Formation Of Multinucleated Giant Cells Research Articles

Intraperitoneal kisspeptin-10 administration induces dose-dependent degenerative changes in maturing rat testes

AimsKisspeptin, a peptide secreted by hypothalamic neurons, is a critical regulator of reproduction and puberty but its role in the regulation of gonadal maturation in sexually immature males is elusive. The present study investigated the effects of 12days of pulsatile kisspeptin administration on gonadotropins and testosterone release and maturation of immature male gonads. Main methodsKisspeptin-10 was administered intraperitoneally at different dosage concentrations (1μg, 1ng, and 10pg) to 5weeks old prepubertal male rats, twice daily for 12days. Plasma LH, FSH and testosterone concentrations were measured through competitive-binding radioimmunoassay. Spermatogenesis was studied mainly at stage VII of the spermatogenic cycle through light and electron microscopy. Key findingsAt the end of the treatments plasma LH and testosterone concentrations were reduced significantly at 1ng and 1μg kisspeptin doses (P<0.05; P<0.01). Type A spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, step 7 spermatids, elongated spermatids and daily sperm production decreased significantly (P<0.05). Sertoli cell efficiency and total support capacity of Sertoli cells were reduced at all doses (P<0.05). Meiotic index decreased (P<0.05) at 1μg dose only, whereas coefficient of mitosis increased at 1ng and 1μg (P<0.01) kisspeptin doses. Histologically, degeneration of seminiferous tubules was evident showing tubular necrosis, multinucleated giant cell formation, intratubular vacuolization, widened lumen and deshaped germ cells. Marked ultrastructural changes characterized by thin basal laminae, enlarged intratubular spaces, abnormal acrosome and disrupted germ cells were noticeable. SignificanceIn conclusion long-term kisspeptin-10 administration negatively regulates gonadal maturation in prepubertal testes.

Effects of gentamicin and gentamicin–RGD coatings on bone ingrowth and biocompatibility of cementless joint prostheses: An experimental study in rabbits

Antimicrobial coatings are of interest as a means to improve infection prophylaxis in cementless joint arthroplasty. However, those coatings must not interfere with the essential bony integration of the implants. Gentamicin–hydroxyapatite (gentamicin–HA) and gentamicin–RGD (arginine–glycine–aspartate)–HA coatings have recently been shown to significantly reduce infection rates in a rabbit infection prophylaxis model. The purpose of the current study was to investigate the in vitro elution kinetics and in vivo effects of gentamicin–HA and gentamicin–RGD–HA coatings on new bone formation, implant integration and biocompatibility in a rabbit model. In vitro elution testing showed that 95% and 99% of the gentamicin was released after 12 and 24h, respectively. The in vivo study comprised 45 rabbits in total, with six animals for each of the gentamicin–HA, gentamicin–RGD–HA group and control pure HA coating groups for the 4week time period, and nine animals for each of the three groups for the 12week observation period. A 2.0mm steel K-wire with one of the coatings under test was placed in the intramedullary canal of the tibia. After 4 and 12weeks the tibiae were harvested and three different areas (proximal metaphysis, shaft area, distal metaphysis) were assessed by quantitative and qualitative histology for new bone formation, direct implant–bone contact and the formation of multinucleated giant cells. The results exhibited high standard deviations in all subgroups. There was a trend towards better bone formation and better direct implant contact in the pure HA coating group compared with both gentamicin coatings after 4 and 12weeks, which was, however, not statistically significant. The number of multinucleated giant cells did not differ significantly between the three groups at both time points. In summary, both gentamicin coatings with 99% release of gentamicin within 24h revealed good biocompatibility and bony integration, which was not statistically significant different compared with pure HA coating. Limitations of the study are the high standard deviation of the results and the limited number of animals per time point.

Multinucleated giant cells with an osteoclast phenotype derived from caprine peripheral blood mononuclear cells

Formation of multinucleated giant cells (MGCs) by macrophage fusion is a typical cytopathic effect of lentiviral replication in caprine monocytes and MGC formation from cultured caprine peripheral blood mononuclear cells (PBMCs) has been considered to be diagnostic for small ruminant lentivirus (SRLV) infection. In this study, formation of MGCs was observed after 7-14 days when PBMCs were cultured from healthy goats free from SRLV infection. These MGCs expressed tartrate-resistant acid phosphatase, calcitonin receptor, integrin αVβ3, cathepsin K and matrix metalloproteinase 9 and were able to resorb bone in vitro in the absence of RANKL and macrophage colony stimulating factor, consistent with an osteoclast phenotype.

Clinical significance of histopathologic and ultrastructural pathologic examination in etiological diagnosis of infantile cholestatic diseases

To study the features of histopathologic and ultrastructural pathologic changes of liver biopsy in patients with infantile cholestatic disease, and to investigate its diagnostic significance combining with the clinical data. Thirty-six children diagnosed as infantile cholestatic disease and received liver biopsy in Chongqing Medical University Children's Hospital from Jun 2007 to Oct 2008 were enrolled and the pathologic and ultrastructural pathologic changes of liver were analyzed. Morphologic changes under light microscope in liver tissues included hepatocyte swelling, hepatocyte denaturation, hepatocyte necrosis, multinucleated giant cell formation, bile duct proliferation, fiber tissues proliferation and inflammatory cells infiltration in liver lobules and portal regions. The characteristics of cholestasis including intralobular cholestasis, acinus formation, feather-like cytoplasmic filaments and bile stasis in bile canaliculi were observed. The morphologic changes of biliary atresia were observed in 7 cases whose image investigations showed no obstruction of biliary tract. Nuclear changes, resolution of cytoplasm, inflammatory cell infiltration, collagen fiber proliferation and increased number of lysosomes were observed under electromicroscope. Two cases of glycogen storage disease, 1 case of Niemann-Pick disease and 1 case of lipid storage disease with unknown cause were confirmed by the combination of histological changes and clinical manifestations. Common pathologic changes of liver tissues existed under light microscope or electroscope. The diagnosis of hereditary metabolic disorders could be made increasingly by application of these two technologies in clinical practice. It is difficult to diagnose biliary atresia in early childhood by image investigations and the pathological changes of liver tissues are helpful.

Methylprednisolone prevents tumour necrosis factor- -dependent multinucleated giant cell formation

Granulomas contain multinucleated giant cells (MGCs), the function of which remains largely unknown. In patients with autoimmune granulomatous disease, the granulomas can be resolved during treatment with glucocorticosteroid (GCS). However, little is known about the influence of GCSs on the formation of MGCs. Monocytes isolated from buffy coats were stimulated with IFN-γ and Concanavalin A to form MGCs either in presence or absence of TNF-α, methylprednisolone (MP), adalimumab or human immunoglobulin G. The concentrations of IL-1β, IL-6, IL-10, IL-12p70 and TNF-α in the culture supernatants were measured after 18 h of stimulation. MP, at a concentration of 0.1 µM, inhibited monocyte fusion (P < 0.05), and abrogated the formation of MGCs completely at higher concentrations. The production of IL-1β, IL-6 and TNF-α that accompanied MGC formation was reduced significantly by 10 µM MP. Recombinant TNF-α, at a concentration of 50 ng/ml, increased the number of formed MGCs and counteracted the inhibitory effect of 0.1 µM MP, while higher concentrations of MP still blocked MGC formation completely. Blockade of TNF-α with adalimumab within 16 h after stimulation significantly reduced the number MGCs formed (P < 0.05). MP inhibits the fusion of monocytes into MGCs, as well as the monocyte production of pro-inflammatory cytokines, which may be an important aspect of its beneficial effect in chronic granulomatous disorders. MCG formation can be promoted by TNF-α and inhibited by TNF-α blockade.

Influence of β-tricalcium phosphate granule size and morphology on tissue reaction in vivo

In this study the tissue reaction to five different β-tricalcium phosphate (β-TCP)-based bone substitute materials differing only in size, shape and porosity was analyzed over 60 days, at 3, 10, 15, 30 and 60 days after implantation. Using the subcutaneous implantation model in Wistar rats both the inflammatory response within the implantation bed and the resulting vascularization of the biomaterials were qualitatively and quantitatively assessed by means of standard and special histological staining methods. The data from this study showed that all investigated β-TCP bone substitutes induced the formation of multinucleated giant cells. Changes in size, shape and porosity influenced the integration of the biomaterials within the implantation bed and the formation of tartrate-resistant acid phosphatase (TRAP)-positive and TRAP-negative multinucleated giant cells, as well as the rate of vascularization. While a high porosity generally allowed cell and fiber in-growth within the center of the bone substitute, a lower porosity resulted in a mosaic-like integration of the materials, with the granules serving as place holders. The number of multinucleated giant cells located in the implantation bed positively correlated with the vascularization rate. These data emphasize that all biomaterials investigated were capable of inducing the formation of TRAP-positive multinucleated giant cells as a sign of biomaterial stability. Furthermore, these cells directly influenced vascularization by secretion of vascular endothelial growth factor (VEGF), as well as other chemokines. Based on these findings, the role of multinucleated giant cells in the foreign body reaction to biomaterials might need to be reconsidered. This study demonstrates that variations in the physical properties of a bone substitute material clearly influence the (extent of the) inflammatory reaction and its consequences.

A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells

We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and α-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 10 6 cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills.

HIV-1 Nef Triggers Macrophage Fusion in a p61Hck- and Protease-Dependent Manner

Macrophages are a major target of HIV-1 infection. HIV-1-infected macrophages form multinucleated giant cells (MGCs) using poorly elucidated mechanisms. In this study, we show that MGC formation was reduced when human macrophages were infected with nef-deleted HIV-1. Moreover, expression of Nef, an HIV-1 protein required in several aspects of AIDS, was sufficient to trigger the formation of MGCs in RAW264.7 macrophages. Among Nef molecular determinants, myristoylation was dispensable, whereas the polyproline motif was instrumental for this phenomenon. Nef has been shown to activate hematopoietic cell kinase (Hck), a Src tyrosine kinase specifically expressed in phagocytes, through a well-described polyproline-SH3 interaction. Knockdown approaches showed that Hck is involved in Nef-induced MGC formation. Hck is expressed as two isoforms located in distinct subcellular compartments. Although both isoforms were activated by Nef, only p61Hck mediated the effect of Nef on macrophage fusion. This process was abolished in the presence of a p61Hck kinase-dead mutant or when p61Hck was redirected from the lysosome membrane to the cytosol. Finally, lysosomal proteins including vacuolar adenosine triphosphatase and proteases participated in Nef-induced giant macrophage formation. We conclude that Nef participates in HIV-1-induced MGC formation via a p61Hck- and lysosomal enzyme-dependent pathway. This work identifies for the first time actors of HIV-1-induced macrophage fusion, leading to the formation of MGCs commonly found in several organs of AIDS patients.

Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro

Granulomatous lymphadenitis is one of the pathognomonic lesions in post-weaning multisystemic wasting syndrome (PMWS)-affected pigs. This unique lesion has not been reported in direct association with viral infection in pigs. The objective of the present study was to evaluate whether porcine circovirus type 2 (PCV2) alone is able to induce functional modulation in porcine monocytic cells in vitro to elucidate its possible role in the development of granulomatous inflammation. It was found that the proliferation activity of blood monocytes (Mo) and monocyte-derived macrophages (MDM) was significantly enhanced by PCV2. During monocyte-macrophage differentiation, the PCV2 antigen-containing rate and formation of multinucleated giant cells (MGC) were significantly increased in MDM when compared to those in Mo. The MDM-derived MGC displayed a significantly higher PCV2 antigen-containing rate than did the mono-nucleated MDM. Supernatants from PCV2-inoculated MDM at 24 h post-inoculation induced an increased tendency of chemotactic activity for blood Mo. At the same inoculation time period, levels of mRNA expression of the monocytic chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1, also significantly increased in PCV2-inoculated MDM. The results suggest that PCV2 alone may induce cell proliferation, fusion, and chemokine expression in swine monocytic cells. Thus, PCV2 itself may play a significant role in the induction of granulomatous inflammation in PMWS-affected pigs.

Cholate-Containing High-Fat Diet Induces the Formation of Multinucleated Giant Cells in Atherosclerotic Plaques of Apolipoprotein E−/− Mice

To determine the role of multinucleated giant cells (MGCs) in cardiovascular diseases. MGCs are a hallmark of giant cell arteritis. They are also described in atherosclerotic plaques from aortic aneurysms and carotid and coronary arteries. Herein, we demonstrate that the cholate-containing Paigen diet yields many MGCs in atherosclerotic plaques of apolipoprotein E-/- mice. These mice revealed a 4-fold increase in MGC numbers when compared with mice on a Western or Paigen diet without cholate. Most of the MGCs stained intensively for cathepsin K and were located at fibrous caps and close to damaged elastic laminae, with associated medial smooth muscle cell depletion. During in vitro experiments, MGCs demonstrated a 6-fold increase in elastolytic activity when compared with macrophages and facilitated transmigration of smooth muscle cells through a collagen-elastin matrix. An elastin-derived hexapeptide (Val-Gly-Val-Ala-Pro-Gly [VGVAPG]) significantly increased the rate of macrophage fusion, providing a possible mechanism of in vivo MGC formation. Comparable to the mouse model, human specimens from carotid arteries and aortic aneurysms contained cathepsin K-positive MGCs. Apolipoprotein E-/- mice fed a Paigen diet provide a model to analyze the tissue-destructive role of MGCs in vascular diseases.

Spontaneous Formation of Multinucleated Giant Cells with Bone Resorbing Activity by Long Culture of Human Peripheral Blood CD14-Positive MonocytesIn Vitro

Several reports indicate that multinucleated giant cells that derived from human peripheral blood CD14-positive monocytes have osteoclastic properties, and although the mechanism is not completely understood, the authors have previously demonstrated that spontaneous osteoclastogenesis from monocytes can occur. Here, the authors investigated the effect of detachment and long-term cultures in this process. When monocytes were incubated for 2 weeks, spontaneous formation of polykaryocytes was rarely observed. In addition, when monocytes precultured for 2 weeks were detached by a cell scraper and further subcultured, almost all cells died. Surprisingly, when monocytes were incubated for 8 weeks without any pro-osteoclastogenic factors and without detachment, the authors observed the spontaneous formation of tartrate-resistant acid phosphatase (TRAP)-positive polykaryocytes that were able of lacunae resorption. These findings indicate that cell adhesion is a prerequisite for differentiation and survival of CD14-positive monocytes, and that a long incubation period spontaneously induces multinucleation and bone-resorbing activity of monocytes, even in the absence of osteoclastogenesis-stimulating factors.

Thalidomide dramatically improves the symptoms of early‐onset Sarcoidosis/Blau syndrome: Its possible action and mechanism

Early-onset sarcoidosis (EOS), which occurs in children younger than 5 years of age, is associated with granulomatous lesions and a sporadic genetic mutation of the nucleotide-binding oligomerization domain 2 that causes constitutive NF-kappaB activation. The symptoms of EOS can be uncontrollable, progressive, and associated with profound complications. However, appropriate therapy is still under investigation. The aim of this study was to assess the efficacy of thalidomide in patients with severe EOS, based on etiology supporting an initial role of NF-kappaB in activation of this disease. Thalidomide was given to 2 patients with EOS (a 16-year-old girl and an 8-year-old boy) at an initial dosage of 2 mg/kg/day, and the dosage was increased if necessary. To elucidate the mechanism of the drug, peripheral blood monocytes were isolated from the patients and stimulated with cytokines (macrophage colony-stimulating factor, tumor necrosis factor alpha, and interleukin-4), and their ability to form multinucleated giant cells (MGCs) and osteoclasts was measured. Both patients showed dramatic improvement of their clinical symptoms (alleviation of fever and optic nerve papillitis, achievement of a response according to the American College of Rheumatology Pediatric 50 and Pediatric 70 criteria) and laboratory findings. Monocytes from patients with EOS had a greater ability to survive and induce MGCs and osteoclasts than those from healthy control subjects. The formation of MGCs and osteoclasts was inhibited by the presence of thalidomide. The ability of thalidomide to improve clinical symptoms and laboratory findings in patients with EOS indicates a central role for NF-kappaB activity in this disorder. Inhibition of IKK might be a pharmacologic action by which thalidomide down-regulates NF-kappaB signaling. Thalidomide may be an effective medication in patients with severe complications of EOS, including ocular involvement.

Pulmonary exposures to Sepiolite nanoclay particulates in rats: Resolution following multinucleate giant cell formation

Sepiolite is a magnesium silicate-containing nanoclay mineral and is utilized as a nanofiller for nanocomposite applications. We postulated that lung exposures to Sepiolite clay samples could produce sustained effects. Accordingly, the pulmonary and extrapulmonary systemic impacts in rats of intratracheally instilled Sepiolite nanoclay samples were compared with quartz or ultrafine (uf) titanium dioxide particle-types at doses of 1 mg/kg or 5 mg/kg. All particulates were well characterized, and dedicated groups were evaluated by bronchoalveolar lavage, lung cell proliferation, macrophage functional assays and full body histopathology at selected times postexposure (pe). Bronchoalveolar lavage results demonstrated that quartz particles produced persistent, dose-dependent lung inflammatory responses measured from 24 h through 3 months pe. Exposures to uf TiO 2 particles or Sepiolite samples produced transient neutrophilic responses at 24-h pe; however, unlike the other particle-types, Sepiolite exposures produced macrophage-agglomerates or multinucleate giant cells at 1 week, 5 weeks and 3 months pe. In vitro alveolar macrophage functional studies demonstrated that mononuclear cells recovered from quartz but not Sepiolite or uf TiO 2-exposed rats were deficient in their chemotactic capacities. Moreover, lung parenchymal cell proliferation rates were increased in rats exposed to quartz but not Sepiolite or uf TiO 2 particles. Histopathological evaluation of lung tissues revealed that pulmonary exposures to Sepiolite nanoclay or quartz samples produced inflammation in centriacinar regions at 24-h pe but the effects decreased in severity over time for Sepiolite and increased for quartz-exposed rats. The quartz-induced lesions were progressive and were characterized at 3 months by acinar foamy alveolar macrophage accumulation and septal thickening due to inflammation, alveolar Type II cell hyperplasia and collagen deposition. In the Sepiolite nanoclay group, the finding of multinucleated giant cell accumulation associated with minor collagen deposition in acinar regions was rarely observed. Exposures to ultrafine TiO 2 produced minimal effects characterized by the occurrence of phagocytic macrophages in alveolar ducts. Full body histopathology studies were conducted at 24 h and 3 months post particle exposures. Histopathological evaluations revealed minor particle accumulations in some mediastinal or thoracic lymph nodes. However, it is noteworthy that no extrapulmonary target organ effects were observed in any of the particle-exposed groups at 3 months postexposure.

Burkholderia mallei Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages

Burkholderia mallei is a facultative intracellular pathogen that causes severe disease in animals and humans. Recent studies have shown that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for survival in a hamster model of glanders. To better understand the role of T6SS-1 in the pathogenesis of disease, studies were initiated to examine the interactions of B. mallei tssE mutants with RAW 264.7 murine macrophages. Results obtained by utilizing modified gentamicin protection assays indicated that although the tssE mutants were able to survive within RAW 264.7 cells, significant growth defects were observed in comparison to controls. In addition, analysis of infected monolayers by differential interference contrast and fluorescence microscopy demonstrated that the tssE mutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy, tssE mutants were shown to undergo escape from lysosome-associated membrane protein 1-positive vacuoles. Curiously, however, following entry into the cytosol, the mutants exhibited actin polymerization defects resulting in inefficient intra- and intercellular spread characteristics. Importantly, all mutant phenotypes observed in this study could be restored by complementation. Based upon these findings, it appears that T6SS-1 plays a critical role in growth and actin-based motility following uptake of B. mallei by RAW 264.7 cells.

Complex Regulation of Tartrate-resistant Acid Phosphatase (TRAP) Expression by Interleukin 4 (IL-4)

Interleukin 4 (IL-4) inhibits receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast formation and functional activity in a STAT6-dependent manner. IL-4 down-regulates expression of tartrate-resistant acid phosphatase (TRAP) in mature osteoclasts. To determine whether IL-4 regulates TRAP promoter activity, RAW264.7 cells were transfected with a TRAP promoter-luciferase reporter. Treatment with IL-4 alone modestly enhanced TRAP luciferase activity. However, IL-4 suppressed the ability of RANKL to up-regulate TRAP-luciferase activity, suggesting that IL-4 has multiple effects on TRAP transcription. IL-4 also reduced the RANKL-induced association of RNA polymerase II with the TRAP gene in osteoclasts. The TRAP promoter contains a STAT6-binding motif, and STAT6 bound to the endogenous TRAP promoter after IL-4 treatment. To determine the impact of STAT6 binding, we transfected cells with STAT6VT, a constitutively active STAT6 mutant. STAT6VT alone up-regulated TRAP-luciferase activity; this effect was abrogated by mutating the STAT6 binding site in the minimal TRAP promoter. STAT6VT did not inhibit the potent up-regulation of TRAP promoter activity caused by overexpression of NFATc1, PU.1, and microphthalmia transcription factor, downstream targets of macrophage colony-stimulating factor and RANKL. IL-4 down-regulated the expression of c-Fos and NFATc1 in mature osteoclasts. Knockdown of NFATc1 by short interfering RNA caused TRAP expression to be down-regulated, and ectopic expression of NFATc1 abrogated the IL-4-induced down-regulation of TRAP. These results suggest that STAT6 plays two distinct roles in TRAP expression. The IL-4-induced activation of STAT6 mediates suppression of the RANKL-induced TRAP promoter activity indirectly by inhibiting NFATc1 expression. However, in the absence of RANKL and osteoclast differentiation, STAT6 binds the TRAP promoter after IL-4 treatment and directly enhances TRAP expression.

Dominant role of CD47–thrombospondin-1 interactions in myeloma-induced fusion of human dendritic cells: implications for bone disease

AbstractLytic bone disease in myeloma is characterized by an increase in multinucleate osteoclasts in close proximity to tumor cells. However, the nature of osteoclast precursors and the mechanisms underlying multinuclearity are less understood. Here we show that culture of myeloma cell lines as well as primary myeloma cells with human dendritic cells (DCs) but not monocytes or macrophages leads to spontaneous cell-cell fusion, which then leads to the facile formation of multinucleate bone-resorbing giant cells. Osteoclastogenesis is cell contact dependent, leading to up-regulation of thrombospondin-1 (TSP-1) in DCs. Disruption of CD47–TSP-1 interaction by TSP-1–blocking antibodies or down-regulation of CD47 on tumor cells by RNA interference abrogates tumor-induced osteoclast formation. Blockade of CD47–TSP-1 interactions also inhibits receptor activator for nuclear factor κB ligand- and macrophage colony-stimulating factor–induced formation of osteoclasts from human monocytes. Further, TSP-1 blockade attenuates hypercalcemia induced by parathyroid hormone in vivo. These data point to a role for CD47–TSP-1 interactions in regulating cell-fusion events involved in human osteoclast formation. They also suggest that DCs, known to be enriched in myeloma tumors, may be direct precursors for tumor-associated osteoclasts. Disruption of CD47–TSP-1 interactions or preventing the recruitment of DCs to tumors may provide novel approaches to therapy of myeloma bone disease and osteoporosis.

Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex

BackgroundEctromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium.ResultsWe show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection.ConclusionEctromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

Multi-nucleated giant cell formation from human cord blood monocytesin vitro, in comparison with adult peripheral blood monocytes

Multi-nucleated giant cells (MGCs; Langhans-type cell), formed from macrophage fusion, are recognized as a hallmark histological feature in chronic inflammation. However, their precise pathological role is still poorly understood, especially for microorganism pathogens in the neonatal immune system, which are capable of surviving intracellularly in phagocytes. To conduct a partial evaluation of the monocyte function of neonates, we investigated the ability of human cord blood monocytes to form MGCs in vitro by stimulating various cytokines and comparing them with adult peripheral blood monocytes. Monocytes from cord blood and adult peripheral blood were isolated and cultured for 14 days with cytokines known to induce MGC in vitro. The fusion index in experiments with a combination of interleukin (IL)-4 and macrophage colony-stimulating factor (M-CSF) and a combination of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly lower in cord blood than in adult blood monocytes (P = 0.0018 and P = 0.0141, respectively). The number of nuclei per MGC was significantly lower in cord blood than in adult blood monocytes in experiments with IL-4 alone, the combination of IL-4 and M-CSF, and the combination of IL-4 and GM-CSF (P < 0.0001). These results suggest the possibility that the susceptibility of newborns to mycobacterium infection is due partly to impaired MGC formation.

Development of a platelet-activating factor antagonist for HIV-1 associated neurocognitive disorders

The neuroregulatory activities of PMS-601, a platelet activating factor antagonist, were investigated in laboratory and animal models of HIV-1 encephalitis (HIVE). For the former, PMS-601 reduced monocyte-derived macrophage pro-inflammatory secretions, multinucleated giant cell (MGC) formation, and neuronal loss independent of antiretroviral responses. PMS-601 treatment of HIVE severe combined immunodeficient mice showed reduced microgliosis, MGCs and neurodegeneration. These observations support the further development of PMS-601 as an adjunctive therapy for HIV-1 associated neurocognitive disorders.

Distinct roles for tetraspanins CD9, CD63 and CD81 in the formation of multinucleated giant cells

Members of the tetraspanin superfamily of proteins are implicated in a variety of complex cell processes including cell fusion. However, the contribution of individual tetraspanins to these processes has proved difficult to define. Here we report the use of recombinant extracellular regions of tetraspanins to investigate the role of specific members of this family in the fusion of monocytes to form multinucleated giant cells (MGC). In contrast to their positive requirement in sperm-egg fusion, previous studies using antibodies and knockout mice have indicated a negative regulatory role for tetraspanins CD9 and CD81 in this process. In an in vitro model of fusion using human monocytes, we have confirmed observations that antibodies to CD9 and CD81 enhance MGC formation; however, in contrast to previous investigations, we found that all members of a panel of antibodies to CD63 inhibited fusion. Moreover, recombinant proteins corresponding to the large extracellular domains (EC2s) of CD63 and CD9 inhibited MGC formation, whereas the EC2s of CD81 and CD151 had no effect. The potent inhibition of fusion and binding of labelled CD63 EC2 to monocytes under fusogenic conditions suggest a direct interaction with a membrane component required for fusion. Our findings indicate that the tetraspanins CD9, CD63 and CD81 are all involved in MGC formation, but play distinct roles.