S.C. van Buul-Offers

Short-term glucocorticoid treatment of piglets causes changes in growth plate morphology and angiogenesis

OBJECTIVEnGlucocorticoid treatment of children often leads to growth retardation, and the precise target(s) in the growth plate responsible for this effect are unknown. Angiogenesis is an important part of the endochondral ossification process, and VEGF expressed in the growth plate is essential for proper angiogenesis to occur. Since glucocorticoid treatment down-regulates VEGF expression in cultured chondrocytes, we hypothesized that in vivo glucocorticoid treatment could result in VEGF down-regulation in the growth plate and disturbed angiogenesis, thus contributing to the growth retardation.nnnDESIGNnWe treated 6-week-old prepubertal piglets (10 kg) for 5 days with prednisolone (50 mg/day). Tibial growth plate sections were studied for apoptosis and the expression of VEGF protein and mRNA and MMP-9 protein. Capillaries in the metaphysis were visualized by CD31 immunostaining. Growth plate morphology (width of various zones) was determined by interactive measurements on hematoxylin/eosin stained sections and apoptotic cells were detected by TUNEL assay.nnnRESULTSnIn the prednisolone-treated animals, the total width of the growth plate decreased to 81% of controls (P<0.02), which was explained by a decrease of the width of the proliferative zone to 73% (P<0.05). The treatment had no effect on the orderly organization of the chondrocyte columns. In the growth plates of control animals, apoptosis was shown in 5.8% of the hypertrophic chondrocytes and was limited to the terminal hypertrophic chondrocytes. In prednisolone-treated animals, 40.5% of the hypertrophic chondrocytes was apoptotic (P<0.02), with apoptotic chondrocytes also appearing higher in the hypertrophic zone. We observed fewer capillaries and loss of their parallel organization in the metaphysis in the prednisolone-treated animals. The capillaries were shorter and chaotic in appearance. In contrast to controls, in prednisolone-treated animals VEGF mRNA and protein could not be detected in the hypertrophic zone of the growth plate. Trabecular bone length in the primary spongiosa was also diminished by the treatment. No changes were observed in the expression pattern of MMP-9, a matrix metalloproteinase, which is also important for angiogenesis and bone formation.nnnCONCLUSIONSnThese results indicate that short-term glucocorticoid treatment of growing piglets severely disturbs the width of the growth plate, apoptosis of chondrocytes, VEGF expression by hypertrophic chondrocytes, the normal invasion of blood vessels from the metaphysis to the growth plate and bone formation at the chondro-osseous junction. These effects could alter the dynamics of endochondral ossification and thus contribute to glucocorticoid-induced growth retardation.

A novel specific bioassay for the determination of glucocorticoid bioavailability in human serum

objective Some patients develop side‐effects even on relatively low doses of topically administered glucocorticoids (GCs), while others appear to be less sensitive to GCs. We have developed and validated a bioassay which can measure glucocorticoid bioavailability directly from small amounts of human serum to help elucidate underlying mechanisms.

Plasma Levels of Insulin-Like Binding Protein-2 in Prepubertal Short Children and Its Diagnostic Value in the Evaluation of Growth Hormone Deficiency

Aim: This study was designed to investigate whether determination of plasma insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) levels could be of benefit in the evaluation of childhood growth hormone (GH) deficiency (GHD). Method: A retrospective analysis was performed on 91 prepubertal children referred for investigation of short stature. Maximal GH levels in plasma after provocative stimuli were between 1.0 and 93.0 mU/l, 6 subjects exhibiting peak values of <5 mU/l. Initially a GH peak of 20 mU/l was used as a cutoff limit to define GHD and idiopathic short stature (ISS) patients. The results of GH provocative tests were compared to age- and gender-based standard deviation scores (SDS) of plasma IGFBP-2, IGF-I, IGFBP-3 and the molar ratios of the latter two to IGFBP-2. The respective normative range values for these parameters were determined in plasma samples from 353 healthy children (i.e. 171 girls, 182 boys). Results: Circulating IGFBP-2 levels did not correlate with height SDS, height velocity SDS or the peak GH levels after provocative stimuli. A weak negative relationship was found between IGFBP-2 and IGF-I. Plasma levels of IGFBP-2 in GHD patients were higher than those of ISS children, who had normal levels. Although at the optimal cutoff point of –0.71 SDS 91.5% of the GHD patients were identified correctly, a substantial proportion (71.9%) of the ISS subjects also had IGFBP-2 levels above this limit. The use of various combinations of IGFBP-2, IGF-I, IGFBP-3 and the derived ratios only slightly improved the diagnostic efficiency as compared to the results of the individual tests. Neither IGFBP-2 nor the IGFBP-3/IGFBP-2 and IGF-I/IGFBP-2 ratios were found to be related to the short- (1 year) or long-term (3 years) growth response to GH therapy. Conclusion: It is concluded that none of the tests investigated, either alone or in various combinations, are reliable in either predicting the peak GH level after provocative stimuli in prepubertal short children or in predicting their growth response to GH.

Expression of recombinant human insulin-like growth factor I in mammalian cells

In order to develop a suitable mammalian expression system for human insulin-like growth factors (hIGFs) and mutant IGFs, we have constructed several artificial IGF genes, based on a cDNA encoding the IGF-I precursor (153 amino acids). Transient expression experiments using mouse Ltk- cells revealed that the IGF-I gene constructs were efficiently expressed when placed under control of the SV40 Early promoter (SV40E). This resulted in the synthesis and secretion of IGF-I receptor-reactive products. Constructs encoding an IGF-I precursor with a truncated signal peptide of 25 amino acids under control of SV40E promoter or the inducible Drosophila heat shock hsp70 promoter, were used to establish stably transformed CHOdhfr- and mouse L cells. Clones secreting IGF-I were identified by an IGF-I-specific radioreceptor assay. Immunoblot analysis of conditioned media from these clones resulted in the specific precipitation of a protein of 7 kDa identical in size to native IGF-I purified from human serum. After optimization of the expression conditions, the stable cell lines secrete 0.5-2 microgram/10(6) cells of IGF-I. The biological activity of the secreted recombinant IGF-I was shown by its ability to stimulate DNA synthesis in human MCF-7 cells. The results described in this paper indicate that a mammalian expression system, employing CHOdhfr- or L cells, is a useful system for the synthesis of biological active IGF-I.

Molecular aspects of the human somatomedins.

The primary structure of the somatomedins (SM) IGF-I and IGF-II has been known for some years. Both from isolation of the SMs from plasma and from the study of cDNAs it has become evident that there are more SMs, needing further study. In plasma the SMs are present in larger molecules of two size-classes: 150 kD and 40 kD, behaving as specific SM-binding proteins. The possibility that such protein classes are heterogeneous and also contain precursor molecules undergoing proteolytic processing is considered. The somatomedin genes are discontinuous and span large regions of the human genome.

Excessive growth in a child with craniopharyngioma and growth hormone deficiency

In a 5-year-old boy presenting with clumsiness and excessive growth, a large craniopharngioma was diagnosed. Biochemically, there was a deficiency of growth hormone, a hypothalamic hypothyroidism and hypocorticalism, a thyroxine binding globulin elevation, an abnormal gonadotropin secretion and a mild hyperprolactinaemia. After removal of the tumour growth stopped almost completely. Plasma insulin-like growth factor (IGF)-I was in the lower normal range. Plasma IGF-II decreased after tumour removal. It is speculated that the tumour produced a growth factor causing excessive growth.

109 PLASMA INSULIN-LIKE GROWTH FACTOR (IGF)-I AND II LEVELS AND BIOASSAYABLE SOMATOMEDIN ACTIVITY IN CHILDREN WITH CEREBRAL CIGANTISM (SOTOS SYNDROME)

Cerebral gigantism (Sotos syndrome) is characterized by a large birth size, excessive statural growth, advanced bone age, mental retardation and dysmorphic features. In this study 30 plasma samples of 14 children with Sotos syndrome were assayed for IGF-I and II and bioassayable somatomedin activity (SM-act). Immunoreactive IGF-I was determined in unextracted plasma and compared to the Nichols Institute references. Immunoreactive IGF-II was measured with a nonequilibrium RIA, using the tyrosylated eight amino acid C-peptide region of IGF-II (CP-II) and an antiserum against this fragment (kindly donated by Dr. R. Hintz). SM-act was determined with the porcine cartilage bioassay. All but 3 values of IGF-I were within the ± 2 SD range for age. When the data were expressed as a Z-score for age, mean IGF-I decreased from -0.1 (range -1.0 to +1.0, n=6) at 0-3 years to -1.1 (range -2.0 to 0.3, n=7) at 3-5 years. IGF-II levels were generally within the reference range. SM-act was usually low between 1 and 5 years of age (mean -2.2 SD, range -5.0 to 3.6, n=11) and in the lower normal range thereafter. These IGF-I and SM-act results are in contrast to the data in constitutional tall stature. The relatively low somatomedin levels between 1 and 5 years of age concur with the deceleration of growth which is usually seen after the first year of life in children with Sotos syndrome.

The effect of hGH, thyroxine and semipurified somatomedin (SM) on cellular growth of some organs of the Snell dwarf mouse

Growth of the hypopituitary Snell mouse is partially restored by administration of hGH, thyroxine and SM. In order to investigate whether these preparations induce cell division and/or hypertrophy DNA and protein of liver,kidney and spleen were measured after treatment and contrasted with normal and dwarfed controls. Results of the latter are as follows:SM induces cell division but only little cell enlargement in the liver and the spleen, as do hGH and T4. Cell division of the kidney is barely affected by SM and hGH, but markedly by T4, whereas all preparations induce hypertrophy of this organ.

Primary sequences of insulin-like growth factors 1 and 2 isolated from porcine plasma

Insulin-like growth factors 1 and 2 were purified from porcine plasma. In addition to the determination of their isoelectric points, the primary structures of both proteins were determined, using low microgram quantities of protein, by the versatile combination of time-of-flight plasma desorption mass spectrometry and automated Edman degradation. Porcine insulin-like growth factor 1 was shown to be homologous to both human and bovine proteins; the type 2 growth factor showed one mutation to both human and bovine type 2 proteins.