Preclinical and clinical in vitro in vivo correlation of an hGH dextran microsphere formulation.

PURPOSE: To investigate the in vitro in vivo correlation of a sustained release formulation for human growth hormone (hGH) based on hydroxyethyl methacrylated dextran (dex-HEMA) microspheres in Pit-1 deficient Snell dwarf mice and in healthy human volunteers. MATERIALS AND METHODS: A hGH-loaded microsphere formulation was developed and tested in Snell dwarf mice (pharmacodynamic study) and in healthy human volunteers (pharmacokinetic study). RESULTS: Single subcutaneous administration of the microspheres in mice resulted in a good correlation between hGH released in vitro and in vivo effects for the hGH-loaded microsphere formulation similar to daily injected hGH indicating a retained bioactivity. Testing the microspheres in healthy volunteers showed an increase (over 7-8 days) in hGH serum concentrations (peak concentrations: 1-2.5 ng/ml). A good in vitro in vivo correlation was obtained between the measured and calculated (from in vitro release data) hGH serum concentrations. Moreover, an increased serum concentration of biomarkers (insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3) was found again indicating that bioactive hGH was released from the microspheres. CONCLUSIONS: Good in vitro in vivo correlations were obtained for hGH-loaded dex-HEMA microspheres, which is an important advantage in predicting the effect of the controlled drug delivery product in a clinical situations.

Differential patterns of insulin-like growth factor-I and -II mRNA expression in medulloblastoma.

Insulin-like growth factors (IGFs) play an important role in tumour growth and development. We hypothesized that this is also the case for medulloblastomas, which are highly malignant cerebellar brain tumours usually occurring in children. In these tumours the expression patterns of IGF-I and -II mRNA were studied. Tumour specimens obtained from 12 children and two adults at diagnosis were hybridized in situ with digoxigenin-labelled cRNA probes for hIGF-I and hIGF-II mRNAs. In all cases, tumour cells showed abundant expression of IGF-I mRNA. Nine of the 14 tumours showed variable but significant IGF-II expression. In these tumours, the hybridization signal almost exclusively colocalized with a subpopulation of Ki-M1P positive cells that were identified as ramified microglia (RM) cells. In the five tumours without IGF-II expression, microglia/brain macrophages with a more rounded amoeboid-like morphology predominated. RM cells in normal cerebellar tissues, residing abundantly in areas of the white and, to a less extent, in the grey matter, were IGF-II mRNA-negative. These RM cells showed a thinner and more extensively branched appearance and were more evenly distributed than those encountered in medulloblastoma. Probably, during the transformation from the resting ramified towards the amoeboid morphology (or vice versa) IGF-II mRNA expression is only temporarily induced. The physiological meaning of the induction of IGF-II mRNA expression by these cells in medulloblastoma remains unclear but any IGF-II peptide synthesized could exert unfavourable mitogenic and antiapoptotic effects on adjacent tumour cells. However, in this relatively small number of cases we could not find any indications for a relationship between clinical characteristics of the various cases and the extent of IGF-II mRNA expression.

Overexpression of human IGF-II mRNA in the brain of transgenic mice modulates IGFBP-2 gene expression in the medulla oblongata.

The insulin-like growth factors, IGF-I and IGF-II, and their binding proteins play an important role in the growth and development of the central nervous system. In the brain, colocalization of IGFs and IGFBPs often occurs, suggesting that IGFBPs can modulate IGF action. In one strain of our human (h)IGF-II transgenic mice, which carry an hIGF-II transgene driven by the H-2Kb promoter, we found overexpression of hIGF-II in the brain, as measured by Northern blot analysis. To clarify the localization and influence of the hIGF-II transgene on different components of the GH-IGF axis in the brain, we studied the expression pattern of the hIGF-II transgene, endogenous IGF-I and IGF-II, and IGFBP-2, -3 and -5 in the brain of prepubertal 4-week-old mice, using nonradioactive in situ hybridization. We found that the hIGF-II transgene is exclusively expressed in neurons of the piriform cortex, the cerebral cortex, the medulla oblongata and the granular layer of the cerebellum. In general, this pattern is comparable to the expression pattern of endogenous IGF-I, with a few exceptions: there is no expression of IGF-I in the granular layer of the cerebellum, whereas the Purkinje cells of the cerebellum and thalamus both express IGF-I but no hIGF-II transgene. This hIGF-II transgene expression pattern contrasts markedly with endogenous IGF-II expression, which is mainly located in nonneuronal cells such as the meninges and choroid plexus, and in some nuclei of the medulla oblongata. The hIGF-II transgene affects neither endogenous IGF-I and IGF-II expression, nor the expression of IGFBP-3, which is located in the choroid plexus. Although the hIGF-II transgene is expressed in neuronal structures similar to IGF-I and IGFBP-5, it is not able to regulate IGFBP-5 expression, as has previously been reported for IGF-I. In the medulla oblongata, the IGFBP-2 expression level showed 10-fold upregulation by the transgene, suggesting a modulating role for IGFBP-2 at the hIGF-II transgene action in this region.

Plasma insulin-like growth factors (IGFs), IGF-Binding proteins (IGFBPs), acid-labile subunit (ALS) and IGFBP-3 proteolysis in individuals with clinical characteristics of Sotos syndrome.

OBJECTIVE: Sotos syndrome is an overgrowth syndrome of poorly understood aetiology. We investigated whether this syndrome is related to alterations in plasma insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), acid-labile subunit (ALS) and serum IGFBP-3 proteolysis. DESIGN: Based on clinical criteria, 32 patients with clinical characteristics of Sotos syndrome (median age 8.4 years, range 1.8-48.4) were categorised into three groups: typical (n = 10, group 1), dubious (n = 12, group 2) and atypical (n = 10, group 3). Blood samples were obtained from 29 patients. MEASUREMENTS: Plasma IGF-I, IGF-II, E-II (pro-IGF-II and E-domain fragments), IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6 and ALS were measured by specific radioimmunoassays (RIAs). Except for E-II immunoreactivity, the concentrations were compared with those of age references, and expressed as standard deviation scores (SDS). IGFBP-3 proteolysis was assessed by incubation of serum with [125I]-IGFBP-3, followed by gel electrophoresis and was then compared with that in normal serum and third trimester pregnancy serum. RESULTS: Patients in group 1 showed significantly reduced plasma levels of IGF-II (median -0.9 SDS; p = 0.01), IGFBP-4 (-0.5 SDS; p = 0.02) and IGFBP-3 (-1.0 SDS; p = 0.01). Mean IGFBP-3 proteolysis was higher than in normal standard serum (61% vs 37%; p < 0.01) but lower than in third trimester pregnancy serum (94%; p < 0.01). Plasma IGF-I showed a tendency towards low values (median -0.9 SDS; p = 0.09), IGFBP-6 and ALS a tendency towards elevated levels (median values +0.8 SDS; p = 0.07 and +2.3 SDS; p = 0.09), and IGFBP-2 was normal. The mean value of E-II immunoreactivity was 8.7 nmol/l, similar to that in pooled normal plasma (8.6 nmol/l). Plasma and serum parameters in groups 2 and 3 were similar to reference values with the exception of plasma IGFBP-3 (in groups 2 and 3 median < or = -1.1 SDS; p < or = 0.02) and ALS (in group 3 median +1.3 SDS; p < 0.01). CONCLUSIONS: Patients with typical Sotos syndrome show low plasma IGF-II, IGFBP-3, IGFBP-4, and increased proteolysis of IGFBP-3 in serum. The extent to which these findings are associated with the pathophysiology of Sotos syndrome remains uncertain.

A comparison of in vitro bioassays to determine cellular glucocorticoid sensitivity.

An altered cellular glucocorticoid (GC) sensitivity is associated with several pathophysiological conditions such as asthma, diabetes, or rheumatoid arthritis. Several bioassays have been developed and employed to assess cellular GC sensitivity of peripheral blood mononuclear cells (PBMC), but correlations between these have rarely been investigated. We have compared four mitogen-based assays and an FK506 binding protein 51 (FKBP51) mRNA induction assay, using ten controls and a GC-resistant patient. The mitogen-based assays were performed using either diluted whole blood or isolated PBMC, and showed relatively large assay variations for the parameters maximal effect and half-maximal effect concentration. The FKBP51 assay showed smaller intra-assay and within-individual variation compared with the mitogen-based assays. The whole blood-based mitogen assays and the FKBP51 assay clearly discriminated the GC-resistant patient from the controls but, in contrast to expectations, both PBMC-based mitogen assays did not. The GC-induced FKBP51 mRNA increase in PBMC may be an alternative to determine an altered individual GC sensitivity with several advantages as compared with mitogen-based assays, such as the use of unstimulated PBMC, and a better intra- and inter-individual reproducibility.

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

OBJECTIVE: Glucocorticoid 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. DESIGN: We 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. RESULTS: In 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. CONCLUSIONS: These 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. METHODS: We have stably transfected the human embryonic kidney cell line HEK293 with a plasmid expressing the glucocorticoid receptor, and a plasmid containing the luciferase gene preceded by three concatenated steroid response elements, bringing luciferase expression under control of the liganded glucocorticoid receptor. RESULTS: The assay, with an intra- and interassay coefficient of variance (CV) better than 10%, showed the expected difference in potency between different GCs (fluticasone propionate > budesonide > dexamethasone > hydrocortisone). No cross-reactivity was detected with other steroid hormones such as progesterone, testosterone and oestradiol. The bioassay easily detects the rise and subsequent fall of bioavailable GCs in human serum following ingestion of only 0.5 mg dexamethasone, and clearly reflects the diurnal cortisol rhythm. Moreover, systemic availability following inhalation of 2 x 250 micro g fluticasone propionate using a pressure dose inhaler could be demonstrated. CONCLUSIONS: This assay can be used to determine levels of bioavailable GCs in serum, both endogenous and administered, and thus may help in optimizing treatment regimens. The small amount of serum needed to perform an analysis makes this assay applicable even to infants.

Short-term glucocorticoid treatment of prepubertal mice decreases growth and IGF-I expression in the growth plate.

The insulin-like growth factor (IGF) system is an important mediator of postnatal longitudinal growth, and the growth inhibiting effects of glucocorticoid (GC) treatment are suggested to be due to impaired action of the IGF system. However, the precise changes of the IGFs and the IGF-binding proteins (IGFBPs) in the growth plate, occurring upon short-term GC treatment have not been characterized. Prepubertal mice treated daily with dexamethasone (DXM) for 7 days, showed significant growth inhibition of total body length and weight and weight of the liver, thymus and spleen, whereas the weight of the kidneys was not affected. Analysis of the tibial growth plate showed that the total growth plate width significantly decreased to 84.5% of control values, caused by a significant decrease in the proliferative zone. The number of proliferating cell nuclear antigen (PCNA)-positive chondrocytes in the proliferative zone decreased significantly (to 40%) and TUNEL staining showed a significant 1.6-fold increase in apoptotic hypertrophic chondrocytes. In the growth plates, both IGF-I and IGF-II, as well as IGFBP-2 mRNAs were detected, mainly in the proliferative and prehypertrophic zones. DXM treatment significantly decreased the number of chondrocytes expressing IGF-I, whereas the number of chondrocytes expressing IGF-II and IGFBP-2 were not affected. The decrease in IGF-I expression in the growth plate indicates that GC treatment affects IGF-I at the local level of the growth plate, which could contribute to the GC-induced growth retardation.

IGF and IGF-binding protein expression in the growth plate of normal, dexamethasone-treated and human IGF-II transgenic mice.

Glucocorticoid (GC) treatment in childhood can lead to suppression of longitudinal growth as a side effect. The actions of GCs are thought to be mediated in part by impaired action of the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins (IGFBP-1 to -6). We have studied the effects of GCs on IGF and IGFBP expression at the local level of the growth plate, using non-radioactive in situ hybridization. We treated 3-week-old normal mice for 4 weeks with dexamethasone (DXM). We also treated human IGF-II (hIGF-II) transgenic mice in order to investigate whether IGF-II could protect against the growth retarding effect of this GC. DXM treatment resulted in general growth retardation in both mice strains, however, only in normal mice was tibial length decreased. In both normal and hIGF-II trangenic mice, the total width of the growth plate was not affected, whereas the width of the proliferative zone decreased as a result of the DXM treatment. Additionally, only in normal mice, the width of the hypertrophic zone thickened. Only expression of IGF-I, IGF-II and IGFBP-2 could be detected in the growth plates of 7-week-old normal mice. IGFBP-1, -3, -4, -5 and -6 mRNAs were not detected. DXM treatment of normal mice induced a significant 2.4-fold increase in the number of cells expressing IGF-I mRNA, whereas IGF-II and IGFBP-2 mRNA levels were not affected. In hIGF-II transgenic mice, IGF-I mRNA levels were significantly increased, while endogenous IGF-II and IGFBP-2 mRNAs were unaffected, compared to normal animals. DXM treatment of the hIGF-II transgenic mice induced a further increase of IGF-I mRNA expression, to a similar extent as in DXM-treated normal mice. The increase of IGF-I due to DXM treatment in normal mice might be a reaction in order to minimize the GC-induced growth retardation. Another possibility could be that the increase of IGF-I would contribute to the GC-induced growth retardation by accelerating the differentiation of chondrocytes, resulting in accelerated ossification. In the growth plates of hIGF-II transgenic mice, the higher basal level of IGF-I, might be responsible for the observed partial protection against the adverse effects of GCs on bone.

Dexamethasone-induced growth inhibition of porcine growth plate chondrocytes is accompanied by changes in levels of IGF axis components.

High (pharmacological) doses of glucocorticoids inhibit the proliferation of growth plate chondrocytes, which leads to one of the side-effects of these steroids, namely suppression of longitudinal growth. Growth inhibition by glucocorticoids is thought to be mediated in part by impaired action of components of the IGF axis, which are important for chondrocyte regulation and hence for longitudinal growth. The aim of the present study was to determine whether glucocorticoid-induced growth retardation involves changes in IGF axis components. Chondrocytes were isolated from epiphyseal growth plates of neonatal piglets and treated with pharmacological doses of dexamethasone (DXM) for 24 h to study glucocorticoid-induced growth retardation. Under IGF-I-supplemented (10 nM) culture conditions, IGF-binding proteins (IGFBPs)-2, -4 and -5 were secreted by the growth plate chondrocytes and IGFBP-2 protein and mRNA levels were decreased by the DXM treatment, whereas IGFBP-4 and -5 were not affected. Proliferation of the chondrocytes, as measured by [(3)H]thymidine incorporation, was 3.5-fold higher in serum-supplemented medium in contrast to IGF-I-supplemented (10 nM) medium. In the presence of serum, DNA synthesis was significantly inhibited by 50-63% when treated with 100 nM DXM, which was prevented by the glucocorticoid-receptor antagonist Org34116. mRNA levels of IGF axis components were determined using Northern blot analysis. IGFBP-2 to -6 were expressed in the chondrocytes, IGFBP-1 was absent and both IGF-I and IGF-II, and the type I and type II IGF receptors were expressed. Treatment with DXM (100 nM) resulted in a 2-fold increase in mRNA levels of both IGFBP-5 and the type I IGF receptor, whereas IGFBP-2 mRNA levels decreased by 55%, in concert with the decrease in protein level observed under IGF-I-supplemented culture conditions. The changes in mRNA levels due to the DXM treatment were prevented by the glucocorticoid receptor antagonist. Our data show that exposure to pharmacological doses of DXM results in inhibition of proliferation and changes in components of the IGF axis, IGFBP-2 and -5 and the type I IGF receptor, suggesting a role for these components in glucocorticoid-induced growth retardation at the local level of the growth plate.

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.

The role of the IGF axis in IGFBP-1 and IGF-I induced renal enlargement in Snell dwarf mice.

Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is generally believed to inhibit IGF action in the circulation. In contrast, IGFBP-1 has been reported to interact with cell surfaces and enhance IGF-I action locally in some tissues. Renal IGFBP-1 levels are found elevated in various conditions characterized by renal growth (e.g. diabetes mellitus, hypokalemia). To test whether IGFBP-1 is a renotropic factor, IGFBP-1 was administered alone or in combination with IGF-I to Snell dwarf mice, an in vivo model without compensatory feedback effects on growth hormone (GH) secretion. In three control groups of Snell dwarf mice, placebo, GH or IGF-I was administered. Compared with placebo, kidney weight increased in all treated groups, however, with different effects on kidney morphology. Administration of IGF-I, alone or in combination with IGFBP-1, tended to increase glomerular volume, while no changes were seen in the other groups. Administration of IGFBP-1 or IGFBP-1+IGF-I both caused dilatation of the thin limbs of Henle's loop, while GH or IGF-I administration had no visible effect. Furthermore, IGF-I administration resulted in an increased mean number of nuclei per cortical area and renal weight, whereas GH, IGF-I+IGFBP-1 or IGFBP-1 caused a decreased renal nuclei number. In situ hybridization and immunohistochemistry showed specific changes of the renal IGF system expression patterns in the different groups. Particularly, IGFBP-1 administration resulted in extensive changes in the mRNA expression of the renal IGF system, whereas the other administration regimen resulted in less prominent modifications. In contrast, administration of IGFBP-1 and IGFBP-1+IGF-I resulted in identical changes in the protein expression of the renal IGF system. Our results indicate that IGFBP-1, alone or in combination with IGF-I, demonstrated effects on the renal tubular system that differ from the effects of IGF-I.

Plasma levels of insulin-like growth factor binding protein-4 (IGFBP-4) under normal and pathological conditions.

OBJECTIVE: Insulin-like growth factor binding protein-4 (IGFBP-4) belongs to a family of six structurally related IGF-binding proteins that are involved in the modulation of the biological effects of the IGFs. In order to obtain more insight into the clinical significance and regulation of IGFBP-4 in vivo we determined the levels of this protein by a specific radioimmunoassay in the human circulation under normal and various pathological conditions. DESIGN AND PATIENTS: Selected human biological fluids and plasma samples from 804 normal healthy males and females, ranging from 0 to 78 years of age, were analysed. In addition, plasma samples from patients with several disorders (i.e. hypothyroidism, hyperthyroidism, GH-deficiency, acromegaly, cancer, chronic renal failure corticosteroid-treatment) were investigated. MEASUREMENTS: A specific RIA for hIGFBP-4 was developed, using a rabbit polyclonal antibody raised against a synthetic peptide containing amino acids 80-103 of the mature hIGFBP-4 sequence. RESULTS: In normal individuals circulating IGFBP-4 levels in males did not change with age. For females the values tended to increase slightly in older age. Overall, the mean +/- SD for males and females (189 +/- 83 microg/l and 193 +/- 72 microg/l, respectively) were not different. Normative range values of IGFBP-4 correlated weakly with those of IGF-II (r = 0.31, P < 0.001). Neither hypothyroidism nor hyperthyroidism appeared to influence circulating IGFBP-4 levels since the levels were within the normal range. Both GH status and pharmacological doses of glucocorticoids, as employed in various chronic diseases, did not seriously affect plasma IGFBP-4 either. Under conditions with increased circulating PTH levels, i.e. dialysed adult patients with chronic renal failure (CRF) and subjects with hyperparathyroidism, a weak positive relationship was noted between the plasma contents of IGFBP-4 and PTH. An excess of IGFBP-4 was found in plasma of both nondialysed and dialysed prepubertal growth retarded children with chronic renal failure (CRF) (mean SDS: 10.75 and 5.78, respectively). IGFBP-4 levels were inversely related to glomerular filtration rate. Similar results were obtained for dialysed adults with CRF. In a group of CRF children who had undergone renal transplantation, circulating IGFBP-4 levels were markedly lower (mean SDS: 3.75). There was no evidence for an increased secretion of IGFBP-4 in the circulation of most of the cancer patients with solid tumours. Several children with acute lymphoblastic leukaemia, however, showed elevated plasma IGFBP-4 levels (mean SDS: 1.27). The presence of IGFBP-4 could also be demonstrated in other human biological fluids. The highest amounts were found in amniotic fluid (391-717 microg/l) and follicular fluid (249-500 microg/l). CONCLUSIONS: Measurement of plasma IGFBP-4 has been shown so far to be of minor clinical relevance. However, the results indicate that different concentration gradients between plasma and various other body fluids may exist. Therefore, it may well be that certain pathophysiological stimuli induce significant alterations in the local turnover rate of IGFBP-4 but that they are not reflected by changes in the circulating levels. The possibility of quantifying IGFBP-4 by RIA will facilitate further in vitro and in vivo studies on its regulation and function in humans.

Differential regulation of IGF-binding proteins in rabbit costal chondrocytes by IGF-I and dexamethasone.

Cartilage is a primary target tissue for the IGFs. The mitogenic activity of these peptides is regulated by a family of high-affinity IGF-binding proteins (IGFBP-1 to -6). We characterized the IGFBPs produced by cultured chondrocytes derived from rib cartilage of prepubertal rabbits. Culture medium, which had been conditioned by these cells for 48 h showed bands of 22 kDa, 24 kDa and a 31/32 kDa doublet by Western ligand blotting with [(125)I]IGF-II. When the cells were grown in the presence of increasing amounts of IGF-I or IGF-II, the 31/32 kDa doublet increased in intensity (reaching a plateau of about 11-fold stimulation between 2 and 10 nM IGF-I). The 22 kDa and 24 kDa bands increased only slightly while a 26 kDa band became faintly visible. By Western immunoblotting the 31/32 kDa doublet was identified as IGFBP-5. An IGF-I analog with reduced affinity for IGFBPs, Long-R3 IGF-I, also induced IGFBP-5, while insulin was less effective (2.2-fold stimulation at 10 nM). IGF-I protected IGFBP-5 against proteolytic degradation by conditioned medium. IGF-I also enhanced the level of IGFBP-5 mRNA. LY294002, a specific inhibitor of the intracellular signaling molecule phosphatidylinositol 3-kinase, inhibited stimulation of IGFBP-5 by IGF-I. Dexamethasone suppressed IGFBP-5 (by 70% at 20 nM) but, at the same time, a 39/41 kDa doublet (presumably IGFBP-3) was induced. IGFBP-5 has been shown in several cell types to enhance the mitogenic activity of IGF-I. IGFBP-3 generally acts as a growth inhibitor. Therefore, the differential effects of dexamethasone on these regulatory proteins could account, at least in part, for the growth-arresting effect of this glucocorticoid.

Human insulin-like growth factor (IGF) binding protein-1 inhibits IGF-I-stimulated body growth but stimulates growth of the kidney in snell dwarf mice.

The actions of insulin-like growth factor-I (IGF-I) are modulated by IGF binding proteins (IGFBPs). The effects of IGFBP-1 in vivo are insufficiently known, with respect to inhibitory or stimulatory actions on IGF-induced growth of specific organs. Therefore, we studied the effects of IGFBP-1 on IGF-I-induced somatic and organ growth in pituitary-deficient Snell dwarf mice. Human GH, IGF-I, IGFBP-1, and a preequilibrated combination of equimolar amounts of IGF-I and IGFBP-1 were administered sc during 4 weeks. Treatment with IGF-I alone induced a significant increase in body length (108% of control) and weight (112%) as well as an increase in weight of the submandibular salivary glands (135%), kidneys (124%), femoral muscles (111%), testes (129%), and spleen (126%) compared with saline-treated controls. IGFBP-1 alone induced a significant increase in weight of the kidneys (152% of control). Coadministration of IGF-I with IGFBP-1 neutralized the stimulating effects of IGF-I on body length and weight as well as on the femoral muscles and testes. In contrast, the weights of the submandibular salivary glands (143%) were not significantly different from those of IGF-I-treated animals, whereas the weights of the kidneys (171%) and spleen (156%) were significantly increased compared with IGF-I-treated mice. The effect of IGFBP-1 plus IGF-I on kidney weight was not significantly greater than the effect of IGFBP-1 alone. Western ligand blotting showed induction of the IGFBP-3 doublet as well as IGFBPs with molecular masses of 24 kDa, most probably IGFBP-4, by human GH, IGF-I alone, and IGF-I in combination with IGFBP-1. Our data show that coadministration of IGFBP-1 inhibits IGF-I-induced body growth of GH-deficient mice but significantly stimulates the growth promoting effects of IGF-I on the kidneys and the spleen. These data warrant further investigation because differences in concentrations of IGFBP-1 occurring in vivo may influence IGF-I-induced anabolic processes.

The effect of dexamethasone on body and organ growth of normal and IGF-II-transgenic mice.

The physiological role of IGF-II remains unclear but there is evidence for a role in postnatal growth, the growth of the thymus and bone homeostasis. Glucocorticoids have many effects that are opposite to the effects of IGF-II such as growth retardation, osteoporosis and thymic involution. We therefore wondered whether IGF-II overexpression in transgenic mice might counteract some of the growth inhibitory effects of the glucocorticoid, dexamethasone (DXM). In a dose-finding study in normal mice, 20 microg DXM/day caused a significant growth delay. The various organs had a different susceptibility to the growth inhibitory effects of DXM. Most affected were thymus and spleen, followed by liver, skeletal muscle and lumbar vertebrae. The weights of the kidney, tibia, and humerus were not significantly diminished. In a second experiment, the effects of DXM in normal and IGF-II-transgenic animals were compared. The IGF-II serum levels in the transgenic animals were more than 40-fold increased compared with control mice and were decreased by 35% in the DXM-treated group. IGF-I serum levels were identical in both mouse strains and rose slightly after DXM administration in controls. Transgenic mice had higher levels of IGF binding protein species of apparent molecular masses of 41.5 kDa, 30 kDa, and 26.5 kDa. DXM reduced the 24 kDa band in both mice strains. In addition it reduced the bands at 38.5 kDa and 26.5 kDa but only in the transgenic animals. The effect of DXM on body growth was similar in normal and IGF-II-transgenic mice. The weight reduction of the various organs caused by DXM was similar in both types of mice except for the skeleton. The weight of the tibia and the humerus were significantly higher in the DXM-treated transgenic mice. In conclusion, we speculate that overexpression of IGF-II in mice partially protects bone from the osteopenic effects of glucocorticoids.

Insulin-like growth factor (IGF) II induced changes in expression of IGF binding proteins in lymphoid tissues of hIGF-II transgenic mice.

Overexpression of human insulin-like growth factor II (IGF-II) in transgenic mice does not result in increased overall body growth. The IGF-II overexpression, however, specifically causes growth of the thymus and not of the spleen. We address the question whether the observed differences in growth induction in lymphoid tissues by IGF-II can be related to differences in local IGF binding protein (IGFBP) production, using nonradioactive in situ hybridization and Northern blot analysis. IGFBP-2, -4, and -5 are expressed in both lymphoid tissues of normal mice. The spleen additionally expresses IGFBP-3 and IGFBP-6. IGFBP-1 expression was not detected. Although the expression pattern of the IGFBPs did not change upon IGF-II overexpression, the level of expression changed in a specific manner for each IGFBP. In both the thymus and the spleen of transgenic mice, IGFBP-2 and -5 gene expression was slightly increased, whereas the level of IGFBP-4 expression was not altered. In the spleen, IGFBP-6 expression was not altered by IGF-II overexpression, whereas IGFBP-3 expression was strongly increased. The differences in IGFBP expression, and the difference in response of these IGFBPs to IGF-II overexpression in thymus and spleen suggests an important role of these proteins in growth regulation of both lymphoid tissues. We speculate that an increase of IGFBP-3 expression together with changes in expression of other IGFBPs, inhibits IGF-II stimulated growth in the spleen by an autocrine-/paracrine pathway.

Circulating levels of human insulin-like growth factor binding protein-6 (IGFBP-6) in health and disease as determined by radioimmunoassay.

OBJECTIVE: Insulin-like growth factor binding protein-6 (IGFBP-6) is a relatively unknown member of a family of six specific structurally related IGF binding proteins which are involved in the modulation of the biological effects of the IGFs. A distinctive property of IGFBP-6 is its preferential affinity for IGF-II relative to IGF-I. In order to obtain more insight into the clinical significance and regulation of circulating levels of IGFBP-6 we developed a specific radioimmunoassay (RIA) for this protein. DESIGN AND PATIENTS: Selected human biological fluids and plasma from 847 normal subjects were analysed. In addition, plasma samples from patients with different disorders (i.e. GH-deficiency, acromegaly, cancer, corticosteroid-treated children suffering from different kinds of severe illness and chronic renal failure) were investigated. MEASUREMENTS: The IGFBP-6 assay is competitive, utilizing a rabbit polyclonal antibody raised against a synthetic peptide comprising amino acids 90-118 of the hIGFBP-6 sequence and an additional tyrosine residue. It is calibrated against recombinant human (rh)IGFBP-6. The 125I tracer is prepared by iodination of the synthetic peptide. There is no significant cross-reactivity with other IGFBPs and no interference with the IGFs. RESULTS: Extensive normative range values for IGFBP-6 were determined using 847 plasma samples from normal males and females, ranging from 0 to 75 years of age. IGFBP-6 levels increased gradually (about two-fold) with age. In childhood the plasma levels of IGFBP-6 in females tended to be slightly higher than those for males. For the adult population the reverse was observed. Overall, the mean +/- SD value for males was higher than that for females (149 +/- 57 vs. 139 +/- 45 micrograms/l, P < 0.004). GH status did not appear to influence IGFBP-6 level since normal levels were found for both untreated acromegalic patients and GH-deficient subjects. GH treatment of the latter group of patients did not alter IGFBP-6 in plasma. Pharmacological doses of glucocorticosteroids affected circulating IGFBP-6 levels only slightly. IGFBP-6 levels in plasma samples derived both from children with acute lymphoblastic leukaemia and from patients with various types of solid neoplasms were generally within the normal range. In contrast, plasma samples from four of six patients with non-islet cell tumour induced hypoglycaemia (NICTH) showed elevated concentrations of IGFBP-6 (SDS > 2.9). An excess of IGFBP-6 was also found in plasma of both dialysed and non-dialysed prepubertal growth retarded children with chronic renal failure (CRF) (mean SDS: 23.0 and 9.3, respectively). IGFBP-6 levels were inversely correlated with glomerular filtration rate. In a group of CRF patients who underwent renal transplantation circulating IGFBP-6 levels were markedly lower (mean SDS: 4.6). The presence of IGFBP-6 could also be demonstrated in several other human biological fluids. Low amounts were detected in saliva (3-12 micrograms/l) and breast milk (6-45 micrograms/l) while the levels in amniotic fluid and follicular fluid were comparable with those determined in normal plasma. The IGFBP-6 content of cerebrospinal fluid (CSF) ranged between 25 and 87 micrograms/l, which is rather high in relation to the relatively low concentration of total protein in this body fluid. CONCLUSIONS: Measurements of IGFBP-6 have been shown so far to be of relatively minor clinical relevance. The exceptions are chronic renal failure patients and subjects with large tumours and non-islet cell tumour induced hypoglycaemia who may exhibit elevated circulating levels of this IGFBP. The physiological significance of this observation remains to be elucidated. The possibility of quantifying IGFBP-6 by specific RIA will facilitate further in vitro and in vivo studies of its regulation and function in man.

The role of growth hormone and insulin-like growth factors in the immune system.

Growth hormone (GH) and insulin-like growth factor I (IGF-I) can modulate the development and function of the immune system. In this chapter, we present data on the expression of receptors for GH and IGFs and the in vitro and in vivo effects of these proteins. We show that expression of GH and IGFs in the immune system opens up the possibility that these proteins are not only involved in endocrine control of the immune system but can also play a role as local growth and differentiation factors (cytokines). Endocrine control of GH could be direct or mediated via endocrine or autocrine/paracrine IGF-I. In addition, GH can act as an autocrine or paracrine factor itself. Furthermore, IGF-I in the immune system has been shown to be regulated by cytokines, such as interleukin-1 and interferon-gamma, alluding to a cytokine-like function of IGF-I. In addition to data on the function of GH and IGF-I in the immune system, we present new findings which imply a possible function of IGF-II and IGF-binding proteins.

Insulin-like growth factor-binding protein-3 protease activity in Snell normal and Pit-1 deficient dwarf mice.

Partial proteolysis of insulin-like growth factor-binding protein-3 (IGFBP-3) lowers its affinity for IGFs. Presumably, this leads to destabilization of the ternary IGF-IGFBP-3-acid-labile subunit complex in the circulation and an increased bioavailability of IGFs. We investigated the effect of GH on IGFBP-3 proteolysis by comparing serum from normal mice and GH-deficient dwarf mice. While normal mouse serum degraded 125I-IGFBP-3, this activity declined with age. In contrast, serum from dwarf mice displayed strong proteolytic activity at all ages tested (up to 10 weeks). In dwarf mice of 4 weeks and older, this activity could not be inhibited by EDTA and 1,10-phenanthroline, indicating the presence of a divalent cation-independent protease. Prolonged treatment with GH (4 weeks) did not decrease the overall potency of the serum to degrade IGFBP-3, but partially restored the ability of EDTA to inhibit IGFBP-3 protease activity. GH deficiency therefore appears to induce a new kind of IGFBP-3 protease. Similarly, serum from hypophysectomized rats displayed enhanced IGFBP-3 protease activity compared with control rat serum. These results suggest that a protease induced under conditions of severe GH deficiency may contribute to making IGFs optimally available to the tissues.