Recombinant Growth Research Articles

The passage of granulocyte-macrophage colony-stimulating factor across the human placenta perfused in vitro

The purpose of this study was to investigate the placental passage of granulocyte-macrophage colony-stimulating factor in a placental perfusion model ex vivo. In an open system, 11 placentas were perfused on both the maternal and the fetal side immediately after delivery. Granulocyte-macrophage colony-stimulating factor was added to the maternal perfusion medium in concentrations from 10-55 micrograms/mL. Maternal and fetal samples were taken, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was measured by enzyme-linked immunosorbent assay. Accumulation of granulocyte-macrophage colony-stimulating factor in the fetal circuit averaged 2.42% of the concentration added initially to the arterial portion of the maternal circuit. There is only low transfer of GM-CSF across the fetal membranes. This finding is particularly remarkable in view of recently published results suggesting that administration of recombinant granulocyte growth factors to pregnant women with imminent preterm delivery helps prevent neonatal sepsis.

Characterization of Chemotactic Migration and Growth Kinetics of Canine Knee Ligament Fibroblasts

Migration and proliferation of ligament fibroblasts are essential for the healing of ligament injuries. This study was designed to evaluate the migration of intraarticular (anterior and posterior cruciate) and extraarticular (medial and lateral collateral) ligament fibroblasts in response to cytokines and to determine the effect of cell passage on cell proliferation. Recombinant human platelet-derived growth factor, hepatocyte growth factor/scatter factor, and bone morphogenic protein-2 stimulated the migration of all ligament cells in a dose-dependent manner, with optimal migration at 10 ng/ml. Recombinant human epithelial growth factor preferentially stimulated the migration of intraarticular ligament fibroblasts, whereas recombinant human interleukin-1 was more effective with extraarticular ligament fibroblasts. Recombinant human insulin-like growth factor-1, insulin-like growth factor-2, transforming growth factor-beta, and fibroblast growth factor had no significant effect on the migration of ligament-derived fibroblasts. These data suggest that specific cytokines stimulate the migration of knee ligament fibroblasts and provide a rationale for possible therapeutic approaches to optimize ligament healing. Fibroblasts derived from the anterior cruciate ligament have been shown to proliferate at a slower rate than those derived from the medial collateral ligament. We have extended these observations and have demonstrated that fibroblasts from both the posterior and anterior cruciate ligaments proliferate at a slower rate than lateral and medial collateral ligament-derived fibroblasts. The differences between the growth rates of intraarticular and extraarticular fibroblasts become insignificant with serial passaging of the cells.

In vitro transduction of human osteoblast cell populations with retroviral vectors

The involvement of cytokines in degeneration and inflammation of human tissue is well established. Interleukin-1 (IL-1) is a major agent in the pathophysiology of periarticular bone resorption in rheumatoid arthritis and in osteoporosis. Because the use of recombinant cytokines and growth factors is limited due to their short half lives, techniques are needed to get a permanent release of these therapeutic proteins. The rational of this study was to show that retroviral transduction of human osteoblastic cells is possible in vitro using the marker gene LacZ and the potentially therapeutic gene encoding for human interleukin-1 receptor antagonist protein (IL-1Ra). Different transduction techniques were combined to improve the rate of transduction in vitro. Osteoblastic cells were isolated from human spongious bone and cultured in vitro. The beta-galactosidase (LacZ) gene and the cDNA of IL-1Ra were introduced into the isolated cells by retrovirus mediated gene transfer. LacZ activity was determined by Xgal staining, IL-1Ra was measured quantitatively by ELISA. The transfer of retroviral IL-1Ra led to IL-1Ra expression of 8614 to 10,089 pg IRAP/50,000 cells/48 h. By combining different techniques to improve transduction, the X-gal staining established a rate of transduction of 60%. Our results demonstrate that retroviral transduction of human osteobalstic cells is possible in vitro, and leads to high levels of the synthesized transgene product. The rate of retroviral transduction can be accelerated in vitro.

Effects of a synthetic growth hormone-releasing peptide on growth and carcass traits of pigs

The current study was to determine the effects of growth hormone-releasing peptide ([His1, Lys6]-GHRP) on growth performance and carcass traits in pigs, and to determine whether the results were comparable to those of recombinant porcine growth hormone (r-PGH) treatment. Thirty Landrace and Yorkshire pigs (40–45 kg) were randomly divided into three groups, each group consisted of two boars, five gilts and three barrows. Peptides were administered daily by intramuscular injection with the following schemes and dosages: Group A ([His1, Lys6]-GHRP, 200 μg/day in 0·5 ml saline), Group B (r-PGH, 3 mg/day in 0·6 ml saline), and Group C (sterilized physiological saline, 0·5 ml/day). [His1, Lys6]-GHRP caused significant improvements both in pig growth and carcass traits. The average daily gain (ADG) and feed efficiency of the [His1, Lys6]-GHRP treated group were higher than those of the control, but less than with the r-PGH treatment. The net increase in the ADG by [His1, Lys6]-GHRP was c. 14%, and was 23% by r-PGH (both P<0·01). The feed efficiency was improved 17% by [His1, Lys6]-GHRP (P<0·01) and 20% by r-PGH (P<0·01). On the other hand, the back-fat thickness in both [His1, Lys6]-GHRP and r-PGH treated groups (1·76±0·041 cm and 1·72±0·040 cm, respectively) was considerably less (P<0·05) than that of the control group (1·81±0·040 cm). In addition, the lean cuts percentage and the rib longissimus muscle area were comparable in the [His1, Lys6]-GHRP and r-PGH treatments (lean cuts percentage: 57±2·0% and 57±1·5%, respectively; rib longissimus muscle area: 32·8±0·91 cm2 and 32·9±1·10 cm2, respectively), and both were significantly (P<0·05) greater than those of the control group (lean cuts percentage: 53±1·5%; rib longissimus muscle area: 28·3±1·61 cm2). In conclusion, [His1, Lys6]-GHRP appeared to be a promising tool for controlling carcass fat in pigs.

Inhibition of stem cell factor- and nerve growth factor-induced morphological change by wortmannin in mast cells.

Recombinant murine stem cell factor (rmSCF) or recombinant murine nerve growth factor (rmNGF) induced the morphological change of large numbers of rat peritoneal mast cells (RPMC). We investigated the role of phosphatidylinositol 3'-kinase (PI3-kinase) in receptors-mediated morphological change in RPMC. Exposure of RPMC to PI3-kinase inhibitor, wortmannin, before the addition of rmSCF and rmNGF antagonized those factors-induced morphological change. These results suggest that the Pl3-kinase is involved in the signal transduction pathway responsible for morphological change following stimulation of rmSCF and rmNGF and that wortmannin blocks these responses.

Synthesis of mini-proinsulin precursors using N-termini of human TNF ? as fusion partners in recombinant Escherichia coli

Synthesis of human mini-proinsulin precursors was investigated in controlled fed-batch cultures at high cell concentrations of recombinant Escherichia coli. Transcription of the recombinant gene was controlled by a T7 promoter system. The human mini-proinsulin was prepared by substituting a C-chain peptide of natural proinsulin with a peptide sequence of only nine amino acids. The reduced size of fusion proinsulin and hence the increased purity of human insulin in the recombinant product may contribute to increasing the fermentation yield of human insulin. Three precursors (T1-, T2-, and T3-M2PI) were constructed by utilizing the N-terminus residues of human tumor necrosis factor α as fusion partners. The T2 precursor was most soluble in the cytoplasm, and exerted the most inhibitory effect on recombinant cell growth. In the production of T2-M2PI, significant amounts of undesirable metabolic by-products (acetate and ammonia) accumulated in the culture broth even at very low specific cell growth rate. The major portion of all synthesized precursors aggregated to insoluble inclusion bodies but the protein aggregates were easily converted to monomers in the presence of the anionic detergent (SDS) without using any reducing agent. With the expression of T1-M2PI, growth inhibition was minimal, and the maximum volumetric yield of mini-proinsulin (M2PI) in fermentation cultures was at the highest level among the synthesized precursors.

A model of consumers' risk perceptions toward recombinant bovine growth hormone (rbGH): the impact of risk characteristics.

This study estimates the effect risk characteristics, described as outrage factors by Hadden, have on consumers' risk perceptions toward the food-related biotechnology, recombinant bovine growth hormone (rbGH). The outrage factors applicable to milk from rbGH treated herds are involuntary risk exposure, unfamiliarity with the product's production process, unnatural product characteristics, lack of trust in regulator's ability to protect consumers in the marketplace, and consumers' inability to distinguish milk from rbGH treated herds compared to milk from untreated herds. An empirical analysis of data from a national survey of household food shoppers reveals that outrage factors mediate risk perceptions. The results support the inclusion of outrage factors into the risk perception model for the rbGH product, as they add significantly to the explanatory power of the model and therefore reduce bias compared to a simpler model of attitudinal and demographic factors. The study indicates that outrage factors which have a significant impact on risk perceptions are the lack of trust in the FDA as a food-related information source, and perceiving no consumer benefits from farmers' use of rbGH. Communication strategies to reduce consumer risk perceptions therefore could utilize agencies perceived as more trustworthy and emphasize the benefits of rbGH use to consumers.

Intracranial delivery of recombinant nerve growth factor: release kinetics and protein distribution for three delivery systems.

Three different polymeric delivery systems, composed of either poly(ethylene-co-vinyl acetate) (EVAc) or poly(lactide-co-glycolide) (PLGA), were used to administer recombinant human nerve growth factor (rhNGF) intracranially in rats. The delivery systems were characterized with respect to release kinetics, both in the brain and in well-stirred buffer solutions. During incubation in buffered saline, the delivery systems released rhNGF in distinct patterns: sustained (EVAc), immediate (PLGA1) and delayed (PLGA2). One 10-mg delivery system was implanted in each rat and an ELISA technique was used to determine the amount of rhNGF in 1-mm coronal brain slices produced immediately after removal of the delivery system. High levels of rhNGF (as high as 60,000 ng in a brain slice of approximately 50 microliters) were recovered from the brain tissue at 1, 2, and 4 weeks after implantation. With all three delivery systems, the amount of rhNGF in each brain slice decreased exponentially with distance from the implant site: the distance over which concentration decreased by 10-fold was 2-3 mm for all delivery systems. When rhNGF release was moderate (10 to 200 ng rhNGF/day), the total amount of rhNGF in the brain increased linearly with release rate, suggesting an overall rate of rhNGF elimination of 0.4 hr-1 or a half-life of 1.7 hr. With higher release rates (500 to 50,000 ng rhNGF/day), total amounts of rhNGF in the brain were considerably higher than anticipated based on this rate of elimination. Polymeric controlled release can provide high, localized doses of rhNGF in the brain. All of the experimental data were consistent with penetration of rhNGF through the brain tissue with a diffusion coefficient approximately 8 x 10(-7) cm2/s, which is approximately 50% of the diffusion coefficient in water.

Thyroid hormones down-regulate thyrotropin β mRNA level in vivo in the turbot (Psetta maxima)

Thyroid stimulating hormone (TSH) is a vertebrate pituitary heterodimeric hormone that stimulates the thyroid gland to produce the thyroid hormones, T3 and T4. We report here the cloning, by PCR on reverse-transcribed pituitary RNAs, of a 180 bp fragment of the cDNA encoding TSH β subunit in the turbot (Psetta maxima). The deduced amino acid sequence displayed 66 and 75% identity with the corresponding sequence from the European eel (Anguilla anguilla) and the rainbow trout (Oncorchyncus mykiss), respectively. This cDNA was then used as a probe for densitometric analysis of individual pituitary Northern blots. TSH β mRNA levels were quantified in turbot where circulating thyroid hormones were modified by dietary treatments or hormone supplementation. Recombinant rainbow trout growth hormone had no effect on circulating thyroid hormone levels or on pituitary TSH β mRNA level. In turbot fed heat-treated rapeseed meal, plasma T4 levels were lowered and TSH β mRNA increased more than two fold. In contrast, when turbot were fed a standard fish-meal supplemented with T3, circulating T3 levels were elevated and there was a dramatic decrease in TSH β mRNA level. It is concluded that both thyroid hormones are able to down-regulate TSH β mRNA level in vivo in the turbot. These results are discussed in the context of the evolution of the TH feed-back on TSH production.

Isolation and characterization of a novel bladder cancer cell line: inhibition by epidermal growth factor.

A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells' proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.

Systemic Effects of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor in Combination with Recombinant Murine Granulocyte Colony-Stimulating Factor in a Murine Model of Myelosuppression

Systemic Effects of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor in Combination with Recombinant Murine Granulocyte Colony-Stimulating Factor in a Murine Model of Myelosuppression

Growth hormone replacement in patients with Langerhan’s cell histiocytosis

OBJECTIVESTo assess the impact of growth hormone on growth and the underlying disease in children with growth hormone deficiency as a result of Langerhan’s cell histiocytosis.STUDY DESIGNRetrospective analysis of data...

Effects of Synovex-S and recombinant bovine growth hormone (Somavubove) on growth responses of steers: III. Muscle growth and protein responses.

We conducted this study to determine whether the growth responses of specific skeletal muscles in crossbred beef steers were differentially affected by treatment with recombinant bovine growth hormone (Somavubove, SbV, .1 mg/kg BW, i.m., daily), Synovex-S (200 mg progesterone + 20 mg 17-beta estradiol benzoate, SYN, ear implant), or a combination of the two. Starting body weights of steers averaged 182+/-1.8 kg. Five steers were used at this average BW to obtain data on weight and composition of individual muscles at d 0, and 20 other steers were assigned in equal numbers to control (C, no implant and placebo daily injection), SYN, SbV, and SYN + SbV treatment groups. After 56 d of treatment with placebo or growth promoters, complete rectus femoris (RF), triceps brachii (TB), supraspinatus (SS), psoas major (PM), and semitendinosus (ST) muscles were dissected, weighed, and then ground for determination of moisture, total protein, and fat. To calculate the average daily muscle wet weight, protein, and fat gains, the initial weight, protein content, and fat content of a muscle were subtracted from those obtained at slaughter and the difference divided by 56. Muscle weight was increased over C in TB and SS by SYN (P < .1); in TB by SbV (P < .09); and in RF (P < .05), TB (P < .03), and SS (P < .03) by SYN + SbV. Overall average daily wet tissue gain was increased over C by SbV + SYN (P < .05) in RF, TB, and SS. Average daily protein gain in RF and TB was increased by SYN (P < .1), SbV (P < .06), and SYN + SbV (P < .01) over that calculated for C. For RF, TB, and SS, average daily protein gain was greater (P < . 1) in SbV + SYN than that obtained with SbV or SYN alone. These data suggest that administration of growth promoters, such as somatotropin and Synovex, to cattle differentially affects growth characteristics in certain muscles and can have additive effects on protein gain when used together.

Topical Platelet-Derived Growth Factor for Diabetic Foot Ulcers

A topical gel formulation of recombinant human platelet-derived growth factor (becaplermin) was recently approved in the U.S. to promote healing of

Clinical Trial: Hematopoietic Progenitor Cell Transplantation in Breast Cancer: Current Status and Future Directions

Breast cancer remains the second leading cause of cancer death despite numerous advances in medical science. In vitro, preclinical, and clinical trials have shown that chemotherapy dose intensity is an important component of therapy. Many clinical trials addressing the use of high-dose chemotherapy and hematopoietic cellular rescue have been conducted over the past decade. Early trials undertaken in heavily pretreated patients who had metastatic disease were associated with high treatment-related mortality rates; good response rates were noted but overall survivals were short. Subsequent technological advances, including the use of recombinant hematopoietic growth factors and peripheral blood progenitor cells as the source of cellular rescue, have dramatically lowered the morbidity and mortality of the procedure, as well as shortened hospital stay and markedly reduced cost. As a result, the high-dose chemotherapy approach has been used earlier in the disease course, both in patients with metastatic disease who were responding and in the adjuvant setting in patients at high risk for relapse. Results of many of these phase II trials are extremely encouraging, and phase III prospective, randomized trials comparing auto-transplant to conventional approaches are currently under way. This review discusses past, current, and future initiatives of this modality. Included is a discussion of new preparative regimens, the addition of agents such as biochemical modifiers to enhance antitumor activity, and issues regarding timing of auto-transplant, stem cell technology, use of allogeneic stem cells, and posttransplantation therapies.

Purification and characterization of recombinant bovine growth hormone produced in Escherichia coli

Using the pUBJ10 plasmid containing the modified bovine growth hormone (bGH) cDNA, large production has been attempted in E. coli BL21 strain. The bGH was highly expressed upto the level of 35% of total cell proteins by IPTG induction and temperature shift to 40°C. The recombinant bGH (rbGH) was isolated from inclusion bodies by solubilization in 10 M urea and followed by DEAE-TOYOPEARL 650C ion exchange and Sephadex G-100 column chromatography. The pUBJ10-derived bGH was eluted at 25.28 min similar to the standard bGH (at 25.18 min) by reverse-phase HPLC. The analysis of N-terminal amino acid showed that the mature bGH has glutamic acid as a first amino acid in agreement with DNA sequencing data. The biological activity was indirectly measured by radioreceptor assay and compared with a pituitary-derived bGH.

Recombinant insulin-like growth factor I normalizes expression of renal glucose transporters in diabetic rats.

By immunocytochemistry we have studied the effect of recombinant human insulin-like growth factor I (rhIGF-I) on expression of renal GLUT-1, -2, and -5 in rats with streptozotocin (STZ)-induced diabetes. In the renal tubules of these rats, expression of GLUT-1 was reduced and that of GLUT-2 was increased. GLUT-1 expression was restored, and GLUT-2 expression was normalized by 2-wk administration of rhIGF-I. We have shown that GLUT-5 was expressed at the brush-border membrane of the proximal convoluted tubules (PCT) of the cortex and at the glomerular mesangial cells (GMC) in normal rat kidney. In the diabetic rats, GLUT-5 expression was increased at both sites, along with an increase of GLUT-2 expression at the basolateral membrane of PCT, and was decreased to normal level at both sites by treatment with rhIGF-I. Thus, like GLUT-2, GLUT-5 is suggested to regulate glucose reabsorption in PCT. The relationship between overexpression of GLUT-5 in GMC and accumulation of sorbitol and advanced glycosylation end products are discussed. Regulation of GLUT expression may play an important role on renal glucose homeostasis.

Use of Hematopoietic Growth Factors in the Neonatal Intensive Care Unit

Recombinant hematopoietic growth factors have emerged as valuable treatments for a variety of medical conditions. Recently, their applications have reached the neonatal intensive care unit, where they offer new therapeutic options for problems as common as anemia of prematurity, or as catastrophic as neonatal sepsis. When facing bacterial infection, it is known that newborn infants are capable of increasing their serum G-CSF concentrations. However, their response does not reach the concentrations that adults are able to achieve, and frequently neutropenia complicates the picture of neonatal sepsis. Although Phase III clinical trials are still in progress, published animal studies, case reports, and Phase I trials suggest that neonates with a variety of neutropenias experience a rapid elevation in their blood neutrophil concentration following administration of rG-CSF, without significant adverse effects. Although many factors contribute to the development of the “anemia of prematurity,” one of the major factors is the inability of preterm infants to generate an erythropoietin (Epo) response appropriate to their degree of anemia. On the basis of this fact, administration of rEpo to preterm neonates to treat or to prevent the anemia of prematurity has been the subject of multiple clinical studies, and it is now clear that rEpo administration to this population can indeed result in lower transfusion requirements, with only occasional and mild adverse effects. Neonatal thrombocytopenia is also a frequent clinical problem, which in most patients develops without a clear underlying cause. Recent studies, quantifying circulating megakaryocyte progenitors in the peripheral blood of thrombocytopenic neonates, suggest that impaired megakaryocytopoiesis may be the main underlying mechanism of many cases of thrombocytopenia. On the basis of this finding, it is tempting to speculate that recombinant thrombopoietin, the newly discovered physiological stimulator of platelet production, will be of clinical relevance in the treatment of thrombocytopenic neonates.

Recombinant insulinlike growth factor-I as therapy in states of altered carbohydrate homeostasis

Recombinant insulinlike growth factor-I as therapy in states of altered carbohydrate homeostasis

Effects of Recombinant Cytokines on Colony Formation by Irradiated Human Cord Blood CD34 + Hematopoietic Progenitor Cells

The role of recombinant hematopoietic growth factors in radiation repair has become a subject of increasing interest in both clinical and basic radiobiology. Combinations of cytokines such as hepatocyte growth factor, interleukin (IL)-3, IL-11, kit ligand, GM-CSF and erythropoietin were used to study the in vitro radiation dose response of human cord blood CD34+ hematopoietic progenitor cells using clonogenic survival assays. CD34+ cells were isolated by immunomagnetic selection and irradiated at 8 cGy/min. Irradiated cells were plated in methylcellulose with or without added cytokines, and hematopoietic colonies including CFU-GM, BFU-E and CFU-GEMM were scored on day 14. The radiation response characteristics of BFU-E and CFU-GEMM were similar for all culture conditions tested. The D0 values for BFU-E ranged between 1.29 and 2.40 Gy and n between 1.0 and 1.4. The D0 values for CFU-GEMM ranged from 86 cGy to 2.02 Gy and n between 1.0 and 1.5. The D0 for CFU-GM grown without added factors was 1.03 Gy. With single cytokine stimulation (IL-3, IL-11 or varying concentrations of HGF), D0 values ranged from 1.11 to 1.44 Gy. With the combination of IL-3, GM-CSF, kit ligand and HGF, D0 values were not significantly altered and ranged between 1.61 and 2.60 Gy. In contrast, the combination of IL-11 and HGF produced an increase in the shoulder of the radiation survival curve (n = 3.35). No increase in the shoulder was detected for any of the other conditions tested (n = 1.0-1.7). Thus the combination of HGF and IL-11 increased the radiation survival of hematopoietic progenitor cells forming CFU-GM. Understanding the mechanism by which combinations of early-acting growth factors support postirradiation recovery of primitive clonogenic hematopoietic cells may be relevant to the design of clinical protocols for improving hematopoietic recovery after total-body irradiation.