Outcome of bone marrow transplantation in acquired and inherited aplastic anaemia in the Republic of Ireland.

BACKGROUND: Severe Aplastic Anaemia (SAA) and Fanconi Anaemia (FA) are rare haematological disorders characterised by pancytopenia and bone marrow hypoplasia. AIMS: We performed a retrospective study of all patients who underwent BMT for SAA and FA at St James's Hospital, Dublin, and at OLHSC, Crumlin, between 1985 and 2002. METHODS: The medical records of 63 patients, 50 with acquired SAA and 13 with FA, were reviewed. RESULTS: The median age at the time of transplant was 14 years (range 3-43 years). The actuarial survival (OS) (n = 63) was 76% at 17 years. The transplant related mortality (TRM) was 22% (n = 14). The most common cause of death was infection (46%). The survival was significantly better in patients receiving their transplant after 1995 (p = 0.002). Outcome was superior in those receiving less than 20 red cell transfusions prior to transplant: OS 91% (< 20 Units) versus 62% (> or = 20 Units). CONCLUSIONS: These national results are comparable to those of published international series and support the use of BMT in the treatment of SAA and FA. The known adverse effect of prior transfusion was confirmed.

The intermediate CCU admission: a preliminary study.

In the current health care era, increasing emphasis is being placed on cost reduction. Admitting only high-risk patients to coronary care units (CCU) may reduce hospital costs and charges without adverse clinical outcomes. Recently, guidelines published by the Agency for Healthcare Policy and Research (AHCPR) on suggest that intermediate-risk patients be admitted to an intermediate CCU (ICCU), but the safety and appropriateness of this approach has not been prospectively evaluated. The authors hypothesized that admitting intermediate-risk patients with to an ICCU would be cheaper than admitting to a CCU with comparable safety supporting AHCPR guidelines. To evaluate this, a retrospective cohort study was conducted. Two hundred forty-three intermediate-risk patients consecutively admitted to the CCU (n = 134) and admitted to the ICCU (n = 109) between June 1, 1992 and April 1, 1994 were compared using AHCPR definitions of intermediate risk and a previously published risk prediction model to exclude both very low- and high-risk patients. Extensive demographic, clinical, and diagnostic testing, and treatment, procedural, and outcome data were collected by a trained nurse data collector at the time of admission. Fifty-nine percent of all study patients had at least two coronary risk factors. Twenty-one percent had diabetes. Ninety-eight percent had at least one AHCPR intermediate risk factor for cardiac complications. The two groups (CCU versus ICCU) were quite similar in baseline characteristics: men (56 versus 55%), age (57 +/- 17 versus 60 +/- 17 years), diabetes (22 versus 20%), previous myocardial infarction (30 versus 36%), previous coronary artery surgery (21 versus 21%), and rest pain (78 versus 66%). The use of coronary angiography (44 versus 52%), angioplasty (24 versus 21%), and coronary artery surgery (13 versus 11%) were also similar. The incidence of myocardial infarction or death was similar (3 versus 5%), and length of stay was also similar between groups (6.7 +/- 4.2 versus 6.5 +/- 4.1 days), but cost was less for patients admitted to the ICCU ($13,481 +/- 9,450 versus $10,619 +/- 8,732, P < 0.015). These preliminary data suggest intermediate-risk patients, as identified by AHCPR guidelines, can be treated in an ICCU at lower cost than in a CCU, with reasonable safety. A small incidence of myocardial infarction in ICCU-admitted patients occurs, requiring availability of cardiac resuscitation and continued monitoring of electrocardiographic and enzymatic abnormalities. Admission to ICCU poses no barrier to recommended patient evaluation and management.

Validated risk stratification model accurately predicts low risk in patients with unstable angina.

BACKGROUND: In the mid 1990s, two unstable angina risk prediction models were proposed but neither has been validated on separate population or compared. OBJECTIVES: The purpose of this study was to compare patient outcome among high, medium and low risk unstable angina patients defined by the Agency for Health Care Policy and Research (AHCPR) guideline to similar risk groups defined by a validated model from our institution (RUSH). METHODS: Four hundred sixteen patients consecutively admitted to the hospital with unstable angina between January 1, 1995, and December 31, 1997, were prospectively evaluated for risk factors. The presence of major adverse events such as myocardial infarction (MI), death and heart failure was assessed for each patient by chart review. RESULTS: The composite end point of heart failure, MI or death occurred in 3% and 5% of the RUSH and AHCPR low risk categories, respectively, and in 8% and 10% of AHCPR and RUSH high risk categories, respectively. Recurrent ischemic events were best predicted by the RUSH model (high: 24% vs. medium: 12% and low: 10%, p = 0.029), but not by the AHCPR model (high: 14% vs. medium: 13% and low: 9%, p = 0.876). The RUSH model identified five times more low risk patients than the AHCPR model. CONCLUSIONS: Both models identify patients with low and high event rates of MI, death or heart failure. However, the RUSH model allowed for five times more patients to be candidates for outpatient evaluation (low risk) with a similar observed event rate to the AHCPR model; also, the RUSH model more successfully predicted ischemic complications. We conclude that the RUSH model can be used clinically to identify patients for early noninvasive evaluation, thereby improving cost effectiveness of care.

Functional assessment of surface loops: deletion of eukaryote-specific peptide inserts in thymidylate synthase of Saccharomyces cerevisiae.

The primary structure is known for at least 29 thymidylate synthases and the crystal structure is known for several from both prokaryotes and eukaryotes. All these are markedly similar making thymidylate synthase one of the most highly conserved enzymes known. There are, however, two surface loops, one near the active site and the other near the dimer interface, which exist in distinctly prokaryotic and eukaryotic versions. Specifically, in eukaryotes these two surface loops have small peptide inserts conserved in size and partly conserved in sequence, that are not present in the prokaryotic thymidylate synthases. To address the possibility that these inserts provide eukaryote-specific functions the Saccharomyces cerevisiae loops were individually modified to mimic their prokaryotic counterparts. Altering the surface loop near the active site increased Km for the nucleotide substrate and decreased apparent Vmax. Mutant variants with alterations in the other surface loop were unable to dimerize. Therefore these surface loops have acquired, perhaps by way of the eukaryotic inserts, characteristics that are important for catalytic activity and quaternary structure respectively.

Expression of trimeric human dUTP pyrophosphatase in Escherichia coli and purification of the enzyme.

In order to rapidly purify human dUTPase, a cDNA fragment that encodes the enzyme was subcloned and expressed using the Escherichia coli plasmid vector pGEX2T. The resulting plasmid expressed high levels of a glutathione S-transferase-dUTPase fusion protein following induction with IPTG. Affinity chromatography was used to purify the fusion protein, and dUTPase was then released from the fusion protein by thrombin treatment. The purified dUTPase has two additional vector-encoded residues at the amino terminus (gly-ser), but they have no apparent effect on the activity of the enzyme since the recombinant dUTPase has catalytic properties similar to those reported for dUTPase purified from human cells (32.3 U/mg, kcat = 25 s-1, Km = 2.6 microM). Enzyme activity was inhibited by 5-mercuri-dUTP and was shown to be sensitive to EDTA. Periodate-oxidized UTP had no effect on the activity of the enzyme, and dTTP caused only slight inhibition. The results of gel filtration experiments are consistent with a homotrimeric subunit composition for dUTPase. The ability to purify human dUTPase from E. coli should allow further characterization of the enzyme and provide material for the screening of potentially useful inhibitors.

Pyoderma gangrenosum, polycythemia rubra vera, and the development of leukemia.

A patient with long-standing, well-controlled polycythemia rubra vera developed recurrent episodes of bullous pyoderma gangrenosum followed by the transformation of his hematologic disease into a rapidly progressive acute myeloid leukemia. This case, together with previously described patients, indicates that the appearance of bullous pyoderma gangrenosum in a patient with polycythemia rubra vera is often of ominous prognostic significance.

Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center.

Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of the serine proteases tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). To systematically investigate the roles of the reactive center P1 and P1' residues in PAI-1 function, saturation mutagenesis was utilized to construct a library of PAI-1 variants. Examination of 177 unique recombinant proteins indicated that a basic residue was required at P1 for significant inhibitory activity toward uPA, whereas all substitutions except proline were tolerated at P1'. P1Lys variants exhibited lower inhibition rate constants and greater sensitivity to P1' substitutions than P1Arg variants. Alterations at either P1 or P1' generally had a larger effect on the inhibition of tPA. A number of variants that were relatively specific for either uPA or tPA were identified. P1Lys-P1'Ala reacted 40-fold more rapidly with uPA than tPA, whereas P1Lys-P1'Trp showed a 6.5-fold preference for tPA. P1-P1' variants containing additional mutations near the reactive center demonstrated only minor changes in activity, suggesting that specific amino acids in this region do not contribute significantly to PAI-1 function. These findings have important implications for the role of reactive center residues in determining serine protease inhibitor (serpin) function and target specificity.

Determination of the physical structure of biological materials at biosensor interfaces by techniques of increasing magnification from microscopic to molecular scale.

Chemical selectivity of biosensors is derived from biological materials interfaced to the surface of transducing devices. Molecular recognition events lead to macroscopic function suitable for analytical measurements. The structure-function relationships of biochemical species at interfaces must be established to characterize and optimize biosensor operation. The techniques of ellipsometry, fluorescence microscopy, electron microscopy, and scanning tunneling microscopy are used to investigate the structure of monolayers and multilayers of proteins and lipids at interfaces that are prepared by Langmuir-Blodgett techniques and by self-assembly from bulk solution. The relative merits and limitations of the measurement techniques in the determination of aspects of interfacial structure are considered.

The prevention of adsorption of interferents to radiolabelled protein by Tween 20.

Radiolabels are often used to quantitatively determine the amount of protein immobilized on chromatographic supports, immunochemical plates and biosensor surfaces. Bovine serum albumin (BSA) was chosen as a model protein for quantitative deposition studies. BSA was radioiodinated (125I-) or fluorescently labelled (fluorescein), then incubated with the following surfaces: quartz, quartz derivatized by 3-aminopropyltriethoxysilane (Qz-APTES), and Qz-APTES reacted with glutaraldehyde or tresyl chloride. The amounts of BSA immobilized to the different surfaces were compared using data from radioactivity and fluorescence assays. Irreproducible results were obtained with radioiodinated BSA due to adsorption/desorption behaviour of an unidentified radioactive species. When the non-ionic detergent Tween 20 was added to the protein/surface incubation mixture, radiolabelled BSA gave reproducible protein binding results which agreed with fluorescent protein binding patterns. The effect of Tween 20 was due to either the binding to BSA displacing the interferent and/or the solubilization of the interferent.

Fluorescence wavelength, intensity and lifetime for multidimensional transduction of selective interactions of acetylcholine receptor by lipid membranes.

Concurrent analysis of the fluorescence intensity, at different emission wavelengths, of lipid vesicles containing acetylcholine receptor (AChR) labelled with a nitrobenzoxadiazole (NBD) moiety shows that selective interactions with the agonist carbamylcholine can be detected reproducibly by a self-calibration method with muM detection limits. Concurrent analysis of the fluorescence intensity and lifetime of the new probe 4-dicyanomethylene-1,2,3,4-tetrahydromethylquinoline (DCQ) shows that general alterations of lipid membrane structure induced by temperature variation in the head-group region of lipid vesicles can be determined. A general approach to detection of selective interactions is introduced by observation of fluorescence intensity and lifetime changes of the probe NBD-phosphatidyl ethanolamine dispersed in lipid membranes containing unlabelled AChR. Detection and differentiation of selective interactions between carbamylcholine and the antagonist alpha-bungarotoxin are possible by correlation with intensity and lifetime at different emission wavelengths.

Chemical transduction with fluorescent lipid membranes using selective interactions of acetylcholine receptor with agonist/antagonist and acetylcholine with substrate.

Alterations in the physical structure of vesicles and monolayers of phospholipids and soybean lecithin were monitored by measurement on the average fluorescence intensity changes from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-a-phosphatidyl ethanolamine (NBD-PE) located in the lipid matrices. This probe was intimately dispersed at a concentration of 1-2 mol-% in lipid membranes and had an emission sensitive to local environmental structure. Alterations in the structure of soybean lecithin vesicles were induced by the selective interaction of acetylcholine receptor with the agonist carbamylcholine and the antagonist alpha-bungarotoxin. Structural changes in vesicles with a 7:3 mole ratio of dipalmitoylphosphatidyl choline to dipalmitoylphosphatidic acid were observed for selective interactions between acetylcholinesterase and acetylcholine. Enhancement of fluorescence emission from the lipid membranes provided transduction of the selective binding events of the receptor and enzyme. A maximum sensitivity of about a 30% enhancement per micromole of carbamylcholine and a detection limit for the toxin of 10 nM were observed for the receptor. Fluorescence microscopy was used to establish that protein could be incorporated in monolayer lipid membranes and to provide information about potential mechanisms of fluorescence enhancement. These studies show that lipid membranes containing NBD-PE can be used as generic transducers of protein-ligand interactions.

Selective electrochemical biosensors from state-switching of bilayer and monolayer lipid membranes by lectin-polysaccharide complexes.

Interaction of the lectin concanavalin A with the polysaccharide glycogen can provide rapid spontaneous transients of the surface potential at bilayer and monolayer lipid membranes. The selective binding process can cause large, rapid potassium ion current fluctuations across bilayer membranes in a manner that is periodic and reproducible. The frequency of these transient ion current signals was shown to be related to sub-nanomolar concentrations of the reactive agents in aqueous solution. The physical mechanism responsible for ion current modulation was investigated by fluorescence methods using lipid vesicles, by the thermal dependence of the potassium ion current across planar bilayers and by pressure-area and dipolar potential measurements of lipid monolayers at an air-water interface. The mechanism is primarily associated with physical perturbations of lipid membranes by lectin-polysaccharide aggregates, resulting in the formation of localised domains of variable electrostatic potential and conductivity.

In vivo probes: problems and perspectives.

Devices constructed for potential use as invasive bioprobes incorporate a selective receiving site for molecular or ionic recognition, and a transducer which is capable of translating a perturbation of physical chemistry of the determinant-site reaction (interaction) into a usable signal. Four types are envisioned--implants for general hospital use, transient-use probes to replace classical blood tests, short-term implantable probes and the long-term variety. Performance criteria are selectivity, sensitivity, fast response, site-reversible, small, rugged, inexpensive, biocompatible, calibratible, facile use by non-expert personnel and ease of telemetry. These demands, not surprisingly, create enormous challenges to the sensor specialist. With respect to biocompatibility the sensor must not be involved in infection, clot formation or antigenic response, and, furthermore, protein adsorption, etc., which can affect the sensor response should be avoided. Calibration remains a problem of monumental proportions. Many devices drift from calibrated levels even in in vitro experiments, let alone in the implanted milieu. One solution has been to carry out on-line switching between patient blood and standard solutions. However, this type of approach leaves a lot to be desired with respect to portability. Another method which is attracting increasing attention is the chemometric or artificial intelligence system involving compensation by multi-sensor array configurations. Sensitivity and limit-of-detection have attracted little research due to the overwhelming nature of other difficulties. In the present paper we evaluate a number of these technical problems and discuss the architecture of devices that are currently available. Finally, some thoughts as to priorities for re-directing sensor research in the bioprobe area are presented.

The multiple assignment: an effective alternative for laboratory experiences.

The purpose of the study was to test out the efficacy of both the Traditional and the Multiple Assignment approaches to the laboratory experiences of students of nursing. The subjects were 22 students enrolled in the first quarter of an Associate Degree Nursing Program and placed in either the multiple or the traditional laboratory assignment. Results showed the students in the peer experience to be superior in overall performance on tests of Nursing Knowledge, Concept Usage and Self-Esteem. It was concluded that the Multiple Assignment approach to laboratory experiences is a viable and useful method for the education of student nurses.