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Biomedical subjects

C F Bennett

Publications and source records attributed to C F Bennett.

At least 55 records · Page 3Linked to original sources
PubMed1998

Structural requirements for cationic lipid mediated phosphorothioate oligonucleotides delivery to cells in culture.

A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes prepared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligonucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cationic lipids synthesized with dimyristyl (Cl4:0) and dioleyl (C18:1) alkyl chains were equally effective. Activity of cationic lipids containing saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C14:0) was more effective than dipalmityl (C16:0) lipid, which was more effective than distearyl (C18:0). The phase transition temperature of cationic lipids containing saturated aliphatic chains was 56 degrees C for the distearyl lipid, 42 degrees C for the dipalmityl lipid and 24 degrees C for the dimyristyl lipid. Cationic lipids with dioleyl alkyl chains required DOPE for activity, with optimal activity occurring at 50 mole%. In contrast, a dimyristyl containing cationic lipid did not require DOPE to enhance the activity of ISIS 1570. Formulation with different phosphatidylethanolamine derivatives, revealed that optimal activity was obtained with DOPE. These studies demonstrate that several cationic lipid species enhance the activity of phosphorothioate antisense oligonucleotides and provide further information on the mechanism by which cationic lipids enhance the activity of phosphorothioate oligodeoxynucleotides.

Base Sequence↗
PubMed1997

In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.

Animals↗
PubMed1997

2'-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells.

Little is known about the mechanisms that account for inhibition of gene expression by antisense oligonucleotides at the level of molecular cell biology. For this purpose, we have selected potent 2'-O-(2-methoxy)ethyl antisense oligonucleotides (IC50 = 2 and 6 nM) that target the 5' cap region of the human intercellular adhesion molecule 1 (ICAM-1) transcript to determine their effects upon individual processes of mRNA metabolism in HUVECs. Given the functions of the 5' cap structure throughout mRNA metabolism, antisense oligonucleotides that target the 5' cap region of a target transcript have the potential to modulate one or more metabolic stages of the message inside the cell. In this study we found that inhibition of protein expression by these RNase H independent antisense oligonucleotides was not due to effects on splicing or transport of the ICAM-1 transcript, but due instead to selective interference with the formation of the 80 S translation initiation complex. Interestingly, these antisense oligonucleotides also caused an increase in ICAM-1 mRNA abundance in the cytoplasm. These results imply that ICAM-1 mRNA turnover is coupled in part to translation.

Cells, Cultured↗
PubMed1997

Characterization and modulation of immune stimulation by modified oligonucleotides.

Single-stranded oligodeoxynucleotides (ODNs) were tested for their ability to stimulate NK cells isolated from murine spleens to lyse target cells. Various sequences were evaluated, some of which have been shown previously to exhibit pharmacologic activity in murine model systems. It was confirmed that the CpG motif was stimulatory only in specific sequence contexts, and we found that phosphorothioate backbones were, in general, less stimulatory than phosphodiester backbones. In addition, this stimulation could be reduced by methylating the cytosine of the CpG and eliminated by modifying all of the cytosines contained in an ODN with methyl, bromo, or iodo modifications to the 5 position of the cytosine ring. These results were compared with the ability of a subset of these ODN sequences to stimulate B cell proliferation in vitro. In this comparison, phosphorothioate backbones were found to be required, and the context of the CpG motif was found to be less critical for activation. Finally, one of the most potent ODNs was shown to activate NK and B lymphocytes when administered in vivo.

Animals↗
PubMed1997

An ICAM-1 antisense oligonucleotide prevents and reverses dextran sulfate sodium-induced colitis in mice.

Mice treated p.o. with 5% dextran sodium sulfate develop a mild to moderate colitis characterized by focal areas of inflammation and crypt abscesses. Immunohistological analysis of colons from dextran sodium sulfate-treated mice revealed an increased expression of intercellular adhesion molecule 1 (ICAM-1) and infiltration of lymphocyte function antigen 1-positive cells. A murine-specific antisense oligonucleotide, ISIS 3082, was used to determine the role of ICAM-1 expression in the development of colitis. Prophylactic treatment of dextran sodium sulfate-treated mice with ISIS 3082 reduced the clinical signs of colitis in a dose-dependent manner, with maximal effects occurring at a dose of 1 mg/kg/day. Reductions in ICAM-1 immunostaining and infiltrating leukocytes were observed in colons of animals treated with 1 mg/kg ISIS 3082. Scrambled control oligonucleotides failed to modify the course of the disease. The ICAM-1 oligonucleotide also diminished the clinical severity of colitis in mice with established colitis. The toxicity of ISIS 3082 was assessed in normal CD-1 mice by administering the oligonucleotide intravenously every other day for 2 weeks. At pharmacologically relevant doses of ISIS 3082 (1 and 10 mg/kg), there were no signs of toxicity with respect to body and organ weights, clinical chemistry or hematology. At a dose of oligonucleotide 20- to 100-fold greater than maximal pharmacological doses, the oligonucleotide produced an increase in liver and spleen weights; a mild chronic inflammation in liver, lung and lymph nodes; monocytosis and an elevation of serum liver transaminases. These data suggest that an antisense oligonucleotide that reduces ICAM-1 expression could be effective in the therapy of inflammatory bowel disease in humans and that such an oligonucleotide would be safe at pharmacologically relevant doses.

Animals↗
PubMed1997

Immune stimulation--a class effect of phosphorothioate oligodeoxynucleotides in rodents.

Treatment of rodents with phosphorothioate oligodeoxynucleotides induces a form of immune stimulation characterized by splenomegaly, lymphoid hyperplasia, hypergammaglobulinemia and mixed mononuclear cellular infiltrates in numerous tissues. Immune stimulation was evaluated in mice with in vivo and in vitro studies using a review of historical data and specific in vivo and in vitro studies. All phosphorothioate oligodeoxynucleotides evaluated induced splenomegaly and B-lymphocyte proliferation. Splenomegaly and B-lymphocyte proliferation increased with dose or concentration of oligodeoxynucleotide. Splenomegaly appeared to occur, at least in part, as a result of stimulation of B-lymphocyte proliferation. There were differences with respect to degree or potency of immune stimulation by different oligodeoxynucleotides. The rank order potencies for B-lymphocyte proliferation in vitro and splenomegaly correlated well for the oligodeoxynucleotides tested. Particular oligodeoxynucleotide sequence motifs or palindromes have been demonstrated to affect in vitro cell proliferation. Inclusion of a 5'-AACGTT-3' palindrome in a phosphorothioate oligodeoxynucleotide sequence significantly enhanced the potency. While inclusion of this palindrome or a CpG motif alone may contribute to the immune stimulation, these palindromes and motifs were clearly not the sole factor required for immune stimulation. Several phosphorothioate oligodeoxynucleotides that did not contain a CpG motif still induced immune stimulation in mice. The immune stimulation induced by phosphorothioate oligodeoxynucleotides was an effect of this class of compounds to which rodents are acutely sensitive.

Adjuvants, Immunologic↗
PubMed1997

Cellular distribution of phosphorothioate oligodeoxynucleotides in normal rodent tissues.

The distribution of intravenously injected phosphorothioate oligodeoxynucleotides (P = S ODN) was studied in vivo in rodent tissues using three histologic methods: immunohistochemistry with a monoclonal antibody that recognizes P = S ODN ISIS 2105; direct fluorescence microscopy of P = S ODN ISIS 2105 conjugated to rhodamine; and autoradiography of 14C-labeled P = S ODN ISIS 2302. All three methods gave the same pattern of oligonucleotide distribution, and the intensity of the histologic signal agreed with previously published pharmacokinetic data on the relative concentration of P = S ODN in different organs. Proximal tubule cells in the kidney and Kupffer and endothelial cells in the liver were among the most heavily labeled with P = S ODN at all doses and time-points. Connective tissues proper, such as the lamina propria and submucosa of the intestine and the dermis and subcutaneous layer of the skin, were also labeled, whereas the P = S ODN signal was weak or negative in epithelial and muscle cells in the skin and intestine. At 2 hours postinjection, P = S ODN were clearly detectable in the extracellular matrix in loose and dense connective tissues, although by 24 hours, the label was predominantly intracellular. Large, nucleated cells in red marrow, and the connective tissues around bone and skeletal muscle cells and lining the knee joint, were positive for oligonucleotide, whereas P = S ODN were not detected in erythrocytes, cartilage, compact bone, and skeletal muscle. In spleen, white pulp was negative for P = S ODN, whereas cells surrounding the sinusoids and nucleated cells in the red pulp were strongly positive for P = S ODN. Our results provide specific information on the tissue and cellular localization of P = S ODN within organs in vivo. The data presented will be used as a reference for studies of P = S ODN distribution in diseased tissues and the distribution of modified oligonucleotides. Furthermore, because our results indicate which cell types are likely to be affected by antisense oligonucleotides, they can be used to guide future in vivo applications of the technology.

Animals↗
PubMed1996

Altered mRNA splicing and inhibition of human E-selectin expression by an antisense oligonucleotide in human umbilical vein endothelial cells.

We have characterized the mechanism of action of an antisense oligodeoxynucleotide (ASO) targeting human endothelial leukocyte adhesion molecule, E-selectin. ISIS 4730, a 20-base ASO designed to be complementary to a region in the 3'-untranslated region (3'-UTR) of human E-selectin, is a potent and specific inhibitor of both mRNA and protein expression in human umbilical vein endothelial cells. Following treatment with ISIS 4730, a lower molecular weight mRNA (3300 bases) species was detected by Northern blot analysis with a corresponding decrease in the mature E-selectin transcript (3875 bases). The ASO-induced low molecular weight mRNA is stable and remains in the nucleus. We demonstrate that ISIS 4730 targets E-selectin pre-mRNA in the nucleus and promotes cleavage of the pre-mRNA at the hybridization site, resulting in prevention of splicing of the last intron. The change in molecular weight of the E-selectin transcript is the result of loss of the 3'-UTR due to ASO-mediated RNA cleavage and retention of the last intron. Cleavage of the E-selectin pre-mRNA appears to be due to endogenous RNase H or a related enzyme activity.

Alternative Splicing↗
PubMed1996

Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.

Group II PLA2 has been implicated in inflammatory processes in both man and other animals and has been shown to be involved in inflammatory conditions, such as arthritis and sepsis. Transgenic mice expressing the human group II PLA2 gene have been generated using a 6.2-kb genomic fragment. These mice express the group II PLA2 gene abundantly in liver, lung, kidney, and skin, and have serum PLA2 activity levels approximately eightfold higher than nontransgenic littermates. The group II PLA2 transgenic mice reported here exhibit epidermal and adnexal hyperplasia, hyperkeratosis, and almost total alopecia. The chronic epidermal hyperplasia and hyperkeratosis seen in these mice is similar to that seen in a variety of dermatopathies, including psoriasis. However, unlike what is seen with these dermatopathies, no significant inflammatory-cell influx was observed in the skin of these animals, or in any other tissue examined. These mice provide an important tool for examining group II PLA2 expression, and for determining the role of group II PLA2 in normal and disease physiology. They serve as an in vivo model for identifying inhibitors of group II PLA2 activity and gene expression.

Animals↗
PubMed1996

Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice.

This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM-1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/1 (75%). No inhibition was observed in the ICAM-1 mutant compared to wild-type mice. These data show that antisense oligonucleotides targeted to ICAM-1 inhibit the endotoxin-induced upregulation of ICAM-1 in the lung and are as effective as anti-ICAM-1 antibodies in preventing neutrophil emigration. The incomplete inhibition by either antisense oligonucleotides or antibodies suggests that alternative adhesion pathways that do not require ICAM-1 are important in neutrophil emigration in the lungs. The disparity in the role of ICAM-1 when evaluated using antisense or antibodies compared to mutant mice suggests that either these inhibitors are exerting additional effects on endothelial cells other than blockade of ICAM-1 or mutant mice have upregulated the ICAM-1-independent pathways to compensate for the long-term loss of ICAM-1.

Animals↗
PubMed1996

Inhibition of interleukin-1 type I receptor expression in human cell-lines by an antisense phosphorothioate oligodeoxynucleotide.

A 20 mer oligodeoxynucleotide designed to hybridize to specific 3'-untranslated sequences in the type I receptor IL-1r mRNA was identified which inhibited the expression of both IL-1r mRNA and protein in human A549 lung carcinoma cells and human dermal fibroblasts. The oligodeoxynucleotide exhibited an IC50 value of approximately 100 nM in both cell lines and reduced IL-1r mRNA expression for up to 48 h. Multiple scrambled control oligonucleotides were without effect on IL-1r mRNA expression. Treatment of A549 cells with this oligodeoxynucleotide (but not scrambled controls) inhibited the IL-1 stimulated expression of the cell adhesion molecule ICAM-1 but was without effect on the TNF-alpha induction of this molecule. This study demonstrates that phosphorothioate oligodeoxynucleotides can selectively inhibit IL-1r expression leading to a reduction in IL-1 dependent gene expression.

Carcinoma↗
PubMed1996

Progress in antisense oligonucleotide therapeutics.

The past several years have seen substantial progress in the development of antisense oligonucleotides as pharmacological tools and as therapeutic agents. With properly designed and executed experiments, it has been possible to demonstrate selective inhibition of gene expression, owing to an antisense mechanisms of action both in cell culture-based experiments and in vivo. As the field has matured, it has also realized that oligonucleotides can produce a variety of effects in cell culture and in vivo that cannot be ascribed to an antisense mechanism of action. Nevertheless, with proper controls it has been possible to demonstrate that the pharmacological activity of an oligonucleotide is consistent with an antisense mechanism of action. The pharmacokinetic properties of first-generation phosphorothioate oligodeoxynucleotides and their toxicological limitations have been characterized in rodents, primates, and humans. Finally, it has been demonstrated that medicinal chemistry can improve the properties of oligonucleotides, as several modifications have been identified that have increased potency, altered pharmacokinetic properties and potentially decreased toxicological liabilities.

Animals↗
PubMed1995

Taxonomy and phylogeny of industrial solvent-producing clostridia.

We performed a systematic study of 55 solvent-producing clostridial strains, the majority of which are currently classified as Clostridium acetobutylicum strains, by using a combination of biotyping and DNA fingerprint analysis. The biotyping procedures used included rifampin susceptibility testing, bacteriocin typing, and bacteriophage typing. The 55 strains examined exhibited a good correlation between their biotypes and DNA fingerprints, which allowed us to divide them into nine groups. The DNA fingerprints of the nine groups differed markedly, but within each group the DNA fingerprints exhibited a high level of similarity. To determine the phylogenetic relationships of the nine groups, we performed a 16S rRNA gene sequence analysis. The results of a comparative analysis of the partial sequence corresponding to positions 830 to 1383 (Escherichia coli numbering) of the 16S rRNA gene indicated that the nine biotype groups could be assembled into four taxonomic groups. The complete 16S rRNA sequences of strains representing these groups were determined. Our phylogenetic analysis revealed that the amylolytic type strain C. acetobutylicum ATCC 824 (taxonomic group I) was only distantly related to the saccharolytic strains belonging to taxonomic groups II, III, and IV (levels of sequence similarity, 90 to 90.5%). The strains belonging to taxonomic groups II, III, and IV, represented by C. acetobutylicum NCP 262, "Clostridium saccharoperbutylacetonicum" N1-4, and C. acetobutylicum NCIMB 8052T (T = type strain), respectively, were closely related (levels of sequence similarity, 98.2 to 98.9%). C. acetobutylicum NCIMB 8052T exhibited a level of similarity of 100% with the type strain of Clostridium beijerinckii. Reclassification of the saccharolytic solvent-producing strains is necessary, and possible names for the four taxonomic groups are discussed.

Bacterial Typing Techniques↗