Experimental arthritis: effect on growth parameters and total skeletal calcium

Aim of the study was to investigate the possible mechanisms leading to stunted growth and osteoporosis in experimental arthritis. Fourty-two female rats of 7-8 weeks of age were randomly assigned to three groups of 14 animals each: (a) controls; (b) adjuvant-inoculated (AA); and (c) adjuvant-inoculated rats receiving 10 mg cyclosporin A (CsA) orally for 30 days. Biological parameters studied were: hindpaw swelling; vertebral length progression expressed as Delta increments between days 1 and 30 as a parameter of skeletal growth, and estimation of total skeletal mineral content by dual energy X-ray absorptiometry (n=10 each group) on day 30. Endocrine parameters measured were pulsatile release of growth hormone (rGH) on day 30 following jugular cannulation and measurement of insulin-like growth factor (IGF-1) in pooled plasma from rGH profiles. Results can be summarized as follows: Untreated AA rats exhibited local signs of inflammation in comparison with controls (hindpaw diameter 8.1-8.9 mm vs. 5.3-5.6 mm in controls). Treatment with CsA normalized this parameter (4.9-5.6 mm). Vertebral growth was significantly retarded in AA rats in comparison with controls (214+/-32 vs. 473+/-33 microm; p<0.001). Administration of CsA normalized vertebral size increment with a clear tendency to overgrowth (523+/-43 microm, n.s.). There was also a marked reduction in total skeletal mineral content in diseased (AA) rats as compared to controls (5.8+/-0.1 vs. 7.5+/-0.1g [OH-apatite]; p<0.001), and a moderate but significant increment above controls in the group receiving CsA (8.0+/-0.1 vs. 7.5+/-0.1g [OH-appatite]; p<0.04). Integrated rGH profiles exhibited a significant fall in arthritic rats and were completely restored to normal under CsA treatment. A trend toward higher rGH values was observed in the latter group (2908+/-554 in AA vs. 8317+/-1492 ng/ml/240 min in controls; p<0.001, and 10940+/-222 ng/ml/240 min, n.s. in the CsA group). There was a good correlation between skeletal growth and rGH pulsatility (r=0.81; p<0.001). IGF-1 followed a similar pattern (630+/-44 in AA vs. 752+/-30 ng/ml in controls; p<0.04, and 769+/-59 ng/ml in the CsA group, n.s. vs. controls). Thus, a clear tendency to skeletal overgrowth following treatment was observed in agreement with the hormonal data. It can therefore be concluded that, in experimental arthritis, attenuated GH-spiking and reduced circulating IGF-1 appear to be causally related to growth retardation, probably mimicking signs and symptoms observed in juvenile arthritis. Therapy with CsA is followed by normalization of hormonal and biological parameters accompanied by a catch up phenomenon in skeletal growth which is also observed clinically in juvenile arthritis. Generalized osteopenia is a prominent feature seemingly connected with the growth abnormalities as they parallel each other during the evolution of the disease and respond equally to therapy.

Calcium channel TRPV6 is involved in murine maternal-fetal calcium transport

Maternal-fetal calcium (Ca(2+)) transport is crucial for fetal Ca(2+) homeostasis and bone mineralization. In this study, the physiological significance of the transient receptor potential, vanilloid 6 (TRPV6) Ca(2+) channel in maternal-fetal Ca(2+) transport was investigated using Trpv6 knockout mice. The Ca(2+) concentration in fetal blood and amniotic fluid was significantly lower in Trpv6 knockout fetuses than in wildtypes. The transport activity of radioactive Ca(2+) ((45)Ca) from mother to fetuses was 40% lower in Trpv6 knockout fetuses than in wildtypes. The ash weight was also lower in Trpv6 knockout fetuses compared with wildtype fetuses. TRPV6 mRNA and protein were mainly localized in intraplacental yolk sac and the visceral layer of extraplacental yolk sac, which are thought to be the places for maternal-fetal Ca(2+) transport in mice. These expression sites were co-localized with calbindin D(9K) in the yolk sac. In wildtype mice, placental TRPV6 mRNA increased 14-fold during the last 4 days of gestation, which coincides with fetal bone mineralization. These results provide the first in vivo evidence that TRPV6 is involved in maternal-fetal Ca(2+) transport. We propose that TRPV6 functions as a Ca(2+) entry pathway, which is critical for fetal Ca(2+) homeostasis.

Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a (45)Ca(2+) uptake assay (7.11 +/- 1.93 versus 3.61 +/- 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuria.

Inhibition of FcepsilonRI-dependent mediator release and calcium flux from human mast cells by sialic acid-binding immunoglobulin-like lectin 8 engagement

BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

PTH and 1.25 vitamin D response to a low-calcium diet is associated with bone mineral density in renal stone formers

BACKGROUND: Renal calcium stones and hypercalciuria are associated with a reduced bone mineral density (BMD). Therefore, the effect of changes in calcium homeostasis is of interest for both stones and bones. We hypothesized that the response of calciuria, parathyroid hormone (PTH) and 1.25 vitamin D to changes in dietary calcium might be related to BMD. METHODS: A single-centre prospective interventional study of 94 hyper- and non-hypercalciuric calcium stone formers consecutively retrieved from our stone clinic. The patients were investigated on a free-choice diet, a low-calcium diet, while fasting and after an oral calcium load. Patient groups were defined according to lumbar BMD (z-score) obtained by dual X-ray absorptiometry (group 1: z-score <-0.5, n = 30; group 2: z-score -0.5-0.5, n = 36; group 3: z-score >0.5, n = 28). The effect of the dietary interventions on calciuria, 1.25 vitamin D and PTH in relation to BMD was measured. RESULTS: An inverse relationship between BMD and calciuria was observed on all four calcium intakes (P = 0.009). On a free-choice diet, 1.25 vitamin D and PTH levels were identical in the three patient groups. However, the relative responses of 1.25 vitamin D and PTH to the low-calcium diet were opposite in the three groups with the highest increase of 1.25 vitamin D in group 1 and the lowest in group 3, whereas PTH increase was most pronounced in group 3 and least in group 1. CONCLUSION: Calcium stone formers with a low lumbar BMD exhibit a blunted response of PTH release and an apparently overshooting production of 1.25 vitamin D following a low-calcium diet.

Mineralocorticoid receptor is essential for corticosteroid-induced up-regulation of L-type calcium currents in cultured neonatal cardiomyocytes

Despite the fact that mineralocorticoid receptor (MR) antagonist drugs such as spironolactone and eplerenone reduce the mortality in heart failure patients, there is, thus far, no unambiguous demonstration of a functional role of MR in cardiac cells. The aim of this work was to investigate the activation pathway(s) mediating corticosteroid-induced up-regulation of cardiac calcium current (ICa). In this study, using neonatal cardiomyocytes from MR or glucocorticoid receptor (GR) knockout (KO) mice, we show that MR is essential for corticosteroid-induced up-regulation of ICa. This study provides the first direct and unequivocal evidence for MR function in the heart.

Ridge augmentation and maxillary sinus grafting with a biphasic calcium phosphate: histologic and histomorphometric observations

OBJECTIVES: This retrospective study reports on histologic and histomorphometric observations performed on human biopsies harvested from sites augmented exclusively by biphasic calcium phosphate [BCP: hydroxyapatite (HA)/ tricalcium phosphate (TCP) 60/40] and healed for a minimum of 6 months. MATERIALS AND METHODS: Five patients benefited from three augmentation regimens (i.e.: one-stage lateral augmentation; two-stage lateral augmentation; and two-stage sinus grafting). In all patients, a degradable collagen membrane served as a cell-occlusive barrier. Core biopsies were obtained from lateral as from crestal aspects 6-10 months after augmentation surgeries. For histologic and histomorphometric evaluations, the non-decalcified tissue processing was performed. RESULTS: The histological examination of 11 biopsies showed graft particles frequently being bridged by the new bone, and a close contact between the graft particles and newly formed bone was seen in all samples. The mean percentages of newly formed bone, soft tissue compartment, and graft material were 38.8% (+/-5.89%), 41.75% (+/-6.08%), and 19.63% (+/-4.85%), respectively. Regarding bone-to-graft contact values, the percentage of bone coverage of graft particles for all biopsies ranged from 27.83% to 80.17%. The mean percentage of bone coverage was 55.39% (+/-13.03%). CONCLUSIONS: Data from the present study demonstrated osteoconductivity scores for the BCP material (HA/TCP 60/40) in patients resembling those previously shown for grafting materials of xenogenic and alloplastic origin.

Comparison of calcium analysis, longitudinal microradiography and profilometry for the quantitative assessment of erosion in dentine

Erosion of dentine causes mineral dissolution, while the organic compounds remain at the surface. Therefore, a determination of tissue loss is complicated. Established quantitative methods for the evaluation of enamel have also been used for dentine, but the suitability of these techniques in this field has not been systematically determined. Therefore, this study aimed to compare longitudinal microradiography (LMR), contacting (cPM) and non-contacting profilometry (ncPM), and analysis of dissolved calcium (Ca analysis) in the erosion solution. Results are discussed in the light of the histology of dentine erosion. Erosion was performed with 0.05 M citric acid (pH 2.5) for 30, 60, 90 or 120 min, and erosive loss was determined by each method. LMR, cPM and ncPM were performed before and after collagenase digestion of the demineralised organic surface layer, with an emphasis on moisture control. Scanning electron microscopy was performed on randomly selected specimens. All measurements were converted into micrometres. Profilometry was not suitable to adequately quantify mineral loss prior to collagenase digestion. After 120 min of erosion, values of 5.4 +/- 1.9 microm (ncPM) and 27.8 +/- 4.6 microm (cPM) were determined. Ca analysis revealed a mineral loss of 55.4 +/- 11.5 microm. The values for profilometry after matrix digestion were 43.0 +/- 5.5 microm (ncPM) and 46.9 +/- 6.2 (cPM). Relative and proportional biases were detected for all method comparisons. The mineral loss values were below the detection limit for LMR. The study revealed gross differences between methods, particularly when demineralised organic surface tissue was present. These results indicate that the choice of method is critical and depends on the parameter under study.

[Casein phosphopeptide--amorphous calcium phosphate (CPP-ACP) and its effect on dental hard tissues]

Dental products with casein phosphopeptide--amorphous calcium phosphate-nanocomplexes (CPP-ACP) are used in several tooth products (toothpastes, chewing gums, mouthrinses) and are as well used in dental filling material. CPP-ACP containing products are supposed to enhance remineralisation of dental hard tissues und thus might play a major role in prevention and therapy of initial caries or erosively dissolved enamel. Furthermore, also in hypersensitive teeth and even cases of hyposalivation, CPP-ACP containig products are supposed to improve the clinical condition. This article aims at three goals: point out the evolvement of CPP-ACP out of milk casein; description of possible biochemical effects of CPP-ACP on dental hard tissues; critical review of the current literature.