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M W Reed

Publications and source records attributed to M W Reed.

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

Mast cell stabilization prevents ethanol-induced rat gastric mucosal injury: mechanisms of protection.

INTRODUCTION: We previously demonstrated that 60% ethanol increased macromolecular leakage and induced lesion formation in areas of permanent flow stasis within gastric mucosal vessels. Mast cells and their mediators have been implicated in acute mucosal injury. Fluorescent in vivo microscopy was used to assess the effects of ketotifen, a mast cell stabilizer, and pyrilamine, a histamine (H1)-receptor antagonist, on ethanol-induced rat gastric mucosal injury. METHODS: Experiments were carried out on anaesthetized rats pretreated orally with ketotifen (1 mg/kg) or pyrilamine (30 mg/kg). Fluorescein isothiocyanate-bovine serum albumin (FITC-BSA; 0.2 mL/100 g), a marker for quantitating macromolecular leakage was administered intra-arterially. Ethanol (60%) or distilled water was applied topically to the gastric mucosa. Macromolecular leakage of FITC-BSA, vessel diameters and leucocyte activity were quantified using image analysis. RESULTS: Pretreatment with ketotifen or pyrilamine, followed by ethanol, caused no change in macromolecular leakage compared with controls. Both compounds prevented blood flow stasis in all areas and no lesion formation was observed. However, increased leucocyte activity and increases in vessel diameter were observed following pretreatment with ketotifen and pyrilamine, respectively. CONCLUSIONS: The data suggest that vasoactive substances released from mast cells may be involved in the aetiology of ethanol-induced gastric mucosal damage. The prevention of these normal physiological responses to injury may lead to the employment of other microcirculatory mechanisms of defence.

Animals↗
PubMed2000

L-Arginine protects and exacerbates ethanol-induced rat gastric mucosal injury.

BACKGROUND AND AIMS: We have previously demonstrated that 60% ethanol (EtOH) increases macromolecular leakage and induces focal lesion formation in areas of permanent flow stasis within gastric mucosal vessels. Nitric oxide (NO) may prevent lesion formation by inhibiting leakage. This study used fluorescent in vivo microscopy to investigate (i) whether L-arginine (NO precursor) prevented EtOH induced injury and (ii) the mechanisms of protection. METHODS: Experiments were carried out on anaesthetized rats (hypnorm/diazepam) receiving either intra-arterial fluorescein isothiocyanate-bovine serum albumin (0.2 mL/100 g), a marker for quantitating leakage, or Acridine red (0.1 mL/100 g) which labels leukocytes. Animals then received 100, 300 or 500 mg/kg L-arginine (i.a.) followed by 60% EtOH or distilled water, topically applied to the gastric mucosa (n = 6 for each group). Vessel diameter, macromolecular leakage of labelled albumin from post capillary venules (PCV) and capillaries and leukocyte activity were quantitated using image analysis. RESULTS: L-Arginine (100 mg/kg) did not increase vessel diameter or prevent EtOH-induced lesion formation and leakage. Both 300 mg/kg and 500 mg/kg alone induced significant and sustained increases in PCV diameter after 15 (P< 0.01) and 5 min (P< 0.001), respectively. Lesion formation was only prevented by 300 mg/kg L-arginine, whereas 500 mg/kg exacerbated haemorrhagic lesion formation over the entire exposed mucosa. Neither 300 mg/kg nor 500 mg/kg L-arginine prevented leakage following EtOH. No leukocyte activity was observed following EtOH with or without L-arginine pretreatment. CONCLUSION: L-Arginine (300 mg/kg) prevented lesion formation. The mechanism of protection probably involved the increased blood flow in the dilated PCV and not the inhibition of leakage. The combined effects of EtOH and the possible high NO levels exacerbate gastric mucosal damage despite the increases observed in vessel diameter.

Animals↗
PubMed2000

Aminolevulinic acid-induced photodynamic therapy: safe and effective ablation of dysplasia in Barrett's esophagus.

Photodynamic therapy (PDT) is a treatment in which cell damage is achieved by the action of light on a photosensitizer. The aim of this study was to assess the value of PDT in the treatment of Barrett's esophagus with low-grade dysplasia (LGD). Forty patients with dysplastic Barrett's esophagus were given aminolevulinic acid (ALA) 30 mg/kg orally followed 4 h later by laser endoscopy. Follow-up endoscopy and biopsies were performed at 1, 6 and 12 months. A macroscopic response was seen in 33 out of 40 patients, with a median decrease in columnar epithelial area of 30% (range 0-90%). Post-treatment biopsies showed no dysplasia in 39 out of 40 patients, with LGD remaining in one case. This was maintained at 6 and 12 months. There were no significant side-effects. This study demonstrates that ALA-induced PDT provides safe and effective ablation therapy for Barrett's esophagus and is particularly useful against dysplasia.

Aminolevulinic Acid↗
PubMed2000

Trafficking of 'immune' CD4(+)/CD8(+)T-lymphocytes into the RENCA tumour microcirculation in vivo in mice.

RENCA-IL-2 (Murine Renal Cell Carcinoma transfected with murine IL-2 gene) cells were rejected by immunocompetent (but not T-cell deficient) Balb/c mice, which developed 'immunity' to subsequent parental RENCA tumour cell challenge. Splenocytes adoptively transferred this immunity. CD4(+)and CD8(+)T-lymphocytes prepared from the spleens of 'tumour immune' mice were evaluated for their ability to traffic into the tumour environment using an in vivo model that enables visualization of events within the microvasculature. RENCA cells were implanted into the mouse cremaster muscle and the trafficking of syngeneic lymphocyte subpopulations, derived from naive and 'immune' animals, into both the RENCA tumour and the surrounding normal cremaster muscle microcirculation was measured by in vivo microscopy. Fluorescently labelled CD4(+)and CD8(+)T lymphocytes taken from the spleens of naive mice or mice previously immunized with RENCA-IL-2 were injected systemically into tumour-bearer mice. Naive effector cells migrated to, and flowed through both the tumour and the normal microcirculation, with negligible adhesion. However we observed the selective recruitment, localization and arrest of immune CD4(+)and CD8(+)T lymphocytes (P< 0.05) into the tumour microcirculation, and in some instances the subsequent extravasation of cells into the tumour interstitium. Lymphocyte rolling by 'immune' CD4(+)and CD8(+)T-cells in the tumour microcirculation was greatly reduced, suggesting impaired adhesion molecule expression on the tumour endothelium. This study clearly demonstrates, by direct in vivo microscopy assessment, the localization of effector cells, CD4(+)and CD8(+)lymphocytes into tumours.

Adenocarcinoma↗
PubMed2000

Distribution of DNA strand breaks produced by iodine-123 and indium-111 in synthetic oligodeoxynucleotides.

Antigene radiotherapy, a procedure based on delivery of short-range Auger-electron-emitting radioisotopes to target genes via sequence-specific triplex-forming oligonucleotides, has been successfully demonstrated in vitro using the well-studied radionuclide 125I. To proceed with in vivo trials, Auger electron emitters with shorter half-lives than 125I are required. Here we report a study of the efficiency and distribution of sequence-specific DNA strand breaks produced by decay of 123I and mIIn. 123I and 111In were introduced into triplex-and duplex-forming oligodeoxyribonucleotides (ODNs) through carbohydrate linkers of various lengths. Labeling with radioiodine was performed through tributylstannylbenzamide intermediates while 111In was attached via DTPA. The Auger-emitter-labeled ODNs were hybridized to a single-stranded DNA target, to form duplexes. After decay accumulation, the target DNA samples were assayed for strand breaks using a sequencing gel-electrophoresis technique. For the first time, we observed footprints of DNA strand breaks produced by 123I and 111In. Most of the breaks were located within 10 nucleotides from the decay site. The yield of strand breaks per decay varies; decay of 111In breaks DNA almost 10 times more effectively than decay of 123I. Both 123I and 111In are less effective in breaking DNA strands than 121I, which reflects the higher total energy of the Auger decay process of 125I.

Binding Sites↗
PubMed2000

Targeting the human mdr1 gene by 125I-labeled triplex-forming oligonucleotides.

Antigene radiotherapy is our approach to targeting specific sites in the genome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as 125I, with the sequence-specific action of triplex-forming oligonucleotides (TFO). As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line. Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method. 125I-TFO were delivered into KB-V1 cells with several delivery systems. DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization. Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence. We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells. Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus. The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Photodynamic therapy for dysplastic Barrett's oesophagus: a prospective, double blind, randomised, placebo controlled trial.

BACKGROUND AND AIMS: Photodynamic therapy (PDT) is a treatment in which cell damage is achieved by the action of light on a photosensitizing agent. We have assessed the potential use of PDT in the ablation of Barrett's oesophagus. METHODS: Thirty six patients with dysplastic Barrett's oesophagus receiving acid suppression medication with omeprazole were randomised to receive oral 5-aminolaevulinic acid (ALA) 30 mg/kg or placebo, followed four hours later by laser endoscopy. Follow up endoscopy was performed at one, six, 12, and 24 months. RESULTS: Of 18 patients in the ALA group, a response was seen in 16 (median decrease in area in the treated region 30%; range 0-60%). In the placebo group, a decrease in area of 10% was observed in two patients with no change in 16 (median 0%; range 0-10%; treatment v placebo, p<0.001). No dysplasia was seen in the columnar epithelium within the treatment area of any patient in the PDT group. However, in the placebo group, persistent low grade dysplasia was found in 12 patients (p<0.001). There were no short or long term major side effects. The effects of treatment were maintained for up to 24 months. CONCLUSIONS: This is the first randomised controlled trial of PDT for Barrett's oesophagus. It demonstrates that ALA induced PDT can provide safe and effective ablation of low grade dysplastic epithelium.

Adult↗
PubMed2000

Proteolysis in human breast cancer.

BACKGROUND: The process of proteolysis is important at several stages of the metastatic cascade. A balance between the expression of the genes encoding endogenous proteinases and inhibitors exists and when the production of proteinases exceeds that of inhibitors proteolysis occurs. AIMS: To determine whether differences in the profile and activity of proteinases and inhibitors exist within breast tumour tissue (n = 51), surrounding background breast tissue (n = 43), normal breast tissue from breast reduction mammoplasty operations (n = 10), and cells of the breast cancer cell line, MCF-7. METHODS: Proteinase (matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, urokinase-type plasminogen activator (uPA), and tissue-type PA (tPA)) and inhibitor (tissue inhibitor of metalloproteinases; TIMP-1 and TIMP-2) expression and proteinase activity were compared using substrate zymography, western blotting, immunohistochemistry, and quenched fluorescent substrate hydrolysis. RESULTS: The presence of all proteinases and inhibitors was greater in breast tumour tissue when compared with all other types of breast tissue (p < 0.05). The activity of total MMPs as determined by quenched fluorescent substrate hydrolysis was also greater in breast tumours (p < 0.05). CONCLUSIONS: There is increased proteolysis in human breast tumours when compared with other breast tissues.

Analysis of Variance↗
PubMed2000

DNA cleavage by 111In-labeled oligodeoxyribonucleotides.

UNLABELLED: We studied the fine structure of DNA damage produced by the decay of 111In incorporated into duplex and triplex DNA strands to evaluate the usefulness of this radionuclide for sequence-specific DNA cleavage. METHODS: Oligodeoxyribonucleotides (ODNs) were prepared with 111In attached by diethylenetriaminepentaacetic acid (DTPA) at the 5' end or 3' end through a long chemical linker or to an internal nucleotide position through a short linker. Subsequent formation of DNA duplexes and triplexes was confirmed by gel electrophoresis. The 111In-induced breaks were assayed in denaturing polyacrylamide gel electrophoresis with a single-nucleotide resolution. RESULTS: 111In-labeled oligonucleotides of high specific activity (740-1554 TBq/mmol) were synthesized. The presence of the bulky 111In-DTPA group did not impede duplex or triplex formation. Localized DNA breaks were observed in all duplexes and triplexes formed. The majority of DNA breaks in duplex formations were located within +/- 10 nucleotides from the site of attachment of the 111In-bearing linker. The yield of DNA breaks per decay was 0.38 in a duplex with internally modified ODNs. This is nearly 2 times less than the yield of DNA breaks in the same duplex with 1251 attached through the same linker. The yield of DNA breaks in the pyrimidine and purine strands of DNA triplexes with 111In attached to the triplex-forming ODNs through the linkers of different length varied from 0.05 to 0.10. The distribution of DNA breaks was wider in comparison with the duplex experiment. The lower yields of breaks per 111In decay compared with 125I may be not only the result of lower deposited energy but also of the ionic repulsion of the negatively charged 111In-DTPA group from the DNA strands. CONCLUSION: We have shown that decay of 111In produces highly localized DNA breaks. 111In introduced into triplex- and duplex-forming ODNs through hydrocarbon linkers produces sequence-specific DNA strand breaks with an efficiency nearly comparable with that of 1251. These findings are supportive of our proposed use of 111In-ODNs for gene-specific radiotherapy.

DNA↗
PubMed1999

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides.

Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.

Alkylating Agents↗
PubMed1999

Common duct diameter as an independent predictor of choledocholithiasis: is it useful?

We have evaluated the value of ultrasonographic measurement of common duct diameter in isolation from other predictors of choledocholithiasis. A cohort of 223 patients who did not have a history of jaundice or pancreatitis and had normal liver function tests underwent pre-operative ultrasound of the common hepatic duct and then had elective (open) cholecystectomy with operative cholangiography. One hundred and ninety-one (86%) of all patients had a normal (< or = 5 mm) common duct and of these only 12 (6%) had common duct calculi. Of the 32 patients who had a common duct diameter greater than 5 mm on pre-operative ultrasonography, 12 (37.5%) patients had ductal calculi. A pre-operatively dilated duct on ultrasonography has a high chance of containing stones and should be considered as a strong indication for pre-operative bile duct imaging or operative cholangiography.

Adult↗
PubMed1999

Proteolysis in human breast and colorectal cancer.

Proteolysis occurs when proteinase activity exceeds inhibitor activity. Proteolysis is normally tightly regulated and is involved in cancer invasion and metastasis. The aim of this study was to compare proteolysis in breast and colorectal cancer. Proteinase and inhibitor expression were analysed in paired tumour and normal tissue samples from 43 breast and 24 colorectal cancer patients using substrate zymography, Western blotting and quenched fluorescence substrate hydrolysis. The expression of the latent forms of matrix metalloproteinase-2 (MMP-2), MMP-3 and MMP-9, urokinase plasminogen activator (uPA), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 expression were observed in both tumour and normal tissue samples from breast and colorectal tissue; however, expression was greater in the tumour tissue. Expression of active MMP-2 and MMP-9 and the total MMP activity were greater in tumour compared to normal samples in both tissues (P < 0.05). The expression of all proteinases and total MMP activity was greater in colorectal tissue than breast tissue samples. Breast and colorectal cancer demonstrated different proteinase profiles, however proteolysis in both tissues was greater in tumour tissue than normal tissue.

Blotting, Western↗
PubMed1999

Ablation treatment for Barrett oesophagus: what depth of tissue destruction is needed?

AIM: To establish the depth of Barrett's columnar epithelium and normal squamous oesophageal epithelium, in order to determine the depth of destruction required in ablation treatment for Barrett oesophagus. METHODS: Histological specimens from 100 cases of Barrett oesophagus and 100 samples of normal squamous oesophageal epithelium were studied. Using a system of multiple measurements until the change in cumulative mean values varied by less than 5%, the overall mean and normal range of depth was calculated for each type of epithelium. RESULTS: Barrett columnar epithelium is minimally thicker (mean (SEM) 0.50 (0.004) mm; range 0.39 to 0.59 mm) than normal squamous epithelium (0.49 (0.003) mm; 0.42 to 0.58 mm), although this difference is probably too small to be of clinical relevance. CONCLUSIONS: Although there are numerous clinical reports of various methods of ablation treatment for Barrett oesophagus, little attention has been paid to the depth of tissue destruction required. This is the first study to look specifically at this issue, and it provides information on the necessary depth of epithelial ablation.

Barrett Esophagus↗
PubMed1999

Proteolysis in colorectal cancer.

BACKGROUND: The process of metastasis is complex, involving many interrelated stages, including proteolysis. Proteolysis occurs in both normal and pathological processes and involves the breakdown of the extracellular matrix and/or basement membrane by proteolytic enzymes. Normally, proteolysis is tightly controlled by specific endogenous proteinase inhibitors. However, in certain disease processes, including cancer, controlled but abnormal proteolysis seems to occur. Proteinases involved in tumour invasion and metastasis include the matrix metalloproteinases (MMPs) and the serine proteinases. AIMS: To gain a greater understanding of the proteolytic process occurring in colorectal cancer and to determine which, if any, proteinases are upregulated. METHODS: The synthesis of proteinases and their inhibitors was compared in paired tumour and normal tissue samples from patients with colorectal cancer (n = 24). Substrate zymography was used to determine the synthesis of MMPs (MMP-2, MMP-9, and MMP-3) and the plasminogen activators (urokinase and tissue-type plasminogen activators); enzyme linked immunosorbent assays (ELISAs) were used to determine the concentrations of MMP-1 and tissue inhibitor of metalloproteinase 1 (TIMP-1); and the technique of quenched fluorescence substrate hydrolysis was performed to determine the total MMP activity of each sample. RESULTS: In general, both proteinase and inhibitor expression was greater in the tumour tissue when compared with the corresponding normal colorectal tissue. The amount of active MMPs was greater in the tumour tissue. CONCLUSIONS: The increased extracellular proteinase concentrations and activity may encourage tumour invasion and metastasis. This study points to MMP-9 as being of potential major importance in the development of this form of cancer.

Colorectal Neoplasms↗
PubMed1999

The management of women with breast symptoms referred to secondary care clinics in Sheffield: implications for improving local services.

Information was collected about 302 women referred for breast symptoms and seen in surgical outpatient or outreach clinics during one month at two hospitals in Sheffield. Three-quarters of the women (n = 244) were referred to specialist breast clinics, 22% (n = 70) were referred to general surgical clinics and 3% (n = 6) were referred to outreach clinics. The ages of the women ranged from 16 to 85 years with a mean and median age of 45 years. Some 200 women (66%) presented with a lump or lumpiness, 42 women (14%) presented with pain, 29 women (10%) had a skin and/or nipple problem, and the remaining 31 women (10%) were concerned about their family history or reported other symptoms. A total of 23 women (8%) were diagnosed as having cancer, 180 (60%) were diagnosed as having benign breast disease, and 99 (33%) were diagnosed as normal. Of the 23 women with cancer, 22 were over 40 years of age; 21 women presented with a lump, one presented with pain, and one presented with metastatic disease. The time required to reach a final clinical diagnosis varied from the same day as the clinic visit to 35 weeks, with a median time of 3 weeks. Surgeons assessed the appropriateness of GPs' referrals for 257 cases and judged that 122 (47%) could have been managed by a GP. The implications of the findings for the organisation of specialist outpatient clinics are discussed, and a categorisation of women as either urgent or routine cases is suggested.

Adolescent↗
PubMed1998

Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.

Alkylating Agents↗