Is BaF3 bioassay useful to identify patients with bioinactive growth hormone?

We analyzed the ability of the BaF3 cell line bioassay to select patients with biologically inactive GH. We first evaluated the biological response of the Ba/F3-hGHR cells to rhGH additional doses from 10 to 5000 pg/ml. The concentration points corresponding to the linear part of the curve were selected. We then analyzed a group of sera, diluted like the standard, including the entire range of GH concentrations that can be analyzed by bioassay. The serum/standard area below the curve ratio was calculated. Serum GH immunoactivity determined by IMMULITE/GH bioactivity ratios was calculated. Our experimental data showed that GH-bioactivity/GH-immunoactivity ratios below 0.303 are indicative of a bioinactive GH molecule. This bioassay would recognize only extreme cases of GH bioinactivity, and it would not be a useful tool in the search for patients with altered forms of GH.

The shortness of Pygmies is associated with severe under-expression of the growth hormone receptor.

The stature is a highly heritable trait controlled by genetic and environmental factors. The African Pygmies represent a paradigmatic example of non-disease-related idiopathic short stature (ISS), showing a similar endocrine profile of Caucasian individuals with ISS. Pygmy children show normal anthropometric and endocrine parameters until puberty, while adult Pygmies show normal baseline and post-stimulation serum growth hormone (GH) levels but low values of baseline serum GH-binding protein (GHBP) and insulin-like growth factor-I (IGF-I). This discrepancy suggests a defective response to GH occurring in adulthood since Pygmies lack both the pubertal serum IGF-I surge and the growth spurt. However, sequencing of the key genes of the GH-IGF-I axis failed to identify Pygmy specific variants or haplotypes. We therefore aimed at assessing whether the quantitative gene expression profile of two key genes of the GH-IGF-I axis, GH and GHR, was also similar in low-stature and normal stature populations. We showed that the GH gene expression is 1.8-fold reduced and the GH receptor (GHR) gene expression is 8-fold reduced in adult Pygmies in comparison with sympatric adult Bantu, and that this reduction is not associated with sequence variants of the GHR gene. The marked decrease of the GHR expression in Pygmies is associated with reduced serum levels of the IGF-I and GHBP. Our results, documenting a markedly reduced GHR gene expression in adult Pygmies, could contribute to elucidate the mechanisms involved in ISS in Caucasoid subjects.

Effect of human recombinant growth hormone therapy on circulating levels of erythropoietin and granulocyte-colony stimulating factor in short children.

Several reports suggest a role of growth hormone (GH) in the regulation of the haematopoietic system, as regards the normal differentiation and function of blood cells. The aim of this study was to evaluate the influence of rhGH therapy on erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF) levels in 18 prepubertal short children with idiopathic GH deficiency (GHD) (n = 8) or without GHD (n = 10), during the first year of treatment. In non-GHD children Epo levels significantly decreased and G-CSF levels increased from basal to 12 months of therapy, whereas in GHD children they did not change significantly. Circulating levels of G-CSF are significantly lower in GHD than in non-GHD children. In non-GHD children the number of red blood cells, haemoglobin and haematocrit values significantly increased after 1 year of rhGH treatment. rhGH therapy influences Epo and G-CSF levels in short non-GHD children, while it shows no effects in GHD children.

Extracellular vesicles derived from mesenchymal cells: perspective treatment for cutaneous wound healing in pediatrics.

AIM: We evaluated the effects of the intradermal injection of extracellular vesicles (EVs) derived from adipose stem cells (ASC-EVs) and bone marrow cells (BM-EVs) in an experimental cutaneous wound repair model. METHODS: Mesenchymal stem cells (MSCs) were in vitro expanded from adipose (ASC) or BM tissues (BM-MSC) of rabbits. EVs were separated from the supernatants of confluent ASC and BM-MSCs. Two skin wounds were induced in each animal and treated with MSC or EV injections. Histological examination was performed postinoculation. RESULTS: EV-treated wounds exhibited a better restoration compared with the counterpart MSC treatment. ASC-EV-treated wounds were significantly better than BM-EVs (p = 0.036). CONCLUSION: EV topical inoculation provides restored architecture during cutaneous wound healing and represents a promising solution for regenerative medicine in children.

CPAM type 2-derived mesenchymal stem cells: Malignancy risk study in a 14-month-old boy.

INTRODUCTION: The association between congenital pulmonary airway malformations (CPAM) and malignancy is reported in the literature. Interactions between the tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. We characterized MSCs isolated from CPAM lesions in order to define potential malignancy risks. METHODS: CPAM II pulmonary tissue was used for MSC expansion; a "healthy" lung section from the same child was used as a comparator. Morphology, immunophenotype, differentiation and immunological capacity, proliferative growth, gene signature telomerase activity, and in vivo tumorigenicity in nude mice were evaluated. RESULTS: MSCs were successfully isolated and propagated from CPAM tissue. CPAM-MSCs presented the typical MSC morphology and phenotype, while exhibiting high proliferative capacity, reaching confluence at a median time of 5 days as well as differentiation capabilities. CPAM-MSCs at early passages were not neoplastic and chromosomally normal, even though unbalanced chromosomal rearrangements were noted by molecular karyotype. CONCLUSIONS: CPAM-MSCs exhibited specific features similar to tumor derived MSCs. Whilst there was no evidence of malignant transformation in the cystic tissue, our results provide evidence that this abnormal tissue has malignant potential. MSCs are considered important players in the tumor microenvironment and they have been closely linked to regulation of tumor survival, growth, and progression. Thus, early lesion resection also in asymptomatic patients might be indicated to exclude that the microenvironment may be potentially permissive to cancer development.

Serum cytokine levels in GH-deficient children during substitutive GH therapy.

OBJECTIVE: The aim of the present study was to investigate the effect of exogenously administered human GH (hGH) on serum levels of interleukin (IL)-4, IL-6, IL-12 and tumour necrosis factor (TNF)-alpha in GH-deficient (GHD) children. DESIGN AND METHODS: We evaluated 13 short prepubertal GHD children, aged between 2 and 13 years, and 13 age-matched healthy subjects as controls. Circulating cytokine values were evaluated in basal conditions in all children, and 6 and 24 h following the 1st hGH injection (0.23 mg/kg per week), and then after 3 months of hGH treatment in GHD patients. Serum levels of IL-4, IL-6, IL-12 and TNF-alpha were measured by commercially available ELISAs. RESULTS: No significant differences were found between controls and GHD children in basal values of serum IL-4, IL-6, IL-12 and TNF-alpha (P > 0.05 by Mann-Whitney U test). Analysis of cytokine levels during hGH treatment showed significant changes over time in TNF-alpha and IL-6 levels (P = 0.0014 and P = 0.00 024 respectively), with the more pronounced effect observed at 6 h following the first administration of hGH (i.e. increase in IL-6 (Wilcoxon matched pairs test, P = 0.0015) and TNF-alpha levels (P = 0.0015)). No significant changes over time were observed in IL-4 and IL-12 serum levels. CONCLUSIONS: In vivo release of the pro-inflammatory cytokines IL-6 and TNF-alpha can be affected by hGH treatment in GHD children, suggesting a direct effect of GH on the immune function.

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.

Stimulating effect of growth hormone on cytokine release in children.

OBJECTIVE: The aim of the present study was to investigate the effect of exogenously administered GH on serum levels of interleukin (IL)-1beta, IL-2, IL-12, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma and their relation with IGF-I levels in normal short stature children. DESIGN AND METHODS: 23 short prepubertal non GH-deficient children (10 females and 13 males) whose mean+/-s.d. chronological age was 11.95+/-1.85 Years (from 8.80 to 14.89 Years), and mean+/-s.d. bone age was 10.48+/-2.44 Years, were evaluated during a somatomedin generation test (human GH 0.1 IU/kg per day for 4 days) to exclude a partial GH resistance as the cause of short stature; 34 sex- and age-matched healthy subjects were studied as controls. Circulating cytokine values were measured in basal conditions in all children, and 12 h following the 4th GH subcutaneous injection in the 23 short children only. RESULTS: No significant differences were found between short children and controls in basal values of serum IGF-I (192.1+/-18.3 and 198.2+/-28.2 ng/ml respectively). In short subjects there was a significant increase in serum IGF-I levels after the 4th GH injection (from 192.1+/-18.3 ng/ml, i.e. -1.16+/-0.16 standard deviation score (SDS) to 338.2+/-27.1 ng/ml, i.e. 0.14+/-0.17; P<0.00001). No significant differences were found between short children and controls in basal concentrations of serum INF-gamma (19+/-4 and 26+/-5 mIU/ml respectively), IL-1alpha (24.950+/-3.613 and 20.896+/-2.778 pg/ml respectively), IL-2 (3.945+/-1.209 and 4.794+/-0.562 pg/ml respectively), IL-12 (1.093+/-0.269 and 1.976+/-0.596 pg/ml respectively), and TNF-alpha (1.794+/-0.559 and 2.188+/-0.346 pg/ml respectively). Likewise, a significant increase was found in serum INF-gamma (before 19+/-4 and after four GH injections 185+/-57 mIU/ml respectively; P<0.008), IL-1beta (24.950+/-3.613 to 43.339+/-5.431 pg/ml respectively; P<0.0001), IL-2 (3.945+/-1.209 to 9.165+/-2.331 pg/ml respectively; P<0.003), IL-12 (1.093+/-0.269 to 3.724+/-0.637 pg/ml respectively; P<0.0007) and TNF-alpha (1.794+/-0.559 to 9.266+/-3.066 pg/ml respectively; P<0.01). CONCLUSIONS: Cytokine release can be affected by short-term GH administration in normal children indicating a direct influence of GH on the immune system.

Effect of growth hormone (GH) on the immune system.

A growing body of evidence indicates a bi-directional relationship between the neuroendocrine system and immune functions. It is well known that lymphoid organs such the thymus, the spleen and peripheral blood produce growth hormone (GH) and GH receptor is expressed on different subpopulations of lymphocytes. Many in vitro and in animal studies demonstrate an important role of GH in immunoregulation. GH stimulates T and B cells proliferation and immunoglobulin synthesis, enhances the maturation of myeloid progenitor cells and is also able to modulate cytokine response. However, in humans GH deficiency (GHD) is not usually associated with immunodeficiency and only minor abnormalities of immune function have been reported, as compared to those observed in GHD animals. It is possible that in humans the GH produced locally in the immune system compensates for the lack of endocrine GH. In this review the main actions of GH on the immune system in vitro, in animal models and in humans are summarized.

Secular trends in growth of African Pygmies and Bantu.

OBJECTIVE: The aim of this study was to investigate whether a secular trend in growth occurred during the last century in Pygmies from Cameroon (West Pygmies) and in Bantu rural farmers, the latter being studied to serve as controls. DESIGN: The evolution in height of West Pygmies and Bantu farmers from 1911 to 2006 was evaluated using data from the literature as well as data gathered by our research team during an expedition to Cameroon in 2006. RESULTS: During the last century, no secular trend in west Pygmies is apparent, as height changed from 151 cm to 155 cm in males and from 143 cm to 146 cm in females. A small though significant (p=0.026), increment (about 2 cm) was observed only in female subjects during the last ten years. By contrast, Bantu heights show a significant change from 1943 to 2006 for both males (from 159 cm to 172 cm; p=0.025) and females (from 148 cm to 160 cm; p=0.029). CONCLUSIONS: Over the last century, the Bantu population exhibited a significant secular trend for height, whereas West Pygmies did not increase their linear growth. The lack of secular trend in Pygmies possibly suggests that their stature reflects adaptation to the forest lifestyle. We may hypothesize that not only environmental but epigenetic factors have also contributed to their growth potential.

Role of adiponectin and leptin on body development in infants during the first year of life.

BACKGROUND: The control of growth and nutritional status in the foetus and neonate is a complex mechanism, in which also hormones produced by adipose tissue, such as adiponectin and leptin are involved. The aim of this study was to evaluate levels of adiponectin, leptin and insulin in appropriate (AGA) and small for gestational age (SGA) children during the 1st year of life and to correlate these with auxological parameters. METHODS: In 33 AGA and 29 SGA infants, weight, length, head circumference, glucose, insulin, adiponectin and leptin levels were evaluated at the second day of life, and at one, six and twelve months, during which a portion of SGA could show catch-up growth (rapid growth in infants born small for their gestational age). RESULTS: Both total and isoform adiponectin levels were comparable between AGA and SGA infants at birth and until age one year. These levels significantly increased from birth to the first month of life and then decreased to lower values at 1 year of age in all subjects. Circulating leptin concentrations were higher in AGA (2.1 +/- 4.1 ng/ml) than in SGA neonates (0.88 +/- 1.03 ng/ml, p < 0.05) at birth, then similar at the 1st and the 6th month of age, but they increased in SGA from six months to one year, when they showed catch-up growth. Circulating insulin levels were not statistically different in AGA and SGA neonates at any study time point. Insulin levels in both AGA and SGA infants increased over the study period, and were significantly lower at birth compared to one, six and 12 months of age. CONCLUSIONS: During the first year of life, in both AGA and SGA infants a progressive decrease in adiponectin levels was observed, while a difference in leptin values was correlated with the nutritional status.

Relationship between thymulin and growth hormone secretion in healthy human neonates.

The relationship among circulating values of growth hormone (GH), thymulin, and zinc in 19 healthy human neonates at birth and at the 4th month of age, and in their respective mothers, was investigated. Cytofluorimetric analysis on some CD antigen markers was conducted on cord blood and peripheral blood mononuclear cells. Active thymulin and zinc plasma levels increased in newborns in comparison with their mothers. In neonates serum GH levels increased with a significant decline later. The expression of CD molecules from newborns at birth and from infants at the 4th month of age was inversely correlated with active thymulin, zinc, and GH levels, whereas CD4 antigen marker was positively correlated with the same parameters at the 4th month of life. A novel interrelationship among active thymulin, zinc, and GH exists from the early up to the late phase of newborn life, in which maternal zinc, via lactation, may be involved.

Response to long-term growth hormone therapy in short children with reduced GH bioactivity.

BACKGROUND/AIMS: The aim of the present study was to investigate whether short children with normal growth hormone (GH) immunoreactivity, but reduced bioactivity (bioinactive GH) could benefit from rhGH treatment as GH deficient (GHD) patients. METHODS: We evaluated 12 pre-pubertal children (8 M, 4 F), with GH deficiency-like phenotype showing normal serum GH peak levels (>10 ng/ml), measured by immunofluorimetric assay (IFMA-GH), in contrast with a reduced GH bioactivity (bio-GH), evaluated using the Nb(2) cells. We also evaluated 15 age-matched GHD pre-pubertal children (11 M, 4 F) with serum GH peak <5 ng/ml. Both groups were treated with rhGH therapy at the dose of 0.23 mg/kg/week s.c. RESULTS: Serum bio-GH/IFMA-GH ratio at peak time for each patient during the provocative test was significantly lower in bioinactive GH than in GHD children (0.29 vs. 2.05, p = 0.00001). Recombinant human GH therapy induced a significant (p < 0.001) increase in growth rate in both groups during the first 2 years. In the third year of treatment, while growth rate in GHD children is maintained, in bioinactive GH patients it decreases remaining, however higher compared to the pre-treatment one. CONCLUSIONS: Short rhGH therapy given to selected bioinactive GH children improve growth rate and might result in greater final adult height.

Macrophage migration inhibitory factor in patients with juvenile idiopathic arthritis.

OBJECTIVE: To evaluate serum and synovial fluid (SF) levels of macrophage migration inhibitory factor (MIF) and in vitro MIF production by peripheral blood mononuclear cells (PBMCs) in patients with juvenile idiopathic arthritis (JIA). METHODS: Serum, SF, and culture supernatant levels of MIF were measured by enzyme-linked immunosorbent assay. Production of MIF by PBMCs was investigated by culturing PBMCs in the absence or presence of 2 different concentrations of concanavalin A. RESULTS: Serum MIF levels were increased in patients with JIA, and the highest levels were present in patients with systemic-onset JIA. In systemic-onset JIA, serum levels of MIF correlated with the persistence of systemic features and the number of active joints. PBMCs from patients with systemic-onset JIA, when cultured under unstimulated conditions or at suboptimal stimulation, released higher amounts of MIF compared with those from patients with oligoarticular-onset JIA or healthy controls. MIF levels in the SF of patients with systemic-onset JIA were significantly higher than those in patients with oligoarticular-onset JIA. In individual joints, in both systemic-onset JIA and oligoarticular-onset JIA, SF MIF levels were inversely correlated with the duration of the clinical remission induced by intraarticular administration of triamcinolone hexacetonide. CONCLUSION: MIF appears to be a relevant cytokine in the pathogenesis of JIA, particularly in systemic-onset JIA.