Abnormal thymic microenvironment in insulin-like growth factor-II transgenic mice.

OBJECTIVES: Intrathymic T cell differentiation is driven by the thymic microenvironment, a tridimensional network of cells and extracellular matrix (ECM). Previous data showed that lymphoid and microenvironmental compartments are under the control of hormones and growth factors. We then attempted to define if insulin-like growth factor-II (IGF-II) was also involved in such a control. METHODS: We used IGF-II transgenic (Tg) mice and studied their thymic microenvironment by immunohistochemistry. Moreover, we evaluated thymocytes in terms of their ability to adhere to thymic epithelial cells and to migrate through epithelial cells and ECM. RESULTS: Transgenic IGF-II expression results in abnormalities of the thymic epithelium. Terminal differentiation of thymic epithelial cells (TEC) is modified, with the appearance of large clusters of cells immunoreactive to the monoclonal antibody KL1, which specifically recognizes highly differentiated TEC. Accordingly, treatment of cultured TEC with exogenous IGF-II induces the appearance of KL1+ cells and increases TEC proliferation. IGF-II Tg animals exhibit increased serum levels of the TEC-derived hormone thymulin. These effects were seen even when the IGF-II transgene was inserted in dwarf mice. Moreover, deposition of fibronectin and laminin is also enhanced in IGF-II Tg mouse thymus and in IGF-II-treated TEC cultures. Furthermore, ECM-mediated interactions between thymocytes and TEC are affected by exogenous IGF-II, as exemplified by the enhancement of thymocyte adhesion to TEC monolayers and thymocyte migration in thymic nurse cell complexes. CONCLUSIONS: Our data indicate that IGF-II pleiotropically affects the thymic epithelium, both in vivo and in vitro, and that some of these changes may have consequences on thymocyte/TEC interactions.

Thyroid hormone, but not parathyroid hormone, partially restores glucocorticoid-induced growth retardation.

Growth retardation is a serious side effect of long-term glucocorticoid (GC) treatment. In order to prevent or diminish this deleterious effect, a combination therapy including growth hormone (GH), a stimulator of bone growth, is often recommended. Parathyroid hormone (PTH) and thyroid hormone (T(4)) are important hormonal regulators of bone growth, and might also be helpful anabolic agents for counteracting the negative effects of GCs. Therefore, we studied the interaction of GCs in combination with a single dose of either PTH or T(4) on GC-induced growth retardation. Dexamethasone (Dex) treatment of mice for four weeks induced a significant growth inhibition of body length and weight and weights of several organs. PTH or T(4) alone did not affect the normal growth pattern. However, T(4) could partially restore the Dex-induced growth inhibition, whereas PTH could not. Although PTH did not affect total body growth, it did affect the height of the proliferative zone, which could be counteracted by Dex. This contrasts with T(4) treatment alone or in combination with Dex, which both resulted in a disturbed morphology of the growth plate. IGF-I mRNA, one of the mediators of longitudinal bone growth, was present in proliferative and hypertrophic chondrocytes. However, its expression was not affected by any of the treatments. In conclusion, T(4) but not PTH can partially counteract the effects of Dex on general body growth, with possible implications for future treatments of GC-induced growth retardation. Additionally, both T(4) and PTH, alone or in combination with Dex, have differential effects on the morphology of the growth plate.

An in vitro bioassay to determine individual sensitivity to glucocorticoids: induction of FKBP51 mRNA in peripheral blood mononuclear cells.

Individual variation in sensitivity to glucocorticoids (GCs) poses a dilemma to the clinician. Currently available assays to determine individual sensitivity to GCs either seem imprecise, or they are based on mitogen-activated lymphocytes, although mitogens themselves may affect cellular GC sensitivity. To avoid these disadvantages, we developed an assay based on the GC-induced accumulation of the 51kDa FK506 binding protein (FKBP51) mRNA in unstimulated peripheral blood mononuclear cells (PBMC), measured using real time PCR. Of several family members tested, only FKBP51 transcript levels showed to be GC-inducible. Furthermore, our bioassay was not affected by progesterone, estradiol, and testosterone. Immunological stimulation of PBMC using tetanus toxoid did not affect bioassay results, and isolated T- and B-lymphocytes showed a similar response to GC stimulation. The intra- and inter-assay variations were 10.6 and 15.9%, respectively. Our bioassay confirms previous reports that a wide variation in GC sensitivity exists in the normal population, yet is able to clearly discriminate a patient with familial GC hyposensitivity from controls. Our bioassay may be suitable to assess altered individual GC sensitivity, and the small amount of PBMC needed for a determination makes this assay easily applicable in a pediatric setting.

Effects of allopurinol and deferoxamine on reperfusion injury of the brain in newborn piglets after neonatal hypoxia-ischemia.

The hypothesis was tested that treatment with allopurinol, a xanthine oxidase inhibitor, or deferoxamine, a chelator of nonprotein-bound iron, preserved cerebral energy metabolism, attenuated development of edema, and improved histologic outcome in the newborn piglet at 24 h after hypoxia-ischemia. Thirty-two newborn piglets were subjected to 1 h of hypoxia-ischemia by occluding both carotid arteries and reducing the fraction of inspired oxygen; five newborn piglets served as sham-operated controls. The depth of hypoxia-ischemia was controlled by phosphorous magnetic resonance spectroscopy. Upon reperfusion and reoxygenation, piglets received vehicle (n= 12), allopurinol (30 mg/kg/d, n = 10), or deferoxamine (12.5 mg/kg/d, n = 10). The cerebral energy status was determined with phosphorous magnetic resonance spectroscopy. The presence of vasogenic edema was assessed by T2-weighted magnetic resonance imaging. Brain cell injury was assessed with caspase-3 activity, histology, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end (TUNEL)-labeling. At 24 h after hypoxia-ischemia, the phosphocreatine/inorganic phosphate ratios were significantly decreased in vehicle-treated, but not in allopurinol- or deferoxamine-treated piglets. Water T2 values were significantly increased at 24 h after hypoxia-ischemia in cerebral cortex, thalamus, and striatum of vehicle-treated piglets, but not in allopurinol- and deferoxamine-treated piglets. No differences in caspase-3 activity, histologic outcome, or TUNEL-labeling were demonstrated between the three treatment groups. We suggest that allopurinol and deferoxamine may have an additional value in the treatment of perinatal hypoxia-ischemia with other neuroprotective agents or in combination with hypothermia.

Dexamethasone differentially inhibits thyroxine- or growth hormone-induced body and organ growth of Snell dwarf mice.

Supraphysiological doses of glucocorticoids cause growth retardation in both animals and humans. Many studies have addressed the interaction of glucocorticoids with the GH/IGF system, but little is known about the effect of glucocorticoids on T(4)-stimulated growth. The Snell dwarf mouse is deficient in GH, thyroid-stimulating hormone, and prolactin and therefore allows the study of the effect of glucocorticoids on the growth induced by GH and T(4) without their mutual interaction. Four weeks of treatment with T(4) (1 micro g/d) or human GH (50 mU/d) equally increased nose-tail length (3.1 +/- 0.1 cm and 3.0 +/- 0.2 cm, respectively). Dexamethasone (DXM) had much less impact on T(4)-stimulated growth than on GH-induced growth (T(4) + DXM: 2.4 +/- 0.1 cm vs. GH+ DXM: 1.4 +/- 0.1 cm). Similar data were obtained for body weight gain. T4 and GH had a different effect on the weight of various organs: GH caused a higher increase in liver and lumbar vertebrae weight, and T(4) was a better stimulator for kidney (P < 0.05), thymus, and spleen growth. Remarkably, T(4)-stimulated growth of the organs was less affected by DXM than GH-induced organ growth. GH even potentiated the growth inhibition by DXM in the thymus and tibia. In conclusion, T(4)-stimulated growth in Snell dwarf mice is less affected by DXM than growth stimulated by GH

Glucocorticoid-induced increase in lymphocytic FKBP51 messenger ribonucleic acid expression: a potential marker for glucocorticoid sensitivity, potency, and bioavailability.

To reduce the side effects of corticosteroid treatment, the administered dose of glucocorticoids (GCs) should be kept to a minimum while preserving therapeutically needed intracellular levels. Currently available assays to determine individual sensitivity to GCs are either imprecise or based on inhibition by GCs of lymphocyte proliferation following stimulation with phytohemagglutinin or other mitogens, which may influence the GC signal transduction pathway. Using the human lymphoblastoid cell line IM-9 as a model system, we studied whether the GC-induced increase of the mRNA encoding the 51-kDa FK506-binding protein (FKBP51) could be used for the development of a novel assay, ultimately to be used in native human peripheral blood mononuclear cells (PBMCs). GC addition to IM-9 cells resulted in a dose-dependent increase of FKBP51 mRNA levels within 2 h, followed by a further increase until 24 h. Northern blot analysis and real-time PCR yielded similar results. Coincubation of GCs with the GC receptor antagonist ORG 34116 or the protein synthesis inhibitor cycloheximide suggested a direct, GC receptor-mediated up-regulation of FKBP51 gene transcription. Expected differences in potency among different GCs could be readily demonstrated in this system. Extending our observations in IM-9 lymphoblasts to normal PBMCs, we found a dose-dependent increase of FKBP51 mRNA on ex vivo incubation of native human PBMCs with GCs, with a sensitivity of about 10(-9) M for dexamethasone. Moreover, dexamethasone ingestion increased FKBP51 mRNA in PBMCs in vivo, extending the use of this assay to the measurement of GC bioavailability. Finally, using this method we were able to demonstrate partial GC-insensitivity in a 6-month-old infant suffering from congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. We conclude that the induction of FKBP51 mRNA by GCs may be a suitable marker to assess individual GC sensitivity, the in vitro measurement of GC potency, and the in vivo determination of GC bioavailability.

Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes.

Vascular endothelial growth factor (VEGF) plays an essential role in angiogenesis in the growth plate and ultimately in regulating endochondral ossification. Since longitudinal bone growth is often disturbed in children who are treated with glucocorticoids, we investigated the effects of dexamethasone on VEGF expression by epiphyseal chondrocytes. Cells were cultured from tibial growth plates of neonatal piglets. Using Northern blotting and RT-PCR techniques, the chondrocyte-specific markers aggrecan, collagen II and CD-RAP were detected. Also the glucocorticoid receptor (GR) was expressed. VEGF protein secreted from these cells was examined by ELISA and Western immunoblotting. The VEGF(121) and VEGF(165) isoforms were detected in the supernatant. As determined by RT-PCR, all three major mRNA splice variants were produced, including the species encoding VEGF(189). Dexamethasone (100 nM) inhibited both protein and mRNA expression by approximately 45%. Hydrocortisone (cortisol) and prednisolone also inhibited VEGF secretion, but they were less active than dexamethasone. The inhibitory actions of dexamethasone were almost completely blocked by the GR antagonist Org34116, indicating that the GR mediates these actions. Degradation of the VEGF mRNA was not accelerated by dexamethasone. Therefore, a transcriptional mechanism seems likely. Downregulation of this important growth factor could lead to disruption of the normal invasion of blood vessels in the growth plate, which could contribute to disturbed endochondral ossification and growth.

Neuroprotection by selective nitric oxide synthase inhibition at 24 hours after perinatal hypoxia-ischemia.

BACKGROUND AND PURPOSE: Perinatal hypoxia-ischemia is a major cause of neonatal morbidity and mortality. Until now no established neuroprotective intervention after perinatal hypoxia-ischemia has been available. The delay in cell death after perinatal hypoxia-ischemia creates possibilities for therapeutic intervention after the initial insult. Excessive nitric oxide and reactive oxygen species generated on hypoxia-ischemia and reperfusion play a key role in the neurotoxic cascade. The present study examines the neuroprotective properties of neuronal and inducible but not endothelial nitric oxide synthase inhibition by 2-iminobiotin in a piglet model of perinatal hypoxia-ischemia. METHODS: Twenty-three newborn piglets were subjected to 60 minutes of hypoxia-ischemia, followed by 24 hours of reperfusion and reoxygenation. Five additional piglets served as sham-operated controls. On reperfusion, piglets were randomly treated with either vehicle (n=12) or 2-iminobiotin (n=11). At 24 hours after hypoxia-ischemia, the cerebral energy state, presence of vasogenic edema, amount of apparently normal neuronal cells, caspase-3 activity, amount of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)-positive cells, and degree of tyrosine nitration were assessed. RESULTS: A 90% improvement in cerebral energy state, 90% reduction in vasogenic edema, and 60% to 80% reduction in apoptosis-related neuronal cell death were demonstrated in 2-iminobiotin-treated piglets at 24 hours after hypoxia- ischemia. A significant reduction in tyrosine nitration in the cerebral cortex was observed in 2-iminobiotin-treated piglets, indicating decreased formation of reactive nitrogen species. CONCLUSIONS: Simultaneous and selective inhibition of neuronal and inducible nitric oxide synthase by 2-iminobiotin is a promising strategy for neuroprotection after perinatal hypoxia-ischemia.

Antibodies directed against the E region of pro-insulin-like growth factor-II used to evaluate non-islet cell tumor-induced hypoglycemia.

BACKGROUND: Detection of incompletely processed precursor forms of insulin-like growth factor-II ("big" IGF-II) in plasma is essential for both the diagnosis and follow-up of non-islet cell tumor-induced hypoglycemia (NICTH) and may be relevant to other diseases as well. RIA using an antibody raised against a synthetic peptide consisting of the first 21 amino acids of the E domain [E(68-88)] of human pro-IGF-II cannot distinguish between E-peptide-containing big IGF-II and cleaved E domain or fragments. We therefore developed and validated an ELISA that specifically detects big IGF-II in plasma. METHODS: The ELISA used a solid-phase antibody to E(68-88) and a liquid-phase monoclonal hIGF-II antibody. Pro-IGF-II purified from normal human plasma was used as a calibrator. Acid Sep-Pak C(18) extracts of plasma from NICTH patients were analyzed, and the results were compared with those obtained for plasma samples from healthy individuals. In addition, blood specimens derived from dialyzed patients with chronic renal failure, which contained relatively high concentrations of cleaved E domain or fragments, were studied. The results were validated by acid Sephadex G-50 gel filtration. RESULTS: Results from this ELISA indicated that the concentration of big IGF-II in NICTH plasma was higher (mean +/- SD, 22.6 +/- 9.4 nmol/L) than in normal plasma (3.8 nmol/L). Conversely, the concentrations in pooled CRF plasma (2.0 +/- 0.8 nmol/L) were low. Antibodies directed against either E(68-88) or E(13-134) of pro-IGF-II could be used to detect these peptides in tumor tissue by immunohistochemistry. CONCLUSIONS: The possibility of quantifying pro-IGF-II by ELISA in plasma represents a potentially useful tool for the diagnosis and follow-up of NICTH and should facilitate further in vitro and in vivo studies on its regulation and function in humans.

Effects of selective nitric oxide synthase inhibition on IGF-1, caspases and cytokines in a newborn piglet model of perinatal hypoxia-ischaemia.

Selective inhibition of neuronal and inducible nitric oxide synthase (NOS) with 2-iminobiotin previously showed a reduction in brain cell injury. In the present study, we investigated the effects of 2-iminobiotin treatment on insulin-like growth factor-1 (IGF-1) expression, caspase activity and cytokine expression in a newborn piglet model of perinatal hypoxia-ischaemia. Newborn piglets were subjected to 1 h of hypoxia-ischaemia and were treated intravenously with vehicle or 2-iminobiotin. Vehicle-treated piglets showed reduced IGF-1 mRNA expression and increased caspase-3 activity and DNA fragmentation. 2-Iminobiotin treatment, administered immediately upon reperfusion, prevented these observations. No differences in caspase-8 and -9 activity and cytokine [interleukin (IL)-1alpha/beta, IL-6, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta] mRNA expression were demonstrated between vehicle- and 2-iminobiotin-treated piglets at 24 h following hypoxia-ischaemia. IGF-1 mRNA correlated inversely with caspase-3 and transferase-mediated dUTP-biotin in situ nick end labelling score in the cortex, but positively with caspase-8. Cytokine mRNA did not correlate with IGF-1 mRNA, caspase-3 activity or DNA fragmentation. The present results indicate that the previously demonstrated neuroprotective effect of 2-iminobiotin treatment after perinatal hypoxia-ischaemia coincided with a preservation of the endogenous IGF-1 production and reduced caspase-3 activity, but not with a significant decrease in cytokine production.