Delivery Of Factors Research Articles

Cell-mediated delivery of neurotrophic factors and neuroprotection in the neostriatum and substantia nigra

Cell-mediated delivery of neurotrophic factors and neuroprotection in the neostriatum and substantia nigra

Polymer-encapsulated cells genetically modified to secrete human nerve growth factor promote the survival of axotomized septal cholinergic neurons.

Effective treatments for neurodegenerative disorders are limited by our inability to alter the progression of the diseases. A number of proteins have specific neuroprotective activities in vitro; however, the delivery of these factors into the central nervous system over the long term at therapeutic levels has been difficult to achieve. BHK cells engineered to express and release human nerve growth factor were encapsulated in an immunoisolation polymeric device and transplanted into both fimbria-fornix-lesioned rat brains and naive controls. In the lesioned rat brain, chronic delivery of human nerve growth factor by the encapsulated BHK cells provided nearly complete protection of axotomized medial septal cholinergic neurons. Human nerve growth factor continued to be released by encapsulated cells upon removal from the aspirative site after 3 weeks or from normal rat striatum after 3 and 6 months in vivo. Long-term encapsulated cell survival was confirmed by histologic analysis. This encapsulated xenogeneic system may provide therapeutically effective amounts of a number of neurotrophic factors, alone or in combination, to virtually any site within the body.

Delivery of growth factor to wounds using a genetically engineered biological bandage.

Increasing the rate of wound healing of acute wounds and promoting the closure of chronic ulcers is an important goal in wound therapy. Growth factors have been shown to facilitate this process; however, the systems described for growth factor delivery are not ideal. In the present report we demonstrate the feasibility of a new method of delivering growth factors to the wound site using a genetically engineered biological bandage. The bandage consists of keratinocytes (SCC-13 cells) that are engineered by gene transfer to produce high levels of bovine growth hormone (bGH). bGH was selected for these studies because it can be easily distinguished from rat and human growth hormone in wound fluids and culture medium. The bGH-producing cells are contained and maintained in serum-free medium inside an envelope composed of a low protein binding, 0.2 micron pore size, polysulfone membrane. The genetically engineered cells cannot escape from the bandage, but the bGH is freely released into the surrounding culture medium. When placed onto a full-thickness, surgically generated wound on rats, the cells within the bandage continue to produce and release bGH into the wound for at least 3 days. This system is a safe and reliable way of providing real-time delivery of any desired biomolecule into the wound site.

Use of endocrine therapy to study the biology of breast cancer

Drug resistance is the main reason for therapeutic failure and death in breast cancer. In vitro investigations have identified some mechanisms that may be responsible for resistance to chemotherapeutic agents in breast cancer, but we know little about the mechanisms responsible for resistance to endocrine treatment in receptor-positive tumors. Accordingly, a major task for future studies is to explore the mechanisms of both primary and acquired resistance to endocrine treatment as well as to chemotherapy in breast cancer. Drug resistance may be related to alterations in endocrine as well as paracrine delivery of certain growth factors or hormones, but it may also be caused by alterations in drug disposition. Primary resistance may be evaluated by comparing the response to drug treatment with the expression of different biochemical parameters in the tumor before treatment. To explore the mechanisms of acquired drug resistance requires study of the changes in biological parameters developing over time during drug therapy. Also, it may be of interest to use certain drugs (like a 'classical' anti-estrogen and a steroidal anti-estrogen) in a particular sequence to explore the possibility of cross-resistance (or lack of it) between particular treatment modalities. Possible implications for the design of future trials in breast cancer treatment are discussed.

Restoration of juvenile baseline growth hormone secretion with preservation of the ultradian growth hormone rhythm by continuous delivery of growth hormone-releasing factor.

The ability of continuously delivered GH-releasing factor (GRF) to enhance GH secretion while maintaining the normal ultradian GH rhythm was investigated. Synthetic human GH-releasing factor (hGRF(1-44)NH2) was continuously infused for 4 days by means of i.v. catheters to 11-week-old broiler chickens. At this age, overall endogenous GH secretion is low, and baseline GH is barely detectable. Six birds per treatment received vehicle (control), 0.324 mg hGRF(1-44)NH2/kg body weight per day (low dose) or 3.24 mg hGRF(1-44)NH2/kg body weight per day (high dose). After 4 days of GRF conditioning, concurrent with continued GRF infusion, serial blood samples were removed via atrial catheters at 15-min intervals for 6 h and GH plasma profiles determined. High dose GRF significantly increased GH plasma concentrations over tenfold compared with controls; however, most of this increase reflected an increase in basal GH, which was reinstated to juvenile baseline levels. Augmentation of pulse amplitude above this increased baseline was not proportionately as high, and failed to reach juvenile levels. The ultradian rhythm of GH was not altered by continuous GRF administration. Both low and high dose GRF treatments resulted in significant enlargement of the anterior pituitary gland. Total pituitary GH mRNA levels, although elevated over twofold by GRF treatment, were not significantly different from controls. Measures of plasma GH magnitude (overall and baseline mean, and peak amplitude) were significantly correlated with pituitary GH mRNA for control birds, but were not correlated for GRF treatments. Feed intake was markedly depressed (33%) on the high dose GRF treatment, in conjunction with total inhibition of body weight gain over the 4-day period of administration. Longitudinal bone growth and width of the epiphyseal growth plate were also significantly reduced by high dose GRF treatment, probably reflecting the reduced level of nutrient intake, despite high circulating concentrations of GH.

Controlled Release of Fibroblast Growth Factor: Activity in Cell Culture

ABSTRACTBasic fibroblast growth factor is a multi-potent cell regulatory factor that stimulates proliferation and angiogenesis. Controlled studies of basic fibroblast growth factor in animals have been hindered by the instability of this protein. In addition, many cells appear to require the continuous addition of basic fibroblast growth factor for optimal growth and function in culture. A system for the sustained delivery of active basic fibroblast growth factor might provide both a means to conduct log-term studies on activity and provide a practical alternative to multiple growth factor additions to cell cultures. Basic fibroblast growth factor was incorporated into standard polymer matrices, but the released growth factor had lost over 99% of its bioactivity. Loss of basic fibroblast growth factor activity was found to result from both physical inactivation and adsorption of the protein to surfaces. These problems were avoided by incorporating the growth factor into calcium cross-linked alginate microspheres containing heparinsepharose beads. Basic fibroblast growth factor was incorporated into these microspheres with 71% efficiency and active growth factor was released with predictable kinetics for up to 7 weeks. Release from these microspheres was controlled by the amount of heparin within the device and could be manipulated by simply altering the heparin content during fabrication. Alginate/heparin-sepharose microspheres were placed into growing cultures of bovine aortic endothelial cells and no cytotoxic effects were observed. Furthermore, microspheres containing growth factor provided long-term stimulation of cell proliferation and maintenance of endothelial cell morphology.

Chronic intracerebroventricular CRF infusion attenuates ACTH-corticosterone release

Bolus intracerebroventricular delivery of corticotropin-releasing factor (CRF) elicits acute responses of both the pituitary-adrenal axis and the sympathetic nervous system. We examined whether these stresslike responses could be maintained over a period of days by central delivery of CRF in nonstressed rats, as would be predicted if this peptide participates in the central nervous system regulation of chronic stress. CRF (4.3 or 21.5 micrograms/day) was continuously delivered into the cerebral ventricle via Alzet minipumps. In contrast to saline-infused controls, rats receiving CRF exhibited elevated excretions of corticosterone, norepinephrine, and urea nitrogen for several days. Thereafter, an attenuation of CRF responsiveness occurred when corticosterone excretion returned to basal levels despite continued central CRF infusion. However, CRF delivered intravenously during attenuation stimulated adrenocorticotropic hormone and corticosterone secretion, implicating a hypothalamic rather than pituitary locus for central CRF resistance. The present data do not permit a conclusion on whether the attenuation of the CRF response with time is the result of an ultrashort-loop negative-feedback mechanism or CRF receptor desensitization.

Use of osmotic pumps for subcutaneous infusion of growth hormone-releasing factors in steers and wethers.

Osmotic pumps were evaluated for 7-d delivery of growth hormone-releasing factor (GRF). In Exp. 1, 12 steers weighing 253 kg received hGRF(1-29)NH2 in H2O at rates of 0, 3, 30 and 300 pmol.h-1.kg-1. Pumps were implanted s.c. on d 0 and removed at 1200 on d 7. Blood samples were drawn at 20-min intervals from 0800 to 1200 on d -1, 1, 3, 5, 7 and 9. Growth hormone levels were not altered by GRF treatment (P greater than .05). Solubility and volume limitations render hGRF(1-29)NH2 delivery via osmotic pumps problematical. Flow rate and duration of release of dimethyl sulfoxide (DMSO):H2) (1:1) from osmotic pumps incubated in vivo and in vitro were found to be consistent with manufacturer's specifications. Two hGRF(1-29) analogues, Ro23-7863 and 4SG-29, were dissolved in DMSO:H2O. In Exp. 2, six 222-kg steers had pumps implanted and blood samples were taken as in Exp. 1. Three steers received each analogue at a rate of 300 pmol.h-1.kg-1. Analogues had similar GH-releasing ability and GH levels differed (P less than 0.001) among days, being approximately fourfold higher on d 3, 5 and 7 than on d -1, 1 and 9. Residual analogue solutions retained full bioactivity after 7-d implantation, and in vitro biopotencies of Ro23-7863 and 4SG-29 were similar (Exp. 3). In Exp. 4, 15 wethers (means = 31.3 kg) received osmotic pumps delivering 0, 3, 15, 75 and 300 pmol.h-1.kg-1 Ro23-7863 in DMSO:H2O for 7 d. Lambs were bled at 0800 and 1400 from d -1 to 8. The latter two doses increased (P less than .01) mean GH levels 2.7- and 4.3-fold over those in control animals during the treatment period. Results demonstrate that increased GH secretion can be elicited in steers and wethers for 1 wk by continuous s.c. infusion of GRF analogues utilizing osmotic pumps.

Secretion of a hyperemia-inducing moiety by mitogen or glycogen stimulated mononuclear inflammatory cells of sheep and rabbit.

A nonlymphokine mediator of hyperemia has been shown to be secreted by both rabbit peritoneal exudate cells stimulated with 5% glycogen and by concanavalin A treated cells from afferent lymph of sheep. This mediator is not stored in an active form in cells, but is either activated or synthesized de novo in response to antigenic or mitogenic stimuli. Secretion of the mediator is inhibited by culturing cells in the presence of dexamethasone but not indomethacin. However, expression of the activity is inhibited by pretreatment of the assay animals with indomethacin, suggesting a two-step induction of hyperemia. A similar mediator was found to be secreted by sheep and rabbit alveolar lavage cells, and rat peritoneal exudate cells. The presence of this hyperemia-inducing activity from diverse species suggests a fundamental role in controlling blood flow. Alteration of blood flow may modulate delivery of blood-borne cells and factors to sites of chronic inflammation.