Search

To determine the influence of high tibial osteotomy on subchondral bone marrow edema in medial osteoarthritis of the varus knee, full leg-length radiographs and magnetic resonance imaging were performed in 20 patients (20 knees) before surgery, 1 year postoperatively, and at a mean of 7 years postoperatively. The extent of bone marrow edema in the medial compartment was quantified with magnetic resonance imaging in two planes using the formula for a prolate ellipsoid as follows: length x width x depth x pi/6. We used the Japanese Orthopaedic Association knee score for clinical evaluation. At the last followup, all knees with valgus alignment (10/10) showed reduced edema. In contrast, bone marrow edema increased or remained unchanged in four of 10 knees with neutral or varus alignment. The percentage of satisfactory results was 100% (10/10) in valgus knees and only 30% (3/10) in neutral or varus knees. Extent of bone marrow edema at the followup correlated with the mechanical axis and knee score. Because of the prognostic value of bone marrow abnormalities in the medial compartment observed on magnetic resonance imaging, early lateral closing wedge osteotomy should be considered in patients with varus malalignment and bone marrow edema even in mild cases of medial osteoarthritis. [ABSTRACT FROM AUTHOR]

The Istanbul Protocol provides a scheme for giving evidence of signs of torture. This scheme does not conform with the principles of logical inference, revolving as it does round the concept of 'consistency'. The shortcomings of the Protocol are explained using the evidence given in the recent case of KV(Sri Lanka) and the logical approach to such evidence explained., (© The Author(s) 2019. Published by Oxford University Press.)

Background: Several single-nucleotide polymorphisms (SNPs) in the TNC gene have recently been found to be associated with degenerative rotator cuff tears., Hypothesis: Exonic SNPs in the TNC gene are related to the risk for a failure to heal after rotator cuff repair., Study Design: Case-control study; Level of evidence, 3., Methods: A total of 302 patients from the Vienna area and European Caucasian ancestry underwent mini-open rotator cuff repair for a full-thickness superior or posterosuperior tear and were assessed for the integrity of the repair 1 year postoperatively with a real-time 7.5- to 10-MHz ultrasound linear array transducer. Outcomes were classified as intact (complete footprint coverage), small (<200 mm 2 ), or large (≥200 mm 2 ) recurrent defect. Patients were genotyped for 15 previously identified risk SNPs within a 49-kbp segment of the TNC gene with the KASP genotyping technology or the Ion-Torrent Personal Genome Machine System., Results: All recurrent defects were atraumatic failures, and the overall failure rate was 39.7%. Of the traditional risk factors, only the initial tear size was significantly associated with a failure to heal. In a multinomial logistic regression model, the T allele at rs1138545 [C>T] was protective for a large recurrent defect (odds ratio = 0.16; 95% CI, 0.09-0.31). The role of rs1138545 was further backed by haplotype analysis, which showed that the combination of the C allele at rs1138545 [C>T], the A allele at rs2104772 [A>T], and the G allele at rs10759752 [A>G] formed the risk-related haplotype [CAG]. The CAG haplotype was associated with large recurrent defects ( P < .0001; haplotype frequency, 0.394; haplotype score, 4.518). Exonic marker rs1138545 transcribed into all isoforms of the TNC protein, whereas exonic marker rs2104772, which has been associated with Achilles tendinopathy before, transcribed only into large isoforms of the TNC protein., Conclusion: Recurrent defects after rotator cuff repairs are clinically relevant, and a heritable component of the disorder is plausible on the basis of a genetic association with 8 TNC variants. Characterization of TNC sequences that favor tendon healing will help engineer new products in regenerative medicine.

Managing context information in forensic casework aims to minimize task-irrelevant information while maximizing the task-relevant information that reaches the examiner. A design and implementation of context information management (CIM) is described for forensic firearms examination. Guided by a taxonomy of different sources of context information, a flow-chart was constructed that specifies the process of casework examination and context information management. Due to the risk of bias, another examiner may need to be involved when context information management is unsuccessful. Application of context information management does not make a subjective examination objective, but can limit the risks of bias with a minimal investment of time and resources., (Copyright © 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.)

In 2010, the Netherlands Forensic Institute (NFI) and the University of Amsterdam (UvA) started a series of tests for the NFI's Firearms Section. Ten cartridge case and bullet comparison tests were submitted by various external parties as regular cases and mixed in the flow of real cases. The results of the tests were evaluated with the VU University Amsterdam (VUA). A total of twenty-nine conclusions were drawn in the ten tests. For nineteen conclusions the submitted cartridge cases or bullets were either fired from the questioned firearm or from one and the same firearm, in tests where no firearm was submitted. For ten conclusions the submitted cartridge cases or bullets were either fired from another firearm than the submitted one or from several firearms, in tests where no firearm was submitted. In none of the conclusions misleading evidence was reported, in the sense that all conclusions supported the true hypothesis. This article discusses the design considerations of the program, contains details of the tests, and describes the various ways the test results were and could be analyzed., (Copyright © 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.)

Measuring the performance of forensic evaluation methods that compute likelihood ratios (LRs) is relevant for both the development and the validation of such methods. A framework of performance characteristics categorized as primary and secondary is introduced in this study to help achieve such development and validation. Ground-truth labelled fingerprint data is used to assess the performance of an example likelihood ratio method in terms of those performance characteristics. Discrimination, calibration, and especially the coherence of this LR method are assessed as a function of the quantity and quality of the trace fingerprint specimen. Assessment of the coherence revealed a weakness of the comparison algorithm in the computer-assisted likelihood ratio method used., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Matching DNA profiles of an accused person and a crime scene trace are one of the most common forms of forensic evidence. A number of years ago the so-called 'DNA controversy' was concerned with how to quantify the value of such evidence. Given its importance, the lack of understanding of such a basic issue was quite surprising and concerning. Deriving the equation for the likelihood ratio of a DNA database match in a much more direct and simple way is the topic of this paper. As it is much easier to follow it is hoped that this derivation will contribute to the understanding., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

In the investigation of arson, evidence connecting a suspect to the fire scene may be obtained by comparing the composition of ignitable liquid residues found at the crime scene to ignitable liquids found in possession of the suspect. Interpreting the result of such a comparison is hampered by processes at the crime scene that result in evaporation, matrix interference, and microbial degradation of the ignitable liquid. Most commonly, gasoline is used as a fire accelerant in arson. In the current scientific literature on gasoline comparison, classification studies are reported for unevaporated and evaporated gasoline residues. In these studies the goal is to discriminate between samples of several sources of gasoline, based on a chemical analysis. While in classification studies the focus is on discrimination of gasolines, for forensic purposes a likelihood ratio approach is more relevant. In this work, a first step is made towards the ultimate goal of obtaining numerical values for the strength of evidence for the inference of identity of source in gasoline comparisons. Three likelihood ratio methods are presented for the comparison of evaporated gasoline residues (up to 75% weight loss under laboratory conditions). Two methods based on distance functions and one multivariate method were developed. The performance of the three methods is characterized by rates of misleading evidence, an analysis of the calibration and an information theoretical analysis. The three methods show strong improvement of discrimination as compared with a completely uninformative method. The two distance functions perform better than the multivariate method, in terms of discrimination and rates of misleading evidence., (Copyright © 2014 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.)

Making changes or additions to written entries in a document can be profitable and illegal at the same time. A simple univariate approach is first used in this paper to quantify the evidential value in color measurements for inks on a document coming from a different or the same source. Graphic, qualitative discrimination is then obtained independently by applying color deconvolution image processing to document images, with parameters optionally optimized by support vector machines (SVM), a machine learning method. Discrimination based on qualitative results from image processing is finally compared to the quantitative results of the statistical approach. As color differences increase, optimized color deconvolution achieves qualitative discrimination when the statistical approach indicates evidence for the different source hypothesis., (Copyright © 2012 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.)

More than just being a substrate, paper can also provide evidence for the provenance of documents. An earlier paper described a method to compare paper structure, based on the Fourier power spectra of light transmission images. Good results were obtained by using the 2D correlation of images derived from the power spectra as a similarity score, but the method was very computationally intensive. Different comparison algorithms are evaluated in this paper, using information theoretical criteria. An angular invariant algorithm turned out to be as effective as the original one but 4 orders of magnitude faster, making the use of much larger databases possible., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In response to genotoxic stress, the p53 tumor suppressor induces target genes for cell cycle arrest, apoptosis, and DNA repair. Although p53 is the most commonly mutated gene in all human cancers, it is only mutated in about 20% of breast cancers. 70% of all breast cancer cases are estrogen receptor (ER)-positive and express ERα. ER-positive breast cancer generally indicates good patient prognosis and treatment responsiveness with antiestrogens, such as tamoxifen. However, ER-positive breast cancer patients can experience loss or a reduction in ERα, which is associated with aggressive tumor growth, increased invasiveness, poor prognosis, and loss of p53 function. Consistent with this, we found that p53 is a target gene of ERα. Specifically, we found that knockdown of ERα decreases expression of p53 and its downstream targets, MDM2 and p21. In addition, we found that ERα activates p53 transcription via binding to estrogen response element half-sites within the p53 promoter. Moreover, we found that loss of ERα desensitizes, whereas ectopic expression of ERα sensitizes, breast cancer cells to DNA damage-induced growth suppression in a p53-dependent manner. Altogether, this study provides an insight into a feedback loop between ERα and p53 and a biological role of p53 in the DNA damage response in ER-positive breast cancers.

This is a discussion of a number of issues that arise from the recent judgment in R v T [1]. Although the judgment concerned with footwear evidence, more general remarks have implications for all disciplines within forensic science. Our concern is that the judgment will be interpreted as being in opposition to the principles of logical interpretation of evidence. We reiterate those principles and then discuss several extracts from the judgment that may be potentially harmful to the future of forensic science. A position statement with regard to evidence evaluation, signed by many forensic scientists, statisticians and lawyers, has appeared in this journal [2] and the present paper expands on the points made in that statement., (Copyright © 2011 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.)

We report simulations of electrochemical generation-collection experiments in which the generator is a small disc producing a specified time-dependent flux of the analyte and the collector is a large planar electrode which collects the analyte at the mass transport-controlled rate. This geometry corresponds to many experiments in bioelectrochemistry where a relatively large sensor is used to detect the products of a cell's metabolism at low concentration. In particular, our simulations are motivated by attempts to understand our results on the detection of the superoxide radical anion burst generated by osteoclasts (bone-resorbing cells) in response to various stimuli. Superoxide is present at low levels and disproportionates in aqueous media; however, the homogeneous kinetics are included in our simulations and the results show that it is possible to estimate the magnitude of the flux of superoxide produced by the cells and to accurately determine the time-dependence of the flux in response to stimuli such as injection of parathyroid hormone, vitamin D(3) and pertussis toxin. In all these cases, the superoxide anion flux was successfully modeled as uniform across the cell surface with time-dependence of the form j(0)e(-k(d)t) + j(∞). j(∞) is the sustained flux of superoxide and the first-order rate constant k(d) and the magnitude j(0) describe the transient component of the flux. The simulations indicate that for cell-electrode gaps D approximately < √(D/k(d)), where D is the diffusion coefficient, the value of k(d) can be accurately extracted from the time-dependence of the collector current without detailed knowledge of parameters which are hard to measure during the experiment, e.g., the cell radius a and cell-electrode separation d. In the case of parathyroid hormone, the first-order rate constant describing the decay of the transient component was k(d) = 1.8 ± 0.8 × 10(-1) s(-1), but much slower decays were observed in response to pertussis toxin (k(d) = 1.5 ± 0.5 × 10(-2) s(-1)) and vitamin D(3) (k(d) = 1.1 ± 0.5 × 10(-3) s(-1)).

In this study we explore the inference of identity of source using a two-dimensional feature vector. As an example, we study the use of the Bayesian framework for the estimation of the value of evidence of color measurements for identity of source of blue ballpoint pen inks. Univariate as well as bivariate analyses are carried out for color data that was acquired with a flatbed scanner. While this might not be the best method to discriminate inks, we will use it as an example to estimate what the value of the evidence is, however low or high it may be. It is hoped that this exercise is instructional, as a similar approach can readily be applied in other situations.

A method for the comparison of paper structure using light transmission images and frequency analysis was developed. The resolution of the light transmission images and the algorithm for the feature extraction were greatly improved to enhance the visibility of peaks in the 2D power spectrum that results from frequency analysis. A comparison method based on correlation measures how well the spectra match as a function of the orientation of the paper, yielding an objective and quantitative measure of similarity between 0 and 1. A technical validation was carried out with 25 different papers showing the potential of this method with common copy papers. Finally, the method was applied in a case.

Nanocrystals of various inorganic materials are being considered for application in the life sciences as fluorescent labels and for such therapeutic applications as drug delivery or targeted cell destruction. The potential applications of the nanoparticles are critically compromised due to the well-documented toxicity and lack of understanding about the mechanisms involved in the intracellular internalization. Here intracellular internalization and toxicity of alkyl-capped silicon nanocrystals in human neoplastic and normal primary cells is reported. The capped nanocrystals lack cytotoxicity, and there is a marked difference in the rate and extent of intracellular accumulation of the nanoparticles between human cancerous and non-cancerous primary cells, the rate and extent being higher in the malignant cells compared to normal human primary cells. The exposure of the cells to the alkyl-capped nanocrystals demonstrates no evidence of in vitro cytotoxicity when assessed by cell morphology, apoptosis, and cell viability assays. The internalization of the nanocrystals by Hela and SW1353 cells is almost completely blocked by the pinocytosis inhibitors filipin, cytochalasin B, and actinomycin D. The internalization process is not associated with any surface change in the nanoparticles, as their luminescence spectrum is unaltered upon transport into the cytosol. The observed dramatic difference in the rate and extent of internalization of the nanocrystals between malignant and non-malignant cells therefore offers potential application in the management of human neoplastic conditions.

Alkyl-capped silicon nanocrystals can be dispersed in aqueous media by shaking or stirring their solutions in organic solvents (DMSO, ether, THF) with excess water. THF is the most straightforward choice with which to prepare stable aqueous dispersions, because the nanocrystals are very soluble in THF and it is also miscible with water. As little as 0.01% v/v tetrahydrofuran is sufficient. DMSO and ether were the preferred choices for subsequent staining of live cells because THF shows some acute toxicity even when very dilute. The luminescence intensity of the aqueous dispersions is linear in particle concentration and independent of pH over the range 5-9. The sols retain their photoluminescence and are stable against flocculation for at least 6 months.

In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3' untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3' UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either pan-HDAC or HDAC6-selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14-fold to 31-fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent pan-HDAC inhibitors, these HDAC6-selective inhibitors produced dose-dependent growth arrest of ERBB2-overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6-selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies.

Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3'-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'-untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function.

Purpose: We report on 24 cases of transient bone marrow edema syndrome in 18 patients who underwent core decompression of the knee., Methods: Diagnosis was made with the use of radiographs, magnetic resonance imaging (MRI), and core biopsy testing. Arthroscopic surgery and core decompression were carried out in all patients, and MRI was performed again, 5 years after surgery was performed., Results: Medial and lateral femoral condyles were affected in 15 and 7 knees, respectively. In all, 6 patients presented with bilateral involvement of the knees (migrating transient bone marrow edema syndrome). Two of these patients had affections of the medial and lateral compartments within the same knee at different times, consistent with intra-articular regional bone marrow edema syndrome. Core biopsy specimens showed areas of bone marrow edema and vital trabeculae covered by osteoblasts and osteoid seams. Resolution of symptoms and normalization of MRI findings occurred in all patients within 12 weeks after surgery., Conclusions: Migrating bone marrow edema was found in a high percentage (33%) of patients at 5-year follow-up; however, all patients were clinically asymptomatic, and signal alterations on MRI had resolved completely. The high incidence of migrating bone marrow edema, the lack of osteonecrotic regions in our specimens, and the fact that none of these cases progressed to spontaneous osteonecrosis seem to further support the contention that bone marrow edema syndrome of the knee is a distinct entity., Level of Evidence: Level II, diagnostic study; development of diagnostic criteria on the basis of consecutive patients and with universally applied reference gold standard.

The limitations of the examination of indented writing impressions using electrostatic detection are often paper related. Paper types such as glossy paper, paper of high basis weight, and lithography or gravure-printed papers often give rise to problems resulting in a decrease in sensitivity or a lack of detection altogether. In this paper, a novel technique for the examination of indented writing is presented, which is in a sense complimentary to the technique of electrostatic detection as it is especially suitable for glossy-coated and printed paper types and can in some instances also deal with paper types of higher basis weight. Indented writing grooves will normally contain more particles than the surrounding nonindented areas due to damage of the surface layer resulting in a build-up of filler powder. The method presented uses black gelatine lifter slabs to lift the paper dust image off the surface of the paper. This image can quite easily be photographed using near-to-coaxial lighting. The gelatine lifting method outperforms oblique lighting for the detection of indented writing and is almost as sensitive as electrostatic detection if compared on the types of paper where both perform well. The main advantage of this new technique is, however, that it is especially suitable for those types of paper where electrostatic detection fails and is therefore a welcome addition to the range of methods available to a forensic document examiner for the examination of indented writing.

Improved understanding of the molecular mechanisms by which small-molecule inhibitors of histone deacetylases (HDAC) induce programs, such as cellular differentiation and apoptosis, would undoubtedly assist their clinical development as anticancer agents. As modulators of gene transcript levels, HDAC inhibitors (HDACi) typically affect only 5% to 10% of actively transcribed genes with approximately as many mRNA transcripts being up-regulated as down-regulated. Using microRNA (miRNA) array analysis, we report rapid alteration of miRNA levels in response to the potent hydroxamic acid HDACi LAQ824 in the breast cancer cell line SKBr3. Within 5 hours of exposure to a proapoptotic dose of LAQ824, significant changes were measured in 40% of the >60 different miRNA species expressed in SKBr3 cells with 22 miRNA species down-regulated and 5 miRNAs up-regulated. To explore a potential functional link between HDACi induced mRNA up-regulation and miRNA down-regulation, antisense experiments were done against miR-27a and miR-27b, both abundantly expressed and down-regulated in SKBr3 cells by LAQ824. Correlating a set of genes previously determined by cDNA array analysis to be rapidly up-regulated by LAQ824 in SKBr3 with a database of potential 3' untranslated region miRNA binding elements, two genes containing putative miR-27 anchor elements were identified as transcriptionally up-regulated following miR-27 antisense transfection, ZBTB10/RINZF, a Sp1 repressor, and RYBP/DEDAF, an apoptotic facilitator. These findings emphasize the importance of post-transcriptional mRNA regulation by HDACi in addition to their established effects on promoter-driven gene expression.

In forensic image processing, it is often important to be able to separate a feature from an interfering background or foreground, or to demonstrate colors within an image to be different from each other. In this study, a color deconvolution algorithm that could accomplish this task is described, and it is applied to color separation problems in document and fingerprint examination. Subtle color differences (sometimes invisible to the naked eye) are found to be sufficient, which is demonstrated successfully for several cases where color differences were shown to exist, or where colors were removed from the foreground or background. The software is available for free in the form of an Adobe Photoshop-compatible plug-in.

Objective: The aim of this study was to evaluate bone metabolism in patients with spontaneous osteonecrosis (ON) of the medial femoral condyle., Method: In 22 consecutive patients, undergoing total knee arthroplasty, biochemical markers of bone metabolism were measured in aspirates from cancellous bone and in samples obtained simultaneously from peripheral blood. Specimens of the medial femoral condyle were available for histologic examination and the lesion size, assessed on radiographs, was compared with the results from bone turnover measurements. Twenty patients with osteoarthritis (OA) of the knee served as a control. Bone-specific alkaline phosphatase (bone ALP), osteocalcin (OC), procollagen type I N-terminal propeptide (PINP), and C-terminal cross-linking telopeptide (ICTP) were studied., Results: Mean serum levels of analytes were not different in patients with ON and OA. The serum concentrations averaged 16.2 vs 13.3 ng/mL (OC), 10.2 vs 12.1 ng/mL (bone ALP), 4.6 vs 4.1 ng/mL (ICTP), and 33.2 vs 40.4 ng/mL (PINP) in patients with ON and OA, respectively. In samples obtained from cancellous bone, mean concentrations of all markers were elevated significantly when compared to serum levels. The mean marker concentrations in samples obtained from cancellous bone were 33.8 vs 43.3 ng/mL (OC), 34.6 vs 37.3 ng/mL (bone ALP), 64.8 vs 36.1 ng/mL (ICTP, P=0.02), and 208.0 vs 176.2 ng/mL (PINP) in patients with ON and OA, respectively. The lesion size was at mean 440.5+/-275.8mm(2) in knees with ON and did not correlate with either serum or bone concentrations of all markers tested (P>0.1)., Conclusion: The marked elevation of markers in samples obtained from cancellous bone pointed at increased turnover in both diseases when compared to healthy individuals. In line with histologic findings of necrosis of subchondral bone, focal degradation of collagen type I was more pronounced in knees with ON. Mean serum concentrations of all markers, however, were not different from healthy individuals and thus did not provide any useful clue in the diagnosis spontaneous ON.

The goal of this study was to determine whether human osteoblasts might harbor the hepatitis C virus. We tested for positive-strand and negative-strand (replicative) hepatitis C virus RNA by reverse transcriptase-polymerase chain reaction, by in situ reverse transcriptase-polymerase chain reaction for intracellular localization of the hepatitis C virus, and by amplicon sequencing in in vitro differentiated mature osteoblasts from STRO-1+ osteoprogenitor cells from patients with chronic hepatitis C and from healthy individuals. We only detected the hepatitis C virus genome in STRO-1+ cells and mature osteoblasts from carriers with chronic hepatitis C, and we found hepatitis C virus negative strands expressed sporadically in these patients. Using in situ hepatitis C virus reverse transcriptase-polymerase chain reaction, we determined that the percentage of infected carrier osteoblasts ranged from 8.0-15.3%. These data provide evidence of hepatitis C virus presence and replication in human osteoprogenitors and osteoblasts, which may have important implications for bone allograft processing.

We compared type I collagen degradation using serum cross-linking C-terminal telopeptide (ICTP) in 18 patients with rapidly destructive osteoarthrosis and in 20 patients with slowly progressive osteoarthrosis of the hip. The diagnosis was established by clinical examination and radiographic evaluation. Total hip arthroplasty was performed in all patients. Serum levels of ICTP, bone-specific alkaline phosphatase, osteocalcin and N-terminal propeptide were studied. Patients with rapidly destructive osteoarthrosis had higher mean (SD) serum ICTP levels than patients with slowly progressive osteoarthrosis [13.2 (5.6) versus 3.7 ng/ml (1.4), p=0.001] whereas no significant difference of all other markers was seen between the groups. Elevation of ICTP levels correlated significantly with decreased joint-space width assessed by radiographs of the hip (p=0.01). Our data suggest that rapidly destructive hip osteoarthrosis is associated with elevated serum ICTP levels, reflecting increased collagen type I degradation.

The aim of this study was to evaluate bone metabolism in patients with bone marrow edema syndrome of the hip. In 37 consecutive patients undergoing core decompression of the femoral head, biochemical markers of bone metabolism were measured in aspirates from cancellous bone and in samples obtained simultaneously from peripheral blood. The diagnosis was made by means of radiographs, magnetic resonance imaging (MRI), and core biopsy specimens. Undecalcified microtome section were available for histopathological evaluation. Bone specific alkaline phosphatase (bone ALP), osteocalcin (OC), procollagen Type I N-terminal propeptide (PINP), and C-terminal cross-linking telopeptide (ICTP) were studied. Mean serum levels of analytes were 13.1 ng/mL (OC), 11.2 ng/mL (bone ALP), 4.7 ng/mL (ICTP), and 38.8 ng/mL (PINP). In samples obtained from cancellous bone, mean concentrations of all markers were elevated significantly. The mean bone to serum ratios for bone ALP and OC were 14.1 (P=0.005) and 4.1 (P=0.002), respectively. For collagen Type I metabolites, bone to serum ratios averaged 16.3 (P=0.001) for ICTP and 9.6 (P=0.001) for PINP. Markers of bone formation correlated with each other in serum as well as in aspirates from cancellous bone. Elevation of all markers in aspirates from cancellous bone pointed at increased bone turnover, which correlated with histopathological findings of irregularly woven bone, osteoid seams, and lining cells. Mean serum concentrations of all markers, however, were not different from healthy individuals and thus did not provide any useful clue in the diagnosis of this disease. The lack of osteonecrotic regions in our specimens, the marked increase of bone turnover in samples obtained from edematous lesions, and the fact that none of the patients developed osteonecrosis of the femoral head so far seem to further support the contention that transient bone marrow edema syndrome of the hip is a distinct clinical entity.

Painful pathological fractures of the femoral neck and the subtrochanteric region of the femur are reported in two women originating from India. After exclusion of renal or intestinal causes, laboratory data on bone metabolism, scintigraphic and radiographic examinations were characteristic for the presence of secondary hyperparathyroidism. Based on vitamin deficiency and low calcium absorption, disturbed mineralization of bone and increased osteoclastic resorption have apparently led to osteomalacia and subsequent fracturing. Fracture localization necessitated surgical fixation in one patient; conservative treatment including protected weightbearing was effective in the other women. After supplementation of calcium and vitamin D3, levels of parathyroid hormone and scintigraphic alterations returned to normal in both patients. In these two cases, pathological fractures of the hip could be attributed to the presence of secondary hyperparathyroidism based on decreased cutaneous vitamin D synthesis.

Parameters of hypofibrinolysis and thrombophilia were assessed in 20 consecutive patients with bone marrow edema sydrome of the hip who lacked typical risk factors for osteonecrosis. Twenty healthy subjects, matched for age and gender, served as the control subjects. In patients with bone marrow edema syndrome, elevated levels of lipoprotein(a) and plasminogen activator inhibitor were found in nine (45%) and two patients (10%), respectively. Elevation of lipoprotein(a) was present in two patients in the control group; however, plasminogen activator inhibitor levels were normal in this group. Mean levels of lipoprotein(a) were 34.8 mg/dL in the patients with bone marrow edema versus 13.8 mg/dL in the control subjects. Mean concentrations of plasminogen activator inhibitor, apolipoproteins, lipid metabolism parameters, and indicators of thrombophilia did not differ in both groups. Pulsed field gel electrophoresis and Southern blots confirmed the presence of pathologic allelotypes in three patients with elevated levels of lipoprotein(a) who had a familial occurrence of bone marrow edema syndrome. These findings also underline a fundamental role of hypofibrinolysis, mediated by elevated levels of lipoprotein(a) or plasminogen activator inhibitor, or both, in the former idiopathically considered etiology of the bone marrow edema syndrome of the hip.

In 27 patients undergoing arthroscopy of the knee for treatment of meniscal diseases, biochemical markers of bone metabolism were measured in cancellous bone, and levels were compared with concentrations obtained from peripheral blood. Bone-specific alkaline phosphatase, osteocalcin, and collagen Type I metabolites (procollagen Type I N-terminal peptide and carboxy-terminal cross-linked telopeptide) were studied simultaneously in serum and in the distal femur using a radioimmunoassay. Although levels of bone-specific alkaline phosphatase and osteocalcin did not differ between serum and cancellous bone, concentrations of collagen Type I metabolites were elevated significantly in healthy cancellous bone. The close correlations between bone and serum concentrations confirmed accuracy of results obtained from cancellous bone. The mean bone-to-serum ratio for alkaline phosphatase and osteocalcin was 1.1 and 1.2, respectively. Collagen Type I metabolite ratios of 2.2 (for carboxy-terminal cross-linked telopeptide) and 2.3 (for procollagen Type I N-terminal peptide) indicate that these markers are formed locally and then released into the circulation. Bone seems to be a major contributor of collagen Type I metabolites to the serum pool.

Osteoclast resorptive activity occurs despite the presence of extremely high levels of ionized calcium ([Ca2+]) within the osteoclast hemivacuole, which is generated as a by-product of its resorptive activity. Previous in vitro observations have shown that increases in extracellular [Ca2+] ([Ca2+]e) in the surrounding medium can inhibit the osteoclast resorptive activity. Therefore, it has been suggested that the osteoclast acts as a "sensor" for [Ca2+]e, and that high [Ca2+]e leads to an increase in intracellular [Ca2+] ([Ca2+]i), thereby inhibiting osteoclasts in a negative feedback manner. In this report we have carried out an experimental and theoretical analysis of calcium disposal during osteoclast activity to evaluate how in vitro models relate to in vivo osteoclast activity, where it is possible that high [Ca2+]e may be present in the hemivacuole but not over the nonresorbing surface of the cell. Scanning electrochemical microscopy (SECM) studies of [Ca2+] and superoxide anion (O2.-) generation by bone-resorbing osteoclasts on the surface of a bovine cortical bone slice were compared with microspectofluorometric measurements of the levels of [Ca2+]i in single osteoclasts and the effect of [Ca2+]i on various aspects of osteoclast function. The generation of O2.- by the osteoclasts has been shown to be positively correlated with osteoclast resorptive function and can therefore serve as an index of acute changes in osteoclast activity. The SECM of bone-resorbing osteoclasts at the surface of a bone slice revealed a continuous steady-state release of Ca2+. Even after prolonged incubation lasting 3 h the near-surface [Ca2+]e in the solution above the cell remained <2 mM. The SECM real-time measurement data were consistent with the osteoclast acting as a conduit for continuous Ca2+ disposal from the osteoclast-bone interface. We conclude that the osteoclast distinguishes [Ca2+]e in the hemivacuole and in the extracellular fluid above the cell which we denote [Ca2+]e. We found that an increase in [Ca2+]i may be associated with activation; inhibition; or be without effect on O2.- generation, bone-matrix, or bone resorption. Similarly, osteoclast adhesion and bone-resorbing activity was affected by [Ca2+]e' but showed no correlation with [Ca2+]i. The data suggest the existence of functional compartmentalization of [Ca2+]i within the osteoclast, where elevated calcium may have an inhibitory, excitatory, or no effect on the overall osteoclast activity while exerting a selective effect on different functional modalities. These observations lead to the conclusion that far from being inhibited by Ca2+ generated, the osteoclast by virtue of the observed functional compartmentalization is highly adapted at carrying out its activity even when the level of [Ca2+] in resorptive lacunae is elevated.

Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one of the two heads of unphosphorylated HMM binds to actin in the presence of ADP, and the heads have different affinities for ADP. This asymmetry suggests that phosphorylation alters the mechanical coupling between the heads of HMM. A model that incorporates strain between the two heads is proposed to explain the data, which have implications for how one head of a motor protein can gate the response of the other.

There is controversy whether bone marrow edema syndrome represents a distinct transient disease or reflects an early reversible phase of spontaneous osteonecrosis of the hip. Hypofibrinolysis on the basis of elevated plasma levels of plasminogen activator inhibitor or lipoprotein(a) or both has been reported to favor the development of bone marrow edema syndrome and nontraumatic avascular necrosis. The current authors report on the familial occurrence of transient bone marrow edema syndrome of the hip in three female family members. Plasma concentrations of lipoprotein(a) were elevated in all three patients, whereas serum levels of plasminogen activator inhibitor were within normal ranges. This first familial description of transient bone marrow edema syndrome of the hip strongly suggests that a genetically determined elevation of lipoprotein(a) may be an important predisposing factor in these patients.

The mode of action of forskolin is of clinical and scientific interest since forskolin has been shown to have potentially therapeutic bone anti-resorptive and anti-hypertensive properties. Forskolin is thought to inhibit the bone resorptive activity of osteoclasts by elevating cytosolic cAMP and to mimic as well as augment the anti-resorptive effect of calcitonin (CT). Other studies have found that forskolin has a dose-dependent dual effect in mouse calavaria, stimulating bone resorption at low doses and having an inhibitory effect at high doses. However, the acute effect of forskolin on osteoclast functional modality has never been studied. The present investigation examined the effect of low (1 mM) and high doses (10 mM) of forskolin on superoxide anion (O2-) generation in isolated bone-resorbing rat osteoclasts. Forskolin was found to have a bimodal cAMP-independent effect on O2- generation, being stimulatory at a low dose and having an inhibitory effect at a higher dose. These findings also suggest that CT-induced inhibition of O2- generation in the osteoclasts is likely to be mediated by cAMP-independent pathways, perhaps involving [Ca2+]i modulation.

We have investigated the possible acute effect of steroid hormones, including 1alpha,25 dihydroxycholecalciferol (1alpha,25(OH)2D3) and estradiol, on the generation of superoxide anion (O2*-) in bone resorbing osteoclasts. Evidence is presented demonstrating acute non-genomic stimulatory action of 1alpha,25(OH)2D3 on the production of free radicals by rat osteoclasts cultured on calcified matrix. The increase in O2*- production was observed in the range of 6-10 s (n = 5) following exposure of enriched osteoclasts to 1alpha,25(OH)2D3 and was found to be transient with the peak response being in the range of 5-45 s (n = 5). The decline in the transient was much slower than the elevation, time for the decay being in the range 1-5 min (n = 5) and remained above the levels present prior to the addition. The exposure of the osteoclast to dexamethasone was found to have no effect on O2*- generation, whilst estradiol was found to be inhibitory. The mode of stimulation and the kinetics of the transients of O2*- in the bone resorbing osteoclasts produced by 1alpha,25(OH)2D3 were similar to that of parathyroid hormone (PTH) and pertussis. The exposure of the bone resorbing osteoclasts to cholera toxin was found to have no effect, suggesting that the stimulatory action is unlikely to be mediated via cAMP elevation. The importance of these observations is discussed in the context of calcium homeostasis and bone physiology.

We have screened the mitochondrial genome of 15 men with symptomatic vertebral fractures (median age 62 years, range 27-72 years) and 17 male control subjects (median age 61 years, range 40-73 years) for the presence of mitochondrial DNA (mtDNA) deletions in peripheral monocyte DNA. Polymerase chain reaction analysis provided evidence of a common age-related (4.9 kb) mtDNA deletion situated between nucleotides 8470 and 13.460 of the genomic sequence in 5 of the 17 controls (29%) and 9 of the 15 patients (60%) investigated. Southern blotting and polymerase chain reaction revealed a novel 3.7 kb deletion in 2 patients. One of the affected patients, a 27-year-old man with severe osteoporosis (lumbar spine bone mineral density (BMD) 0.381 g/cm(2); Z-score -6.45) was found to harbor deletion in almost 50% of the mitochondria. The patient had a blood lactic acid level (4.6 nM) that was over 3 times the upper reference range (0-1.3 mM), thus confirming the presence of systemic oxidative stress. Further analysis by modified primer shift polymerase chain reaction showed the 5' breakpoint to be between the nucleotides 10.63 kb and 10.80 kb of the mtDNA. The second patient harboring the 3.7 kb deletion was older (62 years) with less severe osteoporosis (lumbar spine BMD 0.727/cm(2); Z-score -2.58) and the proportion of affected mitochondria was lower (25%). The significance of these findings is discussed and the possible relation between oxidative stress and accelerated bone loss is examined.