Propofol hemisuccinate suppression of experimental autoimmune encephalomyelitis.

Propofol hemisuccinate is a prodrug water soluble form of the lipophilic, phenolic compound propofol (2,6-di-isopropylphenol), that is the active ingredient in the widely used anesthetic agent Diprovan. Propofol binds to GABA(A) receptors but also has a phenolic structure that confers antioxidant properties to the molecule. The effects of propofol hemisuccinate in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses and time regimes. Propofol hemisuccinate, 100 mg/kg given three times a day from day 7 or day 12 until day 16 after disease initiation, significantly reduced maximal EAE score. Histology studies supported the clinical findings demonstrating reduction in the inflammatory response in the lumbar spinal cord in animals treated with propofol hemisuccinate. Decreased levels of nitrotyrosine and unchanged levels of induced nitric oxide synthase suggest propofol hemisuccinate crossed the blood brain barrier and exerted its effects by lowering reactive oxygen species levels. The results suggest that propofol hemisuccinate may provide an alternative mode of treatment for acute exacerbations of multiple sclerosis.

Gene therapy progress and prospects: recent progress in transgene and RNAi expression cassettes.

Plasmid expression cassette design must include a thoughtful analysis of potentially every nucleotide comprising a covalently closed circular or end-protected linear DNA. This review will discuss recent studies in unraveling the mechanisms of postdelivery gene silencing, codon optimization and promoter identification. The recent discovery of potent RNA interference (RNAi) mechanisms for sequence-specific gene silencing has also invoked a great deal of interest in development of expression cassettes that can produce double-stranded RNA molecules for RNAi. Expression cassettes based on both RNA polymerase II and polymerase III transcription units that generate double-stranded RNA molecules for RNAi will also be discussed.

Gene therapy strategies for the treatment of chronic viral hepatitis.

Chronic viral hepatitis is a major clinical problem, with over half a billion persons infected worldwide. Current therapies, principally treatment with recombinant IFN-alpha protein, have limited benefit. Recent studies suggest that gene-based expression of IFN-alpha is a possible therapeutic alternative that may improve the effectiveness of treatment. Gene delivery to the liver and consequent IFN-alpha expression therein, has the potential to concentrate the protein at the target organ and provide more continuous exposure to the therapeutic agent. Other potential gene and nucleic acid therapeutics for viral hepatitis are also being investigated. Key to the deployment of these future therapies is a suitable method of gene delivery. Although recombinant viral vector systems, such as adenovirus, are currently the most effective means of gene delivery to the liver, their use presents many concerns. These include immune and inflammatory reactions to the viral vector and possible adverse interactions between the recombinant virus and the pre-existing viral infection. Non-viral gene delivery systems would be a preferred treatment modality. The efficiency of current non-viral systems is not adequate for systemically administered liver gene therapy. However, recent use of membrane permeabilisation techniques has shown that high efficiency non-viral gene transfer agents are possible. The future coupling of these improved delivery systems with gene- or nucleic acid-based therapeutics currently in development holds out great promise for new generations of antihepatitis therapies.

Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation, regeneration, and trauma.

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up- or down-regulated during two phases of the retinoic acid-induced differentiation of the embryonal carcinoma line Ntera-2. To identify genes involved in neuronal regeneration we performed three GEM experiments, which included changes in gene expression in rat dorsal root ganglia during the healing of experimentally injured sciatic nerve, in regenerating neonatal opossum spinal cord, and during lipopolysaccharide stimulation of primary cultures of rat Schwann cells. Finally we have monitored genes involved in the recovery phase of the inflammatory disease of the rat spinal cord, experimental allergic encephalomyelitis, as well as those responsible for protection from oxidative stress in a glutamate-resistant rat hippocampal cell line. Analysis of the results of the approximately 70,000 data points collected is presented.

Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.

Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer/promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15-20 microgram/ml of canine factor IX was detected in the plasma of mice injected with 5.6 x 10(11) particles of an AAV vector for >5 months. The activated partial thromboplastin time of the treated mice was fully corrected to higher than normal levels. Liver-specific expression of canine factor IX was confirmed by immunofluorescence staining, and secreted factor IX protein was identified in the mouse plasma by Western blotting. All treated mice survived the tail clip test without difficulty. Thus, a single intraportal injection of a recombinant adeno-associated virus vector expressing factor IX successfully cured the bleeding disorder of hemophilia B mice, proving the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.

Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions.

The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1. High-level secretion was also observed for the hybrid chain as homodimers (V[HB]C[L]::V[HB]C[L]), which is not observed for chimeric chains consisting of a heavy chain variable region and light chain constant region, i.e. V[H]C[L] homodimers or single chains are not secreted. This indicates that regions within the variable domain, required for secretion of light chains, reside outside of the hypervariable regions (CDRs) and that the heavy chain CDRs and supporting residues do not prevent secretion. These results demonstrate the possibility of designing small, single-domain molecules possessing a given binding activity which may be secreted at high levels from mammalian cells.

Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A.

While the gene delivery vehicle is critical for the efficacy of human factor VIII gene therapy, optimization of the potency and duration of the factor VIII gene that is delivered is equally important in light of the poor transcription and translation characteristics of this gene. We discuss here a systematic approach to optimization of factor VIII complementary DNA expression by analysis of specific elements engineered into the transcription unit and other positions in the expression plasmid. Within the transcription unit we have engineered different 5' and 3' sequence modifications and tested them for factor VIII expression in human liver cells. These changes incorporate liver-specific promoter and enhancer sequences and regulatory elements affecting RNA export. Specifically, the thyroid hormone-binding globulin promoter and alpha 1 microglobulin/bikunin enhancer were tested and a synthetic 5' intron was compared to a 3' post-transcriptional regulatory element on factor VIII expression levels. For translation optimization, a leader sequence was designed to be of optimum length, have no RNA secondary structure and contain the optimal translation initiation sequence. Finally, we discuss areas for plasmid optimization, which include removal of near-consensus splicing sequences, the inclusion of strong transcription termination elements and the use of autonomous replicating plasmid sequences for episomal maintenance and enhanced plasmid retention for duration of gene expression.

A COOH-terminal peptide confers regiospecific orientation and facilitates atomic force microscopy of an IgG1.

An antibody (IgG1) was designed for oriented adherence to a metal-containing surface. This was achieved by adding a metal-chelating peptide, (CP = His-Trp-His-His-His-Pro), to the COOH-terminus of the heavy chain through genetic engineering. Electroporation of the engineered heavy chain gene into cells expressing the complimentary light chain yielded colonies secreting an intact antibody containing the metal-chelating peptide (IgG1-CP) which had high affinity for a nickel-loaded iminodiacetate column. Purified IgG1-CP was bound to nickel-treated mica and imaged by atomic force microscopy (AFM). Antibody lacking the COOH-terminal metal binding peptide failed to produce discernible AFM images. The AFM images of individual IgG1-CP molecules and their calculated dimensions demonstrated that regiospecific binding and uniform orientation of the antibody was imparted by the peptide. The ability to stably orient macromolecules in their native state to a surface may be used advantageously to visualize them.

Species specificity of iron delivery in hybridomas.

Studies with Human X Human (H X H), Human X Mouse (H X M), and Mouse X Mouse (M X M) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that H X H hybridomas do not respond to bovine transferrin a+ concentrations up to 100 micrograms/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. H X M and M X M hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on H X H hybridomas but was ineffective on H X M hybridomas. This demonstrated the functionality of the human transferrin receptor in H X H hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in H X M hybridomas. H X H and H X M hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. M X M hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.

Association of thrombin-antithrombin III complex with vitronectin in serum.

Purification of vitronectin by identical procedures from serum instead of plasma results in the coisolation of an additional protein component with mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 82 kDa. We show that this component is the thrombin-antithrombin III complex based on the following evidence. Similar to a complex constructed using purified thrombin and antithrombin III, the 82-kDa component has a reduced molecular size of 69 kDa if it is not boiled prior to SDS-PAGE. Upon prolonged boiling in SDS it dissociates into 56- and 32-kDa components which co-migrate in SDS-PAGE with purified antithrombin III and thrombin, respectively. The 82- and 56-kDa components react with an antiserum against antithrombin III, and an antiserum prepared against the 82-kDa complex reacts with purified antithrombin III. Thrombin-antithrombin III complex, from either serum or recalcified clotted plasma, bound to vitronectin immobilized on Sepharose or plastic. However, purified antithrombin III which had not reacted with thrombin lacked affinity for vitronectin as did antithrombin III from citrated plasma. Purified antithrombin III acquired affinity for immobilized vitronectin if it was complexed with thrombin or was modified by radioiodination. Binding of vitronectin to antithrombin III coated on plastic was demonstrated using enzyme-linked immunosorbent assay. These results demonstrate that vitronectin binds thrombin-antithrombin III complexes through a cryptic site in antithrombin III which can be exposed when antithrombin III is radioiodinated, bound to plastic, or complexed with thrombin. Since vitronectin can interact with cells, the binding of vitronectin to the thrombin-antithrombin III complex may serve to facilitate the interaction of this complex with cell surfaces.

Adhesion of platelets to laminin in the absence of activation.

The binding of platelets to components in the subendothelial matrix is an initial event in hemostasis and thrombosis. The glycoprotein components of the matrix are considered important in this interaction. Of these, collagen binds and activates platelets and induces their aggregation. In this study we demonstrate that substrate-bound laminin causes time- and concentration-dependent adherence of human platelets to the substrate. The binding of platelets to laminin was found to be similar in some respects, but different in others, to their binding to surfaces coated with fibronectin or collagen. The binding of platelets to laminin or fibronectin was not associated with their activation under conditions in which type I collagen activates the platelets as measured by [14C]serotonin secretion. Platelets bound to laminin and fibronectin differed in their appearance; they remained rounded on laminin whereas they flattened completely on fibronectin. Binding of platelets to fibronectin, but not laminin, is inhibited by a recently described peptide (Pierschbacher, M., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) containing the cell-attachment tetrapeptide sequence of fibronectin, which suggests that separate receptors exist for laminin and fibronectin. These studies establish laminin as a platelet-binding protein and suggest that laminin can contribute to the adhesiveness of exposed tissue matrices to platelets. Since laminin and fibronectin do not activate platelets, whereas collagen does, and laminin differs from fibronectin in that it does not induce spreading of the attached platelets, all three proteins appear to confer different signals to the platelets. Some of these may be related to platelet functions other than those necessary for the formation of a hemostatic plug.

Permissive effect of insulin on the adenosine 3',5'-monophosphate-dependent up-regulation of low density lipoprotein receptors and the stimulation of steroid release in bovine adrenal cortical cells.

Bovine adrenal cortical cells growth on extracellular matrix-coated dishes in the presence of F-12 medium supplemented with high density lipoprotein (30 micrograms protein/ml), insulin (50 ng/ml), transferrin (1 microgram/ml), and fibroblast growth factor (100 ng/ml) expressed high affinity low density lipoprotein (LDL) receptors (Kd = 2.0 X 10(-8) M) present at a density of 3 X 10(4) receptors/cell. The density of LDL receptors per cell could be increased 3-fold (9 X 10(4) receptors/cell) by incubating the cells for 24 h with cholera toxin (CT; 10 ng/ml), but not with insulin (100 ng/ml). This correlated with increased LDL internalization (10-fold) and degradation (5-fold). 11-Deoxycortisol (11 DOC) release was increased by 2.7-fold. The addition of insulin (100 ng/ml) together with CT (10 ng/ml) markedly potentiated the effects of CT on LDL binding, internalization, and degradation as well as on steroid release. Preincubation (24 h) of the cells with insulin (100 ng/ml) together with CT resulted in a 12.7-fold increase in high affinity LDL receptor density (3.6 X 10(5) receptors/cell), while LDL internalization and degradation were increased by 45- and 22-fold, respectively. This correlated with a 9.6-fold increase in the 11 DOC released into the medium. The effects of insulin on the induction of high affinity LDL receptors as well as increased internalization and degradation of [125I]LDL were both time and concentration dependent in cultures exposed to CT. Secretion of 11 DOC paralleled in a time-dependent manner the amount of internalized [125I]LDL. Exposure of the cells to chloroquine (100 microM) resulted in a 95% inhibition of [125I]LDL degradation correlating with an 80% decrease in 11 DOC released in cells preexposed to CT and insulin. The results of these studies suggest that in bovine adrenal cortex cells grown in chemically defined medium, insulin modulates steroidogenic cAMP-induced response by increasing both the LDL receptor pathway activity and 11 DOC release.

Factors involved in supporting the growth and steroidogenic functions of bovine adrenal cortical cells maintained on extracellular matrix and exposed to a serum-free medium.

Bovine adrenal cortex cells maintained on extracellular matrix (ECM)-coated dishes will proliferate actively when serum is replaced by HDL (25 micrograms protein/ml), insulin (10 ng/ml), and FGF (100 ng/ml). The cells have an absolute requirement for HDL in order to survive and grow. The omission of insulin, FGF, or both results in a slower growth rate and lower final cell density of the cultures. A requirement for transferrin (1 microgram/ml) becomes apparent only when cells have been grown for at least four generations in the absence of serum. Early passage (P1-P3) bovine adrenal cortex cells cultured in serum-free medium responded to ACTH (10(-8)M) with increased 11-deoxycortisol production; this effect was not observed in later passage cells (P7-P15). The cells' ability to utilize LDL-derived cholesterol and to respond to db cAMP (1mM) by increased steroid release was preserved in cells cultured for over 60 generations in the serum-free medium. HDL, although also able to increase steroid production in early-passage cultures exposed to ACTH or to ACTH and dibutyryl cyclic AMP (db cAMP), was 10 fold less potent than LDL. It did not support steroidogenesis in cultures not exposed to these trophic agents. The life span of bovine adrenal cortex cells grown in the serum-free medium on fibronectin (FN)- versus ECM-coated dishes was compared. Cells seeded in serum-containing medium and grown in serum-free medium had a life span of 34 versus 60 generations when maintained on fibronectin- or ECM-coated dishes, respectively. Cells seeded in the complete absence of serum in the serum-free medium on ECM- or fibronectin-coated dishes could be passaged for 26 or 13 generations, respectively. While FGF was an absolute requirement for cells cultured on fibronectin-coated dishes, it was not required when cells were maintained on ECM. These observations demonstrate the influence of the ECM not only in promoting cell growth and differentiation but also on the life span of cultured cells.

Control of proliferation of human fetal adrenal cells in vitro.

The influence of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on the proliferation of cultured human fetal adrenal cells has been examined. Separated human definitive zone and fetal zone adrenal cells plated at low density in the presence of 10% serum and maintained on plastic culture dishes proliferated slowly. If the cultures were exposed to either FGF or EGF, the growth rate of the cells from each zone increased significantly. Half-maximal stimulation of cell proliferation for both zones occurred at a concentration of 3 X 10(-11) M for EGF and 8 X 10(-9) M for FGF. In addition, 125I-labeled EGF binding to both definitive and fetal zone cells demonstrated high affinity (Kd = 10(-9) M). To investigate the influence of an extracellular matrix (ECM) on cell proliferation, separated fetal adrenal cells maintained on plastic culture dishes were compared with cells maintained on a recently described ECM prepared from bovine corneal endothelial cells. Fetal adrenal cells maintained on the ECM had a significantly higher growth rate than cells maintained on plastic alone. These results demonstrate 1) the mitogenic role of EGF and FGF for human fetal adrenal cells, and 2) that the type of substrate upon which fetal adrenal cells are maintained has a profound influence on their proliferation.

Kinetics of cAMP inhibition of DNA synthesis in bovine adrenocortical cells.

cAMP-treated bovine adrenocortical cells are arrested in the G1 phase of the cell cycle. Removal of serum also arrests bovine adrenocortical cells in G1. In the presence of cAMP, serum and fibroblast growth factor stimulate increases in medium cell volume, but DNA synthesis is not initiated. Under these conditions cAMP increases steroidogenic capacity 7- to 10-fold as assessed by metabolism of pregnenolone to fluorogenic steroids. When the kinetics of entry of cells into S phase are quantitated, serum- and FGF-treated cells initiate DNA synthesis at an exponential rate after a 12-h lag. In contrast when cAMP is removed, cells immediately initiate DNA synthesis without a lag at a similar exponential rate (6.3 and 5.3% of the cells entering S/h). In the presence of growth factors, cAMP-treated bovine adrenocortical cells are thus hypertrophied with increased steroidogenic capacity, but are reversibly arrested at the G1/S boundary. These findings suggest that cAMP arrests cell replication by mechanisms distinct from those of serum deprivation.

Do plasma and serum have different abilities to promote cell growth?

The abilities of plasma and serum to support the growth of vascular smooth muscle cells maintained on uncoated tissue culture dishes or dishes coated with an extracellular matrix (ECM) have been compared. Vascular smooth muscle cells maintained on plastic dishes and exposed to plasma proliferate poorly; when exposed to serum they proliferate actively. Addition of fibroblast growth factor (FGF) incrases the growth rate of the cultures in both cases. In contrast, when vascular smooth muscle cells are maintained on an ECM, they proliferate equally well exposed to either plasma or serum. Because the cultures had an average doubling time (15 hr) that was already at a minimum, FGF no longer had an effect on vascular smooth muscle cell proliferation. These results raise the possibility that the lack of response of vascular smooth muscle cells, as well as that of other cell types in vitro, to plasma factors is not an intrinsic property of the cells but is rather due to the substrate upon which the cells rest. Because cells maintained on an ECM respond to plasma factors, it is likely that the close contact of the cells with the ECM restores their sensitivity to physiological factors present in plasma.

Angiotensin stimulation of bovine adrenocortical cell growth.

Factors controlling proliferation of adrenocortical cells have been studied in monolayer cultures of bovine adrenocortical cells. Angiotensin II stimulated cell proliferation and [3H]thymidine incorporation into DNA with a half-maximal effective concentration of 0.96 +/- 0.27 nM. Similar sensitivity to angiotensin III with reduced sensitivity to angiotensin I and tetradecapeptide renin substrate was observed. Although sensitivity to angiotensin II was equivalent to that for fibroblast growth factor (1.5 nM half-maximal effective concentration), maximal effects of angiotensin were less than for fibroblast growth factor and serum. High concentrations of insulin (1-10 micrometer) also stimulated [3H]thymidine incorporation into DNA and cell proliferation. [Sar1,Ile5,Ile8]Angiotensin II, a competitive antagonist of angiotensin II, blocked angiotensin II stimulation of DNA synthesis but did not affect fibroblast growth factor and insulin stimulation of DNA synthesis. Corticotropin (ACTH) blocked the stimulatory effects of both angiotensin II and fibroblast growth factor. The dose-response curves for angiotensin II stimulation of steroidogenesis were similar to those for stimulation of [3H]thymidine incorporation into DNA. Among the seven cell types examined, only adrenocortical cells responded to angiotension II with stimulation of DNA synthesis.