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Biomedical subjects

M Schleef

Publications and source records attributed to M Schleef.

18 recordsLinked to original sources
PubMed2000

Effect of glycoprotein IIb/IIIa receptor blockade with abciximab on clinical and angiographic restenosis rate after the placement of coronary stents following acute myocardial infarction.

OBJECTIVES: In the Intracoronary Stenting and Antithrombotic Regimen-2 trial (ISAR-2), we sought to investigate the effect of abciximab on angiographic and clinical restenosis after stenting following acute myocardial infarction (AMI). We also intended to assess the impact of abciximab on clinical outcome in this setting. BACKGROUND: It is unclear whether abciximab reduces neointima formation after stenting. Such an effect may be particularly prominent in thrombus-containing lesions. METHODS: Patients undergoing stenting within 48 h after onset of AMI were randomly assigned to receive either standard-dose heparin or abciximab plus reduced-dose heparin. Of 401 patients randomized, 366 without 30-day adverse events were eligible for six-month angiographic follow-up. Scheduled angiography was performed in 80% of these patients. RESULTS: By 30 days, the composite clinical end point of death, reinfarction, and target lesion revascularization (TLR) was reached in 5.0% of the abciximab group and in 10.5% of the control group (p = 0.038). At one year, absolute reduction in the composite clinical end point by abciximab was still 5.7% but had lost its statistical significance. Our primary end point, late lumen loss, was 1.26+/-0.85 mm with abciximab and 1.21+/-0.74 mm with standard heparin (p = 0.61), and binary angiographic restenosis rates were 31.1% and 30.6%, respectively (p = 0.92). CONCLUSIONS: In patients undergoing stenting following AMI, abciximab exerted beneficial effects by substantially reducing the 30-day rate of major adverse cardiac events. During one-year follow-up, there was no additional benefit from a reduction in TLR nor did abciximab reduce angiographic restenosis.

Abciximab↗
PubMed2000

Assessment of gunshot bullet injuries with the use of magnetic resonance imaging.

BACKGROUND: Magnetic resonance imaging (MRI) is rarely used for preoperative assessment of shotgun injuries because of concerns of displacing the possibly ferromagnetic foreign body within the surrounding tissue. METHODS: A total of 56 different projectiles underwent MRI testing for ferromagnetism and imaging quality in vitro and in pig carcasses with a commercially available 1.5-MRI scan. Image quality was compared with that of computed tomographic scans. RESULTS: Projectiles with ferromagnetic properties can be distinguished easily from nonferromagnetic ones by pretesting the motion of an identical projectile within the MRI coil. When ferromagnetic projectiles were excluded, MRI yielded the more precise images compared with other imaging techniques. Projectile localization and associated soft tissue injuries were visualized without artifacts in all cases. CONCLUSIONS: When ferromagnetic foreign bodies are excluded by pretesting their properties within the MRI with a comparative projectile, MRI portends an excellent imaging procedure for assessing the extent of injury and planning the removal by surgery.

Animals↗
PubMed2000

Plasmid DNA for pharmaceutical applications.

With the advent and progress of recombinant DNA technology into a variety of fields such as medical therapy, preventive or curative vaccination or the induction of regeneration, the demand for large quantities of highly purified DNA is increasing. Traditional methods of purifying plasmids usually require sophisticated methodology if the DNA is to be separated from RNA and other contaminating organic components. In particular, methods for obtaining supercoiled covalently closed circular (CCC) plasmid DNA in pure form, cope with the requirement that other plasmid topologies also produced have to be separated from the final product. The innovative technology of capillary gel electrophoresis (CGE) contributes a sensitive tool to the short list of applicable quality control assays for clinical grade plasmid DNA.

Biotechnology↗
PubMed1999

Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis.

Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates. Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution. Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE). The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found. The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks. Additionally, the different isoforms can now be quantified by CGE. Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification. CGE also offers high resolution and the amount of DNA required is very low. Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation.

Chromatography, High Pressure Liquid↗
PubMed1998

In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen.

DNA immunization offers a novel means to induce humoral and cellular immunity in inbred or in outbred animals. Here we have tested the efficiency of genetic immunization with hepatitis B virus (HBV) envelope-based vectors. In naive primates, injection of a plasmid DNA encoding HBV envelope proteins induced an HBV-specific cytotoxic response and appearance of potentially protective anti-HBs antibodies. Moreover, intramuscular and intradermal injections of a DNA expression vector encoding an epitope of the human immunodeficiency virus envelope fused to the surface protein of the hepatitis B virus (HBsAg) induced strong humoral and cytotoxic responses to antigenic determinants of both viruses in mice and nonhuman primates alike. In addition, in protein-primed Rhesus monkeys B-cell memory was successfully boosted by DNA injection of hybrid vectors and animals subsequently developed a multispecific cellular response. This suggests that DNA-based immunization could be used to boost efficiently and broaden the immune response in individuals immunized with conventional vaccines, regardless of their genetic variability. These results also indicate that it might be possible to rationally design HBsAg-based expression vectors to induce multispecific immune responses for vaccination against hepatitis B and other pathogens.

Animals↗
PubMed1996

Comparison of plasmid DNA preparation methods for direct gene transfer and genetic immunization.

Plasmid DNA is widely used for direct gene transfer in animals to study gene therapy, gene regulation, drug delivery and genetic immunization. Here we compare cesium chloride and anion-exchange purified plasmid DNA for direct gene transfer in mouse muscle and show no differences in efficiency of transfection with reporter genes or in humoral response to DNA-based immunization.

Animals↗
PubMed1995

DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects of the humoral response mimic hepatitis B viral infection in humans.

Intramuscular injection of plasmid DNA expression vectors encoding the three envelope proteins of the hepatitis B virus (HBV) induced humoral responses in C57BL/6 mice specific to several antigenic determinants of the viral envelope. The first antibodies appeared within 1-2 weeks after injection of DNA and included antibodies of the IgM isotype. Over the next few weeks, an IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. Peak IgG titers were reached by 4-8 weeks after a single DNA injection and were maintained for at least 6 months without further DNA injections. The antibodies to the envelope proteins reacted with group- and subtype-specific antigenic determinants of the HBV surface antigen (HBsAg). Expression vectors encoding the major (S) and middle (preS2 plus S) envelope proteins induced antibodies specific to the S protein and preS2 domain, and preS2 antibodies were prominent at early time points. In general, the expression vectors induced humoral responses in mice that mimic those observed in humans during the course of natural HBV infection.

Animals↗
PubMed1995

DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid.

Vectors expressing the first 58 amino acids of the hepatitis C virus (HCV) nucleocapsid alone or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) were constructed. Intramuscular immunization of BALB/c mice with the chimeric constructs in the form of naked DNA elicited humoral responses to antigens from both viruses within 2 to 6 weeks postinjection. No anti-HCV responses were obtained in mice immunized with the vector expressing the HCV sequence in the nonfusion context. Sera from chimera-injected mice specifically recognized both HCV capsid and HBV surface antigens in enzyme-linked immunosorbent assay and immunoblot testing. Anti-HCV serum titers formed plateaus of approximately 1:3,000; these remained stable until the end of the study (18 weeks postinfection). Anti-HBV immune responses were found to be lower in the chimera-injected animals (< 200 mIU/ml) than in those immunized with the native HBV vector (> 2,000 mIU/ml). This is the first report of the use of DNA-based immunization for the generation of immune responses to an HCV protein. In addition, these findings show that it is possible to elicit responses to viral epitopes from two distinct viruses via DNA immunization with chimeric vectors.

Animals↗
PubMed1994

Direct gene transfer in skeletal muscle: plasmid DNA-based immunization against the hepatitis B virus surface antigen.

Direct gene transfer by intramuscular injection of plasmid DNA encoding an antigenic protein may be used for the purpose of immunization. DNA-based immunization may be of value for basic immunological research and vaccine development. Several factors influence the uptake and expression of plasmid DNA in skeletal muscle, which in turn influence the immune response to the expressed protein. Physical barriers and other factors may impede diffusion of the DNA within the muscle tissue or its entry into the muscle fibres. Although the efficiency of gene transfer in normal mouse muscle is low (< 100 fibres per injection site), a humoral response to the hepatitis B surface antigen (HBsAg) is obtained after expression of a transferred gene. Direct gene transfer is ten times more efficient in regenerating than in normal mouse muscle. DNA-based immunization in such regenerating muscles results in an earlier and stronger humoral response to HBsAg than is seen in normal mature muscle. A needleless jet injection system (Biojector) is able to deliver DNA into normal muscle in rats and rabbits such that a substantial immune response is obtained.

Animals↗
PubMed1994

Exploring the mammalian neuromuscular system by analysis of mutations: spinal muscular atrophy and myotonia.

Any biological structure can be studied using mutations that interfere either with its emergence or its function. We investigate spontaneous and induced mutations in the mouse that affect neuromuscular development and function. The wobbler mouse (phenotype WR, genotype wr/wr) suffers from muscular atrophy because of the degeneration of 20-40% of the motoneurones; it is also unable to produce functional spermatozoa. As a step towards positional cloning of the wr gene, we have mapped the locus to proximal chromosome 11, thus excluding CNTF and its receptor as candidates, and suggesting the closely-linked Rab 1 gene encoding a GTP-binding protein as a possibility. In the case of the adr (arrested development of righting response) mouse, which shows hyperexcitability of mature muscle fibres due to a reduction of the 'dampening' function of chloride conductance at resting potential, we have shown that the defect is in the chloride channel gene adr/Clc-1 on chromosome 6. This allowed us to predict via synteny the chromosomal location of human Thomsen's and Becker's myotonias as close to the TCRB gene on human chromosome 7q. The combination of these approaches with gene-targeting approaches will allow genetic analysis of the establishment and structure of the neuromuscular system.

Animals↗
PubMed1994

Subtractive cDNA cloning as a tool to analyse secondary effects of a muscle disease. Characterization of affected genes in the myotonic ADR mouse.

In myotonic ADR mice that are homozygous for a defect in the muscular chloride channel gene adr/Clc-1, the hyperexcitability of fast muscles is associated with secondary changes in gene expression and fibre type composition. cDNA clones derived from a set of genes down regulated in fast muscles of the myotonic ADR mouse were isolated by a subtractive cloning procedure. A total of 1200 clones were analysed for high expression in fast muscle of wild type and low expression in mutant mouse. Differential transcript levels were verified by northern blot hybridizations. The identities of the corresponding transcripts were determined by sequencing as myosin heavy chain IIB, alpha-tropomyosin, troponin C, a Ca2+ ATPase and parvalbumin mRNAs. Of these, mRNAs for parvalbumin and myosin heavy chain IIB were drastically downregulated in myotonic muscle (to < 10% of control). A full length cDNA clone for skeletal muscle alpha-tropomyosin was homologous to the mouse fibroblast tropomyosin isoform 2, except for the portion encoding the alpha-tropomyosin specific amino acids 258-284. A cDNA derived from the 1100 nucleotide parvalbumin transcript was cloned and the sequence for the as yet unknown 3' extended trailer, generated by alternative polyadenylation, was determined.

Animals↗
PubMed1993

Chromosomal localization and genomic cloning of the mouse alpha-tropomyosin gene Tpm-1.

In vertebrates, the alpha-tropomyosin gene, Tpm-1, codes for at least 9 tropomyosin isoforms that are expressed by tissue-specific alternative splicing. Using interspecies backcrosses, we have localized Tpm-1 on mouse chromosome 9, cen-Cyp1a2-Tpm-1-Mod-1-Mylc-Scn5a, near the d-se region. The restriction fragment length variant used for chromosomal assignment, as well as other restriction fragments hybridizing to a 3'-specific alpha-tropomyosin cDNA probe in genomic Southern blots, was investigated by cloning 17.5-kb of Tpm-1. The same restriction patterns were observed, proving the identity of the mapped and the cloned gene. The identity was supported by sequencing the 3' end of the gene.

Animals↗
PubMed1992

The structure of the mouse parvalbumin gene.

Parvalbumin (PV) is a calcium-binding protein of the EF-hand family, expressed mainly in fast contracting/relaxing muscles of vertebrates. We have isolated five overlapping genomic PV clones which overall span 28 kilobase pairs (kb) around the Pva locus on mouse Chromosome (Chr) 15. The positions of four introns were determined by DNA sequencing. They interrupt the coding sequences at positions corresponding to those in rat and human PV genes. The transcription start site, 25 bp downstream from the TATA-box, was mapped by oligonucleotide primer extension on poly(A)(+)-RNA. The analysis of 0.4 kb promoter sequence of the mouse PV gene revealed CCAAT- and TATA-box sequences and a 59 bp GC-rich stretch between positions -59 and -118. Similar motifs have been found in the parvalbumin genes of rat and human. A perfect 11-bp repeat upstream to positions -149 and -163 respectively is homologous only to the rat promoter. These results will be related to tissue and species differences in PV expression.

Amino Acid Sequence↗