Deleting Suppressor of Cytokine Signaling-3 in chondrocytes reduces bone growth by disrupting mitogen-activated protein kinase signaling.

OBJECTIVE: To investigate the impact of deleting Suppressor of Cytokine Signaling (SOCS)-3 (SOCS3) in chondrocytes during murine skeletal development. METHOD: Mice with a conditional Socs3 allele (Socs3fl/fl) were crossed with a transgenic mouse expressing Cre recombinase under the control of the type II collagen promoter (Col2a1) to generate Socs3Δ/Δcol2 mice. Skeletal growth was analyzed over the lifespan of Socs3Δ/Δcol2 mice and controls by detailed histomorphology. Bone size and cortical bone development was evaluated by micro-computed tomography (micro-CT). Growth plate (GP) zone width, chondrocyte proliferation and apoptosis were assessed by immunofluorescence staining for Ki67 and TUNEL. Fibroblast growth factor receptor-3 (FGFR3) signaling in the GP was assessed by immunohistochemistry, while the effect of SOCS3 overexpression on FGFR3-driven pMAPK signaling in HEK293T cells was evaluated by Western blot. RESULTS: Socs3Δ/Δcol2 mice of both sexes were consistently smaller compared to littermate controls throughout life. This phenotype was due to reduced long bone size, poor cortical bone development, reduced Ki67+ proliferative chondrocytes and decreased proliferative zone (PZ) width in the GP. Expression of pMAPK, but not pSTAT3, was increased in the GPs of Socs3Δ/Δcol2 mice relative to littermate controls. Overexpression of FGFR3 in HEK293T cells increased Fibroblast Growth Factor 18 (FGF18)-dependent Mitogen-activated protein kinase (MAPK) phosphorylation, while concomitant expression of SOCS3 reduced FGFR3 expression and abrogated MAPK signaling. CONCLUSION: Our results suggest a potential role for SOCS3 in GP chondrocyte proliferation by regulating FGFR3-dependent MAPK signaling in response to FGF18.

Parthenolide inhibits pro-inflammatory cytokine production and exhibits protective effects on progression of collagen-induced arthritis in a rat model.

OBJECTIVES: Progressive destruction of synovial joint cartilage and bone occurs in pathological conditions such as rheumatoid arthritis (RA) because of the overproduction of pro-inflammatory cytokines and activation of nuclear factor kappa B (NF-κB). Through the screening of NF-κB inhibitors by a luciferase reporter gene assay, we identified parthenolide (PAR) as the most potent NF-κB inhibitor, among several PAR analogue compounds. This study was undertaken to determine whether PAR inhibits pro-inflammatory cytokine production, cartilage degradation, and inflammatory arthritis. METHOD: The mRNA levels of pro-inflammatory cytokines were examined by real-time polymerase chain reaction (PCR). Proteoglycan content and release were determined by measuring glycosaminoglycan (GAG) levels using the dimethylmethylene blue (DMMB) dye-binding assay. The potential role of PAR in treatment of arthritis was studied using a collagen-induced arthritis (CIA) model. RESULTS: We established that PAR, as a prototype compound, suppressed lipopolysaccharide (LPS)- and tumour necrosis factor (TNF)-α-induced increases in matrix metalloproteinase (MMP)-1, MMP-3, inducible nitric oxide synthase (iNOS), and interleukin (IL)-1ß mRNA in chondrocytes. In addition, PAR prevented proteoglycan degradation triggered by pro-inflammatory cytokines. PAR treatment at the onset of CIA symptoms significantly reduced synovitis, inflammation, and pannus formation scores. Reduced synovial inflammation after PAR treatment was also reflected in significantly less bone erosion and cartilage damage. CONCLUSIONS: These data indicate a protective effect of PAR on the catabolic insults of pro-inflammatory cytokines on chondrocyte metabolism and GAG release in vitro and in CIA. PAR had anti-inflammatory and structure-modifying effects on experimental arthritis, suggesting that PAR may be useful as a potential alternative or adjunct therapy for inflammatory arthritis.

Exploring the experiences of parents of Autistic children when attending the diagnostic imaging department for an X-ray examination.

INTRODUCTION: Autism is a neuro-developmental condition which affects the social-emotional skills, behaviour, language, communication skills and flexibility of thoughts of an individual and their sensory processing. This can result in Autistic service users finding it difficult to navigate current healthcare provision and cope with the unpredictable environment. This paper explores the experiences of parents of Autistic children when attending the diagnostic imaging department for an X-ray examination. METHODS: A cross sectional, mixed methods approach was adopted and the initial phase consisting of an online survey for parents to complete is the subject of this paper. The quantitative data was analysed using descriptive statistics and cross comparison between questions was also completed. Thematic analysis was taken to analyse the data from the two open questions at the end of the survey. RESULTS: The online survey results are presented in this paper under four key themes; waiting times and environment, forms of communication, lack of understanding of staff regarding Autism and preparation for the X-ray examination. CONCLUSION: The overall rating of the parents' experience whilst in the X-ray/diagnostic imaging department was positive, however there are several areas which received low scores which need further attention. These were waiting areas, waiting times, staff development and patient preparation. IMPLICATIONS FOR PRACTICE: The development of more inclusive waiting areas is needed, more effective lines of communication between staff to expedite the patient journey where possible, staff development of both radiographers and also support staff and the review of design of more accessible and inclusive patient information.

Systematic review of wastewater surveillance of antimicrobial resistance in human populations.

OBJECTIVES: We systematically reviewed studies using wastewater for AMR surveillance in human populations, to determine: (i) evidence of concordance between wastewater-human AMR prevalence estimates, and (ii) methodological approaches which optimised identifying such an association, and which could be recommended as standard. We used Lin's concordance correlation coefficient (CCC) to quantify concordance between AMR prevalence estimates in wastewater and human compartments (where CCC = 1 reflects perfect concordance), and logistic regression to identify study features (e.g. sampling methods) associated with high agreement studies (defined as >70% of within-study wastewater-human AMR prevalence comparisons within ±10%). RESULTS: Of 8,867 records and 441 full-text methods reviewed, 33 studies were included. AMR prevalence data was extractable from 24 studies conducting phenotypic-only (n = 7), genotypic-only (n = 1) or combined (n = 16) AMR detection. Overall concordance of wastewater-human AMR prevalence estimates was reasonably high for both phenotypic (CCC = 0.85 [95% CI 0.8-0.89]) and genotypic approaches (CCC = 0.88 (95% CI 0.84-0.9)) despite diverse study designs, bacterial species investigated and phenotypic/genotypic targets. No significant relationships between methodological approaches and high agreement studies were identified using logistic regression; however, this was limited by inconsistent reporting of study features, significant heterogeneity in approaches and limited sample size. Based on a secondary, descriptive synthesis, studies conducting composite sampling of wastewater influent, longitudinal sampling >12 months, and time-/location-matched sampling of wastewater and human compartments generally had higher agreement. CONCLUSION: Wastewater-based surveillance of AMR appears promising, with high overall concordance between wastewater and human AMR prevalence estimates in studies irrespective of heterogenous approaches. However, our review suggests future work would benefit from: time-/location-matched sampling of wastewater and human populations, composite sampling of influent, and sampling >12 months for longitudinal studies. Further research and clear and consistent reporting of study methods is required to identify optimal practice.

Severe developmental bone phenotype in ClC-7 deficient mice.

Bone development is dependent on the functionality of three essential cell types: chondrocytes, osteoclasts and osteoblasts. If any of these cell types is dysfunctional, a developmental bone phenotype can result. The bone disease osteopetrosis is caused by osteoclast dysfunction or impaired osteoclastogenesis, leading to increased bone mass. In ClC-7 deficient mice, which display severe osteopetrosis, the osteoclast malfunction is due to abrogated acidification of the resorption lacuna. This study sought to investigate the consequences of osteoclast malfunction on bone development, bone structure and bone modeling/remodeling in ClC-7 deficient mice. Bones from wildtype, heterozygous and ClC-7 deficient mice were examined by bone histomorphometry and immunohistochemistry. ClC-7 deficient mice were found to have a severe developmental bone phenotype, characterized by dramatically increased bone mass, a high content of cartilage remnants, impaired longitudinal and radial growth, as well as lack of compact cortical bone development. Indices of bone formation were reduced in ClC-7 deficient mice; however, calcein labeling indicated that mineralization occurred on most trabecular bone surfaces. Osteoid deposition had great regional variance, but an osteopetrorickets phenotype, as observed in oc/oc mice, was not apparent in the ClC-7 deficient mice. A striking finding was the presence of very large abnormal osteoclasts, which filled the bone marrow space within the ClC-7 deficient bones. The development of these giant osteoclasts could be due to altered cell fate of the ClC-7 deficient osteoclasts, caused by increased cellular fusion and/or prolonged osteoclast survival. In summary, malfunctional ClC-7 deficient osteoclasts led to a severe developmental bone phenotype including abnormally large and non-functional osteoclasts. Bone formation paremeters were reduced; however, bone formation and mineralization were found to be heterogenous and continuing.

Overexpression of DeltaFosB transcription factor(s) increases bone formation and inhibits adipogenesis.

Members of the AP-1 family of transcription factors participate in the regulation of bone cell proliferation and differentiation. We report here a potent AP-1-related regulator of osteoblast function: DeltaFosB, a naturally occurring truncated form of FosB that arises from alternative splicing of the fosB transcript and is expressed in osteoblasts. Overexpression of DeltaFosB in transgenic mice leads to increased bone formation throughout the skeleton and a continuous post-developmental increase in bone mass, leading to osteosclerosis. In contrast, DeltaFosB inhibits adipogenesis both in vivo and in vitro, and downregulates the expression of early markers of adipocyte differentiation. Because osteoblasts and adipocytes are thought to share a common precursor, it is concluded that DeltaFosB transcriptionally regulates osteoblastogenesis, possibly at the expense of adipogenesis.

Computational model of an infant brain subjected to periodic motion simplified modelling and Bayesian sensitivity analysis.

Non-accidental head injury in infants, or shaken baby syndrome, is a highly controversial and disputed topic. Biomechanical studies often suggest that shaking alone cannot cause the classical symptoms, yet many medical experts believe the contrary. Researchers have turned to finite element modelling for a more detailed understanding of the interactions between the brain, skull, cerebrospinal fluid (CSF), and surrounding tissues. However, the uncertainties in such models are significant; these can arise from theoretical approximations, lack of information, and inherent variability. Consequently, this study presents an uncertainty analysis of a finite element model of a human head subject to shaking. Although the model geometry was greatly simplified, fluid-structure-interaction techniques were used to model the brain, skull, and CSF using a Eulerian mesh formulation with penalty-based coupling. Uncertainty and sensitivity measurements were obtained using Bayesian sensitivity analysis, which is a technique that is relatively new to the engineering community. Uncertainty in nine different model parameters was investigated for two different shaking excitations: sinusoidal translation only, and sinusoidal translation plus rotation about the base of the head. The level and type of sensitivity in the results was found to be highly dependent on the excitation type.

Astrocytic responses to DNA delivery using nucleofection.

Nucleofection is a powerful non-viral transfection technique that can deliver plasmid DNA with high efficiency to cells that are traditionally difficult to transfect. In this study, we demonstrate that nucleofection of astrocytes grown in primary cell culture resulted in 76 ± 9% transfected cells and low cytotoxicity. However, the nucleofected astrocytes showed a reduced re-attachment to the growth media when replated and subsequent impairment of proliferation. This led to substantially decreased cell densities during the initial 72 h following transfection. Furthermore, these cells were less efficient at producing wound closure in a scratch model of injury. Nucleofection also resulted in the generation of a small proportion of polynucleated cells. The findings demonstrate that nucleofection provides a valuable technique for delivering DNA to astrocytes in culture. However, considerable care is needed in designing and interpreting such studies because of long-lasting changes induced in key properties of these cells by the nucleofection process.

Communication between ephrinB2 and EphB4 within the osteoblast lineage.

Members of the ephrin and Eph family are local mediators of cell function through largely contact-dependent processes in development and in maturity. Production of ephrinB2 mRNA and protein are increased by PTH and PTHrP in osteoblasts. Both a synthetic peptide antagonist of ephrinB2/EphB4 receptor interaction and recombinant soluble extracellular domain of EphB4 (sEphB4), which is an antagonist of both forward and reverse EphB4 signaling, were able to inhibit mineralization and the expression of several osteoblast genes involved late in osteoblast differentiation. The findings are consistent with ephrinB2/EphB4 signaling within the osteoblast lineage having a paracrine role in osteoblast differentiation, in addition to the proposed role of osteoclast-derived ephrinB2 in coupling of bone formation to resorption. This local regulation might contribute to control of osteoblast differentiation and bone formation at remodeling sites, and perhaps also in modeling.

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

Disrupted type II collagenolysis impairs angiogenesis, delays endochondral ossification and initiates aberrant ossification in mouse limbs.

Cartilage remodelling and chondrocyte differentiation are tightly linked to angiogenesis during bone development and endochondral ossification. To investigate whether collagenase-mediated cleavage of the major cartilage collagen (collagen II) plays a role in this process, we generated a knockin mouse in which the mandatory collagenase cleavage site at PQG775↓776LAG, was mutated to PPG775↓776MPG (Col2a1Bailey). This approach blocked collagen II cleavage, and the production of putative collagen II matrikines derived from this site, without modifying matrix metalloproteinase expression or activity. We report here that this mouse (Bailey) is viable. It has a significantly expanded growth plate and exhibits delayed and abnormal angiogenic invasion into the growth plate. Deeper electron microscopy analyses revealed that, at around five weeks of age, a small number of blood vessel(s) penetrate into the growth plate, leading to its abrupt shrinking and the formation of a bony bridge. Our results from in vitro and ex vivo studies suggest that collagen II matrikines stimulate the normal branching of endothelial cells and promote blood vessel invasion at the chondro-osseous junction. The results further suggest that failed collagenolysis in Bailey leads to expansion of the hypertrophic zone and formation of a unique post-hypertrophic zone populated with chondrocytes that re-enter the cell cycle and proliferate. The biological rescue of this in vivo phenotype features the loss of a substantial portion of the growth plate through aberrant ossification, and narrowing of the remaining portion that leads to limb deformation. Together, these data suggest that collagen II matrikines stimulate angiogenesis in skeletal growth and development, revealing novel strategies for stimulating angiogenesis in other contexts such as fracture healing and surgical applications.

Biomechanical testing of the calcified metacarpal articular surface and its association with subchondral bone microstructure in Thoroughbred racehorses.

BACKGROUND: Palmar/plantar osteochondral disease (POD) and third metacarpal/-tarsal condylar fractures are considered fatigue injuries of subchondral bone (SCB) and calcified cartilage due to repetitive high loads in racehorses. In combination with adaptive changes in SCB in response to race training, the accumulation of SCB fatigue is likely to result in changes of joint surface mechanical properties. OBJECTIVES: To determine the spatial relationship and correlation of calcified articular surface biomechanical properties with SCB microstructure and training history in the distal palmar metacarpal condyle of Thoroughbred racehorses. STUDY DESIGN: Cross-sectional study. METHODS: Third metacarpal condyles were examined from 31 Thoroughbred horses with micro-computed tomography (microCT). Hyaline cartilage was removed and reference point indentation (RPI) mechanical testing of the calcified articular surface was performed. Training histories were obtained from trainers. The association among indentation distance increase (IDI, an inverse RPI measure of toughness), and microCT and training variables was assessed using a mixed-effects generalised linear model. RESULTS: Untrained horses had higher IDI than horses that had commenced training (P<0.001). Death as a result of musculoskeletal bone fatigue injury (P = 0.044) and presence of POD (P = 0.05) were associated with higher IDI. The microCT variables connectivity density and trabecular pattern factor were positively (P = 0.002) and negatively (P<0.001) correlated with IDI respectively. MAIN LIMITATIONS: The application of RPI to the calcified articular surface is novel and there is a potential for measurement variability with surface unevenness. CONCLUSION: Commencement of race training is associated with altered material properties of the calcified articular surface in horses. Reduced articular surface material properties can also be detected in horses that have fatigue injuries of the distal metacarpus and at other sites in the skeleton. Measures of SCB connectivity and trabecular surface shape may be more important determinants of resistance to failure of the calcified articular surface than traditional measures such as SCB volume and density.

Bone homeostasis in growth hormone receptor-null mice is restored by IGF-I but independent of Stat5.

Growth hormone (GH) regulates both bone growth and remodeling, but it is unclear whether these actions are mediated directly by the GH receptor (GHR) and/or IGF-I signaling. The actions of GH are transduced by the Jak/Stat signaling pathway via Stat5, which is thought to regulate IGF-I expression. To determine the respective roles of GHR and IGF-I in bone growth and remodeling, we examined bones of wild-type, GHR knockout (GHR(-/-)), Stat5ab(-/-), and GHR(-/-) mice treated with IGF-I. Reduced bone growth in GHR(-/-) mice, due to a premature reduction in chondrocyte proliferation and cortical bone growth, was detected after 2 weeks of age. Additionally, although trabecular bone volume was unchanged, bone turnover was significantly reduced in GHR(-/-) mice, indicating GH involvement in the high bone-turnover level during growth. IGF-I treatment almost completely rescued all effects of the GHR(-/-) on both bone growth and remodeling, supporting a direct effect of IGF-I on both osteoblasts and chondrocytes. Whereas bone length was reduced in Stat5ab(-/-) mice, there was no reduction in trabecular bone remodeling or growth-plate width as observed in GHR(-/-) mice, indicating that the effects of GH in bone may not involve Stat5 activation.

Activated parathyroid hormone/parathyroid hormone-related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone.

Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. To assess the role of the PTH/PTH-related protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansen's metaphyseal chondrodysplasia. In these transgenic mice, osteoblastic function was increased in the trabecular and endosteal compartments, whereas it was decreased in the periosteum. In trabecular bone of the transgenic mice, there was an increase in osteoblast precursors, as well as in mature osteoblasts. Osteoblastic expression of the constitutively active PPR induced a dramatic increase in osteoclast number in both trabecular and compact bone in transgenic animals. The net effect of these actions was a substantial increase in trabecular bone volume and a decrease in cortical bone thickness of the long bones. These findings, for the first time to our knowledge, identify the PPR as a crucial mediator of both bone-forming and bone-resorbing actions of PTH, and they underline the complexity and heterogeneity of the osteoblast population and/or their regulatory microenvironment.

Mechanisms involved in skeletal anabolic therapies.

Since parathyroid hormone (PTH) is the only proven anabolic therapy for bone, it becomes the benchmark by which new treatments will be evaluated. The anabolic effect of PTH is dependent upon intermittent administration, but when an elevated PTH level is maintained even for a few hours it initiates processes leading to new osteoclast formation, and the consequent resorption overrides the effects of activating genes that direct bone formation. Identification of PTH-related protein (PTHrP) production by cells early in the osteoblast lineage, and its action through the PTH1R upon more mature osteoblastic cells, together with the observation that PTHrP+/- mice are osteoporotic, all raise the possibility that PTHrP is a crucial paracrine regulator of bone formation. The finding that concurrent treatment with bisphosphonates impairs the anabolic response to PTH, adds to other clues that osteoclast activity is necessary to complement the direct effect that PTH has in promoting differentiation of committed osteoblast precursors. This might involve the generation of a coupling factor from osteoclasts that are transiently activated by receptor activator of nuclear factor-kappaB ligand (RANKL) in response to PTH. New approaches to anabolic therapies may come from the discovery that an activating mutation in the LRP5 gene is responsible for an inherited high bone mass syndrome, and the fact that this can be recapitulated in transgenic mice, whereas inactivating mutations result in severe bone loss. This has focused attention on the Wnt/frizzled/beta-catenin pathway as being important in bone formation, and proof of the concept has been obtained in experimental models.

Increased substance P immunoreactivity and edema formation following reversible ischemic stroke.

Previous results from our laboratory have shown that neurogenic inflammation is associated with edema formation after traumatic brain injury (TBI). This neurogenic inflammation was characterized by increased substance P (SP) immunoreactivity and could be attenuated with administration of SP antagonists with a resultant decrease in edema formation. Few studies have examined whether neurogenic inflammation, as identified by increased SP immunoreactivity, occurs after stroke and its potential role in edema formation. The present study examines SP immunoreactivity and edema formation following stroke. Experimental stroke was induced in halothane anaesthetized male Sprague-Dawley rats using a reversible thread model of middle cerebral artery occlusion. Increased SP immunoreactivity at 24 hours relative to the non-infarcted hemisphere was observed in perivascular, neuronal, and glial tissue, and within the penumbra of the infarcted hemisphere. It was not as apparent in the infarct core. This increased SP immunoreactivity was associated with edema formation. We conclude that neurogenic inflammation, as reflected by increased SP immunoreactivity, occurs following experimental stroke, and that this may be associated with edema formation. As such, inhibition of neurogenic inflammation may represent a novel therapeutic target for the treatment of edema following reversible, ischemic stroke.

Hand volume estimates based on a geometric algorithm in comparison to water displacement.

Assessing changes in upper extremity limb volume during lymphedema therapy is important for determining treatment efficacy and documenting outcomes. Although arm volumes may be determined by tape measure, the suitability of circumference measurements to estimate hand volumes is questionable because of the deviation in circularity of hand shape. Our aim was to develop an alternative measurement procedure and algorithm for routine use to estimate hand volumes. A caliper was used to measure hand width and depth in 33 subjects (66 hands) and volumes (VE) were calculated using an elliptical frustum model. Using regression analysis and limits of agreement (LOA), VE was compared to volumes determined by water displacement (VW), to volumes calculated from tape-measure determined circumferences (VC), and to a trapezoidal model (VT). VW and VE (mean +/- SD) were similar (363 +/- 98 vs. 362 +/-100 ml) and highly correlated; VE = 1.01VW -3.1 ml, r=0.986, p<0.001, with LOA of +/- 33.5 ml and +/- 9.9 %. In contrast, VC (480 +/- 138 ml) and VT (432 +/- 122 ml) significantly overestimated volume (p<0.0001). These results indicate that the elliptical algorithm can be a useful alternative to water displacement when hand volumes are needed and the water displacement method is contra-indicated, impractical to implement, too time consuming or not available.

Quantitative and functional characterization of muscarinic receptor subtypes in insulin-secreting cell lines and rat pancreatic islets.

Expression of muscarinic receptors in rat islets, RINm5F cells, and INS-1 cells was established by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by RNase protection. Both methods indicated that m3 and m1 receptors were expressed approximately equally in the various cellular preparations and to a much greater extent than the m5 subtype. However, the cell lines, especially RINm5F cells, expressed less of a given receptor subtype than did islets. Immunohistochemistry indicated that m3 receptors were expressed throughout the islet core. Binding studies using the radiolabeled muscarinic receptor antagonist QNB demonstrated a maximal binding capacity of INS-1 cells of 23.0+/-2.9 fmol/mg protein. Functional analyses were undertaken using INS-1 cells stably transfected with either m1 or m3 receptor cDNAs. Overexpression of either receptor did not affect basal responses but markedly enhanced maximal responses to the muscarinic receptor agonist carbachol. Although maximal hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) was twofold greater in m1-transfectants as compared with m3-transfectants, cell lines overexpressing either receptor gave essentially equivalent secretory responses to a full range of carbachol doses. The results demonstrate that both m1 and m3 muscarinic receptors are well expressed in pancreatic beta-cells, functionally linked to signaling pathways, and capable of initiating insulin secretion with equal potencies.

Human and murine osteocalcin gene expression: conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo.

Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6+/-3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2+/-0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2+/-7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5+/-0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly upregulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.

Foot volume estimates based on a geometric algorithm in comparison to water displacement.

Assessing lower extremity limb volume and its change during and after lymphedema therapy is important for determining treatment efficacy and documenting outcomes. Although leg volumes may be determined by tape measure and other methods, there is no metric method to routinely assess foot volumes. Exclusion of foot volumes can under- or overestimate therapeutic progress. Our aim was to develop and test a metric measurement procedure and algorithm for practicing therapists to use to estimate foot volumes. The method uses a caliper and ruler to measure foot dimensions at standardized locations and calculates foot volume (VM) by a mathematical algorithm. VM was compared to volumes measured by water displacement (Vw) in 30 subjects (60 feet) using regression analysis and limits of agreement (LOA). Vw and VM (mean +/- sd) were similar 857 +/- 150 ml vs. 859 +/- 154 ml, and were highly correlated VM = 1.00Vw + 1.67 ml, r = 0.965, p < 0.001. The LOA for absolute volume differences and percentages were respectively +/- 79.6 ml and +/- 9.28 %. These results indicate that this metric method can be a useful alternative to water displacement when foot volumes are needed, but the water displacement method is contraindicated, impractical to implement, too time consuming or is not available.