Effects of human growth hormone administration on growth rate and growth-stimulating activity of serum, measured by lymphocyte bioassay in hypopituitary children

The acute effect of human growth hormone (hGH) upon the serum bioassayable growth-stimulating activity was compared to the long-term effects of hGH on growth rate in two groups of hypopituitary patients aged 2-18 years. 12 patients had complete GH deficiency with GH peak below 3.5 ng/ml at two stimulation tests. 15 others, having both GH peaks below 8 ng/ml and at least one above 3.5 ng/ml, were considered as having partial GH deficiency. The growth-stimulating activity of serum was measured by its effect at concentrations 0.03 to 1.25% upon thymidine incorporation into lectin-activated normal human lymphocytes, named thymidine activity (TA). In patients with complete GH deficiency, the pre-treatment TA was positively correlated with the peak response of GH to stimulation tests. The increase of TA after 3-4 days of hGH treatment was positively correlated with the pretreatment TA level, and negatively correlated with the peak GH level. The effect of a 6-12 months therapeutic course of hGH upon the growth rate was positively correlated with the acute increase of TA. No such correlations were found in patients with partial GH deficiency. Many works have discussed the relationship of acute somatomedin responses and long-term clinical results of treatment with hGH in GH-deficient children. The present data, using a highly sensitive bioassay of serum stimulating activity, suggest that the degree of GH deficiency is an important factor to be considered. The response of GH-dependent serum growth factors to acute treatment with hGH could have more predictive value in cases with total lack of growth hormone than in cases of partial deficiency.     

Effects of human growth hormone on immune functions: in vitro studies on cells of normal and growth hormone-deficient children

We have studied the in vitro effects of human growth hormone on cell surface markers and mitogenic responses of peripheral blood lymphocytes (PBL) of normal and growth hormone-deficient children before, during and after treatment with growth hormone. Growth hormone resulted in a decrease in B cell expression but it did not affect expression of T cell subsets. Growth hormone depressed the proliferation of PBL of normal and untreated growth hormone-deficient children. The proliferative responses to phytohemagglutinin (PHA) versus PHA with growth hormone were not statistically different, though the responses of most normal and on treatment children were diminished by the addition of growth hormone. PBL derived from growth hormone-deficient children during treatment with human growth hormone exhibited significantly greater spontaneous proliferation then the PBL of normal children. Growth hormone further significantly enhanced their proliferation. PHA and PHA with growth hormone resulted in significantly greater proliferation of these patients' PBL when compared to those of normal children. We demonstrated that human growth hormone had substantial in vitro effects on immune functions. These effects, some of which depend on the treatment status of the children, may need to be considered in the clinical use of human growth hormone.     

Spontaneous prolactin secretion in growth hormone-deficient children.

OBJECTIVE: To establish the spontaneous nocturnal prolactin (PRL) release in relation to growth hormone (GH)-deficient children and idiopathic short-stature children (ISS). METHODS: A total of 32 prepubertal children (11 girls, 21 boys) aged between 3 and 12 years were studied retrospectively and sorted according to diagnosis: idiopathic GH deficiency (GHD, n = 9), neurosecretory deficiency of GH secretion (NSD, n = 10) and ISS (n = 13). Nocturnal spontaneous hormone secretion was studied by intermittent venous sampling. Secretion profiles and copulsatility were analyzed using Pulsar and AnCoPuls software. RESULTS: (median, range in mug/l): Children with GHD and NSD had significantly lower GH and area-under-the-curve (AUC) levels than normal children (p < 0.001), whereas ISS children showed normal values. In contrast, prolactin levels were significantly higher (p < 0. 05) in children with GHD and NSD (11.1, 4.9 - 13.0 and 10.3, 8. 8 - 19. 6, respectively) compared to the ISS children (8.0, 4.9 - 13.0). In addition, prolactin AUC and peak height were higher (p < 0.05) in GH-deficient patients, whereas all other secretion parameters were the same. Correlation and copulsatility analysis revealed no evidence for a direct relation between PRL and GH secretion. CONCLUSIONS: PRL secretion is significantly higher in children with GHD and NSD compared to ISS children but PRL and GH show no copulsatile secretion pattern.     

Growth hormone receptors in lymphocytes of growth hormone-deficient children

Human growth hormone was injected intravenously into 18 growth hormone-deficient children and growth hormone binding sites in lymphocytes were investigated. Fresh circulating lymphocytes had a low initial value for the binding of growth hormone to solubilized receptors (3.45 +/- 1.46%) but after growth hormone injection, the binding rapidly increased to 14.8 +/- 4.2% at 2 1/2 h and 8.7 +/- 1.8% at 5 h. The sharp increase in binding is due to increase in the number of binding sites. Two control children who received chorionic gonadotropin had no change in lymphocyte growth hormone receptors. The methodological differences between the present study and previous attempts to identify human growth receptors in lymphocytes were (1) lymphocytes were separated and disrupted with Triton X-100 as quickly as possible (to avoid error from receptor leaking out of the cell) and (2) the receptors were assayed at 2 1/2 h after growth hormone administration (previous studies were 12-24 h later). One possible explanation for the data is that growth hormone receptor from liver is taken up by lymphocytes and rapidly released again, thus, contributing to the hormonal receptor economy in humans.     

Evaluation of cutaneous modifications in seventy-seven growth hormone-deficient children

Cutaneous parameters such as dermal thickness, stiffness, elasticity, skin surface lipid and hydration were evaluated using noninvasive methods in 77 growth hormone-deficient (GHD) children before replacement therapy and in 70 non-GHD children. We showed that in GHD children, dermis was thinner (0.70 +/- 0.10 vs. 0.80 +/- 0.10 mm, p < 0.0001 for prepubertal children and 0.81 +/- 0.10 vs. 0.94 +/- 0.11 mm, p < 0.0001 for pubertal children), stiffer (178.5 +/- 57.3 vs. 113.09 +/- 37 kPa, p < 0.0001 for prepubertal children and 172.5 +/- 61.7 vs. 117.3 +/- 42.5 kPa for pubertal children, p < 0.001) and less elastic (0.44 +/- 0.09 vs. 0.39 +/- 0.06 (nonelasticity index), p < 0.01 for prepubertal children and 0.39 +/- 0.05 vs. 0.33 +/- 0.04, p < 0.001 for pubertal children) compared to controls. Fourteen GHD children were re-evaluated after 1 year of GH treatment: dermal thickness and skin stiffness were significantly improved (p < 0.001 and p < 0.05 respectively) while elasticity was not modified. During the same period, 11 controls did not show any significant cutaneous modification. IGF-1 values, but not IGFBP-3 values, correlated positively with dermal thickness in GHD children, before and after 1 year of GH treatment. To conclude, GHD children exhibited specific cutaneous modifications. In a subset of GHD children, we showed that these modifications were influenced by GH treatment. More extensive studies are needed to see if these changes correlated with other GH effects.     

Medium-term cardiovascular effects of high-dose growth hormone treatment in growth hormone-deficient children

AIM: To investigate the possible cardiac morphofunctional alterations inducd by prolonged and high-dose GH therapy in a group of 14 children with isolated GH deficiency. PATIENTS AND METHODS: Patients were evaluated at phase 1, after 1.1 +/- 0.6 years of treatment with GH 0.93 +/- 0.13 U/kg/week, and at phase 2, after 5.5 +/- 2.1 years of therapy 0.89 +/- 0.11 U/kg/week. At each phase left ventricular volume, mass and systolic function were evaluated by two-dimensional guided M-mode echocardiography; left ventricular diastolic function was assessed by PW-Doppler sampling of transmitral flow. RESULTS: Phase 1: diastolic blood pressure was lower (p < 0.05) and fractional shortening was not adequate for the level of afterload (stress shortening index p < 0.05) in patients compared to controls. Phase 2: diastolic blood pressure was lower (p < 0.01) and mass and mass/volume ratio were increased (mass index p < 0.05, mass/ volume ratio p < 0.05) in patients compared to controls. The increased mass/volume ratio, together with the normal systolic blood pressure, explains the reduction in peak systolic stress (p < 0.005). Among the parameters of left ventricular diastolic function, the peak E velocity/total area under mitral valve tracing and the area under E velocity/total area under mitral value tracing ratios were significantly decreased (p < 0.05). CONCLUSION: After a mean period of 5 years on high-dose GH treatment in GH-deficient children, subclinical morphofunctional alterations in the left ventricle were found.     

Growth hormone receptor structure in adipocytes from normal and growth hormone-deficient rats

Using cross-linking techniques, we compared the properties of the growth hormone (GH) receptor in freshly isolated adipocytes from normal rats, from GH deficient rats, and in preincubated adipocytes from normal rats. Bound [125I]iodo-hGH was cross-linked to adipocytes with disuccinimidyl suberate, and membrane proteins labelled with [125I]iodo-hGH were visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. All of the adipocytes tested exhibited a prominent Mr = 134,000 band and additional less intense bands in the presence of reductant. No significant differences in the overall banding pattern of membrane proteins were evident in reducing or nonreducing gels, using adipocytes from rats made GH deficient by hypophysectomy or by treatment with antibodies against rat GH, or in fresh and preincubated cells from normal rats. Taken together with binding studies, these findings suggest that differences in the ability of GH to stimulate glucose oxidation in rat adipose tissue probably involve differences distal to the GH receptor.     

Increased insulin receptor binding in erythrocytes from growth hormone-deficient children

Erythrocytes from growth hormone-deficient children (GHd-children) (n=10) showed a statistically significant increase in insulin binding at low unlabeled insulin concentrations, together with a threefold decrease in apparent receptor affinity, as compared to control children (C) (n=11). Scatchard analysis of the binding data using the two-site model revealed that both the receptor concentration R₁ [GHd-children 0.10±0.01 ng/ml and C 0.03±0.002 ng/ml] and the dissociation constant K_D1 [GHd-children (0.48±0.05)×10^–9M and C (0.19±0.01)×10^–9M] for high affinitylow capacity sites were significantly increased in erythrocytes from GHd-children, while neither receptor concentrations (R₂) nor the dissociation constant (K_D2) for low affinity-high capacity sites proved to be altered. These events were accompanied by a normal sensitivity to insulin as well as glucose tolerance in the GHd-group. The meaning of the increased insulin binding with normal insulin sensitivity in GH-deficiency is discussed.     

Pubertal growth spurt induced by human chorionic gonadotropin in hypogonadotropic growth hormone-deficient children

Eight hypogonadotropic growth hormone-deficient children were treated with human chorionic gonadotropin (HCG) while they continued to receive a fixed dose of HGH for a one year period. They were observed for changes in somatomedin C (IGF-I) and height increase velocity. Mean somatomedin C was 0.79 +/- 0.30 U/ml in normal prepubertal children (N = 7) and 0.78 +/- 0.31 U/ml in prepubertal normal short children (N = 22). At pubertal stage 3, somatomedin C was 2.21 +/- 1.23 and 2.05 +/- 0.44 U/ml in normals (N = 5) and in normal short children (N = 7), respectively. When 3000-5000 units/week of HCG were given to each of the 8 hypogonadotropic growth hormone-deficient children who were receiving HGH at a mean dose of 0.33 +/- 0.05 IU/kg/week, testosterone increased from less than 0.3 ng/ml to more than 5 ng/ml at 6 months in 3 cases and at 12 months in 2 cases, while the testosterone concentration was less than 3.5 ng/ml in the remaining 3 cases. The rate of height increase rose significantly (p less than 0.001) from 5.2 +/- 1.0 to 9.3 +/- 1.4 cm/year mimicking the normal pubertal growth spurt. However, the mean somatomedin C concentration was 0.44 +/- 0.23 before therapy, 0.33 +/- 0.30 at 6 months and 0.31 +/- 0.14 U/ml at 12 months after the start of HCG therapy. It is concluded that the pubertal growth spurt induced by HCG in hypogonodotropic GH-deficient male children is not mediated by the increase in somatomedin C production.     

Effect of growth hormone therapy on the proinflammatory cytokine profile in growth hormone-deficient children

The aim of the present study was to establish whether growth hormone (GH) treatment in vivo affects pro-inflammatory cytokine production by resting or in vitro, activated, cultured, peripheral blood mononuclear cells (PBMC) from children with complete growth hormone deficiency (GHD). We evaluated 11, pre-pubertal children (6 males and 5 females) with GHD, aged between 6 and 14 years, and 9, age- and sex-matched healthy subjects were studied as controls (CTRLs). Freshly isolated PBMC were cultured for 4 or 24 h in X-VIVO medium in the presence or absence of 0.01 microg/mL lipopolysaccharide for the determination of TNF-alpha and IL-6 production; alternatively, cells were incubated 24 h in X-VIVO medium with or without 25 microg/mL Concanavalin A for IFN-gamma production. Cytokines were measured in the cell supernatants by enzyme-linked immunosorbent assay kits. The results of the present study provide evidence that spontaneous and/or mitogen-induced, in vitro PBMC production of pro-inflammatory cytokines is lower in GHD children than in healthy, age-matched individuals (p<0.05 by the Mann-Whitney U-test). After 3 months of GH therapy, cytokine production was significantly (p<0.05 by the Wilcoxon test) increased, but was still lower than in healthy controls. It is reasonable to speculate that severe GH deficiency can cause alterations in the pro-inflammatory cytokine-induced immune response in humans, and that GH treatment can ameliorate this important immunological function.     

Mutagenicity and DNA- modifying activity: A comparison of two microbial assays

Aspects of the Salmonella mutagenesis and Escherichia coli DNA polymerase deficient (pol A1^?) assay procedures for detecting environmental mutagens are discussed.The chief limitation of the pol A1^?1- assay involves substances that do not diffuse rapidly in agar. This problem can be overcome by performing the test in suspension. A simple procedure for accomplishing this is described.Although the Salmonella assay is more flexible, under routine conditions it does not respond to several classes of substances which give positive responses in the pol A1^?1 system.For optimal testing, it is recommended that the two microbial assays be used in tandem.The DNA-modifying properties of povidone-iodine for eukaryotic and prokaryotic cells are described. Even though this substance does not display mutagenic properties in the standard Salmonella assay, it does so in suspension culture. The basis of the mutagenic and DNA-modifying properties of povidone-iodine appears to involve the iodination of the cystosine residue of DNA.     

The distribution of serum albumin in human normal and degenerate articular cartilage

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymatic methods. With naturally occuring cartilage large a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partitiion coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

The distribution of serum albumin in human normal and degenerate articular cartilage.

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymic methods. With naturally occurring cartilage a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partition coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

Serum insulin-like growth factor I levels in growth hormone-deficient adults: influence of sex steroids

Measurement of serum insulin-like growth factor I (IGF-I) concentrations remains the single most important tool in the evaluation of growth hormone (GH) replacement in GH-deficient adults, and the therapeutic goal is to maintain the level within the age-adjusted normal range. In healthy adults, IGF-I levels do not differ between males and females, whereas spontaneous GH secretion is approximately twofold higher in females. Untreated GH-deficient women exhibit lower IGF-I levels compared with men, and the increase in serum IGF-I during GH replacement is also significantly less. Put together, these data suggest resistance to GH in women, which in healthy individuals is compensated for by increased GH secretion. Administration of oral oestrogen in healthy post-menopausal women suppresses hepatic IGF-I production and increases pituitary GH release, and oral oestrogen replacement in women with GH deficiency lowers IGF-I concentrations and increases the amount of GH necessary to obtain IGF-I target levels during treatment. These data clearly suggest that hepatic suppression of IGF-I production by oestrogen subserves the gender difference in GH sensitivity, but it is also likely that sex steroids may interact with the GH/IGF axis at further levels. There is also circumstantial evidence to indicate that testosterone stimulates IGF-I production, and it is speculated that a certain threshold level of androgens is essential to ensure hepatic IGF-I production. Whether these data should translate into earlier discontinuation of oestrogen replacement therapy in adult women with hypopituitarism merits consideration.     

Kinesiological surface electromyography in normal children: range of normal activity and pattern analysis.

To document the range of activity and patterns of normal surface kinesiological electromyography in normal children, 87 children ranging from age 3 to 18 years who were developing normally were evaluated at free walking speed. Analysis was performed on 6307 gait cycles from 11 different muscles with custom software. We devised an algorithm to do a computer-based KEMG curve pattern analysis to sort out curves that did not match the physiologic muscle activity pattern, We also devised a combined preset amplitude and statistics-based criteria to satisfactorily determine the onset/cessation of KEMG activity. The measurements of timing and duration of activity, body height and weight, body mass index, cadence, stride length, and age were obtained for statistical analysis. From the pattern recognition, the medial and lateral hamstrings, gluteus maximus and medius, and gastrocnemius muscles had the highest percentage of clinically relevant curves. In most muscle groups, The KEMG curves had an average background activity of about 11-15% of maximum amplitude. The surface KEMG curves from normal subjects have 12.7% cycles that did not match their physiologic activity pattern. The timing and duration of KEMG activity was poorly or only fairly correlated with age, body height, body weight, and body mass index. The ensemble average of the linear envelope KEMG curves of each muscle could be applied as a normal database in a clinical gait analysis laboratory focusing on pediatric patients.