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Biomedical subjects

E Uhlmann

Publications and source records attributed to E Uhlmann.

At least 19 recordsLinked to original sources
PubMed2005

Quantification of oligodeoxynucleotides in human plasma with a novel hybridization assay offers greatly enhanced sensitivity over capillary gel electrophoresis.

Capillary gel electrophoresis using UV detection (CGE-UV) has been used to quantify oligodeoxynucleotides (ODN) in human plasma. Although the sensitivity of this method is adequate to detect antisense ODN, which are administered in daily doses up to 10 mg/kg, CGE-UV is not sensitive enough to detect the much lower quantities of ODN administered for other purposes, such as immune stimulation by CpG ODN. We have developed a very sensitive colorimetric hybridization assay that increases the sensitivity of detection by more than four logs compared with CGE-UV. The hybridization assay uses sequence-specific capture and detection ODN probes complementary to portions of the ODN sequence. Herein we provide a prototype for assay development and validation using a 24- mer immunostimulatory phosphorothioate ODN. Probes were locked nucleic acids (LNA), resulting in increased sensitivity and specificity. The linear range of the assay is 7.8-1000 pg/ml, with a 7.8 pg/ml lower limit of quantification (LLOQ) and a detection limit of 2.8 pg/ml. This translates to detection of 40 attamoles. Intraassay and interassay precision were < or =5.0% CV and < or =12.9% CV, respectively, for quality control samples. The assay is suitable for a variety of matrices, including monkey and rat plasma, allowing application to toxicokinetic samples. The methodology is highly specific, with the ability to distinguish almost all single-base mismatched ODN. The assay detects 100% of the parent as well as some metabolites up to N-4, which are known to be the primary metabolites forming in the first hours after in vivo administration and are physiologically active with in vitro assays.

Animals↗
PubMed2001

Bile acid-oligodeoxynucleotide conjugates: synthesis and liver excretion in rats.

The synthesis of bile acid oligodeoxynucleotide conjugates via the 3-OH group of the bile acids is described. When used in vivo in rats, covalent conjugation of an oligodeoxynucleotide via a linker to cholic acid resulted in an increased biliary excretion of bile acid-oligodeoxynucleotide conjugates compared to unconjugated oligodeoxynucleotides.

Animals↗
PubMed2001

The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma: use of telomerase to obtain stable populations of cells from benign neoplasms.

Angiomyolipomas are benign tumors of the kidney derived from putative perivascular epithelioid cells, that may undergo differentiation into cells with features of melanocytes, smooth muscle, and fat. To gain further insight into angiomyolipomas, we have generated the first human angiomyolipoma cell line by sequential introduction of SV40 large T antigen and human telomerase into human angiomyolipoma cells. These cells show phenotypic characteristics of angiomyolipomas, namely differentiation markers of smooth muscle (smooth muscle actin), adipose tissue (peroxisome proliferator-activator receptor gamma, PPARgamma), and melanocytes (microophthalmia, MITF), thus demonstrating that a single cell type can exhibit all of these phenotypes. These cells should serve as a valuable tool to elucidate signal transduction pathways underlying renal angiomyolipomas.

Actins↗
PubMed2001

Differential cellular expression of neurotrophins in cortical tubers of the tuberous sclerosis complex.

Neurotrophins and their receptors modulate cerebral cortical development. Tubers in the tuberous sclerosis complex (TSC) are characterized histologically by disorganized cortical cytoarchitecture and thus, we hypothesized that expression of neurotrophin mRNAs and proteins might be altered in tubers. Using in situ transcription and mRNA amplification to probe cDNA arrays, we found that neurotrophin-3 (NT3) and trkB mRNA expression were reduced whereas neurotrophin-4 (NT4) and trkC mRNA expression were increased in whole tuber sections. Alterations in mRNA abundance were defined in single microdissected dysplastic neurons (DNs) and giant cells (GCs). NT3 mRNA expression was reduced in GCs and trkB mRNA expression was reduced in DNs. NT4 mRNA expression was increased in DNs and trkC mRNA expression was increased in both DNs and GCs. In three patients, TSC2 locus mutations were confirmed and the mean tuberin mRNA expression levels was reduced across all nine cases. Consistent with these observations, NT3 mRNA expression was reduced but trkC mRNA expression was increased in vitro in human NTera2 neurons (NT2N) transfected with a tuberin antisense construct that reduced tuberin expression. Western analysis of tuber homogenates and computer-assisted densitometry of immunolabeled sections confirmed the neurotrophin mRNA expression data in whole sections and single neurotrophin immunoreactive cells. We conclude that alterations in NT4/trkB and NT3/trkC expression may contribute to tuber formation during brain development as downstream effects of the hamartin and tuberin pathway in TSC.

Adolescent↗
PubMed2001

Oligonucleotide technologies: synthesis, production, regulations and applications. 29-30th November 2000, Hamburg, Germany.

The two-day meeting 'Oligonucleotide technologies', organised by IBC Global Conferences Limited and chaired by M Gait (Medical Research Council, UK), was the first event of this type in Europe. It covered a wide range of oligonucleotide-based approaches useful for the identification and validation of new therapeutic targets as well as for the creation of novel oligonucleotide-based drugs. Recent progress in the preclinical and clinical development of new oligonucleotide-based therapeutics as well as new platforms and systems for DNA microarrays, allowing massively parallel gene expression studies, were discussed. Furthermore, new approaches for overcoming challenges in the large scale synthesis and functionalisation of oligonucleotides were revealed. The following meeting highlights are focussed on contributions devoted to the development of novel oligonucleotide-based drugs, such as antisense oligonucleotides, immunostimulatory CpG oligonucleotides and spiegelmers. Drug candidates discussed here are related to various indications, including cancer, infectious diseases and asthma. Besides highlighting of specific clinical candidates, emphasis is put on the basic principles of oligonucleotide-based drugs and approaches towards the improvement of their properties.

Antineoplastic Agents↗
PubMed2000

An antisense oligonucleotide targeting the alphaV integrin gene inhibits adhesion and induces apoptosis in breast cancer cells.

The aim of this study was to show the anti-adhesive potential of an antisense oligodeoxynucleotide (ODN) approach when designed to suppress the cellular function of the alphaV integrin subunit in breast cancer cells. The alphaV integrins play major roles in favouring breast cancer spreading. In this study, we inhibited alphaV subunit synthesis in the human breast carcinoma cell line, MDA-MB231, by a partially phosphorothioated antisense oligodeoxynucleotide (5543-ODN). The alphaV antisense 5543-ODN reduced alphaV, but not actin, mRNA transcription and protein expression by 55% and 65% respectively (1 microM, 72 h). Control sense and mismatch reagents were inactive. The antisense, but not the sense and mismatch, 5543-ODN induced dose- and time-dependent inhibition of MDA-MB231 adhesion to serum, vitronectin, fibrinogen and fibronectin substrates but was inactive on adhesion to laminin. Thus, the alphaV integrin was located in adhesion structures, which were disrupted by treatment with the alphaV antisense 5543-ODN. Antisense treated cells also showed evidence of programmed cell death with the appearance of apoptotic bodies. MDA-MB231 cells express a mutant form of the pro-apoptotic factor p53; however, no changes in the expression of p53 were observed by Western blotting. Immunofluorescence did reveal an increased nuclear translocation of p53 suggesting activation of the protein, but such a translocation did not lead to significant changes in either the expression of the cyclin dependent kinase inhibitor, p21(WAF1/CIP1) the cell survival factor Bcl-2 or the pro-apoptotic factor Bax.

Antigens, CD↗
PubMed2000

Inhibition of Ras p21 synthesis by antisense undecamers with uniform and specifically arranged phosphorothioate linkages.

The design of chimeric oligodeoxynucleotides (ODNs) in which certain phosphodiester linkages are replaced by phosphorothioate (PS) aims to decrease non-sequence-specific effects of uniform PS ODNs and to preserve the PS-provided protection against exo- and endonucleases. This study has, for the fist time, directly compared the differences in nuclease resistance, cellular uptake, antisense potency and sequence specificity of PS and end-capped, pyrimidine-protected (PPS) undecamer ODNs, that are complementary to the initiation codon region of human Ha-ras mRNA. At concentrations above 5 microM, both PS and PPS undecamers were moderately and equally stable for over 48 h in complete medium with RS485 cells overexpressing Ha-ras. They were completely stable at 0.4 microM when complexed with Lipofectin reagent that enhanced cellular uptake up to 9-fold. Both the antisense PPS and PS undecamers produced well-defined inhibition of Ras p21 synthesis in both cell-free and cell-based assays. However, non-sequence-specific effects of the uniform phosphorothioates were still significant. In contrast, the antisense PPS undecamer, when delivered to RS485 cells with Lipofectin reagent, inhibits human Ras p21 synthesis by more than 90% at a concentration of 3.2 microM, while the effect of controls with inverted, mismatched or scrambled sequence was minimal (5% or less) on p21 synthesis and RS485 cell growth.

Cell Line↗
PubMed1999

Synthesis of a monocharged peptide nucleic acid (PNA) analog and its recognition as substrate by DNA polymerases.

The preparation of a novel phosphoramidite monomer based on thyminyl acetic acid coupled to the secondary nitrogen of 2-(2-amino-ethylamino)ethanol is described. This monomer can be used to attach a deoxynucleotide to the carboxy terminus of a PNA oligomer by solid-phase synthesis. The resulting PNA primer is recognized as a substrate by various DNA polymerases.

Base Sequence↗
PubMed1999

Oligodeoxynucleotide targeted to the alphav gene inhibits alphav integrin synthesis, impairs osteoclast function, and activates intracellular signals to apoptosis.

The alphav integrin subunit is highly expressed in osteoclasts where it dimerizes with beta1 and beta3 subunits to form receptors for vitronectin and bone sialoproteins. Inhibition of osteoclast adhesion and function has previously been achieved by alphavbeta3 antibodies or Arg-Gly-Asp-containing peptides which have the disadvantages of blocking a single receptor type, or of being rather nonspecific, respectively. Here we show that alphav integrin expression in rabbit osteoclasts can be inhibited by partially phosphorothioated antisense oligodeoxynucleotide (ODN) spanning the adenine-uracil-guanine (AUG) translational start site of the human/rabbit alphav gene, a procedure which offers the advantage of affecting all the alphav receptors with high efficiency. The alphav antisense ODN caused a dose-dependent, substrate-specific reduction of osteoclast adhesion and bone resorption. Control ODNs, such as sense, inverted, and mismatch, were without effect, providing evidence of specificity of the antisense reagent. It is likely as a consequence of loss of substrate interaction, the antisense ODN induced osteoclast retraction and apoptosis, increase of the cyclin/cyclin-dependent kinase complex inhibitor p21WAF1/CIP1, and inhibition of the cell survival gene, bcl-2. Although the expression of the cell death-promoting gene, bax, remained unchanged, a reduction of the bcl-2/bax ratio, known to underlie the intracellular signal to apoptosis, was observed. This finding led us to hypothesize that these changes could provide a link between reduction of alphav synthesis and osteoclast programmed death. In conclusion, this study provides novel insights into the use of alphav antisense ODN as an efficacious mechanism for blocking osteoclast function and underscores for the first time the involvement of integrins in bone cell apoptosis. In vivo studies may verify potential application of this ODN as alternative therapy for bone diseases.

Animals↗
PubMed1998

Optimization of the binding properties of PNA-(5')-DNA chimerae.

The synthesis and evaluation of PNA-(5')-DNA chimerae containing either a 5'-amide (i.e. 1a), a 5'-phosphodiester (i.e. 1b) or 5'-phosphonate linkages (i.e. 1c,d) at the junction site are described. The 5'-linkages could be installed using either 5'-amino-5'-deoxythymidine phosphoramidite 2, O-[2-(2-aminoethyl)-(thymin-1-ylacetyl)amino]ethyl phosphoramidite 3, N-(2-aminoethyl)-N-(thymin-1-ylacetyl)aminomethyl phosphonate 4 or N-(2-aminoethyl)-N-(allyloxycarbonyl)aminomethyl phosphonate 5 as building blocks, respectively. It is shown that PNA-(5')-DNA of type 1a-c have a higher binding affinity with complementary RNA than native DNA, and that the antisense activity is mainly due to RNase H.

Chimera↗
PubMed1997

Sequence-specific antiproliferative effects of antisense and end-capping-modified antisense oligodeoxynucleotides targeted against the 5'-terminus of basic-fibroblast-growth-factor mRNA in coronary smooth muscle cells.

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells, has been shown to play a fundamental role in the pathogenesis of arteriosclerosis and restenosis by stimulating the proliferation of vascular smooth muscle cells. We found that partially phosphorothioate-modified 15-residue antisense oligodeoxynucleotides complementary to bFGF mRNA at 0.1-2.0 microM block growth and division of cultured human and bovine coronary smooth muscle cells in a dose-dependent manner. The effect is sequence specific at low (0.1-0.5 microM) nontoxic concentrations. It is associated with inhibition of expression of pericellular and intracellular bFGF, with a decreased de novo synthesis of bFGF and is partly reversible by the addition of exogenous (recombinant) bFGF. The antisense effect lasts 48-72 h and diminishes thereafter. If the antisense oligodeoxynucleotide medium is replaced by an oligonucleotide-free medium after 24 h, the [3H]thymidine incorporation rate returns to control levels. Under the same conditions, the corresponding sense oligodeoxynucleotide exerts negligible nonspecific inhibitory actions. The antiproliferative potency of the 15-residue antisense oligodeoxynucleotide is markedly enhanced by adding 3-4 nonbase-pairing guanosine residues at the 5'- and 3'-termini of the 15-residue antisense oligonucleotide. The data implicate bFGF in the process of smooth muscle cell proliferation and an effective and specific antiproliferative potency of bFGF-specific antisense oligonucleotides. The results point to possible new therapeutic strategies for the use of antisense methodology in the suppression of post-angioplasty restenosis.

Animals↗
PubMed1997

Modified antisense oligonucleotides directed against tumor necrosis factor receptor type I inhibit tumor necrosis factor alpha-mediated functions.

Tumor necrosis factor alpha (TNF alpha), a polypeptide produced by activated macrophages, is a highly pleiotropic cytokine which elicits inflammatory and immunological reactions. The binding of TNF alpha to tumor necrosis factor receptor type I (TNFRI) is considered the initial step responsible for some of the multiple biological functions mediated by TNF alpha. The role of TNF alpha as an inflammatory mediator through human TNFRI makes TNFRI an attractive target for intervention in both acute and chronic inflammatory diseases. In this study, we have identified partial phosphorothioate oligodeoxyribonucleotides (ODNs) containing C-5 propynyl or hexynyl derivatives of 2'-deoxyuridine which specifically inhibited TNFRI and subsequently inhibited the functions of TNF alpha mediated through TNFRI. The most active ODNs were directed against the 3'-poly adenylation signal site on the TNFRI mRNA, and in a cellular assay, gene-specific antisense inhibition occurred in a dose-dependent fashion at submicromolar concentrations, in the presence of Cellfectin. The inhibition of gene expression correlated with the binding affinity of the ODN for the target mRNA. The ODNs lowered TNFRI protein levels and TNF alpha-mediated functions by specifically reducing levels of TNFRI mRNA. These anti-TNFRI ODNs offer a novel approach for controlling biological functions of TNF alpha and may be useful as human therapeutic agents for treating diseases in which TNF alpha has been implicated.

Antigens, CD↗
PubMed1997

Inhibition of carrageenan-induced spinal c-Fos activation by systemically administered c-fos antisense oligodeoxynucleotides may be facilitated by local opening of the blood-spinal cord barrier.

Proto-oncogenes of the fos and jun family are rapidly expressed in the central nervous system following various stimuli. Proto-oncogene encoded nuclear proteins such as c-Fos or c-Jun act as transcription factors that may link neuronal excitation to changes in target gene expression. However, the precise in vivo functions of proto-oncogenes in neuroplasticity are still poorly understood. In the present study the effect of systemically administered c-fos antisense oligodeoxynucleotides (ODNs) on c-Fos and dynorphin protein levels in rat L4 spinal cord has been investigated by immunohistochemistry during carrageenan-induced hindpaw inflammation. Continuous infusion of terminal-phosphorothioated c-fos antisense ODNs by subcutaneously implanted miniosmotic pumps for 3 days sequence-specifically suppressed c-Fos protein expression in dorsal horn neurons by about 50%, while the increase in c-Jun immunopositive nuclei was not affected. Digital image analysis revealed a concomitant decrease in spinal dynorphin immunoreactivity. Moreover, 48 hr after carrageenan injection into one hindpaw plasma protein extravasation was observed in numerous blood vessels in the ipsilateral dorsal horn using intravenously administered Evans Blue. Our results provide further evidence that c-Fos may contribute to the regulation of spinal dynorphin gene expression following noxious stimulation. The local increase in blood-spinal cord barrier permeability during sustained peripheral inflammation may permit penetration of hydrophilic antisense ODNs into the central nervous system.

Animals↗
PubMed1997

Nuclease stability as dominant factor in the antiviral activity of oligonucleotides directed against HSV-1 IE110.

The anti-herpes simplex virus type 1 (anti-HSV-1) efficacy of a series of oligonucleotides was determined as a function of their chemical structure. All oligonucleotides consisted of the same sequence directed against the translation initiation codon of the HSV-1 immediate early gene. The oligonucleotides were modified with phosphorothioate linkage patterns according to various protection strategies against nucleolytic degradation. We show that nuclease resistance is the dominant factor that determines the antiviral efficacy in this series. A minimal protection strategy, consisting of end-capping and pyrimidine protection, has proven to be particularly useful, because it not only yields nuclease-resistant oligonucleotides but also minimizes non-sequence-specific effects.

Animals↗
PubMed1997

Synthesis and binding properties of oligodeoxynucleotides containing phenylphosphon(othio)ate linkages.

A method for the synthesis of chimeric oligodeoxynucleotides comprised of phosphodiester and phenylphosphonate [3'-O-P(= O)(C6H5)-O-5'] or phenylphosphono-thioate [3'-O-P(= S)(C6H5)-O-5'] linkages has been developed. Synthesis was performed using suitably protected nucleoside phenylphosphonamidites as building blocks following an adjusted solid-phase phosphoramidite synthesis protocol. The new oligodeoxy-nucleotide analogues were characterized by electrospray ionization- and matrix-assisted laser desorption mass spectrometry, as well as by 31P NMR spectroscopy. Additionally, their binding properties to complementary oligodeoxynucleotides has been studied.

Chromatography, High Pressure Liquid↗
PubMed1997

Studies on the mechanism of stabilization of partially phosphorothioated oligonucleotides against nucleolytic degradation.

The use of chimeric oligonucleotides (ODN), in which certain phosphodiester internucleoside linkages are replaced by phosphorothioate (PS) linkages to provide protection against degradation by nucleases, is gaining increasing attention because of their significantly decreased propensity for nonantisense effects as compared with uniformly PS-modified ODN. We have recently reported that partially PS-modified ODN, in which end- capping is used to prevent hydrolysis by exonucleases in combination with PS protection at internal pyrimidine residues which are the major sites of endonuclease degradation, are surprisingly stable in serum. The present study investigates an additional role of the backbone modification in the stabilization of ODN against nucleolytic degradation. We show that the stability of an unmodified ODN in fetal bovine serum is significantly enhanced in the presence of PS-modified ODN. The magnitude of stabilization is strongly dependent on the type and degree of backbone modification. The observed effect is stronger for PS-modified ODN than for methylphosphonate (MP)-modified ODN and increases as the number of PS linkages in the ODN increases. Thus, nuclease stability of partially PS-modified ODN is not only caused by direct prevention of nuclease attack at the phosphate center but is additionally supported by interference of the nucleases with the PS groups of ODN, resulting in decreased degradation. As the degree of many nonantisense effects caused by ODN, such as protein interactions and B cell stimulation, is dependent on the backbone modification, our results may have implications for the use of non-ODN nuclease inhibitors to reduce undesirable side effects.

Animals↗