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Biomedical subjects

D Reilly

Publications and source records attributed to D Reilly.

At least 55 records · Page 3Linked to original sources
PubMed1996

Phase I trial of the thymidylate synthase inhibitor AG331 as a 5-day continuous infusion.

AG331 (N6-[4-(morpholinosulfonyl)benzyl]-N6-methyl-2, 6-diaminobenz-[c,d]-indole glucuronate) is a lipophilic thymidylate synthase inhibitor with activity in solid tumor models. On the basis of preclinical data supporting regimens of frequent drug administration, we performed a Phase I trial of AG331 as a 5-day continuous infusion repeated every 3 weeks. Twenty-nine patients were entered at doses ranging from 25 to 1000 mg/m2/day. The major side effects were mild to moderate fatigue, nausea, vomiting, diarrhea, and fever. At doses >/=400 mg/m2, acute reversible elevation of bilirubin, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyltranspeptidase was observed. All patients who received >/=600 mg/m2/day experienced elevated alanine aminotransferase. Elevated liver function tests were evident by day 3 of the infusion and had resolved by day 8 in the majority. This toxicity was dose limiting at 1000 mg/m2/day, at which dose two of two patients developed grade 4 reversible hyperbilirubinemia in addition to the enzyme elevations. Serum and urine samples were analyzed by a novel high-pressure liquid chromatography method for the determination of the pharmacokinetics of AG331. Over the 50-1000 mg/m2/day dose range, mean total clearance ranged from 11.6 to 30.0 liters/h/m2, and volume of distribution at steady state ranged from 279.5 to 758.7 liters/m2. These parameters were dose independent over the dose range tested. The harmonic mean terminal half-life of AG331 was 20.2 h. Less than 5% of an AG331 dose is eliminated unchanged in the urine. Both the administered dose and exposure to the drug were related to the changes in bilirubin and aminotransferase blood levels. Evidence for inhibition of thymidylate synthase was obtained at doses ranging from 100 to 1000 mg/m2 in seven patients; plasma deoxyuridine concentrations at end-infusion were 1.8-3.8-fold higher than pretreatment values. Because of the nature of toxicity on this schedule, more extensive Phase II evaluation is not recommended, although an AG331 dose of 800 mg/m2/day for 5 days is tolerable. Exploration of less frequent dose administration is under way.

Adult↗
PubMed1994

Is evidence for homoeopathy reproducible?

We tested, under independent conditions, the reproducibility of evidence from two previous trials that homoeopathy differs from placebo. The test model was again homoeopathic immunotherapy. 28 patients with allergic asthma, most of them sensitive to house-dust mite, were randomly allocated to receive either oral homoeopathic immunotherapy to their principal allergen or identical placebo. The test treatments were given as a complement to their unaltered conventional care. A daily visual analogue scale of overall symptom intensity was the outcome measure. A difference in visual analogue score in favour of homoeopathic immunotherapy appeared within one week of starting treatment and persisted for up to 8 weeks (p = 0.003). There were similar trends in respiratory function and bronchial reactivity tests. A meta-analysis of all three trials strengthened the evidence that homoeopathy does more than placebo (p = 0.0004). Is the reproducibility of evidence in favour of homoeopathy proof of its activity or proof of the clinical trial's capacity to produce false-positive results?

Adult↗
PubMed1994

Urokinase plasminogen activator as a prognostic marker in different subgroups of patients with breast cancer.

BACKGROUND: Urokinase plasminogen activator (uPA) is a serine protease involved in cancer invasion and metastasis. Previously, uPA was shown to be an independent prognostic marker in breast cancer. The aim of this study were to evaluate uPA as a prognostic marker in different subgroups of patients with breast cancer. METHODS: Urokinase plasminogen activator was assayed by enzyme-linked immunosorbent assay in detergent extracts of human breast tumors. RESULTS: Using both disease free interval (DFI) and overall survival (OS) as end points, uPA was an indicator of prognosis in the following groups of patients: those with positive axillary nodes, those who were estrogen receptor (ER)-positive, women younger than 50 years of age, and women older than 50 years of age. For patients with negative axillary lymph nodes, uPA was a significant prognostic marker using DFI as an end point and was almost statistically significant (P = 0.055) using OS as end point. In patients who were ER-negative, uPA levels showed no significant relationship with patient outcome. CONCLUSIONS: Urokinase plasminogen activator is a significant prognostic marker in most of the major subgroups of patients with breast cancer and may be a marker for patients with negative axillary lymph nodes.

Animals↗
PubMed1994

The solution structure of melanoma growth stimulating activity.

The solution structure of melanoma growth stimulating activity (MGSA), a dimeric chemokine consisting of 73 residues per monomer, has been determined using two-dimensional homonuclear and three-dimensional heteronuclear NMR spectroscopy. Structure calculations were carried out using a hybrid distance geometry-simulated annealing approach with the programs DGII and X-PLOR. The structure is based on a total of 2362 experimental restraints, comprising 2150 NOE-derived distance restraints (2076 unambiguous intrasubunit restraints, 60 unambiguous intersubunit restraints, and 14 ambiguous restraints with potential contributions from both intra- and intersubunit NOEs), 84 distance restraints for 42 backbone hydrogen bonds, and 128 torsion angle restraints. The ambiguous distance restraints were treated using a target function which accounts for both intra- and intermolecular contributions to the NOE intensity. A total of 25 structures were calculated, with the backbone (N, C alpha, C) atomic r.m.s. distribution about the mean coordinates for residues 8 to 69 being 0.44(+/- 0.10) A for the dimer and 0.34(+/- 0.07) A for the individual monomers. The N- and C-terminal residues (1 to 7 and 70 to 73, respectively) are disordered. The overall structure of the MGSA dimer is similar to that reported previously for the NMR and X-ray structures of interleukin-8 (IL-8), and consists of a six-stranded antiparallel beta-sheet packed against two C-terminal antiparallel alpha-helices. A best fit superposition of the NMR structure of MGSA on the X-ray and NMR structures of IL-8 yields backbone atomic r.m.s. differences of 0.99 and 1.28 A, respectively for individual monomers, and 1.08 and 1.82 A, respectively for the dimers (using MGSA residues 8 to 14 and 19 to 69). In general, the MGSA structure resembles the IL-8 X-ray structure more than it does the IL-8 NMR structure. At the tertiary (monomer) level the two main differences between the MGSA solution structure and IL-8 NMR structure involve the loops between residues 14 to 19 and between residues 30 to 38. At the quaternary (dimer) level the difference results from differing angles between the beta-strands which form the dimer interface, and is manifest as a different interhelical separation (distance of closest approach between the two helices is 15.3 A in the IL-8 NMR structure and 11.7 (+/- 0.4) A in the MGSA structure).

Amino Acids↗
PubMed1994

Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa.

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.

Anticoagulants↗
PubMed1994

A novel isotope labeling protocol for bacterially expressed proteins.

A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).

Bacteria↗
PubMed1993

Processing and characterization of human proguanylin expressed in Escherichia coli.

Guanylin is a 15-amino acid peptide hormone that was originally isolated from the jejunum of the rat small intestine and shown to be an endogenous activator of the intestinal heat-stable enterotoxin receptor-guanylyl cyclase. Guanylin is synthesized as a 115-amino acid prohormone, proguanylin, which is processed at a site yet to be determined, into a C-terminal bioactive fragment(s). In order to examine the processing of proguanylin in vitro, we have generated large quantities of the properly folded prohormone by constructing an expression vector that directs its secretion into the periplasmic space of Escherichia coli. The bacterially expressed human proguanylin was then processed to smaller C-terminal fragments by protease digestion. Digestion with trypsin or lysine-C generated C-terminal peptides of different length, which have been purified and characterized. Guanylin-22 and guanylin-32 have binding affinities and biological activities similar to guanylin-15, while guanylin-63 and the entire proguanylin have only minimal bioactivity. Circular dichroism spectroscopy reveals that proguanylin is a stably folded protein containing mostly beta-sheet and beta-turn structure.

Amino Acid Sequence↗
PubMed1993

1H assignment and secondary structure determination of human melanoma growth stimulating activity (MGSA) by NMR spectroscopy.

The solution structure of melanoma growth stimulating activity (MGSA) has been investigated using proton NMR spectroscopy. Sequential resonance assignments have been carried out, and elements of secondary structure have been identified on the basis of NOE, coupling constant, chemical shift, and amide proton exchange data. Long-range NOEs have established that MGSA is a dimer in solution. The secondary structure and dimer interface of MGSA appear to be similar to those found previously for the homologous chemokine interleukin-8 [Clore et al. (1990) Biochemistry 29, 1689-1696]. The MGSA monomer contains a three stranded anti-parallel beta-sheet arranged in a 'Greek-key' conformation, and a C-terminal alpha-helix (residues 58-69).

Amino Acid Sequence↗
PubMed1993

Purification, receptor binding analysis, and biological characterization of human melanoma growth stimulating activity (MGSA). Evidence for a novel MGSA receptor.

Human melanoma growth stimulating activity (MGSA) is a mitogenic factor first identified in the conditioned media of human melanoma cells. Structurally, MGSA belongs to a superfamily of proteins that includes interleukin-8 (IL-8) and platelet factor 4. These proteins are involved in inflammatory processes, and an understanding of their mechanism of action should provide insight into their pathophysiology. In this study, we report the high level expression of recombinant human MGSA in Escherichia coli. The structure was confirmed by mass spectrometry and NH2-terminal amino acid sequencing. Receptor binding studies were carried out in a human melanoma cell line, Hs294T, and in U937 cells. Direct binding experiments with 125I-MGSA in Hs294T cells have allowed us to identify a novel MGSA receptor in these cells, with a KD of 3.9-4.25 nM and approximately 52,960-67,758 binding sites/cell. These MGSA-binding sites were specific and could not be displaced by unlabeled IL-8. The MGSA receptor in these cells is biologically active, and the addition of ligand induces cellular proliferation in a dose-dependent manner. In U937 cells, unlabeled IL-8 and MGSA were able to completely displace radiolabeled IL-8. Scatchard analysis of the displacement binding data was consistent with binding to a single class of binding sites, and the calculated KD values were 2.4 +/- 0.6 nM for IL-8 and 3.2 +/- 0.80 nM for MGSA. Treatment of U937 cells with IL-8 or MGSA produced a rapid increase in Ca2+ flux; however, subsequent incubation with either ligand failed to produce any further Ca2+ flux. The IL-8 receptor in U937 cells was covalently labeled with 125I-IL-8 to reveal a protein with a molecular mass of 69 kDa.

Amino Acid Sequence↗
PubMed1993

Adventures in hemostasis. Desmopressin in cardiac surgery.

Desmopressin acetate (1-deamino-8-D-arginine vasopressin [DDAVP]) improves hemostasis in hemophilia A and von Willebrand's disease and in some platelet disorders. In complex cardiac operations, excluding simple coronary artery bypass graft procedures, we found that desmopressin reduced blood loss by 40% and the need for transfusion by 34%. Conflicting reports followed. Future trials should emphasize patients with excessive bleeding. A possible post-desmopressin prothrombotic state was studied after hip replacement surgery. The incidence of deep vein thrombosis associated with warfarin sodium therapy was the same as that associated with desmopressin plus warfarin therapy. No desmopressin-induced thrombotic tendency was detected. A trend toward reduced blood loss with desmopressin was not significant. During cardiac catheterization, the plasma von Willebrand factor level was correlated with hemodynamic variables, including pulmonary vascular resistance, pulmonary arterial pressure, and (inversely) with cardiac index. von Willebrand factor concentration was highest in mitral stenosis. The relationship of these factors to the response to desmopressin remains to be defined.

Aged↗
PubMed1993

Two- to four-year experience with cemented, press-fit, and porous coated applications of the profile total hip system.

The clinical outcome and radiographic results of 90 cemented profile stems, 59 press-fit profile stems and 20 porous-coated profile stems implanted between October, 1987 and December, 1989 were analyzed. All total hip replacements were performed by the same group of surgeons. All used a cementless porous ingrowth acetabular component, and all femoral components were of the same geometrical design. Clinical results were evaluated using the Hip Society/SICOT/AAOS consensus format and were recorded prospectively preoperatively and at 6 months, 1 year, 2 years, 3 years and 4 years postoperatively. Radiographs were evaluated for component migration, subsidence, and cortical and cancellous biologic response as well as zonal analysis of radiolucent lines, using the Muller THR template. The functional results were similar for all three groups with no significant differences at 2 years postoperatively with respect to pain with activity, limp, or support used. However, patient satisfaction tended to be higher for cemented stems than for press-fit or porous-coated stems. Radiographic analysis showed press-fit stems to have a higher incidence (29.5%) of femoral component subsidence (mean 5.7 mm). None of the porous-coated stems subsided. Press-fit and porous-coated stems also demonstrated a higher rate of cortical hypertrophy at mid- and distal stem levels in comparison to cemented components. Press-fit application of the femoral component gives satisfactory clinical results, and the addition of proximal porous coating may confer additional advantages to the technique. Cemented stems enjoy the highest level of patient satisfaction and the most predictable objective clinical results.

Adult↗
PubMed1992

Type-1 plasminogen activator inhibitor in human breast carcinomas.

We have investigated the occurrence of type-1 inhibitor of plasminogen activators (PAI-1) in human breast tumors. PAI-1 levels, measured by enzyme-linked immunosorbent assay, were significantly higher in malignant breast carcinomas (n = 178) than in benign breast tumors (n = 25). The levels of PAI-1 were found to be correlated with those of urokinase-type plasminogen activator (u-PA). The presence of PAI-1 in tumor extracts was also demonstrated by immunoblotting analysis. Immunohistochemical investigations by the use of monoclonal and polyclonal antibodies showed that PAI-1 was mostly localized in the tumor islands, associated with the tumor cells; in addition, it was present in vessel walls and in normal duct epithelia, but absent from the stroma. Analysis of RNA extracted from tumors by polymerase chain reaction revealed the presence of PAI-1 mRNA. We conclude that PAI-1 is present in human breast carcinoma cells, and that it is--at least partially-- produced locally, either by the cancer cells or by other cells in the tumors. We have previously demonstrated that a high level of u-PA in human breast carcinomas is associated with poor prognosis. These results, combined with our present findings, present 2 possibilities: either the cancer cells need PAI-1 in order to utilize the u-PA-mediated pathway of plasminogen activation for invasion and metastasis; or PAI-1 represents a defense mechanism against tumor invasion.

Animals↗
PubMed1992

Establishing a drug and alcohol service in an Australian rural area.

The response to drug and alcohol problems in a small Australian rural community is described. The town of Nimbin, the centre of this community, is characterized by alternative life-styles. The area has received considerable publicity over illicit drug use, particularly marijuana-growing and a recent increase in injecting drug usage. The town has one medical practice with two general practitioners and this centre has been actively involved in the development of drug and alcohol services. This paper describes the development of a drug and alcohol service from the perspective of a local general practitioner. Reference is made to problems of community resistance, hospital issues and the steps taken to acquire knowledge and skills in the management of patients presenting to the practice. The importance of a multidisciplinary team approach in the development of services which included a needle and syringe exchange and a methadone treatment programme is emphasized.

Journal Article↗