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.

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.

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.

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.

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.

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.

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.

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.

How are osteoclasts induced to resorb bone?

Although much is known about how osteoclasts are formed, we know little about how they are activated, or how they recognize bone as the substrate appropriate for resorption. Bone mineral is considered to be essential to this recognition process, but a "mineral receptor" has never been identified. Recently, we found that resorptive behavior, as judged by the formation of ruffled borders and actin rings, occurs on ordinary tissue culture substrates if they are first coated with vitronectin. Similarly, vitronectin-coated substrates induce osteoclasts to secrete tartrate-resistant acid phosphatase and to form podosome belts, and to make resorption trails in the protein that coat the substrate. The same applies to bone mineral, which only induces resorptive behavior if coated with vitronectin. In contrast, fibronectin has none of these effects, despite inducing adhesion and spreading. It appears that osteoclasts recognize bone as the substrate appropriate for resorption through the high affinity of vitronectin-receptor ligands for bone mineral.

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 young man with hyperimmunoglobulin-E syndrome and IgA and IgG deficiencies.

Hyperimmunoglobulin E syndrome (HIES) with recurrent infection is a rare primary immunodeficiency characterized by the clinical triad of recurrent staphylococcal abscesses, cyst-forming pneumonia and an elevated serum immunoglobulin (Ig)E level. We report an 18-year-old man with recurrent chest infections, skin infections and dermatitis. On examination, he had the characteristic facies of HIES: high arched palate, webbing between his thumb and index finger bilaterally, and extensive scarring from multiple staphylococcal skin abscesses. He had an elevated IgE level of 14 300 kU/L. IgA and IgG deficiencies were also identified, which are rare associations of this syndrome and complicated the patient's treatment. The coexistence of HIES, IgA and IgG deficiencies has, to our knowledge, not been reported previously in the literature.

Mitf-PU.1 interactions with the tartrate-resistant acid phosphatase gene promoter during osteoclast differentiation.

It has been postulated that the transcription factors micropthalmia associated factor (Mitf) and PU.1 interact with the tartrate-resistant acid phosphatase (TRAP) gene promoter and activate TRAP gene expression in osteoclasts. However, studies on the interaction of these factors with the TRAP promoter employing nuclear extracts from osteoclasts and osteoclast precursors have not been reported. We therefore treated murine mononuclear phagocyte cells with various cytokines to generate cultures of osteoclasts and macrophagic cells with high or low potential to form osteoclasts. The presence of Mitf and PU.1 in nuclear extracts from these cultures and the ability of these factors to bind to the TRAP promoter was then assessed. We demonstrate that Mitf and a related factor, TFE3, are present in nuclear extracts from all cultures and bind the TRAP promoter. While PU.1 is present in nuclear extracts from all cultures, it does not significantly interact with a putative binding site in the TRAP promoter. These results suggest Mitf and PU.1 interactions with the TRAP promoter are not responsible for the specific activation of TRAP gene expression in osteoclasts.

Aberrant monocyte prostaglandin synthase 2 (PGS2) expression in type 1 diabetes before and after disease onset.

METHODS: We examined monocyte prostaglandin synthase 2 (PGS2/COX2) expression in individuals at risk for or with type 1 diabetes including: (i) 58 established type 1 and 2 diabetic patients; (ii) 34 autoantibody positive (AA+) children and adults; (iii) 164 infants and young children with insulin-dependent diabetes mellitus (IDDM) susceptibility human leukocyte antigen (HLA) alleles; and (iv) 37 healthy control individuals, over a 5-yr period. RESULTS: Established type 1 diabetic patients (1 month to 30+ yr post-disease onset) had significantly higher PGS2 expression than healthy controls; by contrast, insulin-treated type 2 diabetic patients had significantly lower PGS2 expression than healthy controls. Longitudinal studies of AA+ subjects at risk for type 1 diabetes indicated that 73% (11/15) of individuals who developed this disease during the study period expressed high levels of PGS2 prior to or after onset. We also found high level PGS2 expression in genetically at-risk infants and young children that correlated with having a first-degree relative with type 1 diabetes, but not with age, gender, or HLA genotype. In this population, high level PGS2 expression coincided with or preceded autoantibody detection in 30% (3/10) of subjects. CONCLUSIONS: These findings suggest that high level monocyte PGS2 expression, although subject to fluctuation, is present in at-risk subjects at an early age and is maintained during progression to and after type 1 diabetes onset.

Use of videotape to assess mobility in a controlled randomized crossover trial of physiotherapy in chronic multiple sclerosis.

OBJECTIVES: To determine to what degree assessment of mobility based on comparison of videotape recordings before and after courses of physiotherapy in patients with chronic multiple sclerosis (MS) is reliable, correlates with 'live' assessments and indicates benefit. DESIGN: Prospective data collection within a randomized crossover controlled trial of physiotherapy at home, as an outpatient, or 'no therapy' in 40 patients. SETTING: Hospital outpatients: outpatient and home physiotherapy. OUTCOMES: Mobility change based on a comparison of short video recordings before and after each treatment period was scored independently by two physiotherapists blinded to therapy type and other measures of outcome. Scores were compared with changes in the Rivermead Mobility Index (RMI) and other indices assessed by a physiotherapist in the patient's home. RESULTS: The two video observers agreed substantially on patient outcome. Changes in walking based on video correlated with RMI for home treatment (r = 0.41, p = 0.008) but not for hospital or no treatment periods (r = 0.14 and 0.15): video changes correlated with the 'live' assessor's global change score inconsistently ('no therapy' r = 0.48, p = 0.002, hospital r = 0.30, p = 0.06 and home r = 0.17, p = 0.30 treatment periods). Based on video data alone, improved mobility was evident following home therapy for only one observer but not for the other or the averaged scores of both. CONCLUSION: There was substantial agreement between two observers deciding on change in mobility based on independent blinded evaluation of short video sequences. However the correlations of these with 'live' assessments were variable. Physiotherapy had a less clear benefit on mobility based on video analysis alone compared with 'live' assessments. The study highlights the need for more objective measures of habitual mobility over longer periods.

Early detection of cytomegalovirus (CMV) infection in bone marrow transplant patients by reverse transcription-PCR for CMV spliced late gene UL21.5: a two site evaluation.

BACKGROUND: Bone marrow transplant (BMT) patients at risk of developing cytomegalovirus (CMV) pneumonitis are identified routinely by the early detection of virus in blood. For early diagnosis of CMV infection, the RNA-based approach demonstrates advantages when compared with the current CMV antigen and DNA detection methods. OBJECTIVES: We have evaluated our previously developed reverse transcription-polymerase chain reaction (RT-PCR) to a spliced late CMV gene (SLG; J. Virol. Methods 56 (1996), 139) to monitor CMV infection in BMT patients at two clinical sites. The diagnostic value of the SLG RT-PCR was compared with the routine CMV antigen and DNA detection methods. STUDY DESIGN: Weekly blood samples from BMT patients were tested for CMV during the first 3 months post-transplant. The qualitative SLG RT-PCR, semiquantitative DNA PCR, and viral antigen tests were compared. The RNA and DNA PCR results were analysed in terms of their temporal relationship and consistency of CMV detection and compared with CMV infection diagnosed by viral antigen tests. RESULTS: Of the 101 BMT recipients studied, 25 developed CMV antigenemia and/or DNAemia resulting in symptomatic infection in two patients. All CMV PCR-positive patients were either CMV seropositive pretransplant or received marrow from seropositive donor. The highest incidence of CMV infection was seen in seropositive recipients (R+) irrespective of the donor's status. Detection of CMV infection by SLG RNA preceded CMV DNA detection by 0-2 weeks (median 1 week) and CMV antigen detection by 0-8 weeks (median 3 weeks). Once detected, the SLG RNA remained consistently positive before antiviral treatment was commenced. Both the SLG RNA and CMV DNA detection methods had the same clinical sensitivity, specificity, positive and negative predictive values of 100, 94, 80 and 100%, respectively. CONCLUSIONS: The RT-PCR for SLG RNA proved to be the earliest indicator of CMV infection in BMT patients demonstrating a sustained pattern of CMV detection during the 3 months post-transplant period. Although very similar in its diagnostic performance to CMV DNA PCR the SLG RNA RT-PCR does not require quantitation and provides an efficient and ongoing indication of active CMV infection.

Requirement for CD28 co-stimulation is lower in SHP-1-deficient T cells.

This study provides biochemical and functional evidence pertaining to the role of the intracellular protein tyrosine phosphatase, SHP-1, in influencing thresholds for TCR activation. Although the loss of SHP-1 in thymocytes from motheaten mice had minimal effects on the initial rise of cytosolic Ca(2+) concentration following TCR triggering, the post-stimulation equilibrium levels of Ca(2+) were consistently elevated. In keeping with a SHP-1 effect on PLCgamma function, IP3 generation was increased in SHP-1 deficient thymocytes. Importantly, we demonstrate that loss of SHP-1 results in a relaxation of the normally stringent co-stimulatory requirements for IL-2 production. SHP-1 deficient single-positive CD4(+) thymocytes revealed a significantly enhanced capacity to produce IL-2 in response to anti-CD3 stimulation alone. In contrast, the simultaneous triggering of CD3 and CD28 was required for equivalent IL-2 production in control single-positive CD4(+) thymocytes. Furthermore, SHP-1 deficient thymocytes generated an increased and prolonged proliferative response to anti-CD3 stimulation alone. In addition, the simultaneous triggering of CD28 and CD3 resulted in equivalent proliferative responses in SHP-1-deficient and control thymocytes, suggesting that a strong co-stimulatory signal is able to override the effect of SHP-1 loss on TCR hyperresponsiveness. Collectively, these results suggest that SHP-1, rather than acting directly on TCR signaling, may indirectly raise thresholds for TCR triggering by modulating co-stimulatory signals.

The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell-cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via beta-catenin. We also show that the interaction of p85 and beta-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.

FLT3 ligand can substitute for macrophage colony-stimulating factor in support of osteoclast differentiation and function.

Although bone resorption and osteoclast numbers are reduced in osteopetrotic (op/op) mice, osteoclasts are nevertheless present and functional, despite the absence of macrophage colony-stimulating factor (M-CSF). This suggests that alternative factors can partly compensate for the crucial actions of M-CSF in osteoclast induction. It was found that when nonadherent bone marrow cells were incubated in RANKL with Flt3 ligand (FL) without exogenous M-CSF, tartrate-resistance acid phosphatase (TRAP)-positive cells were formed, and bone resorption occurred. Without FL, only macrophagelike TRAP-negative cells were present. Granulocyte-macrophage CSF, stem cell factor, interleukin-3, and vascular endothelial growth factor could not similarly replace the need for M-CSF. TRAP-positive cell induction in FL was not due to synergy with M-CSF produced by the bone marrow cells themselves because FL also enabled their formation from the hemopoietic cells of op/op mice, which lack any M-CSF. FL appeared to substitute for M-CSF by supporting the differentiation of adherent cells that express mRNA for RANK and responsiveness to RANKL. To determine whether FL can account for the compensation for M-CSF deficiency that occurs in vivo, FL signaling was blockaded in op/op mice by the injection of soluble recombinant Flt3. It was found that the soluble receptor induced a substantial decrease in osteoclast number, strongly suggesting that FL is responsible for the partial compensation for M-CSF deficiency that occurs in these mice.