Dissecting the heterogeneity of craniofacial lesions in patients with fibrous dysplasia/McCune-Albright Syndrome.

Fibrous dysplasia/McCune-Albright Syndrome (FD/MAS) frequently involves the craniofacial skeleton. Craniofacial fibrous dysplasia lesions exhibit diverse imaging characteristics on multimodality evaluation, utilizing radiographs, computed tomography (CT), magnetic resonance imaging (MRI), and 18F-sodium fluoride positron emission tomography (18F-NaF PET). A multimodal imaging classification of craniofacial fibrous dysplasia lesions may offer clinical insights into the types of lesions that are (1) prone to progression, (2) amenable to intervention (i.e., pharmacological or surgical), or (3) associated with symptoms such as pain. In this prospective, preliminary single site study of 15 patients with FD/MAS, the heterogeneity of craniofacial lesions (N = 35) was assessed using a combination of 18F-NaF PET, MRI, and CT. A k-means clustering algorithm was used to categorize lesions based on imaging characteristics. Clustering analysis revealed three types of lesion based on the magnitude of the regional 18F-NaF standardized uptake values (SUV), signal intensities on T1-weighted and fluid-sensitive sequences, and appearance on CT (lucent, sclerotic, and/or ground glass). This preliminary study provides a foundation for future longitudinal natural history or treatment studies, where the prognostic value of baseline craniofacial fibrous dysplasia imaging characteristics and clinical symptomatology can be further evaluated.

Mutations in NOTCH2 in patients with Hajdu-Cheney syndrome.

UNLABELLED: The Hajdu-Cheney syndrome is a very rare disease that affects several organ system, leading to severe osteoporosis and other abnormalities. We describe clinical and genetic findings of nine patients with this disease. INTRODUCTION: The Hajdu-Cheney syndrome (HCS) is a rare autosomal dominant disorder characterized by severe osteoporosis, acroosteolysis of the distal phalanges, renal cysts, and other abnormalities. Recently, heterozygous mutations in NOTCH2 were identified as the cause of HCS. METHODS: Nine patients with typical presentations of HCS took part in this study: five affected patients from two small families and four sporadic cases. Peripheral blood DNA was obtained and exome sequencing performed in one affected individual per family and in all four sporadic cases. Sanger sequencing confirmed mutations in all patients. RESULTS: One of the identified mutations was introduced in a plasmid encoding NOTCH2. Wild-type and mutant NOTCH2 were transiently expressed in HEK293 cells to assess intracellular localization after ligand activation. Deleterious heterozygous mutations in the last NOTCH2 exon were identified in all patients; five of the six mutations were novel. CONCLUSION: Consistent with previous reports, all mutations are predicted to result in a loss of the proline/glutamic acid/serine/threonine sequence, which harbors signals for degradation, therefore suggesting activating mutations. One of the six mutations furthermore predicted disruption of the second nuclear localization signal of NOTCH2, but the mutant revealed normal nuclear localization after transfection, which is consistent with the proposed gain-of-function mechanism as the cause of this autosomal dominant disease. Our findings confirm that heterozygous NOTCH2 mutations are the cause of HCS and expand the mutational spectrum of this disorder.

The autosomal dominant hypophosphatemic rickets (ADHR) gene is a secreted polypeptide overexpressed by tumors that cause phosphate wasting.

The gene mutated in autosomal dominant hypophosphatemic rickets (ADHR), a phosphate wasting disorder, has been identified as FGF-23, a protein that shares sequence homology with fibroblast growth factors (FGFs). Patients with ADHR display many of the clinical and laboratory characteristics that are observed in patients with oncogenic hypophosphatemic osteomalacia (OHO), a disorder thought to arise by the secretion of a phosphate wasting factor from different mesenchymal tumors. In the present studies, we therefore investigated whether FGF-23 is a secreted factor and whether it is abundantly expressed in OHO tumors. After transient transfection of OK-E, COS-7, and HEK293 cells with the plasmid encoding full-length FGF-23, all three cell lines efficiently secreted two protein species into the medium that were approximately 32 and 12 kDa upon SDS-PAGE and subsequent Western blot analysis using an affinity-purified polyclonal antibody to FGF-23. Furthermore, Northern blot analysis using total RNA from five different OHO tumors revealed extremely high levels of FGF-23 mRNA, and Western blot analysis of extracts from a sixth tumor detected the 32 kDa FGF-23 protein species. In summary, FGF-23, the gene mutated in ADHR, is a secreted protein and its mRNA is abundantly expressed by several different OHO tumors. Our findings indicate that FGF-23 may be a candidate phosphate wasting factor, previously designated "phosphatonin".

Extracts from tumors causing oncogenic osteomalacia inhibit phosphate uptake in opossum kidney cells.

In oncogenic osteomalacia (OOM), a tumor produces an unknown substance that inhibits phosphate reabsorption in the proximal tubules. This causes urinary phosphate wasting and, as a consequence, hypophosphatemic osteomalacia. To characterize this poorly understood biological tumor activity we generated aqueous extracts from several OOM tumors. Extracts from three of four tumors inhibited, dose- and time-dependently, (32)P-orthophosphate uptake by opossum kidney (OK) cells; maximum inhibition was about 45% of untreated control. Further characterization revealed that the factor is resistant to heat and several proteases, and that it has a low molecular weight. The tumor extracts also stimulated cAMP accumulation in OK cells, but not in osteoblastic ROS 17/2.8 and UMR106 cells, or in LLC-PK1 kidney cells expressing the parathyroid hormone (PTH)/PTH-related peptide receptor or the PTH-2 receptor. HPLC separation of low molecular weight fractions of the tumor extracts revealed that the flow-through of all three positive tumor extracts inhibited (32)P uptake and stimulated cAMP accumulation in OK cells. Additionally, a second peak with inhibitory activity on phosphate transport, but without cAMP stimulatory activity, was identified in the most potent tumor extract. We have concluded that several low molecular weight molecules with the ability to inhibit phosphate transport in OK cells can be found in extracts from OOM tumors. It remains uncertain, however, whether these are related to the long-sought phosphaturic factor responsible for the phosphate wasting seen in OOM patients.

PTH secretion in patients with chronic renal failure assessed by a modified CiCa clamp method: effects of 1-year calcitriol therapy.

BACKGROUND: Secondary hyperparathyroidism (secondary HPT) in patients with chronic renal failure (CRF) is characterized by parathyroid gland hyperplasia and an intrinsic defect in the recognition of parathyroid hormone (PTH) secretion. Conflicting results have been reported regarding the set point for calcium-regulated PTH release and its modification by calcitriol therapy in hemodialysis patients. Additionally, the effect of calcitriol on the calcium/PTH relationship in predialysis CRF patients with early secondary HPT has not been investigated. Our objective in this controlled study was to investigate the calcium/PTH relationship and to determine the calcium set point in patients with early stages of CRF before and after a 1-year treatment with calcitriol and in normal volunteers. METHODS: Nine patients with an early stage of CRF (GFR between 20 and 50 ml/min x 1.73 m2 b.s.) aged 35-77 years and 13 healthy volunteers (HV) aged 26-60, years were included in the study. All participants were investigated by sequential lowering and raising of serum calcium levels comprising the following phases: blood-ionized calcium (Ca2+) was lowered by about 0.2 mmol/l (3 steps), steady-state hypocalcemia of Ca2+ 0.2 mmol/l below the baseline (step 4), stop of the infusion for 5 minutes (step 5), Ca2+ was raised to about 0.2 mmol/l above baseline (steps 6 and 7), and a steady state hypercalcemia of Ca2+ 0.2 mmol/l above baseline (step 8). Ionized calcium and intact PTH (iPTH) were measured at 30 time points during 240 minutes. The calcium set point was determined using the classical 4-parameter model. The CiCa clamp test was performed before and after a 1-year treatment with 0.5 microg of calcitriol thrice weekly. RESULTS: No differences in the set point were observed between HV and CRF patients with early secondary HPT. Four of 9 patients responded to calcitriol treatment with a decrease in basal serum iPTH levels ("responders"). There was no difference between renal function (GFR 18 +/- 6 vs. 17 +/- 8 ml/min x 1.73 m2 b.s.), set point (Ca2+ 1.07 +/- 0.13 vs. 1.07 +/- 0.06 mmol/l) and suppressibility of PTH secretion (PTHmin% 7.3 +/- 1.6 vs. 8.2 +/- 2.9) in responders vs non-responders, nor did these values change after treatment with calcitriol. PTHmin% decreased significantly in the whole group after treatment (10.4 +/- 8.5 vs. 7.8 +/- 2.4). CONCLUSIONS: Although the calcium set point was not different in predialysis CRF patients with early secondary HPT compared to HV, calcitriol treatment improved the calcium-related suppression of PTH secretion (PTHmin%).

[Parathyroid hormone receptors: from cloning to physiological, physiopathological and clinical implications]. / Récepteurs de l'hormone parathyroïdienne: du clonage aux implications physiologiques, physiopathologiques et cliniques.

It has long been known that parathyroid hormone (PTH) exerts its effects on target tissues via its binding to a membrane receptor. Recently, several types of PTH receptors have been identified. The first receptor which has been cloned and well characterized is "PTH/PTHrP receptor-1". It is activated not only by PTH, but also by PTH-related peptide (PTHrP), via a signal transduction system involving G-proteins, adenylate cyclase and phospholipase C. It is expressed in many tissues, in addition to kidney and bone. The results of recent studies are suggestive of the existence of additional PTH receptors. One or several receptors are probably expressed in the keratinocyte and the glomerular podocyte which are not identical with PTH/ PTHrP receptor-1. A third receptor, which has been cloned recently and called "PTH2 receptor", recognizes solely PTH. It is expressed in brain, pancreas, testis and placenta. Its function is unknown. There is also evidence for a fourth receptor, called "C-PTH receptor", recognizing C-terminal PTH fragments which are generally considered to be biologically inactive. The regulation of these receptors is subject to intensive research. Down-regulation of PTH/PTHrP receptor-1 mRNA expression could explain the well-known resistance to the action of PTH in chronic renal failure. In contrast, the receptor mRNA is up-regulated in vitamin D deficiency, despite a similar tissue resistance to PTH. A mutation of PTH/PTHrP receptor-1 causes Jansen-type metaphyseal chondrodysplasia. However, no alteration of the PTH/PTHrP receptor-1 gene structure has been found in type 1b pseudohypoparathyroidism.

Receptors for PTH and PTHrP: their biological importance and functional properties.

The type 1 receptor (PTH1R) for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) is a G protein-coupled receptor that is highly expressed in bone and kidney and mediates in these tissues the PTH-dependent regulation of mineral ion homeostasis. The PTH1R also mediates the paracrine actions of PTHrP, which play a particularly vital role in the process of endochondral bone formation. These important functions, the likely involvement of the PTH1R in certain genetic diseases affecting skeletal development and calcium homeostasis, and the potential utility of PTH in treating osteoporosis have been the driving force behind intense investigations of both the receptor and its peptide ligands. Recent lines of work have led to the identification of constitutively active PTH1Rs in patients with Jansen's metaphyseal chondrodysplasia, the demonstration of inverse agonism by certain ligand analogs, and the discovery of the PTH-2 receptor subtype that responds to PTH but not PTHrP. As reviewed herein, a detailed exploration of the receptor-ligand interaction process is currently being pursued through the use of site-directed mutagenesis and photoaffinity cross-linking methods; ultimately, such work could enable the development of novel PTH receptor ligands that have therapeutic value in treating diseases such as osteoporosis and certain forms of hypercalcemia.

Effects of new vitamin D analogues on parathyroid function in chronically uraemic rats with secondary hyperparathyroidism.

BACKGROUND: Treatment with calcitriol or its analogue, alfacalcidol often leads to hypercalcaemia, hyperphosphataemia or both in patients which chronic renal failure and advanced secondary hyperparathyroidism. We tested three new vitamin D analogues (CB 1093, EB 1213, GS 1725) in an attempt to identify potentially non hypercalcaemic compounds, capable of decreasing plasma parathyroid hormone (PTH) concentration. METHODS: Male Wistar AF rats aged 12-14 weeks were fed a synthetic, phosphate-rich diet and underwent either sham surgery (control) or a standard two-step 5/6th nephrectomy. Four weeks later, renal function was mildly decreased in the latter. Chronic renal failure rats were then divided into six groups, with 8-10 rats in each group. They received daily 1.p. injections, from days 0 to 4, of either placebo, calcitriol, or one of the following three active vitamin D analogues: CB1093, 0.25 micrograms; EB1213, 0.25 or 1.25 micrograms; and GS1725, 0.025 micrograms/kg body weight per day, respectively. Sham-operated rats received no drug. On day 5, arterial blood was sampled and rats were sacrificed. RESULTS: At predefined dosage schedules, all three compounds significantly decreased plasma immunoreactive PTH levels (except EB1213 at low dose). The decrement was somewhat less marked than that obtained with calcitriol, at the dose of 0.25 micrograms/kg b.w. per day. However, calcitriol induced a marked increase in plasma calcium and phosphate concentrations at that dose, whereas vitamin D analogues led to a more modest increase in plasma calcium level, and none to a worsening of hyperphosphataemia. CB1093 treatment was even associated with a significant decrease in plasma phosphate level. CONCLUSION: All three calcitriol analogues tested are promising as non-hypercalcaemic agents in the treatment of uraemic secondary hyperparathyroidism. However, more prolonged administration to uraemic rats of calcitriol analogues with slightly modified dosage schedules and of calcitriol with lower non-hypercalcaemic dose is required for an optimal comparison before considering clinical trials.

Evidence for a ligand interaction site at the amino-terminus of the parathyroid hormone (PTH)/PTH-related protein receptor from cross-linking and mutational studies.

Low resolution mutational studies have indicated that the amino-terminal extracellular domain of the rat parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (rP1R) interacts with the carboxyl-terminal portion of PTH-(1-34) or PTHrP-(1-36). To further define ligand-receptor interactions, we prepared a fully functional photoreactive analog of PTHrP, [Ile5,Bpa23,Tyr36]PTHrP-(1-36)-amide ([Bpa23]PTHrP, where Bpa is p-benzoyl-L-phenylalanine). Upon photolysis, radioiodinated [Bpa23]PTHrP covalently and specifically bound to the rP1R. CNBr cleavage of the broad approximately 80-kDa complex yielded a radiolabeled approximately 9-kDa non-glycosylated protein band that could potentially be assigned to rP1R residues 23-63, Tyr23 being the presumed amino-terminus of the receptor. This assignment was confirmed using a mutant rP1R (rP1R-M63I) that yielded, upon photoligand binding and CNBr digestion, a broad protein band of approximately 46 kDa, which was reduced to a sharp band of approximately 20 kDa upon deglycosylation. CNBr digestion of complexes formed with two additional rP1R double mutants (rP1R-M63I/L40M and rP1R-M63I/L41M) yielded non-glycosylated protein bands that were approximately 6 kDa in size, indicating that [Bpa23]PTHrP cross-links to amino acids 23-40 of the rP1R. This segment overlaps a receptor region previously identified by deletion mapping to be important for ligand binding. Alanine scanning of this region revealed two residues, Thr33 and Gln37, as being functionally involved in ligand binding. Thus, the convergence of photoaffinity cross-linking and mutational data demonstrates that the extreme amino-terminus of the rP1R participates in ligand binding.

Conservative management of biliary obstruction due to Fasciola hepatica.

We report a case of temporary biliary obstruction due to fascioliasis. This case report shows that in Central Europe, fascioliasis is one of the differential diagnoses of abdominal pain, especially if it is associated with eosinophilia. Successful medical treatment is possible even with obstruction of the bile duct.

No generation of bradykinin with a new polyacrylonitrile membrane (SPAN) in haemodialysis patients treated with ACE inhibitors.

BACKGROUND: Anaphylactoid reactions occurring in uraemic patients haemodialysed with polyacrylonitrile haemodialysis (HD) membranes and being treated with ACE inhibitors have been attributed to an excessive generation of bradykinin. METHODS: Here we tested in a prospective trial a new type of polyacrylonitrile membrane (SPAN) with respect to bradykinin generation in nine HD patients receiving either captopril or enalapril. Each patient had three consecutive HD sessions with each of the three tested membranes, high-flux SPAN, high-flux polysulphone (F60) and low-flux Hemophan (GFS Plus 16). RESULTS: No clinical signs of anaphylactoid reactions were observed in any of these patients but the number of patients was relatively small and the duration of exposure to different membranes relatively short. At 5 min after the start of HD session, plasma bradykinin levels were significantly higher in the venous than in the arterial line for all three HD membranes: SPAN, 18.5 +/- 11.9 versus 12.4 +/- 5.3 fmol/ml (P < 0.05); F60, 19.0 +/- 13.8 versus 11.5 +/- 6.5 fmol/ml (P < 0.01); and GFS Plus 16, 39.1 +/- 22.9 versus 15.8 +/- 12.4 fmol/ml (P < 0.005), mean +/- SD respectively. Higher venous line levels were still observed at the 15 and 60 min time points for F60 and GFS Plus 16, but not for SPAN. However, these levels were still insignificant compared to levels measured during episodes of anaphylactic shock from the literature. Plasma histamine and C5a anaphylatoxin levels did not show any increase during HD with SPAN. CONCLUSION: The SPAN membrane did not induce significant bradykinin release in dialysis patients on ACE-inhibitor therapy. It may therefore be used for high-flux dialysis in such patients.

Parathyroidectomy does not prevent the renal PTH/PTHrP receptor down-regulation in uremic rats.

In a recent study we demonstrated that the PTH/PTHrP receptor (PTH-R) mRNA was markedly down-regulated in the remnant kidney of uremic rats with severe secondary hyperparathyroidism. Among the factors potentially implicated in this down-regulation, to date only PTH has been demonstrated to modulate PTH-R expression. Here, we examined the effect of thyroparathyroidectomy (TPTX) on the renal expression of PTH-R in rats with normal renal function or with chronic renal failure (CRF) induced by 5/6 nephrectomy. Four groups of rats were studied: control, TPTX, CRF, and CRF + TPTX. Moderate-degree renal failure was documented by mean (+/- SD) creatinine clearances (microliter/min/100 g body wt) of 259 +/- 40 and 212 +/- 45 in CRF and CRF + TPTX rats, compared with 646 +/- 123 and 511 +/- 156 in control and TPTX rats, respectively. Plasma phosphorus, calcitriol, and ionized calcium were significantly lower in CRF and CRF + TPTX than in control animals. Plasma ionized calcium and calcitriol were also lower in TPTX than in control rats. Plasma PTH levels (pg/ml) were increased in CRF rats (41.8 +/- 29.4), and markedly decreased in TPTX (10.1 +/- 7.8) and CRF + TPTX (8.0 +/- 3.8) rats compared with control rats (21.7 +/- 7.5). Northern blot analysis showed that the level of the steady-state PTH-R mRNA in the kidney of CRF and CRF + TPTX rats was markedly decreased compared with that of control rats, the ratios of PTH-R mRNA/beta-actin mRNA being 0.28 +/- 0.04 and 0.27 +/- 0.03 versus 0.54 +/- 0.05, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The renal PTH/PTHrP receptor is down-regulated in rats with chronic renal failure.

Hypocalcemia, hyperphosphatemia, and resistance to the action of PTH are well characterized features in the setting of advanced chronic renal failure (CRF). Although the underlying mechanisms are ill-understood, clinical and experimental evidence points to both PTH receptor down-regulation and post-receptor abnormalities in their pathogenesis. In the present study we have examined the effect of advanced CRF in rats on the renal expression of PTH/PTHrP receptor (PTH-R). CRF was created by a standard two-step operation (5/6 nephrectomy). Four weeks thereafter, 19 uremic rats were compared with 23 sham-operated rats. Uremic rats had higher mean (+/- SD) plasma creatinine levels than control rats, 164 +/- 107 microM versus 43 +/- 5 microM, respectively. They also had higher plasma phosphorus and iPTH levels, 4.70 +/- 1.71 mM versus 2.59 +/- 0.37 mM and 561 +/- 336 versus 27 +/- 18 pg/ml, respectively. Mean plasma total calcium and blood ionized calcium were significantly lower in uremic than in control rats, 2.13 +/- 0.06 mM versus 2.61 +/- 0.10 mM and 1.07 +/- 0.11 versus 1.31 +/- 0.06 mM, respectively. Mean plasma calcitriol concentration was also significantly lower in uremic than in control rats, 39.8 +/- 14.6 and 80.4 +/- 15.2 pg/ml, respectively. Nine out of the 19 rats were examined for renal PTH-R gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3.

Hypocalcemia and hyperphosphatemia caused by parathyroid hormone (PTH)-resistance are the only discernible abnormalities in pseudohypoparathyroidism type Ib (PHP-Ib). Because mutations in the PTH/PTH-related peptide receptor, a plausible candidate gene, had been excluded previously, we conducted a genome-wide search with four PHP-Ib kindreds and established linkage to a small telomeric region on chromosome 20q, which contains the stimulatory G protein gene. We, furthermore, showed that the genetic defect is imprinted paternally and thus is inherited in the same mode as the PTH-resistant hypocalcemia in kindreds with PHP-Ia and/or pseudo-pseudohypoparathyroidism, two related disorders caused by different stimulatory G protein mutations.