A magnetic resonance imaging based method for measurement of tissue iron concentration in liver arterially embolized with ferrimagnetic particles designed for magnetic hyperthermia treatment of tumors.

Rabbit liver was loaded with ferrimagnetic particles of gamma -Fe2 O3 (designed for magnetic hyperthermia treatment of liver tumors) by injecting various doses of a suspension of the particles into the hepatic artery in vivo. Proton transverse relaxation rate (R(2)) images of the livers in vivo, excised, and dissected were generated from a series of single spin-echo images. Mean R(2) values for samples of ferrimagnetic-particle-loaded liver dissected into approximate 1 cm cubes were found to linearly correlate with tissue iron concentration over the range from approximately 0.1 to at least 2.7 mg Fe/g dry tissue when measured at room temperature. Changing the temperature of ferrimagnetic-particle-loaded samples of liver from 1 degrees C to 37 degrees C had no observable effect on tissue R(2) values. However, a small but significant decrease in R(2) was found for control samples containing no ferrimagnetic material on raising the temperature from 1 degrees C to 37 degrees C. Both chemically measured iron concentrations and mean R(2) values for rabbit livers with implanted tumors tended to be higher than those measured for tumor-free liver. This study indicates that tissue R(2) measurement and imaging by nuclear magnetic resonance may have a useful role in magnetic hyperthermia therapy protocols for the treatment of liver cancer.

The inducible Hsp70 as a marker of tumor immunogenicity.

Growing evidence indicates that the stress response in general and heat shock proteins (Hsps) in particular have a profound impact on tumor immunogenicity. In this study, we show that tumor cells subjected to a nonlethal heat shock stress are unable to form tumors in syngenic mice, whereas they do so in athymic nude mice. Moreover, heat-shocked MethA immunity is tumor specific. Enhancement of T-cell-mediated immunogenicity correlates with the expression of the inducible Hsp70 but not the constitutive Hsc70. These observations have a bearing on the proposed functional role of Hsp-peptide association in antigen processing and presentation by major histocompatibility complex I molecules under normal and stressful conditions.

Cationic lipid gene transfer of an IL-2 transgene leads to activation of natural killer cells in a SCID mouse human tumor xenograft.

Natural killer (NK) cells play an important role in combating infectious and malignant diseases and interleukin-2 (IL-2) has been shown to promote proliferation and activation of NK cells in vitro and in vivo. Here we investigate the effects of local cationic lipid-mediated IL-2 gene transfer on intratumoral accumulation and activation of NK cells in a SCID mouse tumor model. UM449 human melanoma tumors in SCID mice received intratumoral injections of DMRIE/DOPE admixed with VR1103, a DNA plasmid encoding the gene for human IL-2. Dissagregated tumor cells were tested for IL-2 secretion and were characterized using antibodies to asGM1, MAC-1, and F4/80 antigens. Granzyme A, a proteolytic serine esterase, was also measured in tumor cell lysates. IL-2 secretion from tumors injected with VR1103:DMRIE/DOPE peaked at 48 h after injection and fell to baseline levels on day 8. Intratumoral granzyme A activity was significantly increased in tumors injected with IL-2 plasmid:DMRIE/DOPE complexes, but not by an irrelevant plasmid DNA:DMRIE/DOPE control. Importantly, the growth of UM449 tumors was slowed in VR1103:DMRIE/DOPE-injected tumors. These results indicate that local cationic lipid-mediated gene transfer of IL-2 induces activation of intratumoral NK cells and slows tumor growth.

Studies of direct intratumoral gene transfer using cationic lipid-complexed plasmid DNA.

Cationic lipid-mediated gene transfer is a safe and effective means of delivering potent immunomodulatory cytokines directly into tumors. This approach avoids undesirable side effects, including systemic toxicities. To investigate key factors affecting intratumoral (i.t.) gene transfer, cationic lipid-DNA complexes were injected into subcutaneous human melanoma tumors in severe combined immunodeficient mice. Animals received i.t. injections of VR1103, a DNA plasmid encoding the gene for human interleukin-2 (IL-2), either alone or complexed with the cationic lipid N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl) ammonium bromide/dioleoyl phosphatidylethanolamine (DMRIE/DOPE). Tumors were subcultured and supernatants were tested for IL-2 secretion by enzyme-linked immunosorbent assay. IL-2 secretion was consistently higher when lipid:DNA (L:D) complexes were formulated at high L:D ratios (wt/wt), and IL-2 transgene expression increased in a DNA dose-dependent manner. A comparison of naked plasmid and lipid-complexed DNA revealed that lipid complexes were more effective for i.t. gene transfer. Using an enhanced green fluorescent protein reporter plasmid and flow cytometry, i.t. transfection efficiency was 1.74% (+/- 1.08%). Tumor injection technique, including injection volume and location, had a limited impact on i.t. gene transfer. These results indicate that the formulation and dosage of cationic L:D complexes, but not injection technique, play a key role in determining the level of i.t. transgene expression.

Quantitative mapping of transverse relaxivity (1/T(2)) in hepatic iron overload: a single spin-echo imaging methodology.

Recent research into the non-invasive assessment of hepatic iron concentrations using magnetic resonance imaging has shown that the proton transverse relaxivity (1/T(2)) varies linearly with liver iron concentration. However, the development of an image-based system for the assessment of hepatic iron distribution has been confounded by the presence of motion induced artifacts in the T(2)-weighted images. We report on the development of a single spin-echo imaging methodology that enables the generation of transverse relaxivity maps over the liver. A simple smoothing technique is used to accommodate the image intensity perturbations caused by abdominal motion. The relaxivity maps are consistent with the variation of iron concentration throughout the liver. A Parzen density estimate and histogram of the relaxivity distribution are generated to assist in the visual assessment of the degree and variability of T(2) shortening with liver iron loading. It was found that one or two Gaussian functions could be used to characterize the relaxivity distributions with a small number of parameters. We propose that this methodology may be used in the clinical setting to monitor hepatic iron concentrations in the advent of an accurate transverse relaxivity calibration curve.

Construction of new amplifier expression vectors for high levels of IL-2 gene expression.

The success of IL-2 gene therapy in cancer is in part dependent on the development of high level IL-2 gene expression vectors. Currently, expression vectors based on the human cytomegalovirus (CMV) promoter give the highest levels of expression. We have attempted to construct new IL-2 expression vectors to test whether gene expression can be further increased. The first approach was to use the new SR-alpha promoter to control IL-2 gene expression. The second approach was to combine the Tat transcription activator gene and the HIV 1 and 2 promoters in the same construct so that the levels of gene expression can be amplified. Transient transfection results using the human colon cancer cell line SW480 showed that the SR-alpha promoter yields similar levels of activity as the CMV promoter. However, the HIV 1 and 2 promoter-based amplifier constructs produced 11 and 28 times more secreted IL-2 than the CMV promoter control. The augmented activity of the amplifier constructs was dependent on the presence of the Tat gene and the transcriptional units must be placed in the same orientation. Reducing the size of the vectors by elimination of the neomycin selectable marker did not increase the activity of the constructs.

Polycations and cationic lipids enhance adenovirus transduction and transgene expression in tumor cells.

Replication-deficient adenovirus vectors are efficient vehicles for delivering therapeutic genes into mammalian cells. However, the high doses required to produce effective gene transfer in vivo can also cause unwanted cellular toxicity. To improve replication-deficient adenovirus transgene expression while minimizing adverse reactions, we have tested polycationic compounds for their ability to enhance adenovirus adsorption. We demonstrate increased transgene expression after mixing adenovirus preparations with polycations, cationic lipids, and CaCl2 prior to transduction in vitro. An E1-deleted adenovirus vector was admixed with various polycations, and beta-galactosidase (beta-gal) activity was evaluated. The optimal polycation concentrations for augmenting adenovirus-mediated gene transfer were 5-10 microg/mL polybrene, 400 microg/mL protamine sulfate, 10 microg/mL N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP), 2.5 microg/mL Lipofectamine, and 62.5 mM CaCl2. Polycations enhanced beta-gal expression in three of six established cell lines. Similar results were obtained using primary tumor cell cultures, where beta-gal expression was increased 1.5- to 10.7-fold (mean = 3.6) by polybrene, 1.8- to 7.5-fold (mean = 3.4) by DOTAP, and 2.3- to 10.4-fold (mean = 4.8) by protamine sulfate. Adenovirus transduction efficiency in two primary leukemia isolates was improved by 3- and 4.5-fold. We were unable to demonstrate any benefit when adenovirus was admixed with protamine sulfate prior to intratumoral injection in a xenogeneic severe combined immunodeficient mouse melanoma tumor model. Further studies will determine whether polycations can improve intratumoral gene transfer.

Cationic lipid-mediated gene transfer: current concepts.

Infectious disease, heart disease, cancer, autoimmunity, genetic defects and even traumatic injury may someday be treated with gene therapy and gene transfer strategies. The potential impact of this new technology on human disease has produced optimism and expectation for scientists and lay people alike. As more effort is directed at harvesting the potential of this technology it has become clear that the success or failure of gene therapy will hinge on our ability to manipulate and control the process of gene transfer in somatic cells. Today, somatic gene transfer is accomplished using either viral or non-viral gene transfer methods. The benefits and limitations of each system are aggressively being investigated to determine which characteristics are most compatible with safe and reliable gene transfer. Gene transfer with cationic lipid/plasmid DNA complexes (cationic lipoplexes) has become a popular means of delivering therapeutic genes and is being tested in preclinical and clinical trials. Cationic lipoplexes are easy and inexpensive to produce, they are composed of non-toxic and non-immunogenic precursor, and they have the potential of delivering large polynucleotides into somatic cells. Additionally, these reagents are easily manipulated in the laboratory to incorporate novel biological functions or to produce new formulations that can be screened for in vivo gene transfer activity. The last few years has seen many advances in our understanding of molecular and biological factors that influence cationic lipid-mediated gene transfer. In this review we discuss recent developments in the field of cationic lipid-mediated gene transfer with emphasis on in vivo application where possible. We will consider new discoveries concerning the molecular and cellular events that control the uptake, transit and expression of lipoplexes in somatic cells. Recent biodistribution and pharmacokinetic studies and current concepts regarding the toxicity and immunogenicity of cationic lipoplexes will also be discussed. We also survey some of the many preclinical and clinical trials using cationic lipid-mediated gene transfer, with emphasis on cancer applications.

Transfection of primary tumor cells and tumor cell lines with plasmid DNA/lipid complexes.

Cancer vaccines that utilize genetically modified tumor cells require gene transfer methods capable of producing immunostimulatory doses of transgenes from fresh or short-term cultures of human tumor cells. Our studies optimize in vitro transfection of primary tumor cells using cationic lipids and a plasmid encoding the gene for human interleukin-2 (IL-2). Established tumor cell lines produced 10- to 100-fold more IL-2 than did fresh or short-term tumor cultures as measured by enzyme-linked immunoabsorbent analysis. Importantly, transfection of primary tumor cells produced immunostimulatory levels of IL-2 as determined by increased thymidine incorporation by autologous peripheral blood mononuclear cells and lymphokine-activated killer cell activity. IL-2 secretion by tumor cells persisted for at least 30 days post-transfection and was unaffected by freeze thawing or irradiation to 8000 rads. Multiple solid tumor types were successfully transfected, but normal blood mononuclear cells and leukemic blasts were resistant to transfection. Enzyme-linked immunoabsorbent analysis of the amount of IL-2 secreted into the medium by transfected tumor cells correlated with the percentage of tumor cells expressing intracellular IL-2 as measured by flow cytometry. Plasmids utilizing a cytomegalovirus promoter yielded superior transfection efficiencies compared with plasmids containing a Rous sarcoma virus promoter. These results suggest that a clinical vaccine trial using autologous tumor cells genetically modified to secrete IL-2 is feasible in patients with solid tumors.

A novel drug screening assay for papillomavirus specific antiviral activity.

Discovery and development of human papillomavirus (HPV) specific antiviral agents have been hampered by the lack of an in vitro assay permissive to HPV replication. An experimental assay system for monitoring HPV-11 DNA replication has been optimized for use as a papillomavirus antiviral drug screening tool. Cloned HPV DNA was introduced into SCC-4 cells by electroporation and viral DNA replication monitored by Southern blot. Kinetic studies demonstrated an increased HPV genome copy number with time. Viral DNA replicated as episomal, unit length genome and remained episomal after multiple passages. These data suggested the basis for an in vitro replication assay for evaluating the antiviral activity of potential chemotherapeutic agents directly on HPV. This model was used to investigate antiviral activities of current anti-HPV therapies such as 5-fluorouracil (5-FU) and alpha-interferon (alpha-IFN) and potential therapies such as sodium butyrate, 5-bromo-20-deoxyuridine (BrdU) and antisense oligonucleotides. HPV- 11 replication is significantly inhibited by BrdU and sodium butyrate; however 5-FU and alpha-IFN did not give consistent dose response results. Finally, ISIS 2105, a 20-mer phosphorothioate antisense oligonucleotide, which targets HPV-11 E2 gene product, showed potent antiviral activity in this assay with an IC50 of approximately 70 nM.

Measurement of environmental formylmethionyl-peptides.

Formylmethionyl-peptides are naturally occurring, biologically active ligands produced by bacteria. They produce a variety of biological effects including neutrophil chemotaxis, cellular degranulation, oxygen-free radical production, and smooth muscle contraction. Our studies have demonstrated that oxidized and reduced forms of formylmethionyl-leucyl-phenylalanine (fMLP) can be detected in bulk environmental organic dust samples. Organic dust fMLP content may not reflect total formylmethionyl-peptide content and pathological sequelae. Attempts to develop a total formylmethionyl-peptide assay that would reflect its pathological potential have thus far been unsuccessful. Information has been derived concerning the biology of formylmethionyl-peptides from these studies. Chromatographic, radioenzymatic, and radioreceptor-ligand binding studies were performed. High-performance liquid chromatography (HPLC) analysis of synthetic and environmental fMLP demonstrated that fMLP is labile, forming three oxidation products. HPLC is limited by inadequate sensitivity for air sample analysis and the probability of the presence of multiple formylmethionyl-peptides. Deformylases were isolated from Escherichia coli, but their usefulness in a competitive assay to detect formylmethionyl-peptides was limited by specificity differences from that for biological receptors. Receptor binding studies were conducted in an attempt to replace the deformylase with a biological receptor. The receptor binding patterns noted were consistent with the existence of three distinct formylmethionyl-peptide receptor subsets in neutrophils and alveolar macrophages. The plurality of fMLP receptor subtypes interfered with formylmethionyl-peptide measurement in a competitive assay. Formylmethionyl-peptides may contribute to organic dust-induced disease, but better techniques for the assessment of exposure to these agents are needed to properly assess their health impact.

Susceptibility of the anaerobic bacteria, group D streptococci, Enterobacteriaceae, and Pseudomonas to semisynthetic penicillins: carbenicillin, piperacillin, and ticarcillin.

Sodium piperacillin T-1220, a new semisynthetic penicillin, was tested in vitro against 297 clinical isolates of anaerobic bacteria and 669 aerobic bacteria by the conventional agar dilution method and compared with carbenicillin and ticarcillin. At a 100-mug/ml concentration the three drugs showed comparable effectiveness against the anaerobes tested. However, at 20 mug/ml, piperacillin was the most effective drug against Bacteroides fragilis, peptostreptococci, and group D streptococci. At this drug concentration only 48% of the B. fragilis strains exhibited susceptibility to carbenicillin only, 64% exhibited susceptibility to ticarcillin but 90% exhibited susceptibility to piperacillin. Similar findings were observed with peptostreptococci and group D streptococci. On a weight basis piperacillin was statistically shown to be the most effective antibiotic of the three tested against these anaerobes. At 20 mug/ml, piperacillin exhibited a statistically significant difference (P < 0.01) over carbenicillin and ticarcillin for Serratia marcescens, Escherichia coli, Klebsiella species, Klebsiella pneumoniae, Pseudomonas isolates, and Citrobacter diversus. At both 20- and 100-mug/ml concentrations, piperacillin appeared to be the most effective (calculated P < 0.01) upon Klebsiella species, K. pneumoniae, S. marcescens, and C. freundii in activity over ticarcillin and carbenicillin.