[Studies on antiviral compounds. II. The synthesis of acetophenone derivatives].
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Acetophenones↗
Biomedical subjects
L Huang
Publications and source records attributed to L Huang.
Systemic production of IL-12 by naked DNA mediated gene transfer: toxicity and attenuation of transgene expression in vivo.
BACKGROUND: IL-12 is a potent antitumor cytokine for cancer gene therapy. Previously, we demonstrated that single systemic administration of naked DNA (encoding IL-12) could serve as a good model for in vivo evaluation of the antitumor effect of a candidate gene (unpublished data). In the present study, we propose that this gene delivery method could be a very useful model for in vivo evaluation of the toxicity of a given therapeutic gene (using IL-12 as an example). By comparing the toxicities and the effects of initial IL-12 administration on subsequent transgene expression, both IL-12 gene delivery and recombinant murine IL-12 protein (rmIL-12) administration showed similar toxicity profiles. METHODS: Naked DNA encoding murine IL-12 (mIL-12) was delivered into mice by systemic administration. Toxicity profiles of mice treated with DNA or rmIL-12 were compared. RESULTS: Systemic administration of naked DNA encoding mIL-12 resulted in very similar toxicity as rmIL-12 with respect to liver enzyme, hematological and immunological profiles. Repeated injection of mIL-12 gene did not recover a high level of mIL-12 production as the first injection. Moreover, initial mIL-12 administration resulted in inhibition of subsequent reporter gene expression with both viral and non-viral promoters (CMV, human alpha-antitrypsin or chicken beta-actin promoter). This transgene inhibition effect was entirely mediated by IFN-gamma as the transgene expression was fully recovered in IFN-gamma knockout mice. CONCLUSIONS: Systemic IL-12 therapy, with either a protein or gene therapy approach, resulted in comparable liver and systemic toxicities. Refractoriness of mIL-12 production by subsequent administration of mIL-12 gene was observed. The transgene attenuation effect of IL-12 pre-dosing (either by IL-12 or rmIL-12), mediated by IFN-gamma, provided important insights for the design of IL-12 combination gene therapy and the improvement of gene vectors for IL-12 therapy. The present results show that simple injection of naked DNA could serve as a good model for in vivo evaluation of the toxicity of a candidate therapeutic gene.
Animals↗
Improving plasmid DNA-mediated liver gene transfer by prolonging its retention in the hepatic vasculature.
BACKGROUND: Naked DNA is the simplest and safest method to deliver genes to the liver. In this study, we demonstrate that significant gene expression could be achieved in the liver by transiently restricting blood flow through the liver immediately following peripheral intravenous injection of plasmid DNA. METHODS: Mice were intravenously (tail vein) injected with plasmid DNA in 100 microl of saline (0.9% NaCl) immediately followed by 8 s of occlusion of blood flow through the liver. The occlusion of blood flow was performed by using a clip at either the vena cava (VC) or at the portal vein and hepatic artery (PV+HA). Alternatively, the VC was clamped for 4 s followed by clamping the PV+HA for 4 s (VC and PV+HA). RESULTS: Gene transfer to the liver was completed after blood flow through the liver was blocked for as short as 1 s. Up to 560 pg of luciferase protein per mg of extracted protein was observed from the liver after a single injection of 80 microg of plasmid DNA. Gene expression was increased more than 50-fold by the combination of clamping and electroporation. CONCLUSION: This is the first demonstration of gene transfer to the liver via systemic administration without using any carrier system or physical force. Also, the technique provides new insights into the mechanism of hepatic gene transfer.
Animals↗
Immunoliposomes: preparation, properties, and applications.
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Animals↗
High-resolution analysis of engineered type I collagen nanofibers by electron microscopy.
Collagen nanofibers were generated at ambient temperature and pressure by electrospinning a 1 wt% solution of type I collagen and polyethylene oxide. Products were imaged with high-resolution scanning electron microscopy (HRSEM) at medium (approximately 30,000 x) and high magnifications (approximately 100,000 x) and with transmission electron microscopy (TEM). The capacity to produce collagen nanofibers may lead to the generation of extracellular matrix-based fabrics with applications in the fields of wound healing and tissue engineering.
Collagen Type I↗
Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.
A DNA targeted delivery and expression system has been designed based on an N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate, which readily forms a complex with plasmid DNA. Monoclonal antibodies against the cell-surface thrombomodulin conjugated with NPLL were used for targeted delivery of foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line in vitro and to mouse lungs in vivo. In both cases significant amounts of DNA can be specifically bound to the target cells or tissues. Specific gene expression was observed in the treated mouse lung endothelial cells.
Animals↗
Contact-dependent, immunecomplex-mediated lysis of hapten-sensitized liposomes.
Large unilamellar liposomes (d approximately 160 nm) composed of dioleoylphosphatidylethanolamine (DOPE) (80-90%), a negatively charged phospholipid stabilizer (10-20%), and a small amount (0.1-1%) of a haptenated lipid are unusually stable in divalent cation-free isotonic buffer at pH 7.4. The liposomes can be stored under this condition at 4 degrees C for at least 6 months without any detectable leakage of the entrapped fluorescent dye calcein. However, the liposomes undergo a rapid (1 h) aggregation and lysis reaction in the presence of free bivalent anti-hapten antibody. The liposome destabilization was immunospecific in that it did not occur with the normal IgG or in the presence of excess free hapten. Liposome lysis was always accompanied by liposome aggregation. Aggregation and lysis of the liposomes was completed in 5 min if the incubation temperature was raised to 70-80 degrees C. Replacing DOPE with dioleoylphosphatidylcholine in the liposomes did not abolish the liposome aggregation, but no liposome lysis was observed even at 80 degrees C. Since liposome aggregation appeared to be a necessary (but not sufficient) prerequisite for liposome lysis, we have named this new class of liposome "contact-sensitive liposomes." The immunodiagnostic potential of the contact-sensitive liposome was demonstrated with liposomes containing theophylline-DOPE. The aggregation and lysis of the liposomes induced by a monoclonal anti-theophylline antibody could be inhibited by free theophylline at concentrations of therapeutic significance. The observation could be the basis of a homogeneous assay for theophylline.
Antibodies↗
Design, synthesis, and sequence selective DNA cleavage of functional models of bleomycin. 1. Hybrids incorporating a simple offal-complexing moiety of bleomycin and lexitropsin carriers.
The syntheses of functional models for bleomycin, which are composed of a simple analog of the metal-complexing moiety of bleomycin and oligo-N-methylpyrrole peptide DNA-binding moieties, are described. The extent and the relative rate of their cleavage of DNA in the presence of reductants were determined independently by an ethidium binding assay and by agarose gel electrophoresis experiments. The rate of DNA cleavage increases with the number of N-methylpyrrole units in the carrier moiety. The sequence selectivity of DNA cleavage was demonstrated by polyacrylamide sequencing gel electrophoresis of cleavage reactions on two 5'-32P labeled restriction fragments: a 158 bp GC-rich fragment from pBR322 and a 241 bp AT-rich fragment fragment from SV40. Comparison of the sequence selectivity with that of bleomycin A2 indicates that the poly-N-methylpyrrole moiety directs the hybrid compounds to AT-rich sequences of DNA. High-resolution denaturing gel electrophoresis of DNA cleaved by the model compounds reveals that 3' phosphate is produced exclusively, indicating that the chemistry of DNA cleavage differs from that of bleomycin.
Base Sequence↗
Solution structure studies of the cobalt complex of a bleomycin functional model bound to d(CGCAATTGCG)2 by two-dimensional nuclear magnetic resonance methods and restrained molecular dynamics simulation.
The interaction between the cobalt(III) complex of a bleomycin functional model (AMPHIS-NET) and the oligonucleotide d(CGCAATTGCG)2 and the structural features of the 1:1 ligand-DNA complex have been determined by high-resolution two-dimensional nuclear magnetic resonance methods and restrained molecular dynamics calculations. The intermolecular nuclear Overhauser effect (NOE) cross-peaks between ligand protons and the DNA minor groove protons suggest that the cobalt(III) complex of AMPHIS-NET binds in the minor groove of DNA at the central AATT site. The NOE connectivities also clearly indicate that the H8 pyridine proton and the H2 imidazole proton in the metal-binding domain interact with the H4' sugar proton of C19 and the H4' sugar proton of A5, respectively, which defines a structure where the metal binding moiety of Co(III).AMPHIS-NET participates in binding to the DNA and extends into the region two base pairs beyond the central AATT site in the minor groove. This binding model is in accord with the consistently observed nondiffusion DNA cleavage in locations two to three residues beyond the end of AT-rich binding sites induced by the corresponding iron(II) complexes of AMPHIS-NET and other AMPHIS-lexitropsin hybrids of the bleomycin functional model compounds.
Antiviral Agents↗
Serum lipoprotein allotypes in inbred strains of rabbits.
Serum lipoprotein allotypes were examined for 10 males and 10 female rabbits from each of 16 genetically defined strains. The results suggested that the four allotypes, Lpq 1, Lpq2, Lpq3, and Lpq4, were controlled by three closely linked genes, IpqA, IpqB, and IpqC. Gene IpqA, controlling the expression of the Lpq1 and Lpq2 allotypes, and gene IpqB controlling the expression of the Lpq3 allotype, show limited polymorphisms.
Alleles↗
Cholestatic properties and hepatic transport of steroid glucuronides.
In summary, the data suggest that E217G is transported by both MOAT and P-glycoprotein into bile, but that P-glycoprotein serves as the target site for cholestasis. We postulate that this target site may be accessed from either the intracellular compartment or the canaliculus, and that MOAT serves as the major delivery route for E217G to the canaliculus. At low, physiologic concentrations of E217G, MOAT-mediated excretion into bile is a detoxification mechanism, serving to prevent intracellular accumulation of a toxic metabolite. However, following administration of high, cholestatic doses, MOAT-mediated excretion into bile results in very high concentrations in bile, on the other of 2-3 mM (see Fig. 4). It is likely that the hydrophobic nature of E217G allows it to partition from bile into the canalicular membrane, from which it can access P-glycoprotein and thus induce cholestasis. Much work is still needed to validate this model of E217G cholestasis. Definitive evidence of P-glycoprotein-mediated transport of E217G must be obtained in cell lines transfected with P-glycoprotein where MRP is absent. More importantly, the mechanism by which interaction of E217G with P-glycoprotein influences bile flow is unknown. Higgins and colleagues have provided evidence that P-glycoprotein regulates a Cl- channel in a manner analogous to that of CFTR, the cystic fibrosis transmembrane conductance regulator. While Cl- channels have been shown to be important in the regulation of the volume of the hepatocyte in the presence of altered osmotic conditions, a role for this channel in bile flow has not been demonstrated. Nevertheless, these studies implicate a role of P-glycoprotein in the regulation of bile secretion by the liver.
Animals↗
Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission.
To study transmission patterns of Pneumocystis carinii pneumonia (PCP) in persons with AIDS, we evaluated P. carinii isolates from patients in five U.S. cities for variation at two independent genetic loci, the mitochondrial large subunit rRNA and dihydropteroate synthase. Fourteen unique multilocus genotypes were observed in 191 isolates that were examined at both loci. Mixed infections, accounting for 17.8% of cases, were associated with primary PCP. Genotype frequency distribution patterns varied by patients' place of diagnosis but not by place of birth. Genetic variation at the two loci suggests three probable characteristics of transmission: that most cases of PCP do not result from infections acquired early in life, that infections are actively acquired from a relatively common source (humans or the environment), and that humans, while not necessarily involved in direct infection of other humans, are nevertheless important in the transmission cycle of P. carinii f. sp. hominis.
AIDS-Related Opportunistic Infections↗
Issues in the development of gene therapy: preclinical experiments in E1A gene delivery.
We recently completed a phase I trial of E1A gene therapy for patients with advanced breast or ovarian cancers. The trial's rationale was that E1A gene would downregulate HER-2/neu expression by repressing the gene's transcription, thus reversing the malignant phenotype. Our preclinical studies showed that i) transfection capabilities were not reduced in preparing E1A/cationic liposome complexes, ii) samples after ex vivo transfection, and iii) HER-2/neu gene expression could be quantified by quantitative imaging analysis. These results facilitated the translation of our basic research findings into a clinical trial and to obtain final approval from the National Institutes of Health Recombinant DNA Advisory Committee.
Adenovirus E1A Proteins↗
Ultrasound technology: the RADIUS (Routine Antenatal Diagnostic Imaging with Ultrasound) study & national policy.
The RADIUS study opened up controversial discussions of the applications of ultrasound technology in patient management, screening, and outcomes analysis. Advances in technology may ultimately resolve national policy issues.
Biomedical Engineering↗
Apoptosis occurs in isolated and banked primary mouse hepatocytes.
Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.
Animals↗
Liposomal atp or NAD+ protects human endothelial cells from energy failure in a cell culture model of sepsis.
Sepsis depletes intracellular stores of ATP and NAD+, leading to cellular energy failure. Liposome encapsulation improves intracellular delivery of bulky, charged molecules and substrates susceptible to extracellular enzyme degradation. We hypothesized that treatments with liposome encapsulated ATP or NAD+ would protect human endothelial cells exposed to endotoxin (LPS) and interferon-gamma (IFN-gamma) from energy failure. Liposomal ATP and NAD+ were prepared by a modification of the thin film method. Human endothelial cells were exposed to LPS 50 microg/ml and IFN-gamma 50 ng/ml for 72 hours, and liposomal ATP and NAD+ treatments were dosed at 0 and 24 hours. Energy state was determined by rate of mitochondrial respiration as measured by WST-1 assay. Mitochondrial respiration significantly decreased to 57% +/- 3 of control in LPS/IFN-gamma exposed cells after 72 hours. Liposomal ATP (200 microM) and NAD+ (100 microM) completely reversed this respiratory depression while empty liposomes, free ATP (200 microM). and free NAD+ (100 microM) did not. These results support the hypothesis that treatments with liposome encapsulated ATP or NAD+ protect human endothelial cells from energy failure in a cell culture model of sepsis and potentially may provide a novel therapy for use in clinical sepsis.
Adenosine Triphosphate↗
Mutant SV40 large T antigen as a therapeutic agent for HER-2/neu-overexpressing ovarian cancer.
The HER-2/neu gene is frequently amplified and/or its protein product, p185, is overexpressed in a number of human cancers. Overexpression of p185 correlates with poor prognosis and low survival rates in ovarian cancer patients. We previously found that the K1 mutant of SV40 large T antigen inhibits rat neu promoter and suppresses mutation-activated rat neu transformation in mouse fibroblasts. We show here that K1 also inhibits human HER-2/neu promoter in human ovarian cancer cells. To investigate whether K1 can suppress HER-2/neu transformation and thus is a potential therapeutic agent, we used an orthotopic ovarian cancer model in which mice were injected intraperitoneally with HER-2/neu-overexpressing human ovarian cancer cells to induce tumor development. The tumor-bearing mice were then treated with K1-liposome complex weekly. We found that liposome-mediated K1 gene transfer decreased the p185 protein level by K1 expression in these cancer cells and significantly prolonged mice survival; about 40% of these treated mice were alive for more than 1 year without any tumor development. On the other hand, the animals from control groups that did not receive this gene therapy all developed tumors and died within 7 months. The results indicate that liposome-mediated K1 gene transfer is able to suppress tumor development from HER-2/neu-overexpressing ovarian cancer cells in mice.
Animals↗
Efficient lipofection with cisplatin-resistant human tumor cells.
Seven of seven different cisplatin-resistant human tumor cell lines showed elevated lipofection activity as compared with their sensitive parent cells, although the degree of enhancement was not quantitatively correlated with the degree of cisplatin resistance. Enhanced transfection was seen by using the same reporter gene driven by three different promoter/enhancer sequencer or by using different reporter genes driven by the same promoter/enhancer. Cells resistant to actinomycin D, bleomycin, and nitrogen mustards were not more transfectable than the sensitive parent cells. Although the mechanism of enhanced transfection in cisplatin-resistant cells is not known, data indicated that enhanced transcription, multidrug-resistant phenotype, and methallothionein overexpression do not play a role.
Chloramphenicol O-Acetyltransferase↗