Search

The modeling and remodeling of bone requires activation and polarization of osteoclasts, achieved by reorganization of the cytoskeleton. Members of the Rho subfamily of small GTPases, including Cdc42, are known regulators of cytoskeletal components, but the role of these proteins in bone physiology and pathophysiology remains unclear. Here, we examined loss-of-function mice in which Cdc42 was selectively ablated in differentiated osteoclasts and gain-of-function animals wherein Cdc42Gap, a protein that inactivates the small GTPase, was deleted globally. Cdc42 loss-of-function mice were osteopetrotic and resistant to ovariectomy-induced bone loss, while gain-of-function animals were osteoporotic. Isolated Cdc42-deficient osteoclasts displayed suppressed bone resorption, while osteoclasts with increased Cdc42 activity had enhanced resorptive capacity. We further demonstrated that Cdc42 modulated M-CSF-stimulated cyclin D expression and phosphorylation of Rb and induced caspase 3 and Bim, thus contributing to osteoclast proliferation and apoptosis rates. Furthermore, Cdc42 was required for multiple M-CSF- and RANKL-induced osteoclastogenic signals including activation and expression of the differentiation factors MITF and NFATc1 and was a component of the Par3/Par6/atypical PKC polarization complex in osteoclasts. These data suggest that Cdc42 regulates osteoclast formation and function and may represent a promising therapeutic target for prevention of pathological bone loss.

Bone resorption requires the adhesion of osteoclasts to extracellular matrix (ECM) components, a process mediated by the alpha(v)beta(3) integrin. Following engagement with the ECM, integrin receptors signal via multiple downstream effectors, including the integrin-linked kinase (ILK). In order to characterize the physiological role of ILK in bone resorption, we generated mice with an osteoclast-specific Ilk gene ablation by mating mice with a floxed Ilk allele with TRAP-Cre transgenic mice. The TRAP-Cre mice specifically excised floxed alleles in osteoclasts, as revealed by crossing them with the ROSA26R reporter strain. Osteoclast-specific Ilk mutant mice appeared phenotypically normal, but histomorphometric analysis of the proximal tibia revealed an increase in bone volume and trabecular thickness. Osteoclast-specific Ilk ablation was associated with an increase in osteoclastogenesis both in vitro and in vivo. However, the mutant osteoclasts displayed a decrease in resorption activity as assessed by reduced pit formation on dentin slices in vitro and decreased serum concentrations of the C-terminal telopeptide of collagen in vivo. Interestingly, compound heterozygous mice in which one allele of Ilk and one allele of the beta(3) integrin gene were inactivated (ILK(+/-); beta(3) (+/-)) also had increased trabecular thickness, confirming that beta(3) integrin and Ilk form part of the same genetic cascade. Our results show that ILK is important for the function, but not the differentiation, of osteoclasts.

Neuroendocrine tumors are a unique malignant neoplasm that can arise from the respiratory tree. Although well-differentiated bronchial neuroendocrine tumors (also called carcinoid tumors) are reported to account for approximately 25% of all neuroendocrine tumors, they represent only 1% to 2% of all lung cancers. The epidemiology, clinical behavior, and treatment of neuroendocrine carcinoid tumors differ significantly from other lung malignancies. In this article, the recent data regarding these tumors were reviewed with attention to the treatment modalities used. Although conventional cytotoxic therapy has not been reported to demonstrate much promise in this entity over the past 4 decades, newer molecular targeted agents including those that targeted angiogenesis and the mammalian target of rapamycin (mTOR) pathway have shown encouraging results in early phase trials for advanced carcinoid tumors.

The capacity of the osteoclast (OC) to resorb bone is dictated by cytoskeletal organization, which in turn emanates from signals derived from the αvβ3 integrin and c-Fms. Syk is key to these signals and, in other cells, this tyrosine kinase exerts its effects via intermediaries including the SLP adaptors, SLP-76 and BLNK (B cell linker). Thus, we asked whether these two SLP proteins regulate OC function. We find BLNK-deficient OCs are normal, whereas cytoskeletal organization of those lacking SLP-76 is delayed, thus modestly reducing bone resorption in vitro. Cytoskeletal organization and bone resorption are more profoundly arrested in cultured OCs deficient in BLNK and SLP-76 double knockout (DKO) phenotypes. In contrast, stimulated bone resorption in vivo is inhibited ∼40% in either SLP-76−/− or DKO mice. This observation, taken with the fact that DKO OCs are rescued by retroviral transduction of only SLP-76, indicates that SLP-76 is the dominant SLP family member in the resorptive process. We also find SLP-76 is phosphorylated in a Syk-dependent manner. Furthermore, in the absence of the adaptor protein, integrin-mediated phosphorylation of Vav3, the OC cytoskeleton-organizing guanine nucleotide exchange factor, is abrogated. In keeping with a central role of SLP-76/Vav3 association in osteoclastic resorption, retroviral transduction of SLP-76, in which the Vav binding site is disrupted (3YF), fails to normalize the cytoskeleton of DKO OCs and the resorptive capacity of the cells. Finally, c-Fms-activated Syk also exerts its OC cytoskeleton-organizing effect in a SLP-76/Vav3-dependent manner.

Background CT scan measures of emphysema and airway disease have been correlated with lung function in cohorts of subjects with a range of COPD severity. The contribution of CT scan-assessed airway disease to objective measures of lung function and respiratory symptoms such as dyspnea in severe emphysema is less clear. Methods Using data from 338 subjects in the National Emphysema Treatment Trial (NETT) Genetics Ancillary Study, densitometric measures of emphysema using a threshold of −950 Hounsfield units (%LAA-950) and airway wall phenotypes of the wall thickness (WT) and the square root of wall area (SRWA) of a 10-mm luminal perimeter airway were calculated for each subject. Linear regression analysis was performed for outcome variables FEV 1 and percent predicted value of FEV 1 with CT scan measures of emphysema and airway disease. Results In univariate analysis, there were significant negative correlations between %LAA-950 and both the WT ( r = −0.28, p = 0.0001) and SRWA ( r = −0.19, p = 0.0008). Airway wall thickness was weakly but significantly correlated with postbronchodilator FEV 1 % predicted (R = −0.12, p = 0.02). Multivariate analysis showed significant associations between either WT or SRWA (β = −5.2, p = 0.009; β = −2.6, p = 0.008, respectively) and %LAA-950 (β = −10.6, p = 0.03) with the postbronchodilator FEV 1 % predicted. Male subjects exhibited significantly thicker airway wall phenotypes (p = 0.007 for WT and p = 0.0006 for SRWA). Conclusions Airway disease and emphysema detected by CT scanning are inversely related in patients with severe COPD. Airway wall phenotypes were influenced by gender and associated with lung function in subjects with severe emphysema.

Rationale: Little is known about the genetic regulation of granulomatous inflammation in sarcoidosis. Objectives: To determine if tissue gene array analysis would identify novel genes engaged in inflammation and lung remodeling in patients with sarcoidosis. Methods: Gene expression analysis was performed on tissues obtained from patients with sarcoidosis at the time of diagnosis (untreated) (n = 6) compared with normal lung tissue (n = 6). Expression of select genes was further confirmed in lung tissue from a second series of patients with sarcoidosis and disease-free control subjects (n = 11 per group) by semi-quantitative RT-PCR. Interactive gene networks were identified in patients with sarcoidosis using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood, CA) software. The expression of proteins corresponding to selected overexpressed genes was determined using fluorokine multiplex analysis, and immunohistochemistry. Selected genes and proteins were then analyzed in bronchoalveolar lavage fluid in an independent series of patients with sarcoidosis (n = 36) and control subjects (n = 12). Measurements and Main Results: A gene network engaged in Th1-type responses was most significantly overexpressed in the sarcoidosis lung tissues, including genes not previously reported in the context of sarcoidosis (e.g., IL-7). MMP-12 and ADAMDEC1 transcripts were most highly expressed (> 25-fold) in sarcoidosis lung tissues, corresponding with increased protein expression by immunohistochemistry. MMP-12 and ADAMDEC1 gene and protein expression were increased in bronchoalveolar lavage samples from patients with sarcoidosis, correlating with disease severity. Conclusions: Tissue gene expression analyses provide novel insights into the pathogenesis of pulmonary sarcoidosis. MMP-12 and ADAMDEC1 emerge as likely mediators of lung damage and/or remodeling and may serve as markers of disease activity.

Solid rocket motors (SRMs) and liquid rocket engines (LREs) deplete the global ozone layer in various capacities. We estimate global ozone depletion from rockets as a function of payload launch rate and relative mix of SRM and LRE rocket emissions. Currently, global rocket launches deplete the ozone layer ∼0.03%, an insignificant fraction of the depletion caused by other ozone depletion substances (ODSs). As the space industry grows and ODSs fade from the stratosphere, ozone depletion from rockets could become significant. This raises the possibility of regulation of space launch systems in the name of ozone protection. Large uncertainties in our understanding of ozone loss caused by rocket engines leave open the possibility that launch systems might be limited to as little as several tens of kilotons per year, comparable to the launch requirements of proposed space systems such as spaceplanes, space solar power, and space reflectors to mitigate climate change. The potential for limitations on launch syst...

Tumor necrosis factor receptor-associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-κB (RANK) and is an essential component of the signaling complex mediating osteoclastogenesis. However, the osteoclastic activity of TRAF6 is blunted by its association with four and half LIM domain 2 (FHL2), which functions as an adaptor protein in the cytoplasm and transcriptional regulator in the nucleus. We find that TRAF6 also localizes in the nuclei of osteoclasts but not their bone marrow macrophage precursors and that osteoclast intranuclear abundance is specifically increased by RANK ligand (RANKL). TRAF6 nuclear localization requires FHL2 and is diminished in fhl2-/- osteoclasts. Suggesting transcriptional activity, TRAF6 interacts with the transcription factor RUNX1 in the osteoclast nucleus. FHL2 also associates with RUNX1 but does so only in the presence of TRAF6. Importantly, TRAF6 recognizes FHL2 and RUNX1 in osteoclast nuclei, and the three molecules form a DNA-binding complex that recognizes and transactivates the RUNX1 response element in the fhl2 promoter. Finally, TRAF6 and its proximal activator, RANKL, polyubiquitinate FHL2, prompting its proteasomal degradation. These observations suggest a feedback mechanism whereby TRAF6 negatively regulates osteoclast formation by intracytoplasmic sequestration of FHL2 to blunt RANK activation and as a component of a transcription complex promoting FHL2 expression.

SummaryMaintenance of bone mass and integrity requires a tight balance between resorption by osteoclasts and formation by osteoblasts. Exocytosis of functional proteins is a prerequisite for the activity of both cells. In the present study, we show that synaptotagmin VII, a calcium sensor protein that regulates exocytosis, is associated with lysosomes in osteoclasts and bone matrix protein-containing vesicles in osteoblasts. Absence of synaptotagmin VII inhibits cathepsin K secretion and formation of the ruffled border in osteoclasts and bone matrix protein deposition in osteoblasts, without affecting the differentiation of either cell. Reflecting these in vitro findings, synaptotagmin VII-deficient mice are osteopenic due to impaired bone resorption and formation. Therefore, synaptotagmin VII plays an important role in bone remodeling and homeostasis by modulating secretory pathways functionally important in osteoclasts and osteoblasts.

Postnatal bone marrow houses mesenchymal progenitor cells that are osteoblast precursors. These cells have established therapeutic potential, but they are difficult to maintain and expand in vitro, presumably because little is known about the mechanisms controlling their fate decisions. To investigate the potential role of Notch signaling in osteoblastogenesis, we used conditional alleles to genetically remove components of the Notch signaling system during skeletal development. We found that disruption of Notch signaling in the limb skeletogenic mesenchyme markedly increased trabecular bone mass in adolescent mice. Notably, mesenchymal progenitors were undetectable in the bone marrow of mice with high bone mass. As a result, these mice developed severe osteopenia as they aged. Moreover, Notch signaling seemed to inhibit osteoblast differentiation through Hes or Hey proteins, which diminished Runx2 transcriptional activity via physical interaction. These results support a model wherein Notch signaling in bone marrow normally acts to maintain a pool of mesenchymal progenitors by suppressing osteoblast differentiation. Thus, mesenchymal progenitors may be expanded in vitro by activating the Notch pathway, whereas bone formation in vivo may be enhanced by transiently suppressing this pathway.

A tight balance between bone resorption by osteoclasts and bone formation by osteoblasts is required for the maintenance of bone mass and integrity. A net increase in bone resorption over formation results in osteoporosis, a disease associated with significantly morbidity and mortality. Following attachment via the integrin alphavbeta3, osteoclasts degrade bone by generation of the ruffled border, the unique resorptive organelle of the cell. The adherent cell then secretes into the subcellular space protons and acidic proteases. We review here the concepts relating to the mechanisms of regulated secretion and provide preliminary data on the role of one protein important for secretion by osteoclasts.

Glucocorticoid (GC)-induced bone loss is the most common cause of secondary osteoporosis but its pathogenesis is controversial. GCs clearly suppress bone formation in vivo but the means by which they impact osteoblasts is unclear. Because bone remodeling is characterized by tethering of the activities of the two cells, the osteoclast is a potential modulator of the effect of GCs on osteoblasts. To address this issue we compared the effects of dexamethasone on wild-type (WT) osteoclasts with those derived from mice with disruption of the GC receptor in osteoclast lineage cells and found that the bone-degrading capacity of GC-treated WT cells is suppressed. The inhibitory effect of dexamethasone on bone resorption reflects failure of osteoclasts to organize their cytoskeleton in response to M-CSF. Dexamethasone specifically arrests M-CSF activation of RhoA, Rac, and Vav3, each of which regulate the osteoclast cytoskeleton. In all circumstances, mice lacking the GC receptor in osteoclast lineage cells are spared the impact of dexamethasone on osteoclasts and their precursors. Consistent with osteoclasts modulating the osteoblast-suppressive effect of dexamethasone, GC receptor-deficient mice are protected from the steroid's inhibition of bone formation.

The molecular mechanisms by which binding of monocyte/macrophage colony-stimulating factor to its receptor c-Fms promotes replication in primary macrophages are incompletely understood, as all previous studies involved overexpression of receptor mutants in transformed cells not endogenously expressing the receptor. To address this issue we retrovirally expressed, in bone marrow-derived macrophages, a chimeric receptor containing a range of tyrosine to phenylalanine mutations in the c-Fms cytoplasmic tail. We measured incorporation of bromodeoxyuridine as a marker of proliferation and phosphorylation of ERKs, Akt, and the receptor itself. Our data indicate that tyrosine 559 is the major mediator of receptor activation and cell death, intracellular signaling, and cell proliferation and that the tyrosine residues at positions 697 and 807 play lesser roles in these events. Importantly, we find that activation of the ERK and Akt pathways is necessary but not sufficient for induction of macrophage proliferation. Using specific small molecule inhibitors we find that a combination of the Src family kinase, phosphatidylinositol 3-kinase/Akt, phospholipase C, and ERK pathways mediates macrophage proliferation in response to M-CSF.

In this study, we establish that the tyrosine kinase Syk is essential for osteoclast function in vitro and in vivo. Syk(-/-) osteoclasts fail to organize their cytoskeleton, and, as such, their bone-resorptive capacity is arrested. This defect results in increased skeletal mass in Syk(-/-) embryos and dampened basal and stimulated bone resorption in chimeric mice whose osteoclasts lack the kinase. The skeletal impact of Syk deficiency reflects diminished activity of the mature osteoclast and not impaired differentiation. Syk regulates bone resorption by its inclusion with the alpha v beta3 integrin and c-Src in a signaling complex, which is generated only when alpha v beta3 is activated. Upon integrin occupancy, c-Src phosphorylates Syk. Alpha v beta3-induced phosphorylation of Syk and the latter's capacity to associate with c-Src is mediated by the immunoreceptor tyrosine-based activation motif (ITAM) proteins Dap12 and FcRgamma. Thus, in conjunction with ITAM-bearing proteins, Syk, c-Src, and alpha v beta3 represent an essential signaling complex in the bone-resorbing osteoclast, and, therefore, each is a candidate therapeutic target.

This paper describes a vision-based method of guiding autonomous vehicles within crop rows in agricultural fields where the crop rows are challenging to detect or their appearance is not known a-priori. The location of the crop rows is estimated with an SVM regression algorithm using colour, texture and 3D structure descriptors from a forward facing stereo camera pair. Our system rapidly learns a model online with minimal user input, and then uses this model to track crop rows. Results demonstrate our method is able to learn and track a wide variety of crops with an RMS error of less than 3cm. We also present online control results demonstrating our system autonomously steering a robot for 3km.

Background: Long-term fixation of uncemented joint implants requires early mechanical stability and implant osseointegration. To date, osseointegration has been unreliable and remains a major challenge in cementless total knee arthroplasty. We developed a murine model in which an intra-articular proximal tibial titanium implant with a roughened stem can be loaded through the knee joint. Using this model, we tested the hypothesis that intermittent injection of parathyroid hormone (iPTH) would increase proximal tibial cancellous osseointegration. Methods: Ten-week-old female C57BL/6 mice received a subcutaneous injection of PTH (40 μg/kg/day) or a vehicle (n = 45 per treatment group) five days per week for six weeks, at which time the baseline group was killed (n = 6 per treatment group) and an implant was inserted into the proximal part of the tibiae of the remaining mice. Injections were continued until the animals were killed at one week (n = 7 per treatment group), two weeks (n = 14 per treatment group), or four weeks (n = 17 per treatment group) after implantation. Outcomes included peri-implant bone morphology as analyzed with micro-computed tomography (microCT), osseointegration percentage and bone area fraction as shown with backscattered electron microscopy, cellular composition as demonstrated by immunohistochemical analysis, and pullout strength as measured with mechanical testing. Results: Preimplantation iPTH increased the epiphyseal bone volume fraction by 31.6%. When the data at post-implantation weeks 1, 2, and 4 were averaged for the iPTH-treated mice, the bone volume fraction was 74.5% higher in the peri-implant region and 168% higher distal to the implant compared with the bone volume fractions in the same regions in the vehicle-treated mice. Additionally, the trabecular number was 84.8% greater in the peri-implant region and 74.3% greater distal to the implant. Metaphyseal osseointegration and bone area fraction were 28.1% and 70.1% higher, respectively, in the iPTH-treated mice than in the vehicle-treated mice, and the maximum implant pullout strength was 30.9% greater. iPTH also increased osteoblast and osteoclast density by 65.2% and 47.0%, respectively, relative to the values in the vehicle group, when the data at post-implantation weeks 1 and 2 were averaged. Conclusions: iPTH increased osseointegration, cancellous mass, and the strength of the bone-implant interface. Clinical Relevance: Our murine model is an excellent platform on which to study biological enhancement of cancellous osseointegration.

Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated.

This paper presents a framework for synchronising multiple triggered sensors with respect to a local clock using standard computing hardware. Providing sensor measurements with accurate and meaningful timestamps is important for many sensor fusion, state estimation and control applications. Accurately synchronising sensor timestamps can be performed with specialised hardware, however, performing sensor synchronisation using standard computing hardware and non-real-time operating systems is difficult due to inaccurate and temperature sensitive clocks, variable communication delays and operating system scheduling delays. Results show the ability of our framework to estimate time offsets to sub-millisecond accuracy. We also demonstrate how synchronising timestamps with our framework results in a tenfold reduction in image stabilisation error for a vehicle driving on rough terrain. The source code will be released as an open source tool for time synchronisation in ROS.

Osteoclasts are essential for bone dynamics and calcium homeostasis. The cells form a tight seal on the bone surface, onto which they secrete acid and proteases to resorb bone. The seal is associated with a ring of actin filaments. Cortactin, a c-Src substrate known to promote Arp2/3-mediated actin assembly in vitro, is expressed in osteoclasts and localizes to the sealing ring. To address the role of cortactin and actin assembly in osteoclasts, we depleted cortactin by RNA interference. Cortactin-depleted osteoclasts displayed a complete loss of bone resorption with no formation of sealing zones. On nonosteoid surfaces, osteoclasts flatten with a dynamic, actin-rich peripheral edge that contains podosomes, filopodia, and lamellipodia. Cortactin depletion led to a specific loss of podosomes, revealing a tight spatial compartmentalization of actin assembly. Podosome formation was restored in cortactin-depleted cells by expression of wild-type cortactin or a Src homology 3 point mutant of cortactin. In contrast, expression of a cortactin mutant lacking tyrosine residues phosphorylated by Src did not restore podosome formation. Cortactin was found to be an early component of the nascent podosome belt, along with dynamin, supporting a role for cortactin in actin assembly.

Skin and bone — what comes to mind at hearing this phrase? While certainly a metaphor for disease, it also defines two very different tissues, one a flexible and contiguous outer covering, the other a morphologically diverse hard tissue distributed at over 200 sites in the body. As the accompanying series of Reviews highlights, these tissues are indeed diverse, but there are also surprising similarities. Skin is the interface between the internal organs and the environment, and as such plays a crucial role in the body’s defense mechanism. The skin and its many appendages are responsible for functions as diverse as epidermal barrier and defense, immune surveillance, UV protection, thermoregulation, sweating, lubrication, pigmentation, the sensations of pain and touch, and, importantly, the protection of various stem cell niches in the skin. Bone serves a number of purposes: it provides protection for vital organs, a lever for locomotion, a reservoir for calcium, and the site of adult hematopoiesis. The tissue is composed of osteoblasts, osteoclasts, and their individual precursors plus a complex mixture of mesenchymal, myeloid, and lymphoid cells in the marrow space. Finally, the endothelial microenvironment provides nutrition and is a conduit for the influx and emigration of cells that impact bone biology in several important ways. This Review series guides the reader through these various facets of 2 diverse, yet interdependent, tissues.

Studies have shown a potential role for humoral rejection in the evolution of lung graft dysfunction, apparently based on antibodies without human leukocyte antigen specificity. The correlation between extent of C4d deposition with clinical status further illustrates the importance of humoral immunity. Our study examines the potential value of C3d as a further diagnostic adjunct. C3d deposition was examined in lung allograft specimens using frozen tissue indirect direct immunofluorescence (IIF) and avidin biotin immunohistochemical applied to paraffin embedded tissue. Intermediate/extensive amounts of C3d using IIF and immunohistochemical (IH) methodologies correlated with chronic graft dysfunction; IIF C3d deposition was associated with septal and bronchial wall fibrosis (p < 0.0001). Weak/absent amounts of IIF and IH C3d correlated with clinical stability (p < 0.0001). Higher levels of C3d by IH were more sensitive than by IIF as a bronchiolitis obliterans syndrome determinant. C3d and C4d deposition using immunofluorescence and IH were correlated (p < 0.00001). C3d deposition appears prognostically significant. Higher tissue expression of C3d mark chronic graft dysfunction/persistent graft failure following transplantation. The close correlation between C3d and C4d lends credence to the role of humoral allograft rejection as a pulmonary graft dysfunction contributing factor. C3d by IH manifests higher sensitivity but similar specificity compared to C3d by IIF.

Background and Objectives Incorporation of photodynamic therapy (PDT) into the induction therapy regimen utilized for treatment of locally advanced primary non-small cell bronchogenic carcinoma (NSCLC) is explored. Study Design/Materials and Methods We present a retrospective review of 41 patients diagnosed with non-metastatic NSCLC who underwent induction PDT with chemotherapy and/or radiation. Results Fifty percent of patients initially deemed unresectable, were able to undergo definitive surgical resection after trimodality induction therapy. Twenty-seven percent of patients considered to require pneumonectomy were able to have a lobectomy. The pathological stage was less than the preinduction clinical stage in 14 of 22 cases; of which four patients had no residual tumor. There was no 30/90-day postoperative mortality. Mean survival was 35.9 months (lobectomy), 25.5 months (pneumonectomy) and 14.7 months (no surgery). Median survival was 78% (12 months) and 46% (36 months). The main postoperative complication following pneumonectomy was bronchopleural fistula formation. All patients who developed this complication had undergone trimodality induction therapy. Incorporation of PDT into the induction arsenal for patients with loco-regionally advanced NSCLC may be safely performed. Conclusions PDT may define an alternative induction strategy for patients requiring pneumonectomy; further studies exploring the true efficacy of PDT as an induction modality are encouraged. Lasers Surg. Med. © 2006 Wiley-Liss, Inc.

Osteoclasts, the sole bone-resorbing cells, arise by fusion and differentiation of monocyte/macrophage precursors. Matrix degradation requires adhesion of the osteoclast to bone, an integrin alphavbeta3-mediated event that also stimulates signals which polarize the cell and secrete resorptive molecules such as hydrochloric acid and acidic proteases. Two cytokines are necessary and sufficient for osteoclastogenesis, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), both produced by mesenchymal cells in the bone marrow environment. M-CSF promotes survival and proliferation of osteoclast precursors. It also contributes to their differentiation and regulates the cytoskeletal changes that accompany bone resorption. Binding of M-CSF to c-Fms, its receptor, recruits adapter proteins and cytosolic kinases, thereby activating a variety of intracellular signals. We herein review how alphavbeta3 and M-CSF, alone and in concert, impact production, survival, and function of the osteoclast, thereby controlling skeletal mass. Signals from alphavbeta3 and/or c-Fms activate Syk and Vav3, originally defined by their function in lymphoid cells. Genetic depletion of either protein generates a strong bone phenotype, underscoring the promise of osteoimmunobiology.

Accumulating evidence indicates that interaction of stromal cell-derived factor 1 (SDF-1/CXCL12 [CXC motif, ligand 12]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During granulocyte colony-stimulating factor (G-CSF)–induced HPC mobilization, CXCL12 protein expression in the bone marrow decreases. Herein, we show that in a series of transgenic mice carrying targeted mutations of their G-CSF receptor and displaying markedly different G-CSF–induced HPC mobilization responses, the decrease in bone marrow CXCL12 protein expression closely correlates with the degree of HPC mobilization. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Cell sorting experiments showed that osteoblasts and to a lesser degree endothelial cells are the major sources of CXCL12 production in the bone marrow. Interestingly, osteoblast activity, as measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment. However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on osteoblast function. Collectively, these data suggest a model in which G-CSF, through an indirect mechanism, potently inhibits osteoblast activity resulting in decreased CXCL12 expression in the bone marrow. The consequent attenuation of CXCR4 signaling ultimately leads to HPC mobilization.

We show that mice lacking beta3 integrin are protected from OVX-induced bone loss. Using a lentiviral-based strategy to express beta3 mutants in beta3(-/-) mice, we also show that beta3(S752), but not beta3(Y747/Y759), is important for osteoclastic bone resorption in vivo.Mice lacking the beta3 integrin have dysfunctional osteoclasts and therefore accumulate bone mass with age. Thus, the alphavbeta3 integrin is a potential anti-osteoporosis target. Identifying components of the beta3 integrin that determine its function in vivo is essential for therapeutically exploiting the antiresorptive properties of alphavbeta3.We used DXA and histomorphometry to assess bone loss after ovariectomy in wildtype and beta3 integrin null mice. We used lentiviral vectors carrying various human beta3 (hbeta3) integrin constructs to transduce beta3(-/-) bone marrow and reconstituted lethally irradiated beta3(-/-) mice with the transduced marrow. The expressed constructs include the intact integrin and two mutants, namely hbeta3(Y747F/Y759F) and hbeta3(S752P), each of which induces the bleeding dyscrasia, Glanzmann's thrombasthenia, in humans. Two months after transplantation, the expression of hbeta3 was measured by flow cytometry of marrow-derived macrophages. Osteoclast differentiation and function were assessed ex vivo by TRACP and actin-ring staining, respectively. Reconstituted mice were ovariectomized, and bone loss was assessed by DXA, histomorphometry, and serum TRACP5b assay.beta3(-/-) mice are protected from ovariectomy-induced bone loss, showing no difference in BMD compared with sham-operated controls. We successfully expressed hbeta3 integrins in beta3(-/-) hosts using lentiviral transduction of bone marrow. Two months after transplantation, 25-35% of marrow-derived macrophages expressed the hbeta3 constructs. Similar to its effect in vitro, hbeta3(WT) completely rescued the osteoclast and platelet phenotype of beta3(-/-) mice. Whereas platelet function remained deranged in beta3(-/-) mice overexpressing hbeta3(Y747F/Y759F), osteoclast function was fully restored. In contrast, beta3(-/-) mice expressing hbeta3(S752P) continued to exhibit prolonged bleeding times and dysfunctional osteoclasts in vitro and ex vivo. Most importantly, hbeta3(WT) and hbeta3(Y747F/Y759F) transplanted mice underwent equivalent ovariectomy-induced bone loss, whereas, like those bearing the control vector, hbeta3(S752P) transplanted mice were protected.Functional beta3 integrin is required for ovariectomy-induced bone loss. beta3(S752), but not beta3(Y747/Y759), is critical for osteoclast function in vivo.

Primary intrapulmonary nodular amyloidosis is a rare form of immunoglobulin associated amyloidosis, also falling under the alternative appellation of amyloidoma. Although amyloidomas in other organ sites may be reflective of a localized or more generalized plasma cell dyscrasia, in the context of its presentation in the lung the presumptive basis has long been held to be one of chronic inflammation. We encountered 2 patients with nodular amyloidosis in whom the pathologic examination disclosed the basis to be one of a light chain restricted clonal lymphocytic plasma cell infiltrate, although without morphologic features of coexisting pulmonary lymphoma. These 2 patients serve to underscore the potential categorization of some cases of pulmonary nodular amyloidosis as a form of low grade B cell lymphoproliferative disease.

Alpha-1 anti-trypsin (A1AT) deficiency is an inherited enzyme deficiency that manifests with fatal lung and liver complications. In addition to pulmonary and hepatic involvement, the disease has also been linked to an increased incidence of vasculitic syndromes and autoimmune diseases, including Wegener's granulomatosis, microscopic polyarteritis nodosa and Henoch-Schonlein purpura (HSP). HSP, a systemic, small-vessel vasculitis syndrome, is characterized by a non-thrombocytopaenic purpuric rash, arthralgia, abdominal pain and nephritis. Both A1AT deficiency and HSP have been associated with anti-neutrophil cytoplasmic antibodies (ANCA) and anti-endothelial cell antibodies (AECA). We report a case of a 40-year-old man with severe A1AT deficiency, who developed HSP associated with AECA, ANCA and anti-phospholipid antibodies of the immunoglobulin-A isotype.

Cell/matrix detachment is a general inducer of programmed cell death, an event mediated by loss of integrin/ligand association. Because alpha(v)beta3 is the major integrin expressed by the osteoclast, we asked whether its occupancy promotes survival of the resorptive cell. Thus, we generated wild-type preosteoclasts and placed them on selective matrix proteins. Consistent with the posture that alpha(v)beta3 occupancy promotes survival, preosteoclasts plated on native collagen, a matrix not recognized by the integrin, undergo apoptosis 4-fold faster than those on the alpha(v)beta3 ligand, vitronectin. To further explore the role of alpha(v)beta3 in osteoclast apoptosis, wild-type and beta3-/- preosteoclasts were suspended and apoptosis determined, with time. Beta3-/- preosteoclasts, in suspension, undergo a rate of apoptosis only 40-60% of that of their wild-type counterparts, indicating that unoccupied alpha(v)beta3 transmits a positive death signal that we find regulated by caspase-8. Attesting to specificity of the unoccupied integrin-transmitted death signal, apoptosis in the absence of alpha(v)beta3 is mediated by capsase-9. We have shown that the resorptive defect of beta3-/- osteoclasts is rescued by wild-type beta3 cDNA but not by one bearing a S752P mutation. To determine whether the same holds true regarding osteoclast apoptosis, we constructed lentivirus vectors encoding green fluorescent protein, wild-type beta3, or beta3S752P. Once again, native beta3-/- preosteoclasts were protected against apoptosis. Similar to its effect on bone resorption, transduced wild-type beta3 normalizes the apoptotic rate of beta3-/- preosteoclasts. Unexpectedly, however, beta3S752P transductants also die at a rate indistinguishable from wild type. Thus, unoccupied alpha(v)beta3 integrin regulates osteoclast apoptosis via a component of the integrin that is different than that regulating resorption.

Integrin alphavbeta5 is expressed on osteoclast precursors and is capable of recognizing the same amino acid motif as alphavbeta3. Three-month-old beta5(-/-) female OVX mice had increased osteoclastogenesis ex vivo, and microCT assessment of trabecular bone volume was 53% lower than WT-OVX animals. These preliminary data suggest alphavbeta5 integrin's presence on osteoclast precursors may inhibit of osteoclast formation.Osteoclasts are unique resorptive skeletal cells, capable of degrading bone on contact to the juxtaposed matrix. Integrin alphavbeta5 is expressed on osteoclast precursors, structurally similar to alphavbeta3, and capable of recognizing the same amino acid motif. Given the structural relationship and reciprocal regulation of alphavbeta3 and alphavbeta5, the purpose of this study was to evaluate how alphavbeta5 might contribute to osteoclast maturation and activity.Three-month-old wildtype (WT) and beta5(-/-) female mice had ovariectomy (OVX) or sham operations. The osteoclastogenic capacity of marrow-derived precursors, the kinetic, the circulating, and structural parameters of bone remodeling, was determined after 6 weeks of paired feeding.OVX increased osteoclastogenesis ex vivo and in vivo. Osteoclast formation and prolonged pre-osteoclast survival were substantially enhanced in cultures containing beta5(-/-) cells whether obtained from sham-operated or OVX mice. Expression of cathepsin K, beta3 integrin subunit, and calcitonin receptor were accelerated in cultured beta5(-/-)osteoclasts. beta5(-/-) osteoclasts from OVX animals showed a 3-fold enhancement of net resorptive activity, with quantitative muCT showing trabecular bone volume loss after OVX 53% greater in beta5(-/-) OVX compared with similarly treated WT OVX mice (p0.05). alpha5beta3 seems to be an inhibitor of osteoclast formation, in contrast to alphavbeta3. In addition, loss of alphavbeta5 seems to accelerate osteoclast formation in the OVX model. Further examination of alphavbeta5 signaling pathways may enhance our understanding of the activation of bone resorption.

Highly active antiretroviral therapy (HAART), which includes HIV protease inhibitors (PIs), has been associated with bone demineralization. To determine if this complication reflects accelerated resorptive activity, we studied the impact of two common HIV PIs, ritonavir and indinavir, on osteoclast formation and function. Surprisingly, we find that ritonavir, but not indinavir, inhibits osteoclast differentiation in a reversible manner and also abrogates bone resorption by disrupting the osteoclast cytoskeleton, without affecting cell number. Ritonavir given in vivo completely blunts parathyroid hormone-induced osteoclastogenesis in mice, which confirms that the drug is bone sparing. In keeping with its antiresorptive properties, ritonavir impairs receptor activator of nuclear factor kappaB ligand-induced (RANKL-induced) activation of NF-kappaB and Akt signaling pathways, both critical to osteoclast formation and function. In particular, ritonavir is found to inhibit RANKL-induced Akt signaling by disrupting the recruitment of TNF receptor-associated factor 6/c-Src complex to lipid rafts. Thus, ritonavir may represent a bone-sparing PI capable of preventing development of osteopenia in patients currently on HAART.

Background Histoplasma capsulatum is a dimorphic fungus with both hyphal and microconidial elements and is endemic in certain areas of the central United States. Most clinically recognized syndromes are self-limited, presenting in more than 80% of cases as an influenza-like acute pulmonary illness. We encountered four patients with an endobronchial presentation of histoplasmosis masquerading as a bronchogenic malignancy. Methods The patient files from one of the authors (PR) uncovered four patients diagnosed with, and treated for, endobronchial histoplasmosis; all cases were referred for a presumptive diagnosis of endobronchial neoplasia. Results The case study group comprised three women, ages 52, 68, and 81 years, respectively, and one man age 29 years, all presenting with recurrent hemoptysis. An obstructing adherent lesion was apparent on bronchoscopy in three, involving the right upper lobe bronchus in two, and right mainstem bronchus in one. In one patient, blood was observed in the bronchus intermedius; there was no gross lesion. Recurrent hemoptysis necessitated a right upper sleeve lobectomy in one, a right upper lobe lobectomy and right lower wedge resection in one, a right middle lobectomy in one, and wedge resection of the right upper lobe in one. Conclusions Endobronchial histoplasmosis may mimic primary bronchogenic carcinoma, presenting as a fixed obstructing endobronchial lesion associated with hemoptysis; the latter potentially necessitating surgical intervention. Endobronchial histoplasmosis should be considered in the differential diagnosis of obstructing endobronchial lesions associated with hemoptysis, especially when prior and(or) concurrent biopsies fail to disclose malignancy.

Osteoclasts are macrophage derived cells and as such are subject to regulation by molecules impacting other members of the immune system. Dap12 is an adaptor protein expressed by NK cells and B and T lymphocytes. Dap12 also mediates maturation of myeloid cells and is expressed by osteoclasts which are dysfunctional in its absence. We find Dap12-/- osteoclast precursors fail to differentiate, in vitro, and the abnormality is partially rescued by high dose M-CSF. The relative paucity of osteoclast number, even in presence of high dose cytokine, is attended by dampened proliferation of precursor cells and their failure to normally migrate towards the osteoclast-recognized matrix protein, osteopontin. Furthermore, Dap12-/- osteoclasts generated in high dose M-CSF fail to normally organize their cytoskeleton. The incapacity of Dap12 null cells to undergo normal osteoclast differentiation is not due to blunted stimulation of major RANK ligand (RANKL) or M-CSF induced signaling pathways. On the other hand, when plated on osteopontin, Dap12-/- pre-osteoclasts do not activate the tyrosine kinase, Syk, which normally binds to the adaptor protein and transmits downstream signals. Attesting to the importance of the Dap12/Syk complex, Syk deficient macrophages do not undergo normal osteoclastogenesis. Furthermore, the same cells plated onto osteopontin, adhere poorly and fail to phosphorylate c-Src or Pyk2, two kinases central to organization of the osteoclast cytoskeleton.

Endobronchial presentation of Hodgkin lymphoma is exceedingly rare and can be confused clinically with pulmonary small cell carcinoma. Because of the dramatic implications in treatment and prognosis, endobronchial Hodgkin lymphoma, despite its uncommon occurrence, should be considered in the differential of small cell carcinoma and necrotizing vasculitides with pulmonary involvement, especially in a relatively young patient with cough, hemoptysis, atelectasis, and hilar or mediastinal lymphadenopathy. The use of photodynamic laser therapy or stent placement for palliative treatment of life-threatening airway obstruction may be required prior to or as an initial adjunct to tumor-specific therapy.

Animal studies have shown that blockade of complement may reduce the severity of and/or prevent the development of bronchiolitis obliterans syndrome (BOS), suggesting a role for complement activation. We explored the hypothesis that humoral immunity plays a role in the evolution of BOS. Thirteen unilateral lung transplant patients with BOS defined the patient population. Fresh frozen tissue was analyzed for deposition of C1q, C4d, C5b-9 and immunoglobulin (IgG, IgM, IgA). An indirect immunofluorescent assay was also conducted with patient serum against cytospins of the pulmonary endothelium. In each case the biopsies showed a microvascular injury syndrome involving the bronchial wall characterized by one or more of hemorrhage, fibrin deposition, and endothelial cell necrosis. Other features included bronchial epithelial and chondrocyte necrosis. The end-stage lesion was a thinned bronchial epithelial lining mural fibrosis. Immunofluorescent analysis showed deposition of C1q, C3, C4d, C5b-9, and immunoglobulin in the bronchial epithelium, chondrocytes, basement membrane zone of the bronchial epithelium, and bronchial wall microvasculature. The indirect antiendothelial cell antibody assay was positive in all tested. Humoral immunity may play a role in the pathogenesis of BOS; the antigenic targets include the bronchial wall microvasculature, bronchial epithelium, and chondrocytes.

The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

Interstitial lung disease compatible with idiopathic pulmonary fibrosis (IPF) developed in 19 previously healthy patients. Although interstitial and/or honeycomb parenchymal fibrosis was present in all, there were patchy areas of paucicellular septal capillary injury along with corroborative direct immunofluorescent evidence of a humorally mediated microvascular injury syndrome. Significantly elevated factor VIII levels were seen in 17 of 18 patients tested. Antiphospholipids were present in all 18 patients tested, comprising antibodies of phosphatidylethanolamine, beta-2 glycoprotein, phosphatidylcholine, and/or phosphatidylserine. Anti-Ro and/or anti-ribonucleoprotein (RNP) antibodies were seen in 4 patients. Serologic evidence of infection with cytomegalovirus (CMV) was found in 9 patients and parvovirus B19 (B19) in 9 patients; 1 patient was not tested. Molecular studies revealed B19 DNA in 6 of 6 B19-seropositive patients. In situ hybridization studies revealed CMV RNA in pulmonary cells in patients with serologic evidence of active CMV infection despite the absence of cytopathic changes typical of CMV infection. Antiphospholipid antibodies, antiendothelial cell antibodies, and/or endotheliotropic viral infections related to B19 and CMV may be of pathogenetic importance to the evolution of IPF. This report underscores the potential importance of microvascular injury in the evolution of IPF. Idiopathic pulmonary fibrosis (IPF) encompasses a distinctive subgroup of interstitial pneumonias associated with substantial pulmonary fibrosis for which the cause is obscure. 1,2 The current classification scheme recognizes 4 distinct subtypes of IPF: usual interstitial pneumonitis (UIP), desquamative interstitial pneumonitis, acute interstitial pneumonitis, and nonspecific interstitial pneumonitis (NSIP). Most cases fall into the UIP and

Although the histologic characteristics of cryofibrinogenemia have been described in skin lesions, the literature is largely devoid of descriptions of this disorder in other organs. This series is the first to document the histopathologic manifestations of intrapulmonary, intramuscular, and renal cryofibrinogenemia. We describe the histopathologic manifestations of cryofibrinogenemia in 10 cases with manifestations in 4 organ systems: skin in 7 cases, skeletal muscle in 2, lung in 2, and kidney in 1. Irrespective of anatomic site, all lesions showed an occlusive thrombotic diathesis comprising eosinophilic refractile deposits within vessel lumina with extension into the intima, with or without an accompanying characteristic granulomatous vasculitic component. Ultrastructural examination of the renal deposits showed fibrillary material within glomerular capillary lumina with unique morphologic features not previously described. Analysis of plasma from several cases revealed a cold-precipitable protein, which in most cases included a monoclonal paraprotein. The laboratory and histologic distinctions between cryofibrinogenemia and cryoglobulinemia are addressed. We provide guidelines for the proper handling of patient specimens in the workup of cryofibrinogenemia.

The prototranscription factor p100 represents an intersection of the NF-kappaB and IkappaB families, potentially serving as both the precursor for the active NF-kappaB subunit p52 and as an IkappaB capable of retaining NF-kappaB in the cytoplasm. NF-kappaB-inducing kinase (NIK) controls processing of p100 to generate p52, and thus NIK-deficient mice can be used to examine the biological effects of a failure in such processing. We demonstrate that treatment of wild-type osteoclast precursors with the osteoclastogenic cytokine receptor activator of NF-kappaB ligand (RANKL) increases both expression of p100 and its conversion to p52, resulting in unchanged net levels of p100. In the absence of NIK, p100 expression is increased by RANKL, but its conversion to p52 is blocked, leading to cytosolic accumulation of p100, which, acting as an IkappaB protein, binds NF-kappaB complexes and prevents their nuclear translocation. High levels of unprocessed p100 in osteoclast precursors from NIK-/- mice or a nonprocessable form of the protein in wild-type cells impair RANKL-mediated osteoclastogenesis. Conversely, p100-deficient osteoclast precursors show enhanced sensitivity to RANKL. These data demonstrate a novel, biologically relevant means of regulating NF-kappaB signaling, with upstream control and kinetics distinct from the classical IkappaBalpha pathway.

Primary esophageal schwannomas are uncommon. We describe a case of a large asymptomatic primary esophageal schwannoma in a 65-year-old patient. Computed tomography and positron emission tomography revealed an (18)F-fluorodeoxyglucose-avid 11-cm mass arising from the esophagus. A preoperative diagnosis was made via endoscopic ultrasound. The patient underwent a three-field esophagogastrectomy with cervical esophagogastric anastomosis. He remains well and free of recurrence 10 months after treatment.

The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro.

The purpose of this study was to determine the individual and combined effects on periprosthetic cancellous bone of intermittent parathyroid hormone administration (iPTH) and mechanical loading at the cellular, molecular, and tissue levels. Porous titanium implants were inserted bilaterally on the cancellous bone of adult rabbits beneath a loading device attached to the distal lateral femur. The left femur received a sham loading device. The right femur was loaded daily, and half of the rabbits received daily PTH. Periprosthetic bone was evaluated up to 28 days for gene expression, histology, and µCT analysis. Loading and iPTH increased bone mass by a combination of two mechanisms: (1) Altering cell populations in a pro-osteoblastic/anti-adipocytic direction, and (2) controlling bone turnover by modulating the RANKL-OPG ratio. At the tissue level, BV/TV increased with both loading (+53%, p0.05) and iPTH (+54%, p0.05). Combined treatment showed only small additional effects at the cellular and molecular levels that corresponded to a small additive effect on bone volume (+13% compared to iPTH alone, p0.05). This study suggests that iPTH and loading are potential therapies for enhancing periprosthetic bone formation. The elucidation of the cellular and molecular response may help further enhance the combined therapy and related targeted treatment strategies.

The high incidence of fragility fractures in cortico-cancellous bone locations, plus the fact that individual skeletal sites exhibit different responsiveness to load and disease, emphasizes the need to document separately gene expression in cortical and cancellous bone. A further confounding factor is marrow contamination since its high cellularity may effect gene expression measurements. We isolated RNA from cortical and cancellous bone of intact mouse tibiae, and also after marrow removal by flushing or centrifugation. RNA isolated from cancellous bone by each method was sufficient for gene expression analysis. Centrifugation removed contaminating cells more efficiently than flushing, as indexed by histology and decreased expression of Icam4, a highly expressed erythroid gene. In contrast, centrifuged cortical bone had 12- and 13- fold higher expression of the bone-related genes Col1a1 and Bglap, while levels in marrow-free cancellous bone were 30- and 31-fold higher when compared to bone where marrow was left intact. Furthermore, cortical bone had higher expression of Col1a1 and Bglap than cancellous bone. Thus, RNA isolated by this novel approach can reveal site-specific changes in gene expression in cortical and cancellous bone sites.

Estrogen receptor alpha (ERα) has been implicated in bone's response to mechanical loading in both males and females. ERα in osteoblast lineage cells is important for determining bone mass, but results depend on animal sex and the cellular stage at which ERα is deleted. We demonstrated previously that when ERα is deleted from mature osteoblasts and osteocytes in mixed-background female mice, bone mass and strength are decreased. However, few studies exist examining the skeletal response to loading in bone cell-specific ERαKO mice. Therefore, we crossed ERα floxed (ERα(fl/fl)) and osteocalcin-Cre (OC-Cre) mice to generate animals lacking ERα in mature osteoblasts and osteocytes (pOC-ERαKO) and littermate controls (LC). At 10 weeks of age, the left tibia was loaded in vivo for 2 weeks. We analyzed bone mass through micro-CT, bone formation rate by dynamic histomorphometry, bone strength from mechanical testing, and osteoblast and osteoclast activity by serum chemistry and immunohistochemistry. ERα in mature osteoblasts differentially regulated bone mass in males and females. Compared with LC, female pOC-ERαKO mice had decreased cortical and cancellous bone mass, whereas male pOC-ERαKO mice had equal or greater bone mass than LC. Bone mass results correlated with decreased compressive strength in pOC-ERαKO female L(5) vertebrae and with increased maximum moment in pOC-ERαKO male femora. Female pOC-ERαKO mice responded more to mechanical loading, whereas the response of pOC-ERαKO male animals was similar to their littermate controls.

This paper describes a novel vision based texture tracking method to guide autonomous vehicles in agricultural fields where the crop rows are challenging to detect. Existing methods require sufficient visual difference between the crop and soil for segmentation, or explicit knowledge of the structure of the crop rows. This method works by extracting and tracking the direction and lateral offset of the dominant parallel texture in a simulated overhead view of the scene and hence abstracts away crop-specific details such as colour, spacing and periodicity. The results demonstrate that the method is able to track crop rows across fields with extremely varied appearance during day and night. We demonstrate this method can autonomously guide a robot along the crop rows.

The integrin alphavbeta3 has been shown to be tightly linked to progression of human melanoma. In this study, using two clones from the K1735 murine melanoma system, we investigated the role of alphavbeta3 in metastasis. The highly metastatic K1735M2 cells express the alphavbeta3 integrin, whereas the poorly metastatic K1735C23 cells do not. When transduced with the beta3 integrin subunit cDNA, the K1735C23 cells produced lung lesions and, in two animals, cardiac metastases, whereas the parental C23 cells did not. By contrast, transduction of the full-length beta3 integrin antisense DNA into the K1735M2 cells suppressed metastatic colonization. To specifically investigate the activation of beta3 integrin-mediated pathways, the beta3-positive and the beta3-negative K1735 cells were plated onto vitronectin, a major matrix molecule of both primary and metastatic melanomas. Tyr397 of FAK was phosphorylated several times higher in beta3-expressing K1735 melanoma cells than in beta3-negative cells. To determine whether phosphorylation of FAK was associated with K1735 melanoma motility, we expressed the FAK-related non-kinase (FRNK) in the highly metastatic K1735M2 cells. Exogenous expression of FRNK suppressed phosphorylation of FAK at Tyr397 and decreased the invasive ability of these cells. In addition, expression of a constitutively active mutant Src in poorly metastatic K1735C23 cells increased invasion in vitro; whereas expression of a kinase-inactive Src mutant suppressed invasion. Our results suggest that signals initiated by alphavbeta3 promote metastasis in K1735 melanoma cells through the phosphorylation of FAK and activation of Src.