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.

Associations with multiple pituitary hormone deficiency in patients with an ectopic posterior pituitary gland

INTRODUCTION: The presence of an ectopic posterior pituitary gland (EPP) on magnetic resonance imaging (MRI) is associated with hypopituitarism with one or more hormone deficiencies. We aimed to identify risk factors for having multiple pituitary hormone deficiency (MPHD) compared to isolated growth hormone deficiency (IGHD) in patients with an EPP. METHODS: In 67 patients (45 male) with an EPP on MRI, the site (hypothalamic vs. stalk) and surface area (SA) [ x (maximum diameter/2) x (maximum height/2), mm(2)] of the EPP were recorded and compared in patients with IGHD and MPHD in relation to clinical characteristics. RESULTS: In MPHD (n = 32) compared to IGHD (n = 35) patients: age of presentation was younger (1.4 [0.1-10.7]vs. 4.0 [0.1-11.3] years, P = 0.005), major incidents during pregnancy were increased (47%vs. 20%, P = 0.02) as were admissions to a neonatal intensive care unit (NICU) (60%vs. 26%, P = 0.04), whilst EPP SA was lower (12.3 [2.4-34.6]vs. 25.7 [6.9-48.2] mm(2), P < 0.001). In patients with a hypothalamic (n = 56) compared to a stalk sited EPP (n = 11): prevalence of MPHD was greater (55%vs. 9%,P = 0.05) and EPP surface area was smaller (17.3 [2.4-48.2]vs. 25.3 [11.8-38.5] mm(2), P < 0.001). In regression analysis, after adjusting for age, presence of MPHD was associated with: major incidents during pregnancy (RR 6.8 [95%CI 1.2-37.7]), hypothalamic EPP site (RR 10.9 [1.0-123.9]) and small EPP SA (RR 2.5 [1.0-5.0] for tertiles of SA). CONCLUSION: In patients with an EPP, adverse antenatal events, size (small) and position (hypothalamic) of the posterior pituitary gland on MRI were associated with MPHD. These findings suggest that adverse factors during pregnancy may be important for the development of an EPP.

The murine Fgfrl1 receptor is essential for the development of the metanephric kidney

Fgfrl1 is a novel member of the fibroblast growth factor receptor family. Its extracellular domain resembles the four conventional Fgfrs, while its intracellular domain lacks the tyrosine kinase domain necessary for Fgf mediated signal transduction. During embryonic development Fgfrl1 is expressed in the musculoskeletal system, in the lung, the pancreas and the metanephric kidney. Targeted disruption of the Fgfrl1 gene leads to the perinatal death of the mice due to a hypoplastic diaphragm, which is unable to inflate the lungs. Here we show that Fgfrl1-/- embryos also fail to develop the metanephric kidney. While the rest of the urogenital system, including bladder, ureter and sexual organs, develops normally, a dramatic reduction of ureteric branching morphogenesis and a lack of mesenchymal-to-epithelial transition in the nephrogenic mesenchyme result in severe renal dysgenesis. The failure of nephron induction might be explained by the absence of the tubulogenic markers Wnt4, Fgf8, Pax8 and Lim1 at E12.5 of the mutant animals. We also observed a loss of Pax2 positive nephron precursor cells and an increase of apoptosis in the cortical zone of the remnant kidney. Fgfrl1 is therefore essential for mesenchymal differentiation in the early steps of nephrogenesis.

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

BACKGROUND: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006). RESULTS: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1. CONCLUSION: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

Growth after Hypophyseal Stalk Transection and Hypophysectomy in Beef Calves

Growth hormone (GH) is a metabolic hormone that plays an important role in long-bone growth and muscle accretion in mammals. The anterior pituitary gland at the base of the brain is the primary site of GH production and release into the general circulation. Neurons in the arcuate nucleus of the hypothalamus in the lower part of the brain secrete GH-releasing hormone ([GHRH] or factor [GRF]) and GH-release-inhibiting hormone ([GHRIH] or somatostatin [SRIH]) that acutely modulate GH secretion by the pituitary gland. The pituitary gland is connected to the median eminence of the hypothalamus by a stalk (hypophyseal stalk). Complete surgical removal of the pituitary gland (hypophysectomy) arrests growth and greatly impairs metabolism in laboratory and farm animal species. Daily subcutaneous injection of bovine GH (bGH) in immature hypophysectomized rats significantly increased body growth and epiphyseal plate width of the long-bone (tibia) compared with diluent-treated hypophysectomized controls. Growth rate was less, however, in the bGH-treated animals compared with intact controls. In beef calves, hypophysectomy completely arrested body weight gain and long-bone growth. GH is secreted in an episodic pattern in young growing intact calves. Episodic GH secretion was abolished immediately following hypophyseal stalk transection, and basal GH blood concentration was less than in shamoperated controls. Regardless, growth continued in these stalk-transected calves during a 1,008-day period, but at a lower growth rate than seen in the sham-operated controls. At autopsy, pituitary gland weight was greatly decreased in hypophyseal stalktransected compared with sham-operated calves. Thus, in spite of obliterated episodic GH release and decreased basal secretion of GH, the isolated pituitary gland of hypophyseal stalk transected calves continues to secrete sufficient amounts of GH for significant growth and development throughout a long period.

Brain Regulation of Growth in Beef Calves

Small peptide hormones produced in the lower part of the brain (hypothalamus) regulate episodic and basal secretion of hormones from the anterior pituitary gland that affect metabolism and growth in cattle. This study focused on long-term growth in young calves subjected to hypophysectomy (HYPOX), hypophyseal stalk transection (HST), and sham operation control (SOC). Crossbred (Hereford x Aberdeen Angus) and Hereford, and Aberdeen Angus calves were HYPOX (n = 5), HST (n = 5), or SOC (n = 8) at 146 days of age, whereas another group was HST (n = 5) or SOC (n = 7) at 273 days of age. Body weight was determined every 21 days from birth to 1008 days of age. From day 146-1008, growth was arrested (P < 0.001) in HYPOX (0.06 kg/day) compared with SOC (0.50 kg/day) calves. Growth continued but at a significantly lower rate (P < 0.05) in calves HST at 146 days (0.32 kg/day) and 273 days (0.32 kg/day) compared with SOC (0.50 kg/day). Although episodic growth hormone (GH) secretion was abolished and peripheral blood serum GH concentration remained consistently lower in HST calves (2.4 ng/ml) than in the SOC (5.5 ng/ml; P < 0.01), the calves continued to grow throughout 1008 days. Peripheral serum thyroid stimulating hormone (TSH) concentration was less (P < 0.05) in HST compared with SOC calves. There was an abrupt decrease (P < 0.001) in serum thyroxine (T4) (4-fold) and triiodothyronine (T3) (3-fold) concentration after surgery that remained to 360 days in HST compared with SOC calves. At sacrifice, pituitary gland weight was markedly reduced (P < 0.001) in HST (0.18 g/100 kg body weight) compared with SOC (0.55 g/100 kg body weight) calves. Histological examination of pituitary glands from HST calves indicated the persistence of secretory GH and TSH cells in the same areas of the anterior pituitary gland as SOC calves. Coronal sections of the gland revealed GH and TSH secreting cells in HST calves that were similar to the controls. These results indicate that long-term growth continues, but at a slower rate, after hypophyseal stalk transection of immature calves in spite of complete abolition of episodic GH secretion and consistently decreased basal secretion of GH, TSH, T4, and T3 compared with sham-operated animals. Growth was abolished after hypophysectomy of immature calves in which circulating GH and TSH was undetectable.

The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response - Biological and Clinical Aspects

Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

Impact of environmental estrogens on Yfish considering the diversity of estrogen signaling.

Research on endocrine disruption in fish has been dominated by studies on estrogen-active compounds which act as mimics of the natural estrogen, 17β-estradiol (E2), and generally exert their biological actions by binding to and activation of estrogen receptors (ERs). Estrogens play central roles in reproductive physiology and regulate (female) sexual differentiation. In line with this, most adverse effects reported for fish exposed to environmental estrogens relate to sexual differentiation and reproduction. E2, however, utilizes a variety of signaling mechanisms, has multifaceted functions and targets, and therefore the toxicological and ecological effects of environmental estrogens in fish will extend beyond those associated with the reproduction. This review first describes the diversity of estrogen receptor signaling in fish, including both genomic and non-genomic mechanisms, and receptor crosstalk. It then considers the range of non-reproductive physiological processes in fish that are known to be responsive to estrogens, including sensory systems, the brain, the immune system, growth, specifically through the growth hormone/insulin-like growth factor system, and osmoregulation. The diversity in estrogen responses between fish species is then addressed, framed within evolutionary and ecological contexts, and we make assessments on their relevance for toxicological sensitivity as well as ecological vulnerability. The diversity of estrogen actions raises questions whether current risk assessment strategies, which focus on reproductive endpoints, and a few model fish species only, are protective of the wider potential health effects of estrogens. Available - although limited - evidence nevertheless suggests that quantitative environmental threshold concentrations for environmental protection derived from reproductive tests with model fish species are protective for non-reproductive effects as well. The diversity of actions of estrogens across divergent physiological systems, however, may lead to and underestimation of impacts on fish populations as their effects are generally considered on one functional process only and this may underrepresent the impact on the different physiological processes collectively.

Transient Neonatal Zinc Deficiency Caused by a Heterozygous G87R Mutation in the Zinc Transporter ZnT-2 (SLC30A2) Gene in the Mother Highlighting the Importance of Zn (2+) for Normal Growth and Development

Suboptimal dietary zinc (Zn(2+)) intake is increasingly appreciated as an important public health issue. Zn(2+) is an essential mineral, and infants are particularly vulnerable to Zn(2+) deficiency, as they require large amounts of Zn(2+) for their normal growth and development. Although term infants are born with an important hepatic Zn(2+) storage, adequate Zn(2+) nutrition of infants mostly depends on breast milk or formula feeding, which contains an adequate amount of Zn(2+) to meet the infants' requirements. An exclusively breast-fed 6 months old infant suffering from Zn(2+) deficiency caused by an autosomal dominant negative G87R mutation in the Slc30a2 gene (encoding for the zinc transporter 2 (ZnT-2)) in the mother is reported. More than 20 zinc transporters characterized up to date, classified into two families (Slc30a/ZnT and Slc39a/Zip), reflect the complexity and importance of maintaining cellular Zn(2+) homeostasis and dynamics. The role of ZnTs is to reduce intracellular Zn(2+) by transporting it from the cytoplasm into various intracellular organelles and by moving Zn(2+) into extracellular space. Zips increase intracellular Zn(2+) by transporting it in the opposite direction. Thus the coordinated action of both is essential for the maintenance of Zn(2+) homeostasis in the cytoplasm, and accumulating evidence suggests that this is also true for the secretory pathway of growth hormone.

The effect of idursulfase on growth in patients with Hunter syndrome: data from the Hunter Outcome Survey (HOS)

Hunter syndrome (mucopolysaccharidosis type II) is a rare and life-limiting multisystemic disorder with an X-linked recessive pattern of inheritance. Short stature is a prominent feature of this condition. This analysis aimed to investigate the effects of enzyme replacement therapy with idursulfase on growth in patients enrolled in HOS - the Hunter Outcome Survey which is a multinational observational database. As of Jan 2012, height data before treatment were available for 567 of 740 males followed prospectively after HOS entry. Cross-sectional analysis showed that short stature became apparent after approximately 8 years of age; before this, height remained within the normal range. Age-corrected standardized height scores (z-scores) before and after treatment were assessed using piecewise regression model analysis in 133 patients (8-15 years of age at treatment start; data available on ≥ 1 occasion within +/-24 months of treatment start; growth hormone-treated patients excluded). Results showed that the slope after treatment (slope=-0.005) was significantly improved compared with before treatment (slope=-0.043) (difference=0.038, p=0.004). Analysis of covariates (age at treatment start, cognitive involvement, presence of puberty at the start of ERT, mutation type, functional classification), showed a significant influence on growth of mutation type (height deficit in terms of z-scores most pronounced in patients with deletions/large rearrangements/nonsense mutations, p<0.0001) and age (most pronounced in the 12-15-year group, p<0.0001). Cognitive involvement, pubertal status at the start of ERT and functional classification were not related to the growth deficit or response to treatment. In conclusion, the data showed an improvement in growth rate in patients with Hunter syndrome following idursulfase treatment.

Angiotensin receptor agonistic autoantibody is highly prevalent in preeclampsia: correlation with disease severity.

Preeclampsia (PE), a syndrome affecting 5% of pregnancies, characterized by hypertension and proteinuria, is a leading cause of maternal and fetal morbidity and mortality. The condition is often accompanied by the presence of a circulating maternal autoantibody, the angiotensin II type I receptor agonistic autoantibody (AT(1)-AA). However, the prevalence of AT(1)-AA in PE remains unknown, and the correlation of AT(1)-AA titers with the severity of the disease remains undetermined. We used a sensitive and high-throughput luciferase bioassay to detect AT(1)-AA levels in the serum of 30 normal, 37 preeclamptic (10 mild and 27 severe), and 23 gestational hypertensive individuals. Here we report that AT(1)-AA is highly prevalent in PE ( approximately 95%). Next, by comparing the levels of AT(1)-AA among women with mild and severe PE, we found that the titer of AT(1)-AA is proportional to the severity of the disease. Intriguingly, among severe preeclamptic patients, we discovered that the titer of AT(1)-AA is significantly correlated with the clinical features of PE: systolic blood pressure (r=0.56), proteinuria (r=0.70), and soluble fms-like tyrosine kinase-1 level (r=0.71), respectively. Notably, only AT(1)-AA, and not soluble fms-like tyrosine kinase-1, levels are elevated in gestational hypertensive patients. These data serve as compelling clinical evidence that AT(1)-AA is highly prevalent in PE, and its titer is strongly correlated to the severity of the disease.

CART: an Hrs/actinin-4/BERP/myosin V protein complex required for efficient receptor recycling.

Altering the number of surface receptors can rapidly modulate cellular responses to extracellular signals. Some receptors, like the transferrin receptor (TfR), are constitutively internalized and recycled to the plasma membrane. Other receptors, like the epidermal growth factor receptor (EGFR), are internalized after ligand binding and then ultimately degraded in the lysosome. Routing internalized receptors to different destinations suggests that distinct molecular mechanisms may direct their movement. Here, we report that the endosome-associated protein hrs is a subunit of a protein complex containing actinin-4, BERP, and myosin V that is necessary for efficient TfR recycling but not for EGFR degradation. The hrs/actinin-4/BERP/myosin V (CART [cytoskeleton-associated recycling or transport]) complex assembles in a linear manner and interrupting binding of any member to its neighbor produces an inhibition of transferrin recycling rate. Disrupting the CART complex results in shunting receptors to a slower recycling pathway that involves the recycling endosome. The novel CART complex may provide a molecular mechanism for the actin-dependence of rapid recycling of constitutively recycled plasma membrane receptors.

Radiometrical, hormonal and biological correlates of skeletal growth in the female rat from birth to senescence

OBJECTIVE We investigated the skeletal growth profile of female rats from birth to senescence (100weeks) on the basis of sequential radiometrical, hormonal and biochemical parameters. DESIGN Weaning rats entered the study which was divided into two sections: a) sequential measurements of vertebral and tibial growths and bone mineral density (BMD), estimation of mineral content of the entire skeleton (BMC) and chemical analysis of vertebral Ca; and b) determination of basal and pulsatile growth hormone (rGH), insulin-like growth hormone (IGF-I), estradiol (E2), parathyroid hormone (PTH), osteocalcin (OC) and urinary d-pyridinoline (dp) throughout the experimental period. RESULTS Vertebral and tibial growths ceased at week 25 whereas BMD and BMC as well as total vertebral Ca exhibited a peak bone mass at week 40. rGH pulsatile profiles were significantly higher in younger animals coinciding with the period of active growth and IGF-I peaked at 7weeks, slowly declining thereafter and stabilizing after week 60. OC and dp closely paralleled IGF-I coinciding with the period of enhanced skeletal growth, remaining thereafter in the low range indicative of reduced bone turnover. E2 increased during reproductive life but the lower values subsequently recorded were still in the physiological range, strongly suggesting a protective role of this steroid on bone remodeling. PTH followed a similar profile to E2, but the significance of this after completion of growth remains unclear. CONCLUSIONS Mechanisms governing skeletal growth in the female rat appear similar to those in humans. Bone progression and attainment of peak bone mass are under simultaneous control of rGH, IGF-I and calciotropic hormones and are modulated by E2. This steroid seems to protect the skeleton from resorption before senescence whereas the role of PTH in this context remains uncertain.

Biological, diagnostic and therapeutic relevance of the MET receptor signaling in head and neck cancer.

Head and neck cancer constitutes the 6th most common malignancy worldwide and affects the crucial anatomical structures and physiological functions of the upper aerodigestive tract. Classical therapeutic strategies such as surgery and radiotherapy carry substantial toxicity and functional impairment. Moreover, the loco-regional control rates as well as overall survival still need to be improved in subgroups of patients. The scatter-factor/hepatocyte growth factor receptor tyrosine kinase MET is an established effector in the promotion, maintenance and progression of malignant transformation in a wide range of human malignancies, and has been gaining considerable interest in head and neck cancer over the last 15 years. Aberrant MET activation due to overexpression, mutations, tumor-stroma paracrine loops, and cooperative/redundant signaling has been shown to play prominent roles in epithelial-to-mesenchymal transition, angiogenesis, and responses to anti-cancer therapeutic modalities. Accumulating preclinical and translational evidence highly supports the increasing interest of MET as a biomarker for lymph node and distant metastases, as well as a potential marker of stratification for responses to ionizing radiation. The relevance of MET as a therapeutic molecular target in head and neck cancer described in preclinical studies remains largely under-evaluated in clinical trials, and therefore inconclusive. Also in the context of anti-cancer targeted therapy, a large body of preclinical data suggests a central role for MET in treatment resistance towards multiple therapeutic modalities in malignancies of the head and neck region. These findings, as well as the potential use of combination therapies including MET inhibitors in these tumors, need to be further explored.

Targeting class IA PI3K isoforms selectively impairs cell growth, survival, and migration in glioblastoma.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110β expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110β also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.