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Multinucleated Cells (multinucleated + cell)
Kinds of Multinucleated Cells Selected AbstractsThe study of cytopathological aspects induced by human cytomegalovirus infectionDIAGNOSTIC CYTOPATHOLOGY, Issue 5 2004B.S., C.M.I.A.C., Takako Takeuchi C.T. Abstract In cytological examination, human cytomegalovirus (HCMV) infection can not be implied unless typical HCMV-infected cells like owl's-eye cells are present. However, such cells are not always observed in HCMV-infection cases. The aim of our study is to establish the cytopathological features induced by HCMV. In vitro transfection and fluorescence in situ hybridization (FISH) were performed on human embryo lung (HEL) cells. Marked cellular aggregation was observed at 6-hr postinfection (hpi). Multinucleated cells, giant cells, and, particularly, small vacuoles were present in the nuclei or cytoplasm before the appearance of inclusion bodies. However, molding and ground glass in nuclei were absent. Cell clusters displayed round cytoplasm, dispersed later, and showed anisocytosis. All features occurred before 48 hpi when the owl's-eye cell appeared. In FISH, the positive signal highlighted viral particles that became predominant and localized in nuclei. These cytological aspects are dependent on viral replication and contribute to the cytological detection of HCMV infection. Diagn. Cytopathol. 2004;31:289,293. © 2004 Wiley-Liss, Inc. [source] Fine-needle aspiration of subcutaneous panniculitis-like T-cell lymphomaDIAGNOSTIC CYTOPATHOLOGY, Issue 5 2004Frances Manosca M.D. Abstract We report the fine-needle aspiration (FNA) cytology findings of Subcutaneous Panniculitis-like T-cell Lymphoma (SCPTCL) in a 66-year-old woman who presented with a four month history of asymptomatic subcutaneous nodules on her right chest wall and back. An excisional biopsy of the right chest nodules was performed, and the diagnosis of SCPTCL was rendered. On a follow-up visit, several skin lesions were noted throughout her body. A fine-needle aspiration (FNA) of the right inguinal region was performed. The FNA yielded cellular smears, composed mainly of sheets of epithelioid histiocytes and scattered multinucleated cells. However, no distinct granulomas were noted. The background of the cytological smears showed scattered atypical lymphoid cells, some of which displayed nuclear membrane irregularities. To the best of our knowledge, the cytological features on FNA material of SCPTCL have not been described. Diagn. Cytopathol. 2004;31:338,339. © 2004 Wiley-Liss, Inc. [source] Bumetanide, the Specific Inhibitor of Na+ -K+ -2Cl, Cotransport, Inhibits 1,,25-Dihydroxyvitamin D3 -Induced Osteoclastogenesis in a Mouse co-culture SystemEXPERIMENTAL PHYSIOLOGY, Issue 5 2003Hyun-A Lee The Na+ -K+ -2Cl, cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-,B ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 µM bumetanide reduced RANKL mRNA expression induced by 10 nM 1,,25-dihydroxyvitamin D3 (1,,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2 -terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1,,25(OH)2D3 -induced osteoclastogenesis partly via the phosphorylation of JNK. [source] Inhibitors of the PI3-kinase/Akt pathway induce mitotic catastrophe in non-small cell lung cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 5 2006Therese H Hemström Abstract Non-small cell lung cancer cells (NSCLC) are more resistant to anticancer treatment as compared with other types of cancer cells. Recently (Hemström et al., Exp Cell Res 2005;305:200,13) we showed that apoptosis of U1810 NSCLC cells induced by the staurosporine analog PKC 412 correlated with inhibition of Akt and ERK1/2, suggesting the involvement of these kinases in cell survival. Here we investigated the contribution of the PI3-kinase/Akt and MEK/ERK pathways to survival of NSCLC cells. The two signaling pathways were studied by using different combinations of the PI3-kinase inhibitors LY-294002 and wortmannin, the Akt activator Ro 31-8220, the MEK inhibitor PD 98059 and PKC 412. PI3-kinase inhibitors induced apoptosis-like death in U1810 cells. H157 cells in general were relatively resistant to PI3 kinase/Akt inhibitors yet these compounds sensitized cells to the DNA-damaging drug VP-16, while Ro 31-8220 could not. PD 98059 only had a sensitizing effect on H157 cells when combined with PI3-kinase inhibition and VP-16. Morphological data indicated that LY-294002 and PKC 412 induced cell death at anaphase and metaphase, respectively, suggesting death by mitotic catastrophe. Analyzes of cells blocked in G2/M-phase by nocodazol revealed that LY-294002 increased, while PKC 412 decreased histone H3 phosphorylation, suggesting that LY-294002 allowed, while PKC 412 inhibited cells to leave M-phase. Flow cytometric analysis of cell cycle distribution demonstrated that LY-294002 allowed cells to leave G2/M phase, while PKC 412 inhibited cytokinesis, resulting in formation of multinucleated cells. These results indicate that sensitization of NSCLC cells by PI3-kinase inhibition involves interplay between cell cycle regulation, mitotic catastrophe and apoptosis. © 2006 Wiley-Liss, Inc. [source] Histological characteristics of human papilloma-virus-positive and -negative invasive and in situ squamous cell tumours of the penisINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 2 2009Dorrit Krustrup Summary A high prevalence of cervical cancer associated high-risk types of human papillomavirus (hrHPV) has been demonstrated in premalignant and invasive squamous cell lesions of the penis, but large studies correlating histological characteristics with HPV status are few in number. Tumour tissues from 145 patients with invasive (n = 116) or in situ (n = 29) penile squamous cell carcinoma were subjected to systematic histological evaluation and were PCR-tested for 14 hrHPV types and 23 low-risk HPV types. Around half (52%) of invasive and nine-tenths (90%) of in situ lesions were positive for an hrHPV type, of which HPV 16 was by far the predominant type (91% of hrHPV-positive lesions). In relation to histological characteristics, hrHPV positivity was statistically significantly more common in high-grade tumours, lesions dominated by small tumour cells, lesions with a high number of multinucleated cells and mitoses, and lesions with a small amount of parakeratosis. In conclusion, about half of invasive penile squamous carcinomas in this study were hrHPV-positive, most notably to HPV 16, and probably arose through in situ lesions whereas the other half of invasive penile lesions appeared to be unrelated to hrHPV. A number of histological characteristics differed significantly between hrHPV-positive and -negative invasive penile carcinomas. [source] Alteration of RANKL-Induced Osteoclastogenesis in Primary Cultured Osteoclasts From SERCA2+/, Mice,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2009Yu-Mi Yang Abstract RANKL is essential for the terminal differentiation of monocytes/marcrophages into osteoclasts. RANKL induces long-lasting oscillations in the intracellular concentration of Ca2+ ([Ca2+]i) only after 24 h of stimulation. These Ca2+ oscillations play a switch-on role in NFATc1 expression and osteoclast differentiation. Which Ca2+ transporting pathway is induced by RANKL to evoke the Ca2+ oscillations and its specific role in RANKL-mediated osteoclast differentiation is not known. This study examined the effect of a partial loss of sarco/endoplasmic reticulum Ca2+ ATPase type2 (SERCA2) on osteoclast differentiation in SERCA2 heterozygote mice (SERCA2+/,). The BMD in the tibias of SERCA2+/, mice increased >1.5-fold compared with wildtype mice (WT). RANKL-induced [Ca2+]i oscillations were generated 48 h after RANKL treatment in the WT mice but not in the SERCA2+/, bone marrow,derived macrophages (BMMs). Forty-eight hours after RANKL treatment, there was a lower level of NFATc1 protein expression and markedly reduced translocation of NFATc1 into the nucleus during osteoclastogenesis of the SERCA2+/, BMMs. In addition, RANKL treatment of SERCA2+/, BMMs incompletely induced formation of multinucleated cells, leading to reduced bone resorption activity. These results suggest that RANKL-mediated induction of SERCA2 plays a critical role in the RANKL-induced [Ca2+]i oscillations that are essential for osteoclastogenesis. [source] Possible Involvement of I,B Kinase 2 and MKK7 in Osteoclastogenesis Induced by Receptor Activator of Nuclear Factor ,B Ligand,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2002Aiichiro Yamamoto Abstract Recent studies have revealed the essential role of the receptor activator of nuclear factor ,B (NF-,B) ligand (RANKL) in osteoclast differentiation and activation. Adenovirus vector could efficiently transduce genes into RAW264.7 cells, which differentiate into osteoclast-like multinucleated cells in the presence of RANKL. The role of NF-,B and c- jun N-terminal kinase (JNK) activation in RANKL-induced osteoclast differentiation was investigated using an adenovirus vector carrying the dominant negative I,B kinase 2 gene (AxIKK2DN) or dominant negative MKK7 gene (AxMKK7DN). IKK2DN and MKK7DN overexpression in RAW cells specifically suppressed the NF-,B activation and JNK activation in response to RANKL, respectively, without affecting other signaling pathways. Either inhibition of NF-,B or JNK pathways dose-dependently inhibited osteoclast formation induced by RANKL. These results suggest that both NF-,B and JNK activation are independently required for osteoclast differentiation. [source] Optimized transfection of diced siRNA into mature primary human osteoclasts: Inhibition of cathepsin K mediated bone resorption by siRNAJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2005Christina I. Selinger Abstract Osteoclasts are large multinucleated cells responsible for bone resorption. Bone resorption is dependent on the liberation of calcium by acid and protease destruction of the bone matrix by proteinases. The key proteinase produced by the osteoclast is cathepsin K. Targeted knock-down of cathepsin K was performed using small inhibitory RNA (siRNA). siRNA is a method that introduces short double-stranded RNA molecules that instruct the RNA-induced silencing complex (RISC) to degrade mRNA species complementary to the siRNA. Transfection of siRNA by lipid cations allows for short-term inhibition of expression of the targeted gene. We show that transfection of primary human osteoclasts with siRNA to cathepsin K reduces expression by ,60% and significantly inhibits bone resorption with a reduction of both resorption pit numbers (P,=,0.018) and resorbed area (P,=,0.013). We also show that FuGENE 6 is an effective lipid transfection reagent with which to transfect primary human osteoclasts, that does not produce off-target effects. © 2005 Wiley-Liss, Inc. [source] Regulation of osteoclastogenesis and RANK expression by TGF-,1JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2001Tao Yan Abstract Transforming growth factor-, (TGF-,) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-, on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-,B ligand (RANK-L) (50 ng/ml), TGF-,1 (0.01,5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-,1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-,1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-,1 resulted in a significant increase in the percentage of RANK+ RAW cells (P,<,0.05), as well as an increase in the fluorescence intensity per cell (P,<,0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-, directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression. J. Cell. Biochem. 83: 320,325, 2001. © 2001 Wiley-Liss, Inc. [source] Formation of osteoclast-like cells from peripheral blood of periodontitis patients occurs without supplementation of macrophage colony-stimulating factorJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 7 2008Stanley T. S. Tjoa Abstract Aim: To determine whether peripheral blood mononuclear cells (PBMCs) from chronic periodontitis patients differ from PBMCs from matched control patients in their capacity to form osteoclast-like cells. Material and Methods: PBMCs from 10 subjects with severe chronic periodontitis and their matched controls were cultured on plastic or on bone slices without or with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor- ,B ligand (RANKL). The number of tartrate-resistant acid phosphatase-positive (TRACP+) multinucleated cells (MNCs) and bone resorption were assessed. Results: TRACP+ MNCs were formed under all culture conditions, in patient and control cultures. In periodontitis patients, the formation of TRACP+ MNC was similar for all three culture conditions; thus supplementation of the cytokines was not needed to induce MNC formation. In control cultures, however, M-CSF or M-CSF/RANKL resulted in higher numbers compared with cultures without cytokines. Upregulations of osteoclast marker mRNA cathepsin K and carbonic anhydrase II confirmed the osteoclastic character. Bone resorption was only observed when PBMCs were cultured in the presence of M-CSF and RANKL. Conclusion: Our data indicate that PBMCs from periodontitis patients do not need priming by M-CSF to become osteoclast-like cells, suggesting that PBMCs from periodontitis patients are present in the circulation in a different state of activity. [source] Resistance to experimental tumorigenesis in cells of a long-lived mammal, the naked mole-rat (Heterocephalus glaber)AGING CELL, Issue 4 2010Sitai Liang Summary The naked mole-rat (NMR, Heterocephalus glaber) is a long-lived mammal in which spontaneous cancer has not been observed. To investigate possible mechanisms for cancer resistance in this species, we studied the properties of skin fibroblasts from the NMR following transduction with oncogenes that cause cells of other mammalian species to form malignant tumors. Naked mole-rat fibroblasts were transduced with a retrovirus encoding SV40 large T antigen and oncogenic RasG12V. Following transplantation of transduced cells into immunodeficient mice, cells rapidly entered crisis, as evidenced by the presence of anaphase bridges, giant cells with enlarged nuclei, multinucleated cells, and cells with large number of chromosomes or abnormal chromatin material. In contrast, similarly transduced mouse and rat fibroblasts formed tumors that grew rapidly without crisis. Crisis was also observed after > 40 population doublings in SV40 TAg/Ras-expressing NMR cells in culture. Crisis in culture was prevented by additional infection of the cells with a retrovirus encoding hTERT (telomerase reverse transcriptase). SV40 TAg/Ras/hTERT-expressing NMR cells formed tumors that grew rapidly in immunodeficient mice without evidence of crisis. Crisis could also be induced in SV40 TAg/Ras-expressing NMR cells by loss of anchorage, but after hTERT transduction, cells were able to proliferate normally following loss of anchorage. Thus, rapid crisis is a response of oncogene-expressing NMR cells to growth in an in vivo environment, which requires anchorage independence, and hTERT permits cells to avoid crisis and to achieve malignant tumor growth. The unique reaction of NMR cells to oncogene expression may form part of the cancer resistance of this species. [source] Granular cell atypical fibroxanthomaJOURNAL OF CUTANEOUS PATHOLOGY, Issue 4 2005Sarah N. Rudisaile Both neoplasms were solitary, light-tan, dome-shaped papules on sun-exposed areas of the head in two elderly white men. Microscopically, these neoplasms showed a dermal proliferation of pleomorphic granular cells with irregular hyperchromatic nuclei, multinucleated cells, and scattered mitoses. Immunohistochemical stains were positive for CD68 and vimentin and negative for Melan-A or human melanoma black (HMB)-45, S-100 protein, pancytokeratin, and actin, consistent with atypical fibroxanthoma. The differential diagnosis of granular cells in neoplasms containing cytological pleomorphism is challenging in view of the many different neoplasms that may present with granular cytoplasm. These include the conventional granular cell tumor and its malignant form, leiomyoma, leiomyosarcoma, dermatofibroma, dermatofibrosarcoma protuberans, and angiosarcoma. [source] HMB-45 (gp103) and MART-1 expression within giant cells in an atypical fibroxanthoma: a case reportJOURNAL OF CUTANEOUS PATHOLOGY, Issue 3 2004Megan J. Smith-Zagone Background:, Atypical fibroxanthoma (AFX) is a cutaneous tumor that primarily occurs in the sun-damaged skin of the head and neck of adults. It is often a rapidly growing, solitary lesion that may clinically resemble squamous cell carcinoma, malignant melanoma, or lobular hemangioma. The histologic differential diagnosis primarily includes spindle cell squamous carcinoma and spindle cell melanoma, and immunohistochemical studies are often needed to establish the diagnosis. Case report:, We report an unusual case of an AFX with aberrant HMB-45 and MART-1 (melanoma antigen recognized by T cells-1) immunohistochemical expression. Clinical information was obtained. Histologic examination and immunohistochemical studies were performed. Results:, A 54-year-old woman presented with a 1.5 cm posterior scalp lesion, which was excised. Microscopic examination revealed a dermal tumor composed of pleomorphic and spindled cells with numerous giant cells. The tumor cells expressed CD68 but did not express either keratin or S-100. In addition, there was focal gp100 (with HMB-45) and MART-1 expression limited to the large, multinucleated cells with vacuolated cytoplasm. A diagnosis of AFX was subsequently made. Conclusions:, This is the first reported case of an AFX with HMB-45 and MART-1 reactivity. [source] A mutant telomerase defective in nuclear-cytoplasmic shuttling fails to immortalize cells and is associated with mitochondrial dysfunctionAGING CELL, Issue 2 2010Olga A. Kovalenko Summary Telomerase is a reverse transcriptase specialized in telomere synthesis. The enzyme is primarily nuclear where it elongates telomeres, but many reports show that the catalytic component of telomerase (in humans called hTERT) also localizes outside of the nucleus, including in mitochondria. Shuttling of hTERT between nucleus and cytoplasm and vice versa has been reported, and different proteins shown to regulate such translocation. Exactly why telomerase moves between subcellular compartments is still unclear. In this study we report that mutations that disrupt the nuclear export signal (NES) of hTERT render it nuclear but unable to immortalize cells despite retention of catalytic activity in vitro. Overexpression of the mutant protein in primary fibroblasts is associated with telomere-based cellular senescence, multinucleated cells and the activation of the DNA damage response genes ATM, Chk2 and p53. Mitochondria function is also impaired in the cells. We find that cells expressing the mutant hTERT produce high levels of mitochondrial reactive oxygen species and have damage in telomeric and extratelomeric DNA. Dysfunctional mitochondria are also observed in an ALT (alternative lengthening of telomeres) cell line that is insensitive to growth arrest induced by the mutant hTERT showing that mitochondrial impairment is not a consequence of the growth arrest. Our data indicate that mutations involving the NES of hTERT are associated with defects in telomere maintenance, mitochondrial function and cellular growth, and suggest targeting this region of hTERT as a potential new strategy for cancer treatment. [source] NFATc1 and TNF, expression in giant cell lesions of the jawsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 3 2010Fabrķcio Rezende Amaral J Oral Pathol Med (2010) 39: 269,274 Background:, Activation mutations of SH3BP2 gene have been demonstrated in cherubism and central giant cell lesion (CGCL). In the present study we first attempted to investigate the SH3BP2 gene in peripheral giant cell lesion (PGCL). The effect of SH3BP2 gene mutations on the transcription of the downstream genes nuclear factor of activated T cells (NFATc1) and the cytokine tumor necrosis factor-, (TNF-,) was also investigated together with the immunolocalization of NFATc1 protein in a set of cases of PGCL, CGCL and cherubism with and without SH3BP2 mutation. Method:, Fresh samples of five PGCL, five CGCL and one cherubism cases were included in this study. One of the samples of CGCL presented a somatic heterozygous mutation c.1442A>T in exon 11. The cherubism case showed a heterozygotic substitution c.320C>T in both blood and lesion. These mutations were previously published. All coding and flanking regions of the SH3BP2 gene were sequenced in the cases of PGCL. The real-time polymerase chain reaction (RT-PCR) was performed to analyze the transcription of NFATc1 and TNF-, genes. The immunohistochemical analysis of the NFATc1 protein was also performed. Results:, No SH3BP2 gene mutation was found in PGCL. The RT-PCR showed increased expression of NFATc1 and decreased transcription of TNF-, in all the samples. The immunohistochemical analysis of the NFATc1 protein showed a predominant nuclear staining in the multinucleated giant cells. Conclusion:, The development of giant cells lesions of the jaws and cherubism are possibly mediated by overexpression of NFAT in the nucleus of the multinucleated cells. [source] Platelet-rich plasma impairs osteoclast generation from human precursors of peripheral bloodJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2010Elisabetta Cenni Abstract Platelet-rich plasma is used to accelerate bone repair for the release of osteogenic growth factors from activated platelets. To date, the effects on osteoclasts have been only scarcely investigated, even though these cells are crucial for bone remodeling. The aim of this research was the evaluation of the effects of thrombin-activated platelets (PRP) on osteoclastogenesis from human blood precursors. We evaluated both the ability to influence osteoclast differentiation induced by the receptor activator of nuclear factor-kappaB ligand (RANKL), and the ability to induce osteoclast differentiation without RANKL. In both assays, the incubation with PRP supernatant at 10% did not significantly affect the formation of tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells that were able to form the F-actin ring. However, when PRP at 25 and 50% was added to the medium without RANKL, the generation of TRACP-positive multinucleated cells was inhibited. PRP, even at 10%, reduced the osteoclast-mediated bone collagen degradation, suggesting inhibition of osteoclast activation. Similarly, after incubation with PRP supernatant, calcitonin receptor mRNA was lower than the untreated samples. In conclusion, PRP at 10% interfered with the complete differentiation process of human osteoclast precursors. At higher concentration it impaired osteoclast formation also at an early stage of differentiation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:792,797, 2010 [source] Ceramide inhibits development and cytokinesis and induces apoptosis in preimplantation bovine embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2008de Castro e Paula Luiz Augusto Abstract Ceramide is a second messenger induced by various cellular insults that plays a regulatory role in apoptosis. The objective of the present study was to determine whether ceramide signaling can occur in the preimplantation embryo by testing (1) effects of ceramide on development, cytokinesis, and apoptosis and (2) whether heat shock, which can induce apoptosis in embryos, causes activation of neutral or acidic sphingomyelinases responsible for generation of ceramide. Treatment of embryos ,16 cells collected at Day 5 after insemination with 50 µM C(2)-ceramide increased caspase-9 activity and the proportion of blastomeres undergoing apoptosis but did not increase caspase-8 activity. Induction of apoptosis was more extensive when culture with ceramide was for 24 hr than for 9 hr. Ceramide also reduced the proportion of embryos that developed to the blastocyst stage when exposure was for 24 hr. At the two-cell stage, a period in development when apoptosis responses are blocked, culture of embryos with ceramide did not increase caspase-9 activity or the proportion of blastomeres that were apoptotic. However, culture with ceramide for 24 hr reduced cell proliferation and caused an increase in multinucleated cells because of inhibition of cytokinesis. Exposure of Day 5 embryos to a heat shock of 41°C for 15 hr increased neutral sphingomyelinase activity but did not change acid sphingomyelinase activity. In conclusion, ceramide can regulate embryo development and apoptosis in a time and stage-of-development dependent manner and ceramide generation can be activated by cellular insult. Thus, the ceramide signaling pathway is present in the preimplantation embryo. Mol. Reprod. Dev. 75: 1063,1070, 2007. © 2007 Wiley-Liss, Inc. [source] Interleukin-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of nuclear factor of activated T cells c1 and suppressing proximal RANK signalingARTHRITIS & RHEUMATISM, Issue 2 2010George D. Kalliolias Objective Interleukin-27 (IL-27) has stimulatory and regulatory immune functions and is expressed in rheumatoid arthritis (RA) synovium. This study was undertaken to investigate the effects of IL-27 on human osteoclastogenesis, to determine whether IL-27 can stimulate or attenuate the osteoclast-mediated bone resorption that is a hallmark of RA. Methods Osteoclasts were generated from blood-derived human CD14+ cells. The effects of IL-27 on osteoclast formation were evaluated by counting the number of tartrate-resistant acid phosphatase,positive multinucleated cells and measuring the expression of osteoclast-related genes. The induction of nuclear factor of activated T cells c1 (NFATc1) and the activation of signaling pathways downstream of RANK were measured by immunoblotting. The expression of key molecules implicated in osteoclastogenesis (NFATc1, RANK, costimulatory receptors, and immunoreceptor tyrosine,based activation motif,harboring adaptor proteins) was measured by real-time reverse transcription,polymerase chain reaction. Murine osteoclast precursors obtained from mouse bone marrow and synovial fluid macrophages derived from RA patients were also tested for their responsiveness to IL-27. Results IL-27 inhibited human osteoclastogenesis, suppressed the induction of NFATc1, down-regulated the expression of RANK and triggering receptor expressed on myeloid cells 2 (TREM-2), and inhibited RANKL-mediated activation of ERK, p38, and NF-,B in osteoclast precursors. Synovial fluid macrophages from RA patients were refractory to the effects of IL-27. In contrast to the findings in humans, IL-27 only moderately suppressed murine osteoclastogenesis, and this was likely attributable to low expression of the IL-27 receptor subunit WSX-1 on murine osteoclast precursors. Conclusion IL-27 inhibits human osteoclastogenesis by a direct mechanism that suppresses the responses of osteoclast precursors to RANKL. These findings suggest that, in addition to its well-known antiinflammatory effects, IL-27 plays a homeostatic role in restraining bone erosion. This homeostatic function is compromised under conditions of chronic inflammation such as in RA synovitis. [source] Development of an ex vivo cellular model of rheumatoid arthritis: Critical role of cd14-positive monocyte/macrophages in the development of pannus tissueARTHRITIS & RHEUMATISM, Issue 9 2007Toshiko Nozaki Objective To establish an ex vivo cellular model of pannus, the aberrant overgrowth of human synovial tissue (ST). Methods Inflammatory cells that infiltrated pannus tissue from patients with rheumatoid arthritis (RA) were collected without enzyme digestion, and designated as ST-derived inflammatory cells. Single-cell suspensions of ST-derived inflammatory cells were cultured in medium alone. Levels of cytokines produced in culture supernatants were measured using enzyme-linked immunosorbent assay kits. ST-derived inflammatory cells were transferred into the joints of immunodeficient mice to explore whether these cells could develop pannus. CD14 and CD2 cells were depleted by negative selection. Results Culture of ST-derived inflammatory cells from 92 of 111 patients with RA resulted in spontaneous reconstruction of inflammatory tissue in vitro within 4 weeks. Ex vivo tissue contained fibroblasts, macrophages, T cells, and tartrate-resistant acid phosphatase,positive multinucleated cells. On calcium phosphate,coated slides, ST-derived inflammatory cell cultures showed numerous resorption pits. ST-derived inflammatory cell cultures continuously produced matrix metalloproteinase 9 and proinflammatory cytokines associated with osteoclastogenesis, such as tumor necrosis factor ,, interleukin-8, and macrophage colony-stimulating factor. More importantly, transferring ST-derived inflammatory cells into the joints of immunodeficient mice resulted in the development of pannus tissue and erosive joint lesions. Both in vitro development and in vivo development of pannus tissue by ST-derived inflammatory cells were inhibited by depleting CD14-positive, but not CD2-positive, cells from ST-derived inflammatory cells. Conclusion These findings suggest that overgrowth of inflammatory cells from human rheumatoid synovium simulates the development of pannus. This may prove informative in the screening of potential antirheumatic drugs. [source] Olfactomedin 4 promotes S-phase transition in proliferation of pancreatic cancer cellsCANCER SCIENCE, Issue 3 2007Daisuke Kobayashi Induction of olfactomedin 4 (OLFM4GW112/hGC-1) in cancer cells was recently reported to have a novel antiapoptotic action via binding to the potent apoptosis inducer GRIM-19. We sought to clarify undiscovered functions of constitutively expressed OLFM4 in cancer cells. OLFM4 mRNA was highly expressed in pancreatic cancer tissues. In PANC-1 cell cultures, expression was especially elevated during early S phase of the cell cycle. Transduction of small interfering RNA for OLFM4 to decrease mRNA expression caused time-dependent growth inhibition, with typical early S-phase arrest after 6 days. In addition, cell volume increased without increases in multinucleated cells, consistent with premitotic inhibition of DNA synthesis. Inhibition of OLFM4 mRNA expression by small interfering RNA did not promote apoptosis. Taken together, the results indicate that OLFM4 promotes proliferation of PANC-1 cells by favoring transition from the S to G2/M phase. (Cancer Sci 2007; 98: 334,340) [source] Collagen barrier membranes decrease osteoclastogenesis in murine bone marrow culturesCLINICAL ORAL IMPLANTS RESEARCH, Issue 6 2010Hermann Agis Abstract Objective: Collagen barrier membranes (CBM) are used for guided bone regeneration to support the process of graft consolidation. It remains, unknown however, whether CBM can affect the consolidation of bone grafts by controlling the differentiation of progenitor cells into bone-resorbing osteoclasts and bone-forming osteoblasts. Material and Methods: To gain an insight into the underlying mechanisms, we performed in vitro bone marrow cultures on CBM (Bio-Gide®) under conditions that favor osteoclastogenesis and osteoblastogenesis, respectively. Measures of osteoclastogenesis were based on the number of tartrate-resistant acid-phosphatase-positive (TRAP+) multinucleated cells. Resorption assays revealed the activity of mature osteoclasts. Osteoblastogenesis was determined by alkaline-phosphatase activity. Viability was investigated utilizing the MTT assay. Results: Cultivation of murine bone marrow on CBM reduced the number of TRAP+ multinucleated cells compared with cultures on tissue culture plates. Inhibition of osteoclastogenesis was observed on the porous and the dense CBM surfaces. The majority of TRAP+ cells were mononucleated and the decreased osteoclastogenesis was not due to changes in cell viability. Furthermore, CBM are inert regarding the resorptive activity of mature osteoclasts. Moreover, osteoblastogenesis was not reduced when bone marrow cells were grown on the surface of CBM. Conclusions: These in vitro findings demonstrate that CBM can reduce the formation but not the activity of multinucleated osteoclasts. Our data further reveal that the formation of osteogenic cells from their progenitors is not reduced by the CBM. Overall, our results suggest that the beneficial effects of CBM during graft consolidation may involve their direct impact on osteoclastogenesis. To cite this article: Agis H, Magdalenko M, Stögerer K, Watzek G, Gruber R. Collagen barrier membranes decrease osteoclastogenesis in murine bone marrow cultures. Clin. Oral Impl. Res. 21, 2010; 656,661. doi: 10.1111/j.1600-0501.2009.01888.x [source] | |||||||||||||