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Breast cancer associated with osteoclast-like giant cells (OGCs) refers to a morphological pattern of invasive breast carcinoma of non-special type. Their presence is sometimes subtle, but OGCs can be appreciated both histologically and immunohistochemically. The origin of OGCs as well as their implication for prognosis remain debated. We describe the case of a 65-year-old woman, wherein the presence of OGCs in the fine-needle aspiration cytology of a metastatic axillary lymph node suggested the final diagnosis on histology. The differential diagnosis is broad, and here we provide evidence for strict cytological-histological correlation when dealing with unusual breast lesions.

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Equine asthma is currently diagnosed by the presence of increased neutrophil (>5%), mast cell (>2%), and/or eosinophil (>1%) differential cell count. Macrophages are normal resident cells within the alveoli. Their presence in BALF is considered normal, but the clinical implication of the presence of activated or fused macrophages (giant multinucleated cells, GMC) is currently overlooked. We aimed to assess the prevalence, cytological determinants, and clinical significance of increased GMC counts in BALF of 34 asthmatic horses compared to 10 controls. Counts were performed on 15 randomly selected high magnification fields per cytospin slide (40×), and expressed as GMC:single macrophage (GMC:M) ratio. Regression models were used for statistical analysis. GMC was frequently observed in both asthmatic and control horses, with an increased prevalence of equine asthma (p = 0.01). GMC:M ratio was significantly higher in severe vs. mild to moderate equine asthmatic and control horses. In asthmatic horses, an increased GMC:M ratio was significantly associated with BALF mastocytosis (p = 0.01), once adjusting for age and the presence and severity of clinical signs of the horses. Tachypnea was the only clinical sign that tended to be positively associated with GMC:M ratio after adjustment (p = 0.08). In conclusion, our data suggest that a relationship might exist between molecular mechanisms regulating GMC formation and mast cell recruitment in the equine lung. The same mechanisms could lead to tachypnea even in the absence of respiratory effort at rest. We suggest including GMC count in the basic cytological assessment of BALF samples to gain more insights into their role in equine asthma.

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Calcium balance is important in bone homeostasis. The transient receptor potential vanilloid (TRPV) channel is a nonselective cation channel permeable to calcium and is activated by various physiological and pharmacological stimuli. TRPV1 and TRPV4, in particular, have important roles in intracellular Ca2+ signaling and extracellular calcium homeostasis in bone cells. TRPV1 and TRPV4 separately mediate osteoclast and osteoblast differentiation, and deficiency in any of these channels leads to increased bone mass. However, it remains unknown whether bone mass increases in the absence of both TRPV1 and TRPV4. In this study, we used TRPV1 and TRPV4 double knockout (DKO) mice to evaluate their bone mass in vivo, and osteoclast and osteoblast differentiation in vitro. Our results showed that DKO mice and wild type (WT) mice had no significant difference in body weight and femur length. However, the results of dual-energy X-ray absorption, microcomputed tomography, and bone histomorphometry clearly showed that DKO mice had higher bone mass than WT mice. Furthermore, DKO mice had less multinucleated osteoclasts and had lower bone resorption. In addition, the results of cell culture using flushed bone marrow from mouse femurs and tibias showed that osteoclast differentiation was suppressed, whereas osteoblast differentiation was promoted in DKO mice. In conclusion, our results suggest that the increase in bone mass in DKO mice was induced not only by the suppression of osteoclast differentiation and activity but also by the augmentation of osteoblast differentiation and activity. Our findings reveal that both the single deficiency of TRPVs and the concurrent deficiency of TRPVs result in an increase in bone mass. Furthermore, our data showed that DKO mice and single KO mice had varying approaches to osteoclast and osteoblast differentiation in vitro, and therefore, it is important to conduct further studies on TRPVs regarding the increase in bone mass to explore not only individual but also a combination of TRPVs.

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Giant cell tumour of bone (GCTB) is a histologically benign, locally aggressive skeletal lesion with an unpredictable propensity to relapse after surgery and a rare metastatic potential. The microscopic picture of GCTB shows different cell types, including multinucleated giant cells, mononuclear cells of the macrophage-monocyte lineage, and spindle cells. The histogenesis of GCTB is still debated, and morphologic, radiographic or molecular features are not predictive of the clinical course. Characterization of the unexplored cell metabolism of GCTB offers significant clues for the understanding of this elusive pathologic entity. In this study we aimed to characterize GCTB energetic metabolism, with a particular focus on lactate release and the expression of monocarboxylate transporters, to lie down a novel path for understanding the pathophysiology of this tumour. We measured the expression of glycolytic markers (GAPDH, PKM2, MCT4, GLUT1, HK1, LDHA, lactate release) in 25 tissue samples of GCTB by immunostaining and by mRNA and ELISA analyses. We also evaluated MCT1 and MCT4 expression and oxidative markers (JC1 staining and Bec index) in tumour-derived spindle cell cultures and CD14+ monocytic cells. Finally, we quantified the intratumoural and circulating levels of lactate in a series of 17 subjects with GCTB. In sharp contrast to the benign histological features of GCTB, we found a high expression of glycolytic markers, with particular reference to MCT4. Unexpectedly, this was mainly confined to the giant cell, not proliferating cell component. Accordingly, GCTB patients showed higher levels of blood lactate as compared to healthy subjects. In conclusion, taken together, our data indicate that GCTB is characterized by a highly glycolytic metabolism of its giant cell component, opening new perspectives on the pathogenesis, the natural history, and the treatment of this lesion.

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The process of bone formation onto the bone surface using a hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) was investigated. Immersion tests were performed to evaluate the impact of pH on the degradation of the specimens in an aqueous environment. The specimens were soaked in aqueous solutions of pH 4.0, 5.0, and 7.0. Using standardized images, the top-view areas of the specimens were measured. Animal experiments were performed to investigate the bone formation process onto the bone surface. The specimens were placed under the rat calvarial periosteum, and µCT image analysis and histological observation were performed on samples harvested on postoperative Days 3, 5, and 7. In all experiments, ß-tricalciumphosphate (ß-TCP) was adopted as the control. HAp/Col turned to gel in acidic environments below pH 5.0. In contrast to the ß-TCP, the HAp/Col specimens placed under the periosteum expanded and attained a hollow structure with a gel-filled center, accompanied by larger volume of new bone and appearance of TRAP-positive multinucleated cells on postoperative Day 5. Therefore, HAp/Col can enhance bone formation onto the bone surface via induction of TRAP-positive multinucleated cells, and may have clinical applications.

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Some cells cultured in vitro have multiple nuclei. Since cultured cells are used in various fields of science, including tissue engineering, the nature of the multinucleated cells must be determined. However, multinucleated cells are not frequently observed. In this study, a method to efficiently obtain multinucleated cells was established and their morphological properties were investigated. Initially, we established conditions to quickly and easily generate multinucleated cells by seeding a Xenopus tadpole epithelium tissue-derived cell line (XTC-YF) on less and more hydrophilic dishes, and incubating the cultures with medium supplemented with or without Y-27632-a ROCK inhibitor-to reduce cell contractility. Notably, 88% of the cells cultured on a less hydrophilic dish in medium supplemented with Y-27632 became multinucleate 48 h after seeding, whereas less than 5% of cells cultured under other conditions exhibited this morphology. Some cells showed an odd number (three and five) of cell nuclei 72 h after seeding. Multinucleated cells displayed a significantly smaller nuclear area, larger cell area, and smaller nuclear circularity. As changes in the morphology of the cells correlated with their functions, the proposed method would help researchers understand the functions of multinucleated cells.

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The morphotypes of human macrophages (MPh) were studied in the culture on nano-structured biopolymer substrates, made from polyhydroxyalcanoates (PHAs) of five various monomer compositions, followed by the solvent evaporation. Its surface relief, which was further in direct contact with human cells in vitro, was analyzed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). It was shown, that the features of the micro/nano relief depend on the monomeric composition of the polymer substrates. Monocytes (MN) of patients with atherosclerosis and cardiac ischemia, undergoing stenting and conventional anti-atherosclerotic therapy, were harvested prior and after stenting. MN were isolated and cultured, with the transformation into MPh in direct contact with biopolymer culture substrates with different monomer composition and nano-reliefs, and transformed into MPh, in comparison with the same process on standard culture plastic. Sub-populations of cells with characteristic morphology in each phenotypic class were described, and their quantitative ratios for each sample of polymers were counted as an intermediate result in the development of "smart" material for cardiovascular devices. The results obtained allow us to assume, that the processes of MPh differentiation and polarization in vitro depend not only on the features of the micro/nano relief of biopolymer substrates, but also on the initial state of MN in vivo and general response of patients.

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Osteoclasts (OCLs) are multinucleated phagocytes of monocytic origin responsible for physiological and pathological bone resorption including aging processes, chronic inflammation and cancer. Besides bone resorption, they are also involved in the modulation of immune responses and the regulation of hematopoietic niches. Accordingly, OCLs are the subject of an increasing number of studies. Due to their rarity and the difficulty to isolate them directly ex vivo, analyses on OCLs are usually performed on in vitro differentiated cells. In this state, however, OCLs represent a minority of differentiated cells. Since up to date a reliable purification procedure is still lacking for mature OCLs, all cells present in the culture are analyzed collectively to answer OCL-specific questions. With the development of in-depth transcriptomic and proteomic analyses, such global analyses on unsorted cells can induce severe bias effects in further results. In addition, for instance, analysis on OCL immune function requires working on purified OCLs to avoid contamination effects of monocytic precursors that may persist during the culture. This clearly highlights the need for a reliable OCL purification procedure. Here, we describe a novel and reliable method to sort OCLs based on cell multinucleation while preserving cell viability. Using this method, we successfully purified multinucleated murine cells. We showed that they expressed high levels of OCL markers and retained a high capacity of bone resorption, demonstrating that these are mature OCLs. The same approach was equally applied for the purification of human mature OCLs. Comparison of purified OCLs with mononucleated cells or unsorted cells revealed significant differences in the expression of OCL-specific markers at RNA and/or protein level. This exemplifies that substantially better outcomes for OCLs are achieved after the exclusion of mononucleated cells. Our results clearly demonstrate that the in here presented procedure for the analysis and sorting of pure OCLs represents a novel, robust and reliable method for the detailed examination of bona fide mature OCLs in a range that was previously impossible. Noteworthy, this procedure will open new perspectives into the biology of osteoclasts and osteoclast-related diseases.

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Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4+ T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling.

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Osteoporosis is an aging-associated disease requiring better therapeutic modality. Eupatilin is a major flavonoid from Artemisia plants such as Artemisia princeps and Artemisia argyi which has been reported to possess various beneficial biological effects including anti-inflammation, anti-tumor, anti-cancer, anti-allergy, and anti-oxidation activity. Complete blockade of RANK-dependent osteoclastogenesis was accomplished upon stimulation prior to the receptor activator of nuclear factor κB (RANK)-ligand (RANKL) treatment or post-stimulation of bone marrow macrophages (BMCs) in the presence of RANKL with eupatilin. This blockade was accompanied by inhibition of rapid phosphorylation of Akt, GSK3ß, ERK and IκB as well as downregulation of c-Fos and NFATc1 at protein, suggesting that transcriptional suppression is a key mechanism for anti-osteoclastogenesis. Transient reporter assays or gain of function assays confirmed that eupatilin was a potent transcriptional inhibitor in osteoclasts (OC). Surprisingly, when mature osteoclasts were cultured on bone scaffolds in the presence of eupatilin, bone resorption activity was also completely blocked by dismantling the actin rings, suggesting that another major acting site of eupatilin is cytoskeletal rearrangement. The eupatilin-treated mature osteoclasts revealed a shrunken cytoplasm and accumulation of multi-nuclei, eventually becoming fibroblast-like cells. No apoptosis occurred. Inhibition of phosphorylation of cofilin by eupatilin suggests that actin may play an important role in the morphological change of multinucleated cells (MNCs). Human OC similarly responded to eupatilin. However, eupatilin has no effects on osteoblast differentiation and shows cytotoxicity on osteoblast in the concentration of 50 µM. When eupatilin was administered to LPS-induced osteoporotic mice after manifestation of osteoporosis, it prevented bone loss. Ovariectomized (OVX) mice remarkably exhibited bone protection effects. Taken together, eupatilin is an effective versatile therapeutic intervention for osteoporosis via; 1) transcriptional suppression of c-Fos and NFATc1 of differentiating OC and 2) inhibition of actin rearrangement of pathogenic MNCs.

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To delineate the characteristics of nuclear artifacts associated with endoscopic submucosal dissection (ESD), we examined 97 gastric ESD specimens from 79 patients. In 69 of the specimens (71%), multinucleated figures and/or atypical mitotic-like figures, including tripolar-like and bizarre spindles, were found in the peripheral portions close to the marking areas. These nuclear figures were mostly recognizable as artifacts, but were infrequently (13 specimens) accompanied by other nuclear alterations and/or architectural abnormalities, mimicking dysplasia. However, in the deep cut sections, the dysplastic characteristics tended to disappear and coagulative or degenerative findings became more prominent. These nuclear artifacts were not found in 69 age- and gender-matched control gastrectomy specimens without ESD. Multinucleated artifacts were associated with the size of the ESD specimens (P=0.003), frequency of marking (P<0.001) and a history of 'previous' marking 1-6 days prior to ESD (P<0.001); however, they were not associated with age, ESD procedure time, or 'fresh' marking on the day of the ESD. Atypical mitosis-like characteristics were associated with a history of 'fresh' (P=0.007) as well as 'previous' (P=0.002) marking, but not with other variables. Dysplasia-like artifacts were associated with older age only (P=0.031). Follow-up data of all the patients with nuclear artifacts showed no aggressive behavior. Therefore, we concluded that these nuclear changes were ESD-related artifacts. Particularly in older patients, these changes may simulate dysplasia and must be distinguished from true dysplasia or neoplasia.

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OBJECTIVE: LPS can induce differentiation to osteoclast-like cells independent of RANKL. In comparison with RANKL, the effects of Th1 and Th2 cytokines on LPS-induced osteoclastogenesis have not been extensively studied. In this study, we investigated the effects of IFN-γ and IL-4 on RANKL- or LPS-induced osteoclastogenesis. MATERIALS AND METHODS: RAW 264.7 cells were induced to differentiate into osteoclast-like cells by RANKL or LPS, in the absence or presence of IFN-γ or IL-4. The number of TRAP-positive, multinucleated (≥ 3 nuclei) cells (MNCs) was counted. mRNA and protein levels of TRAP and cathepsin K were determined by quantitative RT-PCR and Western immunoblot, respectively. Expression of other genes implicated in osteoclast and macrophage differentiation and inflammation was also quantitated and was subsequently assessed in bone marrow-derived macrophages (BMMs). Phagocytic capacity of differentiated RAW264.7 was investigated by the uptake of pHrodo S. aureus bioparticles conjugates. RESULTS: In contrast to the RANKL-treated cell population that gained more macrophage-like properties at the level of gene and protein expression as well as phagocytosis in the presence of IFN-γ or IL-4, the LPS-induced population gained more osteoclast-like properties by the addition of the same factors. CONCLUSION: These data suggest that the adaptive immune system, through either Th1 or Th2 cytokines, is able to modify the differentiation process of osteoclasts in inflammatory situations. Moreover, the study provides an example of different regulation of osteoclast differentiation during physiological and inflammatory conditions.

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OBJECTIVE: Fusogenic endogenous retroviral syncytin plays an important role in the formation of syncytiotrophoblasts in human placenta. Apart from its expression in placenta, brain and testis, syncytin has also been found in many cancers. Although syncytin has been proposed to serve as a positive prognostic marker in some cancers, the underlying mechanism is unclear. The aim of this study is to evaluate the effects of syncytin expression on the invasive phenotype of melanoma cells. METHODS: The eukaryotic expression plasmid for syncytin-EGFP was constructed and transfected into B16F10 melanoma cells. The effect of syncytin on the invasion potential of tumor cells was evaluated in B16F10 subline cells that stably expressed syncytin-EGFP fusion protein or EGFP alone. RESULTS: The B16F10 sublines that stably expressed syncytin-EGFP or EGFP alone were established respectively and confirmed by immunofluorescent and immunoblotting assay. Syncytin expression in B16F10 cells was associated with decreased cell proliferation, migration and invasion. Multinucleated giant cells that contained as many as five nuclei were induced in syncytin-expressing cells. In addition, syncytin expression did not alter the sensitivity of B16F10 cells to trichosanthin, a toxin that damages syncytiotrophoblasts more efficiently than other tissues. CONCLUSIONS: These results suggest that syncytin expression in some cancers may confine their invasion potential and thus serve as a positive prognostic factor.

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Human mitochondrial ribosomal protein MRPS18-2 (S18-2) is encoded by a cellular gene that is located on the human chromosome 6p21.3. We discovered that overexpression of the S18-2 protein led to immortalization and de-differentiation of primary rat embryonic fibroblasts. Cells showed anchorage-independent growth pattern. Moreover, pathways characteristic for rapidly proliferating cells were upregulated then. It is possible that the S18-2 overexpression induced disturbance in cell cycle regulation. We found that overexpression of S18-2 protein in human cancer cell lines led to an appearance of multinucleated cells in the selected clones.

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Orthodontic tooth movement in response to orthodontic force results from actions of osteoclasts and osteoblasts in the cell level. Convincing evidence has now been provided to support the view that osteoclasts are derived from mononuclear cells that originate in the bone marrow or other hematopoietic organs and they migrate to the bones via vascular routes. Nitric oxide(NO), which accounts for the biological properties of endothelium-derived relaxmg factor(EDRF), is the endogenous stimulator of soluble guanylate cylase. The discovery of the formation of nitric oxide(NO) from L-arginine in mammalian tissues and its biologioal roles has, in the last 7 years, thrown new light onto many areas of research. Data from experiments in vitro showed that N-metyl-L-arginine(L-NMA) and L-nitro-L-arginine(L-NAME) are competitive inhibitors of nitric oxide synthase. This study suggest that the multinucleated cells in our culture have characteristics of osteoclasts and that the potential bone cell activity of nitric oxide in vitro may be mediated in part by stimulation of marrow mononuclear cells to form osteoclast-like cells. Bone marrow cells were obtained from tibia of 19-days old chick embryo. After sacrifice, tibia was quickly dissected and the bone were then split to expose the medullary bone. The cells were attached for 4 hours and the nonadherent cells were collected. Marrow cells were cultured in 96-well plate in medium 199. To examine the number of TRAP-positive multinucleated cells(MNCs), 10(-8) M Vit-D3 and various concentration of L-NMA and L-NAME were added at the beginning of cultures and with each medium change. After 7 days of culture, tartrate-resistant acid phosphatase(TRAP) staining was performed for microscopic evaluation. Cells having more than three nuclei per cell were counted as MNCs. The observed results were as follows; 1. 1,25-dihydroxyvitamine D3 stimulated the osteoclast-like multinucleated cells in cultures of chick embryo bone marrow. 2. Nitric oxide synthase inhibitors(NOSI ; N-NMA, N-NAME) stimulated the osteoclast-like cells in cultures of chick embry bone marrow. 3. 1,25-dihydroxyvltamine D3 and nitric oxide synthase inhibitors did not appear to have additive effect on the generation of TRAP-positive MNCs. These results suggest that nitric oxide synthase inhibitors may stimulate the osteoclast-like multinucleated cell formation and fusion in cultures of chick bone marrow.

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