Endogenous secretory RAGE increases with improvements in body composition and is associated with markers of adipocyte health.

BACKGROUND AND AIMS: The receptor for advanced glycation end products (RAGE) is implicated in obesogenesis. Conversely, soluble RAGE (sRAGE) competitively inhibits RAGE. Our aim was to determine the effects of weight-loss via alternate day fasting (ADF) on sRAGE isoforms and evaluate potential relationships with body composition. METHODS AND RESULTS: 42 obese participants were randomized to control (CON) or ADF. For 24 weeks, the ADF group consumed 25% or 125% of their caloric requirements on alternating days while the CON group did not change their diet. Body fat was measured via DXA, visceral fat (VAT) via MRI and subcutaneous fat (SAT) was derived by subtracting VAT from total fat. sRAGE isoforms were measured via ELISAs. After 24 weeks, ADF -6.8 (-9.5, -3.5)kg (Median, IQR) lost more weight than CON -0.3 (-1.9, 1.0)kg (p < 0.05). The change in endogenous secretory RAGE (esRAGE) was different between ADF 15 (-30, 78)pg/mL and CON -21 (-72, 16)pg/mL after 24 weeks (p < 0.05). To examine the effect of changes in body composition, the cohort was stratified by median weight-, fat-, SAT-, and VAT-loss. The changes in all sRAGE isoforms were different between those above and below median weight-loss (p < 0.05) with sRAGE isoforms tending to decrease in individuals below the median. Changes in total sRAGE and esRAGE were different between individuals above compared to below median fat- and SAT-loss (p < 0.05). Those above median fat-loss increased esRAGE by 29 (-5, 66)pg/mL (p < 0.05). CONCLUSION: Improvements in body composition are related to increased sRAGE isoforms, implicating sRAGE as a potential target for the treatment of obesity. CLINICAL TRIAL REGISTRATION: NCT00960505.

Environmental fate and effect of biodegradable electro-spun scaffolds (biomaterial)-a case study.

Poly-ε-caprolactone (PCL) based medical devices are increasingly produced and thus, their presence in the environment is likely to increase. The present study analysed the biodegradation of PCL electro-spun scaffolds (alone) and PCL electro-spun scaffolds coated with human recombinant (hR) collagen and Bovine Achilles tendon (BAT) collagen in sewage sludge and in soil. Additionally, an eco-toxicological test with the model organism Enchytraeus crypticus was performed to assess environmental hazard of the produced materials in soils. The electro-spun scaffolds were exposed to activated sludge and three different soils for various time periods (0-7-14-21-28-56-180 days); subsequently the degradation was determined by weight loss and microscopical analysis. Although no toxicity occurred in terms of Enchytraeus crypticus reproduction, our data indicate that biodegradation was dependent on the coating of the material and exposure condition. Further, only partial PCL decomposition was possible in sewage treatment plants. Collectively, these data indicate that electro-spun PCL scaffolds are transferred to amended soils.

Neonatal outcomes based on antenatal corticosteroid exposure time for infants delivered between 23 and 34 weeks gestation.

OBJECTIVE: To determine the minimal time interval required after antenatal corticosteroid treatment to see improvement in neonatal outcomes. STUDY DESIGN: A retrospective cohort analysis was performed on all women who delivered an infant between 23 0/7 weeks and 33 6/7 weeks gestational age from January 1, 2009 to August 31, 2013. Maternal data collected: maternal race, parity, mode of delivery, indication for delivery, infant birth weight, antenatal corticosteroid (ACS) administration, and time from ACS until delivery. Neonatal data collected: respiratory distress syndrome (RDS), intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), sepsis, retinopathy of prematurity (ROP), intubation, surfactant administration, length of hospitalization, and mortality. RESULTS: Infants were grouped by ACS exposure time before delivery. Gestational age at delivery was similar between the groups. There was not a statisti- cally significant difference in the rate of RDS between the groups. Infants delivered 24 to 47 hours of ACS had the lowest rates of surfactant, intubation, and IVH. There appears to be a larger impact ofACS on infants delivered at 29 to 34 weeks vs. 23-28 weeks gestation. CONCLUSIONS: Improvement in neonatal outcomes are seen after any amount of ACS exposure but are generally most significant 24 to 47 hours after administration and between 29 to 34 weeks gestation.

Milk fat globule membrane isolated from buttermilk or whey cream and their lipid components inhibit infectivity of rotavirus in vitro.

Milk fat is encapsulated in a milk fat globule membrane (MFGM) that contains bioactive glycoproteins and glycolipids. The MFGM inhibits infectivity of rotavirus (RV), activity that has been attributed to its glycoprotein and carbohydrate components. However, previous studies of proteins and oligosaccharides in the MGFM have not accounted for all the bioactivity associated with the complete MFGM. The lipid fraction of the MFGM accounts for half of its composition by weight, and we postulate that this fraction should be tested by itself to determine if it plays a role in antiviral activity. Herein, the anti-RV activity of an organic extract of MFGM was tested. Natural and whey buttermilk powders containing bovine MFGM enriched in polar lipids were prepared by microfiltration and supercritical fluid extraction treatment to reduce the triglyceride content of the powders. Lipid fractions were then extracted from the MFGM using both single- and dual-phase extraction methods. Whole MFGM and organic extracts were screened in MA-104 cells for anti-infective activity against a neuraminidase-sensitive rotavirus using a focus-forming unit assay. Dose-dependent inhibition was observed for whole buttermilk and cheese whey MFGM against the rotavirus. In general, buttermilk MFGM exhibited greater RV percentage inhibition than cheese whey MFGM. Organic-soluble anti-RV compounds were identified in bovine MFGM. The most active fraction, isolated by dual-phase extraction and iatrobead chromatography, was free of proteins and highly nonpolar. Further separation of this fraction in a less polar solvent (30:1 chloroform:methanol) resolved at least 5 lipid-containing compounds, which likely contribute to the anti-RV activity associated with bovine MFGM. In summary, lipid components associated with MFGM appear to contribute in large part to the anti-RV activity associated with the bovine MFGM.

Translating Dental, Oral, and Craniofacial Regenerative Medicine Innovations to the Clinic through Interdisciplinary Commercial Translation Architecture.

Few university-based regenerative medicine innovations in the dental, oral, and craniofacial (DOC) space have been commercialized and affected clinical practice in the United States. An analysis of the commercial translation literature and National Institute for Dental and Craniofacial Research's (NIDCR's) portfolio identified barriers to commercial translation of university-based DOC innovations. To overcome these barriers, the NIDCR established the Dental Oral Craniofacial Tissue Regeneration Consortium. We provide generalized strategies to inform readers how to bridge the "valley of death" and more effectively translate DOC technologies from the research laboratory or early stage company environment to clinical trials and bring needed innovations to the clinic. Three valleys of death are covered: 1) from basic science to translational development, 2) from translational technology validation to new company formation (or licensing to an existing company), and 3) from new company formation to scaling toward commercialization. An adapted phase-gate model is presented to inform DOC regenerative medicine teams how to involve regulatory, manufacturability, intellectual property, competitive assessments, business models, and commercially oriented funding mechanisms earlier in the translational development process. An Industrial Partners Program describes how to conduct market assessments, industry maps, business development processes, and industry relationship management methods to sustain commercial translation through the later-stage valley of death. Paramount to successfully implementing these methods is the coordination and collaboration of interdisciplinary teams around specific commercial translation goals and objectives. We also provide several case studies for translational projects with an emphasis on how they addressed DOC biomaterials for tissue regeneration within a rigorous commercial translation development environment. These generalized strategies and methods support innovations within a university-based and early stage company-based translational development process, traversing the many funding gaps in dental, oral, and craniofacial regenerative medicine innovations. Although the focus is on shepherding technologies through the US Food and Drug Administration, the approaches are applicable worldwide.

TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts.

TRANCE (tumor necrosis factor-related activation-induced cytokine) is a recently described member of the tumor necrosis factor superfamily that stimulates dendritic cell survival and has also been found to induce osteoclastic differentiation from hemopoietic precursors. However, its effects on mature osteoclasts have not been defined. It has long been recognized that stimulation of osteoclasts by agents such as parathyroid hormone (PTH) occurs through a hormonal interaction with osteoblastic cells, which are thereby induced to activate osteoclasts. To determine whether TRANCE accounts for this activity, we tested its effects on mature osteoclasts. TRANCE rapidly induced a dramatic change in osteoclast motility and spreading and inhibited apoptosis. In populations of osteoclasts that were unresponsive to PTH, TRANCE caused activation of bone resorption equivalent to that induced by PTH in the presence of osteoblastic cells. Moreover, osteoblast-mediated stimulation of bone resorption was abrogated by soluble TRANCE receptor and by the soluble decoy receptor osteoprotegerin (OPG), and stimulation of isolated osteoclasts by TRANCE was neutralized by OPG. Thus, TRANCE expression by osteoblasts appears to be both necessary and sufficient for hormone-mediated activation of mature osteoclasts, and TRANCE-R is likely to be a receptor for signal transduction for activation of the osteoclast and its survival.

Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.

Macrophage colony-stimulating factor (M-CSF) is known to play an important role in osteoclast formation. However, its actions on mature cells have not been fully characterized. We now report that M-CSF dramatically stimulates osteoclastic motility and spreading; osteoclasts responded to a gradient of M-CSF with orientation, and random cell polarization occurred after isotropic exposure. M-CSF also supported the survival of osteoclasts by preventing apoptosis. Paradoxically, M-CSF inhibits bone resorption by isolated osteoclasts. We found that this was effected predominantly by reduction in the number of excavations. Thus, M-CSF showed a propensity to suppress resorption through a reduction in the proportion of cells that were resorbing bone. Our data suggest that apart from the established role of M-CSF in the provision of precursors for osteoclastic induction, a major role for M-CSF in bone resorption is to enhance osteoclastic survival, migration, and chemotaxis. It seems appropriate that during these processes resorptive functions should be suppressed. We suggest that M-CSF continues to modulate osteoclastic activity once osteoclasts are on resorptive sites, through regulation of the balance between resorption and migration, such that not only the quantity, but the spatial pattern of resorption can be controlled by adjacent M-CSF-secreting cells of osteoblastic lineage.

Growing season surface water loading of fecal indicator organisms within a rural watershed.

The loading of microbial contaminants was examined within the Thomas Brook watershed, a 784 ha mixed land-use catchment located in the headwaters of the Cornwallis River drainage basin (Nova Scotia, Canada). The objectives were to: (i) examine spatial and temporal characteristics of fecal bacteria loading during the growing season from five subwatersheds, and (ii) develop areal fecal indicator organism export coefficients for rural landscapes. Fecal coliform, Escherichia coli, total suspended solids (TSS) concentrations and stream flow were monitored at five locations in the watershed over six consecutive growing seasons (May-Oct, 2001-2006). A nested watershed monitoring approach was used to determine bacterial loading from distinct source types (residential vs. agricultural) during both baseflow and stormflow periods. Areal bacterial loading rates increased in each nested watershed moving downstream through the watershed and were highest in the three subcatchments dominated by agricultural activities. Upper watershed bacterial loading throughout the growing season from an agricultural subcatchment (Growing Season Avg 8.92 x 10(10) CFU ha(-1)) was consistently higher than a residential subcatchment (Growing Season Avg 8.43 x 10(9) CFU ha(-1)). As expected, annual average stormflow bacterial loads were higher than baseflow loads, however baseflow loads still comprised between 14 and 35% of the growing season bacterial loads in the five subwatersheds. Fecal bacteria loads were greater during years with higher annual precipitation. A positive linear relationship was observed between E. coli and TSS loading during the 2005 and 2006 growing seasons when both parameters were monitored, indicating that the processes of sediment transport and bacterial transport are linked. It is anticipated that computed areal microbial loading coefficients will be useful in developing watershed management plans. More intensive sampling during stormflow events is recommended for improving these coefficients.

A standardised protocol for the evaluation of small extracellular vesicles in plasma by imaging flow cytometry.

Flow cytometry provides robust, multi-parametric and quantitative information on single cells which also exhibits enormous potential as a tool for small particle characterisation. Small extracellular vesicle (sEV) detection by flow cytometry remains compromised due to the high prevalence of swarm detection, which is defined by the simultaneous illumination of more than one sEV, recorded as a single event. Detection of sEVs by imaging flow cytometry presents a major advantage by having the ability to resolve single particles from swarm detection based on the image features recorded for each event. In this study, we provide a simplified protocol that facilitates the removal of both swarm events and aggregated particles to improve the accuracy of sEV analysis. Our results indicate that imaging flow cytometry should be at the forefront as a robust and sensitive technique for sEV characterisation.

Aberrant prostaglandin synthase 2 expression defines an antigen-presenting cell defect for insulin-dependent diabetes mellitus.

Prostaglandins (PGs) are lipid molecules that profoundly affect cellular processes including inflammation and immune response. Pathways contributing to PG output are highly regulated in antigen-presenting cells such as macrophages and monocytes, which produce large quantities of these molecules upon activation. In this report, we demonstrate aberrant constitutive expression of the normally inducible cyclooxygenase PG synthase 2 (PGS(2)/ COX-2) in nonactivated monocytes of humans with insulin-dependent diabetes mellitus (IDDM) and those with islet autoantibodies at increased risk of developing this disease. Constitutive PGS(2) appears to characterize a high risk for diabetes as it correlates with and predicts a low first-phase insulin response in autoantibody-positive subjects. Abnormal PGS(2) expression in at-risk subjects affected immune response in vitro, as the presence of a specific PGS(2) inhibitor, NS398, significantly increased IL-2 receptor alpha-chain (CD25) expression on phytohemagglutinin-stimulated T cells. The effect of PGS(2) on CD25 expression was most profound in subjects expressing both DR04 and DQbeta0302 high-risk alleles, suggesting that this cyclooxygenase interacts with diabetes-associated MHC class II antigens to limit T-cell activation. These results indicate that constitutive PGS(2) expression in monocytes defines an antigen-presenting cell defect affecting immune response, and that this expression is a novel cell-associated risk marker for IDDM.

Influence of Nonsulfated Polysaccharides on the Properties of Electrospun Poly(lactic-co-glycolic acid) Fibers.

Biomimetic tissue engineering aspires to develop bioinspired implantable devices that would positively interact with the host. Given that glycosaminoglycans are involved in many physiological processes, whereas their deprivation is associated with pathophysiologies, functionalization of implantable devices with natural and/or synthetic carbohydrate moieties is at the forefront of scientific research and industrial innovation. Herein, we venture to assess the influence of various concentrations (0.01%, 0.1%, 1%) of hyaluronic acid and Ficoll on the structural, thermal, biomechanical and biological (human osteoblasts) properties of electrospun poly(lactic-co-glycolic acid) fibers. The addition of hyaluronic acid and Ficoll reduced the fiber diameter, with the 1% hyaluronic acid exhibiting the smallest fibers diameter (p < 0.001). Neither the addition of hyaluronic acid nor the addition Ficoll significantly affected the onset and peak temperatures (p > 0.05). All hyaluronic acid and Ficoll treatments significantly reduced stress at break, strain at break and elastic modulus values (p < 0.001). Hyaluronic acid and Ficoll did not affect osteoblast viability and metabolic activity temperatures (p > 0.05); the 1% hyaluronic acid and Ficoll significantly increased (p < 0.001) osteoblast proliferation at day 21. By day 21, the 1% hyaluronic acid and 1% Ficoll fibers showed the highest alkaline phosphatase activity and calcium deposition. At day 21, osteocalcin was not detected, whereas osteopontin was detected on all samples. Collectively, our data clearly illustrate the biological benefit of nonsulfated polysaccharides as functionalization molecules.

Syndrome of Inappropriate Anti-Diuretic Hormone Secretion Secondary to Strongyloides stercoralis Infection in an Allogeneic Stem Cell Transplant Patient: A Case Report and Literature Review.

Syndrome of inappropriate anti-diuretic hormone (SIADH) has been reported to be associated with systemic Strongyloides stercoralis. Here, we report a case of a stem cell transplant (SCT) recipient who developed severe SIADH secondary to systemic S Stercoralis. The SIADH resolved quickly after treating the systemic S Stercoralis with ivermectin. A systematic review of the literature was performed by PubMed, Scopus, and Cochrane database search. Only eight cases of S Stercoralis in allogeneic SCT recipients have been previously reported. To our knowledge, ours is the first reported case of SIADH secondary to S Stercoralis infection in an allogeneic SCT recipient. Prior to transplantation, even if asymptomatic, patients from endemic regions should be screened with strongyloides immunoglobulin (Ig)G serology. Pretransplantation eosinophilia should be evaluated by screening multiple stool samples for ova and parasites. Transplant candidates with positive serology or stool tests can be treated pretransplantation to eradicate infection. Patients at risk for S Stercoralis who develop nonspecific gastrointestinal complaints, rash, pulmonary infiltrates, or gram-negative bacteremia or meningitis may have S Stercoralis hyperinfection syndrome. Our case indicates that the development of SIADH may be an additional clue to this diagnosis. Appropriate diagnostic studies, including repeat stool and other body fluid sampling, should be expedited and ivermectin therapy initiated rapidly to prevent significant morbidity and mortality.

Fungal Light Sensing at the Bench and Beyond.

Visible light is a pervasive environmental signal that orients most organisms in space and time. For a fungus, the detection of light is facilitated by diverse classes of photoreceptor proteins, which in turn coordinate growth, spore dispersal, stress resistance, primary metabolism, and toxin production. We will first provide a discussion on signal input, focusing on recent insights into how fungal photoreceptors detect and transmit information at the biochemical and molecular levels. We will then pivot our discussion to how light influences fungal behaviors that are of industrial, agricultural, or even medical relevance. Because the light environment can be easily manipulated in many contexts, we will argue that understanding fungal photobiology is both an important basic and applied endeavor.

TGF-α and IL-6 plasma levels selectively identify CML patients who fail to achieve an early molecular response or progress in the first year of therapy.

Early molecular response (EMR, BCR-ABL1 (IS)⩽10% at 3 months) is a strong predictor of outcome in imatinib-treated chronic phase chronic myeloid leukemia (CP-CML) patients, but for patients who transform early, 3 months may be too late for effective therapeutic intervention. Here, we employed multiplex cytokine profiling of plasma samples to test newly diagnosed CP-CML patients who subsequently received imatinib treatment. A wide range of pro-inflammatory and angiogenesis-promoting cytokines, chemokines and growth factors were elevated in the plasma of CML patients compared with that of healthy donors. Most of these normalized after tyrosine kinase inhibitor treatment while others remained high in remission samples. Importantly, we identified TGF-α and IL-6 as novel biomarkers with high diagnostic plasma levels strongly predictive of subsequent failure to achieve EMR and deep molecular response, as well as transformation to blast crisis and event-free survival. Interestingly, high TGF-α alone can also delineate a poor response group raising the possibility of a pathogenic role. This suggests that the incorporation of these simple measurements to the diagnostic work-up of CP-CML patients may enable therapy intensity to be individualized early according to the cytokine-risk profile of the patient.

Identification and characterization of the murine Rag1 promoter.

Rag1 and Rag2 are required for the somatic rearrangement of immunoglobulin genes and T-cell receptor genes and the subsequent development of B and T cells. We describe the pattern of DNase I hypersensitive sites surrounding the Rag1 gene that accompanies mouse B-cell development and show that one of these sites corresponds to the murine Rag1 promoter. Transcription initiates over a 30 bp region, with approximately 70% of the transcripts initiating within a 5 bp region. The promoter contains neither a consensus TATA box nor an initiator, but does contain an AT rich sequence that could serve as a non-consensus TATA box. The Rag1 promoter directs only negligible levels of expression in transient transfection assays, but when combined with a heterologous enhancer, it is capable of driving significant levels of expression in pre-B cells, pre-T cells, and mature B cells. Methylation interference and mutation analysis reveal that the Rag1 promoter contains binding sites for E-box binding proteins, NF-Y proteins, and Ikaros proteins. These findings are discussed with respect to B-cell development and regulation of differential Rag expression by the promoter in pre-B, pre-T, and CNS cells.

Effect of arachidonic acid metabolites on bone resorption by isolated rat osteoclasts.

Arachidonic acid metabolites (eicosanoids) have major effects on bone but their role is unclear. Many are known to stimulate bone resorption in organ culture, but paradoxically, previous work has suggested that at least some of them act as direct inhibitors of osteoclastic function. In an attempt to clarify the role of eicosanoids in bone physiology, we have defined the duration of action and relative potencies of prostaglandin (PG) E1 and E2 and have extended the range of eicosanoids tested on isolated osteoclasts. We have found that PGE1 and PGE2 inhibited bone resorption by isolated osteoclasts for at least 6 h. Inhibition was followed by recovery to control, not supranormal levels. Bone resorption was inhibited in the range 10(-5)-10(-9) M for PGE1 and PGE2, and the rank order as resorption inhibitors was PGE1 greater than 6-keto PGE1 greater than PGE2 greater than PGA2 greater than PGB2. None of the products of lipoxygenase metabolism showed a significant direct effect. The effects of PGE1 and PGE2 were not antagonistic. Prostaglandin production does not seem to be implicated as a second messenger for the action of calcitonin. Although inhibition of osteoclasts by PGs was less prolonged than that observed in the presence of calcitonin, the sensitivity of osteoclasts to inhibition by PGs, and the duration of the effect without subsequent direct stimulation, suggests that inhibition of osteoclastic resorption is a major physiological role of PG production in bone.

Bone matrix stimulates osteoclastic differentiation in cultures of rabbit bone marrow cells.

Cells showing osteoclastic characteristics have not been identified outside bone. Because osteoclasts originate from an extraosseous source, this suggests that identifiable osteoclastic features do not develop until the precursors enter bone, where the local microenvironment may signal osteoclastic differentiation or maturation. We assessed the influence of bone matrix on osteoclastic differentiation by incubating bone marrow cells, after removal of pre-existing osteoclasts, on plastic coverslips or slices of devitalized cortical bone. We found that there was a threefold increase in the number of osteoclast-specific MAb-positive cells on the bone matrix compared with plastic coverslips. The number of MAb-positive cells correlated with the extent of excavation of the surface of the bone slices. Multinuclearity correlated with MAb-positive cell density, and for any given density the proportion of MAb-positive cells that were multinucleate was similar on plastic and bone. We conclude that, in the presence of 1,25-(OH)2 vitamin D3, bone matrix stimulates the generation of osteoclasts but has no demonstrable influence on the fusion of mononuclear osteoclastic precursors.

Automated three-dimensional characterization of osteoclastic resorption lacunae by stereoscopic scanning electron microscopy.

The use of stereoscopic scanning electron microscopy to analyze quantitatively the topography of excavations made by osteoclasts in slices of devitalized cortical bone was evaluated. Using this innovative technique, the need mechanically to tilt the specimen stage to gather three-dimensional information is obviated by instead tilting the electron beam both to produce real-time stereo pairs and to gather measurement data. Based on the comparison of two images of microscopic areas, cross-correlation is used to determine the image shift between the features in the stereo pair. This information is then used dynamically to correct the height of the tilt axis and lens focus in a feedback loop, generating a quantitative measurement of height difference. With this approach, relative heights of individual points, line profiles, area topography maps, and three-dimensional reconstructions of excavations were obtained rapidly and at high resolution. This approach combines the advantage in topographic data acquisition of confocal microscopy with the advantages of the increased resolution and focal depth of scanning electron microscopy (SEM). The technique should facilitate not only the topographic analysis of osteoclastic excavations in bone slices at high resolution but also the three-dimensional analysis of the structure of bone tissue.

The effect of calcium-regulating hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones.

It has previously only been possible to assess osteoclastic bone resorption in intact bone, where other cell types may modify or mediate osteoclastic responses to environmental agents. We have recently developed techniques which enable us to measure bone resorption by osteoclasts extracted from bone and have used these techniques to assess the effects of prostaglandins (PGs) and calcium-regulating hormones on bone resorption by these cells. Osteoclasts were mechanically disaggregated from neonatal rabbit long bones and cultured on slices of devitalized cortical bone for 8 or 24 h. After this time, osteoclasts were associated with the appearance in the scanning electron microscope of characteristic resorption pits, the volume of which was calculated by computer-assisted morphometric and stereophotogrammetric techniques after removal of cells. Salmon calcitonin inhibited osteoclastic bone resorption at concentrations of 1 pg/ml and above, while PTH and 1,25-dihydroxyvitamin D3 were without significant effect. This suggests that the latter hormones do not increase bone resorption in intact bone through a direct effect on osteoclasts. PGI2, PGE1, and PGE2, all of which are known to stimulate resorption when added to intact bone, paradoxically reduced resorption in our cultures. It appears likely that PGs act as direct inhibitors of osteoclastic bone resorption but have an additional effect on other cells in bone, which are induced by PGs to cause osteoclastic stimulation.

Prostaglandin E2 cooperates with TRANCE in osteoclast induction from hemopoietic precursors: synergistic activation of differentiation, cell spreading, and fusion.

It was recently found that osteoblastic cells express TRANCE (tumor necrosis factor-related activation-induced cytokine), a newly identified member of the tumor necrosis factor superfamily, and that expression was increased by calciotropic hormones. Furthermore, soluble recombinant TRANCE induces osteoclast formation and resorption in stroma-free populations of hemopoietic precursor cells. However, overexpression of the decoy receptor osteoprotegerin in vivo shows that there are substantial differences in the sensitivity of different sites to resorption-inhibition, suggesting that either alternative ligands exist or the sensitivity of osteoclasts to TRANCE can be modified by cofactors. We therefore tested the possibility that cofactors might enhance osteoclast formation by TRANCE. We found that the number of tartrate-resistant acid phosphatase-positive and calcitonin receptor-positive cells was increased by a factor of 10 by the presence of PGE2 in the absence of stromal cells. Moreover, although the tartrate-resistant acid phosphatase-positive cells that formed in TRANCE alone were typically mononuclear and poorly spread, the addition of PGE2 induced the formation of large, well spread multinuclear cells. There was an increase in bone resorption that corresponded with the increase in osteoclast number. PGE2 did not synergize with TRANCE for resorption-stimulation in mature cells. 8-Bromo-cAMP showed a similar syngergistic effect on osteoclastic differentiation. Thus, PGE2 appears to stimulate bone resorption through a direct effect on hemopoietic precursors, primarily through a synergistic effect on the ability of TRANCE to induce osteoclastic differentiation.