Body composition in children receiving recombinant human growth hormone after renal transplantation

BACKGROUND: Recombinant human growth hormone (rhGH) is an anabolic hormone promoting protein synthesis in various tissues. Therefore, changes in body composition may be expected during rhGH treatment. METHODS: We studied changes in body composition during two years of rhGH treatment in 21 children after at least one year with a functioning renal transplant. The mean +/- SD age was 12.9+/-2.5 years at the start of rhGH therapy. A whole body, dual energy X-ray absorptiometry (DEXA) exam was performed before the initiation of rhGH therapy (T0), and was repeated at one and two year intervals after initiation of the therapy (T1 and T2, respectively). RESULTS: Lean body mass increased by a median of 0.48 SDS during the first year of treatment (P = 0.022), and the median increase during two years of therapy was 0.36 SDS (P = 0.061). On the contrary, the median fat body mass decreased by 2.17 SDS during the T0 to T1 period (P = 0.04) and by 1.99 SDS during the T0 to T2 period (P = 0.055). The index for fat body mass/lean body mass (FBM/LBM) decreased by a median of 5.3% during T0 to T1 (P < 0.001), however, a slower but still significant decrease by a median of 4.2% was noted at T2 (P < 0.05). Bone mass content did not change significantly during rhGH treatment. The medians in caloric and protein intakes were stable during rhGH treatment. CONCLUSION: A significant increase of lean body mass and a decrease of fat body mass was noted during rhGH therapy in children after renal transplantation.     

Therapy of dilated cardiomyopathy with recombinant human growth hormone

Dilated cardiomyopathy is a disease of the myocardium which is characterized by predominant enlargement of the left ventricle, decreased systolic function, and reduced myocardial wall thickness. The reduction in the number and function of myocardial fibers induces hypertrophy and leads to interstitial fibrosis. Ventricular dilatation and reduced wall thickness contributes to a further increase of the already elevated myocardial wall stress and triggers the continuous decrease of myocardial function. The initial myocyte injury by different and mostly unknown causes leads to progressive myocyte damage resulting in a similar uniform clinical picture independent from the initial myocellular derangement. One of the common pathways may be an inadequate response of the heart to human growth hormone (GH). Several studies support this hypothesis: GH deficiency results in a significant reduction of ventricular wall thickness and myocardial function, and GH-substitution is able to normalize the depressed left ventricular function. In addition, Sacca et al. reported in an uncontrolled study that GH-therapy reduced left ventricular wall stress by 40% in 7 patients with dilated cardiomyopathy. There was a parallel increase of the ejection fraction from 34% to 47%. This improvement was accompanied by a decrease of the functional classification of heart failure from NYHA 2.7 to 1.6. This therapeutic approach to alter the myocyte function directly by growth factors may open a new therapeutic concept in dilated cardiomyopathy. If double blind, randomized studies confirm the results of Sacca's study, a new era of therapeutic options may be available for the treatment of dilated cardiomyopathy.     

Investigation on fouling mechanisms for recombinant human growth hormone sterile filtration

During sterile filtration of recombinant human growth hormone solutions, severe membrane fouling was experienced compared to other protein preparations of significantly higher molecular weights and concentrations. This phenomenon was attributed to rhGH aggregation/adsorption occurring in the filter pore. To better understand this phenomenon, we examined several possible fouling mechanisms: (1) pore constriction, (2) adsorption due to nonspecific binding between protein and the membrane, (3) shear-induced adsorption, (4) hydrophobic interface-induced aggregation/adsorption. The protein solutions were sterily filtered using 0.22 mm filters, and their filtration fluxes were monitored. Filtration on the capillary and the noncapillary filters suggested that constraints by pore constriction and tortuosity played only a limited role. Filtration using filters with different degrees of protein binding tendency suggested that nonspecific adsorption was insignificant. The shear stress acting on the protein during filtration was small. RhGH which was intentionally sheared in a high-speed concentrically rotating device did not aggravate fouling tendency, suggesting that the shear-induced adsorption might not be the major fouling mechanism. The dynamic light scattering data showed a trace amount of rhGH aggregates always present in equilibrium with the hydrophobic (air-water and membrane-water) interface. These aggregates tended to be adsorbed to the membrane, and more aggregates were generated presumably due to the equilibrium between aggregates and protein monomers. This adsorption/aggregation process eventually fouled the membrane. When the hydrophobic interface was occupied by surfactant molecules, the equilibration kinetics ceased to generate aggregates, thereby minimizing membrane fouling. This study clarified the cause of such an unusual fouling phenomenon upon microfiltration.     

A low dose euglycemic infusion of recombinant human insulin-like growth factor I rapidly suppresses fasting-enhanced pulsatile growth hormone secretion in humans

To determine if insulin-like growth factor I (IGF-I) inhibits pulsatile growth hormone (GH) secretion in man, recombinant human IGF-I (rhIGF-I) was infused for 6 h at 10 micrograms.kg-1.h-1 during a euglycemic clamp in 10 normal men who were fasted for 32 h to enhance GH secretion. Saline alone was infused during an otherwise identical second admission as a control. As a result of rhIGF-I infusion, total and free IGF-I concentrations increased three- and fourfold, respectively. Mean GH concentrations fell from 6.3 +/- 1.6 to 0.59 +/- 0.07 micrograms/liter after 120 min. GH secretion rates, calculated by a deconvolution algorithm, decreased with a t 1/2 of 16.6 min and remained suppressed thereafter. Suppression of GH secretion rates occurred within 60 min when total and free IGF-I concentrations were 1.6-fold and 2-fold above baseline levels, respectively, and while glucose infusion rates were < 1 mumol.kg-1.min-1. During saline infusion, GH secretion rates remained elevated. Infusion of rhIGF-I decreased the mass of GH secreted per pulse by 84% (P < 0.01) and the number of detectable GH secretory pulses by 32% (P < 0.05). Plasma insulin and glucagon decreased to nearly undetectable levels after 60 min of rhIGF-I. Serum free fatty acids, beta-hydroxybutyrate, and acetoacetate were unaffected during the first 3 h of rhIGF-I but decreased thereafter to 52, 32, and 50% of levels observed during saline. We conclude that fasting-enhanced GH secretion is rapidly suppressed by a low-dose euglycemic infusion of rhIGF-I. This effect of rhIGF-I is likely mediated through IGF-I receptors independently of its insulin-like metabolic actions.     

Pilot study of the immunologic effects of recombinant human growth hormone and recombinant insulin-like growth factor in HIV-infected patients

OBJECTIVE: To study the immunologic effects of recombinant human growth hormone (rhGH), recombinant human insulin-like growth factor type 1 (rhIGF-1), or the combination, in patients with moderately advanced HIV infection. DESIGN: Randomized but not blinded trial. SETTING: Government medical research center. PATIENTS: Twenty-four HIV-infected patients with CD4 cell counts of 100-400 x 10(6)/l who were receiving nucleoside antiretroviral therapy. INTERVENTIONS: Either rhGH, rhIGF-1, or the combination was administered subcutaneously for 12 weeks. MAIN OUTCOME MEASURES: Immunologic parameters, including T-cell subsets and assays of in vitro interleukin (IL)-2 production in response to antigens and mitogens, and safety profile. RESULTS: Plasma IGF-1 levels were low or low-normal prior to treatment and increased with all three therapies. There were no significant changes in CD4 cell counts, RA/RO CD4 cell subsets, natural killer cell function, immunoglobulin levels, or in vitro IL-2 production in response to mitogen or alloantigens. However, there was an upward trend (and for p18IIIB a statistically significant increase) in the in vitro IL-2 production in response to each of five HIV envelope peptides. Potential toxic effects included fatigue, arthralgia, edema, myalgia, and headache. Patients also were noted to have weight gain averaging 4 kg early in the course of treatment. CONCLUSIONS: These results suggest that treatment with rhGH/rhIGF-1 was reasonably well tolerated and that modest improvement in HIV-specific immune function was attained. Further studies will help clarify the therapeutic potential of rhGH/rhIGF-1 as an immunostimulator in the setting of HIV infection.     

Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants

We demonstrate that a surfactant-stabilized molten globule intermediate exists for recombinant human growth hormone (rhGH), is very hydrophobic, and tends to form aggregates. Characterization of this intermediate included equilibrium denaturation measured by electron paramagnetic resonance (EPR) and CD spectroscopy, assessment of aggregation during refolding, and fluorescence studies of its binding to the hydrophobic probe, 1-anilinonapthalene-8-sulfonate (1,8-ANS). We have found that at 4.5 M guanidinium hydrochloride (GuHCl), a molten globule intermediate of rhGH is stabilized and results in significant aggregation upon refolding. This intermediate is populated by the addition of the nonionic surfactant, Tween. This surfactant also reduces the extent of aggregation during refolding of rhGH from 4.5 M GuHCl. Overall, our studies reveal that rhGH forms a molten globule-like intermediate during folding and this intermediate self-associates. This self-association is reduced upon formation of a Tween-rhGH complex. Tween also binds to the native protein. Thus, nonionic surfactants such as Tween may act like molecular chaperones in facilitating protein folding while not altering the native conformation.     

The effect of recombinant human growth hormone on regulation of growth hormone secretion and blood glucose in insulin-dependent diabetes

The type 1 copper in Pseudomonas aeruginosa azurin was studied by electron paramagnetic resonance (EPR) spectroscopy at low microwave frequencies. Partially resolved ligand hyperfine structure was observed in the perpendicular region of the spectra at both S-band (2.4 GHz) and L-band (1.1 GHz). A trial and error method, requiring several hundred simulations, has been used to simulate the low frequency EPR data and yield an optimum value of 30 MHz for ACUx, more than one half that previously reported. The fit between the simulated and experimental data is sensitive to changes in the Euler angles and, in particular, to the angle alpha which rotates the Cu A-tensor about the z-axis. Thus, the A- and g-tensors for copper in P. aeruginosa azurin do not appear to be coincident. A value for the Euler angle beta of at least 10 degrees does not disturb the fit between the simulated and experimental data. These studies demonstrate the advantage of evaluating EPR parameters from simulations at more than one frequency, especially at low frequencies where ligand superhyperfine structure may be resolved for type 1 copper.     

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone 1(-1) and 7 g fusion glucagon 1(-1). The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e., high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content.     

Effect of excipients on the stability and structure of lyophilized recombinant human growth hormone

We have investigated the effect of mannitol, sorbitol, methyl alpha-D-mannopyranoside, lactose, trehalose, and cellobiose on the stability and structure of the pharmaceutical protein recombinant human growth hormone (rhGH) in the lyophilized state. All excipients afforded significant protection of the protein against aggregation, particularly at levels to potentially satisfy water-binding sites on the protein in the dried state (i.e., 131:1 excipient-to-protein molar ratio). At higher excipient-to-protein ratios, X-ray diffraction studies showed that mannitol and sorbitol were prone to crystallization and afforded somewhat less stabilization than at lower ratios where the excipient remained in the amorphous, protein-containing phase. The secondary structure of rhGH was determined using Fourier transform infrared (FTIR) spectroscopy. rhGH exhibited a decrease in alpha-helix and increase in beta-sheet structures upon drying. Addition of excipient stabilized the secondary structure upon lyophilization to a varying extent depending on the formulation. Samples with a significant degree of structural conservation, as indicated by the alpha-helix content, generally exhibited reduced aggregation. In addition, prevention of protein-protein interactions (indicated by reduced beta-sheet formation) also tended to result in lower rates of aggregation. Therefore, in addition to preserving the protein structure, bulk additives that do not crystallize easily and remain amorphous in the solid state can be used to increase protein-protein distance and thus prevent aggregation.     

High-level production of human growth hormone in Escherichia coli by a simple recombinant process

Diagnoses for autism based on the Autism Diagnostic Interview-Revised (ADI-R) and the Childhood Autism Rating Scale (CARS) were examined for 83 individuals with suspected autism. Agreement between systems reached 85.7%. Participants receiving diagnosis of autism based on only one system were significantly younger in age than individuals receiving diagnoses according to both systems. Individuals who did not receive diagnosis of autism on the ADI-R had lower chronological and mental ages and lower CARS scores compared to individuals who received diagnosis of autism based on the ADI-R. Eighteen females and 18 males were matched to examine possible gender differences. No significant findings were revealed, suggesting that the symptoms of autism according to the ADI-R and CARS do not differ between males and females when matched for chronological and mental ages.     

Improvement of skin flap perfusion by subdermal injection of recombinant human basic fibroblast growth factor

The effect of subcutaneously injected recombinant human basic fibroblast growth factor (bFGF) was studied in an arterial skin flap model on the ear of the hairless mouse. Fifty-three male, hairless mice were randomly assigned to 4 groups and pretreated in two different time intervals with different doses of human bFGF. Microvascular perfusion of the skin flaps was determined over a 5-day period by means of intravital microscopy after intravenous injection of the fluorescence marker fluorescein isothiocyanate-dextran (M(r) 150,000). Human bFGF (2,700 ng) injected 6 days before flap creation could not improve perfusion of the flap (n = 10) when compared with controls. However, when applied 18 days before flap creation (n = 13), the same dose resulted in a significant reduction of nonperfused tissue at day 5 after flap creation (12.3% vs 26.8%, p < 0.01). Eighteen-day pretreatment with 1,200 ng (n = 10) and 480 ng (n = 10) had no significant effect on skin flap perfusion. We conclude, therefore, that successful pretreatment with bFGF for prevention of skin flap necrosis is time and dose dependent.     

Expression of a recombinant human growth hormone binding protein in baculovirus/insect cell system

An eucaryotic recombinant human growth hormone binding protein (rGHBP) was expressed in baculovirus-infected insect cells and purified by affinity chromatography from culture supernatant. This mannose-rich 34-kDa protein specifically bound human growth hormone (hGH) with the same affinity (kDa = 0.42 x 10(-9) M) than the 51.5 kDa GHBP we purified and characterised from human plasma (kDa = 1.1 x 10(-9) M). A high molecular form of the rGHBP was detected by silver-stained SDS-PAGE, Western blot (mAb 263), affinity cross-linking and Western ligand blot with 125I-hGH. Reduction experiments with beta-mercaptoethanol suggested that this form involved a disulfide bound between two rGHBPs.     

Structure and function of bovine growth hormone. Bovine growth hormone as an experimental model for studies of protein-protein interactions

Growth hormone (GH) is a polipeptide that controls the differentiation, growth and metabolism of many cell types, and is secreted from the hypophysis of all vertebrate species tested so far. Despite the overlapping evolutionary, structural, immunological and biological properties, it is well-known that GHs from distinct mammalian species have significant species-specific characteristics. The main purpose of this review is to highlight bovine GH (bGH) structural features related to its species-specific properties. Novel interest in bGH is also aroused by the advent of biotechnological methods for production of recombinant proteins. In fact recombinant bGH will have a great importance in veterinary medicine research and as a 'high tech' drug that needs to be monitored in zootechnical productions.     

Attenuation of skeletal muscle wasting with recombinant human growth hormone secreted from a tissue-engineered bioartificial muscle

Skeletal muscle wasting is a significant problem in elderly and debilitated patients. Growth hormone (GH) is an anabolic growth factor for skeletal muscle but is difficult to deliver in a therapeutic manner by injection owing to its in vivo instability. A novel method is presented for the sustained secretion of recombinant human GH (rhGH) from genetically modified skeletal muscle implants, which reduces host muscle wasting. Proliferating murine C2C12 skeletal myoblasts stably transduced with the rhGH gene were tissue engineered in vitro into bioartificial muscles (C2-BAMs) containing organized postmitotic myofibers secreting 3-5 microg of rhGH/day in vitro. When implanted subcutaneously into syngeneic mice, C2-BAMs delivered a sustained physiologic dose of 2.5 to 11.3 ng of rhGH per milliliter of serum. rhGH synthesized and secreted by the myofibers was in the 22-kDa monomeric form and was biologically active, based on downregulation of a GH-sensitive protein synthesized in the liver. Skeletal muscle disuse atrophy was induced in mice by hindlimb unloading, causing the fast plantaris and slow soleus muscles to atrophy by 21 to 35% ( < 0.02). This atrophy was significantly attenuated 41 to 55% (p < 0.02) in animals that received C2-BAM implants, but not in animals receiving daily injections of purified rhGH (1 mg/kg/day). These data support the concept that delivery of rhGH from BAMs may be efficacious in treating muscle-wasting disorders.     

Use of recombinant human follicle-stimulating hormone for in vitro fertilization-embryo transfer after severe systemic immunoglobulin E-mediated reaction to urofollitropin

OBJECTIVE: To report the successful use of recombinant human FSH in a patient with a history of a severe systemic immunoglobulin E (IgE)-mediated reaction to urofollitropin. SETTING: University hospital. PATIENT: A 38-year-old infertility patient who had experienced a whole-body rash with hives, tachycardia, fever, and other symptoms after urofollitropin administration, with confirmation of IgE-mediated sensitivity to urofollitropin by intradermal skin testing. INTERVENTION: In vitro fertilization-ET after the use of recombinant human FSH to stimulate ovarian follicular development. RESULTS: Establishment of a clinical pregnancy. CONCLUSIONS: Our case provides evidence for the involvement of nongonadotropin proteins as the direct cause of some adverse reactions from conventional urine-derived human FSH preparations.     

Reprioritization of liver protein synthesis resulting from recombinant human growth hormone supplementation in parenterally fed trauma patients: the effect of growth hormone on the acute-phase response

BACKGROUND: One of the major components of the metabolic response to severe trauma is the alteration in concentrations of a large number of plasma proteins referred to as acute-phase proteins (APP). The principle mediators of these liver-synthesized APP are mainly the cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). METHODS: We have measured the plasma levels of IL-6, TNF alpha, and 20 APP in 24 adult, severely injured, hypermetabolic and highly catabolic patients with multiple injuries within 48-60 hours after injury, when they were receiving maintenance fluids without calories or nitrogen, and subsequently during 7 days of total parenteral nutrition with (n = 12) or without (n = 12) recombinant human growth hormone supplementation (rhGH, 0.15 mg/kg/d). RESULTS: Baseline positive APP due to severe trauma include C-reactive protein (CRP), alpha-1 antichymotrypsin, alpha-1 acid glycoprotein, alpha-1 antitrypsin, fibronectin, and factor B. Negative APP include IgG, IgM, complement-3, prealbumin, transferrin, ceruloplasmin, and albumin. Except for CRP, alpha-1 antichymotrypsin, and albumin, all the APP levels increase during 7 days of nutritional support. Plasma levels of cytokines IL-6 and TNF-alpha, although initially markedly increased after injury, decrease with parenteral refeeding. There is a linear correlation between CRP and IL-6 levels and also between the transport proteins prealbumin and transferrin. Trauma-induced increases in CRP and IL-6 levels decreased with nutrition alone, but did not change with rhGH supplementation. An immunosuppressed state of injury is evident from the decreased immunoglobulin levels (IgG, IgM, IgA) in the trauma patients. Total parenteral nutrition alone increases the immunoglobulin levels to normal. However, with adjuvant rhGH, only IgA levels are normalized. CONCLUSIONS: Adjuvant rhGH therapy does not attenuate the reprioritization of acute liver protein synthesis and results in only limited restoration of host defenses. The clinical implications of these findings await further study.     

Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli

A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. Cys182-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (Tv18-19) and the corresponding tryptic peptide from rhGH (T18+19) were identical. Electrospray mass spectrometry (ES MS) of Tv18+19 isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T18+19 from rhGH. A synthetic Tv18+19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragment in ES/MS compared to Tv18+19 isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cys182-SSS-Cys189) bridge not found in the corresponding T18+19 disulfide peptide. Furthermore, the synthetic Tv18+19 co-eluted in RP-HPLC with Tv18+19 isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T18+19 and Tv18+19 peptides were performed. Using these data all protons were assigned. The major chemical shift changes (delta delta > 0.05 ppm) observed were for the beta-protons of Cys182 and Cys189 in Tv18+19 as compared to T18+19. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties.