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Biomedical subjects

C F Bennett

Publications and source records attributed to C F Bennett.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1994

Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities.

Intercellular adhesion molecule-1 (ICAM-1) binds circulating leukocytes through interactions with beta 2 integrins, LFA-1, and macrophage Ag-1. The phosphorothioate antisense oligodeoxynucleotide, IP-3082, specific for ICAM-1 mRNA inhibited ICAM-1, but not vascular cell adhesion molecule-1, mRNA induction and expression of ICAM-1 molecules by mouse endothelioma cells. Scrambled control oligonucleotides were ineffective. Untreated C3H (H-2k) mice rejected C57BL/10 (H-2b) heart allografts with a mean survival time of 7.7 +/- 1.4 days. Administration i.v. of IP-3082 by a 7-day osmotic pump prolonged the survival of heart allografts in a dose-dependent fashion: 1.25 mg/kg, to 11 +/- 0 days; 2.5 mg/kg, to 12 +/- 2.7 days; 5 mg/kg, to 14.1 +/- 2.7 days; and 10 mg/kg, to 15.3 +/- 5.8 days (all p < 0.01). Control IP-1082 (10 mg/kg) was ineffective (7 +/- 0.8 days). Although 7-day anti-LFA-1 mAb (50 micrograms/day; i.p.) prolonged allograft survival to 14.1 +/- 2.7 days, the addition of IP-3082 (5.0 mg/kg x 7 days) induced donor-specific transplantation tolerance (> 150 days). Furthermore, IP-3082 (5 mg/kg x 7 days) acted synergistically with antilymphocyte serum, rapamycin, and brequinar, but not cyclosporin A: a single antilymphocyte serum (0.2 ml) i.p. injection alone prolonged graft survival to 10 +/- 0.5 days (p < 0.01) and in combination with IP-3082 (5 mg/kg), to 32.2 +/- 8.3 days (p < 0.001); rapamycin (0.1 mg/kg x 7 days; i.v.) alone prolonged survival to 13 +/- 7.5 days (p < 0.01), and with IP-3082, to 32.4 +/- 8.9 days (p < 0.03); brequinar (0.5 mg/kg x 7 days; oral gavage) alone to 12 +/- 2.4 days (p < 0.05), and with IP-3082 (5 mg/kg), to 38.8 +/- 30.2 days (p < 0.01); in contrast, cyclosporin A (5 mg/kg x 7 days; i.v.) alone produced graft survival of 9.8 +/- 1.3 days (p < 0.1 and in combination with IP-3082 (5 mg/kg), produced survival of 7.8 +/- 3.5 days (NS). Thus, antisense oligonucleotides may proffer a selective gene-targeted immunosuppressive therapy for organ transplantation.

Animals↗
PubMed1994

Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner.

Animals↗
PubMed1994

Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters.

We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.

Base Sequence↗
PubMed1994

Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides.

In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.

Base Sequence↗
PubMed1994

Lipid membrane permeability of 2'-modified derivatives of phosphorothioate oligonucleotides.

Antisense oligonucleotides have the ability to inhibit gene expression in viral infections, malignancy, and other diseases. Even though much work has been accomplished with oligonucleotides demonstrating in vitro therapeutic effects, little work has been done to address how these molecules gain access to the cell. One of the plausible means of entrance could be through passive diffusion of the oligonucleotides through the cellular lipid bilayer. To enhance membrane permeability of oligonucleotides lipophilic moieties at the 2' position of the ribose ring have been added. To evaluate the effect of this modification, a liposome system was used. The oligonucleotides evaluated were a series composed of poly A 10mers phosphorothioates labeled at the 5' end with fluorescein and modified at the 2' position of the ribose ring with lipophilic alkyl chains ranging from methyl to nonyl. Efflux studies were accomplished by monitoring the appearance of the oligonucleotide in the incubation medium. There were modest but significant differences between the efflux half-life times of the 2'-modified compounds and the control compound. The values ranged from approximately 6 days for the control, unmodified compound to 4.6 days for the propyl modification. The nonyl derivative had a longer efflux half-life time (8.3 days) compared with the control, unmodified phosphorothioate oligonucleotide.

Chemical Phenomena↗
PubMed1994

Enhanced metastatic ability of TNF-alpha-treated malignant melanoma cells is reduced by intercellular adhesion molecule-1 (ICAM-1, CD54) antisense oligonucleotides.

Treating human malignant melanoma cells with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) causes a dose- and time-dependent increase in surface expression of ICAM-1. Increased ICAM-1 expression corresponds to greater binding of human leukocyte functional antigen-1 (CD11a/CD18)-expressing peripheral blood mononuclear cells (PBMC) to C8161 monolayers, suggesting that cytokine-treated melanoma cells would be more metastatic due to PBMC-tumor cell emboli. The purpose of this study was: (1) to test whether TNF-alpha-treated human melanoma cells are indeed more metastatic than untreated C8161 and (2) to determine whether ICAM-1 plays a role in metastasis of C8161. When surface ICAM-1 expression is upregulated, formation of lung metastases in nude mice increases 1.5- to 4-fold (P < 0.05) for human melanoma cell lines C8161, MeWo, and A375. Treatment of C8161 with ICAM-1 phosphorothioate antisense oligonucleotides using cationic lipids results in > 90% inhibition of ICAM-1 surface expression as determined by ELISA and flow cytometry. Antisense ICAM-1-treated cells form 41-64% fewer metastases than sham-treated cells. Metastasis does not increase when antisense-treated melanoma cells are exposed to TNF-alpha. However, treatment of C8161 with antisense 5-lipoxygenase (5-LO) oligonucleotides inhibits metastases 39% in Lipofectin-treated cells, but does not inhibit TNF-alpha-induced upregulation of experimental metastases. Similar experiments were performed to measure PBMC adhesion to antisense oligonucleotide-treated C8161 cells; however, TNF-alpha-inducible increase in adhesion was unaffected by ICAM-1 or 5-LO antisense oligonucleotides. These results demonstrate that ICAM-1 is involved in melanoma metastasis, but probably not at the step of PBMC adhesion to C8161 cells.

Animals↗