Enhanced immunoreceptor tyrosine-based activation motif signaling is related to pathological bone resorption during critical illness.

Prolonged critically ill patients present with distinct alterations in calcium and bone metabolism. Circulating bone formation markers are reduced and bone resorption markers are substantially elevated, indicating an uncoupling between osteoclast and osteoblast activity, possibly resulting in pronounced bone loss, impaired traumatic or surgical fracture healing, and osteoporosis. In addition, we have previously shown that increased circulating osteoclast precursors in critically ill patients result in increased osteoclastogenesis in vitro, possibly through FcγRIII signaling. In the current study, we investigated the effects of sustained critical illness on bone metabolism at the tissue level in a standardized rabbit model of prolonged (7 days), burn injury-induced critical illness. This in vivo model showed a reduction in serum ionized calcium and osteocalcin levels, as is seen in humans. Trabecular area, bone mineral content, and -density were decreased in sick rabbits [by 43% (p<0.01), 31% (p<0.01), and 29% (p<0.05), respectively], as was the trabecular gene expression of osteoblast and angiogenesis markers, indicating decreased bone formation and impaired vascularization. There was no change in the expression of osteoclast differentiation markers from the canonical RANK/RANKL/OPG pathway, however, there was an increase in expression of markers from the non-canonical, immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway, FcγRIII, and DAP12 (148% and 59%, respectively; p<0.01). The current study has shown a detrimental effect of prolonged critical illness on trabecular bone integrity, possibly explained by reduced osteoblast differentiation and angiogenesis, coupled with increased osteoclastogenesis signaling that may be mediated via the non-canonical immunoreceptor tyrosine-based activation motif signaling pathway.

Maternal apelin physiology during rat pregnancy: the role of the placenta.

OBJECTIVE: Apelin is a multifunctional peptide which is catabolized by the angiotensin-converting enzyme-related carboxypeptidase-2 (ACE2). The peptide is well known for its hemodynamic effects and its role in energy and fluid homeostasis. Pregnancy is a state of dramatically altered maternal hemodynamics and metabolism, but the role of apelin is unknown. To gain further insight in apelin physiology, we investigated relative tissue expression, plasma clearance and metabolic pathways of apelin in pregnant rats. METHODS: We measured maternal plasma apelin levels throughout normal rat gestation and examined relative apelin gene expression in several tissues, including the placenta. We documented apelin clearance using radiolabeled apelin and assessed maternal plasma levels in rats that underwent surgical reduction of the fetoplacental mass, thereby further examining the role of the placenta in apelin clearance. Finally, we localized apelin and ACE2 in the placenta and mesometrial triangle using immunohistochemistry. RESULTS: Maternal apelin plasma concentrations dropped by 50% between mid- and late gestation. Apelin expression was comparable between non-pregnant and late-pregnant rats in non-reproductive tissues. The placenta showed low apelin gene expression compared to brain tissue. Apelin clearance was enhanced in term gestation as evidenced by a steeper decline of the slow phase of the elimination curve of radiolabeled apelin. Compared to sham-operated dams, maternal plasma apelin was raised by 23% in late-pregnant rats in which half of the fetoplacental units were removed at day 16 of gestation. ACE2 mRNA expression was detectable in late- but not mid-pregnancy placental tissue; immunohistochemically, ACE2 was primarily localized in the smooth muscle layer of fetal arterioles in the labyrinth. CONCLUSION: Maternal circulating apelin drops considerably between mid- and late- pregnancy owing to faster clearance. The current data suggest a role for placental ACE2 in the accelerated apelin metabolism.

[Electroconvulsive therapy for patients with intracranial aneurysms: a case study and literature review]. / Elektroconvulsietherapie bij patiënten met intracraniële aneurysmata: gevalsbespreking en overzicht van de literatuur.

BACKGROUND: Electroconvulsive therapy (ect) is an effective treatment for severe psychiatric disorders, such as mood disorders and schizophrenia. ect is a safe treatment, even in the presence of somatic comorbidity. There are no absolute contraindications to ect, although a few somatic conditions can constitute an increased risk. ect causes a transient increase in blood pressure and heart rate and an increase in cerebral blood flow. In the presence of intracranial vascular malformations such as aneurysms, these haemodynamic changes can, in theory, increase the risk of bleeding. AIM: To determine the safety of ect for patients with an intracranial aneurysm. METHODS: We performed a Medline-search of articles published from 1967 to 2007 using Mesh-terms 'electroconvulsive therapy', 'central nervous system vascular malformations', 'intracranial arteriovenous malformations' and 'intracranial aneurysm', and the search term 'intracranial vascular malformations'. The literature data was supplemented with a case report concerning a man with bipolar disorder and a treated aneurysm of the internal carotid artery, a ventriculoperitoneal drain and hypertension, who was treated with ect. RESULTS: The literature reported 15 cases in which ect was administered to patients with a treated or untreated aneurysm. In most cases blood-pressure-lowering steps were taken. There were no reports of any complications connected with the presence of the aneurysm. Even in the case described, ect was successful and without complications. CONCLUSION: The presence of intracranial aneurysms is no contraindication to ect. Blood pressure should be carefully monitored. It may be worth considering the use of antihypertensive agents and/or an anaesthetic with blood-pressure-lowering qualities. Prior to the application of ect careful attention should be given to the possible advantages and disadvantages of the treatment, and in addition the psychiatric and somatic state of the patient should be taken into account.

Sequential triage in the first trimester may enhance advanced ultrasound scanning in population screening for trisomy 21.

OBJECTIVE: To design a trisomy 21 screening protocol for sequential triage in the first trimester, and to evaluate whether it reduces the need for advanced ultrasound scanning to such an extent that this could be dealt with by a limited number of well-trained sonographers only. METHODS: Screening results of 31 trisomy 21 affected pregnancies and 16 096 unaffected pregnancies from the first trimester screening program of Algemeen Medisch Laboratorium in Antwerp, Belgium, were used to define high-risk, intermediate-risk and low-risk groups. A serum screening result (age, pregnancy-associated plasma protein-A (PAPP-A) and free beta-human chorionic gonadotropin (beta-hCG)) of >or=1 : 30 and/or a nuchal translucency thickness (NT) measurement of >or= 3.5 mm were classified as high risk. A serum screening result of < 1 : 1000 together with an NT of < 3.5 mm were classified as low risk. Other results were considered intermediate risk, for which further advanced ultrasound screening would be indicated. This protocol was then evaluated prospectively in another population of 13 493 first-trimester pregnancies. RESULTS: Of the total population, 1.9% was identified as being high risk (14 trisomy 21 pregnancies and 222 unaffected pregnancies; prevalence, 1 : 17), 59.6% was identified as being low risk (three trisomy 21 pregnancies and 9615 unaffected pregnancies; prevalence, 1 : 3206) and 38.4% was identified as being intermediate risk (10 trisomy 21 pregnancies and 6190 unaffected pregnancies; prevalence, 1 : 620). A similar distribution was found in the prospective arm of the study. There was no reduction of overall screening performance compared with our current first-trimester combined screening program. The number of intermediate-risk pregnancies was sufficiently low as to enable advanced ultrasound scanning by well-trained sonographers only. CONCLUSION: In population screening for fetal trisomy 21, sequential triage in the first trimester can be achieved using very simple methods. Pregnancies at high or at low risk can be identified easily and the number of pregnancies at intermediate risk can be reduced sufficiently to enable advanced ultrasound scanning by well-trained sonographers only. A prospective study is needed to evaluate the performance of this approach and to compare its results with current combined or integrated screening algorithms.

Audit on nuchal translucency thickness measurements in Flanders, Belgium: a plea for methodological standardization.

OBJECTIVES: To audit nuchal translucency thickness (NT) measurements for fetal aneuploidy screening in Flanders, and to estimate the impact of small variations in NT measurement on the screening result of two first-trimester screening algorithms: maternal age + NT (Algorithm A), and maternal age + NT + pregnancy associated plasma protein-A + free beta-human chorionic gonadotropin (Algorithm B). METHODS: We used the database of first-trimester combined screening, as collected by the General Medical Laboratory AML in Antwerp, Belgium, between 1 January 2001 and 1 April 2004. Audit was performed by establishing a delta-NT distribution curve for one trainee of The Fetal Medicine Foundation (FMF) and for a group of 263 other sonographers, in comparison with the FMF reference values. Risks for fetal aneuploidy were calculated at a cut-off value of 1 : 300 for Algorithm A and 1 : 150 for Algorithm B. These risks were recalculated in both algorithms after a modeled increase of all NT values by 0.1 or 0.2 mm. RESULTS: In a total of 592 measurements performed by the FMF trainee, the 5th, 50th and 95th percentiles of delta-NT measurements were at -0.41, +0.03 and +0.68 mm, respectively. These values were close to the FMF reference values. The screen-positive rate for this set of data was 4.4% (26/592) in both algorithms. For the 12 555 measurements of the 263 other sonographers, the 5th, 50th and 95th percentiles of delta-NT were at -0.81, -0.14 and +0.73 mm, respectively, which clearly indicates underestimation of NT in the lower range. In this set of data the screen-positive rate was 3.5% for both algorithms (439/12 555 for Algorithm A and 436/12 555 for Algorithm B). Also in this group, 5% (59/1186) of negative screening results at maternal age > or = 35 years in Algorithm A became positive after a modeled 0.1-mm increase in NT, whereas this was only in 1.2% (134/11 369) of tests at maternal age < 35 years (P < 0.0001). The overall increase of screen-positive rate in Algorithm A after an NT modification of +0.1 mm was 1.2% (152/12 555), significantly more than in Algorithm B (86/12 555; 0.7%) (P < 0.0001). CONCLUSION: In Flanders, there is a systematic underestimation of NT in comparison with the FMF reference range. Attempts to change these measurements according to the FMF criteria are crucial. This will mainly influence the screening results of women at advanced maternal age and of NT-based algorithms without the use of other parameters.

Intestinal calcium transporter genes are upregulated by estrogens and the reproductive cycle through vitamin D receptor-independent mechanisms.

UNLABELLED: 1alpha,25(OH)2-vitamin D strongly regulates the expression of the epithelial calcium channel CaT1. CaT1 expression is reduced in ERKOalpha mice and induced by estrogen treatment, pregnancy, or lactation in VDR WT and KO mice. Estrogens and vitamin D are thus independent potent regulators of the expression of this calcium influx mechanism, which is involved in active intestinal calcium absorption. INTRODUCTION: Active duodenal calcium absorption consists of three major steps: calcium influx into, transfer through, and extrusion out of the enterocyte. These steps are carried out by the calcium transport protein 1 (CaT1), calbindin-D9K, and the plasma membrane calcium ATPase (PMCA1b), respectively. We investigated whether estrogens or hormonal changes during the female reproductive cycle influence the expression of these genes, and if so, whether these effects are vitamin D-vitamin D receptor (VDR) dependent. MATERIALS AND METHODS: We evaluated duodenal expression patterns in estrogen receptor (ER)alpha and -beta knockout (KO) mice, as well as in ovariectomized, estrogen-treated, pregnant, and lactating VDR wild-type (WT) and VDR KO mice. RESULTS: Expression of calcium transporter genes was not altered in ERKObeta mice. CaT1 mRNA expression was reduced by 55% in ERKOalpha mice, while the two other calcium transporter genes were not affected. Ovariectomy caused no change in duodenal expression pattern of VDR WT and KO mice, whereas treatment with a pharmacologic dose of estrogens induced CaT1 mRNA expression in VDR WT (4-fold) and KO (8-fold) mice. Pregnancy enhanced CaTI expression equally in VDR WT and KO mice (12-fold). Calbindin-D9K and PMCA1b expression increased to a lesser extent and solely in pregnant VDR WT animals. In lactating VDR WT and KO mice, CaT1 mRNA expression increased 13 times, which was associated with a smaller increase in calbindin-D9K protein content and PMCA1b mRNA expression. CONCLUSIONS: Estrogens or hormonal changes during pregnancy or lactation have distinct, vitamin D-independent effects at the genomic level on active duodenal calcium absorption mechanisms, mainly through a major upregulation of the calcium influx channel CaT1. The estrogen effects seem to be mediated solely by ERalpha.

Increased bone formation in mice lacking plasminogen activators.

UNLABELLED: Plasminogen activators tPA and uPA are involved in tissue remodeling, but their role in bone growth is undefined. Mice lacking tPA and uPA show increased bone formation and bone mass. The noncollagenous components of bone matrix are also increased, probably from defective degradation. This study underlines the importance of controlled bone matrix remodeling for normal endochondral ossification. INTRODUCTION: Proteolytic pathways are suggested to play a role in endochondral ossification. To elucidate the involvement of the plasminogen activators tPA and uPA in this process, we characterized the long bone phenotype in mice deficient in both tPA and uPA (tPA-/-:uPA-/-). MATERIALS AND METHODS: Bones of 2- to 7-day-old tPA-/-:uPA-/- and wild-type (WT) mice were studied using bone histomorphometry, electron microscopy analysis, and biochemical assessment of bone matrix components. Cell-mediated degradation of metabolically labeled bone matrix, osteoblast proliferation, and osteoblast differentiation, both at the gene and protein level, were studied in vitro using cells derived from both genotypes. RESULTS: Deficiency of the plasminogen activators led to elongation of the bones and to increased bone mass (25% more trabecular bone in the proximal tibial metaphysis), without altering the morphology of the growth plate. In addition, the composition of bone matrix was modified in plasminogen activator deficient mice, because an increased amount of proteoglycans (2x), osteocalcin (+45%), and fibronectin (+36%) was detected. Matrix degradation assays showed that plasminogen activators, by generating plasmin, participate in osteoblast-mediated degradation of the noncollagenous components of bone matrix. In addition, proliferation of primary osteoblasts derived from plasminogen activator-deficient mice was increased by 35%. Finally, osteoblast differentiation and formation of a mineralized bone matrix were enhanced in osteoblast cultures derived from tPA-/-:uPA-/- mice. CONCLUSIONS: The data presented indicate the importance of the plasminogen system in degradation of the noncollagenous components of bone matrix and suggest that the accumulation of these proteins in bone matrix--as occurs during plasminogen activator deficiency--may in turn stimulate osteoblast function, resulting in increased bone formation.

Thin-layer liquid-based cervical cytology and PCR for detecting and typing human papillomavirus DNA in Flemish women.

The objective of this study was to document the occurrence and to correlate the prevalence of different human papillomavirus (HPV) types with the cytological results on simultaneously performed thin-layer preparations in a large population of Flemish women. During 1 year, 69 290 thin-layer preparations were interpreted using the Bethesda classification system. Using an algorithm for HPV testing based on consensus primers and type-specific PCRs in combination with liquid-based cytology, we determined the occurrence and distribution of 14 different oncogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Reflex HPV testing was performed on cytologically abnormal samples and on an age matched randomly selected control group with normal cervical cytology (n=1351). Correlation between cytology, age and prevalence for the 14 different high-risk HPV types is given. There is a significant increase in predominance of high-risk HPV types, with increasing abnormal cytology. Coinfection with multiple HPV types also increased with cytological abnormalities, and was highest in HSIL (16.7%). In Flanders, HSIL was most often associated with HPV types 16, 33, 35, 31, 18 and 51. Using thin-layer liquid-based cytology and PCR to detect HPV, it is feasible to screen large numbers of women.

Evidence from the aged orchidectomized male rat model that 17beta-estradiol is a more effective bone-sparing and anabolic agent than 5alpha-dihydrotestosterone.

This study was designed to evaluate the impact of estrogen versus androgen action on orchidectomy (ORX)-induced bone loss and associated changes in body composition. During an experimental period of 4 months, aged (12-month-old) ORX rats were treated with 17beta-estradiol (E2; 0.75 microg/day) or different doses of the nonaromatizable androgen 5alpha-dihydrotestosterone (DHT; 45, 75, and 150 microg/day, respectively), via subcutaneous (sc) silastic implants. Low doses of DHT and E2 inhibited the ORX-induced rise of bone turnover markers (serum osteocalcin and urinary deoxypyridinoline [DPD]) to a similar extent. High-dose DHT prevented the ORX-induced decrease of trabecular bone density but had no significant effect on cortical thinning as assessed by peripheral quantitative computed tomography (pQCT). This bone-sparing action of DHT occurred at the expense of hypertrophy of the ventral prostate and seminal vesicles. On the other hand, E2 restored both trabecular bone density and cortical thickness in ORX rats and even prevented age-related bone loss. In contrast to DHT, E2 increased lean body mass and inhibited the ORX-associated increase of fat mass, as measured by DXA. Administration of E2 was associated with increased serum concentrations of insulin-like growth factor (IGF) I and decreased circulating levels of leptin. We conclude that, in the aged ORX rat model, E2 is more effective in preventing ORX-induced bone loss than DHT. Additionally, E2 has anabolic effects on muscle tissue and prevents the ORX-related increase of fat mass. Overall, these data suggest that androgen action on bone and body composition is dependent on stimulation of both androgen receptors (ARs) and estrogen receptors (ERs).

Vitamin D deficiency in guinea pigs: exacerbation of bone phenotype during pregnancy and disturbed fetal mineralization, with recovery by 1,25(OH)2D3 infusion or dietary calcium-phosphate supplementation.

Vitamin D (D) deficiency during human pregnancy appears to disturb fetal growth and mineralization, but fetal development is normal in D-deficient rats and vitamin D receptor gene-ablated mice. We used the guinea pig model to investigate maternal and fetal effects of D deficiency. Pregnant (Pr) and nonpregnant (NPr) animals were fed a D-replete (+D) or D-deficient diet (-D) for 8 weeks. We further studied whether the effects of a -D diet are reversed by continuous 1,25(OH)2D3 infusion (-D+1,25) and/or by a lactose-, Ca- and P-enriched D-deficient diet (-D+Ca/P). Bone analyses included histomorphometry of the proximal tibiae, dual-energy X-ray absorptiometry (DXA), and quantitative computed tomography (QCT) of the femora. Depletion of 25(OH)D3 and 1,25(OH)2D3 levels and the D-deficiency syndrome were more severe in pregnant animals. Indeed, Pr/-D but not NPr/-D guinea pigs were hypophosphatemic, and showed robust increases in growth plate width and osteoid surface and thickness; in addition, bone mineral density on DXA was lower in Pr/-D animals only, which was exclusively in cortical bone on QCT. Bone phenotype was partly normalized in Pr/-D+1,25 and Pr/-D+Ca/P animals. Compared with +D fetuses, -D fetuses had very low or undetectable 25(OH)D3 and 1,25(OH)2D3, were hypercalcemic and hypophosphatemic, and had lower osteocalcin levels. In addition, body weight and total body bone mineral content were 10-15% lower; histomorphometry showed hypertrophic chondrocyte zone expansion and hyperosteoidosis. 1,25(OH)2D3 levels were restored in -D+1,25 fetuses, and the phenotype was partially corrected. Similarly, the fetal +D phenotype was rescued in large part in -D+Ca/P fetuses, despite undetectable circulating 25(OH)D3 and 1,25(OH)2D3. We conclude that pregnancy markedly exacerbates D deficiency, and that augmenting Ca and P intake overrides the deleterious effects of D deficiency on fetal development.

The estrogen receptor ligand ICI 182,780 does not impair the bone-sparing effects of testosterone in the young orchidectomized rat model.

Testosterone (T) can affect bone metabolism not only directly, but also via its metabolites, estrogen or dihydrotestosterone, produced by enzymes present in bone. Therefore, the aim of this study was to investigate whether the high-affinity estrogen receptor ligand ICI 182,780 (ICI) impaired the bone-protective action of T in 3-month-old orchidectomized (Orch) rats, studied during an experimental period of 3 months. As expected, Orch significantly decreased trabecular bone volume in the proximal tibial metaphysis (-52%), as measured by histomorphometry, and had a similar negative effect on volumetric bone mineral density (BMD) in the distal femoral metaphysis (-53%), as assessed by peripheral quantitative computed tomography (pQCT). The loss of bone induced by Orch was completely prevented by T administration. Moreover, the Orch-associated increases of biochemical markers of bone turnover (serum osteocalcin, urinary deoxypyridinoline, and calcium excretion) did not occur when Orch rats received T. Administration of ICI in combination with T did not impair this bone-sparing effect. Cortical bone parameters (as determined by pQCT), body weight gain, and body composition (as measured by dual-energy X-ray absorptiometry) were not affected by T or ICI in combination with T. Furthermore, no differences were observed in serum concentrations of insulin-like growth factor-I or glucose homeostasis. In conclusion, ICI does not impair the long-term bone-protective effects of T in orchidectomized male rats, suggesting that testosterone can mediate its effect on the male skeleton directly via the androgen receptor. The absence of effects on body growth via the growth hormone--insulin-like growth factor-I axis may be a possible explanation for the lack of skeletal effects of this selective estrogen receptor antagonist.

Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects.

Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA(1b)) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH)(2)D(3) injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.

Testosterone prevents orchidectomy-induced bone loss in estrogen receptor-alpha knockout mice.

To examine the role of the estrogen receptor-alpha (ERalpha) during male skeletal development, bone density and structure of aged ERalphaKO mice and wild-type (WT) littermates were analyzed and skeletal changes in response to sex steroid deficiency and replacement were also studied. In comparison to WT, ERalphaKO mice had smaller and thinner bones, arguing for a direct role of ERalpha to obtain full skeletal size in male mice. However, male ERalphaKO mice had significantly more trabecular bone as assessed both by pQCT and histomorphometry, indicating that ERalpha is not essential to maintain cancellous bone mass. Six weeks following orchidectomy (ORX), both WT and ERalphaKO mice showed high-turnover osteoporosis as revealed by increases in serum osteocalcin and decreases in trabecular (-38% and -58% in WT and ERalphaKO, respectively) and cortical bone density (-5% and -4% in WT and ERalphaKO, respectively). Administration of testosterone propionate (T, 5 mg/kg/day) completely prevented bone loss both in ERalphaKO and in WT mice. As expected, estradiol (E2, 60 microg/kg/day) replacement did not prevent cancellous bone loss in ORX ERalphaKO mice. However, E2 stimulated bone formation at the endocortical surface in ORX ERalphaKO, suggesting that osteoblasts may respond to nonERalpha-mediated estrogen action. In conclusion, although functional ERalpha may play a significant role during male skeletal development, this receptor does not seem essential for androgen-mediated skeletal maintenance in older male mice.

Dietary calcium and phosphate restriction in guinea-pigs during pregnancy: fetal mineralization induces maternal hypocalcaemia despite increased 1 alpha,25-dihydroxycholecalciferol concentrations.

Guinea-pig fetuses at term are mineralized to a degree comparable with human fetuses, which makes the guinea-pig an attractive animal model to study maternal-fetal interactions with regard to Ca and phosphate (P) homeostasis. We studied non-pregnant and pregnant (day 57) vitamin D-replete guinea-pigs, fed either a normal guinea-pig chow with 9.6 g Ca/kg and 4.9 g P/kg or a study diet with 2 g Ca/kg and 1 g P/kg (low-Ca-P diet) for 7-8 weeks. Both pregnancy and the low-Ca-P diet decreased plasma concentrations of 25-hydroxycholecalciferol (25(OH)D3), but increased total and free 1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3), strongly suggesting an additive stimulation of 1 alpha-hydroxylase activity. Maternal and fetal 25(OH)D3 and 1,25(OH)2D3 levels were highly correlated (r 0.82 and 0.92 respectively, P < 0.001). Dual-energy absorption X-ray absorptiometry (DXA) showed that both pregnancy and the low-Ca-P diet decreased bone mineral density (BMD) of the maternal femur, particularly at the distal metaphysis. Despite higher 1,25(OH)2D3 concentrations and lower BMD, pregnant animals on the low-Ca-P diet were hypocalcaemic; blood Ca2+ levels were inversely correlated with the number of fetuses in this group (r -0.93, P < 0.001). Fetal growth as well as mineralization (assessed by whole-body and femoral DXA, bone histomorphometry and plasma-bone osteocalcin measurements) were unaltered in the low-Ca-P group. In conclusion, fetal mineralization proceeds normally but induces maternal hypocalcaemia in guinea-pigs with dietary restriction of Ca and P.

Placental growth hormone and IGF-I in a pregnant woman with Pit-1 deficiency.

The respective contributions of pituitary and placental GH to circulating IGF-I in pregnant women have not been well established. We measured the serum concentrations of placental growth hormone (PGH) and IGF-I in a woman with pit-1 deficiency before, during and after pregnancy, resulting in the birth of a healthy child (not pit-1 deficient). Both PGH and IGF-I concentrations were below the assay detection limit before and after pregnancy. During pregnancy, PGH and IGF-I levels increased steadily; the concentrations of PGH and IGF-I in late pregnancy were comparable with levels previously measured in normal pregnancies. PGH and IGF-I concentrations were strongly correlated throughout pregnancy (r = 0.90; P = 0.002). PGH was undetectable in cord serum, whilst the IGF-I concentration was within the normal range. The findings of this case study corroborate the notion that PGH is the prime regulator of maternal serum IGF-I during pregnancy.

Pretreatment with growth hormone-releasing peptide-2 directly protects against the diastolic dysfunction of myocardial stunning in an isolated, blood-perfused rabbit heart model.

Brief coronary occlusion followed by reperfusion leads to reversible myocardial dysfunction (stunning) which can induce irreversible damage of other organ systems. We studied the effects of pretreatment with recombinant human GH (rhGH) and the GH-secretagogue GHRP-2 on myocardial stunning in a blood-perfused isolated rabbit heart model. In a first set of experiments, effects of bolus rhGH administration (3.5 mg/kg) (n = 5) into the aortic root of unpretreated animals were compared with those of saline (n = 6). In a second set, animals were pretreated for 14 days with SC rhGH 3.5 mg/kg x day (n = 9) or 160 microg/kg x day GHRP-2 (n = 8) in two divided doses. Body weight and plasma concentrations of rhGH, rabbit GH (rGH) and IGF-I were determined before and at the end of 14 days pretreatment. Hearts were excised and submitted to 15 min ischemia followed by 80 min reperfusion, after which postischemic recovery was compared with nonischemic hearts mounted into the same system. At study end, all hearts were snap-frozen to examine markers of apoptosis. Circulating levels of rabbit GH (rGH) remained identical in all animals. Pretreatment with rhGH for 14 days induced a 142 +/- 116% rise of serum IGF-I vs. 8 +/- 15% with GHRP-2 (P < 0.001) and increased body weight with 6.8 +/- 2.5% vs. 3.4 +/- 3.3% with GHRP-2 (P = 0.01). A bolus injection of rhGH did not alter myocardial function compared with saline allowing data from these experiments to be pooled into one ischemic control group for further analysis of the effect of pretreatment. No difference in postischemic recovery of left ventricular systolic function among the unpretreated, rhGH pretreated and GHRP-2 pretreated hearts was apparent. At the end of reperfusion, a 3-fold higher end-diastolic pressure (EDP) persisted in the unpretreated and rhGH pretreated hearts compared with the nonischemic hearts. In the GHRP-2 pretreated hearts, EDP decreased to half the pressure observed in unpretreated and rhGH pretreated hearts (all P < or = 0.02), a level which was indistinguishable from that in the non-ischemic hearts, suggesting full postischemic recovery of diastolic function. There were no signs of increased apoptosis in the experimental groups. In conclusion, 14 days pretreatment with GHRP-2, but not rhGH, protected selectively against the diastolic dysfunction of myocardial stunning in this model. This observation may open perspectives for GH-secretagogues as cardioprotective agents.

Skeletal effects of estrogen deficiency as induced by an aromatase inhibitor in an aged male rat model.

Aromatization of androgens into estrogens may be important for maintenance of the male skeleton. To address this hypothesis, we evaluated the skeletal effects of selective estrogen deficiency as induced by the aromatase inhibitor vorozole (Vor), with or without 17beta-estradiol (E(2)) administration (1.35 microg/day), in aged (12-month-old) male rats. A baseline group was killed at the start of the experiment (Base). The control group (Control), the group treated with vorozole alone (Vor), the group treated with E(2) alone (E(2)), or the group with a combination of both (Vor + E(2)) were killed 15 weeks later. Vorozole significantly increased serum testosterone (T) and reduced serum E(2) compared with Control. Body weight gain and serum insulin-like growth factor-I (IGF-I) were also lower in Vor, whereas significant weight loss and decrease of serum IGF-I occurred as a result of E(2) administration. Bone formation as assessed by serum osteocalcin was unaffected but osteoid surface in the proximal metaphysis of the tibia was increased in Vor-treated rats. Bone resorption as evaluated by urinary deoxypyridinoline excretion was increased in Vor. Biochemical parameters of bone turnover were reduced significantly in all E(2) treated rats. Premature closure of the growth plates and decreased osteoid and mineralizing surfaces were also observed in E(2) and Vor + E(2). Apparent bone density of lumbar vertebrae and femur, as measured by dual-energy X-ray absorptiometry (DXA), was significantly reduced in Vor. Vorozole decreased femoral bone density mainly in the distal femur (trabecular and cortical region). This decrease of bone density was not present in E(2) and Vor + E(2). Similar findings were observed when bone density was assessed by peripheral quantitative computed tomography (pQCT); that is, trabecular density of the distal femur, the proximal tibia, and the distal lumbar vertebra were all lower in Vor. This decrease in density was not observed in all E(2)-treated animals. In conclusion, administration of the aromatase inhibitor, vorozole, to aged male rats induces net trabecular bone loss in both the appendicular and axial skeleton, despite a concomitant increase in serum testosterone. E(2) administration is able to prevent this trabecular bone loss in vorozole-treated animals.

Effects of exercise and disuse on bone remodeling, bone mass, and biomechanical competence in spontaneously diabetic female rats.

Diabetes is associated with low bone formation. In this study we investigate the effect of additional or reduced mechanical loading on indices of bone formation and resorption, bone mass, and biomechanical properties in spontaneously diabetic BB rats. Female diabetic (mean age 13 weeks) and age-matched control rats were each allocated to three experimental groups: no-intervention; supervised running exercise program (Ex); and unloading induced by unilateral sciatic neurectomy (USN). The study period was 8 weeks. We measured biochemical parameters of bone formation (plasma osteocalcin) and resorption (urinary deoxypyridinoline [Dpd]); bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) at middiaphyseal and metaphyseal regions of the femur; histomorphometry of the proximal tibial metaphysis (PTM); and biomechanical properties of the femur (neck, diaphysis, and metaphysis) and lumbar vertebra (L-5). In nondiabetic rats, Ex did not affect parameters of bone formation/resorption and BMD, and had little effect on biomechanical properties. USN increased Dpd excretion, whereas there was a decreased trabecular bone formation rate (BFR) on morphometry of PTM in both paralyzed and intact limbs. Compared with intact limbs, paralyzed limbs of USN rats showed decreased trabecular bone volume at the PTM, and decreased BMD and biomechanical properties at the distal femoral metaphysis (DFM) and, to a lesser extent, femoral neck. Diabetic rats of the three experimental groups had low plasma osteocalcin levels and Dpd excretion, as well as low BFR on morphometry. The BMD and biomechanical properties of both femur and L-5 were unchanged in diabetic rats. Diabetic Ex rats, however, showed a lower maximum load and stress at DFM than control Ex rats. Diabetic USN rats showed no increase in Dpd excretion; their paralyzed limbs showed decreased maximum load at DFM, but there was no significant decrease in trabecular bone volume at PTM or BMD at DFM. Thus, the running exercise does not affect low bone formation in diabetic rats; however, trabecular bone loss caused by disuse is less pronounced in diabetic rats, probably as a result of low bone resorption.

An aged rat model of partial androgen deficiency: prevention of both loss of bone and lean body mass by low-dose androgen replacement.

The aim of this study was to evaluate the effects of different doses of androgen replacement, both on body composition and bone, in an aged male orchidectomized rat model. Testosterone was administered by 0.5, 1, and 2.5-cm sc SILASTIC implants (release of, respectively, 11.5, 23, and 55 microg/day) to aged (12 months old, +/- 550 g) male orchidectomized Wistar rats during a 15-week experimental period. T 0.5 only partially prevented decrease of ventral prostate and seminal vesicle weight, compared with an intact group that received an empty implant (Intact). The 1-cm implant (T 1) completely prevented decrease of both seminal vesicles and ventral prostate weight. The 2.5-cm implant (T 2.5) was clearly supraphysiological, as demonstrated by significant hypertrophy of both androgen-sensitive organs. Serum testosterone was lower in T 0.5 and T 1 (0.38 +/- 0.06 ng/ml and 0.92 +/- 0.06 ng/ml, respectively) and higher in T 2.5 (2.4 +/- 0.28. ng/ml), compared with both Intact (1.6 +/- 0.23 ng/ml) and the baseline group(1.6 +/- 0.11 ng/ml). As expected, orchidectomized rats that received an empty SILASTIC implant had significantly lower bone mineral content (-7.9%), apparent density (-5.7%), and lean body mass (-10.8%), as measured by dual-energy x-ray absorptiometry, without significant changes in body weight and fat mass, compared with Intact. Also, cancellous (-50.3%) and cortical (-1.8%) volumetric density, as measured by peripheral quantitative computed tomography, were decreased in the tibia. Bone turnover, as measured by serum osteocalcin and urinary deoxypyridinoline excretion, was increased in orchidectomized rats that received an empty SILASTIC implant. T 0.5 prevented all changes, not only in bone mineral content, density, and turnover but also in lean body mass. Moreover, there were no significant differences, for all these parameters, between the different doses of testosterone replacement. In conclusion, low-dose androgen replacement does not lead to lower bone mineral density, higher bone turnover, and lower lean body mass in aged male rats, whereas complete androgen deficiency does. Therefore, the threshold concentration of testosterone necessary for prevention of both bone and lean body mass loss in aged male rats is clearly lower than for prostate and seminal vesicles.