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Biomedical subjects

S Fletcher

Publications and source records attributed to S Fletcher.

At least 37 records · Page 2Linked to original sources
PubMed2000

T-ceIl recognition of discrete regions of the thrombolytic drug streptokinase.

Streptokinase (SK) is a bacterial protein used clinically as a thrombolytic agent in humans. Administration of SK causes a rapid increase in the frequency of anti-SK T cells and the titre of specific anti-SK antibodies that, on subsequent administration of SK, may neutralize the activity of the drug or elicit allergic-type reactions. By locating and modifying the immunogenic T-cell epitopes within the SK protein, it is possible that an agent with reduced immunogenicity but equal efficacy may be produced. We have investigated the T-cell epitopes within SK using nine non-overlapping, recombinant peptide fragments of SK. We investigated the proliferative T-cell response of peripheral blood mononuclear cells obtained from patients before and 6 days after administration of SK for myocardial infarction. We also examined the response of cultured anti-SK T-cell lines derived from patients 6 days after treatment with SK. Before administration of SK, peripheral blood mononuclear cells from six of nine patients showed a proliferative response to SK. The response was significantly higher 6 days after administration of SK (P = 0.0004). Cultured T-cell lines showed similar proliferative responses to clinical-grade SK and recombinant SK. Marked differences in T-cell responses were apparent in response to each recombinant SK fragment (P = 0.04). The mean proliferative response exceeded background to only two peptides, peptide 2 (P = 0.04) and peptide 3 (P = 0.009). Peptide 3, representing amino acids 100-150 of mature SK, was recognized preferentially in the majority of assays. Marked variation in the T-cell response to SK following treatment with this agent was observed between subjects. Despite these differences, peptides 2 and 3 induced T-cell proliferation at a level significantly above background in the majority of subjects. These epitopes may represent a region of enhanced immunogenicity within SK.

Analysis of Variance↗
PubMed1999

Autoradiographic localization of the [3H]-(S)-zacopride labelled 5-HT3 receptor in porcine brain.

Using the technique of in vitro receptor autoradiography, we have determined the distribution of the [3H]-(S)-zacopride labelled 5-HT3 receptor in porcine brain. Highest densities of 5-HT3 receptor-associated [3H]-(S)-zacopride binding were detected in areas of porcine spinal cord, nodose ganglion, trigeminal nerve nucleus, area postrema and cerebral cortex, with relatively lower levels in other brain regions (e.g. hippocampus, caudate-putamen). The distribution of [3H]-(S)-zacopride binding in porcine forebrain provides further evidence for inter-species differences with respect to the differential expression of the 5-HT3 receptor in the forebrain.

Animals↗
PubMed1999

Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides.

The mdx mouse, which carries a nonsense mutation in exon 23 of the dystrophin gene, has been used as an animal model of Duchenne muscular dystrophy to evaluate cell or gene replacement therapies. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin-positive fibres have been observed in mdx muscle tissue. These dystrophin-positive fibres are thought to have arisen from an exon-skipping mechanism, either somatic mutations or alternative splicing. Increasing the frequency of these fibres may offer another therapeutic approach to reduce the severity of Duchenne muscular dystrophy. Antisense oligonucleotides have been shown to block aberrant splicing in the human beta-globin gene. We wished to use a similar approach to re-direct normal processing of the dystrophin pre-mRNA and induce specific exon skipping. Antisense 2'-O-methyl-oligoribonucleotides, directed to the 3' and 5' splice sites of introns 22 and 23, respectively in the mdx pre-mRNA, were used to transfect myoblast cultures. The 5' antisense oligonucleotide appeared to efficiently displace factors normally involved in the removal of intron 23 so that exon 23 was also removed during the splicing of the dystrophin pre-mRNA. Approximately 50% of the dystrophin gene mRNAs were missing this exon 6 h after transfection of primary mdx myotubes, with all transcripts showing skipping of exon 23 after 24 h. Deletion of exon 23 does not disrupt the reading frame and should allow the synthesis of a shorter but presumably functional Becker-like dystrophin. Molecular intervention at dystrophin pre-mRNA splicing has the potential to reduce the severity of a Duchenne mutation to the milder Becker phenotype.

Alternative Splicing↗
PubMed1999

Effects of oral propylthiouracil treatment on nitric oxide production in rat aorta.

The effects of oral propylthiouracil (PTU) treatment on vascular nitric oxide (NO) production were studied in the rat aorta. Rats were fed a standard low fat diet with or without 0.1% PTU, for 2 or 4 weeks, or for 2 weeks with additional thyroxine injections. Concentration response curves were then constructed to phenylephrine (PE) in both endothelium-intact and denuded aortic rings from these animals and after incubation with 0.1 mM L-N(G)nitroarginine (L-NOARG). In addition, expression of nitric oxide synthase (NOS) was analysed in sections of aorta from PTU-treated and control rats using rabbit polyclonal antibodies to both inducible NOS (iNOS) and endothelial NOS (eNOS). Oral PTU treatment resulted in a significant reduction in both the maximum response (control, 0.53+/-0.02; 2 week PTU, 0.20+/-0.07; 4 week PTU, 0.07+/-0.02 g mg(-1)) and vessel sensitivity (EC50 values: control, 9.10x10(-8)+/-0.67; 2 week PTU, 7.45x10(-7)+/-1.15; 4 week PTU, 9.73x10(-7)+/-0.45 M) to PE in endothelium-intact vessel rings, as compared to controls (P<0.05). Both endothelial removal and incubation with L-NOARG restored the maximum response after 2, but not 4 weeks, although, in general, vessel sensitivity was not altered by either treatment. Vessels from PTU-treated rats given thyroxine injections showed no significant differences between any of the dose response curve parameters. Immunohistochemical analysis suggested that labelling for eNOS may be increased after PTU treatment as compared to control animals, whereas iNOS antibody immunoreactivity was not different between the two groups. These results suggest that the hyporesponsiveness to PE observed after oral PTU treatment is, in part, due to enhanced nitric oxide (NO) production by the endothelium, and demonstrate for the first time that thyroid hormones may play a role in the regulation of eNOS activity in the rat aorta.

Animals↗
PubMed1999

Humoral and cellular immune responses up to 7.5 years after administration of streptokinase for acute myocardial infarction.

AIMS: Administration of streptokinase results in an immunological response which may lead to increased risk of anaphylactic reaction or reduced thrombolytic efficacy on repeat administration. For these reasons current recommendations suggest that streptokinase should not be given up to 1 year after first administration. We sought to define the profile of both the circulating antibody and T-cell response to streptokinase in patients who had received streptokinase up to 7.5 years previously following acute myocardial infarction. METHODS: Neutralizing anti-streptokinase antibody and total anti-streptokinase IgG were measured in 219 patients who had suffered acute myocardial infarction between 12 and 90 months previously and had received streptokinase. T-cell response to streptokinase was assessed by in-vitro proliferation of peripheral blood mononuclear cells (n=234). Data on all parameters were available in 184 patients. Controls (n=22) had suffered acute myocardial infarction between 73 and 84 months previously but had not received thrombolytic therapy. RESULTS: Compared to controls, anti-streptokinase antibodies were elevated at all time periods from 12 to 90 months after streptokinase treatment. Total anti-streptokinase titres showed the expected correlation with neutralizing anti-streptokinase antibodies (P<0.0001). Peripheral blood mononuclear cells showed a vigorous in-vitro proliferative response to streptokinase 6 days after treatment (P=0.05 vs pre-treatment), but this was not detectable at 6 weeks or subsequently. CONCLUSION: There is as yet no evidence of a time limit beyond which administration of streptokinase on a second occasion can be regarded as safe and likely to be effective. Measurement of neutralizing anti-streptokinase or total anti-streptokinase IgG titre appear to provide equivalent information regarding the antibody status of a population. Further studies are required regarding the apparent lack of peripheral blood mononuclear cells responsiveness in patients previously exposed to streptokinase.

Adult↗
PubMed1999

Development of a snapback method of single-strand conformation polymorphism analysis for genotyping Golden Retrievers for the X-linked muscular dystrophy allele.

OBJECTIVE: To develop a snapback method of single-strand conformation polymorphism (SSCP) analysis for genotyping Golden Retrievers for the X-linked muscular dystrophy allele. ANIMALS: 20 Golden Retriever puppies from a colony with X-linked muscular dystrophy. PROCEDURE: DNA spanning the canine dystrophin mutation was amplified by means of a polymerase chain reaction (PCR), using a primer modified to have an additional sequence at the 5' terminus. The primer was designed so that 1 terminus of the single-stranded PCR product could anneal to the normal sequence flanking the region of the mutation in the allele but not in the mutant allele. True disease status of the dogs was determined by means of a PCR and restriction digest protocol. RESULTS: Snapback SSCP analysis allowed for accurate and unambiguous genotyping of unaffected, carrier, and affected dogs, whereas conventional SSCP analysis, using the unmodified primer, did not. Creatine kinase activities measured within 24 hours after birth were not consistent with genotype. CONCLUSION AND CLINICAL RELEVANCE: Snapback SSCP analysis provided a simple, fast, and accurate method for genotyping Golden Retrievers for the mutation known to cause X-linked muscular dystrophy.

Animals↗
PubMed1999

Parallel syntheses of disulfide inhibitors of the thioredoxin redox system as potential antitumor agents.

We have reported previously that unsymmetrical disulfide inhibitors of the human thioredoxin/thioredoxin reductase redox system (hTrx/TR) possess antitumor activity. We have broadened the search for more potent inhibitors and evaluated a large range of mono- and bis-disulfide compounds, prepared using parallel syntheses. Reaction of isothioisourea-HCI salts (R') or bis-salts (R) with aromatic or aryl thiols (R") in wells of 96-well plates produced >450 derivatives with the structures R"SSR' and R"SSRSSR". The excellent yield and purity of the disulfides provided sufficient material for evaluations of enzyme inhibition and cytotoxicity. Selection criteria based on the IC50 values for hTrx/TR inhibition and for cytotoxicities of the disulfides identified agents for subsequent scale-up syntheses and in vivo evaluations of antitumor activity. These scale-up studies confirmed the original activities of agents synthesized in the plates and validated the parallel synthetic approach. Structure-activity information derived from the hTrx/TR IC50 data allow for a number of generalizations. The most potent inhibitors of the Trx system contained two heteroatoms ortho to the disulfide moiety in an aromatic functionality. The thioalkylating moieties had greatest activity with one branch point alpha to the disulfide. In the absence of branching, more potent inhibition was observed with the electron withdrawing functionalities. Bis-disulfides showed patterns of activity which depended on chain length, with optimum activity observed when the disulfide units were separated by 3.9 A, a similar distance to that separating the thioredoxin active site cysteine residues. From the agents selected for scale-up syntheses, three disulfide compounds were studied for their antitumor activity in vivo against human tumor xenografts in scid mice. One of the analogues discovered through the combinatorial syntheses/screening for Trx inhibition, 1-phenylethyl 2-imidazolyl disulfide, N1 (ProlX agent PX-C5), has demonstrated excellent in vivo activity against the MCF-7 human breast cancer and the HL-60 human leukemia, thus validating this approach for novel drug discovery.

Animals↗
PubMed1998

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.

Replication-deficient adenovirus vectors (AdV) have been successfully used to transfer a truncated human dystrophin cDNA to skeletal muscle of dystrophin-deficient mdx mice. A dystrophin-deficient golden retriever dog model (GRMD) has been identified, which, unlike the mouse model, leads to a clinicopathological phenotype similar to that of Duchenne muscular dystrophy (DMD). We show for the first time that high-level dystrophin expression in skeletal muscle of GRMD dogs can be achieved by AdV-mediated gene transfer. However, a humoral and cellular immune response of the host against antigens of viral and transgene origin (similar to that occurring in mdx mice after AdV-mediated dystrophin gene transfer) leads to a decline of dystrophin expression over a 2-month period. Immunosuppression by cyclosporin significantly prolonged transgene expression. The GRMD model may help to solve the open questions pertaining to dystrophin gene transfer such as systemic delivery and improvement of muscle function before human trials for gene replacement therapy in DMD may be considered.

Adenoviridae↗
PubMed1998

Direct dystrophin and reporter gene transfer into dog muscle in vivo.

Bacterial beta-galactosidase cDNA was injected without lipofectin into 41 sites in dog muscle and expression was seen in 22 of them. The cDNA and lipofectin was injected into 35 similar sites and expression was seen in 21. Expression was seen in a maximum of 2.5% of muscle fibers and 23.21% of nonmuscle cells. A total of 106 muscle sites were injected with the minigene with and without lipofectin. In 4 of the 45 sites injected with the minigene without lipofectin human dystrophin was expressed around the periphery of 0.3% of the fibers. Bacterial beta-galactosidase cDNA was injected into the peritoneal cavity of 4 pups, 2 of which also received lipofectin. In all 4, expression was seen in liver, spleen, and mesenteric lymph node. In the 2 pups that received lipofectin, expression was also seen in the diaphragm, intercostal, and abdominal muscles of 1 and in the diagphragm and intercostal muscles of the other. These experiments show that human dystrophin transgene expression can be obtained in dog muscle. However, other methods will be required to increase the degree of expression before gene therapy trials can be undertaken.

Animals↗
PubMed1998

Snapback SSCP analysis: engineered conformation changes for the rapid typing of known mutations.

Several approaches may be applied to detect known mutations, including restriction enzyme cleavage, allele-specific oligonucleotide (ASO) hybridization or amplification, dideoxy fingerprinting, and direct DNA sequencing. All these approaches require several extra steps after PCR and may involve radioactive isotopes, time-consuming hybridization, template purification, or digestion steps. The ease and simplicity of the SSCP test make it a popular choice for mutation detection, but a significant limitation is that some DNA changes will not alter the overall conformation of either single strand and are thus not amenable to SSCP typing. We describe Snapback SSCP to genotype normal and mdx mice (an animal model of Duchenne muscular dystrophy) that previously could not be differentiated by conventional SSCP analysis. A snapback primer was designed with additional bases at the 5' terminus, which were complementary to the normal sequence flanking the mdx mutation and used under the original amplification conditions. Each single strand of these snapback PCR products now had one terminus capable of re-annealing or "snapping back" to the normal sequence but not the mdx mutation. In this manner, a conformation change was engineered into the normal strand that could be readily distinguished from the mdx allele on a SSCP gel. This approach could be applied to the routine screening of other known mutations that are not amenable to detection by simple SSCP analysis.

Animals↗
PubMed1998

Evidence that porcine native 5-HT3 receptors do not contain nicotinic acetylcholine receptor subunits.

The present investigation utilised monoclonal antibodies directed against subunits of the nicotinic acetylcholine receptor in immunoblot and immunoprecipitation studies, which failed to demonstrate that the native 5-hydroxytryptamine3 (5-HT3) receptor complex purified from porcine brain contains the alpha1, alpha3, alpha4, alpha5, alpha7 or beta2 subunits of the nicotinic acetylcholine receptor.

Animals↗
PubMed1998

Desperately seeking subunits: are native 5-HT3 receptors really homomeric complexes?

The 5-HT3 receptor complex is a ligand-gated ion channel, and is therefore likely to comprise multiple subunits in common with other members of this superfamily. To date, however, only one 5-HT3 receptor subunit, plus an alternatively spliced variant, have been identified. In this article, Stephanie Fletcher and Nicholas Barnes review some of the extensive data in the literature that suggest the presence of other 5-HT3 receptor subunits. This is particularly relevant given the recent demonstration that the 5-HT3 receptor purified from pig brain contains a non-5-HT3A-like protein(s).

Allosteric Regulation↗
PubMed1998

Pax7 includes two polymorphic homeoboxes which contain rearrangements associated with differences in the ability to regenerate damaged skeletal muscle in adult mice.

Pax7 is a paired-type homeobox gene which has previously been shown to play an important role in skeletal muscle formation. It is expressed in skeletal muscle of the limbs during embryogenesis and in adulthood. The aims of this study were firstly to determine the degree of polymorphism of Pax7 amongst inbred laboratory mice using Southern blotting and Pax7 regional specific sub-probes. Secondly, functional studies were performed on mice with each of the different structural forms of Pax7 to determine whether they were associated with differences in the ability to regenerate damaged skeletal muscle. Four different allelic forms of Pax7 have now been identified in laboratory mice indicating that the previously reported DNA sequence of Pax7 is not applicable to all laboratory mice. Hybridisation patterns of TaqI digested DNA representing each of the different Pax7 alleles with the Pax7 specific sub-probes suggested that in contrast to previous findings, Pax7 is associated with two highly polymorphic homeoboxes. The presence of two homeoboxes in BALB/c mice has been confirmed by DNA sequencing. Results of functional studies have also shown that the ability to regenerate damaged skeletal muscle in adult mice is strongly associated with the presence of a 0.15-kb TaqI fragment derived from one of the homeoboxes.

Alleles↗
PubMed1998

Assessment of ultrasound guided percutaneous ethanol injection and parathyroidectomy in patients with tertiary hyperparathyroidism.

BACKGROUND: Tertiary hyperparathyroidism continues to cause significant morbidity in patients with chronic renal failure. This is frequently resistant to medical management and may ultimately require a surgical parathyroidectomy. Recent studies have reported upon the technique of percutaneous ethanol ablation for both primary and tertiary hyperparathyroidism. In this study we report on a 5 year experience using ethanol injection and compare the results with surgical parathyroidectomy. METHODS: A prospective study in 39 patients with tertiary hyperparathyroidism, 25 were dialysis dependent and 14 had a functioning renal allograft. Twenty-two patients underwent percutaneous fine needle ethanol injection (PFNEI) and 17 underwent surgical parathyroidectomy. RESULTS: A > 30% reduction in intact parathyroid hormone (iPTH) was achieved in 11 of 22 patients undergoing PFNEI after a mean of 1.8 +/- 1.4 injections per gland. In four patients, symptomatic hyperparathyroidism recurred and they required further PFNEI or surgical parathyroidectomy at 17, 28, 46, and 48 months later. There was no significant reduction in iPTH in 11 patients following PFNEI after a mean of 2.5 +/- 1.3 injections per gland. They all required a subsequent surgical parathyroidectomy for symptomatic hyperparathyroidism. Four patients developed a laryngeal nerve palsy following PFNEI, two of which were permanent. Seventeen patients underwent successful surgical parathyroidectomy as a primary procedure. CONCLUSION: Whilst PFNEI is successful in primary hyperparathyroidism, when typically only one adenoma is present, the effectiveness of PFNEI is unpredictable and the long term results are poor compared with those of surgical parathyroidectomy in tertiary hyperparathyroidism. The procedure is not without complications and makes subsequent surgery more difficult. Therefore it can only be recommended for patients with a known single parathyroid gland such as patients in whom hyperparathyroidism has recurred following a previous surgical subtotal parathyroidectomy and who are unsuitable for further surgery.

Administration, Cutaneous↗