Global miRNA expression and correlation with mRNA levels in primary human bone cells.

MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA-mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA-mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, α1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression.

Podocalyxin is a marker of poor prognosis in colorectal cancer.

BACKGROUND: Over two decades ago, a proposal was that two different colorectal cancer (CRC) entities existed, based on tumour location either proximal (right) or distal (left) of the splenic flexure. Proximal and distal tumours exhibit different clinical, epidemiological, and biological characteristics. Improvement of the prognostic evaluation of CRC requires new molecular markers. Podocalyxin-like 1 (PODXL), an anti-adhesive transmembrane sialomucin, is associated with an aggressive tumour phenotype and poor prognosis. For colorectal cancer, it has been suggested to be a marker of poor prognosis. The aim of this study was to investigate the role of PODXL in CRC by use of a novel monoclonal antibody. METHODS: In 1983-2001, 840 consecutive colorectal cancer patients were treated at Helsinki University Central Hospital, of whom 767 were successfully scored for PODXL immunohistochemical expression from tumour tissue microarrays by use of a novel monoclonal in-house antibody. Associations of PODXL expression and tumour location with other clinicopathological variables were explored by Fisher's exact-test, linear-by- linear association test, and binary logistic regression. Survival analyses were done by the Kaplan-Meier method and Cox proportional hazards model. RESULTS: PODXL protein expression was high in 44 (5.7%) specimens. High expression associated strongly with poor differentiation (p < 0.0001), advanced stage (p = 0.011), and location of the tumour in the right hemicolon (RHC) (p < 0.001). Tumours of the RHC were more poorly differentiated (p < 0.0001) and showed higher PODXL expression (p < 0.001).High PODXL expression associated significantly with higher risk for disease-specific death from CRC (hazard ratio (HR) = 2.00; 95% confidence interval (CI) 1.31-3.06, p = 0.001) and also in the subgroups of left hemicolon (LHC) cancers (HR = 2.60; 95% CI 1.45-4.66, p = 0.001) and rectal cancers (HR = 3.03; 95% CI 1.54-5.60, p = 0.001). Results remained significant in multivariable analysis (respectively, HR = 1.82; 95% CI 1.15-2.86, p = 0.01; HR = 2.59; 95% CI 1.41-4.88, p = 0.002; and HR = 2.69; 95% CI 1.30-5.54, p = 0.007). CONCLUSION: Podocalyxin was an independent factor for poor prognosis in colorectal cancer and in the subgroups of left hemicolon and rectum. This is, to our knowledge, the first evidence of such difference in PODXL expression, its function possibly being dependent upon tumour location.

A comparative study of two PODXL antibodies in 840 colorectal cancer patients.

BACKGROUND: Podocalyxin (PODXL) is a transmembrane sialomucin, whose aberrant expression and/or allelic variation associates with poor prognosis and unfavourable clinicopathological characteristics in different cancers. Membranous expression of PODXL has been suggested to be an independent marker of poor prognosis in colorectal cancer (CRC), and previously by an in-house monoclonal antibody, we showed that also cytoplasmic overexpression of PODXL predicts poor prognosis. The aim of this study was to compare two PODXL antibodies with different epitopes case-by-case in CRC patients. METHODS: Of 840 consecutively operated CRC patients from Helsinki University Central Hospital, PODXL expression by polyclonal HPA 2110 antibody was evaluated from 780. Associations of PODXL expression with clinicopathological parameters and the impact of PODXL expression on survival were assessed. Kappa-value was used to assess the comparability of the two antibodies. RESULTS: Membranous PODXL expression associated with unfavourable clinicopathological parameters and with higher risk for disease-specific death from CRC within 5 years (unadjusted hazard ratio (HR) = 1.90; 95% confidence interval (CI) (1.32-2.75); adjusted HR = 1.64; 95% CI (1.11-2.43)). The comparability of expressions by the two antibodies was low (kappa =0.219, standard error 0.060, p < 0.0001). Combination of two antibodies identified a group of patients with even worse prognosis (unadjusted HR = 6.00; 95% CI (3.27-13.0); adjusted HR = 2.14; 95% CI (1.12-4.07)). CONCLUSION: Membranous expression by the polyclonal PODXL antibody and cytoplasmic overexpression by the monocolonal PODXL antibody are both independent markers of poor prognosis, but they recognise different groups of patients, both of which have poor prognosis. The combined use of the antibodies reveals a group with an even worse prognosis. The biological reasons for the difference between antibodies warrant further studies.

Global analysis of the impact of environmental perturbation on cis-regulation of gene expression.

Genetic variants altering cis-regulation of normal gene expression (cis-eQTLs) have been extensively mapped in human cells and tissues, but the extent by which controlled, environmental perturbation influences cis-eQTLs is unclear. We carried out large-scale induction experiments using primary human bone cells derived from unrelated donors of Swedish origin treated with 18 different stimuli (7 treatments and 2 controls, each assessed at 2 time points). The treatments with the largest impact on the transcriptome, verified on two independent expression arrays, included BMP-2 (t = 2h), dexamethasone (DEX) (t = 24 h), and PGE2 (t = 24 h). Using these treatments and control, we performed expression profiling for 18,144 RefSeq transcripts on biological replicates of the complete study cohort of 113 individuals (n(total) = 782) and combined it with genome-wide SNP-genotyping data in order to map treatment-specific cis-eQTLs (defined as SNPs located within the gene ± 250 kb). We found that 93% of cis-eQTLs at 1% FDR were observed in at least one additional treatment, and in fact, on average, only 1.4% of the cis-eQTLs were considered as treatment-specific at high confidence. The relative invariability of cis-regulation following perturbation was reiterated independently by genome-wide allelic expression tests where only a small proportion of variance could be attributed to treatment. Treatment-specific cis-regulatory effects were, however, 2- to 6-fold more abundant among differently expressed genes upon treatment. We further followed-up and validated the DEX-specific cis-regulation of the MYO6 and TNC loci and found top cis-regulatory variants located 180 kb and 250 kb upstream of the transcription start sites, respectively. Our results suggest that, as opposed to tissue-specificity of cis-eQTLs, the interactions between cellular environment and cis-variants are relatively rare (∼1.5%), but that detection of such specific interactions can be achieved by a combination of functional genomic approaches as described here.

Legg-Calvé-Perthes disease and the risk of ADHD, depression, and mortality.

BACKGROUND AND PURPOSE: Hyperactive behavior pattern (such as attention deficit hyperactivity disorder (ADHD)) is proposed to be present in individuals with Legg-Calvé-Perthes disease (LCPD). We investigated whether individuals with LCPD have a higher risk of ADHD, depression, and mortality. SUBJECTS AND METHODS: We identified 4,057 individuals with LCPD in Sweden during the period 1964-2011. 40,570 individuals without LCPD were randomly selected from the Swedish general population and matched by year of birth, sex, and region (control group). We used Cox proportional hazard regression to estimate the relative risks. RESULTS: Compared to the control group, individuals with LCPD had a raised hazard ratio (HR) of 1.5 (95% CI: 1.2-1.9) for ADHD. The risks were higher for female individuals (HR = 2.1, CI: 1.3-3.5) than for male individuals (HR = 1.4, CI: 1.1-1.8). Individuals with LCPD had a modestly higher hazard ratio for depression (HR = 1.3, CI: 1.1-1.5) than the control group. Furthermore, individuals with LCPD had a slightly higher mortality risk than the control group (HR = 1.2, CI: 1.0-1.4) INTERPRETATION: Individuals with LCPD have a higher risk of ADHD. Hyperactivity could expose the femoral head to higher mechanical stress and contribute to the etiology of LCPD. The higher risk of depression may be due to the burden of LCPD itself or could reflect neurobehavioral aspects of ADHD changing into depression later in life. Individuals with LCPD have a higher mortality risk, with higher risk of suicide and cardiovascular diseases.

Suitability of quality control materials for prostate-specific antigen (PSA) measurement: inter-method variability of common tumor marker control materials.

BACKGROUND: Quality control materials with minimal inter-assay differences and clinically relevant proportions of different molecular forms of the analyte are needed to optimize intra- and inter-laboratory accuracy and precision. METHODS: We assessed if clinically relevant total prostate-specific antigen (tPSA) levels were present in seven commercially available Multi Constituent Tumor Marker Controls (MC-TMC). Further, we determined the concentration of free PSA (fPSA) and calculated the percentage of free PSA (%fPSA) in all materials. Finally, we determined variability of TMC materials across several commonly used PSA platforms. RESULTS: All MC-TMC materials contained at least one concentration of tPSA in normal and pathologic range. Control materials varied in the amount of fPSA and %fPSA, with most controls consisting of fPSA only and only one MC-TMC containing medically relevant levels of around 35% fPSA. Only a minority of MC-TMC materials showed minimal variability across four PSA methods while the majority of PSA controls showed wide inter-method differences. CONCLUSIONS: Use of many commercially available controls for PSA could lead to biased PSA measurements because they contain medically irrelevant proportions of fPSA and show significant variation among different PSA assay platforms.

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material.

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 µl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.

An integration of genome-wide association study and gene expression profiling to prioritize the discovery of novel susceptibility Loci for osteoporosis-related traits.

Osteoporosis is a complex disorder and commonly leads to fractures in elderly persons. Genome-wide association studies (GWAS) have become an unbiased approach to identify variations in the genome that potentially affect health. However, the genetic variants identified so far only explain a small proportion of the heritability for complex traits. Due to the modest genetic effect size and inadequate power, true association signals may not be revealed based on a stringent genome-wide significance threshold. Here, we take advantage of SNP and transcript arrays and integrate GWAS and expression signature profiling relevant to the skeletal system in cellular and animal models to prioritize the discovery of novel candidate genes for osteoporosis-related traits, including bone mineral density (BMD) at the lumbar spine (LS) and femoral neck (FN), as well as geometric indices of the hip (femoral neck-shaft angle, NSA; femoral neck length, NL; and narrow-neck width, NW). A two-stage meta-analysis of GWAS from 7,633 Caucasian women and 3,657 men, revealed three novel loci associated with osteoporosis-related traits, including chromosome 1p13.2 (RAP1A, p = 3.6x10(-8)), 2q11.2 (TBC1D8), and 18q11.2 (OSBPL1A), and confirmed a previously reported region near TNFRSF11B/OPG gene. We also prioritized 16 suggestive genome-wide significant candidate genes based on their potential involvement in skeletal metabolism. Among them, 3 candidate genes were associated with BMD in women. Notably, 2 out of these 3 genes (GPR177, p = 2.6x10(-13); SOX6, p = 6.4x10(-10)) associated with BMD in women have been successfully replicated in a large-scale meta-analysis of BMD, but none of the non-prioritized candidates (associated with BMD) did. Our results support the concept of our prioritization strategy. In the absence of direct biological support for identified genes, we highlighted the efficiency of subsequent functional characterization using publicly available expression profiling relevant to the skeletal system in cellular or whole animal models to prioritize candidate genes for further functional validation.

Analysis of bone mineralization on uncemented femoral stems by [18F]-fluoride-PET: a randomized clinical study of 16 hips in 8 patients.

PURPOSE: We present the first study using fluoride-positron emission CT (F-PET/CT) to analyze mineralization of bone in the femur adjacent to uncemented stems following total hip arthroplasty (THA). We studied patients who were operated bilaterally for osteoarthritis with 2 different stems during the same surgical session. PATIENTS AND METHODS: THA was performed bilaterally during the same surgical session in 8 patients with bilateral osteoarthritis of the hip. An SL-PLUS stem was inserted in one hip and a BetaCone stem was inserted in the contralateral hip, with randomization of side and sequence. A second group of 12 individuals with a normal healthy hip was used as reference for normal bone metabolism. Clinical and radiographic evaluation was performed preoperatively, postoperatively, and at 2 years. We used [18F]-fluoride-PET/CT to analyze bone mineralization adjacent to the stems 1 week, 4 months, and 12 months after surgery. We modified the Polar Map system to fit the upper femur for analysis and presentation of the PET results from 12 regions of interest adjacent to the whole stem. RESULTS: The clinical results were good at 2 years. By radiography, all stems were stable. At PET analyses 1 week after surgery, the activity was higher for the SL-PLUS group than for the BetaCone group. The activity was statistically significantly higher for both stems than the reference values at 4 months, and was most pronounced in the upper femur. At one year, the activity had declined more for the BC group than for the SL group. INTERPRETATION: The bone mineralization activity varied between different regions for the same stem and between different time periods for each group. F-PET/CT is a novel and valuable tool for analysis of bone mineralization patterns around uncemented femoral stems in detail. The combination of PET/CT analysis and the modified Polar Map system may provide a useful tool for future studies of metabolic bone responses to prosthetic implants.

Detection of human papillomavirus oncoprotein E7 in liquid-based cytology.

The selection and characterization of a set of mouse mAbs against high-risk human papillomavirus (HPV) E7 oncoprotein and the development of protocols for immunocytochemistry (ICC) are described here. A large number of antibodies raised towards HPV16 and 18 E7 were tested for high-risk specificity by ELISA using a panel of HPV E7 proteins. Antibodies detecting low-risk E7 were discarded, resulting in 38 high-risk HPV E7-specific antibodies. The corresponding epitopes were mapped using overlapping HPV E7 fragments displayed on phage particles. Functionality in ICC against formalin-fixed cervical cancer cell lines was demonstrated for ten mAbs; their high-risk specificity was confirmed by Western blot analysis and ICC on transiently transformed cells expressing high- or low-risk HPV E7. These mAbs were specific for one or several of the high-risk strains HPV16, 18, 31, 35 and 45. Specific E7 staining of liquid-based cytology (LBC) samples was demonstrated for seven mAbs and optimized protocols were established. The E716-41 and E718-79 mAbs demonstrated particularly strong and specific staining of cells stored in LBC fluid for at least 6 months. It is proposed that the high-risk HPV E7 staining protocols established in this study may have the potential to be included in a complementary test for the detection and identification of malignantly transformed cells, in for example atypical squamous cells of undetermined significance samples.

Population genomics in a disease targeted primary cell model.

The common genetic variants associated with complex traits typically lie in noncoding DNA and may alter gene regulation in a cell type-specific manner. Consequently, the choice of tissue or cell model in the dissection of disease associations is important. We carried out an expression quantitative trait loci (eQTL) study of primary human osteoblasts (HOb) derived from 95 unrelated donors of Swedish origin, each represented by two independently derived primary lines to provide biological replication. We combined our data with publicly available information from a genome-wide association study (GWAS) of bone mineral density (BMD). The top 2000 BMD-associated SNPs (P < approximately 10(-3)) were tested for cis-association of gene expression in HObs and in lymphoblastoid cell lines (LCLs) using publicly available data and showed that HObs have a significantly greater enrichment (threefold) of converging cis-eQTLs as compared to LCLs. The top 10 BMD loci with SNPs showing strong cis-effects on gene expression in HObs (P = 6 x 10(-10) - 7 x 10(-16)) were selected for further validation using a staged design in two cohorts of Caucasian male subjects. All 10 variants were tested in the Swedish MrOS Cohort (n = 3014), providing evidence for two novel BMD loci (SRR and MSH3). These variants were then tested in the Rotterdam Study (n = 2090), yielding converging evidence for BMD association at the 17p13.3 SRR locus (P(combined) = 5.6 x 10(-5)). The cis-regulatory effect was further fine-mapped to the proximal promoter of the SRR gene (rs3744270, r(2) = 0.5, P = 2.6 x 10(-15)). Our results suggest that primary cells relevant to disease phenotypes complement traditional approaches for prioritization and validation of GWAS hits for follow-up studies.

Tissue effect on genetic control of transcript isoform variation.

Current genome-wide association studies (GWAS) are moving towards the use of large cohorts of primary cell lines to study a disease of interest and to assign biological relevance to the genetic signals identified. Here, we use a panel of human osteoblasts (HObs) to carry out a transcriptomic survey, similar to recent studies in lymphoblastoid cell lines (LCLs). The distinct nature of HObs and LCLs is reflected by the preferential grouping of cell type-specific genes within biologically and functionally relevant pathways unique to each tissue type. We performed cis-association analysis with SNP genotypes to identify genetic variations of transcript isoforms, and our analysis indicates that differential expression of transcript isoforms in HObs is also partly controlled by cis-regulatory genetic variants. These isoforms are regulated by genetic variants in both a tissue-specific and tissue-independent fashion, and these associations have been confirmed by RT-PCR validation. Our study suggests that multiple transcript isoforms are often present in both tissues and that genetic control may affect the relative expression of one isoform to another, rather than having an all-or-none effect. Examination of the top SNPs from a GWAS of bone mineral density show overlap with probeset associations observed in this study. The top hit corresponding to the FAM118A gene was tested for association studies in two additional clinical studies, revealing a novel transcript isoform variant. Our approach to examining transcriptome variation in multiple tissue types is useful for detecting the proportion of genetic variation common to different cell types and for the identification of cell-specific isoform variants that may be functionally relevant, an important follow-up step for GWAS.

Metabolic development of necrotic bone in the femoral head following resurfacing arthroplasty. A clinical [18F]fluoride-PET study in 11 asymptomatic hips.

BACKGROUND AND PURPOSE: One concern regarding resurfacing arthroplasty is the viability of the diminished femoral head and the postoperative risk of collapse, or a femoral neck fracture. (18)F-fluoride positron emission tomography (F-PET) enables us to assess bone viability despite there being a covering metal component. By F-PET studies, we recently showed the absence of metabolism in the remaining part of femoral heads, 1-4 years after surgery in 11 of 46 consecutive cases. We now present the further development of bone metabolism in these 11 cases. PATIENTS AND METHODS: 10 patients (11 chips) with previously shown loss of femoral head metabolism were evaluated by radiography and repeated F-PET scans, 3-6.5 years after surgery. The size of the area with low (18)F-fluoride PET uptake in the femoral head was compared to that in earlier PET images. RESULTS: No patients had any clinical symptoms; nor was any necrotic bone area visible in plain radiographs. On F-PET scans, 2 patients showed a diminished area with low uptake, 4 were unchanged, and 5 had enlarged areas. INTERPRETATION: Bone metabolism surrounding a volume of bone with no metabolic activity changes dynamically even 5 years after surgery. The presence of bone with minor uptake of F-tracer, indicating low or no bone metabolism, with further progression in 5 of 11 cases leads us to conclude that resurfacing THA should be used restrictively.

TD-11 workshop report: characterization of monoclonal antibodies to S100 proteins.

Fourteen monoclonal antibodies with specificity against native or recombinant antigens within the S100 family were investigated with regard to immunoreactivity. The specificities of the antibodies were studied using ELISA tests, Western blotting epitope mapping using competitive assays, and QCM technology. The mimotopes of antibodies against S100A4 were determined by random peptide phage display libraries. Antibody specificity was also tested by IHC and pair combinations evaluated for construction of immunoradiometric assays for S100B. Out of the 14 antibodies included in this report eight demonstrated specificity to S100B, namely MAbs 4E3, 4D2, S23, S53, 6G1, S21, S36, and 8B10. This reactivity could be classified into four different epitope groups using competing studies. Several of these MAbs did display minor reactivity to other S100 proteins when they were presented in denatured form. Only one of the antibodies, MAb 3B10, displayed preferential reactivity to S100A1; however, it also showed partial cross-reactivity with S100A10 and S100A13. Three antibodies, MAbs 20.1, 22.3, and S195, were specific for recombinant S100A4 in solution. Western blot revealed that MAb 20.1 and 22.3 recognized linear epitopes of S100A4, while MAb S195 reacted with a conformational dependent epitope. Surprisingly, MAb 14B3 did not demonstrate any reactivity to the panel of antigens used in this study.

Hand injury from powered wood splitters: machine safety, patterns of use and injury events.

INTRODUCTION: The purpose of this study was to describe factors of possible importance for the occurrence of hand injury from powered wood splitters. PATIENTS: Patients were identified by a computerized patient registry. Information was obtained from hospital records, a written questionnaire and a structured telephone interview. RESULTS: Very few splitters were constructed according to European standards. Twenty-one percent of patients injured with wedge splitters thought that having more than one person at the machine was one cause of the accident. Seventy-nine percent of patients injured with screw splitters stated that glove use was one cause of the accident. CONCLUSIONS: The level of safety in wood splitters that cause hand injury is often poor. Having more than one person at the machine during work may contribute to wedge splitter injury. Glove use commonly contributes to screw splitter injury. Prevention should be directed towards unsafe machines and dangerous patterns of use.

Amperometric immunosensor for carcinoembryonic antigen in colon cancer samples based on monolayers of dendritic bipodal scaffolds.

Detection of proteins that signal the presence or recurrence of cancer is a powerful therapeutic tool for effective early diagnosis and treatment. Carcinoembryonic antigen (CEA) has been extensively studied as a tumor marker in clinical diagnosis. We report on the development of an amperometric biosensor for the detection of CEA based on the immobilization of anti-CEA monoclonal antibody on a novel class of bipodal thiolated self-assembled monolayers containing reactive N-hydroxysuccinimide (NHS) ester end groups. The current variations showed a linear relationship with the concentration of CEA over the range of 0-200 ng/mL with a sensitivity of 3.8 nA x mL x ng(-1) and a detection limit of 0.2 ng/mL, which is well below the commonly accepted concentration threshold (5 ng/mL) used in clinical diagnosis. Real time and accelerated stability studies of the reporter antibody under various storage conditions demonstrated that the enzymatic activity and antibody affinity of the conjugate is retained for long periods of time in commercial stabilizing buffers such as StabilGuard Biomolecule Stabilizer, and a prediction of the stability trends was carried out using the kinetic and thermodynamic parameters obtained from the Arrhenius equation. The developed immunosensor as well as a commercially available enzyme-linked immunosorbent assay (ELISA) kit were successfully applied to the detection of CEA in serum samples obtained from colon cancer patients, and an excellent correlation of the levels of CEA measured was obtained. Ongoing work is looking at the incorporation of the developed biosensor into a platform for multiplexed simultaneous detection of several breast cancer related biomarkers.

Expression of markers of activity in cultured human osteoblasts: effects of interleukin-4 and interleukin-13.

Cytokines regulate proliferation, differentiation and activation of osteoblasts. Interleukin-4 (IL-4) and interleukin-13 (IL-13) takes part in this regulation by inhibiting proliferation and by enhancement of interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs). In the present study we have investigated the effects of IL-4 and IL-13 on markers of osteoblastic activity in isolated hOBs. Treatment with either IL-4 or IL-13 (1-100 pM) stimulated the formation of alkaline phosphatase (ALP) dose-dependently, detected by enzyme reaction and histochemistry. IL-4 and IL-13 also induced an increase in the secretion of procollagen type I carboxypeptide (PICP) from cultured hOBs, measured by RIA. Osteocalcin secretion measured by ELISA-technique was unaffected. The rate of mineralization, assessed by von Kossa and Alizarin Red staining, was clearly enhanced in hOBs stimulated by IL-4 or IL-13. In conclusion IL-4 and IL-13 exert multiple effects on osteoblast activity in cultured hOBs. Stimulation of ALP secretion together with enhanced collagen secretion and mineralization suggests that IL-4 and IL-13 also have the capacity to maintain hOBs in a differentiated, productive phase.

A prospective study of cognitive behavioural factors as predictors of pain, disability and quality of life one year after lumbar disc surgery.

PURPOSE: The primary aim of this study was to analyse the predictive value of cognitive and behavioural factors, in relation to pain, disability and quality of life (QoL) one year after lumbar disc surgery. METHOD: The study design was prospective. Fifty-nine patients scheduled for first time lumbar disc surgery were included. Pain, disability, QoL, coping, fear avoidance beliefs, expected outcome and sick leave were assessed preoperatively and 12 months after surgery. Multiple backward stepwise logistic regression analyses were performed to study the contribution of the preoperatively measured independent behavioural/cognitive factors (coping, fear avoidance beliefs and assessed chance to return to work within 3 months) to the dependent variables pain, disability and quality of life at 12 months after surgery. RESULTS: Low expectations on work return within 3 months after surgery was significantly predictive for residual leg pain, odds ratio (OR) = 8.2, back pain, OR = 9.7, disability, OR = 13.8 and sick leave, OR = 19.5. Low QoL, was best predicted by preoperatively high scores on fear avoidance beliefs OR = 6.6 and being a woman OR = 6.0. The regression model explained 26-40% of the variance in pain, disability, QoL and sick leave. CONCLUSIONS: Eliciting patients' expectations on work return after surgery could contribute to early identification of those who run the risk of developing long-term disability and sick-leave.

Bone mineralisation adjacent to cemented and uncemented acetabular cups: analysis by [18F]-fluoride-PET in a randomised clinical trial.

PURPOSE: We present a randomised clinical trial using F-PET/CT to analyse new bone metabolic mineralisation adjacent to acetabular cups following total hip arthoplasty (THA). PATIENTS AND METHODS: THA was performed on 26 patients (26 cases) with hip OA. Patients with hip osteoarthritis (OA) were randomly assigned to operations with cemented or uncemented acetabular components. The contralateral, healthy acetabulum was used as referent for normal bone metabolism. The patients were analysed with radiography, clinical scoring, and F-PET/CT preoperatively, and at 6 weeks and 6 months postoperatively. RESULTS: No major complications were recorded, and clinical results were good in all patients. Radiography showed all cups to be stable. The bone-forming activity, as measured by F-PET/CT, was quantified as standardised uptake values (SUV). The mean SUV was 4.6 (6 weeks) and 3.5 (6 months) around the uncemented cups, and 4.8 and 4.0, respectively, for the cemented cups. Normal healthy bone metabolism in the referent was 2.8 and 2.7 SUV at 6 weeks and 6 months, respectively. P < 0.01 for the cemented group at 6 weeks and 6 months, for the uncemented group only at 6 weeks. INTERPRETATION: An acetabulum affected by OA has elevated SUV activity. Both cemented and uncemented cups had elevated bone metabolic activity at 6 weeks. The raised activity was interpreted as an effect from bone mineralisation secondary to surgical trauma and healing, and to the OA. At 6 months, activity was more normalised for the uncemented group than for the cemented, suggesting healing may terminate faster in the uncemented group. Postoperative bone metabolic activity can be analysed in detail by F-PET/CT.ClinicalTrials.gov Identifier: NCT01623687.

Expression of RANK-ligand in prostate cancer cell lines.

The molecular mediators of bone remodelling, receptor activator of nuclear factor-kappaB ligand (RANKL), receptor activator of nuclear factor-kappaB (RANK) and osteoprotegerine (OPG), are believed to be involved in the cellular mechanisms by which tumours metastasize to bone. RANKL is a potent stimulator of osteoclastic bone resorption and is expressed in a variety of tumour cells. We have investigated if the membrane bound form of RANKL is expressed in prostate cancer cell lines, and whether this expression might be regulated by the presence of human osteoblasts. Three prostate cancer cell lines were co-cultured with human osteoblast-like cells (hOB) and RANKL expression on cell surface was measured by FACS. We found basal expression of RANKL on the cell surface, and in co-culture with hOBs the number of cells expressing RANKL was increased between 2.5 and 4 times. These data suggest a signalling mechanism between bone cells and prostate cancer cells that might increase bone resorption and thereby promote bone metastases.