Osteoclast progenitors from cats with and without tooth resorption respond differently to 1,25-dihydroxyvitamin D and interleukin-6

Both vitamin D and inflammatory cytokines can stimulate osteoclast formation and activity. We studied the effect of 1,25-dihydroxycholecalciferol (1,25(OH)(2)D), and interleukin-6 (IL-6), on the formation and activity of feline osteoclasts, using peripheral blood mononuclear cells (PBMCs) from cats with and without tooth resorption (TR(+) and TR(-)) as a source of osteoclast precursors. The formation of osteoclast-like cells (defined as multinucleated, tartrate-resistant acid phosphatase-positive cells) was assessed at 7 and 14 days. In the presence of M-CSF and RANKL, with and without IL-6, more osteoclasts were formed from TR(-) PBMCs than from TR(+) PBMCs on plastic. More osteoclasts were formed from TR(+) PBMCs on bone slices in the presence of M-CSF/RANKL with 1,25(OH)(2)D. This opposite effect may be due to a higher expression of the vitamin D receptor (VDR) in TR(+) osteoclasts and precursors on bone. Formation of resorption pits was analyzed and confirmed with scanning electron microscopy. In conclusion, we propose that TR(+) PBMCs when cultured on bone are sensitive to 1,25(OH)(2)D, whereas the differentiation of TR(-) PMBCs on bone seem more sensitive to IL-6, suggesting that osteoclast precursors from cats with and without tooth resorption respond differently to osteoclast stimulating factors.     

Expression of receptor activator of nuclear factor kappa-B ligand (RANKL) in neoplasms of dogs and cats

BACKGROUND: Receptor activator of nuclear factor kappa-B (RANK), RANK-ligand (RANKL), and the soluble decoy receptor osteoprotegerin (OPG) form a key axis modulating osteoclastogenesis. In health, RANKL-expressing bone stromal cells and osteoblasts activate osteoclasts through RANK ligation, resulting in homeostatic bone resorption. Skeletal tumors of dogs and cats, whether primary or metastatic, may express RANKL and directly induce malignant osteolysis. HYPOTHESIS: Bone malignancies of dogs and cats may express RANKL, thereby contributing to pathologic bone resorption and pain. Furthermore, relative RANKL expression in bone tumors may correlate with radiographic characteristics of bone pathology. ANIMALS: Forty-two dogs and 6 cats with spontaneously-occurring tumors involving bones or soft tissues were evaluated. METHODS: A polyclonal anti-human RANKL antibody was validated for use in canine and feline cells by flow cytometry and immunocytochemistry. Fifty cytologic specimens were collected from bone and soft tissue tumors of 48 tumor-bearing animals and assessed for RANKL expression. In 15 canine osteosarcoma (OSA) samples, relative RANKL expression was correlated with radiographic characteristics of bone pathology. RESULTS: Expression of RANKL by neoplastic cells was identified in 32/44 canine and 5/6 feline tumor samples. In 15 dogs with OSA, relative RANKL expression did not correlate with either radiographic osteolysis or bone mineral density as assessed by dual energy x-ray absorptiometry. CONCLUSIONS AND CLINICAL IMPORTANCE: In dogs and cats, tumors classically involving bone and causing pain, often may express RANKL. Confirming RANKL expression in tumors is a necessary step toward the rational institution of novel therapies targeting malignant osteolysis via RANKL antagonism.     

Regulation of matrix metalloproteinase-9 protein expression by 1α,25-(OH)2D3 during osteoclast differentiation

To investigate 1α,25-(OH)₂D₃ regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1α,25-(OH)₂D₃ during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1α,25-(OH)₂D₃ inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1α,25-(OH)₂D₃ administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.     

Osteopetrosis in cats

Osteopetrosis was found in two adult cats, radiographically characterised by a ‘marble bone’ appearance of the axial skeleton and of the subchondral bone of all joints, and endosteal thickening of the cortex of long bones. Computer tomography and histological assessment revealed massive endosteal lamellar bone formation. Bone resorption was still apparent, though only atypical mononuclear osteoclasts were seen. Histological findings support the assumption that the disorder was acquired. Severe anaemia was found in one cat. In the other, no clinical abnormalities were present. The condition appears to be different from feline leukaemia virus-induced medullary osteosclerosis described by Hoover and Kociba (1974).     

Immunohistochemical Localization of RANK, RANKL and OPG in Healthy and Arthritic Canine Elbow Joints

Objective— To determine if the receptor activator of nuclear factor-κB–receptor activator of nuclear factor-κB ligand–osteoprotegerin (RANK–RANKL–OPG) system is active in bone remodeling in dogs and, if so, whether differences in expression of these mediators occur in healthy and arthritic joints.
Study Design— Experimental study.
Sample Population— Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease.
Methods— Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints.
Results— All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few.
Conclusions— The RANK–RANKL–OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption.
Clinical Relevance— Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation.     

A clinicopathological study of 52 feline epulides

A retrospective study was performed to characterize 52 new cases of feline epulides between 1995 and 2001, with clinical and pathological results classified according to Head's histopathologic criteria for canine epulides. The incidence of the fibromatous, acanthomatous, ossifying, and giant cell epulis were respectively 57.7% (30/52), 7.7% (4/52), 5.8% (3/52), and 28.8% (15/52). Giant cell epulides presented significant differences in clinical behavior compared with the fibromatous type, including rapid growth (P < .0001), presence of ulcerative changes (P < .01), and rapid recurrence after surgery (P < .01) from which euthanasia was judged necessary in 4 cases. Fifteen giant cell epulides were additionally examined in order to characterize the lesion both histochemically and immunohistochemically and to investigate the origin of the multinucleated giant cells (MGCs). Van Gieson staining showed osteoid and woven bone formation in 11 cases. Both the MGCs and a fraction of the mononuclear cells were positive for vimentin, tartrate-resistant acid phosphatase (TRAP), a commonly accepted marker for osteoclasts, and the polyclonal antibody receptor activator of nuclear factor kappabeta (RANK), a cytokine leading to the differentiation of osteoclast progenitors into mature osteoclasts in presence of its ligand. MGCs were negative for smooth muscle actin, MIB-1, and factor VIII. The giant cell epulis may be a variant of the fibromatous and ossifying epulis in which extensive ulceration and inflammation results in increased osteoclastic activity. The osteoclast-like giant cells are most likely formed from a monocyte/macrophage-like osteoclast precursor that differentiates into osteoclasts under the influence of mononuclear osteoblast-like stromal cells.     

MAPK信号通路介导的自噬调控破骨细胞分化的研究进展

破骨细胞具有骨吸收活性,与骨组织稳态密切相关。丝裂原活化蛋白激酶(MAPK)通路是细胞介导胞内外刺激传导的信号通路,参与细胞的增殖、分化、自噬等多种生理过程。MAPK介导的自噬在调控破骨细胞分化中具有重要作用。探究MAPK的三条经典通路(ERK1/2、JNK及p38 MAPK信号通路)介导的自噬与破骨细胞分化之间的关系,对于寻找与破骨细胞相关的骨代谢疾病的新疗法具有重要意义。     

Inhibitory effects of osteoprotegerin on osteoclast formation and function under serum-free conditions

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor κB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor κB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.     

Combined zoledronic acid and meloxicam reduced bone loss and tumour growth in an orthotopic mouse model of bone‐invasive oral squamous cell carcinoma

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.     

Evaluation of terminal deoxynucleotidyl transferase expression in bone marrow of clinically normal and feline leukemia virus-exposed cats

Bone marrow cells from 8 specific-pathogen-free and 11 feline leukemia virus-exposed cats were examined for the expression of the nuclear antigen terminal deoxynucleotidyl transferase (TdT). Using a standard indirect immunofluorescence technique, feline leukemia-exposed cats had increased expression of TdT in bone marrow aspirates (2.0% to 29.0%) when compared with that in bone marrow cells from specific-pathogen-free cats (2.5% to 6.0%). Seemingly, TdT can be used as an antigenic marker in leukemogenesis of FeLV-infected cats.     

Shifts in circulating lymphocyte subsets in cats with feline infectious peritonitis (FIP): pathogenic role and diagnostic relevance

Cats with feline infectious peritonitis (FIP) are usually lymphopenic and have lymphoid depletion evident in spleen and lymph nodes. In particular, the number of CD4+ lymphocytes in tissues decreases during the evolution of FIP lesions. This decrease is most likely due to increased lymphocyte apoptotic rate. In contrast, cats infected with the Feline Coronavirus (FCoV) develop a follicular hyperplasia in the peripheral lymph nodes. The current study was devised to evaluate the possible pathogenic role of shifts in circulating lymphocyte subsets in FIP. Peripheral blood from cats with FIP was evaluated and compared with peripheral blood from clinically healthy cats living in both FCoV-free and FCoV-endemic catteries. Blood from cats with diseases other than FIP was also examined in order to define the diagnostic relevance of the changes. Lymphocyte subsets were analysed by flow cytometry, using a whole blood indirect immunofluorescence technique and mAbs specific for feline CD5, CD4, CD8, CD21. The results of the current study suggest that cats recently infected with FCoV that do not develop the disease have a transient increase in T cells; cats from groups with high prevalence of FIP have a moderate but persistent decrease in T cell subsets; cats with FIP have a very severe decrease in all the subsets of lymphocytes. Moreover, during FIP many lymphocytes do not express any membrane antigen, most likely due to early apoptosis. Cats with diseases other than FIP also had decreased number of lymphocytes: as a consequence, the diagnostic relevance of these findings is very low. Nevertheless, the lack of flow cytometric changes had a high negative predictive value (NPV), thus allowing to exclude FIP from the list of possible diagnoses in cats with normal cytograms.     

Detection of feline immunodeficiency virus infection in bone marrow of cats.

Natural or experimental feline immunodeficiency virus (FIV) infection in cats is often associated with hematologic abnormalities which are similar to those observed in human immunodeficiency virus (HIV) infected patients. To determine if cells in bone marrow are infected with FIV and whether severity of hematopoietic disorder is correlated with the level of viral infection, bone marrow tissues from ten experimentally and two naturally FIV infected cats were examined by in situ hybridization for presence of FIV RNA. Seven of the 12 FIV infected cats were also naturally or experimentally coinfected with feline leukemia virus (FeLV). FIV RNA was detected mainly in megakaryocytes and unidentified mononuclear cells in the bone marrow of cats that were sick and had marrow hypercellularity and immaturity. These included all cats in the acute phase of FIV infection and two of seven long term FIV infected cats. One long term FIV infected cat with lymphosarcoma was also positive for FIV RNA in bone marrow cells. The other four long term FIV infected cats were relatively healthy, with normal bone marrow morphology, and were negative for FIV infected cells. Bone marrow from three non-infected and two cats infected with FeLV alone were also negative for FIV RNA by in situ hybridization. We concluded that megakaryocytes and mononuclear cells were targets of the viral infection and that the presence of FIV RNA in cells of the bone marrow correlated with marrow hypercellularity and immaturity, and severity of illness.     

Altered surface antigen expression on peripheral blood mononuclear cells in cats infected with feline immunodeficiency virus.

Expression of CD4, CD8, IL-2 receptor alpha chain (IL-2R alpha), and MHC class II (MHC-II) on peripheral blood mononuclear cells were examined in cats infected with feline immunodeficiency virus (FIV). CD4/CD8 T cell ratio in FIV-infected cats was slightly decreased, as compared with that in specific-pathogen-free (SPF) cats. However, there was no statistical differences between them. The number of circulating IL-2R alpha+ cells in FIV-infected cats was higher than that in healthy cats, whereas induction of IL-2R alpha expression by concanavalin A (Con A) stimulation was depressed in FIV-infected cats. By using two-color cytofluorometry, Con A-induced enhancement of IL-2R alpha expression was found to be reduced in both CD4+ and CD8+ populations in PBMC from FIV-infected cats. The circulating MHC-II+ cells were also increased in FIV-infected cats. Furthermore, the induction of IL-2R alpha expression on PBMC after Con A-stimulation significantly depressed by FIV inoculation in vitro. These results suggest that FIV activates PBMC in vivo via direct and/or indirect mechanisms, leading to the unresponsive state of T cells to further stimuli in vitro.     

Stimulation of osteoclast-like cell formation by Pasteurella multocida toxin from hemopoietic progenitor cells in mouse bone marrow cultures.

The effects of purified Pasteurella multocida toxin (PMT) on osteoclast formation from hemopoietic progenitor cells were studied using an in vitro system. Mononuclear adherent mouse bone marrow cells were cultured for 7 or 14 days in the presence of PMT, or 1,25-dihydroxy-vitamin D3, or both. Mononuclear osteoclast-like cells, which are postmitotic osteoclast precursor cells, were identified as tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells possessing calcitonin receptors. Multinucleated osteoclast-like cells were TRAP-positive multinuclear cells with calcitonin receptors. The results demonstrate that, as does 1,25(OH)2D3, Pasteurella multocida toxin stimulates proliferation of adherent bone marrow mononuclear cells (progenitor cells), and their differentiation into postmitotic mononuclear osteoclast precursor cells. It also causes fusion of the latter into multinuclear osteoclasts; however, the number of osteoclasts obtained with PMT is smaller than with 1,25-dihydroxy-vitamin D3.     

破骨细胞相关MicroRNA研究进展

MicroRNA(miRNA)是一种非编码蛋白RNA,其长度约为22nt,广泛存在于真核生物细胞中,在生物的生长、发育和疾病发生等生物学过程中发挥着重要作用。随着对miRNA研究的不断深入,其在骨代谢方面的研究也逐渐展开。作为体内唯一一种负责骨吸收的细胞,破骨细胞(OC)在机体骨组织生理及病理性过程中占有重要地位。OC的形成和分化受多种因素调节,研究发现多种miRNA对OC有着调控作用,如miR-21、miR-422a和miR-148a能够促进OC形成,miR-124、miR-155和miR-503则抑制OC形成,除此以外,某些miRNA还能通过OC调控肿瘤骨转移。对这些机制的研究将从另一个层面揭示代谢性骨病的病因,并对临床治疗提供指导。论文对miRNA在OC形成和分化过程中的作用及机制进行综述。     

Bone-invasive oral squamous cell carcinoma in cats: pathology and expression of parathyroid hormone-related protein

Feline oral squamous cell carcinoma (OSCC) is the most common oral tumor in cats. There is no effective treatment, and the average duration of survival after diagnosis is only 2 months. Feline OSCC is frequently associated with osteolysis; however, the mechanisms responsible are unknown. The objective of this study was to characterize the epidemiology and pathology of bone-invasive OSCC in cats and to determine the expression of select bone resorption agonists. In sum, 451 cases of feline OSCC were evaluated. There was no sex or breed predisposition, although there were more intact cats in the OSCC group compared to the control group. Gingiva was the most common site, followed by the sublingual region and tongue. Cats with lingual OSCC were younger (mean, 11.9 years) compared to cats with gingival OSCC (mean, 13.6 years). In addition to osteolysis, there was periosteal new bone formation, osseous metaplasia of tumor stroma, and direct apposition of OSCC to fragments of bone, suggestive of bone-binding behavior. Eighty-two cases were selected for immunohistochemical detection of parathyroid hormone-related protein (PTHrP). Specimens with osteolysis had increased PTHrP expression and nuclear localization, compared to OSCC without osteolysis. Thirty-eight biopsies of OSCC with osteolysis were evaluated for tumor necrosis factor α expression, and only 4 biopsies had such expression in a small proportion of tumor cells. Increased tumor expression of PTHrP and increased localization of PTHrP to the nucleus were associated with osteolysis and may play an important role in bone resorption and tumor invasion in cats with OSCC.     

Early events in the immunopathogenesis of feline retrovirus infections.

Feline leukemia virus and feline immunodeficiency virus (FIV) are lymphotropic retroviruses that cause a wide range of diseases in domestic cats. Although it is known that both viruses are capable of infecting T lymphocytes and that infected cats are lymphopenic, it was not known how infection with either virus might alter specific lymphocyte subpopulations. Using a panel of monoclonal antibodies to feline lymphocyte subpopulations, we examined, by use of flow cytometric analysis, lymphocyte changes in cats naturally infected with FeLV or FIV and explored the early stages in the immunopathogenesis of experimentally induced infection with these viruses. Both groups of naturally infected cats had T-cell lymphopenia. In the FIV-infected cats, the T-cell decrease was principally attributable to loss of CD4+ cells, whereas CD8+ and B-cell numbers remained normal. This led to inversion of the CD4+ to CD8+ ratio in these cats. In contrast, the T-cell lymphopenia in FeLV-infected cats resulted from decrease in CD4+ and CD8+ cells, which led to a CD4+ to CD8+ ratio within normal limits. Experimentally induced infection with these 2 viruses supported these findings. Infection with FIV induced early (10 weeks after infection), chronic inversion of the CD4+ to CD8+ ratio. In contrast, infection with FeLV did not alter CD4+ to CD8+ ratio in the first 20 weeks after infection.     

Persistent upregulation of MHC class II antigen expression on T-lymphocytes from cats experimentally infected with feline immunodeficiency virus.

A significant elevation in the percentage of CD4+ and CD8+ T-lymphocytes expressing major histocompatibility complex (MHC) Class II antigens was observed in the blood of cats shortly after they were experimentally infected with feline immunodeficiency virus (FIV). In addition to an increase in the relative proportion of T-lymphocytes expressing Class II antigens, there was an increase in the density of Class II antigens on the cell surface. These elevations were still evident at the completion of the 5 month study. A second group of cats that had been infected with FIV for almost 5 years, and with either normal or abnormally low levels of CD4+ T-lymphocytes, had similar elevations in MHC II expression, suggesting that such abnormalities are lifelong. Cats with chronic (2 year) feline leukemia virus (FeLV) infection or dual FIV/FeLV infections also showed similar alterations in MHC II expression on CD4+ and CD8+ T-lymphocytes, suggesting that these alterations were not FIV specific. Feline T-lymphocytes expressed more MHC II antigen and interleukin-2 (IL-2) receptor following stimulation in vitro with conconavalin A and IL-2, demonstrating that feline T-lymphocytes respond to activation signals in a manner similar to T-lymphocytes of other species. However, changes in MHC II expression on T-cells of FIV infected cats were not explainable by viral induced T-cell activation alone, because FIV infected cats with elevated MHC II expression did not have coincident elevations in IL-2 receptor expression.     

Characterization of fibroblast colony-forming units in bone marrow from healthy cats

The adherent cell layer of bone marrow from healthy cats was characterized in vitro, and the mean fibroblast colony-forming unit (CFU-F) was determined. The majority (82%) of the cells in the adherent cell layer were spindle-shaped fibroblastic cells. These cells were weakly positive for acid phosphatase activity and negative for alpha-naphthyl butyrate esterase and alkaline phosphatase activities. They did not phagocytose latex beads. The remaining cells (18%) in the adherent cell layer resembled macrophages. They were strongly positive for acid phosphatase and alpha-naphthyl butyrate esterase activities, and they phagocytosed latex beads. The mean CFU-F per 10(6) mononuclear cells in bone marrow from healthy kittens and adult cats was 62 and 65, respectively. The CFU-F assay was linear over a range of 0.25 to 1.25 x 10(6) bone marrow mononuclear cells cultured. Variation in the feline CFU-F assay was similar to that reported for the human CFU-F assay. Bone marrow collections repeated at 1-month intervals (from the same bone) did not affect CFU-F concentration. A difference was not observed between CFU-F cultured from the feline humerus or femur. Bone marrow adherent cells in cats resembled those described for other species. Results of the feline CFU-F assay were consistent and repeatable and were similar to those reported for other species.