibrosis. Removal of donor BMSCs rescued mice from disease. Freshly isolated PDGFRα+ Sca-1+ BMSCs expressed MHC class II following transplantation and activated host T cells. A decrease in FOXP3+ CD25+ Treg population was observed. T cells proliferated and secreted IL-6 when stimulated with mismatched BMSCs in vitro. Donor T cells were not involved in fibrosis because transplanting T cell-deficient RAG2 knock out mice bone marrow still caused disease. Once initially triggered by mismatched BMSCs, the autoimmune phenotype was not donor BMSC dependent as the phenotype was observed after effector T cells were adoptively transferred into naïve syngeneic mice. Our data suggest that minor antigen mismatched BMSCs trigger systemic fibrosis in this autoimmune scleroderma model. DOI: http://dx.doi.org/10.7554/eLife.09394.001 PMID:26809474

  7. Humanized bone-marrow mouse model as a pre-clinical tool to assess therapy-mediated hematotoxicity

    PubMed Central

    Cai, Shanbao; Wang, Haiyan; Bailey, Barbara; Ernstberger, Aaron; Juliar, Beth E.; Sinn, Anthony L.; Chan, Rebecca J.; Jones, David R.; Mayo, Lindsey D.; Baluyut, Arthur R.; Goebel, W. Scott; Pollok, Karen E.

    2011-01-01

    Purpose Pre-clinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However, one of the challenges in screening anti-cancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example, hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens but in vivo models to assess potential toxicities have not been developed. In this study, a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity. Experimental Design A proof-of-concept, temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing since it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD/SCID/γchainnull mice reconstituted with human hematopoietic cells and the impact of treatment on human hematopoiesis was evaluated. Results The dose-intensive regimen resulted in significant decreases in growth of human-glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow, flow cytometric analyses indicated that the human bone-marrow cells were significantly more sensitive to treatment than the murine bone-marrow cells, and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor, lymphoid, and myeloid). Conclusions The humanized bone-marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anti-cancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation. PMID:21487065

  8. Palmitoyl acyltransferase, Zdhhc13, facilitates bone mass acquisition by regulating postnatal epiphyseal development and endochondral ossification: a mouse model.

    PubMed

    Song, I-Wen; Li, Wei-Ru; Chen, Li-Ying; Shen, Li-Fen; Liu, Kai-Ming; Yen, Jeffrey J Y; Chen, Yi-Ju; Chen, Yu-Ju; Kraus, Virginia Byers; Wu, Jer-Yuarn; Lee, M T Michael; Chen, Yuan-Tsong

    2014-01-01

    ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis. PMID:24637783

  9. Fibrin gel-immobilized primary osteoblasts in calcium phosphate bone cement: in vivo evaluation with regard to application as injectable biological bone substitute.

    PubMed

    Kneser, U; Voogd, A; Ohnolz, J; Buettner, O; Stangenberg, L; Zhang, Y H; Stark, G B; Schaefer, D J

    2005-01-01

    Osteogenic injectable bone substitutes may be useful for many applications. We developed a novel injectable bone substitute based on osteoblast-fibrin glue suspension and calcium phosphate bone cement (BC). Human osteoblasts were isolated from trabecular bone samples and cultured under standard conditions. Osteoblasts were suspended in fibrinogen solution (FS). BC was cured with thrombin solution. 8 x 4 mm injectable bone discs were prepared using silicon molds and a custom-made applicator device. Discs containing BC, BC/FS, or BC/FS/osteoblasts were implanted subcutaneously into athymic nude mice. After 3, 9 and 24 weeks, specimens were explanted and subjected to morphologic and biomechanical evaluation. In vitro fibrin gel-embedded osteoblasts displayed a differentiated phenotype as evidenced by alkaline phosphatase, collagen type 1 and von Kossa stains. A proportion of osteoblasts appeared morphologically intact over a 3-day in vitro period following application into the BC. BC/FS and BC/FS/osteoblast discs were sparsely infiltrated with vascularized connective tissue. There was no bone formation in implants from all groups. However, positive von Kossa staining only in BC/FS/osteoblast groups suggests engraftment of at least some of the transplanted cells. Biomechanical evaluation demonstrated initial stability of the composites. Young's modulus and maximal load did not differ significantly in the BC/FS and BC/FS/osteoblast groups. The practicability of osteoblast-containing injectable bone could be demonstrated. The dense microstructure and the suboptimal initial vascularization of the composites may explain the lack of bone formation. Modifications with regard to enhanced osteoblast survival are mandatory for a possible application as injectable osteogenic bone replacement system. PMID:16046862

  10. Arsenite selectively inhibits mouse bone marrow lymphoid progenitor cell development in vivo and in vitro and suppresses humoral immunity in vivo.

    PubMed

    Ezeh, Peace C; Lauer, Fredine T; MacKenzie, Debra; McClain, Shea; Liu, Ke Jian; Hudson, Laurie G; Gandolfi, A Jay; Burchiel, Scott W

    2014-01-01

    It is known that exposure to As(+3) via drinking water causes a disruption of the immune system and significantly compromises the immune response to infection. The purpose of these studies was to assess the effects of As(+3) on bone marrow progenitor cell colony formation and the humoral immune response to a T-dependent antigen response (TDAR) in vivo. In a 30 day drinking water study, mice were exposed to 19, 75, or 300 ppb As(+3). There was a decrease in bone marrow cell recovery, but not spleen cell recovery at 300 ppb As(+3). In the bone marrow, As(+3) altered neither the expression of CD34+ and CD38+ cells, markers of early hematopoietic stem cells, nor CD45-/CD105+, markers of mesenchymal stem cells. Spleen cell surface marker CD45 expression on B cells (CD19+), T cells (CD3+), T helper cells (CD4+) and cytotoxic T cells (CD8+), natural killer (NK+), and macrophages (Mac 1+) were not altered by the 30 day in vivo As(+3) exposure. Functional assays of CFU-B colony formation showed significant selective suppression (p<0.05) by 300 ppb As(+3) exposure, whereas CFU-GM formation was not altered. The TDAR of the spleen cells was significantly suppressed at 75 and 300 ppb As(+3). In vitro studies of the bone marrow revealed a selective suppression of CFU-B by 50 nM As(+3) in the absence of apparent cytotoxicity. Monomethylarsonous acid (MMA(+3)) demonstrated a dose-dependent and selective suppression of CFU-B beginning at 5 nM (p<0.05). MMA(+3) suppressed CFU-GM formation at 500 nM, a concentration that proved to be nonspecifically cytotoxic. As(+5) did not suppress CFU-B and/or CFU-GM in vitro at concentrations up to 500 nM. Collectively, these results demonstrate that As(+3) and likely its metabolite (MMA(+3)) target lymphoid progenitor cells in mouse bone marrow and mature B and T cell activity in the spleen. PMID:24714590

  11. Arsenite Selectively Inhibits Mouse Bone Marrow Lymphoid Progenitor Cell Development In Vivo and In Vitro and Suppresses Humoral Immunity In Vivo

    PubMed Central

    Ezeh, Peace C.; Lauer, Fredine T.; MacKenzie, Debra; McClain, Shea; Liu, Ke Jian; Hudson, Laurie G.; Gandolfi, A. Jay; Burchiel, Scott W.

    2014-01-01

    It is known that exposure to As+3 via drinking water causes a disruption of the immune system and significantly compromises the immune response to infection. The purpose of these studies was to assess the effects of As+3 on bone marrow progenitor cell colony formation and the humoral immune response to a T-dependent antigen response (TDAR) in vivo. In a 30 day drinking water study, mice were exposed to 19, 75, or 300 ppb As+3. There was a decrease in bone marrow cell recovery, but not spleen cell recovery at 300 ppb As+3. In the bone marrow, As+3 altered neither the expression of CD34+ and CD38+ cells, markers of early hematopoietic stem cells, nor CD45−/CD105+, markers of mesenchymal stem cells. Spleen cell surface marker CD45 expression on B cells (CD19+), T cells (CD3+), T helper cells (CD4+) and cytotoxic T cells (CD8+), natural killer (NK+), and macrophages (Mac 1+) were not altered by the 30 day in vivo As+3 exposure. Functional assays of CFU-B colony formation showed significant selective suppression (p<0.05) by 300 ppb As+3 exposure, whereas CFU-GM formation was not altered. The TDAR of the spleen cells was significantly suppressed at 75 and 300 ppb As+3. In vitro studies of the bone marrow revealed a selective suppression of CFU-B by 50 nM As+3 in the absence of apparent cytotoxicity. Monomethylarsonous acid (MMA+3) demonstrated a dose-dependent and selective suppression of CFU-B beginning at 5 nM (p<0.05). MMA+3 suppressed CFU-GM formation at 500 nM, a concentration that proved to be nonspecifically cytotoxic. As+5 did not suppress CFU-B and/or CFU-GM in vitro at concentrations up to 500 nM. Collectively, these results demonstrate that As+3 and likely its metabolite (MMA+3) target lymphoid progenitor cells in mouse bone marrow and mature B and T cell activity in the spleen. PMID:24714590

  12. Prognostic relevance of disseminated tumor cells in the bone marrow and biological factors of 265 primary breast carcinomas

    PubMed Central

    Schindlbeck, Christian; Kampik, Theresa; Janni, Wolfgang; Rack, Brigitte; Jeschke, Udo; Krajewski, Stan; Sommer, Harald; Friese, Klaus

    2005-01-01

    Introduction The prognostic significance of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has been demonstrated in many studies. Yet, it is not clear which of the primary tumors' biological factors predict hematogenous dissemination. We therefore examined 'tissue micro arrays' (TMAs) of 265 primary breast carcinomas from patients with known bone marrow (BM) status for HER2, Topoisomerase IIα (Top IIa), Ki 67, and p53. Methods BM analysis was performed by cytospin preparation and immunocytochemical staining for cytokeratin (CK). TMAs were examined by immunohistochemistry (IHC) for HER2, Top IIa, Ki 67 and p53, and fluorescence in situ hybridization (FISH) for HER2. Results HER2 (2+/3+) was positive in 35/167 (21%) cases (FISH 24.3%), Top IIa (>10%) in 87/187 (46%), Ki 67 in 52/184 (28%) and p53 (>5%) in 61/174 cases (34%). Of 265 patients, 68 (25.7%) showed DTC-BM with a median of 2/2 × 106 cells (1 to 1,500). None of the examined factors significantly predicted BM positivity. Significant correlation was seen between HER2 IHC and Top IIa (p = 0.06), Ki 67 (p = 0.031), and p53 (p < .001). Top IIa correlated with Ki 67 and p53, and Ki 67 also with p53 (p = 0.004). After a median follow-up of 60.5 months (7 to 255), the presence of DTC-BM showed prognostic relevance for overall survival (p = 0.03), whereas HER2 (IHC, p = 0.04; FISH, p = 0.03) and Ki 67 (p = 0.04) correlated with disease free survival, and HER2 with distant disease free survival (IHC, p = 0.06; FISH, p = 0.05). Discussion The congruence of the examined factors' expression rates indicates a causal line of suppressor, proliferation, and mitosis markers, and growth factor receptors. Hematogenous tumor cell spread seems to be an independent process. The examination of these factors on DTC-BM is the aim of ongoing research. PMID:16457698

  13. Bone marrow-derived macrophages and the CNS: An update on the use of experimental chimeric mouse models and bone marrow transplantation in neurological disorders.

    PubMed

    Larochelle, Antoine; Bellavance, Marc-André; Michaud, Jean-Philippe; Rivest, Serge

    2016-03-01

    The central nervous system (CNS) is a very unique system with multiple features that differentiate it from systemic tissues. One of the most captivating aspects of its distinctive nature is the presence of the blood brain barrier (BBB), which seals it from the periphery. Therefore, to preserve tissue homeostasis, the CNS has to rely heavily on resident cells such as microglia. These pivotal cells of the mononuclear lineage have important and dichotomous roles according to various neurological disorders. However, certain insults can overwhelm microglia as well as compromising the integrity of the BBB, thus allowing the infiltration of bone marrow-derived macrophages (BMDMs). The use of myeloablation and bone marrow transplantation allowed the generation of chimeric mice to study resident microglia and infiltrated BMDM separately. This breakthrough completely revolutionized the way we captured these 2 types of mononuclear phagocytic cells. We now realize that microglia and BMDM exhibit distinct features and appear to perform different tasks. Since these cells are central in several pathologies, it is crucial to use chimeric mice to analyze their functions and mechanisms to possibly harness them for therapeutic purpose. This review will shed light on the advent of this methodology and how it allowed deciphering the ontology of microglia and its maintenance during adulthood. We will also compare the different strategies used to perform myeloablation. Finally, we will discuss the landmark studies that used chimeric mice to characterize the roles of microglia and BMDM in several neurological disorders. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger. PMID:26432480

  14. The Effect of Polymethyl Methacrylate Augmentation on the Primary Stability of Cannulated Bone Screws in an Anterolateral Plate in Osteoporotic Vertebrae: A Human Cadaver Study.

    PubMed

    Rüger, Matthias; Sellei, Richard M; Stoffel, Marcus; von Rüden, Christian

    2016-02-01

    Study Design Cohort study. Objective Expandable anterolateral plates facilitate the reduction of posttraumatic deformities of thoracolumbar spine injuries and are commonly used in cases of unstable injuries or compromised bone quality. In this in vitro study, the craniocaudal yield load of the osseous fixation of an anterior angular stable plate fixation system and the effect of polymethyl methacrylate (PMMA) screw augmentation on the primary stability of the screw-bone interface during kyphosis reduction was evaluated in 12 osteoporotic human thoracolumbar vertebrae. Methods The anterolateral stabilization device used for this study is comprised of two swiveling flanges and an expandable midsection. It facilitates the controlled reduction of kyphotic deformities in situ with a geared distractor. Single flanges were attached to 12 thoracolumbar vertebrae. Six specimens were augmented with PMMA by means of cannulated bone screws. The constructs were subjected to static, displacement-controlled craniocaudal loading to failure in a servohydraulic testing machine. Results The uncemented screws cut out at a mean 393 ± 66 N, whereas the cemented screws showed significantly higher yield load of 966 ± 166 N (p < 0.02). We detected no significant correlation between bone mineral density and yield load in this setting. Conclusion Our results indicate that PMMA augmentation is an effective method to increase two- to threefold the primary stability of the screw-bone interface of an anterolateral spine stabilization system in osteoporotic bone. We recommend it in cases of severely compromised bone quality to reduce the risk of screw loosening during initial kyphosis correction and to increase long-term construct stability. PMID:26835201

  15. The Effect of Polymethyl Methacrylate Augmentation on the Primary Stability of Cannulated Bone Screws in an Anterolateral Plate in Osteoporotic Vertebrae: A Human Cadaver Study

    PubMed Central

    Rüger, Matthias; Sellei, Richard M.; Stoffel, Marcus; von Rüden, Christian

    2015-01-01

    Study Design Cohort study. Objective Expandable anterolateral plates facilitate the reduction of posttraumatic deformities of thoracolumbar spine injuries and are commonly used in cases of unstable injuries or compromised bone quality. In this in vitro study, the craniocaudal yield load of the osseous fixation of an anterior angular stable plate fixation system and the effect of polymethyl methacrylate (PMMA) screw augmentation on the primary stability of the screw–bone interface during kyphosis reduction was evaluated in 12 osteoporotic human thoracolumbar vertebrae. Methods The anterolateral stabilization device used for this study is comprised of two swiveling flanges and an expandable midsection. It facilitates the controlled reduction of kyphotic deformities in situ with a geared distractor. Single flanges were attached to 12 thoracolumbar vertebrae. Six specimens were augmented with PMMA by means of cannulated bone screws. The constructs were subjected to static, displacement-controlled craniocaudal loading to failure in a servohydraulic testing machine. Results The uncemented screws cut out at a mean 393 ± 66 N, whereas the cemented screws showed significantly higher yield load of 966 ± 166 N (p < 0.02). We detected no significant correlation between bone mineral density and yield load in this setting. Conclusion Our results indicate that PMMA augmentation is an effective method to increase two- to threefold the primary stability of the screw–bone interface of an anterolateral spine stabilization system in osteoporotic bone. We recommend it in cases of severely compromised bone quality to reduce the risk of screw loosening during initial kyphosis correction and to increase long-term construct stability. PMID:26835201

  16. Kinetics of in vivo sister chromatid exchange induction in mouse bone marrow cells by alkylating agents: cyclophosphamide

    SciTech Connect

    Charles, J.L.; Jacobson-Kram, D.; Borzelleca, J.F.; Carchman, R.A.

    1983-01-01

    Administration of cyclophosphamide (5, 10, 20 and 25 mg/kg body weight) to male CD-1 mice 2 hr after subcutaneous implantation of a 5-bromo-2'-deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with cyclophosphamide (15 mg/kg body weight) at the time of BrdUrd implantation and 2, 6, 5, and 13 hr post-BrdUrd implantation resulted in the induction of approximately 19 SCE/cell indicating that the bone marrow SCE response was independent of the time of administration. Treatment with cyclophosphamide (15 mg/kg body weight) at 26, 29, 13, and 6 hr prior to BrdUrd implantation resulted in baseline SCE (3.3 SCE/cell) at 26 hr with an increasing number of SCE/cell with decreasing time prior to BrdUrd implantation.

  17. Differential effects of fibromodulin deficiency on mouse mandibular bones and teeth: a micro-CT time course study.

    PubMed

    Goldberg, Michel; Marchadier, Arnaud; Vidal, Catherine; Harichane, Yassine; Kamoun-Goldrat, Agnès; Kellermann, Odile; Kilts, Tina; Young, Marian

    2011-01-01

    Fibromodulin (Fmod) is a keratan sulfate small leucine-rich proteoglycan which is enriched in bones and teeth. In order to determine its functions on bone and tooth mineralization we characterized the phenotype of Fmod-deficient (Fmod-KO) mice using a new-generation microfocus computerized tomography system (micro-CT) and software allowing advanced visualization of 3-D data. Three-week-old and 10- week-old Fmod-KO mandibles and teeth were compared with those of age-matched wild-type (WT) mice. In both young and mature mice the Fmod-KO mandibles were hypomineralized, especially the posterior (proximal) part of the mandible as it appeared to be the main target of the molecule deficiency whereas less extensive alterations were found in the alveolar bone. In transverse sections, larger marrow spaces were observed in the Fmod-KO mice compared with age-matched young or mature WT mice. Quantitative evaluation of the pulp volume of the first molar and 3-D reconstructions suggested that dentinogenesis was diminished in 3-week-old Fmod-KO teeth. In contrast, increased dentin formation was found in 10-week-old Fmod-KO mice and it was accompanied by a reduced pulp volume. Thus, the differential effects of Fmod deficiency on bones and teeth appear to diverge in adult mice. This may result from the previously reported differences in the molecular weight of Fmod in the 2 tissues or from compensatory mechanisms due to the overexpression of DSP and DMP-1 in the dental pulp of Fmod-KO. It is also possible that a single molecule plays diverging roles in a tissue-specific or region-specific manner. PMID:21597266

  18. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    PubMed Central

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-01-01

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis. PMID:25503693

  19. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer.

    PubMed

    Xu, W; Neill, T; Yang, Y; Hu, Z; Cleveland, E; Wu, Y; Hutten, R; Xiao, X; Stock, S R; Shevrin, D; Kaul, K; Brendler, C; Iozzo, R V; Seth, P

    2015-03-01

    In an effort to develop a new therapy for prostate cancer (PCa) bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels and inhibited tumor cell migration. To examine the antitumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum tartrate-resistant acid phosphatase 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for PCa bone metastasis. PMID:25503693

  20. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

    PubMed Central

    Coronado-Cerda, Erika Evangelina; Franco-Molina, Moisés Armides; Mendoza-Gamboa, Edgar; Prado-García, Heriberto; Rivera-Morales, Lydia Guadalupe; Zapata-Benavides, Pablo; Rodríguez-Salazar, María del Carmen; Caballero-Hernandez, Diana; Tamez-Guerra, Reyes Silvestre; Rodríguez-Padilla, Cristina

    2016-01-01

    Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients. PMID:27191003