The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class H (MHC H) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC H chaperone E, and hence in the formation of MHC H-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, II processing and MHC H peptide loading proceeded similarly in all three DC populations. We then analyzed MHC H localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

Persistence of recipient lymphocytes in NOD mice after irradiation and bone marrow transplantation

The non-obese diabetic (NOD) mouse is a unique and invaluable model of autoimmune disease, in particular type I diabetes. Bone marrow transplantation as a therapy for type I diabetes has been explored in NOD mice. NOD mice require higher doses of conditioning irradiation for successful allogeneic bone marrow transplantation, suggesting that NOD hematopoietic cells are radioresistant compared to those of other mouse strains. However, studies of hematopoietic reconstitution in NOD mice are hampered by the lack of mice bearing a suitable cell-surface marker that would allow transferred cells or their progeny to be distinguished. In order to monitor hematopoietic reconstitution in NOD mice we generated congenic NOD mice that carry the alternative allelic form of the pan-leukocyte alloantigen CD45. Following irradiation and congenic bone marrow transplantation, we found that the myeloid lineage was rapidly reconstituted by cells of donor origin but substantial numbers of recipient T lymphocytes persisted even after supra-lethal irradiation. This indicates that radiation resistance in the NOD hematopoietic compartment is a property primarily of mature T lymphocytes. (C) 2004 Elsevier Ltd. All rights reserved.

Maturation temperature and rainfall influence seed dormancy characteristics of annual ryegrass (Lolium rigidum)

The role of temperature and rainfall during seed development in modulating subsequent seed dormancy status was studied for Lolium rigidum Gaud. (annual ryegrass). Climatic parameters relating to geographic origin were compared with annual ryegrass seed dormancy characteristics for seeds collected from 12 sites across the southern Western Australian cropping region. Seed germination was tested soon after collection and periodically during subsequent after-ripening. Temperature in the year of seed development and long-term rainfall patterns showed correlations with aspects of seed dormancy, particularly the proportion of seeds remaining dormant following 5 months of after-ripening. Consequently, for one population the temperature (warm/cool) and water supply (adequate/reduced) during seed development were manipulated to investigate the role of maternal environment in the quantity and dormancy characteristics of seeds produced. Seeds from plants grown at warm temperatures were fewer in number, weighed less, and were less dormant than those from plants grown at cool temperature. Seeds that developed under both cool temperature and reduced moisture conditions lost dormancy faster than seeds from well-watered plants. Seed maturation environment, particularly temperature, can have a significant effect on annual ryegrass seed numbers and seed dormancy characteristics.

Local delivery of EGF-liposome mediated bone modeling in orthodontic tooth movement by increasing RANKL expression

Aims: It has long been demonstrated that epidermal growth factor (EGF) has catabolic effects oil bone. Thus. we examined the role of EGF in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement. Main methods: The maxillary first molars of rats were moved mesially using an orthodontic appliance attached to the maxillary incisor teeth. Rats were randomly divided into 4 groups: (G1) administration of PBS (Phosphate buffer saline Solution (n = 24); (G2) administration of empty liposomes (it = 24): (Q) administration 20 rig of EGF Solution (n = 24): and (G4) 20 ng of EGF-liposomes Solution (it = 24). Each Solution was injected in the mucosa of the left first molar adjacent to the appliance. At days 5, 10, 14 and 2 1 after drug administration. 6 animals of each group were sacrificed. Histomorphometric analysis was used to quantify osteoclasts (Tartrate-resistant acid phosphatase (TRAP) + cells) and tooth movement. Using immunohistochemistry assay we evaluated the RANKL (receptor activator of nuclear factor kappa B ligand) and epidermal growth factor receptor (EGFR) expression. Key findings: The EGF-liposome administration showed an increased tooth movement and osteoclast numbers compared to controls (p<0.05). This was correlated with intense RANKL expression. Both osteoblasts and osteoclasts expressed EGFR. Significance: Local delivery of EGF-liposome stimulates, osteoclastogenesis and tooth movement. (C) 2009 Elsevier Inc. All rights reserved.

Mechanical loading modulates glutamate receptor subunit expression in bone

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-D-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GuR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity. (C) 2005 Elsevier Inc. All rights reserved.

Mechanical vibration preserves bone structure in rats treated with glucocorticoids

Glucocorticoids are an important cause of secondary osteoporosis in humans, which decreases bone quality and leads to fractures. Mechanical stimulation in the form of low-intensity and high-frequency vibration seems to be able to prevent bone loss and to stimulate bone formation. The objective of this study was to evaluate the effects of mechanical vibration on bone structure in rats treated with glucocorticoids. Thirty 3-month-old adult male Wistar rats were randomized to three groups: control (C), glucocorticoid (G), and glucocorticoid with vibration (CV). The G and GV groups received 3.5 mg/kg/day of methylprednisolone 5 days/week for a duration of 9 weeks, and the C group received vehicle (saline solution) during the same period. The CV group was vibrated on a special platform for 30 min per day, 5 days per week during the experiment. The platform was set to provide a vertical acceleration of 1 G and a frequency of 60 Hz. Skeletal bone mass was evaluated by total body densitometry (DXA). Fracture load threshold, undecalcified bone histomorphometry, and bone volume were measured in tibias. Glucocorticoids induced a significantly lower weight gain (-9.7%) and reduced the bone mineral content (-9.2%) and trabecular number (-41.8%) and increased the trabecular spacing (+98.0%) in the G group, when compared to the control (C). Vibration (CV) was able to significantly preserve (29.2%) of the trabecular number and decrease the trabecular spacing (+ 26.6%) compared to the G group, although these parameters did not reach C group values. The fracture load threshold was not different between groups, but vibration significantly augmented the bone volume of the tibia by 21.4% in the CV group compared to the C group. Our study demonstrated that low-intensity and high-frequency mechanical vibration was able to partially inhibit the deleterious consequences of glucocorticoids on bone structure in rats. (C) 2010 Elsevier Inc. All rights reserved.

Coronary Calcification Is Associated With Lower Bone Formation Rate in CKD Patients Not Yet in Dialysis Treatment

Vascular calcification is a strong prognostic marker of mortality in hemodialysis patients and has been associated with bone metabolism disorders in this population. In earlier stages of chronic kidney disease (CKD), vascular calcification also has been documented. This study evaluated the association between coronary artery calcification (CAC) and bone histomorphometric parameters in CKD predialysis patients assessed by multislice coronary tomography and by undecalcified bone biopsy. CAC was detected in 33 (66%) patients, and their median calcium score was 89.7 (0.4-2299.3 AU). The most frequent bone histologic alterations observed included low trabecular bone volume, increased eroded and osteoclast surfaces, and low bone-formation rate (BFR/BS). Multiple logistic regression analysis, adjusted for age, sex, and diabetes, showed that BFR/BS was independently associated with the presence of coronary calcification [p=.009; odd ratio (OR) = 0.15; 95% confidence interval (Cl) 0.036-0.619] This study showed a high prevalence of CAC in asymptomatic predialysis CKD patients. Also, there was an independent association of low bone formation and CAC in this population. In conclusion, our results provide evidence that low bone-formation rate constitutes another nontraditional risk factor for cardiovascular disease in CKD patients. 2010 American Society for Bone and Mineral Research.

Influence of ovariectomy and masticatory hypofunction on mandibular bone remodeling

Introduction: This study was designed to examine the effect of masticatory hypofunction and estrogen deficiency on mandible bone mass and compare this site with spine and femoral bone. Methods: Twenty-four rats were ovariectomized (OVX) or Sham-operated (Sham) and analyzed after feeding with hard diet (Hard) or soft diet (Soft). They were divided into four groups: (GI)Sham-Hard; (GII)OVX-Hard; (GIII)Sham-Soft and (GIV)OVX-Soft. Bone mineral density (BMD) was measured in the spine and femur in the baseline and at the end of the study, and Delta BMD (final BMD - baseline BMD) was calculated. In mandible bone, BMD and histomorphometry were analyzed at the end of the experiment. Results: Sham rats showed higher spine (GI: 13.5%vs GII: 0.74%, P < 0.01; GIII: 10.67%vs GIV: -4.36%, P < 0.001) and femur Delta BMD (GI: 14.43%vs GII: 4.42%, P < 0.01; GIII: 10.58%vs GIV: 0.49%, P < 0.001) than OVX, but no difference was observed in mandible BMD among these groups (P > 0.05). Soft-diet groups showed decreased mandible BMD compared with hard-diet groups (GIV vs GII, P < 0.01; GIII vs GI, P < 0.01). Similarly, mandibular condyle histomorphometry showed that soft-diet groups presented a significant decrease in trabecular thickness and volume (GIV vs GII, P < 0.05; GIII vs GI, P < 0.01) compared with hard diet. Conclusion: Our results suggest that mandibular bone loss resulted from decreased of mechanical loading during mastication, and was not affect by estrogen depletion.

Is Coronary Artery Calcification Associated with Vertebral Bone Density in Nondialyzed Chronic Kidney Disease Patients?

Background and objectives Low bone mineral density and coronary artery calcification (CAC) are highly prevalent among chronic kidney disease (CKD) patients, and both conditions are strongly associated with higher mortality. The study presented here aimed to investigate whether reduced vertebral bone density (VBD) was associated with the presence of CAC in the earlier stages of CKD. Design, setting, participants, & measurements Seventy-two nondialyzed CKD patients (age 52 +/- 11.7 years, 70% male, 42% diabetics, creatinine clearance 40.4 +/- 18.2 ml/min per 1.73 m(2)) were studied. VBD and CAC were quantified by computed tomography. Results CAC > 10 Agatston units (AU) was observed in 50% of the patients (median 120 AU [interquartile range 32 to 584 AU]), and a calcification score >= 400 AU was found in 19% (736 [527 to 1012] AU). VBD (190 +/- 52 Hounsfield units) correlated inversely with age (r = -0.41, P < 0.001) and calcium score (r = -0.31, P = 0.01), and no correlation was found with gender, creatinine clearance, proteinuria, lipid profile, mineral parameters, body mass index, and diabetes. Patients in the lowest tertile of VBD had expressively increased calcium score in comparison to the middle and highest tertile groups. In the multiple logistic regression analysis adjusting for confounding variables, low VBD was independently associated with the presence of CAC. Conclusions Low VBD was associated with CAC in nondialyzed CKD patients. The authors suggest that low VBD might constitute another nontraditional risk factor for cardiovascular disease in CKD. Clin J Am Soc Nephrol 6: 1456-1462, 2011. doi: 10.2215/CJN.10061110

Serum Ferritin Level Remains a Reliable Marker of Bone Marrow Iron Stores Evaluated by Histomorphometry in Hemodialysis Patients

Background and objectives: As well as being a marker of body iron stores, serum ferritin (sFerritin) has also been shown to be a marker of inflammation in hemodialysis (HD) patients. The aim of this study was to analyze whether sFerritin is a reliable marker of the iron stores present in bone marrow of HD patients. Design: Histomorphometric analysis of stored transiliac bone biopsies was used to assess iron stores by determining the number of iron-stained cells per square millimeter of bone marrow. Results: In 96 patients, the laboratory parameters were hemoglobin = 11.3 +/- 1.6 g/dl, hematocrit = 34.3 +/- 5%, sFerritin 609 +/- 305 ng/ml, transferrin saturation = 32.7 +/- 22.5%, and C-reactive protein (CRP) = 0.9 +/- 1.4 mg/dl. sFerritin correlated significantly with CRP, bone marrow iron, and time on HD treatment W = 0.006, 0.001, and 0.048, respectively). The independent determinants of sFerritin were CRP (beta-coef = 0.26; 95% CI = 24.6 to 132.3) and bone marrow iron (beta-coef = 0.32; 95% CI = 0.54 to 2.09). Bone marrow iron was higher in patients with sFerritin >500 ng/ml than in those with sFerritin :5500 ng/ml. In the group of patients with sFerritin :5500 ng/ml, the independent determinant of sFerritin was bone marrow iron (beta-coef = 0.48, 95% CI = 0.48 to 1.78), but in the group of patients with sFerritin >500 ng/ml, no independent determinant of sFerritin was found. Conclusions: sFerritin adequately reflects iron stores in bone marrow of HD patients.

Association of Changes in Bone Remodeling and Coronary Calcification in Hemodialysis Patients: A Prospective Study

Background: Vascular calcification is common and constitutes a prognostic marker of mortality in the hemodialysis population. Derangements of mineral metabolism may influence its development. The aim of this study is to prospectively evaluate the association between bone remodeling disorders and progression of coronary artery calcification (CAC) in hemodialysis patients. Study Design: Cohort study nested within a randomized controlled trial. Setting & Participants: 64 stable hemodialysis patients. Predictor: Bone-related laboratory parameters and bone histomorphometric characteristics at baseline and after 1 year of follow-up. Outcomes: Progression of CAC assessed by means of coronary multislice tomography at baseline and after 1 year of follow-up. Baseline calcification score of 30 Agatston units or greater was defined as calcification. Change in calcification score of 15% or greater was defined as progression. Results: Of 64 patients, 26 (40%) had CAC at baseline and 38 (60%) did not. Participants without CAC at baseline were younger (P < 0.001), mainly men (P = 0.03) and nonwhite (P = 0.003), and had lower serum osteoprotegerin levels (P = 0.003) and higher trabecular bone volume (P = 0.001). Age (P 0.003; beta coefficient = 1.107; 95% confidence interval [Cl], 1.036 to 1.183) and trabecular bone volume (P = 0.006; beta coefficient = 0.828; 95% Cl, 0.723 to 0.948) were predictors for CAC development. Of 38 participants who had calcification at baseline, 26 (68%) had CAC progression in 1 year. Progressors had lower bone-specific alkaline phosphatase (P = 0.03) and deoxypyridinoline levels (P = 0.02) on follow-up, and low turnover was mainly diagnosed at the 12-month bone biopsy (P = 0.04). Low-turnover bone status at the 12-month bone biopsy was the only independent predictor for CAC progression (P = 0.04; beta coefficient = 4.5; 95% Cl, 1.04 to 19.39). According to bone histological examination, nonprogressors with initially high turnover (n = 5) subsequently had decreased bone formation rate (P = 0.03), and those initially with low turnover (n = 7) subsequently had increased bone formation rate (P = 0.003) and osteoid volume (P = 0.001). Limitations: Relatively small population, absence of patients with severe hyperparathyroidism, short observational period. Conclusions: Lower trabecular bone volume was associated with CAC development, whereas improvement in bone turnover was associated with lower CAC progression in patients with high- and low-turnover bone disorders. Because CAC is implicated in cardiovascular mortality, bone derangements may constitute a modifiable mortality risk factor in hemodialysis patients.

RANKL is a mediator of bone resorption in idiopathic hypercalciuria

Background and objectives: This study aimed to determine the expression of osteoprotegerin, receptor activator of nuclear factor kappa B ligand, interleukin-la, transforming growth factor-beta, and basic fibroblast growth factor in stone-forming patients with idiopathic hypercalciuria. Design, setting, participants, & measurements: Immunohistochemical analysis was performed in undecalcified bone samples previously obtained from 36 transiliac bone biopsies of patients who had idiopathic hypercalciuria and whose histomorphometry had shown lower bone volume, increased bone resorption, and prolonged mineralization lag time. Results: Bone expression of receptor activator of nuclear factor kappa B ligand and osteoprotegerin was significantly higher in patients with idiopathic hypercalciuria versus control subjects. Transforming growth factor-beta immunostaining was lower in patients with idiopathic hypercalciuria than in control subjects and correlated directly with mineralization surface. Interleukin-la and basic fibroblast growth factor staining did not differ between groups. Receptor activator of nuclear factor kappa B ligand bone expression was significantly higher in patients who had idiopathic hypercalciuria and exhibited higher versus normal bone resorption. Conclusion: A higher expression of receptor activator of nuclear factor kappa B ligand in bone tissue suggests that increased bone resorption in patients with idiopathic hypercalciuria is mediated by receptor activator of nuclear factor kappa B ligand. Osteoprotegerin bone expression might have been secondarily increased in an attempt to counteract the actions of receptor activator of nuclear factor kappa B ligand. The low bone expression of transforming growth factor-beta could contribute to the delayed mineralization found in such patients.