Natalie Dion

Interferon-γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice

Interferon γ (IFN‐γ) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN‐γ ablation in vivo in C57BL/6 mice expressing the IFN‐γ receptor knockout phenotype (IFNγR1−/−). Compared with their wild‐type littermates (IFNγR1+/+), IFNγR1−/− mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNγR1−/− mice showed a low‐bone‐turnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone‐formation and bone‐resorption markers. Furthermore, administration of IFN‐γ (2000 and 10,000 units) to wild‐type C57BL/6 sham‐operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN‐γ signaling as a potential new anabolic therapeutic target for osteoporosis.

Kinetic evidence that His‐711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state

Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane‐bound Zn‐metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc‐endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site‐directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP‐Gln711 and NEP‐Phe711). Determination of kinetic parameters showed that both mutants had K m values very similar to that of the non‐mutated enzyme but that their k cat values were 25‐fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.

Atypical Femoral Fractures and Bone Turnover

A bone biopsy performed in a woman treated with bisphosphonates who had bilateral atypical femoral fractures did not reveal decreased bone turnover, suggesting the possibility that fractures associated with bisphosphonate use are not due to oversuppression of bone turnover.

Microwave Irradiation of Ethanol-fixed Bone Improves Preservation, Reduces Processing Time, and Allows Both Light and Electron Microscopy on the Same Sample

Methylmethacrylate (MMA) embedding is routinely used for histomorphometry of undecalcified bone preserved by prolonged immersion in ethanol, a procedure that yields poor ultrastructural detail. Because microwave irradiation (MWI) facilitates penetration of fixatives, we have investigated whether it can improve preservation by ethanol. Rat tibiae, some labeled with tetracycline, and a human iliac crest biopsy were immersed in 70% ethanol and dehydrated, both under MWI, for a total processing time of ~7 hr. Controls were not irradiated, and all specimens were embedded in MMA at 4C. They were then processed for histomorphometry, histochemistry, structural analysis, and immunolabeling. The results showed that histological preservation was improved with MWI. Static bone formation and resorption parameters and rate of mineral apposition were similar to those of conventionally processed specimens. Mineral distribution, as visualized by von Kossa staining and backscattered electron imaging, was not affected. Alkaline phosphatase and tartrate-resistant acid phosphatase activity, as well as immunolocalization of bone sialoprotein and osteopontin, were readily visualized. Ultrastructurally, osteopontin exhibited a typical distribution in mineralization foci, between calcified collagen fibrils, and at cement lines. These data show that MWI improves preservation and permits application of a broad spectrum of analytical methodologies on the same bone sample while considerably reducing processing time.

Teriparatide and Bone Turnover and Formation in a Hemodialysis Patient With Low-Turnover Bone Disease: A Case Report

Teriparatide, a recombinant form of parathyroid hormone, is an anabolic agent approved for use in women and men with osteoporosis. However, it is not well studied in people with chronic kidney disease (CKD). We report on a patient with stage 5 CKD treated with dialysis who presented to our clinic with multiple fractures, including bilateral nondisplaced pelvic fractures resulting in chronic pain and interfering with the patients ability to work. Bone histomorphometry demonstrated low-turnover bone disease, and he was treated with 20μg of teriparatide (subcutaneous injection) every morning for 24 months. Within 6 months of initiating therapy, the patients pain resolved and he was able to resume work. Serum calcium and phosphate levels remained within reference ranges throughout his treatment, and he sustained no further fractures. During 24 months of treatment, bone mineral density was maintained at the lumbar spine, and there was an increase of 4% at the femoral neck and total hip. A second transiliac bone biopsy demonstrated improvements in static and dynamic parameters of bone formation. In our patient, 24-month treatment with teriparatide was safe and effective; however, larger studies are needed to determine the efficacy of teriparatide in the dialysis-dependent CKD population.

Characterisation of neprilysin (EC 3.4.24.11) S2′ subsite

Neprilysin is a neutral peptidase that cleaves small peptide substrates on the amino‐side of hydrophobic amino acid residues. In the present study, we have used inhibition of non‐mutated and mutated enzymes with dipeptide inhibitors and hydrolysis of the substrate [Leu5, Arg6]enkephalin in order to evaluate the contribution of the S2′ subsite to substrate and inhibitor binding. Our results suggest that (1) Arg‐102 and Asn‐542 provide major contributions to the interaction of the enzyme with the P2′ residue of the substrate, (2) the S2′ subsite is vast and can accommodate bulky side chains, and (3) Arg‐102 restricts access to the S2′ subsite to some side chains such as arginine.

Impaired rib bone mass and quality in end-stage cystic fibrosis patients

BACKGROUND Advancements in research and clinical care have considerably extended the life expectancy of cystic fibrosis (CF) patients. However, with this extended survival come comorbidities. One of the leading co-morbidities is CF-related bone disease (CFBD), which progresses with disease severity and places patients at high risk for fractures, particularly of the ribs and vertebrae. Evidence that CF patients with vertebral fractures had higher bone mineral density (BMD) than the nonfracture group led us to postulate that bone quality is impaired in these patients. We therefore examined rib specimens resected at the time of lung transplant in CF patients to measure parameters of bone quantity and quality. METHODS In this exploratory study, we analysed 19 end-stage CF and 13 control rib specimens resected from otherwise healthy lung donors. BMD, bone microarchitecture, static parameters of bone formation and resorption and microcrack density of rib specimens were quantified by imaging, histomorphometric and histological methods. Variables reflecting the mineralization of ribs were assessed by digitized microradiography. The degree of bone mineralization (g/cm3) and the heterogeneity index of the mineralization (g/cm3) were calculated for trabecular and cortical bone. RESULTS Compared to controls, CF ribs exhibited lower areal and trabecular volumetric BMD, decreased trabecular thickness and osteoid parameters, and increased microcrack density, that was particularly pronounced in specimens from patients with CF-related diabetes. Static parameters of bone resorption were similar in both groups. Degree of mineralization of total bone, but not heterogeneity index, was increased in CF specimens. CONCLUSION The combination of reduced bone mass, altered microarchitecture, imbalanced bone remodeling (maintained bone resorption but decreased formation), increased microdamage and a small increase of the degree of mineralization, may lead to decreased bone strength, which, when coupled with chronic coughing and chest physical therapy, may provide an explanation for the increased incidence of rib fractures previously reported in this population.

Methods in Bone Histomorphometry for Animal Models

Animal models are essential for preclinical skeletal research, notably in the study of physiopathology of metabolic bone diseases as well as in the evaluation of a test agent for the prevention and treatment of bone diseases such as osteoporosis. However, rat or mouse models do not completely reflect human bone disorders and therapeutic responses, but they provide insight into the understanding of bone pathologies and their treatments.

206 Bone quality at the time of lung transplant in cystic fibrosis patients

Background and Hypothesis As CF patients get older, morbidities such as CF-related bone disease (CFBD), superimpose on the pre-existing complications. CFBD predisposes to fractures, especially of the ribs and vertebrae. Knowing that bone mineral density (BMD) is a poor predictor of fractures in CF, we postulate that alterations in bone quality will likely contribute to their propensity to fracture. Methods In order to assess bone quality in CF patients, a 2 cm segment of rib was recovered during lung transplant (LTx). Controls consisted in similarly aged lung donors (D). Dual-energy X-ray absorptiometry (DXA), micro-CT, bone histomorphometry and microcrack detection were used to assess BMD, microarchitecture, bone remodeling and quality of bone respectively. Results Over 17 CF and 21 donors specimens were analyzed. Patients were stratified according to bisphosphonate (BP) or steroid (GC) treatment at the time of LTx. Ex vivo rib BMD, trabecular BMD and bone volume determined by micro-CT as well as trabecular thickness were significantly reduced in CF, with patients on GC having the lowest values. Despite similar surface occupied by osteoblasts, osteoid parameters (volume, surface and thickness) were lower in CF, regardless of the use of GC or BP. Number of osteoclasts and eroded surfaces were comparable among the groups. Microcracks were 3 to 4 times higher in CF than in D. Of note, the highest microcrack density was found in patients on BP. Conclusion Our data suggest alterations in the bone quality of CF ribs as evidenced by a decrease in osteoblast function and bone formation resulting in the uncoupling of bone remodeling, and the accumulation of microdamages.